AABB Technical Manual 15TH PDF

Technical Manual
15th Edition
Copyright © 2005 by the AABB. All rights reserved.

Other related publications available from the AABB:
Technical Manual and Standards for Blood Banks and

Transfusion Services on CD-ROM
Transfusion Therapy: Clinical Principles and Practice, 2nd Edition

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Transfusion Medicine Self-Assessment and Review

By Pam S. Helekar, MD; Douglas P. Blackall, MD; Jeffrey L. Winters, MD;
and Darrell J. Triulzi, MD
Blood Transfusion Therapy: A Physician’s Handbook, 8th Edition

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Practical Guide to Transfusion Medicine

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By Marian Petrides, MD; Roby Rogers, MD; and Nora Ratcliffe, MD
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AABB ISBN No. 1-56395-196-7

8101 Glenbrook Road Printed in the United States
Bethesda, Maryland 20814-2749
Cataloging-in-Publication Data
Technical manual / editor, Mark E. Brecher. —15th ed.

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

Technical Manual
Program Unit
Chair and Editor
Mark E. Brecher, MD
Associate Editors
Regina M. Leger, MSQA, MT(ASCP)SBB, CQMgr(ASQ)

Jeanne V. Linden, MD, MPH
Susan D. Roseff, MD
Members/Authors
Martha Rae Combs, MT(ASCP)SBB

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

Michael C. Libby, MSc, MT(ASCP)SBB

Acknowledgments
The Technical Manual Program Unit extends special thanks to those volunteers who
provided peer review and made other contributions:
James P. AuBuchon, MD W. John Judd, FIBMS, Arell S. Shapiro, MD

Lucia M. Berte, MA, MIBiol R. Sue Shirey, MS,
MT(ASCP)SBB, DLM, Michael H. Kanter, MD MT(ASCP)SBB
CQA(ASQ)CQMgr Louis M. Katz, MD Bruce Spiess, MD, FAHA
Arthur Bracey, MD Debra Kessler, RN, MS Jerry E. Squires, MD, PhD
Linda Braddy, Thomas Kickler, MD Marilyn J. Telen, MD
MT(ASCP)SBB Karen E. King, MD Susan Veneman,
Donald R. Branch, Joanne Kosanke, MT(ASCP)SBB
MT(ASCP)SBB, PhD MT(ASCP)SBB Phyllis S. Walker, MS,
Ritchard Cable, MD Thomas A. Lane, MD MT(ASCP)SBB
Sally Caglioti, Alan H. Lazarus, PhD Dan A. Waxman, MD
MT(ASCP)SBB German F. Leparc, MD Robert Weinstein, MD
Loni Calhoun, Douglas M. Lublin, MD, Connie M. Westhoff, PhD,
MT(ASCP)SBB PhD MT(ASCP)SBB
Tony S. Casina, Dawn Michelle, Members of AABB com-
MT(ASCP)SBB MT(ASCP)SBB mittees who reviewed
Geoff Daniels, PhD, Kenneth Moise, Jr., MD manuscripts as part of
MRcPath S. Breanndan Moore, MD committee resource
Robertson Davenport, MD Tania Motschman, MS, charges
Richard J. Davey, MD MT(ASCP)SBB, The staff of the Armed
Walter Dzik, MD CQA(ASQ) Services Blood Program
Ted Eastlund, MD Marilyn K. Moulds, Office
Anne F. Eder, MD, PhD MT(ASCP)SBB The staff of the US Food
Ronald O. Gilcher, MD, Nancy C. Mullis, and Drug Administra-
FACP MT(ASCP)SBB tion, Center for
Lawrence T. Goodnough, Scott Murphy, MD Biologics Evaluation
MD Patricia Pisciotto, MD and Research
Linda Hahn, Mark A. Popovsky, MD The staff of the Transplan-
MT(ASCP)SBB, MPM Marion E. Reid, PhD, tation and Transfusion
Heather Hume, MD FIBMS Service, McClendon
Mark A. Janzen, PhD Jennifer F. Rhamy, MBA, Clinical Laboratories,
Susan T. Johnson, MSTM, MA, MT(ASCP), SBB, HP UNC Hospitals
MT(ASCP)SBB Scott D. Rowley, MD
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
T
he 15th edition of the AABB Tech- ence, a source for developing policies and
nical Manual is the first in the procedures, and an educational tool. The
second half century of this publica- Technical Manual is often the first reference
tion. The original Technical Manual (then consulted in many laboratories; thus, it is
called Technical Methods and Procedures) intended to provide the background infor-
was published in 1953 and the 14th edi- mation to allow both students and experi-
tion marked the 50th anniversary of this enced individuals to rapidly familiarize
publication. themselves with the rationale and scientific
Over the years, this text has grown and basis of the AABB standards and current
matured, until today it is a major textbook standards of practice. As in previous edi-
used by students (medical technology and tions, the authors and editors have tried to
residents) and practicing health-care pro- provide both breadth and depth, including
fessionals (technologists, nurses, and phy- substantial theoretical and clinical material
sicians) around the world. Selected editions as well as technical details. Due to space
or excerpts have been translated into limitations, the Technical Manual cannot
French, Hungarian, Italian, Japanese, Span- provide all of the advanced information on
ish, Polish, and Russian. It is one of only any specific topic. However, it is hoped that
two AABB publications that are referenced sufficient information is provided to answer
by name in the AABB Standards for Blood the majority of queries for which individu-
Banks and Transfusion Services (the other als consult the text, or at a minimum, to di-
being the Circular of Information for the rect someone toward additional pertinent
Use of Human Blood Components). All references.
branches of the US Armed Services have Readers should be aware that, unlike
adopted the AABB Technical Manual as most textbooks in the field, this book is
their respective official manuals for blood subjected to extensive peer review (by ex-
banking and transfusion medicine activi- perts in specific subject areas, AABB com-
ties. mittees, and regulatory bodies such as the
The Technical Manual serves a diverse Food and Drug Administration). As such,
readership and is used as a technical refer- this text is relatively unique, and represents
ix

x AABB Technical Manual
a major effort on the part of the AABB to I would like to thank the members of the
provide an authoritative and balanced Technical Manual Program Unit for their
reference source. dedication and long hours of work that
As in previous recent editions, the con- went into updating this edition. I would
tent is necessarily limited in order to retain also like to thank all the AABB committees,
the size of the Technical Manual to that of a the expert reviewers, and the readers who
textbook that can be easily handled. Never- have offered numerous helpful suggestions
theless, readers will find extensive new and that helped to make this edition possible. I
updated information, including expanded would particularly like to thank my three
coverage of quality approaches, apheresis associate editors—Gina Leger, Jeanne Lin-
indications, cellular nomenclature, molec- den, and Sue Roseff—who have provided
ular diagnostics, hematopoietic progenitor countless invaluable hours in the prepara-
cell processing, and transfusion-transmitted tion of this edition. Finally I would like to
diseases. thank Laurie Munk, AABB Publications Di-
Techniques and policies outlined in the rector, whose tireless efforts on behalf of
Technical Manual are, to the best of the the Technical Manual never cease to amaze
Technical Manual Program Unit's ability, in me, and who has made the publication of
conformance with AABB Standards. They this book a pleasure.
are not to be considered the only permissi- This edition is my third and final Techni-
ble way in which requirements of Stan- cal Manual. I served as associate editor for
dards can be met. Other methods, not in- the 13th edition and chief editor for the
cluded, may give equally acceptable results. 14th and 15th editions. It has been an
If discrepancy occurs between techniques honor to help shepherd these editions to
or suggestions in the Technical Manual and fruition and it is my hope that the AABB
the requirements of Standards, authority Technical Manual will continue to be one
resides in Standards. Despite the best ef- of the AABB's premier publications for de-
forts of both the Program Unit and the ex- cades to come.
tensive number of outside reviewers, errors
may remain in the text. As with previous Mark E. Brecher, MD
editions, the Program Unit welcomes sug- Chief Editor
gestions, criticisms, or questions about the Chapel Hill, NC
current edition.

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
Determine Adequate Sample Size and Level of Confidence for
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
xi

xii AABB Technical Manual
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
8. Collection, Preparation, Storage, and Distribution of Components from

Whole Blood Donations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Blood Component Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Prestorage Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

Contents xiii
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
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
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

xiv AABB Technical Manual
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
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
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
16. Platelet and Granulocyte Antigens and Antibodies . . . . . . . . . . . . . . . . 361

Platelet Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Granulocyte Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Contents xv
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
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
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

xvi AABB Technical Manual
Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT

(Excluding Investigation of HDFN) . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Appendix 20-2. Some Drugs Associated with Immune Hemolysis and/or
Positive DATs Due to Drug-Induced Antibodies . . . . . . . . . . . . . . . . . . . 481
Clinical Considerations in Transfusion Practice

21. Blood Transfusion Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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

Contents xvii
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

xviii AABB Technical Manual
Other Nonviral Infectious Complications of Blood Transfusion . . . . . . . . . . . 697

Reducing the Risk of Infectious Disease Transmission . . . . . . . . . . . . . . . . . 699
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
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, and Leb . . . . . . . . . . . . . . . . . . . 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

Contents xix
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

xx AABB Technical Manual
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

Contents xxi
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

Chapter 1: Quality Systems
Chapter 1
1
Quality Systems
A
PRIMARY GOAL OF blood centers ity for the overall quality of the finished
and transfusion services is to pro- product and authority to control the pro-
mote high standards of quality in cesses that may affect this product.4 (See
all aspects of production, patient care, and Code of Federal Regulations quality-related
service. This commitment to quality is re- citations in Appendix 1-2.) Professional and
flected in standards of practice set forth by accrediting organizations, such as the
the AABB.1(p1) A quality system includes the AABB,1 Joint Commission on Accreditation
organizational structure, responsibilities, of Healthcare Organizations (JCAHO),6 Col-
policies, processes, procedures, and re- lege of American Pathologists (CAP),7 and
sources established by the executive man- the Clinical and Laboratory Standards Insti-
agement to achieve quality.1(p1) A glossary of tute (formerly NCCLS),8 have also estab-
quality terms used in this chapter is in- lished requirements and guidelines to ad-
cluded in Appendix 1-1. dress quality issues. The International
The establishment of a formal quality as- Organization for Standardization (ISO)
surance program is required by regulation quality management standards (ISO 9001)
under the Centers for Medicare and Medi- are generic to any industry and describe
2 9
caid Services (CMS) Clinical Laboratory the key elements of a quality system. In
Improvement Amendments (CLIA) and the addition, the Health Care Criteria for Per-
Food and Drug Administration (FDA)3-5 cur- formance Excellence10 published by the
rent good manufacturing practice (cGMP). Baldrige National Quality Program provide
The FDA regulations in 21 CFR 211.22 re- an excellent framework for implementing
quire an independent quality control or quality on an organizational level. The
quality assurance unit that has responsibil- AABB defines the minimum elements that

2 AABB Technical Manual
must be addressed in a blood bank or clude record reviews, monitoring of quality

transfusion service quality system in its indicators, and internal assessments.
Quality System Essentials (QSEs).11 The Quality management considers interre-
AABB QSEs were developed to be compati- lated processes in the context of the organi-
ble with ISO 9001 standards and the FDA zation and its relations with customers and
Guideline for Quality Assurance in Blood suppliers. It addresses the leadership role of
Establishments.5 Table 1-1 shows a compar- executive management in creating a com-
ison of the AABB QSEs and ISO 9001:2000 mitment to quality throughout the organi-
requirements. 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-
Quality Control, Quality passes all of these activities. It ensures the
Assurance, and Quality application of quality principles through-
out the organization and reflects the chang-
Management ing focus of quality efforts from detection
The purpose of quality control (QC) is to to prevention.
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 Quality Concepts
stop until a problem has been resolved
(something is found to be out of control). Juran’s Quality Trilogy
Product QC is performed to determine Juran’s Quality Trilogy is one example of a
whether the product or service meets quality management approach. This model
specifications. Historically, blood banks centers around three fundamental pro-
and transfusion services have employed cesses for the management of quality in
many QC measures as standard practice any organization: planning, control, and
in their operations. Examples include re- improvement.12(p2.5)
agent QC, clerical checks, visual inspec- The planning process for a new product
tions, and measurements such as temper- or service includes activities to identify re-
ature readings on refrigerators and volume quirements, to develop product and pro-
or cell counts performed on finished cess specifications to meet those require-
blood components. ments, and to design the process. During
Quality assurance activities are not tied the planning phase, the facility must per-
to the actual performance of a process. form the following steps:
They include retrospective review and anal- 1. Establish quality goals for the pro-
ysis of operational performance data to de- ject.
termine if the overall process is in a state of 2. Identify the customers.
control and to detect shifts or trends that 3. Determine customer needs and ex-
require attention. Quality assurance pro- pectations.
vides information to process managers re- 4. Develop product and service specifi-
garding levels of performance that can be cations to meet customer, opera-
used in setting priorities for process im- tional, regulatory, and accreditation
provement. Examples in blood banking in- requirements.

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 8.3 Control of nonconforming product

Complications
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 ciencies in the initial planning process; un-
production and delivery, including foreseen factors that are discovered upon
written procedures and resources re- implementation; shifts in customer needs;
quirements. or changes in starting materials, environ-
6. Develop process controls and vali- mental factors, and other variables that af-
date the process in the operational fect the process. Improvements must be
setting. based on data-driven analysis; an ongoing
The results of the planning process are program of measurement and assessment
9
referred to as design output. is fundamental to this process.
Once implemented, the control process
provides a feedback loop for operations
that includes the following:
1. Evaluation of actual performance. Process Approach
2. Comparison of performance to goals.
3. Action to correct any discrepancy In its most generic form, a process in-
between the two. cludes all of the resources and activities
It addresses control of inputs, produc- that transform an input into an output.
tion, and delivery of products and services An understanding of how to manage and
to meet specifications. Process controls control processes in the blood bank or
should put operational staff in a state of transfusion service is based on the simple
self-control such that they can recognize equation:
when things are going wrong, and either
make appropriate adjustments to ensure INPUT à PROCESS à OUTPUT
the quality of the product or stop the pro-
cess. An important goal in quality manage- For example, a key process for donor cen-
ment is to establish a set of controls that ters is donor selection. The “input” in-
ensure process and product quality but that cludes 1) the individual who presents for
are not excessive. Controls that do not add donation and 2) all of the resources re-
value should be eliminated in order to con- quired for the donor health screening.
serve limited resources and to allow staff to Through a series of activities including
focus attention on those controls that are verification of eligibility (based on results
critical to the operation. Statistical tools, of prior donations, mini-physical, and
such as process capability measurement health history questionnaire), an individ-
and control charts, allow the facility to eval- ual is deemed an “eligible donor.” The
uate process performance during the plan- “output” is either an eligible donor who
ning stage and in operations. These tools can continue to the next process (blood
help determine whether a process is stable collection) or an ineligible donor who is
(ie, in statistical control) and whether it is deferred. When the selection process re-
capable of meeting product and service sults in a deferred donor, the resources
specifications.12(p22.19) (inputs) associated with that process are
Quality improvement is intended to at- wasted and contribute to the cost of qual-
tain higher levels of performance, either by ity. One way that donor centers attempt to
creating new or better features that add minimize this cost is to educate potential
value, or by removing existing deficiencies donors before the health screening so that
in the process, product, or service. Oppor- those who are not eligible do not enter the
tunities to improve may be related to defi- selection process.

Strategies for managing a process should product or service. Delivery generally in-
consider all of its components, including its volves interaction with the customer. The
interrelated activities, inputs, outputs, and quality of this transaction is critical to
resources. Supplier qualification, formal customer satisfaction and should not be
agreements, supply verification, and inven- overlooked in the design and ongoing
tory control are strategies for ensuring that assessment of the quality system.
the inputs to a process meet specifications.
Personnel training and competency assess-
ment, equipment maintenance and con- Service vs Production
trol, management of documents and re-
cords, and implementation of appropriate Quality principles apply equally to a
in-process controls provide assurance that broad spectrum of activities, from those
the process will operate as intended. End- involved in processing and production, to
product testing and inspection, customer those involving the interactions between
feedback, and outcome measurement pro- individuals in the delivery of a service.
vide information to help evaluate the qual- However, different strategies may be ap-
ity of the product and to improve the process propriate when there are differing expec-
as a whole. These output measurements and tations related to customer satisfaction.
quality indicators are used to evaluate the Although the emphasis in a production
effectiveness of the process and process process is to minimize variation in order
controls. to create a product that consistently meets
In order to manage a system of pro- specifications, service processes require a
cesses effectively, the facility must under- certain degree of flexibility to address cus-
stand how its processes interact and any tomer needs and circumstances at the
cause-and-effect relationships between time of the transaction. In production,
them. In the donor selection example, the personnel need to know how to maintain
consequences of accepting a donor who is uniformity in the day-to-day operation. In
not eligible reach into almost every other service, personnel need to be able to
process in the facility. One example would adapt the service in a way that meets cus-
be a donor with a history of high-risk be- tomer expectations but does not compro-
havior that is not identified during the se- mise quality. To do this, personnel must
lection process. The donated product may have sufficient knowledge and under-
test positive for one of the viral marker as- standing of interrelated processes to use
says, triggering follow-up testing, look-back independent judgment appropriately, or
investigations, and donor deferral and noti- they must have ready access to higher
fication procedures. Components must be level decision-makers. When designing
quarantined and their discard documented. quality systems for production processes,
Staff involved in collecting and processing it is useful to think of the process as the
the product are at risk of exposure to infec- driver, with people providing the over-
tious agents. Part of quality planning is to sight and support needed to keep it run-
identify those relationships so that quick ning smoothly and effectively. In service,
and appropriate corrective action can be people are the focus; the underlying pro-
taken if process controls fail. It is important cess provides a foundation that enables
to remember that operational processes in- staff to deliver safe and effective services
clude not only product manufacture or ser- that meet the needs of the customers in
vice creation, but also the delivery of a almost any situation.

Quality Management as an Evolving of the relationships and avenues of com-

Science munication between organizational units
and those responsible for key quality
It is important to remember that quality
functions. Each facility may define its
management is an evolving science. The
structure in any format that suits its oper-
principles and tools in use today will
ations. Organizational trees or charts that
change as research provides new knowl-
show the structure and relationships are
edge of organizational behavior, as tech-
helpful.
nology provides new solutions, and as the
The facility must define in writing the
field of transfusion medicine presents
authority and responsibilities of manage-
new challenges. Periodic assessments of the
ment to establish and maintain the quality
quality management systems will help
system. These include oversight of opera-
identify practices that are no longer effec-
tions and regulatory and accreditation
tive or that could be improved through
compliance as well as periodic review and
the use of new technology or new tools.
assessment of quality system effectiveness.
Executive management support for quality
system goals, objectives, and policies is
Practical Application of critical to the success of the program. Man-
Quality Principles agement must participate in the review and
approval of quality and technical policies,
The remainder of this chapter discusses
processes, and procedures.
the elements of a quality system and prac-
The individual designated to oversee the
tical application of quality principles to the
facility’s quality functions must report di-
blood bank and transfusion service envi-
rectly to management. This person has the
ronment. These basic elements include:
responsibility to coordinate, monitor, and
■ Organizational management
facilitate quality system activities and has
■ Human resources the authority to recommend and initiate
■ Customer and supplier relations 5
corrective action when appropriate. The
■ Equipment management designated individual need not perform all
■ Process management of the quality functions personally. Ideally,
■ Documents and records this person should be independent of the
■ Deviations and nonconforming prod- operational functions of the donor center
ucts and services or transfusion service. In small facilities,
■ Monitoring and assessment however, this may not always be possible.
■ Process improvement Depending on the size and scope of the or-
■ Work environment ganization, the designated oversight person
may work in a department (eg, transfusion
Organizational Management service), may have responsibilities covering
The facility should be organized in a man- several areas (eg, laboratory-wide), may
ner that promotes effective implementa- have a staff of workers (eg, quality unit), or
tion and management of its quality system. may be part of an organization-wide unit
The structure of the organization must be (eg, hospital quality management). Individ-
documented and the responsibilities for uals with dual quality and operational re-
the provision of blood, components, pro- sponsibilities should not provide quality
ducts, and services must be clearly de- oversight for operational work they have
fined. These should include a description performed (21 CFR 211.194).

Quality oversight functions may include sible. Policies, processes, and procedures
the following5: must exist to define the roles and responsi-
■ Review and approval of standard op- bilities of all individuals in the development
erating procedures (SOPs) and train- and maintenance of these quality goals.
ing plans. Quality system policies and processes
■ Review and approval of validation should be applicable across the entire facil-
plans and results. ity. A blood bank or transfusion service
■ Review and approval of document need not develop its own quality policies if
control and record-keeping systems. it is part of a larger entity whose quality
■ Audit of operational functions. management system addresses all of the
■ Development of criteria for evaluat- minimum requirements. The quality sys-
ing systems. tem must address all matters related to
■ Review and approval of suppliers. compliance with federal, state, and local
■ Review and approval of product regulations and accreditation standards ap-
specifications, ie, requirements to be plicable to the organization.
met by the products used in the man-
ufacturing, distribution, or transfusion Human Resources
of blood and components.
This element of the quality system is aim-
■ Review of reports of adverse reactions,
ed at management of personnel, includ-
deviations in the manufacturing pro-
ing selection, orientation, training, com-
cess, nonconforming products and ser-
petency assessment, and staffing.
vices, and customer complaints.
■ Participation in decisions to deter-
mine blood and component suitabil- Selection
ity for use, distribution, or recall. Each blood bank, transfusion service, or
■ Review and approval of corrective donor center must have a process to pro-
action plans. vide adequate numbers of qualified per-
■ Surveillance of problems (eg, error sonnel to perform, verify, and manage all
reports, inspection deficiencies, cus- activities within the facility.1(p3),3 Qualifica-
tomer complaints) and the effective- tion requirements are determined based
ness of corrective actions implemented on job responsibilities. The selection pro-
to solve these problems. cess should consider the applicant’s qual-
■ Use of information resources to ifications for a particular position as
identify trends and potential prob- determined by education, training, exper-
lems before a situation worsens and ience, certifications, and/or licensure. For
products or patients are affected. laboratory testing staff, the standards for
■ Preparation of periodic (as specified personnel qualifications must be compat-
by the organization) reports of qual- ible with the regulatory requirements es-
2
ity issues, trends, findings, and cor- tablished under CLIA. Job descriptions are
rective and preventive actions. required for all personnel involved in pro-
Quality oversight functions may be cesses and procedures that affect the
shared among existing staff, departments, quality of blood, components, tissues,
and facilities, or, in some instances, may be and services. Effective job descriptions
contracted to an outside firm. The goal is to clearly define the qualifications, responsi-
provide as much of an independent evalua- bilities, and reporting relationships of the
tion of the facility’s quality activities as pos- position.

Orientation, Training, and Competency To ensure that skills are maintained, the
Assessment facility must have regularly scheduled com-
petence evaluations of all staff whose activ-
Once hired, employees must be oriented ities affect the quality of blood, compo-
to their position and to the organization’s nents, tissues, or services.2,6 Depending
policies and procedures. The orientation upon the nature of the job duties, such as-
program should include facility-specific sessments may include: written evalua-
requirements and an introduction to poli- tions; direct observation of activities; review
cies that address issues such as safety, of work records or reports, computer re-
quality, computers, security, and confi- cords, and QC records; testing of unknown
dentiality. The job-related portion of the samples; and evaluation of the employee’s
orientation program covers the opera- problem-solving skills.5
tional issues specific to the work area. A formal competency plan that includes
Training must be provided for each proce- a schedule of assessments, defined minimum
dure for which employees have responsi- acceptable performance, and remedial
bility. The ultimate result of the orienta- measures is one way to ensure appropriate
tion and training program is to deem new and consistent competence assessments.
employees competent to work independ- Assessments need not be targeted at each
ently in performing the duties and re- individual test or procedure performed by
sponsibilities defined in their job descrip- the employee; instead, they can be grouped
tions. Time frames should be established together to assess like techniques or meth-
to accomplish this goal. Before the intro- ods. Written tests can be used effectively to
duction of a new test or service, existing evaluate problem-solving skills and to rap-
personnel must be trained to perform idly cover many topics by asking one or
their newly assigned duties and must be more questions for each area to be as-
deemed competent. During orientation sessed. For testing personnel, CMS requires
and training, the employee should be that employees who perform testing be as-
given the opportunity to ask questions sessed semiannually during the first year
and seek additional help or clarification. and annually thereafter.2
All aspects of the training must be docu- The quality oversight personnel should
mented and the facility trainer or desig- assist in the development, review, and ap-
nated facility management representative proval of training programs, including the
and the employee should mutually agree criteria for retraining.5 Quality oversight
upon the determination of competence. personnel also monitor the effectiveness of
FDA cGMP training is required for staff the training program and competence eval-
involved in the manufacture of blood and uations and make recommendations for
blood components.4 It should provide staff changes as needed. In addition, JCAHO re-
with an understanding of the regulatory ba- quires the analysis of aggregate compe-
sis for the facility’s policies and procedures tency assessment data for the purpose of
as well as train them in facility-specific ap- identifying staff learning needs.6
plication of the cGMP requirements as de-
scribed in their own written operating pro-
cedures. This training must be provided at Staffing
periodic intervals to ensure that staff re- Management should have a staffing plan
main familiar with regulatory require- that describes the number and qualifica-
ments. tions of personnel needed to perform the

functions of the facility safely and effec- ments. Similarly, the supplier should be
tively. JCAHO requires that hospitals eval- qualified to ensure a reliable source of
uate staffing effectiveness by looking at materials. The facility should clearly de-
human resource indicators (eg, overtime, fine requirements or expectations for the
staff injuries, staff satisfaction) in con- suppliers and share this information with
junction with operational performance staff and the supplier. The ability of sup-
indicators (eg, adverse events, patient’s pliers to consistently meet specifications
6
complaints). The results of this evalua- for a supply or service should be evalu-
tion should feed into the facility’s human ated along with performance relative to
resource planning process along with availability, delivery, and support. Exam-
projections based on new or changing ples of factors that could be considered to
operational needs. qualify suppliers are:
■ Licensure, certification, or accredita-
Customer and Supplier Relations tion.
■ Supply or product requirements.
Materials, supplies, and services used as
■ Review of supplier-relevant quality
inputs to a process are considered “criti-
documents.
cal” if they affect the quality of products
■ Results of audits or inspections.
and services being produced. Examples of
■ Review of quality summary reports.
critical supplies are blood components,
■ Review of customer complaints.
blood bags, test kits, and reagents. Exam-
■ Review of experience with supplier.
ples of critical services are infectious
■ Cost of materials or services.
disease testing, blood component irradia-
■ Delivery arrangements.
tion, transportation, equipment calibration,
■ Financial security, market position,
and preventive maintenance services. The
and customer satisfaction.
suppliers of these materials and services
■ Support after the sale.
may be internal (eg, other departments
A list of approved suppliers should be
within the same organization) or external
maintained, including both primary suppli-
(outside vendors). Supplies and services used
ers and suitable alternatives for contingency
in the collection, testing, processing, pre-
planning. Critical supplies and services
servation, storage, distribution, transport,
should be purchased only from those sup-
and administration of blood, components,
pliers who have been qualified. Once quali-
and tissue that have the potential to affect
fied, periodic evaluation of the supplier’s
quality should be qualified before use and
performance helps to ensure its continued
obtained from suppliers who can meet the
ability to meet requirements. Tracking the
facility’s requirements.1(pp8,9) The quality
supplier’s ability to meet expectations gives
system must include a process to evaluate
the facility valuable information about the
the suppliers’ abilities to meet these re-
stability of the supplier’s processes and its
quirements. Three important elements
commitment to quality. Documented fail-
are supplier qualification; agreements;
ures of supplies or suppliers to meet de-
and receipt, inspection, and testing of in-
fined requirements should result in imme-
coming supplies.
diate action by the facility. These actions
include notifying the supplier, quality over-
Supplier Qualification sight personnel, and management with
Critical supplies and services must be contracting authority, if applicable. Sup-
qualified on the basis of defined require- plies may need to be replaced or quaran-

tined until all quality issues have been re- information that a product may not be
solved. considered safe is discovered, such as
during look-back procedures.
Agreements
Receipt, Inspection, and Testing of
Contracts and agreements define expec- Incoming Supplies
tations and reflect concurrence of the par-
Before acceptance and use, critical mate-
ties involved.1(p8) Periodic review of agree-
rials, such as reagents and blood compo-
ments ensures that expectations of all
nents, must be inspected and tested (if
parties continue to be met. Changes must
necessary) to ensure that they meet speci-
be mutually agreed upon and incorpo-
fications for their intended use.1(pp8,9),4 It is
rated as needed.
essential that supplies used in the collec-
Blood banks and transfusion services
tion, processing, preservation, testing,
should maintain written contracts or agree-
storage, distribution, transport, and ad-
ments with outside suppliers of critical ma-
ministration of blood and components
terials and services such as blood compo-
also meet FDA requirements.
nents, irradiation, compatibility testing, or
The facility must define acceptance cri-
infectious disease marker testing. The out-
teria for critical supplies (21 CFR 210.3) and
side supplier may be another department
develop procedures to control materials
within the same facility that is managed in-
that do not meet specifications to prevent
dependently, or it may be another facility
their inadvertent use. Corrective action
(eg, contract manufacturer). The contract-
may include returning the material to the
ing facility assumes responsibility for the
vendor or destroying it. Receipt and inspec-
manufacture of the product; ensuring the
tion records provide the facility with a
safety, purity, and potency of the product;
means to trace materials that have been
and ensuring that the contract manufac-
used in a particular process and also pro-
turer complies with all applicable product
vide information for ongoing supplier
standards and regulations. Both the con-
qualification.
tracting facility and the contractor are
legally responsible for the work performed
by the contractor. Equipment Management
It is important for the blood bank or Equipment that must operate within de-
transfusion service to participate in the fined specifications to ensure the quality
evaluation and selection of suppliers. They of blood, components, tissues, and ser-
should review contracts and agreements to vices is referred to as “critical” equipment
1(p4)
ensure that all aspects of critical materials in the quality system. Critical equip-
and services are addressed. Examples of is- ment may include instruments, measur-
sues that could be addressed in an agree- ing devices, and computer systems (hard-
ment or a contract include: responsibility ware and software). Activities designed to
for a product or blood sample during ship- ensure that equipment performs as in-
ment; the responsibility of the supplier to tended include qualification, calibration,
promptly notify the facility when changes maintenance, and monitoring. Calibration,
that could affect the safety of blood, com- functional and safety checks, and preven-
ponents, or patients have been made to the tive maintenance must be scheduled and
materials or services; and the responsibility performed according to the manufac-
of the supplier to notify the facility when turer’s recommendations and regulatory

3 2
requirements of the FDA and CMS. Writ- Process Management
ten procedures for the use and control of
Written, approved policies, processes, and
equipment must comply with the manu-
procedures must exist for all critical func-
facturer’s recommendations unless an al-
tions performed in the facility and must
ternative method has been validated by
be carried out under controlled condi-
the facility and approved by the appropri-
tions. Each facility should have a system-
ate regulatory and accrediting agencies.
atic approach for identifying, planning, and
When selecting new equipment, it is im-
implementing policies, processes, and pro-
portant to consider not only the perfor-
cedures that affect the quality of blood,
mance of equipment as it will be used in
components, tissues, and services. These
the facility, but also any supplier issues re-
documents must be reviewed by manage-
garding ongoing service and support. There
ment personnel with direct authority over
should be a written plan for installation,
the process and by quality oversight per-
operational, and performance qualifica-
sonnel before implementation. Changes
tion. After installation, there must be docu-
must be documented, validated, reviewed,
mentation of any problems and the fol-
and approved. Additional information on
low-up actions taken. Recalibration and
policies, processes, and procedures can
requalification may be necessary if repairs
be found in the Documents and Records
are made that affect the critical operating
section.
functions of the equipment. Recalibration
Once a process has been implemented,
and requalification should also be consid-
the facility must have a mechanism to en-
ered when existing equipment is relocated.
sure that procedures are performed as
The facility must develop a mechanism
defined and that critical equipment, re-
to uniquely identify and track all critical
agents, and supplies are used in confor-
equipment, including equipment software
mance with manufacturers’ written instruc-
versions, if applicable. The unique identi-
tions and facility requirements. Table 1-2
fier may be the manufacturer’s serial num-
lists elements that constitute sound process
ber or a facility’s unique identification
control. A facility using reagents, supplies,
number. Maintaining a list of all critical
or critical equipment in a manner that is
equipment helps in the control function of
different from the manufacturer’s direc-
scheduling and performing functional and
tions must have validated such use and
safety checks, calibrations, preventive
may be required to request FDA approval to
maintenance, and repair. The equipment
operate at variance to 21 CFR 606.65(e) if
listing can be used to ensure that all appro-
the activity is covered under regulations for
priate actions have been performed and re-
blood and blood components (21 CFR
corded. Evaluation and analysis of equip-
640.120). If a facility believes that changes
ment calibration, maintenance, and repair
to the manufacturer’s directions would be
data will assist the facility in assessing the
appropriate, it should encourage the man-
suitability of the equipment. They will also
ufacturer to make such changes in the la-
allow for better control in managing defec-
beling (ie, package insert or user manual).
tive equipment and in identifying equip-
ment that may need replacement. When
equipment is found to be operating outside Process Validation
acceptable parameters, the potential effects Validation is used to demonstrate that a
on the quality of products or test results process is capable of achieving planned
must be evaluated and documented. results.9 It is critical to validate processes

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

■ Systematic approach to developing policies, processes, or procedures and controlling changes.
■ Validation of policies, processes, and procedures.
■ Development and use of standard operating procedures.
■ Equipment qualification processes.
■ Staff training and competence assessment.
■ Acceptance testing for new or revised computer software involved in blood bank procedures.
■ Establishment of quality control, calibration, and preventive maintenance schedules.
■ Monitoring of quality control, calibration, preventive maintenance, and repairs.
■ Monitoring and control of production processes.
■ Processes to determine that supplier qualifications and product specifications are maintained.
■ Participation in proficiency testing appropriate for each testing system in place.
■ Processes to control nonconforming materials, blood, components, and products.
in situations where it is not feasible to Validation Plan

measure or inspect each finished product
Validation must be planned if it is to be
or service in order to fully verify confor-
effective. Development of a validation
mance with specifications. However, even plan is best accomplished after obtaining
when effective end-product testing can be an adequate understanding of the system,
achieved, it is advisable to validate impor- or framework, within which the process
tant processes to generate information that will occur. Many facilities develop a tem-
can be used to optimize performance. plate for the written validation plan to
Prospective validation is used for new or ensure that all aspects are adequately ad-
revised processes. Retrospective validation dressed. Although no single format for a
may be used for processes that are already validation plan is required, the following
in operation but were not adequately vali- elements are common to most:
dated before implementation. Concurrent ■ System description
validation is used when required data ■ Purpose/objectives
cannot be obtained without performance ■ Risk assessment
of a “live” process. If concurrent valida- ■ Responsibilities
tion is used, data are reviewed at prede- ■ Validation procedures
fined intervals before final approval for full ■ Acceptance criteria
implementation occurs. Modifications to ■ Approval signatures
a validated process may warrant revalida- ■ Supporting documentation
tion, depending on the nature and extent The validation plan must be reviewed
of the change. It is up to the facility to de- and approved by quality oversight person-
termine the need for revalidation based nel. Staff responsible for carrying out the
on its understanding of how the proposed validation activities must be trained in the
changes may affect the process. process before the plan is implemented. The

results and conclusions of these activities the processes established by the

may be appended to the approved valida- facility and that the output meets
tion plan or recorded in a separate docu- specifications. It evaluates the ade-
ment. This documentation typically contains quacy of equipment for use in a spe-
the following elements: cific process that employs the
■ Expected and observed results facility’s own personnel, procedures,
■ Interpretation of results as accept- and supplies in a normal working
able or unacceptable environment.
■ Corrective action and resolution of
unexpected results
Computer System Validation
■ Conclusions and limitations
■ Approval signatures The FDA considers computerized systems
■ Supporting documentation to include: “hardware, software, periph-
■ Implementation time line eral devices, personnel, and documenta-
When a validation process does not pro- tion.”14 End-user validation of computer
duce the expected outcome, its data and systems and the interfaces between
corrective actions must be documented as systems should be conducted in the envi-
well. The responsible quality oversight per- ronment where it will be used. Testing
sonnel should have final review and ap- performed by the vendor or supplier of
proval of the validation plan, results, and computer software is not a substitute for
corrective actions and determine whether computer validation at the facility. End-
new or modified processes and equipment user acceptance testing may repeat some
may be implemented, or implemented with of the validation performed by the devel-
specified limitations. oper, such as load or stress testing and
verification of security, safety, and control
features, in order to evaluate performance
Equipment Validation under actual operating conditions. In ad-
Validation of new equipment used in a dition, the end user must evaluate the
process should include installation quali- ability of personnel to use the computer
fication, operational qualification, and system as intended within the context of
performance qualification.13 actual work processes. Staff must be able
■ Installation qualification demonstrates to successfully navigate the hardware and
that the instrument is properly in- software interface and respond appropri-
stalled in environmental conditions ately to messages, warnings, and other
that meet the manufacturer’s specifi- functions. Depending upon the nature of
cations. the computer functionality, changes to the
■ Operational qualification demon- computer system may result in changes to
strates that the installed equipment how a process is performed. If this occurs,
operates as intended. It focuses on process revalidation must also be per-
the capability of the equipment to formed. As with process validation, qual-
operate within the established limits ity oversight personnel should review and
and specifications supplied by the approve validation plans, results, and cor-
manufacturer. rective actions and determine whether
■ Performance qualification demon- implementation may proceed with or
strates that the equipment performs without limitations. Facilities that de-
as expected for its intended use in velop their own software should refer to

FDA guidance regarding general princi- performed as intended and information

ples of software validation for additional needed to assess the quality of products
information.15 and services. Together, documents and
records are used by quality oversight per-
Quality Control sonnel to evaluate the effectiveness of a fa-
cility’s policies, processes, and procedures.
QC testing is performed to ensure the
An example of quality system documenta-
proper functioning of materials, equip-
tion is provided in ISO 9001 and includes
ment, and methods during operations.
QC performance expectations and ac- the following items9:
ceptable ranges must be defined and 1. The quality policy and objectives.
readily available to staff so that they will 2. A description of the interactions be-
recognize unacceptable results and trends tween processes.
and respond appropriately. The frequency 3. Documented procedures for the control
for QC testing is determined by the facil- of documents, control of records,
ity in accordance with the applicable control of a nonconforming product,
CMS, FDA, AABB, state, and manufac- corrective action, preventive action,
turer’s requirements. QC results must be and internal quality audits.
documented concurrently with perfor- 4. Records related to the quality sys-
mance.3 Records of QC testing must include tem, operational performance, and
identification of personnel, identification product/service conformance.
of reagent (including lot number, expira- 5. All other documents needed by the
tion dates, etc), identification of equip- organization to ensure the effective
ment, testing date and time (when appli- planning, operation, and control of
cable), results, interpretation, and reviews. its processes.
Unacceptable QC results must be investi- Written policies, process descriptions, pro-
gated and corrective action implemented, cedures, work instructions, labels, forms,
if indicated, before repeating the QC pro- and records are all part of the facility’s doc-
cedure or continuing the operational pro- umentation system. They may be pa-
cess. Specific examples of suggested qual- per-based or electronic. Documents pro-
ity control intervals for blood banks and vide a description or instructions of what is
transfusion services are included in Ap- supposed to happen; records provide evi-
pendix 10 at the end of the book, and in- dence of what did happen. A document
formation regarding methods of quality management system provides assurance
control are found in the methods section that documents are comprehensive, cur-
devoted to QC. rent, and available, and that records are
accurate and complete. A well-structured
document management system links poli-
Documents and Records
cies, process descriptions, procedures, forms,
Documentation provides a framework for and records together in an organized and
understanding and communication through- workable system.
out the organization. Documents describe
the way that processes are intended to
work, how they interact, where they must Documents
be controlled, what their requirements Documents should be developed in a for-
are, and how to implement them. Records mat that conveys information clearly and
provide evidence that the process was provides staff with instructions and forms.

The Clinical and Laboratory Standards In- for use when it is not immediately evident
stitute offers guidance regarding general what information should be recorded or
levels of documentation8 as well as de- how to record it. For quantitative data, the
tailed instructions on how to write proce- form should indicate units of measure.
16
dures. General types of documentation Computer data entry and review screens
are described below. are a type of form. Forms must be designed
Policies. Policies communicate the high- to effectively capture outcomes and sup-
est level goals, objectives, and intent of the port process traceability.
organization. The rest of the organization’s Labels. Blood component labels are a
documentation will interpret and provide critical material subject to the requirements
instruction regarding implementation of of a document management system. Many
these policies. facilities maintain a master set of labels that
Processes. Process documents describe can be used as reference to verify that only
a sequence of actions and identify respon- current approved stock is in use. New label
sibilities, decision points, requirements, stock must be verified as accurate before it
and acceptance criteria. Table 1-3 lists ex- is put into inventory; comparison against a
amples of process documents that might be master label provides a mechanism for ac-
in place to support a quality system. Pro- complishing this. Change control procedures
cess diagrams or flowcharts are often used must be established for the use of on-demand
for this level of documentation. It is helpful label printers to prevent nonconforming
to show process control points on the dia- modification of label format or content.
gram as well as flow of information and hand- Each facility must have a defined process
offs between departments or work groups. for developing and maintaining docu-
Procedures and Work Instructions. ments. It should include: basic elements re-
These documents provide step-by-step di- quired for document formats; procedures
rections on how to perform job tasks and for review and approval of new or revised
procedures. Procedures and work instruc- documents; a method for keeping docu-
tions should include enough detail to per- ments current; control of document distri-
form the task correctly, but not so much as bution; and a process for archiving, pro-
to make them difficult to read. The use of tecting, and retrieving obsolete documents.
standardized formats will help staff know Training must be provided to staff responsi-
where to find specific elements and facili- ble for the content of new or revised docu-
tates implementation and control.1(p66) Proce- ments. Document management systems in-
dures may also be incorporated by refer- clude established processes to:
ence, such as those from a manufacturer’s 1. Verify the adequacy of the document
manual. Relevant procedures must be before approval and issue.
available to staff in each area where the cor- 2. Periodically review, modify, and re-
1(p66),3
responding job tasks are performed. approve as needed to keep documents
Forms. Forms provide a template for current.
capturing data either on paper or electroni- 3. Identify changes and revision status.
cally. These documents specify the data re- 4. Ensure that documents are legible,
quirements called for in SOPs and pro- identifiable, and readily available.
cesses. Forms should be carefully designed 5. Prevent unintended use of outdated
for ease of use, to minimize the likelihood or obsolete documents.
of errors, and to facilitate retrieval of infor- 6. Protect documents from unintended
mation. They should include instructions damage or destruction.

Table 1-3. Examples of Quality System Process Documents
Organization ■ Management review process
Resources ■ Personnel hiring process

■ Training process
■ Competence assessment process
Equipment ■ Equipment management process

■ Installation qualification process
Supplier and Customer Issues ■ Supplier qualification process

■ Contract review process
■ Process for qualification of critical materials
■ Ordering and inventory control of critical materials
■ Receipt, inspection, and testing of incoming critical
materials
Process Control ■ Change control process

■ Validation process
■ Process for acceptance testing of computer software
■ Process for handling proficiency testing
■ Process for handling, storage, distribution, and transport
of blood components
Documents and Records ■ Process for creation and approval of documents

■ Document management process
■ Records management process
Deviations, Nonconformances, and ■ Event management process

Complications ■ Process for handling customer complaints
■ Process for notification of external sources
Assessments ■ Internal audit process

■ Process for quality monitoring
■ Process for handling external assessments
Process Improvement ■ Corrective and preventive action processes
Facilities and Safety ■ Process for handling disasters

■ Employee safety management process

External documents that are incorpo- ■ Creation and identification of records

rated by reference become part of the doc- ■ Protection from accidental or unau-
ument management system and must be thorized modification or destruction
identified and controlled. The facility must ■ Verification of completeness, accu-
have a mechanism to detect changes to ex- racy, and legibility
ternal documents in its system, such as a ■ Storage and retrieval
manufacturer’s package inserts or user ■ Creation of copies or backups
manuals, so that corresponding changes to ■ Retention periods
procedures and forms can be made. When ■ Confidentiality
new or revised policies, process descrip- Record-keeping systems must allow for
tions, procedures, or forms are added to or ready retrieval within time frames estab-
replaced in the facility’s manual, the docu- lished by the facility and must permit trace-
ments must be marked with the effective ability of blood components as required by
date. One copy of retired documents must federal regulations.3 Specific requirements
be retained as defined by existing and ap- for records to be maintained by blood
plicable standards and regulations. banks and transfusion services are included
A master list of all current policies, pro- in the AABB Standards for Blood Banks and
cess descriptions, procedures, forms, and Transfusion Services1(pp69-80) and in 21 CFR
labels is useful for maintaining document 606.160.
control. It should include the document ti- When forms are used for capturing data
tle, the individual or work group responsible or recording steps or test results, the forms
for maintaining it, the revision date and become records. Data must be recorded in
number (if one is assigned), and the area a format that is clear and consistent. The
where it is used. It should also identify the facility must define a process and time
number and location of controlled copies in frames for the record review to ensure ac-
circulation. Copies of documents that will be curacy, completeness, and appropriate fol-
used in the workplace should be identified low-up. It must determine how reports and
and controlled to ensure that none are records are to be archived and define their
overlooked when changes are implemented. retention period. When copies of records
are retained, the facility must verify that the
copy contains complete, legible, and acces-
Records sible content of the original record before
Records provide evidence that critical the original is destroyed.
steps in a procedure have been performed If records are maintained electronically,
appropriately and that products and ser- adequate backup must exist in case of sys-
vices conform to specified requirements. tem failure. Electronic records must be
Review of records is an important tool to readable for the entire length of their reten-
help evaluate the effectiveness of the qua- tion period. Obsolete computer software,
lity system. Records must be created con- necessary to reconstruct or trace records,
currently with the performance of each must be archived appropriately. If the
significant step and clearly indicate the equipment or software used to access ar-
identity of individuals who performed chived data cannot be maintained, the re-
3
each step and when it occurred. The cords should be converted to another for-
quality system must include a process for mat or copied to another medium to
managing records that addresses the fol- permit continued access. Converted data
lowing items: must be verified against the original to en-

sure completeness and accuracy. Electronic ■ Degree of accessibility of records in

media such as magnetic tapes, optical proportion to frequency of their use.
disks, and online computer data storage are ■ Method and location of record stor-
widely used for archiving documents. Re- age related to the volume of records
cords kept in this manner must meet FDA and the amount of available storage
requirements for electronic record-keep- space.
ing.17 Microfilm or microfiche may be used ■ Availability of properly functioning
to archive written records. The medium se- equipment, computer hardware, and
lected should be appropriate for the reten- software to view archived records.
tion requirements. ■ Documentation that microfiched re-
Privacy of patient and donor informa- cords legitimately replace original
tion must be addressed in the quality documents that may be stored else-
system with established policies and proce- where or destroyed.
dures to maintain the security and confi- ■ Retention of original color-coded re-
dentiality of records. Computer systems must cords when only black-and-white re-
be designed with security features to pre- productions are available.
vent unauthorized access and use. This sys- Considerations for electronic records in-
tem may include levels of security defined clude:
by job responsibility and administered by ■ A method of verifying the accuracy
the use of security codes and passwords. of data entry.
Each facility should have a policy for al- ■ Prevention of unintended deletion of
tering or correcting records. A common data or access by unauthorized per-
practice is to indicate the date, the change, sons.
the identity of the person making the ■ Adequate protection against inad-
change, and evidence of review by a re- vertent data loss (eg, when a storage
sponsible person. The original recording device is full).
must not be obliterated in written records; ■ Validated safeguards to ensure that a
it may be crossed out with a single line, but record can be edited by only one
it should remain legible. Electronic records person at a time.
must permit tracking of both original and ■ Security and access of confidential
corrected data and include the date and data.
user identification of the person making A backup disk or tape should be main-
the change. There should be a process for tained in the event of unexpected loss of
controlling changes.1(p10) A method for refer- information from the storage medium.
encing changes to records, linked to the Backup or archived computer records and
original records, and a system for reviewing databases must be stored off-site.1(p68) The
changes for completeness and accuracy are storage facility should be secure and main-
essential. Audit trails for changed data in tain appropriate conditions, in accordance
computerized systems are required by the with the manufacturer’s recommendations
17
FDA. and instructions. An archival copy of the
The following are issues that might be computer operating system and applica-
considered when planning record storage: tions software should be stored in the same
■ Storage of records in a manner that manner.
protects them from damage and from The facility should develop and maintain
accidental or unauthorized destruc- alternative systems to ensure information
tion or modification. access if computerized data are not avail-

able. The backup and recovery procedures ■ Analyze the event to understand root
for computer downtime must be defined, causes.
with validation documentation to show ■ Implement preventive actions as ap-
that the backup system works properly. The propriate on the basis of root-cause
associated processes must be checked peri- analysis.
odically to ensure that the backup system ■ Report to external agencies, when
remains effective. Special consideration required.
should be given to staff competence and Facility personnel should be trained to
readiness to use the backup system. recognize and report such occurrences. De-
To link relevant personnel to recorded pending upon the severity of the event and
data, the facility must maintain a record of risk to patients, donors, and products, as
names, inclusive dates of employment, sig- well as the likelihood of recurrence, investi-
natures, and identifying initials or identifi- gation into contributing factors and under-
cation codes of personnel authorized to lying cause(s) may be warranted. The
create, sign, initial, or review reports and cGMP regulations require an investigation
records. Magnetically coded employee and documentation of the results if a spe-
badges and other computer-related identi- cific event could adversely affect patient
fying methods are generally accepted in lieu safety or the safety, purity, potency, or effi-
of written signatures provided they meet elec- cacy of blood or components.2,3 Tools and
tronic record-keeping requirements. approaches for performing root-cause
analysis and implementing corrective ac-
tion are discussed in the section addressing
process improvement. A summary of the
Deviations and Nonconforming Products event, investigation, and any follow-up
or Services must be documented. Table 1-4 outlines
suggested components of an internal event
The quality system must include a process
report.
for detecting, investigating, and responding
Fatalities related to blood collection or
to events that result in deviations from ac-
transfusion must be reported as soon as
cepted policies, processes, and procedures
possible to the FDA Center for Biologics
or in failures to meet requirements, as de-
Evaluation and Research (CBER) [21 CFR
fined by the donor center or transfusion
606.170(b)]. Instructions for reporting to
service, AABB standards, or applicable re-
CBER are available in published guidance20
gulations.1(p81),3 This includes the discovery
and at http://www.fda.gov/cber/transfu-
of nonconforming products and services
sion.htm. A written follow-up report is sub-
as well as adverse reactions to blood do-
mitted within 7 days of the fatality and
nation and transfusion.1(pp81-85),2 The facility
should include a description of any new pro-
should define how to:
cedures implemented to avoid recurrence.
■ Document and classify occurrences. AABB Association Bulletin #04-06 provides
■ Determine the effect, if any, on the additional information, including a form to
quality of products or services. be used for reporting donor fatalities.
21
■ Evaluate the impact on interrelated Regardless of their licensure and regis-

activities. tration status with the FDA, all donor cen-
■ Implement corrective action, includ- ters, blood banks, and transfusion services
ing notification and recall, as appro- must promptly report biologic product de-
priate. viations (previously known as errors and

18,19
Table 1-4. Components of an Internal Event Report
WHO ■ Identity of reporting individual(s)

■ Identity of individuals involved (by job title) in committing, compounding,
discovering, investigating, and initiating any immediate action
■ Patient or donor identification
■ Reviewer(s) of report
WHAT ■ Brief description of event

■ Effects on and outcome to patient, donor, or blood component
■ Name of component and unit identification number
■ Manufacturer, lot number, and expiration date of applicable reagents and
supplies
■ Immediate action taken
WHEN ■ Date of report

■ Date and time of event occurrence
■ Date and time of discovery
■ Collection and shipping dates of blood component(s)
WHERE ■ Physical location of event

■ Where in process detected
■ Where in process initiated
WHY/HOW ■ Explanation of how event occurred

■ Contributing factors
■ Root cause(s)
FOLLOW-UP ■ External reports or notifications (eg, FDA*, manufacturer, or patient’s

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

accidents) and information relevant to short period involve a particular process or

these events to the FDA 3,22 using Form procedure, that process or procedure should
FDA-3486 when the event: be further investigated. The most useful
■ Is associated with manufacturing (ie, schemes involve use of multiple categories
testing, processing, packing, labeling, for each event, which allow data to be
storing, holding, or distributing). sorted in a variety of ways so that patterns
■ Represents a deviation from current can emerge (see example in Table 1-5).
good manufacturing practice, appli- Such sorting can result in identification of
cable regulations or standards, or situations that require closer monitoring or
established specifications, or is un- of problems needing corrective action. The
expected or unforeseen. extent of monitoring and length of time to
■ May affect the safety, purity, or po- monitor processes will depend on the fre-
tency of the product. quency of the occurrence and the critical
■ Occurs while the facility had control aspects of the occurrences. Reporting and
of or was responsible for the product. monitoring of events are essential problem
■ Involves a product that has left the identification methods for process im-
control of the facility (ie, distributed). provement activities in a quality manage-
There must also be a mechanism to re- ment system.
port medical device adverse events to the Occasionally, the blood bank or transfu-
FDA.23 The JCAHO encourages reporting of sion service may need to deviate from ap-
sentinel events, including hemolytic trans- proved procedures in order to meet the
fusion reactions involving the administra- unique medical needs of a particular pa-
tion of blood or components having major tient. When this situation arises, a medi-
blood group incompatibilities.6 cally indicated exception is planned and
Each facility should track reported events approved in advance by the facility’s medi-
and look for trends. The use of classifica- cal director. The rationale and nature of the
tion schemes may facilitate trend analysis planned exception must be documented.
and typically involves one or more of the Careful consideration should be given to
following categories: the nature of the maintaining a controlled process and to
event, the process (or procedure) in which verifying the safety and quality of the re-
the event occurred, event severity, and sulting product or service. Any additional
causes. If several events within a relatively risk to the patient must be disclosed.
Table 1-5. Example of Event Classification

Event: A unit of Red Blood Cells from a directed donor was issued to an incorrect patient.
■ Classification of event
Type of event – patient
Procedure involved – issuing products
Process involved – blood administration
Product involved – Red Blood Cells
Other factors – directed donor
■ Investigation revealed
Proximate cause – two patients with similar names had crossmatched blood available
Root cause – inadequate procedure for verification of patient identification during issue

Monitoring and Assessment oversight personnel for any deficiencies

noted in the assessment. Quality oversight
The quality system should describe how
personnel should track progress toward im-
the facility monitors and evaluates its
plementation of corrective and preventive
processes. The AABB Standards1(p93) de-
actions and monitor them for effectiveness.
fines assessment as a systematic, inde-
In order to make the best use of these as-
pendent examination that is performed at
sessments, there must be a process to track,
defined intervals and at sufficient fre-
trend, and analyze the problems identified
quency to determine whether actual ac-
so that opportunities for improvement can
tivities comply with planned activities,
be recognized.1(pp86,87) Early detection of trends
are implemented effectively, and achieve
makes it possible to develop preventive ac-
objectives. Evaluations typically include
tions before patient safety or blood compo-
comparison of actual results to expected
nents are adversely affected. Evaluation
results. Depending on the focus, this can
summaries provide information useful in
include evaluation of process outputs (eg,
correcting individual or group performance
results), the activities that make up a pro-
problems and ensuring adequacy of test
cess as well as its outputs, or a group of
methods and equipment. In addition to re-
related processes and outputs (ie, the
view of assessment results, executive man-
system). Types of assessments include ex-
agement must review any associated cor-
ternal assessments, internal assessments,
rective or preventive action.
quality assessments, peer review, and self-
assessments.
Internal Assessments Quality Indicators

Internal assessments may include evalua- Quality indicators are specific perfor-
tion of quality indicator data, targeted au- mance measurements designed to moni-
dits of a single process, or system audits tor one or more processes during a defined
that are broader in scope and cover a set time and are useful for evaluating service
of interrelated processes. These assess- demands, production, adequacy of per-
ments should be planned and scheduled. sonnel, inventory control, and process
The details of who performs the assess- stability. These indicators can be pro-
ments and how they are performed should cess-based or outcome-based. Process-
be addressed. Assessments should cover based indicators measure the degree to
the quality system and major operating which a process can be consistently per-
systems found in the blood bank, transfu- formed. An example of a process-based
sion service, or donor center. indicator is measurement of turnaround
In addition, there must be a process for time from blood component ordering un-
responding to the issues raised as a result of til transfusion. Outcome-based indicators
the assessment, including review processes are often used to measure what does or
and time frames. The results should be does not happen after a process is or is
documented and submitted to manage- not performed. Counting incorrect test
ment personnel with authority over the result reports is an example of such an in-
process assessed as well as to executive dicator. For each indicator, thresholds are
management. Management should develop set that represent warning limits and/or
corrective and preventive action plans with action limits. These thresholds can be de-
input from operational staff and quality termined from regulatory or accreditation

requirements, benchmarking, or inter- ■ Distribution, handling, use, and ad-

nally derived data. ministration of blood components.
Tools frequently used for displaying ■ Evaluating all confirmed transfusion
quality indicator data are run charts and reactions.
control charts. In a run chart, time is plot- ■ Meeting patients’ transfusion needs.
ted on the x-axis and values on the y-axis. ■ Informing patients and physicians
In control charts, the mean of the data and in a timely and confidential manner
upper and lower control limits, which have of possible infectious disease trans-
been calculated from the data, are added to mission.
the chart. Single points outside the upper One method of assessing the blood ad-
and lower control limits result from special ministration process is to observe a prede-
causes. Statistical rules for interpreting con- termined number of transfusions by follow-
secutive points outside 1 standard devia- ing the unit of blood as it is issued for
tion (SD), 2 SD, and 3 SD should be used to transfusion and as it is transfused.25
recognize a process that is out of control; Assessments of transfusion safety policy
the root cause should be determined and and practice may include a review of trans-
corrective action should be initiated if indi- fusion reactions and transfusion-transmit-
cated. ted diseases. The review committee may
monitor policies and practices for notifying
Blood Utilization Assessment recipients of recalled products (look-back
notification) and donors of abnormal test
The activities of blood usage review com-
results. Other assessments important in
mittees in the transfusion setting are an
transfusion practice include the review of
example of internal assessment. Guide-
policies for informed consent, indication
lines are available from the AABB for both
for transfusion, release of directed donor
adult and pediatric utilization review.24-26
units, and outpatient or home transfusion.
Peer review of transfusion practices, re- Additional assessments should include,
quired by the AABB, is also required by where appropriate: therapeutic apheresis,
the JCAHO6 for hospital accreditation, by use of cell-saver devices, procurement and
the CMS 2 for hospitals to qualify for storage of hematopoietic progenitor cells,
Medicare reimbursement, and by some perioperative autologous blood collection,
states for Medicaid reimbursement. procurement and storage of tissue, and
Transfusion audits provide a review of evaluation of evolving technologies and
policies and practices to ensure safe and products. Appendix 1-4 lists blood utiliza-
appropriate transfusions and are based on tion assessment examples.
measurable, predetermined performance
criteria. Transfusion services should inves-
tigate an adequate sampling of cases (eg, External Assessments
5% of the number of cases occurring within External assessments include inspections,
a defined time frame or 30 cases, whichever surveys, audits, and assessments per-
is larger). Audits assess the facility’s perfor- formed by those not affiliated with the or-
mance and effectiveness in: ganization, such as the FDA, AABB, CAP,
■ Blood ordering practices for all cate- or JCAHO. Participation in an external
gories of blood and components. assessment program provides an inde-
■ Minimizing wastage of blood com- pendent, objective view of the facility’s
ponents. performance. External assessors often bring

broad-based experience and knowledge Quality oversight personnel should moni-

of best practices that can be shared. In the tor the proficiency testing program and
preparation phase of scheduled assess- verify that test systems are maintained in
ments, there is typically some data gath- a state of control and that appropriate cor-
ering and information to submit to the or- rective action is taken when indicated.
ganization performing the assessment.
Coordinated scheduling and planning will
Process Improvement
help ensure that adequate time is allotted
for each area to be covered and that ade- Continuous improvement is a fundamen-
quate staff are available to answer ques- tal goal in any quality management sys-
tions and assist in the assessment activi- tem. In transfusion medicine, this goal is
ties. During the assessment phase, it is tied to patient safety goals and expecta-
important to know who is responsible for tions for the highest quality health care.
the assessors or inspectors during the The importance of identifying, investigat-
time they are in the facility. Clear descrip- ing, correcting, and preventing problems
tions of what information can be given to cannot be overstated. The process of de-
these individuals, and in what form, will veloping corrective and preventive action
help the facility through the assessment plans includes identification of problems
or inspection process. After the assess- and their causes, and identification and
ment, identified issues must be addressed. evaluation of solutions to prevent future
Usually a written response is submitted. problems. It must include a mechanism
for data collection and analysis, as well as
follow-up to evaluate the effectiveness of
the actions taken. Statistical tools and
Proficiency Testing for Laboratories their applications may be found in publi-
Proficiency testing (PT) is one means for cations from the AABB and the American
determining that test systems (including Society for Quality.27,28 The JCAHO stan-
methods, supplies, and equipment) are dards for performance improvement are
performing as expected. As a condition outlined in Table 1-6.6
for certification, the CMS requires labora- Corrective action is defined as the action
tories to participate successfully in an ap- taken to eliminate the causes of an existing
proved PT program for each specialty and nonconformance or other undesirable situ-
analyte that they routinely test. When no ation in order to prevent recurrence.1(p94)
approved PT program exists for a particu- Preventive action is defined as the action
lar analyte, the laboratory must have an- taken to eliminate the causes of a potential
other means to verify the accuracy of the nonconformance or other undesirable situ-
2
test procedure at least twice annually. ation in order to prevent occurrence.1(p97)
Proficiency testing must be performed us- Corrective action can be thought of as a re-
ing routine work processes and conditions active approach to reported problems that
if it is to provide meaningful information. includes a preventive component, whereas
Handling and testing of PT samples should preventive action can be thought of as a
be the same as those for patient or donor proactive approach resulting from the anal-
specimens. Supervisory review of the sum- ysis of data and information. In contrast,
mary evaluation report must be documented remedial action is defined as the action
along with investigation and corrective taken to alleviate the symptoms of existing
action for results that are unacceptable. nonconformances or any other undesirable

6
Table 1-6. Applicable JCAHO Performance Improvement Standards
■ Data are collected to measure the performance of potentially high-risk processes, including
blood utilization.
■ Performance data are systematically aggregated and analyzed to determine current performance
levels, patterns, and trends over time.
■ Undesirable patterns and trends in performance are evaluated. All confirmed transfusion
reactions are analyzed.
■ There is a defined process for identification and management of serious adverse events.
Root-cause analysis and corrective action are documented.
■ Information from data analysis is used to improve performance and patient safety and minimize
the risk of serious adverse events.
■ The facility defines and implements a program to proactively identify opportunities for
improvement. Preventive actions are implemented and monitored for effectiveness.
27,28
situation. Remedial action addresses dicators; and external assessments. Active
only the visible indicator of a problem, not monitoring programs may be set up to
the actual cause (see comparisons in Table help identify problem areas. These pro-
1-7). Effective corrective and preventive ac- grams should be representative of the
tions cannot be implemented until the un- facility processes, consistent with organi-
derlying cause is determined and the pro- zational goals, and reflect customer needs.
cess is evaluated in relationship to other Preparation of an annual facility quality
processes. Pending such evaluation, it may report, in which data from all these sources
be desirable to implement interim remedial are collated and analyzed, can be a valu-
action. able tool to identify issues for performance
improvement.
Once identified, problems must be ana-
Identification of Problems and Their Causes lyzed to determine their scope, potential ef-
Sources of information for process im- fects on the quality and operational sys-
provement activities include the follow- tems, relative frequency, and the extent of
ing: blood product and other deviations; variation. This analysis is important to
nonconforming products and services; avoid tampering with processes that are
customer complaints; QC records; profi- showing normal variation or problems with
ciency testing; internal audits; quality in- little impact.
29
Table 1-7. Comparison of Remedial, Corrective, and Preventive Action
Action Problem Approach Outcome
Remedial Existent Reactive Alleviates symptoms

Corrective Existent Reactive Prevents recurrence
Preventive Nonexistent Proactive Prevents occurrence

Identifying underlying causes for an un- impossible, or outside the boundaries of

desirable condition or problem can be ac- the organization. Use of the “repetitive
complished by an individual or a group. why” prevents the mistake of interpreting
The more complex the problem and the an effect as a cause.
more involved the process, the greater the The cause-and-effect diagram, also known
need to enlist a team of individuals and to as the Ishikawa or fish-bone diagram, em-
formalize the analysis. The three most com- ploys a specialized form of brainstorming
monly used tools for identifying underlying that breaks down problems into “bite-size”
causes in an objective manner are process pieces. An example of a cause-and-effect
flowcharting, use of the “repetitive why,” diagram is shown in Fig 1-1. It is a method
and the cause-and-effect diagram. A pro- designed to focus ideas around the compo-
cess flowchart gives a detailed picture of the nent parts of a process, as well as give a pic-
multiple activities and important decision torial representation of the ideas that are
points within the process. By examining generated and their interactions. When us-
this picture, problem-prone areas may be ing the cause-and-effect diagram, one looks
identified. The “repetitive why” is used to at equipment, materials, methods, environ-
work backward through the process. One ment, and human factors. These tools iden-
repeatedly asks the question “why did this tify both active and latent failures. Active
happen?” until: 1) no new information can failures are those that have an immediate
be gleaned, 2) the causal path cannot be adverse effect. Latent failures are those
followed because of missing information, more global actions and decisions with po-
or 3) further investigation is impractical, tential for damage that may lie dormant
Root Cause Analysis of Failed Test Runs
Personnel Procedure
Inadequate training SOP not clear

Specimen suitability not defined
Multi-tasking
Inadequate staffing Inadequate for intended use
Too rushed Not validated
Failed Test Run
Not scheduled
Wrong lot Wrong calibrators Room temperature too hot
used Poor ventilation
Contaminated
Out of calibration Environmental contamination
Cleaning not scheduled
Expired Mechanical failure
Maintenance No SOP
not done
Inadequate cleaning
Reagents, Supplies Equipment Environment
Figure 1-1. Example of a cause-and-effect diagram (SOP = standard operating procedure).

and become evident only when triggered

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

Identification and Evaluation of Solutions
Potential solutions to problems are iden-
tified during the creative phase of process tiveness of the proposed change. Data can
improvement. Brainstorming and process be collected by the methods used initially
flowcharting can be particularly helpful in to identify the problems or by methods
this phase. Possible solutions should be specially designed for the trial. Once solu-
evaluated relative to organizational con- tions have been successfully tested, full im-
straints and narrowed down to those most plementation can occur. Following imple-
reasonable. Individuals who perform the mentation, data should be collected, on at
process are usually the most knowledge- least a periodic basis, to ensure adequate
able about what will work. They should be control of the process.
included when possible solutions are be-
ing considered. Individuals with knowl-
edge of the interrelationships of processes Work Environment
and the more “global” view of the organi-
zation should also be included. Solutions The facility must provide a safe workplace
may fail if representatives with these per- with adequate environmental controls
spectives are not involved. and emergency procedures for the safety
Potential solutions should be tested be- of the employees, donors, patients, and all
fore full implementation, with a clear plan other inhabitants or visitors.1(p88) Proce-
relative to methods, objectives, timelines, dures must be in place to address:
decision points, and algorithms for all pos- ■ General safety
sible results of the trial. Large-scale solu- ■ Disaster preparedness
tions can be tried on a limited basis and ■ Biological safety (blood-borne patho-
expanded if successful; smaller scale solu- gens)
tions can be implemented pending an ef- ■ Chemical safety
fectiveness evaluation. Nonetheless, data ■ Fire safety
should be collected to evaluate the effec- ■ Radiation safety, if applicable

■ Discard of blood, components, and 9. ANSI/ISO/ASQ Q9000-2000 series—quality

management standards. Milwaukee, WI: ASQ
tissue
Quality Press, 2000.
Current good manufacturing practice 10. Baldrige National Quality Program. Health
regulations require quality planning and care criteria for performance excellence.
control of the physical work environment, Gaithersburg, MD: National Institute of Stan-
dards and Technology, 2004 (revised annu-
including: ally).
■ Adequate space and ventilation 11. Quality program implementation. Associa-
■ Sanitation and trash disposal tion Bulletin 97-4. Bethesda, MD: AABB, 1997.
■ Equipment for controlling air quality 12. Juran JM, Godfrey AB. Juran’s quality hand-
book. 5th ed. New York: McGraw-Hill, 1999.
and pressure, humidity, and temper-
13. Food and Drug Administration. Guidance on
ature general principles of process validation. (May
■ Water systems 1, 1987) Rockville, MD: CBER Office of Com-
munication, Training, and Manufacturers
■ Toilet and hand-washing facilities
Assistance, 1987.
An evaluation of the infrastructure and 14. Food and Drug Administration. Glossary of
its limitations before implementation of computerized system and software develop-
procedures or equipment will help to en- ment terminology. (August 1995) Rockville,
MD: Division of Field Investigations, Office of
sure maximum efficiency and safety. A
Regional Operations, Office of Regulatory
more thorough discussion of facilities and Affairs, 1995.
safety can be found in Chapter 2. 15. Food and Drug Administration. Guidance for
industry: General principles of software vali-
dation: Final guidance for industry and FDA
staff. (January 11, 2002) Rockville, MD: CBER
Office of Communication, Training, and
References Manufacturers Assistance, 2002.
16. Clinical laboratory technical procedure man-
1. Silva MA, ed. Standards for blood banks and uals. NCCLS approved guideline. 4th ed.
transfusion services. 23rd ed. Bethesda, MD: (GP2-A4). Wayne, PA: National Committee for
AABB, 2005. Clinical Laboratory Standards, 2002.
2. Code of federal regulations. Title 42 CFR Part 17. Code of federal regulations. Title 21 CFR Part
493. Washington, DC: US Government Print- 11. Washington, DC: US Government Printing
ing Office, 2004 (revised annually). Office, 2004 (revised annually).
3. Code of federal regulations. Title 21 CFR Parts 18. Motschman TL, Santrach PJ, Moore SB. Er-
606, 610, 630, and 640. Washington, DC: US ror/incident management and its practical
Government Printing Office, 2004 (revised application. In: Duckett JB, Woods LL,
annually). Santrach PJ, eds. Quality in action. Bethesda,
4. Code of federal regulations. Title 21 CFR Parts MD: AABB, 1996:37-67.
210 and 211. Washington, DC: US Govern- 19. Food and Drug Administration. Biological
ment Printing Office, 2004 (revised annually). products: Reporting of biological product
5. Food and Drug Administration. Guideline for deviations in manufacturing. Docket No.
quality assurance in blood establishments. 97N-0242. (November 7, 2000) Fed Regist
Docket #91N-0450. (July 11, 1995) Rockville, 2000;65:66621-35.
MD: CBER Office of Communication, Train- 20. Food and Drug Administration. Guidance for
ing, and Manufacturers Assistance, 1995. industry: Notifying FDA of fatalities related to
6. Hospital accreditation standards. Oakbrook blood collection or transfusion. (September
Terrace, IL: Joint Commission Resources, 22, 2003) Rockville, MD: CBER Office of Com-
Inc., 2004. munication, Training, and Manufacturers
7. College of American Pathologists Laboratory Assistance, 2003.
Accreditation Program checklists. Chicago, 21. Reporting donor fatalities. Association Bulle-
IL: College of American Pathologists, 2003. tin #04-06. Bethesda, MD: AABB, 2004.
8. A quality system model for health care; 22. Food and Drug Administration. Draft guid-
NCCLS approved guideline (HS1-A). Wayne, ance for industry: Biological product devia-
PA: National Committee for Clinical Labora- tion reporting for blood and plasma estab-
tory Standards, 2002. lishments. (August 10, 2001) Rockville, MD:

CBER Office of Communication, Training, 26. Strauss RG, Blanchette VS, Hume H. National
and Manufacturers Assistance, 2001. acceptability of American Association of
23. Code of federal regulations. Title 21 CFR Part Blood Banks Hemotherapy Committee guide-
803. Washington, DC: US Government Print- lines for auditing pediatric transfusion prac-
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24. Shulman IA, Lohr K, Derdiarian AK, et al. 27. Anderson TD. Tools for statistical process
Monitoring transfusionist practices: A strat- control. In: Ziebell LW, Kavemeier K, eds.
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sion 1994;34:11-15. trol in blood banking and transfusion medi-
25. Becker J, Blackall D, Evans C, et al for the Sci- cine. Bethesda, MD: AABB Press, 1999:13-48.
entific Section Coordinating Committee. 28. Russell JP, Regel T. After the quality audit. 2nd
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Bethesda, MD: AABB, 2001. 29. Motschman T. Corrective versus preventive
action. AABB News 1999;21(8):5,33.

Appendix 1-1. Glossary of Commonly Used Quality Terms
Calibration Comparison of measurements performed by an instrument to those

made by a more accurate instrument or standard for the purpose of de-
tecting, reporting, and eliminating errors in measurement.
Change control Established procedures for planning, documenting, communicating, and
executing changes to infrastructure, processes, products, or services.
This includes the submission, analysis, decision making, approval, im-
plementation, and postimplementation review of the change. Formal
change control provides a measure of stability and safety and avoids ar-
bitrary changes that might affect quality.
Control chart A graphic tool used to determine whether the distribution of data values
generated by a process is stable over time. A control chart plots a sta-
tistic vs time and helps to determine whether a process is in control or
out of control according to defined criteria, eg, a shift from a central line
or a trend toward upper or lower acceptance limits.
Design output Documents, records, and evidence in any other format used to verify
that design goals have been met. Design output should identify charac-
teristics of a product or service that are crucial to safety and function
and to meeting regulatory requirements. It should contain or make ref-
erence to acceptance criteria. Examples of design output include stan-
dard operating procedures, specifications for supplies, reagents and
equipment, identification of quality control requirements, and the re-
sults of verification and validation activities.
End-product test and Verification through observation, examination, and/or testing that the
inspection finished product or service conforms to specified requirements.
Process capability Ability of a controlled process to produce a service or product that ful-
fills requirements. Also, a statistical measure of the inherent process
variability for a given characteristic relative to design specifications. The
most widely accepted formula for process capability is six sigma.
Process control Activities intended to minimize variation within a process in order to
produce a predictable output that meets specifications.
Qualification Demonstration that an entity is capable of fulfilling specified require-
ments. Verification of attributes that must be met or complied with in
order for a person or thing to be considered fit to perform a particular
function. For example, equipment may be qualified for an intended use
by verifying performance characteristics such as linearity, sensitivity, or
ease of use. An employee may be qualified based on technical, aca-
demic, and practical knowledge and skills developed through training,
education, and on-the-job performance.
Quality assurance Activities involving quality planning, control, assessment, reporting,
and improvement necessary to ensure that a product or service meets
defined quality standards and requirements.

Appendix 1-1. Glossary of Commonly Used Quality Terms (cont’d)
Quality control Operational techniques and activities used to monitor and eliminate
causes of unsatisfactory performance at any stage of a process.
Quality indicators Measurable aspects of processes or outcomes that provide an indica-
tion of the condition or direction of performance over time. Used to
monitor progress toward stated quality goals and objectives.
Quality management The organizational structure, processes, and procedures necessary to
ensure that the overall intentions and direction of an organization’s
quality program are met and that the quality of the product or service is
ensured. Quality management includes strategic planning, allocation of
resources, and other systematic activities such as quality planning, im-
plementation, and evaluation.
Requirement A stated or obligatory need or expectation that can be measured or ob-
served and is necessary to ensure quality, safety, effectiveness, or cus-
tomer satisfaction. Requirements can include things that the system or
product must do, characteristics it must have, and levels of perfor-
mance it must attain.
Specification Description of a set of requirements to be satisfied by a product, mate-
rial, or process indicating, if appropriate, the procedures to be used to
determine whether the requirements are satisfied. Specifications are of-
ten in the form of written descriptions, drawings, professional stan-
dards, and other descriptive references.
Validation Demonstration through objective evidence that the requirements for a
particular application or intended use have been met. Validation pro-
vides assurance that new or changed processes and procedures are ca-
pable of consistently meeting specified requirements before implemen-
tation.
Verification Confirmation, by examination of objective evidence, that specified re-
quirements have been met.

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

21 CFR Citation Topic
606.20 Personnel
606.40 Facilities
606.60 Equipment quality control
606.65 Supplies, reagents
606.100 Standard operating procedures
606.140 Laboratory controls
606.160 Records
606.170 Adverse reactions
606.171 Biological product deviations
211.22 Quality control/quality assurance unit responsibilities
211.25 Personnel qualifications
211.28 Personnel responsibilities
211.160 Laboratory controls
211.192 Production record review
211.194 Laboratory records and reviews

Appendix 1-3. Statistical Tables for Binomial Distribution* Used to Determine

Adequate Sample Size and Level of Confidence for Validation of Pass/Fail Data
Confidence Levels (%) for Percent Conforming
Requirement for Requirement for

% Conforming % Conforming
90% 95% 90% 95%

Sample No. of Sample No. of
Size Failures % Confidence Size Failures % Confidence
10 0 65.1 – 50 0 99.5 92.3

1 26.4 – 1 96.6 72.1
20 0 87.8 64.2 2 88.8 45.9
1 60.8 26.4 3 75.0 –
2 32.3 – 4 56.9 –
30 0 95.8 78.5 5 38.4 –
1 81.6 44.6 60 0 99.8 95.4
2 58.9 – 1 98.6 80.8
3 35.3 – 2 94.7 58.3
40 0 98.5 87.1 3 86.3 35.3
1 91.9 60.1 4 72.9 –
2 77.7 32.3 5 56.3 –
3 57.7 – 6 39.4 –
4 37.1 –
This table answers the question, “How confident am I that [90 or 95]% of all products manufac-
tured will meet specifications if I have tested __ number of samples and found __ number to
be nonconforming (failures)?”
*Data from Reliability Analysis Center, http://rac.alionscience.com/Toolbox/.
(cont’d)


(cont'd)
Minimum Sample Size for Percent Conforming
Requirement for Percent Conforming
90% 95% 99%
Confidence Level Confidence Level Confidence Level
90% 95% 99% 90% 95% 99% 90% 95% 99%

No. of
Failures Sample Size Sample Size Sample Size
0 22 29 45 46 59 90 230 299 459

1 39 47 65 77 94 130 388 467 662
2 53 63 83 106 125 166 526 625 838
3 66 76 98 133 180 198 664 773 1002
4 78 90 113 159 208 228 789 913 1157
5 91 103 128 184 235 258 926 1049 1307
6 104 116 142 209 260 288 1051 1186 1453
7 116 129 158 234 286 317 1175 1312
8 128 143 170 258 310 344 1297 1441
9 140 154 184 282 336 370 1418
10 152 167 197 306 361 397
This table answers the question, “How many samples do I need to test with ___ number of fail-
ures if I want to have [90, 95, or 99] % confidence that [90, 95, or 99]% of all products will
meet specifications?”


(cont'd)
Example of Minimum Sample Size and Number of Failures Allowed to Meet AABB Requirements
for Product Validation and Quality Control
%
Sample Number of Confidence
Product Requirement1 Size
2
Failures Level
Platelets Pheresis At least 90% of units sampled 10 0 65

contain ≥3 × 1011 platelets 10 1 26
and have a pH ≥6.2 at the
end of allowable storage.
Platelets Pheresis, At a minimum, 95% of units 20 0 64
Leukocytes sampled shall contain a re- 20 1 26
Reduced sidual leukocyte count
<5 × 106.
Red Blood Cells At least 95% of units sampled 20 0 64
Pheresis shall have >50 g of hemo- 20 1 26
globin (or 150 mL red cell
volume) per unit.
Granulocytes Prepared by a method known 4 0 68
Pheresis to yield a minimum of 4 1 26
1.0 × 1010 granulocytes in
at least 75% of the units
tested.
1. From Silva MA, ed. Standards for blood banks and transfusion services. 23rd ed. Bethesda, MD: AABB,
2005. Although the AABB Standards does not require a specific confidence level, the facility may use
this as a way to assess the degree of certainty that each product manufactured will meet
specifications.
2. Period of time used to define a population for sampling is determined by the facility. (NOTE: The longer
the period, the more difficult it may be to identify causes of failure, and the more products already in
distribution that may be involved in a recall.)

1,2
Appendix 1-4. Assessment Examples: Blood Utilization
A blood usage review committee should consider the following areas of practice and develop specific
measurements for monitoring blood transfusion processes. Some measurements provide data for
several processes.
Ordering of Appropriate Blood Components
1. Preanalytical errors Errors in specimen collection, verbal orders, transfusion orders.

2. Units transfused Figures for each type of blood component and special preparation.
Use of autologous and directed donor collections. Analyze by clin-
ical service or by prescriber.
3. Patients transfused Total number of patients receiving each of the components or
products listed in item 2.
4. Units transfused Average number of units of each component or product given to
per patient transfused patients receiving that component. May be useful to analyze by di-
agnosis or surgical/medical procedure.
5. Special components Number and relative percent of leukocyte-reduced, irradiated,
prepared and trans- cytomegalovirus-negative units; aliquots prepared and transfused;
fused outpatient and home transfusions.
6. Units returned unused Number and percent of units issued and later returned unused.
Analyze by ward, by clinical service, or by prescriber.
7. Crossmatch-to-trans- Number of units crossmatched divided by the number of units
fusion (C:T) ratio transfused. Analysis could be by institutional total, by emergency
vs routine requests, or by clinical service, surgical procedure, or
prescriber, as needed.
8. Transfusion guidelines Verification that guidelines are current, appropriate for the patient
population being treated, and readily available to physicians.
Distributing, Handling, and Dispensing Blood Components
1. Turnaround time Interval between the time a transfusion request is received and
time the unit is available for transfusion and/or is transported to
the patient’s bedside. May analyze by emergency, routine, or oper-
ative requests.
2. Emergency requests Number and percent may be analyzed by clinical service,
prescriber, diagnosis, or time (week, day, shift).
3. Uncrossmatched units Number and percent of units issued uncrossmatched or with ab-
breviated pretransfusion testing. May analyze by clinical service,
or prescriber.
4. Age distribution Age of units in inventory and crossmatched by ABO and Rh type,
of units age of units when received from the supplier, age at the time of
transfusion, age when returned to the supplier.

1,2
Appendix 1-4. Assessment Examples: Blood Utilization (cont'd)
5. Surgical cancellations Number and percent of cases delayed due to the unavailability of
due to unavailability of blood; number of hours or days of delay, analyzed by surgical
blood procedure and by cause (eg, antibody problem in an individual
patient, general shortage, or shortage of a particular ABO or Rh
type).
6. Significant type Number of Rh-negative patients given Rh-positive red cells or
switches due to platelets; transfusions with ABO-incompatible plasma.
unavailability of blood
7. Outdate rate Number of units outdated (expired unused) divided by the num-
ber of units received; should be monitored for all blood compo-
nents and derivatives. Analysis by ABO and Rh type may prove in-
formative.
8. Wastage rates Number of units wasted due to breakage, improper preparation,
improper handling or storage; units prepared for a patient but not
used; number of units that failed to meet inspection requirements.
9. Adequacy of service Number of orders placed that could be filled as requested; aver-
from the blood age time between the order and receipt of emergency delivery;
supplier number of orders associated with an error such as improper unit
received or units improperly shipped.
10. Compatibility testing Adequacy, currency, and appropriateness of policies and proce-
requirements dures.
11. Quality control policies Percent of records of temperature, equipment, component prepa-
and procedures ration, or testing that are incomplete or have deviations.
Administration of Blood and Components
1. Blood issue/delivery Number of wrong units issued; number of units delivered to

errors wrong patient-care area or improperly transported.
2. Blood administration Adherence to facility-specific requirements when monitoring pa-
policies and tients for signs and symptoms of adverse reactions. Availability of
procedures copies of current policies and procedures and of current Circular
of Information for the Use of Human Blood and Blood Compo-
nents.
3. Blood administration Summary of on-site performance reviews, to include number of
audits deviations by category (eg, identification of patient and donor
unit, documentation and completeness of medical record). May
include audit for documentation of transfusion order and indica-
tion for transfusion or informed consent.
4. Transfusion Review of quality control documentation for equipment, including
equipment blood warmers, infusion pumps, special filters or administration
sets; documentation in the medical record that devices were used;
number of situations where their use was inappropriate.
(cont’d)

1,2
Appendix 1-4. Assessment Examples: Blood Utilization (cont'd)
5. Special transfusion Review of compliance with policies for out-of-hospital transfu-

situations sions and perioperative and postoperative collection of autolo-
gous blood.
Monitoring Transfusion Results
1. Compliance with trans- Number and percent of inappropriate transfusions, as determined

fusion guidelines by the blood usage review committee; analysis of reasons for in-
appropriate transfusion.
2. Transfusion reactions Number and percent of reported transfusion reactions; turn-
around time for complete investigation; documentation of transfu-
sion service and committee review; documentation in the medical
record.
3. Transfusion-trans- Number of cases by infectious agent; turnaround time of investi-
mitted disease gation; completeness of review and recording.
4. Look-back investi- Number of cases by infectious agent; turnaround time of investi-
gations gation; completeness of case-finding, notification, review, and re-
cording.
5. Review of policies Adequacy, currency, and appropriateness of policies and proce-
and procedures dures for detection and reporting of adverse effects of transfu-
sion.
Management Data
1. Workload and produc- Evaluation of activities and efficiency of the laboratory; may be
tivity analyzed by day of week and by shift. Hours worked per unit
transfused or patient transfused may be more valuable as an effi-
ciency measure than data obtained from traditional productivity
calculations.
2. Event reports Number of events dealing with laboratory processes (eg, labeling,
preparation, testing, issue); procedural events in blood adminis-
tration; errors, accidents, and recalls by blood supplier(s).
3. Staff training and Documentation of training and continuing competency of labora-
competency tory and nursing staff to perform transfusion-related procedures
and policies.
1. Comprehensive accreditation manual for hospitals: The official handbook. Oakbrook Terrace, IL: Joint
Commission Resources, Inc., 2002.
2. Becker J, Blackall D, Evans C, et al for the Scientific Section Coordinating Committee. Guidelines for
blood utilization review. Bethesda, MD: AABB, 2001.

Chapter 2: Facilities and Safety
Chapter 2
Facilities and Safety
F
ACILITY DESIGN AND mainte- and the safety of others by adhering to poli-
nance are critical to ensure that op- cies set forth in the facility safety program.
erational needs are met and that The AABB requires accredited blood
the work environment is safe for both banks and transfusion services to plan, im-
staff and visitors. The layout of the physi- plement, and maintain a program to mini-
cal space; management of utilities such as mize risks to the health and safety of do-
water and air ventilation; flow of person- nors, patients, volunteers, and employees
nel, materials, and waste; and ergonomic from biological, chemical, and radiological
factors should all be considered in the fa- hazards.1(p88) Other professional and accred-
cility management plan. iting organizations have similar or more de-
In addition to providing adequate facili- tailed safety program requirements, includ-
ties, the organization must develop and ing the College of American Pathologists
implement a safety program that defines (CAP), the Clinical and Laboratory Stan-
policies and procedures for safe work dards Institute (formerly NCCLS), and the
practices. This includes hazard communi- Joint Commission on Accreditation of
2-5
cation, use of protective equipment, train- Healthcare Organizations (JCAHO).
ing, and competency assessment in accor- Several federal agencies have issued reg-
dance with regulations for emergency and ulations and recommendations to protect
disaster preparedness, chemical hygiene, the safety of workers and the public. Those
blood-borne pathogens, and radiation relevant to health-care settings are listed in
safety when applicable. All employees are Appendix 2-1. The contents of these regula-
responsible for protecting their own safety tions and guidelines are discussed in more
39

detail in each section of this chapter. Blood handling hazardous materials must have
banks and transfusion services should con- ready access to hand-washing sinks. For
sult with state and local agencies as well to certain pieces of heavy equipment, such as
identify any additional safety requirements. irradiators, load-bearing capacity must be
Trade and professional organizations also taken into account.
provide safety recommendations that are Laboratories must be designed with ade-
relevant to blood banks and transfusion quate illumination, electrical power, and
services. These organizations are also listed conveniently located outlets. Emergency
in Appendix 2-1. backup power sources, such as uninter-
ruptible power supplies and backup gener-
ators, should be considered to ensure that
loss of blood products does not occur dur-
Facilities ing power failures. The National Electrical
Facility Design and Workflow Code6 is routinely used as a national guide-
Proper design and maintenance of facili- line for the design of essential electrical dis-
ties and organization of work can reduce tribution systems, with modifications ap-
or eliminate many potential hazards. De- proved by the local building authority having
sign, maintenance, and organization also jurisdiction.
affect efficiency, productivity, error rates, Appropriate systems for heating, ventila-
employee and customer satisfaction, and tion, and air conditioning must be used.
the quality of products and services. State Environmental monitoring systems should
and local building codes should be con- be considered for laboratories that require
sulted in the design planning stages for positive or negative air pressure differen-
architectural safety regarding space, fur- tials to be maintained, or where air filtra-
nishings, and storage. tion systems are used to control particle
During the design phase for a new space, levels. The nationally accepted specifica-
the location and flow of personnel, materi- tions for ventilation are published by the
als, and equipment should be considered American Society of Heating, Refrigerating,
in the context of the processes to be per- and Air-Conditioning Engineers, Inc.7
formed. Adequate space must be allotted
for personnel movement, location of sup-
Housekeeping
plies and large equipment, and private or
“distraction-free” zones for certain manu- The workplace should be kept clean and
facturing tasks (eg, donor interviewing, free of clutter. Work surfaces and equip-
record review, and blood component label- ment should be regularly cleaned and dis-
ing). The facility must be able to accommo- infected. Items that may accumulate dust
date designated “clean” and “dirty” spaces and debris should not be stored above
and provide for controlled movement of clean supplies or work surfaces. Exits and
materials and waste in and out of these ar- fire safety equipment must not be block-
eas so as to avoid contamination. Chemical ed or obstructed in any way. Receptacles
fume hoods and biological safety cabinets and disposal guidelines for nonhazardous
should be located away from drafts and solid waste, biohazardous, chemical, and
high-traffic areas. The number and location radiation waste should be clearly delin-
of eyewashes and emergency showers must eated. Housekeeping responsibilities,
also be considered. Water sources for re- methods, and schedules should be de-
agent preparation must be considered. Staff fined for every work area. Written proce-

Chapter 2: Facilities and Safety 41
dures, initial training, continuing educa- especially, should not be allowed in areas
tion of personnel, and ongoing monitoring where they could be exposed to hazards
of housekeeping effectiveness are essen- and should be closely supervised in those
tial to safe operations. areas where their presence is permitted. Fa-
cilities should consider establishing specific
safety guidelines for visitors with business
Restricted Areas
in restricted areas and documenting that this
Hazardous areas should be clearly and information was received and understood.
uniformly identified with warning signs in
accordance with federal Occupational Mobile Sites
Safety and Health Administration (OSHA)
Mobile blood collection operations can
and Nuclear Regulatory Commission (NRC)
present special problems. An individual
standards so that personnel entering or
trained in safety principles should make an
working around them are aware of exist-
advance visit to the collection site to ensure
ing biological, chemical, or radiation dan-
8-11 that hazards are minimized. All mobile per-
gers. Staff not normally assigned to
sonnel should be trained to recognize un-
these areas should receive adequate train-
safe conditions and understand infection
ing to avoid endangering themselves. Risk
control policies and procedures, but re-
areas can be stratified. For example, “high-
sponsibility for site safety should be as-
risk” areas might include chemical fume
signed to a senior-level employee.
hoods, biological safety cabinets, and stor-
Hand-washing access is essential at all
age areas for volatile chemicals or radio-
collection sites. Carpeted or difficult-to-
isotopes. Technical work areas could be
clean surfaces may be protected with an
considered “moderate risk” and restricted
absorbent overlay with waterproof backing
to laboratory personnel. Administrative
to protect from possible blood spills. Porta-
and clerical areas could be considered
ble screens and ropes are helpful in direct-
“low risk” and not restricted.
ing traffic flow to maintain safe work areas.
Whenever possible, functions not requir-
Food service areas should be physically
ing special precautions should be separated
separated from areas for blood collection
from those performed in restricted areas.
and storage. Blood-contaminated waste must
Every effort should be made to prevent the
be either returned to a central location for
contamination of designated “clean” areas
disposal or packaged and decontaminated
and common equipment. Work area tele-
using thermal (autoclave, incinerator) or
phones can be equipped with speakers to
chemical disinfectant in accordance with
eliminate the need to pick up the receiver.
local regulations for medical wastes. Train-
Computer keyboards and telephones can
ed staff must perform this decontamination
be covered with plastic. They should be
with particular attention paid to cleanup of
cleaned on a regular basis and when visibly
mobile sites after blood collection.
soiled. Employees should remove their per-
sonal protective barriers such as gloves and
laboratory coats and wash their hands with
soap and water when leaving a “contami- Ergonomics
nated” area. Consideration in physical design should
Concerns for safety dictate that there be be given to ergonomics and accommoda-
no casual visitors in areas where laboratory tions for individuals covered under the
hazards may be encountered.12 Children, Americans with Disabilities Act (42 U.S.C.

§ 12101-12213, 1990). Several factors may identifies the applicable regulatory re-
contribute to employee fatigue, musculo- quirements and describes how they will
skeletal disorder syndromes, or injury, in- be met. In general, institutions are re-
cluding the following13: quired to:
■ Awkward postures—positions that ■ Provide a workplace free of recog-
place stress on the body such as nized hazards.
reaching overhead, twisting, bend- ■ Evaluate all procedures for potential
ing, kneeling, or squatting. exposure risks.
■ Repetition—performing the same ■ Evaluate each employment position
motions continuously or frequently. for potential exposure risks.
■ Force—the amount of physical effort ■ Identify hazardous areas or materi-
used to perform work. als with appropriate labels and signs.
■ Pressure points—pressing the body ■ Educate staff, document training,
against hard or sharp surfaces. and monitor compliance.
■ Vibration—continuous or high-intensity ■ Apply Standard Precautions (includ-
hand-arm or whole-body vibration. ing Universal and Blood and Body
■ Other factors—extreme high or low Fluid Precautions) to the handling of
temperatures; lighting too dark or blood, body fluids, and tissues.
too bright. ■ Dispose of hazardous waste appro-
Both the total time per work shift and priately.
the length of uninterrupted periods of work ■ Report incidents and accidents and
can be significant in contributing to prob- provide treatment and follow-up.
lems. Actions to correct problems associ- ■ Provide ongoing review of safety pol-
ated with ergonomics may include: icies, procedures, operations, and
■ Engineering improvements to reduce equipment.
or eliminate the underlying cause, ■ Develop facility policies for disaster
such as making changes to equip- preparedness and response.
ment, workstations, or materials. Safety programs should consider the
■ Administrative improvements, such needs of all persons affected by the work
as providing variety in tasks; adjust- environment. Most obvious is the safety of
ing work schedules and work pace; technical staff, but potential risks for blood
providing recovery or relaxation time; donors, ancillary personnel, volunteers, vis-
modifying work practices; ensuring itors, housekeeping staff, and maintenance
regular housekeeping and maintenance and repair workers must also be evaluated.
of work spaces, tools, and equipment; Appropriate provisions must be applied if
and encouraging exercise. these individuals cannot be excluded from
■ Provision of safety gear such as gloves, risk areas.
knee and elbow pads, footwear, and Laboratories should appoint a safety of-
other items that employees wear to ficer who can provide general guidance and
protect themselves against injury. expertise.3 This individual might develop
the safety program, oversee orientation and
training, perform safety audits, survey work
sites, recommend changes, and serve on or
Safety Program direct the activities of safety committees. It
An effective safety program starts with a is recommended that facilities using haz-
well-thought-out safety plan. This plan ardous chemicals and radioactive materials

appoint a chemical hygiene officer and ra- protect themselves appropriately before
diation safety officer to oversee chemical beginning work with hazardous materials.
and radiation protection programs.8,11 A Supervisors or their designees are res-
general safety officer with sufficient exper- ponsible for documenting the employee’s
tise may fill these roles, or separate officers understanding of and ability to apply safety
may be appointed and program oversight precautions before independent work is
given to a safety committee. permitted. Safety training must precede
There are five basic elements that must even temporary work assignments if signifi-
be addressed for each type of hazard cov- cant potential for exposure exists. Staff who
ered in the safety program: do not demonstrate the requisite under-
1. Training. standing and skills must undergo retrain-
2. Hazard identification and communi- ing. These requirements apply not only to
cation. laboratory staff, but also to housekeeping
3. Engineering controls and personal and other personnel who may come into
protective equipment. contact with hazardous substances or
4. Safe work practices, including waste waste. Table 2-1 lists topics to cover in
disposal. work safety training programs.
5. Emergency response plan.
In addition, management controls should Hazard Identification and Communication
be implemented to ensure that these ele-
Employees must know when they are
ments are in place and effective. Manage-
working with hazardous substances and
ment is responsible for:
must know where they are located in the
1. Developing and communicating the
workplace. Employers are required to pro-
written plan.
vide information about workplace haz-
2. Ensuring implementation and pro-
ards to their employees to help reduce the
viding adequate resources.
risk of occupational illnesses and injuries.
3. Providing access to employee health
This is done by means of signage, labels
services related to prevention strate-
on containers, written information, and
gies and treatment of exposures.
training programs.
4. Monitoring compliance and effec-
tiveness.
5. Evaluating and improving the safety Engineering Controls and Personal
plan. Protective Equipment
Whenever possible, the physical work-
space should be designed to eliminate the
potential for exposure. When this is not
Basic Elements of a Safety Program possible, protective gear must be pro-
vided to protect the employee. Engineer-
Training ing controls are physical plant controls or
Employees must understand the hazards equipment such as sprinkler systems,
in their workplace and the appropriate chemical fume hoods, and needleless sys-
precautions to take in order to manage tems that isolate or remove the hazard from
them safely. The mandate for employee the workplace. Personal protective equip-
training programs is based on good gen- ment (PPE) is specialized clothing or
eral practice as well as OSHA require- equipment worn by an employee for pro-
ments.9-11 All persons must be trained to tection against a hazard, such as gloves,

Table 2-1. Topics to Cover in a Work Safety Training Program

Work safety training programs should ensure that all personnel:
■ Have access to a copy of pertinent regulatory texts and an explanation of the contents.
■ Understand the employer’s exposure control plan and how to obtain a copy of the written plan.
■ Understand how hepatitis and human immunodeficiency virus (HIV) are transmitted and how
often; be familiar with the symptoms and consequences of hepatitis B virus (HBV), hepatitis C
virus (HCV), and HIV infection.
■ Know that they are offered vaccination against HBV.
■ Recognize tasks that pose infectious risks and distinguish them from other duties.
■ Know what protective clothing and equipment are appropriate for the procedures they will
perform and how to use them.
■ Know and understand the limitations of protective clothing and equipment (eg, different types of
gloves are recommended based on the permeability of the hazardous material to be used).
Employers and staff should be forewarned against a false sense of security.
■ Know where protective clothing and equipment are kept.
■ Are familiar with and understand all requirements for work practices specified in standard
operating procedures (SOPs) for the tasks they perform, including the meaning of signs and
labels.
■ Know how to remove, handle, decontaminate, and dispose of contaminated material.
■ Know the appropriate actions to take and the personnel to contact if exposed to blood or other
biological, chemical, or radiological hazards.
■ Know the corrective actions to take in the event of spills or personal exposure to fluids, tissues,
and contaminated sharps, the appropriate reporting procedures, and the medical monitoring
recommended when parenteral exposure may have occurred.
■ Know their right for access to medical treatment and medical records.
masks, and laboratory coats. General Emergency Response Plan

guidance on the use of engineering con-
trols and PPE is included in Appendix 2-2. When engineering and work practice con-
trols fail, employees must know how to re-
spond. The purpose of advance planning
Safe Work Practices is to control the hazardous situation as
Employees must be trained to know how quickly and safely as possible. Regular
to work with hazardous materials in a way testing of the emergency response plan
that protects themselves, their co-workers, will identify areas for improvement and
and the environment. Safe work practices will also build confidence in staff to re-
are defined as tasks performed in a man- spond effectively in a real situation. OSHA
ner that reduces the likelihood of expo- requires a written plan for facilities with
sure to workplace hazards. General rec- more than 10 employees. Verbal commu-
ommendations for safe work practices are nication of the plan is acceptable for 10 or
included in Appendix 2-2. fewer employees.14

Management Controls is reason to believe that exposure levels

routinely exceed the action level.16
Supervisory personnel must monitor safe-
ty practices in their areas of responsibility.
Continuing attention to safety issues should Medical First Aid and Follow-up
be addressed in routine staff meetings When requested by a worker who has sus-
and training sessions. Periodic audits per- tained known or suspected blood expo-
formed by a safety professional help in- sure, monitoring for HBV, hepatitis C virus
crease safety awareness. Management (HCV), and human immunodeficiency vi-
should seek staff input into the design and rus (HIV) antibodies should be provided
improvement of the facility’s safety plan. with appropriate counseling. Informed
The safety program, with its policies, consent is required for this voluntary test-
guidelines, and supporting references to ing; rejection of offered testing must be
regulatory documents, should be detailed documented. The usual schedule would
in a safety manual and made available to all include immediate tests on the worker
personnel at risk. This manual, along with and on the source of the potentially infec-
operational procedures manuals, should be tious material, with follow-up of the work-
reviewed at least annually and updated as er at intervals after exposure.9,10 All aspects
technology evolves and new information of accident follow-up should be appropri-
becomes available. Work sites and safety ately documented.
equipment also should be inspected regu- The Centers for Disease Control and Pre-
larly to ensure compliance and response vention (CDC) has published recommen-
readiness. Checklists are helpful in docu- dations for both pre- and postexposure
menting these audits and assessing safety prophylaxis if the contaminating material is
preparedness. Checklist items and essential HBV-positive or if this information is un-
elements for safety and environmental known.17 Hepatitis B Immune Globulin is
management audits can be obtained from usually given concurrently with hepatitis B
other sources2,3,15 or can be developed inter- vaccine in cases of penetrating injuries.
nally. When administered in accordance with the
manufacturer’s directions, both products
Employee Health Services are very safe and carry no documented risk
for infection with HBV, HCV, or HIV. Post-
Hepatitis Prophylaxis exposure prophylaxis for HIV is continu-
All employees routinely exposed to blood ally evolving; policies are generally based on
must be offered hepatitis B virus (HBV) Public Health Service recommendations17
vaccine if they do not already have HBV- and current standards of practice.
protective antibodies (ie, anti-HBs). OSHA
requires that the vaccine be offered at no Reporting Accidents and Injuries
cost to the employee, and if the employee
When an injury occurs, as much relevant
refuses the vaccine, that the refusal be
information as possible should be docu-
documented.10
mented (see Table 2-2). In addition, the
supervisor should complete any accident
Monitoring Programs reports and investigation forms required
The employer must provide a system for by the institution’s insurer and worker’s
monitoring exposure to certain substances compensation agencies. Medical records
as defined in the OSHA standard if there for individual employees should be pre-

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

■ Name and address of the injured person.
■ Time of the injury (hour, day, month, year).
■ Specified place where the injury occurred.
■ Details of the injured person’s activities at the time of injury.
■ Nature of the injury (eg, bruise, laceration, burn, etc).
■ Part of the body injured (eg, head, arm, leg, etc).
■ Nature of the known or potential agent, in cases of exposure to pathologic organisms or other
hazardous materials.
■ Nature of medical attention or first aid applied in the workplace.
■ Date the injured person stopped work.
■ Date the injured person returned to work.
■ Estimated cost of damage to property or to equipment.
■ Injured person’s statement of the events leading to the injury.
■ Statements from witnesses.
■ Cause of the injury.
■ Corrective action taken or recommendations for corrective action.
served for the duration of employment workers with latex allergies. Adverse reac-
plus 30 years, with few exceptions.17 tions associated with latex and/or powdered
OSHA requires health service employers gloves include contact dermatitis, allergic
with 11 or more workers to maintain re- dermatitis, urticaria, and anaphylaxis.
cords of occupational injuries and illnesses Medical devices that contain latex must
that require care that exceeds the capabili- bear a caution label. The National Insti-
18
ties of a person trained in first aid. Records tute for Occupational Safety and Health
of first aid provided by a nonphysician for offers the following recommendations19:
minor injuries such as cuts or burns do not
■ Make nonlatex gloves available as an
have to be retained. Initial documentation
alternative to latex. Encourage use of
must be completed within 6 days of the in-
nonlatex gloves for activities and
cident. All logs, summaries, and supple-
work environments where there is
mental records must be preserved for at
minimal risk of exposure to infec-
least 5 years beyond the calendar year of
tious materials.
occurrence. Employers must report fatali-
■ If latex gloves are used, provide re-
ties and injuries resulting in the hospital-
duced protein, powder-free gloves.
ization of three or more employees to
18 (Note: This is not a requirement, but
OSHA within 8 hours of the accident.
a recommendation to reduce expo-
sure.)
Latex Allergies ■ Use good housekeeping practices to
With the increased use of gloves, there has remove latex-containing dust from
been a rise in the number of health-care the workplace.

■ Use work practices that reduce the pation and understanding should be docu-
chance of reaction, such as hand wash- mented.
ing and avoiding oil-based hand lo-
tions. Hazard Identification and Communication
■ Provide workers with education pro- Emergency exits must be clearly marked
grams and training materials about with an “EXIT” sign. Additional signage
latex allergy. must be posted along the route of egress
■ Periodically screen high-risk workers to show the direction of travel if it is not
for latex allergy symptoms. immediately apparent. All flammable ma-
■ Evaluate current prevention strategies. terials should be labeled with appropriate
■ If symptoms of latex allergy develop, hazard warnings and flammable storage
avoid direct contact with latex and cabinets should be clearly marked.
consult a physician about allergy
precautions.
Engineering Controls and Personal
Protective Equipment
Fire Prevention Laboratories storing large volumes of
flammable chemicals are usually built
Fire prevention relies on a combination of with 2-hour fire separation walls, or with
facility design based on the National Fire 1-hour separation if there is an automatic
Protection Association (NFPA) Life Safety fire extinguishing system.3 Permanent exit
Code,20 defined processes to maintain fire routes must be designed to provide free
protection systems in good working order, and unobstructed egress from all parts of
and fire safe work practices. The Life the facility to an area of safety. Secondary
Safety Code includes both active and pas- exits may be required for areas larger than
sive fire protection systems (eg, alarms, 1000 square feet; consult local safety au-
smoke detectors, sprinklers, egress lights thority having jurisdiction such as the
and corridors, and fire-rated barriers). local fire marshal and the NFPA. Fire de-
tection and alarm systems should be pro-
Training vided in accordance with federal, state,
Fire safety training is recommended at the and local regulations.
start of employment and at least annually
thereafter. Training should emphasize Safe Work Practices
prevention and an employee’s awareness All fire equipment should be inspected on
of the work environment, including how a regular basis to ensure good working or-
to recognize and report unsafe condi- der. Fire extinguishers should be made
tions, how to report fires, the locations of readily available and staff should be
the nearest alarm and fire containment trained to use them properly. Nothing
equipment and their use, and evacuation should be stored along emergency exit
policies and routes. routes that would obstruct evacuation ef-
All staff are required to participate in fire forts. Exit doors cannot be locked from the
drills at least annually by the CAP and the inside. Housekeeping and inventory man-
JCAHO. 2 , 4 In areas where patients are agement plans should be designed to
housed or treated, the JCAHO requires control the accumulations of flammable
quarterly drills on each shift. Staff partici- and combustible materials stored in the

facility. In areas where sprinkler systems Training

are installed, all items should be stored at
Safety training should be designed to make
least 18 inches below the sprinkler head.
employees aware of electrical hazards as-
Local fire codes may require greater clear-
sociated with receptacles and connectors
ance.
and help them recognize potential prob-
lems such as broken receptacles and con-
Emergency Response Plan
nectors, improper electrical connections,
The fire emergency response plan should damaged cords, and inadequate ground-
encompass both facility-wide and area- ing.
specific situations. It should describe re-
porting and alarm systems; location and Hazard Identification and Communication
use of emergency equipment; roles and
responsibilities for staff during the re- The safety plan should address the proper
sponse; “defend in place” strategies; and use of receptacles and connectors. Equip-
conditions for evacuation, evacuation pro- ment that does not meet safety standards
4,14
cedures, and routes of egress. When a should be marked to prevent accidental use.
fire occurs, the general sequence for im-
mediate response should be to 1) rescue Engineering Controls and Personal
anyone in immediate danger, 2) activate Protective Equipment
the fire alarm system and alert others in OSHA requires that electrical systems and
the area, 3) confine the fire by closing doors equipment be constructed and installed
and shutting off fans or other oxygen in a way that minimizes the potential for
sources if possible, and 4) extinguish the workplace hazards. When purchasing
fire with a portable extinguisher if the fire equipment, the facility should verify that
is small, or evacuate if it is too large to it bears the mark of an OSHA-approved
manage. independent testing laboratory such as
Underwriters Laboratories (UL).22 Ade-
quate working space should be provided
Electrical Safety around equipment to allow easy access
for safe operation and maintenance.
Electrical hazards, including fire and
Ground-fault circuit interrupters should
shock, may arise from use of faulty elec-
be installed in damp or wet areas.
trical equipment, damaged receptacles,
connectors or cords, or unsafe work prac-
tices. Proper use of electrical equipment, Safe Work Practices
periodic inspection and maintenance, Electrical safety practices are focused
and hazard recognition training are es- around 1) proper use of electrical equip-
sential to help prevent accidents that may ment and 2) proper maintenance and re-
result in electric shock or electrocution. pair. Staff should not plug or unplug
The severity of shock depends on the path equipment from an electrical source when
that the electrical current takes through their hands are wet. Overloading circuits
the body, the amount of current flowing with too many devices may cause the cur-
through the body, and the length of time rent to heat the wires to a very high tem-
that it is flowing through the body. Even perature and generate a fire. Damaged re-
low-voltage exposures can lead to serious ceptacles and faulty electrical equipment
injury.21 must be tagged and removed from service

until they have been repaired and check- controls, personal protective equipment,
ed for safety. Flexible cords should be se- and work practice controls to minimize
cured to prevent tripping and should be the risk of exposure. It also requires em-
protected from damage from heavy or ployers to provide hepatitis B vaccination
sharp objects. Slack in flexible cords for staff with occupational exposure, to
should be kept to prevent tension on elec- provide medical follow-up in case of acci-
trical terminals and cords should be dental exposure, and to keep records re-
checked for cut, broken, or cracked insulated to accidents and exposures.
lation. Extension cords should not be
used in lieu of permanent wiring. Standard Precautions
Standard Precautions represent the most
Emergency Response Plan
current recommendations by CDC to re-
When it is not possible to decrease the duce the risk of transmission of blood-
power or disconnect equipment, the power borne and other pathogens in hospitals.
supply should be shut off from the circuit Published in 1996 in the Guidelines for
breaker. If it is not possible to interrupt Isolation Precautions in Hospitals,9 they
the power supply, a nonconductive mate- build on earlier recommendations, in-
rial such as dry wood should be used to cluding Body Substance Isolation (1987),
pry a victim from the source of current.21 Universal Precautions (1986), and Blood
Victims must not be touched directly. and Body Fluid Precautions (1983). The
Emergency first aid for victims of electri- Bloodborne Pathogen Standard refers to
cal shock must be sought. Water-based the use of Universal Precautions; however,
fire extinguishers are not to be used on OSHA recognizes the more recent guide-
electrical fires. lines from the CDC and, in Directive CPL
2-2.69, allows hospitals to use acceptable
alternatives, including Standard Precau-
Biosafety tions, as long as all other requirements in
The blood bank or transfusion service must the standard are met.23
define and enforce measures to minimize Standard Precautions apply to all patient
the risk of exposure to biohazardous ma- care activities regardless of diagnosis where
terials in the workplace. Requirements there is a risk of exposure to 1) blood; 2) all
published by OSHA (Bloodborne Pathogen body fluids, secretions, and excretions, ex-
Standard)10 and recommendations pub- cept sweat; 3) nonintact skin; and 4) mucous
lished by the US Department of Health and membranes.
Human Services9,12 provide the basis for
an effective biosafety plan. Biosafety Levels
Recommendations for biosafety in labo-
Bloodborne Pathogen Standard ratories are based on the potential haz-
The OSHA Bloodborne Pathogen Stan- ards for specific infectious agents and the
12
dard is intended to protect employees in activities performed. They include guid-
all occupations where there is a risk of ex- ance on both engineering controls and
posure to blood and other potentially in- safe work practices. The four biosafety
fectious materials. It requires that the fa- levels are designated in ascending order,
cility develop an Exposure Control Plan with increasing protection for personnel,
and describes appropriate engineering the environment, and the community.

Biosafety Level 1 (BSL-1) involves work knowledge is a first step in planning pro-
with agents of no known or of minimal po- gram content. Workplace volunteers require
tential hazard to laboratory personnel and at least as much safety training as paid
the environment. Activities are usually con- staff performing similar functions.
ducted on open surfaces and no contain-
ment equipment is needed.
Hazard Identification and Communication
Biosafety Level 2 (BSL-2) work involves
agents of moderate potential hazard to per- The facility’s Exposure Control Plan com-
sonnel and the environment, usually from municates the risks present in the work-
contact-associated exposure. Most blood place and describes controls to minimize
bank laboratory activities are considered exposure. BSL-2 through BSL-4 facilities
BSL-2. Precautions described in this section must have a biohazard sign posted at the
will focus on BSL-2 requirements. Labora- entrance when infectious agents are in
tories should consult the CDC or National use. It serves to notify personnel and
Institutes of Health (NIH) guidelines for visitors about the agents used, a point of
precautions appropriate for higher levels of contact for the area, and any special pro-
containment. tective equipment or work practices re-
Biosafety Level 3 (BSL-3) includes work quired.
with indigenous or exotic agents that may Biohazard warning labels must be placed
cause serious or potentially lethal disease on containers of regulated waste; refrigera-
as a result of exposure to aerosols (eg, My- tors and freezers containing blood or other
cobacterium tuberculosis) or by other routes potentially infectious material; and other
that would result in grave consequences to containers used to store, transport, or ship
the infected host (eg, HIV). Recommenda- blood or other potentially infectious mate-
tions for work at BSL-3 are designed to con- rials. Blood components that are labeled to
tain biohazardous aerosols and minimize identify their contents and have been re-
the risk of surface contamination. leased for transfusion or other clinical use
Biosafety Level 4 (BSL-4) applies to work are exempted.
with dangerous or exotic agents that pose
high individual risk of life-threatening dis-
ease from aerosols (eg, agents of hemor-
rhagic fevers, filoviruses). BSL-4 is not
applicable to routine blood-bank-related OSHA requires that hazards be controlled
activities. by engineering or work practices when-
ever possible. Engineering controls for
BSL-2 laboratories include limited access
Training
to the laboratory when work is in progress
OSHA requires annual training for all em- and biological safety cabinets or other
ployees whose tasks carry risk of infec- containment equipment for work that may
10,23
tious exposure. Training programs involve infectious aerosols or splashes.
must be tailored to the target group, both Hand-washing sinks and eyewash stations
in level and content. General background must be available. The work space should
knowledge of biohazards, understanding be designed so that it can be easily cleaned
of control procedures, or work experience and bench-tops should be impervious to
cannot meet the requirement for specific water and resistant to chemicals and sol-
training, although assessment of such vents.

Biological safety cabinets (BSCs) are pri- quire daily cleaning and decontamination.
mary containment devices for handling Obvious spills on equipment or work sur-
moderate- and high-risk organisms. There faces should be cleaned up immediately;
are three types—Class I, II, and III—with routine wipe-downs with disinfectant
Class III providing the highest protection to should occur at the end of each shift or on
the worker. A comparison of the features a regular basis that provides equivalent
and applications for the three classes of safety. Equipment that is exposed to blood
cabinets is provided in Table 2-3.24 BSCs are or other potentially infectious material
not required for Standard Precautions, but must be decontaminated before servicing
centrifugation of open blood samples or or shipping. When decontamination of all
manipulation of units known to be positive or a portion of the equipment is not feasi-
for HBsAg or HIV are examples of blood ble, a biohazard label stating which por-
bank procedures for which a BSC could be tions remain contaminated should be at-
useful. The effectiveness of the BSC is a tached before servicing or shipping.
function of directional airflow inward and
downward, through a high-efficiency filter.
Efficacy is reduced by anything that dis-
rupts the airflow pattern, eg, arms moving Choice of Disinfectants
rapidly in and out of the BSC, rapid move- The Environmental Protection Agency
ments behind the employee using the BSC, (EPA) maintains a list of chemical prod-
downdrafts from ventilation systems, or ucts that have been shown to be effective
open laboratory doors. Care should be antimicrobial disinfectants.27 (See http://
taken not to block the front intake and rear www.epa.gov/oppad001/chemreginex.htm
exhaust grills. Performance should be certi- for a current list.) The Association for Pro-
fied annually.25 fessionals in Infection Control and Epide-
Injuries from contaminated needles and miology also publishes a guideline to as-
other sharps continued to be a major consist health-care professionals in their
cern in health-care settings even after the decisions involving judicious selection
Bloodborne Pathogens Standard went into and proper use of specific disinfectants.28
effect. In 2001, OSHA revised the standard For facilities covered under the Blood-
to include reference to engineered sharps borne Pathogens Rule, OSHA allows the
injury protections and needleless systems.26 use of EPA-registered tuberculocidal dis-
It requires that employers implement ap- infectants, EPA-registered disinfectants
propriate new control technologies and safer that are effective against both HIV and
medical devices in their Exposure Control HBV, and/or a diluted bleach solution to
Plan and that they solicit input from their decontaminate work surfaces.23
employees to identify, evaluate, and select Before selecting a product, workers
engineering and work practice controls. Ex- should consider several factors. Among
amples of safer devices are needleless sys- them are the type of material or surface to
tems and self-sheathing needles in which be treated, the hazardous properties of the
the sheath is an integral part of the device. chemical such as corrosiveness, and the
level of disinfection required. After select-
ing a product, procedures need to be writ-
Decontamination ten to ensure effective and consistent
Reusable equipment and work surfaces cleaning and treatment of work surfaces.
that may be contaminated with blood re- Some factors to consider for effective de-

52
Table 2-3. Comparison of Class I, II, and III Biological Safety Cabinets*
AABB Technical Manual

Main Features Intended Use Common Applications
Class I Unfiltered room air is drawn into the cabi- Personal and environ- To enclose equipment (eg, centrifuges) or
net. Inward airflow protects personnel mental protection procedures that may generate aerosols
from exposure to materials inside the
cabinet. Exhaust is HEPA filtered to pro-
tect the environment. Maintains airflow at
a minimum velocity of 75 linear feet per
minute (lfpm) across the front opening

(face velocity).
Class II (General- Uses laminar flow (air moving at a constant Personal, environmen- Work with microorganisms assigned to
applies to all velocity in one direction along parallel tal, and product pro- biosafety levels 1, 2, or 3
types of Class II lines). Room air is drawn into the front tection Handling of products where prevention of
cabinets) grille. HEPA filtered air is forced down- contamination is critical, such as cell cul-
ward in a laminar flow to minimize ture propagation or manipulation of
cross-contamination of materials in the blood components in an open system
cabinet. Exhaust is HEPA filtered.
Class II, A 75% of air is recirculated after passing See Class II, general See Class II, general
through a HEPA filter. Face velocity = 75
lfpm.
Class II, B1 70% of air exits through the rear grille, is See Class II, general Allows for safe manipulation of small quan-
HEPA filtered, and then discharged from tities of hazardous chemicals and
the building. The other 30% is drawn into biologics
the front grille, HEPA filtered, and
recirculated. Face velocity = 100 lfpm.
Class II, B2 100% of air is exhausted; none is See Class II, general Provides both chemical and biological con-
recirculated. A supply blower draws air tainment. More expensive to operate be-
from the room or outside and passes it cause of the volume of conditioned room
through a HEPA filter to provide the air being exhausted.
downward laminar flow. Face velocity =
100 lfpm.
Class II, B3 Similar in design to Type A, but the system See Class II, general Allows for safe manipulation of small quan-
is ducted and includes a negative pres- tities of hazardous chemicals and
sure system to keep any possible con- biologics
tamination within the cabinet. Face veloc-
ity = 100 lfpm.
Class III Cabinet is airtight. Materials are handled Maximum protection to Work with biosafety level 4 microorganisms
with rubber gloves attached to the front personnel and envi-
of the cabinet. Supply air is HEPA fil- ronment.
tered. Exhaust air is double HEPA filtered
or may have one filter and an air incinera-

tor. Materials are brought in and out of
the cabinet either through a dunk tank or
a double-door pass-through box that can
be decontaminated. Cabinet is kept under
negative pressure.
*Data from the US Department of Health and Human Services. 24
53
contamination include the contact time, ■ Wear a mask and eye protection or a
the type of microorganisms, the presence of face shield during activities that are
organic matter, and the concentration of likely to generate splashes or sprays
the chemical agent. Workers should review of blood, body fluids, secretions, and
the basic information on decontamination excretions.
and follow the manufacturer’s instructions. ■ Wear a gown during activities that
are likely to generate splashes or
Storage sprays of blood, body fluids, secre-
Hazardous materials must be segregated tions, or excretions.
and areas for different types of storage ■ Handle soiled patient-care equipment
must be clearly demarcated. Blood must in a manner that prevents expo-
be protected from unnecessary exposure sures; ensure that reusable equip-
to other materials and vice versa. If trans- ment is not used for another patient
fusion products cannot be stored in a sep- until it has been cleaned and repro-
arate refrigerator from reagents, specimens, cessed appropriately; and ensure
and unrelated materials, areas within the that single-use items are discarded
refrigerator must be clearly labeled, and properly.
extra care must be taken to reduce the ■ Ensure that adequate procedures are
likelihood of spills and other accidents. defined and followed for the routine
Storage areas must be kept clean and or- care, cleaning, and disinfection of
derly; food or drink is never allowed where environmental surfaces and equip-
biohazardous materials are stored. ment.
■ Handle soiled linen in a manner that
Personal Protective Equipment prevents exposures.
■ Handle needles, scalpels, and other
Where hazards cannot be eliminated, OSHA
sharp instruments or devices in a
requires employers to provide appropriate
manner that minimizes the risk of
PPE and clothing, and to clean, launder,
exposure.
or dispose of PPE at no cost to their em-
■ Use mouthpieces, resuscitation bags,
ployees.10 Standard PPE and clothing in-
or other ventilation devices as an al-
clude uniforms, laboratory coats, gloves,
ternative to mouth-to-mouth resus-
face shields, masks, and safety goggles. In-
citation methods.
dications and guidelines for their use are
■ Place in a private room those pa-
discussed in Appendix 2-2.
tients who are at risk of contaminat-
ing the environment or who are not
Safe Work Practices
able to maintain appropriate hy-
Safe work practices appropriate for Stan- giene (eg, tuberculosis).
dard Precautions include the following:
■ Wash hands after touching blood,
body fluids, secretions, excretions, and Laboratory Biosafety Precautions
contaminated items, whether or not Several factors need to be considered when
gloves are worn. assessing the risk of blood exposures among
■ Wear gloves when touching blood, laboratory personnel. Some factors in-
body fluids, secretions, excretions, and clude the number of specimens processed,
contaminated items, and change them personnel behaviors, laboratory tech-
between tasks. niques, and type of equipment.29 The lab-

oratory director may wish to institute (FDA) provides guidance for collecting
BSL-3 practices for procedures that are blood from such “high-risk” donors.30 The
considered to be higher risk than BSL-2. most recent regulations and guidelines
When there is doubt whether an activity is should be consulted for changes or addi-
BSL-2 or BSL-3, the safety precautions for tions.
BSL-3 should be followed. BSL-2 precau-
tions that are applicable to the laboratory Emergency Response Plan
setting are summarized in Appendix 2-3. Blood Spills
Every facility handling blood should be
Considerations for the Donor Room prepared for spills in advance. Table 2-4
The Bloodborne Pathogen Standard ac- lists steps to be taken when a spill occurs.
knowledges a difference between hospital Cleanup is easier when preparation in-
patients and healthy donors, in whom the cludes the following elements:
prevalence of infectious disease markers ■ Design work areas so that cleanup is
is significantly lower. The employer in a relatively simple.
volunteer blood donation facility may de- ■ Prepare a spill kit or cart that con-
termine that routine use of gloves is not tains all necessary supplies and equip-
required for phlebotomy as long as :
10 ment with instructions for their use.
■ The policy is periodically reevaluated. Place it near areas where spills are
■ Gloves are made available to those anticipated.
who want to use them, and use is not ■ Assign responsibility for kit/cart main-
discouraged. tenance, spill handling, record-keep-
■ Gloves are required when an em- ing, and review of significant inci-
ployee has cuts, scratches, or breaks dents.
in skin; when there is a likelihood ■ Train personnel in cleanup proce-
that contamination will occur; while dures and reporting of significant in-
drawing autologous units; while per- cidents.
forming therapeutic procedures;
and during training in phlebotomy. Biohazardous Waste
Procedures used in the donation of blood Medical waste is defined as any waste
should be assessed for risks of biohazard- (solid, semisolid, or liquid) generated in
ous exposures and risks inherent in work- the diagnosis, treatment, or immunization
ing with a donor or patient. Some proce- of human beings or animals in related re-
dures are more likely to cause injury than search, production, or testing of biologics.
others, such as using lancets for finger Infectious waste includes disposable equip-
puncture, handling capillary tubes, crush- ment, articles, or substances that may
ing vials for arm cleaning, handling any un- harbor or transmit pathogenic organisms
sheathed needle, cleaning scissors, and giv- or their toxins. In general, infectious
ing cardiopulmonary resuscitation. waste should either be incinerated or de-
In some instances, it may be necessary contaminated before disposal in a sani-
to collect blood from donors known to pose tary landfill. Blood and components,
a high risk of infectivity (eg, collection of suctioned fluids, excretions, and secre-
autologous blood or Source Plasma for the tions may be carefully poured down a
production of other products such as vac- drain connected to a sanitary sewer if
cines). The Food and Drug Administration state law allows. Sanitary sewers may also

Table 2-4. Blood Spill Cleanup

■ Contain the spill if possible.
■ Evacuate the area for 30 minutes if an aerosol has been created.
■ Post warnings to keep the area clear.
■ Remove clothing if it is contaminated.
■ If the spill occurs in the centrifuge, turn the power off immediately and leave the cover closed
for 30 minutes. The use of overwraps helps prevent aerosolization and helps contain the spill.
■ Wear appropriate protective clothing and gloves. If sharp objects are involved, gloves must be
puncture-resistant, and a broom or other instrument should be used during cleanup to avoid
injury.
■ Use absorbent material to mop up most of the liquid contents.
■ Clean the spill area with detergent.
■ Flood the area with disinfectant and use it as described in the manufacturer’s instructions. Allow
adequate contact time with the disinfectant.
■ Wipe up residual disinfectant if necessary.
■ Dispose of all materials safely in accordance with biohazard guidelines. All blood-contaminated
items must be autoclaved or incinerated.
be used to dispose of other potentially in- Guidelines for Biohazardous Waste Dis-
fectious wastes that can be ground and posal. Employees must be trained before
flushed into the sewer. State and local handling or disposing of biohazardous
health departments should be consulted waste, even if it is packaged. The following
31
about laws and regulations on disposal of disposal guidelines are recommended :
biological waste into the sewer. ■ Identify biohazardous waste consis-
Laboratories should clearly define what tently; red seamless plastic bags (at
will be considered hazardous waste. For ex- least 2 mil thick) or containers carry-
ample, in the blood bank items contami- ing the biohazard symbol are recom-
nated with liquid or semiliquid blood are mended.
biohazardous. Items contaminated with
■ Place bags in a protective container
dried blood are considered hazardous if
with closure upward to avoid break-
there is potential for the dried material to
age and leakage during storage or
flake off during handling. Contaminated
transport.
sharps are always considered hazardous
■ When transported over public roads,
because of the risk for percutaneous injury.
However, items such as used gloves, swabs, the waste must be prepared and
plastic pipettes with excess liquid removed, shipped according to US Department
or gauze contaminated with small droplets of Transportation regulations.
of blood may be considered nonhazardous ■ Discard sharps (eg, needles, broken
if the material is dried and will not be glass, glass slides, wafers from sterile
released into the environment during han- connecting devices) in rigid, punc-
dling. ture-proof, leakproof containers.

■ Put liquids only in leakproof, un- Longer treatment times are needed for
breakable containers. sterilization, but decontamination requires
■ Do not compact waste materials. a minimum of 1 hour. A general rule is to
Storage areas for infectious material process 1 hour for every 10 pounds of waste
must be secured to reduce accident risk. being processed. Usually, decontaminated
Infectious waste must never be placed in laboratory wastes can be disposed of as
the public trash collection system. Most fa- nonhazardous solid wastes. Staff should
cilities hire private carriers to decontami- check with the local solid waste authority to
nate and dispose of infectious or hazardous ensure that the facility is in compliance
waste. Contracts with these companies with the regulations for their area. Waste
should include disclosure of the risks of containing broken glass or other sharp
handling the waste by the facility, and an items should be disposed of in a method
acknowledgment by the carrier that all fed- consistent with policies for the disposal of
eral, state, and local laws for biohazardous other sharp or potentially dangerous mate-
(medical) waste transport, treatment, and rials.
disposal are known and followed.
Treating Infectious or Medical Waste.
Facilities that incinerate hazardous waste
must comply with EPA standards of perfor-
Chemical Safety
mance for new stationary sources and One of the most effective preventive mea-
emission guidelines for existing sources.32 sures a facility can take to reduce hazard-
In this regulation, a hospital/medical/in- ous chemical exposure is to evaluate the
fectious waste incinerator (HMIWI) is any use of alternative nonhazardous chemi-
device that combusts any amount of hospi- cals whenever possible. A review of order-
tal waste or medical/infectious waste. ing practices of hazardous chemicals can
Decontamination of biohazardous waste result in the purchase of smaller quanti-
by autoclaving is another common method ties of hazardous chemicals, thus reduc-
for decontamination/inactivation of blood ing the risk of storing excess chemicals
samples and blood components. The fol- and later dealing with the disposal of
lowing elements are considered in deter- these chemicals.
mining processing time for autoclaving: OSHA requires that facilities using haz-
■ Size of load being autoclaved. ardous chemicals develop a written Chemi-
cal Hygiene Plan (CHP) and that the plan
■ Type of packaging of item(s) being
be accessible to all employees. The CHP
autoclaved.
should outline procedures, equipment,
■ Density of items being autoclaved. personal protective equipment, and work
■ Number of items in single autoclave practices that are capable of protecting em-
load. ployees from hazardous chemicals used in
■ Placement of items in the autoclave, the facility.11,16 This plan must also provide
to allow for steam penetration. assurance that equipment and protective
It is useful to place a biological indicator devices are functioning properly and that
in the center of loads that vary in size and criteria to determine implementation and
contents to evaluate optimal steam pene- maintenance of all aspects of the plan are
tration times. The EPA provides detailed in- in control. Employees must be informed of
formation about choosing and operating all chemical hazards in the workplace and
such equipment.31 be trained to recognize chemical hazards,

to protect themselves when working with also accountable for monitoring and docu-
these chemicals, and where to find infor- menting accidents and initiating process
mation on particular hazardous chemicals. change as needed.
Appendix 2-4 provides an example of a haz-
ardous chemical data safety sheet that may
Training
be used in the CHP. Safety audits and an-
nual reviews of the CHP are important con- Initial training is required for all employ-
trol steps to help ensure that safety prac- ees who may be exposed to hazardous
tices comply with the policies set forth in chemicals—before they begin work in an
the CHP and that the CHP is up to date. area where hazards exist. If an individual
Establishing a clear definition of what has received prior training, it may not be
constitutes hazardous chemicals is some- necessary to retrain them, depending on
times difficult. Generally, hazardous chemi- the employer’s evaluation of the new em-
cals are those that pose a significant health ployee’s level of knowledge. New employee
risk if an employee is exposed to them or training is likely to be necessary regarding
pose a significant physical risk, such as fire such specifics as the location of the rele-
or explosion, if handled or stored improp- vant Material Safety Data Sheets (MSDS),
erly. Categories of health and physical details of chemical labeling, the personal
hazards are listed in Tables 2-5 and 2-6. Ap- protective equipment to be used, and
pendix 2-5 lists examples of hazardous che- site-specific emergency procedures.
micals that may be found in the blood Training must be provided whenever a
bank. new physical or health hazard is introduced
The facility should identify a qualified into the workplace, but not for each new
chemical hygiene officer to be responsible chemical that falls within a particular haz-
for determining guidelines for hazardous ard class.11 For example, if a new solvent is
materials.16 The chemical hygiene officer is brought into the workplace and it has haz-
Table 2-5. Categories of Health Hazards

Hazard Definition
Carcinogens Cancer-producing substances

Irritants Agents causing irritations (edema, burning, etc) to skin or mu-
cous membranes upon contact
Corrosives Agents causing destruction of human tissue at the site of con-
tact
Toxic or highly toxic agents Substances causing serious biologic effects following inhala-
tion, ingestion, or skin contact with relatively small amounts
Reproductive toxins Chemicals that affect reproductive capabilities, including chro-
mosomal damages and effects on fetuses
Other toxins Hepatotoxins, nephrotoxins, neurotoxins, agents that act on the
hematopoietic systems, and agents that damage the lungs,
skin, eyes, or mucous membranes

Table 2-6. Categories of Physical Hazards

Hazard Definition
Combustible or flammable Chemicals that can burn (includes combustible and flammable
chemicals liquids, solids, aerosols, and gases)
Compressed gases A gas or mixture of gases in a container under pressure
Explosives Unstable or reactive chemicals that undergo violent chemical
change at normal temperatures and pressure
Unstable (reactive) chemicals Chemicals that could be self-reactive under conditions of
shocks, pressure, or temperature
Water-reactive chemicals Chemicals that react with water to release a gas that is either
flammable or presents a health hazard
ards similar to existing chemicals for which ports for hazardous chemicals in the facil-
training has already been conducted, then ities, and employee training.
the employer need only make employees Safety materials made available to em-
aware of the new solvent’s hazard category ployees should include:
(eg, corrosive, irritant). However, if the ■ The facility’s written CHP.
newly introduced solvent is a suspected ■ The facility’s written program for
carcinogen and carcinogenic hazard train- hazard communication.
ing has not been provided before, then new ■ Identification of work areas where
training must be conducted for employees hazardous chemicals are located.
with potential exposure. Retraining is ad- ■ Required list of hazardous chemicals
visable as often as necessary to ensure that and their MSDS. (It is the responsi-
employees understand the hazards, partic- bility of the facility to determine
ularly the chronic and specific target-organ which chemicals may present a haz-
health hazards, linked to the materials with ard to employees. This determina-
which they work. tion should be based on the quantity
of chemical used; the physical prop-
Hazard Identification and Communication erties, potency, and toxicity of the
chemical; the manner in which the
Hazard Communication
chemical is used; and the means
Employers must prepare a comprehensive available to control the release of, or
hazard communication program for all exposure to, the chemical.)
areas using hazardous chemicals to com-
plement the CHP and to “ensure that the
hazards of all chemicals produced or im- Hazardous Chemical Labeling and Signs
ported are evaluated, and that information The Hazard Communication Standard re-
concerning their hazards is transmitted to quires manufacturers of chemicals and
employers and employees.” 11 The pro- hazardous materials to provide the user
gram should include labeling hazardous with basic information about the hazards
chemicals, when and how to post warning of these materials through product label-
labels for chemicals, managing MSDS re- ing and Material Safety Data Sheets.11 Em-

ployers are required to provide the follow- Transfer containers used for temporary
ing to employees who are expected to work storage need not be labeled if the person
with these hazardous materials: informa- performing the transfer retains control and
tion about the hazards of the materials, intends them for immediate use. Informa-
how to read the labeling, how to interpret tion regarding acceptable standards for
symbols and signs on the labels, and how hazard communication labeling is provided
to read and use the MSDS. Table 2-7 lists by the NFPA33 and the National Paint and
34
Coatings Association.
the elements to be included in an MSDS.
Signs meeting OSHA requirements must
At a minimum, hazardous chemical con-
be posted in areas where hazardous chemi-
tainer labels must include the name of the
cals are used. Decisions on where to post
chemical, the name and address of the
warning signs are based on the manufac-
manufacturer, hazard warnings, labels,
turer’s recommendations on the chemical
signs, placards, and other forms of warning
hazards, the quantity of the chemical in the
to provide visual reminders of specific haz-
room or laboratory, and the potency and
ards. The label may refer to the MSDS for
toxicity of the chemical.
additional information. Labels applied by
the manufacturer must remain on contain-
ers. The user may add storage requirements
and dates of receipt, opening, and expira-
tion. If chemicals are aliquotted into sec-
Material Safety Data Sheets
ondary containers, the secondary container The MSDS identifies the physical and
must be labeled with the name of the che- chemical properties of a hazardous chem-
mical and appropriate hazard warnings. ical (eg, flash point, vapor pressure), its
Additional information such as precaution- physical and health hazards (eg, potential
ary measures, concentration if applicable, for fire, explosion, signs and symptoms of
and date of preparation are helpful but not exposure), and precautions for safe han-
mandatory. It is a safe practice to label all dling and use. Specific instructions in an
containers with the content, even water. individual MSDS take precedence over
Table 2-7. Required Elements of a Material Safety Data Sheet

■ Identity of product as it appears on label
■ Chemical and common name(s) of all hazardous ingredients
■ Physical/chemical characteristics
■ Fire and explosion hazard data
■ Reactivity data
■ Health hazard data, including primary route(s) of entry and exposure limits
■ Precautions for safe handling and use
■ Exposure control measures
■ Emergency and first aid procedures
■ Manufacturer information, MSDS revision date

generic information in the Hazardous Ma- rupting the airflow and compromising the
terials (HAZMAT) program. containment field.
Employers must maintain copies of the Personal protective equipment that may
required MSDS in the workplace for each be provided depending on the hazardous
hazardous chemical and must ensure that chemicals used includes chemical resistant
they are readily accessible during each gloves and aprons, shatterproof safety gog-
work shift to employees when they are in gles, and respirators.
their work areas. When household con- Emergency showers should be available
sumer products are used in the workplace to areas where caustic, corrosive, toxic,
in the same manner that a consumer would flammable, or combustible chemicals are
use them, ie, where the duration and fre- used.3,36 There should be unobstructed ac-
quency of use (and therefore exposure) are cess, within 10 seconds, from the areas
not greater than those the typical consumer where hazardous chemicals are used. Safety
would experience, OSHA does not require showers should be periodically flushed and
that an MSDS be provided to purchasers. tested for function, and associated floor
However, if exposure to such products ex- drains should be checked to ensure that
ceeds that normally found in consumer ap- drain traps remain filled with water.
plications, then employees have a right to
know about the properties of such hazard- Safe Work Practices
ous chemicals. OSHA does not require or
Hazardous material should not be stored
encourage employers to maintain an MSDS
or transported in open containers. Con-
for nonhazardous chemicals.
tainers and their lids or seals should be
designed to prevent spills or leakage in all
Engineering Controls and Personal reasonably anticipated conditions. Con-
Protective Equipment tainers should be able to safely store the
maximum anticipated volume and should
Guidelines for laboratory areas in which
be easy to clean. Surfaces should be kept
hazardous chemicals are used or stored
clean and dry at all times. When working
must be established. Physical facilities,
with a chemical fume hood, all materials
especially ventilation, must be adequate
should be kept at a distance of at least six
for the nature and volume of work con-
inches behind the face opening; the verti-
ducted. Chemicals must be stored accord-
cal sliding sash should be positioned at
ing to chemical compatibility (eg, corrosives,
the height specified on the certification
flammables, oxidizers, etc) and in mini-
sticker. The airfoil, baffles and rear venti-
mal volumes. Bulk chemicals should be
lation slot must not be blocked. Appendix
kept outside work areas. NFPA standards
2-6 lists specific chemicals and sugges-
and others provide guidelines for proper
3,33,35 tions on how to work with them safely.
storage.
Chemical fume hoods are recommended
for use with organic solvents, volatile liq-
uids, and dry chemicals with a significant Emergency Response
inhalation hazard.3 Although constructed The time to prepare for a chemical spill is
with safety glass, most fume hood sashes before a spill occurs. A comprehensive
are not designed as safety shields. Hoods employee training program should pro-
should be positioned in an area where vide the employee with all tools necessary
there is minimal foot traffic to avoid dis- to act responsibly at the time of a chemi-

cal spill. The employee should know re- The spill may require evacuation of
sponse procedures, be able to assess the the immediate area. The response typ-
severity of a chemical spill, know or be ically comes from outside the imme-
able to quickly look up the basic physical diate release area by personnel trained
characteristics of the chemicals, and know as emergency responders. These
where to find emergency response phone spills include but are not limited to:
numbers. The employee should be able immediate danger to life or health,
to: assess, stop, and confine the spill; ei- serious threat of fire or explosion,
ther clean up the spill or call for a spill and high levels of toxic substances.
clean-up team; and follow up on the re- Appendix 2-8 addresses the manage-
port of the spill. The employee must know ment of hazardous chemical spills. Spill
when to ask for assistance, when to iso- cleanup kits or carts tailored to the specific
late the area, and where to find cleanup hazards present should be available in each
materials. area. These may contain the following: rub-
Chemical spills in the workplace can be ber gloves and aprons, shoe covers, goggles,
categorized as follows37: suitable aspirators, general absorbents,
■ Incidental releases are spills that are neutralizing agents, broom, dust pan, ap-
limited in quantity and toxicity and propriate trash bags or cans for waste dis-
pose no significant safety or health posal, and cleanup directions. Chemical
hazard to the employee. They may absorbents such as clay absorbents or spill
be safely cleaned up by the employ- blankets can be used for cleaning up a
ees familiar with the hazards of the number of chemicals and thus may be eas-
chemical involved in the spill. Waste ier for the employee to use in spill situa-
from the cleanup may be classified tions.
as hazardous and must be disposed With any spill of a hazardous chemical,
of in the proper fashion. Appendix but especially with a carcinogenic agent, it
2-7 describes appropriate responses is essential to refer to the MSDS and to con-
to incidental spills. tact a designated supervisor or designee
■ Releases that may be incidental or trained to handle these spills and hazard-
3
may require an emergency response ous waste disposal. Facility environmental
are spills that may pose an exposure health and safety personnel also can offer
risk to the employees depending assistance. The employer must assess the
upon the circumstances. Consider- extent of the employee’s exposure. After an
ations such as the hazardous sub- exposure, the employee must be given an
stance properties, the circumstances opportunity for medical consultation to
of release, and mitigating factors determine the need for a medical examina-
play a role in determining the ap- tion.
propriate response. The facility’s Another source of a workplace hazard is
emergency response plan should the unexpected release of hazardous vapors
provide guidance in how to deter- into the environment. OSHA has set limits
mine whether the spill is incidental for exposure to hazardous vapors from
or requires an emergency response. toxic and hazardous substances.38 The po-
■ Emergency response releases are tential risk associated with the chemical is
spills that pose a threat to health and determined by the manufacturer and listed
safety regardless of the circum- on the MSDS. See Table 2-8 for a listing of
stances surrounding their release. the limits of exposure.

32
Table 2-8. Regulatory Limits for Exposure to Toxic and Hazardous Vapors
Limit Definition
Permissible exposure The maximum concentration of vapors in parts per million (ppm) that
limit an employee may be exposed to in an 8-hour day/40-hour work week.
Short-term exposure The maximum allowable concentration of vapors that an employee
limit may be exposed to in a 15-minute period, with a maximum of four ex-
posures per day allowed with at least 1 hour between each.
Ceiling limit The maximum concentration of vapors that may not be exceeded in-
stantaneously at any time.
Chemical Waste Disposal Radiation Measurement Units

Most laboratory chemical waste is consid- The measurement unit quantifying the
ered hazardous and is regulated by the amount of energy absorbed per unit mass
EPA through the Resource Conservation of tissue is the Gray (Gy) or rad (radiation
and Recovery Act (42 U.S.C. § 6901 et seq, absorbed dose); 1 Gy equals 100 rads.
1976). This regulation specifies that haz- Dose equivalency measurements are
ardous waste can only be legally disposed more useful than simple energy measure-
of at an EPA-approved disposal facility. ments because they take into account the
Disposal of chemical waste into a sanitary effectiveness of the different types of radia-
sewer is regulated by the Clean Water Act tion to cause biologic effects. The ability of
(33 U.S.C. § 1251 et seq, 1977), and most radiation to cause damage is assigned a
states have strict regulations concerning number called a quality factor (QF). For ex-
disposal of chemicals in the water system. ample, exposure to a given amount of alpha
Federal and applicable state regulations particles (QF = 20) is far more damaging
should be consulted when setting up and than exposure to an equivalent amount of
reviewing facility waste disposal policies. gamma rays (QF = 1). The common unit of
measurement for dose equivalency is the
rem (rad equivalent man). To obtain dose
from a particular type of radiation in rem,
Radiation Safety multiply the number of rad by the quality
Radiation can be defined as energy in the factor (rad × QF = rem). Because the quality
form of waves or particles emitted and factor for gamma rays, x-rays, and most
propagated through space or a material beta particles is 1, the dose in rad is equal
medium. Gamma rays are electromag- to the dose in rem for these types of radia-
netic radiation, whereas alpha and beta tion.
emitters are examples of particulate radi-
ation. The presence of radiation in the
Biologic Effects of Radiation
blood bank, either from radioisotopes
used in laboratory testing or from self- Any harm to tissue begins with the ab-
contained blood irradiators, requires adsorption of radiation energy and subse-
3,39
ditional precautions and training. quent disruption of chemical bonds. Mol-

ecules and atoms become ionized and/or training, warning signs and labels, shipping
excited by absorbing this energy. The “di- and handling guidelines, radiation moni-
rect action” path leads to radiolysis or for- toring, and exposure management. Emer-
mation of free radicals that, in turn, alter gency procedures must be clearly defined
the structure and function of molecules in and readily available to staff.
the cell.
Molecular alterations can cause cellular
or chromosomal changes, depending upon Exposure Limits
the amount and type of radiation energy
The NRC sets standards for protection
absorbed. Cellular changes can manifest as
against radiation hazards arising from li-
a visible somatic effect, eg, erythema. 8
censed activities, including dose limits.
Changes at the chromosome level may
These limits, or maximum permissible
manifest as leukemia or other cancers, or
dose equivalents, are a measure of the ra-
possibly as germ cell defects that are trans-
diation risk over time and serve as stan-
mitted to future generations.
dards for exposure. The occupational total
The type of radiation, the part of the
effective-dose-equivalent limit is 5 rem/
body exposed, the total absorbed dose, and
year. The shallow dose equivalent (skin) is
the dose rate influence biologic damage.
50 rem/year, the extremity dose equiva-
The total absorbed dose is the cumulative
lent limit is 50 rem/year, and the eye dose
amount of radiation absorbed in the tissue.
equivalent limit is 15 rem/year.8,41 Dose
The greater the dose, the greater the poten-
limits to an embryo/fetus must not ex-
tial for biologic damage. Exposure can be
ceed 0.5 rem during the pregnancy.8,41,44
acute or chronic. The low levels of ionizing
Employers are expected not only to main-
radiation likely to occur in blood banks
tain radiation exposure below allowable
should not pose any detrimental risk.40-43
limits, but also to keep exposure levels as
far below these limits as can reasonably be
Regulations achieved.
The NRC controls use of radioactive ma-
terials by establishing licensure require-
ments. States and municipalities may also Radiation Monitoring
have requirements for inspection and/or Monitoring is essential for early detection
licensure. The type of license for using ra- and prevention of problems due to radia-
dioisotopes or irradiators will depend on tion exposure. It is used to evaluate the
the scope and magnitude of the use of ra- environment, work practices, and proce-
dioactivity. Facilities should contact the dures, and to comply with regulations and
NRC and appropriate state agencies for li- NRC licensing requirements. Monitoring is
cense requirements and application as accomplished with the use of dosimeters,
soon as such activities are proposed. bioassay, survey meters, and wipe tests.3
NRC-licensed establishments must have Dosimeters, such as film or thermo-
a qualified radiation safety officer who is re- luminescent badges and/or rings, measure
sponsible for establishing personnel pro- personnel radiation doses. The need for do-
tection requirements and for proper dis- simeters depends on the amount and type
posal and handling of radioactive materials. of radioactive materials in use; the facility
Specific radiation safety policies and proce- radiation safety officer will determine indi-
dures should address dose limits, employee vidual dosimeter needs. Film badges must

be changed at least quarterly and in some signs and labels in use. Instruction in the
instances monthly, protected from high following is also suggested:
temperature and humidity, and stored at ■ NRC regulations and license condi-
work away from sources of radiation. tions.
Bioassay, such as thyroid and whole ■ The importance of observing license
body counting or urinalysis, may be used to conditions and regulations and re-
determine if there is radioactivity inside the porting violations or conditions of
body and, if so, how much. If necessary, unnecessary exposure.
bioassays are usually performed quarterly ■ Precautions to minimize exposure.
and after an incident where accidental in- ■ Interpretation of results of monitor-
take may have occurred. ing devices.
Survey meters are sensitive to low levels ■ Requirements for pregnant workers.
of gamma or particulate radiation and pro- ■ Employees’ rights.
vide a quantitative assessment of radiation ■ Documentation and record-keeping
hazard. They can be used to monitor stor- requirements.
age areas for radioactive materials or wastes, The need for refresher training is deter-
testing areas during or after completion of a mined by the license agreement between
procedure, and packages or containers of the NRC and the facility.
radioactive materials. Survey meters must
be calibrated annually by an authorized
NRC licensee. Selection of appropriate me-
ters should be discussed with the radiation
safety officer. Although self-contained blood irradiators
In areas where radioactive materials are present little risk to laboratory staff and
handled, work surfaces, equipment, and film badges are not required for routine
floors that may be contaminated should be operation, blood establishments with ir-
checked regularly with a wipe test. In the radiation programs must be licensed by
wipe test, a moistened absorbent material the NRC.41
(the wipe) is passed over the surface and The manufacturer of the blood irradiator
then counted for radiation. Kits are avail- usually accepts responsibility for radiation
able for this purpose. In most clinical labo- safety requirements during transportation,
ratories, exposure levels of radiation are installation, and validation of the unit as
well below the limits set by federal and part of the purchase contract. The radiation
state regulations. safety officer can help oversee the installa-
tion and validation processes and confirm
that appropriate training, monitoring sys-
Training
tems, procedures, and maintenance proto-
Personnel who handle radioactive materi- cols are in place before use and reflect the
als or work with blood irradiators must re- manufacturer’s recommendations. Sus-
ceive radiation safety training before be- pected malfunctions must be reported im-
ginning work. This training should cover mediately to defined facility authorities so
an explanation of the presence and po- that appropriate actions can be initiated.
tential hazards of radioactive materials Blood irradiators should be located in
found in the employee’s specific work area, secure areas with limited access so that
general health protection issues, emer- only trained individuals have access. Fire
gency procedures, and radiation warning protection for the unit must also be consid-

ered. Automatic fire detection and control If a spill occurs, contaminated skin sur-
systems should be readily available in the faces must be washed several times and the
immediate area. Blood components that radiation safety officer must be notified im-
have been irradiated are not radioactive mediately for further guidance. Others
and pose no threat to staff or the general must not be allowed to enter the area until
public. emergency response personnel arrive.
Safe Work Practices Waste Management

Each laboratory should establish policies Policies for the disposal of radioactive
and procedures for the safe use of radio- waste, whether liquid or solid, should be
active materials. They should include established, with input from the radiation
requirements for following general labo- safety officer and the disposal contractor,
ratory safety principles, appropriate stor- if an approved company is used.
age of radioactive solutions, and proper Liquid radioactive waste may be col-
disposal of radioactive wastes. Radiation lected into large sturdy bottles labeled with
safety can be improved with the follow- an appropriate radiation waste tag. The
ing: rules for separation by chemical compati-
■ Minimize time of exposure by work- bility apply. Bottles must be carefully stored
ing as efficiently as possible. to protect against spillage or breakage. Dry
■ Maximize distance from the source or solid waste may be sealed in a plastic
of the radiation by staying as far bag and tagged as radiation waste. The iso-
from the source as possible. tope, activity of the isotope, and date that
■ Maximize shielding (eg, by using a the activity was measured should be placed
self-shielded irradiator or by wear- on the bag. Radiation waste must never be
ing lead aprons when working with discharged into the drain system without
certain radioactive materials). These prior approval of the radiation safety offi-
requirements are usually stipulated cer.
in the license conditions.
■ Use good housekeeping practices to
minimize spread of radioactivity to
uncontrolled areas.
Shipping Hazardous
Emergency Response Plan Materials
Radioactive contamination is the dis- Local surface transport of blood specimens,
persal of radioactive material into or onto components, and biohazardous materials
areas where it is not intended; for exam- from one facility (or part thereof ) to an-
ple, the floor, work areas, equipment, other may be made by a local approved
personnel clothing, or skin. The NRC reg- courier service. The safe transport of these
ulations state that gamma or beta radio- materials requires that they be packaged
active contamination cannot exceed 2200 in such a way that the possibility of leak-
2
dpm/100 cm in the posted (restricted) age or other release from the package un-
2
area or 220 dpm/100 cm in an unre- der normal conditions of transport does
stricted area such as corridors; for alpha not occur. See Method 1.1 for detailed
emitters, these values are 220 dpm/100 shipping instructions for diagnostic spec-
cm2 and 22 dpm/100 cm2, respectively.45 imens and infectious substances.

taminated with blood is considered a multi-

Waste Management hazardous waste. The disposal of this waste
Those responsible for safety must be con- poses several problems with transportation
cerned with protecting the environment, from draw sites to a central facility to dis-
as well as staff. Every effort should be posal of the final containers. State and local
made to establish facility-wide programs health departments must be involved in the
to reduce solid wastes, including nonhaz- review of transportation and disposal prac-
ardous and especially hazardous wastes tices where this is an issue, and procedures
(ie, biohazardous, chemical, and radia- must be developed in accordance with
tion wastes). A hazardous waste reduction their regulations as well as those of the US
program instituted at the point of use of Department of Transportation.
the material achieves several goals. It re-
duces the institutional risk for occupa-
tional exposures to hazardous agents and
“cradle to grave” liability for disposal as Disaster Planning
well as enhances compliance with envi- Blood banks and transfusion services should
ronmental requirements to reduce pollu- establish action plans for uncommon but
tion generated from daily operations of potential dangers (eg, floods; hurricanes;
the laboratory.31,46,47 These requirements tornadoes; earthquakes; fires; explosions;
necessitate that a facility minimize pollu- biological, chemical, or radiation emer-
tion of the environment by the “three R’s” gencies; structural collapse; hostage situ-
(reduce, reuse, and recycle). Seeking suit- ations; bomb threats and other acts of ter-
able alternatives to the use of materials rorism; or other events in which mass
that create hazardous waste and separat- casualties might occur). These events re-
ing hazardous waste from nonhazardous quire a plan to ensure the safety of pa-
waste can reduce the volume of hazard- tients, visitors, workers, and the blood
ous waste and decrease costs for its dis- supply. Such disasters may involve the fa-
posal. cility alone, the surrounding area, or both,
A goal of waste management should be and can be categorized by severity level:
to reduce to a minimum the volume of minor impact on normal operations;
hazardous material. Noninfectious waste moderate to substantial reduction in op-
should always be separated from infectious erations; or severe, prolonged loss of op-
waste. Changes in techniques or materials, erations. The JCAHO requires a plan to
which reduce the volume of infectious address four phases of activities: mitiga-
waste or render it less hazardous, should be tion, preparedness, response, and recov-
carefully considered and employees should ery.4 Policies and procedures may address:
be encouraged to identify safer alternatives ■ Notification procedures.
wherever possible. ■ Ongoing communication (ie, com-
Facilities should check with state and lo- mand center).
cal health and environmental authorities ■ Evacuation or relocation.
for current requirements for storage and ■ Isolation or containment.
disposal of a particular multihazardous ■ Personal safety and protection.
waste before creating that waste. If the ■ Provision of additional staffing.
multihazardous waste cannot be avoided, Typically, in a disaster situation, the first
the volume generated should be mini- person who becomes aware of the disaster
mized. In some states, copper sulfate con- takes immediate action and notifies others,

either through an alarm activation system Part 20. Washington, DC: US Government
Printing Office, 2004 (revised annually).
(eg, fire alarm) or by notifying an individual
9. Garner JS, for the CDC Hospital Infection Con-
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tial response steps and contacts the facil- lines for isolation precautions in hospitals.
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80.
phone numbers should be prominently
10. Code of federal regulations. Occupational ex-
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11. Code of federal regulations. Hazard communi-
ducted to ensure appropriate responses cation standard. Title 29 CFR Part 1910.1200.
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workplace factors: A critical review of epide-
made to the disaster plan as needed. How- miologic evidence for work-related musculo-
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Cincinnati, OH: US Department of Health
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Liberman DF, ed. Biohazards management hand-
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book. 2nd ed. New York: Marcel Dekker, Inc, 1995.
47. Clinical laboratory waste management. Ap-
proved Standard Doc GP5-A. Wayne, PA: Na- NIH guide to waste disposal. Bethesda, MD: Na-
tional Committee for Clinical Laboratory tional Institutes of Health, 2003. [Available at http://
Standards, 1993. www.nih.gov/od/ors/ds/wasteguide.]
Prudent practices for handling hazardous chemi-

cals in laboratories. Washington, DC: National
Suggested Reading Academy Press, 1981.
Risk management and safety procedure manual.

CDC Office of Biosafety. Radiation safety manual.
In: Developing a disaster plan. Bethesda, MD:
Atlanta, GA: Centers for Disease Control, 1992.
AABB, 1998.
Disaster operations handbook: Coordinating the
Vesley D, Lauer JL. Decontamination, sterilization,
nation’s blood supply during disasters and biolog-
disinfection and antisepsis. In: Fleming DO, Rich-
ical events. Bethesda, MD: AABB, 2003.
ardson JH, Tulis JJ, Vesley D, eds. Laboratory
Disaster plan development procedure manual. In: safety, principles and practices. 2nd ed. Washing-
Developing a disaster plan. Bethesda, MD: AABB, ton, DC: American Society for Microbiology Press,
1998. 1995:219-37.
Handbook of compressed gases. 3rd ed. Compressed

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

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

Settings
Agency/Organization Reference Title
Federal Regulations and Recommendations

Nuclear Regulatory Com- 10 CFR 20 Standards for Protection Against
mission (NRC) Radiation
Guide 8.29 Instruction Concerning Risks
from Occupational Radiation
Exposure
Department of Labor, Occu- 29 CFR 1910.1030 Occupational Exposure to

pational Safety and Bloodborne Pathogens
Health Administration
(OSHA)
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- 49 CFR 171-180 Hazardous Materials Regulations

tion (DOT)
Environmental Protection EPA Guide for Infectious Waste

Agency (EPA) Management
Centers for Disease Control Guideline for Isolation Precau-

and Prevention (CDC) tions in Hospitals
Food and Drug Administra- 21 CFR 606.40, 606.60, Current Good Manufacturing
tion (FDA) and 606.65 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)

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

Settings (cont'd)
Agency/Organization Reference Title
Trade and Professional Organizations

National Fire Protection NFPA 70 National Electrical Code
Association (NFPA)
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 Hazardous Materials Identifica-
Association tion System (HMIS) Imple-
mentation Manual
International Air Traffic Dangerous Goods Regulations
Association (IATA)

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)

Equipment, and Engineering Controls (cont'd)
■ When cleaning up spills or handling waste materials.

■ When likelihood of exposure cannot be assessed because of lack of experience with a procedure
or situation.
The Occupational Safety and Health Administration (OSHA) does not require routine use of gloves
by phlebotomists working with healthy prescreened donors or the changing of unsoiled gloves
between donors if gloves are worn.1,2 Experience has shown that the phlebotomy process is low
risk because donors have low rates of infectious disease markers. Also, exposure to blood is rare
during routine phlebotomy, and other alternatives can be utilized to provide barrier protection,
such as using a folded gauze pad to control any blood flow when the needle is removed from the
donor’s arm.
The employer whose policies and procedures do not require routine gloving should periodically
reevaluate the potential need for gloves. Employees should never be discouraged from using
gloves, and gloves should always be available.
Guidelines on Use
Guidelines for the safe use of gloves include the following3,4:
■ Securely bandage or cover open skin lesions on hands and arms before putting on gloves.
■ Change gloves immediately if they are torn, punctured, or contaminated; after handling high-risk
samples; or after performing a physical examination, eg, on an apheresis donor.
■ Remove gloves by keeping their outside surfaces in contact only with outside and by turning the
glove inside out while taking it off.
■ Use gloves only where needed and avoid touching clean surfaces such as telephones, door
knobs, or computer terminals with gloves.
■ Change gloves between patient contacts. Unsoiled gloves need not be changed between donors.
■ Wash hands with soap or other suitable disinfectant after removing gloves.
■ Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may
cause “wicking” (ie, the enhanced penetration of liquids through undetected holes in the glove).
Disinfecting agents may cause deterioration of gloves.
■ Use only water-based hand lotions with gloves, if needed; oil-based products cause minute
cracks in latex.
Face Shields, Masks, and Safety Goggles

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

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
3,6
Laboratory areas that contain hazardous chemicals must be equipped with eye wash stations.
Unobstructed access, within 10 seconds from the location of chemical use, must be provided for
these stations. Eye washes must operate so that both of the user’s hands are free to hold open
the eyes. Procedures and indications for use must be posted and routine function checks must be
performed. Testing eye wash fountains weekly helps ensure proper function and flushes out the
stagnant water. Portable eye wash systems are allowed only if they can deliver flushing fluid to
the eyes at a rate of at least 1.5 liters per minute for 15 minutes. They should be monitored 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.
(cont’d)

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 2-3. 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.

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): ____________________________
____________________________________________________________________________

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

Fire:_________________________________________________________________________
Decontamination procedures: ____________________________________________________
Disposal procedures: ___________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

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

Appendix 2-5. Sample List of Hazardous Chemicals in the Blood Bank (cont'd)
Chemical Hazard
Sodium phosphate Irritant, hygroscopic

Sulfosalicylic acid Toxic, corrosive
Trichloroacetic acid (TCA) Corrosive, toxic
Trypsin Irritant, sensitizer
Xylene Highly flammable, toxic, irritant

Appendix 2-6. Specific Chemical Categories and How to Work Safely with These
Chemicals
Chemical
Category Hazard Precautions Special Treatment
Acids, alkalis, Irritation During transport, protect Store concentrated acids in

and corrosive Severe burns large containers with acid safety cabinets
compounds Tissue damage plastic or rubber bucket Limit volumes of concen-
carriers trated acids to 1 liter
During pouring, wear eye Post cautions for materials
protection and chemical- in the area
resistant-rated gloves Report changes in appear-
and gowns as recom- ance (perchloric acid
mended may be explosive if it be-
Always ADD ACID TO comes yellowish or
WATER, never water to brown) to chemical
acid safety officer
When working with large
jugs, have one hand on
the neck and the other at
the base, and position
them away from the face
Acrylamide Neurotoxic Wear chemically rated Store in a chemical cabinet
Carcinogenic gloves
Adsorbed Wash hands immediately
through the after exposure
skin
Compressed Explosive Label as to contents Transport using hand trucks
gases Leave valve safety covers on or dollies
until use Place cylinders in a stand or
Open valves slowly for use secure them to prevent
Label empty tanks falling
Store in well-ventilated 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 Use heavy insulated gloves The tanks should be se-
Severe burns to and goggles when work- curely supported to avoid
skin or eyes ing with liquid nitrogen being tipped over
The final container of liquid
nitrogen (freezing unit)
must be securely sup-
ported to avoid tipping
over

Appendix 2-6. Specific Chemical Categories and How to Work Safely with These
Chemicals (cont'd)
Chemical
Category Hazard Precautions Special Treatment
Flammable Classified accord- Use extreme caution when Make every attempt to re-
solvents ing to flash handling place hazardous materi-
point—see Post NO SMOKING signs in als with less hazardous
MSDS working area materials
Classified accord- Have a fire extinguisher and Store containers larger than
ing to volatility solvent cleanup kit in the 1 gallon in a flammable
room solvent storage room or
Pour volatile solvents under in a fire safety cabinet
suitable hood Ground metal containers by
Use eye protection when connecting the can to a
pouring and chemical-re- water pipe or ground
sistant neoprene gloves connection; if recipient
No flame or other source of container is also metal, it
possible ignition should should be electrically
be in or near areas where connected to the delivery
flammable solvents are container while pouring
being poured
Label as FLAMMABLE

84
Appendix 2-7. Incidental Spill Response*

Chemicals Hazards PPE Control Materials
Acids Severe irritant if inhaled Acid-resistant gloves Acid neutralizers/

Acetic Contact causes burns to skin Apron and coveralls absorbent
Hydrochloric and eyes Goggles and face shield Absorbent boom
Nitric Corrosive Acid-resistant foot Leakproof containers
Perchloric Fire or contact with metal may covers Absorbent pillow
Sulfuric produce irritating or poison- Mat (cover drain)
Photographic ous gas Shovel or paddle

chemicals Nitric, perchloric, and sulfuric
(acid) acids are water-reactive oxi-
dizers
Bases and caustics Corrosive Gloves; impervious apron or Base control/neutralizer
Potassium hydroxide Fire may produce irritating or coveralls Absorbent pillow
Sodium hydroxide poisonous gas Goggles or face shield; impervi- Absorbent boom
Photographic chemicals ous foot covers Drain mat
(basic) Leakproof container
Shovel/paddle
Chlorine Inhalation can cause respiratory Gloves (double set 4H Chlorine control powder
Bleach irritation undergloves and butyl or Absorbent pillow
Sodium hypochlorite Liquid contact can produce irri- nitrile overgloves); Absorbent
tation of the eyes or skin impervious apron Absorbent boom
Toxicity due to alkalinity, possi- or coveralls Drain mat
ble chlorine gas generation, Goggles or face shield Vapor barrier
and oxidant properties Impervious foot covers (neo- Leakproof container
prene boots for emergency Shovel or paddle
response releases)
Self-contained breathing appara-
tus (emergency response
releases)
Cryogenic gases Contact with liquid Full face shield or goggles; neo- Hand truck (to transport cylinder
Carbon dioxide nitrogen can produce prene boots; gloves (insu- outdoors if necessary)
Nitrous oxide frostbite lated to protect from the cold) Soap solution (to check for
Liquid nitrogen Asphyxiation (displaces oxygen) leaks)
Anesthetic effects Putty (to stop minor pipe and
(nitrous oxide) line leaks)
Flammable gases Simple asphyxiate Face shield and goggles; neo- Hand truck (to transport cylinder
Acetylene (displaces air) prene boots; double set of outdoors if needed)
Oxygen gases Anesthetic potential gloves; coveralls with hood Soap solution (to check for
Butane Extreme fire and explosion and feet leaks)

Propane hazard
Release can create an oxy-
gen-deficient
atmosphere
Flammable liquids Vapors harmful if inhaled Gloves (double 4H undergloves Absorbent
Acetone (central nervous system and butyl or nitrile Absorbent boom
Xylene depressants) overgloves); impervious Absorbent pillow
Methyl alcohol toluene Harmful via skin absorption apron or coveralls; goggles or Shovel or paddle (nonmetal,
Ethyl alcohol Extreme flammability face shield; impervious foot nonsparking)
Other alcohols Liquid evaporates to form flam- covers Drain mat
mable vapors Leakproof containers

Formaldehyde and Harmful if inhaled or absorbed Gloves (double set 4H Aldehyde neutralizer/absorbent
glutaraldehyde through skin; undergloves and butyl or Absorbent boom
4% formaldehyde Irritation to skin, eyes, and nitrile overgloves); impervi- Absorbent pillow
37% formaldehyde respiratory tract ous apron or coveralls; gog- Shovel or pallet (nonsparking)
10% formalin Formaldelyde is a suspected gles; impervious foot covers Drain mat
2% glutaraldehyde human carcinogen Leakproof container
Keep away from heat, sparks,
and flame (37% formalde-
hyde)
85
(cont’d)
86
Appendix 2-7. Incidental Spill Response* (cont'd)
Chemicals Hazards PPE Control Materials
Mercury Mercury and mercury vapors are Gloves (double set 4H Mercury vacuum or spill kit
Cantor tubes rapidly absorbed in respira- underglove and butyl or nitrile Scoop
Thermometers tory tract, GI tract, skin overglove); impervious apron Aspirator

Barometers Short-term exposure may cause or coveralls; goggles; imper- Hazardous waste containers
Sphygmomanometers erosion of respiratory/GI vious foot covers Mercury indicator powder
Mercuric chloride tracts, nausea, vomiting, Absorbent
bloody diarrhea, shock, head- Spatula
ache, metallic taste Disposable towels
Inhalation of high concentrations Sponge with amalgam
can cause pneumonitis, chest Vapor suppressor
pain, dyspnea, coughing
stomatitis, gingivitis, and sali-
vation
Avoid evaporation of mercury
from tiny globules by quick
and thorough cleaning
*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 2-8. 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, Isolate the spill area and evacuate everyone from the area surrounding the
and secure the spill except those responsible for cleaning up the spill. (For mercury,
area evacuate within 10 feet for small spills, 20 feet for large spills.) Secure
area.
Have the appropriate See Appendix 2-2 for recommended PPE.
PPE
Contain the spill Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release (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)

Appendix 2-8. Managing Hazardous Chemical Spills (cont'd)
Actions Instructions for Hazardous Liquids, Gases, and Mercury
Disposal Liquids: For material that was neutralized, dispose of it as solid waste. 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.

Chapter 3: Blood Utilization Management
Chapter 3
Blood Utilization
Management
3
T
HE GOAL OF BLOOD utilization not limited to, outdate rates, the frequency
management is to ensure effective of emergency blood shipments, and delays
use of limited blood resources. It in scheduling elective surgery. Inventory
includes the policies and practices related levels should also be reevaluated when-
to inventory management and blood us- ever a significant change is planned or
age review. Although regional blood cen- observed. Examples of significant change
ters and transfusion services approach may include adding more beds; perform-
utilization management from different ing new surgical procedures; or changing
perspectives, they share the common goal practices in oncology, transplantation, neo-
of providing appropriate, high-quality natology, or cardiac surgery.
blood products with minimum waste. This
chapter reviews the elements of utiliza-
tion management, emphasizing the trans-
fusion service. Determining Inventory
Levels
The ideal inventory level provides ade-
Minimum and Ideal quate supplies of blood for routine and
emergency situations and minimizes out-
Inventory Levels dating. Forecasting is an attempt to pre-
Transfusion services should establish both dict future blood product use from data
minimum and ideal inventory levels. In- collected about past usage. The optimal
ventory levels should be evaluated period- number of units to keep in inventory can
ically and adjusted if needed. Important be estimated using mathematical formu-
indicators of performance include, but are las, computer simulations, or empirical
89

calculations. Three less complicated meth- required to be on hand (this may be 3,

ods of estimating minimum inventory are 5, or 7 days depending on the blood
described below. When the minimum in- supplier’s delivery schedule).
ventory level has been calculated, a buffer A transfusion service may find the aver-
margin for emergencies should be added age daily use calculation more helpful
to obtain an ideal inventory level. when blood shipments are made once or
more per day.
Average Weekly Use Estimate
This method gives an estimate of the av- Moving Average Method
erage weekly blood usage of each ABO The moving average method can be use-
group and Rh type. ful in facilities with any level of activity.
1. Collect weekly blood and product 1. Determine the preferred recording
usage data over a 26-week period. period (such as day or week).
2. Record usage by ABO group and Rh 2. Add the number of units used in each
type for each week. period to obtain the total use.
3. Disregard the single highest usage 3. Divide the total number of units by
for each type to correct for unusual the number of recording periods.
week-to-week variation (eg, a large 4. Delete old data as new data are added.
volume used for an emergency). This method tends to minimize variation
4. Total the number of units of each from one period to another.
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). Factors that Affect Outdating
This gives an estimate of the average
Benchmark data on component outdating
weekly blood usage of each ABO
have been published by the National Blood
group and Rh type.
Data Resource Center (NBDRC).1 The data
from this report showed that approxi-
Average Daily Use Estimate mately 2,190,000 total components out-
Facilities that transfuse on a daily basis dated in 2001, a 6.6% decrease from 1999.
may calculate daily blood usage by the Whole-blood-derived platelet concen-
following method. trates accounted for more than half of the
1. Determine the total use over several outdated components (49%), or 1,074,000
months. units. Outdated allogeneic RBCs (directed
2. Divide the total use by the number and nondirected) accounted for 26.8% of
of days in the period covered. all outdated components, or 588,000 units.
3. Determine the percentage of each of The outdate rate for each component is
the blood types used during one or shown in Table 3-1. Outdating continues to
more representative months. be a problem, particularly for autologous
4. Multiply the average blood use per units and platelets. The outdate rate is af-
day by the percentage of blood use fected by many factors other than inven-
by type. tory level (eg, the size of the hospital, the
5. Determine the minimum inventory extent of services provided, the shelf life
level by multiplying the daily use by of the products, the shipping distance
the number of days of blood supply and frequency, and ordering policies).

Chapter 3: Blood Utilization Management 91
Table 3-1. Blood Component Units Processed, Transfused, and Outdated in United
1
States in 2001
Units Units Units Percent

Component Processed Transfused Outdated Outdated
RBCs (allogeneic, 14,259,000 13,361,000 576,000 4.0

nondirected)
RBCs (autologous) 619,000 359,000 263,000 42.5
RBCs (directed) 169,000 95,000 12,000 7.1
Platelets 4,164,000 2,614,000 1,074,000 25.8

(whole-blood-
derived)
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.
In addition to establishing both mini- tion. Technologists generally find it easier

mum and ideal inventory levels, maximum to select, crossmatch, and issue the oldest
inventory levels may assist staff in deter- units first when inventories are arranged by
mining when to arrange for return or trans- expiration date.
fer of in-date products to avoid outdating. Policies on blood selection must be flexi-
Both transfusion services and donor cen- ble, to allow use of fresher blood when indi-
ters should establish record-keeping sys- cated (eg, for infants). Generally, however,
tems that allow personnel to determine the oldest units are crossmatched for patients
number of units ordered and the number of most likely to need transfusion.
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 Improving Transfusion
planning for both transfusion services and Service Blood Ordering
blood centers. Blood centers may send a
predetermined number of units on a regu-
Practices
lar schedule or may keep the transfusion The shelf life decreases each time a unit is
service inventory at established levels by held or crossmatched for a patient who
replacing all units reported as transfused. does not use it. When physicians order more
Optimal inventory management requires blood than needed, it is unavailable for
distribution and transfusion of the oldest other patients, which may increase the
blood first and this requires clearly written outdate rate. Providing testing guidelines,
policies on blood storage and blood selec- such as type and screen (T/S) policies and

maximum surgical blood order schedules Table 3-2. Example of a Maximum

(MSBOS),2 as well as monitoring cross- Surgical Blood Order Schedule
match-to-transfusion (C:T) ratios may be
helpful. A C:T ratio greater than 2.0 usu- Procedure Units*
ally indicates excessive crossmatch re-
General Surgery
quests. In some situations, it may be use- Breast biopsy T/S
ful to determine C:T ratios by service to Colon resection 2
identify areas with the highest ratio. Exploratory laparotomy T/S
Some institutions define those proce- Gastrectomy 2
dures that normally do not use blood in a Laryngectomy 2
“type and screen” guideline. Both the T/S Mastectomy, radical T/S
guideline and the MSBOS use data about Pancreatectomy 4
past surgical blood use to recommend a Splenectomy 2
T/S order or a maximum number of units Thyroidectomy T/S
that should be ordered initially for common
Cardiac-Thoracic
elective surgical procedures. With the
Aneurism resection 6
MSBOS, physicians may order the number Redo coronary artery bypass graft 4
of units believed to be appropriate for the Primary coronary artery bypass graft 2
patient since the MSBOS is intended to be a Lobectomy T/S
guideline for appropriate patient care. Lung biopsy T/S
Some institutions have modified the MSBOS
concept into a “standard” blood order (SBO) Vascular
system for surgical procedures.3 Aortic bypass with graft 4
Ordering guidelines such as those in Ta- Endarterectomy T/S
ble 3-2 are derived by reviewing a facility’s Femoral-popliteal bypass with graft 2
blood use over a suitable period. Conclu-
Orthopedics
sions can then be drawn about the likeli-
Arthroscopy T/S
hood of transfusion and probable blood Laminectomy T/S
use for each surgical procedure. A T/S order Spinal fusion 3
is a recommended SBO for procedures that Total hip replacement 3
require on average less than 0.5 unit of Total knee replacement T/S
blood per patient per procedure. An SBO
often represents the average number of OB-GYN
units transfused for each procedure, where- Abdomino-perineal repair T/S
as the MSBOS often defines the number of Cesarean section T/S
units needed to meet the needs of 80% to Dilation and curettage T/S
Hysterectomy, abdominal/ T/S
90% of patients undergoing a specific surgi-
laparoscopic
cal procedure.3 Hysterectomy, radical 2
An institution’s guidelines must reflect
local patterns of surgical practice and pa- Urology
tient population. These may be compared Bladder, transurethral resection T/S
to published guidelines to ensure that local Nephrectomy, radical 3
practice does not markedly deviate from Radical prostatectomy, perineal 2
generally accepted standards of care. Prostatectomy, transurethral T/S
(Transfusion audits are discussed in Chap- Renal transplant 2
ter 1.) Once the T/S, MSBOS, SBO, or other *Numbers may vary with institutional practice.

schedule is accepted, inventory levels often shortages and unexpected emergencies.

can be reduced. Ordering guidelines should Equally important is specifying the actions
be periodically reviewed to keep pace with to take if transfusion requests cannot im-
changing methods and practices. A change mediately be met.
in the C:T ratio might signal a significant Transfusion services should develop pol-
modification in clinical practice. icies defining the following:
The T/S, MSBOS, or SBO systems are in- ■ When ABO-compatible units may be
tended for typical circumstances. Surgeons given instead of ABO-identical units.
or anesthesiologists may individualize spe- ■ When Rh-positive units may be given
cific requests and override the system to ac- to Rh-negative recipients.
commodate special needs. The transfusion ■ When units crossmatched for a sur-
service must give special consideration to gical procedure may be released be-
patients with a positive antibody screen. fore the standard interval.
The antibody should be identified and, if it ■ If units may be crossmatched for more
is potentially clinically significant, an ap- than one patient at a time.
propriate number of antigen-negative units ■ What resources are available for trans-
should be identified (eg, two, if the original fer of inventory.
order was a type and screen). ■ Mechanisms to notify physicians of
Facilities using the immediate spin or critical blood shortages.
computer crossmatch can provide addi- ■ When cancellation of elective proce-
tional crossmatched units more rapidly if dures should be considered.
required. This capability can allow such fa- ■ Methods to notify staff and patients
cilities to adjust their T/S, MSBOS, and SBO of surgery cancellations.
schedules accordingly. More procedures
can be safely handled as type and screens
and fewer crossmatched units may be nec-
Inventory Counts and Inspection
essary. On-hand units may be counted once or
several times a day to determine ordering
Routine vs Emergency Orders needs; computerized facilities may prefer
to take inventory electronically. Individ-
Transfusion services should establish pro- ual units must be visually inspected for
cedures that define ideal stocking levels for signs of contamination or atypical ap-
each blood type and critical levels at which pearance before issue or shipping. Units
emergency orders are indicated. Transfu- that do not meet inspection criteria must
sion service staff should have institutional be quarantined for further evaluation.
policies identifying the following: An organizational format for storage
■ Who monitors inventory levels? should be established and followed. Unpro-
■ Who is responsible for placing orders? cessed or incompletely processed units,
■ When and how are orders to be autologous units, and unsuitable units
placed (by telephone or facsimile)? must be clearly segregated (quarantined)
■ How are orders documented? from routine stock.4(p15) Most institutions or-
The addresses and telephone numbers of ganize their blood inventory by status
approved blood suppliers and any needed (quarantined, retype unconfirmed, retype
courier or cab services should be immedi- confirmed, available, crossmatched, etc), by
ately available. Transfusion services need to product, by ABO group and Rh type, and,
establish guidelines for handling blood within these categories, by expiration date.

Attention to detail in placing blood into products. The formulas described in the
storage is necessary because a placement beginning of the chapter may be used to
error could be critical if a quarantined unit estimate ideal inventory ranges.
is issued or a group O Rh-positive unit, in- Platelet usage often increases the day af-
correctly placed among group O Rh-nega- ter a holiday because elective procedures
tive units, is issued without careful check- and oncology transfusions will have been
ing in an emergency situation. postponed. Planning ahead to stock plate-
lets helps meet postholiday demand.
Frozen Plasma Products

Special Product Concerns Because plasma components can be stored
Platelets frozen up to 1 year, these inventories are
easier to manage. Optimal inventory lev-
Few articles address the management of
els are determined by assessing statistics
platelet inventory. Optimal levels are diffi-
on patient populations and usage pat-
cult to determine because demand is epi-
terns. Production goals and schedules can
sodic and the shelf life is short. Often, the
then be established. Most centers find it
effective shelf life is 3 days because, of the
best to maintain consistent production
allowable 5 days after phlebotomy, day 1
levels throughout the year, to achieve
may be taken for testing and day 2 for
evenly distributed expiration dates.
shipment. Planning is further complicated
Some facilities prefer to freeze plasma
by requests for special products, such as
from group AB and A donors because these
leukocyte-reduced, crossmatched, HLA-
units will be ABO-compatible with most
matched, or cytomegalovirus (CMV)-sero-
potential recipients. Plasma can be col-
negative platelets.
lected by apheresis to increase general
Platelet inventory management requires
stock and provide for special needs.
good communication and cooperation
Cryoprecipitated AHF is a labor-inten-
among patient care providers, transfusion
sive product to prepare and supplies can-
services, and blood centers. Information
not easily be increased to meet large acute
about patients’ diagnoses and expected
needs. It is prudent to maintain inventories
transfusion schedules helps the blood cen-
at close-to-maximum levels.
ter plan how many platelets to prepare and
which donors to recruit for plateletpheresis.
Transfusion services with low platelet Autologous and Directed Units
use usually order platelets only when they If autologous and directed donor units
receive a specific request. If the transfusion constitute an increasing fraction of inven-
service staff follows daily platelet counts tory, their management becomes a signif-
and special transfusion requirements of icant and controversial issue both for the
known platelet users, they can often antici- intended recipients and for institutions.
pate needs and place orders in advance. An extended discussion of autologous
Transfusion services with high use may find blood collection and transfusion methods
it helpful to maintain platelets in inventory. can be found in Chapter 5.
Transfusion services should define selec- Autologous and directed units should be
tion and transfusion guidelines for ABO stored in separate designated areas in the
group, Rh type, irradiation, CMV serologic blood refrigerator. Such units are often ar-
testing, and leukocyte reduction of platelet ranged alphabetically by the intended re-

cipient’s last name. Available units must be If demand and inventory levels are very
clearly identified and monitored to ensure high, a transfusion service may need to
issue in the proper sequence. Autologous keep separate inventories of these products
blood should always be used first, followed to make them easier to locate and monitor.
by directed donor blood, and, finally, allo- These special units can be rotated into gen-
geneic units from general stock. Policies eral stock as they near their outdate be-
about the reservation period for directed cause they can be given safely to others.
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, References
to potential recipients, and to their physi-
1. Comprehensive report on blood collection
cians. and transfusion in the United States in 2001.
Bethesda, MD: National Blood Data Resource
Special Inventories Center, 2003.
2. Friedman BA, Oberman HA, Chadwick AR, et
Donor centers and transfusion services al. The maximum surgical blood order sched-
are faced with requests for specialty prod- ule and surgical blood use in the United States.
Transfusion 1976;16:380-7.
ucts such as CMV-reduced-risk units,
3. Devine P, Linden JV, Hoffstadter L, et al. Blood
HLA-matched platelets, antigen-matched donor-, apheresis-, and transfusion-related
red cells, or irradiated components. The activities: Results of the 1991 American Asso-
appropriate use of these products is dis- ciation of Blood Banks Institutional Member-
ship Questionnaire. Transfusion 1993;33:779-
cussed in other chapters. Depending on 82.
how and when they were prepared, these 4. Silva MA, ed. Standards for blood banks and
products may have shortened expiration transfusion services. 23rd ed. Bethesda, MD:
dates. AABB, 2005:15.

Chapter 4: Allogeneic Donor Selection and Blood Collection
Chapter 4
Allogeneic Donor Selection

and Blood Collection
B
LOOD CENTERS AND transfusion fortably ventilated, orderly, and clean.
services depend on volunteer do- Personnel should be friendly, understand-
nors to provide the blood neces- ing, professional, and well trained. The area
sary to meet the needs of the patients must provide adequate space for private
they serve. To attract volunteer donors and accurate examinations of individuals
and encourage their continued participa- to determine their eligibility as blood do- 4
tion, it is essential that conditions sur- nors, and for the withdrawal of blood
rounding blood donation be as pleasant, from donors with minimum risk of con-
safe, and convenient as possible. To pro- tamination or exposure to activities and
tect donors and recipients, donors are equipment unrelated to blood collection.
questioned about their medical history
and are given a miniphysical examination Registration
to help blood center staff determine The information obtained from the donor
whether they are eligible donors. The during registration must fully identify the
phlebotomy is conducted carefully to donor and link the donor to existing re-
minimize any potential donor reactions cords.1(p13) Some facilities require photo-
or bacterial contamination of the unit. graphic identification. Current informa-
tion must be obtained and recorded for
each donation. Selected portions of dona-
tion records must be kept indefinitely and
Blood Donation Process must make it possible to notify the donor
The donor area should be attractive, ac- of any information that needs to be con-
1(p69)
cessible, and open at hours convenient veyed. The following information should
for donors. It must be well lighted, com- be included:
97

1. Date and time of donation. expected antibodies. Care should be

2. Name: Last, first (and middle initial taken to be sure that minority popu-
if available). lations understand the medical im-
3. Address: Residence and/or business. portance and scientific applications
4. Telephone: Residence and/or busi- of this information.3,4
ness. 4. Unique characteristics of the donor.
5. Gender. Certain information about the donor
6. Age or date of birth. Blood donors must may enable the blood bank to make
be at least 17 years of age or the age optimal use of the donation. For ex-
stipulated by applicable state law. ample, blood from donors who are
7. A record of reasons for previous de- seronegative for cytomegalovirus
ferrals, if any. Persons who have been (CMV), or who are group O, Rh neg-
placed on a deferral or surveillance ative, is often designated for neona-
list must be identified before any tal patients. The blood center may
unit drawn from them is made avail- specify that blood from these indi-
able for release. Ideally, a donor de- viduals be drawn routinely into col-
ferral registry should be available to lection bags suitable for pediatric
identify ineligible donors before transfusion. Individuals known to
blood is drawn. If such a registry is have clinically significant antibodies
not available, there must be a proce- may be identified so that their blood
dure to review prior donation re- can be processed into components
cords and/or deferral registries be- that contain only minimal amounts
fore releasing the components from of plasma.
quarantine.2 5. A record of special communications
The following information may also be to a donor, special drawing of blood
useful: samples for studies, etc.
1. Additional identification such as so- 6. If the donation is directed to a spe-
cial security or driver’s license num- cific patient, information about when
ber or any other name used by the and where the intended recipient will
donor on a previous donation. The be hospitalized should be obtained.
Social Security Act specifically al- An order from the intended recipi-
lows the use of the social security ent’s physician should be provided
number for this purpose. These data to the blood center staff. The in-
are required for information retrieval tended recipient’s date of birth, so-
in some computerized systems. Iden- cial security number, or other identi-
tification of other names used by a fiers may be required by the transfusion
donor is particularly important to service. If the donor is a blood relative
ensure that the appropriate donor of the intended recipient, this infor-
file is accessed or that a deferral sta- mation must be noted so that cellular
tus is accurate. 1(p43)
components can be irradiated.
2. Name of patient or group to be ac-
knowledged.
3. Race. Although not required, this in-
Information Provided to the Prospective
formation can be particularly useful
Donor
when blood of a specific phenotype All donors must be given educational ma-
is needed for patients who have un- terials informing them of significant risk

Chapter 4: Allogeneic Donor Selection and Blood Collection 99
of the procedure, the clinical signs and abnormal test results are recorded and the
symptoms associated with human immu- donor has been placed on a deferral list.
nodeficiency virus (HIV ) infection and When applicable, the donor must also be
AIDS, high-risk activities for transmission, informed that his or her blood is to be
and the importance of refraining from do- tested with an investigational test such as a
nating blood if they have engaged in these nucleic acid amplification test. The possi-
activities or experienced associated signs bility that testing may fail to identify infec-
or symptoms. Before donating, the pro- tive individuals in an early seronegative
spective donors must document that they stage of infection should be included as
have read the material and have been well.6 The same educational material can
given the opportunity to ask questions be used to warn the prospective donor of
about the information. This information possible reactions and provide suggestions
must include a list of activities defined by for postphlebotomy care.
the Food and Drug Administration (FDA) This information should be presented in
that increase the risk of exposure to HIV. A a way that the donor will understand.5 Pro-
description of HIV-associated clinical signs visions should be made for the hearing- or
and symptoms, including the following, vision-impaired, and interpreters should be
must be provided5: available for donors not fluent in English.
1. Unexplained weight loss. The use of interpreters known to the donor
2. Night sweats. should be discouraged. If such a practice is
3. Blue or purple spots on or under the necessary, a signed confidentiality state-
skin or on mucous membranes. ment should be obtained. In some loca-
4. Swollen lymph nodes lasting more tions, it may be helpful to have brochures
than 1 month. in more than one language. It is also helpful
5. White spots or unusual sores in the to provide more detailed information for
mouth. first-time donors. Information about alter-
6. Temperature greater than 100.5 F for native sites or other mechanisms to obtain
more than 10 days. HIV tests should be available to all prospec-
7. Persistent cough and shortness of tive donors.
breath.
8. Persistent diarrhea.
The donor should be provided with
Donor Selection
information about the tests to be done on
his or her blood, the existence of registries The donor screening process is one of the
of ineligible donors, and regulations or lo- most important steps in protecting the
cal standard operating procedures (SOPs) safety of the blood supply. The process is
that require notification to government intended to identify elements of the med-
agencies of the donor’s infectious disease ical history and behavior or events that
status. The requirement to report positive put a person at risk for transmissible dis-
test results may differ from state to state; ease or at personal medical risk. It is,
they may include HIV, syphilis, and hepati- therefore, imperative that proper guide-
tis testing. Prospective donors must also be lines and procedures be followed to make
informed if there are routine circumstances the donor screening process effective.
in which some tests for disease markers are A qualified physician must determine
not to be performed.1(p15) The donor should the eligibility of donors. The responsibility
be told that he or she will be notified when may be delegated to a designee working

under the physician’s direction after appro- the job. Good interpersonal and public re-
priate training.7 Donor selection criteria are lations skills are essential for job
established through regulations, recom- competency. Because donor center staff are
mendations, and standards of practice. in constant contact with donors, knowl-
When a donor’s condition is not covered or edgeable personnel and effective commu-
addressed by any of these, a qualified phy- nication contribute to positive public
sician should determine the eligibility. perception and to the success of donor
Donor selection is based on a medical screening programs.
history and a limited physical examination
done on the day of donation to determine
Medical History
if giving blood will harm the donor or if
transfusion of the unit will harm a recipi- While the medical history is obtained,
ent.1(p17) The medical history questions (in- some very specific questions are neces-
cluding questions pertaining to risk behav- sary to ensure that, to the greatest extent
ior associated with HIV infections) may be possible, it is safe for the donor to donate
asked by a qualified interviewer or donors and for the blood to be transfused. The in-
may complete their own record, which terviewer should review and evaluate all
must then be reviewed with the donor and responses to determine eligibility for do-
initialed by a trained knowledgeable staff nation and document the decision. To be
member of the donor service according to sure that all the appropriate questions are
local SOPs, state, and FDA approval.5,8 Some asked and that donors are given a consis-
donor centers have instituted an FDA-ap- tent message, use of the most recent FDA-
proved computer-generated questionnaire. approved AABB donor history question-
The interview and physical examination naire is recommended. The most recent
must be performed in a manner that en- FDA-approved version is found on the AABB
sures adequate auditory and visual privacy, Web site. (See Appendices 4-1 through 4-3,
allays apprehensions, and provides time for which were current at the time of this writ-
any necessary discussion or explanation. ing.)
Details explaining a donor’s answers that One area of the medical history—medi-
require further investigation should be doc- cations and drugs taken by the donor—of-
umented by the staff on the donor form. ten requires further investigation. New pre-
Results of observations made when a phys- scription drugs and over-the-counter
ical examination is given and when tests medications enter the marketplace daily,
are performed must be recorded concur- and donors may report use of a drug not
rently. specifically noted in the facility’s SOP man-
Donors must understand the informa- ual. Although there is consensus on those
tion that is presented to them in order to drugs that are always or never a cause for
make an informed decision to donate their deferral, many drugs fall into a category
blood. Effective communication is vital for over which disagreement exists. In these
conveying important information and elimi- cases, the reason for taking the drug (rather
nating ineligible donors from the donor than the drug itself) is usually the cause for
pool. Of equal importance is the training of deferral. Appendix 4-4 lists drugs that many
donor center staff. Screening can be effec- blood banks do consider acceptable with-
tive only if the staff members are proficient out approval from a donor center physician.
in their jobs and understand thoroughly the Prospective donors who have taken iso-
technical information required to perform tretinoin (Accutane) or finasteride (Proscar

or Propecia) within the 30 days preceding results. Counseling or referral must be pro-
donation; dutasteride (Avodart) within the vided for positive HIV test results, or if any
6 months preceding donation; acitretin other medically significant test results have
(Soriatane) within the 3 years preceding do- been detected.
nation; or etretinate (Tegison) at any time
must be deferred.1(p62) The Armed Services Physical Examination
Blood Program Office makes its drug defer- The following variables must be evaluated
ral list available to the public.9 for each donor. The donor center physi-
Deferring or rejecting potential donors cian must approve exceptions to routinely
often leaves those persons with negative acceptable findings. For special donor
feelings about themselves as well as the categories, the medical director may pro-
blood donation process. Donors who are vide policies and procedures to guide de-
deferred must be given a full explanation of cisions. Other donors may require indi-
the reason and be informed whether or vidual evaluation.
when they can return to donate. It may be 1. General appearance: If the donor
prudent to document this notification. looks ill, or is excessively nervous, it
is best to defer the donation.
Confidential Unit Exclusion 2. Weight: No more than 10.5 mL of
Donors may be given the opportunity to whole blood per kilogram of body
indicate confidentially whether their blood weight shall be collected at a dona-
is or is not suitable for transfusion to oth- tion.1(p61) This amount shall include
ers. This should be done by a mechanism samples for testing. If it is necessary
that allows the donor to avoid face-to- to draw a smaller amount than ap-
face admission of risk behaviors. propriate for a standard collection
The donor must be given instructions to container, then the amount of anti-
the effect that he or she may call the blood coagulant in the container must be
bank after the donation and ask that the adjusted appropriately. The formula
unit collected not be used. A mechanism in Table 4-1 may be used to deter-
should exist to allow retrieval of the unit mine the amount of anticoagulant to
without obtaining the donor’s identity (eg, remove. The volume of blood drawn
use of Whole Blood number). must be measured carefully and ac-
If the donor indicates that blood col- curately.
lected should not be used for transfusion, 3. Temperature: The donor’s tempera-
he or she should be informed that the ture must not exceed 37.5 C (99.5 F)
blood will be subjected to testing and that if measured orally, or its equivalent if
there will be notification of any positive measured by another method. Lower
Table 4-1. Calculations for Drawing Donors Weighing Less than 50 kg (110 lb)
A. Volume to draw* = (Donor’s weight in kg/50) × 450 mL

B. Amount of anticoagulant† needed = (A/100) × 14
C. Amount of anticoagulant to remove from collection bag = 63 mL – B
*Approximately 12% of total blood volume.
†
CPD or CPDA-1 solutions for which the desired anticoagulant:blood ratio is 1.4:10.

than normal temperatures are usu- the lower limits of hemoglobin for
ally of no significance in healthy in- accepting allogeneic donors. Indi-
dividuals; however, they should be viduals with unusually high hemo-
repeated for confirmation. globin or hematocrit levels may need
4. Pulse: The pulse rate should be counted to be evaluated by a physician be-
for at least 15 seconds. It should ex- cause the elevated levels may reflect
hibit no pathologic irregularity, and pulmonary, hematologic, or other
the frequency should be between 50 abnormalities. Methods to evaluate
and 100 beats per minute. If a pro- hemoglobin concentration include
spective donor is a known athlete 1) specific gravity determined by cop-
with high exercise tolerance, a pulse per sulfate (see Method 6.1), 2) spec-
rate below 50 may be noted and trophotometric measurement of he-
should be acceptable. A donor cen- moglobin or determination of the
ter physician should evaluate marked hematocrit, or 3) alternate accepted
abnormalities of pulse and recom- methods to rule out erroneous re-
mend acceptance, deferral, or refer- sults that may lead to rejection of a
ral for additional evaluation. donor. Earlobe puncture is not an
5. Blood pressure: The blood pressure acceptable source for a blood sam-
should be no higher than 180 mm Hg ple.1(p61),10
systolic and 100 mm Hg diastolic. Pro- 7. Skin lesions: The skin at the site of
spective donors whose blood pres- venipuncture must be free of lesions.
sure is above these values should not Both arms must be examined for
be drawn without individual evalua- signs of repeated parenteral entry,
tion by a qualified physician. especially multiple needle puncture
6. Hemoglobin or packed cell volume marks and/or sclerotic veins as seen
(hematocrit): Before donation, the with drug use. Such evidence is rea-
hemoglobin or hematocrit must be son for indefinite exclusion of a pro-
determined from a sample of blood spective donor. Mild skin disorders
obtained at the time of donation. or the rash of poison ivy should not
Although this screening test is in- be cause for deferral unless unusu-
tended to prevent collection of blood ally extensive and/or present in the
from a donor with anemia, it does antecubital area. Individuals with
not ensure that the donor has an ad- boils, purulent wounds, or severe
equate store of iron. Table 4-2 gives skin infections anywhere on the
Table 4-2. Minimum Levels of Hemoglobin, Hematocrit, and Red Cell Density for
Accepting an Allogeneic Blood Donor
1
Donor Test Method Minimal Acceptable Value
Hemoglobin 12.5 g/dL

Hematocrit 38%
Copper sulfate 1.053 sp gr

body should be deferred, as should plasma. If I am potentially at risk for

anyone with purplish-red or hemor- spreading the virus known to cause AIDS, I
rhagic nodules or indurated plaques agree not to donate blood or plasma for
suggestive of Kaposi’s sarcoma. transfusion to another person or for further
The record of the physical examination manufacture. I understand that my blood
and the medical history must identify and will be tested for HIV and other disease
contain the examiner’s initials or signature. markers; however, there may be unforeseen
Any reasons for deferral must be recorded circumstances when infectious disease test-
and explained to the donor. A mechanism ing may not be performed. If this testing in-
must exist to notify the donor of clinically dicates that I should no longer donate blood
significant abnormal findings in the physi- or plasma because of the risk of transmitting
cal examination, medical history, or post- an infectious disease, my name will be en-
donation laboratory testing.11 Abnormalities tered on a list of permanently deferred do-
found before donation may be explained nors. I understand that I will be notified of a
verbally by qualified personnel. Test results positive laboratory test result(s). If, instead,
obtained after donation that preclude fur- the results of the testing are not clearly nega-
ther donation may be reported in person, tive or positive, my blood will not be used
by telephone, or by letter. Donors should be and my name may be placed on a deferral
asked to report any illness developing with- list.”
in a few days after donation and, especially, If units are occasionally used for reasons
to report a positive HIV test or the occur- other than transfusion, such as research,
rence of hepatitis or AIDS that develops then the informed consent should address
within 12 months after donation. such occasions.
Donor Consent Special Donor Categories

Written consent that allows donor center Exceptions to the usual eligibility require-
personnel to collect and use blood from ments may be made for special donor cat-
the prospective donor is required.1(p15) The egories:
consent form is part of the donor record 1. Autologous donors: The indications
and must be completed before donation. for collection and variations from
The procedure must be explained in usual donor procedures are discus-
terms that donors can understand, and sed in Chapter 5.
there must be an opportunity for the pro- 2. Hemapheresis: Special requirements
spective donor to ask questions. The and recommendations for cytaphere-
signed donor record or consent form sis donors or for donors in a plasma-
should also indicate that the donor has pheresis program are detailed in
read and understood the information Chapter 6.
about infectious diseases transmissible by 3. Recipient-specific “designated” dona-
transfusion and has given accurate and tions: Under certain circumstances,
truthful answers to the medical history it may be important to use blood or
questions. Wording equivalent in mean- components from a specific donor
ing to the following is suggested: for a specific patient. Examples in-
“I have read and understand the infor- clude the patient with an antibody
mation provided to me regarding the spread to a high-incidence antigen or a
of the AIDS virus (HIV ) by blood and combination of antibodies that makes

it difficult to find compatible blood; hospitals provide this service. The

the infant with neonatal alloimmune selection and testing of directed do-
thrombocytopenia whose mother nors should be the same as for other
can provide platelets; the patient allogeneic donors, although special
awaiting a kidney transplant from a exemptions to the 56-day (or 112
living donor; or the multitransfused days for double red cell donation)
patient whose family members can waiting period between donations
provide components. may be made. Federal regulations
The repeated use of a single donor state that a person may serve as a
to supply components needed for a source of Whole Blood more than
single patient is allowed, provided it once in 8 weeks only if at the time of
is requested by the patient’s physi- donation the donor is examined and
cian and approved by the donor cen- certified by a physician to be in good
ter physician. The donor must meet health. 12 To avoid misunderstand-
all the usual requirements for dona- ings, it is important to establish SOPs
tion, except that the frequency of that define the interval required be-
donation can be as often as every 3 tween collection of the blood and its
days, as long as the predonation he- availability to the recipient; the policy
moglobin level meets or exceeds the about determining ABO group before
minimum value for routine allo- collection; and the policy for releas-
geneic blood donation. ing units for use by other patients.
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-
Collection of Blood
ient must be affixed to the blood or Blood is to be collected only by trained
component bag, and all such units personnel working under the direction of
must be segregated from the normal a qualified licensed physician. Blood col-
inventory. A protocol for handling lection must be by aseptic methods, using
such units must be included in the a sterile closed system. If more than one
SOP manual. skin puncture is needed, a new container
4. Directed donors: The public’s con- and donor set must be used for each addi-
cern about the safety of transfusion tional venipuncture unless the SOP allows
has generated demands from poten- the use of an FDA-approved device to at-
tial recipients to choose the donors tach a new needle while preserving steril-
to be used for their transfusions. ity. The phlebotomist must sign or initial
Several states have laws establishing the donor record, even if the phlebotomy
this as an acceptable procedure that did not result in the collection of a full
must be offered by a donor service unit.
in nonemergency situations, if re-
quested by a potential blood recipi- Blood Containers
ent or ordered by a physician. Despite Blood must be collected into an FDA-ap-
logistic and philosophic problems proved container that is pyrogen-free and
associated with these “directed” do- sterile and contains sufficient anticoagu-
nations, most blood centers and lant for the quantity of blood to be col-

lected. The container label must state the collection of blood. Tubes may be at-
type and amount of anticoagulant and the tached in any convenient manner to
approximate amount of blood collected. the primary bag or integral tubing.
Blood bags may be supplied in packages 4. Recheck all numbers.
containing more than one bag. The manu-
facturer’s directions should be followed for Preparation of the Venipuncture Site
the length of time unused bags may be
Blood should be drawn from a large firm
stored in packages that have been opened.
vein in an area (usually the antecubital
space) that is free of skin lesions. Both
Identification arms must be inspected for evidence of
Identification is essential in each step drug use, skin disease, or scarring. A tour-
from donor registration to final disposi- niquet or a blood pressure cuff inflated to
tion of each component. A numeric or al- 40 to 60 mm Hg makes the veins more
phanumeric system must be used that prominent. Having the donor open and
identifies, and relates to, the source do- close the hand a few times is also helpful.
nor, the donor record, the specimens used Once the vein is selected, the pressure de-
for testing, the collection container, and vice should be released before the skin
all components prepared from the unit. site is prepared.
Extreme caution is necessary to avoid any There is no way to make the veni-
mix-up or duplication of numbers. All re- puncture site completely aseptic, but surgi-
cords and labels should be checked be- cal cleanliness can be achieved to provide
the best assurance of an uncontaminated
fore use for printing errors. If duplicate
unit. Several acceptable procedures exist
numbers are found, they must be re-
(see Method 6.2). After the skin has been
moved and may be investigated to ascer-
prepared, it must not be touched again to
tain the reason for the duplication (eg,
repalpate the vein. The entire site prepara-
supplier error, etc). A record must be kept
tion must be repeated if the cleansed skin is
of all voided numbers.
touched.
Before beginning the collection, the
phlebotomist should:
1. Identify the donor record (at least by Phlebotomy and Collection of Samples
name) with the donor and ask the A technique for drawing a donor unit and
donor to state or spell his or her collecting samples for testing appears in
name. Method 6.3. The unit should be collected
2. Attach numbered labels to the donor from a single venipuncture after the pres-
record and ensure that it matches sure device has again been inflated. Dur-
the blood collection container, ating collection, the blood should be mixed
tached satellite bags, and tubes for with the anticoagulant. The amount of
donor blood samples. Attaching the blood collected should be monitored care-
numbers at the donor chair, rather fully so that the total, including samples,
than during the examination proce- does not exceed 10.5 mL per kilogram of
1(p61)
dures, helps reduce the likelihood of donor weight per donation. When the
identification errors. appropriate amount has been collected,
3. Be sure that the processing tubes are segments and specimen tubes must be
correctly numbered and that they filled. The needle and any blood-contam-
accompany the container during the inated waste must be disposed of safely in

accordance with universal precaution c. Drink more fluids than usual in

guidelines. The needle must not be re- the next 4 hours.
capped unless a safety recapping device is d. Avoid consuming alcohol until
used. Disposal of the needle must be in a something has been eaten.
puncture-proof container. After collec- e. Do not smoke for 30 minutes.
tion, there must be verification that the f. If there is bleeding from the
identifiers on the unit, the donor history, phlebotomy site, raise arm and
and the tubes are the same. Gloves must apply pressure to the site.
be available for use during phlebotomy g. If fainting or dizziness occurs,
and must be worn by phlebotomists when either lie down or sit with the
collecting autologous blood and when in- head between the knees.
dividuals are in training. h. If any symptoms persist, either
telephone or return to the do-
nor center or see a doctor.
Care of the Donor After Phlebotomy
i. Resume all normal activities if
After removing the needle from the vein, asymptomatic. Donors who work
the phlebotomist should: in certain occupations (eg, con-
1. Apply firm pressure with sterile gauze struction workers, operators of
over the point of entry of the needle machinery) or persons working
into vein. (The donor may be in- at heights should be cautioned
structed to continue application of that dizziness or faintness may
pressure for several minutes.) Check occur if they return to work im-
arm and apply a bandage only after mediately after giving blood.
all bleeding stops. j. Remove bandage after a few
2. Have donor remain reclining on the hours.
bed or in the donor chair for a few k. Maintain high fluid intake for
minutes under close observation by several days to restore blood
staff. volume.
3. Allow the donor to sit up under ob- 5. Thank the donor for an important
servation until his or her condition contribution and encourage repeat
appears satisfactory. The donor should donation after the proper interval.
be observed in the upright position All personnel on duty throughout
before release to the observation/re- the donor area, volunteer or paid,
freshment area. Staff should monitor should be friendly and qualified to
donors in this area. The period of ob- observe for signs of a reaction such
servation and provision of refreshment as lack of concentration, pallor, rapid
should be specified in the SOP manual. breathing, or excessive perspiration.
4. Give the donor instructions about Donor room personnel should be
postphlebotomy care. The medical trained and competent to interpret
director may wish to include some instructions, answer questions, and
or all of the following recommenda- accept responsibility for releasing
tions or instructions: the donor in good condition.
a. Eat and drink something before 6. Note on the donor record any ad-
leaving the donor site. verse reactions that occurred. If the
b. Do not leave until released by a donor leaves the area before being
staff member. released, note this on the record.

Adverse Donor Reactions signs of a reaction occur during

the phlebotomy.
Most donors tolerate giving blood very b. If possible, remove any donor
well, but adverse reactions occur occa- who experiences an adverse re-
sionally. Personnel must be trained to rec- action to an area where he or
ognize adverse reactions and to provide she can be attended in privacy.
initial treatment. c. Apply the measures suggested
Donor room personnel should be train- below and, if they do not lead
ed in cardiopulmonary resuscitation (CPR). to rapid recovery, call the blood
Special equipment to handle emergency bank physician or the physician
situations must be available. designated for such purposes.
Syncope (fainting or vasovagal syn- 2. Fainting.
drome) may be caused by the sight of a. Apply cold compresses to the
blood, by watching others give blood, or by donor’s forehead or the back of
individual or group excitement; it may also the neck.
happen for unexplained reasons. Whether b. Place the donor on his or her
caused by psychologic factors or by neuro- back, with their legs raised above
physiologic response to blood donation, the the level of the head.
symptoms may include weakness, sweat- c. Loosen tight clothing.
ing, dizziness, pallor, loss of consciousness, d. Be sure the donor has an ade-
convulsions, and involuntary passage of fe- quate airway.
ces or urine. On occasion, the skin feels e. Monitor blood pressure, pulse,
cold and blood pressure falls. Sometimes, and respiration periodically un-
the systolic blood pressure levels fall as low til the donor recovers.
as 50 mm Hg or cannot be heard with the Note: Some donors who experi-
stethoscope. The pulse rate often slows sig- ence prolonged hypotension may re-
nificantly. This can be useful in distinguish- spond to an infusion of normal sa-
ing between vasovagal attack and cardio- line. The decision to initiate such
genic or hypovolemic shock, in which cases therapy should be made by the do-
the pulse rate rises. This distinction, al- nor center physician either on a case-
though characteristic, is far from abso- by-case basis or in a policy stated in
lute. the facility’s SOP manual.
Rapid breathing or hyperventilation may 3. Nausea and vomiting.
cause the anxious or excited donor to lose a. Make the donor as comfortable
excessive amounts of carbon dioxide. This as possible.
may cause generalized sensations of suffo- b. Instruct the donor who is nau-
cation or anxiety, or localized problems seated to breathe slowly and
such as tingling or twitching. deeply.
The donor center physician must pro- c. Apply cold compresses to the
vide written instructions for handling do- donor’s forehead and/or back
nor reactions, including a procedure for ob- of neck.
taining emergency medical help. Sample d. Turn the donor’s head to the side.
instructions might be as follows: e. Provide a suitable receptacle if
1. General. the donor vomits and have clean-
a. Remove the tourniquet and with- sing tissues or a damp towel
draw the needle from the arm if ready. Be sure the donor’s head

is turned to the side because of self or herself. During severe

the danger of aspiration. seizures, some people exhibit
f. After vomiting has ended, give great muscular power and are
the donor some water to rinse difficult to restrain. If possible,
out his or her mouth. hold the donor on the chair or
4. Twitching or muscular spasms. Ex- bed; if not possible, place the
tremely nervous donors may hyper- donor on the floor. Try to pre-
ventilate, causing faint muscular vent injury to the donor and to
twitching or tetanic spasm of their yourself.
hands or face. Donor room person- b. Be sure the donor has an ade-
nel should watch closely for these quate airway. A padded device
symptoms during and immediately should separate the jaws after
after the phlebotomy. convulsion has ceased.
a. Divert the donor’s attention by c. Notify the donor center physi-
engaging in conversation, to cian.
interrupt the hyperventilation 7. Serious cardiac difficulties.
pattern. a. Call for medical aid and/or an
b. Have the donor cough if he or emergency care unit immediately.
she is symptomatic. Do not give b. If the donor is in cardiac arrest,
oxygen. begin CPR immediately and
5. Hematoma during or after phlebotomy. continue it until help arrives.
a. Remove the tourniquet and the The nature and treatment of all reactions
needle from the donor’s arm. should be recorded on the donor’s record
b. Place three or four sterile gauze or a special incident report form. This should
squares over the venipuncture include a notation of whether the donor
site and apply firm digital pres- should be accepted for future donations.
sure for 7 to 10 minutes, with The medical director should decide what
the donor’s arm held above the emergency supplies and drugs should be in
heart level. An alternative is to the donor area. The distance to the nearest
apply a tight bandage, which emergency room or emergency care unit
should be removed after 7 to 10 heavily influences decisions about neces-
minutes to allow inspection. sary supplies and drugs. Most donor cen-
c. Apply ice to the area for 5 min- ters maintain some or all of the following:
utes, if desired. 1. Emesis basin or equivalent.
d. Should an arterial puncture be 2. Towels.
suspected, immediately with- 3. Oropharyngeal airway, plastic or hard
draw needle and apply firm rubber.
pressure for 10 minutes. Apply 4. Oxygen and mask.
pressure dressing afterwards. 5. Emergency drugs: Drugs are seldom
Check for the presence of a ra- required to treat a donor’s reaction.
dial pulse. If pulse is not palpa- If the donor center physician wishes
ble or is weak, call a donor cen- to have any drugs available, the kind
ter physician. and amount to be kept on hand must
6. Convulsions. be specified in writing. In addition,
a. Call for help immediately. Pre- the medical director must provide
vent the donor from injuring him- written policies stating when and by

whom any of the above medical sup- 12. Code of federal regulations. Title 21 CFR
640.3(f ). Washington, DC: US Government
plies or drugs may be used.
References Suggested Reading

Code of federal regulations. Title 21 CFR 640.3.
Washington, DC: US Government Printing Office,
AABB, 2005.
2004 (revised annually). [History of viral hepatitis
2. Code of federal regulations. Title 21 CFR
before the 11th birthday.]
606.160(e). Washington, DC: US Government
Printing Office, 2004 (revised annually). Food and Drug Administration. Memorandum:
3. Beattie KM, Shafer AW. Broadening the base Revised recommendations for the prevention of
of a rare donor program by targeting minority human immunodeficiency virus (HIV ) transmis-
populations. Transfusion 1986;26:401-4. sion by blood and blood products. (April 23, 1992)
4. Vichinsky EP, Earles A, Johnson RA, et al. Allo- Rockville, MD: CBER Office of Communication,
immunization in sickle cell anemia and Training, and Manufacturers Assistance, 1992.
transfusion of racially unmatched blood. N
Food and Drug Administration. Guidance for in-
Engl J Med 1990;322:1617-21.
dustry: Revised preventive measures to reduce the
5. Food and Drug Administration. Memoran-
possible risk of transmission of Creutzfeldt-Jakob
dum: Revised recommendations for the pre-
disease (CJD) and new variant Creutzfeldt-Jakob
vention of human immunodeficiency virus
disease (nvCJD) by blood and blood products.
(HIV) transmission by blood and blood prod-
( January 9, 2002) Rockville, MD: CBER Office of
ucts. (April 23, 1992) Rockville, MD: CBER Of-
Communication, Training, and Manufacturers
fice of Communication, Training, and Manu-
Assistance, 2002.
facturers Assistance, 1992.
6. Centers for Disease Control. Update: Univer- Infectious disease testing for blood transfusions.
sal precautions for prevention of transmis- NIH Consensus Statement 13:1, January 1995.
sion of human immunodeficiency virus, hep- Bethesda, MD: National Institutes of Health, 1995.
atitis B virus, and other bloodborne pathogens
in health-care settings. JAMA 1988;260:528-31. Kasprisin C, Laird-Fryer B, eds. Blood donor col-
7. Code of federal regulations. Title 21 CFR lection practices. Bethesda, MD: AABB, 1993.
640.4(a). Washington, DC: US Government Linder J, ed. Practical solutions to practical prob-
Printing Office, 2004 (revised annually). lems in transfusion medicine and tissue banking.
8. Food and Drug Administration. Guidance for [Supplement 1 to Am J Clin Pathol 1997;107(4).]
Industry: Streamlining the donor interview Chicago, IL: American Society of Clinical Patholo-
process: Recommendations for self-adminis- gists, 1997.
tered questionnaires. (July 3, 2003) Rockville,
MD: CBER Office of Communication, Train- Schmuñis GA. Trypanosoma cruzi, the etiologic
ing, and Manufacturers Assistance, 2003. agent of Chagas’ disease: Status in the blood sup-
9. Armed Services Blood Program Office. Drugs ply in endemic and nonendemic countries. Trans-
and medications. [Available at http://www. fusion 1991;31:547-57.
tricare.osd.mil/asbpo/librar y/policies/
Smith KJ, Simon TL. Recruitment and evaluation
downloads/medication_list.doc.]
of blood and plasma donors. In: Rossi EC, Simon
10. Newman B. Blood donor suitability and
TL, Moss GS, eds. Principles of transfusion medi-
allogeneic whole blood donation. Transfus
cine. 2nd ed. Baltimore, MD: Williams and Wilkins,
Med Rev 2001;15:234-44.
1995:871-9.
11. Food and Drug Administration. General re-
quirements for blood, blood components, and Tan L, Williams MA, Khan MK, et al. Risk of trans-
blood derivatives; donor notification. Title 21 mission of bovine spongiform encephalopathy to
CFR 630.6. Fed Regist 2001;66:31165-77. humans in the United States. JAMA 1999;281:2330-8.

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

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

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.

Appendix 4-2. Medication Deferral List*

Appendix 4-3. Blood Donor Education Materials*

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
A
UTOLOGOUS BLOOD TRANSFU- depending on the type of surgery, condition
sion is an alternative therapy for of the patient, and technology available.
many patients anticipating trans- Each facility must analyze its own transfu-
fusion. Different categories of autologous sion practices, transfusion practices of
transfusion are: other similarly situated institutions, and its
1. Preoperative collection (blood is drawn own capabilities to determine the appropri-
and stored before anticipated need). ate services to be offered.
2. Perioperative collection and admin- However, it is generally accepted that,
istration. when feasible, the patient should have the
a. Acute normovolemic hemodi- option to use his or her own blood. The US
lution (blood is collected at the Supreme Court has ruled that asymptom-
start of surgery and then in- atic infection with HIV is a disability pro-
fused during or at the end of tected under the Americans with Disabili-
the procedure). ties Act.1 Therefore, if institutions offer 5
b. Intraoperative collection (shed autologous services to any patient, they
blood is recovered from the should consider offering such services to
surgical field or circulatory de- HIV-positive patients.2 Patients who are
vice and then infused). likely to require transfusion therapy and
c. Postoperative collection (blood who also meet the donation criteria should
is collected from drainage de- be told about the options for autologous
vices and reinfused to the pa- transfusion therapies. Patients considering
tient). autologous transfusion therapy should be
Each type of autologous transfusion informed about the risks and benefits of both
practice offers potential benefits and risks the autologous donation and the auto-
117

logous transfusion process. Specific issues In selected patient subgroups, preopera-

unique to the use of autologous transfusion tive collection of autologous blood can sig-
in the anticipated surgical procedure should nificantly reduce exposure to allogeneic
be identified, including the possibility of blood. PAD collections should be considered
administrative error. In addition, patients for patients likely to receive transfusion,
need information about any special fees for such as patients undergoing major ortho-
autologous services, the level of infectious pedic procedures, vascular surgery, and
disease testing that will be performed, and cardiac or thoracic surgery.3,4 The most
the possibility that additional, allogeneic, common surgical procedures for which
units may be used. autologous blood is donated are total joint
replacements.4 Autologous blood should not
be collected for procedures that seldom
(less than 10% of cases) require transfusion,
Preoperative Autologous such as cholecystectomy, herniorrhaphy,
Blood Collection vaginal hysterectomy, and uncomplicated
obstetric delivery.5
Frequently cited advantages and disad-
vantages of preoperative autologous blood
Special Patient Categories
donation (PAD) are summarized in Table
5-1. Candidates for preoperative collec- In special circumstances, preoperative
tion are stable patients scheduled for sur- autologous blood collection can be per-
gical procedures in which blood transfu- formed for patients who would not ordi-
sion is likely. For procedures that are narily be considered for allogeneic dona-
unlikely to require transfusion (ie, a maxi- tion. The availability of medical support is
mal surgical blood ordering schedule important in assessing patient eligibility.
does not suggest that crossmatched blood With suitable volume modification, pa-
be available), the use of preoperative blood rental cooperation, and attention to prep-
collection is not recommended. aration and reassurance, pediatric patients
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 4. Results in wastage of blood that is not
alloantibodies. transfused.
5. Prevents some adverse transfusion reac- 5. Increased incidence of adverse reactions to
tions. autologous donation.
6. Provides reassurance to patients con- 6. Subjects patients to perioperative anemia
cerned about blood risks. and increased likelihood of transfusion.

Chapter 5: Autologous Blood Donation and Transfusion 119
14
can participate in preoperative collection ranted, because blood is so seldom needed.
programs.6 The successful use of autolo- Many centers give serious consideration to
gous blood in a patient with sickle cell autologous collection for women with allo-
disease has been reported,7 and it may be antibodies to multiple or high-incidence
particularly useful for a sickle cell patient antigens, placenta previa, or other conditions
with multiple alloantibodies; however, the placing them at high risk for ante- or intra-
patient may derive greater benefit from partum hemorrhage.5 A policy should be
allogeneic transfusions that provide he- developed for situations in which maternal
moglobin A. Red cells containing hemo- red cells are considered for transfusion to
globin S require special handling during the infant.
the cryopreservation process.8
Patients with significant cardiac disease
are considered poor risks for autologous Voluntary Standards
blood donation. Despite reports of safety in AABB Standards for Blood Banks and Trans-
small numbers of patients who underwent fusion Services offers uniform standards to
autologous blood donation,9 the risks that be followed in determining patient eligi-
are associated with autologous blood dona- bility; collecting, testing, and labeling the
tion10 in these patients are probably greater unit; and pretransfusion testing.15(pp18,39,51)
than the current estimated risks of allo- These AABB standards apply to preopera-
geneic transfusion.11,12 Table 5-2 summa- tive autologous blood collection. Stan-
rizes the contraindications to a patient’s dards for Perioperative Autologous Blood
participation in an autologous blood dona- Collection and Administration have been
tion program.13 established to enhance the quality and
The collection of autologous blood from safety of perioperative autologous trans-
women during routine pregnancy is unwar- fusion activities (intra- and postoperative
blood recovery, perioperative autologous
component production, and intraopera-
Table 5-2. Contraindications to tive acute normovolemic hemodilution).16
Participation in Autologous Blood
Donation Programs Compliance Considerations
1. Evidence of infection and risk of Food and Drug Administration (FDA) re-
bacteremia. quirements have evolved over time. The
2. Scheduled surgery to correct aortic steno- FDA first issued guidance for autologous
sis. blood and blood components in March of
3. Unstable angina. 1989.17 This guidance was clarified in a
4. Uncontrolled seizure disorder. second memorandum issued in February
5. Myocardial infarction or cerebrovascular of 1990.18 Much of the information in pre-
accident within 6 months of donation. vious guidance has been superseded by
6. Patients with significant cardiac or pulmo- regulations. The FDA included requirements
nary disease who have not yet been cleared
regarding autologous blood in regulations
for surgery by their treating physician.
issued June 11, 2001.19,20
7. High-grade left main coronary artery dis-
ease.
8. Cyanotic heart disease. Testing
9. Uncontrolled hypertension. The FDA requires tests for evidence of in-
fection resulting from the following com-

municable diseases: HIV-1, HIV-2, HBV, Special Labeling Considerations

HCV, HTLV-I, and HTLV-II (21 CFR 610.40)
Each autologous unit must be labeled
and a serologic test for syphilis [21 CFR
“Autologous Donor.” Another special label,
610.40(a)(1) and 610.40(I)]. Such tests in-
21
“BIOHAZARD,” is required for any unit
clude nucleic acid tests for HCV and HIV.
that is reactive in the current collection or
Testing of autologous donations is not
reactive in the last 30 days. Autologous
required unless the donations are to be units that are untested must be labeled
used for allogeneic transfusion [21 CFR “DONOR UNTESTED.” If the autologous
610.40(d)(1)]. Autologous donations that unit tested negative within the last 30
are to be shipped to another facility that days, it must be labeled “DONOR TESTED
does allow autologous units to be used for WITHIN THE LAST 30 DAYS” [21 CFR
allogeneic transfusion must be tested [21 610.40(d)(4)].
CFR 610.40(d)(2)]. For autologous dona-
tions shipped to another establishment
that does not allow autologous donations
Shipping
to be used for allogeneic transfusion, the Blood or components (including reactive
first donation in each 30-day period must donations) intended for autologous use
be tested [21 CFR 610.40(d)(3)].15(p34) may be shipped provided that the units
Autologous donations found to be reac- have been tested as required and are la-
tive by a required screening test must be re- beled appropriately [21 CFR 610.40(d)]. If
tested whenever a supplemental (additional, distributed on a common carrier not un-
more specific) test has been approved by der the direct control of the collection fa-
the FDA. At a minimum, the first reactive cility, the transportation of the product
donation in each 30-day period must be must meet provisions for shipping an in-
tested unless a record exists for a positive fectious agent.22
supplemental test result for that donor [21
CFR 610.40(e)(1,2)]. Both the AABB Stan-
dards15(p34) and the FDA requirements [21 Establishing a Preoperative Autologous
CFR 630.6(d)] state that the patient and the Blood Collection Program
patient’s physicians must be notified of any Each blood center or hospital that decides
medically significant abnormalities. to conduct an autologous blood collection
program must establish its own policies,
Donor Deferral processes, and procedures. Guidelines ex-
ist for establishing a new program, moni-
If an autologous donor has a reactive
toring utilization, or improving an exist-
screening test for a communicable dis-
ing one.13,23,24
ease agent or a reactive screening test for
syphilis (21 CFR 610.41), the donor must
be deferred from making future allogeneic Physician Responsibility
donations. Within 8 weeks, the patient A successful autologous program requires
and referring physician must be notified cooperation and communication among
of the reason for deferral, including the all the physicians involved. Responsibility
results of supplemental testing and, where for the health and safety of the patient
appropriate, the types of donation that the during the collection process rests with the
autologous donor should not make in the medical director of the collecting facility;
future (21 CFR 630.6). during the transfusion, responsibility rests

with the patient’s physician and the medi- be drawn, whenever possible, so that the
cal director of the transfusion service. The patient can minimize exposure to allo-
patient’s physician initiates the request geneic blood. However, excessive collection
for autologous services, which must be and/or collection close to the date of sur-
approved by the transfusion service phy- gery increases the patient’s likelihood of re-
sician. There should be a transfusion quiring transfusion. A hospital’s surgical
medicine physician available to help as- blood order schedule can provide estimates
sess patients whose medical history sug- of transfusion levels for specific procedures.
gests a risk for complications if a donor Two-unit collections via an automated red
reaction occurs during blood collection. cell apheresis system may be an option. The
collection of units for liquid blood storage
Supplemental Iron should be scheduled as far in advance of
surgery as possible, in order to allow comp-
The patient should be advised about tak-
ensatory erythropoiesis to minimize anemia.
ing supplemental iron. Ideally, supple-
A schedule for blood collections should
mental iron is prescribed by the requesting
be established with the patient. A weekly
physician before the first blood collection,
schedule is often used. Table 5-3 details the
in time to allow maximum iron intake.
value of beginning autologous blood dona-
Iron-restricted erythropoiesis is one of the
tion early in the known preoperative inter-
limiting factors in collecting multiple
val, in order to allow optimal compensatory
units of blood over a short interval. Oral
erythropoiesis (shown here as equivalent
iron is commonly provided but may be
RBC units).26 Ordinarily, the last collection
insufficient to maintain iron stores.25 The
should occur no sooner than 72 hours be-
dose and administration schedule should
fore the scheduled surgery and preferably
be adjusted to minimize gastrointestinal
longer, to allow time for adequate volume
side effects.
repletion. Programs should notify the re-
questing physician of the total number of
Collection units donated when the requested number
The collection of autologous blood has of units cannot be collected. Each program
many elements in common with collec- should establish a policy regarding re-
tion from regular volunteer donors, but scheduling of surgery beyond the expira-
numerous special considerations exist. tion date of autologous units and whether
Requests for autologous blood collection discarding or freezing the unit are options.
are made in writing by the patient’s physi-
cian; a request form (which may be a sim-
ple prescription or a form designed for Donor Screening
the purpose) is kept by the collecting fa- Because of the special circumstances re-
cility. The request should include the garding autologous blood transfusion,
patient’s name, a unique identification rigid criteria for donor selection are not
number, the number of units and kind of required. In situations where require-
component requested, the date of sched- ments for allogeneic donor selection or
uled surgery, the nature of the surgical collection are not applied, alternative re-
procedure, and the physician’s signature. quirements must be established by the
It is important to establish guidelines for medical director and recorded in the pro-
the appropriate number of units to be col- cedures manual. The hemoglobin con-
lected. A sufficient number of units should centration of the donor should be no less

Table 5-3. Timing and Red Cell Regeneration During Preoperative Autologous
26
Donation
Time from Donation to No. of Mean RBC Units 5% CI
Surgery (days) Patients Regenerated 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
than 11.0 g/dL and the hematocrit, if sub- plasma ratio. Under-collected units (<300
stituted, should be no less than 33%. Indi- mL) may be suitable for autologous use
vidual deviations from the alternate re- with approval of the medical director. For
quirements must be approved by the blood patients weighing <50 kg, there should be
bank medical director, usually in consul- a proportional reduction in the volume of
tation with the donor-patient’s physician. blood collected. Regardless of donor weight,
the volume collected should not exceed
Medical Interview 10.5 mL/kg of the donor’s estimated body
The medical interview should be struc- weight, including the samples for testing.
tured to meet the special needs of autolo-
gous donors. For example, more attention Serologic Testing
should be given to questions about medi- The collecting facility must determine
cations, associated medical illnesses, and ABO and Rh type on all units. Transfusing
cardiovascular risk factors.10 Questions facilities must retest ABO and Rh type on
should elicit any possibility of intermittent units drawn at other facilities, unless the
bacteremia. Because crossover is not rou- collecting facility tests segments from the
tinely permitted, a substantially shortened unit according to AABB Standards.15(p37)
set of interview questions can be used for Testing for ABO and Rh type must be
autologous donations; for example, ques- performed on a properly labeled blood
tions related to donor risks for transfusion- sample from the patient. An antibody
transmitted diseases are not necessary. screen should be performed to provide for
the possible need for allogeneic blood.
Volume Collected
For autologous donors weighing >50 kg, Labeling
the 450-mL collection bag is usually used Units should be clearly labeled with the
instead of the 500-mL bag, in case the do- patient’s name and an identifying num-
nor cannot give a full unit. If a low-volume ber, the expiration date of the unit, and, if
(300-405 mL) unit is collected, the red cells available, the name of the facility where
are suitable for storage and subsequent the patient is to be transfused. The unit
autologous transfusion. The plasma from should be clearly marked “For Autologous
low-volume units cannot be transfused Use Only” if intended for autologous use
because of the abnormal anticoagulant/ only. If components have been prepared,

the container of each component must be place regarding the issue of autologous,
similarly labeled. A biohazard label must allogeneic, and/or directed units to the
be applied when indicated by FDA require- operating room.
ments (see Compliance Considerations).
Labeling requirements for autologous units Records
are detailed in the AABB Standards,15(p51) AABB standards for the proper issue and
which parallels the FDA regulations. return of unused autologous units are the
same as for allogeneic units.15(pp45,71) Re-
Storage cords must be maintained that identify
Collection should be scheduled to allow the unit and all components made from
for the longest possible shelf life for col- it, from collection and processing through
lected units. This increases flexibility for their eventual disposition.
the patient and the collecting facility and
allows time for the patient to rebuild red Adverse Reactions
cell mass during the interval between The investigation of suspected adverse
blood collection and surgery. Liquid stor- transfusion events should be the same for
age is feasible for up to 6 weeks. Some autologous and allogeneic units. Autolo-
programs store autologous units as Whole gous transfusions have a lower risk of in-
Blood for 35 days rather than as RBCs; fectious and immune complications but
Whole Blood is simpler to store, and the carry a similar risk of bacterial contami-
risk of volume overload subsequent to nation, volume overload, and misadminis-
transfusion is low. The collection of autol- tration compared with volunteer allogeneic
ogous units more than 6 weeks before units. For these reasons, autologous blood
scheduled surgery has been described, should not be transfused without a clear
but requires that the red cells be frozen. indication for transfusion.
Although this provides more time for the
donor to recover lost red cell mass, freez- Continuous Quality Improvement
ing and thawing add to the cost of the
Several quality improvement issues have
program, reduce the volume of red cells 5
been identified for PAD practices. The
through processing losses, and compli-
most important indicator for autologous
cate blood availability during the peri-
blood practice is how effectively it reduces
operative period.
allogeneic transfusions to participating
patients. The “wastage” rate of autologous
Transfusion of Autologous Units units for surgical procedures can also be
Autologous transfusion programs should monitored. However, even for procedures
have a system to ensure that if autologous such as joint replacement or radical pros-
blood is available, it be issued and used tatectomy, a well-designed program may
before allogeneic components are given. result in 50% of collected units being un-
5,27
A special “autologous” label may be used used (Fig 5-1). Nevertheless, as much as
with numbering to ensure that the oldest 25% of autologous blood is collected for
units are issued first. Anesthesiologists, procedures that seldom require transfu-
surgeons, and physicians should be edu- sion, such as vaginal hysterectomies and
cated about the importance of selecting normal vaginal deliveries. Up to 90% of
autologous components before allogeneic units collected for these procedures are
units are given, and a policy should be in wasted.14 The additional costs associated

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
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 )
with the collection of autologous units, Evolving Issues in Preoperative

along with advances in the safety of allo- Autologous Services
geneic blood, have altered the cost-effec-
tiveness of PAD in many situations.28 Such Selection of Patients
cost-effectiveness analyses do not consider
an immunomodulatory effect of avoiding Attempts to stratify patients into groups
allogeneic leukocytes, which remains at high and low risk for needing transfu-
29,30
controversial. sion based on the baseline level of hemo-
Criteria can be established to monitor globin and on the type of procedure show
the appropriateness of autologous transfu- some promise. In a Canadian study using
sions. These criteria may be the same as, or a point score system, 80% of patients un-
different from, those established for allo- dergoing orthopedic procedures were
geneic units.24 As with allogeneic blood, identified to be at low risk (<10%) for
transfusion of preoperatively donated autotransfusion, so autologous blood procure-
logous blood carries the same risks associ- ment for these patients would not be rec-
31
ated with administrative error or bacterial ommended. However, one problem with
contamination. Autologous programs should algorithms that consider the estimated
be monitored for unavailability of autolo- blood loss and preoperative hematocrit is that
gous blood when needed, the transfusion blood losses are difficult to measure32,33 or
of allogeneic blood before autologous predict because specific surgical proce-
blood, and identification errors. dures performed even by the same sur-

geon can be accompanied by a wide range sis representing 19% to 26% red cell volume
of blood losses. expansion.39-41 Exogenous (pharmacologic)
erythropoietin therapy to further stimulate
erythropoiesis (up to 50% red cell volume
The Role of Aggressive Phlebotomy and the
expansion39-41) during autologous phlebot-
Use of Erythropoietin
omy has been approved in Canada and Ja-
The efficacy of PAD is dependent on the pan but not in the United States.
42
degree to which the patient’s erythro-

poiesis increases the production of red
cells.25,34-36 The endogenous erythropoietin
Transfusion Trigger
response and compensatory erythropoie-
sis are suboptimal under “standard” con- Disagreement exists about the proper he-
ditions of one blood unit donated weekly. moglobin/hematocrit level (“transfusion
Weekly PAD is accompanied by an 11% trigger”) at which autologous blood should
(with no oral iron supplementation) to 19% be given.23 Autologous blood transfusion
(with oral iron supplementation) expan- is not without risks to the recipient; these
sion in red cell volume over a 3-week pe- include misidentification of patients or
riod, which is not sufficient to prevent in- units, bacterial contamination of stored
creasing anemia in patients undergoing units, and volume overload. The case can
PAD.34,35 If the erythropoietic response to be made that autologous and allogeneic
autologous blood phlebotomy is not able blood transfusion triggers should be simi-
to maintain the patient’s hematocrit level lar because the additional mortality risks
of allogeneic blood now approach the risks
during the donation interval, the dona-
of mortality from administrative errors as-
tion of autologous blood may be harm-
sociated with both autologous and allo-
ful.37 This outcome was confirmed in a
geneic blood.43 Data from a well-designed
study of patients undergoing hysterecto-
clinical trial indicate that even critical care
mies,38 in which it was shown that PAD
patients can tolerate substantial anemia
resulted in perioperative anemia and an
(to hemoglobin ranges of 7 to 9 g/dL) with
increased likelihood of any blood transfu-
no apparent benefit from more aggressive
sion. A published mathematical model37
transfusion therapy.44
illustrates the relationship between antic-
ipated surgical blood losses, the level of
hematocrit that the physician may want
to maintain perioperatively, and the need Cost-Effectiveness
for autologous blood donation for indivi- Although autologous blood collections have
dual patients (Fig 5-2). Models such as become popular, the costs associated with
this may be helpful in designing autolo- their collection are usually higher than
gous procurement programs or monitor- those associated with the collection of
ing their value through quality assurance. allogeneic blood. The continued need for
In contrast to autologous blood donation autologous blood programs has been ques-
under “standard” conditions, studies of “ag- tioned because of the reduced risk of
gressive” autologous blood phlebotomy allogeneic blood transfusions and pressure
(twice weekly for 3 weeks, beginning 25 to to reduce health-care costs.27 Table 5-4 lists
35 days before surgery) have demonstrated suggestions for improving the efficiency
that endogenous erythropoietin levels do of hospital-based autologous blood pro-
increase, along with enhanced erythropoie- grams without sacrificing safety.45

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 )
Preoperative Collection of Components telet-derived growth factors, which have

been reported in small studies to enhance
Some workers believe that preoperative or
tissue repair and wound healing.48,49 Larger
intraoperative collection of platelet-rich
randomized clinical trials are needed to es-
plasma during cardiopulmonary bypass
tablish the clinical efficacy of this product.
surgery may improve hemostasis and de-
Platelet gel is not FDA-approved yet.
crease allogeneic exposures, but others
have found no benefit.46 Preoperative col-
lection of autologous platelets, especially
in cardiac surgery, is often impractical be- Acute Normovolemic
cause patients may be taking antiplatelet
drugs; surgery is often scheduled on an
Hemodilution
emergency basis; and relatively low num- Acute normovolemic hemodilution (ANH)
bers of platelets are harvested. is the removal of whole blood from a pa-
Recently, autologous platelet collection tient, with concurrent restoration of the
using commercial point-of-care collection circulating blood volume with an acellular
systems has been advocated to produce a fluid shortly before an anticipated signifi-
platelet gel for topical use.47 Platelet gel is cant surgical blood loss. To minimize the
created by adding calcium chloride and manual labor associated with hemodilu-
thrombin to a platelet concentrate. The tion, the blood should be collected in
platelet gel serves as a rich source of pla- standard blood bags containing anticoag-

Table 5-4. Suggestions for Making Autologous Blood Transfusion Protocols

Cost-Effective
1. Use standardized indications for preoperative autologous blood collection and transfusion.
2. Streamline the autologous blood donor interview.
3. Discontinue serologic tests for infectious disease markers following autologous blood
collections.
4. Simplify the donation process for uncomplicated patients.
5. Limit the use of frozen blood.
6. Store autologous whole blood, rather than components.
7. Use standardized indications and appropriate technology for intraoperative and postoperative
autologous blood recovery.
8. Share intraoperative blood recovery resources among institutions.
9. Cautiously adopt new research applications for autologous blood techniques.
ulant on a tilt-rocker with automatic cut- Physiologic Considerations

off via volume sensors. Then, the blood is
Conserved Red Cell Mass
stored at room temperature and reinfused
during surgery after major blood loss has The chief benefit of ANH is the reduction
ceased, or sooner if indicated. Simultaneous of red cell losses when whole blood is shed
infusions of crystalloid (3 mL crystalloid perioperatively at lower hematocrit levels
after ANH has been completed.52 Mathe-
for each 1 mL of blood withdrawn) and
matical modeling has suggested that se-
colloid (dextrans, starches, gelatin, albu-
vere ANH to preoperative hematocrit lev-
min, 1 mL for each 1 mL of blood with-
els of less than 20%, accompanied by
drawn) have been recommended.50 substantial blood losses, would be re-
Subsequent intraoperative fluid manage- quired before the red cell volume “saved”
ment is based on the usual surgical require- by ANH would become clinically impor-
ments. Blood units are reinfused in the 53
tant. However, the equivalent of one blood
reverse order of collection. The first unit 54 55
unit can be “saved” by ANH, which ap-
collected, and therefore the last unit trans- proaches the red cell volume expansion
fused, has the highest hematocrit and con- generated by PAD under standard condi-
centration of coagulation factors and plate- tions (Table 5-3).
lets. Although this technique has been
primarily developed and used in Europe,
increasing interest in the United States has Improved Oxygenation
led to data that show promise as an alterna- Withdrawal of whole blood and replace-
tive method of autologous blood procurement with crystalloid or colloid solution
ment.51 Augmented hemodilution (replace- decrease arterial oxygen content, but com-
ment of ANH collected or surgical blood pensatory hemodynamic mechanisms and
lost in part by oxygen therapeutics) has the the existence of surplus oxygen-delivery
advantage of not being limited by anemia. capacity make ANH safe. A sudden de-
Its use is restricted to the investigational crease in red cell concentration lowers
setting until these solutions are approved blood viscosity, thereby decreasing pe-
by the FDA. ripheral resistance and increasing cardiac

output. If cardiac output can effectively require no inventory or testing costs. Be-
compensate, oxygen delivery to the tis- cause the blood never leaves the patient’s
sues at a hematocrit of 25% to 30% is as room, ANH minimizes the possibility of
good as, but no better than, oxygen deliv- an administrative or a clerical error that
ery at a hematocrit of 30% to 35%.56 could lead to an ABO-incompatible blood
transfusion and death, as well as bacterial
Preservation of Hemostasis contamination associated with prolonged
storage at 4 C.
Because blood collected by ANH is stored
at room temperature and is usually re-
turned to the patient within 8 hours of Practical Considerations
collection, there is little deterioration of
The following considerations are impor-
platelets or coagulation factors. The
tant in establishing an ANH program:
hemostatic value of blood collected by
1. Decisions about ANH should be
ANH is of questionable benefit for ortho-
based on the surgical procedure and
pedic or urologic surgery because plasma
on the patient’s preoperative blood
and platelets are rarely indicated in this
volume and hematocrit, target hemo-
setting. Its value in protecting plasma and
dilution hematocrit, and other phys-
platelets from the acquired coagulopathy
iologic variables.
of extracorporeal circulation in cardiac
2. The institution’s policy and proce-
surgery is better established.46,57
dures and the mechanisms for edu-
cating staff should be established
Clinical Studies and periodically reviewed.
Prospective randomized studies in radical 3. There should be careful monitoring
prostatectomy,58 knee replacement,59 and of the patient’s circulating volume
60
hip replacement suggest that ANH can and perfusion status during the pro-
be considered equivalent to PAD as a cedure.
method of autologous blood procure- 4. Blood must be collected in an asep-
ment. Additional, selected clinical trials of tic manner, ordinarily into standard
ANH are summarized in Table 5-5.61-67 Re- blood collection bags with citrate
views68,69 and commentaries70 on the mer- anticoagulant.
its of ANH have been published. However, 5. Units must be properly labeled and
a recently published meta-analysis of 42 stored. The label must contain, at a
clinical trials of ANH found only a modest minimum, the patient’s full name,
benefit with unproven safety.71 When ANH medical record number, date, and
and reinfusion are accomplished in the time of collection, and the statement
operating room by on-site personnel, the “For Autologous Use Only.” Room
procurement and administration costs temperature storage should not ex-
are minimized. Blood obtained during ceed 8 hours. Units maintained at
ANH does not require the commitment of room temperature should be re-
the patient’s time, transportation, costs, infused in the reverse order of col-
and loss of work time that can be associ- lection to provide the maximum
ated with PAD. The wastage of PAD units number of functional platelets and
(approximately 50% of units collected) coagulation factors in the last units
also is eliminated with ANH. Additionally, infused. If more time elapses be-
autologous blood units procured by ANH tween collection and transfusion,

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

Postoperative Allogeneic RBC-Containing
Estimated Blood Loss (mL) Hematocrit (%) Units or Liters Transfused
Chapter 5: Autologous Blood Donation and Transfusion

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
53
Modified from Brecher and Rosenfeld. NR = not reported; NS = not significant.
129
the blood should be stored in a colloid/crystalloid ratios), and full

monitored refrigerator. ANH blood adherence to AABB guidelines. Some
collected from an open system (eg, practical considerations are listed in
from a central venous line or an ar- Table 5-6. Suggested criteria for pa-
terial catheter) may be stored for up tient selection are listed in Table 5-7.
to 8 hours at room temperature or 24
hours in a monitored refrigerator.
Policies, procedures, and guidelines
must be developed for ANH by an
Intraoperative Blood
experienced group of anesthesiolo- Collection
gists in conjunction with the operat- The term intraoperative blood collection
ing room’s nursing staff and the hos- or recovery describes the technique of
pital’s blood bank and transfusion collecting and reinfusing blood lost by a
services.68 These will include indica- patient during surgery. The oxygen-trans-
tions for ANH, monitoring require- port properties of recovered red cells are
ments, endpoints for blood with- equivalent to stored allogeneic red cells.
drawal and transfusion, types and The survival of recovered blood cells ap-
amounts of replacement fluids (ie, pears to be at least comparable to that of
Table 5-6. Practical Considerations for Acute Normovolemic Hemodilution (ANH)

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

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.
72
transfused allogeneic red cells. Intra- Most programs use machines that col-
operative collection is contraindicated lect shed blood, wash it, and concentrate
when certain procoagulant materials (eg, the red cells. This process typically results
topical collagen) are applied to the surgi- in 225-mL units of saline-suspended red
cal field because systemic activation of cells with a hematocrit of 50% to 60%. Pa-
coagulation may result. Microaggregate tients exhibit a level of plasma-free hemo-
filters (40 microns) are used most often globin that is usually higher than after
because recovered blood may contain tis- allogeneic transfusion. Sodium and chlo-
sue debris, small blood clots, or bone ride concentrations are the same as in the
fragments. saline wash solution, and potassium con-
Cell washing devices can provide the centration is low. The infusate contains
equivalent of 12 units of banked blood per minimal coagulation factors and platelets.
hour to a massively bleeding patient.72 Data
regarding adverse events of reinfusion of
recovered blood have been published.73 Air Clinical Studies
embolus is a potentially serious problem.
As with PAD and ANH, collection and re-
Three fatalities from air embolus were re-
ported over a 5-year interval to the New covery of intraoperative autologous blood
York State Department of Public Health, for should undergo scrutiny with regard to
an overall fatality risk of one in 30,000.43 both safety and efficacy. Controlled stud-
Hemolysis of recovered blood can occur ies in cardiothoracic surgery have re-
during suctioning from the surface instead ported conflicting results when transfu-
of from deep pools of shed blood. For this sion requirements and clinical outcome
reason, manufacturers’ guidelines recom- were followed.75,76 Although the collection
mend a maximum vacuum setting of no of a minimum of one blood unit equiva-
more than 150 torr. One study found that lent is possible for less expensive (with
vacuum settings as high as 300 torr could unwashed blood) methods, it is generally
be used when necessary, without causing agreed that at least two blood unit equiva-
74
excessive hemolysis. The clinical impor- lents need to be recovered using a cell-re-
tance of free hemoglobin in the concentra- covery instrument (with washed blood) in
77
tions usually seen has not been established, order to achieve cost-effectiveness. The
although excessive free hemoglobin may value of intraoperative blood collection
indicate inadequate washing. Positive bac- has been best defined for vascular surger-
terial cultures from recovered blood are ies with large blood losses, such as aortic
sometimes observed; however, clinical in- aneurysm repair and liver transplanta-
75 78
fection is rare. tion. However, a prospective randomized

79
trial of intraoperative recovery and re- curs in roller pumps and plastic tubing
infusion in patients undergoing aortic an- make some degree of hemolysis inevitable.
eurysm repair showed no benefit in the High concentrations of free hemoglobin
reduction of allogeneic blood exposure. A may be nephrotoxic to patients with im-
mathematical model of cell recovery sug- paired renal function. Many programs limit
gests that when it is combined with normo- the quantity of recovered blood that may be
volemic anemia, the need for allogeneic reinfused without processing.
transfusion can be avoided—even with
large blood loss, eg, 5 to 10 liters.80 The Practical Considerations
value of this technology may rest on cost Collection and recovery services require
savings and blood inventory consider- the coordinated efforts of surgeons, anes-
ations in patients with substantial blood thesiologists, transfusion medicine spe-
losses. cialists, and specific personnel trained in
the use of special equipment. Equipment
options may include:
Medical Controversies 1. Devices that collect recovered blood
Collection devices that neither concentrate for direct reinfusion.
nor wash shed blood before reinfusion in- 2. Devices that collect recovered blood,
crease the risk of adverse effects. Shed which is then concentrated and wash-
ed in a separate cell washer.
blood has undergone varying degrees of
3. High-speed machines that automat-
coagulation/fibrinolysis and hemolysis,
ically concentrate and wash recov-
and infusion of large volumes of washed
ered red cells.
or unwashed blood has been described in
Some hospitals develop their own pro-
association with disseminated intravascular
81 grams, whereas others contract with outside
coagulation. Factors that affect the degree
services. Each hospital’s needs should dic-
of coagulation and clot lysis include:
tate whether blood collection and recovery
1. Whether the patient had received
are used and how they are achieved.
systemic anticoagulation.
2. The amount and type of anticoagu-
lant used. Processing Before Reinfusion
3. The extent of contact between blood Several devices automatically process re-
and serosal surfaces. covered blood before reinfusion. Vacuum
4. The extent of contact between blood suction and simultaneous anticoagula-
and artificial surfaces. tion are used for collection. To minimize
5. The degree of turbulence during col- hemolysis, the vacuum level should ordi-
lection. narily not exceed 150 torr, although higher
In general, blood collected at low flow rates levels of suction may occasionally be
or during slow bleeding from patients who needed during periods of rapid bleeding.
are not systemically anticoagulated will Either citrate (ACD) or heparin may be
have undergone coagulation and fibrinoly- used as an anticoagulant. Blood is held in
sis and will not contribute to hemostasis a reservoir until centrifuged and washed
upon reinfusion. with a volume of saline that varies be-
The high suction pressure and surface tween 500 and 1500 mL. If not infused im-
skimming during aspiration and the turbu- mediately, the unit must be labeled with
lence or mechanical compression that oc- the patient’s name and identification

number, the date and time collection was Hospitals with collection and recovery
initiated, and the statement “For Autolo- programs should establish written policies
gous Use Only.” and procedures that are regularly reviewed
An alternative approach is to collect by a physician who has been assigned re-
blood in a canister system designed for di- sponsibility for the program. Transfusion
rect reinfusion and then concentrate and medicine specialists should play an active
wash the recovered red cells in a blood role in design, implementation, and opera-
bank cell washer. Intraoperatively collected tion of the program. Written policies must
and recovered blood must be handled in be in place for the proper collection, label-
the transfusion service laboratory like any ing, and storage of intraoperative autolo-
other autologous unit. The unit should be gous blood. Equipment and techniques for
reinfused through a filter. collection and infusion must ensure that
the blood is aseptic. Quality management
should include evaluation of the appropri-
Direct Reinfusion ate use of blood collection and recovery
services and adequate training of person-
Systems are available that collect recovered
nel. Written protocols, procedure logs, ma-
blood and return it directly. These sys-
chine maintenance, procedures for han-
tems generally consist of a suction cathe-
dling adverse events, and documentation
ter attached to a disposable collection bag
are recommended.24
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
Postoperative Blood
preferred in nonwashed systems. Collection
Postoperative blood collection denotes
Requirements and Recommendations the recovery of blood from surgical drains
The AABB requires a process that includes followed by reinfusion, with or without
patient and storage bag identification and processing. In some programs, postopera-
time collected with expiration date.16(p10) tive shed blood is collected into sterile
Units collected intraoperatively should be canisters and reinfused, without process-
labeled with the patient’s first and last ing, through a microaggregate filter. Re-
name, hospital identification number, the covered blood is dilute, partially hemolyzed
date and time of collection and expira- and defibrinated, and may contain high
tion, and the statement “For Autologous concentrations of cytokines. For these
Use Only.”16(p10) reasons, most programs set an upper limit
Conditions for storage and expiration of on the volume (eg, 1400 mL) of unprocessed
autologous components collected in the blood that can be reinfused. If transfusion
operating room are listed in Table 5-8.16(p14) If of blood has not begun within 6 hours of
the blood leaves the patient for washing or initiating the collection, the blood must be
storage in a remote location, there must be discarded. Hospitals should establish
appropriate procedures to ensure proper written policies, procedures, labeling re-
labeling of the blood according to AABB quirements, quality assurance, and review
standards.16(pp9,10) consistent with AABB standards.16(p2)

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

Collection Storage Special
Type Temperature Expiration Conditions
Acute normovolemic Room tempera- 8 hours from start None

hemodilution ture of collection
1-6 C 24 hours from start Storage at 1-6 C shall
of collection begin within 8 hours
of start of collection
Intraoperative blood Room tempera- 4 hours from end of None

recovered with ture collection
processing 1-6 C 24 hours from start Storage at 1-6 C shall
of collection begin within 4 hours
of start of collection
Intraoperative blood Room tempera- 4 hours from end of None

recovered without ture or 1-6 C collection
processing
Shed blood under post- N/A 6 hours from start None

operative or post- of collection
traumatic conditions
with or without pro-
cessing
Non-red-cell component Room tempera- Shall be used before None

preparation ture leaving the oper-
ating room
Clinical Studies in transfusion practices. Modification of

physician transfusion practices may have
The evolution of cardiac surgery has been been an uncredited intervention in these
accompanied by a broad experience in blood conservation studies.
postoperative conservation of blood. Post- In the postoperative orthopedic surgical
operative autologous blood transfusion is setting, several reports have similarly des-
practiced widely, but not uniformly. Pro- cribed the successful recovery and reinfu-
spective and controlled trials have dis- sion of washed87 and unwashed88,89 wound
agreed over the efficacy of postoperative drainage from patients undergoing arthro-
blood recovery in cardiac surgery pat- plasty. Red cells recovered in this setting
ients; at least three such studies have appear to have normal survival in the circu-
demonstrated lack of efficacy,82-84 but at lation.90 The volume of reinfused drainage
85,86
least two studies have shown benefit. blood has been reported to be as much as
The disparity of the results in these stud- 3000 mL and averages more than 1100 mL
ies may be explained, in part, by differences in patients undergoing cementless knee re-

89
placement. Because the red cell content of
the fluid collected is low (hematocrit levels
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transfusion of shed mediastinal blood after Transfusion 2000;40:1063-6.

Chapter 6: Apheresis
Chapter 6
Apheresis
A
PHERESIS, FROM THE Greek
pheresis meaning “to take away,”
Separation Techniques
involves the selective removal of Automated blood processing devices are
blood constituents from blood donors or used for both component preparation and
patients. therapeutic applications of apheresis.
The AABB provides standards1 for volun- Manual apheresis, in which whole blood
tary compliance for apheresis activities. is collected in multiple bags and centri-
The Food and Drug Administration (FDA) fuged offline, requires great care to ensure
has established specific requirements that that the bags are labeled correctly and are
are set forth in the Code of Federal Regula- returned to the correct donor. With the
2
tions for apheresis activities. The American currently available automated technology,
Society for Apheresis (ASFA)3 has published this process is seldom used.
additional guidelines and recommendations.
In addition, hemapheresis practitioner (HP) Separation by Centrifugation
and apheresis technician (AT) certifications In most apheresis instruments, centrifu-
are available through the American Society gal force separates blood into components
of Clinical Pathology Board of Registry. All on the basis of differences in density. A
personnel involved with apheresis activities measured amount of anticoagulant solu-
should be familiar with these sources and tion is added to the whole blood as it is
should have documentation that they are drawn from the donor or patient. The blood 6
qualified by training and experience to per- is pumped into a rotating bowl, chamber,
form apheresis. or tubular rotor in which layering of com-
139

ponents occurs on the basis of their den- depleted plasma along with the cellular
sities. The desired fraction is diverted and components reduces or eliminates the
the remaining elements are returned to the need for replacement fluids. Immuno-
donor (or patient) by intermittent or con- adsorption can be performed online, or
tinuous flow. the plasma can be separated from the cel-
All systems require prepackaged dispos- lular components, passed through an off-
able sets of sterile bags, tubing, and centrif- line column, and then reinfused.
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
Component Collection
method, the centrifuge container is alternately Whenever components intended for trans-
filled and emptied. Most instruments in use fusion are collected by apheresis, the do-
today employ a method that involves the nor must give informed consent. Although
continuous flow of blood through a separa- apheresis collection and preparation pro-
tion chamber. Depending on the procedure cesses are different from those used for
and device used, the apheresis procedure whole-blood-derived components, stor-
time varies from 30 minutes to several hours. age conditions, transportation require-
Each manufacturer supplies detailed in- ments, and some quality control steps are
formation and operational protocols. Each the same. See Chapter 8 for more detailed
facility must have, in a manual readily information. The facility must maintain
available to nursing and technical person- written protocols for all procedures used
nel, detailed descriptions of each type of and must keep records for each procedure
procedure performed, specific for each type as required by AABB Standards for Blood
of blood processor.4 Banks and Transfusion Services.1(p2)
Platelets Pheresis
Separation by Adsorption
Plateletpheresis is used to obtain platelets
Selective removal of a pathologic material from random volunteer donors, from pat-
has theoretical advantages over the re- ients’ family members, or from donors with
moval of all plasma constituents. Centrif- matched HLA or platelet antigen pheno-
ugal devices can be adapted to protocols types. Because large numbers of platelets
that selectively remove specific soluble can be obtained from a single individual,
plasma constituents by exploiting the collection by apheresis reduces the num-
principles of affinity chromatography.5 Se- ber of donor exposures for patients. AABB
lective removal of low-density lipoproteins Standards requires the component to
(LDLs) in patients with familial hypercho- contain at least 3 × 10 platelets in 90% of
11
lesterolemia has been accomplished using sampled units.

1(p31)
When a high yield is
both immunoaffinity (anti-LDL) and che- obtained, the original apheresis unit may
6
mical affinity (eg, dextran sulfate) columns. be divided into multiple units, each of which
Adsorbents such as staphylococcal protein must meet minimum standards inde-
A (SPA), monoclonal antibodies, blood pendently. Some instruments are pro-
group substances, DNA-collodion, and grammed to calculate the yield from the
polymers with aggregated IgG attached donor’s hematocrit, platelet count, height,
can extract antibodies, protein antigens, and weight. For alloimmunized patients
and immune complexes. Returning the who are refractory to random allogeneic

Chapter 6: Apheresis 141
platelets (see Chapters 16 and 21), platelets occur less often among apheresis donors
from an apheresis donor selected on the than among whole blood donors.
basis of a compatible platelet crossmatch Plateletpheresis donors should meet
or matched for HLA antigens may be the usual donor requirements, including he-
only way to achieve a satisfactory post- moglobin or hematocrit level. A platelet
transfusion platelet increment. Within the count is not required before the first
United States, the use of apheresis plate- apheresis collection or if 4 weeks or more
lets has been steadily increasing over the have elapsed since the last procedure. If the
last 25 years. Currently, it is estimated that donation interval is less than 4 weeks, the
77% of therapeutic platelet doses are donor’s platelet count should be above
7
transfused as apheresis platelets. 150,000/µL before subsequent platelet-
pheresis occurs. AABB Standards permits
documentation of the platelet count from a
sample collected immediately before the
Donor Selection and Monitoring
procedure or from a sample obtained either
Plateletpheresis donors may donate more before or after the previous procedure.1(p21)
frequently than whole blood donors but Exceptions to these laboratory criteria should
must meet all other donor criteria. The in- be approved in writing by the apheresis phy-
terval between donations should be at sician. The FDA specifies that the total vol-
least 2 days, and donors should not un- ume of plasma collected should be no more
dergo plateletpheresis more than twice than 500 mL (or 600 mL for donors weighing
in a week or more than 24 times in a more than 175 pounds).8 The platelet count
year.1(pp19,20) If the donor donates a unit of of each unit should be kept on record but
Whole Blood or if it becomes impossible need not be written on the product label.8
to return the donor’s red cells during pla- Some plateletpheresis programs collect
teletpheresis, at least 8 weeks should elapse plasma for use as Fresh Frozen Plasma
before a subsequent plateletpheresis pro- (FFP) in a separate bag during platelet col-
cedure, unless the extracorporeal red cell lection. Apheresis can also be used to col-
volume is less than 100 mL.1(p20) Platelets lect plasma for FFP without platelets, ie,
may be collected from donors who do not plasmapheresis. The FDA has provided
meet these requirements if the compo- guidance with regard to the volume of
nent is expected to be of particular value plasma that is allowed to be collected using
to a specific intended recipient, and if a automated devices.9 A total serum or plasma
physician certifies in writing that the do- protein determination and a quantitative
nor’s health will not be compromised (eg, determination of IgG and IgM (or a serum
an HLA-matched donor). Donors who protein electrophoresis) must be deter-
have taken aspirin-containing medica- mined at 4-month intervals for donors un-
tions within 36 hours of donation are usu- dergoing large-volume plasma collection, if
ally deferred because the platelets ob- the total annual volume of plasma collected
tained by apheresis are often the single exceeds 12 liters (14.4 L for donors weigh-
source of platelets given to a patient. ing more than 175 pounds) or if the donor
Vasovagal and hypovolemic reactions are is a frequent (more often than every 4
rare in apheresis donors, but paresthesias weeks) plasma donor.10 AABB Standards re-
and other reactions to the citrate antico- quires that the donor’s intravascular volume
agulant are common (see Complications, deficit must be less than 10.5 mL per kilo-
later in this chapter). Serious reactions gram of body weight at all times.1(p24)

Laboratory Testing Plasma

Tests for ABO group and Rh type, unex- Apheresis can be used to collect plasma as
pected alloantibodies, and markers for FFP or for Source Plasma for subsequent
transfusion-transmitted diseases must be manufacturing. FDA requirements for
performed by the collecting facility in the plasma collection are different from those
same manner as for other blood compo- for whole blood or plateletpheresis; per-
nents. Each unit must be tested unless the sonnel who perform serial plasmapheresis
donor is undergoing repeated procedures must be familiar with both AABB stan-
to support a single patient, in which case dards and FDA requirements. If plasma is
testing for disease markers need be re- intended for transfusion, testing require-
1(p34)
peated only at 30-day intervals. ments are the same as those for red cell
If red cells are visible in a product, the components. Plasma collected for manu-
hematocrit should be determined. FDA facture of plasma derivatives is subject to
guidelines require that if the component different requirements for infectious dis-
contains more than 2 mL of red cells, a
ease testing.
sample of donor blood for compatibility
A distinction is made between “occa-
testing be attached to the container.8 In
sional plasmapheresis,” in which the donor
some instances, it may be desirable for the
undergoes plasmapheresis no more often
donor plasma to be ABO-compatible with
than once in 4 weeks, and “serial plasma-
the recipient’s red cells—for example, if the
pheresis,” in which donation is more fre-
recipient is a child or an ABO-mismatched
quent than every 4 weeks. For donors in an
allogeneic progenitor cell transplant recipi-
occasional plasmapheresis program, donor
ent. In order to be considered leukocyte-
selection and monitoring are the same as
reduced, apheresis platelets must contain
for whole blood donation. For serial plasma-
less than 5 × 106 leukocytes and must meet
pheresis using either automated instru-
the specifications of the apheresis device
ments or manual techniques, the following
manufacturer. Chapter 8 describes addi-
principles apply:
tional quality control measures that apply
1. Donors must provide informed con-
to all platelet components.
sent. They must be observed closely
during the procedure and emergency
medical care must be available.
Records 2. Red cell losses related to the proce-
Complete records (see Chapter l) must be dure, including samples collected
kept for each procedure. All adverse reac- for testing, must not exceed 25 mL
tions should be documented along with per week, so that no more than 200
the results of their investigation and fol- mL of red cells are removed in 8
low-up. Records of all laboratory findings weeks. If the donor’s red cells cannot
and collection data must be periodically be returned during an apheresis pro-
reviewed by a knowledgeable physician cedure, hemapheresis or whole blood
and found to be within acceptable limits. donation should be deferred for 8
FDA guidelines require review at least weeks.
once every 4 months.8 Facilities must have 3. In manual plasma collection systems,
policies and procedures in place to ensure there must be a mechanism to en-
that donor red cell loss during each pro- sure safe reinfusion of the autologous
cedure does not exceed acceptable limits. red cells. Before the blood container

is separated from the donor for pro- geneic or autologous Red Blood Cell units
cessing, there should be two sepa- every 16 weeks by an automated aphere-
rate, independent means of identifi- sis method. Saline infusion is used to min-
cation, so that both the donor and imize volume depletion, and the proce-
the phlebotomist can ascertain that dure is limited to persons who are larger
the contents are those of the donor. and have higher hematocrits than current
Often, the donor’s signature is one minimum standards for whole blood do-
identifier, along with a unique iden- nors (for males: weight 130 lb, height 5′1″;
tification number. for females: weight 150 lb, height 5′5″;
4. In manual procedures for donors hematocrit 40% for both genders).11
weighing 50 to 80 kg (110-176 lb), no
more than 500 mL of whole blood Granulocytes
should be removed at one time, or The indications for granulocyte transfu-
1000 mL during the session or within sion are controversial (see Chapter 21). A
a 48-hour period. The limits for do- meta-analysis of randomized controlled
nors who weigh more than 80 kg are trials of granulocyte transfusion indicates
600 mL and 1200 mL, respectively. that effectiveness depends on an adequate
For automated procedures, the allow- dose (>1 × 10 10 granulocytes/day) and
able volume has been determined crossmatch compatibility (no recipient
9
for each instrument by the FDA. antibodies to granulocyte antigens). 1 2
5. At least 48 hours should elapse be- There is renewed interest in granulocyte
tween successive procedures; ordi- transfusion therapy for adults because
narily, donors should not undergo much larger cell doses can be delivered
more than two procedures within a when cells are collected from donors who
7-day period. Exceptions are permis- receive colony-stimulating factors.13 Some
sible when plasma is expected to success with granulocyte transfusions has
have special therapeutic value for a been observed in the treatment of septic
single recipient. infants,14 possibly because the usual dose
6. At the time of initial plasmapheresis is relatively larger in these tiny recipients
and at 4-month intervals thereafter and because HLA alloimmunization is ab-
for donors undergoing plasmaphere- sent.
sis more often than once every 4
weeks, serum or plasma must be
Drugs Administered for Leukapheresis
tested for total protein and serum
A daily dose of at least 1 × 10 granulo-
10
protein electrophoresis or quantita-
tive immunoglobulins. Results must cytes is necessary to achieve a therapeutic
be within normal limits.
2 effect.15 Collection of this number of cells
7. A qualified, licensed physician, know- requires administration of drugs or other
ledgeable in all aspects of hema- adjuvants to the donor. The donor’s con-
pheresis, must be responsible for the sent should include specific permission
program. for any drugs or sedimenting agents to be
used.
Hydroxyethyl Starch. A common sedi-
Red Cells menting agent, hydroxyethyl starch (HES),
Both AABB standards and FDA-approved causes red cells to aggregate and thereby
protocols address the removal of two allo- sediment more completely. Sedimenting

agents enhance granulocyte harvest and re- should be ABO-compatible with the re-
sult in minimal red cell content. Because cipient’s plasma and, if more than 2 mL
HES can be detected in donors for as long are present, the component should be
as a year after infusion, AABB Standards re- crossmatched. Ideally, D-negative recipi-
quires facilities performing granulocyte col- ents should receive granulocyte concentrates
lections to have a process to control the from D-negative donors. Leukocyte (HLA)
maximal cumulative dose of any sedi- matching is recommended in alloim-
menting agent administered to the donor munized patients.
1(p24)
within a given interval. Because HES is a
colloid, it acts as a volume expander, and Storage and Infusion
donors who have received HES may experi- Because granulocyte function deteriorates
ence headaches or peripheral edema because during storage, concentrates should be
of expanded circulatory volume. transfused as soon as possible after prep-
Corticosteroids. Corticosteroids can aration. AABB Standards prescribes a
double the number of circulating granulo- storage temperature of 20 to 24 C, for no
cytes by mobilizing them from the marginal longer than 24 hours.1(p57) Agitation during
pool. A protocol using 60 mg of oral predni- storage is probably undesirable. Irradiation
sone as a single or divided dose before do- is required before administration to im-
nation gives superior granulocyte harvests munodeficient recipients and will proba-
16
with minimal systemic steroid activity. Al- bly be indicated for nearly all recipients
ternatively, 8 mg of oral dexamethasone may because of their primary disease. Infusion
be used. Before administration of cortico- through a microaggregate or leukocyte re-
steroids, donors should be questioned about duction filter is contraindicated.
any history or symptoms of hypertension,
diabetes, cataracts,17 and peptic ulcer. Hematopoietic Progenitor Cells
Growth Factors. Recombinant hemato-
Cytapheresis for collection of hematopoietic
poietic growth factors—specifically, granulo-
progenitor cells is useful for obtaining
cyte colony-stimulating factor (G-CSF)—
progenitor cells for marrow reconstitution
can effectively increase granulocyte yields.
in patients with cancer, leukemia in re-
Hematopoietic growth factors alone can re-
mission, and various lymphomas (see
sult in collection of up to 4 to 8 × 1010 granu-
Chapter 25). Cytapheresis procedures can
locytes per apheresis procedure.13 Typical
also be used to collect donor lymphocytes
doses of G-CSF employed are 5 to 10 µg/kg
for infusion as an immune therapy in these
given 8 to 12 hours before collection.18 Pre-
patients (see Chapter 25). The AABB has
liminary evidence suggests that in-vivo re-
published Standards for Cellular Therapy
covery and survival of these granulocytes
Product Services.19 Additional requirements
are excellent and that growth factors are
13 are reviewed in Chapter 25.
well tolerated by donors.
Laboratory Testing Therapeutic Apheresis

Testing for ABO and Rh, alloantibodies, Therapeutic apheresis has been used to
and infectious disease markers on a sam- treat many different diseases. Cells, plasma,
ple drawn at the time of phlebotomy are or plasma constituents may be removed
required. Red cell content in granulocyte from the circulation and replaced by nor-
concentrates is inevitable; the red cells mal plasma, crystalloid, or colloid solu-

tions of starch or albumin. The term tient’s care should establish a treatment
“therapeutic apheresis” is used for the plan and the goal of therapy. The end-
general procedure and the term “thera- point may be an agreed-upon objective
peutic plasma exchange” (TPE) is used for outcome or a predetermined duration for
procedures in which the goal is the re- the therapy, whichever is achieved first. It
moval of plasma, regardless of the solu- is helpful to document these mutually ac-
tion used as replacement. ceptable goals in the patient’s medical re-
The theoretical basis for therapeutic cord. The nature of the procedure, its ex-
apheresis is to reduce the patient’s load of a pected benefits, its possible risks, and the
pathologic substance to levels that will al- available alternatives should be explained
low clinical improvement. In some condi- to the patient by a knowledgeable individ-
tions, replacement with normal plasma is ual, and the patient’s consent should be
intended to supply an essential substance documented. The procedure should be
that is absent. In the absence of the need to performed only in a setting where there is
replace plasma constituents, colloidal solu- ready access to care for untoward reac-
tions and/or saline should be used as re- tions, including equipment, medications,
placement fluids. Other possible outcomes and personnel trained in managing seri-
of therapeutic apheresis include alteration ous reactions.
of the antigen-to-antibody ratio, modifica-
tion of mediators of inflammation or im- Vascular Access
munity, and clearance of immune com-
For most adults needing a limited number
plexes. Some perceived benefit may result
of procedures, the antecubital veins are
from a placebo effect. Despite difficulties in
suitable for removal and return of blood.
documentation, there is general agreement
For critically ill adults and for children,
that therapeutic apheresis is effective treat-
indwelling central or peripheral venous
ment for the conditions listed in Table 6-1
catheters are typically used. Especially ef-
as Category I or Category II.3,20-22
fective are rigid-wall, large-bore, double-
lumen catheters placed in the subclavian,
General Considerations femoral, or internal jugular vein. Cathe-
Appropriate use of therapeutic apheresis ters of the type used for temporary hemo-
requires considerable medical knowledge dialysis allow both removal and return of
and judgment. The patient should be blood at high flow rates. Central catheters
evaluated for treatment by his or her per- can be maintained for weeks if multiple
sonal physician and by the apheresis phy- procedures are necessary. Tunnel catheters
sician. Close consultation between these can be used when long-term apheresis is
physicians is important, especially if the anticipated.
patient is small or elderly, has poor vascu-
lar access or cardiovascular instability, or Removal of Pathologic Substances
has a condition for which apheresis is of During TPE, plasma that contains the
uncertain benefit. The apheresis physi- pathologic substance is removed and a re-
cian should make the final determination placement fluid is infused. The efficiency
about appropriateness of the procedure with which material is removed can be
and eligibility of the patient (see Table estimated by calculating the patient’s
3,20-22
6-1). When therapeutic apheresis is plasma volume and using Fig 6-1. This es-
anticipated, those involved with the pa- timate depends on the following assump-

20
Table 6-1. Indication Categories for Therapeutic Apheresis
Indication
Disease Procedure Category
Renal and metabolic diseases

Antiglomerular basement membrane antibody Plasma exchange I
disease
Rapidly progressive glomerulonephritis Plasma exchange II
Hemolytic uremic syndrome Plasma exchange III
Renal transplantation
Rejection Plasma exchange IV
Sensitization Plasma exchange III
Recurrent focal glomerulosclerosis Plasma exchange III
Heart transplant rejection Plasma exchange III
Photopheresis III
Acute hepatic failure Plasma exchange III
Familial hypercholesterolemia Selective adsorption I
Plasma exchange II
Overdose or poisoning Plasma exchange III
Phytanic acid storage disease Plasma exchange I
Autoimmmune and rheumatic diseases
Cryoglobulinemia Plasma exchange II
Idiopathic thrombocytopenic purpura Immunoadsorption II
Raynaud's phenomenon Plasma exchange III
Vasculitis Plasma exchange III
Autoimmune hemolytic anemia Plasma exchange III
Rheumatoid arthritis Immunoadsorption II
Lymphoplasmapheresis II
Plasma exchange IV
Scleroderma or progressive systemic Plasma exchange III
sclerosis
Systemic lupus erythematosus Plasma exchange III
Lupus nephritis Plasma exchange IV
Psoriasis Plasma exchange IV
Hematolic diseases
ABO-mismatched marrow transplant RBC removal (marrow) I
Plasma exchange II
(recipient)

20
Table 6-1. Indication Categories for Therapeutic Apheresis (cont'd)
Indication
Erythrocytosis or polycythemia vera Phlebotomy I

Erythrocytapheresis II
Leukocytosis and thrombocytosis Cytapheresis I
Thrombotic thrombocytopenia purpura Plasma exchange I
Posttransfusion purpura Plasma exchange I
Sickle cell diseases RBC exchange I
Myeloma, paraproteins, or hyperviscosity Plasma exchange II
Myeloma or acute renal failure Plasma exchange II
Coagulation factor inhibitors Plasma exchange II
Aplastic anemia or pure RBC aplasia Plasma exchange III
Cutaneous T-cell lymphoma Photopheresis I
Leukapheresis III
Hemolytic disease of the fetus and newborn Plasma exchange III
Platelet alloimmunization and refractoriness Plasma exchange III
Immunoadsorption III
Malaria or babesiosis RBC exchange III
AIDS Plasma exchange IV
Neurologic disorders
Chronic inflammatory demyelinating Plasma exchange I
polyradiculoneuropathy
Acute inflammatory demyelinating Plasma exchange I
polyradiculoneuropathy
Lambert-Eaton myasthenia syndrome Plasma exchange II
Multiple sclerosis
Relapsing Plasma exchange III
Progressive Plasma exchange III
Lymphocytapheresis III
Myasthenia gravis Plasma exchange I
Acute central nervous system inflammatory Plasma exchange II
demyelinating disease
Paraneoplastic neurologic syndromes Plasma exchange III
(cont'd)

20
Table 6-1. Indication Categories for Therapeutic Apheresis (cont'd)
Indication
Demyelinating polyneuropathy with IgG and Plasma exchange I

IgA
Sydenham's chorea Plasma exchange II
Polyneuropathy with IgM (with or without Plasma exchange II
Waldenstrom's) Immunoadsorption III
Cryoglobulinemia with polyneuropathy Plasma exchange II
Multiple myeloma with polyneuropathy Plasma exchange III
POEMS syndrome Plasma exchange III
Systemic (AL) amyloidosis Plasma exchange IV
Polymyositis or dermatomyositis Plasma exchange III
Leukapheresis IV
Inclusion-body myositis Plasma exchange III
Leukapheresis IV
Rasmussen's encephalitis Plasma exchange III
Stiff-person syndrome Plasma exchange III
PANDAS Plasma exchange II
Amyotrophic lateral sclerosis Plasma exchange IV
POEMS = polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin lesions; PANDAS = pediat-
ric autoimmune neuropsychiatric disorders; Category I = standard acceptable therapy; Category II = sufficient evidence
to suggest efficacy usually as adjunctive therapy; Category III = inconclusive evidence of efficacy or uncertain risk/bene-
fit ratio; Category IV = lack of efficacy in controlled trials.
tions: 1) the patient’s blood volume does make it necessary to repeat the process.
not change; 2) mixing occurs immedi- Rarely are two or more plasma volumes
ately; and 3) there is relatively little pro- exchanged in one procedure. Although
duction or mobilization of the pathologic larger volume exchange causes greater ini-
material to be removed during the proce- tial diminution of the pathologic sub-
dure. As seen in Fig 6-1, removal is great- stance, overall it is less efficient and re-
est early in the procedure and diminishes quires considerably more time. Larger
progressively during the exchange. Ex- volume exchanges can increase the risk of
change is usually limited to 1 or 1.5 coagulopathy, citrate toxicity, or electro-
plasma volumes, or approximately 40 to lyte imbalance, depending on the re-
60 mL plasma exchanged per kg of body placement fluid.
weight in patients with normal hemato- The rates at which a pathologic sub-
crit and average body size. This maxi- stance is synthesized and distributed be-
mizes the efficacy per procedure but may tween intravascular and extravascular com-

Figure 6-1. The relationship between the volume of plasma exchange and the patient’s original plasma
remaining.
partments affect the outcome of TPE. For Plasma removed during TPE should be
example, the abnormal IgM of Walden- handled carefully and disposed of properly.
strom’s macroglobulinemia is synthesized Such plasma cannot be used for subse-
slowly and remains almost entirely (about quent manufacture of transfusable plasma
75%) intravascular, making apheresis par- derivatives.
ticularly effective in removing it.23 In addi-
tion, a relatively small change in intra-
vascular protein concentration may result Removal of Normal Plasma Constituents
in a large change in blood viscosity. In con- When the quantity of plasma removed
trast, efforts to prevent hydrops fetalis with during TPE exceeds 1.5 times the plasma
intensive TPE to lower the mother’s level of volume, different rates of removal and re-
IgG anti-D have been less successful. This is constitution are observed for different
25
due in part to the fact that about 55% of IgG constituents. In the case of fibrinogen,
is in the extravascular fluid. In addition, the third component of complement (C3),
rapid reduction of IgG may cause antibody and immune complexes, 75% to 85% of the
synthesis to increase rapidly and “rebound” original substance is removed after a 1.5
over pretreatment levels.24 Rebound synthe- plasma-volume procedure. Pretreatment
sis may also complicate TPE treatment of levels are restored in 3 to 4 days. The con-
autoimmune diseases. Immuno-suppressive centrations of electrolytes, uric acid, Fac-
agents such as cyclophosphamide, azathio- tor VIII, and other proteins are less af-
prine, or prednisone may be administered fected by a plasma exchange. A 10% or
to blunt the autoantibody rebound response more decrease in platelet count generally
to apheresis. occurs, with 2 to 4 days needed for a re-

26
turn to pretreatment values. Coagulation plasma, or cryoprecipitate. Because plasma
factors other than fibrinogen generally re- contains citrate, its use may increase the
turn to pretreatment values within 24 risk of citrate toxicity.
hours. Immunoglobulin removal occurs at
about the expected rate of 65% per plasma
volume, but recovery patterns vary for dif- Complications
ferent immunoglobulin classes, depend- With careful patient selection and atten-
ing on intravascular distribution and rates tion to technical details, most therapeutic
of synthesis. (Table 11-3 describes immu- apheresis procedures are completed with-
noglobulin characteristics.) Plasma IgG out complications. Adverse effects of ther-
levels return to approximately 60% of the apeutic apheresis were reported in only
pretreatment value within 48 hours be- 4% of patients in one large study.28 How-
cause of reequilibration with protein in the ever, therapeutic apheresis is often required
extravascular space. These issues are im- for patients who are critically ill and at risk
portant in planning the frequency of ther- for a variety of complications.
apeutic procedures. Weekly apheresis per- Vascular Access. Patients requiring ther-
mits more complete recovery of normal apeutic apheresis have often been subjected
plasma constituents; daily procedures can to multiple venipunctures and achieving
be expected to deplete many normal, as peripheral vascular access may be difficult.
well as abnormal, constituents. Addition- Frequently, special venous access, such as
ally, intensive apheresis reduces the con- placement of an indwelling double-lumen
centration of potentially diagnostic plasma apheresis/dialysis catheter, is required. Ve-
constituents, so blood for testing should be nous access devices may cause further
drawn before TPE. vascular damage, sometimes resulting in
thrombosis. Infrequently, they may result in
severe complications such as pneumo-
Replacement Fluids thorax or perforation of the heart or great
Available replacement solutions include: vessels. 28 Other complications include
crystalloids, albumin solutions, plasma arterial puncture, deep hematomas, and
(FFP, cryosupernatant plasma, or Plasma arteriovenous fistula formation. Bacterial
Frozen within 24 Hours of Collection), and colonization often complicates long-term
HES.27 Table 6-2 presents advantages and placement and may lead to catheter-associ-
disadvantages of each. A combination is ated sepsis, especially in patients who are
often used, the relative proportions being receiving steroids or other immunosup-
determined by the physician on the basis pressants. Inadvertent disconnection of
of the patient’s disease and physical con- catheters may produce hemorrhage or air
dition, the planned frequency of proce- embolism.
dures, and cost. Acute treatment of Alteration of Pharmacodynamics. TPE
immediately life-threatening conditions can lower blood levels of drugs, especially
usually requires a series of daily plasma those that bind to albumin. Apheresis re-
exchange procedures, often producing a duces plasma levels of antibiotics and anti-
significant reduction of coagulation factors. convulsants, but few clinical data exist to
Monitoring the platelet count, prothrom- suggest adverse patient outcomes due to
bin time, activated partial thromboplastin apheresis-associated lowering of drug lev-
time, and fibrinogen level helps deter- els. Nevertheless, the pharmacokinetics of
mine the need for supplemental platelets, all drugs being given to a patient should be

Table 6-2. Comparison of Replacement Fluids

Replacement
Solution Advantages Disadvantages
Crystalloids Low cost 2-3 volumes required

Hypoallergenic Hypo-oncotic
No viral risk No coagulation factors
No immunoglobulins
Albumin Iso-oncotic High cost
No contaminating No coagulation factors
“inflammatory mediators”
No viral risk No immunoglobulins
Hydroxyethyl starch Moderate cost No coagulation factors
Iso-oncotic Long-term residual levels of HES
No contaminating “inflammatory Contraindicated with renal failure
mediators” Possible coagulopathy
Plasma Maintains normal levels of: Viral transmission risk
immunoglobulins Citrate load
complement ABO incompatibility risk
antithrombin Allergic reactions
other proteins Sensitization
considered before starting apheresis and is returned, ionized and bound calcium are
dosage schedules adjusted if necessary. It is removed, and ionized calcium is bound to
prudent to withhold the administration of “calcium-stripped” albumin replacement.
drugs scheduled to be given during or up to Hyperventilation, hypothermia, hypo-
an hour before apheresis until after the pro- magnesemia, and the use of plasma as a re-
cedure has finished. Removal of plasma placement solution exacerbate citrate tox-
cholinesterase may complicate the admin- icity. Hypocalcemia can usually be controlled
istration of paralyzing agents such as by reducing the proportion of citrate or
succinylcholine in the immediate post- slowing the reinfusion rate. If untreated,
exchange period. symptoms may progress to muscle twitch-
Hypocalcemia. Most patients and donors ing, chills, pressure in the chest, nausea,
with normal parathyroid and liver function vomiting, and hypotension. Low ionized
maintain calcium homeostasis during aphere- calcium concentrations can induce severe
sis. However, symptoms of hypocalcemia cardiac arrhythmias. Asking the patient to
related to citrate toxicity are the most com- report any vibrations or tingling sensations
mon adverse effect reported in 3.0% of can help determine the appropriate rein-
therapeutic apheresis procedures in one fusion rate. Extra precautions must be
large study.28 Symptoms of reduced plasma taken in patients who are unable to com-
levels of ionized calcium (perioral pare- municate or who may metabolize citrate
sthesias, tingling, a feeling of vibrations) re- poorly (eg, those with liver failure). Hypo-
flect the rate at which citrate anticoagulant calcemic toxicity can usually be managed

by administering oral calcium carbonate or production may further compromise de-

intravenous calcium.22,23,29 fense mechanisms.
Circulatory Effects. Hypovolemia and Mechanical Hemolysis and Equipment
subsequent hypotension may occur during Failures. Collapsed or kinked tubing, mal-
apheresis, especially when the volume of functioning pinch valves, or improper
extracorporeal blood exceeds 15% of the to- threading of tubing may damage donor or
tal blood volume. Hypotension tends to oc- patient red cells in the extracorporeal cir-
cur in ill children, the elderly, neurology pa- cuit. Machine-related hemolysis was ob-
tients, anemic patients, and those treated served in 0.07% of over 195,000 apheresis
with intermittent-flow devices that have procedures performed in the United King-
large extracorporeal volumes. Continuous- dom.31 Similar rates of hemolysis, 0.06% and
flow devices typically do not require large 0.01%, were reported in response to uniform
extracorporeal volumes but can produce questionnaires regarding therapeutic and
hypovolemia if return flow is inadvertently donor apheresis procedures, respectively.32,33
diverted to a waste collection bag, either Hemolysis can also occur with incom-
through operator oversight or mechanical patible replacement fluids such as D5W
or software failures. Hypovolemia may also (eg, D5W used to dilute 25% albumin) or
be secondary to inadequate volume or pro- ABO-discrepant plasma. The operator
tein replacement. During all procedures, it should carefully observe plasma collection
is essential to maintain careful and contin- lines for pink discoloration suggestive of
uous records of the volumes removed and hemolysis. Other types of equipment fail-
returned. The use of antihypertensive med- ure, such as problems with the rotating
ications, especially angiotensin-converting seal, leaks in the plastic, and roller pump
34
enzyme (ACE) inhibitors combined with al- failure, are rare.
bumin replacement, may also contribute to Allergic Reactions and Respiratory Dis-
hypotensive reactions (see Chapter 27). Pa- tress. Respiratory difficulty during or im-
tients taking agents that inhibit ACE have mediately following apheresis can have
experienced severe hypotensive episodes many causes: pulmonary edema, massive
when treated with SPA columns and with pulmonary embolism, air embolism, ob-
other immunosorbents.30 Patients should not struction of the pulmonary microvascula-
receive these medications for 72 hours be- ture, anaphylactic reactions, and transfusion-
fore undergoing immunoabsorption treat- related acute lung injury.35 Hemothorax or
ment. Because infusion of cold fluids hemopericardium due to vascular erosion
through a central venous catheter may in- by a central venous catheter is typically un-
36,37
duce arrhythmias, some apheresis programs suspected yet may be fatal. Pulmonary
use blood warmers for selected patients. edema that results from volume overload or
Infections. Plasma is the only commonly cardiac failure is usually associated with
used replacement solution with the risk of dyspnea, an increase in the diastolic blood
transmitting infectious viruses. Bacterial pressure, and characteristic chest X-ray
colonization and infection related to re- findings. Acute pulmonary edema can also
peated apheresis usually arise from within arise from damage to alveolar capillary
the vascular catheter. Intensive apheresis membranes secondary to an immune reac-
regimens decrease levels of immunoglobu- tion or to vasoactive substances in FFP or
lins and the opsonic components of com- colloid solutions prepared from human
plement. In addition, immunosuppressive plasma. The use of FFP as a replacement
drugs used to prevent rebound antibody fluid has been associated with complement

activation and with allergic reactions that ate or amplify a placebo effect and bias
produce urticaria, swelling of oral mucosa, the evaluation of clinical improvement.
and bronchospasm; these usually respond For many of the diseases being treated,
to antihistamines and corticosteroids. Hypo- the etiology, pathogenesis, and natural his-
tension and flushing associated with the tory are incompletely understood, and re-
rapid infusion of albumin in patients taking ductions in such measured variables as
ACE inhibitors are discussed in Chapter 27. complement components, rheumatoid fac-
Predominantly ocular (periorbital edema, tor, or immune complexes cannot be corre-
conjunctival swelling, and tearing) reac- lated reliably with changes in disease activ-
tions have occurred in donors sensitized to ity. An example is the use of the erythrocyte
the ethylene oxide gas used to sterilize dis- sedimentation rate (ESR) as an index of dis-
posable plastic apheresis kits.38 ease activity in rheumatoid arthritis. The
Fatalities During Apheresis. Despite the ESR invariably decreases during intensive
fact that patients undergoing therapeutic TPE, but this reflects removal of fibrinogen
apheresis are often critically ill, fatalities and not necessarily a decrease in disease
during apheresis are comparatively rare. activity. For the same reasons, the optimal
Estimates of fatality rates range from 3 in volume and frequency of exchange are of-
10,00039 to 1 in 50040 procedures. Most deaths ten not established. For severe imminently
were due to cardiac arrhythmias or arrest life-threatening disease, when albumin/sa-
during or shortly after the procedure or to line is the replacement fluid, TPE is initially
acute pulmonary edema or adult respira- performed daily. After a few days, the
tory distress syndrome occurring during a fibrinogen or platelet count may be low
procedure. Rare fatalities resulted from enough to significantly increase the risk of
anaphylaxis, vascular perforation, hepatitis, bleeding. Clinical judgment must then be
sepsis, thrombosis, and hemorrhage. exercised to decide whether to proceed
with TPE using clotting factor/platelet
transfusions or to withhold TPE until these
parameters normalize. The conditions dis-
Indications for Therapeutic Apheresis
cussed below are established indications
Although therapeutic apheresis has been for therapeutic apheresis.3,20,21,41,42
used in the treatment of many diseases,
most published studies are case reports or
small uncontrolled series, often providing
insufficient evidence of efficacy. Publica- Hematologic Conditions
tion bias tends to favor positive results, Serum Hyperviscosity Syndrome. Serum
and physicians should avoid subjecting hyperviscosity resulting from multiple
patients to the risks and high costs of myeloma or Waldenstrom’s macroglobu-
apheresis procedures based on marginal linemia can cause congestive heart failure;
clinical studies. Controlled, randomized, reduced blood flow to the cerebral, car-
blinded studies of therapeutic apheresis diac, or pulmonary circulation; or symp-
are difficult to conduct, especially be- toms of headache, vertigo, somnolence, or
cause using sham treatments as a control obtundation. Paraproteins may interfere
is expensive and carries some risk. How- with hemostasis, leading to hemorrhagic
ever, the complicated apheresis instru- symptoms.
ments and associated attention from The presence of hyperviscosity correlates
nursing and medical personnel may cre- only in very general terms with the concen-

tration of paraprotein. Measurement of se- weeks or longer, and leukocyte reduction

rum viscosity relative to water is a simple can be effected with chemotherapy, with or
procedure that provides more objective in- without leukapheresis. Leukapheresis is
formation. For some pathologic proteins, sometimes used to reduce the white cell
serum viscosity is highly temperature de- count to <100,000/µL before the start of
pendent, so serum viscosity should be chemotherapy, to reduce the likelihood of
measured at physiologically relevant tem- tumor lysis syndrome. However, there have
peratures. Normal serum viscosity ranges been no controlled clinical trials to sub-
from 1.4 to 1.8 relative to water. Because stantiate this approach, and it must be rec-
most patients are not symptomatic until ognized that more malignant cells are
their relative serum viscosity is more than present outside the circulation than within
4.0 or 5.0, patients with mild elevations may the bloodstream.
not require treatment. For symptomatic Thrombocythemia. Therapeutic plate-
hyperviscosity, a single apheresis procedure letpheresis is usually undertaken for symp-
is usually highly effective.23 tomatic patients with platelet counts above
Hyperleukocytosis. Leukapheresis is of- 1,000,000/µL. The measured count, by itself,
ten used to treat the dramatically elevated should not determine whether platelet
white cell count that can occur in acute leu- reduction is indicated. In patients with
kemia. Several different thresholds have evidence of thrombosis secondary to
been used: fractional volume of leukocytes thrombocythemia, plateletpheresis can be
(leukocrit) above 10%; total circulating leu- beneficial. The rationale for platelet reduc-
kocytes above 100,000/µL; and circulating tion in bleeding patients with thrombocy-
blasts above 50,000/µL.43 However, the use themia is less clear. There are no accepted
of a single laboratory value as an indication indications for prophylactic plateletphere-
for treatment is an oversimplification. Such sis in asymptomatic patients, although the
factors as erythrocyte concentration, leuke- risk of placental infarction and fetal death
mic cell type, rate at which the count is ris- may justify the procedure in a pregnant
ing, potential obstructions to cerebral or woman with severe thrombocythemia.43
pulmonary blood flow, and the patient’s co- Thrombotic Thrombocytopenic Purpura/
agulation status and general condition must Hemolytic-Uremic Syndrome (TTP/HUS).
be considered. Most leukemic patients with The conditions described as TTP/HUS are
extreme leukocytosis have significant ane- multisystem disorders, in which platelet/fi-
mia. Reduced red cell mass reduces blood brin thrombi occlude the microcirculation.
viscosity, so unless there is an acute need to They are characterized by varying degrees
increase oxygen-carrying capacity, red cells of thrombocytopenia, microangiopathic
should not be transfused until the hypervis- hemolytic anemia, renal dysfunction, neu-
cosity crisis has been resolved.43 rologic abnormalities, and fever. Patients
In some patients with acute blast crisis presenting with fulminant TTP usually have
or in unusual types of leukemia, both the platelet counts below 50,000/µL and lactic
hematocrit and leukocrit are elevated. If dehydrogenase (LDH) levels above 1000
there is evidence of cerebral or pulmonary IU/mL, resulting from systemic ischemia
symptoms, rapid reduction of leukocyte and hemolysis. 44 The peripheral blood
concentration should be considered, al- smear characteristically shows increased
though the efficacy of such leukocyte re- numbers of schistocytes. Evidence for dis-
duction is unproved. More commonly, seminated intravascular coagulation is gen-
however, the white cell count rises over erally absent.

TTP usually develops without obvious ated with disease exacerbation and death,
cause, although episodes may occur after they are usually contraindicated (except in
infections, pregnancy, or use of some com- the presence of life-threatening hemorrhage).
mon drugs such as ticlopidine, or clopido- TPE is typically performed daily for l to 2
grel. Recent reports suggest that it is caused weeks, but the intensity and duration of
by a transient antibody to a protease treatment should be guided by the individ-
(ADAMTS13) that normally cleaves large ual patient’s course. Occasionally, pro-
von Willebrand factor (vWF) multimers. longed courses of treatment are required.
The unusually large vWF multimers avidly Therapeutic plasma exchange has impres-
aggregate circulating platelets, triggering sively improved the survival rate in TTP,
the syndrome.45,46 Increasingly, cases of re- from being almost universally fatal before
current or relapsing TTP are being recognized. 1964 to 80% survival in a recent series.49
HUS is a similar condition that occurs Signs of response to therapy include a ris-
more commonly in children than adults. ing platelet count and reduction of LDH
HUS may follow diarrheal infections with between procedures. As patients recover to
verotoxin-secreting strains of Escherichia near normal LDH and platelet count (100-
coli (strain 0157:H7) or Shigella. Compared 150,000/µL), TPE is discontinued. Some
with patients who have classic TTP, those programs switch from intensive TPE to in-
with HUS have more renal dysfunction and termittent plasma exchange or simple
less prominent neurologic and hematologic plasma infusion, but the efficacy of this ap-
findings. Most patients with HUS do not proach has not been established.52 Despite
have antibody to the vWF protease and the success of TPE, TTP/HUS remains a
have normal concentrations of vWF prote- serious condition. Treatment failures con-
ase. TTP/HUS can occur after treatment with tinue to occur and to cause major organ
certain cytotoxic drugs, including mito- damage or death.
mycin C. A microangiopathic hemolysis Complications of Sickle Cell Disease.
similar to TTP/HUS can occur in organ or Several complications of sickle cell disease
stem cell transplant recipients receiving are syndromes that can be treated by red
cyclosporine and tacrolimus.47,48 Transplant- cell exchange. These conditions include
associated microangiopathic hemolysis ap- stroke or impending stroke, acute chest
pears to be less responsive to therapy and syndrome, and multiorgan failure. Either
probably represents a different disease pro- manual or automated techniques can be
cess.48 used for red cell exchange, but automated
TPE with plasma or Plasma Cryopreci- techniques are faster and better controlled.
pitate Reduced replacement has become The goal is to replace red cells containing
49,50
the treatment of choice for TTP/HUS. hemoglobin S with a sufficient number of
Protease levels have been shown to be sta- red cells containing hemoglobin A so that
ble for at least 2 weeks in citrated plasma the overall proportion of hemoglobin A in
stored at 37 C.51 TPE is now thought to re- the blood is 60% to 80%. Some centers pro-
move both antibody to the protease and vide partially phenotypically matched red
vWF and to replace deficient protease. cells (eg, C, E, and K1) to avoid alloimmuni-
Other largely unproved treatments include zing long-term transfusion recipients to
prednisone, antiplatelet agents, splenec- these antigens. At the end of the procedure,
tomy, vincristine, rituximab, and intrave- the patient’s hematocrit should be no higher
nous immunoglobulin. Because platelet than 30% to 35% to avoid increased blood
transfusions have anecdotally been associ- viscosity. Chronic erythrocytapheresis can be

used to manage iron overload in patients tients who remain untreated for several
requiring long-term transfusion therapy.53 weeks. Recent controlled studies suggest
that intravenous immunoglobulin gives re-
Cryoglobulinemia sults equivalent to five TPE procedures over
a 2-week period.54 A cost-effectiveness anal-
Significant elevations of cryoglobulins may
ysis has suggested that TPE is less costly
cause cold-induced vascular occlusion,
than a course of intravenous immune glob-
abnormalities of coagulation, renal insuf- 55
ulin. Multicenter trials have suggested that
ficiency, or peripheral nerve damage. Re-
TPE, if initiated early, can decrease the pe-
moval of cryoglobulins by apheresis can
riod of minimal sensorimotor function.56
be used to treat acute symptomatic epi-
Patients whose illness is not acute in onset,
sodes, but definitive therapy depends on
is not characteristic of Guillain-Barré syn-
identifying and treating the underlying
drome, or in whom nerve conduction stud-
causative condition.
ies show complete axonal block may have a
poorer prognosis and less response to
Neurologic Conditions apheresis therapy.
Myasthenia Gravis. Myasthenia gravis re- Chronic Inflammatory Demyelinating
sults from autoantibody-mediated block- Polyneuropathy. Chronic inflammatory
ade of the acetylcholine receptor located demyelinating polyneuropathy (CIDP), of-
on the postsynaptic motor endplate of ten seen in HIV patients, is a group of disor-
muscles. Standard treatment includes ste- ders with slow onset and progressive or
roids and acetylcholinesterase inhibitors. intermittent course, characterized by ele-
TPE is used as adjunctive treatment for vated spinal fluid protein, marked slowing
patients experiencing exacerbations not of nerve conduction velocity, and segmen-
controlled by medications and for pa- tal demyelination of peripheral nerves. Var-
tients being prepared for thymectomy. A ious sensorimotor abnormalities result.
typical treatment protocol is five or six Polyneuropathy may also occur in the
TPE procedures over l to 2 weeks. Concur- POEMS syndrome, characterized by poly-
rent immunosuppression to prevent anti- neuropathy, organomegaly, endocrino-
body rebound is recommended. Chronic pathy, MGUS (monoclonal gammopathy of
TPE has been used with some success in a unknown significance), and skin changes.
small number of patients. Corticosteroids are the first-line treatment
Acute Guillain-Barré Syndrome. for CIDP. TPE and intravenous immuno-
Guillain-Barré syndrome is an acute auto- globulin have equivalent efficacy in patients
immune demyelinating polyneuropathy unresponsive to corticosteroids.57
that can produce dramatic paralysis in oth- Polyneuropathy Associated with Mono-
erwise healthy individuals. The cause is un- clonal Gammopathy of Undetermined Sig-
known; many cases appear to follow benign nificance. When polyneuropathy is associ-
viral infections or Campylobacter jejuni inated with monoclonal paraproteins of un-
fection. Most patients recover spontane- certain significance, TPE has been shown to
58,59
ously, but as many as one in six may be- be effective for all variants.
come unable to walk or may develop
respiratory failure requiring ventilatory
support. Early treatment is beneficial for Renal Diseases
patients with rapidly progressive disease. Rapidly progressive glomerulonephritis
The response to therapy is inferior in pa- (RPGN) associated with antibodies to

basement membranes of glomeruli and al- longed reduction in circulating lipids can
veoli, which may result in pulmonary be achieved with repeated TPE, often with
hemorrhage (Goodpasture’s disease), usu- selective adsorption or filtration tech-
ally responds to TPE as an adjunct to im- niques.43 Heterozygous hypercholesterol-
munosuppressive drugs.41 TPE accelerates emia results from several gene defects in
the disappearance of antibodies to base- the LDL receptor. Some patients with het-
ment membranes and improves renal erozygous hypercholesterolemia also de-
function. TPE is particularly effective in velop high levels of cholesterol and are at
halting pulmonary hemorrhage in these increased risk for developing premature
patients, even if renal function does not atherosclerotic heart disease.
completely normalize following treat- Two apheresis systems for selective LDL
ment. Therapeutic apheresis has been removal in patients with homozygous or
used in treating the vasculitis associated heterozygous hypercholesterolemia have
with RPGN and the presence of anti- been cleared by the FDA. The Liposorber
neutrophil cytoplasmic antibody (ANCA- LA-150 (Kaneka Pharma America, New
positive RPGN).60,61 TPE is most effective York, NY) is based on a dextran sulfate ad-
41
in the more severe cases. sorption system. The H.E.L.P. LDL system
Myeloma light chains may be toxic to re- (B. Braun, Melsugen, Germany) is a hepa-
nal tubular epithelium and cause renal fail- rin-induced LDL cholesterol precipitation
ure in up to 10% of cases. TPE is useful as system. The LDL apheresis procedure se-
adjunctive therapy in some myeloma pa- lectively removes apolipoprotein-B-con-
tients with cast nephropathy but is not as- taining cholesterols such as LDL and very
sociated with improved survival.41,62 LDL, sparing high-density lipoprotein
(HDL) cholesterol. This provides an advan-
tage over standard apheresis, which re-
Other Conditions moves all plasma proteins, including the
TPE has been used as adjunctive treatment “protective” HDL. The procedure acutely
for a variety of multisystem diseases. A lowers levels of targeted cholesterols by
combination of steroid, cytotoxic agents, 60% to 70%. Treatment is usually per-
and TPE has been used for severely ill pa- formed every 1 to 2 weeks, and cholesterol-
tients with polyarteritis nodosa, 6 3 al- lowering drugs are generally employed si-
though most rheumatologic conditions multaneously. Several studies have shown
respond poorly to TPE. Clinical trials us- that use of LDL apheresis can achieve sig-
ing standard TPE have not shown benefit nificant lowering of lipids in nearly all pa-
in the treatment of systemic lupus erythe- tients with severe hypercholesterolemia.65,66
matosus, polymyositis, dermatomyositis, or Promising results have also been reported
scleroderma.21 Immunoadsorption col- from a system capable of direct adsorption
umns are of benefit in patients with rheu- of LDL and Lp(a) (DALI) from whole
matoid arthritis refractory to medical blood.67
management.64 Refsum’s Disease (Phytanic Acid Dis-
Homozygous Type II Familial Hyper- ease). Refsum’s disease is a rare inborn er-
cholesterolemia. Homozygous hypercho- ror of metabolism resulting in toxic levels of
lesterolemia, a rare disorder of the receptor phytanic acid, causing neurologic, cardiac,
for low-density lipoproteins, results in se- skeletal, and skin abnormalities.21 TPE is
vere premature atherosclerosis and early useful in conjunction with a phytanic-
death from coronary artery disease. Pro- acid-deficient diet and should be started as

soon as possible, before permanent dam-

age occurs.
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tice. 2nd ed. Bethesda, MD: AABB Press, 2003. fectiveness analysis. J Clin Apheresis 1999;14:
43. Klein HG. Principles of apheresis. In: Ander- 107-13.
son KC, Ness PM, eds. Scientific basis of 56. Guillain-Barré Syndrome Study Group.
transfusion medicine. 2nd ed. Philadelphia: Plasmapheresis and acute Guillain-Barré syn-
WB Saunders, 2000:553-68. drome. Neurology 1985;35:1096-104.
44. Cohen JA, Brecher ME, Bandarenko N. Cellu- 57. vanDoorn PA, Vermeulen M, Brand A. Intra-
lar source of serum lactate dehydrogenase el- venous immunoglobulin treatment in pa-
evation in patients with thrombotic thrombo- tients with chronic inflammatory demye-
cytopenic purpura. J Clin Apheresis 1998;13: linating polyneuropathy. Arch Neurol 1991;
16-9. 48:217-20.
45. Furlan M, Robles R, Galbusera M, et al. von 58. Dyck PJ, Low PA, Windebank AJ, et al. Plasma
Willebrand factor-cleaving protease in throm- exchange in polyneuropathy associated with
botic thrombocytopenic purpura and the monoclonal gammopathy of undetermined
hemolytic-uremic syndrome. N Engl J Med significance. N Engl J Med 1991;325:1482-6.
1998;339:1578-84. 59. Simovic D, Gorson KC, Popper AH. Compari-
46. Tsai H-M, Chun-Yet Lian E. Antibodies to von son of IgM-MGUS and IgG-MGUS polyneuro-
Willebrand factor-cleaving protease in acute pathy. Acta Neurol Scand 1998;97:194-200.
thrombotic thrombocytopenic purpura. N Engl 60. Frasca GM, Zoumparidis NG, Borgnino LC, et
J Med 1998;339:1585-94. al. Plasma exchange treatment in rapidly pro-
47. McLeod BD. Thrombotic microangiopathies gressive glomerulonephritis associated with
in bone marrow and organ transplant pa- anti-neutrophil cytoplasmic autoantibodies.
tients. J Clin Apheresis 2002;17:118-23. Int J Artif Organs 1992;3:181-4.
48. George JN, Li X, McMinn JR, Terrell DR, et al. 61. Pusey CD, Rees AJ, Evans JJ, et al. A random-
Thrombotic thrombocytopenic purpura- ized controlled trial of plasma exchange in
hemolytic uremic syndrome following allo- rapidly progressive glomerulonephritis with-
geneic HPC transplantation: A diagnostic di- out anti-GBM antibodies. Kidney Int 1991;40:
lemma. Transfusion 2004;44:294-304. 757-63.
49. George JN, Rizui MA. Thrombocytopenia. In: 62. Johnson WJ, Kyle RA, Pineda AA, et al. Treat-
Beutler E, Lichtman MA, Coller BS, et al, eds. ment of renal failure associated with multiple
Williams’ hematology. 6th ed. New York: myeloma. Arch Intern Med 1990;150:863-9.
McGraw-Hill, 2001:1495-539. 63. Guillevin L, Lhote F, Leon A, et al. Treatment
50. Rock G, Shumak KH, Sutton DM, et al. Cryo- of polyarteritis nodosa related to hepatitis B
supernatant as replacement fluid for plasma virus with short-term steroid therapy associ-
exchange in thrombotic thrombocytopenic ated with antiviral agents and plasma ex-
purpura. Br J Haematol 1996;94:383-6. changes: A prospective trial in 33 patients J
51. Gerritsen HE, Robles R, Lammle B, Furlan M. Rheumatol 1993;20:289-98.
Partial amino acid sequence of purified von 64. Felson DT, LaValley MP, Baldassare AR, et al.
Willebrand factor-cleaving protease. Blood 2001; The Prosorba column for treatment of refrac-
98;1654-61. tory rheumatoid arthritis: A randomized,
52. Bandarenko N, Brecher ME, and members of double-blind, sham-controlled trial. Arthritis
the US TTP Apheresis Study Group. United Rheum 1999;42:2153-9.
States Thrombotic Thrombocytopenic Pur- 65. Kroon AA, Aengevaeren WRM, van der Werf T,
pura Apheresis Study Group (US TTP ASG): et al. LDL-apheresis atherosclerosis regres-
Multicenter survey and retrospective analysis sion study (LAARS): Effect of aggressive ver-
of current efficacy of therapeutic plasma ex- sus conventional lipid lowering treatment on
change. J Clin Apheresis 1998;13:133-41. coronary atherosclerosis. Circulation 1996;
53. Adans DM, Schultz WH, Ware RE, Kinney TR. 93:1826-35.
Erythrocytapheresis can reduce iron overload 66. Thompson GR, Maher VMG, Matthews S, et
and prevent the need for chelation therapy in al. Familial hypercholesterolemia regression
chronically transfused pediatric patients. J study: A Randomized trial of low-density-li-
Pediatr Hematol Oncol 1996;18:46-50. poprotein apheresis. Lancet 1995;345:811-16.
54. Plasma Exchange/Sandoglobulin Guillain- 67. Bosch T, Lennertz A, Schenzle D, et al. Direct
Barré Syndrome Trial Group. Randomized adsorption of low density lipoprotein and li-
trial of plasma exchange, intravenous immu- poprotein (a) from whole blood: Results of
noglobulin, and combined treatments in the first clinical long-term multicenter study

using DALI apheresis. J Clin Apheresis 2002; 69. Gaddis TG, Guthrie TH, Drew MJ, et al. Treat-
17:161-9. ment of refractory thrombotic thrombocyto-
68. Handelsman H. Office of health technology penic purpura with protein A immuno-
assessment report, No. 7. Protein A columns adsorption. Am J Hematol 1997;55:55-8.
for the treatment of patients with idiopathic 70. Lim HW, Edelson RL. Photopheresis for treat-
thrombocytopenic purpura and other indica- ment of cutaneous T-cell lymphoma. Hema-
tions. Rockville, MD: DHHS, PHS, Agency for tol Oncol Clin North Am 1995;9:1117-26.
Health Care Policy and Research, 1991:1-8.

Chapter 7: Blood Component Testing and Labeling
Chapter 7
7
Blood Component Testing

and Labeling
E
ACH DONOR UNIT must be tested of blood component testing strictly in
and properly labeled before its re- compliance with current instructions pro-
lease for transfusion. Although the vided by the test manufacturers. Testing
scope and characteristics of donor tests must be performed in a planned, orderly
changed with the release of new tests and manner under a quality plan and a writ-
the advent of new regulatory require- ten set of procedures that instruct the
ments, the intent of donor testing remains staff how to perform testing, under what
constant: to enhance the safety of the circumstances additional testing needs to
blood supply. This chapter presents the be done, and what to do if things go
general principles that apply to testing wrong. The facilities and equipment must
and labeling donor blood, and it provides be adequate for the activity being con-
a description of the specific tests that are ducted. Access to the area must be lim-
required or done voluntarily at most ited. The environment must be controlled
blood banks on each donation. Discus- so that temperature specifications for the
sion of the infectious complications of tests will be met, and the test will not be
blood transfusion is found in Chapter 28. adversely affected by the environment.
Other aspects of component preparation The test materials and equipment in use
are covered in Chapter 8. must be those previously approved and
validated by the facility. If a facility uses
reagents or equipment from several dif-
Testing ferent manufacturers, the facility is respon-
sible for documentation that validation of
General Requirements the equipment or reagent combination
Each laboratory needs to develop standard for each test in use has occurred and that
operating procedures for the performance staff have been trained on the most cur-
163

rent applicable instructions. For tests re- most problematic notifications are those in
quired by the Food and Drug Administra- which the donor has a false-positive test re-
tion (FDA)1 and/or AABB Standards for sult. For some analytes [eg, antibodies to
Blood Banks and Transfusion Services, all hepatitis C virus (anti-HCV) or human im-
reagents used must meet or exceed the re- munodeficiency virus, types ½ (anti-
2(p9)
quirements of the FDA. If the manufac- HIV-1/2)], confirmatory or supplemental
turer of a licensed test supplies controls, testing is routinely performed for donor
they must be used for that test. However, counseling purposes and possible donor
if these controls are used for calibration, reentry, if applicable. In the case of a minor,
different controls must be used to verify state and local laws apply.3
test performance. These controls may need
to be purchased separately. The manufac- Required Tests
turer defines acceptable sample (speci-
ABO group and Rh type must be determined
men) requirements and considerations
at each donation. A sample from each do-
that usually include the presence and na-
nation intended for allogeneic use must
ture of anticoagulant, the age of suitable
be tested for the following1,2(p33):
samples, and permissible storage inter-
■ Syphilis
vals and conditions. Tests must be per-
■ Hepatitis B surface antigen (HbsAg)
formed on a properly identified sample
■ HIV nucleic acid (individual or com-
from the current donation. Testing must
bined HIV/HCV assay)
be completed for each blood donation be-
■ HCV nucleic acid (individual or
fore release. Each test result must be re-
combined HIV/HCV assay)
corded concurrently with its observation;
■ Anti-HIV-1
interpretation is to be recorded only when
■ Anti-HIV-2
testing has been completed. Testing re-
■ Antibodies to hepatitis B core anti-
sults must be recorded and records main-
gen (anti-HBc)
tained so that any results can be traced for
■ Anti-HCV
a specific unit and/or component.
■ Antibodies to human T-cell lympho-
The facility should have a policy for noti-
tropic viruses, types I/II (anti-HTLV-
fying donors of positive infectious disease
I/II)
test results. Test results are confidential and
A combination test for anti-HIV-1/2 may
must not be released to anyone (other than
be used. A test for alanine aminotransferase
the donor) without the donor’s written con-
(ALT) is not required by the FDA or the
sent. At the time of donation, the donor
AABB. Recommendations for labeling units
must be told if the policy is to release posi-
associated with an elevated ALT have been
tive test results to state or local public
released by the FDA.4
health agencies, and the donor must agree
to those conditions before phlebotomy. Do-
nors must sign a consent form before they Equipment Requirements
donate blood acknowledging that the facil- All equipment used for testing must be
ity maintains a registry of donors who gave properly calibrated and validated upon
disqualifying donor histories or have posi- installation, after repairs, and periodically.
tive infectious disease results. The donor There must be a schedule for planned
must also be informed if the sample will maintenance. All calibration, mainte-
undergo research testing, including investi- nance, and repair activities must be docu-
gational new drug (IND) protocols. The mented for each instrument. Software

Chapter 7: Blood Component Testing and Labeling 165
used to control the instrument or to inter- nonreactive with anti-D in direct aggluti-
face with the institution’s computer sys- nation tests must be tested by a method
tem must also be properly validated.5 designed to detect weak D. Red cells that
react with anti-D either by direct aggluti-
Records Requirements nation or by the weak D test must be
labeled Rh positive. Red cells that are
Records must show each production step
nonreactive with anti-D by direct aggluti-
associated with each blood component
nation and the weak D test must be la-
from its source to its final disposition.6,7
beled Rh negative. Some of the automated
Records must be kept in a manner that
techniques have sufficient D sensitivity to
protects the identity and personal infor-
obviate the need for a weak D test. These
mation of the donor from discovery by
instruments add reagents, incubate re-
anyone other than the facility doing the
agent and sample appropriately, read the
donor recruitment, qualification, and
reaction, and provide a result ready for in-
blood collection, with the exception of
terpretation. In addition, the automated
government agencies that require certain
devices incorporate positive sample iden-
test-positive results to be reported by law
tification with the use of barcode readers
for public health purposes. Testing records
and use anticoagulated blood so that only
on donor units must be kept in a manner
one tube is needed for both red cell and
that makes it possible to investigate ad-
plasma sampling. See Chapter 13 and
verse consequences to a recipient. In ad-
Chapter 14 for a more complete discus-
dition, donor testing records must be suit-
sion of the principles of ABO and D test-
able for look-back to previously donated
ing.
components when a donor’s blood gives
positive results on a new or improved in-
fectious disease test. Antibody Screening
Previous records of a donor’s ABO and D Blood from donors with a history of trans-
typing results must be reviewed and com- fusion or pregnancy must be tested for
pared with the ABO and D test findings on unexpected antibodies. Because it is usu-
the current donation. This is a very valuable ally impractical to segregate blood that
quality check on both the sample identity should be tested from units that need not
correctness and the operation of the labo- be tested, most blood centers test all do-
ratory. If a discrepancy is found between nor units for unexpected red cell antibod-
any current or historic test required, the ies. Donor serum or plasma may be tested
unit must not be released until there is un- against individual or pooled reagent red
equivocal resolution of the discrepancy.2(p39) cells of known phenotypes. Methods must
be those that demonstrate clinically sig-
ABO and D Testing nificant red cell antibodies. See Methods
Section 3 for antibody detection techni-
Every unit of blood intended for transfu-
2(p32),8 ques and Chapter 19 for a discussion of
sion must be tested for ABO and D.
antibody detection.
The ABO group must be determined by
testing donor red cells with reagent anti-A
and anti-B, and donor serum or plasma Serologic Test for Syphilis
with A1 and B red cells. The Rh type must Serologic testing for syphilis (STS) has been
be determined by testing donor red cells carried out on donor samples for over 50
with anti-D serum. Red cells that are years. Although experimental studies in

the 1980s showed that survival of spiro- tested for routinely are HIV and HCV. Two
chetes at 4 C is dependent on the concen- experimental tests for West Nile virus
tration, it is not known how long the spiro- ( WNV ) are undergoing study nation-
chete (Treponema pallidum) survives at wide.12,13 Nucleic acid testing (NAT) for
refrigerated temperatures in a naturally HBsAg is undergoing clinical trials in
9
infected blood component. The last re- some centers.
ported transfusion transmitted case of In the capture approach frequently used
syphilis was reported in fresh blood com- in assays, serum or plasma is incubated
10
ponents in 1969. with fixed antigen. If present, antibody
The majority of screening tests for syphi- binds firmly to the solid phase and remains
lis in US blood collection centers are micro- fixed after excess fluid is washed away. An
hemagglutinin or cardiolipin-based tests enzyme-conjugated preparation of antigen
that are typically automated. Donor units or antiglobulin is added; if fixed antibody is
testing positive for syphilis (STS) may not present, it binds the labeled antigen or
be used for allogeneic transfusion. Results antiglobulin, and the antigen-antibody-an-
can be confirmed before a donor is noti- tigen (or antiglobulin) complex can be
fied. Volunteer donors are much more quantified by measuring enzyme activity.
likely to have a false-positive test result One assay for anti-HBc uses an indirect
than a true-positive one. capture method (competitive assay), in
which an enzyme-antibody conjugate is
Viral Marker Testing added to the solid-phase antigen along
with the unknown specimen. Any antibody
Two screening methods are widely used present in the unknown specimen will
to detect viral antigens and/or antibodies. compete with the enzyme-conjugated anti-
The first is the enzyme-linked immuno- body and significantly reduce the level of
sorbent assay (EIA). The EIA tests for the enzyme fixed, compared with results seen
viral antigen HBsAg employ a solid sup- when nonreactive material is present. Anti-
port (eg, a bead or microplate) coated gens used in the viral antibody screening
with an unlabeled antiserum against the tests may be made synthetically by recom-
antigen. The indicator material is the binant technology or extracted from viral
same or another antibody, labeled with an particles.
enzyme whose presence can be detected NAT is a powerful but expensive technol-
by a color change in the substrate. If the ogy that reduces the exposure window for
specimen contains antigen, it will bind to HIV and HCV by detecting very low num-
the solid-phase antibody and will, in turn, bers of viral copies after they appear in the
be bound by the enzyme-labeled indica- bloodstream. Primers for HCV and HIV vi-
tor antibody. To screen for viral antibod- ruses are placed in microplate wells, either
ies, ie, anti-HIV-1, anti-HIV-2, anti-HBc, separately or in combination according to
anti-HCV, or anti-HTLV-I/II, the solid the specific test design. In the use of pooled
phase (a bead or microtiter well) is coated sera, 16 to 24 donor samples are mixed and
with antigens prepared from the appro- tested. If viral RNA matching the fragments
priate viral recombinant proteins or syn- already in the well is present in the donor
thetic peptides. The second technology samples, heat-cycling nucleic acid amplifi-
for virus detection is based on nucleic cation using a heat-cycling technique will
acid amplification and detection of viral cause the viral fragments to multiply and
11
nuclear material. The RNA viruses being be easily detectable. The microplate with

the aliquot of pooled sera is placed in the age insert. All results, both the reactive
well with the viral primers and substrate so and the nonreactive, obtained in the run
that if the primers and viral material in the must be declared invalid; all specimens
donor samples are the same, the primer involved must be tested in a new run,
and viral particles will increase geometri- which becomes the initial test of record.13
cally with each cycle. Viral presence can However, if the batch controls are ac-
then be detected reliably. When a pool is ceptable and no error is recognized in test
found to be positive, all the individual sam- performance, the reactive and nonreactive
ples making up the pool are tested sepa- results from the initial run remain as the
rately for the individual viruses HIV and initial test of record for the specimens in-
HCV to find the positive donor sample. The volved. Specimens with reactive results
second major advantage of this test is the must be retested in duplicate, as required
relative lack of false-positive results as long by the manufacturer’s instructions.
as sample and laboratory cross-contamina- Before a test run is invalidated, the prob-
tion are controlled.12 lems observed should be reviewed by a su-
For most of the assays, samples giving pervisor or equivalent, causes should be
nonreactive results on the initial screening analyzed, and corrective action should be
test, as defined by the manufacturer’s pack- taken, if applicable. A record of departure
age insert, are considered negative and from normal standard operating proce-
need not be tested further. Samples that are dures should be prepared, with a complete
reactive on the initial screening test must description of the reason for invalidation
be repeated in duplicate. Reactivity in one and the nature of corrective actions.13
or both of the repeated tests constitutes a
positive result and is considered repeatedly
reactive. All components must be discarded Use of External Controls
in this case. If both the duplicate repeat Other considerations may need to be ad-
tests are nonreactive, the test is interpreted dressed before the invalidation of test re-
as having a negative result. sults when external controls are used.13
Before a donor is designated as antigen- Internal controls are the validation mate-
or antibody-positive, a status that may have rials provided with the licensed assay kit;
significant clinical and social consequences they are used to demonstrate that the test
and cause permanent exclusion from blood performs as expected. External controls
donation, it is important to determine generally consist of at least one positive
whether the screening result is a true- or control and one negative control. If the
false-positive result. negative control from the kit is used to
calculate the assay cutoff, it cannot be
used as an assay control reagent for test-
Invalidation of Test Results ing. An external negative control should
In the course of viral marker testing, it be used in its place (see Table 7-1). Exter-
may be necessary to invalidate test results nal controls are frequently used to dem-
if the test performance did not meet the onstrate the ability of the test to identify
requirements of the manufacturer’s pack- weakly reactive specimens. External con-
age insert (eg, faulty equipment, im- trols are surrogate specimens, either pur-
proper procedure, compromised reagents), chased commercially or developed by the
or if the control results do not meet the institution, that are not a component of
acceptance criteria defined in the pack- the test kit; they are used for surveillance

Table 7-1. Use of External Controls

Used to Calculate
Test Kit Reagents Assay Cutoff? External Controls Required?
Negative control only Yes Yes–negative control
No No
Positive control only Yes Yes–positive control
No No
Both positive and negative Yes Yes–positive and negative
controls controls
of test performance. External controls are state, federal, and international regulations,
tested in the same manner as donor sam- as applicable.
ples to augment blood safety efforts and A facility may invalidate nonreactive test
to alert the testing facility to the possibil- results on the basis of external controls, but
ity of an increasing risk of error. if the assay was performed in accordance
Before being entered into routine use, with manufacturer’s specifications and the
external controls must be qualified, lot by internal controls performed as expected,
lot, because each control lot may vary with external controls cannot be used to invali-
the test kit. One way to qualify an external date reactive test results. The use of external
control is: controls may be more stringent than, but
1. Run the external control for 2 days, must be consistent with, the package in-
four replicates per day, using two to sert’s criteria for rejection of test results.
three different test kit lots. The per- Observation of donor population data,
formance of the external controls such as an unexpectedly increased reactive
must meet specified requirements rate within a test run, may cause non-
before use. reactive results to be considered invalid.
2. If the external controls do meet the The next assay, performed on a single
specifications, the acceptable sam- aliquot from affected specimens, becomes
ple-to-cutoff ratio for the external the initial test of record for those samples
control (eg, within three standard nonreactive in the invalidated run. How-
deviations of the mean) must be ever, reactive results obtained in a run with
determined. an unexpectedly increased reactive rate
Additional qualification testing must be may not be invalidated unless the entire
performed whenever a new lot of test kits run fails to meet the performance criteria
or external controls is introduced. specified in the package insert. Such reac-
When a change of test kit lot occurs, rep- tive results remain the initial test of record.
licates (eg, 20 replicates) of the external The samples must be tested in duplicate as
control should be run with the current kit the repeat test.
lot and the new lot. The new sample-to- External controls may also be used to in-
cutoff ratio and limits should be deter- validate a duplicate repeat test run when an
mined. assay run is valid by test kit acceptance cri-
Users should verify special requirements teria and both the repeated duplicate tests
for external control handling in pertinent are nonreactive. The duplicate samples

may be repeated in duplicate; the second Supplemental Test for EIA-Reactive

duplicate test becomes the test of record. If Anti-HIV-1/2, -HTLV I/II, and -HCV Tests
either of the original duplicate repeat tests
is reactive, the donor(s) must be classified Western blot is the method most fre-
as repeatedly reactive and no further repeat quently used for the confirmation of re-
screening tests should be performed. peatedly reactive anti-HIV EIA tests. The
When samples are reactive on the initial technique separates antigenic viral mate-
screening test, allogeneic donor units must rial into bands according to molecular
be quarantined until the results of dupli- weight. The material is transferred to
cate repeat testing are available. Compo- nitrocellulose membranes. Antibody in
nents associated with repeatedly reactive the test serum reacts with the individual
test results must not be used for allogeneic bands, depending on the specificity.
transfusion. Supplemental or confirmatory Most persons infected with HIV, whether
testing may be performed on samples that asymptomatic or exhibiting AIDS, show
are repeatedly reactive to obtain additional multiple bands, representing antibodies
information for donor counseling and pos- to virtually all of the various gene prod-
sible reentry, depending on the viral marker ucts. A fully reactive test serum should re-
and availability of approved assays. act with the p17, p24, and p55 gag pro-
teins; the p31, p51, and p66 pol proteins;
and the gp41, gp120, and gp160 env
Supplemental Tests: Neutralization
glycoproteins. Western blot results in
In confirmatory antigen neutralization EIA-reactive blood donor samples are
tests (eg, HBsAg), the reactive specimen is classified as positive, negative, or indeter-
incubated with human serum known to minate. Positive results are those with re-
contain antibody specific for the antigen activity to at least two of the following HIV
in question. If incubation causes the posi- proteins: p24, core protein; gp41, trans-
tive reaction to disappear or to diminish membrane protein; and gp120/160, exter-
by at least 50% (or the percentage speci- nal protein and external precursor pro-
fied in the package insert) and all controls tein. Indeterminate results are those with
behave as expected, the presence of anti- other patterns of reactivity.
gen is confirmed and the original result is An immunofluorescence assay (IFA) is
considered a true positive. If incubation used in some blood centers as an alterna-
with a known antibody does not affect tive to Western blot testing. Cells infected
subsequent reactivity, the original reactiv- with virus are fixed on a slide. The sample is
ity is considered a false-positive result. incubated with the fixed cells. Antibody in
Known positive and negative control sam- the sample will bind to the antigen sites on
ples must be tested in parallel with donor the viral particles. The reaction mixture is
or patient samples. Parallel incubations incubated with fluorescent-labeled anti-
must be performed with a preparation human IgG. Following incubation and
known to contain antibody specific for washing, binding of the labeled antihuman
the antigen in question and with a prepa- IgG is read using a fluorescence micro-
ration known to be free of both antigen scope, with subsequent interpretation of
and antibody. If the positive and negative the fluorescence pattern.
control values do not fall within limits Although there are FDA-approved West-
stated in the package insert, the test must ern blot confirmatory tests for anti-HIV, the
be repeated. Western blot test using recombinant DNA

and viral lysate antigens for anti-HTLV-I/II infective antibody-positive units from
has not been approved by the FDA. An ap- noninfective units containing anti-CMV.
propriate supplemental test to confirm a Routine testing for anti-CMV is not re-
reactive anti-HTLV test result is to repeat quired by AABB Standards,2(p43) but, if it is
the test using another manufacturer’s EIA performed, the usual quality assurance
test. If that test is repeatedly reactive, the considerations apply. The most common
result is considered confirmed. If the test is CMV antibody detection methods in use
negative, the anti-HTLV test result is are EIA and latex agglutination. Other
considered a false-positive result. methods, such as indirect hemagglutina-
The FDA has approved a recombinant tion, complement fixation, and immuno-
immunoblot assay (RIBA) system to further fluorescence, are also available.
differentiate anti-HCV EIA repeatedly reac-
tive samples. The RIBA system is based on
the fusion of HCV antigens to human
superoxide dismutase in the screening test
and to a recombinant superoxide dismu- Labeling, Records, and
tase in the confirmatory test to detect non- Quarantine
specific reactions. A positive result requires
reactivity to two HCV antigens and no reac- Labeling
tivity to superoxide dismutase. Reactivity to Labeling is a process that includes a final
only one HCV antigen or to one HCV anti- review of records of donor history, collec-
gen and superoxide dismutase is classified tion, testing, blood component modifica-
as an indeterminate reaction. Results are tion, quality control functions, and any
usually presented as positive, negative, or additional information obtained after do-
indeterminate. As with all procedures, it is nation. This also includes a review of la-
essential to follow the manufacturer’s in- bels attached to the components and checks
structions for classification of test results. to ensure that all labels meet regulatory
The use of nucleic acid amplification test- requirements and are an accurate reflec-
ing is discussed in Chapter 28. tion of the contents of the blood or blood
components.1,8
All labeling of blood components must
Cytomegalovirus Testing
be performed in compliance with AABB
Optional tests may be performed on units Standards2(p12) and FDA regulations. Blood
intended for recipients with special needs. centers and transfusion services must en-
For example, cytomegalovirus (CMV ) sure that labeling is specific and controlled.
testing is a commonly performed optional Before the labeling process begins, there
test. CMV can persist in the tissues and should be a mechanism or procedure in
leukocytes of asymptomatic individuals place that ensures the use of appropriate
for years after initial infection. Blood from labels. This process should include assur-
persons lacking antibodies to the virus ance of acceptable label composition, in-
has reduced risk of transmitting infection spection on receipt, secure storage and dis-
compared with untested (nonleukocyte- tribution of labels, archiving of superseded
reduced) units. Only a small minority of labels, and availability of a master set of la-
donor units with positive test results for bels in use. In addition, procedures should
anti-CMV will transmit infection. However, address generation of labels, changes in la-
there is presently no way to distinguish bels, and modification of labels to reflect la-

bel control of altered or new components. be that the use of standardized computer-
Labels should be checked for the proper generated barcode labels (with better dif-
product code for the component being la- ferentiation between components, prepara-
beled, which is based on collection method, tion methods, and expiration dates) en-
anticoagulant, and modifications to the hances efficiency, accuracy, and ultimately
component during processing. All aspects safety of labeled components. For example,
of labeling (the bag label as well as the Cir- the ISBT 128 label shows conspicuously
cular of Information for the Use of Human that an autologous, biohazard unit is not a
Blood and Blood Components,14 including standard unit by making the blood type a
the label size, type size, wording, spacing, different, smaller size and filling the space
and the base label adhesive) are strictly usually reserved for blood type with a bio-
controlled. hazard symbol. Adherence to the guidelines
ensures compliance with AABB standards2(p12)
and FDA regulations. The United States
ISBT 128
blood banking community has recently
The ISBT 128 labeling system is an inter- been prompted by a general directive from
nationally defined system based on bar- the Secretary of Health and Human Ser-
code symbology called Code 128. It stan- vices and a subsequent proposal from the
dardizes the labeling of blood so that FDA for uniform acceptance of this more
bar-coded labels can be read by blood comprehensive labeling system. Until the
centers and transfusion services around new international guidelines are imple-
the world. The system allows for each mented, the 1985 FDA Uniform Labeling
number assigned to a unit of blood (blood Guideline remains in effect. More informa-
identification number) to be unique. The tion on ISBT 128 is available from the Inter-
unique number will allow tracking of a national Council on Commonality in Blood
unit of blood from donor to recipient, re- Banking Automation at www.iccbba.com.
gardless of where the unit was drawn or
transfused. As outlined in the United States Label Requirements
Industry Consensus Standard for the Uni-
form Labeling of Blood and Blood Compo- The following pieces of information are
nents Using ISBT 128,15 the information required2(p12),16,17 in clear readable letters on
appearing on the label, the location of the a label firmly attached to the container of
label, and the exact wording on the label all blood and component units:
are standardized. ■ The proper name of the component,
ISBT 128 differs from its predecessor, in a prominent position.
which used CODABAR symbology, by in- ■ A unique numeric or alphanumeric
cluding more specific information on the identification that relates the origi-
label. One advantage of the standardized nal unit to the donor and each com-
system is that additional information on ponent to the original unit.
the label allows for better definition of ■ For components, the name, address,
product codes. Other changes include an and FDA license number or registra-
expanded donation identification number tion number (whichever is appropri-
to include the collection facility’s identifica- ate) of the facility that collected the
tion; bar-coded manufacturer’s lot number, blood and/or the component. The la-
bag type, etc; bar-coded expiration date; bel must include the name and loca-
and special testing barcode. A benefit will tion of all facilities performing any

part of component manufacturing. administration, side effects, and

This includes facilities that wash, ir- hazards.
radiate, and reduce leukocytes by fil- ■ Essential instructions or precautions
tration. If a process is performed for use, including the warning that
under contract, and the process is the component may transmit infec-
performed under processes con- tious agents, and the statements: “Rx
trolled by the contracting facility, only” and “Properly Identify In-
only that facility’s name is required tended Recipient.”
in this case. There should not be ■ The appropriate donor classification
more than two alphanumeric identi- statement—“autologous donor,” “paid
fiers on the unit. donor,” or “volunteer donor”—in
■ The expiration dates, including the type no less prominent than that
date and year; if the shelf life is 72 used for the proper name of the
hours or less, the hour of expiration component.
must be stated. ■ Any additives, sedimenting agents,
■ The amount of blood collected. or cryoprotective agents that might
■ The kind and quantity of anticoagu- still be present in the component.
lant (not required for frozen, degly-
cerolized, rejuvenated, or washed red Special Labeling
cells).
■ Cellular blood components issued as
■ For all blood and blood components,
“Leukocytes Reduced” must be la-
including pooled components, the
beled as such.
approximate volume of the compo-
■ The name and final volume of the
nent must appear on the container.
component and a unique identifier
■ Recommended storage temperature.
for a pool must appear on all pooled
■ ABO group and Rh type.
components.
■ Interpretation of unexpected red cell
■ The number of units in a pool and
antibody tests for plasma-containing
their ABO group and Rh type must
components when positive (not re-
be on the label or an attached tie tag.
quired for cryoprecipitate or frozen,
■ Identification numbers of the indi-
deglycerolized, rejuvenated, or washed
vidual units in a pool should not be
RBCs).
on the label but must be in the re-
■ Results of unusual tests or procedures
cords of the facility preparing the
performed when necessary for safe
pool.
and effective use. Routine tests per-
■ Cellular blood components issued as
formed to ensure the safety of the
“CMV negative” must be labeled as
unit need not be on the label if they
such.
are listed in the Circular of Informa-
■ Irradiated blood components must
tion for the Use of Human Blood and
carry the appropriate irradiated label.
Blood Components.14
■ Reference to the Circular of Informa-
tion for the Use of Human Blood and Records
Blood Components,14 which must be Current good manufacturing practice reg-
available for distribution, and con- ulations, as defined by Title 21 CFR Parts
tains information about actions, in- 200 and 600,6,7,16,18 state that master pro-
dications, contraindications, dosage, duction and control records must be a

part of the labeling process. These records Transfer of those results must be
must be described in the facility’s proce- performed by a system that properly
dures. Before labeling, these records must identifies test results to all appropri-
be reviewed for accuracy and complete- ate blood and blood components.
ness. Appropriate signatures and dates ■ Quarantine: that any nonconforming
(either electronic or manual) must docu- unit is appropriately isolated.
ment the review process.
Production Records
Control Records Master production records must be trace-
Control records include but may not be able back to:
■ Dates of all processing or modifica-
limited to:
tion.
■ Donation process: that all questions
■ Identification of the person and equip-
are answered on the donor card,
consent is signed, all prequalifying ment used in the process steps.
■ Identification of batches and in-pro-
tests are acceptable (eg, hemoglo-
bin, blood pressure), and a final re- cess materials used.
■ Weights and measures used in the
view is documented by qualified
supervisory personnel. course of processing.
■ In-laboratory control results (tem-
■ Infectious disease testing: if per-
formed at the collecting facility, that peratures, refrigerator, etc).
■ Inspection of labeling area before
tests have acceptable quality control
and performance; that daily equip- and after use.
■ Results of component yield when
ment maintenance was performed
and was acceptable; and that final applicable.
■ Labeling control.
results are reviewed to identify the
■ Secondary bag and containers used
date and person performing the
review. in processing.
■ Any sampling performed.
■ Donor deferral registry: That the list
■ Identification of person performing
of deferred donors has been checked
to ensure that the donor is eligible. and checking each step.
■ Any investigation made on noncon-
■ Component preparation: that all
blood and blood components were forming components.
■ Results of examinations of all review
processed and/or modified under
controlled conditions of tempera- processes.
ture and other physical require-
ments of each component. Quarantine
■ Transfer of records: if testing is per- Before final labeling, there must be a pro-
formed at an outside facility, that all cess to remove nonconforming blood and
records of that facility are up to date blood components from the labeling pro-
and that the appropriate licensure is cess until further investigation has oc-
indicated. Records, either electronic curred. This process must be validated to
or manual, must transfer data ap- capture and isolate all blood and blood
propriately. All electronically trans- components that do not conform to re-
ferred test records must be transmitted quirements in any of the critical areas of
by a previously validated system. collecting, testing, and processing. This

process must also capture verbal (eg, tele- 10. Chambers RW, Foley HT, Schmidt PJ. Trans-
mission of syphilis by fresh blood compo-
phone calls) information submitted to the
nents. Transfusion 1969;9:32-4.
collection facility after the collection pro- 11. Busch MP, Stramer SL, Kleinman SH. Evolving
cess. All nonconforming units must re- applications of nucleic acid amplification as-
main in quarantine until they are investi- says for prevention of virus transmission by
blood components and derivatives. In:
gated and all issues are resolved. The units Garratty G, ed. Applications of molecular bi-
may then be discarded, labeled as non- ology to blood transfusion medicine. Bethesda,
conforming units (eg, autologous units), MD: AABB, 1997:123-76.
or labeled appropriately for transfusion if 12. Vargo K, Smith K, Knott C, et al. Clinical spec-
ificity and sensitivity of a blood screening as-
the investigation resolved the problems. If say for detection of HIV-1 and HCV RNA.
the nonconformance cannot be resolved Transfusion 2002;42:876-85.
and the units are from an allogeneic do- 13. Food and Drug Administration. Guidance for
nation, they must be discarded. industry. Revised recommendations regard-
ing invalidation of test results of licensed and
510(k)-cleared blood-borne pathogen assays
used to test donors. (July 11, 2001) Rockville,
References ing, and Manufacturers Assistance, 2001.
1. Code of federal regulations. Title 21 CFR 14. AABB, American Red Cross, and America’s
610.40. Washington, DC: US Government Blood Centers. Circular of information for the
Printing Office, 2004 (revised annually). use of human blood and blood components.
2. Silva MA, ed. Standards for blood banks and Bethesda, MD: AABB, 2002.
transfusion services. 23rd ed. Bethesda, MD: 15. Food and Drug Administration. Guidance for
AABB, 2005. Industry: United States industry consensus
3. Dodd RY, Stramer SL. Indeterminate results standard for the uniform labeling of blood
in blood donor testing: What you don’t know and blood components using ISBT 128, Ver-
can hurt you. Transfus Med Rev 2000;14:151-9. sion 1.2.0. (November 28, 1999) Rockville,
4. Food and Drug Administration. Memoran- MD: CBER Office of Communication, Train-
dum: Recommendations for labeling and use ing, and Manufacturers Assistance, 1999.
of units of whole blood, blood components, 16. Code of federal regulations. Title 21 CFR
source plasma, recovered plasma or source 606.210, and 606.211. Washington, DC: US
leukocytes obtained from donors with ele- Government Printing Office, 2004 (revised
vated levels of alanine aminotransferase annually).
(ALT). (August 8, 1995) Rockville, MD: CBER
17. Food and Drug Administration. Guidelines
Office of Communication, Training, and
for the uniform labeling of blood and blood
Manufacturers Assistance, 1995.
components. (August 1985) Rockville, MD:
CBER Office of Communication, Training and
606.60. Washington, DC: US Government
Manufacturers Assistance, 1985.
6. Code of federal regulations. Title 21 CFR 18. Code of federal regulations. Title 21 CFR Part
606.160. Washington, DC: US Government 210, 211 and 606. Washington, DC: US Gov-
Printing Office, 2004 (revised annually). ernment Printing Office, 2004 (revised annu-
7. Code of federal regulations. Title 21 CFR ally).
Printing Office, 2004 (revised annually). Suggested Reading
9. van der Sluis JJ, ten Kate FJ, Vuzevski VD, et
al. Transfusion syphilis, survival of Tre- AABB, American Red Cross, and America’s Blood
ponema pallidum in donor blood. II. Dose Centers. Circular of information for the use of hu-
dependence of experimentally determined man blood and blood components. Bethesda, MD:
survival times. Vox Sang 1985;49:390-9. AABB, 2002.

Chapter 8: Components from Whole Blood Donations
Chapter 8
Collection, Preparation,
Storage, and Distribution of 8
Components from Whole
Blood Donations
D
ONOR CENTERS AND transfusion Blood Component
services share a common goal in
blood component production: to Descriptions
provide a safe and efficacious component Readers should refer to Chapters 21, 23, and
that benefits the intended recipient. To this 24 and the current Circular of Information
end and in keeping with Food and Drug for the Use of Human Blood and Blood
Administration (FDA) current good man- Components 1 for more detailed indica-
ufacturing practice regulations, all pro- tions and contraindications for transfusion.
cesses involved in the collection, testing,
preparation, storage, and transport of blood Whole Blood
and components are monitored for qual- Fresh Whole Blood contains all blood ele-
ity, including procedures, personnel, re- ments plus the anticoagulant-preservative
agents, equipment, and the contents of the in the collecting bag. It is used commonly
components themselves. Processes should as a source for component production.
ensure the potency and purity of the final After 24-hour storage, it essentially be-
product, minimize microbial contamina- comes red cells suspended in a protein
tion and proliferation, and prevent or de- solution equivalent to liquid plasma, with
lay the detrimental physical and chemical a minimum hematocrit of approximately
changes that occur when blood is stored. 33%.
175

Red Blood Cells RBCs can be prepared at any time during

their shelf life, but AS must be added within
Red Blood Cells (RBCs) are units of Whole
the time frame specified by the manufac-
Blood with most of the plasma removed
turer, generally within the first 72 hours of
(see Method 6.4). If prepared from whole
storage. Shelf life at 1 to 6 C storage de-
blood collected into citrate-phosphate-
pends on the anticoagulant-preservative
dextrose (CPD), citrate-phosphate-dextrose-
used and the method of preparation.
dextrose (CP2D), or citrate-phosphate-
dextrose-adenine (CPDA-1), the final
hematocrit must be ≤80%. Additive red Platelets
cell preservative systems consist of a pri- Platelet concentrates (Platelets) are prepared
mary collection bag containing an antico- from units of whole blood that have not
agulant-preservative with at least two sat- been allowed to cool below 20 C. Plate-
ellite bags integrally attached; one is let-rich plasma (PRP) is separated within
empty and one contains an additive solu- 4 hours after completion of the phlebot-
tion (AS). AS contains sodium chloride, omy or within the time frame specified in
dextrose, adenine, and other substances that the directions for the use of the blood col-
support red cell survival and function up lecting, processing, and storage system—
2
to 42 days (see Table 8-1). The volume of typically 8 hours.3 The platelets are concen-
the AS in a 450-mL collection set is 100 trated by an additional centrifugation step
mL and the volume in 500-mL sets is 110 and the removal of most of the superna-
mL. AS is added to the red cells remaining tant plasma. A procedure for preparation
in the primary bag after most of the of platelets from single units of whole
plasma has been removed. This allows blood appears in Method 6.13. The final
blood centers to use or recover a maxi- component should contain resuspended
mum amount of plasma, yet still prepare platelets in an amount of plasma ade-
a red cell component with a final hemato- quate to maintain an acceptable pH; gen-
crit between 55% and 65%, a level that fa- erally, 40 to 70 mL is used. Although not
cilitates excellent flow rates and allows approved in the United States, platelet
easy administration. concentrates are commonly manufac-
Table 8-1. Content of Additive Solutions (mg/100 mL)
AS-1 AS-3 AS-5

(Adsol) (Nutricel) (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

Chapter 8: Components from Whole Blood Donations 177
tured in Europe using buffy coat as an in- See further discussion of additional
termediate product. In this schema, the plasma products later in the chapter.
initial centrifugation is a “hard-spin” in
which the platelets are concentrated in the Cryoprecipitated AHF
buffy coat. The supernatant platelet-poor Cryoprecipitated antihemophilic factor
plasma and the red cells can be expressed (AHF) is the cold-insoluble portion of
using a top-and-bottom device. The buffy plasma that precipitates when FFP is
coats can then be centrifuged in a “soft- thawed between 1 to 6 C. It is essentially a
spin” to remove the white cells or, more concentrate of high-molecular-weight
commonly, pooled before storage (not glycoproteins also known as CRYO. This
currently allowed by the FDA), then soft- component is prepared from a single
spun as a pooled concentrate, with ex- Whole Blood unit collected into CPDA-1,
4-6
pression of the PRP. CPD, or CP2D and suspended in less than
15 mL of plasma. It contains ≥80 IU Factor
Plasma VIII (AHF), >150 mg of fibrinogen, and
most of the Factor XIII originally present
Plasma in a unit of Whole Blood can be
in the fresh plasma. CRYO contains both
separated at any time during storage, up
the procoagulant activity (Factor VIII) and
to 5 days after the expiration date of the
the von Willebrand factor of the Factor
Whole Blood. When stored frozen at –18 C
VIII von Willebrand complex.
or colder, this component is known as
Once separated, CRYO is refrozen within
Plasma and can be used up to 5 years af-
1 hour of preparation and stored at –18 C or
ter the date of collection. If not frozen, it
colder for up to 1 year after the date of phle-
is called Liquid Plasma, which is stored at botomy. See Method 6.11 for a preparation
1 to 6 C and transfused up to 5 days after procedure.
the expiration date of the Whole Blood
from which it was prepared. Plasma Cryoprecipitate Reduced
Fresh Frozen Plasma (FFP) is plasma
prepared from whole blood, either from the If cryoprecipitate has been removed from
primary centrifugation of whole blood into plasma, this must be stated on the label.
red cells and plasma or from a secondary When stored at –18 C or colder, this com-
centrifugation of PRP. The plasma must be ponent has a 12-month expiration date
3
frozen within 8 hours of collection. See from the date of collection.8(p59) This com-
Methods 6.10 and 6.13. ponent is used primarily in the treatment
Blood centers often convert plasma and of thrombotic thrombocytopenic purpura.9
liquid plasma to an unlicensed component,
“Recovered Plasma (plasma for manufac- Granulocytes
ture),” which is usually shipped to a frac- Granulocytes are usually collected by aphere-
tionator and processed into derivatives sis techniques; however, buffy coats har-
such as albumin and/or immune globulins. vested from fresh Whole Blood units can
provide a source (<1 × 10 ) of granulocytes
9
To ship recovered plasma, the collecting fa-
cility must have a “short supply agreement” in urgent neonatal situations. Their effec-
with the manufacturer.7 Because recovered tiveness is controversial, however.10 Granu-
plasma has no expiration date, records for locytes should be transfused as soon as
this component must be retained indefi- possible after collection but may be
nitely. stored at 20 to 24 C without agitation for

up to 24 hours. Arrangements for pre- toward 1 to 6 C unless it is to be used for

collection testing are often useful. This room temperature component production,
product is becoming obsolete. in which case it should be cooled toward,
but not below, 20 C. Chapter 4 discusses
blood collection in detail.
Collection
Anticoagulants and Preservatives
Blood component quality begins with a
healthy donor and a clean venipuncture Whole blood is collected into a bag that
site to minimize bacterial contamination. contains an FDA-approved anticoagulant-
To prevent activation of the coagulation preservative solution designed to prevent
system during collections, blood should clotting and to maintain cell viability and
be collected rapidly and with minimal function during storage. Table 8-2 com-
trauma to tissues. Although the target col- pares some common solutions. Citrate
lection time is usually 4 to 10 minutes, prevents coagulation by chelating cal-
one study has shown platelets and fresh cium, thereby inhibiting the several cal-
frozen plasma to be satisfactory after col- cium-dependent steps of the coagulation
lection times of up to 15 minutes.11 The fa- cascade.
2
cility’s written procedures should be fol- The FDA approves 21-day storage at 1 to
lowed regarding these collection times. 6 C for red cells from whole blood collected
There should be frequent, gentle mixing in CPD and CP2D and 35-day storage for
of the blood with the anticoagulant. If pre- red cells collected in CPDA-1.12 Most blood
storage filtration is not intended after col- centers now collect up to 500 mL ± 50 mL
lection, the tubing to the donor arm may be (450-550 mL) whole blood in bags specifi-
stripped into the primary collection bag, al- cally designed for this larger volume. Blood
lowed to fill, and segmented, so that it will bags intended for a collection volume of
represent the contents of the donor bag for 450 mL ± 45 mL of whole blood (ie, 405-495
compatibility testing. Blood is then cooled mL) contain 63 mL of anticoagulant-pre-
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. The volume of anticoagulant-pre- based on intended use: RBCs, platelets,

servative in 500 mL ±50 mL bags is 70 mL. FFP, Cryoprecipitated AHF, or neonatal
The allowable range of whole blood col- aliquots. Refer to Methods Section 6 for
lected is dependent upon the collection bag specific component preparation proce-
selected and can vary with manufacturer, dures. Whole blood must be separated
but the total amount collected including and prepared components placed into their
testing samples must not exceed 10.5 required storage temperatures within the
mL/kg donor weight per donation. anticoagulant-preservative manufacturer’s
3
If only 300 to 404 mL of blood is col- recommended times of collection. Re-
lected into a blood bag designed for a cords of component preparation should
450-mL8(p28) collection, the red cells may be identify each individual performing a sig-
used for transfusion provided the unit is la- nificant step in processing.
beled “Red Blood Cells Low Volume.” How- Because red cells, platelets, and plasma
ever, other components should not be pre- have different specific gravities, they are
pared from these low-volume units. If a separated using differential centrifugation.13
whole blood collection of less than 300 mL Rotor size, speed, and duration of spin are
is planned, the volume of anticoagulant- critical variables in centrifugation. Method
preservative solution in that bag should be 7.4 describes how to calibrate a centrifuge
reduced proportionately (see Chapter 4 for for platelet separation, but each centrifuge
calculations), to ensure that the correct must be calibrated for optimal speeds and
amount of anticoagulant is used (ratio of spin times for each combination of compo-
anticoagulant: whole blood). nents prepared in like fashion and for each
different type of collection bag. Times in-
Transportation from a Collection Site clude the time of acceleration and “at
Whole blood should be transported from speed,” not deceleration time. Once the op-
the collection site to the component pre- erating variables are identified for compo-
paration laboratory as soon as possible. nent production, timer accuracy, rpm, and
Units should be cooled toward 1 to 6 C temperature (if appropriate), centrifuges
unless platelets are to be harvested, in should be monitored periodically to verify
which case, units should be cooled toward, equipment performance.
but not below, 20 C. The time between Another practical way to assess centrifu-
collection and the separation of compo- gation is to monitor quality control data on
nents must not exceed the time frame components prepared in each centrifuge. If
specified in the directions for use of the component quality does not meet defined
blood collection,3 processing, and storage standards, eg, if platelet concentrate yields
system. are inconsistent, the entire process should
be evaluated. Factors affecting the quality
assessment are the calibration of the centri-
fuge, the initial platelet count on the whole
Prestorage Processing blood donations, storage time, conditions
between blood collection and platelet prep-
Differential Centrifugation aration, sampling technique, and counting
To simplify the separation of whole blood methods.
into its component parts, blood is collected Large centrifuges rotate at high speeds,
into primary bags to which up to three exerting gravitational forces of thousands of
satellite bags are attached.5 Set design is pounds on blood bags. Weaknesses in these

blood bags or the seals between tubing seg- the specific filter in use. This method may
ments can cause rupture and leakage. The be preferable if special units are to be se-
addition of filters for blood sets presents lected for leukocyte reduction.
different challenges for cup insertion. During inline filtration of red cells, plate-
Blood bags may be overwrapped with plas- lets and/or plasma are first removed from
tic bags to contain any leaks. Bags should the whole blood donation and the additive
be positioned so that a broad surface faces solution is transferred to the red cells. The
the outside wall of the centrifuge to reduce red cells are then filtered through an inline
the centrifugal force on blood bag seams. filter into a secondary storage container.
Contents in opposing cups of the centri- This filtration step should occur as early in
fuge must be equal in weight to improve the shelf life as possible and within the al-
centrifuge efficiency and prevent damage to lowed time frame for the specific filter in
the rotor. Dry balancing materials are pref- use.
erable to liquid material. Weighted rubber Leukocyte-reduced platelets may be pre-
discs and large rubber bands are excellent pared from PRP using inline leukocyte re-
and come in several thicknesses to provide duction filtration.14 FFP manufactured using
flexibility in balancing. Swinging centrifuge this intermediate step typically will have a
cups provide better separation between cells leukocyte content of <5 × 106.
and plasma than fixed-angle cups.
Freezing
Testing and Labeling of Donor Units
Acellular Components
Chapter 7 contains detailed information
When stored at –18 C or colder, FFP con-
on testing and labeling blood components.
tains maximum levels of labile and non-
labile clotting factors (about 1 IU per mL)
Filtration and has a shelf life of 12 months from the
During inline filtration of whole blood, the date of collection. FFP frozen and main-
anticoagulated whole blood is filtered by tained at –65 C may be stored up to 7 years.
gravity through an inline leukocyte reduc- See Method 6.10 for preparation details.
tion filter contained in the collection sys- Plasma can be rapidly frozen by placing the
tem. The filtered whole blood may be bag 1) in a dry ice-ethanol or dry ice-anti-
manufactured into leukocyte-reduced freeze bath, 2) between layers of dry ice, 3)
RBCs. Whole blood leukocyte reduction in a blast freezer, or 4) in a mechanical
filters retain the platelets to a variable de- freezer maintained at –65 C or colder.
gree, so platelet concentrates cannot be Plasma frozen in a liquid bath should be
routinely prepared. However, newly de- overwrapped with a plastic bag to protect
signed platelet sparing filters are under the container from chemical alteration.
investigation. This filtration should occur When a mechanical freezer is used, care
within the time specified by the filter must be taken to avoid slowing the freezing
manufacturer. process by introducing too many units at
A leukocyte reduction filter can be at- one time.
tached to a unit of Whole Blood or RBCs It is prudent practice to use a method to
using a sterile connection device. Ideally, facilitate detection of inadvertent thawing
such filtration should occur as early as pos- of plasma during storage, such as:
sible after collection but must conform to 1. Pressing a tube into the bag during
the manufacturer’s recommendations for freezing to leave an indentation that

disappears if the unit thaws. Remove into the cytoplasm. The intracellular cryo-
tube(s). protectant provides an osmotic force that
2. Freezing the plasma bag in a flat, prevents water from migrating outward as
horizontal position but storing it up- extracellular ice is formed. A high concen-
right. Air bubbles trapped along the tration of cryoprotectant prevents forma-
bag’s uppermost broad surface dur- tion of ice crystals and consequent mem-
ing freezing will move to the top if brane damage.15 Glycerol, a trihydric alcohol,
the unit is thawed in a vertical posi- is a colorless, sweet-tasting, syrup-like fluid
tion. that is miscible with water. Pharmacologi-
3. Placing a rubber band around the cally, glycerol is relatively inert.
liquid plasma bag and removing it Nonpenetrating cryoprotective agents
after freezing to create an indenta- (eg, hydroxyethyl starch) are large mole-
tion that disappears with thawing. cules that do not enter the cell. The mole-
Plasma separated and frozen at –18 C cules protect the cells by a process called
between 8 and 24 hours (eg, plasma that “vitrification” because they form a non-
does not meet the stricter time require- crystalline “glassy” shell around the cell.
ments of FFP) may be labeled as “Plasma This prevents loss of water and dehydration
Frozen Within 24 Hours after Phlebotomy.” injury and alters the temperature at which
It contains all the stable proteins found in the solution undergoes transition from
FFP (see Table 21-3). FFP thawing guide- liquid to solid.
lines apply. 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-
Cellular Components tively stockpiled for military mobilization
Frozen storage can significantly extend the or civilian disasters, but the high cost and
shelf life of red cell components. Unfortu- the 24-hour shelf life after deglycerolization
nately, the process can also cause cell dam- make them less useful for routine inventory
age and add considerable expense. management. Recently, an effectively closed
Effects of Freezing and Thawing. When system was approved with a 2-week post-
unprotected cells are frozen, damage may thaw shelf life when the blood is collected
result from cellular dehydration and from in CPDA-1, frozen within 6 days, and stored
mechanical trauma caused by intracellular at –80 C.
ice crystals. At rates of freezing slower than Two concentrations of glycerol have been
10 C/minute, extracellular water freezes be- used to cryopreserve red cells, as shown in
fore intracellular water, producing an osmotic Table 8-3. This chapter and Methods 6.7
gradient that causes water to diffuse from and 6.8 discuss only the high-concentration
inside the cell to outside the cell. This leads glycerol technique used by most blood
to intracellular dehydration. Controlling the banks. Modifications have been developed
freezing rate, however, is not sufficient by for glycerolizing, freezing, storing, thawing,
itself to prevent cellular damage, so cryo- and deglycerolizing red cells and are dis-
protective agents must be used. cussed elsewhere.16 Several instruments are
Cryoprotective agents are classified as available that partially automate glyceroli-
penetrating and nonpenetrating. Penetrat- zation and deglycerolization of red cells.
ing agents such as glycerol are small mole- The manufacturer of each instrument pro-
cules that freely cross the cell membrane vides detailed instructions for its use.

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

High-Concentration Low-Concentration
Consideration Glycerol 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 Critical
refrozen
Type of storage Polyvinyl chloride; Polyolefin
polyolefin
Shipping Dry ice Liquid nitrogen
Special deglycerolizing equipment Yes No
required
Deglycerolizing time 20-40 minutes 30 minutes
Hematocrit 55-70% 50-70%
Blood intended for freezing can be col- glycerolization that uses an 800-mL pri-
lected into CPD, CP2D, or CPDA-1 and stored mary collection container suitable for
as Whole Blood or RBCs (including AS- freezing (see Method 6.8). Because the cyto-
RBCs). Ordinarily, red cells are glycerolized protective agent for freezing is transferred
and frozen within 6 days of collection or re- directly into the primary collection con-
juvenated and frozen up to 3 days after they tainers, there is less chance of contamina-
expire, but RBCs preserved in AS have been tion and/or identification error. In addition,
frozen up to 42 days after collection, with the amount of extracellular glycerol is
adequate recovery.17,18 smaller and it is more efficient to store and
Some glycerolization procedures require ship units prepared by this method.
removal of most of the plasma or additive Storage Bags. Storage bag composition can
from the RBCs; others do not. The concen- affect the freezing process; less hemolysis
tration of glycerol used for freezing is hyper- may occur in polyolefin than in some poly-
tonic to blood. Its rapid introduction can vinyl chloride (PVC) bags. Contact between
cause osmotic damage to red cells, which red cells and the PVC bag surface may cause
manifests as hemolysis only after thawing. an injury that slightly increases hemolysis
Therefore, when initiating the cryopreser- upon deglycerolization. In addition, polyo-
vation process, glycerol should be intro- lefin bags are less brittle at –80 C and less
duced slowly to allow equilibration within likely to break during shipment and han-
the red cells. dling than PVC bags.
The US Department of Defense has Freezing Process. Red cells frozen within
adopted a method for high-concentration 6 days of collection with a final glycerol

concentration of 40% (wt/vol) must be Irradiation

stored at –65 C or colder. Red cells are usu-
Cellular blood components may be irradi-
ally placed in canisters to protect the plastic
ated before storage to prevent transfusion-
bag during freezing, storage, and thawing.
associated graft-vs-host disease (GVHD).
Although up to 18 hours at room tem-
This does not shorten the shelf life of pla-
perature may elapse between glycerolizing
telets or granulocytes, but red cells expire
and freezing without increased postthaw
28 days after irradiation or at the end of
hemolysis, an interval not exceeding 4
16
the storage period, whichever comes first.
hours is recommended. With current 40%
(wt/vol) glycerol methods, controlled rate
freezing is unnecessary; freezing is accom-
Pooling
plished by placing the RBC container into a
–80 C freezer. Sterile connection devices are used to at-
Refreezing Deglycerolized RBCs. It may tach additional bags and compatible tub-
occasionally be desirable to refreeze thawed ing to a blood bag without breaking the
RBC units that have not been used as ex- sterile integrity of the system. The shelf
pected or have been unintentionally thawed. life of components thus prepared is the
Units that were deglycerolized, stored 20 same as those prepared in a closed system
hours at refrigerator temperature, and then except for Pooled Platelets, which expire 4
reglycerolized and refrozen showed no loss hours after pooling. All sterile connection
of adenosine triphosphate (ATP), 2,3- device welds must be inspected for com-
diphosphoglycerate (2,3-DPG), or in-vivo pleteness, integrity, leakage, and air bub-
survival,19 and RBCs subjected three times bles; procedures must address the action
to glycerolizing, freezing, and thawing ex- to take if the weld is not satisfactory. Re-
hibited a 27% loss of total hemoglobin.20 cord-keeping should include documenta-
AABB Standards for Blood Banks and Trans- tion of the products welded, weld quality
fusion Services does not address refreezing control, and lot numbers of disposables.23
thawed units because this should not be
considered a routine practice. If thawed Cryoprecipitated AHF
units are refrozen, the records should docu- Units of CRYO may be pooled before la-
ment the valuable nature of such units and beling, freezing, and storage. If pooled
the reasons for refreezing them. promptly after preparation using aseptic
Freezing of Platelets. Perhaps because of technique and refrozen immediately, the
their greater complexity, platelets appear to resulting component is labeled “Cryopre-
sustain greater injury during cryopreserva- cipitated AHF Pooled,” with the number
tion than red cells, although several proto- of units pooled stated on the label. The
cols have successfully used dimethyl sul- volume of saline, if added to facilitate
foxide as a cryoprotectant. 21,22 Because pooling, must also appear on the label.
postthaw platelet recovery and function are The statement may appear in the Circular
significantly reduced when compared with of Information for the Use of Human Blood
those of liquid-stored platelets, the clinical and Blood Components1 instead of on the
use of cryopreserved platelets is not wide- container label.
spread. The primary use of this procedure The facility preparing the pool must
is to freeze autologous platelets for future maintain a record of each individual donor
use. Platelet cryopreservation is essentially traceable to the unique identifier used for
a research technique. the pooled component.8(p26)

If an open-system pool or component is before blood reaches unacceptable storage

to be stored frozen, it should be placed in temperatures.
the freezer within 6 hours after the seal has Interiors should be clean, adequately
been broken. The AABB and the FDA re- lighted, and well organized. Clearly desig-
quire transfusion within 4 hours for pooled nated and segregated areas are needed for:
thawed components stored at 20 to 24 1) unprocessed blood; 2) labeled blood
C.8(p58) suitable for allogeneic transfusion; 3) re-
jected, outdated, or quarantined blood; 4)
Platelets autologous blood; and 5) biohazardous
autologous blood. Refrigerators used for
Prestorage pooling of PRP whole-blood-
the storage of blood and blood components
derived platelets is possible using a sterile
may also be used for blood derivatives, tis-
connection device and a storage con-
sues, patient and donor specimens, and
tainer suitable for storage of a high-yield
blood bank reagents.
platelet concentrate. Platelet concentrates
Refrigerators for blood storage outside
can be leukocyte-reduced using inline fil-
the blood bank, as may be found in surgical
tration 14 or they can be pooled into a
suites or emergency rooms, must meet
pooling container, then subsequently leu-
these same standards. Temperature records
kocyte-reduced by filtration and stored in
are required at all times when blood is pres-
a storage container. Although current FDA
ent. It is usually most practical to make
requirements limit the dating of these pools
blood bank personnel responsible for mon-
to 4 hours, studies of such prestorage leu-
itoring these refrigerators.
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 Frozen Storage
storage.24 However, the AABB and FDA re-
The FDA licenses Red Blood Cells Frozen
quire transfusion of pooled platelets within
for storage up to 10 years when prepared
4 hours of pooling.
with high glycerol (40% wt/vol) methods.
As indicated earlier, buffy-coat-derived
Units stored for up to 21 years have been
platelets are commonly pooled before stor-
transfused successfully. A facility’s medi-
age with filtration of the PRP after centri-
cal director may wish to extend the stor-
fugation.4-6
age period; however, storage beyond 10
years requires exceptional circumstances.
The distinctive nature of such units and
Storage the reason for retaining them past the
10-year storage period should be docu-
Refrigerated Storage mented. As units are put into long-term
Blood must be stored only in refrigerators storage, many consider it prudent to freeze
that, by design and capacity, maintain the samples of serum or plasma for subse-
required blood storage temperatures of 1 quent testing should new donor screening
to 6 C throughout their interior space. tests be introduced in the future. The type
There must be a system to monitor tem- of any specimen saved, date of collection,
peratures continuously and record them date of freezing, and specimen location, if
at least every 4 hours, and an alarm sys- necessary, should be included in records
tem with an audible signal that activates of frozen blood.

Not all such specimens may meet the duction and a decrease in pH. Elliptical,
sample requirements of new tests. If stored circular, and flat-bed agitators are avail-
samples are not available or inappropriate able for tabletop or chamber use. Ellipti-
for testing, blood centers may attempt to cal rotators are not recommended for use
call the donor back for subsequent testing. with storage bags made of polyolefin
Frozen rare RBCs that have not been tested without plasticizer (PL-732).25
for all required disease markers should be Other components that require 20 to 24
transfused only after weighing the risks and C temperatures, eg, cryoprecipitate, can be
benefits to the patient. The label should in- stored on a tabletop in any room with an
dicate that the unit has not been com- appropriate ambient temperature, pro-
pletely tested and should identify the miss- vided the temperature is recorded every 4
ing test(s) results. hours during storage. Because room tem-
Blood freezers have the same tempera- peratures fluctuate, “environmental” or
ture monitoring and alarm requirements as “platelet chambers” have been developed to
blood refrigerators and must be kept clean provide consistent, controlled room temper-
and well organized. Freezers designated for atures. These chambers are equipped with
plasma storage must maintain tempera- circulating fans, temperature recorders, and
tures colder than –18 C (many function at alarm systems.
–30 C or colder); RBC freezers must main-
tain temperatures colder than –65 C (many Liquid Storage
maintain temperatures colder than –80 C).
Self-defrosting freezers must maintain ac- Red Blood Cells
ceptable temperatures throughout their Biochemical changes occur when red cells
defrost cycle. are stored at 1 to 6 C; these changes, some
Freezer alarm sensors should be accessi- of which are reversible, contribute to the
ble and located near the door, although “storage lesion” of red cells and to a re-
older units may have sensors located be- duction in viability and levels of 2,3-DPG
tween the inner and outer freezer walls affecting oxygen delivery to tissues.26 The
where they are neither apparent nor acces- most striking biochemical changes that
sible. In such cases, the location of the sen- affect stored red cells are listed in Table
sor can be obtained from the manufacturer 8-4, but some of these changes rarely
and a permanent mark placed on the wall have clinical significance, even in mas-
at that location. Clinical engineers may be sively transfused recipients. Hemoglobin
able to relocate the sensor thermocouple becomes fully saturated with oxygen in
for easier use. the lungs but characteristically releases
Liquid nitrogen tanks used for blood only some of its oxygen at the lower oxy-
storage also have alarm system require- gen pressure (pO2) of normal tissues. The
ments. The level of liquid nitrogen should relationship between pO2 and oxygen satu-
be measured and the sensor placed some- ration of hemoglobin is shown by the oxy-
where above the minimum height needed. gen dissociation curve (see Fig 8-1). Re-
lease of oxygen from hemoglobin at a
given pO 2 is affected by ambient pH,
Room Temperature Storage
intracellular red cell levels of 2,3-DPG, and
Platelets require gentle, continuous agita- other variables. High levels of 2,3-DPG in
tion during storage because stationary the red cells cause greater oxygen release
platelets display increased lactate pro- at a given pO2, which occurs as an adap-

186
Table 8-4. Biochemical Changes in Stored Non-Leukocyte-Reduced Red Blood Cells
CPD CPDA-1 AS-1 AS-3 AS-5
Whole Red Blood Whole Red Blood Red Blood Red Blood Red Blood
Variable Whole Blood Blood Cells Blood Cells Cells Cells Cells
Days of Storage 0 21 0 0 35 35 42 42 42
% Viable cells
(24 hours 100 80 100 100 79 71 76 (64-85) 84 80
posttransfusion)
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 100 86 100 100 56 (± 16) 45 (± 12) 60 59 68.5
value)
2,3-DPG (% of initial 100 44 100 100 <10 <10 <5 <10 <5
value)
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.
Figure 8-1. Oxygen dissociation of hemoglobin under normal circumstances and in Red Blood Cells
(RBCs) stored in excess of 14 days. At tissue pO 2 (40 mm Hg), 25% to 30% of the oxygen is normally re-
leased. In stored RBCs, this will decrease to 10% to 15%.
tive change in anemia; lower red cell lev- Red cells lose potassium and gain so-
els of 2,3-DPG increase the affinity of he- dium during the first 2 to 3 weeks of storage
moglobin for oxygen, causing less oxygen at 1 to 6 C because sodium/potassium
release at the same pO2. In red cells stored adenosine triphosphatase, which pumps
in CPDA-1 or in current additive systems, sodium out of red cells and replaces it with
2,3-DPG levels fall at a linear rate to zero potassium, has a very high temperature co-
after approximately 2 weeks of storage. efficient and functions poorly in the cold.
This is caused by a decrease in intra- Supernatant levels of potassium in a unit of
cellular pH caused by lactic acid, which CPDA-1 RBCs have been reported to in-
26
increases the activity of a diphosphatase. crease from 5.1 mmol/L on the day of col-
This causes the dissociation curve to shift lection to 23 mmol/L on day 7 and 75
to the left, resulting in less oxygen release mmol/L on day 35. Intracellular levels of
(Fig 8-1). potassium will be replenished after transfu-
Upon entering the recipient’s circulation, sion.
stored red cells regenerate ATP and 2,3-DPG, Supernatant potassium levels in red cell
resuming normal energy metabolism and components seem high when compared
hemoglobin function as they circulate in with levels in units of Whole Blood of
the recipient. It takes approximately 12 equivalent age. However, the smaller
hours for severely depleted red cells to re- supernatant fluid volumes must be consid-
generate half their 2,3-DPG levels, and ered when determining total potassium
about 24 hours for complete restoration of load. Blood stored at 1 to 6 C for more than
2,3-DPG and normal hemoglobin func- 24 hours has few functional platelets, but
tion.26,27 levels of coagulation Factors II, VII, IX, X,

and fibrinogen are well maintained. Labile cal surrogates for predicting in-vivo via-
33
factors (Factors V and VIII) decrease with bility and function. Attempts to date to
time and are not considered sufficient to define the biochemical nature of the
correct specific deficiencies in bleeding pa- platelet storage lesion have not been con-
tients, although levels of 30% for Factor V clusive. These observed changes may rep-
and 15% to 20% for Factor VIII have been resent a normal aging process, which is
reported in Whole Blood stored for 21 days, attenuated by the lower temperature of
and platelets stored at room temperature storage (20-24 C), rather than the in-vivo
have been shown to have Factor V levels of temperature of 37 C. However, a role for
47% (see Chapter 21) and Factor VIII levels mitochondrial injury as a contributing
of 68% after 72 hours.13 For better preserva- cause of these changes is plausible. Rest-
tion of Factors V and VIII and platelets, ing platelets derive substantial energy
whole blood is separated into its compo- from β-oxidation of fatty acids. Alter-
34
nent parts and the plasma is stored as FFP. ation in mitochondrial integrity would re-
sult in a reduction in carbon flux through
the tricarboxylic acid cycle and require
Platelets
energy metabolism through glycolysis,
Platelets stored in the liquid state at 20 to with increased lactate production. Such a
24 C are suspended in either autologous reduction would compromise the genera-
anticoagulated plasma (United States and tion of efficient ATP and result in a de-
Europe) or in platelet additive solutions crease in the metabolic pool of ATP and,
(Europe). Under these conditions, the therefore, the energy charge of the pla-
current shelf life of the platelets in most 35
telet. This decrease in the energy charge
countries is 5 days (Table 8-5). This time
would be expected to affect membrane
limitation is partly related to concerns
integrity, resulting in a leakage of cyto-
about storage-related deterioration in
plasmic contents, a diminished response
product potency28 and partly to the poten-
to physiologic stimuli, and an inability to
tial for bacteria to grow rapidly in this
repair oxidized membrane lipids, with
temperature range.29-30 With regard to po-
subsequent distortions in platelet mor-
tency, liquid-stored platelets undergo 36
phology.
in-vitro changes, which are related to the
duration of storage and are collectively
known as the platelet storage lesion.31,32
Shelf Life
This is characterized by a change in pla-
telet shape from discoid to spherical; the The maximum allowable storage time for
generation of lactic acid from glycolysis, a blood component held under acceptable
with an associated decrease in pH; the re- temperatures and conditions is called its
lease of cytoplasmic and granule con- “shelf life.” For red cells, the criteria for
tents; a decrease in various in-vitro mea- determining shelf life for an approved an-
sures of platelet function, particularly ticoagulant-preservative require that at
osmotic challenge; shape changes in- least 75% of the original red cells (from a
duced by adenosine diphosphate; and re- normal allogeneic donor) be in the recipi-
duction in in-vivo recovery and survival. ent’s circulation 24 hours after transfusion.
The in-vitro measures are useful measures For other components, their shelf life is
of qualitative potency, but controversy based on functional considerations. Stor-
still exists regarding their utility as practi- age times are listed in Table 8-5.

8(pp53-60)
Table 8-5. Expiration Dates for Selected Blood Components
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 4 hours, unless otherwise specified
System
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)

8(pp53-60)
Table 8-5. Expiration Dates for Selected Blood Components (cont'd)
Category Expiration
FFP Thawed 24 hours

FFP Thawed – Open System 24 hours
Plasma Frozen within 24 hours 12 months
after Phlebotomy
Plasma Frozen within 24 hours 24 hours
after Phlebotomy Thawed
Thawed Plasma <5 days if whole blood derived
24 hours if apheresis
Plasma Liquid 5 days after expiration of RBCs
Plasma Cryoprecipitate 12 months
Reduced Frozen
Plasma Cryoprecipitate 24 hours to 5 days
Reduced Frozen
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.
formed soon after collection; therefore, pre-

Poststorage Processing storage leukocyte reduction is preferred.
39
Additional discussion of some of the fol- There were concerns that early removal
lowing topics can be found in Chapters 21 of leukocytes would allow bacteria, present
and 22. at the time of collection, to proliferate.
However, studies suggest that early removal
(within 24 hours) in the case of RBCs may
Filtration for Leukocyte Reduction
reduce the likelihood of significant bacte-
Only special leukocyte reduction filters rial contamination.40 Bedside filtration, par-
reliably provide the ≥99.9% (log 3) re- ticularly of platelets, may not be as effective
moval needed to meet the 5 × 106 specifi- in preventing reactions in multitransfused
cation.37 Red cell leukocyte reduction fil- patients and is less desirable for this reason
41
ters contain multiple layers of synthetic than prestorage leukocyte reduction. Bed-
nonwoven fibers that retain white cells and side filtration has also caused hypotensive
platelets, allowing red cells to flow through. reactions.42 Cytokines and other substances
Leukocyte reduction filters are commer- that accumulate during storage (particu-
cially available in a number of set config- larly in platelet components) may account
urations to facilitate filtration during the for some failures of bedside filtration to
separation process at the bedside or in the prevent febrile reactions43 (see Chapter 27).
laboratory before issue.38 Intact leukocyte Furthermore, quality control is difficult to
removal efficiency is best when per- attain at the bedside.44

Thawing All pooled CRYO, whether prepared in an

open or a closed system, must be trans-
Thawing FFP
fused within 6 hours after thawing, or 4
FFP is thawed either at temperatures be- hours after pooling, whichever comes
tween 30 and 37 C or in an FDA-cleared first. CRYO may be pooled into one bag
device. 8 ( p 5 9 ) It is then known as “FFP after thawing to simplify transfusion to a
Thawed” and should be transfused imme- patient requiring multiple units. The
diately or stored between 1 and 6 C for no pooled product is assigned a unique pool
more than 24 hours. The expiration date number, but records must document the
and time must be indicated on the label. individual units included. See Method 6.12
FFP thawed in a waterbath should be for guidelines on how to thaw and pool
protected so that entry ports are not con- CRYO for transfusion.
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 Thawing and Deglycerolizing RBCs
water level. Microwave devices should be The protective canister and enclosed frozen
shown not to exceed temperature limits cells may be placed directly in a 37 C dry
and not to damage the plasma proteins, warmer or can be overwrapped and im-
and there should be a warning device to in- mersed in a 37 C waterbath. Units frozen
dicate if the temperature rises unaccept- in the primary collection bag system
ably. As with any device, there should be a 16
should be thawed at 42 C. The thawing
procedure for the quality control of indi- process takes at least 20 to 25 minutes and
cated functions. should not exceed 40 minutes. Gentle agi-
When whole-blood-derived FFP pretation may be used to speed thawing.
pared in a closed system is thawed but not Thawed cells contain a high concentra-
transfused within 24 hours, the label must tion of glycerol that must be reduced grad-
be modified. This product should be la- ually to avoid in-vivo or in-vitro hemolysis.
beled “Thawed Plasma” and can be stored Deglycerolization is achieved by washing
at 1 to 6 C and transfused up to 5 days after the red cells with solutions of decreasing
thawing. It is similar to FFP except for a re- osmolarity. In one procedure (see Method
duction in both Factor V and Factor VIII, 6.9), glycerolized cells are diluted with 150
particularly Factor VIII. mL of 12% saline, then washed with 1 L of
1.6% saline, followed by 1 L of 0.9% saline
Thawing CRYO with 0.2% dextrose. The progressive de-
CRYO is thawed at temperatures between crease in osmolarity of the washing solu-
30 to 37 C for no more than 15 minutes tions causes osmotic swelling of the cells,
[CFR 606.122(n) (4)]. Bags should not reso each solution must be added slowly, with
main at 30 to 37 C once thawed, so that adequate time allowed for mixing and os-
degradation of Factor VIII is minimized. motic equilibration. Any of the commer-
As with FFP, entry ports should be pro- cially available instruments for batch or
tected from water contamination if the continuous-flow washing can be used to
unit is thawed in a waterbath. Single-unit deglycerolize red cells frozen in a high con-
thawed CRYO must be transfused imme- centration of glycerol. Because there are
diately or can be stored at room tempera- many potentially important variations in
ture (20 to 24 C) for no more than 6 hours. deglycerolization protocols for each instru-

ment, personnel in each facility should not quent storage at 1 to 6 C tolerate freezing
only follow the manufacturer’s instructions, with no more detectable damage than
but also validate the local process. The pro- unirradiated cells.46,47
cess selected must ensure adequate re-
moval of cryoprotectant agents, minimal
Irradiation
free hemoglobin, and recovery of greater
than 80% of the original red cell volume af- Blood components that contain viable
ter the deglycerolization process.8(p27) lymphocytes (including red cell, platelet,
When deglycerolization is complete, the and granulocyte components) should be
integrally connected tubing should be filled irradiated to prevent proliferation of trans-
with an aliquot of red cells and sealed in fused T lymphocytes in recipients at risk
such a manner that it can be detached for of acquiring, or from donors at risk of caus-
subsequent compatibility testing. The label ing, GVHD. The AABB and FDA recom-
must identify both the collecting facility mend a minimum 25 Gy dose of gamma
and the facility that prepares the degly- radiation to the central portion of the
cerolized unit if it is different from the col- container, with no less than 15 Gy deliv-
8(p26)
lection facility. ered to any part of the bag.
When glycerolized frozen red cells from Irradiation is accomplished using ce-
persons with sickle cell trait are suspended sium-137 or cobalt-60, in self-contained
in hypertonic wash solutions during de- blood irradiators or hospital radiation ther-
glycerolization and centrifuged, they form a apy machines. More recently, an x-ray de-
jelly-like mass and hemolyze.16 Modified vice has been developed that is capable of
wash procedures using only 0.9% saline adequate dose delivery. Measurement of
with 0.2% dextrose after the addition of dose distribution; verification of exposure
12% saline can eliminate this problem.45 In time, proper mechanical function, and
some cryopreservation programs, dona- turntable rotation; and adjustment of expo-
tions are screened for the presence of he- sure time as the radioactive source decays
moglobin S before being frozen. should be addressed in the facility’s proce-
When glycerolization or deglyceroliza- dures.48 Records must be maintained, and
tion involves entering the blood bag, the all steps, supplies, and equipment used in
system is considered “open” and the result- the irradiation process must be documented.
ing suspension of deglycerolized cells can To confirm the irradiation of individual
be stored for only 24 hours at 1 to 6 C. A units, radiochromic film labels (available
method for glycerolization and degly- commercially) may be affixed to bags be-
cerolization in an effectively closed system fore irradiation. When exposed to an ade-
allows for the resulting suspension of quate amount of radiation, the film portion
deglycerolized red cells to be stored for 2 of the label darkens, indicating that the
weeks at 1 to 6 C. This method allows more component has been exposed to an ade-
effective inventory management of the quate radiation dosage. Because irradiation
deglycerolized RBC units. damages red cells and reduces the overall
When deglycerolized RBCs are stored at viability (24-hour recovery),49 red cell com-
1 to 6 C for periods up to 14 days, the major ponents that have been irradiated expire on
changes observed are increased concentra- their originally assigned outdate or 28 days
tions of potassium and hemoglobin in the from the date of irradiation, whichever co-
supernatant fluid. Red cells that have un- mes first. Platelets sustain minimal damage
dergone gamma irradiation and subse- from irradiation, so their expiration date

50
does not change. Irradiated blood is es- with any visible red cells present in the
sential for patients at risk from transfu- pool. Only one unique number is affixed
sion-associated GVHD, including fetuses to the final component, but records must
receiving intrauterine transfusion, select reflect the pooling process and all units
immunocompetent or immunocompro- included in the pool. This pooled product
mised recipients, recipients who are under- has an expiration time of 4 hours.
going hematopoietic transplantation, recip- Single CRYO units may be pooled after
ients of platelets selected for HLA or platelet thawing and labeled appropriately. The
compatibility, and recipients of donor units AABB and FDA require transfusion of CRYO
from blood relatives. within 4 hours of pooling and subsequent
storage at 20 to 24 C.
Washing
Washing a unit of RBCs with 1 to 2 L of Volume Reduction
sterile normal saline removes about 99% Platelets may be volume-reduced in order
of plasma proteins, electrolytes, and anti- to decrease the total volume of the com-
bodies. Automated and manual washing ponent transfused or partially remove
methods remove some of the leukocytes supernatant substances, such as ABO allo-
in the RBCs, but not enough to prevent antibodies. The former need may arise in
alloimmunization. Up to 20% of the red patients with small intravascular volumes
cell mass may be lost depending on the or those with fluid overload (eg, resulting
protocol used. Washed red cells must be from renal or cardiac failure). The latter
used within 24 hours because preparation need may be better addressed by washing
is usually accomplished in an open sys- (see below). If sterility is broken, the expi-
tem, and removal of the anticoagulant- ration of the product becomes 4 hours. If
preservative solution compromises long- sterility is not broken (eg, a sterile con-
term preservation of cell viability and nection device is used23), removal of the
function. supernatant still reduces glucose avail-
Platelets can be washed with normal sa- ability and buffering capacity, and the
line or saline-buffered with ACD-A or citrate, subsequent storage of the platelets is in a
using manual or automated methods. The suboptimal environment. Transfusion as
procedures may result in a reduction in soon as possible is generally advocated.
radiolabeled recovery (about 33% less), but
not in survival of the washed platelets51; Aliquoting
white cell content is not significantly
Blood components may be aliquoted in
changed. Washed platelets must be used
smaller volumes into other containers in
within 4 hours of preparation.
order to meet the needs of very-low-vol-
ume transfusion recipients or to provide a
Pooling component to meet the needs of patients
When a patient requires multiple units of with fluid overload. The composition of
Platelets, pooling them into a single bag red cell and plasma components is not al-
simplifies issue and transfusion. This prod- tered by aliquoting, unlike volume reduc-
uct should be labeled “Platelets Pooled.” tion. Therefore, the expiration date is not
If Platelets contain a significant number altered if a sterile connection device is
of red cells and ABO groups are mixed, used to perform the aliquoting.23 The shelf
plasma antibodies should be compatible life and viability of platelets, however, are

dependent on the storage bag, plasma components that look abnormal must not
volume, and storage environment. Re- be shipped or transfused.
moving aliquots of platelets from the Deleterious conditions should be sus-
“mother” bag changes the storage envi- pected if 52 : 1) segments appear much
ronment of the platelets remaining in the lighter in color than what is in the bag (for
“mother” bag. If the altered storage envi- AS-RBCs), 2) the red cell mass looks purple,
ronment does not meet the storage bag 3) a zone of hemolysis is observed just
manufacturer’s requirements, the expira- above the cell mass, 4) clots are visible, 5)
tion period of the remaining component blood or plasma is observed in the ports or
should also be modified. at sealing sites in the tubing, or (6) the
plasma or supernatant fluid is murky, pur-
Rejuvenation ple, brown, or red. A green hue from
It is possible to restore levels of 2,3-DPG light-induced changes in bilirubin pig-
and ATP in red cells stored in CPD or ments need not cause the unit to be re-
CPDA-1 solutions by adding an FDA-li- jected. Mild lipemia, characterized by a
censed solution containing pyruvate, milky appearance, does not render a dona-
inosine, phosphate, and adenine (see tion unsuitable provided that all infectious
Method 6.6). RBCs may be rejuvenated af- disease testing can be performed. Grossly
ter 1 to 6 C storage up to 3 days after expi- lipemic specimens are unsuitable.
ration; then, they may be glycerolized and A component that is questioned for any
frozen in the same manner as fresh red reason should be quarantined until a re-
cells. If rejuvenated RBC units are to be sponsible person determines its disposi-
transfused within 24 hours, they may be tion. Evaluation might include inverting the
stored at 1 to 6 C; however, they must be unit gently a few times to mix the cells with
washed before use to remove the inosine, the supernatant fluid because considerable
which might be toxic to the recipient. The undetected hemolysis, clots, or other alter-
blood label and component records must ations may be present in the undisturbed
indicate the use of rejuvenating solutions. 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
Inspection, Shipping, taken, when, and by whom. Units of FFP
Disposition, and Issue and CRYO should be inspected when they
are removed from frozen storage for evi-
Inspection dence of thawing and refreezing and for ev-
Stored blood components are inspected idence of cracks in the tubing or plastic
immediately before issue for transfusion bag. Unusual turbidity in thawed compo-
or shipment to other facilities.8(pp14,15) These nents may be cause for discard.
inspections must be documented; records All platelet components should be in-
should include the date, donor number, spected before release and issue. Units with
description of any abnormal units, the ac- macroscopically visible platelet aggregates
tion taken, and the identity of personnel should not be used for transfusion. Some
involved. Visual inspections cannot al- facilities assess the “swirling” appearance
ways detect contamination or other dele- of platelets by holding platelet bags up to a
terious conditions; nonetheless, blood light source and gently tapping them. This

swirl phenomenon correlates well with pH incidental to routine handling. Refer to

values associated with adequate platelet Chapter 2 for more information on ship-
in-vivo viability.53 Some platelet compo- ping regulations and guidelines.
nents have been noted to contain small Simple exposure to temperatures outside
amounts of particulate matter. These com- the acceptable range does not necessarily
ponents are suitable for use. render blood unsuitable for transfusion. Ex-
Bacterial contamination of transfusion ceptions may be made under unusual cir-
components is rare because of the use of cumstances such as for autologous units or
aseptic technique and screening for bacte- cells of a rare phenotype, but the records
ria in platelets, the availability of closed sys- must document the reasons for preserving
tems for collection and preparation, and the unit, the evaluation of its continued
careful control of storage conditions. Steril- suitability for transfusion, and the identity
ity testing of blood components plays a role of the person responsible for the decision.
in validating initial production processes. If Other factors to consider when assessing
a transfusion component has an abnormal component acceptability after transport in-
appearance, or if an adverse clinical reac- clude the length of time in shipment, mode
tion appears to be related to contaminated of transportation, magnitude of variance
donor blood, culturing should be performed above or below the acceptable range, pres-
and a Gram’s stain should be evaluated. ence of residual ice in the shipping box, ap-
Microbiologists can best advise blood cen- pearance of the unit(s), age of the unit(s),
ter staff on sample requirements and appro- and likelihood of additional storage before
priate test methods for detecting potential transfusion. The shipping facility should be
blood contaminants, including cryophilic mi- notified when a receiving facility observes
croorganisms. Making cultures directly from that acceptable transport temperatures
the contents of the bag and from the recipi- have been exceeded.
ent can provide useful diagnostic information.
Any facility that collected a reportedly
contaminated component should be noti- Whole Blood and RBCs
fied so that donor bacteremia, potentially Liquid Whole Blood and RBCs shipped
inadequate donor arm preparation, or im- from the collection facility to another fa-
proper handling or pooling technique can cility must be transported in a manner
be investigated. The donor’s health should that ensures a temperature of 1 to 10 C.
be reviewed, and other components prepared The upper limit of 10 C can be reached in
from that collection should be withdrawn. 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-
Shipping
monly used for pediatric patients, can
Shipment to areas outside the facility re- warm even more quickly.
quires additional packaging. Transport Wet ice, securely bagged to prevent leak-
containers or coolers and packaging pro- age, is the coolant of choice to maintain re-
cedures must be validated before use to quired temperatures during transport and
verify that they are able to maintain blood shipping. An appropriate volume is placed
components at required temperatures for on top of the units within the cardboard
the intended time and conditions. Con- box or insulated container. Clinical coolant
tainers must also be able to withstand packs or specially designed containers may
leakage, pressure, and other conditions also be used to maintain acceptable trans-

port temperatures. Super-cooled ice (eg, fluctuations in temperatures between –85 C

large blocks of ice stored at –18 C) in con- and –20 C with no significant change in
tact with Whole Blood or RBCs may result in-vitro recovery or 24-hour posttrans-
in temperatures below 1 C, with resultant fusion survival, so transport with dry ice is
hemolysis of the red cells. acceptable. Blood shipments containing
dry ice as a coolant are considered “danger-
Platelets and Granulocytes ous goods,” and special packaging and la-
beling requirements apply (see Chapter 2).
Every reasonable effort must be made to
Shipping boxes containing dry ice must not
ensure that platelets and granulocytes are
be completely sealed so that the carbon di-
maintained at 20 to 24 C during ship-
oxide gas released as the dry ice sublimes
ment. A well-insulated container without
can escape without risk of explosion.
added ice, or with a commercial coolant
designed to keep the temperature at 20 to
24 C, is recommended. Fiber-filled enve- Disposition
lopes or newspaper are excellent insula- Both blood collection sites and transfu-
tors. For very long distances or travel sion services must maintain records on all
times in excess of 24 hours, double-insu- blood components handled so that units
lated containers may be needed. can be tracked from collection to final
disposition. Units of blood that cannot be
Frozen Components released for transfusion should be re-
turned to the provider or discarded as
Frozen components must be packaged for
biohazardous material. The nature of the
transport in a manner designed to keep
problem disqualifying the unit should be
them frozen. This may be achieved by us-
investigated and the results reported to
ing a suitable quantity of dry ice in well-
the blood supplier. Findings may indicate
insulated containers or in standard ship-
a need to improve phlebotomy techni-
ping cartons lined with insulation mate-
ques, donor screening methods, or the
rial, such as plastic air bubble packaging
handling of units during processing, stor-
or dry packaging fragments. The dry ice,
age, or transport.
obtained as sheets, may be layered at the
Disposal procedures must conform to the
bottom of the container, between each
local public health code for biohazardous
layer of frozen components, and on top.
waste. Autoclaving or incineration is rec-
Shipping facilities should determine op-
ommended. If disposal is carried out off-
timal conditions for shipping frozen com-
site, a contract with the waste disposal firm
ponents, which depend on the temperature
must be available and should specify that
requirements of the component, the dis-
appropriate Environmental Protection Agency,
tance to be shipped, the shipping container
state, and local regulations are followed
used, and ambient temperatures to be en-
(see Chapter 2 for the disposal of biohazar-
countered. Procedures and shipping con-
dous waste).
tainers should be validated and periodically
monitored. The receiving facility should al-
ways observe the shipment temperature Issuing from a Transfusion Service
and report unacceptable findings to the Blood transported short distances within
shipping facility. a facility, eg, to the patient care area for
Red cells cryopreserved with high-con- transfusion, requires no special packaging
centration glycerol (40% wt/vol) tolerate other than that dictated by perceived safety

concerns and institutional preferences. tency evaluated. The contents of final

However, blood should never unnecessar- products should be periodically assessed
ily be allowed to reach temperatures out- to make sure that they meet expectations.
side its accepted range. Specific transport How much quality control is performed is
guidelines may be warranted if transport best determined by the institution, with
time is prolonged. input from the compliance officer and con-
Units that have left the control of the sideration of AABB and FDA requirements.
transfusion service or donor center and See Appendix 8-1.
then have been returned must not be reis-
sued for transfusion unless the following Quality Control of Equipment
conditions have been met:
1. The container closure has not been Continuous Temperature Monitoring
penetrated, entered, or modified in Systems
any manner. Most blood refrigerators, freezers, and
2. Red cell components have been main- chambers have built-in temperature mon-
tained continuously between 1 and itoring sensors connected to recording
10 C, preferably 1 to 6 C. Blood cen- charts or digital readout systems for easy
ters and transfusion services usually surveillance. Digital recording devices
do not reissue RBC units that have measure the differences in potential gen-
remained out of a monitored refrigerated by a thermocouple; this difference
erator or a validated cooler for lon- is converted to temperature. Because warm
ger than 30 minutes because, beyond air rises, temperature-recording sensors
that time, the temperature of the should be placed on a high shelf and im-
component may have risen above 10 C. mersed in a volume of liquid not greater
3. At least one sealed segment of inte- than the volume of the smallest compo-
gral donor tubing remains attached nent stored. Either a glass container or a
to the red cell component, if the plastic blood bag may be used. Recording
blood has left the premises of the is- charts and monitoring systems must be in-
suing facility. spected daily to ensure proper function.
4. Records indicate that the blood has When recording charts or tapes are
been reissued and has been inspected changed, they should be dated inclusively
before reissue. (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-
Blood Component Quality ature should be explained in writing on the
Control chart beside the tracing or on another doc-
Ensuring safe and efficacious blood com- ument and should include how the stored
ponents requires applying the principles components were managed. A chart with a
of quality assurance to all aspects of com- perfect circle tracing may indicate that the
ponent collection, preparation, testing, recorder is not functioning properly or is
storage, and transport. All procedures and not sensitive enough to record the expected
equipment in use must be validated be- variations in temperature that occur in any
fore their implementation and periodi- actively used refrigerator.
cally monitored thereafter. Staff must be Blood banks with many refrigerators and
appropriately trained and their compe- freezers may find it easier to use a central

alarm monitoring system that monitors all age temperatures, as long as their accuracy
equipment continuously and simultane- is calibrated against a NIST-certified ther-
ously and prepares a hard copy tape of mometer or a thermometer with a NIST-
temperatures at least once every 4 hours. traceable calibration certificate (see Method
These systems have an audible alarm that 7.2). Of equal importance is that they be
sounds as soon as any connected equip- used as intended, according to the manu-
ment reaches its predetermined tempera- facturer’s recommendations.
ture alarm point and indicates the equip-
ment in question. Blood storage equipment
so monitored does not require a separate Alarm Systems
independent recording chart. To ensure that alarm signals will activate
at a temperature that allows personnel to
take proper action before blood reaches
Thermometers undesirable temperatures, both tempera-
Visual thermometers in blood storage equip- ture of activation and power source are
ment provide ongoing verification of tem- tested periodically. The electrical source
perature accuracy. One should be immersed for the alarm system must be separate
in the container with the continuous mon- from that of the refrigerator or freezer; ei-
itoring sensor. The temperature of the ther a continuously rechargeable battery
thermometer should be compared peri- or an independent electrical circuit served
odically with the temperature on the re- by an emergency generator is acceptable.
cording chart. If the two do not agree Method 7.3.1 provides a detailed proce-
within 2 C, both should be checked dure to test the temperatures of activation
against a thermometer certified by the for refrigerator alarms. Suggestions for
National Institute of Standards and Tech- freezer alarms are in Method 7.3.2 Ther-
nology (NIST) and suitable corrective ac- mocouple devices that function at freezer
tion should be taken (see Method 7.2). (A temperatures are especially useful for de-
2 C variation between calibrated ther- termining the temperature of activation
mometers allows for the variation that with accuracy when sensors are accessible.
may occur between thermometers cali- When they are not, approximate activation
brated against the NIST thermometers.) temperatures can be determined by check-
Thermometers also help verify that the ing a freezer’s thermometer and recording
temperature is appropriately maintained chart when the alarm sounds after it is shut
throughout the storage space. Large refrig- down for periodic cleaning or mainte-
erators or freezers may require several ther- nance. It can also be assessed by placing a
mometers to assess temperature fluctua- water bottle filled with cold tap water
tions. In addition to the one immersed with against the inner freezer wall where the
the continuous monitoring sensor (usually sensor is located.
located on a high shelf), at least one other When the alarm goes off, usually in a
in a similar container is placed on the low- short time, the recording chart can be
est shelf on which blood is stored. The tem- checked immediately for the temperature
perature in both areas must be within the of activation. There must be written in-
required range at all times. structions for personnel to follow when the
Either liquid-in-glass (analog) thermom- alarm sounds. These instructions should
eters or electronic and thermocouple (digi- include steps to determine the immediate
tal) devices can be used for assessing stor- cause of the temperature change and ways

8(p30)
to handle temporary malfunctions, as well mg of fibrinogen. Each pool must have
as steps to take in the event of prolonged a Factor VIII content of at least 80 mg
failure. It is important to list the names of times the number of donor units in the
key people to be notified and what steps pool; for fibrinogen, the content should be
should be taken to ensure that proper stor- 150 mg times the number of donor units.8(p30)
age temperature is maintained for all blood, (See Appendix 8-1.)
components, and reagents.
Quality Control of Red Blood Cells

RBCs prepared without additive solutions
References
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industry: Gamma irradiation of blood and

Appendix 8-1. Component Quality Control

Specifications AABB
Component and Standards* Standards†
Red Blood Cells Hematocrit ≤80% (in all) 5.7.5.1

Red Blood Cells Retain 85% of original red cells, 95% of tested 5.7.5.6
Leukocytes units <5 × 106 leukocytes in the final con-
Reduced tainer
Cryoprecipitated AHF Factor VIII: ≥80 IU/bag (100%) 5.7.5.14
Fibrinogen ≥150 mg/bag (100%)
Platelets ≥5.5 × 1010 platelets per unit and pH ≥6.2 in 5.7.5.16
90% of units tested
Platelets Leukocytes ≥5.5 × 1010 platelets in 75% of units tested, 5.7.5.17
Reduced ≥6.2 pH in 90% of units tested, and
<8.3 × 105 leukocytes in 95% of units tested
Platelets Pheresis ≥3.0 × 1011 platelets in final container of compo- 5.7.5.19
nents tested; and pH ≥6.2 in 90% of units
tested
Platelets Pheresis <5.0 × 106 leukocytes in 95% of components 5.7.5.20
Leukocytes tested and ≥3.0 × 1011 platelets in the final
Reduced container and pH ≥6.2 in 90% tested units
Granulocytes ≥1.0 × 1010 granulocytes in at least 75% of com- 5.7.5.21
Pheresis ponents tested
Irradiated compo- 25 Gy delivered to the central portion of the con- 5.7.4.2
nents tainer; minimum of 15 Gy at any point in the
component
*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
9
P
ROTEINS ARE MACROMOLECULES strands of deoxyribonucleic acid (DNA).
composed of amino acids, the se- DNA is composed of the sugar deoxyribose,
quences of which are determined a phosphate group, the purine bases ade-
by genes. Lipids and carbohydrates are nine (A) and guanine (G), and the pyrimi-
not encoded directly by genes; genetic de- dine bases thymine (T) and cytosine (C).
termination of their assembly and func- The combination of a sugar, a phosphate
tional structures results from the action of group, and a base is called a nucleotide. A
different protein enzymes. Blood group double strand of DNA consists of two com-
antigens can be considered gene prod- plementary (nonidentical) single strands
ucts, either directly, as polymorphisms of held together by hydrogen bonds between
membrane-associated proteins, or indi- specific base pairings of A-T and G-C. The
rectly, as carbohydrate configurations cat- two strands form a double helix configu-
alyzed by glycosyltransferases. ration with the sugar-phosphate back-
bone on the outside and the paired bases
on the inside (see Fig 9-1). DNA synthesis
is catalyzed by DNA polymerase, which
From DNA to mRNA to adds a deoxyribonucleotide to the 3′ end
Protein of the existing chain. The 3′ and 5′ nota-
tion refers to the carbon position of the
Structure of DNA deoxyribose linkage to the phosphate
A gene consists of a specific sequence of group. Phosphate groups bridge the sugar
nucleotides located at a specific position groups between the fifth carbon atom of
(locus) along a chromosome. Each chro- one deoxyribose molecule and the third
mosome consists of long molecules or carbon atom of the adjacent deoxyribose
203

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-
Figure 9-1. Schematic representation of the base pairs the promoter activity and is respon-
1
pairing of double-stranded DNA. sible for the Fy(a–b–) phenotype.
After binding to the promoter, RNA poly-
merase opens up the double helix of a local
molecule and thus create the backbone of region of DNA, exposing the nucleotides on
the DNA strand. DNA polymerase-depend- each strand. The nucleotides of one ex-
ent synthesis always occurs in the direc- posed DNA strand act as a template for com-
tion of 5′ to 3′. plementary base pairing; RNA is synthe-
sized by the addition of ribonucleotides to
DNA Transcription the elongating chain. As RNA polymerase
moves along the template strand of DNA,
Linear sequences of nucleotides along the
the double helix is opened before it and
DNA strands constitute the genes. Genes
closes behind it like a zipper. The process
occupy a constant location (locus) in the
continues, usually 0.5 to 2 kb downstream
DNA of a specific chromosome; the loci of
of the poly-A signal, whereupon the en-
most known genes have been identified due
zyme halts synthesis and releases both the
to the human genome project. For protein
DNA template and the new RNA chain.
synthesis to occur (see Fig 9-2), the infor-
mation encoded in the DNA sequence
must be copied into RNA (transcription)
mRNA Processing
and transported to the cytoplasmic organ- Shortly after the initiation of transcrip-
elles called ribosomes where protein as- tion, the newly formed chain is capped at

Chapter 9: Molecular Biology in Transfusion Medicine 205
Figure 9-2. Model of a nucleotide sequence. The sequence is fictitious.
its 5′ end by the addition of a methylated ies of adenylic acid, called the poly-A tail.
G nucleotide. The 5′ cap is important for The poly-A tail functions in the export of
initiating protein synthesis and possibly mature mRNA from the cell nucleus to the
for protecting the mRNA molecule from cytoplasm, in the stabilization of the mRNA,
degradation during its transport to the cy- and as a ribosomal recognition signal re-
toplasm. At the 3′ end of the mRNA, a quired for efficient translation.
multiprotein cleavage-polyadenylation In eukaryotic cells, the nucleotide se-
complex carries out a two-step process quence of a gene often contains certain re-
that cleaves the new RNA at a specific se- gions that are represented in the mRNA and
quence and then attaches 100 to 200 cop- other regions that are not represented. The
Figure 9-3. The promoter sequence (• ) contains the starting site for RNA synthesis. RNA polymerase
binds to the promoter and opens up a local region of the DNA sequence. One strand of DNA (the lower
one in this figure) acts as a template for complementary base pairing. The RNA polymerase copies the
DNA in a 5′ to 3′ direction until it encounters a stop signal (■). Lowercase letters represent nucleo-
tides in introns; uppercase letters represent coding bases in exons. The sequence is fictitious.

regions of the gene that are represented in pre-RNA is highly regulated during differ-
mRNA are called exons, which specify the entiation and is tissue-specific. For example,
protein-coding sequences and the se- acetylcholinesterase, which bears the Cart-
quences of the untranslated 5′ and 3′ re- wright blood group antigen, is spliced in a
gions. The regions that are not represented manner to produce a glycosylphosphati-
in the mRNA are called intervening se- dylinositol-linked protein in red cells, but it
quences or introns. In the initial transcrip- is a transmembrane protein in nerve cells.2
tion of DNA to RNA, the introns and exons Alternative splicing may also occur in a sin-
are copied in their entirety, and the result- gle tissue type and may result in the pro-
ing product is known as the primary RNA duction of more than one protein from the
transcript or pre-mRNA. Processing occurs same gene; an example of this type is the
while pre-mRNA is still in the nucleus, and production of glycophorins C and D from a
the introns are cut out by a process known single glycophorin C (GPC) gene.3
as RNA splicing (see Fig 9-4).
RNA splicing depends on the presence of
certain highly conserved sequences con-
Translation of mRNA
sisting of GU at the 5′ splice site (donor site)
and AG at the 3′ splice site (acceptor site). The bases within a linear mRNA sequence
Additionally, an adenosine residue within a are read (or translated) in groups of three,
specific sequence in the intron participates called codons. Each three-base combina-
in a complex reaction along with a very tion codes for one amino acid. There are
large ribonucleoprotein complex called the only 20 amino acids commonly used for
spliceosome. The reaction results in cleav- protein synthesis, and there are 64 (4 × 4 ×
age and joining of the 5′ and 3′ splice sites, 4) possible codons. Most amino acids can
with release of the intervening sequence as be specified by each of several different
a lariat. Substitution of any of these highly codons, a circumstance known as “degen-
conserved sequences can result in inaccu- eracy” or “redundancy” in the genetic
rate RNA splicing (see Fig 9-4). Splicing of code. For example, lysine can be specified
Figure 9-4. Substitution of two nucleotides (boldface) in the intron sequences flanking exon 3 of GYPB
prevents normal splicing. Instead, all nucleotides between the 5′ donor site of the second intron and
the 3′ acceptor site of the third intron are excised. Because exon 3 is not translated, it is called a
pseudoexon (Ψ).

by either AAA or AAG. Methionine has at the 5´ and 3´ ends of mRNA, can affect
only the single codon AUG. Three codons gene expression.
(UAA, UGA, and UAG) function as stop sig-
nals; when the translation process en-
counters one of them, peptide synthesis Genetic Mechanisms that
stops.
For the translation of codons into amino Create Polymorphism
acids, cytoplasmic mRNA requires the as- Despite the redundancy inherent in de-
sistance of transfer RNA (tRNA) molecules. generacy of the genetic code, molecular
The tRNA molecules interact with the events such as substitution, insertion, or
mRNA through specific base pairings and deletion of a nucleotide may have far-
bring with them the amino acid specified reaching effects on the protein encoded.
by the mRNA codon. Thus, the amino acids Some of the blood group polymorphisms
are linked in amino to carboxyl peptide observed at the phenotypic level can be
bonds forming a growing polypeptide traced to small changes at the nucleotide
chain. Proteins are synthesized such that level. The sequence in Fig 9-2, which is
the initial amino acid has an unlinked not meant to represent a known sequence,
amine (NH2) group and ends with a termi- can be used to illustrate the effects of
nal carboxylic acid (COOH) group. Protein minute changes at the nucleotide level,
synthesis occurs on ribosomes, which are discussed below.
large complexes of RNA and protein mole-
cules; ribosomes bound to the rough endo-
plasmic reticulum are the site of synthesis Nucleotide Substitution
of membrane and secretory proteins, where- Nucleotide substitutions in the genomic
as free ribosomes are the site of synthesis of DNA can have profound effects on the re-
cytosolic proteins. The ribosome binds to sultant protein. Many blood group anti-
the tRNA and to the mRNA, starting at its 5′ gens are the result of single nucleotide
end (amino terminal). Therefore, protein changes at the DNA level. The nucleotide
synthesis occurs from the amino-terminal changes are transcribed into the RNA,
end toward the carboxyl-terminal end. The which alters the sequence of the codon. In
protein is produced sequentially until a stop some instances, the codon change results
codon is reached, which terminates the in the incorporation of a different amino
translation process and releases the newly acid. For example, a change in the DNA
synthesized protein. sequence from a T to a C at a given posi-
Many of the steps in the pathway of RNA tion would result in an mRNA change
synthesis to protein production are closely from U to G. Any one of three possible
regulated at different levels to control gene outcomes can follow the substitution of a
expression. Control steps include: initiation single nucleotide:
of transcription, proofreading of the tran- 1. Silent mutation. For example, the
scription process, addition of the poly-A tail, substitution of an A with a C in the
transportation of mRNA to the cytosol, ini- third position of the DNA coding
tiation of translation, and elongation of the strand for serine (UCU) in Fig 9-2 also
polypeptide chain. Moreover, tissue-specific codes for serine (UCG). Thus, there
and differentiation or stage-specific tran- would be no effect on the protein
scription factors, as well as hormone re- because the codon would still be
sponse elements and regulatory elements translated as serine.

2. Missense response. The substitution (see Fig 9-4). Because exon 3 is a coding
of a G with an A in the second posi- exon in GYPA but is noncoding in GYPB, it
tion of the DNA coding strand for the is called a pseudoexon in GYPB.
second serine changes the product
of the codon from serine (UCG) to Nucleotide Insertion and Deletion
leucine (UUG). Many blood group Insertion of an entirely new nucleotide re-
polymorphisms reflect a single amino sults in a frameshift, described as +1, be-
acid change in the underlying mole- cause a nucleotide is being added. Nucle-
cule. For example, the K2 antigen has otide deletion causes a –1 frameshift. A
threonine, but K1 has methionine, as peptide may be drastically altered by the
the amino acid at position 193. This insertion or deletion of a single nucleo-
results from a single C to T substitu- tide. For example, the insertion of an A af-
tion in exon 6 of the Kell (KEL) gene.4 ter the second nucleotide of the noncoding
3. Nonsense response. The substitu- DNA strand of Fig 9-2 would change the
tion of a G with a T in the second po- reading frame of the mRNA to UAU CAG
sition of the DNA coding strand for AAG CUG CCC UGG and represent the
serine (UCG) results in the creation of polypeptide isoleucine-valine-phenylala-
the codon (UAG), which is one of the nine-aspartic acid-glycine-threonine.
three stop codons. No protein syn-
thesis will occur beyond this point,
resulting in a shortened or truncated
protein. Depending upon where this Genetic Variability
nonsense substitution occurs, the Gene conversion and crossing over may
synthesized protein may be rapidly occur between homologous genes located
degraded or may retain some func- on two copies of the same chromosome
tion in its abbreviated form. The that are misaligned during meiosis. Exam-
Cromer blood group antigens reside ples of homologous genes encoding blood
on the decay-accelerating factor group antigens are the RHD and RHCE
(DAF) membrane protein. In the null genes of the Rh blood group system and
(Inab) phenotype, a G to A nucleo- GYPA and GYPB, which encode the anti-
tide substitution of the DAF gene gens of the MNS blood group system.
creates a stop codon at position 53,
and the red cell has no DAF expres- Single Crossover
sion.5 A single crossover is the mutual exchange
A nucleotide substitution outside an of nucleotides between two homologous
exon sequence may alter splicing and lead genes. If crossover occurs in a region
to the production of altered proteins, as where paired homologous chromosomes
seen with the altered expression of glyco- are misaligned, two hybrid genes are
phorin B (GPB)6 or in the failure to produce formed in reciprocal arrangement (see Fig
a normal amount of protein, as in the 9-5). The novel amino acid sequences en-
Dr(a–) phenotype of Cromer.7 In GYPB, the coded by the nucleotides at the junction
gene that encodes GPB, substitutions of of the hybrid gene may result in epitopes
two conserved nucleotides required for recognized by antibodies in human se-
RNA splicing and present in the rum and are known to occur in at least
glycophorin A gene result in the excision of three blood group systems: Rh, MNS, and
exon 3 as well as introns 2 and 3 of GYPB Gerbich.

Figure 9-5. Single crossover: exchange of nucleotides between misaligned homologous genes. The
products are reciprocal.
Gene Conversion Molecular Techniques

The process of gene conversion is thought
The development of modern molecular
to consist of crossover, a general DNA re-
techniques has greatly expanded our
combination process, and DNA repair
knowledge of all biologic systems. These
during meiosis. The result is that nucleo-
same techniques are also applicable to the
tides from one homologous gene are in-
diagnosis of disease, the practice of foren-
serted into another gene without recipro-
sic science, the generation of recombi-
cal exchange. At the site of chromosome
nant proteins, and the production of func-
crossover during meiosis, a heteroduplex
tional genes for gene therapy. Many of
joint can form; this is a staggered joint be-
these processes begin with DNA typing
tween nucleotide sequences on two par-
and analysis, techniques that are reviewed
ticipating DNA strands (see Fig 9-6). A
below.
second type of gene conversion occurs
in meiosis when the DNA polymerase
switches templates and copies informa- Isolation of Nucleic Acids
tion from a homologous sequence. This The first step in most molecular biology
event is usually the result of mismatch techniques is the isolation and purifica-
repair; nucleotides removed from one tion of nucleic acid, either DNA or RNA.
strand are replaced by repair synthesis us- For applications of interest to the blood
ing the homologous strand as a template. banking community, the desired nucleic

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.
acid is typically human genomic DNA and cells, and tissues. These kits vary in the
mRNA. Genomic DNA is present in all nu- quantity and quality of the DNA isolated, in
cleated cells and can be isolated from pe- rough proportion to the cost and ease of
ripheral blood white cells or from buccal use of the kit. High-quality DNA is of high
tissue obtained by a simple cheek swab. molecular weight and is relatively free of
Both nucleated cells and reticulocytes are contamination by protein or RNA. DNA pu-
cell-specific sources of mRNA. rity is assessed by the ratio of its optical
Manufacturers offer kits for the isolation density (OD) at 260 nm to that at 280 nm,
of human genomic DNA from whole blood, with the OD 260/280 ratio for pure DNA be-

ing 1.8. Low ratios (<1.6) indicate that the and a reverse primer (3′). These are de-
DNA is contaminated with protein or mate- signed so that one is complementary to
rials used in the isolation procedure, and each strand of DNA, and, together, they
high ratios (>2.0) indicate that the DNA is flank the region of interest. Primers can
contaminated with RNA. If the DNA is pure be designed that add restriction sites to
and of sufficient concentration, it can be the PCR product to facilitate its subse-
quantitated by measurement of the OD at quent cloning or labeled to facilitate its
260 nm. If the DNA is impure or in low con- detection. Labels may incorporate radio-
centration, it is best quantitated by electro- activity, or, more frequently, a nonradio-
phoresis in agarose gel along with DNA active tag, such as biotin or a fluorescent
standards of known concentration, fol- dye. The PCR reaction is catalyzed by one
lowed by visualization of the DNA with of several heat-stable DNA polymerases
ethidium bromide staining. isolated from bacterial species that are na-
For certain molecular biology techniques tive to hot springs or to thermal vents on
such as polymerase chain reaction (PCR), the ocean floor. The thermostability of
the quantity and quality of the genomic these enzymes allows them to withstand
DNA used as starting material are not cru- repeated cycles of heating and cooling.
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 Reaction Procedure
as cloning, larger quantities of high-molec- The amplification technique is simple and
ular-weight DNA are required. requires very little DNA (typically <100 ng
One nucleated cell contains about 6 pg of genomic DNA). The DNA under study
of genomic DNA. Based on an average is mixed together with a reaction buffer,
white cell count of 5000/µL, each milliliter excess nucleotides, the primers, and poly-
of peripheral blood contains about 30 µg of merase (see Fig 9-7). The reaction cocktail
DNA. Commercial DNA isolation kits typi- is placed in a thermocycler programmed
cally yield in excess of 15 µg of DNA per to produce a series of heating and cooling
milliliter of whole blood processed. 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
Polymerase Chain Reaction
into single strands. Subsequent cooling in
The introduction of the PCR technique has the presence of excess quantities of sin-
revolutionized the field of molecular ge- gle-stranded forward and reverse primers
netics.8 This technique permits specific allows them to bind or anneal with com-
DNA sequences to be multiplied rapidly plementary sequences on the single-
and precisely in vitro. PCR can amplify, to stranded template DNA. The specific cool-
a billionfold, a single copy of the DNA se- ing temperature is calculated to be appro-
quence under study, provided a part of the priate for the primers being used. The re-
nucleotide sequence is known. The inves- action mixture is then heated to the
tigator must know at least some of the optimal temperature for the thermo-sta-
gene sequence in order to synthesize DNA ble DNA polymerase, which generates a
oligonucleotides for use as primers. Two new strand of DNA on the single-strand
primers are required: a forward primer (5′) template, using the nucleotides as build-

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 Applications of PCR

denatures newly formed double strands,
Amplification of minute quantities of DNA
and the single-stranded DNA copies serve as
to detectable levels may significantly af-
templates for subsequent synthesis. The
fect the practice of transfusion medicine.
number of DNA copies doubles with each
In screening donor blood for infectious
cycle, such that after 20 cycles, there is a
agents, PCR has become the procedure of
billionfold amplification of the target DNA.
choice and thus eliminate our reliance on
The amplified DNA may be analyzed by
seroconversion, which occurs well after
agarose gel electrophoresis in the presence
exposure to viruses or other pathogens
of ethidium bromide, which binds to DNA
(see Chapter 28). Blood centers have been
and is visible under ultraviolet light. The
using nucleic acid amplification testing
DNA will be present as a single discrete
(NAT) to identify the presence of HIV and
band equivalent in length to the distance
HCV RNA in donor samples; detection of
between the 5′ ends of the primers. The
West Nile virus RNA has been performed
amplified DNA can also be differentiated by
size using capillary electrophoresis. Alter- as an investigational test. Detection of vi-
natively, the DNA sample can be blotted ral RNA involves three steps: extraction,
onto a membrane and hybridized to a la- amplification, and detection. Extraction
beled, allele-specific probe. This is known may occur following centrifugation steps
as a dot blot and is particularly useful when to concentrate the virus and remove con-
multiple samples are being analyzed for the taminants. Otherwise, RNA extraction can
same polymorphism. occur first followed by capture of viral
Variations of the PCR have been devel- RNA onto a molecule that is immobilized
oped to meet specific needs. For instance, on a solid phase or onto magnetic parti-
“long-distance” PCR, which has a mixture cles in solution (this procedure is referred
of thermostable polymerases, can amplify to as target capture). Once immobilized,
much larger targets (up to 40 kilobases in the impurities may be removed by a series
length) than those typically amplified by of wash steps. Before amplification, viral
conventional PCR (up to 2 kilobases in RNA must be converted to DNA; this is ac-
length). It is even possible to perform PCR complished by the enzyme, reverse tran-
in situ, in tissue and cells. A related tech- scriptase (RT). Amplification of the DNA
nology, the ligase chain reaction (LCR), uses then can occur through multiple interme-
a thermostable DNA ligase instead of a diates. In the case of PCR, the amplified
thermostable DNA polymerase. Rather product is DNA (and is synthesized using
than amplification of a DNA target segment thermostable Taq DNA polymerase),
located between two flanking primers using whereas, in the case of transcription-me-
a DNA polymerase as occurs in PCR, in the diated amplification, the amplified prod-
LCR direct primer ligation occurs with no uct is RNA (and is synthesized using T7
amplification of an intervening DNA seg- RNA polymerase) (see Fig 9-8). Detection
ment. Other PCR variations include multi- of the amplified product can occur by
plex PCR, in which multiple independent capture of the amplified DNA on nitro-
segments of DNA are co-amplified in the cellulose or by enzyme immunoassay or
same reaction, and kinetic PCR, in which chemiluminescence. Currently, NAT for
the amplification product is measured in blood donor screening has been imple-
real time in order to quantitate the amount mented for HIV and hepatitis C; NAT for
of starting nucleic acid. hepatitis B is under development. NAT for

other agents (eg, human T-cell lympho- Restriction Fragment Length Polymorphism
tropic virus, hepatitis A virus, parvovirus Analysis
B19) is not widely available.
PCR is being used for prenatal determi- The unique properties of restriction endo-
nation of many inheritable disorders, such nucleases make analysis of restriction
as sickle cell disease, in evaluating hemolytic fragment length polymorphism (RFLP)
disease of the fetus and newborn, to type suitable for the detection of a DNA poly-
fetal amniocytes,9 to quantitate residual morphism. The changes in nucleotide se-
white cells in filtered blood, and for tracing quence described above (substitution, in-
donor leukocytes in transfusion recipients sertion, deletion) can alter the relative
(chimerism). Long-distance PCR is used for locations of restriction nuclease cutting
cloning, sequencing, and chromosome sites and thus alter the length of DNA
mapping, and reverse transcriptase (RT)- fragments produced. RFLPs are detected
PCR is used for studying gene expression using Southern blotting and probe hy-
and cDNA cloning. LCR has special appli- bridization (see Fig 9-9).
cability in transfusion medicine because of The isolated DNA is cleaved into frag-
its powerful ability to detect genetic vari- ments by digestion with one or more re-
ants. In the field of transplantation, PCR us- striction endonucleases. The DNA frag-
ing sequence-specific oligonucleotide ments are separated by electrophoresis
probes or sequence-specific primers is through agarose gel and then transferred
used to determine HLA types (see Chapter onto a nylon membrane or nitrocellulose
17). paper. Once fixed to a nylon membrane or
nitrocellulose paper, the DNA fragments
are examined by application of a probe,
Restriction Endonucleases
which is a small fragment of DNA whose
The discovery of bacterial restriction endo- nucleotide sequence is complementary to
nucleases provided the key technique for the DNA sequence under study. A probe
DNA analysis. These enzymes, found in may be an artificially manufactured oligo-
different strains of bacteria, protect a bac- nucleotide or may derive from cloned com-
teria cell from viral infection by degrading plementary DNA. The probe is labeled with
viral DNA after it enters the cytoplasm. a radioisotope or another indicator that
Each restriction endonuclease recognizes permits visualization of the targeted DNA
only a single specific nucleotide sequence, restriction fragments and is then allowed
typically consisting of four to six nucleo- to hybridize with the Southern blot. Un-
tides. These enzymes cleave the DNA bound excess probe is washed off, and hy-
strand wherever the recognized sequence bridized DNA is visualized as one or more
occurs, generating a number of DNA frag- bands of specific size, dictated by the spe-
ments whose length depends upon the cific nucleotide sequence. If several indi-
number and location of cleavage sites in viduals are analyzed for polymorphism,
the original strand. Many endonucleases several different banding patterns may be
have been purified from different species observed.
of bacteria; the name of each enzyme re- RFLP analysis has been used in gene
flects its host bacterium, eg, Eco RI is iso- mapping and analysis, linkage analysis,
lated from Escherichia coli, Hind III is characterization of HLA genes in transplan-
from Hemophilus influenzae, and Hpa I is tation, paternity testing, and forensic sci-
from Hemophilus parainfluenzae. ence.

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.
Figure 9-8. Transcription-mediated amplification cycle.

Figure 9-9. Southern blotting: a technique for the detection of polymorphism by gel-transfer and hy-
bridization with known probes.
DNA Profiling variation between individuals that the

chances are very low that the same num-
Regions of DNA that show great allelic vari- bers of repeats will be shared by two indi-
ability (“minisatellites” and “microsatel- viduals, even if related. The VNTR and/or
lites”) can be studied by the application of STR patterns observed at four to eight dif-
RFLP mapping and/or PCR analysis (a pro- ferent loci may be unique for an individ-
cess sometimes called DNA profiling, DNA ual and thus constitute a profile or “fin-
typing, or DNA fingerprinting). Minisatel- gerprint” that identifies his or her DNA.
lites or variable number of tandem repeat When DNA profiling was developed,
(VNTR) loci consist of tandem repeats of a testing was performed by RFLP analysis.
medium-sized (6-100 base-pair) sequence, DNA profiling is now increasingly done by
whereas microsatellites or short tandem amplification of selected, informative
repeat (STR) loci consist of tandem re- VNTR and/or STR loci using locus-specific
peats of a short (typically four base pair) oligonucleotide primers, followed by mea-
sequence. These regions are almost al- surement of the size of the PCR products
ways found in the noncoding regions of produced. PCR products can be separated
DNA. Variability stems from differences in by size by electrophoresis through poly-
the number of repeat units contained acrylamide gel and detected by silver stain-
within the fragments. There is so much ing, or, if the PCR products incorporate a

fluorescent tag, by fluorescent detection successfully incorporated the recombinant

systems including those designed for auto- vector will survive to form colonies or
mated DNA sequencing. Comparison of the clones. Each individual vector potentially
VNTR and STR PCR products with a stan- contains a different cDNA sequence. The
dard-size ladder distinguishes the alleles sum of bacterial clones harboring recombi-
present in the sample. nant vectors is called a DNA library. Librar-
DNA profiling is a technique that is ex- ies can be obtained from many commercial
tremely powerful for identifying the source sources or can be produced by the individ-
of human DNA; therefore, it has applica- ual investigator. The library can be probed
tions in forensic and paternity testing, as well through a technique similar to Southern
as in the documentation of chimerism, which blotting, with an oligonucleotide probe
is of special importance in monitoring based on part of a known sequence. Posi-
allogeneic hematopoietic transplantation. 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
DNA Cloning
characterization.
PCR may also be used for analysis of mRNA, The ability to insert genes into the
which is an especially useful source of ge- genomes of virtually any organism, includ-
netic material because only the exons of ing bacteria, plants, invertebrates (such as
the gene are present. By a modification of insects), and vertebrates (such as mam-
PCR called RT-PCR, the single-stranded mals), permits not only gene characteriza-
mRNA is converted to double-stranded tion but also genetic engineering, including
DNA. The enzyme RT is used to generate the production of recombinant proteins (see
a single strand of DNA, which serves as a below) and gene therapy. Although still in
template for a second strand generated by the developmental stages, gene therapy
DNA polymerase. The product is comple- promises to have a role in the management
mentary DNA (cDNA) and it is the DNA of disorders as diverse as inherited genetic
molecule of choice for cloning and se- diseases, human immunodeficiency virus,
quencing. and cancer, and in the development of
In gene cloning, the DNA containing the novel vaccines.10,11
gene of interest is inserted into a vector,
which is a self-replicating genetic element
DNA Sequencing
such as a virus (eg, the bacteriophage lambda
gt11) or a plasmid. Plasmids are small cir- A major worldwide scientific effort called
cular molecules of double-stranded DNA the Human Genome Project has obtained
that occur naturally in bacteria and typi- the complete nucleotide sequence of the
cally confer antibiotic resistance. After the human genome as well as the genomes of
gene has been inserted into the DNA of the several other key organisms. The initiative
vector, the recombinant DNA can be intro- also improved DNA sequencing technol-
duced into a bacterial host where it under- ogy. Realization of both goals has had a
goes replication. Because many vectors positive impact on transfusion practice.
carry genes for antibiotic resistance, this The identification of all human genes12,13
characteristic can be exploited by growing provides a complete blueprint of the pro-
the host bacteria in the presence of the ap- teins that are relevant in transfusion med-
propriate antibiotic; only bacteria that have icine. In turn, this information has helped

in the development of recombinant pro- tissue for all of the genes on the micro-
teins for use as transfusion components and array. Also, microarrays can be used for
in-vitro test reagents. It also plays a role in comparative genomics and genotyping, an
clarifying the disorders that afflict trans- application for blood groups.
fusion recipients.
Advances in DNA sequencing have taken
Recombinant Proteins
the field a long way from the cumbersome
manual techniques common in research The technology to make recombinant
laboratories until recently.14 Automated proteins includes in-vitro systems in bac-
DNA sequencers using laser detection of teria, yeast, insect cells, and mammalian
fluorescently labeled sequencing products cells, as well as in-vivo systems involving
detect all four nucleotide bases in a single transgenic plants and animals.16 First, a
lane on polyacrylamide gel and can be op- source of DNA corresponding in nucleic
timized for specialty applications such as acid sequence to the desired protein is
heterozygote detection and sizing of PCR prepared, typically by cloning the cDNA
fragments. Automated DNA sequencers us- and ligating it into a suitable expression
ing capillary electrophoresis are especially vector. Then, the expression vector con-
useful for the rapid sequencing of short taining the DNA of interest is transfected
DNA templates. DNA sequence can also be into the host cell, and the DNA of interest
obtained using mass spectrometry. Auto- is transcribed under the control of the
mated sequencers will become increasingly vector promoter. Next, the resulting mRNA
common in clinical laboratories as this is translated into protein by the host cell.
technology evolves. If it can be made Posttranslational modifications such as
cost-effective for routine use, then DNA se- the addition of carbohydrates to the new
quencing could become a routine genotyp- protein will be carried out by the host cell.
ing method. If specific posttranslational modifications
required for the new protein’s function can-
not be carried out by the host cell, then it
DNA Microarrays
may be necessary to endow the host cell
The complexity of the human genome re- with additional capabilities; for example, by
quires that the differential expression of cotransfection with the cDNA for a specific
multiple genes be analyzed at once to un- enzyme. In some cases, posttranslational
derstand normal biologic processes as well modification may not be crucial for a re-
as changes in diseases. A powerful tech- combinant protein to be effective; for
nique to accomplish this goal is DNA instance, granulocyte colony-stimulating
15
microarrays or gene chips. In this method, factor (G-CSF) is produced in a nonglyco-
tens of thousands of separate DNA mole- sylated form in E. coli (filgrastim) and in a
cules are spotted or synthesized on a small glycosylated form in yeast (lenograstim).
area of a solid support, often a glass slide. Recombinant proteins are finding multi-
The DNA can be generated by PCR or oligo- ple uses in transfusion medicine, as thera-
nucleotide synthesis. This microarray is peutic agents and vaccine components, in
then probed, in a process analogous to component preparation, in virus diagnosis,
Southern blotting, using cDNA created and in serologic testing. Recombinant hu-
from the total mRNA expressed at a given man erythropoietin,17 G-CSF and GM-CSF,18
time by a cell or tissue. The result is a pic- interferon-alpha, interleukin-2, interleu-
19 20 21
ture of the gene expression profile of the kin-11, and Factors VIII, IX, VII, and VIIa

are all available and finding clinical accep- Recombinant proteins corresponding to
tance. For instance, recombinant erythro- proteins from clinically relevant viruses,
poietin can be used to increase red cell pro- bacteria, and parasites, some of which may
duction in anemic patients before surgery, be transmitted by blood transfusion, may
reducing the need for allogeneic blood.22 It be used as vaccine components29 and as
can also be used to increase the amount of antigens in test kits for the detection of an-
autologous red cells that can be withdrawn tibodies. Cells transfected with appropriate
before surgery from non-anemic patients.23 vectors can be induced to express recombi-
Moreover, it has revolutionized the man- nant proteins on the membrane surface
agement of renal transplant candidates and, as such, may become useful as geneti-
whose kidneys are too impaired to produce cally engineered reagent cells for in-vitro
endogenous erythropoietin. testing.
Recombinant human thrombopoietin
may be of value in augmenting platelet
Gene Therapy
yields from apheresis donors but is unlikely
to significantly reduce the need for platelet Gene therapy refers to the introduction of
transfusions when given to thrombo- nonself genetic material into cells to treat
cytopenic patients.24 In the coagulation or prevent disease. At present, gene ther-
arena, recombinant proteins such as pro- apy is restricted to somatic cells because
tein C, antithrombin, and hirudin are ap- of ethical concerns about the transfer of
proved by the Food and Drug Administra- genes to germ-line cells. More than 3500
tion for use, and tissue factor pathway patients have been administered gene
inhibitor, Factor XIII, and von Willebrand therapy in clinical trials,30 but results over-
factor appear promising. Recombinant all have been disappointing largely due to
myeloid growth factors such as G-CSF are problems with delivery (transfection) of
used to enhance yields of progenitor cells the genetic material (transgene) into cells
during apheresis and to support patients and the limited lifespan of the success-
following chemotherapy and hemato- fully transfected cells.
poietic transplantation.25 There are different types of vectors (gene-
Recombinant proteins can be used as delivery vehicles) that deliver genes to cells,
transfusion components.26 Recombinant including viral vectors (retrovirus, adeno-
human hemoglobin has been produced in virus, adeno-associated virus, vaccinia, her-
a number of in-vitro expression systems pes simplex), naked DNA, and modified DNA.
and in vivo in transgenic swine and may be Genetic material can also be transferred to
useful as a noninfectious blood substitute.27 cells by physical means such as electro-
Recombinant human serum albumin has poration (use of an electric field). As meth-
been produced in yeast. Alphagalactosi- ods for gene delivery improve, it is antici-
dase, an enzyme that is capable of convert- pated that the benefits of gene therapy will
ing group B cells into group O cells, has become more apparent.
been produced in a recombinant form that Gene therapy can be used to replace a
can modify group B units for transfusion to defective gene, leading to increased pro-
group A and O recipients.28 These recombi- duction of a specific protein as in the re-
nant proteins and other products under de- placement of Factor VIII or IX for patients
velopment will undoubtedly affect the vari- with hemophilia,31 or it can be used to
ety of transfusion components that will downregulate (reduce) expression of an un-
become available in the future. desirable gene. The latter can be accom-

plished by the introduction of DNA se- 2. Li Y, Camp S, Taylor P. Tissue-specific expres-

sion and alternative mRNA processing of the
quences (antisense oligonucleotides)
mammalian acetylcholinesterase gene. J Biol
corresponding to the antisense strand of Chem 1993;268:5790-7.
the mRNA, which then interferes with 3. Le Van Kim C, Mitjavila MT, Clerget M, et al.
translation of the mRNA into protein. An ubiquitous isoform of glycophorin C is
produced by alternative splicing. Nucleic Ac-
ids Res 1990;18:3076.
Protein and RNA Targeted Inactivation 4. Lee S, Wu X, Reid M, et al. Molecular basis of
the Kell (K1) phenotype. Blood 1995;85:912-
More recently, novel pharmacologic agents
16.
have been developed that interfere with 5. Lublin DM, Mallinson G, Poole J, et al. Molec-
specific molecules produced by cancer ular basis of reduced or absent expression of
cells or infectious particles. One such ex- decay-accelerating factor in Cromer blood
group phenotypes. Blood 1994;84:1276-82.
ample is imatinib mesylate (Gleevec,
6. Kudo S, Fukuda M. Structural organization of
Novartis Pharmaceuticals, East Hanover, glycophorin A and B genes: Glycophorin B
NJ), which binds specifically to the Bcr- gene evolved by homologous recombination
Abl protein and blocks its tyrosine kinase at Alu repeat sequences. Proc Natl Acad Sci
U S A 1989;86:4619-23.
activity in malignant white cells. It specif-
7. Lubin DM, Thompson ES, Green AM, et al.
ically blocks the binding site for adeno- Dr(a–) polymorphism of decay accelerating
sine triphosphate on the kinase, thus factor. Biochemical, functional, and molecu-
inhibiting its ability to phosphorylate lar characterization and production of allele-
specific transfection. J Clin Invest 1991;87:
intracellular proteins.32 The inactivation 1945-52.
prevents Bcr-Abl-induced malignant cell 8. Erlich HA. Principles and applications of the
proliferation and anti-apoptosis. Normal polymerase chain reaction. Rev Immuno-
genet 1999;1:127-34.
kinase signaling pathways are largely un-
9. Bennett PR, Le Van Kim C, Colin Y, et al. Pre-
affected. natal determination of fetal RhD type by DNA
Another promising therapy is RNA inter- amplification. N Engl J Med 1993;329:607-10.
ference, which has its historical roots as a 10. Friedmann T. Overcoming the obstacles to
gene therapy. Sci Am 1997;276:80-5.
research tool used to characterize the func-
11. Hillyer CD, Klein HG. Immunotherapy and
tion of known genes. RNA interference is gene transfer in the treatment of the oncol-
based on an antiviral mechanism in which ogy patient: Role of transfusion medicine.
dsRNA is delivered to a cell and is subse- Transfus Med Rev 1996;10:1-14.
12. International Human Genome Sequencing
quently processed into small (21-25 bp) in-
Consortium. Initial sequencing and analysis
terfering RNA (siRNA) molecules. The siRNA of the human genome. Nature 2001;409:860-
molecules silence the expression of a target 921.
gene in a sequence-specific manner. More 13. Venter JC, Adams MD, Myers EW, et al. The
sequence of the human genome. Science
important, RNA interference has potential 2001;291:1304-51.
as a therapeutic strategy to silence cancer-re- 14. Griffin HG, Griffin AM. DNA sequencing. Re-
lated genes or infectious diseases like viral cent innovations and future trends. Appl Bio-
hepatitis.33 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,
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1. Tournamille C, Colin Y, Cartron JP, Le Van animal bioreactors. Transfus Med Rev 1996;
Kim C. Disruption of a GATA motif in the 10:131-43.
Duffy gene promoter abolishes erythroid 17. Cazzola M, Mercuriali F, Brugnara C. Use of
gene expression in Duffy-negative individu- recombinant human erythropoietin outside
als. Nat Genet 1995;10:224-8. the setting of uremia. Blood 1997;89:4248-67.

18. Ganser A, Karthaus M. Clinical use of hema- ands on transfusion medicine. Transfus Med
topoietic growth factors. Curr Opin Oncol 1996; Rev 1997;11:243-55.
8:265-9. 35. Barbara JA, Garson JA. Polymerase chain re-
19. VanAken WG. The potential impact of recom- action and transfusion microbiology. Vox
binant factor VIII on hemophilia care and the Sang 1993;64:73-81.
demand for blood and blood products. Trans- 36. Power EG. RAPD typing in microbiology—a
fus Med Rev 1997;11:6-14. technical review. J Hosp Infect 1996;34:247-
20. White GC II, Beebe A, Nielsen B. Recombi- 65.
nant factor IX. Thromb Haemost 1997;78: 37. Larsen SA, Steiner BM, Rudolph AH, Weiss JB.
261-5. DNA probes and PCR for diagnosis of para-
21. Lusher JM. Recombinant factor VIIa (Novo- sitic infections. Clin Microbiol Rev 1995;8:1-
Seven) in the treatment of internal bleeding 21.
in patients with factor VIII and IX inhibitors. 38. Weiss JB. DNA probes and PCR for diagnosis
Haemostasis 1996;26(Suppl 1):124-30. of parasitic infections. Clin Microbiol Rev
22. Braga M, Gianotti L, Gentilini O, et al. Ery- 1995;8:113-30.
thropoietic response induced by recombi-
39. Majolino I, Cavallaro AM, Scime R. Peripheral
nant human erythropoietin in anemic cancer
blood stem cells for allogeneic transplanta-
patients candidate to major abdominal sur-
tion. Bone Marrow Transplant 1996;18(Suppl
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2):171-4.
23. Cazenave JP, Irrmann C, Waller C, et al. Epo-
40. Gretch DR. Diagnostic tests for hepatitis C.
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Hepatology 1997;26:43S-7S.
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scheduled for orthopaedic or cardiovascular 41. Pena SD, Prado VF, Epplen JT. DNA diagnosis
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25. Ketley NJ, Newland AC. Haemopoietic growth cally important microorganisms by use of PCR.
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27. Kumar R. Recombinant hemoglobins as 44. Hyland CA, Wolter LC, Saul A. Identification
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28. Lenny LL, Hurst R, Zhu A, et al. Multiple-unit 1995;9:289-301.
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ically converted from group B to group O: Re-
port on the end of phase 1 trials. Transfusion
1995;35:899-902.
29. Ellis RW. The new generation of recombinant Suggested Reading
viral subunit vaccines. Curr Opin Biotechnol
1996;7:646-52. Denomme G, Lomas-Francis C, Storry RJ, Reid ME.
30. Mountain A. Gene therapy: The first decade. Approaches to molecular blood group genotyping
Trends Biotechnol 2000;18:119-28. and their applications. In: Stowell C, Dzid W, eds.
31. Kaufman RJ. Advances toward gene therapy Emerging technologies in transfusion medicine.
for hemophilia at the millennium. Hum Gene Bethesda, MD: AABB Press, 2003:95-129.
Therapy 1999;10:2091-107.
32. Druker BJ, Tamura S, Buchdunger E, et al. Ef- Garratty G, ed. Applications of molecular biology
fects of a selective inhibitor of the Abl tyro- to blood transfusion medicine. Bethesda, MD:
sine kinase on the growth of Bcr-Abl positive AABB, 1997.
cells. Nat Med 1996;2:561-6. Lewin B. Genes VII. Oxford: Oxford University Press,
33. Radhakrishnan SK, Layden TJ, Gartel AL. RNA 2000.
interference as a new strategy against viral
hepatitis. Virology 2004;323:173-81. Sheffield WP. Concepts and techniques in molecu-
34. Farese AM, Schiffer CA, MacVittie TJ. The im- lar biology—an overview. Transfus Med Rev 1997;
pact of thrombopoietin and related mpl-lig- 11:209-23.

Appendix 9-1. Molecular Techniques in Transfusion Medicine
Technique Applications Examples References
PCR Infectious disease Viruses, bacteria, 35

testing parasites 36
37, 38
Polymorphism detec- HLA, blood group 1, 39
tion antigens
Prenatal detection Rh 9
Recombinant proteins/ Therapy Erythropoietin, 17, 18, 34

DNA cloning thrombopoietin,
G-CSF
Apheresis G-CSF 39
Infectious disease Viral testing 40
testing
Recombinant Coagulation factors 19
components
Hemoglobin 27
Component processing Alpha-galactosidase 28
Vaccine production Hepatitis, malaria, HIV 27-29
DNA profiling Human identification Chimerism, forensic 41

science
Bacterial identification Bacterial typing 42
DNA sequencing Polymorphism detec- HLA 14

tion, heterozygote
detection
Phage display/ Monoclonal antibody Anti-D 43

repertoire cloning production
RFLP Polymorphism HLA, blood group 44

detection 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
L
ANDSTEINER’S DISCOVERY OF of a cell. This nuclear material is called
the ABO blood group system dem- chromatin and is primarily made up of DNA
onstrated that human blood ex- (see Chapter 9). When a cell divides, the
pressed inheritable polymorphic structures. chromatin loses its homogenous appear-
Shortly after the discovery of the ABO sys- ance and forms a number of rod-shaped
tem, red cells proved to be an easy and ac- organelles called chromosomes. Encoded 10
cessible means to test for blood group in the chromatin or chromosomal DNA
polymorphisms in individuals of any age. are units of genetic information called
As more blood group antigens were de- genes. The genes are arranged in a spe-
scribed, blood group phenotyping pro- cific order along a chromosome with the
vided a wealth of information about the precise gene location known as the locus.
polymorphic structures expressed on pro-
teins, glycoproteins and glycolipids on
red cells, and the genetic basis for their Chromosomes
inheritance. 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-
Basic Principles half of each pair inherited from each par-
Inheritance of transmissible characteris- ent). Twenty-two of the pairs are alike in
tics or “traits,” including blood group an- both males and females and are called
tigens, forms the basis of the science of autosomes; the sex chromosomes, XX in
genetics. The genetic material that deter- females and XY in males, are the remain-
mines each trait is found in the nucleus 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- Figure 10-1. Diagram of Giemsa-stained normal
human chromosome 7. With increased resolution
activation is randomly determined but (left to right), finer degrees of banding are evi-
once the decision is made, the choice is dent. Bands are numbered outward from the
permanent. This process is termed lyoni- centromere (c), which divides the chromosome
into p and q arms.1
zation.
Mitosis and Meiosis mation of the original parent cell (Fig

Cells must replicate their chromosomes 10-2).
as they divide so that each daughter cell Meiosis occurs only in primordial cells
receives the full complement of genetic destined to mature into haploid gametes.
information. Cell division is of two kinds: Diploid cells that undergo meiosis give rise
mitosis and meiosis. Mitosis is the pro- to haploid gametes (sperm and egg cells with
cess whereby the body grows or replaces only 23 chromosomes). Hence, somatic
dead or injured somatic cells. This process cells divide by mitosis, giving rise to diploid
consists of five stages: prophase, pro- cells that have a 2N chromosome comple-
metaphase, metaphase, anaphase, and ment. Gametes formed following meiosis
telophase. The end result after cytokinesis are haploid, with a 1N chromosome com-
is two complete daughter cells, each with plement. It is during meiosis that genetic
a nucleus containing all the genetic infor- diversity occurs. One type of diversity is the

Chapter 10: Blood Group Genetics 225
Figure 10-2. The two types of cell division are mitosis and meiosis.
independent assortment of maternal and leles. The ISBT terminology distinguishes

paternal homologous chromosomes that between the alleles for blood group anti-
occurs during division I of meiosis. By the gens (ie, the genetic polymorphisms) and
end of meiosis, gametes are formed with an the antigens that they encode. For exam-
assortment of maternal and paternal chro- ple, the major antigens of the ABO system
mosomes. The other type of diversity oc- are A, B, and O, yet the alleles are A1, B1,
curs when homologous pairs of chromo- and O1. In the Kell system, two alleles, K
somes line up during the first prophase. and k, determine the K and k antigens, re-
The homologous chromosomes genetically spectively. Individuals who have identical
recombine in a process called chromo- alleles at a given locus on both chromo-
somal crossing over (see below). Therefore, somes are homozygous for the allele (eg,
not only are the chromosomes shuffled A1/A1 or K/K or k/k). In the heterozygous
during meiosis, but also portions of chro- condition, the alleles present at the par-
mosomes are recombined to shuffle the ticular locus on each chromosome are
genes of an individual chromosome. nonidentical (eg, A1/O1 or A1/B1 or K/k). Ta-
ble 10-1 summarizes genotypes and chro-
mosomal locations for the 29 blood group
antigen systems.
Genetics and Heredity Individuals who are homozygous for an
Alleles allele in some blood group systems may
Alternative forms of genes, any one of have more antigen expressed on their red
which may occupy a single locus on ho- cells than persons who are heterozygous for
mologous chromosomes, are called al- that allele. For example, red cells from a

Table 10-1. Chromosomal Locations of Human Blood Group System Genes*
Gene(s)
ISBT Designation
System ISBT No. Symbol (ISGN) Location
ABO 001 ABO ABO 9q34.2

MNS 002 MNS GYPA, GYPB, GYPE 4q28.2-q31.1
P 003 P1 P1 22q11.2-qter
Rh 004 RH RHD, RHCE 1p36.13-p34.3
Lutheran 005 LU 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 XG Xp22.32
Scianna 013 SC SC 1p34
Dombrock 014 DO DO 12p12.3
Colton 015 CO AQP1 7p14
Landsteiner-Wiener 016 LW LW 19p13.3
Chido/Rodgers 017 CH/RG C4A, C4B 6p21.3
H 018 H FUT1 19q13.3
Kx 019 XK 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
1 2 3
*Modified from Zelinski ; Garratty et al ; and Denomme et al.

person whose phenotype is Jk(a+b–) have a Mutations may result in the creation of
“double dose” of the Jka allele and, as a re- new polymorphisms associated with the
sult, express more Jka antigen on the red altered gene. Figure 10-3 illustrates how
cell surface than an individual whose phe- mutations in the genes that code for the
notype is Jk(a+b+) (a single dose of the Jka MNS blood group antigens have resulted
allele). The difference in amount of antigen in the creation of various low-incidence
expressed on the red cell membrane be- MNS system antigens.
tween a homozygous and a heterozygous
phenotype can often be detected serologi-
cally and is termed the dosage effect. For Allele (Gene) Frequencies
example, some anti-Jka sera may give the
following pattern of reactivity: 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-
Phenotype of RBC Donor
cus within a given population at a given
Antibody Jk(a+b–) Jk(a+b+)
time. This frequency can be calculated
Anti-Jka 3+ 2+
from phenotype frequencies observed
within a population. The sum of allele fre-
Dosage effect is not seen with all blood
quencies at a given locus must equal 1.
group antigens or even with all antibodies
of a given specificity. Antibodies that typi-
cally demonstrate dosage include those in
the Rh, MNS, Kidd, and Duffy blood group The Hardy-Weinberg Law
systems. The Hardy-Weinberg law is based on the
Alleles arise by genetic changes at the assumption that genotypes are distrib-
DNA level and may result in a different ex- uted in proportion to the frequencies of
pressed phenotype. Some of the changes individual alleles in a population and will
found among blood group alleles may re- remain constant from generation to gen-
sult from: eration if the processes of mutation, mi-
■ Missense mutations (a single nucle- gration, etc do not occur. For example, the
otide substitution leading to the Kidd blood group system is basically a
coding of a different amino acid) two-allele system (Jka and Jkb; the silent Jk
■ Nonsense mutations (a single nucle- allele is extremely rare) that can be used
otide substitution leading to the to illustrate the calculation of gene fre-
coding of a stop codon) quencies. This calculation uses the Hardy-
■ Mutations in motifs involved in tran- Weinberg equation for a two-allele sys-
scription tem. If p is the frequency of the Jka allele
■ Mutations leading to alternate RNA and q is the frequency of the Jkb allele,
splicing then the frequencies of the three combi-
■ Deletion of a gene, exon, or nucleo- nations of alleles can be represented by
tide(s) the equation p2 + 2pq + q2 = 1 where:
■ Insertion of an exon or nucleotide(s) a
p = frequency of Jk allele
■ Alternate transcription initiation site
q = frequency of Jkb allele
■ Chromosome translocation p
2
= frequency of
a
Jk /Jk
a
genotype
■ Gene conversion or recombination 2pq = frequency of
a
Jk /Jk
b
genotype
2 b b
■ Crossing over q = frequency of Jk /Jk genotype

Figure 10-3. How crossover, recombination, and nucleotide substitution (nt subs) result in variations
of genes producing glycophorin A and B. The changes are associated with the presence of various
low-incidence MNS system antigens. (Modified from Reid.4 )
Using the observation that 77% of indi- p+q = 1

a
viduals within a population express Jk anti- p = 1–q
gen on their red cells, then:
p = 1 – 0.48
2
p + 2pq = frequency of p = 0.52 (allele frequency of Jka)
persons who
Once the allele frequencies have been
are Jk(a+) and
calculated, the number of Jk(b+) individu-
carry the Jka allele als (both homozygous and heterozygous)
= 0.77 can be calculated as:
2 2
q = 1 – (p + 2pq) = frequency of
persons who 2pq + q2 = frequency of Jk(b+)
are Jk(a–) = 2 (0.52 × 0.48) + (0.48)2
(homozygous for = 0.73
the Jkb allele)
If both anti-Jka and anti-Jkb sera are avail-
q2 = 1 – 0.77 = 0.23
able, allele frequencies can be determined
q = 0.23 more easily by direct counting. As shown in
q = 0.48 (allele Table 10-2, the random sample of 100 peo-
b a b
frequency of Jk ) ple tested for Jk and Jk antigens possess a
total of 200 alleles at the Jk locus (each per-
Because the sum of frequencies of both son inherits two alleles, one from each par-
alleles must equal 1.00, ent). There are two Jka alleles inherited by

Table 10-2. Gene Frequencies in the Kidd Blood Group System Calculated Using
Direct Counting Method*
Gene Frequencies (%)

No. of No. of
Phenotype Individuals Kidd Genes Jka Jk>
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 Jk allele.
each of the 28 individuals who phenotype pass to different gametes. In blood group
as Jk(a+b–), for a total of 56 alleles. There genetics, this can be illustrated by the in-
are 49 Jka alleles in the individuals who are heritance of the ABO alleles (Fig 10-4). In
Jk(a+b+), for a total of 105 alleles or a gene this example, the members of the paren-
frequency of 0.52 (105 ÷ 200). The fre- tal generation (P1) are homozygous for an
quency of Jkb is 95 ÷ 200 = 0.48. A allele and an O allele. All members of
The Hardy-Weinberg law is generally the first filial generation (F1) will be het-
used to calculate allele and genotype fre- erozygous (A/O) but will still express the
quencies in a population when the fre- blood group A antigen (O is a silent al-
quency of one genetic trait (eg, antigen lele). If an F1 individual mates with an A/O
phenotype) is known. However, it relies on genotypic individual, the resulting prog-
certain assumptions: no mutation; no mi- eny [termed the second filial generation:
gration (in or out) of the population; lack of (F2)] will blood group as A (either hetero-
selective advantage/disadvantage of a par- zygous or homozygous) or group O. If the
ticular trait; and a large enough population F1 individual mated with a heterozygous
so that chance alone cannot alter an allele group B person (B/O), the offspring could
frequency. If all of these conditions are have the blood group A, B, AB, or O.
present, the gene pool is in equilibrium and
allele frequencies will not change from one Independent Assortment
generation to the next. If these assumptions
do not apply, changes in allele frequencies Mendel’s law of independent assortment
may occur over a few generations and can states that genes determining various
explain many of the differences in allele traits are inherited independently from
frequencies between populations. each other. For example, if one parent is
group A (homozygous for A) and K+k+,
and the other parent is group B (homozy-
Segregation gous for B) and K–k+ (homozygous for k),
The term segregation refers to the con- all the F1 children would be group AB; half
cept that the two members of a single would be K+k+ and half K–k+ (Fig 10-5). A
gene pair (alleles) are never found in the second filial generation could manifest
same gamete but always segregate and any of the following phenotypes: group A,

Figure 10-4. Mendel’s law of independent segregation demonstrated by the inheritance of ABO genes.
K+k+; group AB, K+k+; group B, K+k+; matids is termed crossing over (Fig 10-6).
group A, K–k+; group AB, K–k+; group B, Genes close together on a chromosome
K–k+. The proportions would be tend to be transmitted together during
1:2:1:1:2:1. these recombinations and their alleles,
Independent assortment applies if the therefore, do not segregate independently.
genes are on different chromosomes or on Sometimes, the linkage is very tight so that
distant portions of the same chromosome. recombination rarely occurs. The strength
One exception to this rule is that closely of linkage can be used as a unit of mea-
linked genes on the same chromosome do surement to estimate the distance between
not sort independently but often remain to- different loci. This type of analysis can
gether from one generation to another. This help in identifying, mapping, and diag-
observation is termed linkage. nosing the genes responsible for certain
inherited diseases.
Linkage The demonstration of linkage between
Genetic linkage is defined as the tendency the gene controlling ABH secretion (Se) and
for alleles close together on the same the expression of Lutheran blood group an-
chromosome to be transmitted together. tigens (Lua, Lub) was the first recognized ex-
5
During mitosis, each pair of homologous ample of autosomal linkage in humans.
chromosomes undergoes a series of re- Analysis of this relationship also provided
combinations. The resultant reciprocal the first evidence in humans of recombina-
exchange of segments between the chro- tion due to crossing-over and helped dem-

Figure 10-5. Mendel’s law of independent assortment demonstrated by the inheritance of ABO and Kell
genes.
onstrate that crossing-over occurs more be the product of the frequencies of the in-
often in females than in males. dividual alleles. However, the frequencies
observed are not those expected:
Linkage Disequilibrium
Expected Observed
When two loci are closely linked, alleles at Frequency Frequency
those loci tend to be inherited together MS = 0.53 × 0.33 = 0.17 0.24
and are said to constitute a haplotype. Ms = 0.53 × 0.67 = 0.36 0.28
Again, the close linkage between the loci NS = 0.47 × 0.33 = 0.16 0.08
controlling expression of M and N and of Ns = 0.47 × 0.67 = 0.31 0.40
S and s is an example of linkage disequi- Total 1.00 1.00
librium. The approximate frequencies of
each of the four alleles are: This is an example of linkage disequilib-
rium: the tendency of specific combina-
M = 0.53 S = 0.33 tions of alleles at two or more linked loci
N = 0.47 s = 0.67 to be inherited together more frequently
than would be expected by chance.
If the alleles of the M, N, S, and s anti- Another commonly cited example of
gens segregated independently, the ex- linkage disequilibrium occurs in the HLA
pected frequency of each haplotype would system (see Chapter 17). The combination

Figure 10-6. Very closely linked loci are rarely affected by crossing over so that alleles of those loci
are inherited together (N and S, M and s in the example shown). Loci on the same chromosome that
are not closely linked (the Ss locus and the Zz locus shown) can demonstrate crossing over. Crossing
over is one kind of recombination. It occurs between homologous chromatids during meiosis, resulting
in segregation of alleles on the same chromosome.
of HLA-A1 with HLA-B8 occurs in some then the frequencies of the combination
populations approximately five times more should be the product of the frequency of
frequently than would be expected based each allele:
on the frequencies of the individual alleles,
an example of positive linkage disequilib- YZ 0.53 × 0.3 = 0.16
rium. Linkage disequilibrium may be posi- Yz 0.53 × 0.7 = 0.37
tive or negative, and it may indicate a selec- yZ 0.47 × 0.3 = 0.14
tive advantage of one haplotype over another.
yz 0.47 × 0.7 = 0.33
Over many generations, the alleles of even
Total 1.00
closely linked loci may reach equilibrium
and associate according to their individual In such a case, the alleles are in linkage
frequencies in the population. equilibrium because they are inherited
When there is linkage equilibrium, the independently.
alleles at two loci associate with frequen-
cies that reflect their individual frequencies.
For example, if alleles in the population
have the following frequencies: Patterns of Inheritance
Dominant and Recessive Traits
Y 0.53 Z 0.30 Traits are the observed expression of genes.
y 0.47 z 0.70 A trait that is observable when the deter-
Total 1.00 1.00 mining allele is present is called domi-

nant; when different alleles on homologous (frequency: <1:10,000) and display the trait,
chromosomes each produce an observable the parents are often blood relatives [Fig
trait, the term co-dominant is used. A re- 10-7(B)]. Recessive traits may remain unex-
cessive trait is observable only when the pressed for many generations, so that the
allele is not paired with a dominant allele appearance of a rare recessive trait does not
(two recessive alleles are present). De- necessarily imply consanguinity, although
scribing traits as dominant and recessive family ethnicity and geographic origin may
depends on the method used to detect gene be informative. A higher frequency for a re-
products. Observable traits are called phe- cessive allele indicates the less likelihood of
notypes. Thus, blood group antigen typing consanguinity. Traits inherited in either
using antisera identifies a phenotype. In autosomal dominant or autosomal reces-
some cases, genotypes may be inferred sive fashion typically occur with equal fre-
from the phenotype, especially when quency in males and females.
family studies are performed, but geno-
types are not usually determined directly Sex-Linked Dominant or Co-dominant
by typing red cells. Trait
A male always receives his single X chro-
Autosomal Dominant Trait mosome from his mother. The predomi-
An autosomal dominant trait shows a nant feature of X-linked inheritance, of
characteristic pattern of inheritance. The either dominant or recessive traits, is ab-
trait appears whenever an individual pos- sence of male-to-male (father-to-son)
sesses the allele. Figure 10-7(A) presents a transmission of the trait. Because a male
pedigree showing the pattern of auto- passes his X chromosome to all his dau-
somal dominant inheritance. Typically, ghters, all daughters of a man expressing
each person with the trait has at least one a dominant X-linked trait also possess the
parent with the trait, continuing back- allele and the trait. If a woman expresses a
ward through generations. dominant trait, but is heterozygous, each
child, male or female, has a 50% chance of
Autosomal Recessive Trait inheriting that allele and thus the trait
[Fig 10-7(C)]. If the mother possesses the
People who exhibit a recessive trait are
determining allele on both X chromo-
homozygous for the encoding allele. Their
somes, all her children will express the
parents may or may not express the trait.
trait. X-linked dominant traits tend to ap-
However, parents who lack the trait must
pear in each generation of a kindred, but
be carriers, ie, heterozygotes for an allele
without male-to-male transmission. A
whose presence is not phenotypically
sex-linked dominant trait of interest in
apparent.
blood group genetics is the Xg blood
If the frequency of the variant allele is
group system.
low, the recessive trait will be rare and gen-
erally will occur only in members of one
generation, not in preceding or successive Sex-Linked Recessive Trait
generations unless consanguineous mating Hemophilia A provides a classic example
occurs. Blood relatives are more likely to of X-linked recessive inheritance [Fig
carry the same rare allele than unrelated 10-7(D)]. Males inherit the trait from car-
persons from a random population. When rier mothers or, very rarely, from a mother
offspring are homozygous for a rare allele who is homozygous for the allele and

Figure 10-7. Four pedigrees showing different patterns of inheritance.
therefore expresses the trait. In the mat- males will be seen because the likelihood
ing of a normal male and a carrier female, increases that an affected male will mate
one half of the male offspring are affected with a carrier female and produce daugh-
and one half of the females are carriers. ters, half of whom will be homozygous for
Among the children of an affected male the abnormal allele.
and a female who lacks the determining
allele, all sons are normal and all daugh- Blood Group Co-dominant Traits
ters are carriers. Blood group antigens, as a rule, are ex-
If the recessive X-linked allele is rare, the pressed as co-dominant traits: heterozy-
trait will be exhibited almost exclusively in gotes express the products of both alleles.
males. If the X-linked allele occurs more If an individual’s red cells type as both K+
frequently in the population, affected fe- and k+, the K/k genotype may be inferred.

Figure 10-8. Inheritance and co-dominant expression of Kidd blood group antigens.
2 2
Figure 10-8 shows the inheritance patterns transferases. Similarly, in an A /O person, A
of the two active alleles of the Kidd blood is dominant to O. The O allele codes for a
a b
group system (Jk and Jk ) and the co- specific protein, but this protein (trans-
dominant phenotypic expression of the ferase) is nonfunctional. The presence of
two respective antigens Jka and Jkb. ABO genes can be demonstrated by molec-
In the ABO system, the situation is more ular techniques (see Chapter 13).
complex. The genes of the ABO system do
not code for membrane proteins but con- Chromosomal Assignment
trol production of enzymes termed glyco-
syltransferases. These enzymes add specific The loci of all major blood group genes
sugars to a precursor structure on the red have been mapped to one or another of
cell membrane, resulting in specific antigen the 22 pairs of autosomes, as shown in Ta-
expression. In an A1/A2 heterozygote, the ble 10-1. The Xg and XK loci are the only
phenotype is A1; the presence of the A2 al- blood group genes mapped to the X chro-
1
lele cannot be inferred. Although the A al- mosome.
2
lele appears dominant to that of the A al- Interaction among alleles or the prod-
lele by simple cell typing, techniques that ucts of different genes may modify the ex-
identify the specific transferases reveal that pression of a trait. The terms “suppressor”
an A1/A2 heterozygote does generate the and “modifier” are used to describe genes
products of both alleles, ie, both A1 and A2 that affect the expression of other genes;

however, the mechanism of these postu- The frequency of Jk(a–) individuals should
lated gene interactions is not always fully be 23%. If blood is needed for a patient
understood. Some observations in blood with anti-Jka, 23% or approximately one in
group serology have been explained by four ABO-compatible units of blood should
gene interaction: weakening of the D anti- be compatible.
gen expression when the C allele is present
in cis (on the same chromosome) or in Calculations for Combined Phenotypes
6
trans (on the paired chromosome), and the
If a patient has multiple blood group anti-
suppression of Lutheran antigen expression
bodies, it may be useful to estimate the
by the dominant modifier gene, In(Lu).7
number of units that will have to be tested
When products of two different genes
in order to find units of blood negative for
are important in the sequential develop-
all the antigens. For example, if a patient
ment of a biochemical end product, the
has anti-c, anti-K, and anti-Jka, how many
gene interaction is called epistasis. Failure
ABO-compatible units of blood would
to express A or B antigens if H substance
have to be tested to find 4 units of the ap-
has not first been produced (absence of the
propriate phenotype?
H gene) is an example of epistasis. A muta-
tion database of gene loci encoding com-
Phenotype Frequency (%)
mon and rare blood group antigens has
c– 20
been established (Blood Group Antigen
K– 91
Mutation Database) and is available on the
Jk(a–) 23
Internet (see http://www.bioc.aecom.yu.
edu/bgmut/index.htm).
To calculate the frequency of the com-
bined phenotype, the individual frequen-
cies are multiplied because the phenotypes
Population Genetics are independent of one another. Thus, the
Some understanding of population genet- proportion of persons who are c– is 20%. Of
ics is essential for parentage testing and the 20% of c– individuals, 91% are K–;
helpful in such clinical situations as pre- hence, 18% (0.20 × 0.91 = 0.18) are c– and
dicting the likelihood of finding blood K–. Of this 18% of c–K– individuals, 23%
compatible with a serum that contains will be Jk(a–); therefore, only 4% of individ-
multiple antibodies. Calculations use uals will have c–K–Jk(a–) blood (0.2 ×
published phenotype frequencies. 0.91 × 0.23 = 0.04). Therefore, of 100 units
tested, 4 compatible units should be found.
Phenotype Frequencies Calculations such as this influence deci-
sions about asking for assistance from the
The frequencies of blood group pheno-
local blood supplier or reference laboratory
types are obtained by testing many ran-
when trying to find compatible blood for an
domly selected people of the same race or
alloimmunized patient.
ethnic group and observing the propor-
tion of positive and negative reactions
with a specific blood group antibody. In a Parentage Testing
blood group system, the sum of pheno- Blood group antigens, many of which are
type frequencies should equal 100%. For expressed as co-dominant traits with sim-
example, in a Caucasian population, 77% of ple Mendelian modes of inheritance, are
randomly selected individuals are Jk(a+). useful in parentage analyses. If one as-

sumes maternity and that test results are When the alleged father cannot be ex-
accurate, paternity can be excluded in cluded from paternity, it is possible to cal-
either of two ways: culate the probability of paternity. The
1. Direct exclusion of paternity is es- probability that the alleged father transmit-
tablished when a genetic marker is ted the paternal obligatory genes is com-
present in the child but is absent pared with the probability that any other
from the mother and the alleged fa- randomly selected man from the same eth-
ther. Example: nic/racial population could have transmit-
ted the genes. The result is expressed as a
Blood Group Phenotype likelihood ratio (paternity index) or as a
Child Mother Alleged Father percentage (posterior probability of pater-
B O O nity given some prior probability). Methods
for parentage analysis often include the
The child has inherited a B gene, which study of many genetic systems other than
could not be inherited from either the red cell blood groups [ie, HLA and short
mother or the alleged father, assuming that tandem repeat (STR) systems]. Many par-
neither the mother nor the alleged father is entage testing laboratories employ the STR
of the rare Oh phenotype. Based on the phe- method of DNA analysis (see Chapter 9) as
notypes of mother and child, the B gene a means of evaluating cases of disputed
must have been inherited from the biologic parentage. The AABB has developed stan-
father and is called a paternal obligatory gene. dards for laboratories that perform parent-
2. Exclusion is indirect when the child age studies.8
lacks a genetic marker that the alleged
father (given his observed pheno- Chimerism
type) must transmit to his offspring.
Example: A chimera is one whose cells are derived
from more than one distinct zygotic line.
Blood Group Phenotype Although rare, this may occur when an
Child Mother Alleged Father anastomosis occurs within the vascular
Jk(a+b–) Jk(a+b–) Jk(a–b+) tissues of twin embryos, or when two fer-
tilized zygotes fuse to form one individ-
In this case, the alleged father is presum- ual. This condition, although not heredi-
ably homozygous for Jkb and should have tary, leads to dual (multiple) phenotypic
b
transmitted Jk to the child. populations of cells within one individual.
Direct exclusion is more convincing than Blood types of such rare individuals may
indirect exclusion when trying to establish demonstrate a mixed-field appearance,
parentage. Apparent indirect exclusion can with distinct populations of cells of the
sometimes result from the presence of a si- person’s true genetic type, as well as cells
lent allele. In the example above, the al- of the implanted type. Chimeras also
leged father could have one silent allele demonstrate immune tolerance: a geneti-
(Jk), which was transmitted to the child. cally group O person with implanted A
a
The child’s genotype could be Jk Jk instead cells does not produce anti-A. More com-
a a
of the far more common Jk Jk . Interpreta- monly, chimeras are artificial and arise
tion of phenotypic data must take into ac- from the transfer of actively dividing cells,
count all biologic and analytic factors eg, through hematopoietic transplanta-
known to influence results. tion (see Chapter 25).

system, the assumed amorphic gene

Blood Group Nomenclature is called Lu, not lu, whereas the
The terminology and notations for blood amorph in the Lewis system is le.
group systems embody many inconsis- 6. Numeric terminology was intro-
tencies because blood group serologists duced for some blood group sys-
failed to follow conventions of classic tems, resulting in mixtures of letters
Mendelian genetics. Listed below are a and numbers for antigen designa-
a
few examples of the confusion engen- tions, eg, K, Kp , and K11.
dered by many decades of uncoordinated Colloquial use of these terminologies,
scientific publications. even in some published articles and texts,
1. An allele that determines a dominant has compounded their improper use. Early
trait often is signified by a capital model computers or printers also did not
letter; one that determines a reces- easily accept certain terminologies (eg, su-
sive trait is denoted by both lower- perscripts, subscripts, unusual fonts).
case letters. The A and B co-domi- In recent years, concerted attempts have
nant genes of the ABO system are been made to establish rational, uniform
signified by a capital letter. The O criteria for the notations used to designate
gene is also given a capital but does phenotype, genotype, and locus informa-
not present as a dominant trait. tion for blood group systems. Issitt and
Without prior knowledge, it would Crookston9 and Garratty et al2 presented
be impossible for one to recognize guidelines for the nomenclature and termi-
that these notations represent allelic nology of blood groups. The International
products in a blood group system. Society of Blood Transfusion (ISBT) Work-
2. Some co-dominant traits have been ing Party on Terminology for Red Cell Sur-
designated with capital letters and face Antigens has provided a standardized
allelic relationships with lowercase system for classifying blood group antigens
letters; for example, K and k of the (see Appendix 6). Similar international
Kell blood group system and C and c committees have established principles for
of the Rh system. assigning nomenclature of the hemoglo-
3. Some co-dominant traits have iden- bins, immunoglobulin allotypes, histo-
tical base symbols but different su- compatibility antigens, clusters of differen-
perscript symbols, such as Fya and tiation, STR sequences, and other serum
b a b
Fy (Duffy system) and Lu and Lu protein and red cell enzyme systems. Al-
(Lutheran system). though many of the older terminologies
4. In some allelic pairs, the lower fre- must be retained to avoid even further
quency antigen is expressed with an confusion, common conventions now exist
“a” superscript (Wra has a lower fre- for correct usage.
b
quency than Wr ). In other allelic The ISBT terminology for red cell anti-
pairs, the “a” superscript denotes the gens was devised as a numeric nomencla-
higher incidence antigen (Coa has a ture suitable for computerization. A six-
b
higher frequency than Co ). digit designation indicates each blood
5. Some authors have denoted the ab- group specificity. The first three numbers
sence of a serologic specificity with a identify the blood group system and the
base symbol devoid of superscripts, last three numbers identify the individual
and others use a lowercase version specificity. This numeric terminology is de-
of the base symbol. In the Lutheran signed mainly for computer databases and

is not necessarily intended to supplant more 3. When antigen phenotypes are ex-
common usage. pressed using single letter designa-
For ISBT classification, each defined tions, results are usually written as +
blood group system must be genetically or –, set on the same line as the let-
distinct. Assignment of antigens to a spe- ter(s) of the antigen: K+ k–.
cific blood group system is dependent on 4. To express phenotypes of antigens
genetic, serologic, and/or biochemical rela- designated with a superscript letter,
tionships. Gene cloning has made the task that letter is placed in parentheses
of assignment more definitive and has al- on the same line as the symbol de-
lowed some designations previously un- fining the antigen: Fy(a+) and Fy(a–).
proved by traditional family studies (ie, the 5. For antigens designated by num-
expansion of the Diego system to include a bers, the symbol defining the system
number of low-incidence antigens). is notated in capital letters followed
Some antigens, however, have not yet by a colon, followed by the number
been proven to be part of a recognized sys- representing the antigen tested. Plus
tem. Collections (termed the 200 series) are signs do not appear when test re-
apparently related sets of antigens for which sults are positive (K:1), but a minus
definitive genetic information is lacking. sign is placed before negative test re-
Other isolated antigens of high (901 series) sults: K:1, K:–1. If tests for several an-
or low (700 series) incidence are listed to- tigens in one blood group have been
gether until genetic information becomes done, the phenotype is designated
available. In recent years, the number of by the letter(s) of the locus or blood
antigens in these three series has dramati- group system followed by a colon,
cally declined as further genetic and bio- followed by antigen numbers sepa-
chemical data allow reassignment. rated by commas: K:–1,2,–3,4. Only
antigens tested are listed; if an anti-
body defining a specific antigen was
Correct Terminology
not tested, the number of the anti-
The following are accepted conventions gen is not listed: K:–1,–3,4.
for expressing red cell antigen pheno- Although numeric terminology has been
types and genotypes.2 devised for various systems and antigens, it
1. Genes encoding the expression of should not be assumed that it must replace
blood group antigens are written in conventional terminology. The use of con-
italics (or underlined if italics are not ventional antigen names is also acceptable.
available). If the antigen name in- In some systems, notably Rh, multiple ter-
cludes a subscript (A1), generally the minologies exist and not all antigens within
encoding gene is expressed with a the system have names in each type.
superscript (A1).
2. Antigen names designated by a su-
a
perscript or a number (eg, Fy , Fy:1) References
are written in normal (Roman) script. 1. Zelinski T. Chromosomal localization of hu-
Numeric designations are written on man blood group genes. In: Silberstein LE, ed.
the same line as the letters. Super- Molecular and functional aspects of blood
group antigens. Bethesda, MD: AABB, 1995:
script letters are lowercase. (Some
41-73.
exceptions occur, based on historic 2. Garratty G, Dzik W, Issitt PD, et al. Terminol-
usage: hrS, hrB.) ogy for blood group antigens and genes—his-

torical origins and guidelines in the new mil- characters. Acta Path Microbiol Scand 1951;
lennium. Transfusion 2000;40:477-89. 28:207-10.
3. Denomme G, Lomas-Francis C, Storry JR, Reid 6. Araszkiewicz P, Szymanski IO. Quantitative
ME. Approaches to blood group molecular studies on the Rh-antigen D effect of the C
genotyping and its applications. In: Stowell C, gene. Transfusion 1987;27:257-61.
Dzik W, eds. Emerging diagnostic and thera- 7. Crawford NM, Greenwait TJ, Sasaki T. The
peutic technologies in transfusion medicine. phenotype Lu(a–b–) together with unconven-
Bethesda, MD: AABB Press, 2003: 95-129. tional Kidd groups in one family. Transfusion
4. Reid ME. Molecular basis for blood groups and 1961;1:228-32.
functions of carrier proteins. In: Silberstein 8. Gjertson D, ed. Standards for parentage test-
LE, ed. Molecular and functional aspects of ing laboratories. 6th ed. Bethesda, MD: AABB,
blood group antigens. Bethesda, MD: AABB, 2004.
1995:75-125. 9. Issitt PD, Crookston MC. Blood group termi-
5. Mohr J. A search for linkage between the Lu- nology: Current conventions. Transfusion 1984;
theran blood group and other hereditary 24:2-7.

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: The inactivation of one of the female X chromosomes during
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 11: Immunology
Chapter 11
Immunology
T
HE IMMUNE RESPONSE is a tive, the host may be susceptible to a wide
highly evolved innate and adap- variety of infectious agents or proliferation
tive system that is fundamental for of malignant cells. The ultimate goal of im-
survival. It has a sophisticated ability to mune activity is to maintain this delicate
distinguish self from nonself and provides balance.
a memory bank that allows the body to
rapidly respond to recurring foreign or-
ganisms. A healthy immune response can Immune Response
recognize foreign material or pathogens The immune response can be classified
that invade the body and can initiate a se- into two categories: the innate response
ries of events to eliminate these pathogens and the adaptive (acquired) response. In-
with minimal or no prolonged morbidity nate responses are indiscriminate: the
to the host. same mechanisms can be deployed against 11
The numerous components of the im- invasive organisms or harmful stimuli. In
mune system work in delicate balance to contrast, the adaptive response recognizes
ensure a state of health. This may include specific features of the harmful stimuli
destruction of abnormal/malignant cells, and provides a customized response based
removal of harmful bacteria or viruses, on previous experiences (Fig 11-1). The
and/or an inflammatory response to pro- adaptive response is a late evolutionary
mote healing. If the immune system be- development, found only in vertebrates.
comes hyperreactive, the body may attack Innate immunity, on the other hand, uses
its own tissue or organs (autoimmune dis- universal properties and processes such as
ease), or allergies may develop. If hyporeac- epithelial barriers, proteolytic enzymes,
243

Figure 11-1. Examples of the factors used in innate and adaptive immunity and examples of the two
types of immunity.
cellular phagocytosis, and inflammatory these receptors have been grouped into a
reactions. It is important to note that in- single superfamily of molecules. Examples
nate and adaptive immunity are comple- of these receptors include: immunoglobu-
mentary—not mutually exclusive—immune lins, T-cell receptor (TCR), major histo-
responses. Regardless of the classification, compatibility complex (MHC) Class I and
the immune response reflects the complex Class II molecules, and receptors for growth
interaction of cells, tissues, organs, and factors and cytokines. There are several
soluble factors. Appendix 11-1 describes hundred members of the IgSF (ie, immu-
some frequently used immunology terms. noglobulin receptors, integrins, etc).2
Immunoglobulin Superfamily Major Histocompatibility Complex

The MHC is a large cluster of genes. In
Molecules that belong to the immuno- humans, the MHC includes the genes of
globulin superfamily (IgSF) of receptors the HLA system and genes that encode
play a critical role in the recognition of other proteins such as tumor necrosis fac-
foreign antigens. Antigen recognition is tor ( TNF), some complement compo-
accomplished through receptors that are nents, and some heat shock proteins.3 The
found in a soluble form and on the sur- genes that encode MHC molecules are lo-
face of lymphoid cells. Figure 11-2 illus- cated on the short arm of chromosome 6.
trates the similar structure of some of There are three classes of these molecules.
these immunology receptors. The overall
structure of these receptors is very simi-
lar, and it has been proposed that they MHC Class I Molecules
arose from a common ancestral gene. The MHC Class I molecules are found on al-
basic structure is similar to the domains most all cells in the body. The molecules
of the immunoglobulin molecule; hence, are defined by three major Class I genes

Chapter 11: Immunology 245
1
Figure 11-2. Structures of some receptors found on various cells in the immune system.
designated HLA-A, -B, and -C. Hence, sion and function on the cell surface. The
each individual will express six distinct antigen-binding groove of the Class I mole-
Class I HLA molecules: two HLA-A, two cule is formed by the α1 and α2 domains of
HLA-B, and two HLA-C. Each locus has the heavy chain. The structure of the anti-
many alleles; more than 50 have been degen-binding groove consists of a platform
fined for HLA-A; more than 75 for HLA-B; made up of eight parallel β strands that is
and more than 30 for HLA-C. supported by two α helices.4 The peptides
The structure of a Class I HLA molecule displayed in the antigen-binding groove are
is illustrated in Fig 11-2. Each molecule 8 to 12 amino acids long and represent hy-
contains a heavy chain (45 kDa) and a drolyzed proteins that have been synthe-
smaller (12 kDa) peptide chain called sized within the antigen-presenting cell;
β2-microglobulin. The heavy chain has a cy- hence, they are referred to as endogenous
toplasmic tail, a transmembrane region, antigens. The endogenous source of pro-
and three extracellular immunoglobu- teins indicates that the genes encoding the
lin-like domains. β2-microglobulin is nonprotein also must reside in the cell. These
covalently associated with the heavy chain proteins could be the product of host genes
and is not a transmembrane protein. This including tumorigenic genes or genes from
protein is required for Class I MHC expres- viruses or intracellular bacteria.

MHC Class II Molecules cules expressed on cells and components

of the blood and lymphoid organs. Over
There are three major Class II gene loci:
200 CD markers have been described to
HLA-DR, -DQ, and -DP. As with the Class I 3
date. Some of the major CD markers on
molecules, there are many different al-
cells of the immune system are summa-
leles that could occupy each locus. Each
rized in Table 11-1.
individual expresses four DR molecules, four
DQ molecules, and two DP molecules, for
a total of 10 forms of Class II HLA mole- Cell Adhesion Molecules
cules. For normal immune function to occur,
Class II molecules are heterodimeric leukocytes must be able to attach to extra-
structures consisting of a heavy α chain (30 cellular matrices and each other. Three
to 34 kDa) and a light β chain (26 to 29 families of adhesion molecules facilitate
kDa). Each chain has a cytoplasmic tail, a this attachment process: selectins, inte-
transmembrane region, and an extracellu- grins, and IgSF adhesion molecules. The
lar portion. There are two immunoglobu- members of the selectin family (L-selec-
lin-like domains on each chain (α1 and α2 tin, E-selectin, and P-selectin) are found
and β1 and β2). The α2 and β2 domains for on leukocytes and participate in the pro-
each gene have a constant structure; the α1 cess of leukocyte rolling along the vascu-
and β1 domains are diverse. The antigen- lar endothelium. The integrins ( VAL,
binding groove is located within the α1, β1 LFA-1, and MAC-I) and the IgSF adhesion
domains and is similar in structure to the molecules (ICAMs, VCAMs, LFA-2, and
Class I molecules. However, the groove is LFA-3) are required to stop leukocyte roll-
larger, accommodating peptides that are 12 ing and mediate leukocyte aggregation
to 20 amino acids in length. Class II mole- and transendothelial migration.6 Most ad-
cules are expressed on monocytes, macro- hesion molecules have CD designations
phages, dendritic cells, and B lymphocytes. (eg, LFA-2 is CD2 and LFA-3 is CD58).7
The antigenic peptides displayed in the
groove of Class II HLA molecules come Signal Transduction
from proteins that have been phagocytosed
Signal transduction is the process of send-
or endocytosed by antigen-presenting cells.
ing signals between or within cells that
These proteins are termed exogenous anti-
results in the initiation or inhibition of
gens and include most bacteria, parasites,
gene transcription. Cell surface activation
and viral particles released from other
signals associated with the immune re-
cells.5
sponse are initiated by extracellular inter-
action of various ligands and receptors.
MHC Class III Molecules
Many of these ligands and receptors have
There are approximately 20 genes in the CD designations. Any defect or deficiency
Class III region of the MHC. These genes in the signal transduction process can have
code for proteins of the complement sys- significant consequences on the normal
tem and proinflammatory molecules such functioning of the immune system. For
as TNF. example, severe combined immunodefi-
ciency disease can result from a defi-
Cluster of Differentiation (CD) Molecules ciency of Jak3, which impairs signal
The CD designation is a nomenclature transduction of a specific cytokine recep-
system used to describe numerous mole- tor subunit.7

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

Designation Population 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 NK cells This is a sheep-cell rosette receptor;
thymocytes activation and adhesion function
(LFA-2)
CD3 T lymphocytes —— Functions as a signal transduction
complex
CD4 Developing and mature thymocytes T helper cells and some macrophages Adhesion molecule that mediates MHC
and on 2/3 of peripheral T cells restriction; signal transmission; HIV
receptor
CD5 Pan-T marker, from late cortical stage B cells of chronic lymphocytic leuke- Function unknown; possibly involved
mia; possibly long-lived in costimulatory effects of
autoreactive B cells cell-to-cell adhesion
CD8 Developing and mature thymocytes None Adhesion molecule that mediates MHC
and cytotoxic T lymphocytes restriction; signal transmission
Chapter 11: Immunology

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)
247
248
Table 11-1. Some Major CD Antigens on Cells of the Immune System (cont’d)
CD Cell Other Cells

Designation Population 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, This is a receptor for C3b (CR4)
dendritic cells
CD45 Immature and mature B and T cells All cells of hematopoietic origin except Also called leukocyte common antigen
red cells (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 Activated hematopoietic cells; prolifer- This is the transferrin receptor
cells ating cells of other somatic lines;
reticulocytes
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.
cells are the precursors of the cells that

Organs of the Immune make antibody (plasma cells). T cells con-
System sist of subpopulations that either help in
Numerous organs are involved in the im- antibody formation (helper T cells), kill
mune system. The central organs include target cells (cytotoxic T cells), induce in-
the marrow, liver, and thymus. Peripheral flammation (delayed hypersensitivity T
organs are the lymph nodes and spleen. cells), or inhibit the immune response
The gastrointestinal tract-associated lym- (regulatory T cells).
phoid tissue and bronchus-associated
lymphoid tissue also play an important
B Lymphocytes
role involving both central and peripheral
functions. 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,
Cells of the Immune System which are produced and transported to
The primary cells involved in the adaptive the membrane surface. When the immu-
immune system are lymphocytes—B cells, noglobulin receptor binds to a specific
T cells, and antigen-presenting cells. antigen, the cell is stimulated to divide
Other cells such as macrophages are in- and differentiate into a plasma cell. The
volved in the induction of the immune re- plasma cell can secrete a soluble form of
sponse, and both macrophages and poly- the immunoglobulin “receptor” known as
morphonuclear leukocytes participate in antibody. The antibody secreted is specific
various inflammation responses associated for the same antigen that interacted with
with the immune response. All of these cells the cell-bound immunoglobulin receptor.
are of hematopoietic origin (ie, marrow- The B-cell receptor (BCR) has one addi-
derived) and are formed from a single pro- tional heavy chain domain (CH4) compared
genitor called a pluripotent hematopoietic to secreted immunoglobulin. This domain
stem cell. As illustrated in Fig 11-3, the is required to anchor the receptor to the cell
pluripotent stem cell can give rise to two membrane. Several accessory molecules on
lineages: myeloid and lymphoid. Granulo- the surface of B cells are closely associated
cytes (neutrophils, basophils, and eosino- with the BCR and are important for signal
phils), platelets, mast cells, red cells, and transduction. Some of these accessory mol-
macrophages arise from myeloid progeni- ecules include Igα, Igβ, MHC Class II mole-
tors. T and B lymphocytes are formed from cules, complement receptors, and some CD
lymphoid progenitors. Dendritic cells and markers (see Table 11-2). All of these com-
natural killer (NK) cells are also derived ponents are part of the BCR antigen com-
from the pluripotent stem cell; however, plex (some of these structures are illustra-
their precise origin is unknown. ted in Fig 11-2).
The challenge for the body is to have B
Lymphocytes lymphocytes that can recognize each of the
The two major lineages of lymphocytes thousands of different foreign antigens en-
are B cells and T cells. B cells are derived countered over a lifetime. The immune sys-
from the marrow and T cells are derived tem has a unique approach to ensure this
from T-cell precursors produced in the diversity. The human genome is known to
marrow, which migrate to the thymus. B contain approximately 40,000 genes. The B

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
Igα Transport and assemble IgM monomers in
the cell membrane
Igβ Accessory 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
cells in the body probably make more than Several genes code for the heavy chain
100 million different antibody proteins that and light chain that make up the immuno-
are expressed as immunoglobulin recep- globulin receptor on B cells. The loci for
tors. Because the human body does not genes that code for the heavy chain are on
have enough genes to code for the millions chromosome 14 and the loci that code for
of different foreign proteins that the im- the light chains are on chromosome 2 (κ
mune system must have the capability to light chain) or chromosome 22 (λ light
recognize, a process called gene rearrange- chain). Three gene loci contribute to the di-
ment is used to create the required diver- versity of the immunoglobulin receptor: V
9
sity. (variable), D (diversity), and J (joining). The

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 Because the formation of the B-cell im-
of the immunoglobulin receptor and de- munoglobulin receptor occurs through
fines the expression of isotypes. The C region random rearrangement, some of the recep-
does not affect antigen binding but codes tors produced will react to the body’s own
for the biologic functions associated with cells. To prevent autoimmune disease, the
the immunoglobulin such as complement body must eliminate or downregulate these
activation and binding to specific receptors. B cells. This is accomplished through a pro-
Isotype switching (changing from IgM or cess of negative selection.12 When a B cell is
IgD to one of the other isotypes) is a T-cell- formed, it encounters large quantities of self
dependent process that occurs at the DNA antigen. If a B cell binds strongly to self an-
level. The process of isotype switching al- tigen, the immunoglobulin receptor sends
lows the specificity of antibody molecule to a signal that activates enzymes within the
be maintained regardless of the heavy chain cell to cleave nuclear DNA. This causes the
isotype.9,10 Table 11-3 summarizes the bio- cell to die, a process termed apoptosis (pro-
logic properties of the different immuno- grammed cell death). Only 25% of the B cells
globulin isotypes. that mature in the marrow reach the circu-
At the pre-B-cell stage of development, lation. The majority of B cells undergo
one heavy chain gene is randomly selected apoptosis. This process results in B cells
from each of the four segments (VH, DH, JH, that have low affinity to self antigen but still
4
and CH). There are over 10 possible combi- bind to foreign antigens that enter the
nations because of the large number of body. When mature B cells enter the pe-
possible genes at each segment. The light ripheral blood circulation, they bind for-
chain gene has only three segments (VL, JL, eign antigens that are specific for their im-
and CL), and one gene is selected from each munoglobulin receptors. When specific
of these segments in a process similar to antigen binds to the immunoglobulin re-
the heavy chain gene selection. This pro- ceptor, a signal occurs causing the recep-
cess results in over 1000 possible light chain tor/antigen complex to be internalized. In-
combinations. When the heavy chain and side the cell, the antigen is degraded into
light chains are combined, approximately small peptides that bind to MHC Class II
10 million different combinations could be molecules within the cell. This MHC-pep-
formed, each one representing an immu- tide complex is transported to the outer
noglobulin receptor with unique antigen membrane of the cell where it can interact
specificity. In addition to gene rearrange- with the TCR. This interaction signals the
ment, several other processes contribute to cell to produce various cytokines, causing
this diversity. These processes include so- the B cell to proliferate into a memory cell
matic mutations that occur at the time of or a plasma cell. The antibodies produced
B-cell activation, combinatorial shuffling by a plasma cell are always of the same im-
that occurs when the heavy and light munoglobulin class. However, each time
chains are assembled, and the addition of B-cell proliferation occurs, somatic muta-
random DNA bases to the end of the genes tions result in slight differences in the bind-
during the joining process. The combina- ing affinity of the immunoglobulin recep-
tion of all of these processes ensures that B tor. Because immunoglobulin receptors
cells have immunoglobulin receptors spe- with the highest binding affinity will be the
cific for any foreign antigen that could be ones most likely to encounter antigen, this
encountered (approximately 1011 antigen process preferentially results in prolifera-
specificities).9-11 tion of B cells with the highest affinity for

Table 11-3. Characteristics and Biologic Properties of Human Immunoglobulins
Class IgG IgA IgM IgD IgE
Structure
H-chain isotype γ α µ δ ε
Number of subclasses 4 2 1 ? ?
L-chain, types κ,λ κ,λ κ,λ κ,λ κ,λ
Molecular weight (daltons) 150,000 180,000- 900,000 180,000 200,000
500,000
Exists as polymer No Yes Yes No No
Electrophoretic mobility γ γ between γ between γ fast γ
and β and β
Sedimentation constant 6-7S 7-15S 19S 7S 8S
(in Svedberg units)
Gm allotypes (H chain) + 0 0 0 0
Km allotypes (Kappa L chain: + + + ? ?
formerly Inv)
Am allotypes 0 + 0 0 0
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 45 42 76 75 51
in intravascular space)
Present in epithelial secretions No Yes No No No
Antibody activity Yes Yes Yes Probably Yes
no
Serologic characteristics Usually Usually Usually ? ?
nonagglu- nonagglu- agglu-
tinating tinating tinating
Fixes complement Yes No Yes No No
Crosses placenta Yes No No No No
antigen. This preferential selection is termed sence of specific CD markers on the cell.
a focused immune response.5,13,14 Cytotoxic T cells are positive for the sur-
face marker CD8 and negative for CD4
T Lymphocytes and make up approximately one-third of
There are two major types of T cells: cyto- the circulating T cells in the peripheral
toxic T cells and helper T cells. These two blood. Helper T cells are CD4 positive and
cell types can be differentiated by the pre- CD8 negative and represent approximately

two-thirds of the circulating T cells. Helper The TCR is noncovalently associated with
T cells recognize antigen presented by the CD3 complex, which is made up of three
Class II HLA molecules, whereas cytotoxic pairs of dimers. This CD3 complex is re-
T cells recognize antigen in the context of sponsible for signal transduction once pep-
Class I HLA molecules. Approximately 5% tide is recognized by the TCR.16
of peripheral blood T cells are negative for Recognition by Cytotoxic T Cells.
both CD4 and CD8. Cytotoxic T cells recognize peptides associ-
T cells go through a process of positive ated with Class I MHC molecules. As dis-
and negative selection in the thymus dur- cussed previously, these peptides may be
ing T-cell ontogeny. If a T cell is able to rec- derived from self proteins or proteins from
ognize self MHC antigens, it survives (posi- intracellular viruses or microbes. The
tive selection) and migrates to the medulla TCR-2 receptor on the cytotoxic T cell rec-
of the thymus. In the medulla, the T cells ognizes the peptide-MHC Class I molecule
undergo a process (negative selection) that in combination with a co-receptor (CD8) on
deletes T cells with high affinity for self the T cell. These interactions signal the cell
MHC antigens. T cells that fail to recognize to produce proteins (eg, perforin) that dis-
self MHC antigens undergo apoptosis. The rupt the integrity of the target cell mem-
primary goal is to select T cells that recog- brane, resulting in cell death. During this
nize self MHC molecules that have foreign process, cytokines [TNF-α and interferon-γ
peptides in their groove. The process is so (IFNγ)] are also produced. These cytokines
exquisite that approximately 10% of T cells prevent replication of virus that may be shed
have the ability to react with foreign MHC from the cell during cell death; hence, the
complexes, which forms the major basis of infection is stopped through these processes.
transplantation rejection. In the end, only Although the process is an extremely effec-
5% of the cells in the thymus survive both tive mechanism for killing cells infected with
positive and negative selection and become virus, the process can be harmful to the host.
mature T cells.5,13,15 The cytokines produced to prevent viral rep-
The receptor on the T cell that is respon- lication can cause adverse effects including
sible for MHC/peptide recognition is the TCR the damage or destruction of healthy host
(see Fig 11-2). There are two major types of tissue. Liver damage associated with hepa-
TCRs: those that express α and β chains or γ titis B infection is an example of morbidity
and δ chains as part of the TCR complex. caused by the cytotoxic T-cell response.5,11
Approximately 90% of all T cells bear α, β Stimulation of B Cells. B-cell activation
chains. Each transmembrane chain has two can occur through activation by T cells or by
domains (one variable, the other constant). a mechanism independent of T-cell interaction.
These chains are produced through a pro- These two mechanisms are described below.
cess of gene rearrangement in a manner T-Cell-Dependent Stimulation. Helper
similar to MHC and immunoglobulin re- T-cell receptors recognize foreign peptides
ceptors. The number of possible TCR α and in the antigen-binding groove of Class II
β specificities is estimated at 1015. The TCRs MHC molecules in combination with CD4.
bearing γ and δ are expressed on 5% to 15% This TCR-CD4 interaction with MHC Class
of T cells, predominantly by those T cells in II upregulates the expression of CD80/86
the mucosal endothelium. These T cells ap- on the surface of antigen-specific B cells.
pear to play an important role in protecting CD80/86 reacts with the ligand CD28 on
the mucosal surfaces of the body from for- the T-cell surface, causing upregulation of
eign bacteria. the CD40 ligand (CD40L), which engages

with CD40 on B cells. This cascade of sig- viral proteins are expressed on the surface
nals is important for cytokine production, of an infected cell, usually as a result of
which results in the isotype switch response viral budding. The proteins are recog-
by the B cell. The production of interleukin nized as foreign by the immune system
(IL)-4 causes B cells to switch from IgM to and antibodies are produced, which later
IgG4 and IgE. The production of transform- bind to the viral proteins expressed on the
ing growth factor β (TGF-β) and IL-10 infected cell. NK cells recognize the pres-
causes the B cell to switch to IgA1 and IgA2. ence of antibody bound on the surface of
If there is an absence or impaired function the cells via their Fcγ receptors. The NK
of the ligand interaction isotype, switching cells produce perforins, which cause lysis
can be affected. For example, if the CD40L- of the virus-infected cells by a mechanism
CD40 interaction is impaired, isotype that is not entirely understood.5
switching will not occur and only IgM anti-
body is produced. This clinical situation is
Phagocytic Cells
termed hyper-IgM immune deficiency.7
T-Cell-Independent Stimulation. B cells Phagocytes include cells such as monocytes
can be activated to produce antibodies and polymorphonuclear granulocytes
by polysaccharides, lipopolysaccharides, (eosinophils, basophils, and neutrophils).
and polymeric proteins independent of Some monocytes migrate into tissues
T-cell interaction. The B cells react directly (liver, lungs, spleen, kidney, lymph nodes,
with these molecules, producing a rapid and brain) and become tissue macrophages.
immune response to pathogens. However, The polymorphonuclear granulocytes are
there are disadvantages to this mechanism: rapidly produced and live only for a short
the process is ineffective for the production time (several days). The neutrophil is the
of memory B cells; antibody affinity matu- most abundant granulocyte. These cells
ration is poor; and isotype switching is not respond to chemotactic agents such as
induced. The T-cell-independent process complement fragments and cytokines,
can also cause antibody production by B causing them to migrate to the site of in-
cells whose immunoglobulin is specific for flammation. Eosinophils represent only
antigens other than those found on the 2% to 15% of the white cells and play an
pathogen; hence, both protective antibodies important role in regulating the inflam-
and autoantibodies may be produced. When matory response by releasing an antihis-
autoantibodies are produced through this tamine. These cells may also play a role in
mechanism, transient clinical symptoms of phagocytosing and killing microorgan-
autoimmune disease may occur.7 isms. Basophils make up less than 0.2% of
the total leukocyte pool. These cells also
respond to chemotactic factors and are
NK Cells involved in the allergic response. Histori-
NK cells do not express T-cell receptors or cally, this network of phagocytic cells was
B-cell receptors and represent approxi- called the reticuloendothelial system but is
mately 10% of the lymphocyte popula- now termed the mononuclear phagocytic
tion. These cells have the ability to kill system.
some cells infected with viruses and some The ability of cells to phagocytose is ac-
tumor cells. NK cells do this by a mecha- complished through the presence of recep-
nism termed antibody-dependent cellular tors on the cell membrane. There are many
cytotoxicity or ADCC. ADCC occurs when types of receptors including: mannose-fucose

receptors that bind to sugars on the surface of the immunoglobulin molecule are held
of microorganisms, Fc receptors that bind together by disulfide bonds. The polypep-
to IgG, and complement receptors. A sum- tide chains (both heavy and light) are
mary of some membrane receptors found looped, forming globular structures called
on macrophages and monocytes is found the immunoglobulin domain. On each
in Table 11-2. The internalization and pro- chain, there is a variable domain in which
cessing of particulate matter occur through the amino acid sequence is diverse, giving
the production of enzymes (peroxidase and the immunoglobulin its specificity. The epi-
acid hydrolases).5 Under optimal cytokine topes expressed in this region are termed
conditions, macrophages and monocytes idiotypes. The remaining domains on both
can become formal antigen-presenting the heavy and light chains are called con-
cells; therefore, their ability to ingest and stant domains and have similar amino acid
process foreign molecules is an essential sequences, depending on the isotype. The
part of the adaptive immune response. 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-
Soluble Components of the pendently. The biologic functions of the
Immune Response molecule are associated with the constant
domains on the heavy chain. These func-
There are three major soluble components
tions include: placental transfer, macro-
of the immune response: immunoglobu-
phage binding, and complement activa-
lins, complement, and cytokines.
tion.4
Immunoglobulins
Interchain Bonds
Immunoglobulins are the proteins that can
Each light chain is joined to one heavy chain
be cell bound and serve as antigen recep-
by a disulfide bond. One or more disulfide
tors on B cells (see section on B lympho-
bonds link the two heavy chains at a point
cytes) or can be secreted in a soluble form
between CH1 and CH2, in an area of con-
as antibodies. The molecular development
siderable flexibility called the hinge re-
of the immunoglobulin molecule is dis-
gion. It is these interchain disulfide bonds
cussed in the section on B lymphocytes.
that are the target of reducing agents used
The structures of the different types of
to produce “chemically modified” anti-D
immunoglobulin are discussed below.
reagent.
Each monomeric immunoglobulin mol-
ecule consists of two identical heavy chains
and two identical light chains. The heavy Fab and Fc Fragments
chains consist of approximately 450 amino Polypeptides can be cleaved at predictable
acids with a molecular weight of approxi- sites by proteolytic enzymes. Much infor-
mately 50 to 77 kDa. There are five heavy mation about immunoglobulin structure
chain classes termed isotypes. They include and function is derived from the study of
mu (µ), gamma (γ), alpha (α), delta (δ), and cleavage fragments generated by papain
epsilon (ε). The light chains are smaller (ap- digestion of Ig molecules. Papain cleaves
proximately 210 amino acids; molecular the heavy chain at a point just above the
weight 25 kDa) and can be either kappa (κ) hinge, creating three separate fragments.
or lambda (λ). The heavy and light chains Two are identical, each consisting of one

Figure 11-4. The basic four-chain immunoglobulin unit. Idiotypic specificity resides in the variable do-
mains of heavy and light chains (VH and VL ). Antigen-binding capacity depends on intact linkage be-
tween one light chain (VL and CL ) and the amino-terminal half of one heavy chain (VH and CH 1), the Fab
fragments of the molecule. Disulfide bonds in the hinge region join carboxy-terminal halves of both
heavy chains (CH 1 and CH 3, plus CH 4 for and heavy chains), to form the Fc fragment. (Used with per-
mission from Goldsby et al. 8 )
light chain linked to the N-terminal half exist only in the monomeric form; there
of one heavy chain; the other fragment are no polymeric forms of these Ig classes.
consists of the C-terminal halves of the The IgM synthesized by unstimulated B
heavy chains, still joined to one another cells and expressed on the membrane as
by the hinge-region disulfide bonds. The the immunoglobulin receptor is expres-
two identical N-terminal fragments, sed in the monomeric form. As men-
which retain the specificity of the anti- tioned previously, the µ heavy chain has
body, are called Fab fragments. The joined four constant domains in the membrane
C-terminal halves of the heavy chains form of IgM. The fourth domain allows
constitute a nonantibody protein frag- the IgM monomer to bind to the cell
ment capable of crystallization, called the membrane as the immunoglobulin recep-
Fc fragment. tor. Following clonal expansion and differ-
entiation to a plasma cell, the activated
Immunoglobulin Polymers cell produces µ chains with one less con-
Disulfide bonds may also join some Ig stant domain. While still in the plasma-
monomeric units to one another to form cell cytoplasm, five IgM monomers are
larger polymeric molecules; only IgM and joined by the formation of disulfide bonds
IgA can form polymers. IgG, IgE, and IgD between the CH3 domains and CH4 do-

5
mains. The result is a pentamer that is multivalency, IgM molecules readily bind to
secreted to the exterior of the cell and antigens on particulate surfaces, notably
constitutes the form in which IgM accu- those on red cells or microorganisms. IgM
mulates in body fluids. antibodies can crosslink cells expressing a
Secreted IgA exists in both monomeric specific antigen, forming a clump of cells
and polymeric forms. Monomeric forms (agglutination). Although extremely useful
predominate in the bloodstream, but dimers as a laboratory endpoint, agglutination
and trimers that are secreted by B cells in probably plays a relatively minor role in
mucosal surfaces and exocrine tissue are biologic events.
the biologically active form. The most important biologic effect of IgM
is its ability to activate the complement
Other Chains cascade, which enhances inflammatory
and phagocytic defense mechanisms and
Pentameric IgM and the dimers and tri-
may produce lysis of antigen-bearing cells.
mers of IgA contain a 15-kDa polypeptide
IgG. Immunoglobulin G exists only as a
called the J chain. Before the polymer
monomer and accounts for 75% to 80% of
leaves the plasma-cell cytoplasm, this
the immunoglobulins present in serum. It
chain attaches to the terminal constant
is equally distributed in the intravascular
domain of two adjacent monomers. No
and extravascular compartments. In vivo,
matter how many monomers constitute
cells or particles coated with IgG undergo
the polymer, there will be only one J chain.
markedly enhanced interactions with cells
Its function is not fully understood.
that have receptors for the Fc portion of γ
The polymeric Ig molecules present in
chains, especially neutrophils and macro-
epithelial secretions also exhibit a subunit
phages.
called the secretory component. The secre-
IgG molecules can be classified into four
tory component appears to protect the bio-
subclasses: IgG1, IgG2, IgG3, and IgG4.
logically important surface antibodies from
Structurally, these subclasses differ primar-
proteolysis in the enzyme-rich secretions of
ily in the characteristics of the hinge region
respiratory and alimentary tracts.
and the number of inter-heavy-chain
disulfide bonds. Biologically, they have sig-
nificantly different properties. IgG3 has the
Individual Immunoglobulin Classes greatest ability to activate complement, fol-
IgM. IgM is the first Ig class produced by lowed by IgG1 and, to a much lesser extent,
the maturing B cell. It is the first to appear IgG2. IgG4 is incapable of complement ac-
in the serum of maturing infants and the tivation. IgG1, IgG3, and IgG4 readily cross
first to become detectable in a primary the placenta. IgG1, IgG2, and IgG4 have a
immune response. Secreted pentameric half-life of 23 days, significantly longer than
IgM normally constitutes 5% to 10% of the that of other circulating immunoglobulins;
immunoglobulin in normal serum. Very however, the half-life of IgG3 is only slightly
few of these large molecules diffuse into longer than IgA and IgM. IgG1 and IgG3
interstitial fluid. readily interact with the Fc receptors on
Although the five monomers comprise phagocytic cells, but IgG4 and IgG2 have
10 antigen-combining sites, only five sites low affinity for these receptors with the ex-
are readily available to combine with most ception that IgG2 has an affinity similar to
antigens; hence, IgM is described as penta- IgG1 and IgG3 for an allotype of Fcγ recep-
valent. Because of their large size and tor IIa.

IgA. Although there is a large body con- proteins that act in a cascading manner—
tent of IgA (10% to 15% of serum immuno- similar to the coagulation, fibrinolytic,
globulin concentration), relatively little is and kinin systems—to produce numerous
found in the blood. Most of the IgA exists in biologic effects. The participating pro-
mucosal secretions. Secretory IgA protects teins remain inactive until an event initi-
the underlying epithelium from bacterial ates the process, following which the
and viral penetration. Polymeric IgA is product of one reaction becomes the cata-
thought to combine with environmental lyst for the next step (see Fig 11-5). Each
antigens, forming complexes that are elimi- evolving enzyme or complex can act on
nated as surface secretions are excreted. multiple substrate molecules, creating the
This process may be important in control- potential for tremendous amplification of
ling the development of hypersensitivity. an initially modest or localized event.
The heavy chain of IgA has no comple- Complement has three major roles: pro-
ment-binding site; hence, IgA cannot acti- motion of acute inflammatory events; alter-
vate complement through the classical ation of surfaces so that phagocytosis is en-
pathway. IgA can activate complement hanced; and the modification of cell mem-
through the alternative pathway (see be- branes, which leads to cell lysis. These ac-
low). tions cause destruction of bacteria, protect
IgE. The concentration of serum IgE is against viral infection, eliminate protein
measured in nanograms, compared with complexes, and enhance development of
milligram levels for other immunoglobu- immune events. However, activation of
lins. Even when patients with severe aller- complement can also initiate inflammatory
gies have markedly elevated serum concen- and immune processes that may harm the
trations of IgE, the absolute level is minimal host and mediate destruction of host cells,
compared with other immunoglobulins. especially those in the blood.
17
Most IgE is present as monomers tightly Different mechanisms exist for activat-
bound to the membrane of basophilic ing the complement cascade: the classical
granulocytes or mast cells. IgE is responsible pathway, which is initiated by interaction
for immediate hypersensitivity events, such between an antibody and its antigen, and
as allergic asthma, hay fever, and systemic the alternative pathway, which is usually not
anaphylactic reactions. Although IgE ap- antibody mediated.
pears to be involved in reactions to proto-
zoal parasites, no specific protective mecha-
nisms have been identified.
The Classical Pathway
IgD. Serum contains only trace amounts
of IgD. Most IgD exists as membrane im- For the classical pathway of complement
munoglobulin on unstimulated B cells. The activation to occur, an immunoglobulin
function of IgD is unknown, but it may be must react with target antigen. Combina-
important for lymphocytic differentiation, tion with antigen alters the configuration
which is triggered by antigen binding, and of the Fc portion of the immunoglobulin,
in the induction of immune tolerance. rendering accessible an area in one of the
heavy-chain constant domains that inter-
acts with the C1 component of comple-
Complement
ment. C1 can combine only with Ig mole-
Complement is the term applied to a sys- cules having an appropriate heavy-chain
tem of 25 to 30 serum and membrane configuration. This configuration exists in

Classical Pathway
Figure 11-5. Diagram illustrating the activation of complement by the classical and alternative path-
ways. (Used with permission from Heddle. 1 )
the µ heavy chain and in the γ chains in tional change, resulting in activation of two
IgG subclasses 1, 2, and 3. C1r molecules that then cleave two C1s
The C1 component of complement atta- molecules into activated C1s (a strong
ches to activation sites on the Fc portion of serine esterase). The C1r, C1s, and C1q
two or more Ig monomers. The pentameric complex is stabilized by Ca2+ ions. In the ab-
2+
IgM molecule provides an abundance of sence of Ca , the complex dissociates.
closely configured Fc monomers; hence, a Thus, chelating agents often used in the
single IgM molecule can initiate the com- laboratory, such as citrate and oxalate anti-
plement cascade. For IgG antibodies to ac- coagulants, prevent the stabilization of the
tivate the sequence, two separate molecules C1 complex and subsequent activation of
3
must attach to antigen sites close together. complement proteins.
Hence, complement activation by IgG anti- Activated C1s works on two substrates.
bodies depends not only on the concentra- C1s cleaves C4 into two fragments: a large
tion and avidity of the antibody, but also on fragment (C4b) and a smaller fragment
the topography of the antigen. (C4a) that has modest anaphylatoxic activ-
Circulating C1 is a macromolecule con- ity. Most of the C4b generated is inacti-
sisting of three distinct proteins (C1q, C1r, vated; however, some C4b binds to the cell
and C1s). When an antigen-antibody reac- surface and acts as a binding site for C2.
tion occurs, two or more chains of C1q at- C1s also cleaves the bound C2, releasing a
tach to the CH2 domain of IgG or the CH3 fragment called C2b. The C2a fragment that
domain of IgM. This causes a conforma- remains bound to C4 forms an activated

complex C4b2a, also known as C3 con- vated; however, if C3b binds to a foreign
vertase. Each C3 convertase can cleave more surface such as a bacteria cell wall, the acti-
than 200 native C3 molecules, splitting the vation of the complement cascade can be
molecule into two fragments. A small frag- accelerated.
ment (C3a) that has anaphylatoxic activity When C3b binds to a cell surface, factor
is released into the plasma; the larger frag- B is bound to give C3bB. Factor D can also
ment (C3b) attaches to proteins and sugars react with this cell-bound substrate, releas-
on the cell surface. ing the small Ba fragment leaving cell-
The classical pathway and the alternative bound C3bBb. This complex will dissociate
pathway are both means of generating a C3 unless it is stabilized by properdin, result-
convertase. Once C3 has been cleaved, the ing in the complex C3bBbP. Like the C4b2a
same events occur in the two pathways. complex of the classical pathway, C3bBbP
is capable of converting more C3 into C3b.
In summary, both the classical and alterna-
The Alternative Pathway tive pathways result in C3b generation. The
The alternative pathway allows comple- C3 convertase of the classical pathway is
ment activation in the absence of an anti- C4b2a, whereas the C3 convertases of the
gen/antibody interaction. This pathway is alternative pathway are C3iBb in the fluid
a first-line antimicrobial defense for ver- phase and C3bBbP when cell bound.
tebrates and a mechanism whereby pre-
vertebrates can enhance their inflamma-
tory effectiveness. The alternative pathway The Membrane Attack Complex
is a surface-active phenomenon that can The final phase of activation is called the
be triggered by such initiators as dialysis membrane attack complex and can occur
membranes; the cell wall of many bacte- once C3b has been cleaved through either
ria, yeasts, and viruses; protein com- the classical or alternative pathway. C3
plexes, including those containing anti- convertase cleaves C5 into two fragments:
bodies that do not bind complement; a small peptide (C5a) having potent ana-
anionic polymers such as dextran; and phylatoxin activity and a larger fragment
some tumor cells. The alternative path- (C5b). The C5b fragment binds C6, C7,
way of complement activation can also C8, and up to 14 monomers of C9, result-
occur spontaneously in the plasma at a ing in a lytic hole in the membrane. Small
slow but steady rate (tickover activation) amounts of lysis can occur when C8 is
(see Fig 11-5). bound; however, the binding of C9 facili-
Four proteins participate in the alterna- tates cell lysis.
tive pathway: factor B, factor D, properdin The binding of C3b to a cell membrane
(factor P), and C3. Fluid-phase C3 under- is the pivotal stage of the pathway. The
goes continuous but low-level spontaneous cell-bound C3b can proceed to activate the
cleavage, resulting in C3i that is rapidly in- membrane attack complex (C5-C9) and
activated by fluid-phase control proteins. If cause cell lysis; alternatively, inhibitors may
C3i encounters factor B, a complex called stop the activation sequence, leaving the
C3iB is formed and additional interactions cell coated with C3b. Factor I is an inhibitor
can occur. Factor D acts on bound factor B, that can cleave cell-bound C3b, leaving
generating a C3iBb complex capable of iC3b on the membrane. These two sub-
cleaving C3 into C3a and C3b. Most of the components of C3 (C3b, iC3b) can facilitate
C3b generated in the fluid phase is inacti- phagocytosis by acting as opsonins. How-

ever, C3b can be further cleaved, leaving a decay and destruction of convertases, and
small fragment called C3dg. It is the C3dg control of membrane attack complexes.19
molecule that is detected by the anti- C3d
component in anticomplement reagents Physiologic Effects of Complement
used for the direct antiglobulin test. Activation
Opsonization. Neutrophils and macro-
Complement Receptors phages phagocytose any particle that pro-
Some phagocytic cells have receptors that trudes from the surface of a cell or micro-
can bind to C3 on the cell. Four different organism with no regard to the nature of
complement receptors have been identi- the material. Phagocytosis is more intense
fied on phagocytic cells: CR1, CR2, CR3, if the particle adheres firmly to the mem-
and CR4.18 brane of the phagocytic cell. To achieve
CR1. CR1 is found on a variety of cells. adherence, phagocytic cells have various
On red cells and platelets, the CR1 receptor receptor molecules such as the Fc recep-
plays an important role in clearing immune tors for certain immunoglobulins and
complexes. On phagocytic cells and B lym- receptors for C3b. The enhancement of
phocytes, it is an opsonic receptor that is phagocytosis resulting from antibody or
involved in lymphocyte activation. CR1 also complement coating of cells is called
plays a regulatory role in complement acti- opsonization.
vation by assisting factor I in cleaving C3b Anaphylatoxins Promote Inflammation.
into iC3b and C3dg. Complement fragments C3a and C5a are
CR2. CR2 is found on B cells, some epi- anaphylatoxins and they play an important
thelial cells, and follicular dendritic cells. It role in acute inflammation. These ana-
plays an important role in mediating B-cell phylatoxins bind to receptors on mast cells
activation. It is also the receptor for inter- and basophils, causing them to release his-
feron α and the Epstein-Barr virus. tamine and other biologic response modifi-
CR3. CR3 is found on cells of the myeloid ers that can be associated with anaphylaxis.
lineage. CR3 mediates phagocytosis of parti- More frequently, anaphylatoxins affect vas-
cles coated with iC3b and is also an impor- cular permeability, membrane adhesion
tant adhesion molecule capable of binding properties, and smooth-muscle contraction
to certain types of bacteria and yeast. that constitute a large part of the acute in-
CR4. CR4 is found on both lymphoid flammatory response. C5a and C3a also
and myeloid cells. Its function is not well cause neutrophils and macrophages to mi-
characterized, but it appears to have op- grate to the site of complement activation.
sonic activity for iC3b, and it plays a role in
adhesion.
Cytokines
Regulation of Complement Activation Throughout this chapter, inference has
There is a need to control or regulate the been made to a number of soluble media-
enzyme and activation factors of the com- tors termed cytokines. Cytokines are a
plement cascade. The regulatory actions diverse group of intracellular signaling
of these control proteins prevent damage peptides and glycoproteins that have mo-
to host tissue. These control systems lecular weights ranging from 6,000 to
(Table 11-4) act by several different mecha- 60,000 daltons. Each cytokine is secreted
nisms: direct inhibition of serine proteases, by particular cell types in response to dif-

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

Complement Control Proteins Function
Inhibition of Serine Protease

C1 inhibitor A serine protease inhibitor that binds and acti-
vates Clr and Cls
Decay/Destruction of Convertases
Factor I plus C4 binding protein (C4-bp) Catabolizes C4 in the fluid phase
C4-bp Causes dissociation of C2a from C4b2a
Decay accelerating factor Inhibits the binding of C2 to C4b
(DAF, CD55) 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)
ferent stimuli and has been implicated in sion reactions, immunomodulation, stem
a wide variety of important regulatory bi- cell collection, etc) (see Chapter 27).
ologic functions such as inflammation,
tissue repair, cell activation, cell growth,
fibrosis, and morphogenesis. One cytokine
can have several different functions, de-
Immunology Relating to
pending on the type of cell to which it Transfusion Medicine
binds. Several hundred different cytokines Several examples of how the immune sys-
have been described. Some of the cyto- tem relates to situations encountered in
kines involved in immune functions are transfusion medicine are described below,
1,5
summarized in Table 11-5. Cytokines including red cell alloimmunization, pla-
have been implicated in a variety of clini- telet alloimmunization, immune-medi-
cally important factors of transfusion ated red cell destruction, and reagent an-
medicine (hemolytic and febrile transfu- tibody production.

Table 11-5. Summary of Some Cytokines Involved in the Immune System, Their
1
Site of Production, and Function
Cytokine Produced by Primary Functions
Interleukins
IL-1 Many cells (eg, APCs, T-cell activation, neutrophil
endothelial cells, B cells, activation, stimulates
fibroblasts) marrow, pyrogenic, acute-
phase protein synthesis
IL-2 Activated TH cells T-cell growth, chemotaxis,
macrophage activation
IL-4 Activated TH cells B-cell activation, B-cell
differentiation, T-cell
growth, TH2 differentiation
IL-6 Many cells (eg, T cells, B-cell differentiation,
APCs, B cells, fibroblasts, pyrogenic, acute-phase
and endothelial cells) protein synthesis
IL-8 Many cells (eg, macrophages Inflammation, cell migration/
and endothelial cells) 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 Neutrophil activation,
lymphocytes pyrogenic, acute-phase
protein synthesis
Interferon γ (IFNγ) T cells Phagocyte activation
Transforming growth Various cells Stimulates connective tissue growth
factor-β (TGF-β) and collagen formation, inhibi-
tory function
Colony-stimulating Various cells Growth and activation of phagocytic
factors (GM-CSF, cells
M-CSF, G-CSF)
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 tively, the process of antigen presentation

and immune recognition of foreign HLA
The mechanism of red cell alloimmuniza-
peptides can occur by the recipient’s own
tion is not well understood. When allo-
immune system. This alternative form of
geneic red cells are transfused, some of
allorecognition is termed “indirect” and is
the red cells fragment as they age or when
the classical alloimmune response seen for
they pass through the spleen, thus releas-
most foreign antigens. Leukocyte reduc-
ing membrane-bound red cell proteins
tion appears to be effective in reducing
into the bloodstream as the fragments de-
HLA alloimmunization because antigen
grade. Both formal antigen-presenting
presentation by donor leukocytes is re-
cells and B lymphocytes can present anti-
duced and the amount of HLA Class I an-
gen either as a primary immune response
tigens transfused is greatly reduced.
(formal antigen presentation by dendritic
cells) or in the secondary immune re-
sponse by B cells. Immune-Mediated Red Cell Destruction
The activation of complement and/or the
HLA Alloimmunization to Platelets presence of IgG on the red cell surface can
Leukocyte reduction of red cells and pla- trigger red cell destruction by predomi-
6
telets to a threshold of 5 × 10 per product nantly two mechanisms: intravascular
has been shown to reduce HLA alloim- hemolysis and extravascular red cell de-
munization, possibly by the following struction (Fig 11-7). Intravascular hemolysis
mechanism. Donor leukocytes express occurs when complement is activated,
both Class I and Class II MHC antigens. resulting in activation of the membrane
Some of the donor Class II MHC antigens attack complex. As the integrity of the
will contain peptides that originated from membrane is disrupted, hemoglobin is re-
the MHC Class I antigens on the donor’s leased into the plasma. Free hemoglobin
cells. Most of the transfusion recipient’s T binds to haptoglobin and is excreted in
cells recognize self-MHC-carrying foreign the urine. The heme portion of hemoglo-
peptides. However, as mentioned previ- bin binds to albumin, forming methemal-
ously, a small percentage of T cells (<10%) bumin, which can be visually detected in
are able to recognize foreign-MHC-carry- plasma by a brownish green discolor-
ing foreign peptides. When platelets are ation. At times, the inhibitors of comple-
transfused, the recipient’s T helper cells ment stop the cascade, leaving C3b on the
recognize the foreign MHC Class II com- red cells. This complement fragment has
plex on the donor leukocytes. If a signal chemotactic activity for phagocytic cells
occurs, the T cells activate recipient B and these cells have receptors for C3b.
cells, which have also bound donor MHC C3b-coated red cells adhere to phagocytic
Class I antigen fragments to their Ig re- cells but are relatively ineffective at trig-
ceptor, resulting in cell proliferation and gering phagocytosis. Enzymes can cleave
MHC Class I (HLA) antibody production the cell-bound C3b, leaving a small frag-
(Fig 11-6). In this case, the donor’s leuko- ment (C3dg) present on the cells. C3dg-
cytes serve as the antigen-presenting sensitized red cells survive normally be-
cells. This major mechanism is termed cause phagocytic cells do not have recep-
“direct allorecognition” because the recip- tors for C3dg. If IgG is present on the red
ient’s T cells are directly stimulated by do- cells, binding to Fcγ receptors will occur,
nor antigen-presenting cells.20 Alterna- resulting in phagocytosis. If both IgG and

Figure 11-6. Diagram illustrating the major mechanism of HLA alloimmunization due to leukocytes
present in platelet transfusion. (Used with permission from Heddle. 1 )
C3b are present on the red cells, clearance ques in an attempt to purify the resulting
of the cells may be enhanced, probably sera. Monoclonal antibody production
because of the chemotactic function of provided an alternative to human and an-
C3b and its adherence capability. imal sources of these proteins. Using this
approach, a single B-cell clone is propa-
Reagent Antibodies gated in cell culture and the supernatant
Heterogeneous antibodies are not opti- fluid from the culture contains antibody
mal as reagents for use in serologic testing of a single specificity. However, there are
because they can vary in concentration, problems with this approach. Normal B
serologic properties, and epitope recogni- cells reproduce themselves only a limited
tion and can contain other antibodies of number of times; hence, the cultured cell
unwanted specificity. The ideal serum for lines survive only a short time.
21
serologic testing is a concentrated sus- In 1976, Köhler and Milstein provided a
pension of highly specific, well-character- solution to this problem. Plasma cells of
ized, uniformly reactive, immunoglobulin normal antibody-producing capacity were
molecules. Until the 1970s, the only way fused to neoplastic plasma cells of infinite
to obtain reagents was to immunize ani- reproductive capacity (ie, myeloma cells).
mals or humans with purified antigens and Techniques had previously been developed
then perform time-consuming and some- that cause cell membranes to merge, allow-
times unpredictable separation techni- ing the cytoplasm and the nucleus of two

*Ineffective mediator of phagocytosis.

†
Due to the chemotactic ability of C3b, extravascular destruction may be enhanced when both IgG and C3b are present on the
red cells.
Figure 11-7. Summary of intravascular and extravascular red cell destruction.
different kinds of cells to fuse into a single act more strongly than immune-serum
cell. These plasma cell/myeloma cell hy- preparations when tested against cells with
brids can be maintained in cell culture for weakly expressed antigens.
prolonged periods, producing large quanti- Phage technology is under investigation
ties of the selected antibody. as an approach for producing genetically
The exquisite specificity of monoclonal engineered antibodies for a variety of thera-
antibodies is both an advantage and a dis- peutic treatments,22 as well as for use as
23
advantage for reagent use. An antibody that typing reagents.
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-
References
1. Heddle NM. Overview of immunology. In:
tion. Thus, reagent preparations typically Reid ME, Nance SJ, eds. Red cell transfusion.
used in the laboratory are blends of several A practical guide. Totowa, NJ: Humana Press,
different monoclonal products, thereby in- 1998:13-37.
creasing the range of variant phenotypes 2. Barclay AN. Membrane proteins with immu-
noglobulin-like domains—a master super-
that the antiserum can identify. Single or family of interaction molecules. Semin
blended monoclonal preparations often re- Immunol 2003;15:215-3.

3. Marsh SG, Albert ED, Bodmer WF, et al. No- 21. Köhler G, Milstein C. Derivation of specific
menclature for factors of the HLA system, 2002. antibody-producing tissue culture and tumor
Hum Immunol 2002;63:1213-68. lines by cell fusion. Eur J Immunol 1976;6:
4. Santos-Aguado J, Barbosa JA, Biro A, Stro- 511-19.
minger JL. Molecular characterization of se- 22. Marks C, Marks JD. Phage libraries—a new
rologic recognition sites in the human HLA- route to clinically useful antibodies. N Engl J
A2 molecule. J Immunol 1988;141:2811-18. Med 1996;335:730-3.
5. Roitt I, Brostoff J, Male D. Immunology. 5th 23. Siegel DL. Phage display tools for automated
ed. St. Louis: Mosby, 1998. blood typing (abstract). Transfusion 2004;
6. Ono SJ, Nakamura T, Miyazaki D, et al. 44(Suppl):2A.
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.
Suggested Reading
8. Goldsby RA, Kindt TJ, Osborne BA. Kuby im- Abbas AK, Lichtman AH, Pober JS, eds. Cellular
munology. 4th ed. New York: WH Freeman and molecular immunology. 4th ed. Philadelphia:
and Company, 2000. WB Saunders, 2000.
9. Tonegawa S. Somatic generation of antibody
Anderson KC, Ness PM, eds. Scientific basis of
diversity. Nature 1983;302:575-81.
transfusion medicine. 2nd ed. Philadelphia: WB
10. Alt FW, Backwell TK, Yancopoulos GD. Devel-
Saunders, 1999.
opment of the primary antibody repertoire.
Science 1987;238:1079-87. Barclay AN, Brown MH, Law SKA, et al. The leuko-
11. Janeway CA Jr. How the immune system rec- cyte antigen factsbook. 2nd ed. San Diego, CA: Ac-
ognizes invaders. Sci Am 1993;269(3):73-9. ademic Press, 1997.
12. Russell DM, Dembic Z, Morahan G, et al. Pe-
ripheral deletion of self-reactive B cells. Na- Carroll MC. The role of complement and comple-
ture 1991;354:308-11. ment receptors in induction and regulation of im-
13. Weissman IL, Cooper MD. How the immune munity. Annu Rev Immunol 1998;16:545-68.
system develops. Sci Am 1993;269(3):64-71. Janeway CA. Immunobiology: The immune system
14. Paul WE. Infectious diseases and the immune in health and disease. 5th ed. New York: Garland
system. When bacteria, viruses and other Publishing, 2001.
pathogens infect the body, they hide in differ-
ent places. Sci Am 1993;269(3):90-7. Marchalonis JJ, Schluter SF, Bernstein RM, et al.
15. Marrack P, Lo D, Brinster R, et al. The effects Phylogenetic emergence and molecular evolution
of thymus environment on T cell develop- of the immunoglobulin family. Adv Immunol 1998;
ment and tolerance. Cell 1988;53:627-34. 70:417-506.
16. Clevers H, Alarcon B, Willeman T, Terhorst C.
Muller D. The molecular biology of autoimmunity.
The T cell receptor/CD3 complex: A dynamic
Immunol Allerg Clin North Am 1996;16:659-82.
protein ensemble. Annu Rev Immunol 1988;
6:629-62. Paul WE. Fundamental immunology. 5th ed. Phila-
17. Sakamoto M, Fujisawa Y, Nishioka K. Physio- delphia: Lippincott Williams & Wilkins, 2003.
logic role of the complement system in host
defense, disease, and malnutrition. Nutrition Paul W, Raghavan M, Bjorkman PJ. Fc receptors
1998;14:391-8. and their interactions with immunoglobulins.
18. Roitt I. Essential immunology. 8th ed. Oxford: Annu Rev Cell Dev Biol 1996;12:181-220.
Blackwell Scientific Publications, 1994. Stites DP, Terr AI, Parslow TG, eds. Medical immuno-
19. Devine DV. The regulation of complement on logy. 10th ed. Stamford, CT: Appleton & Lange, 2001.
cell surfaces. Transfus Med Rev 1991;5:123-31.
20. Semple JW, Freedman J. Recipient antigen- Vamvakas EC, Blajchman MA, eds. Immunomodu-
processing pathways of allogeneic platelet latory effects of blood transfusion. Bethesda, MD:
antigens: Essential mediators of immunity. AABB Press, 1999.

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

Adhesion molecule: Any of the many membrane Immune system: A collective term for all the
molecules expressed on white cells and en- cells and tissues involved in immune activity.
dothelial cells that allow cells to come into It includes, in addition to lymphocytes and
close apposition with each other. cells of monocyte/macrophage lineage, the
Allotypic: Variations in the amino acid structure thymus, lymph nodes, spleen, marrow, por-
of heavy and light chains unrelated to anti- tions of the liver, and the mucosa-associated
body specificity. Present in some but not all lymphoid tissue.
members of the species. Immunogen: A material capable of provoking an
Antibody: Immunoglobulin secreted by the immune response when introduced into an
plasma-cell progeny of B lymphocytes after immunocompetent host to whom it is for-
stimulation by a specific immunogen. Immu- eign.
noglobulin molecules on the surface of un- Ligand: A molecule, either free in a fluid milieu
stimulated lymphocytes serve as antigen or present on a membrane, whose three-di-
receptors. mensional configuration allows it to form a
tightly fitting complex with a cell-surface
Antigen: Any material capable of specific combi-
molecule (its receptor) of complementary
nation with antibody or with cell-surface re-
shape.
ceptors of T lymphocytes. Often used as a
synonym for “immunogen,” although some MHC Class I molecules: Heterodimeric membrane
antigens that react with products of the im- proteins determined by genes in the MHC,
mune response are not capable of eliciting consisting of a highly polymorphic α chain
an immune response. linked noncovalently with the nonpolymorphic
β 2 -microglobulin chain; these molecules
Antigen-presenting cell: A cell capable of incor-
present antigen to CD8+ T cells and are the
porating antigenic epitopes into MHC Class II
site of HLA antigens of the HLA-A, HLA-B,
molecules and displaying the epitope-MHC
and HLA-C series.
complex on its membrane.
MHC Class II molecules: Heterodimeric mem-
Clone: A population of genetically identical cells
brane proteins determined by genes in the
derived from successive divisions of a single
MHC, consisting of two transmembrane
progenitor cell.
polypeptide chains; these molecules present
Cytokine: A low-molecular-weight protein, se- antigen to CD4+ T cells and exhibit the
creted from an activated cell, that affects the HLA-DP, HLA-DQ, and HLA-DR series of an-
function or activity of other cells. tigens.
Epitope: The small portion of an immunogen, Phagocytosis: The process whereby macro-
usually 5 to 15 amino acids or 3 to 5 glyco- phages and granulocytes ingest particulate
sides, that combines specifically with the an- material present in the surrounding milieu
tigen receptor of a T or B lymphocyte. and subject it to intracellular alteration.
Idiotype: The molecular configuration unique to Receptor: A cell-membrane protein molecule
the variable portion of an antigen-receptor whose three-dimensional configuration al-
molecule, reflecting the DNA rearrangement lows it to form a tightly fitting complex with
occurring in earliest lymphocyte differentia- another molecule (called its ligand) of com-
tion and conferring upon the cell its specific- plementary shape.
ity of antigen recognition.

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection
Chapter 12
Red Cell Antigen-Antibody

Reactions and Their
Detection
D
EMONSTRATION OF RED cell an- glutination or to otherwise measure the
tigen-antibody reactions is key to reaction.
immunohematology. The combi- Hemolysis is the rupture of red cells with
nation of antibody with antigen may pro- release of intracellular hemoglobin. In-vitro
duce a variety of observable results. In antibody-mediated hemolysis depends on
blood group serology, the most com- activity of the membrane attack unit of
monly observed reactions are agglutina- complement. Hemolysis does not occur if
tion, hemolysis, and precipitation. the antigen and antibody interact in serum
Agglutination is the antibody-mediated that lacks complement, or in plasma in
clumping of particles that express antigen which the anticoagulant has chelated the
on their surface. Agglutination of red cells cations (calcium and magnesium) neces-
occurs because antibody molecules bind to sary for complement activation. In tests for
antigenic determinants on multiple adja- antibodies to red cell antigens, hemolysis is
cent red cells, linking them together to a positive result because it demonstrates
form a visible aggregate. Agglutination is the union of antibody with antigen that ac-
the endpoint for most tests involving red tivates the complement cascade. (The ac-
cells and blood group antibodies and is the tions of complement are described in
primary reaction type discussed in this Chapter 11.) Pink or red supernatant fluid
chapter. In some tests, antibody directly in a test system of serum and red cells is an
bridges the gap between adjacent cells; in important observation that may indicate
others, antibody molecules attach to, but that antigen-antibody binding has taken 12
do not agglutinate, the red cells, and an ad- place. Some antibodies that are lytic in vitro
ditional step is needed to induce visible ag- (eg, anti-Vel, anti-Lea, and anti-Jka) may
271

cause intravascular hemolysis in a transfu- Stage One: Sensitization

sion recipient. Initially, the antigen and antibody must
Precipitation is the formation of an in- come together and interact in a suitable
soluble, usually visible, complex when solu- spatial relationship. The chance of associ-
ble antibody reacts with soluble antigen. ation between antibody and antigen can
Such complexes are seen in test tubes as a be enhanced in a number of ways, such as
sediment or ring and in agar gels as a white agitation or centrifugation, or by varying
line. Precipitation is the endpoint of proce- the relative concentration of antibody and
dures such as immunodiffusion and im- antigen. As shown in Fig 12-1, antibody
munoelectrophoresis. and antigen must complement each other
Precipitation may not occur, even when with both a structural (steric) and a chem-
soluble antigen and its specific antibody ical fit.
are present. Precipitation of the antigen-an- For sensitization to occur, a noncovalent
tibody complex requires that antigen and chemical bond must form between antigen
antibody be present in optimal propor- and antibody. The forces holding antigens
tions. If antibody is present in excess, too and antibodies together are generally weak
few antigen sites exist to crosslink the mol- (compared with covalent bonds that hold
ecules and a lattice structure is not formed. molecules together) and are active only
Antigen-antibody complexes form but do over a very short distance. The antigen-an-
not accumulate sufficiently to form a visi- tibody combination is reversible, and ran-
ble lattice. This phenomenon is called a dom bonds are constantly made and bro-
prozone. ken until a state of equilibrium is attained.
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- Figure 12-1. Antigen-antibody “goodness of fit.”
tween the sensitized red cells to form the For maximum complementarity, both structural
fit and complementary distribution of chemical
lattice that constitutes agglutination. Vari- groups must be achieved. (A) Good structural fit
ous factors affect these two stages and with complementary chemical attraction.
can be manipulated to enhance (or de- (B) Chemical groups are complementary, but
crease) agglutination. The effects of en- structural fit is poor. (C) Good structural fit, but
chemical groupings are not attractive and may
hancement techniques on the two stages repel each other. (Reprinted with permission
1
cannot always be clearly differentiated. from Moore.2 )

Chapter 12: Red Cell Antigen-Antibody Reactions and Their Detection 273
Chemical Bonding
All chemical reactions are reversible. Anti-
Various types of chemical bonds are re-
gen (Ag)-antibody (Ab) reactions may be
sponsible for the binding of antibody to expressed as
antigen, including hydrogen bonds, hy-
drophobic bonds, electrostatic or ionic Ag + Ab→ AgAb
←
bonds, and van der Waals forces. These
types of chemical bonds are relevant to
The reaction proceeds until a state of
immunohematology because different types
equilibrium is reached. This is controlled
of bonds have different thermodynamic
by the rate constants of association (ka)
characteristics; they are either exothermic and dissociation (kd).
or endothermic. Thermodynamic charac- ka
teristics, in turn, may affect the serologic Ag + Ab→ AgAb

←
phenomena observed in the test system. kd
For example, carbohydrate antigens tend

By the law of mass action, the speed of
to form exothermic hydrogen bonds with
the reaction is proportional to the concen-
the antibody-combining site, so the bond
trations of the reactants and their prod-
is stronger at lower temperatures. In con- uct. The equilibrium constant (Ko) is a
trast, hydrophilic bonds formed with pro- function of these intrinsic association
tein antigens are endothermic, so these constants for the antibody being tested.
bonds are enhanced at higher reaction [AbAg] k a
temperatures. = =Ko
[Ab][Ag] k d
Equilibrium (Affinity) Constant of the

Antibody Figure 12-2. The law of mass action and the equi-
librium constant.
The equilibrium constant or affinity con-
stant (Ko) of a reaction is determined by
the relative rates of association and disso- conditions such as the temperature at
ciation (see Fig 12-2). For each antigen- which the reaction occurs, the pH and ionic
antibody reaction, the Ko varies. The Ko re- strength of the suspending medium, and
flects the degree to which antibody and the relative antigen-to-antibody concentra-
antigen associate and bind to one another tions. In laboratory tests that use agglutina-
(“goodness of fit”) and the speed of the re- tion as an endpoint, altering the physical
action. The higher the Ko value, the better conditions of the system can increase or
the association or “fit.” When the Ko is decrease the test’s sensitivity.
large, the reaction occurs more readily
and is more difficult to dissociate; such Temperature
antibodies may have a greater clinical im- Most blood group antibodies react within
portance. When the Ko is small, a higher restricted temperature ranges. Typically,
ratio of antibody to antigen may be re- these antibodies fall into two broad cate-
quired for detection. gories: those optimally reactive at “cold”
The degree of antigen-antibody “fit” is temperatures (eg, 4 to 25 C) and those op-
influenced by the type of bonds predomi- timally reactive at “warm” temperatures
nating. Hydrophobic bonds are usually as- (eg, 30 to 37 C). Antibodies that react in
sociated with higher Ko than hydrogen vitro only at temperatures below 30 C
bonds. The Ko is also affected by physical rarely cause destruction of transfused an-

tigen-positive red cells and are generally tachment, 30 to 60 minutes of incubation at

considered clinically insignificant. Many 37 C is adequate to detect most clinically
of these “cold-reactive” antibodies have been significant antibodies. For some weakly re-
found to be IgM, whereas their “warm-reactive antibodies, association may not
active” counterparts have often been found reach equilibrium at 30 minutes and ex-
to be IgG. This has led to the mistaken tending the incubation time may increase
conclusion by many that antibody class sensitivity of the test. Prolonging the incu-
determines the temperature of bonding bation time beyond 60 minutes has few dis-
(and clinical significance). Instead, how- advantages except for the delay before re-
ever, the temperature of optimal antigen- sults are available.
antibody reactivity has more to do with Incubation time at 37 C can usually be
the type of reaction and the chemical na- reduced to 10 to 15 minutes if a low-ionic-
ture of the antigen than with the antibody strength saline (LISS) solution is used (in-
class. Carbohydrate antigens are more cluding LISS additive solutions). The use of
commonly associated with “cold-reactive” water-soluble polymers such as polyethyl-
antibodies and protein antigens with ene glycol (PEG) can also reduce the neces-
“warm-reactive” antibodies. sary incubation time, although for different
reasons. (See section on Enhancement of
pH Antibody Detection and Method Section 3.)
Changes in pH can affect electrostatic
bonds. For most clinically significant Ionic Strength
+ –
blood group antibodies, optimal pH has In normal saline, Na and Cl ions cluster
not been determined but is assumed to around and partially neutralize opposite
approximate the physiologic pH range. charges on antigen and antibody mole-
Occasional antibodies, notably some ex- cules. This hinders the association of anti-
amples of anti-M, react best at a lowered body with antigen. By lowering the ionic
pH. For most routine testing, a pH around strength of the reaction medium, how-
7.0 should be used. Stored saline often has ever, this shielding effect can be weak-
a pH of 5.0 to 6.0. Buffered saline is an al- ened and electrostatic attractions enhanced.
ternative and may be particularly helpful Reducing the salt concentration of the se-
3
in solid-phase testing. rum-cell system increases the rate at
which antibody and antigen come into
proximity and may increase the amount
Incubation Time of antibody bound. Extending the incuba-
tion time in LISS systems may result in a
The time needed to reach equilibrium dif-
loss of sensitivity.4 (See section on LISS and
fers for different blood group antibodies.
LISS Additives.)
Significant variables include temperature
requirements, immunoglobulin class, and
specific interactions between antigen Antigen-Antibody Proportions
configuration and the Fab site of the anti- An excess of antigen to antibody should
body. The addition of enhancement agents result in increased antibody uptake. For
to the system can decrease the incubation inhibition or adsorption tests, such an ex-
time needed to reach equilibrium. cess of antigen is desirable. For most red
For saline systems in which antiglobulin cell tests, however, antigen excess reduces
serum is used to demonstrate antibody at- the number of antibody molecules bound

per red cell, limiting their ability to agglu- The bridges formed between antibodies
tinate. Antibody excess is, therefore, de- interlinked to antigen sites on adjacent red
sirable in most routine test systems. A cells usually result from chance collision of
commonly used ratio in red cell serology the sensitized cells. Under isotonic condi-
is 2 drops of serum to 1 drop of a 2% to tions, red cells cannot approach each other
5% red cell suspension. If the antibody is closer than a distance of 50 to 100 Å.1 IgG
weakly reactive, increasing the quantity of molecules characteristically fail to bridge
antibody present can increase the test’s this distance between red cells and cause
sensitivity. Very rarely, significant anti- sensitization without lattice formation. For
body excess may inhibit direct agglutina- larger, multivalent IgM molecules, however,
tion, producing a prozone phenomenon direct agglutination occurs easily. The loca-
comparable to what occurs with precipi- tion and density of antigen sites on the cells
tation reactions. Usually, however, in- may also allow some IgG antibodies to
creasing antibody concentration en- cause direct agglutination; A, B, M, and N
hances the sensitivity of agglutination antigens, for example, are on the outer
tests. Reducing the concentration of red edges of red cell glycoproteins and have rel-
cells from 5% to 2% or 3% doubles the se- atively high densities, allowing IgG antibod-
rum-to-cell ratio, as does adding 4 drops ies to crosslink.
of serum to the standard cell suspension. Red cells suspended in saline have a net
Sometimes, it is useful to increase the vol- negative charge at their surface and there-
ume of serum to 10 or even 20 drops, par- fore repel one another. Negatively charged
ticularly during an investigation of a molecules on the red cell membrane cause
hemolytic transfusion reaction in which mutual repulsion of red cells. This repul-
routine testing reveals no antibody. Alter- sion may be decreased by various labora-
ations in the volume of serum or plasma tory manipulations and by inherent or al-
significantly affect the ionic strength of tered red cell membrane characteristics.
test systems in which LISS has reduced Various strategies are used to overcome
the dielectric constant, so procedures this repulsion and to enhance agglutina-
must be modified so that the appropriate tion. Centrifugation physically forces the
ratio of serum to LISS is maintained. cells closer together. The indirect anti-
Chapters 18 and 19 give more details globulin test (IAT) uses antiglobulin serum
about antibody detection and pretrans- to crosslink the reaction. Other methods in-
fusion testing. clude reducing the negative charge of sur-
face molecules (eg, proteolytic enzymes),
reducing the hydration layer around the cell
Stage Two: Agglutination (eg, albumin), and introducing positively
charged macromolecules (eg, Polybrene®)
1
Once antibody molecules attach to anti- that aggregate the cells.
gens on the red cell surface, the sensitized
cells must be linked into a lattice. This al-
Inhibition of Agglutination
lows visualization of the reaction. The size
and physical properties of the antibody In agglutination inhibition tests, the pres-
molecules, the concentration of antigen ence of either antigen or antibody is de-
sites on each cell, and the distance be- tected by its ability to inhibit agglutina-
tween cells all have an effect on the devel- tion in a system with known reactants
opment of agglutinates. (see Chapter 19). For example, the saliva

from a secretor contains soluble blood molecules from polysaccharide chains.

group antigens that combine with anti-A, Sialic acid is a major contributor to the net
-B, or -H. The indicator system is a stan- negative charge at the red cell surface. Any
dardized dilution of antibody that aggluti- mechanism that reduces the net charge
nates the corresponding cells to a known should enhance red cell agglutination, and
degree. If the saliva contains blood group red cells pretreated with proteolytic en-
substance, incubating saliva with anti- zymes often show enhanced agglutination
body will wholly or partially abolish ag- by IgG molecules.
glutination of cells added to the incu-
bated mixture. The absence of expected
Polyethylene Glycol
agglutination indicates the presence of
soluble antigen in the material under test. PEG is a water-soluble linear polymer
Agglutination of the indicator cells is a used as an additive to potentiate anti-
negative result. gen-antibody reactions.5 It 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
Enhancement of Antibody closer proximity, resulting in increased
Detection cell-antibody collisions and subsequent
antibody binding. Multiple studies have
Albumin Additives shown that PEG increases the detection of
potentially clinically significant antibod-
Although used routinely for many years as ies and decreases the detection of clini-
an enhancement medium, albumin itself cally insignificant antibodies.6
probably does little to promote antibody Anti-IgG is usually the antihuman globu-
uptake (Stage 1). Much of the enhance- lin (AHG) reagent of choice with PEG test-
ment effect attributed to albumin may be ing, to avoid false-positive reactions with
due to its attributes as a low-ionic-strength some polyspecific AHG reagents. Commer-
buffer. Albumin may influence agglutina- cially available PEG reagents may be pre-
tion by reducing the repulsion between pared in a LISS solution. PEG can be used
cells, thus predisposing antibody-coated in tests with eluates, as well as with serum
cells to agglutinate. Bovine serum albu- or plasma. PEG can enhance warm-reactive
min is available as solutions of 22% or autoantibodies and thus may be advanta-
30% concentration. geous in detecting weak serum autoanti-
bodies for diagnostic purposes. On the
Enzymes other hand, this enhancement may be dis-
The proteolytic enzymes used most often advantageous when trying to detect allo-
in immunohematology laboratories are antibodies in the presence of autoanti-
bromelin, ficin, papain, and trypsin. While bodies. In such cases, testing the serum by
enhancing agglutination by some anti- LISS or a saline IAT may allow for the detec-
bodies, the enzymes destroy certain red tion or exclusion of alloantibodies without
cell antigens, notably M, N, S, Fya, and Fyb. interference from autoantibodies.
Proteolytic enzymes reduce the red cell Centrifugation of PEG with test serum
surface charge by cleaving polypeptides and red cells before washing should be
containing negatively charged sialic acid avoided because the nonspecific aggregates

generated by PEG may not disperse. After ment of antibodies that did not produce
incubation with PEG, test cells should be agglutination. This test uses antibody to
washed immediately in saline for the human globulins and is known as the
antiglobulin test. antiglobulin test. It was first used to dem-
Precipitation of serum proteins when onstrate antibody in serum, but later the
PEG is added has been reported; this prob- same principle was used to demonstrate
lem appears to be related to elevated serum in-vivo coating of red cells with antibody
globulin levels.6 This problem becomes ap- or complement components. As used in
parent when the IgG-coated red cells are immunohematology, antiglobulin testing
nonreactive. At least four washes of the red generates visible agglutination of sensi-
cells at the antiglobulin phase, with agita- tized red cells. The direct antiglobulin test
tion, will ensure that the red cells are fully (DAT) is used to demonstrate in-vivo sen-
resuspended and will serve to prevent this sitization of red cells. An IAT is used to
problem from occurring. demonstrate in-vitro reactions between
red cells and antibodies that sensitize, but
LISS and LISS Additives do not agglutinate, cells that express the
LISS (approximately 0.03 M) greatly in- corresponding antigen.
creases the speed of antibody sensitiza-
tion of red cells, compared with normal Principles of the Antiglobulin Test
saline (approximately 0.17 M). To prevent
All antibody molecules are globulins. Ani-
lysis of red cells at such a low ionic strength,
mals injected with human globulins pro-
a nonionic substance such as glycine is
duce antibody to the foreign protein. Af-
incorporated in the LISS.
ter the animal serum is adsorbed to remove
Most laboratories use a LISS additive re-
unwanted agglutinins, it will react specifi-
agent, rather than LISS itself. These com-
mercially available LISS additives may con- cally with human globulins and can be
tain albumin in addition to ionic salts and called AHG serum. AHG sera with varying
buffers. LISS solutions increase the rate of specificities can be produced, notably,
antibody association (Stage 1) when vol- anti-IgG and antibodies to several com-
ume proportions are correct. (See Methods plement components. Hybridoma tech-
3.2.2 and 3.2.3.) Increasing the volume of niques are used for the manufacture of
serum used in a test will increase the ionic most AHG. These techniques are more
strength of the test; hence, any alteration in fully described in Chapter 11.
prescribed volumes of serum used requires Anti-IgG combines mainly with the Fc
adjustment of the LISS volume or omission portion of the sensitizing antibody mole-
of LISS. For this reason, the use of LISS for cules, not with any epitopes native to the
routine titration studies and for some other red cell (see Fig 12-3). The two Fab sites of
tests is problematic. When LISS is used as the AHG molecule form a bridge between
an additive reagent, the manufacturer’s in- adjacent antibody-coated cells to produce
structions must be followed. 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
The Antiglobulin Test bound globulin.
7
In 1945, Coombs, Mourant, and Race de- AHG will react with human antibodies
scribed procedures for detecting attach- and complement molecules that are bound

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
Figure 12-3. The antiglobulin reaction. Anti-
human IgG molecules are shown reacting with has occurred.
the Fc portion of human IgG coating adjacent red Methods have been developed that obvi-
cells (eg, anti-D coating D-positive red cells). ate the need to wash coated red cells before
adding antiglobulin reagent. Column ag-
to red cells or are present, free, in serum. glutination technology, described later in
Unbound globulins may react with AHG, this chapter, is an example. It uses a micro-
causing false-negative antiglobulin tests. column filled with mixtures of either glass
Unless the red cells are washed free of un- beads or gel, buffer, and sometimes re-
bound proteins before addition of AHG se- agents and can be used for direct or indi-
rum, the unbound globulins may neutralize rect antiglobulin procedures. Density barri-
AHG and cause a false-negative result. ers allow separation of test serum (or plasma)
from red cells, making a saline washing
phase unnecessary.
Direct Antiglobulin Testing
The DAT is used to demonstrate in-vivo
coating of red cells with antibodies or Antiglobulin Reagents
complement, in particular IgG and C3d. Monospecific antibodies to human globu-
Washed red cells from a patient or donor lins can be prepared by injecting animals
are tested directly with AHG reagents (see with purified IgG, IgA, IgM, C3, or C4.
Method 3.6). The DAT is used in investi- Such sera require adsorption to remove
gating autoimmune hemolytic anemia unwanted (eg, heterophile) antibodies
(AIHA), drug-induced hemolysis, hemolytic from the monospecific AHG reagent.
disease of the fetus and newborn, and These animal-made antisera are poly-
alloimmune reactions to recently trans- clonal in nature. Monospecific mono-
fused red cells. clonal reagents can also effectively be
prepared from hybridomas (see Chapter
11). Monospecific animal or hybridoma-
Indirect Antiglobulin Testing derived antibodies can be combined into
In indirect antiglobulin procedures, se- reagent preparations containing any de-
rum (or plasma) is incubated with red sired combination of specificities, or dif-

ferent clones all recognizing the same an- tine compatibility tests and antibody de-
tigen specificity can be combined into a tection. These reagents contain antibody
single reagent. Thus, reagents may be to human IgG and to the C3d component
polyclonal, monoclonal, blends of mono- of human complement. Other comple-
clonal, or blends of monoclonal and poly- ment antibodies may be present, includ-
clonal antibodies. ing anti-C3b, -C4b, and -C4d. Currently
The Food and Drug Administration available, commercially prepared, poly-
(FDA) has established definitions for a vari- specific antiglobulin sera contain little, if
8
ety of AHG reagents, as shown in Table 12-1. any, activity against IgA and IgM heavy
Antisera specific for other immunoglobulins chains. However, some reagents may re-
(IgA, IgM) or subclasses (IgG1, IgG3, etc) exact with IgA or IgM molecules because the
ist but are rarely standardized for routine polyspecific mixture may react with lambda
test tube methods and must be used with and kappa light chains, which are present
rigorous controls. in immunoglobulins of all classes.
Because most clinically significant anti-
Polyspecific AHG bodies are IgG, the most important func-
Polyspecific AHG reagents are used for tion of polyspecific AHG, in most procedures,
DATs and, in some laboratories, for rou- is detection of IgG. The anticomplement
Table 12-1. Antihuman Globulin Reagents
Antibody Designation on
Container Label Definition*
(1) Anti-IgG, -C3d; Contains anti-IgG and anti-C3d (may contain other
Polyspecific 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 Contains only antibodies reactive against human gamma
chains 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.
8
*As defined by the FDA.

component has limited usefulness in cross- bound to red cells by cold-reactive anti-
matching and in antibody detection be- bodies that are not clinically significant.
cause antibodies detectable only by their
ability to bind complement are quite rare. Anti-C3b, -C3d
Anti-C3d activity is important, however, for
Anti-C3b, -C3d reagents prepared by ani-
the DAT, especially in the investigation of
mal immunization contain no activity
AIHA. In some patients with AIHA, C3d
against human immunoglobulins and are
may be the only globulin detectable on
used in situations described for anti-C3d.
their red cells.9
This type of anti-C3d characteristically re-
acts with C3b and possibly other epitopes
present on C3-coated red cells. Murine
Monospecific AHG Reagents monoclonal anti-C3b, -C3d reagent is a
blend of hybridoma-derived antibodies.
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, Role of Complement in Antiglobulin
and anti-C4d, but these products are not Reactions
generally available. If the DAT with a Complement components may attach to
polyspecific reagent reveals globulins on red cells in vivo or in vitro by one of two
red cells, monospecific AHG reagents are mechanisms:
used to characterize the coating proteins. 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-
Anti-IgG ment activation by the antigen-anti-
Reagents labeled “anti-IgG” contain no body complex.
anticomplement activity. The major com- 2. Immune complexes, not specific for
ponent of anti-IgG is antibody to human red cell antigens, may be present in
gamma heavy chains, but unless labeled plasma and may activate comple-
as “heavy-chain-specific,” these reagents ment components that adsorb onto
may exhibit some reactivity with light red cells in a nonspecific manner.
chains, which are common to all immu- Attachment of complement to the
noglobulin classes. An anti-IgG reagent membrane of cells not involved in
not designated “heavy-chain-specific” the specific antigen-antibody reac-
must be considered theoretically capable tion is often described as “innocent
of reacting with light chains of IgA or IgM. bystander” complement coating.
A positive DAT with such an anti-IgG does Red cells coated with elements of the
not definitively prove the presence of IgG, complement cascade may or may not un-
although it is quite rare to have an in-vivo dergo hemolysis. If the cascade does not go
coating with IgA or IgM in the absence of to completion, the presence of bound early
IgG. Many workers prefer anti-IgG over components of the cascade can be detected
polyspecific AHG in antibody detection by anticomplement reagents. The compo-
and compatibility tests because anti-IgG nent most readily detected is C3 because
AHG does not react with complement several hundred C3 molecules may be

bound to the red cell by the attachment of from the cells, leaving complement
only a few antibody molecules. C4 coating components firmly bound to the red
also can be detected, but C3 coating has cell membrane. The component
more clinical significance. usually detected by AHG reagents is
C3d.
4. Immune complexes that form in the
plasma and bind weakly and non-
Complement as the Only Coating Globulin specifically to red cells may cause
Complement alone, without detectable im- complement coating. The activated
munoglobulin, may be present on washed complement remains on the red cell
red cells in certain situations. surface after the immune complexes
1. IgM antibodies reacting in vitro oc- dissociate. C3 remains as the only
casionally attach to red cell antigens detectable surface globulin.
without agglutinating the cells, as is
seen with IgM antibodies to Lewis
antigens. IgM coating is difficult to IgG-Coated Cells
demonstrate in AHG tests, partly be-
cause IgM molecules tend to disso- The addition of IgG-coated cells to nega-
ciate during the washing process tive antiglobulin tests (used to detect IgG)
and partly because polyspecific AHG is required for antibody detection and
contains little if any anti-IgM activ- crossmatching procedures. 1 2 ( p 3 3 ) These
ity. IgM antibodies may activate sensitized red cells should react with the
complement, and the IgM reactivity antiglobulin sera, verifying that the AHG
can be demonstrated by identifying reagent was functional. Reactivity with
the several hundred C3 molecules IgG-sensitized cells demonstrates that, in-
bound to the cell membrane near deed, AHG was added and it had not been
the site of antibody attachment. neutralized. Tests need to be repeated if
2. About 10% to 20% of patients with the IgG-coated cells are not reactive.
warm AIHA have red cells with a pos- Testing with IgG-sensitized cells does
itive DAT due to C3 coating alone.10 not detect all potential failures of the
No IgG, IgA, or IgM coating is de- antiglobulin test.13-15 Partial neutralization of
monstrable with routine procedures, the AHG may not be detected at all, partic-
although some specimens may be ularly if the control cells are heavily coated
coated with IgG at levels below the with IgG. Errors in the original test, such as
detection threshold for the DAT. omission of test serum, improper centri-
3. In cold agglutinin syndrome, the fuge speed, or inappropriate concentra-
cold-reactive autoantibody can react tions of test red cells, may yield negative
with red cell antigens at tempera- test results but positive results with control
tures up to 32 C.11 Red cells passing cells. Oversensitized control cells may ag-
through vessels in the skin at this glutinate when centrifuged.
temperature become coated with Complement-coated cells can also be
autoantibody, which activates com- prepared and are commercially available.
plement. If the cells escape hemoly- They can be used in some cases to control
sis, they return to the central circula- tests with complement-specific reagents.
tion, where the temperature is 37 C, Some sources of error in antiglobulin
and the autoantibody dissociates tests are listed in Tables 12-2 and 12-3.

Table 12-2. Sources of Error in Antiglobulin Testing—False-Negative Results
Neutralization of Antihuman Globulin (AHG) Reagent

■ Failure to wash cells adequately to remove all serum/plasma. Fill tube at least ¾ full of saline for
each wash. Check dispense volume of automated washers.
■ If increased serum volumes are used, routine wash may be inadequate. Wash additional times or
remove serum before washing.
■ Contamination of AHG by extraneous protein. Do not use finger or hand to cover tube.
Contaminated droppers or wrong reagent dropper can neutralize entire bottle of AHG.
■ High concentration of IgG paraproteins in test serum; protein may remain even after multiple
washes.13
Interruption in Testing
■ Bound IgG may dissociate from red cells and either leave too little IgG to detect or may
neutralize AHG reagent.
■ Agglutination of IgG-coated cells will weaken. Centrifuge and read immediately.
Improper Reagent Storage

■ AHG reagent may lose reactivity if frozen. Reagent may become bacterially contaminated.
■ Excess heat or repeated freezing/thawing may cause loss of reactivity of test serum.
■ Reagent red cells may lose antigen strength on storage. Other subtle cell changes may cause
loss of reactivity.
Improper Procedures
■ Overcentrifugation may pack cells so tightly that agitation required to resuspend cells breaks up
agglutinates. Undercentrifugation may not be optimal for agglutination.
■ Failure to add test serum, enhancement medium, or AHG may cause negative test.
■ Too heavy a red cell concentration may mask weak agglutination. Too light suspension may be
difficult to read.
■ Improper/insufficient serum:cell ratios.
Complement
■ Rare antibodies, notably some anti-Jka, -Jkb, may only be detected when polyspecific AHG is
used and active complement is present.
Saline
■ Low pH of saline solution can decrease sensitivity.3 Optimal saline wash solution for most
antibodies is pH 7.0 to 7.2.
■ Some antibodies may require saline to be at specific temperature to retain antibody on the cell.
Use 37 C or 4 C saline.

Table 12-3. Sources of Error in Antiglobulin Testing—False-Positive Results
Cells Agglutinated Before Washing

■ If potent agglutinins are present, agglutinates may not disperse during washing. Observe cells
before the addition of antihuman globulin (AHG) or use control tube substituting saline for AHG;
reactivity before the addition of AHG or in saline control invalidates AHG reading.
Particles or Contaminants
■ Dust or dirt in glassware may cause clumping (not agglutination) of red cells. Fibrin or
precipitates in test serum may produce cell clumps that mimic agglutination.
Improper Procedures
■ Overcentrifugation may pack cells so tightly that they do not easily disperse and appear positive.
■ Centrifugation of test with polyethylene glycol or positively charged polymers before washing
may create clumps that do not disperse.
Cells That Have Positive Direct Antiglobulin Test (DAT)

■ Cells that are positive by DAT will be positive in any indirect antiglobulin test. Procedures for
removing IgG from DAT-positive cells are given in Methods 2.13 and 2.14.
Complement
■ Complement components, primarily C4, may bind to cells from clots or from CPDA-1 donor
segments during storage at 4 C and occasionally at higher temperatures. For DATs, use red cells
anticoagulated with EDTA, ACD, or CPD.
■ Samples collected in tubes containing silicone gel may have spurious complement attachment.14
■ Complement may attach to cells in specimens collected from infusion lines used to administer
dextrose-containing solutions. Strongest reactions are seen when large-bore needles are used or
when sample volume is less than 0.5 mL.15
Other Methods to Detect Solid-Phase Red Cell Adherence Tests

Solid-phase microplate techniques use
Antigen-Antibody Reactions immobilized antigen or antibody. In a di-
The following methods represent alterna- rect test, antibody is fixed to a microplate
tives to traditional tube testing and use of well and red cells are added. If the cells
antiglobulin serum. Some methods do express the corresponding antigen, they
not allow detection of both IgM and IgG will adhere across the sides of the well; if
antibodies and may not provide informa- no antigen-antibody reaction occurs, the
tion on the phase and temperature of re- red cells pellet to the bottom of the well
activity of antibodies that is obtained when centrifuged.16 An indirect test uses
when traditional tube tests are used. red cells of known antigenic composition

bound to a well. The test sample is added Column Agglutination Technology

to the red-cell-coated wells and allowed
Various methods have been devised in
to react with the cells, after which the
which red cells are filtered through a col-
plates are washed free of unbound pro-
umn containing a medium that separates
teins. The indicator for attached antibody
selected red cell populations. As commer-
is a suspension of anti-IgG-coated red
cially prepared, the systems usually em-
cells. The reaction is positive if the indica-
ploy a card or strip of microtubes, rather
tor cells adhere across the sides of the than conventional test tubes. They allow
well. If they pellet to the bottom when simultaneous performance of several tests.
centrifuged, it demonstrates that no anti- Usually, a space or chamber at the top of
gen-antibody reaction has occurred (see each column is used for red cells alone or
Fig 12-4).17 In the indirect test, isolated to incubate red cells and serum or plasma.
membrane components (eg, specific pro- As the cells pass through the column
teins), rather than intact cells, can be af- (usually during centrifugation), the column
fixed to the microwell. Solid-phase at- medium separates agglutinated from
tachment of antigen or antibody is an unagglutinated red cells, based on aggre-
integral part of other tests, such as the gate size.17 Alternatively, in some tests,
monoclonal antibody-specific immobili- specific antisera or proteins can be in-
zation of erythrocyte antigens (MAIEA) cluded in the column medium itself; cells
assay, discussed below. Solid-phase sys- bearing a specific antigen are selectively
tems have also been devised for use in de- captured as they pass through the me-
tecting platelet antibodies and in tests for dium. When the column contains anti-
syphilis, cytomegalovirus, and hepatitis B globulin serum or a protein that specifi-
surface antigen.17-20 cally binds immunoglobulin, the selected
Figure 12-4. An indirect solid-phase test. A monolayer of red cells is affixed to a microwell (1). Test
serum is added. If antibody (2) is present, it binds to antigens on the affixed red cells (3). Indicator red
cells coated with IgG and anti-IgG (4) are added. The anti-IgG portion binds to any antibody attached to
the fixed red cells (5). In a positive test, the indicator red cells are effaced across the microwell. In a
negative test, the indicator cells do not bind but pellet to the center of the well when centrifuged. Weak
reactions give intermediate results.

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,
the need for saline washing for anti-
21
globulin tests can be eliminated.
Typically in column tests, negative test
cells pellet to the bottom of the column. In
positive tests, the cells are captured at the
top, or in the body, of the column. An 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
Figure 12-5. Gel test.
cases, documented by photocopying. Col-
umn tests generally have sensitivity similar
formance of multiple tests, using solid-
to LISS antiglobulin methods, but such
phase, gel-column, and/or microtiter
tests have reportedly performed less well in
plate technology. The systems are con-
detecting weak antibodies, especially those
trolled by a computer program and posi-
in the ABO system.22
tive sample identity can be ensured by
In 1986, Lapierre et al developed a pro-
barcode technology. The test system’s
cess leading to a technology that uses a col-
computer may be integrated into a labo-
umn of gel particles.23 As commercially pre-
ratory’s information system for results re-
pared, the gel test uses six microtubes
porting. Automated test systems may be
instead of test tubes, contained in what is
particularly useful in institutions per-
called a card or strip. The gel particles func-
forming large volumes of patient or donor
tion as filters that trap red cell agglutinates
testing.
when the cards are centrifuged. Gels con-
taining antiglobulin serum are used to cap-
ture sensitized, but unagglutinated, cells. Immunofluorescence
Gels with various antisera can be used for Immunofluorescence testing allows iden-
phenotyping cells (see Fig 12-5). tification and localization of antigens
In another column agglutination tech- inside or on the surface of cells. A fluoro-
nology, a column of glass microbeads in a chrome such as fluorescein or phyco-
diluent is used instead of gel. As with the erythrin can be attached to an antibody
gel test, the beads may either entrap agglu- molecule, without altering its specificity
tinated cells or antisera, such as anti-IgG, or its ability to bind antigen. Attachment
can be added to the diluent.24 of fluorescein-labeled antibody to cellular
antigen makes the antibody-coated cells
appear brightly visible yellow-green or
Automated Testing Platforms
red (depending on the fluorochrome).
Several automated devices have been de- Immunofluorescent antibodies can be
veloped for the detection of antigen-anti- used in direct or indirect procedures, with
body reactions. All steps in the testing the fluorescence analogous to agglutination
process, from sample aliquotting to reas an endpoint. In a direct test, the fluores-
porting results, are performed by the sys- cein-labeled antibody is specific for a single
tem. These test systems permit the per- antigen of interest. In an indirect test,

fluorescein-labeled antiglobulin serum is antibody also attached). Conjugated anti-

added to cells that have been incubated human antibody is added, which reacts
with an unlabeled antibody of known spec- with the bound human antibody and
ificity. Immunofluorescent techniques were gives an ELISA-readable reaction. Thus
initially used to detect antigens in or on far, this method has been used primarily
lymphocytes or in tissue sections. More re- to isolate specific membrane structures
cently, immunofluorescent antibodies have for blood group antigen studies.26,27
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 References
cell-bound IgG, and to distinguish homozy- 1. van Oss CJ. Immunological and physio-
gous from heterozygous expression of chemical nature of antigen-antibody interac-
tions. In: Garratty G, ed. Immunobiology of
blood group antigens.25 transfusion medicine. New York: Marcel
Dekker, Inc., 1994:327-64.
Enzyme-Linked Immunosorbent Assay 2. Moore BPL. Antibody uptake: The first stage
of the hemagglutination reaction. In: Bell CA,
Enzyme-linked immunosorbent assays ed. A seminar on antigen-antibody reactions
(ELISAs) are used to measure either anti- revisited. Arlington, VA: AABB, 1982:47-66.
3. Rolih S, Thomas R, Fisher E, Talbot J. Anti-
gen or antibody. Enzymes such as alkaline body detection errors due to acidic or un-
phosphatase can be bound to antibody buffered saline. Immunohematology 1993;
molecules without destroying either the 9:15-18.
antibody specificity or the enzyme activ- 4. Jørgensen J, Nielsen M, Nielsen CB, Nørmark
J. The influence of ionic strength, albumin
ity. ELISAs have been used to detect and and incubation time on the sensitivity of the
measure cell-bound IgG and to demon- indirect Coombs’ test. Vox Sang 1980;36:186-
strate fetomaternal hemorrhage. When 91.
5. Nance SJ, Garratty G. Polyethylene glycol: A
red cells are examined, the test often is
new potentiator of red blood cell antigen-an-
called an enzyme-linked antiglobulin test tibody reactions. Am J Clin Pathol 1987;87:
(ELAT). 633-5.
6. Issitt PD, Anstee DJ. Applied blood group se-
rology. 4th ed. Durham, NC: Montgomery
Monoclonal Antibody-Specific Scientific Publications, 1998:47-8.
Immobilization of Erythrocyte Antigens 7. Coombs RRA, Mourant AE, Race RR. A new
Assay test for the detection of weak and “incom-
plete” Rh agglutinins. Br J Exp Pathol 1945;
In the MAIEA assay, red cells are incu- 26:255-66.
bated with two antibodies. One contains 8. Code of federal regulations. Title 21 CFR
human alloantibody to a blood group an- Printing Office, 2004 (revised annually).
tigen and the other is a nonhuman (usu- 9. Packman CH. Acquired hemolytic anemia
ally mouse monoclonal) antibody that re- due to warm-reacting autoantibodies. In:
acts with a different portion of the same Beutler E, Lichtman MA, Coller BS, et al, eds.
Williams’ hematology. 6th ed. New York:
membrane protein. The red cells are lysed McGraw Hill, 2001:639-48.
and the membrane solubilized, then 10. Sokol RJ, Hewitt S, Stamps BK. Autoimmune
added to a microwell coated with goat haemolysis: An 18-year study of 865 cases re-
ferred to a regional transfusion centre. Br
antimouse antibody. This antibody then
Med J 1981;282:2023-7.
captures the mouse antibody attached to 11. Packman CH. Cryopathic hemolytic syn-
the membrane protein (with the human dromes. In: Beutler E, Lichtman MA, Coller

BS, et al, eds. Williams’ hematology. 6th ed. 21. Malyska H, Weiland D. The gel test. Lab Med
New York: McGraw-Hill, 2001:649-55. 1994;25:81-5.
12. Silva MA, ed. Standards for blood banks and 22. Phillips P, Voak D, Knowles S, et al. An expla-
transfusion services. 23rd ed. Bethesda, MD: nation and the clinical significance of the
AABB, 2005. failure of microcolumn tests to detect weak
13. Ylagen ES, Curtis BR, Wildgen ME, et al. In- ABO and other antibodies. Transfus Med
validation of antiglobulin tests by a high ther- 1997;7:47-53.
mal amplitude cryoglobulin. Transfusion 23. Lapierre Y, Rigal D, Adam J, et al. The gel test:
1990;30:154-7. A new way to detect red cell antigen-antibody
14. Geisland JR, Milam JD. Spuriously positive di- reactions. Transfusion 1990;30:109-13.
rect antiglobulin tests caused by silicone gel. 24. Reis KJ, Chachowski R, Cupido A, et al. Col-
Transfusion 1980;20:711-13. umn agglutination technology: The antiglo-
15. Grindon AJ, Wilson MJ. False-positive DAT bulin test. Transfusion 1993;33:639-43.
caused by variables in sample procurement. 25. Garratty G, Arndt P. Applications of flow cyto-
Transfusion 1981;21:313-14. fluorometry to transfusion science. Transfu-
16. Rolih SD, Eisinger RW, Moheng JC, et al. Solid sion 1994;35:157-78.
phase adherence assays: Alternatives to con- 26. Petty AC. Monoclonal antibody-specific im-
ventional blood bank tests. Lab Med 1985;16: mobilisation of erythrocyte antigens (MAIEA).
766-70. A new technique to selectively determine an-
17. Walker P. New technologies in transfusion tigenic sites on red cell membranes. J Immunol
medicine. Lab Med 1997;28:258-62. Methods 1993;161:91-5.
18. Plapp FV, Sinor LT, Rachel JM, et al. A solid 27. Petty AC, Green CA, Daniels GL. The mono-
phase antibody screen. Am J Clin Pathol 1984; clonal antibody-specific antigens assay
82:719-21. (MAIEA) in the investigation of human red-
19. Rachel JM, Sinor LT, Beck ML, Plapp FV. A cell antigens and their associated membrane
solid-phase antiglobulin test. Transfusion proteins. Transfus Med 1997;7:179-88.
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.

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens
Chapter 13
13
ABO, H, and Lewis Blood

Groups and Structurally
Related Antigens
T
HE ABO, AS well as the H, Lewis, I, found that serum from group A individu-
and P, blood group antigens reside als agglutinated the red cells from group B
on structurally related carbohydrate individuals, and, conversely, the serum from
molecules. The antigens arise from the group B individuals agglutinated group A
action of specific glycosyltransferases that red cells. A and B were thus the first red
add individual sugars sequentially to sites cell antigens to be discovered. Red cells
on short chains of sugars (oligosaccha- that were not agglutinated by the serum
rides) on common precursor substances. In- of either the group A or group B individu-
teractions of the ABO, Hh, Sese, and Lele als were later called group O; the serum
gene products affect the expression of the from group O individuals agglutinated the
ABO, H, and Lewis antigens. Refer to Ap- red cells from both group A and group B
pendix 6 for ISBT numbers and nomencla- individuals. Von Decastello and Sturli in
ture for the blood groups. 1902 discovered the fourth group, AB.
4
The importance of Landsteiner’s discov-

ery is the recognition that antibodies to A
and B antigens are present when the cor-
The ABO System responding antigen is missing. Routine ABO
The ABO system was discovered when Karl typing procedures developed from these and
5
Landsteiner recorded the agglutination of later studies.
human red cells by the sera of other indi- The ABO antigens and antibodies remain
viduals in 19001 and, in the following year, the most significant for transfusion practice.
detailed the patterns of reactivity as three It is the only blood group system in which
types, now called groups A, B, and O.2,3 He the reciprocal antibodies (see Table 13-1)
289

Table 13-1. Routine ABO Typing
Reaction of Cells Reaction of Serum Interpre- Incidence (%) in

Tested with Tested Against tation US Population
ABO
Anti-A Anti-B A1 Cells B Cells O Cells Group Whites Blacks
0 0 + + 0 0 45 49
+ 0 0 + 0 A 40 27
0 + + 0 0 B 11 20
+ + 0 0 0 AB 4 4
+ = agglutination; 0 = no agglutination.
are consistently and predictably present in at the cellular level to form the H antigen
the sera of most people who have had no on red cells. The amorph, h, is very rare.
exposure to human red cells. Due to these The active allele at the Se locus, Se, pro-
antibodies, transfusion of ABO-incompati- duces a transferase that also acts to form H
ble blood may cause severe intravascular antigen, but primarily in secretions such
hemolysis as well as the other manifesta- as saliva.6 Eighty percent of individuals are
tions of an acute hemolytic transfusion re- secretors. The amorphic allele is se. The
action (see Chapter 27). Testing to detect ABO enzymes produced by H and Se alleles are
incompatibility between a recipient and the both fucosyltransferases, but they have
donor is the foundation on which all pre- slightly different activity. H antigen on red
transfusion testing is based. cells and in secretions is the substrate for
the formation of A and B antigens.
Genetics and Biochemistry There are three common alleles at the
The genes for all of the carbohydrate anti- ABO locus on chromosome 9, A, B, and O.7
gens discussed in this chapter encode spe- The A and B alleles encode glycosyltrans-
cific glycosyltransferases, enzymes that ferases that produce the A and B antigens
transfer specific sugars to the appropriate respectively; the O allele does not encode a
carbohydrate chain acceptor; thus, the an- functional enzyme.8 The red cells of group
tigens are indirect products of the genes. O individuals lack A and B antigens but carry
Genes at three separate loci (H, Se, and an abundant amount of H antigen, the un-
ABO) control the occurrence and the loca- converted precursor substance on which A
tion of the A and B antigens. The H and Se and B antigens are built.
(secretor) loci, officially named FUT1 and The carbohydrate chains (oligosaccha-
FUT2, respectively, are on chromosome rides) that carry ABH antigens can be at-
19 and are closely linked. Each locus has tached to either protein (glycoprotein),
two recognized alleles, one of which has sphingolipid (glycosphingolipid), or lipid
no demonstrable product and is consid- (glycolipid) carrier molecules. Glycopro-
ered an amorph. The active allele at the H teins and glycosphingolipids carrying A or
locus, H, produces a transferase that acts B antigens are integral parts of the mem-

Chapter 13: ABO, H, and Lewis Blood Groups and Structurally Related Antigens 291
branes of red cells, epithelial cells, and galactosamine (or GalNAc) to H to make A
endothelial cells (Fig 13-1) and are also antigen on red cells. The B allele encodes
present in soluble form in plasma. Glyco- galactosyltransferase that adds D-galactose
proteins secreted in body fluids such as sa- (or Gal) to H to make B antigen. Group AB
liva contain molecules that may, if the per- individuals have alleles that make transfer-
son possesses an Se allele, carry A, B, and H ases to add both GalNAc and Gal to the pre-
antigens. A and B antigens that are unat- cursor H antigen. Attachment of the A or B
tached to carrier protein or lipid molecules immunodominant sugars diminishes the se-
are also found in milk and urine as free oli- rologic detection of H antigen so that the ex-
gosaccharides. pressions of A or B antigen and of H antigen
The transferases encoded by the A, B, H, are inversely proportional. Rare individuals
and Se alleles add a specific sugar to a pre- who lack both H and Se alleles (genotype hh
cursor carbohydrate chain. The sugar that and sese) have no H and, therefore, no A or B
is added is referred to as immunodominant antigens on their red cells or in their secre-
because when it is removed from the structions (see Oh phenotype below). However, H,
ture, the specific blood group activity is A, and B antigens are found in the secretions
lost. The sugars can be added only in a se- of some hh individuals who appear, through
quential manner. H structure is made first, family studies, to possess at least one Se allele
then sugars for A and B antigens are added (see para-Bombay phenotype below).
to H. The H and Se alleles encode a fuco- The oligosaccharides to which the A or B
syltransferase that adds fucose (Fuc) to the immunodominant sugars are attached may
precursor chain; thus, fucose is the im- exist as simple repeats of a few sugar mole-
munodominant sugar for H (see Fig 13-2). cules linked in linear fashion, or as part of
The A allele encodes N-acetyl-galactosa- more complex structures, with many sugar
minyltransferase that adds N-acetyl-D- residues linked in branching chains. Differ-
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.)

terminal disaccharide; there are at least six

of these types of disaccharide linkages.9
Type 1 chains and Type 2 chains differ in
the linkage of the terminal Gal to GlcNAc
disaccharide (see Fig 13-3). Type 1 A, B, and
H structures are present in secretions, plasma,
and endodermally derived tissues. They are
not synthesized by red cells but are 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
10
cells from group A individuals. They are
synthesized by the addition of Gal to the
terminal GalNAc Type 2 A chains, thus
forming Type 3 H; Type 3 H chains are sub-
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).
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.
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
Figure 13-3. Type 1 and 2 oligosaccharide chains
carbohydrate chains that are characterized differ only in the linkage between the GlcNAc and
by different linkages and composition of the the terminal Gal.

Figure 13-4. Type 3 A antigen structure.
Active alleles H and Se, of the FUT1 and ies frequently produce weaker reactions
FUT2 genes, encode fucosyltransferases with with red cells from newborns than with
a high degree of homology. The enzyme red cells from adults. Although A and B
produced by H acts primarily on Type 2 antigens can be detected on the red cells
chains, which are prevalent on the red cell of 5- to 6-week-old embryos, A and B anti-
membranes. The enzyme produced by Se gens are not fully developed at birth, pre-
prefers, but is not limited to, Type 1 chains sumably because the branching carbohy-
and acts primarily in the secretory glands. drate structures develop gradually. By 2 to
4 years of age, A and B antigen expression
ABO Genes at the Molecular Level is fully developed and remains fairly con-
11 stant throughout life.
Yamamoto et al have shown that A and B
alleles differ from one another by seven
nucleotide differences, four of which re-
sulted in amino acid substitutions at posi- Subgroups
tions 176, 235, 266, and 268 in the protein
ABO subgroups are phenotypes that differ
sequence of the A and B transferases. Re-
in the amount of antigen carried on red
cent crystallography of A- and B-transfer-
cells and, for secretors, soluble antigen
ases demonstrated the role of these criti-
12 present in the saliva. Subgroups of A are
cal amino acids in substrate recognition.
more commonly encountered than sub-
The initial O allele examined had a single
groups of B. The two principal subgroups
nucleotide deletion that resulted in a frame
of A are A1 and A2. Red cells from A1 and A2
shift and premature stop codon resulting in
persons both react strongly with reagent
the predicted translation of a truncated (ie,
anti-A in direct agglutination tests. The
inactive) protein. Subsequently, other O al-
serologic distinction between Al and A2
leles have been identified as well as muta-
cells can be determined by testing with
tions of A and B alleles that result in weak-
anti-A1 lectin (see anti-A1 below). There is
ened expression of A and B antigens (reviewed
both a qualitative and quantitative differ-
elsewhere).13,14 The A2 allele encodes a pro-
ence between A1 and A2.15 The A1-trans-
tein with an additional 21 amino acids.
ferase is more efficient at converting H
substance into A antigen and is capable of
Antigens making the repetitive Type 3 A structures.
Agglutination tests are used to detect A and There are about 10.5 × 105 A antigen sites
on adult A1 red cells, and about 2.21 × 10
5
B antigens on red cells. Reagent antibod-

9
A antigen sites on adult A2 red cells. Ap- by anti-A,B from group O persons. Ax red
proximately 80% of group A or group AB cells may react with some monoclonal anti-A
individuals have red cells that are aggluti- reagents, depending on which monoclonal
nated by anti-A1 and thus are classified as antibody is selected for the reagent. Ael red
A1 or A1B. The remaining 20%, whose red cells are not agglutinated by anti-A or
cells are strongly agglutinated by anti-A but anti-A,B of any origin, and the presence of
not by anti-A1, are called A2 or A2B. Rou- A antigen is demonstrable only by adsorp-
tine testing with anti-A1 is unnecessary for tion and elution studies. Subgroups of B are
donors or recipients. even less common than subgroups of A.
Subgroups weaker than A2 occur infre- Molecular studies have confirmed that A
quently and, in general, are characterized and B subgroups are heterogeneous, and the
by decreasing numbers of A antigen sites serologic classification does not consistently
on the red cells and a reciprocal increase in correlate with genomic analysis; multiple
H antigen activity. Subgroups are most of- alleles yield the same weakened phenotype,
ten recognized when there is a discrepancy and, in some instances, more than one
between the red cell (forward) and serum phenotype has the same allele.16
(reverse) grouping. Generally, classification
of weak A subgroups (A3, Ax, Am, Ael) is based
Antibodies to A and B
on the:
1. Degree of red cell agglutination by Ordinarily, individuals possess antibodies
anti-A and anti-A1. directed toward the A or B antigen absent
2. Degree of red cell agglutination by hu- from their own red cells (see Table 13-1).
man and some monoclonal anti-A,B. This predictable complementary relation-
3. Degree of red cell agglutination by ship permits ABO testing of sera as well as
anti-H (Ulex europaeus). of red cells (see Methods 2.2 and 2.3). One
4. Presence or absence of anti-A1 in the hypothesis for the development of these
serum. antibodies is based on the fact that the
5. Presence of A and H substances in configurations that confer A and B anti-
the saliva of secretors. genic determinants also exist in other bio-
6. Adsorption/elution studies. logic entities, notably bacteria cell walls.
7. Family (pedigree) studies. Bacteria are widespread in the environ-
Identification of the various A subgroups ment, and their presence in intestinal
is not routinely done. The serologic classifi- flora, dust, food, and other widely distrib-
cation of A (and B) subgroups was devel- uted agents ensures a constant exposure
oped using human polyclonal anti-A, of all persons to A-like and B-like antigens.
anti-B, and anti-A,B reagents. These re- Immunocompetent persons react to the
agents have been replaced by murine environmental antigens by producing an-
monoclonal reagents, and the reactivity is tibodies to those that are absent from
dependent upon which clone(s) is selected their own systems. Thus, anti-A is pro-
by the manufacturer. There are, however, duced by group O and group B persons
some characteristics that should be noted. and anti-B is produced by group O and
A3 red cells give a characteristic mixed-field group A persons. Group AB people, hav-
pattern when tested with anti-A from group ing both antigens, make neither antibody.
B or O donors. Ax red cells are characteristi- This “environmental” explanation for the
cally not agglutinated by human anti-A emergence of anti-A and anti-B remains a
from group B persons but are agglutinated hypothesis that has not been proven.

Time of Appearance to red or when the cell button is absent or

reduced in size. Hemolysis must be inter-
Anti-A and anti-B produced by an infant
preted as a positive result. Because the
can generally be detected in serum after 3
hemolysis is complement-mediated, it will
to 6 months of life. Most of the anti-A and
not occur if plasma is used for testing, or if
anti-B present in cord blood are of ma-
reagent red cells are suspended in solutions
ternal origin, acquired by the placental
that contain EDTA or other agents that pre-
transfer of maternal IgG; occasionally, in-
vent complement activation.
fants can be found who produce these
17(p124)
antibodies at the time of birth. Thus,
anti-A and anti-B detected in the sera of Reactivity of Anti-A,B (Group O Serum)
newborns or infants younger than 3 to 6 Serum from a group O individual contains
months cannot be considered valid. Anti- an antibody designated as anti-A,B because
body production increases, reaching the it reacts with both A and B red cells, and
adult level at 5 to 10 years of age, and de- the anti-A and anti-B cannot be separated
clines later in life. Elderly people usually by differential adsorption. In other words,
have lower anti-A and anti-B levels than after adsorption of group O serum, an elu-
young adults. ate prepared from the group A or group B
adsorbing cells reacts with both A and B
Reactivity of Anti-A and Anti-B test cells. Saliva from a secretor of either A
or B substance inhibits the activity of
IgM is the predominant immunoglobulin
anti-A,B against A or B red cells, respec-
class of anti-A produced by group B indi-
tively.
viduals and anti-B produced by group A
individuals, although small quantities of
IgG antibody are also present. IgG is the Anti-A1
dominant class of anti-A and anti-B of group Anti-A1 occurs as an alloantibody in the
O serum.17(p124) Because IgG readily crosses serum of 1% to 2% of A2 individuals and
15
the placenta and IgM does not, group A or 25% of A 2 B individuals. Sometimes,
B infants of group O mothers are at higher Anti-A1 can also be found in the sera of in-
risk for ABO hemolytic disease of the fetus dividuals with other weak subgroups of A.
and newborn (HDFN) than the infants of Anti-A1 can cause discrepancies in ABO
group A or B mothers; but severe HDFN testing and incompatibility in cross-
can also occur in infants of group A and matches with A1 or A1B red cells. Anti-A1
group B mothers. usually reacts better or only at tempera-
Both IgM and IgG anti-A and anti-B pref- tures well below 37 C and is considered
erentially agglutinate red cells at room tem- clinically insignificant unless there is re-
perature (20-24 C) or below and efficiently activity at 37 C. When reactive at 37 C, only
activate complement at 37 C. The comple- A2 or O red cells should be used for trans-
ment-mediated lytic capability of these an- fusion.
tibodies becomes apparent if serum testing In simple adsorption studies, the anti-A
includes an incubation phase at 37 C. Sera of group B serum appears to contain sepa-
from some people will cause hemolysis of rable anti-A and anti-A1. Native group B se-
ABO-incompatible red cells at tempera- rum agglutinates A1 and A2 red cells; after
tures below 37 C. Hemolysis due to ABO adsorption with A2 red cells, group B serum
antibodies should be suspected when the reacts only with A1 red cells. If further tests
supernatant fluid of the serum test is pink are performed, however, the differences in

A antigen expression between A1 and A2 red but are helpful in resolving typing discrep-
cells appear to be quantitative rather than ancies (see below). The use of anti-A,B may
qualitative.9 not have the same benefit in detecting weak
A reliable anti-A1 reagent from the lectin subgroups when testing with monoclonal
of Dolichos biflorus is commercially avail- reagents (depending on the clones used) as
able or may be prepared (see Method 2.10). when human polyclonal reagents were in
The raw plant extract will react with both A1 use. Many monoclonal ABO typing re-
and A2 red cells, but an appropriately diluted agents have been formulated to detect
reagent preparation will not agglutinate A2 some of the weaker subgroups. Manufac-
cells and thus constitutes an anti-A1. 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-
Routine Testing for ABO
crepancy (eg, the absence of expected se-
Routine testing for determining the ABO rum antibodies) usually distinguishes these
group consists of testing the red cells with specimens from group O specimens.
anti-A and anti-B (cell or forward type) The A2 red cells are intended to facilitate
and testing the serum or plasma with A1 the recognition of anti-A1. Because most group
and B red cells (serum or reverse type). A specimens do not contain anti-A1, routine
Both red cell and serum testing are reuse of this reagent is not necessary.
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 Discrepancies Between Red Cell and
are confirmation testing of the ABO type of Serum Tests
donor units that have already been la-
beled and testing blood of infants less Table 13-1 shows the results and interpre-
than 4 months of age; in both of these in- tations of routine red cell and serum tests
stances, only ABO testing of red cells is re- for ABO. A discrepancy exists when the
quired. results of red cell tests do not agree with
Anti-A and anti-B typing reagents agglu- serum tests. When a discrepancy is en-
tinate most antigen-positive red cells on di- countered, the discrepant results must be
rect contact, even without centrifugation. recorded, but interpretation of the ABO
Anti-A and anti-B in the sera of some pa- group must be delayed until the discrep-
tients and donors are too weak to aggluti- ancy has been resolved. If the specimen is
nate red cells without centrifugation or pro- from a donor unit, the unit must not be
longed incubation. Serum tests should be released for transfusion until the discrep-
performed by a method that will adequa- ancy has been resolved. When the blood
tely detect the antibodies—eg, tube, micro- is from a potential recipient, it may be
plate, or column agglutination techniques. necessary to administer group O red cells
Procedures for ABO typing by slide, tube, of the appropriate Rh type before the in-
and microplate tests are described in Meth- vestigation has been completed. It is im-
ods 2.1, 2.2, and 2.3. portant to obtain sufficient pretransfusion
Additional reagents, such as anti-A,B for blood samples from the patient to com-
red cell tests and A2 and O red cells for serum plete any additional studies that may be
tests, are not necessary for routine testing required.

Red cell and serum test results may be Specimen-Related Problems in Testing Red
discrepant because of intrinsic problems Cells
with red cells or serum, or technical errors.
Discrepancies may be signaled either be- ABO testing of red cells may give unex-
cause negative results are obtained when pected results for many reasons.
positive results are expected, or positive re- 1. Red cells from individuals with vari-
sults are found when tests should have been ant A or B alleles may carry poorly
negative (see Table 13-2). expressed antigens. Antigen expres-
Table 13-2. Possible Causes of ABO Typing Discrepancies

Category Causes
Red cell weak/ ABO subgroup
missing reactivity Leukemia/malignancy
Transfusion
Intrauterine fetal transfusion
Transplantation
Excessive soluble blood group substance
Extra red cell Autoagglutinins/excess protein coating red cells

reactivity 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 Recent transfusion

reactivity Transplantation
Fetomaternal hemorrhage
Twin or dispermic (tetragametic) chimerism
Serum weak/missing Age related (<4-6 months old, elderly)

reactivity 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 8. Red cells of individuals with the
cells of some persons with leukemia acquired B phenotype typically ag-
or other malignancies. glutinate strongly with anti-A and
2. A patient who has received red cell weakly with anti-B, and the serum
transfusions or a marrow transplant contains strong anti-B. The acquired
may have circulating red cells of more B phenotype arises when microbial
than one ABO group and constitute deacetylating enzymes modify the A
a transfusion or transplantation chi- antigen by altering the A-determin-
mera (see Mixed-Field Agglutination ing sugar (N-acetylgalactosamine)
below). so that it resembles the B-determin-
3. Exceptionally high concentrations of ing galactose. The acquired B phe-
A or B blood group substances in the nomenon is found most often in in-
serum can combine with and neutra- dividuals with the A1 phenotype.
lize reagent antibodies to produce an 9. Inherited or acquired abnormalities
unexpected negative reaction against of the red cell membrane can lead to
serum- or plasma-suspended red cells. what is called a polyagglutinable state.
4. A patient with potent autoagglutinins The abnormal red cells can be unex-
may have red cells so heavily coated pectedly agglutinated by human re-
with antibody that the red cells ag- agent anti-A, anti-B, or both because
glutinate spontaneously in the pres- human reagents will contain anti-
ence of diluent, independent of the bodies to the so-called cryptantigens
specificity of the reagent antibody. that are exposed in polyagglutinable
5. Abnormal concentrations of serum states. In general, monoclonal anti-A
proteins or the presence in serum of and anti-B reagents will not detect
infused macromolecular solutions may polyagglutination.
cause the nonspecific aggregation of
serum-suspended red cells that sim-
ulates agglutination. Specimen-Related Problems in Testing
6. Serum- or plasma-suspended red cells Serum or Plasma
may give false-positive results with ABO serum/plasma tests are also subject
monoclonal reagents if the serum or to false results.
plasma contains a pH-dependent 1. Small fibrin clots that may be mis-
autoantibody.19,20 Serum- or plasma- taken for agglutinates may be seen
suspended red cells may also give in ABO tests with plasma or incom-
discrepant results due to an antibody pletely clotted serum.
21
to a reagent dye/constituent or to 2. Negative or weak results are seen in
proteins causing rouleaux. serum tests from infants under 4 to 6
7. Red cells of some group B individu- months of age. Serum from newborns
als are agglutinated by a licensed is not usually tested because anti-
anti-A reagent that contains a partic- bodies present are generally passively
ular murine monoclonal antibody, transferred from the mother.
MHO4. These group B individuals had 3. Unexpected absence of ABO aggluti-
excessively high levels of B allele- nins may be due to the presence of
specified galactosyltransferase, and an A or B variant.
the designation B(A) was given to 4. Patients who are immunodeficient
this blood group phenotype.22 due to disease or therapy may have

such depressed immunoglobulin levels 10. Recent infusion of intravenous im-

that there is little or no ABO aggluti- mune globulin that may contain ABO
nin activity. Samples from elderly isohemagglutinins can cause unex-
patients whose antibody levels have pected reactions.
declined with age or from patients
whose antibodies have been greatly Mixed-Field Agglutination
diluted by plasma exchange proce-
Occasional samples are encountered that
dures may also have unexpectedly weak
contain two distinct, separable populations
agglutinins.
of red cells. Usually, this reflects the re-
5. If the patient has received a marrow
cent transfusion of group O red cells to a
transplant of a dissimilar ABO group,
non-group-O recipient or receipt of a
serum antibodies will not agree with
marrow transplant of an ABO group dif-
red cell antigens. For example, a group
ferent from the patient’s own. Red cell
A individual who receives group O
mixtures also occur in a condition called
marrow may have circulating group
blood group chimerism, resulting either
O red cells and produce only anti-B in
from intrauterine exchange of erythro-
the serum. Refer to Chapter 25 for more
poietic tissue by fraternal twins or from
information on the effects of ABO-
mosaicism arising through dispermy. In all
mismatched transplants.
such circumstances, ABO red cell tests may
6. Cold allo- or autoantibodies that re-
give a mixed-field pattern of agglutina-
act at room temperature can react
tion. Mixed-field reactions due to transfu-
with one or both reverse grouping
sion last only for the life of the transfused
cells. For example, if the patient has
red cells. After hematopoietic transplanta-
a room-temperature-reactive anti-M,
tion, the mixed-field reaction usually dis-
it may cause an unexpected reaction
appears when the patient’s own red cells
with M-positive A1 and/or B reagent
are no longer produced. Persistent mixed-
red cells.
cell populations do occur in some marrow
7. Antibodies to constituents of the dil-
recipients. Mixed-field reactions that arise
uents used to preserve reagent A1 and
through blood group chimerism may per-
B red cells can agglutinate the cells
sist throughout the life of the individual.
independent of ABO antigens and
For more information regarding the trans-
antibodies.
fusion and evaluation of hematopoietic
8. Abnormal concentrations of proteins,
transplant patients, refer to Chapters 21
altered serum protein ratios, or the
and 25.
presence of high-molecular-weight
plasma expanders can cause non-
specific red cell aggregation or roule- Technical Errors
aux that is difficult to distinguish from Technical errors leading to ABO discrep-
true agglutination. Rouleaux forma- ancies include:
tion is easily recognized on micro- 1. Specimen mix-up.
scopic examination if the red cells 2. Red cell suspensions are too heavy
assume what has been described as or too light.
a “stack of coins” formation. 3. Failure to add reagents.
9. Recent transfusion with plasma com- 4. Missed observation of hemolysis.
ponents containing ABO agglutinins 5. Failure to follow manufacturer’s in-
may cause unexpected reactions. structions.

6. Under- or overcentrifugation of tests. cells probably comes from a group A per-

7. Incorrect interpretation or recording son, even though the red cells are not
of test results. agglutinated by anti-A or anti-B. The fol-
lowing procedures can be used to en-
Resolving ABO Discrepancies hance the detection of weakly expressed
antigens.
The first step in resolving an apparent se-
1. Incubate washed red cells with anti-A,
rologic problem should be to repeat the
anti-B, and anti-A,B for 15 minutes
tests on the same sample. If initial tests
at room temperature to increase the
were performed on red cells suspended in
association of antibody with antigen.
serum or plasma, the testing should be re-
Incubating the test system for 15 to
peated after washing the red cells several
30 minutes at 4 C may further en-
times with saline. Washing the red cells
hance antibody attachment. An inert
can eliminate many problems for red cell
(eg, 6% albumin) or autologous con-
typing that are associated with plasma
trol for room temperature and 4 C
proteins. If the discrepancy persists, the
tests is recommended. The manufac-
following initial steps can be incorporated
turer’s directions for any reagent
into the investigation.
should be consulted for possible
1. Obtain the patient’s diagnosis, his-
comments or limitations.
torical blood group, and history of
2. Treat the patient’s red cells with a
previous transfusions, transplanta-
proteolytic enzyme such as ficin,
tion, and medications.
papain, or bromelin. Enzyme treat-
2. Review the results of the antibody
ment increases the antigen-antibody
detection test against group O red cells
reaction with anti-A or anti-B. In
and autologous red cells to detect
some instances, reactions between
possible interference from allo- or
reagent antibody and red cells ex-
autoantibodies.
pressing antigens will become de-
3. Obtain a new blood specimen and test
tectable at room temperature within
the new sample if a discrepancy due
30 minutes if enzyme-treated red
to a contaminated specimen is sus-
cells are employed. Enzyme-treated
pected.
group O red cells and an autologous
In addition to a discrepancy between the
control must be tested in parallel as
red cell and serum tests, an ABO discrep-
a control for the specificity of the
ancy may also exist when the observed re-
ABO reaction. The manufacturer’s
activity is not in agreement with a previous
directions should be consulted for
type on record. The first step in resolving
possible comments or limitations.
this type of discrepancy is to obtain a new
3. Incubate an aliquot of red cells at
blood specimen.
room temperature or at 4 C with
anti-A or anti-B (as appropriate) to
Resolving Discrepancies Due to Absence of adsorb antibody to the correspond-
Expected Antigens ing red cell antigen for subsequent
The cause of a discrepancy can some- elution (see Method 2.4). Group A or
times be inferred from the strength of the B (as appropriate) and O red cells
reactions obtained in red cell or serum should be subjected to parallel ad-
tests. For example, serum that strongly sorption and elution with any re-
agglutinates group B red cells but not A1 agent to serve as positive and nega-

tive controls. Anti-A1 lectin should Resolving Discrepancies Due to Unexpected

not be used for adsorption/elution Red Cell Reactions with Anti-A and Anti-B
studies because in a more concen-
trated form, such as an eluate, it may Red cell ABO tests sometimes give unex-
react nonspecifically with red cells. pected positive reactions. For example,
Test the eluate against group A1, B, reagent anti-A may weakly agglutinate red
and O cells. Unexpected reactivity in cells from a sample in which the serum
control eluates invalidates the re- gives reactions expected of a normal
sults obtained with the patient’s red group B or O sample. The following para-
cells. This indicates that the adsorp- graphs describe some events that can
tion/elution procedure was not per- cause unexpected reactions in ABO typing
formed correctly, another antibody is tests and the steps that can be taken to
present (ie, in a polyclonal reagent), identify them.
or the specificity of a monoclonal re- B(A) Phenotype. When cells react weakly
agent is not distinct enough for the with monoclonal anti-A and strongly with
reagent to be used by this method. anti-B, and the serum reacts with A1 red
4. Test the saliva for the presence of H cells, but not B cells, the B(A) phenotype
and A or B substances (see Method should be suspected. Verification that the
2.5). Saliva tests help resolve ABO anti-A reagent contains the discriminating
discrepancies only if the person is a MHO4 clone confirms the suspicion. B(A)
secretor. This may be surmised from red cells can show varying reactivity with
the Lewis phenotype but may not be anti-A; the majority of examples react
known until after the saliva testing is weakly, and the agglutinates are fragile and
complete. See the discussion on Lewis easily dispersed, although some examples
antigens. have reacted as strongly as 2+.22 Sera from
these individuals agglutinate both A1 and A2
cells. Except for newborns and immuno-
Resolving Discrepancies Due to Absence of compromised patients, serum testing should
Expected Antibodies distinguish this phenomenon from the AB
1. Incubate the serum with A1 and B red phenotype in which a subgroup of A is ac-
cells for 15 to 30 minutes at room companied by anti-A 1 . Testing with an
temperature. If there is still no reac- anti-A without the MHO4 clone should re-
tion, incubate at 4 C for 15 to 30 solve the discrepancy. The recipient can be
minutes. It is recommended to in- considered a group B.
clude an autocontrol and group O Acquired B Phenotype. Red cells aggluti-
red cells for room temperature and 4 nated strongly by anti-A and weakly by
C testing to control for reactivity of anti-B and a serum containing strong
common cold autoagglutinins. anti-B suggest the acquired B state. The ac-
2. Treat the A1 and B reagent cells with quired B phenomenon is found most often
a proteolytic enzyme such as ficin, in individuals with the A1 phenotype; a few
papain, or bromelin. Enzyme-treated examples of A2 with acquired B have been
group O and autologous red cells found. Most red cells with acquired B anti-
must be tested in parallel as a con- gens react weakly with anti-B, but occa-
trol for reactivity. The manufacturer’s sional examples are agglutinated quite
directions should be consulted for strongly. Behavior with monoclonal anti-B
possible comments or limitations. reagents varies with the particular clone

used. Acquired B antigens had been ob- but, sometimes, the sensitized red cells also
served with increased frequency in tests with agglutinate in ABO reagents with protein
certain FDA-licensed monoclonal anti-B concentrations of 6% to 12%. Methods 2.12
blood grouping reagents containing the ES-4 or 2.14 may be used to remove much of this
clone,23 but the manufacturers have lowered antibody from the red cells so that the cells
the pH of the anti-B reagent so the fre- can be tested reliably with anti-A and anti-B.
quency of the detection of acquired B is Red cells from a specimen containing cold-
similar to polyclonal anti-B or have discon- reactive IgM autoagglutinins may autoag-
tinued the use of that clone. To confirm that glutinate in saline tests. Incubating the cell
group A red cells carry the acquired B struc- suspension briefly at 37 C and then wash-
ture: ing the cells several times with saline
1. Check the patient’s diagnosis. Ac- warmed to 37 C can usually remove the an-
quired B antigens are usually associ- tibodies. If the IgM-related agglutination is
ated with tissue conditions that al- not dispersed by this technique, the red cells
low colonic bacteria to enter the can be treated with the sulfhydryl com-
circulation, but acquired B antigens pound dithiothreitol (DTT) (see Method 2.11).
have been found on the red cells of
apparently normal blood donors.23
2. Test the patient’s serum against auto- Resolving Discrepancies Due to Unexpected
logous red cells or known acquired B Serum Reactions
cells. The individual’s anti-B will not The following paragraphs describe some
agglutinate his or her own red cells events that can cause unexpected or erro-
or red cells known to be acquired B. neous serum test results and the steps that
3. Test the red cells with monoclonal can be taken to resolve them.
anti-B reagents for which the manu- 1. Reactivity of the A 1 reagent cells
facturer’s instructions give a detailed when anti-A is strongly reactive with
description. Unlike most human poly- the red cells suggests the presence of
clonal antibodies, some monoclonal anti-A1 in the serum of an A2 or A2B
antibodies do not react with the ac- individual. To demonstrate this as
quired B phenotype; this information the cause of the discrepancy:
may be included in the manufac- a. Test the red cells with anti-A1
turer’s directions. lectin to differentiate group A1
4. Test the red cells with human anti-B from A2 red cells.
serum that has been acidified to pH b. Test the serum against several
6.0. Acidified human anti-B no lon- examples of each of the follow-
ger reacts with the acquired B anti- ing: A1, A2, and O red cells. Only
gen. if the antibody agglutinates all
Antibody-Coated Red Cells. Red cells A1 red cells and none of the A2
from infants with HDFN or from adults suf- or O red cell samples can it be
fering from autoimmune or alloimmune called anti-A1.
conditions may be so heavily coated with 2. Strongly reactive cold autoaggluti-
IgG antibody molecules that they agglutinins, such as anti-I, anti-IH, anti-IA,
nate spontaneously in the presence of re- and anti-IB, can agglutinate red cells
agent diluents containing high protein con- of adults, including autologous cells
centrations. Usually, this is at the 18% to and reagent red cells, at room tem-
22% range found in some anti-D reagents, perature (20-24 C). With few excep-

tions, agglutination caused by the cold tigen and use them for serum
autoagglutinin is weaker than that testing.
caused by anti-A and anti-B. The fol- b. Raise the temperature to 30 to
lowing steps can be performed when 37 C before mixing the serum
such reactivity interferes to the point and cells, incubate for 1 hour, and
that the interpretation of serum tests perform a “settled” reading (ie,
is difficult. without centrifugation). If the
a. Warm the serum and reagent red thermal amplitude of the allo-
cells to 37 C before mixing and antibody is below the tempera-
testing. Incubate at 37 C for 1 ture at which anti-A and anti-B
hour and perform a “settled” react, this may resolve the dis-
reading (ie, observe for aggluti- crepancy.
nation without centrifugation). c. If the antibody detection test is
Rare weakly reactive examples negative, test the serum against
of IgM anti-A or anti-B may not several examples of A1 and B
be detected by this method. red cells. The serum may con-
b. Remove the cold autoagglutinin tain an antibody directed against
from the serum using a cold an antigen of low incidence,
autoadsorption method as de- which will be absent from most
scribed in Method 4.6. The ad- randomly selected A1 and B red
sorbed serum can then be tested cells.
against A1 and B reagent red cells. 4. Sera from patients with abnormal
3. Unexpected alloantibodies that react concentrations of serum proteins, or
at room temperature, such as anti-P1 with altered serum protein ratios, or
or anti-M, may agglutinate the red who have received plasma expand-
cells used in serum tests if the cells ers of high molecular weight can ag-
carry the corresponding antigen. One gregate reagent red cells and mimic
or more of the reagent cells used in agglutination. Some of these sam-
the antibody detection test may also ples cause aggregation of the type
be agglutinated if the serum and cell described as rouleaux. More often,
mixture was centrifuged for either a the aggregates appear as irregularly
room temperature or 37 C reading; a shaped clumps that closely resemble
rare serum may react with an anti- antibody-mediated agglutinates. The
gen of low incidence on the serum results of serum tests can often be
testing cells that is not present on cells corrected by diluting the serum 1:3
used for antibody detection. Steps to in saline to abolish its aggregating
determine the correct ABO type of sera properties or by using a saline re-
containing cold-reactive alloantibodies placement technique (see Method
include: 3.4).
a. Identify the room temperature
alloantibody, as described in
Chapter 19, and test the re-
agent A1 and B cells to determine The H System
which, if either, carries the cor- On group O red cells, there is no A or B
responding antigen. Obtain A1 antigen, and the membrane expresses
and B red cells that lack the an- abundant H. Because H is a precursor of A

and B antigens, A and B persons have less Para-Bombay Phenotype

H substance than O persons. The amount
The para-Bombay phenotype designation,
of H antigen detected on red cells with the Ah, Bh, and ABh, is classically used for indi-
anti-H lectin Ulex europaeus is, in order of viduals who are H-deficient secretors, ie,
diminishing quantity, O>A2>B>A2B>A1>A1B. those who have an inactive H-transferase
Occasionally, group A1, A1B, or (less com- but have active Se-transferase. The red
monly) B individuals have so little uncon- cells lack serologically detectable H anti-
verted H antigen on their red cells that they gen but carry small amounts of A and/or
produce anti-H. This form of anti-H is gen- B antigen (sometimes detectable only by
erally weak, reacts at room temperature or adsorption/elution tests), depending on
below, and is not considered clinically sig- the individual’s alleles at the ABO locus.
nificant. Individuals of the rare Oh pheno- Tests with anti-A or anti-B reagents may
type (see below), whose red cells lack H, or may not give weak reactions, but the
have a potent and clinically significant cells are nonreactive with anti-H lectin or
alloanti-H in their serum (in addition to with anti-H serum from Oh persons. Indi-
anti-A and anti-B). viduals with the para-Bombay phenotype
have a functional Se allele and thus will
Oh Phenotype express A, B, and H antigens in their
The term Oh or Bombay phenotype has plasma and secretions. The sera of Ah and
been used for the very rare individuals Bh people contain anti-H and/or anti-IH
whose red cells and secretions lack H, A, in addition to the expected anti-A or
and B antigens and whose plasma con- anti-B.
tains potent anti-H, anti-A, and anti-B.6 H-deficient secretors may also be group
This phenotype was first discovered in O. These individuals will have traces of H
Bombay, India. The phenotype initially antigen, but no A or B antigen, on their red
mimics normal group O but becomes ap- cells and only have H in their secretions.
parent when serum from the Oh individ- In 1994, Kelly et al reported the molecu-
lar bases for the Bombay and para-Bombay
ual is tested against group O red cells, and 24
phenotypes. Many mutations at the H lo-
strong immediate-spin agglutination
cus have subsequently been associated
and/or hemolysis occurs. The anti-H of
with H-deficiency.
an Oh person reacts over a thermal range
of 4 to 37 C with all red cells except those
of other Oh people. Oh persons must be
transfused only with Oh blood because The Lewis System
their non-red-cell-stimulated antibodies a
The Lewis system antigens, Le and Le ,
b
rapidly destroy cells with A, B, or H anti- result from the action of a glycosyltrans-
gens. If other examples of Oh red cells are ferase encoded by the Le allele that, like
available, further confirmation can be ob- the A, B, and H glycosyltransferases, adds
tained by demonstrating compatibility of a sugar to a precursor chain. Lea is pro-
the serum with Oh red cells. At the geno- duced when Le is inherited with sese and
typic level, the Oh phenotype arises from Leb is produced when Le is inherited with
the inheritance of hh at the H locus and at least one Se allele. When the silent or
sese at the Se locus. Because the Se allele is amorphic allele le is inherited, regardless
necessary for the formation of Leb, Oh red of the secretor allele inherited, no Lea or
b a b
cells will be Le(a+b–) or Le(a–b–). Le is produced. Thus, Le and Le are not

a b
antithetical antigens produced by alleles; no secretion of Le or Le and the red cells
rather, they result from the interaction of will have the Le(a–b–) phenotype.
independently inherited alleles. Table 13-3 shows phenotypes of the Lewis
The Lewis antigens are not intrinsic to system and their frequencies in the popula-
red cells but are expressed on glycosphing- tion. Red cells that type as Le(a+b+) are rare
olipid Type 1 chains adsorbed from plasma in people of European and African origin
onto red cell membranes. Plasma lipids ex- but are relatively common in persons of
change freely with red cell lipids. Asian origin, due to a fucosyltransferase en-
coded by a variant secretor allele that com-
Gene Interaction and the Antigens petes less efficiently with the Le fuco-
9
The synthesis of the Lewis antigens is de- syltransferase.
pendent upon the interaction of two dif-
ferent fucosyltransferases: one from the Lewis Antibodies
Se locus and one from the Lewis locus. Lewis antibodies occur almost exclusively
Both enzymes act upon the same precur- in the sera of Le(a–b–) individuals, usually
sor Type 1 substrate chains. The fuco- without known red cell stimulus. Those
syltransferase encoded by the Le allele at- individuals whose red cell phenotype is
taches fucose in α(1→4) linkage to the Le(a–b+) do not make anti-Lea because
subterminal GlcNAc; in the absence of the small amounts of unconverted Le a are
transferase from the Se allele, this config- present in their saliva and plasma. It is
a
uration has Le activity. This product can- most unusual to find anti-Leb in the sera
b b
not be further glycosylated. Le occurs of Le(a+b–) individuals, but anti-Le may
when the Type 1 precursor is converted to exist along with anti-Lea in the sera of
Type 1 H by the fucosyltransferase from Le(a–b–) individuals. Lewis antibodies are
the secretor allele, and subsequently acted often found in the sera of pregnant women
upon by the fucosyltransferase from the who transiently demonstrate a Le(a–b–)
Le allele. This Leb configuration has two phenotype. The Lewis antibodies, however,
9 b
fucose moieties. Thus, Le reflects the are almost always IgM and do not cross
presence of both the Le and Se alleles. Le the placenta. Because of this and because
without Se results in Lea activity only; Se Lewis antigens are poorly developed at
with the amorphic allele le will result in birth, the antibodies are not associated
Table 13-3. Phenotypes in the Lewis System and Their Incidence

Reactions with Anti- Adult Phenotype Incidence %
a b
Le Le Phenotype Whites Blacks
+ 0 Le(a+b−) 22 23
0 + Le(a−b+) 72 55
0 0 Le(a−b−) 6 22
+ + Le(a+b+) Rare Rare
+ = agglutination; 0 = no agglutination.

with HDFN. Lewis antibodies may bind blood for the presence of Lewis antigens
complement, and fresh serum that con- before transfusion or when crossmatch-
tains anti-Lea (or infrequently anti-Leb) ing for recipients with Lewis antibodies;
may hemolyze incompatible red cells in red cells that are compatible in tests at 37
vitro. Hemolysis is more often seen with C can be expected to survive normally in
enzyme-treated red cells than with un- vivo.25,26
treated red cells.
Most Lewis antibodies agglutinate sa- Lewis Antigens in Children
line-suspended red cells of the appropriate
Red cells from newborn infants usually do
phenotype. The resulting agglutinates are a
not react with either human anti-Le or
often fragile and are easily dispersed if red b
anti-Le and are considered to be Le(a–b–).
cell buttons are not resuspended gently af-
Some can be shown to carry small amounts
ter centrifugation. Agglutination sometimes
of Le a when tested with potent mono-
is seen after incubation at 37 C, but rarely a
clonal anti-Le reagents. Among children,
of the strength seen in tests incubated at
the incidence of Le(a+) red cells is high
room temperature. Some examples of
and that of Le(b+) red cells low, reflecting
anti-Lea, and less commonly anti-Leb, can be
a greater production of the Le allele-spe-
detected in the antiglobulin phase of test-
cific transferase in infants; the Se allele-
ing. Sometimes this reflects complement
specific transferase is produced in lower
bound by the antibody if a polyspecific re-
levels. The phenotype Le(a+b+) may be
agent (ie, containing anticomplement) is
transiently observed in children as the Se
used. In other cases, antiglobulin reactivity
allele transferase levels increase toward
results from an IgG component of the anti-
adult levels. Reliable Lewis typing of young
body.
children may not be possible because test
Sera with anti-Leb activity can be divided
reactions may not reflect the correct phe-
into two categories. The more common type
notype until approximately 2 to 3 years of
reacts best with Le(b+) red cells of group O
age.25
and A2; these antibodies have been called
bH
anti-Le . Antibodies that react equally well
with the Leb antigen on red cells of all ABO
phenotypes are called anti-LebL. The I/i Antigens and
Antibodies
Transfusion Practice
Cold agglutinins with I specificity are fre-
Lewis antigens readily adsorb to and elute quently encountered in sera of normal in-
from red cell membranes. Transfused red dividuals. The antibody is usually not
cells shed their Lewis antigens and assume clinically significant and reacts with all
the Lewis phenotype of the recipient red cells except cord cells and the rare i
within a few days of entering the circula- adult phenotype. I and i antigens, however,
tion. Lewis antibodies in a recipient’s se- are not antithetical but are expressed in a
rum are readily neutralized by Lewis reciprocal relationship. At birth, infant red
blood group substance in donor plasma. cells are rich in i; I is almost undetectable.
For these reasons, it is exceedingly rare for Thus, for practical purposes, cord cells are
Lewis antibodies to cause hemolysis of considered to be I–, i+. During the first 2
transfused Le(a+) or Le(b+) red cells. It is years of life, I antigen gradually increases
not considered necessary to type donor at the expense of i. The red cells of most

adults are strongly reactive with anti-I nin at 4 C with a titer of <64. Agglutination
and react weakly or not at all with anti-i. with adult red cells and weaker or no ag-
Rare adults have red cells that carry high glutination with cord cells is the classic
levels of i and only trace amounts of I; reactivity. Some stronger examples agglu-
these red cells have the i adult phenotype. tinate cells at room temperature; others
There is no true I– or i– phenotype. may react only with the strongest I+ red
The I and i antigens on red cells are in- cells and give inconsistent reactions. Incu-
ternal structures carried on the same glyco- bating tests in the cold enhances anti-I re-
proteins and glycosphingolipids that carry activity and helps to confirm its identity;
H, A, or B antigens and in secretions on the albumin and testing enzyme-treated red
same glycoproteins that carry H, A, B, Lea, cells also enhance anti-I reactivity.
b
and Le antigens. The I and i antigens are Autoanti-I assumes pathologic signifi-
located closer to the membrane than the cance in cold agglutinin syndrome (CAS),
terminal sugars that determine the ABH in which it behaves as a complement-bind-
antigens. The i structure is a linear chain of ing antibody with a high titer and high ther-
at least two repeating units of N-acetyl- mal amplitude. The specificity of the auto-
galactosamine [Galβ(1→4)GlcNAcβ(1→3)]. antibody in CAS may not be apparent when
On the red cells of adults, many of these the undiluted sample is tested (I adult and
linear chains are modified by the addition cord cells may react to the same strength
of branched structures consisting of GlcNAc even at room temperature); titration studies
in a β(1→6) linkage to a galactose residue and/or thermal amplitude studies may be
internal to the repeating sequence. The necessary to define the specificity (see
branching configuration confers I specific- Chapter 20). Anti-I is often made by pa-
ity.27,28 Different examples of anti-I appear to tients with pneumonia due to Mycoplasma
recognize different portions of the branch- pneumoniae. These patients may experience
ed oligosaccharide chain. As branching oc- transient hemolytic episodes due to the an-
curs and as the sugars for H, A, and B anti- tibody.
gens are added, access of anti-i and anti-I Autoanti-i is less often implicated in
may be restricted.25 symptomatic disease than anti-I. On rare
The I antigen, together with the i anti- occasions, anti-i may be seen as a relatively
gen, used to comprise the Ii Blood Group weak cold autoagglutinin reacting preferen-
Collection in the ISBT nomenclature. Re- tially at 4 C. Anti-i reacts strongest with
cent cloning of the gene that encodes the cord and i adult red cells, and weakest with
transferase responsible for converting i ac- I adult red cells. Patients with infectious
tive straight chains into I active branched mononucleosis often have transient but
chains and identification of several muta- potent anti-i.
tions responsible for the rare i adult pheno- Table 13-4 illustrates the serologic be-
type have caused the I antigen to be as- havior of anti-I and anti-i at 4 C and 22 C.
signed to the new I Blood Group System; Reaction strengths should be considered
29
the i antigen remains in the Ii collection. relative; clear-cut differences in reactivity
between the two are seen only with weaker
examples of the antibodies. Titration stud-
Antibodies to I/i
ies may be needed to differentiate strong
Anti-I is a common, benign autoantibody examples of the antibodies.
found in the serum of many normal healthy Serum containing anti-I or anti-i is some-
individuals that behaves as a cold aggluti- times reactive at the antiglobulin phase of

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
4C I adult 4+ 0-1+
i cord 0-2+ 3+
i adult 0-1+ 4+
22 C I adult 2+ 0
i cord 0 2-3+
i adult 0 3+
testing when polyspecific antihuman glob- all, with group A1 cells of adults or cord
ulin is used. Such reactions rarely indicate cells of any group. Anti-IH should be sus-
antibody activity at 37 C. Rather, comple- pected when serum from a group A pa-
ment components are bound when serum tient causes direct agglutination of all
and cells interact at lower temperatures; cells used for antibody detection but is
during the 37 C incubation, the antibody compatible with all or most group A do-
dissociates but the complement remains nor blood. Other examples of complex re-
bound to the red cells. Thus, the anti- activity include anti-IA, -IP1, -IBH, -ILebH,
globulin phase reactivity is usually due to and -iH.
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- The P Blood Group and
antibodies. Cold autoadsorption to remove Related Antigens
the autoantibody from the serum may be
necessary for stronger examples; cold auto- The P blood group has traditionally con-
k
adsorbed serum or plasma can also be used sisted of the P, P1, and P antigens, and
in ABO typing (see Method 4.6). The pre- later Luke (LKE). However, the biochemis-
warming technique can also be used once try and molecular genetics, although not
the reactivity has been confirmed as cold yet completely understood, make it clear
autoantibody (see Method 3.3). that at least two biosynthetic pathways
and genes at different loci are involved in
the development and expression of these
Complex Reactivity
antigens. Thus, in ISBT nomenclature, the
Some antibodies appear to recognize I de- P antigen is assigned to the new globoside
terminants with attached H, A, or B im- (GLOB) blood group system, the P1 anti-
munodominant sugars. Anti-IH occurs gen is assigned to the P blood group system,
quite commonly in the serum of A1 indi- and Pk and LKE remain in the globoside
29
viduals; it reacts stronger with red cells collection of antigens. For simplicity,
that have high levels of H as well as I (ie, these antigens are often referred to as the
group O and A2 red cells) and weaker, if at P blood group.

The first antigen of the P blood group Biochemistry and Genetics

was discovered by Landsteiner and Levine
The P blood group antigens, like the ABH
in 1927, in a series of animal experiments
antigens, are sequentially synthesized by
that led also to the discovery of M and N.
the addition of sugars to precursor chains.
Originally called P, the name of the antigen
The different oligosaccharide determi-
was later changed to P1. The designation P
nants of the P blood group antigens are
has since been reassigned to an antigen
present on almost all human red cells. The shown in Fig 13-5. All the antigens are ex-
Pk antigen is also present on almost all hu- clusively expressed on glycolipids on hu-
30
man red cells, but it is not readily detected man red cells, not on glycoproteins. The
k
unless P is absent, eg, in the rare P1 or P2
k precursor of P1 can also be glycosylated to
phenotypes. The null phenotype, p, is very Type 2H chains, which carry ABH antigens.
rare. LKE antigen is present on almost all There are two distinct pathways for the
red cells except those of the rare phenotypes synthesis of the P blood group antigens as
p or Pk and in about 2% of P+ red cells. shown in Fig 13-6. The common precursor
is lactosylceramide, also known as cera-
mide dihexose or CDH. One pathway re-
sults in the formation of paragloboside and
Common and Rare Phenotypes
P1. Paragloboside is also the Type 2 precur-
There are two common phenotypes asso- sor for ABH antigens. The other pathway
ciated with the P blood group, P1 and P2, results in the formation of the globoside se-
k k
and three rare phenotypes, p, P1 , and P2 , ries of antigens: Pk, P, and LKE.
as shown in Table 13-5. The P1 phenotype The genes encoding the glycosyltrans-
describes those red cells that react with ferases that are responsible for synthesizing
k
anti-P1 and anti-P; red cells that do not re- P from lactosylceramide and for converting
k
act with anti-P1, but do react with anti-P, P to P were cloned in 2000. Several muta-
k
are of the P2 phenotype. When red cells tions that result in the p and P phenotypes
31-33
are tested only with anti-P1 and not with have been identified. The genetic rela-
k
anti-P, the phenotype should be written as tionship between P1, P and P is still not un-
P1+ or P1–. derstood. Red cells of the p phenotype are
Table 13-5. Phenotypes of the P Blood Group and Related Antigens

Reactions with Anti- Phenotype Incidence (%)
k k
P1 P P P1 +P+P Phenotype Whites Blacks
+ + 0 + P1 79 94
0 + 0 + P2 21 6
0 0* 0 0 p
+ 0 + + P1k All extremely rare
k
0 0 + + P2
*Usually negative, occasionally weakly positive.

Lactosylceramide (CDH) Galβ(1→4)Glc-Cer

k
P Globotriosylceramide (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
P1 Galactosylparagloboside 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.
k
P1– in addition to being P– and P –; the P1– reported to cause HDFN. Only rarely has
status of p red cells cannot be explained. it been reported to cause hemolysis in
The P1 gene is located on chromosome 22 vivo.17(p139),34
and the P gene is located on chromosome 3. The strength of the P1 antigen varies widely
among different red cell samples, and anti-
gen strength has been reported to diminish
when red cells are stored. These character-
Anti-P1 istics sometimes create difficulties in iden-
tifying antibody specificity in serum with a
The sera of P 1 – individuals commonly positive antibody screen. An antibody that
contain anti-P1. If sufficiently sensitive reacts weakly in room temperature testing
techniques are applied, it is likely that can often be shown to have anti-P1 specific-
anti-P1 would be detected in the serum of ity by incubation at lower temperatures or
25
virtually every person with P1– red cells. by the use of enzyme-treated red cells.
The antibody reacts optimally at 4 C but Hydatid cyst fluid or P1 substance derived
may occasionally be detected at 37 C. from pigeon eggs inhibits the activity of
Anti-P1 is nearly always IgM and has not been anti-P1. Inhibition may be a useful aid to
Lactosylceramide (CDH)
Lactotriaosylceramide Pk antigen
Globotriosylceramide (CTH)
Paragloboside P antigen
(type 2 precursor) Globoside
P1 antigen LKE
Figure 13-6. Biosynthesis of P blood group antigens.

antibody identification, especially if anti-P1 fectiosum (Fifth disease). Individuals of p

is present in a serum with antibodies of phenotype who lack globoside are natu-
other specificities. rally resistant to infection with this patho-
39
gen.
Rare Antibodies
Alloanti-P, found as a naturally occurring
potent hemolytic antibody in the sera of References
P1k and P2k individuals, reacts with all red
1. Landsteiner K. Zur Kenntnis der antifer-
cells except those of the rare p and Pk phe- mentativen, lytischen und agglutinierenden
notypes. Anti-P can be IgM or a mixture of Wirkungen des Blutserums und der Lymph.
IgM and IgG. Anti-PP1Pk, formerly called Zbl Balk 1900;27:367.
a 2. Landsteiner K. Uber Agglutinationserschers-
anti-Tj , is produced by individuals of the
chein ungen normalen menschlichen Blutes.
p phenotype without red cell stimulation Wien Klin Wochenschr 1901;14:1132-4.
and reacts with all red cells except those 3. Garratty G, Dzik W, Issitt PD, et al. Terminol-
of the rare p phenotype. Anti-PP1Pk can be ogy for blood group antigens and genes—his-
torical origins and guidelines in the new mil-
separated into its components (anti-P, lennium. Transfusion 2000;40:477-89.
anti-P1, and anti-Pk) through adsorptions. 4. Von Decastello A, Sturli A. Yber due usiaglutinie
These components can be IgM and/or im Serumgesunder und lronker Menschen.
IgG, react over a broad thermal range, and Munchen Med Wochenschr 1902;26:1090-5.
5. Watkins WM. The ABO blood group system:
can efficiently bind complement, which Historical background. Transfus Med 2001;
make them potent hemolysins. Anti- 11:243-65.
PP1Pk has caused hemolytic transfusion 6. Oriol R, Candelier JJ, Mollicone R. Molecular
reactions and, occasionally, HDFN.
17(p139) genetics of H. Vox Sang 2000;78(Suppl 2):105-
8.
There is an association between both 7. Yamamoto F. Molecular genetics of ABO. Vox
anti-P and anti-PP1Pk and spontaneous Sang 2000;78(Suppl 2):91-103.
35,36
abortions occurring early in pregnancy 8. Yamamoto F. Molecular genetics of the ABO
histo-blood group system. Vox Sang 1995;69:
Autoanti-P associated with paroxysmal
1-7.
cold hemoglobinuria is a cold-reactive IgG 9. Daniels G. Human blood groups. 2nd ed. Ox-
autoantibody that is described as a biphasic ford: Blackwell Science, 2002.
hemolysin.17(pp220-1) The antibody typically 10. Clausen H, Levery SB, Nudelman E, et al. Re-
petitive A epitope (type 3 chain A) defined by
does not react in routine test systems, but is
group A 1 -specific monoclonal antibody
demonstrable only by the Donath-Land- TH-1: Chemical basis of qualitative A1 and A2
steiner test (see Chapter 20). distinction. Proc Natl Acad Sci U S A 1985;82:
1199-203.
11. Yamamoto F, Clausen H, White T, et al. Mo-
P Antigens as Receptors for Pathogens lecular genetic basis of the histo-blood group
ABO system. Nature 1990;345:229-33.
The P blood group antigens are receptors
12. Patenaude SI, Seto NOL, Borisova SN, et al.
for several pathogens. P, P1, Pk, and LKE The structural basis for specificity in human
are receptors for uropathogenic Esche- ABO(H) blood group biosynthesis (letter).
k
richia coli; P and P1 are receptors for tox- Nat Struct Biol 2002;9:685-90.
13. Lee AH, Reid ME. ABO blood group system: A
ins from enterohemorrhagic E. coli37; and review of molecular aspects. Immunohema-
the meningitis-causing bacterium Strep- tology 2000;16:1-6.
tococcus suis binds to Pk antigen.38 The P 14. Olsson ML, Chester MA. Polymorphism and
recombination events at the ABO locus: A
antigen (globoside) has also been shown
major challenge for genomic ABO blood
to serve as a receptor for erythrovirus (par- grouping strategies. Transfus Med 2001;11:
vovirus) B19, which causes erythema in- 295-313.

15. Reid ME, Lomas-Francis C. The blood group tigens: Vancouver report. Vox Sang 2003;84:
antigen factsbook. 2nd ed. San Diego, CA: Ac- 244-7.
ademic Press, 2004. 30. Yang Z, Bergstrom J, Karlsson KA. Glycopro-
16. Olsson ML, Irshaid NM, Hosseini-Maaf B, et teins with Galα4Gal are absent from human
al. Genomic analysis of clinical samples with erythrocyte membranes, indicating that
serologic ABO blood grouping discrepancies: glycolipids are the sole carriers of blood group
Identification of 15 novel A and B subgroup P activities. J Biol Chem 1994;269:14620-4.
alleles. Blood 2001;98:1585-93. 31. Hellberg A, Poole J, Olsson ML. Molecular ba-
17. Mollison PL, Engelfriet CP, Contreras M. sis of the globoside-deficient Pk blood group
Blood transfusion in clinical medicine. 10th phenotype. J Biol Chem 2002;277;29455-9.
ed. Oxford: Blackwell Scientific Publications, 32. Furukawa K, Iwamura K, Uchikawa M, et al.
1997. Molecular basis for the p phenotype. J Biol
18. Silva MA, ed. Standards for blood banks and Chem 2000;275:37752-6.
transfusion services. 23rd ed. Bethesda, MD: 33. Koda Y, Soejima M, Sato H, et al. Three-base
AABB, 2005. deletion and one-base insertion of the α(1,4)
19. Spruell P, Chen J, Cullen K. ABO discrepancies galactosyltransferase gene responsible for the
in the presence of pH-dependent autoagglu- p phenotype. Transfusion 2002;42:48-51.
tinins (abstract). Transfusion 1994;34(Suppl): 34. Arndt PA, Garratty G, Marfoe RA, Zeger GD.
22S. An acute hemolytic transfusion reaction
20. Kennedy MS, Waheed A, Moore J. ABO dis- caused by an anti-P1 that reacted at 37 C.
crepancy with monoclonal ABO reagents Transfusion 1998;38:373-7.
caused by pH-dependent autoantibody. Im- 35. Shirey RS, Ness PM, Kickler TS, et al. The as-
munohematology 1995;11:71-3. sociation of anti-P and early abortion. Trans-
21. Garratty G. In vitro reactions with red blood fusion 1987;27:189-91.
cells that are not due to blood group antibod- 36. Cantin G, Lyonnais J. Anti-PP1Pk and early
ies: A review. Immunohematology 1998;14:1- abortion. Transfusion 1983;23:350-1.
11. 37. Spitalnik PF, Spitalnik SL. The P blood group
22. Beck ML, Yates AD, Hardman J, Kowalski MA. system: Biochemical, serological, and clinical
Identification of a subset of group B donors aspects. Transfus Med Rev 1995;9:110-22.
reactive with monoclonal anti-A reagent. Am 38. Haataja S, Tikkanen K, Liukkonen J, et al.
J Clin Pathol 1989;92:625-9. Characterization of a novel bacterial adhe-
23. Beck ML, Kowalski MA, Kirkegaard JR, Korth sion specificity of Streptococcus suis recogniz-
JL. Unexpected activity with monoclonal anti-B ing blood group P receptor oligosaccharides.
reagents (letter). Immunohematology 1992;8: J Biol Chem 1993;268:4311-17.
22. 39. Brown KE, Hibbs JR, Gallinella G, et al. Resis-
24. Kelly RJ, Ernst LK, Larsen RD, et al. Molecular tance to parvovirus B19 infection due to lack
basis for H blood group deficiency in Bombay of virus receptor (erythrocyte P antigen). N
(Oh) and para-Bombay individuals. Proc Natl Engl J Med 1994;330:1192-6.
Acad Sci U S A 1994;91:5843-7.
26.
Scientific Publications, 1998.
Waheed A, Kennedy MS, Gerhan S, Senhauser
Suggested Reading
DA. Success in transfusion with cross- Chester MA, Olsson ML. The ABO blood group
match-compatible blood. Am J Clin Pathol gene: A locus of considerable genetic diversity.
1981;76:294-8. Transfus Med Rev 2001;15:177-200.
27. Yu L-C, Twu Y-C, Chang C-Y, Lin M. Molecu-
lar basis of the adult I phenotype and the Daniels G. Human blood groups. 2nd ed. Oxford:
gene responsible for the expression of the hu- Blackwell Science Publications, 2002.
man blood group I antigen. Blood 2001;98:
Hanfland P, Kordowicz M, Peter-Katalinic J, et al.
3840-5.
Immunochemistry of the Lewis blood-group sys-
28. Yu L-C, Twu Y-C, Chou M-L, et al. The molec-
tem: Isolation and structure of Lewis-c active and
ular genetics of the human I locus and mo-
related glycosphingolipids from the plasma of
lecular background explain the partial asso-
blood-gro up O L e(a–b–) nonsecretors. Arch
ciation of the adult I phenotype with congenital
Biochem Biophys 1986;246:655-72.
cataracts. Blood 2003;101:2081-8.
29. Daniels GL, Cartron JP, Fletcher A, et al. Inter- Issitt PD, Anstee DJ. Applied blood group serology.
national Society of Blood Transfusion Com- 4th ed. Durham, NC: Montgomery Scientific Pub-
mittee on terminology for red cell surface an- lications, 1998.

Judd WJ. Methods in immunohematology. 2nd ed. challenge for genomic ABO blood grouping strate-
Durham, NC: Montgomery Scientific Publications, gies. Transfus Med 2001;11:295-313.
1994.
Palcic MM, Seto NOL, Hindsgual O. Natural and
Lee AH, Reid ME. ABO blood group system: A re- recombinant A and B gene encoded glycosyltrans-
view of molecular aspects. Immunohematology ferases. Transfus Med 2001;11:315-23.
2000;16:1-6.
Rydberg L. ABO-incompatibility in solid organ
Morgan WTJ, Watkins WM. Unraveling the bio- transplant. Transfus Med 2001;11:325-42.
chemical basis of blood group ABO and Lewis an-
tigenic specificity. Glycoconj J 2000;17:501-30. Watkins WM. The ABO blood group system: His-
torical background. Transfus Med 2001;11:243-65.
Olsson ML, Chester MA. Polymorphism and re-
combination events at the ABO locus: A major Yamamoto F. Cloning and regulation of the ABO
genes. Transfus Med 2001;11:281-94.

Chapter 14: The Rh System
Chapter 14
14
The Rh System
T
HIS CHAPTER USES the DCE no- the serum of a woman whose fetus had
menclature—a modification of the hemolytic disease of the fetus and new-
nomenclature originally proposed born (HDFN) and who experienced a
by Fisher and Race,1 which has been able hemolytic reaction after transfusion of
to accommodate our present understand- her husband’s blood. In 1940, Landsteiner
ing of the genetics and biochemistry of this 3
and Wiener described an antibody ob-
complex system. The Rh-Hr terminology tained by immunizing guinea pigs and
of Wiener is presented only in its histori- rabbits with the red cells of Rhesus mon-
cal context, as molecular genetic evidence keys; it agglutinated the red cells of ap-
does not support Wiener’s one-locus theory. proximately 85% of humans tested, and
they called the corresponding determi-
nant the Rh factor. In the same year, Le-
4
The D Antigen and Its vine and Katzin found similar antibodies
in the sera of several recently delivered
Historical Context women, and at least one of these sera
gave reactions that paralleled those of the
Discovery of D animal anti-Rhesus sera. Also in 1940,
5
The terms “Rh positive” and “Rh negative” Wiener and Peters observed antibodies of
refer to the presence or absence of the red the same specificity in the sera of persons
cell antigen D. The first human example whose red cells lacked the determinant
of the antibody against the antigen later and who had received ABO-compatible
called D was reported in 1939 by Levine transfusions in the past. Later evidence
and Stetson2; the antibody was found in established that the antigen detected by
315

animal anti-Rhesus and human anti-D Although Rh antigens are fully expressed
were not identical, but, by that time, the at birth with antigen detection as early as 8
Rh blood group system had already re- weeks’ gestation,10 they are present on red
ceived its name. Soon after anti-D was cells only and are not detectable on plate-
discovered, family studies showed that lets, lymphocytes, monocytes, neutrophils,
the D antigen is genetically determined; or other tissues.11,12
transmission of the trait follows an auto-
somal dominant pattern.
Clinical Significance
Genetic and Biochemical
After the A and B antigens, D is the most
Considerations
important red cell antigen in transfusion Attempts to explain the genetic control of
practice. In contrast to A and B, however, Rh antigen expression were fraught with
persons whose red cells lack the D anti- controversy. Wiener13 proposed a single
gen do not regularly have anti-D. Forma- locus with multiple alleles determining
tion of anti-D results from exposure, surface molecules that embody numerous
through transfusion or pregnancy, to red antigens. Fisher and Race14 inferred from
cells possessing the D antigen. The D an- the existence of antithetical antigens the
tigen has greater immunogenicity than existence of reciprocal alleles at three in-
other red cell antigens; it is estimated that dividual but closely linked loci. Tippett’s
6,7
30% to 85% of D– persons who receive a prediction15 that two closely linked struc-
D+ transfusion will develop anti-D. There- tural loci on chromosome 1 determine
fore, in most countries, the blood of all re- the production of Rh antigens has been
cipients and all donors is routinely tested shown to be correct.
for D to ensure that D– recipients are
identified and given D– blood. RH Genes
Two highly homologous genes on the
short arm of chromosome 1 encode the
Other Rh Antigens nonglycosylated polypeptides that ex-
press the Rh antigens (Fig 14-1).16,17 One
By the mid-1940s, four additional anti- gene, designated RHD, determines the
gens—C, E, c, and e—had been recog- presence of a membrane-spanning pro-
nized as belonging to what is now called tein that confers D activity on the red cell.
the Rh system. Subsequent discoveries In Caucasian D– persons, the RHD gene is
have brought the number of Rh-related deleted; the D– phenotype in some other
antigens to 49 ( Table 14-1), many of populations (persons of African descent,
which exhibit both qualitative and quan- Japanese, and Chinese) is associated with
titative variations. The reader should be an inactive, mutated, or partial RHD
aware that these other antigens exist (see gene.18 The inactive RHD gene or pseudo-
the suggested reading list), but, in most gene (RHD ) responsible for the D– phe-
transfusion medicine settings, the five notype in some Africans has been de-
principal antigens (D, C, E, c, e) and their scribed.19
corresponding antibodies account for the The RHCE gene determines the C, c, E,
vast majority of clinical issues involving and e antigens; its alleles are RHCe, RHCE,
the Rh system. RHcE, and RHce.20 Evidence derived from

Chapter 14: The Rh System 317
Table 14-1. Antigens of the Rh Blood Group System and Their Incidence
Incidence (%)* Incidence (%)*

Numeric Antigen Numeric Antigen
Designation Name White Black Overall Designation Name 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 Cw 2 1 Rh40 Tar <0.01
Rh9 Cx <0.01 Rh41 Ce-like 70
s
Rh10 V 1 30 Rh42 Ce <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 hrs 98 Rh47 Dav >99.9
Rh20 VS <0.01 32 Rh48 JAL <0.01
Rh21 CG 68 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 Goa 0 <0.01 Rh56 CENR <0.01
Rh31 hrB 98
8,9
*Incidence in White and Black populations where appropriate.
21
transfection studies indicates that both that, modeling studies suggest, traverse
C/c and E/e reside on a single polypeptide the red cell membrane 12 times and dis-
product. play only short exterior loops of amino
acids (Fig 14-1). The polypeptides are fatty
Biochemical and Structural Observations acylated and, unlike most blood-group-
The predicted products of both RHD and associated proteins, carry no carbohy-
RHCE are proteins of 417 amino acids drate residues.

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.
Considerable homology exists between phology changes in Rhnull syndrome (see

the products of RHD and RHCE; the prod- later in this chapter), their function remains
ucts of the different alleles of RHCE are unknown. There is evidence, however, that
even more similar.18 C and c differ from one the RhAG protein plays a role in ammo-
another in only four amino acids, at posi- nium transport.24,25
tions 16, 60, 68, and 103, of which only the
difference between serine and proline at
103 appears to be critical. The presence of
proline or alanine at position 226 distin-
Rh Terminology
guishes E from e. The D polypeptide, by Three systems of nomenclature were de-
contrast, possesses 32 to 35 amino acids veloped to convey genetic and serologic
that will be perceived as foreign by D– information about the Rh system before
individuals. the recent advances in our understanding
Within the red cell membrane, the Rh of the genetics.
polypeptides form a complex with the Rh-
associated glycoprotein (RhAG), which has System Terminology
37% sequence homology with the Rh poly- The Rh-Hr terminology derives from
peptides but is encoded by the RHAG gene Wiener,13 who believed the RH gene prod-
on chromosome 6 (Fig 14-1).22 uct to be a single entity he called an
The study of Rhnull red cells, which lack all agglutinogen. An agglutinogen was char-
Rh antigens, reveals that this complex (Rh acterized by numerous individual speci-
proteins and RhAG) is essential for expres- ficities, called factors, that were identified
sion of other membrane proteins. Rhnull cells by specific antibodies. This theory was in-
lack LW antigens, are negative for Fy5 of the correct, but for the designation of pheno-
Duffy system, and have weakened expres- type, particularly in conversation, many
sion of the antigens carried on glycophorin serologists use a shorthand system based
B (S, s, and U).23 Although Rh/RhAG pro- on Wiener’s Rh-Hr notation. The pheno-
teins play a structural role in the red cell type notations convey haplotypes with the
membrane as evidenced by red cell mor- single letters R and r. R is used for haplo-

types that produce D, r for haplotypes that -c, and -e. Routine pretransfusion studies
do not produce D. Subscripts or, occa- include only tests for D. Other reagents
sionally, superscripts indicate the combi- are used principally in the resolution of
nations of other antigens present. For ex- antibody problems or in family studies.
ample, R 1 indicates DCe haplotype; R 2 The assortment of antigens detected on a
indicates DcE; r indicates dce; R0 indicates person’s red cells constitutes that person’s
Dce; and so on (Table 14-2). Rh phenotype. Table 14-3 shows reaction
CDE terminology was introduced by patterns of cells tested with antibodies to
Fisher and Race,1 who postulated three sets the five antigens and the probable Rh
of closely linked genes (C and c, D and d, phenotype in modified Wiener terminol-
and E and e). Both gene and gene product ogy.
have the same letter designation, with ital-
ics used for the name of the gene. A modi-
fied CDE terminology is now commonly
used to communicate research and sero-
Serologic Testing for Rh
logic findings. Rosenfield and coworkers
26
Antigen Expression
proposed a system of nomenclature based
on serologic observations. Symbols were Expression of D
not intended to convey genetic informa- D– persons either lack RHD, which en-
tion, merely to facilitate communication of codes for the D antigen, or have a non-
phenotypic data. Each antigen is given a functional RHD gene. Most D– persons
number, generally in the order of its discov- are homozygous for RHce, the gene en-
ery or its assignment to the Rh system. Ta- coding c and e; less often they may have
ble 14-1 lists the Rh system antigens by RHCe or RHcE, which encode C and e or c
number designation, name, and incidence. and E, respectively. The RHCE gene,
which produces both C and E, is quite
Determining Phenotype rare in D– or D+ individuals.
In clinical practice, five blood typing re- The D genotype of a D+ person cannot
agents are readily available: anti-D, -C, -E, be determined serologically; dosage studies
are not effective in showing whether an in-
dividual is homozygous or heterozygous for
Table 14-2. The Principal RH Gene
RHD. Using serologic tests, RHD genotype
Complexes and the Antigens Encoded
can be assigned only by inference from the
antigens associated with the presence of D.
Genes Antigens Pheno- Molecular techniques, however, allow the
Haplotype Present Present type
determination of D genotype.19,27,28
R1 RHD,RHCe D,C,e R1 Interaction between genes results in so-
called “position effect.” If the interaction is
R2 RHD,RHcE D,c,E R2
between genes or the product of genes on
Ro RHD,RHce D,c,e Ro
the same chromosome, it is called a cis ef-
Rz RHD,RHCE D,C,E Rz fect. If a gene or its product interacts with
r′ RHCe C,e r′ one on the opposite chromosome, it is
r″ RHcE c,E r″ called a trans effect. Examples of both ef-
r RHce c,e r fects were first reported in 1950 by Lawler
ry RHCE C,E ry and Race,29 who noted as a cis effect that the
E antigen produced by DcE is quantitatively

Table 14-3. Determination of Likely Rh Phenotypes from the Results of Tests with
the Five Principal Rh Blood Typing Reagents
Reagent
Antigens Probable
Anti-D Anti-C Anti-E Anti-c Anti-e Present Phenotype
+ + 0 + + D,C,c,e R1r
+ + 0 0 + D,C,e R1R1
+ + + + + D,C,c,E,e R1R2
+ 0 0 + + D,c,e R0R0/R0r
+ 0 + + + D,c,E,e R2r
+ 0 + + 0 D,c,E R2R2
+ + + 0 + D,C,E,e R1Rz
+ + + + 0 D,C,c,E R2Rz
+ + + 0 0 D,C,E RzRz
0 0 0 + + c,e rr
0 + 0 + + C,c,e r′r
0 0 + + + c,E,e r″r
0 + + + + C,c,E,e r′r″
weaker than E produced by cE. They noted genes differs from one geographic group
as trans effects that both C and E are to another. For example, a White person
weaker when they result from the genotype with the phenotype Dce would probably
DCe/DcE than when the genotypes are be Dce/ce, but, in a Black person, the ge-
DCe/ce or DcE/ce, respectively. notype could as likely be either Dce/Dce or
Dce/ce. Table 14-4 shows the incidence of
Expression of C, c, E, e D, C, E, c, and e antigens in White and
Black populations.
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- Gene Frequency
gens. If the red cells express both C and c
The phenotype DCcEe (line 3 of Table
or both E and e, it can be assumed that
14-3) can arise from any of several geno-
the corresponding genes are present in the
types. In any population, the most proba-
individual. If the red cells carry only C or
ble genotype is DCe/DcE. Both these
c, or only E or e, the person is assumed to
haplotypes encode D; a person with this
be homozygous for the particular allele.
phenotype will very likely be homozygous
for the RHD gene, although heterozygous
Ethnic Origin for the RHCE gene (Ce/cE). Some less
Ethnic origin influences deductions about likely genotypes could result if the person
genotype because the incidence of Rh is heterozygous at the D locus (for exam-

ple, DCe/cE, DcE/Ce, or DCE/ce), but these sumptions regarding the most probable ge-
are uncommon in all populations. Table notype rest on the incidence of antigenic
14-4 gives the incidence of the more com- combinations determined from population
mon genotypes in D+ persons. The figures studies in different ethnic groups. Infer-
given are for Whites and Blacks. The ab- ences about genotype are useful in popu-
sence of the RHD gene is uncommon in
lation studies, paternity tests, and in pre-
other ethnic groups.
dicting the Rh genes transmitted by the
husband/partner of a woman with Rh an-
Determining Genotype
tibodies (Table 14-4).
Identifying antigens does not always al- Molecular techniques are now available
low confident deduction of genotype. Pre- that can determine Rh genotype. Determi-
Table 14-4. Incidence of the More Common Genotypes in D+ Persons*
Likelihood of Zygosity for D (%)
Genotype Incidence (%) Homo- Hetero- Homo- Hetero-
Antigens Mod.
Present DCE 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
2
DcE/Ce Rr 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 manufacturers and to know the character-
reaction methods can be made using DNA istics of the product being used.
harvested from white cells or amnio-
cytes19,27,28 or from noncellular fetal DNA in Quantitative Weak D
30
maternal plasma. Rarely, DNA genotype
In the majority of cases, this form of the
results will disagree with serologic findings.
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-
Weak D rane or cytoplasmic location of the amino
acid changes in the altered D protein does
Most D+ red cells show clear-cut macro-
not result in the loss of D epitopes; thus,
scopic agglutination after centrifugation
the production of alloanti-D as in the par-
with reagent anti-D and can be readily
tial D phenotype (see partial D) would not
classified as D+. Red cells that are not im-
be expected.31,32 Weak D expression is fairly
mediately or directly agglutinated cannot
common in Blacks, often occurring as part
as easily be classified. For some D+ red
of a Dce haplotype. Genes for weak D ex-
cells, demonstration of the D antigen re-
pression are less common in Whites and
quires incubation with the anti-D reagent
may be seen as part of an unusual DCe or
or addition of antihuman globulin (AHG)
DcE haplotype.
serum after incubation with anti-D [indi-
Red cell samples with a quantitative
rect antiglobulin test (IAT)]. These cells are
weak D antigen either fail to react or react
considered D+, even if an additional step
very weakly in direct agglutination tests with
in testing is required.
most anti-D reagents. However, the cells will
In the past, red cells that required addi-
react strongly by an IAT.
tional steps for the demonstration of D
Red cells from some persons of the ge-
were classified as Du. The term Du is no lon-
notype Dce/Ce have weakened expression
ger considered appropriate; red cells that
of D, a suppressive effect exerted by RHC in
carry weak forms of D are classified as D+
the trans position to RHD on the opposite
and should be described as “weak D.” Im-
chromosome. Similar depression of D can
provement of polyclonal reagents and the
be seen with other RHD haplotypes accom-
more widespread use of monoclonal anti-D
panied by RHCe in trans position. Many of
reagents have resulted in the routine detec-
the weak D phenotypes due to position ef-
tion of some D+ red cells that would have
fect that were reported in the early litera-
been considered weak D when tested with
ture appear as normal D.
less sensitive polyclonal reagents. Addition-
ally, monoclonal anti-D may react by direct
agglutination with epitopes of D that had Partial D
previously required more sensitive test The concept that the D antigen consists of
methods or, occasionally, may fail to react multiple constituents arose from observa-
with some epitopes of the D antigen. Con- tions that some people with D+ red cells
versely, some monoclonal anti-D may react produced alloanti-D that was nonreactive
by direct testing with rare D epitopes that with their own cells. Most D+ persons who
were not detected with polyclonal reagents produce alloanti-D have red cells that re-
(eg, DHAR and Crawford). It is important to act strongly when tested with anti-D. But
realize that anti-D reagents differ among some, especially those of the DVI pheno-

type, give weaker reactions than normal D– recipients is not recommended due to
D+ red cells or react only in the AHG test. the fact that some weak or partial D red
Red cells lacking components of the D an- cells could elicit an immune response to
tigen have been referred to in the past as D.37 Weak forms of the D antigen, how-
“D mosaic” or “D variant.” Current termi- ever, seem to be less immunogenic than
nology more appropriately describes these normal D+ blood; transfusion of a total of
red cells as “partial D.” 68 units of blood with weak D to 45 D– re-
Categorization of partial D phenotypes cipients failed to stimulate production of
was performed by cross-testing red cells a single example of anti-D.38
with alloanti-D produced by D+ persons. Hemolytic transfusion reactions and HDFN
The four categories initially described by due to weak D red cells were reported in
Wiener have been expanded considerably the early literature, but it is probable that,
over the years. Tests of many monoclonal with currently available reagents, the re-
anti-D reagents with red cells of various D sponsible cells would have been considered
categories suggest that the D antigen com- D+.39
prises numerous epitopes. Partial D pheno-
types can be defined in terms of their D
33 Significance of Weak/Partial D in
epitopes. Tippett et al established at least
Recipients
10 epitopes but point out that the D anti-
gen is not large enough to accommodate The transfusion recipient whose red cells
more than eight distinct epitopes and there test as weak D is sometimes a topic of de-
must be considerable overlap between them. bate. Most of these patients can almost al-
A current model describes 30 epitopes,34 ways receive D+ blood without risk of im-
thus demonstrating the dynamic nature of munization, but if the weak D expression
the D-epitope model as it is revised due to reflects the absence of one or more D epi-
variation in reagents and techniques. Dogma topes, the possibility exists that transfu-
regarding the existence of many discrete sion of D+ blood could elicit the produc-
epitopes is giving way to a model that is more tion of alloanti-D. This is especially true in
topographic in nature, with the fit of anti- persons of the DVI phenotype. The same
body to antigen described as a “footprint.”35 possibility exists, however, for persons
Molecular studies have elucidated the whose partial D red cells react strongly
genetic mechanisms behind many of the with anti-D reagents. AABB Standards for
partial D phenotypes and have shown that Blood Banks and Transfusion Services36(p48)
the phenotypes arise as the result of ex- only requires recipients’ specimens to be
change between the RHCE gene and the RHD tested with anti-D by direct agglutination.
9,18
gene or from single-point mutations. The test for weak D is not required. Cur-
rently available, licensed anti-D reagents
are sufficiently potent that most patients
Significance of Weak/Partial D in Blood
with weak D are found to be D+. The few
Donors
patients classified as D–, whose D+ status
AABB Standards for Blood Banks and is only detectable by an IAT, can receive
36(p32)
Transfusion Services requires donor D – b l o o d w i t h o u t p r o b l e m s. So m e
blood specimens to be tested for weak ex- serologists consider this practice wasteful
pression of D and to be labeled as D+ if of D– blood and prefer to test potential re-
the test is positive. Transfusion of blood cipients for weak D, then issue D+ blood
with weak expression of the D antigen to when indicated.

If D+ blood is given to recipients of the ditional to those defined as D, C, and e.

weak D phenotype, it is important to safe- These include Ce (rhi), a cis product that
guard against careless or incorrect interpre- almost always accompanies C and e when
tation of tests. D– recipients erroneously they are encoded by the same haplotype.
classified as D+, possibly due to a positive di- The Ce antigen is absent from red cells on
rect antiglobulin test (DAT) causing a false- which the C and e were encoded by differ-
positive test for weak D, run the risk of im- ent haplotypes, for example, in a person
munization to D if given D+ blood. Individu- of the genotype DCE/ce. Similar cis prod-
als whose weakly expressed D antigen is de- uct antigens exist for c and e determined
tectable only by an IAT will be classified as by the same haplotype (the antigen called
D– recipients if an IAT is not performed. ce or f ), for c and E (cE), and for C and E
However, if they donate blood subsequently, (CE).
they will be classified as D+ at the time of Although antibodies directed at cis prod-
blood donation. Personnel in blood centers uct antigens are encountered infrequently,
and transfusion services should be prepared it would not be correct to consider them
to answer questions from puzzled donors or rare. Such antibodies may be present but
their physicians. This can present special unnoticed in serum containing antibodies
problems in autologous donations, when the of the more obvious Rh specificities; only
D– patient’s own blood is labeled as D+. In adsorption with red cells of selected pheno-
this case, confirmation of the patient’s D sta- types would demonstrate their presence.
tus by the IAT resolves the apparent discrep- Anti-f (ce) may be present, for example, as a
ancy between recipient and donor types. 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
Other Rh Antigens given by anti-c and anti-e because all red
Numbers up to 56 have been assigned to cells that react with anti-f will express both
Rh red cell antigens (see Table 14-1); some c and e. A person with the genotype
of the numbers are now obsolete because DCe/DcE who makes anti-f may receive c–
antigens have been rescinded or reas- or e– red cells because cells of either phe-
signed. Of the currently included 49, most notype would also be f–.
beyond D, C, c, Cw, E, and e and their cor- Anti-Ce is frequently the true specificity
responding antibodies are encountered of the apparent anti-C that a DcE/DcE per-
much less frequently in routine blood son produces after immunization with C+
transfusion therapy. blood. This knowledge can be helpful in es-
tablishing an individual’s Rh genotype. If
Cis Product Antigens anti-Ce is the predominant specificity in a
The membrane components that exhibit reagent anti-C, the individual whose weak
Rh activity have numerous possible anti- C antigen resulted from a DCE haplotype
genic subdivisions. Each gene or gene may be mistyped unless test methods and
complex determines a series of interre- control red cells are chosen carefully.40
lated surface structures, of which some
portions are more likely than others to
The G Antigen and Cross-Reactions
elicit an immune response. The poly-
peptides determined by the genes in the The G antigen results from serine at posi-
haplotype DCe express determinants ad- tion 103 of the Rh polypeptides and is en-

41
coded by either RHD or RHCE. As a re- antithetical antigens at specific surface
sult, the G antigen is almost invariably sites. Antigens that behave as if they have
present on red cells possessing either C or an antithetical relationship to C/c or E/e
D. Antibodies against G appear superfi- have been found, mainly in Whites. The
w
cially to be anti-C+D, but the anti-G activ- most common is C , but the relationship
ity cannot be separated into anti-C and is phenotypic only because Cw and Cx are
anti-D. The fact that G appears to exist as antithetical to the high-incidence antigen,
an entity common to C and D explains the MAR. Variant forms of the e antigen have
fact that D– persons immunized by C–D+ been identified, for example, hrS or hrB an-
red cells sometimes appear to have made tigens (Rh19 and Rh31, respectively). Per-
S B
anti-C as well as anti-D, and why D– persons who are e+ and hr – and/or hr – are
sons who are exposed to C+D– red cells found more frequently in Black popula-
develop antibodies appearing to contain tions.9 The absence of hrB is associated in
an anti-D component. Differentiation of most cases with the presence of the VS
anti-D, -C, and -G is not necessary in the antigen.44
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 Rhnull Syndrome and Other
specificities to determine the need for Rh
42
Deletion Types
immune globulin prophylaxis. Differen-
tial adsorption and elution studies to dis-
Rhnull
tinguish anti-D, -C, and -G are outlined by
Issitt and Anstee.
43 The literature reports at least 43 persons
Rare red cells have been described that in 14 families whose red cells appear to
G
possess G but lack D and C. The r pheno- have no Rh antigens; others are known
type is found mostly in Blacks; generally, but have not been reported. The pheno-
the G antigen is weakly expressed and is as- type, described as Rhnull, is produced by at
sociated with the presence of the VS anti- least two different genetic mechanisms.
G
gen. The r phenotype has been described In the more common regulator type of
in Whites but is not the same as r in
G Rhnull, mutations occur in the RHAG gene
Blacks. Red cells also exist that express par- that result in the complete absence of the
tial D but lack G entirely, for example, per- core Rh complex (Rh polypeptides and
sons of the DIIIb phenotype.39 RhAG) that is necessary for the expression
of Rh antigens.18 Such persons transmit
normal RHD and RHCE genes to their off-
Variant Antigens
spring.
Although red cells from most people give The other form of Rhnull, the amorph
straightforward reactions with reagent type, has a normal RHAG gene; however,
anti-D, anti-C, anti-E, anti-c, and anti-e there is a mutation in each RHCE gene to-
sera, some cells give atypical reactions gether with the common deletion of RHD
and other seemingly normal red cells (as in D– individuals).18 The amorph type of
stimulate the production of antibodies Rhnull is considerably rarer than the regula-
that do not react with red cells of com- tor type. Parents and offspring of this type
mon Rh phenotypes. It has been conve- of Rhnull are obligate heterozygotes for the
nient to consider C and c, and E and e as amorph.

Red Cell Abnormalities will demonstrate their presence. As in

Rhnull, hemolytic anemia is a feature of the
Whatever the genetic origin, red cells lack-
ing Rh/RhAG proteins have membrane Rhmod condition. It may be appropriate to
abnormalities and a compensated hemoly- think of the two abnormalities as being
tic anemia. The severity of hemolysis and essentially similar, differing only in de-
resulting anemia varies among affected gree. As in the regulator type of Rhnull, a
persons, but stomatocytosis, shortened mutation in RHAG was shown to result in
18
red cell survival, absence of LW and Fy5 the Rhmod phenotype.
antigens, and variably altered activity of S,
s, and U have been consistent features. Deleted Phenotypes
Rare RH haplotypes encode D antigen but
fail to encode some or all of the CE anti-
Serologic Observations gens. Some examples of deleted phenotypes
A few Rhnull individuals were recognized include D– – , D••, DCw–, and Dc–. These
because their sera contained Rh antibod- rare phenotypes have been shown to arise
ies. Some came to light, however, when from replacement of large portions of RHCE
routine Rh phenotyping of their red cells with RHD.18 Red cells that lack C/c and/or
revealed the absence of any Rh antigens. E/e antigens may show exceptionally
In three cases, the discovery resulted from strong D activity, an observation by which
deliberate testing for Rh antigens in pa- such red cells have sometimes been rec-
tients with morphologically abnormal red ognized during routine testing with anti-D.
cells and hemolytic anemia. Immunized The D– – phenotype may be identified in
Rhnull people have produced antibodies the course of studies to investigate an un-
varying in specificity from apparently expected antibody. Such persons may have
straightforward anti-e or anti-C to several alloantibody of complex specificity be-
examples that reacted with all red cells cause the person’s red cells lack all the
tested except those from other Rhnull peo- epitopes expressed on the RhCE poly-
ple. This antibody, considered to be “anti- peptide. Antibody with Rh17 (Hro) speci-
total Rh,” has been given the numerical ficity is often made by persons of this rare
designation anti-Rh29. phenotype, although some sera have been
reported to contain apparently separable
specificities, such as anti-e.
Rhmod The D•• phenotype is similar to D– –, ex-
The Rhmod phenotype represents less com- cept that the D antigen is not elevated to
plete suppression of Rh antigen expres- the same degree. D•• red cells may be ag-
sion. Unlike Rhnull red cells, those classi- glutinated weakly by some examples of sera
fied as Rhmod do not completely lack Rh from immunized Rh-deletion persons if the
and LW antigens. Rhmod red cells show serum sample contains anti-Rh47 in addi-
39
much reduced and sometimes varied action to anti-Rh17. Rh47 is a high-inci-
tivity, depending on the Rh system genes dence antigen encoded by the RHCE gene
the individual possesses and on the po- and is absent from other deleted pheno-
tency and specificity of the antisera used types (eg, D– –, DCw– , Dc–). Another distin-
in testing. Sometimes, the Rh antigens guishing characteristic of D•• red cells is
have sufficiently weakened expression that they possess the low-incidence antigen
that only adsorption-elution techniques Rh37 (Evans).

Rh Antibodies Anti-D in D+ Individuals

Alloanti-D may be produced in D+ indi-
Most Rh antibodies result from exposure viduals with partial D phenotype (see Par-
to human red cells through pregnancy or tial D), although not all persons who are
transfusion. Occasionally, Rh antibodies D+ and produce what appears to be anti-D
(eg, anti-E, anti-Cw) are naturally-occur- should be assumed to have epitope-defi-
ring. D is the most potent immunogen, cient red cells. Weakly reactive anti-LWab
a
followed by c and E. Although a few exam- or anti-LW may react with D+ cells but
ples of Rh antibodies behave as saline ag- not with D– cells. A D+ person whose an-
glutinins, most react best in high-protein, tibody is a weakly reactive anti-LWa may
antiglobulin, or enzyme test systems. Even be indistinguishable on initial serologic
sera containing potent saline-reactive testing from an individual with a partial D
anti-D are usually reactive at higher dilu- antigen who has made anti-D to missing
tions in antiglobulin testing. Some work- epitopes. (See the section on the LW sys-
ers find enzyme techniques especially use- tem in Chapter 15.) Anti-LW can be differ-
ful for detecting weak or developing Rh entiated from anti-D by testing the anti-
antibodies. body with 0.02M DTT-treated red cells
Antibody usually persists for many years. (Method 3.10); the LW antigen is de-
If serum antibody levels fall below detect- stroyed by sulfhydryl reagents, whereas D
able thresholds, subsequent exposure to the is unaffected.
antigen characteristically produces a rapid
secondary immune response. With exceed- Concomitant Antibodies
ingly rare exceptions, Rh antibodies do not
bind complement, at least to the extent rec- Some Rh antibodies tend to occur in con-
ognizable by techniques currently used. As cert. For example, the DCe/DCe person
a result, primarily extravascular hemolysis, manifesting immune anti-E almost cer-
instead of intravascular hemolysis, occurs tainly has been exposed to c as well as E.
in transfusion reactions involving Rh anti- Anti-c may be present in addition to
bodies. anti-E, although substantially weaker and
possibly undetectable at the time the
anti-E is found, and transfusion of seem-
Dosage Effect
ingly compatible E–c+ blood may elicit an
Anti-D seldom shows any difference in re- immediate or delayed reaction. Generally,
activity between red cells from individu- it is not a sound practice to select donor
als homozygous or heterozygous for RHD, blood that is negative for antigens absent
but D expression seems to vary somewhat from the recipient’s red cells when anti-
with the accompanying alleles of the ge- body has not been detected, but some
notype. For example, red cells from a practitioners feel that the DCe/DCe recipi-
DcE/DcE individual carry more D antigen ent with anti-E is a case that merits avoid-
sites than red cells from a DCe/DCe per- ance of c+ blood as well.45 Anti-E less con-
son and may show higher titration scores sistently accompanies anti-c because the
with anti-D. Dosage effects can some- patient can easily have been exposed to c
times be demonstrated with some anti- without being exposed to E. There is little
bodies directed at the E, c, and e antigens clinical value in pursuing anti-E in serum
and, occasionally, at the C antigen. known to contain anti-c because the vast

majority of c– donor blood will be nega- can cause the red cells to aggregate and be
tive for the E antigen. misinterpreted as agglutination. There are
also greater biohazardous risks associated
with increased potential for spillage of the
specimen during manipulation. Represen-
Rh Typing tative procedures for tube, slide, and micro-
Routine Rh typing for donors and patients plate tests are given in Methods 2.6, 2.7,
involves only the D antigen. Tests for the and 2.8.
other Rh antigens are performed when Techniques to demonstrate weak D are
identifying unexpected Rh antibodies, ob- required by AABB Standards only for donor
taining compatible blood for a patient blood or for testing blood from neonates
with an Rh antibody, investigating parent- born to Rh-negative women to determine
age or other family studies, selecting a Rh immune globulin candidacy. 36(pp32,48)
panel of phenotyped cells for antibody When there is an indication to test for weak
identification, or evaluating whether a D, an IAT should be performed (Method
person is likely to be homozygous or het- 2.9). A reliable test for weak D expression
erozygous for RHD. cannot be performed on a slide.
In finding compatible blood for a recipi-
ent with a comparatively weak Rh antibody,
High-Protein Reagents
tests with potent blood typing reagents
more reliably confirm the absence of anti- Some anti-D reagents designated for use
gen than mere demonstration of a compat- in slide, rapid tube, or microplate tests
ible crossmatch. Determination of the pa- contain high concentrations of protein
tient’s Rh phenotype may help confirm the (20-24%) and other macromolecular addi-
antibody specificity and indicate which tives. Such reagents are nearly always pre-
other Rh antibodies could also be present. pared from pools of human sera and give
rapid reliable results when used in accor-
Routine Testing for D dance with manufacturers’ directions.
High-protein levels and macromolecular
Until recently, high-protein anti-D re-
additives may cause false-positive reac-
agents of human polyclonal origin that
tions. A false-positive result could cause a
were suitable for slide, tube, or microplate
D– patient to receive D+ blood and be-
tests were used for most routine testing.
come immunized. An appropriate control
More recently, monoclonal anti-D re-
tested according to the manufacturer’s di-
agents have become widely available.
rections must be performed.
Tests may employ red cells suspended in
saline, serum, or plasma, but test condi-
tions should be confirmed by reading the Control for High-Protein Reagents
manufacturer’s directions before use. Pro- Manufacturers offer their individual dilu-
cedures for microplate tests are similar ent formulations for use as control re-
to those for tube tests, but very light sus- agents. The nature and concentration of
pensions of red cells are used. additives differ significantly among re-
Slide tests produce optimal results only agents from different manufacturers and
when a high concentration of red cells and may not produce the same pattern of
protein are combined at a temperature of false-positive reactions. If red cells exhibit
37 C. A disadvantage of the slide test is aggregation in the control test, the results
evaporation of the reaction mixture, which of the anti-D test cannot be considered

valid. In most cases the presence or ab- 3. Red cells possessing weakly expres-
sence of D can be determined with other sed D antigen may not react well
reagents, as detailed later in this chapter. within the 2-minute limit of the slide
test or upon immediate centrifuga-
tion in the tube test.
Misleading Results with High-Protein

Low-Protein Reagents
Reagents
The low-protein Rh reagents in current
False Positives. The following circum-
use are formulated predominantly with
stances can produce false-positive red cell
monoclonal antibodies. Immunoglobu-
typing results.
lin-coated red cells can usually be suc-
1. Cellular aggregation resulting from
cessfully typed with low-protein Rh re-
immunoglobulin coating of the pa-
agents that contain saline-agglutinating
tient’s red cells or serum factors that
antibodies.
induce rouleaux will give positive re-
sults in both the test and the control
tubes. Serum factors can be elimi- Monoclonal Source Anti-D
nated by thoroughly washing the red Monoclonal anti-D reagents are made
cells (with warm saline if cold agglu- predominantly from human IgM antibod-
tinins are present or suspected) and ies, which require no potentiators and ag-
retesting. If the cells in the control glutinate most D+ red cells from adults
test remain unagglutinated and the and infants in a saline system. Monoclonal
anti-D test gives a positive result, the anti-D reagents usually promote reactions
red cells are D+. If agglutination still stronger than those with polyclonal IgG
occurs in the control tube, the most reagents, but they may fail to agglutinate
likely explanation is immunoglobu- red cells of some partial D categories.
lin coating of the red cells, which Adding small amounts of IgG anti-D to
should then be tested with low-pro- the monoclonal IgM antibodies provides
tein reagents. a reagent that will react with weak or par-
2. Red cell aggregation, simulating ag- tial D red cells in antiglobulin tests. Cer-
glutination, may occur if red cells and tain rare D+ red cells (DHAR, DVa, DVc,
anti-D are incubated too long and Rh43+) may react at immediate spin with
excessive evaporation occurs during some of these blended reagents, but, with
the slide test. It is important to fol- other reagents, these same cells may be
low the manufacturer’s recommen- nonreactive in an IAT or reactive only in
dations to interpret the test within an IAT.
the recommended period. Licensed monoclonal/polyclonal or
False Negatives. The following circum- monoclonal/monoclonal blends can be
stances can produce false-negative red used by all routine typing methods and are
cell typing results. as satisfactory as high-protein reagents in
1. Too heavy a red cell suspension in an IAT for weak D. False-negative findings
the tube test or too weak a suspen- can result, however, if tests using mono-
sion in the slide test may weaken ag- clonal reagents are incubated in excess of a
glutination. manufacturer’s product directions. These re-
2. Saline-suspended red cells must not agents, prepared in a low-protein medium,
be used for slide testing. can be used to test red cells with a positive

DAT, provided those tests are not subjected fant’s red cells may be so heavily coated
to an IAT. with antibody that all antigen sites are oc-
cupied, leaving none available to react
Control for Low-Protein Reagents with a low protein antibody of appropri-
ate specificity. This “blocking” phenome-
Most monoclonal blended reagents have
non should be suspected if the infant’s
a total protein concentration approximat-
cells have a strongly positive DAT and are
ing that of human serum. False-positive
not agglutinated by a low protein reagent
reactions due to spontaneous aggregation
of the same specificity as the maternal an-
of immunoglobulin-coated red cells occur
tibody.
no more often with this kind of reagent
Anti-D is the specificity responsible for
than with other saline-reactive reagents.
nearly all cases of blocking by maternal an-
False-positive reactions may occur in any
tibody. It is usually possible to obtain cor-
saline-reactive test system if the serum
rect typing results with a low protein anti-D
contains cold autoagglutinins or a protein
after 45 C elution of the maternal antibody
imbalance causing rouleaux and the red
from the cord blood red cells. (See Method
cells are tested unwashed. It is seldom
2.12.) Elution liberates enough antigen sites
necessary to perform a separate control
to permit red cell typing, but it must be per-
test. Absence of spontaneous aggregation
formed cautiously because overexposure to
can usually be demonstrated by observ-
heat may denature or destroy Rh antigens.
ing absence of agglutination by anti-A
and/or anti-B in the cell tests for ABO. For
red cell specimens that show agglutina- Tests for Antigens Other than D
tion in all tubes (ie, give the reactions of Reagents are readily available to test for
group AB, D+), a control should be per- the other principal Rh antigens: C, E, c,
formed as described by the reagent man- and e. These are formulated as either low-
ufacturer; this is not required when do- protein (usually monoclonal or mono-
nors’ cells are tested. In most cases, a clonal/polyclonal blends) or high-protein
suitable control is a suspension of the pa- reagents. High-protein reagents of any
tient’s red cells with autologous serum or specificity have the same problems with
with 6% to 8% bovine albumin, although false-positive results as high-protein anti-D
exceptions have been noted.46 If the test is and require a comparable control test
one of several performed concurrently performed concurrently and under the
and in a similar manner, any negative re- same conditions. Observation of a nega-
sult serves as an adequate control. For ex- tive result in the control test for anti-D
ample, a separate control tube would be may not properly control the tests for
required only for a red cell specimen that
other Rh antigens because results with
gives positive reactions with all the Rh
anti-D are usually obtained after immedi-
reagents (ie, is typed as D+C+E+c+e+).
ate centrifugation; tests for the other Rh
antigens are generally incubated at 37 C
Testing for D in Hemolytic Disease of the before centrifugation.
Fetus and Newborn Rh reagents may give weak or negative
Because red cells from an infant suffering reactions with red cells possessing variant
from HDFN are coated with immunoglob- antigens. This is especially likely to happen
ulin, a low protein reagent is usually nec- if anti-e is used to test the red cells from
essary for Rh testing. Occasionally, the in- Blacks, among whom variants of e are rela-

9
tively common. It is impossible to obtain sources. Replicate testing is not an
anti-e reagents that react strongly and con- absolute safeguard, however, because
sistently with the various qualitative and reagents from different manufactur-
quantitative variants of e. Variable reactivity ers may not be derived from different
with anti-C reagents may occur if the DCE sources.
or CE haplotypes are responsible for the ex- 3. Polyagglutinable red cells may be ag-
pression of C on red cells. Variant E and c glutinated by any reagent containing
antigens have been reported but are con- human serum. Although antibodies
siderably less common. that agglutinate these surface-altered
Whatever reagents are used, the manu- red cells are present in most adult
facturer’s directions must be carefully fol- human sera, polyagglutinins in re-
lowed. The IAT must not be used unless the agents very rarely cause problems.
manufacturer’s instructions state explicitly Aging, dilution, and various steps in
that the reagent is suitable for this use. The the manufacturing process tend to
pools of human source sera (nonmono- eliminate these predominantly IgM
clonal) used to prepare reagents for the antibodies.
other Rh antigens may contain anti- 4. Autoagglutinins and abnormal pro-
globulin-reactive, “contaminating” speci- teins in the patient’s serum may cause
ficities. Positive and negative controls false-positive reactions when un-
should be tested; red cells selected for the washed red cells are tested.
positive control should have a single dose 5. Reagent vials may become contami-
of the antigen or be known to show weak nated with bacteria, with foreign
reactivity with the reagent. substances, or with reagent from an-
other vial. This can be prevented by
Additional Considerations in Rh Testing the use of careful technique and the
The following limitations are common to periodic inspection of the vials’ con-
all Rh typing procedures, including those tents. However, bacterial contamina-
performed with high-protein reagents. tion may not cause recognizable tur-
bidity because the refractive index of
bacteria is similar to that of high-
False-Positive Reactions
protein reagents.
The following circumstances can produce
false-positive red cell typing results.
1. The wrong reagent was inadvertently False-Negative Reactions
used. The following circumstances can produce
2. An unsuspected antibody of another false-negative red cell typing results.
specificity was present in the human 1. The wrong reagent was inadvertently
source reagent. Antibodies for anti- used.
gens having an incidence of less than 2. The reagent was not added to the tube.
1% in the population may occasion- It is good practice to add serum to
ally be present and cause false-posi- all the tubes before adding the red
tive reactions, even when the manu- cells and any enhancement medium.
facturer’s directions are followed. 3. A specific reagent failed to react with
For crucial determinations, many a variant form of the antigen.
workers routinely perform replicate 4. A reagent that contains antibody di-
tests using reagents from different rected predominantly at a cis-prod-

uct Rh antigen failed to give a reli- 9. Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. London: Academic
ably detectable reaction with red
Press, 2004.
cells carrying the individual antigens 10. Gemke RJBJ, Kanhai HHH, Overbeeke MAM,
as separate gene products. This oc- et al. ABO and Rhesus phenotyping of fetal
curs most often with anti-C sera. erythrocytes in the first trimester of preg-
nancy. Br J Haematol 1986;64:689-97.
5. The manufacturer’s directions were 11. Dunstan RA, Simpson MB, Rosse WF. Erythro-
not followed. cyte antigens on human platelets. Absence of
6. The red cell button was shaken so Rh, Duffy, Kell, Kidd, and Lutheran antigens.
roughly during resuspension that
12. Dunstan RA. Status of major red cell blood
small agglutinates were dispersed. group antigens on neutrophils, lymphocytes
7. Contamination, improper storage, and monocytes. Br J Haematol 1986;62:301-9.
or outdating cause antibody activity 13. Wiener AS. Genetic theory of the Rh blood
types. Proc Soc Exp Biol Med 1943;54:316-19.
to deteriorate. Chemically modified 14. Fisher RA, Race RR. Rh gene frequencies in
IgG antibody appears to be particu- Britain. Nature 1946;157:48-9.
larly susceptible to destruction by 15. Tippett P. A speculative model for the Rh
blood groups. Ann Hum Genet 1986;50:241-7.
proteolytic enzymes produced by cer-
16. Colin Y, Chérif-Zahar B, Le Van Kim C, et al.
tain bacteria. Genetic basis of RhD-positive and RhD-nega-
tive blood group polymorphisms as deter-
mined by southern analysis. Blood 1991;78:
2747-52.
17. Arce MA, Thomson ES, Wagner S, et al. Mo-
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1962;2:287-312. rology. 4th ed. Durham, NC: Montgomery
27. Wagner FF, Flegel WA. RHD gene deletion oc- Scientific Press, 1998:350-3.
curred in the Rhesus box. Blood 2000;95: 44. Reid ME, Storry JR, Issitt PD, et al. Rh haplo-
3662-8. types that make e but not hrB usually make
28. Chiu RW, Murphy MF, Fidler C, et al. Determi- VS. Vox Sang 1997;72:41-4.
nation of RhD zygosity: Comparison of a 45. Shirey RS, Edwards RE, Ness PM. The risk of
double amplification refractory mutation alloimmunization to c (Rh4) in R1R1 patients
system approach and a multiplex real-time who present with anti-E. Transfusion 1994;34:
quantitative PCR approach. Clin Chem 2003; 756-8.
47:667-72. 46. Rodberg K, Tsuneta R, Garratty G. Discrepant
29. Lawler SD, Race RR. Quantitative aspects of Rh phenotyping results when testing IgG-
Rh antigens. Proceedings of the International sensitized rbcs with monoclonal Rh reagents
Society of Hematology 1950:168-70. (abstract). Transfusion 1995;35(Suppl):67S.
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- Suggested Reading
lar basis of weak D phenotypes. Blood 1999;
93:385-93. Agre P, Cartron JP. Molecular biology of the Rh an-
32. Wagner FF, Frohmajer A, Ladewig B, et al. tigens. Blood 1991;78:551-3.
Weak D alleles express distinct phenotypes. Avent ND, Reid ME. The Rh blood group system: A
Blood 2000;95:2699-708. review. Blood 2000;95:375-87.
33. Tippett P, Lomas-Francis C, Wallace M. The
Rh antigen D: Partial D antigens and associ- Cartron JP. Defining the Rh blood group antigens.
ated low incidence antigens. Vox Sang 1996; Blood Rev 1994;8:199-212.
70:123-31.
Daniels G. Human blood groups. 2nd ed. Oxford:
34. Scott ML. Section 1A: Rh serology. Coordina-
Blackwell Scientific Publications, 2002.
tor’s report. 4th International Workshop on
Monoclonal Antibodies Against Human Red Issitt PD. An invited review: The Rh antigen e, its
Cell Surface Antigens, Paris. Transfus Clin Biol variants, and some closely related serological ob-
2002;9:23-9. servations. Immunohematology 1991;7:29-36.
35. Chang TY, Siegel DL. Genetic and immuno-
logical properties of phage-displayed human Issitt PD. The Rh blood group. In: Garratty G, ed.
anti-Rh(D) antibodies: Implications for Rh(D) Immunology of transfusion medicine. New York:
epitope topology. Blood 1998;91:3066-78. Marcel Dekker, 1994:111-47.
Issitt PD. The Rh blood group system 1988: Eight
new antigens in nine years and some observations
AABB, 2005.
on the biochemistry and genetics of the system.
37. Flegel WA, Khull SR, Wagner FF. Primary
Transfus Med Rev 1989;3:1-12.
anti-D immunization by weak D type 2 RBCs.
Transfusion 2000;40:428-33. Issitt PD, Anstee DJ. Applied blood group serology.
38. Schmidt PJ, Morrison EG, Shohl J. The antige- 4th ed. Durham, NC: Montgomery Scientific Press,
nicity of the Rho Du blood factor. Blood 1962; 1998.
20:196-202.
39. Daniels G. Human blood groups. 2nd ed. Ox- Lomas-Francis C, Reid ME. The Rh blood group
ford: Blackwell Scientific Publications, 2002. system: The first 60 years of discovery. Immuno-
40. Van Loghem JJ. Production of Rh agglutinins hematology 2000;16:7-17.
anti-C and anti-E by artificial immunization Race RR, Sanger R. Blood groups in man. 6th ed.
of volunteer donors. Br Med J 1947;ii:958-9. Oxford: Blackwell Scientific Publications, 1968.
41. Faas BHW, Beckers EAM, Simsek S, et al. In-
volvement of Ser103 of the Rh polypeptides Reid ME, Ellisor SS, Frank BA. Another potential
in G epitope formation. Transfusion 1996;36: source of error in Rh-Hr typing. Transfusion 1975;
506-11. 15:485-8.

Reid ME, Lomas-Francis C. The blood group anti- Vengelen-Tyler V, Pierce S, eds. Blood group sys-
gen factsbook. 2nd ed. London: Academic Press, tems: Rh. Arlington, VA: American Association of
2004. Blood Banks, 1987.
Sonneborn H-H, Voak D, eds. A review of 50 years White WD, Issitt CH, McGuire D. Evaluation of the
of the Rh blood group system. Biotest Bulletin 1997; use of albumin controls in Rh typing. Transfusion
5(4):389-528. 1974;14:67-71.

Chapter 15: Other Blood Groups
Chapter 15
Other Blood Groups

15
T
HERE ARE MANY antigens on red (ISBT ) working party on blood group
1-3
cells in addition to the ones men- terminology, and their gene location.
tioned in previous chapters. These Additional information on ISBT terminol-
antigens are grouped into blood group ogy for all the antigens mentioned in this
systems, collections, and a series of inde- chapter can be found in Appendix 6. Table
pendent antigens, composed mostly of 15-1 shows the serologic behavior and
antigens of low or high incidence. A blood characteristics of the major blood group
group system is a group of one or more antibodies derived from human sources.
antigens governed by a single gene locus The major systems will be discussed first
or by a complex of two or more closely in the chapter, followed by the other blood
linked homologous genes that have been group systems, collections, and independ-
ent high-incidence and low-incidence an-
shown to be phenotypically and geneti-
tigens. In each grouping, the order will
cally related to each other and genetically
reflect the ISBT number order.
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, Distribution of Antigens
there is insufficient information or data that Antigens present in almost all persons are
shows them to be a distinct blood group known as high-incidence antigens, where-
system genetically independent from other as antigens found in very few persons are
blood group systems. Table 10-1 lists the termed very low-incidence antigens. The
blood group systems, as defined by the frequency of these high- or low-incidence
International Society of Blood Transfusion antigens may also differ by ethnic group.
335

Table 15-1. Serologic Behavior of the Principal Antibodies of Different Blood Group
Systems
Associated
Saline Albumin Papain/Ficin with
In-Vitro
Antibody Hemolysis 4 C 22 C 37 C AHG 37 C 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-Lua 0 Some Most Few Few Few Few No No
Anti-Lub 0 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-Kpa 0 Some Some Most Some Most Yes Yes
Anti-Kpb 0 Few Few Most Some Most Yes Yes
Anti-Jsa 0 Few Few Most Few Most Yes Yes
Anti-Jsb 0 0 0 Most Few Most Yes Yes
Anti-Lea Some Most Most Some Some Some Most No Rare
Anti-Leb Some Most Most Some Some Some Most No No
Anti-Fya 0 Rare Rare Most 0 0 Yes Yes
Anti-Fyb 0 Rare Rare Most 0 0 Mild Yes
Anti-Jka Some Few Few Most Some Most Mild Yes
Anti-Jkb Some Few Few Most Some Most Mild Yes
Anti-Xga 0 Few Few Most 0 0 No report
Anti-Dia 0 Some Some Most Some Some Yes Yes
Anti-Dib 0 Most Some Some Yes Yes
Anti-Yta 0 0 0 Most 0 Some No Yes
Anti-Ytb 0 All No report
Anti-Doa 0 0 0 Some Some Most No Yes
Anti-Dob 0 All All All No Yes
Anti-Coa 0 0 0 Some Some Most Yes Yes
Anti-Cob 0 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.

Chapter 15: Other Blood Groups 337
Antigens that occur as codominant traits, molecules or hybrid molecules of the two
such as Jka and Jkb, may have a variable in- proteins. The M, N, S, s, and U antigens
cidence and may differ in ethnic groups. are the most important antigens of the MNS
For an illustration, the Duffy glycoprotein system with regard to transfusion medi-
is known to be a receptor for the parasite cine. They have also been important to
Plasmodium vivax, one of the causative our understanding of biochemistry and
agents of malaria. In West Africa, where genetics. The M and N antigens are lo-
malaria is endemic, the Fy(a–b–) red cell cated on glycophorin A (GPA). The S, s,
phenotype, very rare in Whites, occurs with and U antigens are located on glycophorin
an incidence of greater than 80%. B (GPB). Table 15-2 shows the frequencies
Each of the known antigens described of the common phenotypes of the MNS
in this chapter was initially identified system. There is considerable linkage dis-
through the detection of its specific anti- equilibrium between M,N and S,s due to
body in a serum. Tables listing phenotype the gene location on the chromosome.
frequencies among Whites and Blacks in the For example, the gene complex producing
US population are given throughout this N with s is much more common than that
chapter. Frequencies among other groups producing N with S. The MNSs genes
in the population are not given because GYPA and GYPB are in very close proxim-
data are scanty and wide differences be- ity on chromosome 4.4 See the section be-
tween groups of diverse Asian, South Ameri- low on Genes Encoding Glycophorins and
can, or Native American origins make gen- Chapters 9 and 10 for more information
eralizations about phenotypes inappropriate. 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
MNS System exposed to U+ red cells.
M, N, S, s, and U Antigens Low-Incidence Antigens of the MNS System

The MNS system is a complex system of The MNS system includes several low-inci-
over 40 antigens carried on two glycophorin dence antigens. Recent biochemical data
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- called the Miltenberger system, based on
cidence determinants to one or more amino reactivity with selected sera. As more anti-
acid substitutions, variation in the extent gens have been identified and knowledge of
or type of glycosylation, or the existence the genetic events that give rise to these
of a hybrid sialoglycoprotein (SGP). novel antigens has increased, it is clear that
the Miltenberger subsystem is outdated.
Genes Encoding Glycophorins These antigens, such as Mia, Vw, Hil, etc,
should be considered glycophorin variants.
The genes encoding the MNS system anti-
gens are located on chromosome 4 at
4q28-q31. The gene that encodes GPA is Biochemistry of the MNS System
called GYPA and the gene that encodes
Antigens of the MNS system are carried
GPB is GYPB. The similarities in amino
on GPA and GPB, which are single-pass trans-
acid sequences of GPA and GPB suggest
membrane glycoproteins. The carboxy (C)
that both genes derive from a common
terminal of each glycophorin extends into
ancestral gene. GYPA and GYPB consist of
the cytoplasm of the red cell, and a hydro-
seven and five exons, respectively. The
phobic segment is embedded within the
genes share >95% identity. Although the
lipid bilayer. An amino (N) terminal seg-
genes are highly homologous, GYPB re-
ment extends into the extracellular envi-
sults in a shorter protein because a point
ronment. The molecules are sensitive to
mutation at the 5′ splicing site of the third
cleavage at varying positions by certain
intron prevents transcription of exon 3,
proteases (see Fig 15-1).
called pseudo exon 3. Following the ho-
There are approximately 1,000,000 cop-
mologous sequences, GYPA and GYPB dif-
ies of GPA per red cell. M and N blood
fer significantly in the 3′ end sequences.
group antigen activity resides on the extra-
cellular segment, a sequence of 72 amino
Hybrid Molecules acids with carbohydrate side chains at-
Pronounced SGP modifications occur in tached within the first 50 residues of the
hybrid molecules that may arise from un- amino terminal. When GPA carries M anti-
equal crossing over or gene conversion gen activity (GPAM), the first amino acid res-
between GYPA and GYPB. Hybrid SGPs idue is serine and the fifth is glycine. When
may carry the amino-terminal portion of it carries N antigen activity (GPAN), leucine
GPA and the carboxy-terminal portion of and glutamic acid replace serine and gly-
GPB, or vice versa. Other hybrids appear cine at positions one and five, respectively
as a GPB molecule with a GPA insert or a (see Fig 15-1).
GPA molecule with a GPB insert. The low- Red cells that lack most or all of GPA are
incidence antigens Hil (MNS20), St a described as En(a–). These rare En(a–) indi-
(MNS15), Dantu (MNS25), and Mur viduals may produce antibodies (collec-
(MNS10), among others, are associated with tively called anti-Ena) that react with vari-
hybrid SGPs. Some variants are found in ous portions of the extracellular part of the
specific ethnic groups. For example, the glycoprotein. Some En(a–) persons may
Dantu antigen occurs predominantly in produce an antibody against an antigen
b
Blacks, although the antigen is of low inci- called Wr that is part of the Diego blood
b
dence. group system. Wr arises from an interac-
Many of the MNS low-incidence anti- tion between GPA and the anion exchange
gens were categorized into a subsystem molecule, AE-1 (also known as band 3).5

determine M, N, S, and s are given. indicates an O -linked oligosaccharide side chain, !

Figure 15-1. Schematic diagram of glycophorin A and glycophorin B. The amino acid sequences that
indicates
an N -linked polysaccharide side chain. Approximate locations of protease cleavage sites are indi-
cated. (Courtesy New York Blood Center.)
GPB is a smaller protein than GPA, and should be considered clinically significant.
there are fewer (approximately 200,000) Some S–s– red cells also have a variant GPB.
copies per red cell. GPB carries S, s, and U
antigens. GPB that expresses S activity has
The Effect of Proteolytic Enzymes on MNS
methionine at position 29; GPB with s ac-
Antigens
tivity has threonine at that position (see Fig
15-1). The N-terminal 26 amino acids of Proteolytic enzymes, such as ficin or papain,
N
GPB are identical to the sequence of GPA , cleave red cell membrane SGPs at well-
which accounts for the presence of an N defined sites. Reactivity with anti-M and
antigen (known as ‘N’) on all red cells of anti-N is abolished by commonly used
normal MNS types. Red cells that lack GPB enzyme techniques. The effect of different
altogether lack not only S, s, and U activity enzymes on the expression of MNS sys-
but also ‘N’. Immunized individuals of the tem antigens reflects the point at which
rare M+N–S–s–U– phenotype can produce the particular enzyme cleaves the anti-
a potent anti-N (anti-U/GPB) that reacts gen-bearing SGP and the position of the
with all red cells of normal MNS types, antigen relative to the cleavage site (see
whether N-positive or N-negative, and Fig 15-1). Sensitivity of the antigens to

proteases may help in the identification because these people lack or possess an
of antibodies to M and N antigens, but the altered form of GPB.
effects of proteases on tests for the S and s
antigens are more variable. In addition, Antibodies to S, s, and U
the S antigen is sensitive to trace amounts Unlike anti-M and anti-N, antibodies to S,
of chlorine bleach.6 s, and U usually occur following red cell
immunization. All are capable of causing
MNS System Antibodies hemolytic transfusion reactions (HTRs)
The antibodies most commonly encoun- and HDFN. Although a few saline-reactive
tered are directed at the M, N, S, and s an- examples have been reported, antibodies
tigens. to S, s, and U are usually detected in the
antiglobulin phase of testing. Most, but
Anti-M not all, investigators 7 have found that
papain or ficin destroys the reactivity of
Anti-M is detected frequently as a saline
S+ red cells with anti-S. Depending on the
agglutinin if testing is done at room tem-
enzyme solution used, the reactivity of
perature. Anti-M is often found in the sera
anti-s with s+ cells can be variable.8(p477)
of persons who have had no exposure to
Most examples of anti-U react equally
human red cells. Although M antibodies
with untreated and enzyme-treated red
are generally thought to be predominantly
cells, but there have been examples of
IgM, many examples that are partly or
broadly reactive anti-U, which detect an
wholly IgG are frequently found. However,
enzyme-sensitive determinant.
these antibodies are rarely clinically sig-
Anti-U is rare but should be considered
nificant. Some examples of anti-M cause
when serum from a previously transfused
stronger agglutination if the pH of the test
or pregnant Black person contains anti-
system is reduced to 6.5 and when testing
body to a high-incidence antigen.
red cell samples possessing a double-dose
expression of the M antigen. Examples that
react at 37 C or at the antiglobulin phase
Antibodies to Low-Incidence Antigens
of testing should be considered potentially There are many examples of antibodies to
g
significant. Compatibility testing per- low-incidence antigens, such as anti-M
W
formed by a strictly prewarmed method or anti-V , in the MNS blood group sys-
(see Method 3.3) should eliminate the re- tem. Many of these antibodies occur as a
activity of most examples of anti-M. In a saline agglutinin in sera from persons who
few exceptional cases, anti-M detectable have no known exposure to human red
at the antiglobulin phase has caused cells. The rarity of these antigens makes it
hemolytic disease of the fetus and newborn unlikely that the antibodies will be de-
(HDFN) or hemolysis of transfused cells. tected if present.
Anti-N
Anti-N is comparatively rare. Examples are Kell System
usually IgM and typically appear as weakly
reactive cold agglutinins. Some powerful and Kell System Antigens
potentially significant IgG examples have Kell system antigens are expressed on the
been observed in a few persons of the rare red cell membrane in low density and are
phenotypes M+N–S–s–U– and M+N–S–s–U+w weakened or destroyed by treatment with

reducing agents and with acid. The anti- same chromosome. Kp a is an antigen
a
gens are carried on one protein and en- found predominantly in Whites, and Js is
coded by a single gene. For an in-depth found predominantly in Blacks. The
review, see Lee, Russo, and Redman.9 a
haplotype producing K and Kp has also
not been found. Table 15-3 shows some
K and k phenotypes of the Kell system. The table
also includes K o , a null phenotype in
The K antigen was first identified in 1946
because of an antibody that caused HDFN. which the red cells lack all of the antigens
The allele responsible for the K antigen is of the Kell system. Several high-incidence
present in 9% of Whites and approxi- antigens were assigned to the Kell system
mately 2% of Blacks. The existence of the because the identifying antibodies were
expected allele for k was confirmed when found to be nonreactive with Ko red cells.
an antithetical relationship was estab- For simplicity, various Kell antigens of
lished between K and the antigen de- high and low incidence have not been
tected by anti-k. Anti-k reacts with the red included in the table.
cells of over 99% of all individuals.
Phenotypes with Depressed Kell Antigens
Other Kell Blood Group Antigens Kmod is an umbrella term used to describe
Other antithetical antigens of the Kell sys- phenotypes characterized by weak ex-
tem include Kpa, Kpb, and Kpc; Jsa and Jsb; pression of Kell system antigens. Adsorp-
K11 and K17; and K14 and K24. Not all tion/elution tests are often necessary for
theoretically possible genotype combina- their detection. The K mod phenotype is
tions have been recognized in the Kell thought to arise through several different
system. For example, Kp a and Js a have point mutations of the KEL gene. Red cells
never been found to be produced by the of persons with some Gerbich negative
Table 15-3. Some Phenotypes and Frequencies in the Kell System
Reactions with Anti- Frequency (%)

a b a b
K k Kp Kp Js Js Phenotype 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
0 0 0 0 0 0 K0 Exceedingly rare

phenotypes also exhibit depressed Kell ture. Kell antigens are also destroyed by
phenotypes (see the Gerbich System). treatment with EDTA-glycine acid.
Persons of the Ge:–2,–3 and Ge:–2,–3,–4 The function of the Kell protein is un-
(Leach) phenotypes have depression of at known, but it has structural similarities to a
least some Kell system antigens. family of zinc-binding neutral endopep-
The presence of the Kpa allele weakens tidases. It has most similarity with the
the expression of other Kell antigens when common acute lymphoblastic leukemia
in cis position. For example, the k antigen antigen (CALLA or CD10), a neutral endo-
of Kp(a+) red cells reacts more weakly than peptidase on leukocytes.8(pp647-648)
expected and, when tested with weaker ex-
amples of anti-k, may be interpreted as k–.
Kx Antigen, the McLeod Phenotype, and
Their Relationship to the Kell System
Biochemistry of the Kell System
Although the Kell system locus is on the
The Kell system antigens are carried on a long arm of chromosome 7 and the Kx lo-
93-kD single-pass red cell membrane pro- cus (XK) is on the Xp21 region of the X
tein. Kell system antigens are easily inac- chromosome, evidence suggests that the
tivated by treating red cells with sulfhy- Kell and Kx proteins form a covalently
8
dryl reagents such as 2-mercaptoethanol linked complex on normal red cells. On
(2-ME), dithiothreitol (DTT), or 2-amino- red cells that carry normal expressions of
ethylisothiouronium bromide (AET ). Kell antigens, Kx appears to be poorly ex-
Such treatment is useful in preparing red pressed. It is believed that this finding
cells that artificially lack Kell system anti- represents steric interference by the Kell
gens to aid in the identification of Kell-re- glycoprotein in the approach of anti-Kx to
lated antibodies. Treatment with sulfhydryl its antigen. Red cells of Ko individuals re-
reagents may impair the reactivity of act strongly with anti-Kx. Similarly, the re-
other antigens (LWa, Doa, Dob, Yta, and oth- moval or denaturation of Kell glycopro-
ers). Thus, although treatment with these teins with AET or DTT renders the cells
reagents may be used in antibody prob- strongly reactive with anti-Kx.
lem solving, specificity must be proven by It is believed that the presence of the
other means. As expected, Kell system an- glycoprotein on which Kx is carried is es-
tigens are also destroyed by ZZAP, a mix- sential for the antigens of the Kell system to
ture of DTT and enzyme (see Methods attach to or be expressed normally on red
3.10, 4.6, and 4.9). This susceptibility to cells. Therefore, a lack of Kx is associated
sulfhydryl reagents suggests that disulfide with poor expression of Kell system antigens.
bonds are essential to maintain activity of Red cells that lack Kx exhibit not only
the Kell system antigens. This hypothesis markedly depressed expression of Kell sys-
has been supported by the biochemical tem antigens but also shortened survival,
characterization of Kell proteins deduced reduced deformability, decreased perme-
from cloned DNA10; they exhibit a number ability to water, and acanthocytic morphol-
of cysteine residues in the extracellular ogy. This combination or group of red cell
region. Cysteine readily forms disulfide abnormalities is called the McLeod pheno-
bonds, which contribute to the folding of type, after the first person in whom these
a protein. Antigens that reflect protein observations were made. Persons with McLeod
conformation will be susceptible to any red cells also have a poorly defined abnor-
agent that interferes with its tertiary struc- mality of the neuromuscular system, char-

acterized by persistently elevated serum serologic characteristics similar to anti-K

levels of the enzyme creatine phospho- but occurs much less frequently. Only about
kinase and, in older people, disordered one person in 500 lacks the k antigen and
muscular function. The McLeod phenotype finding compatible blood is correspond-
arises through deletion and mutations of ingly more difficult.
the XK locus of chromosome X.
In a few instances, the McLeod pheno- Other Kell System Antibodies
type has been found in patients with chronic a b a b
Anti-Kp , anti-Kp , anti-Js , and anti-Js are
granulomatous disease (CGD), in which
all much less common than anti-K but
granulocytes exhibit normal phagocytosis
show similar serologic characteristics and
of microorganisms but an inability to kill
are considered clinically significant. Any of
ingested pathogens. The McLeod pheno-
them may occur following transfusion or
type associated with CGD appears to result
fetomaternal immunization. Antibody fre-
from deletion of a part of the X chromo-
quency is influenced by the immunogeni-
some that includes the XK locus as well as
city of the particular antigen and by the
the gene responsible for X-linked CGD.
distribution of the relevant negative phe-
notypes among transfusion recipients and
Kell System Antibodies positive phenotypes among donors. In
Black patients frequently transfused with
Anti-K and Anti-k phenotypically matched blood, usually
from other Black donors, anti-Jsa is rela-
Because the K antigen is strongly immuno-
tively common. This is due to the approx-
genic, anti-K is frequently found in sera
imate 20% incidence of the Jsa antigen in
from transfused patients. Rare examples
the Black population (see Table 15-3). Ordi-
of anti-K have appeared as a saline agglu-
narily, however, these antibodies are rare.
tinin in sera from subjects never exposed
Assistance from a rare donor file is usually
to human red cells. Most examples are of
needed to find compatible blood for patients
immune origin and are reactive in anti-
immunized to the high-incidence antigens
globulin testing; some bind complement.
Kpb and Jsb. Anti-Ku is the antibody char-
Some workers have observed that exam-
acteristically seen in immunized Ko per-
ples of anti-K react less well in tests that in-
sons. It has been reported to cause a fatal
corporate low-ionic-strength-saline (LISS)
HTR,12 and it appears to be directed at a
solutions (notably the Polybrene test) than
single determinant because it has not been
in saline tests or tests that include albumin.
separable into other Kell specificities.
Others, however, have not shown differ-
However, antibodies to other Kell system
ences in antibody reactivity, testing many
antigens may be present in serum contain-
examples of anti-K in low ionic systems.
ing anti-Ku. Some people of the Kmod pheno-
Anti-K has caused HTRs on numerous oc-
type have made a Ku-like antibody.
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 Duffy System
macrophages in the fetal liver.11
Because over 90% of donors are K–, it is Duffy System Antigens
a b
not difficult to find compatible blood for The antigens Fy and Fy are encoded by a
patients with anti-K. Anti-k has clinical and pair of codominant alleles at the Duffy

a b
(FY) locus on chromosome 1. Anti-Fy and undetected unless potent anti-Fy is used in
b
anti-Fy define the four phenotypes observed testing. The Fy5 antigen appears to be de-
in this blood group system, namely: fined by an interaction of the Duffy and Rh
Fy(a+b–), Fy(a+b+), Fy(a–b+), and Fy(a–b–) gene products because it is not expressed
(see Table 15-4). In Whites, the first three on Rhnull red cells. The Fy6 antigen has been
phenotypes are common and Fy(a–b–) in- described only by murine monoclonal anti-
dividuals are extremely rare. However, the bodies and is not present on red cells that
incidence of the Fy(a–b–) phenotype among are Fy(a–b–) and Fy:–3,–5.8(p448)
Blacks is 68%.
The Duffy gene encodes a glycoprotein Biochemistry of the Duffy System
that is expressed in other tissues, including
the brain, kidney, spleen, heart, and lung. In red cells, the Duffy gene encodes a multi-
The Fy(a–b–) individual can be the result of pass membrane glycoprotein. The antigens
the FyFy genotype or null phenotype. How- Fya, Fyb, and Fy6 are located on the N-ter-
ever, in many Black Fy(a–b–) individuals, minal of the Duffy glycoprotein and are
the transcription in the marrow is pre- sensitive to denaturation by proteases such
vented and Duffy protein is absent from the as ficin, papain, and α-chymotrypsin, un-
red cells. These individuals have an allele like Fy3 or Fy5. Fy3 has been located on
that is the same in the structural region as the last external loop of the Duffy glyco-
the Fyb gene that prevents the transcrip- protein. It is unaffected by protease treat-
8(p439)
tion. However, the Duffy protein is ex- m e n t (reviewed in Pierce and Mac-
pressed normally in nonerythroid cells of pherson).14 The glycoprotein is the recep-
these persons.13 Other Fy(a–b–) individuals tor for the malarial parasite Plasmodium
either appear to have a total absence or vivax, and persons whose red cells lack
markedly altered Duffy glycoprotein.8(pp457-458) Fya and Fyb are resistant to that form of
This affects other cell lines and tissues, not the disease. In sub-Saharan Africa, nota-
only the red cells. Those individuals who bly West Africa, the resistance to P. vivax
have absent or altered glycoprotein can malaria conferred by the Fy(a–b–) pheno-
make anti-Fy3, which will react with cells type may have favored its natural selec-
that are Fy(a+) and/or Fy(b+). tion, and most individuals are Fy(a–b–).
A rare inherited form of weak Fyb called The Duffy gene has been cloned and
13
x
Fy has been described and is probably due the Duffy glycoprotein has been identified
x
to a point mutation. The Fy antigen may go as an erythrocyte receptor for a number of
Table 15-4. Phenotypes and Frequencies in the Duffy System
Reactions with Anti- Adult Phenotype Frequency (%)

a b
Fy Fy Phenotype 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

15
chemokines, notably interleukin-8. Be- reported. It reacted with red cells of the
cause chemokines are biologically active Fy(a–b–) phenotype and with some Fy(a+b–)
molecules, it has been postulated that Duffy and Fy(a–b+) red cells from Blacks but not
acts as a sponge for excess chemokines, with Fy(a+b+) red cells, suggesting reactiv-
without ill effect on the red cells. ity with a putative product of the Fy gene.
However, different reference laboratories
obtained equivocal results and evidence for
Duffy System Antibodies
the existence of the Fy4 antigen is weak.
a
Anti-Fy is quite common and may cause Anti-Fy5 is similar to anti-Fy3, except
HDFN and HTRs. Anti-Fyb is rare and gen- that it fails to react with Rhnull red cells that
b
erally is weakly reactive. Anti-Fy can cause express Fy3 and is nonreactive with cells
rare mild HDFN and has been responsible from Fy(a–b–) Blacks. It may react with the
for mostly mild HTRs. Both antibodies are red cells from Fy(a–b–) Whites. This pro-
usually IgG and react best by antiglobulin vided a previously unrecognized distinction
testing. The glycoprotein that expresses between the Fy(a–b–) phenotype so com-
the antigens is cleaved by most proteases mon in Blacks and the one that occurs, but
8(p447)
used in serologic tests, so anti-Fya and very rarely, in Whites.
b
anti-Fy are usually nonreactive in en- Anti-Fy6 is a murine monoclonal anti-
zyme test procedures. body that describes a high-incidence anti-
a
Weak examples of anti-Fy or anti-Fy
b
gen in the same region as Fya and Fyb. The
may react only with red cells that have a antibody reacts with all Fy(a+) and/or
double dose of the antigen. In Whites, red Fy(b+) red cells, is nonreactive with Fy(a–b–)
cells that express only one of the two anti- red cells, but, unlike anti-Fy3, is nonreac-
gens are assumed to come from persons tive with enzyme-treated red cells.
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
Kidd System
a b
reaction with antibodies that show dosage. Jk and Jk Antigens
a b
For example, the patient typing Fy(a+b–) The Jk and Jk antigens are located on the
a
may be Fy Fy. urea transporter, encoded by the HUT 11
Anti-Fy3 was first described in the serum gene on chromosome 18. Jk(a–b–) red cells,
of a White person of the Fy(a–b–) pheno- which lack the JK protein, are more resis-
type and is directed at the high-incidence tant to lysis by 2M urea.16 Red cells of nor-
antigen Fy3. The only cells with which it is mal Jk phenotype swell and lyse rapidly in
nonreactive are Fy(a–b–). Unlike Fya and a solution of 2M urea.
b
Fy , the Fy3 antigen is unaffected by prote- The four phenotypes identified in the Kidd
ase treatment, and anti-Fy3 reacts well with system are shown in Table 15-5. The
enzyme-treated cells positive for either Fya Jk(a–b–) phenotype is extremely rare, ex-
b
or Fy . Anti-Fy3 is rare but is sometimes cept in some populations of Pacific Island
made by Black Fy(a–b–) patients lacking origin. Two mechanisms have been shown
17
Fy3 who have been immunized by multiple to produce the Jk(a–b–) phenotype. One is
transfusions. the homozygous presence of the silent Jk
Two other rare antibodies have been de- allele. The other is the action of a dominant
scribed, both reactive with papain-treated inhibitor gene called In(Jk). The dominant
red cells. One example of anti-Fy4 has been suppression of Kidd antigens is similar to

Table 15-5. Phenotypes and Frequencies in the Kidd System

Reactions with Anti- Phenotype Frequency (%)
a b
Jk Jk Phenotype 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
the In(Lu) suppression of the Lutheran sys- Kidd system antibodies occasionally
tem. cause HDFN, but it is usually mild. These
antibodies are notorious, however, for their
Kidd System Antibodies involvement in severe HTRs, especially de-
layed hemolytic transfusion reactions
Anti-Jka and Anti-Jkb (DHTRs). DHTRs occur when antibody de-
a
Anti-Jk was first recognized in 1951 in the velops so rapidly in an anamnestic re-
serum of a woman who had given birth sponse to antigens on transfused red cells
to a child with HDFN. Two years later, that it destroys the still-circulating red cells.
anti-Jkb was found in the serum of a pa- In many cases, retesting the patient’s pre-
tient who had suffered a transfusion reac- transfusion serum confirms that the anti-
tion. Both antibodies react best in antibody was undetectable in the original tests.
globulin testing, but saline reactivity is
sometimes observed in freshly drawn Anti-Jk3
specimens or when antibodies are newly Sera from some rare Jk(a–b–) persons
forming. Both anti-Jka and anti-Jkb are of- have been found to contain an antibody
ten weakly reactive, perhaps because, that reacts with all Jk(a+) and Jk(b+) red
sometimes, they are detected more readily cells but not with Jk(a–b–) red cells. Al-
through the complement they bind to red though a minor anti-Jka or anti-Jkb com-
cells. Some examples may become unde- ponent is sometimes separable, most of
tectable on storage. Other examples may the reactivity has been directed at an anti-
react preferentially with red cells from gen called Jk3, which is present on both
homozygotes. Jk(a+) and Jk(b+) red cells.
Some workers report no difficulties in
detecting anti-Jka and anti-Jkb in low ionic
tests that incorporate anti-IgG. Others find
that an antiglobulin reagent containing an Other Blood Group Systems
anticomplement component may be im- So far, this chapter has been devoted to
portant for the reliable detection of these blood group systems of red cell antigens
inconsistently reactive antibodies. Stronger of which the principal antibodies may be
reactions may be obtained with the use of seen fairly frequently in the routine blood
polyethylene glycol (PEG) or enzyme- typing laboratory. The other blood group
treated red cells in antiglobulin testing. systems listed in Table 10-1 will be re-

viewed here briefly; the interested reader Lu16, Lu17, and Lu20) has been assigned
should refer to other texts and reviews for to the Lutheran system because the corre-
greater detail. sponding antibodies do not react with
Lu(a–b–) red cells of any of the three ge-
Lutheran System netic backgrounds. Two low-incidence
antigens, Lu9 and Lu14, have gained ad-
Lua and Lub Antigens mission to the Lutheran system because
The phenotypes of the Lutheran system, of their apparent antithetical relationship
a b
as defined by anti-Lu and anti-Lu , are to the high-incidence antigens Lu6 and
shown in Table 15-6. The Lu(a–b–) pheno- Lu8, respectively.
type is very rare and may arise from one Aua (Lu18), an antigen of relatively high
of three distinct genetic circumstances incidence [90% of all populations are
14
(reviewed in Pierce and Macpherson ). In Au(a+)] and its antithetical partner, Aub
the first, a presumably amorphic Lu- (Lu19), present in 50% of Whites and 68%
theran gene (Lu) is inherited from both of Blacks, have been shown to belong to the
parents. In the second and most com- Lutheran system.18,19
mon, the negative phenotype is inherited
as a dominant trait attributed to the inde- Biochemistry of the Lutheran System
pendently segregating inhibitor gene,
Lutheran antigens are carried on a glyco-
In(Lu), which prevents the normal expres-
protein bearing both N-linked and O-linked
sion of Lutheran and certain other blood
oligosaccharides. This protein exists in
group antigens (notably P1, I, AnWj, Ina,
b two forms and has been shown to have a
and In ). The third Lu(a–b–) phenotype is
role in cell adhesion. The antigens are de-
due to an X-borne suppressor, recessive in
stroyed by trypsin, α-chymotrypsin, and
its effect.
sulfhydryl-reducing agents. 20 These re-
sults and results of immunoblotting ex-
Other Lutheran Blood Group Antigens periments suggest the existence of inter-
A series of high-incidence antigens (Lu4, chain or intrachain disulfide bonds. Tests
Lu5, Lu6, Lu7, Lu8, Lu11, Lu12, Lu13, performed with monoclonal anti-Lub sug-
b
gest that the number of Lu antigen sites
per red cell is low, approximately 600-
1600 per Lu(a+b+) red cell and 1400-3800
Table 15-6. Phenotypes and Frequencies
in the Lutheran System in Whites* per Lu(a–b+) red cell.21
The Lu and Se (secretor) loci were shown
Reactions with to be linked in 1951, the first recorded ex-
Anti- Phenotype ample of autosomal linkage in humans.
Frequency The two loci have been assigned to chro-
a b
Lu Lu Phenotype (%) mosome 19. The gene encoding the Lu
glycoproteins has been cloned.22
+ 0 Lu(a+b–) 0.15
+ + Lu(a+b+) 7.5
Lutheran System Antibodies
0 + Lu(a–b+) 92.35 a
The first example of anti-Lu (-Lu1) was
0 0 Lu(a–b–) Very rare found in 1945 in a serum that contained
*Insufficient data exist for the reliable calculation of fre- several other antibodies. Anti-Lu a and
b
quencies in Blacks. anti-Lu are not often encountered. They

are most often produced in response to HTR or HDFN. Anti-Wrb is a rarely en-
pregnancy or transfusion but have oc- countered antibody that may be formed
curred in the absence of obvious red cell by rare Wr(a+b–) and some En(a–) indi-
stimulation. Lutheran antigens are poorly viduals. Anti-Wrb may recognize an en-
developed at birth. It is not surprising that zyme-resistant or enzyme-sensitive anti-
anti-Lua has not been reported to cause 23
gen. It should be considered to have
HDFN; neither has it been associated with potential to destroy Wr(b+) red cells.8(p589)
HTRs. Anti-Lub has been reported to shor-
ten the survival of transfused red cells but Cartwright System
causes no, or at most very mild, HDFN.
Cartwright Antigens
Most examples of anti-Lua and some anti-
Lub will agglutinate saline-suspended red The Yt (Cartwright) blood group system
a b
cells possessing the relevant antigen, char- consists of two antigens, Yt and Yt (see
acteristically producing a mixed-field ap- Table 15-7). A gene on chromosome 7 en-
pearance with small to moderately sized, codes the antigens. The Yt antigens are lo-
loosely agglutinated clumps of red cells cated on red cell acetylcholinesterase
24
interspersed among many unagglutinated (AChE), an enzyme important in neural
red cells. transmission, but the function of which is
unknown on red cells. Enzymes have a
Diego System variable effect on the Yta antigen but 0.2M
DTT appears to destroy the Yta antigen ex-
Diego System Antigens pression.
The Diego system consists of two inde-
pendent pairs of antithetical antigens, Cartwright Antibodies
called Dia/Dib and Wra/Wrb. The system a
Some examples of anti-Yt are benign. A
also contains a large number of low-inci- a
few cases of anti-Yt have shown acceler-
dence antigens as seen in Appendix 6.
ated destruction of transfused Yt(a+) red
The antigens are located on AE-1 (band
cells. Prediction of the clinical outcome by
3), which is encoded by a gene on chro-
the monocyte monolayer assay has proved
mosome 17. The Dia and Dib antigens are
successful.25,26 Anti-Yta is not known to cause
useful as anthropologic markers because b
HDFN. Anti-Yt is rare and has not been
the Dia antigen is almost entirely confined
implicated in HTR or HDFN.
to populations of Asian origin and Native
North and South Americans, in which the
Xg System
incidence of Dia can be as high as 54%.8(p583)
a b
The Wr and Wr antigens are located on Xga Antigen
AE-1 in close association with GPA. Wrb ex- In 1962, an antibody was discovered that
pression is dependent upon the presence of identified an antigen more common among
GPA (see MNS Blood Group System). women than among men. This would be
expected of an X-borne characteristic be-
Diego System Antibodies cause females inherit an X chromosome
a
Anti-Di may cause HDFN or destruction from each parent, whereas males inherit
b
of transfused Di(a+) red cells. Anti-Di is X only from their mother. The antigen was
rare but clinically significant. Anti-Wra is named Xga in recognition of its X-borne
fairly common and can occur without red manner of inheritance. Table 15-8 gives
cell alloimmunization. It is a rare cause of the phenotype frequencies among White

Table 15-7. Phenotype Frequencies in Other Blood Group Systems with

Co-Dominant Antithetical Antigens
Phenotype
Frequency in
System Reactions with Anti- Phenotype Whites (%)*
Yt Yta Ytb
+ 0 Yt(a+b–) 91.9
+ + Yt(a+b+) 7.9
0 + Yt(a–b+) 0.2
Dombrock Doa Dob
+ 0 Do(a+b–) 17.2
+ + Do(a+b+) 49.5
0 + Do(a–b+) 33.3
Colton Coa Cob
+ 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 Ina Inb
+ 0 In(a+b–) Very rare
+ + In(a+b+) <1
0 + In(a–b+) >99
Diego Dia Dib
+ 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.
males and females. Enzymes, such as pa- linkage with the Xg locus has been dem-
pain and ficin, denature the antigen. The onstrated for few traits to date.
gene encoding Xga has been cloned.27,28
Scianna System
Xga Antibody
a Scianna Antigens
Anti-Xg is an uncommon antibody that
usually reacts only in antiglobulin testing. Five antigens—Sc1, Sc2, Sc3, Rd, and
Anti-Xga has not been implicated in HDFN STAR—are recognized as belonging to the
a
or HTRs. Anti-Xg may be useful for trac- Scianna blood group system. Scianna an-
ing the transmission of genetic traits asso- tigens are expressed by the red cell adhe-
ciated with the X chromosome, although sion protein ERMAP.3 Sc1 is a high-inci-

Table 15-8. Frequencies of the Xg(a+) Dombrock Blood Group System

and Xg(a–) Phenotypes in White Males Dombrock Antigens
and Females
Initially, this blood group system consisted
a b
Phenotype Frequency (%) of Do and Do , with the phenotype fre-
quency as shown in Table 15-7. The dis-
Phenotype Males Females covery that red cells negative for the high-
a 29
incidence antigen Gy were Do(a–b–) has
Xg(a+) 65.6 88.7 led to the recent expansion of the Dombrock
Xg(a–) 34.4 11.3 blood group system. Three high-incidence
Frequencies are based on the combined results of testing antigens are Gya, Hy, and Joa. The interre-
nearly 7000 random blood samples from populations of lationship of the phenotypes is shown in
Northern European origin. There are insufficient data for Table 15-9.30 Gy(a–) red cells represent the
reliable calculation of frequencies in Blacks.
null phenotype. The Gy(a+w), Hy–, and
Jo(a–) phenotypes have been found exclu-
dence antigen, whereas Sc2 occurs very sively in Blacks. The Dombrock antigens
infrequently. Sc1 and Sc2 behave as prod- are located on a glycoprotein of 46 to 58
ucts of allelic genes (see Table 15-7). Sc3 kD, the function of which is unknown.
is thought to be present on the red cells of
any individual who inherits a functional Dombrock Antibodies
Sc1or Sc2 gene, analogous to Fy3. Rd and a b
Anti-Do and anti-Do are uncommon an-
STAR are low-incidence antigens recently tibodies and are usually found in sera
assigned to the Scianna system. The anti- containing multiple red cell antibodies.
gens are resistant to enzymes routinely This can make the detection and identifi-
used in blood group serology. The gene cation of anti-Doa and anti-Dob difficult.
encoding the Scianna antigens is located a
Anti-Do has caused HDFN and HTR.
31
on chromosome 1. b
HDFN due to anti-Do has not been re-
ported, but examples have caused HTR.
Scianna Antibodies Antibodies to Gya, Hy, and Joa may cause
The antibodies are rare. Anti-Sc1 has not shortened survival of transfused anti-
been reported to cause HTR or HDFN. gen-positive red cells or mild HDFN. Re-
Anti-Sc2 has caused positive DATs in the activity of Dombrock antibodies may be
neonate but no clinical HDFN. enhanced by papain or ficin treatment of
Table 15-9. Relationship of the Dombrock Blood Group Antigens

a b a a
Phenotype Do Do Gy Hy Jo
Normal Do(a+b–) + 0 + + +
Normal Do(a+b+) + + + + +
Normal Do(a–b+) 0 + + + +
Gy(a–) 0 0 0 0 0
Hy– 0 (+) (+) 0 (+)
Jo(a–) (+) (+) + (+) 0
(+) = weak antigen expression.

red cells but is weakened or destroyed by Table 15-10. Phenotypes and

sulfhydryl reagents. Frequencies in the LW System in
Whites
Colton System
Reactions with
Colton Antigens Anti- Phenotype
a
The Colton system consists of Co , a high- Frequency
a b
incidence antigen; Cob, a low-incidence LW LW Phenotype (%)
antigen; and Co3, an antigen (like Fy3)
+ 0 LW(a+b–) >99%
considered to be the product of either the
Coa or Cob gene. The antigens are products + + LW(a+b+) <1%
of a gene on chromosome 7. The pheno- 0 + LW(a–b+) Very rare
type frequencies in Whites are shown in 0 0 LW(a–b–) Very rare
Table 15-7. The Colton antigens have
been located on membrane protein CHIP
mosome 19 and has been cloned. The
28 (Aquaporin), which functions as the
glycoprotein it encodes has homology
red cell water transporter.32 33
with cell adhesion molecules.
Red cells from persons of the Rhnull phe-
Colton Antibodies
a
notype are LW(a–b–). It appears that the LW
Anti-Co has been implicated in HTRs and glycoprotein requires an interaction with
HDFN.8 Anti-Cob has caused an HTR and Rh proteins for expression, although the
mild HDFN. Enzyme treatment of red basis of this interaction is not clear. (For a
cells enhances reactions with the Colton review of the evolution of the LW system,
antibodies. see Storry.34)
LW System
LW Antibodies
LW Antigens a
Anti-LW has not been reported to cause
Table 15-10 shows the LW phenotypes and HTRs or HDFN and both D+ and D–
their frequencies. The antigens are dena- LW(a+) red cells have been successfully
tured by sulfhydryl reagents, such as DTT, transfused into patients whose sera con-
and by pronase but are unaffected by tained anti-LWa. Reduced expression of
papain or ficin. There are reported cases LW antigens can occur in pregnancy and
of both inherited and acquired LW(a–b–) some hematologic diseases. LW antibod-
individuals. ies may occur as an autoantibody or as an
apparent alloantibody in the serum of
Association with Rh ab
such individuals. Anti-LW has been re-
a
LW is more strongly expressed on D+ ported in LW(a–b–) individuals as well as
than D– red cells. However, the gene en- in patients with suppressed LW antigens.19
coding the LW antigens is independent of
the genes encoding the Rh proteins. Ge- Chido/Rodgers System
netic independence was originally estab-
lished through the study of informative Chido/Rodgers Antigens
families in which LW has been shown to The Chido (Ch) and Rodgers (Rg) antigens
segregate independently of the RH genes. are high-incidence antigens present on
The LW gene has been assigned to chro- the complement component C4. The an-

tigens are not intrinsic to the red cell. In or by inhibition with pooled plasma from
antigen-positive individuals, the antigens antigen-positive individuals. (See Method
are adsorbed onto red cells from the 3.9.) The antibodies are nonreactive with
plasma through an attachment mecha- enzyme-treated red cells.
nism that remains unclear.35 Ch has been
subdivided into six antigens and Rg into
two antigens. A ninth antigen, WH, re-
Gerbich System
quires the interaction of Rg1 and Ch6 for Gerbich Antigens
expression. C4 is encoded by two linked
genes, C4A and C4B, on chromosome 6. The Gerbich system includes eight anti-
Existing sera are poorly classified, but gens, of which three (Ge2, Ge3, and Ge4)
a
the phenotype frequency may be consid- are of high incidence and five (Wb, Ls ,
a a
ered as in Table 15-11. The antigens are de- An , Dh , and GEIS) are of low incidence.
stroyed by papain/ficin treatment but unaf- Several phenotypes that lack one or more
fected by DTT/AET treatment. of the high-incidence antigens are shown
in Table 15-12; all are rare. Red cells with
Chido/Rodgers Antibodies the Gerbich or Leach phenotype have a
Antibodies to Ch and Rg are generally be- weakened expression of some Kell system
a a a
nign but may be a great nuisance in sero- antigens. Ge2, Ge4, Wb, Ls , An , Dh , and
logic investigations. Rapid identification GEIS are destroyed by papain and ficin,
is possible using red cells coated with C4 but Ge3 resists protease treatment.
Table 15-11. Some Blood Group Antigens with Phenotypic Relationships

Approximate Frequency (%)
Antigens Phenotypes Whites Blacks
Chido (Ch) and Rodgers (Rg) Ch+,Rg+ 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 Csa is not part of the Knops blood group system, there is a phenotypic association between Yk a and Csa.

Table 15-12. Ge– Phenotypes

Phenotype Antibody Produced
Ge: –2,3,4 (Yus type) Anti-Ge2

Ge:–2,–3,4 (Gerbich type) Anti-Ge2 or -Ge3
Ge:–2,–3,–4 (Leach type) Anti-Ge2, -Ge3, or -Ge4
The antigens of the Gerbich blood group incidence antigens and three low-inci-
system are carried on glycophorin C (GPC) dence antigens (see Table 15-13). Tca is
and glycophorin D (GPD). GPC carries Ge3 antithetical to the low-incidence antigen
and Ge4, whereas GPD carries Ge2 and Tcb in Blacks and to Tcc in Whites. WESb is
Ge3. Ana is carried on an altered form of the high-incidence antigen antithetical to
GPD. Dha and Wb are located on altered WESa. Cra, Dra, Esa, UMC, GUTI, SERF, and
a
forms of GPC. Ls is found on an altered ZENA are not associated with low-inci-
36
form of GPC and GPD. The proteins are dence antigens. IFC is absent only in the
the product of a single gene, GYPC, on null phenotype (Inab).
chromosome 2. GPC is approximately four The antigens are located on the comple-
times more abundant than GPD. The ment regulatory protein called decay-accel-
mechanism whereby these two proteins are erating factor (DAF). The protein is en-
derived from a single gene involves an al- coded by DAF, one gene of the regulators of
ternative initiation site in the gene. GPC complement activation (RCA) complex, on
and GPD interact directly with protein chromosome 1. The antigens are on leuko-
band 4.1 in the membrane skeleton. It is cytes, platelets, and trophoblasts of the pla-
clear that the interaction is important in centa as well as in soluble form in the se-
maintaining cell shape because deficien- rum/plasma and urine.37 The antigens are
cies of either band 4.1 or GPC/D cause not affected by ficin or papain. DTT or AET
elliptocytosis.36 may weaken the antigens but do not com-
pletely destroy them.37
Gerbich Antibodies
The antibodies that Ge-negative individu- Cromer Antibodies
als may produce are shown in Table 15-12; Antibodies to antigens of the Cromer sys-
they may be immune or occur without red tem are immune-mediated and extremely
a
cell stimulation. Anti-Ge is usually IgG uncommon. Most examples of anti-Cr ,
b a
but may have an IgM component. The -WES , and -Tc have been found in the
clinical significance of the antibodies is sera of Black individuals. Anti-GUTI was
variable. Antibodies to the Gerbich anti- found in a Canadian of Chilean ancestry.37
gens may be a rare cause of HDFN. The clinical significance of the antibodies
is variable, and some examples cause de-
Cromer System creased survival of transfused red cells.
The antibodies will not cause HDFN be-
Cromer Antigens cause the placenta tissue is a rich source
A total of 13 antigens have been assigned of DAF, which is thought to adsorb the
to the Cromer blood group system: 10 high- maternal antibodies.37

Table 15-13. Antigens of High and Low that the variable reactivity of anti-CR1-re-
Incidence in the Cromer Blood Group lated sera is a direct reflection of the
System number of CR1 sites that exhibit both size
and expression polymorphisms and vary
Antigen Incidence (%) widely among individuals. The antibodies
are of no clinical significance.
Cra >99
Tca >99
Indian System
Tcb <1 a b
In and In are located on CD44, a protein
Tcc <1
of wide tissue distribution with the char-
Dra >99 acteristics of a cell adhesion molecule. In
a
Esa >99 b
is a low-incidence antigen, and In is of
IFC >99 high incidence (see Table 15-7). Inb shows
WESa <1 reduced expression on Lu(a–b–) red cells
WESb >99 of the In(Lu) type but is normally ex-
pressed on Lu(a–b–) red cells from per-
UMC >99
sons homozygous for the amorph or pos-
GUTI >99
sessing the X-borne suppressor gene. The
SERF >99 antigens are destroyed by papain and ficin
ZENA >99 as well as by reducing agents such as 0.2
M DTT. There are few data on the clinical
significance of the corresponding antibod-
Knops System ies.
Knops Antigens Other Blood Group Systems

Most of the eight Knops system antigens
a b a b a a
(Kn , Kn , McC , McC , Sl , Yk , Vil, and Sl3)
Ok System
have been located on the C3b/C4b recep- The Ok system consists of a single high-
a
tor (CR1), the primary complement re- incidence antigen, Ok . The few rare
ceptor on red cells. A gene on chromo- Ok(a–) individuals to date have been Jap-
a a a a
some 1 encodes CR1. Kn , McC , Sl , Yk , anese. Proteases do not seem to weaken
and Sl3 are high-incidence antigens. Kn ,
b
expression of Oka in routine agglutination
b a
McC , and Vil are low-incidence antigens. tests. Anti-Ok reacts optimally by an indi-
Some variation in frequency is observed rect antiglobulin test and appears to be
between the red cells of Whites and Blacks clinically significant in transfusion ther-
(see Table 15-11). apy, causing rapid destruction of Ok(a+)
The Knops system antigens are not dered cells.
stroyed by ficin or papain but may be weak-
ened or destroyed by DTT or AET. Raph System
The Raph system consists of a single anti-
Knops Antibodies gen, MER2. Anti-MER2 has been reported
The antibodies commonly show variable in three Israeli Jews. All three were on re-
weak reactivity in the antiglobulin phase nal dialysis, raising the possibility that an-
of testing but may continue to react even tibody production may be associated with
at high dilutions. Moulds et al38 have shown kidney disease. The MER2 antigen has

been detected on the red cells of 92% of Cost

those tested. The MER2 antigen is sensi-
Csa and Csb are all that remain of this col-
tive to DTT but is not affected by treat-
lection after the Knops antigens were
ment with ficin, papain, or chloroquine.
identified on CR1. Cs a occurs in a fre-
The antibodies have been IgG and some
quency greater that 98% in most popula-
have bound complement. To date, there b
tions, whereas Cs appears in about 34%
has been no information on whether these
of the population.19 There is, however, an
antibodies are capable of causing HTR or a
unexplained connection between the Yk
HDFN. a
and Cs antigens, such that red cells nega-
tive for one antigen are often weak or neg-
John Milton Hagen System ative for the other. (See Table 15-11.) The
The John Milton Hagen (JMH) antigen is antigens are not destroyed by ficin, papain,
a
carried on a GPI-linked CD108 glycopro- or DTT. Anti-Cs behaves similarly to anti-
tein. JMH antigen decreases over time. The bodies produced to the Knops system an-
JMH– phenotype can be transient. The tigens and is not considered clinically sig-
JMH– phenotype can be acquired or in- nificant.
herited. The antigen is destroyed or al-
tered by ficin or papain treatment and by Er
AET or DTT treatment. The Er collection consists of two antigens,
Antibodies that react with JMH show which give rise to four phenotypes: Er(a+b–),
variation in reactivity and are usually weak. Er(a+b+), Er(a–b+), and Er(a–b–). Era is a
Autoanti-JMH can often be found in older high-incidence antigen present on the red
people, along with an acquired absent or cells of >99% of all individuals, but Erb has
weak JMH antigen expression. The anti- a prevalence of less than 1%. The presence
body is not routinely considered capable of of a silent third allele, Er, is thought to ac-
HTR or HDFN. count for the Er(a–b–) phenotype, as de-
monstrated by family studies. The antigens
GIL System are not destroyed by ficin, papain, or DTT
but are destroyed by EDTA-glycine acid.
There is one antigen of high frequency, GIL,
in this system. GIL is located on aquaporin
Vel
3 (AQP3), which is a glycerol transporter.
The antibody has not been reported to The Vel collection was recently created to
cause HDFN or HTR. 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-
Blood Group Collections ment. It is well developed at birth, but anti-
gen expression is variable.8 Despite its oc-
In addition to the blood group systems, currence after known immunizing stimuli,
there are collections of antigens that ex- anti-Vel is most commonly IgM. It has been
hibit shared characteristics but do not as reported to range from causing only a posi-
yet meet the criteria for blood group sys- tive DAT in the neonate to causing severe
19
tem status defined by the ISBT. They in- HDFN. It has been implicated in HTRs.
clude Cost (ISBT 205), Er (ISBT 208), and Anti-Vel binds complement, and in-vitro
Vel (ISBT 211). hemolysis of incompatible red cells is often

seen when testing freshly drawn serum

Table 15-14. Some Antigens of High
containing this antibody. Reactivity of
Incidence Not Assigned to a Blood
anti-Vel is usually enhanced by enzyme
Group System or Collection
treatment of red cells expressing the anti-
gen. Name Symbol
August Ata
Langereis Lan
Sid Sda
High-Incidence Red Cell Duclos
Antigens Not Assigned to a Jra
Blood Group System or Emm
AnWj
Collection
PEL
Table 15-14 lists the antigens of high inci- MAM
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 a
Jr Antigen
lacking that antigen. For this reason, anti- a
bodies directed at high-incidence antigens Jr is a high-incidence antigen that is re-
are rarely encountered. The antibodies sistant to enzyme treatment and to 0.2 M
corresponding to these antigens usually DTT treatment. The Jr(a–) phenotype is more
react best by antiglobulin testing. commonly found in Japanese individuals
but has been found in other populations
as well.8(pp805-806) Anti-Jra has been shown to
Lan Antigen
cause reduced red cell survival. 8(pp805-806)
a
Lan is a high-incidence antigen that is re- Other examples of anti-Jr have shown lit-
sistant to enzyme-treatment and to 0.2 M tle or no clinical significance in HDFN or
DTT treatment. A weak form of the Lan HTR.8(p806),39
antigen has been reported.19 Anti-Lan is
characteristically IgG, may bind comple- AnWj Antigen
ment, and may cause HTRs. Cases of
AnWj is a high-incidence antigen that is
HDFN due to anti-Lan have been mild
resistant to enzyme treatment but weak-
even though the Lan antigen is present on
ened by 0.2 M DTT treatment.19 The anti-
cord red cells.19
gen is carried on CD44, which also carries
a the Indian blood group system antigens.
At Antigen The AnWj antigen is weakened on the red
a
At is a high-incidence antigen that is re- cells from individuals with the In(Lu)
sistant to enzyme treatment and to 0.2 M gene (see section on Lutheran System).
DTT treatment. The At(a–) phenotype has Some patients with Hodgkin’s disease
been found only in Black individuals.19 may experience a long-term suppression
a
Anti-At is characteristically IgG. The anti- of the AnWj antigen.8(pp783-784) The AnWj an-
body appears to cause only moderate tigen is the receptor for Haemophilus
HTRs and no clinical HDFN.19 influenzae. 8(pp784-785) Anti-AnWj has been

implicated in severe HTRs but not in HDFN

because the antigen is not present on cord
Low-Incidence Red Cell
red cells. Antigens Not Assigned to a
Blood Group System or
a
Sd Antigen Collection
a
Sd is an antigen of fairly high incidence, Many independent low-incidence red cell
widely distributed in mammalian tissues antigens have been recognized in addition
and body fluids. The antigen is variably to a growing number that have been as-
expressed on the red cells of Sd(a+) indi- signed to the MNS, Rh, and Diego systems.
a
viduals. Sd expression may diminish dur- Table 15-15 lists those that have been
ing pregnancy and the Sd(a–) phenotype studied and shown to be inherited in a
is observed in 30% to 75% of pregnant dominant manner. Antibodies specific for
women. The antigen is not present on these low-incidence antigens react with
cord cells. The strongest expression of Sda so few random blood samples that they
has been observed on polyagglutinable virtually never cause difficulties in select-
red cells of the Cad phenotype. The fre- ing blood for transfusion but may be im-
quency of Sd(a–) blood is considered to plicated in some rare cases of HDFN. The
be around 9%, but weakly positive reac- antibodies are of interest to the serologist,
tions are often difficult to distinguish from however, because of the unexpectedly
negative ones. high incidence with which they occur,
Anti-Sda can be reactive by antiglobulin often without an identifiable antigenic
testing. Microscopic examination of posi- stimulus.
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-
Table 15-15. Antigens of Low Incidence
jority of the examples of anti-Sda are IgM,
Not Assigned to a Blood Group System
the wide use of anti-IgG means that many
or Collection
examples of anti-Sda are no longer de-
tected. Anti-Sda is not considered to be clin- Batty (By) Livesay (Lia)
ically significant. However, there have been
Biles (Bi) Milne
reported cases of HTRs with red cells with a
strong Sda expression.8(pp816-817) Box (Bx ) Oldeide (Ola)
a
The immunodominant sugar of Sd is Christiansen (Chra) Peters (Pta)
N-acetylgalactosamine (GalNAc), also the HJK Rasmussen (RASM)
immunodominant sugar of the A blood
HOFM Reid (Rea)
group antigen and of the Tamm-Horsfall
glycoprotein, found in human and guinea JFV REIT
pig urine. Anti-Sda activity can be inhibited JONES SARA
by incubation with urine from guinea pigs Jensen (Je ) a
Torkildsen (Toa)
or from Sd(a+) humans. See Method 3.11
for the performance of a urine neutraliza- Katagiri (Kg)
tion of anti-Sda. The antigens occur with a frequency of 1 in 500 or less.

strengths are observed when a single se-

Antibodies to Low-Incidence rum containing “anti-Bg” is tested with
Antigens different Bg+ red cells. Reactivity is most
commonly observed in antiglobulin test-
Antibodies to low-incidence antigens have
ing, but highly potent anti-Bg sera may di-
sometimes been implicated in transfusion
rectly agglutinate red cells with an unusu-
reactions and HDFN. These antibodies
ally strong expression of the Bg antigens.
are usually encountered by chance, when
Confident and precise classification of
the red cells used for antibody detection
reactivity is made difficult by the weak ex-
or selected for crossmatching happen to
pression of these antigens on some red cells
carry the corresponding antigen.
and by multiple specificities among differ-
Antibodies to low-incidence antigens may
ent examples of the Bg antibodies. These
also be present as unsuspected contaminants
antibodies may also occur as unsuspected
in blood typing reagents prepared from hu-
contaminants in human source blood typ-
man serum and may cause false-positive
ing sera, where they may cause false-posi-
test results if the red cells tested carry the
tive reactions with cells having unusually
antigen. Testing with reagents from differ-
strong expression of the corresponding Bg
ent manufacturers may not eliminate this
antigen. Bg-related antigens are denatured
error because it is not uncommon for a sin-
by chloroquine diphosphate or a solution
gle individual with an uncommon antibody
of glycine-HCl/EDTA.
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 References
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ogy for blood group antigens and genes—his-
by antiglobulin testing, even if there has
torical origins and guidelines in the new mil-
been no exposure to red cell immunization. lennium. Transfusion 2000;40:477-89.
It is common for several low-incidence an- 2. Daniels GL, Anstee DJ, Cartron JP, et al. Ter-
tibodies to occur together in a single serum; minology for red cell surface antigens. ISBT
Working Party Oslo Report. International So-
multiple specificities are especially likely in ciety of Blood Transfusion. Vox Sang 1999;77:
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ditions. 3. Daniels GL, Cartron JP, Fletcher A, et al. Inter-
national Society of Blood Transfusion Com-
mittee on terminology for red cell surface an-
Bg (Bennett-Goodspeed) Antigens tigens: Vancouver Report. Vox Sang 2003;84:
244-7.
Antibodies directed at certain leukocyte 4. Race RR, Sanger R. Blood groups in man. 6th
antigens sometimes cause confusing reed. Oxford: Blackwell Scientific Publications,
actions in serologic tests with red cells. At 1975.
5. Bruce LJ, Ring SM, Anstee DJ, et al. Changes
least three separate specificities have been in the blood group Wright antigens are asso-
given names as Bg antigens: Bga corresponds ciated with a mutation at amino acid 658 in
b
to HLA-B7; Bg corresponds to HLA-B17; human erythrocyte band 3: A site of interac-
c tion between band 3 and glycophorin A un-
and Bg corresponds to HLA-A28. A fourth der certain conditions. Blood 1995;85:299-
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tion of the S antigen by Clorox (abstract).
HLA-A10. The so-called Bg antigens are
Transfusion 1983;23:410.
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with the result that reactions of differing ficin-treated human red cells. In: Abstracts of

volunteer papers. 30th Annual Meeting of the 22. Crew VK, Green C, Daniels G. Molecular
American Association of Blood Banks. Wash- bases of the antigens of the Lutheren blood
ington, DC: American Association of Blood group system. Transfusion 2003;43:1729-37.
Banks, 1977:36. 23. Storry JR, Reid ME, Chiofolo JT, et al. A new
8. Issitt PD, Anstee DJ. Applied blood group se- Wr(a+b–) Proband with anti-Wrb recognizing
rology. 4th ed. Durham, NC: Montgomery a ficin sensitive antigen (abstract). Transfu-
Scientific, 1998. sion 2001;41(Suppl):23S.
9. Lee S, Russo D, Redman CM. The Kell blood 24. Spring FA. Characterization of blood-group-
group system: Kell and XK membrane proactive erythrocyte membrane glycoproteins
teins. Semin Hematol 2000;37:113-21. with human antisera. Transfus Med 1993;3:
10. Zelinski T, Coghlan G, Myal Y, et al. Genetic 167-78.
linkage between the Kell blood group system 25. Eckrich RJ, Mallory DM. Correlation of mono-
and prolactin-inducible protein loci: Provi- cyte monolayer assays and posttransfusion
sional assignment of KEL to chromosome 7. survival of Yt(a+) red cells in patients with
Ann Hum Genet 1991;55:137-40. anti-Yt a (abstract). Transfusion 1993;33
11. Daniels G, Hadley A, Green CA. Causes of fe- (Suppl):18S.
tal anemia in hemolytic disease due to anti-K 26. Garratty G, Arndt P, Nance S. The potential
(letter). Transfusion 2003;43:115-16. clinical significance of blood group alloanti-
bodies to high frequency antigens (abstract).
12. Lin M, Wang CL, Chen FS, et al. Fatal hemoly-
Blood 1997;90(Suppl):473a.
tic transfusion reaction due to anti-Ku in a
K null patient. Immunohematol 2003;19:19- 27. Ellis NA, Ye T-Z, Patton S, et al. Cloning of
21. PBDX, a MIC2-related gene that spans the
pseudoautosomal boundary on chromosome
13. Chaudhuri A, Polyakova J, Zbrezezna V, et al.
Xp. Nat Genet 1994;6:394-9.
Cloning of glycoprotein D cDNA which en-
28. Ellis NA, Tippett P, Petty A, et al. PBDX is the
codes the major subunit of the Duffy blood
XG blood group gene. Nat Genet 1994;8:285-
group system and the receptor for the Plasmo-
90.
dium vivax malaria parasite. Proc Natl Acad
29. Banks JA, Parker N, Poole J. Evidence to show
Sci U S A 1993;90:10793-7.
that Dombrock antigens reside on the Gya/
14. Pierce SP, Macpherson CR, eds. Blood group
Hy glycoprotein. Transfus Med 1992;(Suppl)
systems: Duffy, Kidd and Lutheran. Arlington,
1:68.
VA: American Association of Blood Banks,
30. Scofield TL, Miller JP, Storry JR, et al. Evi-
1988.
dence that Hy– RBCs express weak Joa anti-
15. Horuk R, Chitnis C, Darbonne W, et al. A re- gen. Transfusion 2004;44:170-2.
ceptor for the malarial parasite Plasmodium
31. Judd WJ, Steiner EA. Multiple hemolytic
vivax: The erythrocyte chemokine receptor.
transfusion reactions caused by anti-Do a .
Science 1993;261:1182-4.
16. Heaton DC, McLoughlin K. Jk(a–b–) red blood 32. Smith BL, Preston GM, Spring F, et al. Human
cells resist urea lysis. Transfusion 1982;22:70-1. red cell aquaporin CHIP. J Clin Invest 1994;94:
17. Mougey R. The Kidd blood group system. In: 1043-9.
Pierce SR, Macpherson CR, eds. Blood group 33. Bailly P, Hermand P, Callebaut I, et al. The LW
systems: Duffy, Kidd and Lutheran. Arlington, blood group glycoprotein is homologous to
VA: American Association of Blood Banks, intercellular adhesion molecules. Proc Natl
1988:53-71. Acad Sci U S A 1994;91:5306-10.
18. Zelinski K, Kaita H, Coghlan G, Philipps S. As- 34. Storry JR. Review: The LW blood group sys-
signment of the Auberger red cell antigen tem. Immunohematology 1994;8:87-93.
polymorphism to the Lutheran blood group 35. Moulds JM, Laird-Fryer B, eds. Blood groups:
system: Genetic justification. Vox Sang 1991; Chido/Rodgers, Knops/McCoy/York and Cro-
61:275-6. mer. Bethesda, MD: American Association of
19. Reid ME, Lomas-Francis C. The blood group Blood Banks, 1992.
antigen factsbook. 2nd ed. San Diego, CA: Ac- 36. Reid ME, Spring FA. Molecular basis of glyco-
ademic Press, 2004. phorin C variants and their associated blood
20. Daniels G. Effect of enzymes on and chemical group antigens. Transfus Med 1994;4:139-46.
modifications of high-frequency red cell anti- 37. Storry JR, Reid ME, The Cromer blood group
gens. Immunohematology 1992;8:53-7. system: A review. Immunohematology 2002;
21. Merry AH, Gardner B, Parsons SF, Anstee DJ. 18:95-101.
Estimation of the number of binding sites for 38. Moulds JM, Moulds JJ, Brown M, Atkinson JP.
a murine monoclonal anti-Lu b on human Antiglobulin testing for CR1-related (Knops/
erythrocytes. Vox Sang 1987;53:57-60. McCoy/Swain-Langley/York) blood group an-

tigens: Negative and weak reactions are betan nationalities. Immunohematology 2003;19:
caused by variable expression of CR1. Vox Sang 22-5.
1992;62:230-5.
39. Kwon M, Ammeus M, Blackall D. A Japanese Lögdberg L, Reid M, Miller J. Cloning and genetic
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clinical significance despite multiple incom- and antigens. Transfus Med Rev 2002;16:1-10.
patible transfusions (abstract). Transfusion Pogo AO, Chaudhuri A. The Duffy protein: A ma-
2001;41(Suppl):58S. larial and chemokine receptor. Semin Hematol
2000;37:122-9.
Reid ME. The Dombrock blood group system: A re-

view. Transfusion 2003;43:107-14.
Suggested Reading
Reid ME, Rios M, Yazdanbakhsh K. Applications of
Akane A, Mizukami H, Shiono H. Classification of molecular biology techniques to transfusion med-
the standard alleles of the MN blood group sys- icine. Semin Hematol 2000;37:166-76.
tem. Vox Sang 2000;79:183-7.
Reid ME, Storry JR. Low-incidence MNS antigens
Liu M, Jiang D, Liu S, et al. Frequencies of the ma- associated with single amino acid changes and their
jor alleles of Diego, Dombrock, Yt, and Ok blood susceptibility to enzyme treatment. Immunohema-
group systems in the Chinese, Han, Hui, and Ti- tology 2001;17:76-81.

Chapter 16: Platelet and Granulocyte Antigens and Antibodies
Chapter 16
Platelet and Granulocyte

Antigens and Antibodies
A
NTIBODIES REACTIVE WITH an- observed to be essentially platelet spe-
tigens expressed on platelets and cific.
leukocytes are assuming increas-
ing importance. Some blood group anti-
ABH Antigens
gens are shared by red cells, white cells,
and platelets; others are specific to cer- The ABH antigens expressed on platelets 16
tain cell types. This chapter discusses an- are a combination of structures intrinsic
tibodies directed at antigens expressed on to the plasma membrane and those ad-
platelets and neutrophils, with an empha- sorbed from the plasma. The amount of
sis on those specific for these cells. HLA ABH antigen present on platelets is quite
antibodies and antigens are covered more variable from individual to individual,
fully in Chapter 17. 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
Platelet Antigens “high-expresser” form of glycosyltrans-
ferase in their sera. Although platelets are
Antigens Shared with Other Tissues often transfused without regard to ABO
Platelets express a variety of antigenic compatibility, in some cases, ABO anti-
markers on their surface. Some of these bodies (particularly IgG antibodies of
antigens are shared with other cell types, high titer in group O recipients) may react
as in the case of ABH antigens and HLA with platelets carrying large amounts of A
antigens, which are shared with virtually or B antigen.1 High-expresser platelets are
all nucleated cells in the body. Others are particularly vulnerable to this type of im-
361

mune destruction. ABO antibodies in re- principal cause of primary HLA alloim-
cipients may also cause reduced survival munization.
of ABO-incompatible platelets from nor-
mal-expresser phenotype donors, causing
Platelet Transfusion Refractoriness
occasional patients to exhibit refractori-
ness to platelet transfusions on this basis. A less-than-expected increase in platelet
a
Other red cell antigens—including Le , count occurs in about 20% to 70% of
b 2
Le , Ii, and P as well as the Cromer antigens multitransfused thrombocytopenic pa-
associated with decay accelerating fac- tients,6 and patients treated for malignant
tor3—are also found on platelets, but there hematopoietic disorders are particularly
is no evidence that antibodies to these anti- likely to become refractory to platelet
gens significantly reduce platelet survival in transfusions. A widely accepted definition
vivo. 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
HLA Antigens
study, two consecutive 1-hour posttrans-
HLA antigens are found on the surfaces of fusion platelet corrected count increments
(CCI) of less than 5000 platelets × m body
2
both platelets and white cells (see Chap-
ter 17). In fact, platelets are the major surface area/µL indicated refractoriness.7
source of Class I HLA antigens in whole Others have used less stringent criteria
blood. 2 Recent evidence indicates that (eg, three platelet transfusions over a
most Class I HLA molecules on platelets 2-week period that yield inadequate post-
are integral membrane proteins, and transfusion platelet counts).8,9 Response is
smaller amounts may be absorbed from often determined by calculating either a
surrounding plasma.3 CCI or a posttransfusion platelet recovery
HLA alloantibodies do not occur natu- (PPR) between 10 and 60 minutes after
rally, arising only after sensitization by transfusion (see Table 16-1). Responses of
pregnancy or blood transfusion. Studies of 7500 platelets × m2 body surface area/µL
HLA alloimmunization in patients trans- or 20% can be considered acceptable from
fused with platelets document the develop- the CCI or the PPR calculation, respec-
ment of antibodies within 3 to 4 weeks after tively.
primary exposure and as early as 4 days af- Alloimmune platelet refractoriness is
ter secondary exposure in patients previ- most often the result of HLA sensitization
4
ously transfused or pregnant. The likeli- and can be diagnosed by demonstration of
hood of HLA alloimmunization by significant levels of HLA antibodies. Patient
transfusion in patients not previously sensi- serum is tested against a panel of lympho-
tized is variable,4,5 and the risk of HLA cytes (or synthetic beads bearing Class I an-
alloimmunization appears to be related to tigens) that represent most of the Class I
the underlying disease as well as to the HLA specificities in the population. A
immunosuppressive effects of treatment panel-reactive antibody (PRA) score of 20%
regimens. Platelets carry Class I HLA anti- or higher is evidence that HLA sensitization
gens but lack Class II antigens, which are may be contributing to the platelet refrac-
necessary for primary sensitization. There- toriness (see Chapter 17).
fore, exposure to leukocytes expressing Although platelet alloimmunization is
HLA antigens during transfusion is the one cause of refractoriness, there are multi-

Chapter 16: Platelet and Granulocyte Antigens and Antibodies 363
Table 16-1. Determination of Response to Transfused Platelets

Calculation of Corrected Count Increment (CCI)
CCI = Body Surface Area (m2) × Platelet Count Increment × 1011

No. of Platelets Transfused
EXAMPLE: If 4 × 10 platelets are transfused to a patient whose body surface area is 1.8 m and
11 2
the increase in posttransfusion platelet count is 25,000/µL, then:

1.8 m 2 × 25,000 / µL × 1011
CCI = = 11,250 platelets × m 2 / µL
4 × 10 11
Calculation of Posttransfusion Platelet Recovery (PPR)

Estimated Total Blood Volume* × Platelet Count Increment
PPR(%) =
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:
70 kg × 70 mL / kg × 25,000 plts / µL × 10 3
PPR = = 30.6%
4 × 1011 platelets
ple, nonimmune reasons why transfused apheresis platelets matched to the patient’s
12
platelets may not yield the expected in- HLA type. A disadvantage is that a pool of
crease in platelet count [eg, sepsis, dissemi- several thousand HLA-typed potential
nated intravascular coagulation (DIC), or apheresis donors is necessary to find suffi-
the administration of certain drugs]. Some cient HLA-compatible matches.13 Moreover,
of the most commonly cited nonimmune donor selection on the basis of HLA type
causes of platelet refractoriness are listed in can lead to the exclusion of donors with
Table 16-2. A study of patients undergoing HLA types different from that of the recipi-
marrow transplant suggested that pa- ent but potentially effective if the recipient
tient-related variables such as total body ir- is alloimmunized to other antigenic deter-
radiation, advanced disease status, and liver minants.14 For patients who are likely to re-
dysfunction are important predictors of quire multiple platelet transfusions, HLA
poor platelet count increments as well.10,11 typing should be performed in advance of a
Even when possible immune causes of re- planned course of treatment.
fractoriness are identified, nonimmune fac- It is important to understand the degree
tors are often simultaneously present. of match that may be provided (see Table
Several strategies may be considered 16-3). Platelets received following a request
when selecting platelets for patients with for “HLA-matched” platelets are typically
immune-mediated refractoriness. When the closest match obtainable within the
antibodies to HLA antigens are demon- constraints of time and donor availability.
strated, a widely used approach is to supply In one study,15 43% of platelets provided as

Table 16-2. Some Nonimmune Causes crossmatching assay. This approach can be
of Platelet Refractoriness used to predict and, therefore, avoid subse-
quent platelet transfusion failures.17 The
Massive bleeding solid-phase red cell adherence test (SPRCA)
Fever is the most widely used method for platelet
Sepsis
crossmatching, and test results are reason-
Splenomegaly (splenic sequestration)
ably predictive of posttransfusion platelet
Disseminated intravascular coagulation
Allogeneic transplantation counts.18-20 Compared with HLA matching,
Poor storage of platelets before transfusion crossmatching can prove both more conve-
Effects of drugs (may include immune nient and economically advantageous. It
mechanisms) avoids exclusion of HLA-mismatched but
Intravenous amphotericin B compatible donors and has the added ad-
Thrombotic thrombocytopenic purpura 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-
HLA-matched were relatively poor grade B cessful, particularly when patients are
or C matches. The most successful re- highly alloimmunized (PRA >50%). In these
sponses occur with the subset of grade A instances, finding sufficient compatible
and B1U or B2U HLA matches, but mis- units may be problematic, and selection of
matches for some antigens (B44, 45) that HLA-matched platelets may be more prac-
are poorly expressed on platelets can be tical. Although the incidence of platelet-
useful. According to AABB Standards for specific antibodies causing patients to be
Blood Banks and Transfusion Services,16(p43) refractory to most or all attempted platelet
HLA-matched platelets should be irradi- transfusions is very small, this possibility
ated to prevent transfusion-associated should be investigated when most of the at-
graft-vs-host disease. tempted crossmatches are positive. If pla-
A second approach to provide effective telet-specific antibodies are present, donors
platelets is to use a pretransfusion platelet of known platelet antigen phenotype or
Table 16-3. Degree of Matching for HLA-Matched Platelets

Examples of Donor
Match Phenotypes for a Recipient
Grade Description 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 expanded. Although this strategy may
share the patient’s phenotype, should be prove useful in selecting platelet donors for
tested. refractory patients, it has not yet been eval-
An alternative approach to supplying uated in a clinical trial for this purpose and
HLA-compatible transfusions is to deter- remains to be validated for this indication.
mine the specificity of the patient’s HLA an-
tibodies and select donors whose platelets
Prevention of Platelet Alloimmunization
lack the antigens with which the antibodies
react. This is termed the antibody specific- Once refractoriness resulting from plate-
8
ity prediction (ASP) method. One study let alloimmunization is established, it is
compared the effectiveness of transfused very difficult, if not impossible, to reverse.
platelets selected by the ASP method with Therefore, in addition to developing meth-
those selected on the basis of HLA match- ods of selecting compatible platelet do-
ing, platelet crossmatching, or on a random nors for the refractory patient, several
basis.8 Platelets selected by the ASP method strategies have been evaluated to prevent
were equally effective as those selected by alloimmunization to platelets from occur-
HLA matching or by crossmatching, and ring in the first place. They include reduc-
superior to randomly selected platelets. In tion in the number of leukocytes in the
addition, from a file of HLA-typed donors, platelet products and ultraviolet B (UVB)
many more potential donors were identi- irradiation. The report of the Trial to Re-
fied by the ASP method than were available duce Alloimmunization to Platelets (TRAP)
using traditional HLA matching criteria, Study Group7 indicated that use of either
making the acquisition of compatible leukocyte-filtered or UVB-irradiated blood
platelets for alloimmunized refractory pa- components reduced the incidence of HLA
tients much more feasible. antibody generation from 45% to between
A further refinement of HLA matching 17% and 21%. The incidence of platelet
was proposed by Duquesnoy.21 A computer- refractoriness was reduced from 16% to
ized algorithm—HLA Matchmaker, available between 7% and 10%. Although a rela-
at http://tpis.upmc.edu/tpis/HLAMatchmaker— tionship had been reported between allo-
is employed for evaluation of the molecular immunization and the number of donor
similarities and differences between HLA exposures in one report,22 a second study23
Class I epitopes. First developed to aid in found no relationship between the num-
locating compatible organs for alloim- ber of donor exposures and the rate or se-
munized prospective renal transplant pa- verity of alloimmunization. The TRAP
tients, the strategy is based on the concept study found that leukocyte reduction, not
that immunogenic epitopes are repre- the number of donor exposures, was sig-
sented by amino acid triplets on exposed nificant in modifying the rate of allo-
parts of protein sequences of the Class I immunization. Thus, leukocyte-reduced,
alloantigens that are accessible to allo- pooled, whole-blood-derived platelets
antibodies. Using this scheme, many Class I appear to be clinically equivalent to
HLA antigens classified as mismatches to a apheresis platelets, at least in terms of re-
patient’s HLA type have no incompatible ducing primary alloimmunization.24
exposed amino acid triplets and, therefore, Antibodies to HLA antigens may be de-
would not be expected to elicit an antibody tected by lymphocytotoxicity tests or by
response. The pool of potentially compati- many of the platelet antibody tests dis-
ble HLA-selected donors is thereby greatly cussed below. Lymphocytotoxicity tests de-

tect complement-binding antibodies capa- their chief clinical importance remains

ble of killing lymphocytes. One strategy for linked to their presence on platelets. Of
managing patients who are receiving multi- the dozens of recognized platelet mem-
ple platelet transfusions and who have de- brane glycoproteins, at least five [GPIa, Ib
veloped clinical refractoriness is to test for (alpha and beta), IIb, IIIa, and CD109] are
the presence of HLA antibodies using a polymorphic and have been demon-
lymphocytotoxicity antibody screen against strated to be alloimmunogenic.28 In addi-
a panel of lymphocytes representing most tion, rare individuals who lack a sixth
of the Class I HLA antigens present in the membrane glycoprotein, GPIV (CD36),
donor population. Reactivity to greater may become sensitized to this antigen.29
than 20% of the cells in the panel (PRA Approximately 3% to 5% of individuals of
>20%) indicates that HLA sensitization may Asian or African ethnicity lack GPIV on
be at least a contributing cause of the their platelets30 and can become immu-
31
platelet refractoriness. Newer, more sensi- nized by transfusion or pregnancy. Al-
tive HLA antibody detection techniques though antibodies to these various mem-
such as the Flow PRA 25 (One Lambda, brane glycoproteins may be associated, in
Canoga Park, CA) have been adapted to a rare instances, with refractoriness to pla-
PRA result format, and some HLA testing telet transfusions, alloantibodies to plate-
laboratories use these methods instead of let-specific antigens are more often asso-
the traditional lymphocytotoxicity test. ciated with the alloimmune syndromes
The laboratory detection of lymphocyto- posttransfusion purpura (PTP) and neo-
toxic antibodies does not necessarily indi- natal alloimmune thrombocytopenia
cate that the patient will experience re- (NAIT).
duced survival of transfused platelets. Several antigen systems on platelets are
Moreover, HLA antibodies may disappear now recognized32 (Table 16-4).27,33 The no-
from the patient’s plasma despite contin- menclature adopted by the International
ued exposure through transfusions.26 A Society of Blood Transfusion classifies the
fuller discussion of HLA antibody and anti- systems numerically according to the date
gen testing can be found in Chapter 17. of publication and alphabetically to reflect
their frequency in the population.34 As with
red cells, different terminologies for platelet
Platelet-Specific Alloantigens
antigens often coexist. The first recognized
To date, 22 platelet-specific alloantigens antigen,35 Zwa, is now designated HPA-1a of
have been characterized as to their local- the HPA-1 system. The HPA-1a antigen is
ization to platelet surface glycoprotein often better known as PlA1. HPA-1a is pres-
structures, quantification of their density ent on the platelets of about 98% of persons
on the platelet surface, and determination of European ethnicity and anti-HPA-1a
of DNA polymorphisms in genes encod- (anti-PlA1) is the most frequently encoun-
27
ing for them (see Table 16-4). Several tered clinically significant platelet-specific
others have been described serologically, antibody in this population. Its antithetical
but genetic polymorphisms underlying antigen, HPA-1b (PlA2), occurs in 27% of this
them have not yet been determined.28 The population.
term “platelet specific,” is a misnomer for The HPA-1a and HPA-1b alleles reside on
some of these markers because they may the platelet membrane glycoprotein GPIIIa.
be found on other types of cells as well Patients with Glanzmann’s thrombasthenia
(especially endothelial cells). However, Type I, a disorder of platelet function, lack

Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*
HPA System Antigens (Familiar Phenotypic Amino Acid Alloimmune
Name Names) Frequencies GP Location Substitution Syndromes
POLYMORPHISMS OF GLYCOPROTEIN IIIa
HPA-1 HPA-1a, (PlA1, Zwa) 98% IIIa

Leu↔Pro33 NAIT, PTP

HPA-1b, (Pl , Zw )
A2 b
27% IIIa
HPA-4 HPA-4a (Pena, Yukb)† 99.9% IIIa
Chapter 16: Platelet and Granulocyte Antigens and Antibodies

Arg↔Gln143 NAIT, PTP
HPA-4b (Pen , Yuk )
b a
<1% IIIa
HPA-6 HPA-6bw (Caa, Tua) <1% IIIa Arg↔Gln489 NAIT
HPA-7 HPA-7bw (Mo) <1% IIIa Pro↔Ala407 NAIT
a
HPA-8 HPA-8bw (Sr ) <1% IIIa Arg↔Cys636 NAIT
a
HPA-10 HPA-10bw (La ) <1% IIIa Arg↔Gln62 NAIT
a
HPA-11 HPA-11bw (Gro ) <1% IIIa Arg↔His633 NAIT
a
HPA-14 HPA-14bw (Oe ) <1% IIIa Lys611 Deleted NAIT
a
HPA-16 HPA-16bw (Duv ) <1% IIIa Ile↔Thr140 NAIT
POLYMORPHISMS OF GLYCOPROTEIN IIb
HPA-3 HPA-3a (Baka, Leka) 85% IIb

IIe↔Ser843 NAIT, PTP
HPA-3b (Bak , Lek )
b b
63% IIb
HPA-9 HPA-9bw (Maxa) 0.6% IIb Val↔Met837 NAIT
367
(cont’d)
368
Table 16-4. Alloantigenic Polymorphisms of Platelet Glycoproteins that Have Been Implicated in Alloimmune Syndromes*
(cont’d)
HPA System Antigens (Familiar Phenotypic Amino Acid Alloimmune

Name Names) Frequencies GP Location Substitution Syndromes
POLYMORPHISMS OF GLYCOPROTEIN Ia
HPA-5 HPA-5a (Brb, Zavb) 99% Ia

a a
glu↔Lys505 NAIT, PTP
HPA-5b (Br , Zav ) 20% Ia
HPA-13 HPA-13bw (Sita) <0.2% Ia Thr↔Met799 NAIT
POLYMORPHISMS OF GLYCOPROTEIN Ib
HPA-2 HPA-2a (Kob, Sibb) 99% Ib alpha

a a
Thr↔Met145 NAIT
HPA-2b (Ko , Sib ) 15% Ib alpha
HPA-12 HPA-12bw (Iya) 0.3% Ib beta Gly↔Glu15 NAIT
OTHER PROBABLE PLATELET ALLOANTIGEN SPECIFICITIES
HPA-15 HPA-15a (Govb) 80% CD109

a
Tyr↔Ser703 NAIT, PTP
HPA-15b (Gov ) 60% CD109
*Modified from Kroll.27 GP = glyprotein; NAIT = neonatal alloimmune thrombocytopenia; PTP = posttransfusion purpurpa.
this glycoprotein and, therefore, do not ex- often paired (eg, Ia/IIa, IIb/IIIa, or Ib/IX),
press HPA-1 antigens. The HPA-1 polymor- referring to the alpha and beta chains in
phism arises by the substitution of a single each complex. GPIb/IX is a leucine-rich
base pair (leucine in HPA-1a and proline in membrane glycoprotein that serves as a re-
HPA-1b) at amino acid position 33 of the ceptor for von Willebrand factor on plate-
protein’s DNA coding sequence. GPIIIa is lets. The Ia/IIa and IIb/IIIa complexes are
also the carrier of HPA-4, -6, -7, -8, -10, -11, members of a broadly distributed family of
-14, and -16 antigens. Alleles in each of adhesion molecules called integrins. Inte-
these systems also arise as a result of single grins are essential for platelet adhesion and
amino acid substitutions at different posi- aggregation because the molecules serve as
tions. The HPA-2 antigen system is situated receptors for ligands such as fibrinogen
on GPIb alpha; the HPA-3 system on GPIIb; (IIb/IIIa), von Willebrand factor (Ib/IX), and
and the HPA-5 system on GPIa.36,37 collagen (Ia/IIa). When present on other
On the platelet membrane, most of the cells, the glycoprotein pairings may differ.
glycoproteins that carry these “platelet-spe- For example, on platelets, GPIIIa is nor-
cific” antigens are present as heterodimeric mally paired with GPIIb. On endothelial
compounds, ie, each consists of two differ- cells, fibroblasts, and smooth muscle, how-
38
ent glycoprotein molecules (see Fig 16-1 ). ever, GPIIIa is paired with a different
Therefore, platelet glycoprotein names are glycoprotein. Thus, these cells share the
Figure 16-1. Schematic diagram of platelet glycoprotein complex IIb/IIIa. Dots and letters (Yuk, Oe,
Pl a , Ca, Gro, Sr, Mo, Bak, Max) designate positions and names of recognized allotypic epitopes. The
molecular regions where autoepitopes have been recognized are indicated by brackets. 38

HPA alloantigens found on the GPIIIa mol- nied by clinically severe reactions) are de-
ecule, but not those found on the GPIIb stroyed. Transfusion of antigen-negative
molecule. platelets may be of value during the acute
CD109 is an exception to the hetero- phase of PTP; however, such platelets
dimeric rule, occurring as a monomeric have a reduced in-vivo survival.42 Plasma-
structure on the platelet membrane. This p h e re s i s — o n c e t h e t re a t m e n t o f
GPI-linked protein is found on activated T choice—has largely been supplanted by
cells, cultured endothelial cells, several tu- the use of intravenous immune globulin
mor cell lines, as well as platelets.39 Two (IGIV ). The mechanism by which these
a
alloantigens designated HPA-15wb (Gov ) treatments are efficacious is unknown.
b
and HPA-15wa (Gov ) have been localized Platelet antibody assays usually reveal a
to platelet CD109.39 Unlike most platelet- serum antibody with HPA-1a specificity.
specific alloantigens, both alleles are highly Typing of the patient’s platelets after re-
expressed [0.53% (Govb) and 0.47% (Gova)] covery will document a HPA-1a-negative
in persons of European ethnicity (Table phenotype or analogous typing for other
16-4). Sensitization to Gov alloantigens has platelet-specific antigen systems. Follow-
been associated with platelet refractoriness, ing recovery, future transfusions should
NAIT, and PTP, albeit usually together with be provided using washed antigen-nega-
other alloantibodies to platelet antigens.40 tive RBC units if possible. Washed RBC
units may offer some protection against
Clinical Importance of Platelet-Specific recurrence, although at least one case of
Antigens and Antibodies PTP caused by antibody to HPA-5b was
Neonatal Alloimmune Thrombocytopenia precipitated by transfusion of a washed
RBC unit.43
Neonatal alloimmune thrombocytopenia
(variously abbreviated NAIT, NATP, etc) is
described in Chapter 23. Testing for Platelet-Specific Antigens and
Antibodies
Posttransfusion Purpura Clinically useful platelet antibody assays
Posttransfusion purpura (PTP) is charac- emerged later than serologic assays to di-
terized by the development of dramatic, agnose immunologic disorders involving
sudden, and self-limiting thrombocytopenia red cells. This is mainly because it is diffi-
5 to 10 days after a blood transfusion in a cult to separate platelets from whole blood
patient with a history of sensitization by specimens and to distinguish antibody-
pregnancy or transfusion. Coincident with dependent endpoints from nonspecific
the thrombocytopenia is the development changes that occur in platelets under as-
of a potent platelet-specific alloantibody say conditions. Three types of platelet an-
in the patient’s serum, usually anti-HPA- tibody detection methods have been de-
1a. Other specificities have been implicated, veloped44 (Table 16-5). The earliest were
almost always associated with antigens Phase I assays that involved mixing pa-
on GPIIb/IIIa.37,41 PTP differs from transfu- tient serum with normal platelets and
sion reactions caused by red cell antibod- used platelet function-dependent end-
ies because the patient’s own antigen- points such as alpha granule release, ag-
negative (usually HPA-1a-negative) plate- gregation, or agglutination. Phase II tests
lets as well as any transfused antigen-pos- measured either surface or total platelet-
itive platelets (which may be accompa- associated immunoglobulin on patient

Table 16-5. Platelet Antibody Assays

Phase I Assays
Platelet aggregation
Inhibition of platelet aggregation
Inhibition of clot retraction
Inhibition of platelet migration
Complement fixation
Platelet factor 3 release
51
Chromium release
14
C-Serotonin release
Phase II Assays
Detection of platelet surface-associated immunoglobulin
■ Platelet suspension immunofluorescence test (PSIFT)
■ Flow cytometry
125 125
■ Radioimmunoassay ( I staphylococcal Protein A, I antihuman immunoglobulin, polyclonal or
monoclonal)
■ Antiglobulin consumption (two-stage assay)
■ Solid-phase red cell adherence
Detection of total platelet-associated immunoglobulin
■ Nephelometry
■ Electroimmunoassay
■ Radial immunodiffusion
Phase III Assays

Monoclonal antibody immobilization of platelet antigens (MAIPA)
Antigen capture ELISA (ACE)
Modified antigen capture ELISA (MACE)
Immunobead assay
Immunoblotting
platelets or on normal platelets after sen- significant platelet alloantibodies.45 A modi-

sitization with patient serum. Phase III fication of MPHA, the SPRCA, is widely
solid-phase assays were developed in used.18 In this assay, intact platelets are im-
which the binding of antibodies to iso- mobilized in the round-bottom wells of a
lated platelet surface glycoproteins is de- microtiter tray and are sensitized with anti-
tected. The test methods are examples of body to be detected. After washing, detec-
Phase I, II, and III assays; variations of each tor red cells previously coated with an anti-
test method are also used. Lymphocyto- body specific for human immunoglobulin
toxicity tests are discussed in Chapter 17. are added. After incubation from several
Mixed Passive Hemagglutination Assay hours to overnight, the tray is subjected to a
(MPHA). A Phase II assay used for the de- slow centrifugation and examined visually.
tection of platelet-specific antibodies as If antibody is bound to the immobilized
well as for platelet crossmatching is the platelets, the indicator red cells fail to form
MPHA. Shibata et al were the first to use a compact button in the center of the well
this method to detect and identify clinically because they are evenly distributed like a

“carpet” over the antibody-coated platelets. fore, binding of the labeled probe can be
In a negative reaction, a red cell button forms assumed to be via its F(ab’)2 or antigen-spe-
in the center of the well. A limitation of the cific end. A second fluorescent label [eg,
MPHA assay is that it fails to distinguish phycoerythrin (PE)] can be attached to an
platelet-specific from non-platelet-specific antihuman IgM probe to detect IgM plate-
antibodies. A modification of the MPHA as- let antibodies. Because FITC and PE fluo-
say, the Capture-P (Imucor Gamma, Norcross, resce with peak light intensities at different
GA), is available as a commercial kit and is wavelengths when exposed to the mono-
most often marketed for platelet cross- chromatic argon laser in the flow cytometer
matching.18 SPRCA testing may be modified (520-nm green light and 580-nm reddish-
by treatment of target platelets with chloro- orange light, respectively), cells labeled
quine or acid,46,47 which disrupts the Class I with FITC can be distinguished from those
HLA heavy chain-peptide-β2-microglobulin labeled with PE. Both anti-IgG and anti-
trimolecular complex. This modifies anti- IgM labeled with different fluorochromes
genic epitopes, reducing the binding of can be added to the same tube with washed
specific antibodies directed against HLA on sensitized platelets for the simultaneous
platelets. However, strong HLA antibodies detection of antiplatelet IgG and IgM.
may still bind, giving the impression that Flow cytometry has proven to be a very
the antibody is directed to non-HLA anti- sensitive method for detection of alloanti-
gens. bodies. The assay is capable of detecting
Flow Cytometry. Another example of a very small numbers of antibody molecules
Phase II assay is platelet antibody detection bound to platelets as is the case with allo-
using immunofluorescence. Originally a antibodies specific for antigens of the
slide-based method,48 the technique now HPA-5 (Br) system having only 1000 to 2000
uses flow cytometry to detect platelet-reac- sites per platelet. Moreover, some alloanti-
tive antibody in patient sera that binds to bodies that are specific for labile epitopes
intact platelets.49 In the assay, washed plate- that are unreliably detected in Phase III as-
lets are sensitized with patient or control says can be detected on intact platelets
serum for up to 60 minutes, usually at room using flow cytometry.
temperature. The platelets are then washed Because the target platelets used in the
repeatedly to remove nonspecific immunoassay are intact, flow cytometry does not
globulins, and platelet-bound antibodies differentiate between platelet-specific (ie,
are detected with a fluorescent-labeled platelet glycoprotein directed) and non-
(usually fluorescein isothiocyanate, FITC) platelet-specific antibodies. Examples of
polyclonal or monoclonal antibody specific the latter are HLA and ABO antibodies. This
for human immunoglobulin. The platelets is an advantage when the method is used to
are analyzed in the flow cytometer and re- detect antibodies that will affect the success
sults can be expressed as a ratio of the of a platelet transfusion, and, for this rea-
mean or peak channel fluorescence of nor- son, flow cytometry has been advocated as
mal platelets sensitized with patient serum a platelet crossmatching method. However,
over that of normal platelets incubated in when used to investigate cases of suspected
normal serum. In order to prevent nonspe- NAIT or PTP, the method has a potential
cific binding of the immunoglobulin probe drawback—the more relevant platelet-spe-
via Fc receptors on the target platelets, the cific antibodies characteristic in these dis-
probe antibodies are enzyme treated to re- eases can be obscured by non-platelet-spe-
move the Fc end of the molecule. There- cific reactivity.

Monoclonal Antibody-Specific Immobi- such as restriction fragment length poly-

lization of Platelet Antigen (MAIPA). An ex- morphism (RFLP) analysis and sequence-
ample of a Phase III assay is the MAIPA,50-52 specific oligonucleotide hybridization, have
perhaps the most widely used assay to de- been developed.56,57 All of these techniques
tect platelet-specific antibodies. The assay are reliable, but they are also laborious and
requires the use of murine monoclonal an- time-consuming. For this reason, PCR
tibodies (MoAbs) that recognize the target genotyping with sequence-specific primers
antigens of interest but do not compete (SSP) appears to be much more practical to
with the human antibody being detected. use.58,59 In a recent workshop, SSP-PCR was
In the assay, target platelets are simulta- the most common and reliable method of
neously sensitized with patient serum and determining platelet antigens,60 making it
a murine MoAb recognizing the desired tar- feasible for genotyping HPAs independent
get molecule on the platelet surface. After of the patient’s platelet count and of rare
the initial sensitization step, platelets are typing sera.61
washed and solubilized in a nonionic de-
tergent. After centrifugation to remove
cytoskeletal fragments, an aliquot of the Autoimmune Platelet Disorders
supernatant lysate is added to wells of a
microtiter tray containing immobilized Idiopathic (Autoimmune)
goat antibody specific for mouse IgG. The Thrombocytopenic Purpura
MoAb is thereby captured and the platelet Autoantibodies directed against platelet
surface glycoprotein with its bound human antigens may result in thrombocytopenia.
antibody is immobilized. After a wash step, Chronic idiopathic thrombocytopenic
the human antibody is detected with an purpura (ITP), most often a disease in
enzyme-labeled goat antihuman immuno- adults, is characterized by an insidious
globulin probe. onset and moderate thrombocytopenia
There are several other versions of Phase that may exist for months to years before
III assays in use today, including the anti- diagnosis. Females are twice as likely to
gen capture enzyme-linked immunosorbent be affected as males. Spontaneous remis-
assay (ACE), the modified antigen capture sions are rare, and treatment is usually
ELISA (MACE),53,54 and the commercially required to raise the platelet count. First-
55
available GTI PAKPLUS (GTI, Waukesha, line therapy consists of steroids, high-
WI). Each relies on MoAbs to immobilize dose IGIV or Rh immunoglobulin (RhIG),
only the glycoproteins of interest, thereby followed by splenectomy in nonresponders.
reducing or eliminating interfering reactions Many other therapies have been used in
due to non-platelet-specific antibodies, es- patients who fail to respond to splenec-
pecially anti-HLA, which, if present, is de- tomy. Results have varied. Chronic auto-
tected only in wells containing pools of im- immune thrombocytopenia may be idio-
mobilized HLA Class I antigens. pathic or associated with other diseases
Platelet Typing Using Molecular Meth- (eg, HIV infection, malignancy, other au-
ods. Molecular typing by polymerase chain toimmune conditions). Acute ITP is mainly
reaction (PCR) is available for many platelet a childhood disease characterized by abrupt
antigens. Because immunophenotyping is onset of severe thrombocytopenia and
limited by the shortage of characterized bleeding symptomatology, often after a
typing antisera and by low platelet counts, viral infection. The majority of cases re-
several DNA-based HPA typing techniques, solve spontaneously over a 2- to 6-month

period. If treatment is required, IGIV or have limited usefulness in patients who

RhIG infusions are usually effective in have very low platelet counts that prevent
raising the platelet count. Steroids are adequate numbers of platelets to be collected
used less often because of serious side ef- for use in the tests.
fects in children. Splenectomy, if used, is One commercially available Phase III
reserved for those children whose disease test, the GTI PAKAUTO,65 uses eluates pre-
is severe and lasts longer than 6 months, pared from washed patient platelets. The
similar to chronic ITP in adults. eluates are tested against a panel of MoAb-
immobilized platelet GP complexes, and
antibody binding is detected using an en-
Testing for Platelet Autoantibodies zyme-linked antihuman immunoglobulin
Numerous Phase I, II, and III platelet anti- probe. In the indirect phase of the assay,
body assays have been developed to de- patient plasma is tested against the same
tect relevant autoantibodies in ITP pa- glycoprotein panel. In general, plasma anti-
tients. Although many tests have been bodies are detected less often than anti-
demonstrated to be quite sensitive, par- bodies in the eluates. ITP patients may
ticularly in detecting total or cell surface have antibodies that are reactive with one
platelet-associated immunoglobulins (Phase or several GP targets. To date, there is no
II assays), 62 none has been sufficiently correlation between the specificity of auto-
specific to be particularly useful in either antibodies in ITP and disease severity.
the diagnosis or management of ITP. The
American Society of Hematology’s prac-
Drug-Induced Immune Platelet Disorders
tice guidelines for ITP state that serologic
testing is unnecessary, assuming the clini- Thrombocytopenia associated with specific
cal findings are compatible with the diag- drugs is not uncommon. Drugs often im-
nosis.63 However, platelet antibody tests plicated include quinidine/quinine, sulfa
may be helpful in the evaluation of patients drugs, heparin, and colloidal gold. Both
suspected of having ITP when other, non- drug-dependent and drug-independent
immune causes may be present. antibodies may be produced. Drug-inde-
The goal of serologic testing in ITP is to pendent antibodies, although stimulated
detect autoantibody bound to the patient’s by drugs, do not require the continued
own platelets with or without demonstra- presence of the drug to react with plate-
tion of similar reactivity in the patient’s lets and are serologically indistinguish-
plasma. Most of the newer assays offered able from other platelet autoantibodies.
for evaluation of patients suspected of hav- (Unlike typical autoimmune thrombocyto-
ing ITP are Phase III assays, designed to de- penia, these antibodies are transient ex-
tect immunoglobulin binding to platelet- cept when caused by therapy with gold,
specific epitopes found on platelet glyco- which is excreted very slowly.) Drug-de-
protein complexes GPIIb/IIIa, GPIa/IIa, pendent antibodies result when a drug
and/or GPIb/IX. combines with platelets in such a way as to
These solid-phase GP-specific assays ap- create neoantigens to which antibodies are
pear to have improved specificity in distin- formed. The drug must be present for the
guishing ITP from nonimmune thrombo- antibody to react. These antibodies can
cytopenia when compared to Phase II assays, cause a thrombocytopenia of sudden and
but this is often balanced by a decrease in rapid onset, usually resolving when the
sensitivity.64 Moreover, all of these methods drug is discontinued.

Testing for Drug-Dependent Platelet- tibodies have been detected and confirmed
Reactive Antibodies by flow cytometry in a large platelet immu-
nology reference laboratory.
Serology. Virtually any platelet serology Flow cytometry has its limitations, as do
test that is used to detect platelet-bound other antibody detection methods, in de-
immunoglobulin can be modified for use tecting drug-dependent antibodies. For
in the detection of drug-dependent pla- many drugs, the optimal concentration to
telet-reactive antibodies. In performing demonstrate in-vitro binding of antibody
drug-dependent antibody testing, it is es- has not been determined. Probably the
sential to establish the proper positive most extensively studied drugs in this re-
67
and negative controls for the assay. Each gard are quinine or quinidine. Another
serum or plasma sample suspected of con- cause of poor sensitivity is the weak bind-
taining drug-dependent antibody must be ing of drug to platelets, leading to rapidly
tested against normal target platelets in declining numbers of drug molecules on
the presence and absence of drug. More- the platelet surface once drug is removed
over, at least one normal serum should be from the environment of the platelet. It is
tested with and without drug to control therefore important to maintain a critical
for any possible drug-related platelet ef- concentration of drug in all washing buffers
fect that does not require specific anti- before addition of probe at the end of an
body. Finally, a positive control serum assay.66 Yet another potential reason for in-
known to be reactive with the drug being sensitivity is that a patient may not be sen-
assayed should be tested with and with- sitized to the native drug but, rather, to a
out drug to complete the evaluation. A metabolite of the drug. Antibodies depend-
positive result must show that the serum ent on metabolites of acetaminophen and
is positive against normal target platelets sulfamethoxazole have been reported.68,69
in the presence of drug and not without A number of other assays have been
drug, and that the drug did not non- adopted for the detection of drug-depend-
specifically cause a positive result in the ent platelet antibodies. Among these are
target platelet. Likewise, the positive con- the SPRCA.70,71 Phase I assays have also been
trol must be positive with the drug and modified for detection of heparin-depend-
negative without it. ent platelet antibodies (see below). In some
Flow Cytometry. The flow cytometry test cases, determination of the specific glyco-
can be readily adapted to detect both IgG protein to which antibody is directed by
and IgM drug-dependent platelet antibod- Phase III assays may provide useful clinical
53,66
ies. In this modification, fluorescence of information. For example, drug-dependent
normal platelets sensitized with the pa- antibody to GPIb/IX was associated with a
tient’s serum in the presence of drug can be more acute, but reversible, quinine-in-
compared with that of the patient’s sample duced thrombocytopenia, whereas anti-
without drug or to a normal serum with body to GPIIb/IIIa was associated with a more
drug to determine relative intensity of la- prolonged course.72
beling. Flow cytometry has proven to have
superior sensitivity to other assays for de-
tection of quinine-quinidine- and sulfona- Heparin-Induced Thrombocytopenia
mide-dependent platelet-reactive antibod- Two types of heparin-induced thrombo-
ies.53 Table 16-6 shows other agents for cytopenia (HIT) have been recognized.
which drug-dependent platelet-reactive an- Type I, of nonimmune origin, presents with

Table 16-6. Drugs Confirmed to Elicit Drug-Dependent Platelet-Reactive Antibodies

In Vitro Using Flow Cytometry Testing
abciximab heparin ranitidine
acetaminophen ibuprofen rifampin
carbamazepine levofloxacin sulfamethoxazole
ceftazidime loracarbef sulfisoxazole
ceftizoxime naproxen suramin
ceftriaxone orbofiban tirofiban
ciprofloxacin oxaliplatin trimethoprim
eptifibatide phenytoin vancomycin
esomeprazole propoxyphene xemilofiban
fentanyl quinidine
fexofenadine quinine
mild transient thrombocytopenia within ischemia, deep venous thrombosis, or

minutes to several days after heparin ex- ischemia of other organs. The thrombotic
posure but generally resolves despite on- complications may force limb amputation
going heparin therapy and is not clinically or may prove fatal.
important. In contrast, immune, or Type Thrombosis or an unexplained decrease
II, HIT may lead to life- and limb-threat- in platelet count while on heparin therapy
ening thrombotic complications and requires should raise concern about HIT. Heparin,
careful evaluation and management of af- including heparin flushes and heparin-
flicted patients. coated catheters, should be discontinued,
The exact incidence of immune HIT is and the patient should be evaluated for
unknown, but it may develop in up to 3% of laboratory evidence of HIT and signs of
patients treated with unfractionated hepa- thrombosis. In mild-to-moderate thrombo-
rin. Low-molecular-weight heparin is less cytopenia, monitoring of platelet counts
likely to be associated with either antibody and observation may be sufficient, but be-
production or thrombocytopenia.73 Bovine cause of the high risk of thrombosis, treat-
heparin appears somewhat more likely to ment with alternative anticoagulants is
cause HIT than porcine heparin.74 A reduc- generally recommended. Warfarin should
tion in baseline platelet count by at least be avoided in the early treatment of HIT
50% occurs generally within 5 to 14 days af- because it does not prevent thrombosis in
ter primary exposure and sooner after sec- this setting and may provoke limb-threat-
ondary exposure to the drug. The platelet ening venous gangrene by reducing levels
count is often less than 100,000/µL and of naturally occurring anticoagulants faster
usually recovers within 5 to 7 days upon than it reduces activated coagulation fac-
discontinuation of heparin. tors. However, warfarin can and should be
About 30% of patients with HIT, or ap- used after the patient is anticoagulated
proximately 0.9% of patients who receive with alternative drugs. Suitable anticoagu-
heparin, develop thrombosis, which can lants approved by the Food and Drug Ad-
occur in the arterial, venous, or both sys- ministration (FDA) include hirudin (a natu-
tems.75,76 Patients may develop cardiovascu- ral thrombin inhibitor) and argatroban.
lar problems, myocardial infarction, limb Selected patients may also benefit from

thrombolectomy or thrombolytic therapy. does not differentiate IgG, IgM, and IgA
Platelet transfusion should be avoided, antibodies that bind to the PF4-heparin
given that bleeding is a rare complication in complex.
HIT, and administration of platelets may The 14C-serotonin release assay (SRA) is
precipitate thrombosis.77-79 an example of a Phase I assay for detection
Heparin forms a complex with platelet of heparin-dependent antibodies.83 Normal,
factor 4 (PF4), a tetrameric protein released fresh target platelets are incubated with
14
from platelet alpha granules. Antibodies C-serotonin that is taken up into the
(IgG, IgM, and some IgA) form against vari- dense granules of the platelets. Then, target
ous epitopes on this complex and attach to platelets are exposed to patient serum in
platelet Fcγ IIa receptors, whereby platelets the presence of low and high concentra-
become activated. The antibody may also tions of heparin. Release of at least 20% of
bind to the complexes at other sites, nota- the radioactive label at the low dose of hep-
bly on endothelial cells. Thus, HIT might arin and inhibition of this release at the
involve activation and damage not only of high dose confirms the presence of hepa-
platelets but also of endothelium, causing rin-dependent antibodies. Other functional
increased susceptibility to thrombosis. This tests used to detect heparin-dependent an-
new understanding of the mechanism of tibodies include the heparin-induced
heparin antibodies is exploited by ELISA platelet aggregation test and the heparin-
tests in which microwells are coated with induced platelet activation test.
the complexes rather than with the plate- The PF4 ELISA and the SRA are both
lets themselves.80 more sensitive and specific than the plate-
let aggregation test for the detection of hep-
arin-dependent platelet antibodies in pa-
Testing for Heparin-Dependent Antibodies tients for whom there is clinical suspicion
The PF4 ELISA is an example of a Phase of HIT.83-85 However, in asymptomatic pa-
III assay for HIT. Target complexes of PF4 tients receiving heparin or in those who
and heparin or heparin-like molecules are have not yet received the drug, neither test
immobilized on a solid phase. To perform is sufficiently predictive of HIT to warrant
the test, patient serum is added to premade its use in screening.
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- Granulocyte Antigens
pendent antibody binds to the complexes Analogous to platelet alloantigens, neu-
and is detected via enzyme-conjugated trophil alloantigens are implicated in
antihuman immunoglobulin. An optical clinical syndromes including neonatal
density value above 0.4 in the PF4-PVS alloimmune neutropenia (NAN), transfu-
well that is inhibited by high-dose hepa- sion-related acute lung injury (TRALI),
rin confirms the presence of a heparin- immune neutropenia after marrow trans-
dependent antibody in the patient’s sam- plantation, refractoriness to granulocyte
ple. Although IgG antibodies are the most transfusion, and chronic benign autoim-
clinically relevant antibodies causing this mune neutropenia of infancy. A neutrophil
81
syndrome, occasional patients with HIT equivalent of PTP has not been described.
appear to have only non-IgG (IgM or IgA) To date, seven neutrophil alloantigens have
antibodies.82 The PF4 ELISA detects but been described, including localization to

neutrophil surface glycoprotein structures has been identified; therefore, the existence
and, in some cases, determination of DNA of a second allele to HNA-2a (NB1) is un-
polymorphisms in genes encoding for them proven.88 HNA-2a has been associated with
(see Table 16-7). TRALI and NAN. The DNA sequence of the
The first granulocyte-specific antigen, NB1(HNA-2a) gene has been determined as
NA1 (HNA-1a), was described in 1966 by have the molecular polymorphisms associ-
Lalezari and Bernard.86 HNA-1a and its anti- ated with HNA-1a and HNA-1b and HNA-1c
thetical antigen, HNA-1b, are present on on the gene for FcγRIII. Therefore, genotyp-
FcγRIIIb. Antibodies to HNA-1a and -1b ing for these specificities can be performed
have been implicated in TRALI, NAN, and on genomic DNA using PCR-SSP.89 Reduced
autoimmune neutropenia of infancy. About expression of granulocyte antigens occurs in
0.1% of individuals of European ethnicity paroxysmal nocturnal hemoglobinuria,
have neutrophils with no detectable chronic myelogenous leukemia, and in pre-
FcγRIIIb (NAnull). The FcγRIII protein also mature infants.
carries the neutrophil alloantigen SH Additional antigens on granulocytes are
(HNA-1c).87 NB1 (HNA-2a) is found on an- shared with other cells and are not granulo-
other granulocyte surface glycoprotein, cyte-specific. These include 5b (HNA-3a),
CD177, the function of which is still unde- MARTa (HNA-4a), and ONDa (HNA-5a). The
termined. HNA-2a has been reported to HNA-3a is located on a 70- to 95-Kd protein
have an allele, NB2, but the product of this that has not yet been cloned. HNA-3a is also
gene cannot be reliably identified with expressed on the surface of lymphocytes.
alloantisera, and no MoAb specific for NB2 The antibodies directed at this antigen are
Table 16-7. Neutrophil Alloantigens

Antigen Frequency (%)
Antigen HNA Glycoprotein

System Allele Designation White Black Location
Neutrophil-specific
NA NA1 HNA-1a 46 46 FcγRIIIb (CD16)
NA NA2 HNA-1b 88 84 FcγRIIIb (CD16)
SH SH HNA-1c 5 22 FcγRIIIb (CD16)
NB NB1 HNA-2a 97 CD177
Neutrophil-nonspecific
5 5b HNA-3a 97 70-95 kD GP
a
MART MART HNA-4a 99 CD11b
a
OND OND HNA-5a 99 CD11a

usually agglutinins; they occasionally occur casionally be life-threatening because of

in women after pregnancy and may be as- increased susceptibility to infection. Man-
sociated with febrile transfusion reactions. agement with antibiotics, IGIV, granulo-
Potent anti-HNA-3a agglutinins in trans- cyte colony-stimulating growth factor,
fused plasma have been responsible for fa- and/or plasma exchange may be helpful.
90-92 a a
tal TRALI. MART and OND , both high-
incidence antigens, are also present on
monocytes and lymphocytes. MARTa has TRALI
been localized to the alpha M chain TRALI is an acute, often life-threatening
(CD11b) of the C3bi receptor (CR3) and re- reaction characterized by respiratory dis-
sults from a single nucleic acid substitu- tress, hypo- or hypertension, and non-
a
tion. MART has been recently reported to cardiogenic pulmonary edema that oc-
93 a
cause NAN. OND is expressed on the al- curs within 6 hours of a transfusion of a
pha L integrin unit, leukocyte function an- plasma-containing blood component.
tigen-1 (CD11a) and also results from a sin- TRALI has been reported to be induced by
gle nucleotide substitution. This marker, neutrophil antibodies, although more re-
found in a chronically transfused aplastic cent reports are more likely to implicate
anemia patient, has not been reported to antibodies to both Class I and II HLA anti-
94
be associated with clinical disease. gens. In TRALI, the causative antibodies
are most often found in the plasma of the
Clinical Syndromes in Which blood donor (see Chapters 17 and 27). Re-
Granulocyte-Specific Alloantigens Are cent reports have postulated that another
Implicated etiology for TRALI is possible. This theory
suggests that two events must coincide
Nonhemolytic, febrile transfusion reac-
for TRALI to occur: 1) the patient must
tions are often associated with granulo-
have a predisposing clinical condition
cyte antibodies. Although such reactions
that releases cytokines or other factors
are more often caused by antibodies to
that prime neutrophils, causing adher-
Class I HLA antibodies directed at Class I
ence to endothelium, and 2) the patient
epitopes present on granulocytes, granu-
must receive a transfusion of biologically
locyte-specific antibodies have been asso-
active lipids (which also stimulate neutro-
ciated with clinical syndromes similar to
phils) from stored blood components.95 In
those seen with antibodies to red cell and
one study, neutrophil antibodies were de-
platelet antigens.
tected in only three of 10 incidents of
TRALI, and none of the donors had HLA
Neonatal Alloimmune Neutropenia antibodies.
NAN is caused by maternal antibodies Autoimmune neutropenia, usually oc-
against alloantigens of fetal neutrophils; curring in adults, may be idiopathic or may
the most frequent specificities seen are occur secondary to such diseases as rheu-
against NA1, NA2, and NB1 antigens (see matoid arthritis, systemic lupus erythe-
Table 16-7). NAN most often occurs in matosus, or bacterial infections. In autoim-
women of the neutrophil alloantigen phe- mune neutropenia of infancy, usually
notypes NA1/NA1 and NA2/NA2; it may occurring in children between the ages of 6
also occur in women of the rare NAnull months to 2 years, the autoantibody has a
phenotype who lack the FcγRIII protein. neutrophil antigen specificity (usually
The neutropenia in all these cases can oc- HNA-1a and -1b) in about half the cases.

The condition is usually self-limiting (with sions: A prospective study of 50 patients. Br J

Haematol 1992;81:395-400.
recovery usually in 7-24 months) and the
6. Dzik WH. Leukoreduced blood components:
condition is relatively benign and manage- Laboratory and clinical aspects. In: Simon
able with antibiotics.96 Drug-dependent an- TL, Dzik WH, Snyder EL, et al, eds. Rossi’s
tibodies can also cause neutropenia. principles of transfusion medicine. 3rd ed.
Philadelphia, PA: Lippincott Williams and
Wilkins, 2002:270-87.
Testing for Granulocyte Autoantibodies 7. The Trial to Reduce Alloimmunization to
Platelets Study Group. Leukocyte reduction
Tests for granulocyte antibodies are not and ultraviolet B irradiation of platelets to
widely performed, although the implica- prevent alloimmunization and refractoriness
tion of neutrophil antibodies as a cause of to platelet transfusions. N Engl J Med 1997;
337:1861-9.
TRALI has increased the demand for this
8. Petz LD, Garratty G, Calhoun L, et al. Select-
laboratory resource. Agglutination tests ing donors of platelets for refractory patients
performed in tube, capillary, or micro- on the basis of HLA antibody specificity.
plate formats use heat-inactivated serum Transfusion 2000;40:1446-56.
9. Koerner TAW, Vo TL, Wacker KE, Strauss RG.
in the presence of EDTA and require fresh The predictive value of three definitions of
granulocytes. Immunofluorescence tests, platelet transfusion refractoriness (abstract).
read with either a fluorescence micro- Transfusion 1988;28:33S.
scope or a flow cytometer, are also used 10. Ishida A, Handa M, Wakui M, et al. Clinical
factors influencing post-transfusion platelet
and are capable of detecting granulocyte- increment in patients undergoing hema-
bound antiglobulin. A combination of topoietic progenitor cell transplantation—a
agglutination and immunofluorescence prospective analysis. Transfusion 1998;38:
839-47.
tests is beneficial.97 Other methods include 11. Bishop J, McGrath K, Wolf M, et al. Clinical
chemiluminescence and a MoAb-specific factors influencing the efficacy of pooled
immobilization of granulocyte antigens platelet transfusions. Blood 1988;71:383-7.
(MAIGA) assay, similar to the MAIPA as- 12. Moroff G, Garratty G, Heal JM, et al. Selection
of platelets for refractory patients by HLA
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its ability to differentiate readily between Transfusion 1992;32:633-40.
HLA and granulocyte-specific antibodies. 13. Bolgiano DC, Larson EB, Slichter SJ. A model
to determine required pool size for HLA-
typed community donor apheresis programs.
14. Duquesnoy RJ, Filip DJ, Rodey G, et al. Suc-
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on behalf of the International Society of treated with low molecular weight heparin or
Blood Transfusion. Vox Sang 2001;80:72-8. unfractionated heparin. N Engl J Med 1995;
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75. Nand S, Wong W, Yuen B, et al. Heparin-in- NA2 and SH are located in two closely linked
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Am J Hematol 1997;56:12-6. allele in the Danish population. Transfusion
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arin-induced thrombocytopenia: Towards Determination of neutrophil antigen gene
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Chapter 17: The HLA System
Chapter 17
The HLA System
T
HE HLA SYSTEM includes a com- The HLA antigen molecules play a key
plex array of genes and their pro- role in antigen presentation. Immunologic
tein products. HLA antigens con- recognition of differences in HLA antigens
tribute to the recognition of self and is probably the first step in the rejection of
nonself, to the immune responses to anti- transplanted tissue. The HLA system is sec-
genic stimuli, and to the coordination of ond in importance only to the ABO anti-
cellular and humoral immunity. The HLA gens in influencing the long-term survival
genes, which are located in the major of transplanted solid organs and is of para-
histocompatibility complex (MHC) on the mount significance in hematopoietic pro-
short arm of chromosome 6, code for genitor cell (HPC) transplantation. HLA an-
glycoprotein molecules found on cell sur- tigens and antibodies are also important in
face membranes. Class I molecules are such complications of transfusion therapy
found on the surface of platelets and of all as platelet refractoriness, febrile non-
nucleated cells of the body. Mature red hemolytic transfusion reactions (FNHTRs),
cells usually lack HLA antigens demon- transfusion-related acute lung injury (TRALI), 17
strable by conventional methods, but nu- and posttransplant and posttransfusion
cleated immature erythroid cells express graft-vs-host disease (GVHD).
them. MHC Class II antigens are restricted Studies correlating HLA polymorphisms
to a few cell types; the most important are with susceptibility and disease resistance
B lymphocytes, macrophages, and den- began soon after serologic techniques for
dritic cells. Other terms that have been HLA Class I typing were developed. Histori-
applied to antigens of the HLA system are: cally, HLA antigen typing has been of value
major histocompatibility locus antigens, in parentage testing and in forensic investi-
transplantation antigens, and tissue anti- gations. Molecular analysis of the HLA re-
gens. gion permits selection of more closely
385

matched donors for HPC transplantation, polymorphic system of genes described in

for investigation of disease associations, humans.
and for anthropologic population studies. The HLA-A, HLA-B, and HLA-C genes
Because of the polymorphic nature of the encode the corresponding Class I antigens
HLA genes, a complex nomenclature has A, B, and C. The HLA-DR, HLA-DQ, and
been developed to refer to the unique allele HLA-DP gene cluster codes for the synthe-
sequences based on the relationship of the sis of correspondingly named Class II anti-
allele to the serologic specificity of the cor- gens. Located between the Class I and Class
responding antigen.1 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
Genetics of the Major cytokine (tumor necrosis factor). This re-
Histocompatibility Complex gion is referred to as MHC Class III.
Class I and II HLA antigens are cell sur-

face glycoproteins that are products of Organization of HLA Genetic Regions
closely linked genes mapped to the p21.3 The HLA Class I region contains, in addi-
band on the short arm of chromosome 6 tion to the classical genes HLA-A, HLA-B,
(Fig 17-1). This genomic region is called and HLA-C, other gene loci designated
the MHC and is usually inherited en bloc HLA-E, HLA-F, HLA-G, HFE, HLA-J, HLA-K,
as a haplotype. Each of the several loci HLA-L, MICA, and MICAB. These latter
has multiple alleles with codominant ex- genes encode the nonclassical or Class Ib
pression of the products from each chro- HLA proteins, characterized by limited
mosome. The HLA system is the most polymorphism and low levels of expres-
Figure 17-1. (A) The major histocompatibility complex located on the short arm of chromosome 6. The
centromere is to the left. The key Class I, II, and III genetic loci are shown. The Class III region 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.

Chapter 17: The HLA System 387
2
sion. Some Class I genes express non- gene and B5 gene (if present) express
functional proteins or are not able to ex- HLA-DR51. The HLA-DQ1 through DQ9
press a protein. Genes unable to express a antigens are expressed on the glycoproteins
functional protein product are termed coded by the DQA1 and DQB1 genes in the
pseudogenes and presumably represent DQ gene cluster. Many of the other genes of
an evolutionary dead end. HLA-E regu- the DQ cluster are probably pseudogenes. A
lates natural killer cells. HLA-G is ex- similar organization is found in the HLA-
pressed by the trophoblast and may be in- DP gene cluster.
volved in the development of maternal The MHC Class III region contains four
immune tolerance of the fetus. Hereditary complement genes, whose alleles are gen-
hemochromatosis (HH), an iron overload erally inherited together as a unit, termed a
disorder with a 10% carrier frequency in complotype. There are more than 10 differ-
Northern Europeans, is associated with ent complotypes inherited in humans. Two
two missense mutations in a Class I-like of the Class III genes, C4A and C4B, code
gene.3 The gene conferring HH was ini- for variants of the C4 molecule. These vari-
tially named HLA-H; however, the HLA-H ants have distinct protein structure and
designation had already been assigned to function; the C4A molecule (if present) car-
an HLA Class I pseudogene by the World ries the Rodgers antigen and the C4B mole-
Health Organization (WHO) Nomencla- cule (if present) carries the Chido antigen,
ture Committee.4 The gene conferring HH both of which are adsorbed onto the red
is now called HFE. Class I molecules are cells of individuals who possess the gene.
also located outside the MHC, such as
CD1, which can present nonprotein anti- Patterns of Inheritance
gens (such as lipids) to T cells.
Although the organization of the MHC is
The genomic organization of the MHC
complicated, its inheritance follows the
Class II region (HLA-D region) is more
established principles of genetics. Every
complex. An MHC Class II molecule con-
person has two different copies of chro-
sists of a noncovalent complex of two struc-
mosome 6 and, thus, possesses two HLA
turally similar chains, the α-chain and the
haplotypes, one from each parent. An in-
β-chain. Both of these chains are encoded
dividual’s haplotype is typically determined
within the MHC. The polymorphism of
by typing multiple family members from
HLA Class II molecules results from differ-
different generations and observing which
ences in both the α-chain and the β-chain;
alleles are inherited together. The ex-
this depends on the Class II isoform. For
pressed gene products constitute the phe-
example, with HLA-DR, the α-chain is
notype, which can be determined for an
monomorphic, but the β-chain is very poly-
individual by typing for the HLA antigens.
morphic. Multiple loci code for either alpha
Because the HLA genes are autosomal
or beta chains of the Class II MHC proteins.
and codominant, the phenotype repre-
Different haplotypes have different num-
sents the combined expression of both
bers of Class II genes and pseudogenes.
haplotypes. Figure 17-2 illustrates inheri-
The proteins coded by the DRA gene and
tance of haplotypes.
the DRB1 gene result in HLA-DR1 through
HLA-DR18. The products of the A gene and
the B3 gene (if present) express HLA-DR52; Finding HLA-Identical Siblings
those of the A gene and the B4 gene (if pres- Each child inherits one copy of chromo-
ent) express HLA-DR53; and those of the A some 6 from each parent; hence, one MHC

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 and b = A3,Cw7,B7,DR15. Offspring of a single
mating pair must have one of only four possible combinations of haplotypes, assuming there has been
no crossing-over.
haplotype is inherited from each parent. Having two siblings provides a 44% chance
Because each parent has two different and three siblings a 58% chance that one
copies of chromosome 6, four different sibling will be HLA-identical. No matter
combinations of haplotypes are possible how many siblings are available for typing
in the offspring (assuming no recombina- (aside from identical twins), the probabil-
tion). This inheritance pattern is impor- ity will never be 100% for finding an HLA-
tant in predicting whether family members identical sibling.
will be compatible donors for transplan-
tation. The chance that two siblings will
be HLA-identical is 25%. The chance that Absence of Antigens
any one patient with “n” siblings will have Usually, both copies of the genes within
at least one HLA-identical sibling is 1-(3/4)n. the MHC are expressed as antigens; how-

ever, in certain individuals, only one anti- ered and characterized. As of 2004, there
gen can be identified. This may occur if were 309 HLA-A, 563 HLA-B, and 368
the individual is homozygous for the al- DRB1 alleles. In reality, many HLA haplo-
lele, or if appropriate antisera are not types are overrepresented compared with
available to type the individual’s antigen what would be expected if the distribution
(referred to as a blank allele). Very rarely, of HLA genes were random. The phenom-
the absence of an antigen can result from enon of linkage disequilibrium accounts
a null allele. A null allele is characterized for the discrepancy between expected and
by substitutions within the coding region observed HLA haplotype frequencies.
of the gene that prevent the expression of Expected frequencies for HLA haplo-
a functional protein at the cell surface. Such types are derived by multiplication of the
inactivation of a gene may be caused by frequencies of each allele. For example, in
nucleotide substitutions, deletions, or in- individuals of European ancestry, the over-
sertions, which lead to a premature cessa- all frequency of the gene coding for HLA-A1
tion in the antigen’s synthesis. When reis 0.15 and that for HLA-B8 is 0.10; there-
ferring to phenotypes, a blank is often fore, 1.5% (0.15 × 0.10) of all HLA haplo-
written as “x” (for A locus), “y” (for B lo- types in this population would be expected
cus), or “–” (for any locus) (eg, A1,x;B7,40 to contain genes coding for both HLA-A1
or A1,–;B7,40). Family studies must be per- and HLA-B8 if they were randomly distrib-
formed to determine the correct genotype. uted. The actual frequency of the A1 and B8
combination, however, is 7% to 8% in this
Crossing-Over population. Certain allelic combinations
occur with increased frequency in different
The genes of the HLA region occasionally
racial groups and constitute common haplo-
demonstrate chromosome crossover, in
types in those populations. These are called
which segments containing linked genetic
ancestral haplotypes because they appear
material are exchanged between the two
to be inherited from a single common an-
chromosomes during meiosis or gameto-
cestor. The most common ancestral haplo-
genesis (see Fig 10-6). These recombinants
type in Northern Europeans, the A1, B8,
are then transmitted as new haplotypes to
DR3, DQ2 haplotype, includes both Class I
the offspring. Crossover frequency is in
and Class II regions. It is unclear whether
part related to the physical distance be-
ancestral haplotypes represent relatively
tween genes. For example, the HLA-A,
young haplotypes that have not had suffi-
HLA-B, and HLA-DR loci are close to-
cient time to undergo recombination, or
gether, with 0.8% crossover between the A
whether they are old haplotypes that are re-
and B loci and 0.5% between the B and
sistant to recombination because of selec-
DR loci. In family studies and in parent-
tion. Linkage disequilibrium in the HLA
age testing, the possibility of recombina-
system is important in studies of parent-
tion must be considered.
age because haplotype frequencies in the
relevant population make the transmis-
Linkage Disequilibrium sion of certain gene combinations more
The MHC system is so polymorphic that, likely than others. Linkage disequilibrium
theoretically, the number of possible uni- also affects the likelihood of finding suit-
que HLA phenotypes is greater than the able unrelated donors for HLA-matched
global human population. Moreover, new platelet transfusions and for HPC trans-
HLA alleles are constantly being discov- plantation.

and consist of two chains: a glycoprotein

Biochemistry, Tissue heavy chain (45,000 daltons) encoded on
Distribution, and Structure the short arm of chromosome 6 and, as a
The HLA antigens are cell surface glyco- light chain, the β2-microglobulin molecule
proteins. HLA Class I molecules contain (12,000 daltons) encoded by a gene on
one copy of two polypeptides: a heavy chromosome 15. The heavy chain pene-
chain, which is attached to the mem- trates the cell membrane. β2-microglobu-
brane, and a light chain, which is called lin is not attached to the cell membrane;
β2-microglobulin. The HLA Class II mole- it associates with the heavy chain via the
cules are composed of one copy each of latter’s nonvariable (α3) domain but is not
an α-chain and a β-chain, both of which covalently bound to it (see Fig 17-3). The
are attached to the cell surface. HLA anti- external portion of the heavy chain con-
gens are divided into Class I and Class II sists of three amino acid domains (α1, α2,
based on their function, tissue distribu- and α3), of which the outermost α1 and
tion, and biochemistry. α2 domains contain the polymorphic re-
gions conferring the HLA antigen speci-
Characteristics of Class I and Class II ficity.
Antigens Class I molecules are found on platelets
Class I antigens (HLA-A, -B, and -C) have and on most nucleated cells in the body,
a molecular weight around 57,000 daltons with some exceptions such as neurons, cor-
Figure 17-3. Stylized diagram of Class I and Class II MHC molecules showing α and β polypeptide
chains, their structural domains, and attached carbohydrate units.

neal epithelium, trophoblast, and germinal ing immune reactivity.5 Levels of soluble
cells. Only vestigial amounts remain on HLA increase with infection [including hu-
mature red cells, with certain allotypes man immunodeficiency virus (HIV)], in-
better expressed than others. These Class I flammatory disease, and transplant rejec-
polymorphisms were independently recog- tion, and decline with progression of
nized as red cell alloantigens by serologists malignancy. Levels of soluble HLA in blood
and were designated as Bennett-Good- components are proportionate to the num-
speed (Bg) antigens. The specificities called ber of residual donor leukocytes and to the
a b c
Bg , Bg , and Bg are identified as HLA-B7, length of storage.6 Soluble HLA in blood
HLA-B17, and HLA-A28, respectively. Plate- components may be involved in the im-
lets express primarily HLA-A and HLA-B munomodulatory effect of blood transfu-
antigens. HLA-C antigens are present at sion.
very low levels and Class II antigens are
generally not expressed at all on platelets. Configuration
Class II antigens (HLA-DR, -DQ, and A representative three-dimensional struc-
-DP) have a molecular weight of approxi- ture of these molecules can be obtained
mately 63,000 daltons and consist of two by X-ray crystallographic analysis of puri-
structurally similar glycoprotein chains (α fied HLA antigens. The outer domains,
and β), both of which traverse the mem- which contain the regions of amino acid
brane (see Fig 17-3). The extramembranous variability and the antigenic epitopes of
portion of each chain has two amino acid the molecules, form a structure known as
domains, of which the outermost domain the “peptide-binding groove.” Alleles de-
contains the variable regions of the Class II fined by polymorphisms in the HLA gene
alleles. The expression of Class II antigens sequences have unique amino acid se-
is more restricted than that of Class I. Class quences and, therefore, form unique
II antigens are expressed constitutively on binding grooves, each able to bind differ-
B lymphocytes, monocytes, and cells de- ent classes of peptides. The peptide-bind-
rived from monocytes such as macro- ing groove is critical for the functional as-
phages and dendritic cells, intestinal epi- pects of HLA molecules (see section on
thelium, and early hematopoietic cells. Biologic Function).
There is also constitutive expression of
Class II antigens on some endothelial cells,
especially those lining the microvasculature.
However, in general, endothelium, particu- Nomenclature
larly that of larger blood vessels, is negative An international committee sponsored by
for Class II antigen expression, although its the World Health Organization estab-
presence can be induced (for instance, by lishes the nomenclature of the HLA sys-
interferon-gamma during immune activa- tem. It is updated regularly to incorporate
tion). T lymphocytes are negative for Class new HLA alleles. HLA antigens are desig-
II antigen expression but become positive nated by a number following the letter
when activated. Class II antigens are ex- that denotes the HLA series (eg, HLA-A1
pressed abnormally in autoimmune dis- or HLA-B8). Previously, antigenic speci-
ease and on some tumor cells. ficities that were not fully confirmed car-
Soluble HLA Class I and Class II antigens ried the prefix “w” (eg, HLA-Aw33). When
shed from cells are found in blood and identification of the antigen became de-
body fluids and may play a role in modulat- finitive, the WHO Nomenclature Commit-

tee dropped the “w” from the designation. collective term for a group of HLA anti-
The Committee meets regularly to update gens that exhibit such cross-reactivity is
nomenclature by recognizing new speci- cross-reactive group (CREG).
ficities or genetic loci. The “w” prefix is no
longer applied in this manner and is now “Public” Antigens
used only for the following: 1) Bw4 and
In addition to splits and CREGs, HLA pro-
Bw6, to distinguish these “public” antigens
teins have reactivity that is common to
from other B locus alleles; 2) all C locus
many different HLA specificities. Called
specificities, to avoid confusion with
“public” antigens, these common amino
members of the complement system; and
acid sequences appear to represent the
3) Dw and DP specificities that were de-
less variable portion of the HLA molecule.
fined by mixed lymphocyte reactions and
Two well-characterized “public” antigens,
primed lymphocyte typing. The numeric
HLA-Bw4 and HLA-Bw6, are found in the
designations for the HLA-A and HLA-B
HLA-B series. The Bw4 antigen is also
specificities were assigned based on the
found on some A locus molecules. “Pub-
order of their discovery.
lic” antigens are clinically important be-
cause patients exposed to foreign HLA
Splits
antigens via pregnancy, transfusion, or
Refinement of serologic methods permit- transplantation can make antibodies to
ted antigens previously believed to repre- them. A single antibody, when directed
sent a single specificity to be “split” into against a “public” antigen, can resemble
specificities that were serologically (and, the sum of multiple discrete alloanti-
later, genetically) distinct. The designa- bodies.
tion for an individual antigen that is a
split of an earlier recognized antigen of-
ten includes the number of the parent an- Nomenclature for HLA Alleles
tigen in parentheses, eg, HLA-B44 (12).
As nucleotide sequencing is used to in-
Shared Determinants vestigate the HLA system, increasing
numbers of HLA alleles are being identi-
As the specificity of HLA typing sera im- fied, many of which share a common se-
proved, it was found that some epitopes, rologic phenotype. The minimum re-
the part of the antigen that binds anti- quirement for designation of a new allele
body, were common to several antigens. is the sequence of exons two and three for
Antibodies to these epitopes identified HLA Class I and exon two for HLA Class II
antigens that constituted a group, of which (DRB1). These exons encode the variable
the individual members could be identi- amino acids that confer HLA antigen spe-
fied by antisera with more restricted ac- cificity. A uniform nomenclature has been
tivity. adopted that takes into account the locus,
the major serologic specificity, and the al-
Cross-Reactive Groups lele determined by molecular typing tech-
In addition to “splits,” HLA antigens and niques. For example, isoelectric focusing,
antigen groups may have other epitopes amino acid sequencing, and nucleotide
in common. Antibodies that react with sequencing have identified several unique
these shared determinants often cause variants of HLA-DR4. The first HLA-DR4
cross-reactions in serologic testing. The variant is designated DRB1*0401, indicat-

ing the locus (DR), the protein (β1 chain), peptide antigens. T lymphocytes interact
an asterisk to represent that an allele with peptide antigen only when the T-cell
name follows, the major serologic speci- receptor (TCR) for antigen engages both
ficity (04 for HLA-DR4), and the allele an HLA molecule and the antigenic pep-
number (variant 01). For Class I alleles, the tide contained with its peptide-binding
name of the locus, for example HLA-B, is groove. This limitation is referred to as
followed by an asterisk and then a num- “MHC restriction.”7
ber of digits. The first two digits corre- In the thymus, T lymphocytes whose
spond to the serologic specificity of the TCRs bind to a self HLA molecule are se-
antigen. The third and fourth digits are lected (positive selection), with the excep-
used to list the subtypes, numbers being tion of those whose TCRs also bind to a
assigned in the order in which the DNA peptide derived from a self antigen, in
sequences have been determined. There- which case they are deleted (negative selec-
fore, B*2704 represents the HLA-B locus, tion). Some self-reactive T cells escape neg-
with a serologic specificity of B27, and ative selection, however. If not functionally
was the fourth allele described in this inactivated, for instance, by the mechanism
family (see Table 17-1). Finally, the no- of anergy, these self-reactive T cells may be-
menclature can accommodate alleles come involved in an autoimmune process.
with silent mutations, ie, those that have (See Chapter 11.)
different DNA sequences but identical
amino acid sequences and null alleles.
Role of Class I
Class I molecules are synthesized, and
peptide antigens are inserted into the
Biologic Function peptide-binding groove, in the endo-
The essential function of the HLA system plasmic reticulum. Peptide antigens that
is self/nonself discrimination. Discrimina- fit into the Class I peptide-binding groove
tion of self from nonself is accomplished are typically eight or nine amino acids in
by the interaction of T lymphocytes with length and are derived from proteins that
Table 17-1. HLA Nomenclature
Number of
Genetic Antigenic Identified Polypeptide
Locus Specificity Allele Alleles Location
HLA-A A1 to A80 A*0101 to *8001 207 a

HLA-B B7 to B81 B*0702 to *8301 412 a
HLA-C Cw1 to Cw10 Bw*0102 to *1802 100 a
DRA DR1 to DR18 DRA*0101 to *0102 2 a
DRB1 DRB1*0101 to *1608 271 β1
DQA1 DQ1 to DQ9 DQA1*0101 to *0601 20 a
DQB1 DQB1*0501 to *0402 45 β1
DPA1 DPw1 to DPw6 DPA1*0103 to *0401 19 a1
DPB1 DPB1*0101 to *8901 93 β1

are made by the cell (endogenous pro- by endocytosis (exogenous proteins). Ex-
teins). These endogenous proteins, which ogenous proteins, which may be normal
may be normal self proteins, altered self self proteins or proteins derived from
proteins such as those found in cancer pathogens such as bacteria, are degraded
cells, or viral proteins such as those found to peptides by enzymes in the endosomal
in virus-infected cells, are degraded in the pathway. Class II molecules are then
cytosol by a large multifunctional prote- transported to the cell surface where they
ase (LMP) and transported to the endo- are available to interact with CD4-positive
plasmic reticulum by a transporter associ- T lymphocytes, which secrete immuno-
ated with antigen processing (TAP). The stimulatory cytokines in response. This
LMP and TAP genes are both localized to mechanism is especially important for the
the MHC. production of antibodies.
Class I molecules are transported to the
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-
Detection of HLA Antigens
tigenic peptide in the context of the specific and Alleles
Class I molecule displaying it, then this Methods for the detection of HLA antigens
binding activates the cytotoxic properties of and alleles fall into three groups: molecu-
the T cell, which will then attack the cell, lar (DNA-based), serologic, and cellular
characteristically eliciting an inflammatory assays. Detailed procedures of commonly
response. The presentation of antigen by used assays are provided in the current
Class I molecules is especially important in edition of the American Society for Histo-
a host’s defense against viral pathogens and compatibility and Immunogenetics Labo-
against malignant transformation. Tumor ratory Manual. Depending on the clinical
cells that do not express Class I escape this situation, a particular HLA antigen detec-
immune surveillance. tion or typing method may be preferable
(Table 17-2).
Role of Class II
DNA-Based Assays
Class II molecules, like Class I molecules,
are synthesized in the endoplasmic retic- DNA-based typing has several advantages
ulum, but peptide antigens are not in- over serologic and cellular assays: high
serted into the peptide-binding groove sensitivity and specificity; small sample
here. Instead, an invariant chain (Ii) is in- volumes; decreased turnaround time, for
serted. The Class II-invariant chain mole- some methods, as short as a few hours;
cule is transported to an endosome where and absence of the need for cell surface
the invariant chain is removed by a spe- antigen expression or cell viability. Al-
cialized Class II molecule called DM though serologic methods can readily dis-
(whose locus is also localized to the MHC). tinguish only about 21 serologic specifi-
A Class II antigenic peptide is then in- cities, high-resolution DNA-based methods
serted into the peptide-binding groove. can detect up to 368 alleles.
Peptide antigens that fit into the Class II
peptide-binding groove are typically 12 to Polymerase Chain Reaction Testing
25 amino acids in length and are derived Polymerase chain reaction (PCR) technol-
from proteins that are taken up by the cell ogy allows amplification of large quanti-

Table 17-2. HLA Typing Methods and Appropriate Applications

Method Clinical Application Resolution
SSP (PCR) Solid organ, related and unrelated HPC Serologic to allele level,
transplantation higher resolution with
large number of primers
DNA sequencing Unrelated HPC transplantation, resolu- Allele level
tion of typing problems with other
methods, characterization of new
alleles
Forward SSOP Solid organ and HPC transplantation Serologic to allele level
hybridization (can accommodate high-volume
testing)
Reverse SSOP Solid organ, related and unrelated HPC Serologic, higher resolution
hybridization transplantation with larger number of
probes
Microlympho- Solid organ transplantation, evaluation Serologic specificity
cytotoxicity of platelet refractoriness, HLA typing
(Class I only) of platelet recipients
and platelet donors
ties of a particular target segment of geno- typed and highly specific information ob-
mic DNA. Low- to intermediate-resolution tained. Disadvantages include potential
typing detects the HLA serologic equiva- difficulty in interpretation of results and the
lents with great accuracy; eg, it distin- need to use multiple filters and perform
guishes DR15 from DR16, whereas high- multiple amplifications and subsequent
resolution typing distinguishes individual hybridizations. A variation of this tech-
alleles, eg, DRB1*0101 from DRB1*0102. nique, the reverse line or dot blot, elimi-
Several PCR-based methods have been nates the need for multiple filters and hy-
developed, of which two general approaches bridizations by incorporation of a label
are described below. (such as biotin) into the PCR product dur-
Oligonucleotide Probes. The first tech- ing its amplification from genomic DNA.
nique uses sequence-specific oligonucleo- The PCR product is then hybridized to a
tide probes (SSOPs) and is known as PCR- membrane containing all the relevant
SSO, PCR-SSOP, or allele-specific oligo- SSOPs and its pattern of hybridization with
nucleotide (ASO) hybridization.8 A PCR the SSOPs revealed by detection of the in-
product amplified from genomic DNA is corporated label. In the past few years, a
applied to a membrane or filter to which new method for establishing HLA geno-
the labeled SSOPs are hybridized. These types that features arrays of oligonucleotide
short DNA probes will hybridize with the probes on a solid phase has begun to ap-
complementary sequences and identify pear in HLA typing laboratories. The
groups of alleles or individual alleles. Ad- microbead array assay is an SSOP method
vantages are that all Class II loci can be for HLA-A, -B, and -DR antigen level typing.

This technique also has the ability for low- HLA sera of known specificities are
to-intermediate resolution DNA-based tis- placed in wells of a microdroplet test plate.
sue typing, with a reduction in sample pro- A suspension of lymphocytes is added to
cessing time. each well. Rabbit complement is then
Sequence-Specific Primers. A second added and, if sufficient antibody has bound
major technique uses sequence-specific to the lymphocyte membranes, the com-
primer pairs (SSPs) that target and amplify plement cascade will be activated through
a particular DNA sequence.9 This method the membrane attack complex, leading to
requires the performance of multiple PCR lymphocytotoxicity. Damage to the cell
reactions in which each reaction is specific membrane can be detected by the addition
for a particular allele or group of alleles. Di- of dye: cells that have no attached antibody,
rect visualization of the amplified alleles is no activated complement, and no damage
seen after agarose gel electrophoresis. Be- to the membrane keep the vital dyes from
cause SSPs have such specific targets, pres- penetrating; cells with damaged mem-
ence of the amplified material indicates branes allow the dye to enter. The cells are
presence of the corresponding allele(s). The examined for dye exclusion or uptake un-
pattern of positive and negative PCR ampli- der phase contrast microscopy. If a fluores-
fications is examined to determine the HLA cent microscope is available, fluorescent vi-
alleles present. Primer pair sets are avail- tal dyes can also be used.
able that can determine the full HLA-A, -B, Because HLA-DR and HLA-DQ antigens
-C, -DR, -DQ, and -DP type. are expressed on B cells and not on resting
T cells, typing for these antigens usually re-
Sequence-Based Typing quires that the initial lymphocyte prepara-
High-resolution nucleic acid sequencing tion be manipulated before testing to yield
of HLA alleles generates allele-level se- an enriched B-cell population. This is typi-
quences that are used to characterize new cally accomplished by the use of magnetic
allele(s). With the ever-increasing avail- beads, to which monoclonal antibodies to
ability and ease of use of automated se- B cells have been bound.
quencers, sequence-based typing has be- The interpretation of serologic reactions
come a routine HLA typing method in requires skill and experience. Control wells
some HLA laboratories. of known reactivity and careful quality con-
trol of reagents are required, especially for
Serologic Assays the activity of the complement used to in-
duce lymphocytotoxicity. In addition, anti-
Lymphocytotoxicity gen assignments can be made only on the
The microlymphocytotoxicity test can be basis of results obtained with multiple anti-
used to detect HLA-A, -B, -C, -DR, and sera because few reagent antisera have suf-
-DQ antigens. Lymphocytes are used for ficient monospecific reliability to be used
testing because they are readily obtained alone. The extreme polymorphism of the
from anticoagulated peripheral blood HLA system, the variation in antigen fre-
and, unlike granulocytes, give reproduc- quencies among different racial groups, the
ible results. Lymphocytes obtained from reliance on biologic antisera and living tar-
lymph nodes or spleen may also be used. get cells, and the complexities introduced
HLA typing sera are obtained primarily by splits, CREGs, and “public” antigens all
from multiparous women. Some mouse contribute to difficulties in accurate sero-
monoclonal antisera are also available. logic HLA typing.

Antibodies in Patients The HLA System and

Microlymphocytotoxicity testing can be
used to test serum specimens against se-
Transfusion
lected target cells. This is routinely done HLA system antigens and antibodies play
in HLA crossmatching, which consists of important roles in a number of transfu-
testing serum from a potential recipient sion-related events. They include allo-
against unfractionated lymphocytes (or immunization and platelet refractoriness,
fractionated T and B lymphocytes) from a FNHTR, TRALI, and posttransfusion GVHD.
potential donor. A variation of the micro- HLA antigens are highly immunogenic. In
lymphocytotoxicity test, which uses an anti- response to pregnancy, transfusion, or
globulin reagent, is one of the methods transplantation, immunologically normal
used to increase sensitivity. Flow cyto- individuals are more likely to form anti-
metry is also used as an independent bodies to HLA antigens than to any other
method to increase the sensitivity of the antigen system.
crossmatch.
Testing the patient’s serum against a Platelet Refractoriness
panel of 30 to 60 or more different target The incidence of HLA alloimmunization
cells can assess the extent of HLA allo- and platelet refractoriness among pa-
immunization. The percent of the panel tients receiving repeated transfusions of
cells to which the recipient has formed cellular components is 20% to 71%.10 The
cytotoxic antibodies is referred to as the refractory state exists when transfusion of
panel reactive antibody (PRA) level. Deter- suitably preserved platelets fails to in-
mination of PRA can be useful in the inves- crease the recipient’s platelet count. Pla-
tigation of FNHTRs, in the workup of telet refractoriness may be due to clinical
platelet refractoriness, and in following pa- factors such as sepsis, high fever, dissemi-
tients who are awaiting cadaver solid organ nated intravascular coagulopathy, medi-
transplants. This “HLA antibody screen” not cations, hypersplenism, complement-me-
only detects the presence of HLA antibod- diated destruction, or a combination of
ies but also may allow their specificity to be these, or it may have an immune basis.
determined. The presence of HLA antibod- (See Chapter 16 for more information
ies can also be demonstrated by using an about platelets.)
enzyme-linked immunosorbent assay with
solid-phase HLA antigens or by flow cyto- Antibody Development
metric analysis using antigen-coated beads.
Antibodies against HLA antigens usually
cause immune-mediated platelet refrac-
Cellular Assays toriness, but antibodies to platelet-spe-
Historically, the mixed lymphocyte cul- cific or ABH antigens may also be in-
ture (MLC) (also called mixed leukocyte volved. HLA alloimmunization can follow
culture, mixed lymphocyte reaction, or pregnancy, transfusion, or organ trans-
MLR) was used to detect genetic differ- plantation because the foreign antigens
ences in the Class II region. In the MLR, are the donor MHC antigens themselves.
lymphocytes from different individuals A common example of this is the develop-
are cultured together and have the oppor- ment of HLA antibodies directed against
tunity to recognize foreign HLA-D region Class I antigens that occurs with transfu-
antigens and to respond by proliferating. sion of platelets, which express only Class

I antigens. The presence, in the trans- based on matching for “public” speci-
fused component, of leukocytes bearing ficities rather than cross-reactive private
Class I and II antigens elicits alloimmuni- antigens. Obtaining an adequate number
zation. The likelihood of immunization of readily available HLA-typed donors can
can be lessened with leukocyte-reduced prove difficult; it has been estimated that
blood components, or by treatment with a pool of 1000 to 3000 or more donors
ultraviolet light, which alters the co- would be needed to provide the transfu-
stimulatory molecules or impairs anti- sion requirements of most HLA-allo-
gen-presenting cell activity. The threshold immunized patients.11 Use of single anti-
level of leukocytes required to provoke a gen beads to identify HLA antibody
primary HLA alloimmune response is un- specificities precisely can allow a better
clear and probably varies among different selection of donors who have acceptable
recipients. Some studies have suggested mismatched antigens.12
that 5 × 106 leukocytes per transfusion In the past, it was recommended that
may represent an immunizing dose. In patients who were at risk of becoming
patients who have been previously sensi- alloimmunized and refractory be serologi-
tized by pregnancy or transfusion, expo- cally Class I HLA-typed early in the course
sure to even lower numbers of allogeneic of their illness, when enough lymphocytes
cells is likely to provoke an anamnestic were present in the peripheral blood to ob-
antibody response. tain a reliable HLA type. Intensive chemo-
therapy makes it very difficult to obtain
enough cells for such typing. More recently,
Finding Compatible Donors with the advent of molecular typing tech-
The HLA antibody response of transfused niques, a patient’s HLA alleles can be deter-
individuals may be directed against indi- mined using genomic DNA isolated from
vidual specificities present on donor cells very small numbers of white cells or even
or against “public” alloantigens. Precise nonblood tissue (eg, buccal swabs).
characterization may be difficult. An over- HLA-alloimmunized patients often re-
all assessment of the degree of HLA allo- spond to crossmatch-compatible platelets
immunization can be obtained by mea- selected using patient serum and samples
suring the PRA of the recipient’s serum. of apheresis platelets in a platelet antibody
Platelet-refractory patients with a high assay. Crossmatching techniques may as-
PRA are broadly alloimmunized and may sess compatibility for both HLA and plate-
be difficult to support with platelet trans- let-specific antibodies.13 These histocom-
fusions. HLA-matched platelets, obtained patible platelet components are further
by plateletpheresis, benefit some, but not discussed in Chapter 21.
all, of these refractory patients. Because
donors with a four-antigen match for an
Febrile Nonhemolytic Transfusion
immunized recipient are hard to find, strat-
Reactions
egies for obtaining HLA-matched platelets
vary. Selection of partially mismatched HLA antibodies, as well as granulocyte
donors, based on serologic cross-reactive and platelet-specific antibodies, have
groups, has been emphasized, but such been implicated in the pathogenesis of
donors may fail to provide an adequate FNHTRs. The recipient’s antibodies, re-
transfusion response in vivo. An alterna- acting with transfused antigens, elicit the
tive approach to the selection of donors is release of cytokines (eg, interleukin-1) ca-

pable of causing fever. Serologic investi- tween donor and recipient. The observa-
gation, if undertaken, may require multi- tion of posttransfusion GVHD with the use
ple techniques and target cells from a of fresh blood components from blood
number of different donors (see Chapter relatives has highlighted the role of the
27). HLA system in GVHD.
Figure 17-4 illustrates the conditions for
Transfusion-Related Acute Lung Injury increased risk of GVHD. The parents have
one HLA haplotype in common. Each child,
In TRALI, a transfusion reaction that is
therefore, has a one in four chance of inher-
being recognized with increasing fre-
iting the same haplotype from each parent,
quency, acute noncardiogenic pulmonary
and Child #1 is homozygous for the shared
edema develops in response to transfusion.
parental HLA haplotype. Transfusion of
Pathogenesis appears to reflect the pres-
blood from this person to an unrelated re-
ence of HLA antibodies in donor blood,
cipient who did not have this haplotype
which react with and fix complement to
would have no untoward consequences. If,
granulocytes of the recipient, leading to
however, Child #1 were a directed donor for
severe capillary leakage and pulmonary
the relatives heterozygous for that haplo-
edema. Rarely, HLA antibodies of the re-
type (both parents and Child #3), the recipi-
cipient react with transfused leukocytes
ent would not recognize any foreign anti-
from the donor (see Chapter 27). Cases of
gens on the transfused lymphocytes and
TRALI have been reported that appear to
would not eliminate them. The donor cells,
be caused by donor antibodies against
however, would recognize the recipient’s
Class II antigens in recipients. Because
foreign HLA antigens, would become acti-
Class II antigens are not expressed on
vated, proliferate, and attack the host. To
neutrophils, an alternate explanation for
avoid this situation, it is recommended that
activation of neutrophils in these instances
all cellular components known to be from
is required. One hypothesis is that Class II
blood relatives be irradiated before transfu-
antigens on the recipient’s pulmonary
sion. Other specially chosen donor units,
macrophages are targeted by these com-
such as HLA-matched platelets, may also
plement-activating antibodies. Subse-
present an increased risk of posttransfusion
quent release of cytokines and chemokines
GVHD. Rarely, transfusion-associated GVHD
results in the recruitment and activation
has occurred after the transfusion of blood
of neutrophils in the lungs.14
from an unrelated donor.15
Chimerism is also proposed to be re-
Chimerism and Posttransfusion sponsible for the maintenance of tolerance
Graft-vs-Host Disease in some organ transplant recipients and
16
Chimerism refers to the presence of do- for the maintenance of HLA sensitization.17
nor cells in the recipient. Persistent chi- It has been postulated that scleroderma is a
merism after blood transfusion may lead form of GVHD resulting from chimeric cells
to the development of GVHD in the recip- derived from fetal cells transferred across
ient. The development of posttransfusion the placenta during pregnancy.18
GVHD depends on several factors: the de-
gree to which the recipient is immuno-
Hemolytic Transfusion Reactions
compromised; the number and viability
of lymphocytes in the transfused compo- HLA incompatibility has rarely been im-
nent; and the degree of HLA similarity be- plicated as a cause of shortened red cell

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.
survival in patients with antibodies to HLA Hematopoietic Progenitor Cell Transplants

a b
antigens such as Bg (B7), Bg (B17), and
c
Bg (A28) that are expressed, although It has long been recognized that disparity
weakly, on red cells (see Chapter 15). Such within the HLA system represents an im-
an incompatibility may not be detected portant barrier to successful HPC trans-
with conventional pretransfusion testing. 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
HLA Testing and recipients of allogeneic HPCs, despite
immunosuppressive conditioning.
Transplantation Candidate donors and recipients are
typed for their HLA-A, -B, -C, -DR and -DQ
HLA testing is an integral part of organ alleles. The goal is to match, as closely as
transplantation. The extent of testing dif- possible, the alleles of the prospective do-
fers for different types of transplants. Or- nor and recipient at the HLA-A, -B, and
gan transplantation is discussed in great- -DRB1 loci, with the optimal match being
er detail in Chapter 26. an allele-level match.20 Some transplant

programs additionally match for HLA-C jection and delayed graft function, both of
and -DQ alleles. Molecular HLA typing is which are strong predictors of chronic re-
performed on samples from both the donor jection and long-term allograft survival.24 In
and recipient for optimal assessment of patients undergoing cadaveric kidney re-
Class I and II region compatibility. Al- transplantation, the 7-year graft survival
though HLA-identical sibling donors re- rate using the T-cell flow crossmatch to se-
main the best choice for HPC transplanta- lect the donor kidney was comparable to
tion, there is increasing use of unrelated that of patients undergoing primary cada-
donors identified by searching the file of 5 veric transplantation (68% vs 72%) and was
million HPC donors listed in the National significantly better than that of regraft pa-
Marrow Donor Program’s registry of volun- tients for whom only the antiglobulin
teer donors. The use of umbilical cord lymphocytotoxicity crossmatch was used
25
blood stem cells and hematopoietic stem (45%). Because HLA antibody responses
cell grafts that have undergone T-cell deple- are dynamic, the serum used for the cross-
tion may allow greater donor-recipient mismatch is often obtained within 48 hours of
21,22
matches. surgery and is retained in the frozen state
for any required subsequent testing. An in-
compatible crossmatch with unfractionated
Kidney and Pancreas Transplants
or T lymphocytes is a contraindication to
ABO compatibility is the most important kidney transplantation. The significance of
factor determining the immediate survival a positive B-cell crossmatch is unclear.
of kidney transplants. Because ABH anti- Serum from a patient awaiting cadaver-
gens are expressed in varying amounts on donor kidney transplant surgery is tested at
all cells of the body, transplanted ABO-in- regular intervals for the degree of alloim-
compatible tissue comes into continuous munization by determining the PRA. In ad-
contact with the recipient’s ABO antibod- dition, many laboratories identify the
ies. Of particular importance is the ex- specificities of HLA alloantibodies formed.
pression of ABH antigens on vascular en- If an antibody with a defined HLA specific-
dothelial cells because the vascular supply ity is identified in a recipient, it is a com-
in the transplant is a common site for re- mon practice to avoid the corresponding
jection. antigen when allocating a deceased donor
Both the recipient and the donor are or- allograft. The serum samples used for peri-
dinarily tested for ABO, HLA-A, -B, and -DR odic PRA testing are usually frozen. The
antigens. HLA-C and -DQ testing is also samples with the highest PRA are often
usually performed. Before surgery, a major used, in addition to the preoperative sam-
crossmatch of recipient serum against do- ple, for pretransplant crossmatching. The
nor lymphocytes is required. ASHI Stan- necessity of a prospective crossmatch for
dards for Histocompatibility Testing23 re- recipients with no evidence of HLA sensiti-
quire that the crossmatch be performed zation has been questioned. Prompt trans-
using a method more sensitive than routine plantation with reduced cold ischemia time
microlymphocytotoxicity testing, such as for the renal allograft may provide greater
prolonged incubation, washing, augmenta- benefit to the patient than prospective
tion with antihuman globulin reagents, or crossmatching, provided 1) a very sensitive
flow cytometry. Flow cytometry is the most method for antibody detection, such as
sensitive method and is especially useful flow cytometry, has been used26,27 and 2) it is
because it can best predict early acute re- absolutely certain that the patient has had

no additional sensitizing event (ie, immuni- with graft survival after heart transplanta-
zation or transfusions within 2 weeks be- tion, but prospective HLA matching has
fore or any time since that serum was been difficult to implement.31
screened).
The approach to kidney transplants us-
ing living donors is different. In the past,
when several prospective living donors
Parentage and Other
were being considered, MLC testing be- Forensic Testing
tween the recipient and the donors was HLA typing (particularly DNA-based HLA
sometimes performed, but it is rarely per- typing) has proven useful in forensic test-
formed today. HLA matching of recipients ing. In parentage testing, HLA typing
with kidney donors (both living and cada- alone can exclude about 90% of falsely ac-
veric donor) contributes to long-term allo- cused males. With the addition of red cell
graft survival by decreasing the likelihood antigen typing, the exclusion rate rises to
of chronic rejection. In 1996, the projected 95% and exceeds 99% when typing for red
20-year allograft survival rates were 57% for cell enzymes and serum proteins is in-
two-haplotype-matched sibling donors, cluded. Haplotype frequencies, rather
30% for one-haplotype-matched parental than gene frequencies, are used in these
donors, and 18% for cadaver donors.28 For calculations because linkage disequilib-
cadaver donors with no mismatches with rium is so common in the HLA system. It
the recipient for HLA-A, -B, and -DR, the is important, however, to keep in mind
projected 20-year allograft survival was the racial differences that exist in HLA
40%. Surprisingly, projected survival for haplotype frequencies; recombination
allografts from living, unrelated donors is events must also be considered.
similar to those from parental donors.29 Re- Other useful DNA-based assays for fo-
cently, the 1-year survival rates for grafts rensic testing are detection of alleles with
from living and cadaver renal donors were variable numbers of tandem repeats and al-
93.9% and 87.7% respectively, and the half- leles with variation in the number of short
lives of living-donor and cadaver-donor re- tandem repeats, which assess other poly-
nal allografts were 21.6 and 13.8 years, re- morphic, non-HLA genetic regions. DNA-
spectively.30 based assays allow identification of individ-
uals on the basis of extremely small sam-
Other Solid Organ Transplants ples of fluid or tissue, such as hairs, epithe-
lial cells, or semen.
For liver, heart, lung, and heart/lung trans-
plants, ABO compatibility remains the
primary immunologic system for donor
selection, and determining pretransplant HLA and Disease
ABO compatibility between donor and re- For some conditions, especially those be-
cipient is mandatory. HLA-A, -B, and -DR lieved to have an autoimmune etiology,
testing of potential recipients is required, an association exists between HLA phe-
and the transplant crossmatch must be notype and occurrence of clinical disease
32-34
available before transplantation when the (see Table 17-3). HLA-associated dis-
recipient has demonstrated presensitiza- eases have several features in common.
tion, except for emergency situations. They are known or suspected to be inher-
Levels of HLA compatibility do correlate ited, display a clinical course with acute

Table 17-3. HLA-Associated Diseases

32-37
Disease HLA RR
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.
exacerbations and remissions, usually have plete, often giving false-negative and
characteristics of autoimmune disorders, false-positive results. The association of
and the exact cause is unknown. Evidence HLA-B27 and ankylosing spondylitis in
has been accumulating that implicates those of European ancestry is instructive.
the HLA molecules themselves in disease The test is highly sensitive; more than 90%
susceptibility. For instance, resistance to of such patients with ankylosing spondylitis
cerebral malaria results from a strong possess the HLA-B27 antigen. On the other
cytotoxic T-cell response to particular ma- hand, specificity is low; only 20% of individ-
larial peptides that are restricted by (fit uals with the B27 antigen will develop an-
into the peptide-binding grooves of ) two kylosing spondylitis. A second condition,
specific HLA molecules.35 Another mecha- narcolepsy, is strongly associated with the
nism that could lead to the association of HLA allele DQB1*0602.37 As with the case of
HLA phenotype and disease is the pres- HLA-B27 and ankylosing spondylitis, over
ence of a Class I or II heterodimer en- 90% of individuals with narcolepsy are pos-
coded by a specific allele that preferen- itive for HLA-DQB1*0602, but only a minor-
tially presents autoantigens to the T-cell ity of individuals with this marker develop
receptor. the disease.
The ancestral haplotype A1, B8, DR3, The degree of association between a
DQ2 discussed previously (under Linkage given HLA type and a disease is often de-
Disequilibrium) is associated with suscepti- scribed in terms of relative risk (RR), which
bility to Type 1 diabetes, lupus, celiac dis- is a measure of how much more frequently
ease, common variable immunodeficiency a disease occurs in individuals with a spe-
and IgA deficiency, myasthenia gravis, and cific HLA type when compared to individu-
also with an accelerated course of HIV in- als not having that HLA type. Calculation of
fection, likely due to the presence of multi- RR is usually based on the cross-product
36
ple genes. However, HLA typing has only ratio of a 2 × 2 contingency table. However,
limited value in assessing risk for most dis- because the HLA system is so highly poly-
eases because the association is incom- morphic, there is an increased possibility of

finding an association between an HLA an- 11. Bolgiano DC, Larson EB, Slichter SJ. A model
to determine required pool size for HLA-
tigen and a disease by chance alone. There-
typed community donor apheresis programs.
fore, calculation of RR for HLA disease as- Transfusion 1989;29:306-10.
sociations is more complex and is typically 12. Pei R, Lee JH, Shih NJ, et al. Single human
done by Haldane’s modification of Woolf’s leukocyte antigen flow cytometry beads for
accurate identification of human leukocyte
formula.38-39 The RR values for some dis- antigen antibody specificities. Transplanta-
eases associated with HLA are shown in Ta- tion 2003;75:43-9.
ble 17-3. 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
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14-7. high-risk acute leukemia with T cell depleted
8. Cao K, Chopek M, Fernandez-Vina MA. High stem cells from related donors with one fully
and intermediate resolution DNA typing sys- mismatched HLA haplotype. N Engl J Med
tems for class I HLA-A, -B, -C genes by hy- 1998;339:1186-93.
bridization with sequence-specific oligo- 23. Standards for histocompatibility testing. Mt.
nucleotide probes (SSOP). Rev Immunogenet Laurel, NJ: American Society for Histo-
1999;1:177-208. compatibility and Immunogenetics, 1998.
9. Welsh K, Bunce M. Molecular typing for the 24. Utzig MJ, Blumke M, Wolff-Vorbeck G, et al.
MHC with PCR-SSP. Rev Immunogenet 1999; Flow cytometry cross-match: A method for
1:157-76. predicting graft rejection. Transplantation
10. Dzik WH. Leukoreduced blood components: 1997;63:551-4.
Laboratory and clinical aspects. In: Simon 25. Bryan CF, Baier KA, Nelson PW, et al. Long-
TL, Dzik WH, Snyder EL, et al, eds. Rossi’s term graft survival is improved in cadaveric
principles of transfusion medicine. 3rd ed. renal retransplantation by flow cytometric
Baltimore, MD: Lippincott Williams and crossmatching. Transplantation 2000;66:
Wilkins, 2002:270-87. 1827-32.

26. Taylor CJ, Smith SI, Morgan CH, et al. Selec- 36. Price P, Witt C, Allcock R, et al. The genetic
tive omission of the donor crossmatch before basis for the association of the 8.1 ancestral
renal transplantation: Efficacy, safety, and ef- haplotype (A1, B8, DR3) with multiple im-
fects of cold storage time. Transplantation munopathological diseases. Immunol Rev
2000;69:719-23. 1999;167:257-74.
27. Gebel HM, Bray RA. Sensitization and sensi- 37. Pelin Z, Guilleminault C, Risch N, et al. HLA-
tivity: Defining the unsensitized patient. DQB1*0602 homozygosity increases relative
Transplantation 2000;69:1370-4. risk for narcolepsy but not for disease sever-
28. Terasaki PI, Cho Y, Takemoto S, et al. Twenty- ity in two ethnic groups. US Modafinil in
year follow-up on the effect of HLA matching Narcolepsy Multicenter Study Group. Tissue
on kidney transplant survival and prediction Antigens 1998;51:96-100.
of future twenty-year survival. Transplant 38. Haldane JBS. The estimation and significance
Proc 1996;28:1144-5. of the logarithm of a ratio of frequencies. Ann
29. Terasaki PI, Cecka JM, Gjertson DW, Takemoto Hum Genet 1955;20:309-11.
S. High survival rates of kidney transplants
39. Woolf B. On estimating the relation between
from spousal and living unrelated donors. N
blood groups and disease. Ann Hum Genet
Engl J Med 1995;333:333-6.
1955;19:251-3.
30. Hariharan S, Johnson CP, Bresnahan BA, et al.
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- Suggested Reading
plantation. J Heart Lung Transplant 1999;18:
226-30.
ASHI Clinical Affairs Committee. Guidelines for
32. Thorsby E. Invited anniversary review: HLA
clinical histocompatibility practice. Mt. Laurel,
associated diseases. Hum Immunol 1997;53:
NJ: American Society for Histocompatibility and
1-11.
Immunogenetics, 1999.
33. Pile KS. HLA and disease associations. Pa-
thology 1999;31:202-12. Phelan DL, Mickelson EM, Noreen HS, et al. ASHI
34. Howell WM, Jones DB. The role of human laboratory manual. 4th ed. Mt. Laurel, NJ: Ameri-
leukocyte antigen genes in the development can Society for Histocompatibility and Immuno-
of malignant disease. J Clin Pathol Mol Pathol genetics, 2001.
1995;48:M302-6.
35. Hill AV. The immunogenetics of resistance to Standards for histocompatibility testing. Mt. Lau-
malaria. Proc Assoc Am Physicians 1999;111: rel, NJ: American Society for Histocompatibility
272-7. and Immunogenetics, 1998.

Chapter 18: Pretransfusion Testing
Chapter 18
Pretransfusion Testing
T
HE PURPOSE OF pretransfusion ■ Confirmation of the ABO group of
testing is to select blood compo- red cell components.
nents that will not cause harm to ■ Confirmation of the Rh type of Rh-
the recipient and will have acceptable negative red cell components.
survival when transfused. If performed ■ Selection of components of ABO
properly, pretransfusion tests will confirm group and Rh type appropriate for
ABO compatibility between the compo- the recipient.
nent and the recipient and detect most ■ Performance of a serologic or com-
clinically significant unexpected antibod- puter crossmatch.
ies. ■ Labeling of products with the recipi-
The AABB Standards for Blood Banks ent’s identifying information.
and Transfusion Services1 requires that the
following procedures be performed before
blood components are issued for transfu-
sion: Transfusion Requests
■ Positive identification of the recipi- Requests for transfusion may be submit-
ent and the recipient’s blood sample. ted electronically or on paper and must
■ ABO group and Rh typing of the re- contain sufficient information for positive
cipient’s blood. recipient identification. Standards1(p36) re-
■ Red cell antibody detection tests for quires two independent identifiers to
clinically significant antibodies us- identify the patient. These identifiers
ing the recipient’s serum or plasma. could be the patient’s first and last names,
■ Comparison of current findings on an identification number unique to that 18
the recipient’s sample with records individual, a birth date, or other identify-
of previous results. ing system. Other information necessary
407

to process the request includes identifica- tems vary in design: color-coded numbers
tion of the component needed, the quan- on wristbands, tubes, and units; a wristband
tity, any special requests such as irradia- with an embosser for label printing; and a sys-
tion, gender and age of the recipient (42 tem for barcoding that provides positive sam-
CFR 493.1241), and the name of the re- ple and patient identification.
sponsible physician. The diagnosis and Hospital policies generally require phle-
the recipient’s history of transfusion and botomists to collect blood specimens only
pregnancy may be helpful in problem- from patients who have an attached patient
solving. Each facility should have a writ- identification wristband. However, in some
ten policy defining request acceptance circumstances, it may not be possible for
criteria. Blood requests that lack the re- the patient to wear an identification wrist-
quired information, are inaccurate, or are band, and an alternative means of positive
illegible should not be accepted.1(p36) Tele- patient identification may be needed. The
phoned requests are acceptable in urgent use of wristbands is difficult when the pa-
situations but should be documented, for tient has total body burns, when the patient
example, in a telephone log; a subsequent is an extremely premature infant, or when
request as a written authorization is to be the wristband is inaccessible during sur-
made within 30 days.2 gery. Some facilities allow identifying infor-
mation to be placed on a patient’s ankle or
forehead. Intraoperative patient identifica-
Patient Identification
tion procedures may allow the use of an al-
Collection of a properly labeled blood ternative identification process in lieu of an
sample from the intended recipient is inaccessible wristband. It is important to
critical to safe blood transfusion. Most remind clinical personnel that transfusions
hemolytic transfusion reactions result should not be administered to a patient
from errors in sample or patient identifi- who lacks positive identification.
cation.3,4 The person drawing the blood When the patient’s identity is unknown,
sample must identify the intended recipi- an emergency identification method may
ent in a positive manner. Each facility be used. This patient identification must be
must develop and implement policies and attached to the patient and affixed or repro-
procedures for patient identification and duced on blood samples. This identifica-
specimen collection. tion must be cross-referenced with the pa-
Most hospitals identify patients with an tient’s name and hospital identification
identification wristband. Ideally, this wrist- number or code when they become known.
band is placed on the patient before speci- When hospitals allow the use of confiden-
men collection and remains on the patient tial or alias names, the facility must have
until discharge. The same identifying infor- policies and procedures that govern their
mation on the specimen tube submitted for use.
testing will be used to label blood compo- Outpatients may be identified with the
nents and this information will be com- use of a patient wristband for the purpose
pared against the patient’s wristband at the of blood sample collection. Alternative
time of transfusion. Some hospitals use an methods of positive patient identification
internally generated or commercially avail- include a driver’s license or other photo-
able identification band with a substitute or graphic identification. Whenever possible,
additional “blood bank number” as the the patient should be asked to state his or
unique patient identifier. Commercial sys- her name and to provide confirmation of

Chapter 18: Pretransfusion Testing 409
birth date and address. If a discrepancy is cation may be placed on the label of the
noted, the sample must not be collected tube, placed on the requisition, or docu-
until the patient’s identity has been clari- mented in a computer system.
fied.
The identification of specimens used for Confirming Sample Identity in the
preadmission testing must meet the same Laboratory
requirements as those used for inpatient
When a sample is received in the labora-
transfusion—there must be no doubt about
tory, a trained member of the staff must
the identity of the specimen and the pa-
confirm that the information on the label
tient. With the advent of patient admission
and on the transfusion request is identi-
on the same day as surgery, hospitals have
cal. If there is any doubt about the iden-
devised several mechanisms to identify pa-
tity of the patient, a new sample must be
tients when specimens have been collected 1(p37)
obtained. It is unacceptable for any-
several days or weeks before surgery. One
one to correct identifying information on
option is to require that the patient wear an
an incorrectly labeled sample. Each labo-
identification wristband. Other facilities use
ratory should establish policies and pro-
a unique number on specimens and on a
cedures that define identifying information
patient identification form that the patient
and describe how to document receipt of
must provide on the day of surgery in order
mislabeled specimens.
for the preadmission specimen to be valid
for transfusion.5 An alternative procedure is
to place on the patient’s medical record the
wristband used during specimen collec-
tion. This wristband would be attached to
Blood Sample
the patient upon arrival on the day of sur- Pretransfusion testing may be performed
gery after proper identification of the pa- on either serum or plasma. Plasma may be
tient.6 the preferred specimen for some methods
Regardless of the system used, it must be such as tests using gel technology. Incom-
well known to all those who collect blood pletely clotted blood samples may cause
specimens and be followed routinely. small fibrin clots that trap red cells into
Ideally, patient identification procedures aggregates that could resemble aggluti-
should be used as a matter of course to nates. Plasma collected from patients with
identify patients for all treatment methods, high levels of fibrinogen or patients with
not solely transfusions. dysproteinemia may demonstrate rouleaux.
Rouleaux formation can be mistaken for
agglutination. Clotting may be incomplete
Sample Labeling
in specimens not intended to be anti-
Before leaving the patient, the phleboto- coagulated, such as patients who have been
mist must label the blood sample tubes treated with heparin. Adding thrombin or
with two independent patient identifiers protamine sulphate to the sample usually
and the date of collection. Either hand- corrects the problem.
written or imprinted labels may be used It is permissible to collect blood from an
as long as the information on the label is infusion line. To avoid interference from re-
identical to that on the wristband and sidual intravenous fluid, the tubing should
request. There must be a mechanism to be flushed with saline, and 5 mL or a vol-
identify the phlebotomist1(p37); this certifi- ume of blood approximately twice the fluid

volume in the line should be withdrawn (day 3). The 3-day requirement applies
and discarded before sample collection.7 only to patients who have been transfused
or pregnant within the last 3 months, but
Appearance of Sample many laboratories prefer to standardize
their operations by setting a 3-day limit
The appearance of the serum or plasma
on all specimens used for pretransfusion
may create difficulties in detecting antibody-
testing.
induced hemolysis. Whenever possible, a
Each institution should have a policy that
hemolyzed sample should be replaced with
defines the length of time samples may be
a new specimen. Test results observed with
used. Testing of stored specimens should
lipemic serum can be difficult to evaluate.
be based on the specimen storage limita-
On occasion, it may be necessary to use
tions in the reagent manufacturer’s infor-
hemoglobin-tinged or lipemic serum or
mation circulars. Lack of appropriate stor-
plasma. If hemolyzed samples are used, it
age space may also limit the length of time
should be noted in the patient testing re-
specimens are stored.
cords to differentiate hemolysis as a result
of an antigen-antibody reaction. Each in-
Retaining and Storing Blood Samples
stitution should have a procedure describ-
ing the indications for using hemolyzed The recipient’s blood specimen and a
and lipemic specimens. sample of the donor’s red cells must be
stored at refrigerator temperature for at
Age of Sample least 7 days after each transfusion.1(p37) Do-
nor red cells may be from the remainder
Blood samples intended for use in cross- of the segment used in the crossmatch or
matching should be collected no more a segment removed before issuing the
than 3 days before the intended transfu- blood. If the opened crossmatch segment
sion unless the patient has not been preg- is saved, it should be placed in a tube la-
nant or transfused within the preceding 3 beled with the unit number and sealed or
months. If the patient’s transfusion or stoppered. Keeping the patient’s and do-
pregnancy history is uncertain or unavail- nor’s samples allows repeat or additional
able, compatibility tests must be per- testing if the patient experiences adverse
formed on blood samples collected within effects.
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 Serologic Testing
pregnancy may stimulate production of The patient’s ABO group and Rh type must
unexpected antibodies. Because it is not be determined in order to transfuse ABO-
possible to predict whether or when such and Rh-compatible components. Stan-
antibodies will appear, a 3-day limit has dards1(pp37,38) requires that the red cells of
been selected as an arbitrary interval ex- the intended recipient be typed for ABO
pected to be both practical and safe. It is and Rh and the serum/plasma be tested
short enough to reflect acute changes in for expected and unexpected antibodies
immunologic status but long enough to before components containing red cells
allow the results of preadmission testing are issued for transfusion. There should
completed on Friday (day 0) to be used be written procedures for exceptions dur-
for surgical cases performed on Monday ing emergencies. When only plasma and

platelets or Cryoprecipitated AHF are be- harm to recipients with the weak D pheno-
ing infused, historical testing information type. Omitting the test for weak D prevents
in the patient’s record may be used. Refer misinterpretations arising from the presence
to Table 18-1 for selection of the ABO types of a positive direct antiglobulin test (DAT).
for red cell and plasma transfusion when However, some transfusion services test pa-
ABO-identical products are not available. tient pretransfusion specimens for weak D
in order to identify patients who could be
ABO Grouping and Rh Typing of the given Rh-positive blood components, thus
Recipient reserving the Rh-negative components for
To determine the ABO group of the recipi- patients who are D–. Routine testing for
ent, red cells must be tested with anti-A other Rh antigens is not required.
and anti-B, and the serum or plasma with
A1 and B red cells. The techniques used Detecting Unexpected Antibodies to Red
and interpretation of the results are de- Cell Antigens
scribed in Chapter 13. Any discrepant re- Before deciding upon routine procedures
sults should be resolved before blood is for antibody detection, the blood bank di-
given. If transfusion is necessary before rector must approve which antibodies are
resolution, the patient should receive group considered potentially clinically signifi-
O red cells. cant. In general, an antibody is consid-
The patient’s red cells must be tested ered potentially clinically significant if an-
with anti-D, with suitable observations or tibodies of that specificity have been
controls to avoid a false-positive interpreta- associated with hemolytic disease of the
tion. Chapter 14 contains a more extensive fetus and newborn, a hemolytic transfusion
discussion of Rh typing reagents, appropri- reaction, or notably decreased survival of
ate control techniques, and weak D types. If transfused red cells. Antibodies reactive at
problems in D typing arise, the patient 37 C and/or in the antiglobulin test are
should be given Rh-negative blood until the more likely to be clinically significant
problem has been resolved. Testing a recipi- than cold-reactive antibodies.8
ent’s red cells for weak D is not necessary Numerous serologic techniques have
because giving Rh-negative cells causes no been developed that are suitable for detec-
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 oratory should use the same interpreta-
12 and Chapter 19). Goals in providing tions and notations and be consistent in
compatible blood for a recipient are to: grading (eg, 0-4+) reactions. Some labora-
■ Detect as many clinically significant tories prefer to use a numeric scoring (eg,
antibodies as possible. 0-12) system to indicate reaction strength.
■ Detect as few clinically insignificant Refer to Method 1.8 for grading and scor-
antibodies as possible. ing. An optical aid such as a concave mir-
■ Complete the procedure in a timely ror enhances visualization in reading tube
manner. tests. Microscopic observation is not rou-
Standards1(p38) requires that tests for unex- tinely recommended in manufacturer’s in-
pected antibodies use unpooled reagent serts for enhancement media. A micro-
red cells in a method that detects clinically scope can be useful in distinguishing
significant antibodies and includes an anti- rouleaux from true agglutination. Micro-
globulin test preceded by incubation at 37 C. scopic reading may also allow for the
Each negative antiglobulin test must be detection of specific patterns of aggluti-
followed by a control system of IgG-sensi- nation that are characteristic of some an-
a
tized cells (check cells). If alternative proce- tibodies. For example, anti-Sd typically
dures are used, there must be documenta- produces small refractile agglutinates in a
tion of equivalent sensitivity, and the sea of free red cells, giving the appearance
manufacturer’s specified controls must be of a mixed-field appearance. If gel or solid
used. phase is used, the manufacturer’s direc-
The method chosen should have suffi- tions must be followed for reading and in-
cient sensitivity to detect very low levels of terpreting positive and negative reactions.
antibody in a recipient’s serum. Transfusion
of antigen-incompatible red cells to a recip-
ient with a weakly reactive antibody may Autologous Control
result in rapid anamnestic production of An autologous control or DAT is not re-
antibody, with subsequent red cell destruc- quired as a part of pretransfusion testing.
tion. The same antibody detection proce- It is of limited value, even for patients
dure may be used for all categories of spec- who have recently been transfused, and
imens, including pretransfusion and prenatal should be used only when antibody iden-
tests on patients and screening of donor tification is required.
blood. Once a procedure has been adopted,
the method must be described in the facil-
Practical Considerations
ity’s standard operating procedures manual.
Antibody detection tests may be performed
in advance of, or together with, a cross-
Reading and Interpreting Reactions
match between the patient’s serum/plasma
In serologic testing, the hemolysis or ag- and donor red cells. Performing antibody
glutination that constitutes the visible end- detection tests before crossmatching per-
point of a red cell antigen-antibody inter- mits early recognition and identification
action must be observed accurately and of clinically significant antibodies. This
consistently. The strength of agglutination allows for the selection of the appropriate
or degree of hemolysis observed with each crossmatch procedure and acquisition of
cell sample should be recorded immedi- units with special antigen blood types (eg,
ately after reading. All personnel in a lab- e– units).

Comparison with Previous Records unexpected antibody if the patient’s anti-

body detection test is negative.10,11
Results of ABO and Rh tests on a current
The potential benefits of omitting a rou-
specimen must be compared with previ-
tine antiglobulin crossmatch include de-
ous transfusion service records if there has
creased turnaround time, decreased work-
been prior testing during the past 12
load, reduced reagent costs, and more effective
months, and the comparison must be doc-
use of blood inventory. Omitting the anti-
umented.1(p39) Errors in identification and/
globulin phase of the crossmatch for patients
or testing may be detected when discrep-
who meet the criteria must be in approved
ancies are found between previous and
written standard operating procedures. The
current ABO and Rh results.
methods used for serologic crossmatching
Records are also to be reviewed for the
may be the same as those used for red cell
presence of clinically significant red cell an-
antibody detection or identification or they
tibodies, for difficulties in testing, for the
may be different. For example, gel methods
occurrence of significant adverse reactions,
may be used for both antibody detection
and for special transfusion requirements.1(p39)
and identification, but the crossmatch may
Clinically significant red cell alloantibodies
be performed using a tube test.
may become undetectable in a recipient’s
serum over time. Between 30% and 35% of
antibodies become undetectable within 1 Repeat Testing of Donor Blood
year and nearly 50% become undetectable A serologic test to confirm the ABO group
after 10 or more years.9 of all RBC units and the Rh type of RBC
units labeled as Rh-negative must be per-
formed before transfusion. Confirmatory
Crossmatching Tests testing for weak D is not required. This
confirmatory testing is to be performed
Unless there is an urgent need for blood, a
on a sample obtained from an attached
crossmatch must be performed for red
segment. Any discrepancies are to be re-
cell transfusion. The crossmatch shall use
solved before the unit is issued for trans-
procedures to demonstrate ABO incom-
fusion.1(p37)
patibility and clinically significant anti-
bodies to red cell antigens. Blood lacking
the relevant antigens is to be selected for Suggested Procedures for Routine
transfusion when a patient has clinically Crossmatching
significant antibody identified currently Red cells used for crossmatching must be
or historically, even though the antibody obtained from a segment of tubing origi-
is presently nonreactive.1(pp39,40) The cross- nally attached to the blood container. For
match shall include the antiglobulin test. routine tube testing, the cells may be
When no clinically significant antibodies washed and resuspended to 2% to 5% in
are detected in current antibody screen- saline. Washing the donor’s red cells re-
ing tests and there is no record of previ- moves small fibrin clots and some cold
1(p40)
ous detection of such antibodies, then agglutinins that may interfere with inter-
only a method to detect ABO incompati- pretation of results. Because the ratio of
bility, such as an immediate-spin or com- serum to cells markedly affects the sensi-
puter crossmatch, is required. It is very tivity of agglutination tests, it is important
rare for the antiglobulin phase of the to stay within the 2% to 5% cell suspen-
crossmatch to detect a clinically significant sion range or as specified by the manufac-

turer’s instructions. For example, if too many compatible blood can be safely released
red cells are present, weak antibodies may after an immediate-spin or computer
be missed because too few antibody mol- crossmatch, if the antibody screen is neg-
ecules bind to each cell. Many laborato- ative and there is no history of clinically
rians find that a 2% to 3% concentration significant antibodies. However, if the anti-
yields the best results. For tests using col- body screen is positive, the antibody(ies)
umn (gel) or solid-phase microplate sys- must be identified and antigen-negative
tems, follow the manufacturer’s directions. units for the clinically significant antibod-
The simplest serologic crossmatch method ies identified must be available for use if
is the immediate-spin saline technique, in needed.
which serum is mixed with saline-sus-
pended red cells at room temperature and
the tube is centrifuged immediately. The Routine Surgical Blood Orders
immediate-spin crossmatch method is de- Blood ordering levels for common elective
signed to detect ABO incompatibilities be- procedures can be developed from previ-
tween donor red cells and recipient serum. ous records of blood use. Because surgical
It can be used as the sole crossmatch requirements vary among institutions,
method only if the patient has no present routine blood orders should be based on
or previous clinically significant antibodies. local transfusion utilization patterns. The
Because the testing is performed at room surgeons, anesthesiologists, and the med-
temperature, antibodies such as anti-M, -N, ical director of the blood bank should
and -P1 may be detected that were not ob- agree on the number of units required for
served if antibody detection tests omitted each procedure. See Chapter 3 for a more
room-temperature testing. A sample imme- detailed discussion of blood ordering pro-
diate-spin crossmatch technique is de- tocols. Routine blood order schedules are
scribed in Method 3.1. An antiglobulin successful only when there is cooperation
crossmatch procedure that meets the re- and confidence among the professionals
quirements of Standards for all routine situ- involved in setting and using the guidelines.
ations is described in Method 3.2. Once a surgical blood ordering schedule
has been established, the transfusion ser-
vice routinely crossmatches the predeter-
Type and Screen (Antibody Detection Test) mined number of units for each patient un-
Type and screen is a policy in which the dergoing the designated procedures. Routine
patient’s blood sample is tested for ABO, orders may need to be modified for pa-
Rh, and unexpected antibodies, then stored tients with anemia, bleeding disorders, or
in the transfusion service for future cross- other conditions in which increased blood
match if a unit is needed for transfusion. use is anticipated. As with other circum-
Crossmatched blood is not labeled and stances that require rapid availability of
reserved for patients undergoing surgical blood, the transfusion service staff must be
prepared to provide additional blood if the
procedures that rarely require transfu-
need arises.
sion. The blood bank must have enough
donor blood available to meet unexpected
needs of patients undergoing operations Computer Crossmatch
under a type and screen policy. If transfu- When no clinically significant antibodies
sion becomes necessary, ABO- and Rh- have been detected by antibody screening

and history review, it is permissible to omit Compatibility Testing for Neonates Less
the antiglobulin phase of the crossmatch than 4 Months of Age
and perform only a procedure to detect
Requirements for compatibility testing for
ABO incompatibility. Computerized match-
neonates less than 4 months of age are
ing of blood can be used to fulfill the re-
discussed in Chapter 24. An initial pre-
quirement, provided that the following
1(pp40,41)
transfusion specimen must be obtained
conditions have been met :
from the infant to determine ABO and Rh
■ The computer system has been vali-
type. For ABO typing, testing the cells
dated, on site, to ensure that only
with anti-A and anti-B is the only test re-
ABO-compatible whole blood or red
quired. Serum or plasma from either the
cells have been selected for transfu-
infant or the mother may be used to de-
sion.
tect unexpected red cell antibodies and
■ Two determinations of the recipi-
for crossmatching. The infant’s serum
ent’s ABO group as specified in Stan-
need not be tested for ABO antibodies un-
dards are made, one on a current
less a non-group-O infant is to receive
specimen and a second one by one
non-group-O cells that are incompatible
of the following methods: by retest-
with passively acquired anti-A or anti-B.
ing the same sample, by testing a
This antibody most often comes from the
second current sample, or by com-
mother but can be present from group O
parison with previous records.
RBCs or plasma from incompatible plate-
■ The computer system contains the
lets. Test methods in this case are to in-
unit number, component name,
clude an antiglobulin phase using either
ABO group, and Rh type of the com-
donor or reagent A1 or B red cells. If no
ponent; the confirmed donor unit
clinically significant unexpected antibod-
ABO group; two unique recipient
ies are present, it is unnecessary to cross-
identifiers; and the recipient’s ABO
match donor red cells for the initial or
group, Rh type, and antibody screen
subsequent transfusions. Repeat testing
results.
may be omitted for infants less than 4
■ A method exists to verify correct en-
months of age during any one hospital
try of data before the release of
admission as long as they are receiving
blood components.
only group O cells.1(p42)
■ 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- Interpretation of Antibody
tion of the blood group confirmatory Screening and Crossmatch
test, and to ABO incompatibility be-
tween recipient and donor unit. Results
Butch et al12,13 and Safwenberg et al14 have Most samples tested have a negative anti-
described in detail a model computer cross- body screen and are crossmatch-compati-
match system. Potential advantages of a ble with units selected. A negative anti-
computer crossmatch include decreased body screen, however, does not guarantee
workload, reduced sample volume required that the serum does not have clinically
for testing, reduced exposure of personnel significant red cell antibodies, only that it
to blood specimens, and better use of blood contains no antibodies that react with the
inventory. screening cells by the techniques employed.

Furthermore, a compatible crossmatch ■ A final check of records maintained

does not guarantee normal red cell sur- in the blood bank for each unit of
vival. blood or component must include:
Table 18-2 reviews the possible causes of 1. Two independent identifiers,
positive pretransfusion tests. Most of what one of which is usually the pa-
is known about red cell antigen and anti- tient’s name.
body reactions comes from work per- 2. The recipient’s ABO group and
formed in tube testing. This information is Rh type.
not necessarily applicable to antibody de- 3. The donor unit or pool identifi-
tection tests using other technologies such cation number.
as solid-phase microtiter plates or column 4. Donor’s ABO and, if required,
technologies. Spurious or unexpected reac- Rh type.
tions in these other technologies may have 5. The interpretation of the cross-
the same or other causes than those seen in match tests (if performed).
the tube tests. Depending on the antigen/ 6. The date and time of issue.
antibody reaction strength and the testing 7. Special transfusion requirements
conditions, not all of the scenarios will (eg, cytomegalovirus-reduced-
result in positive tests. risk, irradiated, or antigen-neg-
The cause of the serologic problems ative).
should be identified before transfusion. ■ There must be a process to confirm
Chapter 19 reviews techniques for problem that the identifying information, the
resolution. If the patient is found to have request, the records, and the blood
clinically significant antibodies, units is- or component are in agreement and
sued for transfusion should be nonreactive that discrepancies have been re-
for such antigens when tested with licensed solved before issue.
reagents (see Selection of Units, Other Additional records that may be of use in-
Blood Groups). When the antibody identi- clude those that identify the person issuing
fied is considered clinically insignificant the blood and the person to whom the
(eg, anti-A1, -M, -N, -P1, -Lea, and/or -Leb) blood was issued or the destination of the
15
and is not reactive at 37 C, random units unit.
may be selected for crossmatch. 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-
Labeling and Release of per form. Records must contain the identi-
Crossmatched Blood at the fication of the person(s) performing the test
and, if blood is issued before the resolution
Time of Issue of compatibility problems, the final status
1(p44)
Standards requires that the following of the serologic findings.
activities take place at the time of issue: There should be a system to ensure that
■ A tag or label indicating the recipi- the proper blood component is issued for
ent’s two independent identifiers, the intended patient. Before issuing a unit
donor unit number, and compatibil- of blood or component, personnel must
ity test interpretation, if performed, ensure that the product is acceptable for
must be attached securely to the use, the unit is checked to make sure it
blood container. does not have an abnormal color or ap-

Table 18-2. Causes of Positive Pretransfusion Tests*
Negative Antibody Screen, Incompatible Immediate-Spin Crossmatch

■ Donor red cells are ABO-incompatible, caused by error in selecting donor unit, patient
specimen, or labeling the donor unit.
■ Donor red cells are ABO-incompatible, caused by failure to detect weak expressions of antigens.
■ Donor red cells are polyagglutinable.
■ Anti-A1 in the serum of an A2 or A2B individual.
■ Other alloantibodies are reactive at room temperature (eg, anti-M).
■ Rouleaux formation.
■ Cold autoantibodies (anti-I), especially if immediate spin is not tested on antibody detection
screen.
Negative Antibody Screen, Incompatible Antiglobulin Crossmatch

■ Donor red cells have a positive direct antiglobulin test.
■ Antibody reacts only with red cells having strong expression of a particular antigen either
because of dosage effect (eg, Rh, Kidd, Duffy, and MN antigens) or because of intrinsic variation
in antigen strength (eg, P1).
■ Antibody reacts with a low-incidence antigen present on the donor red cells.
■ Passively transferred antibody is present—significant levels of circulating anti-A or -B may
be present after infusion of ABO-incompatible platelets to a recipient.
Positive Antibody Screen, Compatible Crossmatches

■ Auto-anti-IH (-H).
bH
■ Anti-Le .
■ Antibodies are dependent on reagent cell diluent.
■ Antibodies demonstrating dosage effect and red cells of the unit are from heterozygotes
(ie, express a single dose of antigen).
Positive Antibody Screen, Incompatible Crossmatches, Negative Auto Control

■ Alloantibody(ies).
■ Unexpected interactions with reagent red cells.
Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control, Negative

Direct Antiglobulin Test
■ Antibody to ingredient in enhancement media.
Positive Antibody Screen, Incompatible Crossmatches, Positive Auto Control

■ Alloantibody is present and patient is experiencing either a delayed serologic or hemolytic
transfusion reaction.
■ Passively transferred alloantibody from a derivative reactive with the recipient’s cells (eg,
intravenous immune globulin).
■ Cold-reactive autoantibody.
■ Warm-reactive autoantibody.
■ Rouleaux formation.
■ Reagent-related problems.
*Causes are dependent upon serologic methods used.

pearance, the container is not leaking, and red cells transfused, it may be desirable to
the product is not outdated. administer Rh Immune Globulin to a D–
Final identification of the recipient and patient given Rh-positive blood.16
the blood container rests with the trans-
fusionist, who must identify the patient and Other Blood Groups
donor unit and certify that identifying in-
formation on forms, tags, and labels is in Antigens other than ABO and D are not
agreement (see Chapter 22). 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-
Selection of Units body is weakly reactive or no longer de-
monstrable, a licensed reagent should be
ABO Compatibility used to confirm that the donor units are
Whenever possible, patients should re- antigen negative. If there is an adequate
ceive ABO-identical blood; however, it quantity of the patient’s serum, or if an-
may be necessary to make alternative se- other patient’s serum with the same anti-
lections. If the component to be trans- body specificity is available, and that anti-
fused contains 2 mL or more of red cells, body reacts well with antigen-positive red
the donor’s red cells must be ABO-com- cells, that serum may be used to screen
patible with the recipient’s plasma.1(p40) Be- for antigen-negative units. Those units
cause plasma-containing components found to be antigen negative must be
can affect the recipient’s red cells, the confirmed with a licensed reagent, when
ABO antibodies in transfused plasma available. When licensed reagents are not
should be compatible with the recipient’s available (eg, anti-Lan or anti-Yta), expired
red cells when feasible. Requirements for reagents or stored serum specimens from
components and acceptable alternative patients or donors can be used, provided
choices are summarized in Table 18-1. that controls tested on the day of use are
acceptable (see the Food and Drug Ad-
ministration Compliance Program Guid-
Rh Type ance Manual, Chapter 42, Blood and Blood
Rh-positive blood components should Products). When crossmatch-compatible
routinely be selected for D+ recipients. units cannot be found, the medical direc-
Rh-negative units will be compatible but tor should be involved in the decision to
should be reserved for D– recipients. D– transfuse the patient. (See Chapter 19 and
patients should receive red-cell-contain- Chapter 20 for additional information on
ing components that are Rh-negative to issuing crossmatch-incompatible units.)
avoid immunization to the D antigen. Oc- Antigen-negative units are not usually
casionally, ABO-compatible Rh-negative provided for the patient who has antibod-
components may not be available for D– ies that are not clinically significant.
recipients. In this situation, the blood Sometimes, problems associated with
bank physician and the patient’s physi- crossmatching units for patients with
cian should weigh alternative courses of these antibodies may be avoided by alter-
action. Depending on the childbearing ing the serologic technique used for the
potential of the patient and the volume of crossmatch.

Blood Administered in Urgent Situations patibility testing was not complete

at the time of issue.
When blood is urgently needed, the pa-
3. Begin compatibility tests and com-
tient’s physician must weigh the risk of
plete them promptly (for massive
transfusing uncrossmatched or partially
transfusion, see below). If incompat-
crossmatched blood against the risk of
ibility is detected at any stage of test-
delaying transfusion until compatibility
ing, the patient’s physician and the
testing is complete. Ideally, a transfusion
transfusion service physician should
service physician should provide consul-
be notified immediately.
tation. The risk that the transfused unit
might be incompatible may be judged to
be less than the risk of depriving the pa- Massive Transfusion
tient of oxygen-carrying capacity of that Massive transfusion is defined as infusion,
transfusion. See Chapter 21. within a 24-hour period, of a volume of
blood approximating the recipient’s total
Required Procedures blood volume. Exchange transfusion of an
infant is considered a massive transfusion.
When blood is released before pretrans-
Following massive transfusion, the pre-
fusion testing is complete, the records
transfusion sample no longer represents
must contain a signed statement of the
the blood currently in the patient’s circula-
requesting physician indicating that the
tion and its use for crossmatching has lim-
clinical situation was sufficiently urgent
ited benefit. It is only important to confirm
to require release of blood. 1(p46) Such a
ABO compatibility of units administered
statement does not absolve blood bank
subsequently. The blood bank director may
personnel from their responsibility to is-
implement a more limited pretransfusion
sue properly labeled donor blood that is
testing protocol to be used in these situa-
ABO-compatible with the patient. When
tions. This protocol should be in writing to
urgent release is requested, blood bank
ensure consistent application by all labora-
personnel should:
tory personnel.
1. Issue uncrossmatched blood, which
should be:
a. Group O Red Blood Cells if the Blood Administered After
patient’s ABO group is unknown. Non-Group-Specific Transfusion
It is preferable to give Rh-nega- Transfusion services sometimes release
tive blood if the recipient’s Rh units for transfusion during emergencies
type is unknown, especially if the before they receive a sample for blood
patient is female with the po- typing. When it arrives, the sample is a
tential to bear children. pretransfusion specimen. In most cases,
b. ABO and Rh compatible, if there the sample is tested and units of that ABO
has been time to test a current group are issued for transfusion without
specimen. Previous records must concern for anti-A and/or anti-B remain-
not be used, nor should infor- ing from the initial emergency-release
mation be taken from other re- units. Because most donor units are RBCs
cords such as cards, identifica- with comparatively little supernatant
tion tags, or driver’s license. plasma, or RBCs (Additive Solution Added)
2. Indicate in a conspicuous fashion on with even less residual plasma, the risks
the attached tag or label that com- involved in following this practice are

minimal. For example, the patient may 6. AuBuchon JP. Blood transfusion options: Im-
proving outcomes and reducing costs. Arch
exhibit a transient positive DAT.
Pathol Lab Med 1997;121:40-7.
In some cases, when large volumes of 7. Procedures for the collection of diagnostic
red cells are transfused, or when young blood specimens by venipuncture. 3rd ed.
children or infants receive transfusions, NCCLS document H3-A2, approved standard.
Villanova, PA: National Committee for Clini-
passively acquired ABO antibodies may be cal Laboratory Standards, 1991.
detected,17 and it may be appropriate to 8. Issitt PD, Anstee DJ. Applied blood group se-
demonstrate compatibility of red cells of rology. Durham, NC: Montgomery Scientific
the patient’s original ABO group with a Publications, 1998:873-905.
9. Ramsey G, Smietana SJ. Long term follow-up
freshly drawn serum specimen. If the cross- testing of red cell alloantibodies. Transfusion
match is incompatible because of ABO an- 1994;34:122-4.
tibodies, transfusion with red cells of the al- 10. Oberman HA. The present and future cross-
ternative group should be continued. match. Transfusion 1992;32:794-5.
11. Meyer EA, Shulman IA. The sensitivity and
If the change in blood type involves only specificity of the immediate-spin crossmatch.
the Rh system, return to type-specific blood Transfusion 1989;29:99-102.
is simple because antibodies are unlikely to 12. Butch SH, Judd WJ, Steiner EA, et al. Elec-
be present in the plasma of either the recip- tronic verification of donor-recipient com-
patibility: The computer crossmatch. Trans-
ient or the donor. If a patient has received fusion 1994;34:105-9.
blood of an Rh type other than his or her 13. Butch SH, Judd WJ. Requirements for the
own before a specimen has been collected computer crossmatch (letter). Transfusion 1994;
34:187.
for testing, it may be difficult to determine
14. Safwenberg J, Högman CF, Cassemar B. Com-
the correct Rh type. If there is any question puterized delivery control a useful and safe
about the recipient’s D type, Rh-negative complement to the type and screen compati-
blood should be transfused if possible. The bility testing. Vox Sang 1997;72:162-8.
use of Rh Immune Globulin prophylaxis 15. Shulman IA, Petz LD. Red cell compatibility
testing: Clinical significance and laboratory
should be considered when Rh-positive methods. In: Petz LD, Swisher SN, Kleinman
components are transfused to Rh-negative S, eds. Clinical practice of transfusion medi-
patients. See Chapter 21. cine. 3rd ed. New York: Churchill Livingstone,
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.
References 17. Garratty G. Problems associated with pas-
sively transfused blood group alloantibodies.
1. Silva MA, ed. Standards for blood banks and Am J Clin Pathol 1998;109:169-77.
AABB, 2005.
Printing Office, 2004 (revised annually). Suggested Reading
3. Sazama K. Reports of 355 transfusion associ-
ated deaths: 1976 through 1985. Transfusion Beck ML, Tilzer LL. Red cell compatibility testing:
1990;30:583-90. A perspective for the future. Transfus Med Rev 1996;
4. Linden JV, Wagner K, Voytovich AE, Sheehan 10:118-30.
J. Transfusion errors in New York State: An
Brecher ME. Collected questions and answers. 7th
analysis of 10 years’ experience. Transfusion
ed. Bethesda, MD: AABB, 2001:49-56.
2000;40:1207-13.
5. Butch SH, Stoe M, Judd WJ. Solving the same- Butch SH for the Scientific Section Coordinating
day admission identification problem (ab- Committee. Guidelines for implementing an elec-
stract). Transfusion 1994;34(Suppl):93S. tronic crossmatch. Bethesda, MD: AABB, 2003.

Butch SH, Oberman HA. The computer or elec- of erroneous blood grouping of blood bank speci-
tronic crossmatch. Transfus Med Rev 1997;11:256- mens. Transfusion 1997;37:1169-72.
64.
Padget BJ, Hannon JL. Variations in pretransfusion
Frohn C, Dumbgen L, Brand JM, et al. Probability practices. Immunohematology 2003;19:1-6.
of anti-D development in D– patients receiving D+
Rossmann SN for the Scientific Section Coordinat-
RBCs. Transfusion 2003;43:893-8.
ing Committee. Guidelines for the labeling of spec-
Garratty G. How concerned should we be about imens for compatibility testing. Bethesda, MD:
missing antibodies to low-incidence antigens? AABB, 2002.
(editorial) Transfusion 2003;43:844-7.
Schonewille H, van Zijl AM, Wijermans PW. Im-
Issitt PD. From kill to overkill: 100 years of (per- portance of antibodies against low-incidence RBC
haps too much) progress. Immunohematology 2000; antigens in complete and abbreviated cross-
16:18-25. matching. Transfusion 2003;43:939-44.
Lumadue JA, Biyd JS, Ness PM. Adherence to a strict

specimen-labeling policy decreases the incidence

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens
Chapter 19
19
Initial Detection and

Identification of
Alloantibodies to Red Cell
Antigens
R
ED CELL ALLOANTIBODIES other or passenger lymphocytes in transplanted
than naturally occurring anti-A or organs or hematopoietic progenitor cells.
-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- Significance of
bodies can be found in 0.3% to 38% of the Alloantibodies
population.1,2 Alloantibodies react only with Alloantibodies to red cell antigens may be
allogeneic red cells, whereas red cell auto- initially detected in any test that uses se-
antibodies react with the red cells of the rum or plasma (eg, ABO test, antibody de-
antibody producer. Immunization to red tection test, crossmatch) or in an eluate
cell antigens may result from pregnancy, prepared from red cells coated with allo-
transfusion, transplantation or from in- antibody. Once an antibody is detected, its
jections with immunogenic material. In specificity should be determined and its
some instances, no specific immunizing clinical significance assessed.
event can be identified. These naturally A clinically significant red cell antibody,
occurring antibodies are a result of expo- although difficult to define, could be char-
sure to environmental, bacterial, or viral acterized as an antibody that shortens the
antigens that are similar to blood group survival of transfused red cells or has been
antigens. Also, antibodies detected in se- associated with hemolytic disease of the fe-
rologic tests can be passively acquired. tus and newborn (HDFN). The degree of
These antibodies may be acquired from clinical significance varies with antibodies
injected immunoglobulins, donor plasma, of the same specificity. Some antibodies
423

cause destruction of incompatible red cells similar. Antibody identification methods

within hours or even minutes, others de- can be more focused and based on the re-
crease the survival by only a few days, and activity patterns seen in the antibody de-
some cause no discernible red cell destruc- tection test.
tion. Antibodies of some specificities are Each facility should establish which
known to cause HDFN, whereas others may techniques for antibody detection and
cause a positive direct antiglobulin test identification will be employed routinely.
(DAT) in the fetus without clinical evidence Sometimes, it is valuable to develop flow-
of HDFN. charts to clearly guide the technologist
Reported experience with other exam- through the process of selecting additional
ples of antibody with the same specificity techniques to identify antibody specifici-
can be used in assessing clinical signifi- ties. This approach is helpful in expediting
cance. Table 15-1 summarizes the expected the identification process and minimizing
reactivity and clinical significance of com- unnecessary testing.
monly encountered alloantibodies. Daniels
et al3 have published a review of these and Specimen Requirements
other specificities. For some antibodies, few
Either serum or plasma may be used for
or no data exist, and decisions must be
antibody detection and identification.
based on the premise that clinically signifi-
Plasma is not suitable for detecting com-
cant antibodies are usually those active at
plement-activating antibodies. A 5- to
37 C and/or by an indirect antiglobulin test
10-mL aliquot of whole blood usually
(IAT). It is not true, however, that all anti-
contains enough serum or plasma for
bodies active in vitro at 37 C and/or by an
identifying simple antibody specificities;
IAT are clinically significant.
more may be required for complex studies.
Antibodies encountered in pretransfu-
When autologous red cells are studied,
sion testing should be identified to assess
the use of a sample anticoagulated with
the need to select antigen-negative red cell
EDTA avoids problems associated with
components for transfusion. Patients with
the in-vitro uptake of complement com-
clinically significant antibodies should,
ponents by red cells, which may occur in
whenever practical, receive red cells that
clotted samples.
have been tested and found to lack the cor-
responding antigen. In prenatal testing, the
specificity and immunoglobulin class of an Medical History
antibody influence the likelihood of HDFN. It is useful to know a patient’s clinical di-
The results of antibody identification tests agnosis, history of transfusions or preg-
on donor blood may be used to character- nancies, and recent drug therapy when
ize the units for labeling before transfusion performing an antibody identification.
and to procure blood typing reagents or For example, in patients who have had re-
teaching samples. 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-
General Procedures ing may be required (see Method 2.15).
The techniques employed for antibody de- Other special procedures may be required
tection and antibody identification are for patients with autoantibodies.

Chapter 19: Initial Detection and Identification of Alloantibodies to Red Cell Antigens 425
Reagents tions exists for each of many antigens. To

be functional, a reagent red cell panel must
Antibody Detection Red Cells
make it possible to identify with confidence
Group O red cells suitable for antibody those clinically significant alloantibodies
screening are commercially available and that are most frequently encountered, such
are offered as sets of either two or three as anti-D, -E, -K, and -Fya. The phenotypes
vials of single-donor red cells. Pooled an- of the reagent red cells should be distrib-
tibody detection cells (usually from two uted such that single specificities of the
donors) can only be used in testing serum common alloantibodies can be clearly
samples from donors. identified and most others excluded. Ide-
The decision to use two or three cells in ally, the pattern of reactivity for most exam-
an antibody detection test should be based ples of single alloantibodies will not overlap
on circumstances in each individual labo- with any other; eg, all of the K+ samples
ratory. The reagent red cells are selected to should not be the only ones that are also
express the antigens associated with most E+. It may also be valuable to include red
commonly encountered antibodies. Re- cell samples with a double dose of the anti-
agent cells licensed by the Food and Drug gen in question for antibodies that fre-
Administration (FDA) for this purpose must quently show dosage. To lessen the possi-
express the following antigens: D, C, E, c, e, bility that chance alone has caused an
M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and apparently definitive pattern, there must be
Jkb.4 Some weakly reactive antibodies react a sufficient number of red cell samples that
only with red cells from donors who are ho- lack, and sufficient red cell samples that ex-
mozygous for the genes controlling the express, most of the antigens listed in Table
pression of these antigens, a serologic phe- 19-1.
nomenon called dosage. Antibodies in the Commercially prepared panels are gen-
Rh, Duffy, MNS, and Kidd systems most erally issued every 2 to 4 weeks. Each panel
commonly demonstrate dosage. Reagent contains different red cell samples with dif-
red cells should be refrigerated when not in ferent antigen patterns, so it is essential to
use and should not be used for antibody use the phenotype listing sheet that comes
detection beyond their expiration date. with the panel in use. Commercial cells
usually come as a 2% to 5% suspension in a
Antibody Identification Panels preservative medium that can be used di-
Identification of an antibody to red cell an- rectly from the vial. Washing is generally
tigen(s) requires testing the serum against unnecessary unless the media in which the
a panel of selected red cell samples with reagent cells are suspended are suspected
known antigen composition for the major of interfering with alloantibody identifica-
blood groups. Usually, they are obtained tion.
from commercial suppliers, but institutions Panel cells should not be used beyond
may assemble their own by using red cells the expiration date; however, this is not always
from local sources. Panel cells are (except practical. Most serologists use in-date reagent
in special circumstances) group O, allow- cells for initial antibody identification pan-
ing serum of any ABO group to be tested. els and, if necessary, use expired reagent
Each cell of the panel is from a different cells for exclusion or confirmation of speci-
individual. The cells are selected so that, ficity. Each laboratory must establish and
taking all the cells into account, a distinc- validate a policy for the use of expired re-
tive pattern of positive and negative reac- agent cells.5

426
Table 19-1. A Reagent Red Cell Panel for Alloantibody Identification
Rh Kell Duffy Kidd P Lewis MNS

Sample Rh
# Phenotype C Cw c D E e K Fya Fyb Jka Jkb P1 Le a Le b M N S s
1 r′r + 0 + 0 0 + 0 + 0 + + + 0 + + + 0 +
2 R1w + + 0 + 0 + + + + 0 + + + 0 + + + +
3 R1 + 0 0 + 0 + 0 + + + + 0 0 + + 0 + 0
4 R2 0 0 + + + 0 0 0 + 0 + + + 0 0 + 0 +
5 r″r 0 0 + 0 + + 0 + + 0 + 0 0 + + + + 0
6 r 0 0 + 0 0 + 0 0 + + 0 + 0 0 + + 0 +
7 r 0 0 + 0 0 + + 0 + + 0 + 0 + + 0 + 0
8 r 0 0 + 0 0 + 0 + 0 0 + + + 0 0 + 0 +
9 r 0 0 + 0 0 + 0 0 + + 0 0 0 + 0 + + 0
10 R0 0 0 + + 0 + 0 0 0 + + + 0 0 + + + +
+ Denotes presence of antigen; 0 denotes absence of antigen.
Antiglobulin Reagents pressed on recently transfused red cells may

have circulating donor red cells coated
To detect clinically significant antibodies,
with alloantibodies, resulting in a positive
most antibody identification tests include
autocontrol. Because this result may be
an antiglobulin phase. Either polyspecific
misinterpreted as being due to autoanti-
or IgG-specific antiglobulin reagents may
body, a detailed history of recent transfu-
be used. Polyspecific reagents may detect,
sions should be obtained for all patients
or detect more readily, antibodies that bind
with a positive DAT or positive auto-
complement. This may be of value in the
control.
detection of certain Kidd antibodies.6 Al-
The autologous control, in which serum
though this may be advantageous in some
and autologous cells undergo the same test
instances, many serologists prefer to use
conditions as serum and reagent cells, is
IgG-specific reagents to avoid unwanted
not the same as a DAT. Incubation and the
reactivity resulting from in-vitro comple-
presence of enhancement reagents may
ment binding by cold-reactive antibodies.
cause reactivity in the autologous control
that is only an in-vitro phenomenon. If the
Enhancement Media
autocontrol is positive in the antiglobulin
Although the test system may consist solely phase, a DAT should be performed. If the
of serum and red cells (reagent red cells as DAT is positive, elution studies should be
provided by the manufacturer or saline- considered if the patient has been recently
suspended red cells), most serologists use transfused, if there is evidence of immune
some type of enhancement medium. Many hemolysis, or if the results of serum studies
different media are available, including prove inconclusive. A reactive DAT may
low-ionic-strength saline (LISS), polyeth- also indicate the presence of autoantibody.
ylene glycol (PEG), and 22% to 30% albu- If autoantibody is detected in the serum,
min. For the initial antibody identifica- adsorption studies may be necessary to
tion panel, most laboratories use the detect coexisting alloantibodies.
same enhancement method used in their
routine antibody detection tests. Addi-
tional enhancement techniques may be
employed for more complex studies. En- Basic Antibody Identification
hancement techniques are discussed later Techniques
in this chapter. For initial panels, it is common to use the
same methods and test phases used in the
Autologous Control (Autocontrol) antibody detection test or crossmatch.
It may be helpful to know how a serum Some serologists may choose to include
under investigation reacts with autologous an immediate centrifugation reading and/
red cells. This helps determine whether or a room temperature incubation and
alloantibody, autoantibody, or both are reading without adding an enhancement
present. Serum that reacts only with the medium. This may enhance the detection
reagent red cells usually contains only of certain antibodies (anti-M, -N, -P1, -I,
a b
alloantibody, whereas reactivity with both -Le , or -Le ) and may help to explain re-
reagent and autologous red cells suggests actions detected at other phases. Many in-
the presence of autoantibody or autoanti- stitutions omit these steps to avoid finding
body plus alloantibody. However, a pa- antibodies that react only at lower tem-
tient with alloantibodies to antigens ex- peratures and have little or no clinical sig-

nificance. Test observation after 37 C in- be a more complex process combining

cubation may detect some antibodies (eg, technical knowledge and intuitive skills.
potent anti-D, -K, or -E) that can cause di- Panel results generally will include both
rect agglutination of red cells. Other anti- positive and negative results at different
a a
bodies (eg, anti-Le , -Jk ) may be detected phases of testing, each of which should be
by their lysis of antigen-positive red cells explained by the final conclusion. Deter-
during the 37 C incubation. Some serolo- mination of the patient’s red cell pheno-
gists believe that because clinically signif- type and the probability of antibody spec-
icant antibodies will be detected with the ificity can also play roles in the final
IAT, the reading after 37 C can be safely interpretation.
omitted. This omission will lessen the de-
tection of unwanted positive reactions re- Positives and Negatives
sulting from clinically insignificant cold-
Both positive and negative reactions are
reactive auto- and alloantibodies.7,8
important in antibody identification. Pos-
The phenotype of the reactive antibody
itive reactions indicate the phase and
detection cells will provide clues to the
strength of reactivity (see Method 1.8 for
specificity or help exclude specificities. This
grading agglutination), which can suggest
information is useful for selecting cells that
certain specificities. Positive reactions also
would be most informative in additional
can be compared to the antigen patterns
testing.
expressed by the panel cells to help assign
If the patient has previously identified
specificity. Single alloantibodies usually
antibodies, this may affect panel selection.
yield definite positive and negative reac-
For example, if the patient is known to have
tions that create a clear-cut pattern with
anti-e, it will not be helpful to test the se-
antigen-positive and -negative reagent
rum against a panel of 10 red cell samples,
red cell samples. For example, if a serum
nine of which are e+. Testing a panel of se-
reacts only with cells 4 and 5 of the re-
lected e– red cell samples will better reveal
agent red cell panel shown in Table 19-1,
any newly formed antibodies.
anti-E is very likely present. Both reactive
Sometimes, the patient’s phenotype in-
samples express E and all nonreactive
fluences the selection of reagent cells. For
samples lack E.
example, if the patient is D– and the serum
Negative reactions are important in anti-
is reactive with D+ cells in the screening
body identification because they allow ten-
test, an abbreviated panel or select cell panel
tative exclusion of antibodies to antigens
of D– red cell samples may be tested. This
expressed on the nonreactive cells. Exclu-
can both confirm the presence of anti-D
sion of antibodies is an important step in
and demonstrate the presence or absence
the interpretation process and must be per-
of additional antibodies, while minimizing
formed to ensure proper identification of
the amount of testing required.9
all the antibodies present.
Exclusion or “Crossing Out”

Interpreting Results
A widely used first approach to the inter-
Antibody screening results are interpreted pretation of panel results is to exclude
as positive or negative based on the pres- specificities based on nonreactivity with
ence or absence of reactivity (eg, aggluti- the serum tested. Such a system is some-
nation). Interpretation of panel results can times referred to as a “cross-out” or “rule-

out” method. Once results have been re- pression of an antigen (see Variations in
corded on the worksheet, the antigen pro- Antigen Expression).
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, Probability
the presence of the corresponding anti-
To ensure that an observed pattern is not
body may be, at least tentatively, excluded.
the result of chance alone, conclusive an-
Many technologists will cross out that an-
tibody identification requires serum to be
tigen from the listing on the panel sheet
tested against sufficient reagent red cell
to facilitate the process. After all antigens
samples that lack, and that express, the
present on that cell have been crossed off,
antigen that corresponds to the apparent
interpretation proceeds with the other non-
specificity of the antibody.
reactive cells and additional specificities
A standard approach (based on Fisher’s
are excluded. In most cases, this process
exact method10) has been to require, for
will leave a group of antibodies that still
each specificity identified, three antigen-
have not been excluded.
positive cells that react and three anti-
Next, the cells reactive with the serum
gen-negative cells that fail to react. This
are evaluated. The pattern of reactivity for
standard is not always possible, but it works
each nonexcluded specificity is compared
well in practice, especially if cells with
to the pattern of reactivity obtained with
strong antigen expression are available. A
the test serum. If there is a pattern that
somewhat more liberal approach is derived
matches exactly, that is most likely the
from calculations by Harris and Hochman,11
specificity of the antibody in the serum.
whereby minimum requirements for a
However, if there are remaining speci-
probability (p) value of 0.05 are met by hav-
ficities that have not been excluded, addi-
ing two positive and three negative cells, or
tional testing may be needed to eliminate
one positive and seven negative cells (or
remaining possibilities and to confirm the
the reciprocal of either combination). Com-
specificity identified. This requires testing
parative p values are shown in Table 19-2.
the serum against cells selected for specific
The use of two positive and two negative
antigenic characteristics. For example, this
cells is also an acceptable approach for an-
approach could be employed if the pattern
tibody confirmation.12 Additional details on
of positive reactions exactly fits anti-Jka, but
calculating probability may be found in the
anti-K and anti-S have not been excluded.
suggested readings by Race and Sanger,
Then serum should be tested against se-
Menitove, and Kanter. The possibility of
lected cells, ideally with the following phe-
false-negative results with antigen-positive
notypes: Jk(a–), K–, S+; Jk(a–), K+, S–; and
cells must be considered as well as unex-
Jk(a+), K–, S–. The reaction pattern with
pected positives, ie, false-positive results
these cells should both confirm the pres-
a due either to the presence of an additional
ence of anti-Jk and include or exclude
antibody specificity or an error in the pre-
anti-K and anti-S.
sumptive antibody identification.
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 Phenotype of Autologous Red Cells
was completed based on the nonreactivity Once an antibody has been tentatively
of cells with weaker (eg, heterozygous) ex- identified in a serum, it is often helpful to

Table 19-2. Probability Values
10 11
No. Tested No. Positive No. Negative p (Fisher ) p (Harris and Hochman )
5 3 2 0.100 0.035
6 4 2 0.067 0.022
6 3 3 0.050 0.016
7 5 2 0.048 0.015
7 4 3 0.029 0.008
8 7 1 0.125 0.049
8 6 2 0.036 0.011
8 5 3 0.018 0.005
8 4 4 0.014 0.004
9 8 1 0.111 0.043
9 7 2 0.028 0.008
9 6 3 0.012 0.003
10 9 1 0.100 0.039
10 8 2 0.022 0.007
10 7 3 0.008 0.002
10 6 4 0.005 0.001
10 5 5 0.004 0.001
demonstrate the presence or absence of specimen to be phenotyped. Phenotyping

the corresponding antigen on the autolo- results on posttransfusion samples can be
gous red cells. For example, if serum from misleading, however, and should be inter-
an untransfused individual appears to preted with caution.13 If there is little uncer-
contain anti-Fya but the autologous red tainty about antibody identification, exten-
cells have a negative DAT and type as sive efforts to separate and type the patient’s
Fy(a+), the data are clearly in conflict and own red cells are not necessary. Compatible
further testing is indicated. antiglobulin crossmatch,14(p40) of antigen-
Determination of the patient’s pheno- negative donor units provides additional
type can be difficult if the patient has been confirmation of antibody specificity. Defini-
transfused recently, generally within 3 tive testing can be performed on the pa-
months. If a pretransfusion specimen is tient’s red cells after a sufficient period
available, these red cells should be used to without red cell transfusion. In a chroni-
determine the phenotype. Alternatively, the cally transfused patient, definitive testing
patient’s own red cells can be separated can be performed after an interval during
from the transfused red cells and then which only antigen-negative blood has
typed (see Methods 2.15 and 2.16). The use been given. Any antigen-positive red cells
of potent blood typing reagents, appropri- detected after prolonged transfusion of an-
ate controls, and observation for mixed- tigen-negative blood would presumably be
field reactions often allow an unseparated the patient’s own.

recognize dosage. Many antibodies to an-

Complex Antibody Problems tigens in the Rh, Duffy, MNS, and Kidd
Not all antibody identifications are sim- systems have this trait.
ple. The exclusion procedure does not al-
ways lead directly to an answer and addi- Variation in Adults and Infants
tional approaches may be required. Figure a a
Some antigens (eg, I, P1, Le , and Sd ) are
19-1 shows some approaches to identify-
expressed to varying degrees on red cells
ing antibodies in a variety of situations
from different adult donors. This varia-
when the autocontrol is negative. Addi-
tion is unrelated to zygosity; however, the
tional approaches may be needed if the
antigenic differences can be demon-
autocontrol is positive; they are discussed
strated serologically. Certain antibodies
later in this chapter.
(eg, anti-I, -Lea) demonstrate weaker reac-
tivity with cord red cells than with red
Variations in Antigen Expression cells from adults (see Table 19-3).
For a variety of reasons, antibodies do not
always react with all cells positive for the Changes with Storage
corresponding antigen. Basic interpreta-
Blood group antibodies may give weaker
tion by exclusion, as described previously,
reactions with stored red cells than with
may result in a given specificity being ex-
fresh red cells. Some antigens (eg, Fya, Fyb,
cluded because the sample is nonreactive a a 17
M, P1, Kn /McC , Bg) deteriorate during
with an antigen-positive red cell sample,
storage more rapidly than others and the
despite the presence of the antibody.
rate varies among red cells from different
Technical error, weak antibody reactivity,
donors. Because red cells from donors are
and variant or weak antigenic expression
often fresher than commercial reagent cells,
are all possible causes. Therefore, when-
some antibodies give stronger reactions
ever possible, antibody specificities should
with suspensions of donor cells than with
be excluded only on the basis of cells
reagent cells. Frozen storage of red cells
known to bear a strong expression of the
may result in antigen deterioration that
antigen. Enhancement techniques often help
can cause misleading antibody identifica-
resolve problems associated with varia-
tion results.
tions in antigen expression (see Methods
The pH or other characteristics of stor-
3.2.2, 3.2.3, 3.2.4, 3.5.5, and 3.5.6).
age media can affect the rate of antigen de-
terioration.17,18 For example, Fya and Fyb an-
Zygosity tigens may be weakened when the cells are
Reaction strength of some antibodies may stored in a suspending medium of low pH
vary from one red cell sample to another and low ionic strength. Alternatively, cer-
due to a phenomenon known as dosage, tain antibodies may demonstrate stronger
in which antibodies react preferentially or weaker reactions with red cells from dif-
with red cells from persons homozygous ferent manufacturers using different sus-
for the gene that determines the antigen pending media. The age and nature of the
(ie, possessing a “double dose” of the anti- specimen must also be considered when
gen). Red cells from individuals heterozy- typing red cells. Antigens on cells from clot-
gous for the gene may express less antigen ted samples tend to deteriorate faster than
and may react weakly or be nonreactive. antigens on cells collected in citrate antico-
Alloantibodies vary in their tendency to agulants such as ACD or CPD. Red cells in

432
15
Figure 19-1. Approaches for identifying antibodies (modified from Brendel ).
Table 19-3. Antigen Expression on Cord Red Blood Cells*

Expression Antigens
Negative Lea, Leb, Sda, Ch, Rg, AnWj

Weak I, H, P1, Lua, Lub, Yta, Vel, Bg, McCa, Yka, S1a, Csa, Hy, Gy, Joa, Doa, Dob, Fy3
Strong i, LWa, LWb
16
*Modified from Reid.
donor units collected into these anticoagu- If all the reactive cells are Jk(b+), but not
lants generally retain their antigens through- all the Jk(b+) cells react, the reactive ones
out the standard shelf life of the blood com- might all be Jk(a–b+), with a double-dose
ponent. EDTA samples up to 14 days old expression of the antigen. Enhancement
are suitable for antigen typing; however, the techniques, such as enzymes, LISS, or PEG,
manufacturer’s instructions should be con- may then help demonstrate reactivity with
sulted when using commercial typing re- all the remaining Jk(b+) cells. Typing the
19
agents. patient’s cells to confirm they lack the cor-
responding antigen can also be very help-
No Discernible Specificity ful.
Factors other than variation in antigen ex-
pression may contribute to difficulty in Inherent Variability
interpreting results of antibody identifica- Nebulous reaction patterns that do not
tion tests. If the reactivity obtained with appear to fit any particular specificity are
the serum is very weak and/or if the cross- characteristic of antibodies, such as anti-
out process has excluded all likely speci- Bga, that react with HLA antigens on red
ficities, alternative approaches to inter- cells. These antigens vary markedly in
pretation should be used. their expression on red cells from differ-
ent individuals. Rarely, a pattern of clear-
Antigens Present in Common cut reactive and nonreactive tests that
cannot be interpreted is due to the incor-
Instead of excluding antibodies to anti-
rect typing of reagent red cells. If the cell
gens on nonreactive cells, one can ob-
is from a commercial source, the manu-
serve what antigens are common to the
facturer should be notified immediately
reactive cells. For example, if the cells re-
of the discrepancy.
acting at room temperature are all P1+, yet
not all the P1+ cells react, the antibody
could be an anti-P1 that does not react Unlisted Antigens
with cells having a weaker expression of Sometimes a serum sample reacts with an
the antigen. (Sometimes, such cells are antigen not routinely listed on the antigen
marked on the panel sheet as “+w.”) With profile supplied by the reagent manufac-
this in mind, one could use a method to turer; Ytb is one example. Even though se-
enhance anti-P1, such as testing at colder rum studies yield clear-cut reactive and
temperatures. nonreactive tests, anti-Ytb may not be sus-

pected. In such circumstances, it is useful pattern, it is helpful to see if the pat-

to ask the manufacturer for additional tern matches any two combined
phenotype information. If the appropri- specificities. For example, if the reac-
ate blood typing reagent is available, reactive cells (see Table 19-1) are num-
tive and nonreactive red cell samples, as bers 2, 4, 5, and 7, none of the speci-
well as the autologous red cells, can be ficities remaining after crossing-out
tested. These problems often have to be exactly fits that pattern, but if both K
referred to an immunohematology refer- and E are considered together, a pat-
ence laboratory. tern is discerned. Cells 2 and 7 react
because of anti-K, cells 4 and 5 be-
ABO Type of Red Cells Tested cause of anti-E. If the typing pat-
terns for no two specificities fit the
A serum sample may react with many or
reaction pattern, the possibility of
all of the group O reagent red cell sam-
more than two antibodies must be
ples, but not with red cells of the same
considered. The more antibodies a
ABO phenotype as the autologous red
serum contains, the more complex
cells. This occurs most frequently with
the identification and exclusion of
anti-H, -IH, or -LebH. Group O and A2 red
specificities will be, but the basic
cells have large amounts of H antigen; A1
process remains the same.
and A1B red cells express very little H (see
2. Reactivity is present at different test
Chapter 13). Sera containing anti-H or -IH
phases.
react strongly with group O reagent red
When reactivity occurs at several
cell samples, but autologous A1 or A1B red
phases, each phase should be evalu-
cells or donor cells used for crossmatch-
ated separately. The pattern seen at
ing may be weakly reactive or nonreac-
room temperature may indicate a
tive. Anti-LebH reacts strongly with group
different specificity from the pattern
O, Le(b+) red cells, but reacts weakly or
of antiglobulin results. It is also
not at all with Le(b+) red cells from A1 or
helpful to look at variability in the
A1B individuals. Such antibodies should
strength of reactions seen at each
be suspected when the antibody screen,
phase of testing. Table 15-2 provides
which uses group O red cells, is strongly
information on the characteristic re-
reactive, but serologically compatible A1
activity phase of several antibodies.
or A1B donor samples can be found with-
3. Unexpected reactions are obtained when
out difficulty.
attempts are made to confirm the spec-
ificity of a suspected single antibody.
Multiple Antibodies If a serum suspected of containing
When a serum contains two or more alloanti-e reacts with additional samples
antibodies, it may be difficult to interpret that are e–, another antibody may be
the results of testing performed on a sin- present or the suspected antibody
gle panel of reagent red cells. The pres- may not be anti-e. Testing a panel of
ence of multiple antibodies may be sug- selected e– red cell samples may
gested by a variety of test results. help indicate an additional specific-
1. The observed pattern of reactive and ity.
nonreactive tests does not fit that of a 4. No discernible pattern emerges.
single antibody. When the exclusion When uniform or variable reaction
approach fails to indicate a specific strengths are observed, and dosage or

other variation in antigen strength does types and by testing the patient’s autologous
not provide an explanation, addi- red cells with sera known to contain anti-
tional approaches and methods of bodies to high-incidence antigens. Knowing
testing are indicated. Some helpful the race or ethnic origin of the antibody
steps include: producer can help in selecting additional
a. If strong positive results were tests to be performed. Cells that are null
obtained, use the exclusion for all antigens in a system (eg, Rhnull or Ko)
method with nonreactive cells or modified red cells (eg, dithiothreitol-
to eliminate some specificities treated cells, see Method 3.10) can help
from initial consideration. limit possible specificities to a particular
b. If weak or questionable positive blood group.
results were obtained, test the se- If cells negative for particular high-inci-
rum against cells carrying a strong dence antigens are not available, cells posi-
expression of antigens corre- tive for lower-incidence alleles can some-
sponding to any suspected spe- times be helpful. Weaker reactivity with
cificities and combine this with Co(a+b+) cells when compared with com-
methods to enhance reactivity. mon Co(a+b–) cells, for instance, might
c. If the patient has not been re- suggest anti-Coa. Antibodies to high-inci-
cently transfused, type the pa- dence antigens may be accompanied by
tient’s red cells and eliminate other antibodies to common antigens,
from consideration specificities which can make identification much more
that correspond to antigens on difficult. Because the availability of cells
the autologous cells. negative for high-incidence antigens is lim-
d. Use methods to inactivate cer- ited, it may be necessary to refer specimens
tain antigens on the red cells, suspected of containing antibodies to high-
eg, enzyme treatment to render incidence antigens to an immunohemato-
cells negative for antigens such logy reference laboratory.
as Fya, Fyb, and S.
e. Use adsorption/elution methods
to separate antibodies. Serologic Clues
f. Enhance antibody reactivity by Knowledge of the serologic characteristics
using a more sensitive method of particular antibodies to high-incidence
(eg, PEG). These and other meth- antigens can help in identification.
ods that may be helpful are dis- 1. Reactivity in tests at room tempera-
k
cussed below. ture suggests anti-H, -I, -P1, -P, -PP1P
a a
(-Tj ), -LW (some), -Ge (some), -Sd ,
or -Vel.
Antibodies to High-Incidence Antigens
2. Lysis of reagent red cells when test-
If all reagent red cell samples are reactive, ing with fresh serum is characteristic
but the autocontrol is nonreactive, an of anti-Vel, -P, - PP1Pk, and -Jk3. It is
alloantibody to a high-incidence antigen also seen with some examples of
should be considered, especially if the anti-H and -I.
strength and test phase of reactions are 3. Reduced or absent reactivity in en-
uniform for all cells tested. Antibodies to zyme tests occurs with anti-Ch, -Rg,
high-incidence antigens can be identified -Inb, -JMH, or -Ge2 and is seen with
a
by testing red cells of selected rare pheno- some examples of anti-Yt .

4. Weak nebulous reactions in the anti- from alloantibody. Chapter 15 discusses

globulin phase are often associated additional serologic characteristics of an-
with anti-Kna, -McCa, -Yka, and -Csa. tibodies reacting with high-incidence red
Complement-binding autoantibodies, cell antigens.
such as anti-I or anti-IH, give similar
results when polyspecific antiglobu- Antibodies to Low-Incidence Antigens
lin reagents are used.
Reactions between a serum sample and a
5. Antibodies such as anti-U, -McCa,
single donor or reagent red cell sample
-Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and -Ata
may be caused by an antibody to a low-
should be considered if the serum is a
incidence antigen, such as anti-Wr . If red
from a Black individual because the
cells known to carry low-incidence anti-
antigen-negative phenotypes occur
gens are available, the serum can be
almost exclusively in Blacks. Individ-
tested against them, or the one reactive
uals with anti-Kpb are almost always
red cell sample can be tested with known
White. Anti-Dib is usually found among
examples of antibodies to low-incidence
Asian, South American Indians, and
antigens. A single serum often contains
Native American populations.16(pp526,527)
multiple antibodies to low-incidence an-
tigens; therefore, the expertise and re-
Interpreting a Positive DAT sources of an immunohematology refer-
When a patient produces antibody di- ence laboratory may be required to confirm
rected to a high-incidence antigen after the suspected specificities.
transfusion, the posttransfusion red cells
may have a positive DAT, and both serum
Serologic Strategies
and eluate may react with all cells tested.
Because this pattern of reactivity is identi- If an antibody to a low-incidence antigen
cal to that produced by many warm-reac- is suspected, transfusion should not be
tive autoantibodies that may also appear delayed while identification studies are
after transfusion, these two scenarios can undertaken. If an antibody in the serum
be very difficult to differentiate. A post- of a pregnant woman is thought to be di-
transfusion alloantibody to a high-inci- rected against a low-incidence antigen,
dence antigen would be expected to pro- testing the father’s red cells can predict
duce a DAT of mixed-field appearance (ie, the possibility of incompatibility with the
some cells agglutinated among many fetus, and identifying the antibody is un-
unagglutinated cells) because only the necessary. If a newborn has a positive
transfused red cells would be coated with DAT, testing of the mother’s serum or an
antibody. In practice, however, weak sen- eluate from the infant’s cells against the
sitization and mixed-field sensitization father’s red cells (assuming they are ABO-
can be difficult to differentiate. If a pre- compatible) can implicate an antibody to
transfusion red cell sample is not available, a low-incidence antigen as the probable
it may be helpful to use cell separation cause; identifying the antibody is usually
procedures to isolate autologous cells for of little importance.
testing. Performing a DAT on autologous Some reference laboratories do not attempt
cells and/or testing the posttransfusion to identify antibodies to low-incidence an-
serum with DAT-negative autologous cells tigens because they are often only of aca-
may help to distinguish autoantibody demic interest. Identification may be made

when time permits and suitable reagents Most of these anomalous reactions are
are available. in-vitro phenomena and have no clinical
significance in transfusion therapy other
Unexpected Positive Results than causing laboratory problems that de-
lay needed transfusions. They rarely cause
When a serum reacts with a panel cell
erroneous interpretations of ABO typing
designated as positive for a low-incidence
that could endanger the patient. For a more
antigen, further testing to exclude the an-
detailed discussion, see the suggested read-
tibody is usually unnecessary. For every
ing by Garratty.
antigen of low incidence represented on a
panel, there are many more that are not
represented and are also not excluded by Ingredients in the Preservative Solution
routine testing. Reactivity against low-in- Antibodies that react with an ingredient
cidence antigens is not uncommon; al- in the solution used to preserve reagent
though the antigens are rare, antibodies red cells (eg, chloramphenicol, neomycin,
against some of the low-incidence anti- tetracycline, hydrocortisone, EDTA, sodium
gens are much less rare. Presumably, the caprylate, or various sugars) may aggluti-
testing is being performed because the nate cells suspended in that solution. Re-
serum contains some other antibody and activity may occur with cells from several
reactivity with the cell expressing the commercial sources or may be limited to
low-incidence antigen is a coincidental cells from a single manufacturer. The
finding. This may complicate interpreta- autologous control is often nonreactive,
tion of the panel results but rarely re- unless the suspension of autologous red
quires confirmation of antibody specific- cells is prepared with the manufacturer’s
ity or typing of donor blood to ensure the red cell diluent or a similar preservative.
absence of the antigen. If typing is desired, Such reactions can often be circumvented
a negative crossmatch with the patient’s by washing the reagent cells with saline
serum is sufficient demonstration that the before testing. The role of the preservative
antigen is absent. Many antibodies to can often be confirmed by adding the me-
low-incidence antigens are reactive only dium to the autologous control and con-
at temperatures below 37 C and are of verting a nonreactive test to a positive test.
doubtful clinical significance. In some cases, however, washing the re-
When the serum reacts only with red agent cells does not circumvent reactivity
cells from a single donor unit or reagent and the resolution may be more complex.
cell, the other possibilities to consider are
that the reactive donor red cells are ABO- Ingredients in Enhancement Media
incompatible, have a positive DAT, or are
Antibodies reactive with ingredients in
polyagglutinable.
other reagents, such as commercially pre-
pared LISS additives or albumin, can cause
Antibodies to Reagent Components and agglutination in tests using reagent, donor,
Other Anomalous Serologic Reactions and/or autologous red cells. Ingredients that
Antibodies to a variety of drugs and addi- have been implicated include parabens (in
tives can cause positive results in antibody some LISS additives), sodium caprylate (in
detection and identification tests. The some albumins), and thimerosal (in some
mechanisms are probably similar to those LISS/saline preparations). Antibody to in-
discussed in Chapter 20. gredients in enhancement media may be

suspected if the autologous control is or shows only weak reactivity, an eluate may
positive but the DAT is negative. Omitting demonstrate more potent autoantibody.
the enhancement medium will usually A negative DAT but a positive auto-
circumvent this reactivity. control by an IAT is unusual and may indi-
In some cases, antibodies dependent upon cate antibody to a reagent constituent
reagent ingredients will show blood group causing in-vitro reactivity with all cells, in-
specificity, eg, paraben-dependent anti-Jka, cluding the patient’s own. It may also indi-
caprylate-dependent anti-c. The auto- cate the presence of warm autoantibodies
control may be reactive if the patient’s own or cold autoagglutinins such as anti-I, -IH,
red cells carry the antigen, but the DAT or -Pr reacting by IAT when enhancement
should be negative. media are used.
Cold Autoantibodies. Potent cold auto-
agglutinins that react with all cells, includ-
ing the patient’s own, can create special
Problems with Red Cells
problems, especially when reactivity per-
The age of the red cells can cause anoma- sists at temperatures above room tempera-
lous serologic reactions. Antibodies exist ture. Cold autoagglutinins may be benign
that react only with stored red cells; they or pathologic. (See Chapter 20 for a more
can cause agglutination of reagent red cells detailed discussion.)
by all techniques and enhanced reactivity There are different approaches to testing
in tests with enzyme-treated red cells. Such a serum with a potent cold agglutinin. One
reactivity is not affected by washing the approach is to determine if the thermal
red cells, and the autocontrol is usually amplitude is high enough (usually 30 C or
nonreactive. No reactivity will be seen in above) that the antibody has clinical signifi-
tests on freshly collected red cells, ie, from cance. For identification purposes and de-
freshly drawn donor or autologous blood termination of thermal amplitude, in-vitro
samples. autoadsorption of the serum must be avoided
by keeping the freshly collected blood
warm (37 C) until the serum is separated.
The Patient with a Positive Autocontrol For purposes of detecting potentially clini-
cally significant antibodies, methods that
No Recent Transfusions circumvent the cold autoantibody are com-
Reactivity of serum with the patient’s own monly used.
cells may indicate the presence of auto- Procedures for the detection of alloanti-
antibody (see Chapter 20). If this reactiv- bodies in the presence of cold-reactive auto-
ity occurs at room temperature or below, antibodies are discussed in Chapter 20 and
the cause is often anti-I or another cold include:
autoagglutinin. Reactivity of the auto- 1. Prewarmed techniques, in which red
control in the antiglobulin phase usually cells and serum to be tested, and sa-
signifies a positive DAT and the possibility line used for washing, are incubated
of autoantibody. If, in addition, the serum at 37 C before they are combined (see
reacts with all cells tested, autoadsorption Method 3.3).
or other special procedures may be nec- 2. The use of anti-IgG rather than poly-
essary to determine whether autoantibody specific antiglobulin serum.
in the serum is masking any significant 3. Cold autoadsorption, to remove auto-
alloantibodies. If the serum is not reactive antibodies but not alloantibodies.

4. Adsorption with rabbit red cells. considered. Detection of masked alloanti-

Dealing with Warm Autoantibodies. Pa- bodies may require allogeneic adsorptions.
tients with warm-reactive autoantibody Accurate phenotyping of red cells may
present in their sera create a special prob- be difficult if the DAT is reactive in any pa-
lem because the antibody reacts with virtu- tient, whether or not there has been recent
ally all cells tested. If such patients are to be transfusion. A positive DAT will cause the
transfused, it is important to detect any cells to be reactive in any test requiring the
clinically significant alloantibodies that the addition of antiglobulin serum and with
autoantibody may mask. Techniques are some reagent antibodies (notably those in
discussed in Chapter 20 and Methods 4.9, the Rh system) in a high protein medium.
4.10, 4.11, and 4.12. With rare exception, most monoclonal re-
Reactivity of most warm-reactive auto- agents not tested by an IAT can give valid
antibodies is greatly enhanced by such phenotyping results despite a positive DAT.20
methods as PEG and enzymes, and to lesser
extent by LISS and albumin. It may be ad- Immunohematology Reference
vantageous to perform antibody detection Laboratories
tests without the enhancement media usu-
When antibody problems cannot be re-
ally employed. If tests are nonreactive, the
solved or when rare blood is needed, im-
same procedure can be used for compati-
munohematology reference laboratories
bility tests, without the need for adsorptions.
can provide consultation and assistance
through their access to the American Rare
Recent Transfusions Donor Program (see Method 3.13).
If the autocontrol is positive in the anti-

globulin phase, there may be antibody-
coated cells in the patient’s circulation, Selecting Blood for
causing a positive DAT, which may show
mixed-field reactivity. Elution may be help-
Transfusion
ful, especially when tests on serum are Once an antibody has been identified, it is
inconclusive. For example, a recently trans- important to decide its clinical signifi-
fused patient may have a positive auto- cance. Antibodies reactive at 37 C and/or
control and serum that reacts weakly with by IAT are potentially clinically significant
most but not all Fy(a+) red cells. It may be and those reactive at room temperature
possible to confirm anti-Fya specificity by and below are not; however, there are
elution, which concentrates into a small many exceptions. For example, anti-Ch,
fluid volume the immunoglobulin mole- anti-Rg, and many of the Knops and Cost
cules present in small numbers on the red antibodies have little or no clinical effect
cells in the whole blood sample. It is rare despite reactivity by an IAT. Anti-Vel, -P,
for transfused cells to make the autocontrol and -PP1Pk (-Tja) may react only at cold
positive at other test phases, but it can oc- temperatures yet may cause red cell des-
cur, especially with a newly developing or truction in vivo. Comparison with docu-
cold-reactive alloantibody. mented cases in the literature and con-
If the positive DAT does not have a sultation with immunohematology refer-
mixed-field appearance and, especially, if ence laboratories should provide guidance
the serum is reactive with all cells tested, about previous examples of similar speci-
the possibility of autoantibody should be ficities.

Phenotyping Donor Units Reagents prepared in-house from sera

that meet these dilution criteria can be
Whenever possible, red cell units selected used.
for transfusion to a patient with a poten-
tially clinically significant antibody should Source of Antibodies
be tested and found to be negative for the
When selecting units for patients with clini-
appropriate antigen. Even if the antibody
cally significant antibodies, some serolo-
is no longer detectable, the red cells of all
gists recommend typing the chosen units
subsequent transfusions to that patient
with antibodies from two different sources,
should lack the antigen, to prevent a sec-
but others consider it unnecessary, espe-
ondary immune response. The transfu-
cially when potent reagents are available.
sion service must maintain records of all
Different lots of antibody from the same
patients in whom significant antibodies
manufacturer and even reagents from dif-
have been previously identified.14(pp38,72) An
ferent manufacturers may not have been
antiglobulin crossmatch procedure is re- prepared from different source material
quired if the serum contains, or has previ- because manufacturers often share the
ously contained, a significant antibody. same resources.
A potent example of the antibody should
be used to identify antigen-negative blood.
Labeling Units
Often, this is a commercial antiserum, but
to save expensive or rare reagents, units can If a donor unit from a blood establishment
first be tested with the patient’s serum. The is to be labeled with the results of special
absence of antigen, in nonreactive units, can antigen typing, use of licensed (commer-
then be confirmed with the commercial re- cial) reagents is preferred. If no licensed
agent. If the antibody is of unusual specific- reagent is available, the unit may be la-
ity or one for which commercial reagents beled with appropriate wording (eg,
are not available, a stored sample from the “Tested and found to be negative for XX
sensitized patient can be used to select antigen using unlicensed typing rea-
units for transfusion at a later time, espe- gents”).22 Except for results of ABO and D
cially if the patient’s later specimens lose typing, there is no requirement that re-
reactivity. If a patient’s serum is to serve as a sults of antigen typing appear on the label
typing reagent, it should be well character- of donor units. The establishment may
ized and retain its reactivity after storage, use a tie tag attached to the unit for the
and appropriate negative and weak-posi- additional labeling.
tive controls should be used at the time of
testing. The FDA has established the follow- When to Test
ing criteria for licensing some reagents21: For certain antibody specificities, typing
a a
1. Anti-K, anti-k, anti-Jk , anti-Fy , and of donor units may not be necessary and
w
anti-C : dilution of 1:8 to give at least the patient’s serum can be used to select
1+ reaction. serologically compatible red cells. This is
2. Anti-S, anti-s, anti-P1, anti-M, anti-I, especially true for antibodies that charac-
anti-c (saline), anti-e (saline), and teristically react below 37 C (eg, anti-M,
anti-A1: dilution of 1:4 to give at least -N, -P1, -Lea, -Leb, -A1) and do not ordi-
1+ reaction. narily exhibit an anamnestic response to
3. Most other specificities: undiluted, the transfusion of antigen-positive red
must give at least a 2+ reaction. cells.

It is rarely necessary to provide anti- measure survival of 1 mL or less of infused

gen-negative donor units as a prophylactic cells. Flow cytometry can also be used to
measure for patients whose cells lack an measure the survival of infused cells, but a
antigen but who do not have detectable an- larger aliquot of red cells (about 10 mL) is
tibody. However, special consideration is generally required. Small aliquots of incom-
sometimes given to certain Rh antibodies. patible cells may have a faster rate of de-
When a patient of the R1R1 phenotype has struction than an entire unit of red cells.
anti-E detected in the serum, some workers
suggest that donor blood be negative for
both the E and c antigens,23 based on the
When Blood of Rare Type Is Needed
assumption that the stimulus to produce
the anti-E may also have stimulated an Blood of a rare type includes not only units
anti-c or anti-cE that remains undetected negative for high-incidence antigens but
by routine tests. For an R2R2 patient with also blood negative for a combination of
demonstrated anti-C, the use of C–, e– do- common antigens. When a patient has
nor blood may be considered. When an multiple antibodies, it can be helpful to
antibody has not been specifically demon- determine the frequency of compatible
strated, but cannot conclusively be ex- donors. To calculate this, the frequency of
cluded, it may be appropriate to transfuse random donors negative for one antigen
blood that lacks the antigen. must be multiplied by the frequency of
donors negative for each of the other anti-
gens. For example, if a serum contains
Tests to Predict Clinical Significance
anti-c, -Fya, and -S, and, among random
Certain laboratory procedures have been donors, 18% are c–, 34% are Fy(a–), and
used to predict the significance of partic- 45% are S–, the frequency of compatible
ular antibodies. The monocyte monolayer units would be: 0.18 × 0.34 × 0.45 = 0.028.
assay, which quantifies rosetting and/or If the patient is group O, then, because
phagocytosis of antibody-sensitized red 45% of random donors are group O, 1.3%
cells, can be used to predict the in-vivo (0.028 × 0.45) of random donors would be
clinical significance of some antibodies. compatible with the patient’s serum. If
The test for antibody-dependent cellular any of these three antibodies occurred
cytotoxicity (ADCC), which measures lysis singly, finding compatible blood would
of antibody-coated cells, and the chemi- not be too difficult. Clearly, when all three
luminescence assay, which measures the antibodies are present, a large number of
respiratory release of oxygen radicals after random donors would be necessary to
phagocytosis of antibody-coated cells, provide even one unit. The preceding cal-
have been helpful in predicting in-vivo culation uses frequencies in populations
antibody reactivity, particularly for sever- of European ethnicity. If the donor popu-
ity of HDFN. For cold-reactive antibodies, lation is predominantly of a different ori-
in-vitro thermal amplitude studies can gin, frequencies for that group, if avail-
predict the likelihood of in-vivo problems. able, should be used.
In-vivo tests may also be used to evalu- When units of rare (<1 in 5000) or un-
ate significance of a given antibody. The common (<1 in 1000) type are needed, the
most common technique is infusion of American Rare Donor Program can be very
radiolabeled, antigen-positive red cells, helpful. This program, which can be ac-
usually tagged with 51Cr. It is possible to cessed only by personnel of an accredited

immunohematology reference laboratory, be performed? A primary antibody re-

can identify blood suppliers known either sponse will produce detectable antibody
to have units available (usually frozen red as early as 7 to 10 days but typically over a
cells) or to have eligible donors who may be period of 2 weeks to several months. A
asked to donate (see Method 3.13). secondary immune response produces
Family members offer another potential detectable antibody in a shorter time, as
source of rare blood donors. Siblings are of- early as 2 to 7 days and usually within 20
ten the best source of serologically compat- days. Shulman24 found that, in a small
ible blood for patients with multiple anti- number of patients, “new” antibodies could
bodies or antibodies to high-incidence be detected within 1 to 2 days after trans-
antigens. The absence of high-incidence fusion. AABB Standards for Blood Banks
antigens usually reflects inheritance of the and Transfusion Services14(p38) requires that,
same rare blood group gene from each par- for a patient who has been pregnant or re-
ent, and offspring of the same parents are ceived red cells within the preceding 3
far more likely to have the same two rare months, antibody detection and compati-
genes than someone in the general popula- bility tests must be performed on a speci-
tion. In most cases, blood from the patient’s men obtained within 3 days of the next
parents or children (and some siblings) will scheduled transfusion. The transfusion
carry only a single dose of the relevant anti- service may consider testing a fresher
gen; if transfusion is essential, and there is specimen if clinical evidence suggests
no alternative to giving incompatible blood, failure of recently transfused red cells to
these heterozygous donors would be con- survive as expected.
sidered preferable to random donors. Occa- If a patient has previously identified clin-
sionally, blood from a parent or child also ically significant antibodies, antigen-nega-
lacks the high-incidence antigen. tive red cells must be selected for all future
In HDFN or other alloantibody-associ- transfusions, even if the antibodies are no
ated problems in infants, the mother, if ABO longer detectable. In addition, an antiglo-
compatible, is often the logical donor. If the bulin crossmatch must be performed using
mother’s red cells are transfused, it is helpful antigen-negative red cells.
to retain the plasma for use as a rare reagent. It is rarely necessary to repeat identifica-
If the clinical situation allows, autologous tion of known antibodies. AABB Standards
transfusion should be considered for patients states that in patients with previously iden-
for whom compatible blood is difficult to tified antibodies, methods of testing shall
find. For some patients with multiple anti- be those that identify additional clinically
bodies for whom autologous transfusion is significant antibodies.14(p38) Each laboratory
not an option, it may be necessary to deter- should define and validate methods for the
mine whether any of the antibodies is likely detection of additional antibodies in these
to be significantly less destructive than the patients. Depending on the specificity of
others and, in a critical situation, give blood the known antibody, repeated testing of the
incompatible for that particular antigen. patient’s serum against routine antibody
detection cells is often not informative. It is
more useful to test against cells negative for
Frequency of Antibody Testing
the antigen(s) to which the patient has anti-
Once an antibody has been identified in a body and positive for other major antigens.
patient’s serum, how frequently should This allows detection of most additional
antibody detection and identification tests antibodies that might develop. Usually, ap-

propriate cells can be selected from avail- pension of test cells and, more commonly,
able red cell panels. Selection of test cells the use of commercially available low-
may be simplified if the patient’s cells are ionic-strength additive media. The use of a
known to express a given antigen. The se- LISS additive requires no preparatory
lected cells need not be positive for that an- stages, but care should be taken to adhere
tigen because the corresponding antibody closely to the manufacturer’s product
would not be anticipated. insert to ensure that the appropriate pro-
portion of serum to LISS is achieved.
Commercially prepared LISS additives
may include other enhancement compo-
Selected Serologic nents besides low-ionic-strength saline.
Procedures Commercially prepared PEG additives are
Many techniques and methods may be also available and may contain additional
useful in antibody identification. Some of enhancing agents. Because LISS and PEG
the methods given here are used routinely enhance autoantibody activity, their use
by many laboratories; others are alterna- may create problems with certain sam-
25,26
tives that may apply only in special cir- ples.
cumstances. It is important to remember
that no single method is optimal for de- Enzyme Techniques
tecting all antibodies in all samples. Any
laboratory performing antibody detection Treatment of red cells with proteolytic en-
or identification should have standard pro- zymes enhances their reactivity with anti-
cedures for routine testing and have access bodies in the Rh, P, I, Kidd, Lewis, and
to at least some alternative approaches. some other blood group systems and si-
Additional procedures are available in a multaneously destroys or weakens reac-
variety of references (see Suggested Read- tivity with other antibodies, most notably
ing). those in the Duffy and MNS systems (see
Table 19-4). The clinical significance of
antibodies that react only with enzyme
Enhancement Techniques techniques is questionable. The literature
indicates that “enzyme-only” antibodies
When a pattern of weak reactions fails to 28
may have no clinical significance. Proce-
indicate specificity, or when the presence
dures for the preparation and use of pro-
of an antibody is suspected but cannot be
teolytic enzyme solutions are given in
demonstrated, use of the following proce-
Methods 3.5 through 3.5.6.
dures may be helpful. An autologous con-
trol should be included with each test
performed. Temperature Reduction
Some alloantibodies (eg, anti-M, -N, -P1,
LISS and PEG a b
-Le , -Le , -A1) that react at room tempera-
The rationale for these procedures and ture react better at lower temperatures;
some technical details are discussed in specificity may be apparent only below 22
Chapter 12 and Method 3.2. Each may be C. An autocontrol is especially important
used to enhance reactivity and reduce infor tests at cold temperatures because many
cubation time. LISS methods include the sera also contain anti-I or other cold-re-
use of low-ionic-strength saline for resus- active autoantibodies.

Increased Serum-to-Cell Ratio the only cells agglutinated are M+N–,

modifying the serum to a pH of 6.5 may
Increasing the volume of serum incubated
reveal a definitive pattern of anti-M reac-
with a standard volume of red cells may
tivity. The addition of one volume of 0.1 N
enhance the reactivity of antibodies pres-
HCl to nine volumes of serum brings the
ent in low concentration. One acceptable
pH to approximately 6.5. The acidified se-
procedure is to mix 5 to 10 volumes of se-
rum should be tested against known M–
rum with one volume of a 2% to 5% saline
cells as a control for nonspecific aggluti-
suspension of red cells and incubate for
nation. Similarly, some examples of anti-P
60 minutes at 37 C; periodic mixing dur-
may benefit from a lower pH.30
ing incubation promotes contact between
Low pH, however, significantly decreases
red cells and antibody molecules. It is
reactivity of some antibodies.31 If unbuffered
helpful to remove the serum before wash-
saline used for cell suspensions and for
ing the red cells for the antiglobulin test
washing has a pH much below 6.0, anti-
because the standard three or four washes
bodies in the Rh, Duffy, Kidd, and MNS sys-
may be insufficient to remove all the un-
tems may lose reactivity. Use of phos-
bound immunoglobulin present in the
phate-buffered saline (see Method 1.7) can
additional volume. Additional washes are
control pH and enhance detection of anti-
not recommended because bound anti- 32
bodies poorly reactive at a lower pH.
body molecules may dissociate. Increas-
ing the serum-to-red cell ratio is not
appropriate for tests using a low-ionic- Techniques to Isolate, Remove, or
strength medium or requiring specific Depress Antibody Reactivity
proportions of serum and additive. It is sometimes useful to decrease or elim-
inate the reactivity of an antibody. This
Increased Incubation Time can be done by inhibiting the antibody with
For most antibodies, a 15-minute incuba- specific substances, by physically remov-
tion period is insufficient to achieve equi- ing immunoglobulin molecules, or by re-
librium and the observed reactions may moving (or weakening) corresponding anti-
be weak, particularly in saline or albumin gens from the red cells. Such methods can
media. Extending incubation to 30 to 60 help confirm suspected specificities and pro-
minutes may improve reactivity and help mote identification of additional antibodies.
clarify the observed pattern of reactions.
Extended incubation may have a nega- Inhibition Tests
tive effect when LISS or PEG are used. If in- Soluble forms of some blood group anti-
cubation exceeds the recommended times gens exist in such body fluids as saliva,
for these methods, antibody reactivity may urine, or plasma, or can be prepared from
be lost. Care must be taken to use all re- other sources. These substances can be used
agents according to the manufacturer’s di- to inhibit reactivity of the corresponding
rections. antibody. If, for example, a suspected
anti-P1 does not give a definitive aggluti-
Alteration of pH nation pattern, loss of reactivity after ad-
Decreasing the pH of the reaction system dition of soluble P 1 substance strongly
to 6.5 enhances the reactivity of certain suggests that this is the specificity. A par-
antibodies, notably some examples of allel dilution control with saline is essen-
anti-M.29 If anti-M is suspected because tial.

Inhibition can also be used to neutralize 4. Chido and Rodgers substances. Ch

antibodies that mask the concomitant pres- and Rg antigens are epitopes of the
ence of nonneutralizable antibodies. The fourth component of human com-
following soluble blood group substances plement (C4).34,35 Anti-Ch and -Rg re-
can be used in antibody identification tests: act by an IAT with the trace amounts
1. Lewis substances. Lea and/or Leb sub- of C4 present on normal red cells. If
stances are present in the saliva of red cells are coated in vitro with ex-
persons who possess the Le gene. Lea cess C4,36 these antibodies may cause
substance is present in the saliva of direct agglutination. A useful test to
Le(a+b–) individuals, and Le(a–b+) identify anti-Ch and -Rg is by the in-
persons have both Lea and Leb sub- hibition of the antibodies with plasma
stances in their saliva (see Method from Ch+, Rg+ individuals (see Method
2.5). Commercially prepared Lewis 3.9).
substance is also available. 5. Blood group sugars. Sugars that cor-
2. P1 substance. Soluble P1 substance is respond to the immunodominant
present in hydatid cyst fluid and can configurations of A, B, H, and some
be prepared from pigeon egg whites. other red cell structures can be used
P1 substance is available commercially. to inhibit antibodies. Inhibiting anti-A
a a
3. Sd substance. Soluble Sd blood group or -B may allow a serum to be tested
substance is present in various body against non-group-O cells.
fluids; the most abundant source is
urine.33 To confirm anti-Sda specific- Inactivation of Blood Group Antigens
ity in a serum sample, urine from a Certain blood group antigens can be de-
known Sd(a+) individual (or a pool stroyed or weakened by suitable treatment
of urine specimens) can be used to of the cells (see Table 19-4). Modified cells
inhibit reactivity. Urine known to lack can be useful both in confirming the pres-
Sda substance, or saline, should be used ence of suspected antibodies and in de-
as a negative control (see Method 3.11). tecting additional antibodies. This can be
Table 19-4. Alteration of Antigens by Various Agents*

†
Agent Antigens Usually Denatured or Altered
Proteolytic enzymes‡ M, N, S, Fya, Fyb, Yta, Ch, Rg, Pr, Tn, Mg, Mia/Vw, Cla, Jea, Nya,
JMH, some Ge, Inb
DTT Yt , JMH, Kna, McCa, Yka, LWa, LWb, all Kell, Lutheran, Dombrock,
a
and Cromer blood group antigens

ZZAP (a combination of Alteration of all the antigens listed above
DTT and proteolytic
enzymes)
27
*Modified from Wilkinson.
†
Some antigens listed may be weakened rather than completely denatured. Appropriate controls should be used with
modified cells.
‡
Different proteolytic enzymes may have different effects on certain antigens.

especially helpful if the antigen is one of Adsorption techniques are useful in such
high incidence and antigen-negative cells situations as:
are rare. 1. Separating multiple antibodies pres-
Proteolytic enzymes are commonly used ent in a single serum.
to alter red cell antigens. Ficin, papain, 2. Removing autoantibody activity to
trypsin, and bromelin, the enzymes most permit detection of coexisting allo-
frequently used, remove antigens such as antibodies.
M, N, Fya, Fyb, Xga, JMH, Ch, and Rg (see Ta- 3. Removing unwanted antibody (often
ble 19-4). Depending on the specific enzyme anti-A and/or anti-B) from serum that
and method used, other antigens may be contains an antibody suitable for re-
altered or destroyed. Antigens inactivated agent use.
by one proteolytic enzyme will not neces- 4. Confirming the presence of specific
sarily be inactivated by other enzymes. antigens on red cells through their
Sulfhydryl reagents such as 2-amino- ability to remove antibody of corre-
ethylisothiouronium bromide (AET) or sponding specificity from previously
dithiothreitol (DTT) (see Method 3.10) can characterized serum.
be used to weaken or destroy antigens in the 5. Confirming the specificity of an anti-
Kell system and some other anti-gens.37-39 body by showing that it can be ad-
ZZAP reagent, which contains proteolytic sorbed only to red cells of a particu-
40
enzyme and DTT, denatures antigens sen- lar blood group phenotype.
sitive to DTT (eg, all Kell system antigens) Adsorption serves different purposes in
in addition to enzyme-sensitive antigens different situations; there is no single pro-
(see Method 4.9). Glycine-HCl/EDTA treat- cedure that is satisfactory for all purposes.
ment of red cells also destroys Bg and Kell A basic procedure for an antibody adsorp-
system antigens. However, with the exception can be found in Method 3.12. The
tion of Era antigen,41 other antigens outside usual serum-to-cell ratio is one volume of
the Kell system that are often destroyed by serum to an equal volume of washed red
sulfhydryl reagents remain intact (see Meth- cells. To enhance antibody uptake, the pro-
ods 2.14 and 4.2). Chloroquine diphosphate portion of antigen can be increased by us-
can be used to weaken the expression of ing a larger volume of cells. The incubation
Class I HLA antigens (Bg antigens) on red temperature should be that at which the
cells.42 Chloroquine treatment also weakens antibody is optimally reactive. Pretreating
some other antigens, including Rh antigens red cells with a proteolytic enzyme may en-
(see Method 2.13). hance antibody uptake and reduce the
number of adsorptions required for com-
plete removal of antibody. Because some
antigens are destroyed by proteases, anti-
Adsorption
bodies directed against these antigens will
Antibody can be removed from a serum not be removed by enzyme-treated red
sample by adsorption to red cells carrying cells.
the corresponding antigen. After the anti- In separating mixtures of antibodies, the
body attaches to the membrane-bound selection of red cells of the appropriate
antigens and the serum and cells are sep- phenotype is extremely important and de-
arated, the specific antibody remains at- pends on the object of the separation. If
tached to the red cells. It may be possible none of the antibodies in the serum has
to harvest the bound antibody by elution. been identified, weakly reactive cells may

be used, on the assumption that they are presence of stromal debris may in-
reactive with only a single antibody. The terfere with the reading of tests. Careful
phenotype of the person producing the an- technique and strict adherence to
tibody gives a clue to what specificities protocols should eliminate such prob-
might be present, and cells intended to lems.
separate those particular antibodies can be 2. Incomplete washing. The sensitized
chosen. If one or more antibodies have red cells must be thoroughly washed
been identified, cells lacking those antigens before elution to prevent contami-
are usually chosen so that only one anti- nation of the eluate with residual se-
body is removed. Adsorption requires a rum antibody. If it is known that the
substantial volume of red cells. Vials of re- serum does not contain antibody,
agent red cells usually will not suffice, and saline washing may not be necessary.
blood samples from staff members or donor Six washes with saline are usually
units are the most convenient sources. adequate, but more may be needed
if the serum contains a high-titer an-
tibody. To determine the efficacy of
Elution the washing process, supernatant
Elution frees antibody molecules from fluid from the final wash phase should
sensitized red cells. Bound antibody may be tested for antibody activity and
be released by changing the thermody- should be nonreactive.
namics of antigen-antibody reactions, by 3. Binding of proteins to glass surfaces.
neutralizing or reversing forces of attrac- If the eluate is prepared in the same
tion that hold antigen-antibody complexes test tube that was used during the
together, or by disturbing the structure of sensitization phase (eg, in an ad-
the antigen-antibody binding site. The sorption/elution process), antibody
usual objective is to recover bound anti- nonspecifically bound to the test
body in a usable form. tube surface may dissociate during
Various elution methods have been de- the elution. Similar binding can also
scribed. Selected procedures are given in occur from a whole blood sample if
Methods 4.1 through 4.5. No single method the patient has a positive DAT and
is best in all situations. Use of heat or free antibody in the serum. To avoid
freeze-thaw elution is usually restricted to such contamination, the washed red
the investigation of HDFN due to ABO in- cells should be transferred into a clean
compatibility because these elution proce- test tube before the elution procedure
dures rarely work well for antibodies out- is begun.
side the ABO system. Acid or organic solvent 4. Dissociation of antibody before elu-
methods are used for elution of warm-reaction. IgM antibodies, such as anti-A
tive auto- and alloantibodies. or -M, may spontaneously dissociate
Technical factors that influence the suc- from the cells during the wash phase.
cess of elution procedures include: To minimize this loss of bound anti-
1. Incorrect technique. Such factors as body, cold (4 C) saline can be used
incomplete removal of organic solvents for washing. Although this is not a
or failure to correct the tonicity or concern with most IgG antibodies,
pH of an eluate may cause the red some low-affinity IgG antibodies can
cells used in testing the eluate to also be lost during the wash phase. If
hemolyze or to appear “sticky.” The such antibodies are suspected, wash-

ing with cold LISS instead of normal and treated serum can be used for further
saline may help maintain antibody testing. Unmodified red cells are generally
association. used for adsorption and subsequent elu-
5. Instability of eluates. Dilute protein tion; elution from enzyme- or ZZAP-
solutions, such as those obtained by treated cells may create technical prob-
elution into saline, are unstable. Elu- lems.
ates should be tested as soon after
preparation as possible. Alternatively,
Use of Sulfhydryl Reagents
bovine albumin may be added to a
final concentration of 6% w/v and the Sulfhydryl reagents, such as DTT and
preparation stored frozen. Eluates can 2-mercaptoethanol (2-ME), cleave the
also be prepared directly into anti- disulfide bonds that join the monomeric
body-free plasma, 6% albumin, or a subunits of the IgM pentamer. Intact 19S
similar protein medium instead of IgM molecules are cleaved into 7S subunits,
into saline. which have altered serologic reactivity.43
Elution techniques are useful for: The interchain bonds of 7S Ig monomers
1. Investigation of a positive DAT (see are relatively resistant to such cleavage
Chapter 20). (see Chapter 11 for the structure of immu-
2. Concentration and purification of noglobulin molecules). Sulfhydryl re-
antibodies, detection of weakly ex- agents are used to diminish or destroy
pressed antigens, and identification IgM antibody reactivity. DTT also destroys
of multiple antibody specificities. certain red cell antigens. The applications
Such studies are used in conjunction of DTT and 2-ME in immunohematology
with an appropriate adsorption tech- include:
nique, as described above and in 1. Determining the immunoglobulin
Method 2.4. class of an antibody (see Method 3.8).
3. Preparation of antibody-free red cells 2. Identifying specificities in a mixture
for use in phenotyping or autologous of IgM and IgG antibodies, particu-
adsorption studies. Procedures used larly when an agglutinating IgM an-
to remove cold- and warm-reactive tibody masks the presence of IgG
autoantibodies from red cells are antibodies.
discussed in Method 4.6 and Method 3. Determining the relative amounts of
4.9, and a discussion of autologous IgG and IgM components of a given
adsorption of warm-reactive auto- specificity (eg, anti-A or -B).
antibodies appears in Chapter 20. 4. Dissociating red cell agglutinates
caused by IgM antibodies (eg, the
spontaneous agglutination of red
Combined Adsorption-Elution cells caused by potent autoantibodies)
Combined adsorption-elution tests can be (see Method 2.11).
used to separate mixed antibodies from a 5. Dissociating IgG antibodies from red
single serum, to detect weakly expressed cells using a mixture of DTT and a
antigens on red cells, or to help identify proteolytic enzyme (ZZAP reagent)
weakly reactive antibodies. The process (see Method 4.9).
consists of first incubating serum with se- 6. Converting nonagglutinating IgG an-
lected cells, then eluting antibody from tibodies into direct agglutinins. 44
the adsorbing red cells. Both the eluate Commercially prepared, chemically

modified, blood typing reagents for Most weakly reactive antibodies

use in rapid saline tube, slide, or lose reactivity when diluted even
microplate tests have been manu- modestly, but some antibodies that
factured in this manner (see Chapter give weak reactions when undiluted
12). continue to react at dilutions as high
7. Destroying selected red cell antigens as 1 in 2048. Such antibodies include
(eg, those of the Kell, Dombrock, anti-Ch, -Rg, -Csa, -Yka, -Kna, -McCa,
Cartwright, and LW systems) for use -JMH, and other specificities. When
in antibody investigations (see Method weak reactions are observed in indi-
3.10). rect antiglobulin tests, titration may
be used to indicate specificity within
this group. Not all antibodies of the
Titration
specificities mentioned demonstrate
The titer of an antibody is usually deter- such “high titer, low avidity” charac-
mined by testing serial twofold dilutions teristics. Thus, although demonstra-
of the serum against selected red cell tion of these serologic characteristics
samples. Results are expressed as the re- may help point to certain specifici-
ciprocal of the highest serum dilution that ties, failure to do so does not elimi-
shows macroscopic agglutination. Titra- nate those possibilities. Antibodies
tion values can provide information about of other specificities may sometimes
the relative amount of antibody present in react at high titers. Details of titra-
a serum, or the relative strength of anti- tion are given in Method 3.7 and
gen expression on red cells. Method 3.9.
Titration studies are useful in the follow- 3. Separating multiple antibodies. Titra-
ing situations: tion results may suggest that one an-
1. Prenatal studies. When the antibody tibody reacts at higher dilutions than
is of a specificity known to cause HDFN another. This information can allow
or its clinical significance is un- the serum to be diluted before test-
known, the results of titration stud- ing against a cell panel, effectively
ies may contribute to the decision removing one antibody and allowing
about performing invasive proce- identification of the other.
dures, eg, amniocentesis (see Chap-
ter 23 and Method 5.3).
2. Antibody identification. Some anti- Other Methods
bodies that agglutinate virtually all Methods other than traditional tube tech-
reagent red cell samples may pro- niques may be used for antibody identifi-
duce an indication of specificity by cation. Some are especially useful for
demonstrating reactivity of different identifying individual antibody speci-
strength with different samples in ti- ficities, for dealing with small volumes of
tration studies. For example, potent test reagents, for batch testing, or for use
autoanti-I may react in the undi- with automated systems. Such methods
luted state with both adult and cord include testing in capillary tubes, micro-
red cells, but titration may reveal re- plates, or by solid phase; enzyme-linked
activity at a higher dilution with immunosorbent assays; and column ag-
adult I+ red cells than with cord red glutination (eg, gel techniques). Other
cells. methods useful in laboratories with spe-

cialized equipment include radioimmuno- 15. Brendel WL. Resolving antibody problems.
In: Pierce SR, Wilson JK, eds. Approaches to
assay, immunofluorescence (including flow
serological problems in the hospital transfu-
cytometric procedures), immunoblotting, sion service. Arlington, VA: AABB, 1985:51-72.
and immunoelectrode biosensoring. Some 16. Reid ME, Lomas-Francis C. The blood group
of these methods are discussed in Chap- antigen factsbook. 2nd ed. New York: Academic
Press, 2004.
ter 12.
Scientific Publications, 1998.
18. Malyska H, Kleeman JE, Masouredis SP, et al.
References Effects on blood group antigens from storage
1. Giblett ER. Blood group alloantibodies: An at low ionic strength in the presence of
assessment of some laboratory practices. neomycin. Vox Sang 1983;44:375-84.
Transfusion 1977;17:299-308. 19. Westhoff CM, Sipherd BD, Toalson LD. Red cell
2. Walker RH, Lin DT, Hatrick MB. Alloimmuni- antigen stability in K3EDTA. Immunohema-
zation following blood transfusion. Arch tology 1993;9:109-11.
Pathol Lab Med 1989;113:254-61. 20. Rodberg K, Tsuneta R, Garratty G. Discrepant
3. Daniels G, Poole J, deSilva M, et al. The clini- Rh phenotyping results when testing IgG-
cal significance of blood group antibodies. sensitized rbcs with monoclonal Rh reagents
Tranfus Med 2002;12:287-95. (abstract). Transfusion 1995;35(Suppl):67.
4. Code of federal regulations. Title 21 CFR Part 21. Code of federal regulations. Title 21 CFR Part
660.33. Washington, DC: US Government 660.25. Washington, DC: US Government
Printing Office, 2004 (revised annually). Printing Office, 2004 (revised annually).
5. Standards Source – 4.3.2.1. ( January 2004) 22. Food and Drug Administration. Compliance
Bethesda, MD: AABB, 2004. program guidance manual. Chapter 42; in-
6. Howard JE, Winn LC, Gottlieb CE, et al. Clini- spection of licensed and unlicensed blood
cal significance of anti-complement compo- banks, brokers, reference laboratories, and
nent of antiglobulin antisera. Transfusion contractors—program 7342.001. Attachment
1982;22:269-72. C—Product testing system, blood grouping
7. Judd WJ, Fullen DR, Steiner EA, et al. Revisit- and typing (ABO and Rh), and compatibility
ing the issue: Can the reading for serologic retesting. Rockville, MD: CBER Office of Com-
activity following 37 C incubation be omitted? munication, Training, and Manufacturers As-
Transfusion 1999;39:295-9. sistance, 2003. [Available at http://www.fda.
8. Issitt PD. Antibody screening: Elimination of gov/cber/cpg.htm.]
another piece of the test (editorial). Transfu-
23. Shirey RS, Edwards RE, Ness PM. The risk of
sion 1999;39:229-30.
alloimmunization to c (Rh4) in R1R1 patients
9. Shulman IA, Calderon C, Nelson JM, Nakayama
who present with anti-E. Transfusion 1994;34:
R. The routine use of Rh-negative reagent red
756-8.
cells for the identification of anti-D and the
24. Shulman IA. Controversies in red blood cell
detection of non-D red cell antibodies. Trans-
compatibility testing. In: Nance SJ, ed. Immune
fusion 1994;34:666-70.
destruction of red blood cells. Arlington, VA:
10. Fisher RA. Statistical methods and scientific
AABB, 1989:171-99.
inference. 2nd ed. Edinburgh, Scotland: Oli-
ver and Boyd, 1959. 25. Reisner R, Butler G, Bundy K, Moore SB.
11. Harris RE, Hochman HG. Revised p values in Comparison of the polyethylene glycol anti-
testing blood group antibodies. Transfusion globulin test and the use of enzymes in anti-
1986;26:494-9. body detection. Transfusion 1996;36:487-9.
12. Kanter MH, Poole G, Garratty G. Misinterpre- 26. Issitt PD, Combs MR, Bumgarner DJ, et al.
tation and misapplication of p values in anti- Studies of antibodies in the sera of patients
body identification: The lack of value of a p who have made red cell autoantibodies.
value. Transfusion 1997;37:816-22. Transfusion 1996;36:481-6.
13. Reid ME, Oyen R, Storry J, et al. Interpreta- 27. Wilkinson SL. Serological approaches to
tion of RBC typing in multi-transfused pa- transfusion of patients with allo- or auto-
tients can be unreliable (abstract). Transfu- antibodies. In: Nance SJ, ed. Immune de-
sion 2000;40 (Suppl):123. struction of red blood cells. Arlington, VA:
14. Silva MA, ed. Standards for blood banks and AABB, 1989:227-61.
transfusion services. 23rd ed. Bethesda, MD: 28. Issitt PD, Combs MR, Bredehoeft SJ, et al.
AABB, 2005. Lack of clinical significance of “enzyme-only”

red cell alloantibodies. Transfusion 1993;33:

284-93. Suggested Reading
29. Beattie KM, Zuelzer WW. The frequency and
properties of pH-dependent anti-M. Transfu- Boorman KE, Dodd BE, Lincoln PJ. Blood group
sion 1965;5:322-6. serology. 6th ed. Edinburgh, Scotland: Churchill
30. Judd WJ. A pH dependent autoagglutinin with Livingstone, 1988.
anti-P specificity. Transfusion 1975;15:373-6.
31. Bruce C, Watt AH, Hare V, et al. A serious Crookston MC. Soluble antigens and leukocyte re-
source of error in antiglobulin testing. Trans- lated antibodies. Part A. Blood group antigens in
fusion 1986;26:177-81. plasma: An aid in the identification of antibodies.
In: Dawson RD, ed. Transfusion with “crossmatch
32. Rolih S, Thomas R, Fisher F, Talbot J. Antibody
incompatible” blood. Washington, DC: AABB, 1975:
detection errors due to acidic or unbuffered
20-5.
saline. Immunohematology 1993;9:15-8.
33. Morton J, Pickles MM, Terry AM. The Sd a Daniels G. Human blood groups. 2nd ed. Oxford,
blood group antigen in tissues and body flu- England: Blackwell Scientific Publications, 2002.
ids. Vox Sang 1970;19:472-82.
34. O’Neil GJ, Yang SY, Tegoli J, et al. Chido and Engelfriet CP, Overbeeke MAM, Dooren MC, et al.
Rodgers blood groups are distinct antigenic Bioassays to determine the clinical significance of
components of human complement, C4. Na- red cell antibodies based on Fc receptor-induced
ture 1978;273:668-70. destruction of red cells sensitized by IgG. Transfu-
sion 1994;14:617-26.
35. Tilley CA, Romans DG, Crookston MC. Local-
ization of Chido and Rodgers to the C4d frag- Garratty G. In-vitro reactions with red blood cells
ment of human C4 (abstract). Transfusion that are not due to blood group antibodies: A re-
1978;18:622. view. Immunohematology 1998;14(1):1-11.
36. Judd WJ, Kreamer K, Moulds JJ. The rapid
identification of Chido and Rodgers antibod- Issitt PD, Anstee DJ. Applied blood group serology.
ies using C4d-coated red blood cells. Transfu- 4th ed. Durham, NC: Montgomery Scientific Pub-
sion 1981;21:189-92. lications, 1998.
37. Advani H, Zamor J, Judd WJ, et al. Inactiva-
Johnson ST, Rudmann SV, Wilson SM, eds. Sero-
tion of Kell blood group antigens by 2-amino-
logic problem-solving strategies: A systematic ap-
ethylisothiouronium bromide. Br J Haematol
proach. Bethesda, MD: AABB, 1996.
1982;51:107-15.
38. Branch DR, Muensch HA, Sy Siok Hian AL, Judd WJ. Elution of antibody from red cells. In:
Petz LD. Disulfide bonds are a requirement Bell CA, ed. A seminar on antigen-antibody reac-
for Kell and Cartwright (Yta) blood group an- tions revisited. Washington, DC: AABB, 1982:175-
tigen integrity. Br J Haematol 1983;54:573-8. 221.
39. Moulds J, Moulds MM. Inactivation of Kell
blood group antigens by 2-amino-ethylisothio- Judd WJ. Methods in immunohematology. 2nd ed.
uronium bromide. Transfusion 1983;23:274-5. Durham, NC: Montgomery Scientific Publications,
1994.
40. Branch DR, Petz LD. A new reagent (ZZAP)
having multiple applications in immuno- Kanter MH. Statistical analysis. In: Busch MP, Brecher
hematology. Am J Clin Pathol 1982;78:161-7. ME, eds. Research design and analysis. Bethesda,
41. Liew YW, Uchikawa M. Loss of Era antigen in MD: AABB, 1998:63-104.
very low pH buffers. Transfusion 1987;27:442-3.
42. Swanson JL, Sastamoinen R. Chloroquine Mallory D, ed. Immunohematology methods and
stripping of HLA A,B antigens from red cells procedures. Rockville, MD: American Red Cross,
(letter). Transfusion 1985;25:439-40. 1993.
43. Freedman J, Masters CA, Newlands M, et al. Marsh WL, Reid ME, Kuriyan M, et al. A handbook
Optimal conditions for use of sulphydryl of clinical and laboratory practices in the transfu-
compounds in dissociating RBC antibodies. sion of red blood cells. Moneta, VA: Moneta Medi-
Vox Sang 1976;30:231-9. cal Press, 1993.
44. Romans DG, Tilley CA, Crookston MC, et al.
Conversion of incomplete antibodies to di- Menitove JE. The Hardy-Weinberg principle: Se-
rect agglutinins by mild reduction. Evidence lection of compatible blood based on mathematic
for segmental flexibility within the Fc frag- principles. In: Fridey JL, Kasprisin CA, Chambers
ment of immunoglobulin G. Proc Natl Acad LA, Rudmann SV, eds. Numbers for blood bankers.
Sci U S A 1977;74:2531-5. Bethesda, MD: AABB, 1995:1-11.

Mollison PL, Engelfriet CP, Contreras M. Blood Rolih S. A review: Antibodies with high-titer, low-
transfusion in clinical medicine. 10th ed. London: avidity characteristics. Immunohematology 1990;
Blackwell Scientific Publications, 1997. 6:59-67.
Race RR, Sanger R. Blood groups in man. 6th ed. Telen MJ. New and evolving techniques for anti-
Oxford, England: Blackwell Scientific Publications, body and antigen identification. In: Nance ST, ed.
1975. Alloimmunity: 1993 and beyond. Bethesda, MD:
AABB, 1993:117-39.
Reid ME, Lomas-Francis C. The blood group anti-
gen factsbook. 2nd ed. New York: Academic Press,
2004.

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction
Chapter 20
The Positive Direct

Antiglobulin Test and 20
Immune-Mediated Red
Cell Destruction
or recipients of high-dose intravenous
T
HE DIRECT ANTIGLOBULIN test
(DAT) is generally used to determine gammaglobulin,1,2 or modification of
if red cells have been coated in vivo the red cell membrane by certain
with immunoglobulin, complement, or both. drugs, eg, some cephalosporins.
A positive DAT, with or without shortened 7. Red-cell-bound complement. This may
red cell survival, may result from: be due to complement activation by
1. Autoantibodies to intrinsic red cell alloantibodies, autoantibodies, drugs,
antigens. or bacterial infection.
2. Alloantibodies in a recipient’s circu- 8. Antibodies produced by passenger
lation, reacting with antigens on re- lymphocytes in transplanted organs
3
cently transfused donor red cells. or hematopoietic components.
3. Alloantibodies in donor plasma, A positive DAT does not necessarily
plasma derivatives, or blood frac- mean that a person’s red cells have short-
tions that react with antigens on the ened survival. Small amounts of both IgG
red cells of a transfusion recipient. and complement appear to be present on
4. Alloantibodies in maternal circula- all red cells. A range of 5 to 90 IgG mole-
tion that cross the placenta and coat cules/red cell4 and 5 to 40 C3d molecules/
5
fetal red cells. red cell appears to be normal on the red
5. Antibodies directed against certain cells of healthy individuals.
drugs that bind to red cell mem- The DAT can detect a level of 100 to 500
branes (eg, penicillin). molecules of IgG/red cell and 400 to 1100
6. Nonspecifically adsorbed proteins, molecules of C3d/red cell, depending on
including immunoglobulins, associ- the reagent and technique used. Positive
ated with hypergammaglobulinemia DATs without clinical manifestations of im-
453

mune-mediated red cell destruction are re- most laboratories. If cord blood samples
ported in 1 in 1000 up to 1 in 14,000 blood are to be tested, it is appropriate to use
donors and 1% to 15% of hospital patients.4 anti-IgG only because hemolytic disease of
Most blood donors with positive DATs the fetus and newborn (HDFN) results from
appear to be perfectly healthy, and most the fetal red cells becoming sensitized with
patients with positive DATs have no obvi- maternally derived IgG antibody and com-
ous signs of hemolytic anemia, although plement activation rarely occurs.3
some may show slight evidence of in-
creased red cell destruction.4,6(p222) Elevated The Pretransfusion DAT and the
levels of IgG or complement have been Autologous Control
noted on the red cells of patients with sickle Neither the AABB, in the Standards for
cell disease, β-thalassemia, renal disease, Blood Banks and Transfusion Services,8 nor
multiple myeloma, autoimmune disorders, any other accrediting agency requires a DAT
AIDS, and other diseases with elevated se- or an autologous control (autocontrol) as
rum globulin or blood urea nitrogen (BUN) part of pretransfusion testing. Studies
levels with no clear correlation between a have shown that eliminating the DAT/
positive DAT and anemia.1,2,7 Interpretation autocontrol portion of routine pretransfu-
of positive DATs should include the pa- sion testing carries minimal risk.9
tient’s history, clinical data, and the results
of other laboratory tests.
Evaluation of a Positive DAT
Extent of Testing
The Direct Antiglobulin Test Clinical considerations should dictate the
The principles of the DAT are discussed in extent to which a positive DAT is evalu-
Chapter 12. Although any red cells may be ated. Dialogue with the attending physi-
tested, EDTA-anticoagulated blood sam- cian is important. Interpretation of the
ples are preferred to prevent in-vitro fixa- significance of serologic findings requires
tion of complement. If red cells from a knowledge of the patient’s diagnosis; re-
clotted blood sample have a positive DAT cent drug, pregnancy, and transfusion
due to complement, the results should be history; and information on the presence
confirmed on cells from a freshly col- of acquired or unexplained hemolytic
lected or EDTA-anticoagulated specimen anemia. The results of serologic tests alone
if those results are to be used for diagnos- are not diagnostic; their significance must
tic purposes. be assessed in conjunction with clinical
Most DATs are initially performed with information and such laboratory data as
a polyspecific antihuman globulin (AHG) hematocrit, bilirubin, haptoglobin, and
reagent capable of detecting both IgG and reticulocyte count. When investigating a
C3d (see Method 3.6). If positive, tests with transfusion reaction, performance of the
specific anti-IgG and anticomplement re- DAT on postreaction specimens is part of
agents may be appropriate. Occasionally, the initial transfusion reaction investiga-
polyspecific AHG reagents react with cell- tion. The DAT may be positive if sensi-
bound proteins other than IgG or C3d (eg, tized red cells have not been destroyed or
IgM, IgA, or other complement compo- negative if hemolysis and rapid clearance
nents); specific reagents to distinguish have occurred. Positive DAT results should
these proteins are not readily available in be further evaluated. The patient’s history

Chapter 20: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction 455
is important in interpreting a posttransfu- persist for more than 300 days fol-
sion reaction positive DAT (see Chapter 27). lowing a transfusion reaction, which
Answers to the following questions may is far longer than the transfused cells
help decide what investigations are appro- would be expected to survive, sug-
priate: gesting that autologous as well as
1. Is there any evidence of in-vivo red transfused red cells are sensitized
cell destruction? Reticulocytosis, following a transfusion reaction.10,11 A
spherocytes observed on the periph- mixed-field appearance in the post-
eral blood film, hemoglobinemia, transfusion DAT may or may not be
hemoglobinuria, decreased serum observed.
haptoglobin, and elevated levels of 3. Is the patient receiving any drugs, such
serum unconjugated bilirubin or as cephalosporins, procainamide, intra-
lactate dehydrogenase (LDH), espe- venous penicillin, or -methyldopa?
cially LDH1, may be associated with Cephalosporins are associated with
increased red cell destruction. If an positive DATs; the second- and third-
anemic patient with a positive DAT generation cephalosporins can be as-
does show evidence of hemolysis, sociated with immune red cell destruc-
testing to evaluate a possible im- tion.12 In one study, 21% of patients
mune etiology is appropriate. IF receiving procainamide developed a
THERE IS NO EVIDENCE OF IN- positive DAT (three of whom had evi-
CREASED RED CELL DESTRUC- dence of hemolytic anemia).13 A high in-
TION, NO FURTHER STUDIES ARE cidence (39%) of positive DATs has been
NECESSARY, unless the patient needs reported in patients taking Unasyn.14
transfusion and the serum contains Although not commonly seen in recent
incompletely identified unexpected years, approximately 3% of patients re-
antibodies to red cell antigens. ceiving intravenous penicillin, at very
2. Has the patient been recently trans- high doses, and 15% to 20% of patients
fused? Many workers routinely at- receiving α-methyldopa will develop a
tempt to determine the cause of a positive DAT. However, fewer than 1%
positive DAT when the patient has of those patients who develop a posi-
received transfusions within the pre- tive DAT have hemolytic anemia. Posi-
vious 3 months because the first in- tive DATs associated with other drugs
dication of a developing immune re- are rare. If a positive DAT is found in a
sponse may be the attachment of patient receiving such drugs, the at-
antibody to recently transfused red tending physician should be alerted so
cells. Antibody may appear as early that appropriate surveillance for red
as 7 to 10 days (but typically 2 weeks cell destruction can be maintained. If
to several months) after transfusion red cell survival is not shortened, no
in primary immunization and as further studies are necessary.
early as 2 to 7 days (but usually with- 4. Has the patient received marrow, pe-
in 20 days) in a secondary response; ripheral blood stem cells, or an organ
these alloantibodies could shorten the transplant? Passenger lymphocytes of
survival of red cells already trans- donor origin produce antibodies di-
fused or given subsequently. rected against ABO or other antigens
Studies have shown that the posi- on the recipient’s cells, causing a pos-
tive DAT and reactive eluates can itive DAT.3

5. Is the patient receiving IGIV or intra- See Appendix 20-1 for an example of an
venous RhIG? Immune Globulin, In- algorithm for investigating a positive DAT
travenous (IGIV) may contain ABO (excluding investigation of HDFN).
antibodies, anti-D, or, sometimes, other
antibodies. Intravenous Rh Immune
Globulin (RhIG) causes Rh-positive Elution
patients to develop a positive DAT.15 Elution frees antibody from sensitized red
6. Is the patient septic? Complement cells and recovers antibody in a usable form.
activation can occur in septic patients, Details of eluate preparation are given in
leading to intravascular hemolysis. Chapter 19 and in Methods 4.1 through
This is most often seen in cases of 4.5. Commercial elution kits are also
polyagglutination resulting from or- available. Table 20-1 lists the advantages
ganisms that produce neuraminidase. 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
Serologic Studies 2.11, 2.12, 2.13, and 2.14) remove anti-
Three investigative approaches are help- body but leave the cells sufficiently intact
ful in the evaluation of a positive DAT. to allow testing for various antigens or for
1. Test the DAT-positive red cells with use in adsorption procedures. Some anti-
anti-IgG and anti-C3d reagents to gens may be altered by elution, however,
characterize the types of proteins and appropriate controls are essential.
coating the red cells. In cases of HDFN or hemolytic transfu-
2. Test the serum/plasma to detect and sion reactions, specific antibody (or anti-
identify clinically significant antibodies) is usually detected in the eluate,
bodies to red cell antigens. which may or may not be detectable in the
3. Test an eluate (see Methods 4.1 through serum. In the case of transfusion reactions,
4.5) prepared from the coated red newly developed antibodies initially detect-
cells with a panel of reagent red cells able only in the eluate are usually detect-
to define whether the coating protein able in the serum after about 14 to 21 days.
has red cell antibody activity. When Eluate preparation from the patient’s red
the only coating protein is comple- cells often concentrates antibody activity
ment, eluates are frequently nonreac- and may facilitate identification of weakly
tive. However, an eluate from the pa- reactive serum antibodies.
tient’s red cells coated only with When the eluate reacts with all the cells
complement should be tested if there tested, autoantibody is the most likely ex-
is clinical evidence of antibody-me- planation, especially if the patient has not
diated hemolysis. The eluate prepa- been recently transfused. WHEN NO UN-
ration can concentrate small amounts EXPECTED ANTIBODIES ARE PRESENT IN
of IgG that may not be detectable on THE SERUM, AND IF THE PATIENT HAS
direct testing using routine methods. NOT BEEN RECENTLY TRANSFUSED, NO
Results of these tests combined with FURTHER SEROLOGIC TESTING OF AN
the patient’s history and clinical data AUTOANTIBODY IS NECESSARY.
should assist in classification of the A nonreactive eluate prepared from IgG-
problems involved. coated red cells may have several causes.

Table 20-1. Antibody Elution Techniques

Method Advantages Disadvantages
Heat (56 C) Good for ABO-HDFN; quick and Poor recovery of other blood
easy group allo- and
autoantibodies
Freeze-thaw Good for ABO-HDFN; quick Poor recovery of other blood
method; requires small vol- group allo- and
ume of red cells autoantibodies
Cold acid Quick and easy; adequate for Possible false-positive elution
most warm auto- and (see Leger et al16)
alloantibodies; commercial
kits available
Digitonin acid Nonhazardous; good recovery of Time-consuming washing of
most antibodies stroma
Dichloromethane/ Noncarcinogenic, nonflamma- Vapors harmful
Methylene chloride ble; good for IgG auto- and
alloantibodies
17 18
Compiled from Judd and South et al.
One cause may be that the eluate was not hospital patients with a positive DAT have a
tested against cells positive for the corre- nonreactive eluate. It is suggested that at
sponding antigen, notably group A or least one contributing factor to these posi-
group B cells, or antigens of low incidence, tive DAT results is nonspecific uptake of
which are absent from most reagent cell proteins on the red cells, which occurs in
panels. If a non-group-O patient has re- patients with elevated gamma globulin lev-
ceived plasma containing anti-A or anti-B els.1,2
(as in transfusion of group O platelets), and Reactivity of eluates can be enhanced by
the recipient appears to have immune testing them against enzyme-treated cells
hemolysis, the eluate can be tested against or by the use of enhancement techniques
A and/or B cells. If the expected ABO anti- such as polyethylene glycol (PEG). Anti-
bodies are not detected, other causes of the body reactivity can be increased by the use
positive DAT should be sought. It may be of a concentrated eluate, either by alter-
appropriate to test the eluate against red ation of the fluid-to-cell ratio or by use of
cells from recently transfused donor units, commercial concentration devices. Wash-
which could have caused immunization to ing the red cells with low-ionic-strength sa-
a rare antigen, or, in HDFN, against cells line (LISS) or cold wash solutions may pre-
from the father, from whom the infant may vent the loss of antibody while the cells are
have inherited a rare gene. Pursuing the being prepared for elution.
cause of a nonreactive eluate for patients Certain elution methods give poor re-
with no evidence of hemolysis is usually sults with certain antibodies. When eluates
not indicated. Toy et al1 showed that 79% of are nonreactive yet clinical signs of red cell

destruction are present, elution by a differ- serum bilirubin, but not by hemoglobinemia
ent method may be helpful. If both serum and hemoglobinuria. As a description of
and eluate are nonreactive at all test phases in-vitro antibody reactivity, hemolysis or
and if the patient has received high-dose lysis of the red cells with release of free he-
intravenous penicillin or other drug ther- moglobin to the surrounding media is both
apy, testing to demonstrate drug-related obvious and rare.
antibodies should be considered. 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-
Immune-Mediated body AIHA ( WAIHA), cold agglutinin
Hemolysis syndrome (CAS), mixed-type AIHA, parox-
Immune-mediated hemolysis (immune ysmal cold hemoglobinuria (PCH), and
hemolysis) is the shortening of red cell DAT-negative AIHA. Not all cases fit neatly
survival by the product(s) of an immune into these categories. Drugs (discussed in a
response. If marrow compensation is ade- later section of this chapter) may also in-
quate, the reduced red cell survival may duce immune hemolysis. The prevalence of
not result in anemia. Immune hemolysis each type can vary depending on the pa-
is only one cause of hemolytic anemia, tient population studied. Table 20-3 shows
and many causes of hemolysis are unre- the serologic characteristics of the autoim-
lated to immune reactions. The serologic mune and drug-induced hemolytic ane-
investigations carried out in the blood bank mias.
do not determine whether a patient has a DATs performed with IgG- and C3-spe-
“hemolytic” anemia. The diagnosis of cific AHG reagents as well as the serum and
hemolytic anemia rests on clinical find- eluate studies described earlier can be used
ings and such laboratory data as hemo- to help classify AIHAs. Three additional pro-
globin or hematocrit values; reticulocyte cedures may be useful: a cold agglutinin ti-
count; red cell morphology; bilirubin, ter and thermal amplitude studies (Meth-
haptoglobin, and LDH levels; and, some- ods 4.7 and 4.8) and the Donath-Landsteiner
times, red cell survival studies. The sero- test for PCH (Method 4.13).
logic findings help determine whether the The binding of antibody to red cells does
hemolysis has an immune basis and, if so, not, in itself, damage the cells. It is the phe-
what type of immune hemolytic anemia is nomena that the bound antibody-antigen
present. This is important because the complex promotes that may eventually
treatment for each type is different. damage cells. These include complement
The terms hemolysis and hemolytic are binding, adherence to Fc receptors on
frequently used to indicate both intravas- macrophages leading to phagocytosis, and
cular and extravascular red cell destruction; cytotoxic lysis. The IgG subclass of bound
however, this may be misleading. In-vivo antibody may be significant. IgG1 is the
lysis of cells and release of free hemoglobin subclass most commonly found, some-
within the intravascular compartment (ie, times alone but often in combination with
resulting in hemoglobinemia and hemoglo- other subclasses. The other IgG subclasses
binuria) is uncommon and, often, dramatic. occur more often in combination with
Extravascular hemolysis, which is more other subclasses than alone. In general,
common, is characterized by an increase in IgG3 antibodies have the most destructive

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
b. secondary (to such conditions as lymphoma, mycoplasma pneumonia, infectious
mononucleosis)
3. Mixed-type AIHA
b. secondary (to such conditions as SLE, lymphoma)
4. Paroxysmal cold hemoglobinuria
b. secondary (to such conditions as syphilis, viral infections)
5. DAT-negative AIHA
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
effects, followed by IgG1. IgG2 antibodies Warm Antibody Autoimmune Hemolytic

are associated with less destruction and Anemia
IgG4 with little to no destruction.
The most common type of AIHA is associ-
The number of antibody molecules per
ated with warm-reactive (37 C) antibod-
red cell also plays a role. The number of an-
ies. Typical serologic findings are described
tibody molecules on the red cells of appar-
below.
ently healthy blood donors with positive
DATs (<200 molecules/red cell) is far less
than that usually seen in patients with DAT
AIHA.4,5 Some patients with apparent im- When IgG-specific and complement-spe-
mune hemolysis may have negative DATs. cific AHG reagents are used, three pat-

460
Table 20-3. Serologic Findings in Immune Hemolytic Anemias

WAIHA CAS Mixed-Type AIHA PCH Drug-Induced
Percent of cases 48%19 to 70%3 16%3 to 32%19 7%19 to 8%20 Rare in adults; 32% in 12%3 to 18%19
children21
DAT IgG: C3 only: IgG + C3: C3 only: IgG:
20%3 to 66%19 91%19 to 98%3 71%19 to 100%3 94%19 to 100%3 94%19
IgG + C3: C3: IgG + C3:

24%19 to 63%3 13%3 6%19
C3:
7%19 to 14%3
Immuno- IgG (sometimes IgA or IgM IgG, IgM IgG IgG
globulin type IgM, rarely alone)
Eluate IgG antibody Nonreactive IgG antibody Nonreactive IgG antibody
Serum 57% react by saline-IAT; IgM agglutinating IgG IAT-reactive antibody IgG biphasic hemolysin IgG antibody similar to
13% hemolyze antibody; titer usually plus IgM (Donath-Landsteiner WAIHA3
enzyme-treated RBCs >1000 at 4 C; usually agglutinating antibody)3
at 37 C; 90% react at 30 C in antibody, usually
agglutinate enzyme- albumin; monoclonal react at 30-37 C in
treated RBCs at 37 C; antibody in chronic saline; high titer at
30% agglutinate disease3 4 C (classic CAS) or
untreated RBCs at low titer (<64) at
20 C; rarely 4 C3,19,20,22
agglutinate untreated
RBCs at 37 C3
Specificity Rh specificity; other Usually anti-I but can be Usually specificity is Anti-P (nonreactive with Specificity often Rh
specificities have anti-i; rarely anti-Pr23 unclear19,20,22 p and Pk RBCs) related23
been reported* Can be anti-I, -i, or other
cold agglutinin
specificities
*See text.
terns of reactivity may be found: coating treated cells or when PEG is used. The
with IgG alone, with complement alone, eluate will usually have no serologic activ-
or with both. In approximately 1% of cases, ity if the only protein coating the red cells
the DAT will be positive with a poly- is complement components. Occasion-
specific AHG reagent but negative with ally, antibody not detected by the DAT will
IgG- and complement-specific AHG re- be detected in the eluate, possibly due to
agents. Some of these may be due to at- the concentrating effect of eluate prepa-
tachment of IgM or IgA alone if reactivity ration.
with these immunoglobulins has not been
excluded by the manufacturer.3,19
Specificity of Autoantibody
The specificity of autoantibodies associ-
Serum
ated with WAIHA is complex. In routine
Autoantibody in the serum typically is IgG tests, all cells tested are usually reactive.
and reacts by indirect antiglobulin testing Some autoantibodies that have weaker or
against all cells tested.3 If the autoanti- negative reactivity with cells of rare Rh
body has been adsorbed by the patient’s phenotypes, such as D– – or Rhnull, appear
red cells in vivo, the serum may contain
to have broad specificity in the Rh system.
very little free antibody. The serum will
Apparent specificity for simple Rh antigens
contain antibody after all the specific an-
(D, C, E, c, e) is occasionally seen, either
tigen sites on the red cells have been oc-
as the sole autoantibody or as a predomi-
cupied and no more antibody can be
nant portion, based on stronger reactivity
bound in vivo. In such cases, the DAT is
with cells of certain phenotypes. Such re-
usually strongly positive. Approximately
activity is often termed a “relative” speci-
50% of patients with WAIHA have serum
ficity. Such relative specificity in a serum
antibodies that react with untreated sa-
may be mistaken for alloantibody, but cells
line-suspended red cells. When testing
negative for the apparent target antigen
with PEG, enzyme-treated red cells, or
can adsorb and remove the “mimicking”
solid-phase methods, over 90% of these 23,24
specificity.
sera can be shown to contain autoantibody.
Unusual Specificities. Apart from Rh
Approximately one-third of patients with
specificity, warm autoantibodies with many
WAIHA have cold-reactive autoagglutinins
other specificities have been reported, eg,
demonstrable in tests at 20 C, but cold ag-
specificities in the LW, Kell, Kidd, Duffy, and
glutinin titers at 4 C are normal. The pres-
Diego systems.24 Dilution and selective ad-
ence of this cold agglutinin does not
sorption of eluates may uncover specificity
mean the patient has CAS in addition to
or relative specificity of autoantibodies. Pa-
WAIHA.3
tients with autoantibodies of Kell, Rh, LW,
Ge, Sc, Lu, and Lan specificities may have
Eluate depressed expression of the respective anti-
The presence of the IgG autoantibody on gen and the DAT may be negative or very
24
the red cells may be confirmed by elution weakly positive.
at least upon initial diagnosis and/or at Practical Significance. Tests against red
pretransfusion testing. (See Methods 4.1, cells of rare phenotype and by special tech-
4.2, and 4.5.) Typically, the eluate reacts niques have limited clinical application. In
with virtually all cells tested, with reactiv- rare instances of WAIHA involving IgM ag-
ity enhanced in tests against enzyme- glutinins, determining autoantibody speci-

ficity may help differentiate such cases may depress compensatory erythropoiesis.
from typical CAS.25 It is rarely, if ever, neces- Destruction of transfused cells may increase
sary to ascertain autoantibody specificity in hemoglobinemia and hemoglobinuria. In pa-
order to select antigen-negative blood for tients with active hemolysis, transfused red
transfusion. If apparent specificity is di- cells may be destroyed more rapidly than the
rected to a high-incidence antigen (eg, patient’s own red cells. In rare cases, this may
anti-U), or when the autoantibody reacts promote hypercoagulability and dissemi-
with all red cells except those of a rare Rh nated intravascular coagulation (DIC). Trans-
phenotype (eg, D– –, Rhnull), compatible do- fusion reactions, if they occur, may be diffi-
nor blood is unlikely to be available and cult to investigate.
there is little point in determining specific- Transfusion in WAIHA. Transfusion is
ity. Such blood, if available, should be especially problematic for patients with
reserved for alloimmunized patients of that rapid in-vivo hemolysis, who may present
uncommon phenotype. with a very low hemoglobin level and hypo-
Transfusion-Stimulated Autoantibodies. tension. Reticulocytopenia may accompany
Transfusion itself may lead to the produc- a rapidly falling hematocrit, and the patient
tion of autoantibodies that may persist and may exhibit coronary insufficiency, conges-
cause positive DATs for some time after tive heart failure, cardiac decompensation,
transfusion yet not cause obvious red cell or neurologic impairment. Under these cir-
destruction. Such cell-bound autoantibodies cumstances, transfusion is usually required
sometimes display blood group specificity as a lifesaving measure. The transfused
(eg, E, K, Jka). The positive DAT may persist cells may support oxygen-carrying capacity
long after transfused red cells should have until the acute hemolysis diminishes or
disappeared from the circulation, appar- other therapies can effect a more lasting
ently adsorbed to the patient’s own anti- benefit. These patients represent a signifi-
gen-negative red cells.11 cant challenge because serologic testing
may be complex while clinical needs are
acute.
Transfusion of Patients with Warm-Reactive Transfusion should not be withheld
Autoantibodies solely because of serologic incompatibility.
Inherent Risks. Patients with warm-reac- The volume transfused should usually be
tive autoantibodies range from those with the smallest amount required to maintain
no apparent decreased red cell survival to adequate oxygen delivery, not necessarily
those with life-threatening anemia. Pa- to reach an arbitrary hemoglobin level. Vol-
tients with little or no evidence of signifi- umes of about 100 mL may be appropriate.3
cant in-vivo red cell destruction tolerate The patient should be carefully monitored
transfusion quite well. throughout the transfusion.
When autoantibody is active in serum, it Transfusion in Chronic WAIHA. Most
may be difficult to exclude the presence of patients with WAIHA have a chronic stable
alloantibodies, which increases the risk of anemia, often at relatively low hemoglobin
an adverse reaction. Transfusion may stim- levels. Those with hemoglobin levels above
ulate alloantibody production, complicat- 8 g/dL rarely require transfusion, and many
ing subsequent transfusions. Transfusion patients with levels of 5 g/dL (or even
may intensify the autoantibody, inducing lower) can be managed with bed rest and
or increasing hemolysis and making sero- no transfusions. Transfusion will be re-
logic testing more difficult. Transfusion quired if the anemia progresses or is ac-

companied by such symptoms as severe preferentially with e+ red cells), the use of
angina, cardiac decompensation, respira- blood lacking the corresponding antigen is
tory distress, and cerebral ischemia. debatable. It may be undesirable to expose
Most patients with chronic anemia due the patient to Rh antigens absent from
to WAIHA tolerate transfusion without autologous cells, especially D and espe-
overt reactions, even though the transfused cially in females who may bear children
cells may not survive any better than their later, merely to improve serologic compati-
own. Because transfusion may lead to cir- bility testing with the autoantibody (eg,
culatory overload or to increased red cell de- when a D– patient has autoanti-e).
struction, the decision to transfuse should In many cases of WAIHA, no autoanti-
be carefully considered. A few patients body specificity is apparent. The patient’s
without acute hemolysis have had severe serum reacts with all red cell samples to the
hemolytic reactions after transfusion. This same degree or reacts with red cells from
may be due to the sudden availability of different donors to varying degrees for rea-
large volumes of donor cells and the expo- sons seemingly unrelated to Rh phenotypes.
nential curve of decay, by which the num- Even if specificity is identified, the exotic
ber of cells hemolyzed is proportional to cells used for such identification are not
the number of cells present.3,6(pp230,369) available for transfusion. The most impor-
Selecting Blood. If the decision is made tant consideration in such cases is to ex-
to transfuse, selection of appropriate donor clude the presence of clinically important
blood is essential. It is important to deter- alloantibodies before selecting either pheno-
mine the patient’s ABO and Rh type and, if typically similar or dissimilar, crossmatch-
time permits, to detect potentially clinically incompatible red cells for transfusion. In
significant alloantibodies. Adsorption and extremely rare cases in which there is se-
other special techniques described later in vere and progressive anemia, it may be es-
this chapter can greatly reduce the risk of sential to transfuse blood that does not re-
undetected alloantibodies but may be act with the patient’s autoantibody.
time-consuming. If clinically significant Frequency of Testing. Although AABB
8(p38)
alloantibodies are present, the transfused Standards requires that a sample be
cells should lack the corresponding anti- tested every 3 days, some serologists con-
gen(s). tend that, in these difficult cases, the con-
If the autoantibody has apparent and tinued collection and testing (to include
relatively clear-cut specificity for a single antibody investigation) of patient samples
antigen (eg, anti-e) and there is active on- are unnecessary.26 Others disagree with that
going hemolysis, blood lacking that antigen opinion. In studies of patients with WAIHA,
may be selected. There is evidence that, in there was 12% to 40% alloimmunization,
some patients, such red cells survive better with many alloantibodies developing after
than the patient’s own red cells.6(p230),20 In the recent transfusions.27,28 These two papers
absence of hemolysis, autoantibody speci- offer methods to assist in the detection of
ficity is not important, although donor units alloantibodies in the presence of autoanti-
negative for the antigen may be chosen be- bodies. For patients with previously identi-
cause this is a simple way to circumvent the fied clinically significant antibodies, Stan-
autoantibody and detect potential alloanti- dards8(p38) requires that methods of testing
bodies. If the autoantibody shows broader shall be those that identify additional clini-
reactivity, reacting with all cells but show- cally significant antibodies. It is the exclu-
ing some relative specificity (eg, it reacts sion of newly formed alloantibodies that is

of concern. Autoantibodies that react with Serum

all reagent red cells, even weakly, are capa-
Warm IgM autoagglutinins are typically
ble of masking alloantibody reactivity; the
weak and sometimes are enhanced in the
serologic reactivity is not necessarily addi-
presence of albumin or when the serum is
tive.29 Due to the presence of autoantibodies,
acidified.33 Occasionally, optimal reactiv-
all crossmatches will be incompatible. This
ity is between 20 C and 30 C, rather than
is unlike the case of clinically significant
37 C. These antibodies have low or negli-
alloantibodies, where a compatible cross-
gible antibody titers; a 4 C titer of <64 eas-
match with antigen-negative red cells can
ily differentiates this IgM warm antibody
be obtained. Monitoring for evidence of red
from those seen in CAS.
cell destruction due to alloantibodies is dif-
ficult in patients who already have AIHA;
the patient’s own red cells and transfused Eluate
red cells will have shortened survival. In pa- IgM agglutinins are often detected in an
tients who have autoantibodies without eluate when inspected at the agglutinin
hemolytic anemia, transfused red cells phase before proceeding to the antiglo-
should have normal survival. bulin test.
An alternative transfusion management
protocol proposed by one group uses pro-
Cold Agglutinin Syndrome
phylactic antigen-matched units for pa-
tients with warm autoantibodies where fea- Cold agglutinin syndrome (also called cold
sible, in combination with streamlined hemagglutinin disease, CHD) is the hemoly-
adsorption procedures.30 Such a protocol tic anemia most commonly associated with
depends on the ability to maintain an cold-reactive autoantibodies and accounts
adequate inventory of antigen-negative for approximately 16% to 32% of all cases
units.31 of immune hemolysis.3,19 (See Table 20-3.)
It occurs as an acute or chronic condition.
IgM Warm AIHA The acute form is often secondary to
lymphoproliferative disorders (eg, lym-
AIHA associated with IgM agglutinins that
phoma) or Mycoplasma pneumoniae in-
react at 37 C is unusual but is character-
fection. The chronic form is often seen in
ized by severe hemolysis.25,32-34 The prog-
elderly patients, sometimes associated
nosis for these patients is poor.
with lymphoma, chronic lymphocytic
leukemia, or Waldenstrom’s macroglo-
DAT bulinemia. Acrocyanosis and hemoglo-
The patient’s red cells are typically spon- binuria may occur in cold weather. CAS is
taneously agglutinated, requiring disrup- often characterized by rapid agglutina-
tion of the IgM agglutinin by dithiothrei- tion, at room temperature, of red cells in
tol in order to obtain accurate DAT (and an EDTA specimen. Clumping of red cells
ABO/Rh) results. Complement is usually may be obvious in such a sample, some-
detected on the red cells. In one series, times so strong that the cells appear to be
IgG was detected in 17% of cases and IgM clotted. Problems with ABO and Rh typing
in 28%. By a more sensitive flow cyto- and other tests are not uncommon. Main-
metric method, red cell-bound IgM was taining the EDTA specimen at 37 C and
detected on 82% of patients’ red cells not washing the red cells with 37 C saline is
reacting with anti-IgM by tube DAT.33 usually necessary to disperse the cold

autoagglutinin before performing ABO and min). Hemolytic activity against untreated
Rh typing and the DAT. red cells can be demonstrated sometimes
at 20 to 25 C, and, except in rare cases
DAT with Pr specificity, enzyme-treated red
cells are hemolyzed in the presence of ad-
Complement is the only protein detected
equate complement.
on the red cells in almost all cases. If other
Determination of the true thermal am-
proteins are detected, a negative control
plitude or titer of the cold autoagglutinin
for the DAT, eg, 6% to 10% albumin,
requires that the specimen be collected and
should be tested to ensure that the cold
maintained strictly at 37 C until the serum
autoagglutinin is not causing a false-posi-
and cells are separated, to avoid in-vitro
tive test.
autoadsorption. Alternatively, plasma can
The cold-reactive autoagglutinin is usu-
be used from an EDTA-anticoagulated
ally IgM, which binds to red cells in the
specimen that has been warmed for 10 to
comparatively low temperature of the pe-
15 minutes at 37 C (with repeated mixing)
ripheral circulation and causes comple-
and then separated from the cells, ideally at
ment components (C3 and C4 in particular)
37 C. This should release autoadsorbed an-
to attach to the red cells. As the red cells cir-
tibody back into the plasma.
culate to warmer areas, the IgM dissociates,
In chronic CAS, the IgM autoagglutinin
but the complement remains. Red-cell-
is usually a monoclonal protein with kappa
bound C3b can react with the CR1 or CR3
light chains. In the acute form induced by
receptors of macrophages in the reticulo-
Mycoplasma or viral infections, the anti-
endothelial system. More of the red cell de-
body is polyclonal IgM with normal kappa
struction occurs in the liver. Regulatory
and lambda light-chain distribution. Rare
proteins convert the bound C3 and C4 to
examples of IgA and IgG cold-reactive
C3dg and C4d, and it is the anti-C3d com-
autoagglutinins have also been described.
ponent of polyspecific AHG reagents that
accounts for the positive DAT. The presence
of C3dg alone does not shorten red cell sur- Eluate
vival because macrophages have no C3dg Elution is seldom necessary in obvious
or C3d receptors. cases of CAS. If the red cells have been
collected properly and washed at 37 C,
Serum there will be no immunoglobulin on the
cells and no reactivity will be found in the
IgM cold-reactive autoagglutinins associ-
eluate.
ated with immune hemolysis usually re-
act ≥30 C and have a titer ≥1000 when
tested at 4 C; they rarely react with sa- Specificity of Autoantibody
line-suspended red cells above 32 C. If The autoantibody specificity in CAS is
30% bovine albumin is included in the re- usually of academic interest only. CAS is
action medium, 100% and 70% of clini- most often associated with antibodies with
cally significant examples will react at 30 I specificity.3,23 Less commonly, i specific-
35
C or 37 C, respectively. Occasionally, ity is found, usually associated with infec-
pathologic cold agglutinins will have a tious mononucleosis.3 On rare occasions,
lower titer (ie, <1000), but they will have a cold-reactive autoagglutinins with Pr or
high thermal amplitude (ie, reactive at 30 other specificities are seen3,23 (see Method
C with or without the addition of albu- 4.7). Dilution of the serum may be neces-

sary to demonstrate specificity of very transfused, rabbit red cells may be used to
high-titer antibodies. remove autoanti-I and -IH from sera36; clini-
Autoantibody specificity is not diagnos- cally significant alloantibodies, notably
tic for CAS. Autoanti-I may be seen in anti-B, -D, -E, Vel, and others, have been re-
healthy subjects as well as patients with moved by this method.37,38 A preparation of
CAS. The nonpathologic forms of auto- rabbit red cell stroma is commercially avail-
anti-I, however, rarely react to titers above able. Alternatively, allogeneic adsorption
64 at 4 C, and are usually nonreactive with studies at 4 C can be performed as for
I– (i cord and i adult) red cells at room tem- WAIHA (see below).
perature. In contrast, the autoanti-I of CAS
may react quite strongly with I– red cells in Mixed-Type AIHA
tests at room temperature, and equal or Although about one-third of patients with
even stronger reactions are observed with WAIHA have nonpathologic IgM antibod-
I+ red cells. Autoanti-i reacts in the oppo- ies that react to high titer at low tempera-
site manner, demonstrating stronger reac- ture, another group of patients with
tions with I– red cells than with red cells WAIHA have cold agglutinins that react at
that are I+. Procedures to determine the or above 30 C. This latter group is referred
titers and specificities of cold-reactive auto- to as “mixed-type” AIHA and can be sub-
antibodies are given in Method 4.6 and divided: patients with high titer, high
Method 4.7. thermal amplitude IgM cold antibodies
Pretransfusion Testing. Antibody detec- (the rare WAIHA plus classic CAS) and pa-
tion tests should be performed in ways that tients with normal titer (<64 at 4 C), high
minimize cold-reactive autoantibody activ- thermal amplitude cold antibodies.19,20,22,39
ity yet still permit detection of clinically sig- Patients with mixed-type AIHA often
nificant alloantibodies. The use of albumin present with hemolysis and complex se-
and other potentiators may increase the re- rum reactivity present in all phases of
activity of the autoantibodies. To avoid the testing. Typical serologic findings are de-
detection of bound complement, most scribed below.
serologists use an IgG-specific reagent,
rather than a polyspecific AHG serum. Ad-
DAT
ditionally, a prewarming technique may be
used (see Method 3.3). When the patient has WAIHA plus classic
Adsorption Procedures. When cold-re- CAS, both IgG and C3 are usually detect-
active autoantibody reactivity continues to able on the patient’s red cells. When the
interfere with antibody detection tests (eg, cold agglutinin has a normal titer, but
when performed strictly at 37 C), cold high thermal amplitude (greater than or
autoadsorption studies (see Method 4.6) equal to 30 C), IgG and/or C3 may be de-
can be helpful. One or two cold auto- tectable on the red cells.3
adsorptions should remove enough auto-
antibody to make it possible to detect Serum
alloantibodies at 37 C that were otherwise Both warm-reactive IgG autoantibodies
masked by the cold-reactive autoantibody; and cold-reactive, agglutinating IgM auto-
many cold autoadsorptions would be re- antibodies are present in the serum. These
quired to remove enough of the cold-reac- usually result in reactivity at all phases of
tive autoantibody for room temperature testing, with virtually all cells tested. The
testing. If the patient has been recently IgM agglutinating autoantibody(ies) re-

acts at 30 C or above. If adsorption studies DAT

are done to detect alloantibodies, it may
PCH is caused by an IgG complement-fix-
be necessary to perform adsorptions at both
ing antibody, but, as with IgM cold-reac-
warm and cold temperatures.
tive autoagglutinins, it reacts with red
cells in colder areas of the body (usually
the extremities), causes C3 to bind irre-
Eluate
versibly to red cells, and then the anti-
A suitably prepared eluate will contain a body dissociates from the red cells as the
warm-reactive IgG autoantibody. blood circulates to warmer parts of the
body. Red cells washed in a routine man-
ner for the DAT are usually coated only
Specificity of Autoantibodies with complement components, but IgG
The unusual cold-reactive IgM agglutinat- may be detectable on cells that have been
ing autoantibody can have specificities washed with cold saline and tested with
3
typical of CAS (ie, I or i) but often has no cold anti-IgG reagent. Keeping the system
apparent specificity.19,20,22 The warm-reac- nearer its optimal binding temperature
tive IgG autoantibody often appears sero- allows the cold-reactive IgG autoantibody
logically indistinguishable from specifi- to remain attached to its antigen.
cities encountered in typical WAIHA.
Serum
Transfusion in Mixed-Type AIHA
The IgG autoantibody in PCH is classically
If blood transfusions are necessary, the
described as a biphasic hemolysin because
considerations in the selection of blood
binding to red cells occurs at low temper-
for transfusion are identical to those de-
atures but hemolysis does not occur until
scribed for patients with acute hemolysis
the coated red cells are warmed to 37 C.
due to WAIHA (see above).
This is the basis of the diagnostic test for
the disease, the Donath-Landsteiner test
Paroxysmal Cold Hemoglobinuria
(see Method 4.13). The autoantibody may
The rarest form of DAT-positive AIHA is agglutinate normal red cells at 4 C but
PCH. In the past, it was characteristically rarely to titers greater than 64. Because the
associated with syphilis, but this associa- antibody rarely reacts above 4 C, the se-
tion is now unusual. More commonly, PCH rum is usually compatible with random
presents as an acute transient condition donor cells by routine crossmatch proce-
secondary to viral infections, particularly dures and pretransfusion antibody detec-
in young children. In such cases, the tion tests are usually nonreactive.
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 Eluate
none of 531 adults having well-defined Because complement components are
immune hemolytic anemias had Donath- usually the only globulins present on cir-
Landsteiner hemolysins, whereas 22 of 68 culating red cells, eluates prepared from
(32%) children were shown to have Donath- red cells of patients with PCH are almost
Landsteiner hemolysins.21 always nonreactive.

Specificity of Autoantibody not causing the positive results.3,40 There

may be too few antibody molecules on
The autoantibody of PCH has most fre-
the cell for detection by routine methods
quently been shown to have P specificity,
but enough to be demonstrable by meth-
reacting with all red cells by the Donath-
ods such as flow cytometry, enzyme-
Landsteiner test (including the patient’s
linked antiglobulin tests, solid phase,
own red cells) except those of the very
PEG, direct Polybrene, column agglutina-
rare p or Pk phenotypes. Exceptional ex-
tion, or concentrated eluate.
amples with other specificities have been
described.6(p221),23
Nonroutine Reagents
Transfusion in PCH The causative antibody may be IgM or IgA
Transfusion is rarely necessary for adult not detected by routine AHG reagents.
patients with PCH, unless their hemolysis Anti-IgG, anti-C3d, and the combined
is severe. In children, especially under age anti-C3b, -C3d reagents are the only li-
6, the thermal amplitude of the antibody censed products available in the United
tends to be much wider than in adults States for use with human red cells. AHG
and hemolysis more brisk, so transfusion reagents that react with IgA, IgM, or C4
may be required as a lifesaving measure. are available commercially but have been
Although there is some evidence that p prepared for use with endpoints other
red cells survive better than P+ (P1+ or P1–) than agglutination. These must be used
red cells,6(p221) the prevalence of p blood is cautiously and their hemagglutinating re-
approximately 1 in 200,000 and the urgent activity carefully standardized by the
need for transfusion usually precludes at- user.3 Quality control must be rigorous
tempts to obtain this rare blood. Transfu- because agglutination with AHG reagents
sion of random donor blood should not is more sensitive than precipitation; a se-
be withheld from PCH patients whose rum that appears to be monospecific by
need is urgent. Red cells negative for the P precipitation tests may react with several
antigen should be considered only for different proteins when used in agglutina-
those patients who do not respond ade- tion tests.
quately to random donor blood.3
Antigen Depression
DAT-Negative AIHA Patients with autoantibodies of Kell, Rh,
Clinical evidence of hemolytic anemia is LW, Ge, Sc, Lu, and Lan specificities may
present in some patients whose DAT is non- have depressed red cell expression of the
reactive. Frequently, autoantibody cannot respective antigens. When this occurs, an-
be detected in either eluate or serum. tibody may be detected in the serum and
There may be several reasons the DAT is eluate, but the DAT may be negative or very
negative. The autoantibody may be IgA or weakly positive. This may provide in-vivo
IgM.3,40 Antibodies with low binding affin- protection of autologous cells. Donor cells
ity may dissociate from the red cells dur- of common specific antigen type may be
ing saline washing of the cells for the DAT. destroyed, but cells lacking the corre-
Washing with ice cold (eg, 4 C) LISS or sa- sponding antigen (usually high-incidence)
line may help retain antibody on the cells; may survive well. When the autoantibody
a control (eg, 6-10% albumin) is necessary subsides, autologous cells again express
to confirm that cold autoagglutinins are normal amounts of antigen.6(p228),24

control test is nonreactive, the results ob-

Serologic Problems with tained with anti-A and anti-B are usually
Autoantibodies valid. If autoagglutination still occurs, it
may be necessary to treat the red cells
In pretransfusion tests on patients with auto-
with sulfhydryl reagents.
antibodies, the following problems may
Because cold-reactive autoagglutinins
arise:
are almost always IgM and sulfhydryl re-
1. Cold-reactive autoantibodies can cause
agents denature IgM molecules, reagents
autoagglutination, resulting in erro-
such as 2-mercaptoethanol (2-ME) or
neous determinations of ABO and
dithiothreitol (DTT) can be used to abolish
Rh type.
autoagglutination (see Method 2.11). Treat-
2. Red cells strongly coated with globu-
ing the red cells with ZZAP reagent as in the
lins may undergo spontaneous ag-
preparation for adsorptions can also be used
glutination with high-protein, anti-
(see Method 4.10). Appropriate controls are
Rh, blood-typing reagents, and occa-
essential for all tests.
sionally even with low-protein re-
When the serum agglutinates group O
agents.41
reagent red cells, the results of serum tests
3. The presence of free autoantibody in
may be unreliable. Repeating the tests us-
the serum may make antibody de-
ing prewarmed serum and group A, B, and
tection and crossmatching tests dif-
O red cells at 37 C will often resolve any dis-
ficult to interpret. If time permits, the
crepancy, but weak anti-A and/or -B in
presence or absence of unexpected,
some patients’ sera may not react at 37 C.
clinically important alloantibody(ies)
Alternatively, adsorbed serum (either auto-
should be determined (see Methods
adsorbed or adsorbed with allogeneic
4.9 through 4.12) before blood is trans-
group O red cells) can be used. Because
fused.
rabbit red cells express a B-like antigen,
Although resolving these serologic prob-
sera adsorbed with rabbit red cells or
lems is important, delaying transfusion in
stroma may not contain anti-B, and sera
the hope of finding serologically compati-
adsorbed in this manner should not be
ble blood may cause greater danger to the
used for ABO serum tests.
patient in some cases. Only clinical judg-
ment can resolve this dilemma; therefore,
dialogue with the patient’s physician is im- Resolution of Rh Problems
portant.
Autoagglutination of red cells by cold- or
spontaneous agglutination of red cells by
Resolution of ABO Problems warm-reactive autoantibodies may also
There are several approaches to the reso- cause discrepant Rh typing. The same
lution of ABO typing problems associated procedures described for the resolution of
with cold-reactive autoagglutinins. Often, ABO problems, with the exception of us-
it is only necessary to maintain the blood ing ZZAP-treated red cells, may be useful.
sample at 37 C immediately after collec- Also, IgG antibody can be dissociated from
tion and to wash the red cells with warm the cells by treatment with chloroquine
(37 C) saline before testing. It is helpful to diphosphate (Method 2.13), or by glycine-
perform a parallel control test, using 6% HCl/EDTA (Method 2.14), methods that
to 10% bovine albumin in saline, to deter- leave red cells intact for subsequent typ-
mine if autoagglutination persists. If the ing. IgM-coated cells can be treated with

sulfhydryl reagents (such as 2-ME or DTT, cedures involve adsorption, the principles
Method 2.11) to circumvent autoagglu- of which are discussed in Chapter 19. Two
tination and spontaneous agglutination. widely used approaches are discussed be-
low.
Detection of Alloantibodies in the
Presence of Warm-Reactive Autologous Adsorption
Autoantibodies
In a patient who has not been recently
If the patient who has warm-reactive transfused, autologous adsorption (see
autoantibodies in the serum needs trans- Method 4.9) is the best way to detect allo-
fusion, it is important to evaluate the antibodies in the presence of warm-reac-
possible simultaneous presence of allo- tive autoantibodies. The adsorbed serum
antibodies to red cell antigens. Some can be used in the routine antibody de-
alloantibodies may make their presence tection procedure.
known by reacting more strongly or at dif- Autoadsorption generally requires some
ferent phases than the autoantibody, but initial preparation of the patient’s red cells.
quite often studies may not suggest the At 37 C, in-vivo adsorption will have oc-
29
existence of masked alloantibodies. It is curred and all antigen sites on the patient’s
helpful to know which of the common red own red cells may be blocked. It may be
cell antigens are lacking on the patient’s necessary, therefore, to remove autoanti-
red cells, to predict which clinically signif- body from the red cells to make sites avail-
icant alloantibodies the patient may have able for adsorption. A gentle heat elution at
produced or may produce. Antigens ab- 56 C for 5 minutes can dissociate some of
sent from autologous cells could well be the bound IgG. This can be followed by
the target of present or future alloanti- treatment of the autologous red cells with
bodies. When the red cells are coated with proteolytic enzymes to increase their ca-
IgG, antiglobulin-reactive reagents can- pacity to adsorb autoantibody. Treatment
not be used to test IgG-coated cells unless of the red cells with ZZAP, a mixture of
the IgG is first removed (see Methods 2.13 papain or ficin and DTT (see Method 4.9)
and 2.14). Low-protein antisera (eg, mono- accomplishes both of these actions in one
clonal reagents) that do not require an step; the sulfhydryl component makes the
antiglobulin test may be helpful in typing IgG molecules more susceptible to the pro-
the DAT-positive red cells. Cell separation tease and dissociates the antibody mole-
procedures (see Methods 2.15 and 2.16) cules from the cell. Multiple sequential
may be necessary if the patient has been autoadsorptions with new aliquots of red
transfused recently. cells may be necessary if the serum con-
Methods to detect alloantibodies in the tains high levels of autoantibody. Once the
presence of warm-reactive autoantibodies autoantibody has been removed, the ad-
attempt to remove, reduce, or circumvent sorbed serum is examined for alloantibody
the autoantibody. Antibody detection meth- activity.
ods that use PEG, enzymes, column aggluti- If the patient is to be transfused, it can
nation, or solid-phase red cell adherence be advantageous to collect and save addi-
generally enhance autoantibodies. Testing tional aliquots of pretransfusion cells, to be
LISS- or saline-suspended red cells may used for later adsorptions.
avoid autoantibodies but allow detection of Autologous adsorption is not recom-
most significant alloantibodies. Other pro- mended for patients who have been re-

cently transfused, because they may have adsorptions because the adsorbing cells
an admixture of transfused red cells that will almost invariably express the antigen
might adsorb alloantibody. Red cells nor- and adsorb the alloantibody along with
mally live for about 110 to 120 days. In pa- autoantibody.
tients with AIHA, autologous and trans- Patient’s Phenotype Unknown. When
fused red cells can be expected to have the patient’s phenotype is not known,
shortened survival. However, determining group O red cell samples of three different
how long transfused red cells remain in cir- Rh phenotypes (R1R1, R2R2, and rr) should
culation in patients who need repeated be selected (see Method 4.10). One should
transfusions is not feasible. It has been lack Jka and another Jkb. If treated with
demonstrated that very small amounts ZZAP, these cells would also lack all anti-
(<10%) of antigen-positive red cells are ca- gens of the Kell system and enzyme-sensi-
pable of removing alloantibody reactivity in tive antigens (see Table 19-3). If ZZAP is not
in-vitro studies42; therefore, it is recom- available, cells treated only with proteolytic
mended to wait for 3 months after transfu- enzyme can be used, but at least one of the
sion before autologous adsorptions are per- adsorbing cells must be K– because Kell
formed. system antigens will not be destroyed. Un-
treated cells may be used, but antibody
may be more difficult to remove and the
adsorbing cells must, at a minimum, in-
Allogeneic Adsorption clude at least one negative for the S, s, Fya,
b
The use of allogeneic red cells for adsorp- Fy , and K antigens in addition to the Rh
tion may be helpful when the patient has and Kidd requirements above.
been recently transfused or when insuffi- Each aliquot may need to be adsorbed
cient autologous red cells are available. two or three times. The fully adsorbed
The goal is to remove autoantibody and aliquots are tested against reagent red cells
leave the alloantibody in the adsorbed se- known either to lack or to carry common
rum. The adsorbing cells must not have antigens of the Rh, MNS, Kidd, Kell, and
the antigens against which the alloanti- Duffy blood group systems. If an adsorbed
bodies react. Because alloantibody speci- aliquot is reactive, that aliquot (or an addi-
ficity is unknown, red cells of different tional specimen similarly adsorbed) should
phenotypes will usually be used to adsorb be tested to identify the antibody. Adsorb-
several aliquots of the patient’s serum. ing several aliquots with different red cell
Given the number of potential alloanti- samples provides a battery of potentially
bodies, the task of selecting cells may ap- informative specimens. For example, if the
pear formidable. However, the selected aliquot adsorbed with Jk(a–) red cells sub-
cells need only demonstrate those few allo- sequently reacts only with Jk(a+) red cells,
antibodies of clinical significance likely to the presence of alloanti-Jka can confidently
be present. These include the common Rh be inferred.
antigens (D, C, E, c, and e), K, Fya and Fyb, Patient’s Phenotype Known. If the pa-
Jka and Jkb, and S and s. Cell selection is tient’s Rh and Kidd phenotypes are known
made easier by the fact that some antigens or can be determined, adsorption can be
can be destroyed by appropriate treatment performed with a single sample of allo-
(eg, with enzymes) before use in adsorption geneic ZZAP-treated red cells of the same
procedures. Antibodies to high-incidence Rh and Kidd phenotypes as the patient (see
antigens cannot be excluded by allogeneic Method 4.11).

Problems Encountered. Occasionally Detection of Alloantibodies in the

autoantibody will not be removed by three Presence of Cold-Reactive Autoantibodies
sequential adsorptions. Further adsorptions
Cold-reactive autoagglutinins rarely mask
can be done, but multiple adsorptions have
clinically significant alloantibodies if se-
the potential to dilute the serum. If the ad-
rum tests are conducted at 37 C and if
sorbing cells do not appear to remove the
IgG-specific reagents are used for the
antibody, the autoantibody may have an
antiglobulin phase. In rare instances, it
unusual specificity that does not react with
may be necessary to perform autoadsorp-
the cells used for adsorption. For example,
a tion at 4 C (see Method 4.6). Achieving the
autoantibodies with Kell, LW, or En FS
complete removal of potent cold-reactive
specificity would not be removed by ZZAP-
autoagglutinins is very time-consuming
treated cells (see Table 19-3 for a list of anti-
and is usually unnecessary. Removal of
gens altered by various agents).
sufficient cold autoagglutinins may be fa-
cilitated by treating the patient’s cells with
enzymes or ZZAP before adsorption.
Autoantibodies Mimicking Alloantibodies
Sometimes, autoantibodies have patterns
of reactivity that are easily mistaken for
alloantibody. For example, the serum of a Drug-Induced Immune
D– patient may have apparent anti-C and
-e reactivity. The anti-C reactivity may re-
Hemolytic Anemia
flect warm-reactive autoantibody even if Drugs sometimes induce the formation of
the patient’s cells lack C. The autoantibody antibodies, either against the drug itself
nature of the reactivity can be demon- or against intrinsic red cell antigens, that
strated by autologous and allogeneic ad- may result in a positive DAT, immune red
sorption studies. In this case, the appar- cell destruction, or both. Some of the an-
ent alloanti-C would be adsorbed by C– tibodies produced appear to be depend-
red cells, both autologous and allogeneic. ent on the presence of the drug (ie, drug
This is quite unlike the behavior of a true dependent) for their detection or destruc-
alloanti-C, which would be adsorbed only tive capability, whereas others do not (ie,
by C+ red cells. In one study,43 the serum drug independent). In some instances, a
prepared from an initial autoadsorption reactive DAT may result from nonim-
would often retain autoantibodies that munologic effects of the drugs. Drugs that
mimicked alloantibodies in addition to have been reported to cause hemolytic
the true alloantibody(ies) present. Serum anemia and/or a positive DAT are listed in
prepared from an initial alloadsorption Appendix 20-2.
most often contains only alloantibodies.
The differences in the auto- or alloanti- Theories of the Immune Response and
body nature of specificities detected in Drug-Dependent Antibodies
the autoadsorbed serum as compared to Numerous theories have been suggested
the alloadsorbed serum reflect an ineffi- to explain how drugs induce immune re-
ciency of autologous adsorption. This is sponses and what relation such responses
primarily due to limited volumes of auto- may have to the positive DAT and immune-
logous cells available for removing all mediated cell destruction observed in some
autoantibody reactivity from the serum.43 patients. For many years, drug-associated

positive DATs were classified into four Serologic and Clinical Classification
mechanisms: drug adsorption (penicil-
lin-type), immune complex formation, Drug-induced antibodies can be classi-
autoantibody production, and nonspe- fied into three groups according to their
cific adsorption. Such classification has clinical and serologic characteristics.3 In
been useful, but many aspects lacked de- one group, the drug binds firmly to the
finitive proof. In addition, some drugs cell membrane and antibody is appar-
created immune problems involving as- ently largely directed against the drug it-
pects of more than one mechanism. More self. This was called the drug adsorption
recent theories, still unproven, tend to- mechanism. Antibodies to penicillin are
44-48
ward a more comprehensive approach. the best described of this group.
Most drugs are probably capable of The second group of drug-dependent
binding loosely, or firmly, to circulating antibodies reacts with drugs that do not
cells, which can lead to an immune re- bind well to the cell membrane (eg, quini-
sponse. Figure 20-1 illustrates this concept. dine, ceftriaxone). The reactive mechanism
Antibodies can be formed to the drug itself of these antibodies was previously thought
or to the drug plus membrane components. to be due to drug/antidrug immune com-
When an antibody is formed to the drug plex formation, but the theory has never
plus membrane components, the antibody been proven.48 Antibodies in this group may
may recognize primarily the drug or primar- cause acute intravascular hemolysis and
ily the membrane. One or all three of these may be difficult to demonstrate serologi-
antibody populations may be present. cally. Testing for this type of drug antibody
Figure 20-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by
Habibi as cited by Garratty 23 ). The thicker darker lines represent antigen-binding sites on the F(ab) 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” positive DAT; only occasionally will these
method. patients develop hemolytic anemia.6(p231) A
Antibodies of the third group (eg, methyl- possible mechanism for the positive DAT is
dopa, procainamide, and fludarabine) have given in Fig 20-2. The penicillin becomes
serologic reactivity independent of the drug, covalently linked to the red cells in vivo. If
despite the fact that it was the drug that the patient has antibodies to penicillin, they
originally induced the immune response. bind to the penicillin bound to the red cells.
Serologically, they behave as autoantibodies. The result is that the penicillin-coated red
cells become coated with IgG. If cell destruc-
tion occurs, it takes place extravascularly,
Drug-Dependent Antibodies Reactive with probably in the same way that red cells
Drug-Treated Red Cells: Penicillin-Type coated with IgG alloantibodies are de-
Antibodies stroyed. Intravascular hemolysis is rare.
The clinical and laboratory features of drug- Many cephalosporins, which are related
induced immune hemolytic anemia oper- to penicillins, behave in a similar manner.
ating through this mechanism are: The cephalosporins are generally classified
1. The DAT is strongly positive due to by “generations,” based on their effective-
IgG coating. Complement coating may ness against gram-negative organisms (see
also be present. Table 20-4). Approximately 4% of patients
2. Antibody eluted from the patient’s receiving first- or second-generation cephalos-
red cells reacts with drug-treated red porins develop a positive DAT.48 Dramati-
cells but not with untreated red cells. cally reduced red cell survival has been
3. The serum contains a high-titer IgG associated with second- and third-genera-
antibody (especially when the target tion cephalosporins.12,47,49-53 The prevalence
is penicillin or cefotetan) reactive with and severity of cephalosporin-induced im-
the drug-treated red cells but not with mune red cell destruction appear to be in-
the untreated red cells, unless the creasing.3
patient also has alloantibodies to red
cell antigens.
4. For penicillin, the hemolysis-induc-
ing dose is millions of units daily for Drug-Dependent Antibodies Reacting by the
a week or more; for other drugs, eg, “Immune Complex” Mechanism
cefotetan, a single 1 to 2 g dose has been Many drugs have been reported as caus-
implicated in immune hemolysis. ing hemolytic anemia by this mechanism.
5. Hemolysis develops gradually but Some of the second- and third-generation
may be life-threatening if the etiol- cephalosporins react by this mechanism;
ogy is unrecognized and drug ad- anti-ceftriaxone has been detected only
ministration is continued. by the immune complex method.49 The
6. Discontinuation of the drug is usu- following observations are characteristic:
ally followed by increased cell sur- 1. Complement may be the only globu-
vival, although hemolysis of decreas- lin easily detected on the red cells,
ing severity may persist for several but IgG may be present.
weeks. 2. The serum antibody can be either
Approximately 3% of patients receiving IgM or IgG, or IgM with IgG.
large doses of penicillin intravenously (ie, 3. A drug (or metabolite) must be pres-
millions of units per day) will develop a ent in vitro for demonstration of the

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.
antibody in the patient’s serum. An- Drug-Independent Antibodies: Autoantibody

tibodies may cause hemolysis, ag- Production
glutination, and/or sensitization of Some drugs induce autoantibodies that
red cells in the presence of the drug. appear serologically indistinguishable
4. The patient need only take a small from those of WAIHA. Red cells are coated
amount of the drug. with IgG, and the eluate as well as the se-
5. Acute intravascular hemolysis with rum react with virtually all cells tested in
hemoglobinemia and hemoglobinuria the absence of the drug. Blood group spe-
is the usual presentation. Renal fail- cificity has been demonstrated at times,
ure is quite common. similar to that seen in AIHA. The antibody
6. Once antibody has been formed, se- has no in-vitro activity with the drug, di-
vere hemolytic episodes may recur rectly or indirectly.
after exposure to very small quanti- The best studied of such cases are those
ties of the drug. induced by α-methyldopa. A closely related

Table 20-4. Some Cephalosporins drug, L-dopa, has been implicated, as have
several drugs unrelated to α-methyldopa,
Generic Name Trade Name* including procainamide, nonsteroidal
anti-inflammatory drugs (eg, mefenamic
First Generation acid), second- and third-generation cep-
cefadroxil Duricef halosporins, and fludarabine. In some
cases, drug-dependent antibodies are also
cefazolin Ancef, Kefzol,
Zolicef present.
Proof that a drug causes autoantibody
cephalexin Keflex
production is difficult to obtain. Sufficient
cephalothin Keflin evidence would include: demonstration
cephapirin Cefadyl that autoantibody production began after
drug administration; resolution of the im-
cephradine Anspor
mune process after withdrawal of the drug;
and recurrence of hemolytic anemia or
Second Generation autoantibodies if the drug is readmini-
cefaclor Ceclor stered. The last requirement is crucial and
the most difficult to demonstrate.
cefamandole Mandol
cefmetazole Zefazone
Nonimmunologic Protein Adsorption
cefonicid Monocid
The positive DAT associated with some
cefotetan Cefotan drugs is due to a mechanism independent
cefoxitin Mefoxin of antibody production. Hemolytic ane-
cefuroxime Zinacef, Kefurox, mia associated with this mechanism oc-
Ceftin curs rarely.
Cephalosporins (primarily cephalothin)
cefuroxime axetil Ceftin
are the drugs with which this was originally
associated. Red cells coated with cephalo-
Third Generation thin (Keflin) and incubated with normal
plasma will adsorb albumin, IgA, IgG, IgM,
cefixime Suprax
and C3 in a nonimmunologic manner. If
cefoperazone Cefobid this occurs, a positive indirect antiglobulin
cefotaxime Claforan test will be seen with AHG reagents.
ceftazidime Fortaz, Ceptaz, Other drugs that may cause nonimmuno-
Pentacef, Tazicef, logic adsorption of proteins and a positive
Tazidime DAT include diglycoaldehyde, suramin,
ceftizoxime Ceftizox cisplatin, clavulanate (in Timentin and
Augmentin), sulbactam in Unasyn,54 and
ceftriaxone Rocephin tazobactam (in Tazocin and Zosyn).
55,56
Fourth Generation Laboratory Investigation of Drug-Induced

cefepime Maxipime Antibodies
The drug-related problems most com-
*Several forms are marketed under other trade names. monly encountered in the blood bank are
This list is intended to be informative, not inclusive.
those associated with a positive DAT. Typ-

ical DAT results are shown in Table 20-3. During the testing of cefotetan-treated red
Recent red cell transfusions and/or dra- cells, a 1 in 100 dilution of the patient’s se-
matic hemolysis may result in a weak DAT rum should be tested because it has been
by the time hemolysis is suspected. shown that some normal sera appear to
The patient’s serum should be tested for contain “naturally occurring” antibodies to
unexpected antibodies by routine proce- cefotetan, some of which still react weakly at
dures. If the serum does not react with un- a 1 in 20 dilution.57,58 Cefotetan antibodies
treated red cells, the tests should be re- associated with drug-induced immune
peated against ABO-compatible red cells in hemolytic anemia have very high anti-
the presence of the drug(s) suspected of globulin titers (4000 to 256,000).49
causing the problem. Techniques are given Two other observations have been made
in Method 4.14 and Method 4.15. regarding the testing of cefotetan antibod-
If the drug has already been reported as ies: 1) the last wash from the eluate prepa-
causing hemolytic anemia, testing methods ration may react with cefotetan-treated red
may be available in the case reports. If such cells (possibly due to the high-titer anti-
information is not available, an initial bodies and/or the antibody affinity), and 2)
screening test can be performed with a so- drug-independent antibodies may be detected
lution of the drug at a concentration of ap- in the serum and eluate and hemolysis may
proximately 1 mg/mL in phosphate-buf- be inadvertently attributed to idiopathic
49
fered saline at a pH optimal for solubility of WAIHA.
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
References
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an eluate from the patient’s red cells can be associated with positive direct antiglobulin
tested against the drug-coated red cells. tests in pretransfusion patients: A case con-
This is the method of choice when penicil- trol study. Vox Sang 1985;49:215-20.
2. Heddle NM, Kelton JG, Turchyn KL, Ali MAM.
lin or cephalosporins are thought to be im- Hypergammaglobulinemia can be associated
plicated. Results definitive for a penicil- with a positive direct antiglobulin test, a non-
lin-induced positive DAT are reactivity of reactive eluate, and no evidence of hemolysis.
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fusion 1986;26:167-70. ciated with IgM warm autoantibodies (ab-
19. Sokol RJ, Hewitt S, Stamps BK. Autoimmune stract). Blood 2001;98(Suppl 1):61a.
haemolysis: An 18 year study of 865 cases re- 34. Nowak-Wegrzyn A, King KE, Shirey RS, et al.
ferred to a regional transfusion centre. Br Med Fatal warm autoimmune hemolytic anemia
J 1981;282:2023-7. resulting from IgM autoagglutinins in an in-
20. Shulman IA, Branch DR, Nelson JM, et al. Au- fant with severe combined immunodefi-
toimmune hemolytic anemia with both cold ciency. J Pediatr Hematol Oncol 2001;23:250-
and warm autoantibodies. JAMA 1985;253: 2.
1746-8. 35. Garratty G, Petz LD, Hoops JK. The correla-
21. Göttsche B, Salama A, Mueller-Eckhardt C. tion of cold agglutinin titrations in saline and
Donath-Landsteiner autoimmune hemolytic albumin with haemolytic anemia. Br J Haematol
anemia in children. A study of 22 cases. Vox 1975;35:587-95.
Sang 1990;58:281-6. 36. Marks MR, Reid ME, Ellisor SS. Adsorption of
22. Sokol RJ, Hewitt S, Stamps BK. Autoimmune unwanted cold autoagglutinins by formalde-
haemolysis. Mixed warm and cold antibody hyde-treated rabbit erythrocytes (abstract).
type. Acta Haematol 1983;69:266-74. Transfusion 1980;20:629.
23. Garratty G. Target antigens for red-cell- 37. Dzik W, Yang R, Blank J. Rabbit erythrocyte
bound autoantibodies. In: Nance SJ, ed. Clin- stroma treatment of serum interferes with
ical and basic science aspects of immuno- recognition of delayed hemolytic transfusion
hematology. Arlington, VA: AABB, 1991:33-72. reactions (letter). Transfusion 1986;26:303-4.

38. Mechanic SA, Maurer JL, Igoe MJ, et al. Anti- 53. Viraraghavan R, Chakravarty AG, Soreth J.
Vel reactivity diminished by adsorption with Cefotetan-induced haemolytic anaemia. A re-
rabbit RBC stroma. Transfusion 2002;42: view of 85 cases. Adv Drug React Toxicol Rev
1180-3. 2002;21:101-7.
39. Garratty G, Arndt PA, Leger RM. Serological 54. Garratty G, Arndt PA. Positive direct antiglo-
findings in autoimmune hemolytic anemia bulin tests and haemolytic anemia following
(AIHA) associated with both warm and cold therapy with beta-lactamase inhibitor con-
autoantibodies (abstract). Blood 2003;102 taining drugs may be associated with non-
(Suppl 1):563a. immunologic adsorption of protein onto red
40. Garratty G. Autoimmune hemolytic anemia. blood cells. Br J Haematol 1998;100:777-83.
In: Garratty G, ed. Immunobiology of transfu- 55. Broadberry RE, Farren TW, Kohler JA, et al.
sion medicine. New York: Marcel Dekker, Haemolytic anaemia associated with Tazobac-
1994:493-521. tam (abstract). Vox Sang 2002;83(Suppl 2):
41. Garratty G, Postoway N, Nance SJ, Brunt DJ. 227.
Spontaneous agglutination of red cells with a 56. Arndt PA, Leger RM, Garratty G. Positive di-
positive direct antiglobulin test in various rect antiglobulin tests and haemolytic anae-
media. Transfusion 1984;24:214-7. mia following therapy with the beta-lacta-
42. Laine EP, Leger RM, Arndt PA, et al. In vitro mase inhibitor, tazobactam, may also be
studies of the impact of transfusion on the associated with non-immunologic adsorp-
detection of alloantibodies after autoadsorption of protein onto red blood cells (letter).
tion. Transfusion 2000;40:1384-7. Vox Sang 2003;85: 53.
43. Issitt PD, Combs MR, Bumgarner DJ, et al. 57. Arndt P, Garratty G. Is severe immune hemoly-
Studies of antibodies in the sera of patients tic anemia, following a single dose of cefo-
who have made red cell autoantibodies. tetan, associated with the presence of “natu-
Transfusion 1996;36:481-6. rally-occurring” anti-cefotetan? (abstract)
44. Salama A, Mueller-Eckhardt C. Immune-me- Transfusion 2001;41(Suppl):24S.
diated blood cell dyscrasias related to drugs. 58. Arndt PA. Practical aspects of investigating
Semin Hematol 1992;29:54-63. drug-induced immune hemolytic anemia
45. Petz LD, Mueller-Eckhardt C. Drug-induced due to cefotetan or ceftriaxone—a case study
immune hemolytic anemia. Transfusion approach. Immunohematology 2002;18:27-
1992;32:202-4. 32.
46. Shulman NR, Reid DM. Mechanisms of drug-
induced immunologically mediated cyto-
penias. Transfus Med Rev 1993;7:215-29.
47. Christie DJ. Specificity of drug-induced im-
mune cytopenias. Transfus Med Rev 1993;7:
Suggested Reading
230-41. Dacie J. Historical review. The immune haemolytic
48. Garratty G. Drug-induced immune hemolytic anaemias: A century of exciting progress in under-
anemia. In: Garratty G, ed. Immunobiology of standing. Br J Haematol 2001;114:770-85.
transfusion medicine. New York: Marcel
Dekker, 1994:523-51. Engelfriet CP, Overbeeke MAM, von dem Borne
49. Arndt PA, Leger RM, Garratty G. Serology of AEGKr. Autoimmune hemolytic anemia. Semin
antibodies to second- and third-generation Hematol 1992;29:3-12.
cephalosporins associated with immune
Garratty G. Novel mechanisms for immune destruc-
hemolytic anemia and/or positive direct
tion of circulating autologous cells. In: Silberstein
antiglobulin tests. Transfusion 1999;39:1239-
LE, ed. Autoimmune disorders of blood. Bethesda,
46.
MD: AABB, 1996:79-114.
50. Gallagher NI, Schergen AK, Sokol-Anderson
ML, et al. Severe immune-mediated hemolytic Garratty G. Autoantibodies induced by blood
anemia secondary to treatment with cefote- transfusion (editorial). Transfusion 2004:445-9.
tan. Transfusion 1992;32:266-8.
51. Garratty G, Nance S, Lloyd M, Domen R. Fatal Mack P, Freedman J. Autoimmune hemolytic ane-
immune hemolytic anemia due to cefotetan. mia: A history. Transfus Med Rev 2000;14:223-33.
Transfusion 1992;32:269-71. Petz LD, Garratty G. Immune hemolytic anemias.
52. Stroncek D, Procter JL, Johnson J. Drug-in- 2nd ed. Philadelphia: Churchill Livingstone, 2004.
duced hemolysis: Cefotetan-dependent
hemolytic anemia mimicking an acute Petz LD. A physician’s guide to transfusion in auto-
intravascular immune transfusion reaction. immune haemolytic anaemia. Br J Haematol 2004;
Am J Hematol 2000;64:67-70. 124:712-16.

480
Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT (Excluding Investigation of HDFN)

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 Antineoplastic DI
(chlorodeoxyadenosine)
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)

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 H2 receptors) ?
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
3,44-48
Mechanisms listed are based on descriptions in the literature.
DAT = Direct antiglobulin test.
DD-DA = Drug-dependent. Drug adsorbed onto red cells; antibody reacts with drug on cells.
DD-IC = Drug-dependent. “Immune complex mechanism.” Requires drug, serum, and red cells for serologic
demonstration. For most of these drugs, there are only single or very few case reports.
DI = Drug-independent. Associated with autoantibodies similar to those in AIHA. Drug not required for in-vitro
demonstration. Mechanisms of autoantibody production may vary.
NIA = Nonimmunologic adsorption of proteins.
? = Mechanism unclear or unknown.

Chapter 21: Blood Transfusion Practice
Chapter 21
Blood Transfusion Practice

21
T
HE DECISION TO transfuse, like sue demands; transfusion to replace red
any other therapeutic decision, cells destined for destruction in hemoly-
should be based on the risks, ben- tic disease of the newborn is discussed in
efits, and alternatives of treatment. Unfor- Chapter 24. Because demand for oxygen
tunately, data regarding the indications varies greatly among different individuals
for transfusion are frequently not available in different clinical circumstances, mea-
and recipients run the risk of both over- surement of only the hematocrit or the
transfusion and undertransfusion. Trans- hemoglobin concentration (“the hemo-
fusions based solely on laboratory test globin”) cannot accurately assess the need
1-3
“triggers,” in particular, are problematic. for transfusion.
Consensus statements on the use of blood
components such as those produced by
the National Institutes of Health (NIH)
Normal Oxygen Supply and Demand
help guide therapy but cannot substitute
for clinical judgment. 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
Red Blood Cell Transfusion rest. The oxygen content of blood (mL
Physiologic Principles O2/mL blood) is determined by the hemo-
The primary indication for transfusion of globin, the binding coefficient of hemo-
Red Blood Cells (RBCs) is to restore or main- globin for oxygen, the oxygen saturation
tain oxygen-carrying capacity to meet tis- of hemoglobin, and a small quantity of
483

oxygen dissolved in the plasma. This is O2 supply (Calculation assumes a hemo-

described as: globin of 140 g/L and a pO2 of 100)
= Cardiac output × O2 contentarterial
O2 content = (Hb × 1.39 × %sat) = 5 L/minute × [(140 × 1.39 × 100%)
+ (pO2 × 0.003) + (100 × 0.003)]
= 5 L/minute × 200 mL O2/L
Tissue oxygen consumption is calculated = 1000 mL O2/minute
as the difference between oxygen delivery
in the arterial blood and oxygen return by O2 consumption = Cardiac output × (O2
the venous blood: contentarterial – O2 contentvenous)
= 5 L/minute × (200 mL O2/L –
150 mL O2/L)
O2 consumption = Cardiac output ×
= 5 L/minute × 50 mL O2/L =
Hb × 1.39 × (%satarterial – %satvenous)/100
250 mL O2/minute
which is expressed as Compensation for Anemia

(mL O2/minute) = The above equations demonstrate that any
decrease in oxygen content due to anemia
L/minute × g/L × mL O2/g
can be compensated for by an increase in
The oxygen saturation of arterial and ve- cardiac output.1,2 This occurs because of
nous hemoglobin varies with the partial increased cardiac work and also because
pressure of oxygen. Under normal circum- anemia decreases blood viscosity, and
stances the pO2 falls from 100 mm Hg in the thus peripheral vascular resistance. The
arteries to 40 mm Hg in the veins as the tis- increase in oxygen supply provided by in-
sues extract oxygen, and hemoglobin satu- creased cardiac output is augmented by
ration falls from near 100% in the arteries to increased oxygen extraction. Oxygen ex-
approximately 75% in the veins; thus, the traction is augmented acutely by a de-
oxygen extraction ratio is 0.25. That is, the crease in tissue oxygen tension, acting at
hemoglobin “gives up” only 25% of its oxy- the steep portion of the hemoglobin-oxy-
gen. When tissue demand for oxygen in- gen dissociation curve1 (see Fig 8-1), and
creases or the supply of oxygen decreases, by acidosis, which promotes oxygen dis-
the tissues extract a greater fraction of oxy- sociation from hemoglobin. Over time, an
gen from the plasma and from hemoglobin; increase in red cell 2,3-diphosphogly-
this results in a lower venous pO2 and de- cerate (2,3-DPG) concentration also has a
creased oxygen saturation of the venous significant positive effect on oxygen un-
blood. Studies in primates suggest that a loading.
critical point of limited oxygen delivery is
reached when the oxygen extraction ratio Measuring the Adequacy of Oxygen Supply
approaches twice normal or 0.50.2 As shown above, multiple factors deter-
Under normal resting conditions, the mine oxygen delivery to the tissues, ex-
body has a large reserve of oxygen supply cept at the lower extremes. Therefore,
relative to demand. In the average adult, measurements in addition to the hemo-
approximately 1000 mL/minute is available globin must be used to guide most trans-
to the tissues and only 250 mL/minute is fusion decisions. The adequacy of the
consumed as follows: oxygen supply depends on the partial

Chapter 21: Blood Transfusion Practice 485
pressure of inspired oxygen, gas exchange Assuming that oxygen extraction remains
in the lungs, the patient’s cardiac perfor- constant, oxygen delivery could be normal-
mance, hemoglobin, oxygen-hemoglobin ized by an increase in the hemoglobin con-
affinity, and current oxygen demand; all centration to 9 g/dL, or by an increase in
but the oxygen-hemoglobin affinity are the cardiac index to 7 L/minute/m 2. A
subject to substantial variation. For pa- smaller increase in hemoglobin would
tients in an intensive care unit or in the blunt the required increase in cardiac in-
operating room, direct measurement of dex.
the cardiac output and mixed venous oxy-
gen tension, in association with hemoglo-
bin level, may serve as more physiologic Red-Cell-Containing Components
guides for transfusion decisions than he- Whole Blood
moglobin alone. Nonetheless, most such
Whole Blood provides oxygen-carrying
decisions will continue to be made based
capacity, stable coagulation factors (the
on hemoglobin and standard clinical as-
concentrations of Factors V and VIII de-
sessment.
crease during storage), and blood volume
Determinants of cardiac performance
expansion. Thus, it is useful for patients
include the patient’s intravascular volume,
with concomitant red cell and volume
the anemia-related reduction in peripheral
deficits, such as actively bleeding pa-
vascular resistance, the presence of coro-
tients, and will help support coagulation
nary artery disease or other forms of heart
in appropriate clinical settings such as
disease, and the patient’s age. Tissue oxy-
liver transplantation.4 In fact, Whole Blood
gen debt results when oxygen demand ex-
is rarely available for allogeneic transfu-
ceeds supply; tissues convert to anaerobic
sion; RBCs and asanguinous solutions
metabolism and produce increased quanti-
have become the standard for most cases
ties of lactic acid. Metabolic acidosis, in
of active bleeding in trauma and surgery,
turn, impairs cardiac performance, further
with supplementation of hemostatic ele-
decreasing perfusion and tissue oxygen de-
ments as needed. The major use of Whole
livery, leading to greater tissue hypoxia in a
Blood in the United States today is for
vicious cycle.
autologous transfusion (see Chapter 5).
Treating Inadequate Oxygen Supply Red Blood Cells

RBC transfusion is the most direct means Red cell components are indicated for the
of raising the hemoglobin concentration. treatment of patients who require an in-
Other ways to improve oxygen supply rel- crease in oxygen-carrying capacity and
ative to demand include increasing tissue red cell mass.5 The transfusion of red cells
perfusion (maximizing cardiac perfor- increases oxygen-carrying capacity with
mance), increasing oxygen saturation less expansion of blood volume per unit
with supplemental oxygen or mechanical than Whole Blood. This is important for
ventilation, and decreasing tissue oxygen patients who are at risk for circulatory
demands with bed rest, antipyretics, and overload (eg, neonates or patients with
avoidance of hypertension. congestive heart failure). In a typical adult
For example, a patient with a hemoglo- in the absence of bleeding, hemolysis, or
bin of 6 g/dL and a cardiac index of 5 L/ major fluid shifts, one RBC unit is ex-
2
minute/m has decreased oxygen delivery. pected to raise the hemoglobin concen-

tration by approximately 1 g/dL, or the lutions take precedence over restoration

hematocrit by 3%. of oxygen-carrying capacity and should
be started immediately. In situations of
Selection of Whole Blood and Red Cell acute bleeding, guidelines suggest trans-
Components fusing patients who have lost 30% to 40%
Whole Blood must be ABO identical. RBC of their blood volume in conjunction with
units need not be ABO identical but must other measures to correct and maintain
be compatible with ABO antibodies in the total blood volume.3,6 Patients with car-
recipient’s plasma. Rh-negative recipients diac or other disease may require replace-
should receive Rh-negative Whole Blood ment sooner. Healthy resting adults have
or red cell components. In cases of been demonstrated to tolerate acute iso-
trauma or massive transfusion, it may be volemic hemodilution to hemoglobin
necessary to use Rh-positive components, concentrations as low as 5 g/dL without
as discussed below. See Table 21-1 for demonstrating evidence of inadequate
10
blood group selection in red cell transfu- oxygenation. In patients who refuse
sion. Selection of RBC units for patients transfusion on religious grounds and un-
with blood group alloantibodies is dis- dergo surgery, mortality increases pro-
cussed in Chapter 19. gressively below postoperative hemoglo-
bin levels of 5 or 6 g/dL, particularly for
11
Indications for Transfusion those with cardiovascular disease.
Perioperative transfusion accounts for
Several organizations have published guide- 12
3,6,7 55% to 65% of red cell component use.
lines for RBC transfusion. Reviews of
Randomized trials have demonstrated the
indications for transfusion are also avail-
safety of a “transfusion trigger” of 8 g/dL of
able.1,8,9
hemoglobin in patients undergoing cardio-
vascular surgery,13,14 orthopedic surgery,15
Blood Loss and Perioperative Transfusion and acute gastrointestinal bleeding. Even
16
For actively bleeding patients, the goal of before most of this evidence was available,
initial treatment should be to prevent the an NIH consensus conference on periopera-
7
development of hypovolemic shock by tive red cell transfusion emphasized that a
stopping the bleeding and restoring intra- hemoglobin of 10 g/dL was inappropriate
vascular volume. Efforts to restore volume as a guideline or transfusion trigger in the
by the infusion of crystalloid or colloid so- perioperative setting and suggested a he-
Table 21-1. Suggested ABO Group Selection Order for Transfusion of RBCs
Component ABO Group

Recipient
ABO Group 1st Choice 2nd Choice 3rd Choice 4th Choice
AB AB A B O
A A O
B B O
O O

moglobin of 7 g/dL as a level at which count for a large proportion of those re-
13
transfusion was frequently required in oth- ceiving RBC units. In a prospective ran-
erwise healthy individuals with acute ane- domized trial of red cell transfusion, ane-
mia. Factors to be considered in making an m i c b u t e u vo l e m i c p a t i e n t s i n t h e
individual transfusion decision included intensive care unit (ICU) were assigned to
“the duration of the anemia, the intravas- either “restrictive” or “liberal” transfusion
cular volume, the extent of the operation, regimens that maintained the hemoglo-
the probability of massive blood loss, and bin between 7 and 9 g/dL or between 10
17
the presence of coexisting conditions such and 12 g/dL, respectively. The mortality
as impaired pulmonary function, inade- rate during hospitalization (but not at 30
quate cardiac output, myocardial ischemia, days) was significantly lower in the re-
or cerebrovascular or peripheral circulatory strictive-strategy group (22.3% vs 28.1%, p
disease.”7 The guidelines also emphasized = 0.05). No difference in mortality rate
that transfusion does not improve wound was seen among all patients with clini-
healing, which depends on pO2 rather than cally significant cardiac disease. One third
total oxygen content of the blood. of the restrictive group avoided transfu-
A guideline by an American Society of sion, and total red cell use was half that of
3
Anesthesiologists task force cited a hemo- the liberal group. These authors con-
globin below 6 g/dL as “almost always” included that a restrictive strategy of red
dicating transfusion, a hemoglobin above cell transfusion is at least as effective as,
10 g/dL as rarely indicating transfusion, and possibly superior to, a liberal transfu-
and the range in between as the realm of sion strategy in critically ill patients, with
clinical judgment. A task force of the Col- the possible exception of the subset of pa-
lege of American Pathologists reached a tients with acute myocardial infarction
similar conclusion and proposed several (MI) and unstable angina. Reanalysis of
objective measures that might indicate red the patients in this study with cardiovas-
cell transfusion for hemoglobin levels in the cular disease showed a trend, albeit not
range of 6 to 10 g/dL, including tachycardia statistically significant, toward increased
or hypotension in the face of normovole- mortality in the restrictive group among
18
mia, a mixed venous pO2 of <25 torr, an ox- patients with MI and unstable angina.
ygen extraction ratio >50%, or a total oxy- A retrospective study of a large number
gen consumption of <50% of baseline.6 of elderly (>65 years old) patients hospital-
In the past, there was some concern that ized with acute MI divided into groups ac-
transfusions of a single RBC unit were likely cording to admission hematocrit compared
to represent unnecessary intervention. How- 30-day mortality rates in patients who re-
ever, if transfusion of a single unit will ceived transfusion and those who did not.19
achieve the desired clinical outcome, then Transfusion appeared beneficial in patients
only one unit should be transfused. Trans- with a hematocrit <30%. This result per-
fusing additional units in this setting will sisted when data were adjusted for multiple
increase the risk of transfusion without any clinical and institutional factors.
additional benefit. Patients with chronic anemia tolerate a
low hemoglobin better than those with
acute anemia because of cardiovascular
Anemia compensation and increased oxygen ex-
Among medical patients, those with car- traction. Moreover, patients at bed rest
diovascular and malignant diseases ac- who are not febrile, who do not have con-

gestive heart failure, and who are not cant prolongation of the bleeding time to
hypermetabolic have low oxygen require- major life-threatening hemorrhage. Pla-
ments and may tolerate anemia remark- telet plug formation results from the com-
ably well. However, the high oxygen needs bined processes of adhesion, activation and
of cardiac muscle may precipitate angina release, aggregation, and procoagulant
in patients with cardiac disease and ane- activity.22 Platelet adhesion to damaged
mia. A hemoglobin concentration of 8 g/ endothelium is mediated largely by the
dL adequately meets the oxygen needs of von Willebrand factor (vWF), which binds
most patients with stable cardiovascular to the surface glycoprotein (GP) receptor
disease. GPIb-IX-V complex. The process of acti-
Although it is desirable to prevent un- vation and release causes a dramatic
necessary transfusions, anemic patients change in platelet shape, with extension
who are symptomatic should receive ap- of pseudopod-like structures, a change in
propriate treatment. Anemia may cause the binding properties of membrane acti-
symptoms of generalized weakness, head- vation proteins, secretion of internal gran-
ache, dizziness, disorientation, breathless- ule contents, and activation of several
ness, palpitations, or chest pain, and signs metabolic pathways. These changes have
include pallor (not cyanosis) and tachycar- many effects, including the recruitment of
dia. Elwood and coworkers20 could not cor- additional platelets, which aggregate with
relate symptoms with the hemoglobin level the help of fibrinogen or vWF binding to
in patients with chronic iron deficiency platelet surface glycoproteins. Finally, the
anemia and a hemoglobin as low as 8 g/dL. platelet membrane procoagulant activity
A study of the use of erythropoietin demon- localizes and directs formation of fibrin.
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
Assessing Platelet Function
known to be transfusion dependent should Decreased platelet numbers may result
be maintained at a level that prevents from decreased production, increased de-
symptoms by establishing a transfusion struction, or splenic sequestration. Plate-
schedule and then adjusting it as needed. let function may be adversely affected by
such factors as drugs, liver or kidney dis-
ease, sepsis, fibrin(ogen) degradation
products, cardiopulmonary bypass, and
Platelet Transfusion primary marrow disorders. Platelet hemo-
Physiologic Principles stasis is assessed by the medical history,
Hemostasis occurs in four phases: the physical examination, and laboratory
vascular phase, the formation of a platelet tests including platelet count and bleed-
plug, the development of fibrin clot on ing time or in-vitro platelet function as-
the platelet plug, and the ultimate lysis of says (eg, PFA100). Patients with inadequate
the clot. Platelets are essential to the for- platelet number or function may demon-
mation of the primary hemostatic plug strate petechiae, easy bruising, mucous
and provide the surface upon which fibrin membrane bleeding, nose and gum bleed-
forms. Deficiencies in platelet number ing, and hematuria. Preprocedure platelet
and/or function can have unpredictable counts have predicted bleeding in some
23,24 25-27
effects that range from clinically insignifi- studies but not in others.

Bleeding time measures both the vascu- Chapter 16. Two consecutive poor re-
lar phase and the platelet phase of hemo- sponses suggest platelet refractoriness.
stasis. Although the bleeding time may be a
useful diagnostic test in the evaluation of Platelet Components
patients with known or suspected abnor-
Platelets
malities of platelet function, it is a poor pre-
28
dictor of surgical bleeding and is not a re- A single unit of Platelets prepared from an
liable indicator of the need for platelet individual unit of Whole Blood may be
transfusion therapy.29 The in-vitro measure- adequate for transfusion to neonates or
ment of platelet function is useful but, like infants, but, for adults, 4 to 6 units are or-
the bleeding time, is a poor predictor of dinarily pooled for transfusion to achieve
bleeding.30 a dose greater than 3.0 × 1011 platelets.
This should increase the platelet count by
30,000 to 60,000/µL.
Platelet Life Span and Kinetics Platelets Pheresis

Platelets normally circulate with a life span Units of platelets prepared by apheresis
31
of 10.5 days, and platelets that have been technology (“single-donor platelets”) have
properly collected and stored have a near a platelet content similar to that of pooled
normal residual mean life span of 4 to 5 platelets from four to six donors and, de-
days when reinfused into the original do- pending on the equipment used, may
nor. Conditions that shorten platelet life have a reduced leukocyte content. The
span include splenomegaly, sepsis, drugs, fact that a single such unit can provide an
disseminated intravascular coagulation entire transfusion facilitates provision of
(DIC), auto- and alloantibodies, endothe- compatible platelets to recipients who are
lial cell activation, and platelet activation refractory because of alloimmunization.
(eg, cardiopulmonary bypass or intra-aor- Transfusion of recipients who are refrac-
tic balloon pumps). Because a relatively tory to platelet transfusions is discussed
constant number of platelets (7,000- in Chapter 16.
10,000/µL/day) are consumed by routine
plugging of minor endothelial defects, the Selection of Platelets
fraction of the circulating platelet pool re-
quired for maintenance functions increases ABO Matching
as the total number of platelets declines. Because ABO antigens are present on the
Therefore, the life span of native and platelet surface, recovery of group A pla-
transfused platelets decreases with pro- telets transfused into group O patients is
gressive thrombocytopenia. 3 1 The re- somewhat decreased,32 but this effect is
sponse to platelet transfusion is best as- not usually clinically significant (see
sessed by observing whether bleeding Chapter 16). Transfusion of ABO-incom-
stops and by measuring the posttransfu- patible plasma present in platelet compo-
sion platelet increment. The posttransfu- nents may also result in a blunted post-
33
sion increment is generally measured transfusion platelet count increment.
between 10 minutes and 1 hour after Hemolysis occurs rarely in this setting but
completion of the transfusion and is exit frequently causes a positive direct
pressed as a corrected count increment antiglobulin test (DAT), which may in-
(CCI) or percent recovery, as outlined in crease costs and charges to the patient for

serologic investigation. Moreover, a retro- istered. If hematoma formation is an is-

spective analysis suggested that survival sue, an intravenous form of RhIG is avail-
after marrow transplantation was signifi- able. A full dose of RhIG, which is consid-
cantly reduced in patients who received ered immunoprophylactic for up to 15 mL
substantial amounts of ABO-incompati- of Rh-positive red cells, should protect
ble plasma from platelet transfusion, 34 against the red cells in a minimum of 30
and it has been suggested that infusion of units of Rh-positive Platelets or 7 units of
soluble A and B antigen in platelet or Rh-positive Platelets Pheresis.
plasma components may have a similar
adverse effect mediated by immune com- Therapeutic Platelet Transfusion
plex function.35 Therefore, it may be pru-
Significant bleeding due to thrombo-
dent to use ABO-matched platelets, par-
cytopenia or abnormal platelet function is
ticularly for patients requiring repeated
an indication for “therapeutic” platelet
transfusions. However, urgently needed
transfusion.37 The decision to transfuse
transfusions should not be delayed in or-
platelets depends on the cause of bleed-
der to obtain them. ing, the patient’s clinical condition, and
For infants, it is desirable to avoid ad- the number and function of the circulat-
ministration of plasma that is incompatible ing platelets.3,38-40 Platelet transfusions are
with the infant’s red cells; if platelets con- most likely to be of benefit when thrombo-
taining compatible plasma are not avail- cytopenia is the primary hemostatic de-
able, the plasma can be reduced (see fect. The goal is to maintain counts
Method 6.15). This is rarely necessary in >50,000/µL. Other blood components
adults or older children, although signifi- may also be required in patients with
cant hemolysis has been reported after multiple defects. Bleeding due to the de-
transfusion of group O Platelets Pheresis fects in platelet function that follow
with high-titer anti-A or B.36 If transfused cardiopulmonary bypass surgery or to the
ABO antibodies are detectable in the recipi- ingestion of aspirin-containing com-
ent, it may become necessary to use group pounds, glycoprotein IIb/IIIa antagonists
O RBC units. (eg, abciximab), and P2 inhibitors (eg,
clopidigrel and ticlopidine) often re-
Matching for Rh sponds to platelet transfusion. Other de-
fects such as those found in uremia or von
The D antigen is not detectable on plate-
Willebrand disease respond less well be-
lets, and posttransfusion survival of plate-
cause the transfused platelets tend to ac-
lets from Rh-positive donors is normal in
quire the same defect.
recipients with anti-D. However, platelet
components contain small numbers of
red cells so Rh-negative individuals may Prophylactic Platelet Transfusion
become alloimmunized by platelet com- Indications for prophylactic platelet
ponents from Rh-positive donors. For im- transfusion are more controversial than
munocompetent normal Rh-negative fe- for therapeutic rationales. A threshold of
males of childbearing potential, it is 20,000/µL or less for patients with chemo-
especially desirable to avoid administra- therapy-induced thrombocytopenia has
tion of platelets from Rh-positive donors; been used by many physicians, but pro-
however, if this is unavoidable, Rh Im- spective randomized trials have shown
mune Globulin (RhIG) should be admin- that a threshold of 10,000/µL in stable pa-

tients is equally safe and results in signifi- prophylaxis was ineffective. Although it en-
cant decreases in platelet usage. 41-43 A dorsed the logic of prophylactic platelet
higher transfusion trigger is often used for transfusion for thrombocytopenic patients
patients with fever, evidence of rapid con- undergoing surgery, the NIH consensus
38
sumption, high white cell counts, coagu- panel suggested that such transfusions
38
lation defects, and intracranial lesions. were most appropriate for patients in
In contrast, many stable thrombocyto- whom hemorrhage could not be observed
penic patients can tolerate platelet counts or in whom it occurred at a site where it
as low as 5000/µL.41 could be critical in small amounts (eg, in
Despite the widespread use of prophy- the central nervous system). Published
lactic platelet transfusions, few studies have guidelines3,39 suggest a platelet transfusion
documented their clinical benefit. One trigger of 50,000/µL for most major surgery,
study comparing patients given prophylac- with counts “near” 100,000/µL possibly re-
tic transfusions with patients transfused quired for patients undergoing neurosur-
only for clinically significant bleeding dem- gery or ophthalmic procedures.39 Prophy-
onstrated a significant decrease in bleed- lactic platelet transfusion may also be
ing, but the number of patients in the study useful for patients who are having surgery
39
was too small to show a difference between and who have a platelet function defect,
the groups in overall survival or in deaths including that due to treatment with
44 47
due to bleeding. Of interest, the prophy- abciximab.
lactic group received twice as many platelet
transfusions, and there was a suggestion Refractoriness to Platelet Transfusion
that refractoriness developed more often in
Platelet refractoriness, defined as a poor
this group. This observation raises the ca-
increment following a dose of platelets, can
veat that prophylactic platelet transfusion
result from either immune or nonimmune
may be most relevant to patients in whom
mechanisms and is discussed in detail in
thrombocytopenia is expected to be a tem-
Chapter 16 and other reviews.48,49 The anti-
porary condition.38 If thrombocytopenia or
bodies that cause immune refractoriness
platelet dysfunction will be prolonged, the
may have either allo- or autoreactivity, with
development of refractoriness may limit the
alloantibodies most commonly directed
response to platelets, particularly if im-
against Class I HLA antigens. Autoantibodies
mune function is normal as it is in patients
occur in immune thrombocytopenic pur-
with aplastic anemia or congenital thrombo-
pura (ITP) (see Chapter 16). Nonimmune
cytopathies.
causes of the refractory state include infec-
Patients with severe preoperative thrombo-
tion, splenomegaly, drugs (particularly
cytopenia are generally assumed to benefit
amphotericin B), and accelerated platelet
from prophylactic platelet transfusion, but
consumption (see Table 16-3).
this has not been demonstrated in experi-
mental studies. The threshold for such pro-
phylaxis is typically set at platelet counts Contraindications to Platelet Transfusion
3
between 50,000 and 100,000/µL. Prophy- There are several conditions for which
lactic transfusion of platelets has been in- platelet transfusions may be requested
vestigated in circumstances in which but are contraindicated. Relative contra-
thrombocytopenia is expected to develop indications include conditions in which
intraoperatively, either because of dilution45 the likelihood of benefit is remote; trans-
46
or cardiopulmonary bypass ; in both cases, fusion in this setting merely wastes a valu-

able component. Examples include pro- 2. Fever for 24 to 48 hours, positive

phylactic platelet transfusions in stable bacterial or fungal blood cultures, or
patients with ITP50 or platelet refractoriness. progressive parenchymal infection
For ITP patients undergoing splenectomy, unresponsive to appropriate antibi-
transfusion of platelets should be delayed otic therapy.
until the vascular pedicle is clamped. 3. Myeloid hypoplasia.
Platelet transfusion should be avoided for 4. A reasonable chance for recovery of
patients with thrombotic thrombocyto- marrow function.
penic purpura (TTP) or active heparin-in- Patients with documented granulocyte
duced thrombocytopenia except in life- or dysfunction, such as those with chronic
organ-threatening hemorrhage. These granulomatous disease, may also be candi-
conditions are associated with platelet dates to receive granulocyte transfusions
thrombi, and major thrombotic compli- during life-threatening episodes of infec-
cations may follow platelet transfusions.51 tion or while awaiting hematopoietic pro-
genitor cell transplantation.
Other Considerations
Granulocyte Transfusion Granulocyte components contain signifi-
The use of granulocyte transfusions for cant amounts of red cells, which must be
adult recipients is rare. New antibiotics, crossmatch compatible and Rh specific,
adverse effects attributable to granulocyte particularly for females with childbearing
transfusions, the advent of recombinant potential. Granulocytes should be irradi-
growth factors, and difficulty demonstrat- ated to avoid the risk of graft-vs-host dis-
ing efficacy have contributed to this de- ease (GVHD). If cytomegalovirus (CMV)
cline. Nevertheless, in selected patients, transmission is an issue, its risk can be re-
transfused granulocytes may produce duced by the use of a CMV-seronegative
clinical benefits,52 particularly with the donor (leukocyte reduction filters are
larger granulocyte doses available from contraindicated). For alloimmunized re-
donors treated with granulocyte colony- cipients, donors should be matched by HLA
stimulating factor. 53 Attention to HLA typing or leukocyte crossmatching.52,54
compatibility is also required for allo-
immunized recipients.54 The preparation,
storage, and pretransfusion testing of Special Cellular Blood
granulocytes are discussed in Chapter 6,
and their use in neonates is discussed in Components
Chapter 24. Leukocyte Reduction
The approximate leukocyte content of com-
Indications and Contraindications mon blood components is summarized in
The goals of granulocyte transfusion should Table 21-2.55-59 Leukocyte reduction has
be clearly defined before a course of ther- been used for some time for select groups
apy is initiated. In general, the patient of patients. Current federal guidelines57,58
should meet the following minimum con- and AABB Standards for Blood Banks and
59(pp25,28,29,31,32)
ditions: Transfusion Services define a
1. Neutropenia (granulocyte count less leukocyte-reduced component as one
than 500/µL). with <5 × 106 residual donor leukocytes

Table 21-2. Approximate Leukocyte 2. CMV transmission.60

Content of Blood Components (per 3. HLA alloimmunization that may lead
Unit)
55-59 to patients becoming refractory to
platelet transfusions.61
Whole Blood 109 Controversial and unproven indications
RBCs 108 for leukocyte reduction include:
1. Reduction of immunomodulation that
RBCs Washed 107
may lead to an increased risk of can-
RBCs Deglycerolized 106-107 cer recurrence or bacterial infections.62
RBCs Leukocytes Reduced <5 × 106 2. Reduction in the risk of prion disease.
(by filtration)* 3. Reduction in the risk of Yersinia entero-
Platelets Pheresis 106-108 colitica contamination of RBC units.63
Platelets 107 Many studies have investigated the pos-
sibility that leukocyte reduction can reduce
Platelets Pheresis <5 × 106
the incidence of clinical outcomes due to
Leukocytes Reduced*
transfusion-related immunomodulation,
Platelets Leukocytes Reduced <8.3 × 105 but the results are contradictory.62 One such
Platelets Pooled <5 × 106 proposal was that savings related to re-
Leukocytes Reduced duced immunomodulation could offset the
FFP Thawed <0.6 × 106 - costs of leukocyte reduction, but this was
1.5 × 107 not demonstrable in a large prospective
*Leukocyte reduction with third-generation leukocyte ad- randomized trial.64 Nonetheless, several
sorption filter. countries have converted to a leukocyte-re-
duced blood supply, and this subject re-
mains controversial.65
per final product (this includes RBCs;
Platelets Pheresis; and pooled Platelets). Irradiation
requires <8.3 × 10
59(p31) 5
AABB Standards
Irradiation of a cellular blood component
leukocytes in Platelets Leukocytes Re-
is the only accepted method to prevent
duced, which are prepared from a single
GVHD. GVHD has been reported after
unit of Whole Blood, to achieve the re-
transfusion of leukocyte-reduced compo-
quirement for a pool of 6 platelet units.
nents.66 For more details on GVHD and in-
Draft guidance from the Food and Drug
dications for irradiation, see Chapter 27.
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- Replacement of Coagulation
pean guidelines define leukocyte-reduced Factors
components as those with <1 × 10 resid-
6
ual leukocytes per unit and require that Physiologic Principles

there should be no more than a 10% fail- Coagulation results from a complex but
ure rate in the process. ordered enzyme cascade occurring on the
Published data demonstrate that leuko- surface of platelets and cells that express
cyte reduction reduces the risk of: tissue factor (see Fig 21-1). The coagula-
1. Febrile nonhemolytic reactions (see tion cascade is typically divided into the
Chapter 27). intrinsic and the extrinsic pathways, the

Roberts HR, Monroe DM III, Hoffman M. Molecular biology and biochemistry of the coagulation factors and pathways of
hemostasis. In: Beutler E, Lichtman MA, Coller BS, et al. Williams’ hematology. 6th ed. New York: McGraw-Hill, 2001:1409-34.
Figure 21-1. (A) The classic cascade model of coagulation reactions was based on in-vitro 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 )
in-vitro activity of which can be measured Monitoring Hemostasis

by the activated partial thromboplastin
time (aPTT) and prothrombin time (PT), The PT, aPT T, and measurement of
respectively, but, in vivo, the cascades are fibrinogen level are commonly used to
interdependent.67 The central procoagu- monitor coagulation. Results should be
lant enzyme is thrombin, which is acti- interpreted with four considerations in
vated by both pathways. mind: 1) mild prolongations of the PT or
Minimal levels of coagulation factors (see aPTT occur before the residual factor con-
Table 21-3) are required for normal forma- centration falls below the level normally
tion of fibrin and hemostasis, so normal needed for hemostasis; 2) conversely, the
plasma contains coagulation factors in ex- PT and aPTT are relatively insensitive to
cess, a reserve that usually allows patients low fibrinogen levels; 3) significant defi-
to tolerate replacement of one or more ciencies of coagulation factors (or the
blood volumes of red cells and crystalloid presence of coagulation factor inhibitors)
without needing Fresh Frozen Plasma (FFP). cause clearly prolonged values for the PT
Patients with liver disease have less physio- or aPTT; and 4) an infusion of FFP that in-
logic reserve and are more susceptible to creases the concentration of factors by 20%
dilutional coagulopathy. will have a far greater impact on a greatly

Table 21-3. Coagulation Factors
% of Normal
In-vivo In-vitro, 4 C Needed for % In-vivo
Factor Name Half-life Half-life Hemostasis 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 18-24 hours >21 days 15-40 20 See Table 21-6
component, Christmas factor
X Stuart-Prower factor 20-42 hours >21 days 10-40 50-95 10-20 units/kg body weight
XI Plasma thromboplastin 40-80 hours >28 days 20-30 90 10-20 mL/kg body weight
antecedent (PTA)
Chapter 21: Blood Transfusion Practice

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.
495
prolonged PT or aPTT than on a mildly Plasma derivatives are concentrates of

prolonged PT or aPTT. For example, the specific plasma proteins from large pools of
infusion of two units of FFP in a patient plasma or cryoprecipitate. Cohn fraction-
with a PT of 14.5 seconds (normal: 11 to ation, which relies on the precipitation of
13 seconds) is unlikely to provide any clin- various plasma proteins in cold ethanol-
ical benefit and is also unlikely to correct water mixtures, was developed during
the PT to the normal range. World War II and is still used with some
Guidelines typically cite a PT 1.5 times modifications.68 After fractionation, deriva-
3
the upper limit of normal or the midpoint tives undergo further processing to purify
of the normal range39 and an aPTT 1.5 times and concentrate the proteins and inactivate
the upper limit of normal3,39 as thresholds at contaminating viruses. Virus-inactivation
which therapeutic or prophylactic replace- procedures include heat treatment, micro-
ment may be indicated in an appropriate filtration, the use of chemical solvents and
clinical setting. Of note, however, studies detergents, and affinity column purifica-
have consistently shown that the PT and tion. Factors VIII, IX, VIIa, and anti-
aPTT, even when elevated to this degree, thrombin are also produced by recombi-
have little predictive value for bleeding nant DNA technology. These products
complications of invasive procedures in- appear to be efficacious and are not known
cluding paracentesis or thoracentesis,25 liver to carry any infectious risk.
biopsy,26,27 angiography,23 or central venous The specific activity (factor units/mg
catheter placement.24 protein) of presently available concentrates
has been dramatically increased in concen-
trates prepared with affinity columns or by
recombinant technology. Moreover, HIV,
Components and Products Available for
HBV, and HCV transmission appear to be
Coagulation Factor Replacement
absent in patients with hemophilia treated
The plasma components that are avail- exclusively with new preparations.69 Unfor-
able differ according to the timing of tunately, their cost has also increased due
freezing and/or thawing and variations in to the increased complexity of manufactur-
preparation (see Chapter 8). FFP contains ing and the protein losses resulting from
all the clotting factors, including labile extensive manipulation. Coagulation factor
Factors V and VIII. Other forms of plasma concentrates are supplied in lyophilized
have lower levels of labile factors but form and the factor activity is stated on the
could substitute for FFP for most of the label.69 Currently available products for re-
indications for which the latter is trans- placement of Factors VIII and IX are listed
fused. Plasma Cryoprecipitate Reduced in Table 21-4.
has a decreased content of vWF and is
used specifically for treatment of TTP (see
Chapter 6). Pooled Plasma, Solvent/De-
Selection of ABO-Compatible Plasma
tergent-Treated is no longer available in
the United States. Because of its long shelf life, group-spe-
Cryoprecipitated AHF (CRYO) is a con- cific or compatible plasma (see Table
centrate of high-molecular-weight plasma 21-5) is typically available. (Note that
proteins that precipitate in the cold, includ- platelet transfusions usually contain a
ing vWF, Factor VIII, fibrinogen, Factor XIII, volume of plasma equivalent to one unit
and fibronectin (see Chapter 8). of FFP, and limitations in availability may

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- Alphanate Factor VIII replacement Contains vWF
fied
Factor VIII, immunaffinity Hemofil-M Factor VIII replacement
purified
Monarc M Factor VIII replacement
Monoclate-P Factor VIII replacement
Factor VIII, recombinant Kogenate FS Factor VIII replacement Contains trace amounts
Helixate FS of human albumin
Recombinate Factor VIII replacement
(Bioclate)
ReFacto Factor VIII replacement Does not contain human
albumin
Factor VIII, porcine Hyate C Factor VIII inhibitor tx.
Factor IX, affinity AlphaNine-SD Factor IX replacement
purified
Factor IX, immunaffinity Mononine Factor IX replacement
purified
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 Contains Factor II,
Factor VIII inhibitor tx. Factor X, some
Warfarin reversal Factor VII
Profilnine SD Factor IX replacement Contains Factor II,
Factor X, some
Factor VII
Proplex T Factor IX and Factor VII Contains Factor II,
replacement Factor VII, and
Factor VIII inhibitor tx. Factor X
Factor IX complex, Autoplex-T Factor VIII inhibitor tx. Contains Factor lIa,
activated Factor VIIa, and
Factor Xa
FEIBA VH Factor VIII inhibitor tx. Contains Factor IIa,
Factor VIIa, and
Factor Xa
Factor VIIa, NovoSeven Factor VIII inhibitor tx. Does not contain human
recombinant albumin

Table 21-5. Suggested ABO Group Selection Order for Transfusion of Plasma
Component ABO Group
Recipient
ABO Group 1st Choice 2nd Choice 3rd Choice 4th Choice
AB AB (A) (B) (O)
A A AB (B) (O)
B B AB (A) (O)
O O A B AB
(Blood groups in parentheses represent choices with incompatible plasma, listed in “least incompatible” order.)
require infusion of incompatible plasma weeks, so deficiency may occur in hospi-

in this context.) talized patients unable to tolerate normal
food intake. Absorption of vitamin K re-
Indications for FFP quires precursor metabolism by bacteria
in the intestine and the action of bile salts;
Guidelines exist for the appropriate use of
therefore, deficiencies can occur with anti-
FFP.3,39,70 FFP is the only approved compo-
biotic use, obstructive jaundice, and fat
nent for clinically significant deficiency of
malabsorption syndromes.
Factors II, V, X, and XI. Plasma is most of-
Vitamin K depletion or warfarin usually
ten used in patients with multiple factor
cause a prolongation of the PT that is out of
deficiencies, including those with liver
proportion to the aPTT because Factor VII,
disease, dilutional and consumption
which has the shortest half-life of the vita-
coagulopathy, or a need for rapid reversal
min-K-dependent factors, has little effect
of warfarin treatment. It is of limited clini-
on the aPTT. Deficiency of vitamin K is best
cal benefit in patients with inhibitors to
managed by treatment of the underlying
any coagulation factor. Plasma Cryopreci-
condition and by administration of vitamin
pitate Reduced may be more effective
K.67 If liver function is adequate, coagulation
than FFP for some patients receiving
factors will return to effective levels in
plasma exchange treatment for TTP or
about 12 hours.
hemolytic-uremic syndrome71 (see Chap-
Although most patients with vitamin K
ter 6).
deficiency do not require plasma, plasma
transfusion is occasionally needed to treat
Vitamin K Deficiency and Warfarin Reversal active bleeding or to prepare for emergency
The most common cause of multiple co- invasive procedures.67 Transfusion of 10 to
agulation factor abnormalities among 15 mL of plasma per kg of body weight will
hospitalized patients is deficiency of the generally achieve hemostatic coagulation
vitamin-K-dependent factors due to treat- factor levels in patients with warfarin-in-
ment with the vitamin K antagonist duced coagulopathy. One form of Factor IX
warfarin (Coumadin) or nutritional defi- complex concentrate is licensed for warfa-
67
ciency. Vitamin K is a fat-soluble vitamin rin reversal (see Table 21-4) but carries a sig-
required for hepatocellular synthesis of nificant risk of thrombotic complications and
coagulation Factors II, VII, IX, and X, as is rarely used for this indication. Concurrent
well as the anticoagulant proteins C and vitamin K supplementation should also be
S. Body stores of vitamin K last only 2 given unless only transient correction is de-

sired. Although the International Normalized coagulopathy is overdependence on the PT.

Ratio can provide useful information about Again, a normal PT is rarely, if ever, re-
response to therapy, the need for additional quired for the cessation of serious bleeding,
treatment should be guided by the clinical re- and the goal of FFP therapy in severe liver
sponse and not by the results of laboratory disease should be to correct or prevent
tests. As discussed above, it is rarely neces- bleeding complications. If FFP is to be used
sary to correct the PT or aPTT to normal to prophylactically before an invasive proce-
achieve adequate hemostasis. dure, it should be given immediately before
the procedure.
Patients with liver disease may also have
Liver Disease abnormalities of platelet plug formation
Patients with liver disease have multiple and fibrinolysis. In addition, severe spleno-
derangements that contribute to an in- megaly may impair the response to platelet
creased bleeding tendency. These abnor- transfusions. Platelet function in some pa-
malities include portal hypertension and tients with liver disease can be enhanced by
engorgement of systemic collateral ve- administration of 1-deamino-8-D-arginine
nous shunts; splenomegaly with second- vasopressin (desmopressin, DDAVP).73 Cryo-
ary thrombocytopenia; decreased synthe- precipitated AHF should be given if there is
sis of all coagulation factors except Factor severe hypofibrinogenemia or bleeding re-
VIII; dysfibrinogenemia; decreased clear- lated to dysfibrinogenemia. The increase in
ance of fibrin, fibrinogen degradation systemic fibrinolysis associated with severe
products, and fibrinolytic activators; and liver disease may not respond to FFP alone,
decreased synthesis of inhibitors of the and antifibrinolytic agents in combination
fibrinolytic system. As in vitamin K defi- with plasma therapy can be useful in these
ciency, the short half-life of Factor VII patients (see below).
causes the PT to be prolonged more than
the aPTT, and, for the same reason, FFP
infusion corrects the PT for only about 4 Dilutional Coagulopathy
hours. 72 Because the defect in hepato- Massive blood loss and replacement with
cellular disease is in primary protein syn- crystalloid and/or colloid solutions may
thesis, supplemental vitamin K will not produce a dilutional coagulopathy,74 but
usually correct the abnormality. However, most patients can tolerate loss and re-
because it is one of the few treatable causes placement of at least one blood volume
of coagulopathy in liver disease, a trial of without developing impaired hemo-
replacement vitamin K may be indicated. stasis.75 Shock accompanying traumatic
FFP corrects coagulation factor deficien- hemorrhage also contributes to the coa-
cies found in severe liver disease, but is of- gulopathy (see Chapter 27). In the setting
ten used inappropriately. The most com- of trauma, thrombocytopenia generally
mon error is to attribute all bleeding to develops before plasma clotting factors are
coagulopathy and to give systemic treat- diluted to the point of causing impaired
ment when the cause is localized bleeding. hemostasis, and adequate platelet re-
For example, bleeding at the operative site placement generally has priority.75 However,
after cardiac surgery usually responds in elective surgical patients, coagulation
better to local hemostatic measures than to factor deficits may predominate.74 FFP may
intravenous infusion of FFP. A second com- be beneficial if the PT and/or aPTT are
mon error in treating liver-associated greater than 1.5 times normal.3,39 If surgi-

38,70,75
cal hemostasis has not been achieved and enhance wound healing. Transfusing
significant continued bleeding is expected, plasma for volume expansion carries a
FFP may be indicated.39 risk of infectious disease transmission and
Patients undergoing plasmapheresis other transfusion reactions (eg, allergic)
without plasma replacement develop a va- that can be avoided by using crystalloid or
riety of coagulation factor deficits, particu- colloid solutions. Plasma is also not a
larly hypofibrinogenemia, depending on suitable source of immunoglobulins for
the volume and frequency of the ex- patients with severe hypogammaglobu-
76
changes. Although these changes can be linemia because more effective prepara-
striking, most authors have concluded that tions exist (immunoglobulin for intrave-
routine supplements with FFP in patients nous or intramuscular use).
with normal liver function are unnecessary, FFP is often given prophylactically to pa-
particularly for alternate-day regimens. tients with mild to moderate prolongation
of the PT or aPTT before invasive proce-
Other Conditions dures, but there is little or no evidence that
this prevents bleeding complications. Be-
Plasma exchange is lifesaving in TTP (see
cause these tests do not accurately predict
Chapter 6). FFP may be an adjunct to treat-
the risk of bleeding when mildly prolonged,
ment of DIC. Hereditary angioneurotic
there is little logic for a transfusion in-
edema results from a congenital defi-
tended to “improve” the results.
ciency of C1-esterase inhibitor, an inhibi-
tory protein that regulates complement
activation. Patients with this condition
develop localized edema and may experi- Cryoprecipitated AHF
ence lifethreatening obstruction of the Transfusion
upper respiratory tract following comple-
CRYO is the only concentrated fibrinogen
ment activation. FFP or Liquid Plasma
product currently available for systemic
contain normal levels of C1-esterase in-
use, and intravenous supplementation of
hibitor and FFP transfusion appeared to
fibrinogen is its primary clinical use, par-
prevent attacks at the time of oral surgery
ticularly in DIC. A second major use has
in one study.77 There are rare anecdotal re-
been in patients with severe von Wille-
ports of exacerbation of angioneurotic
brand disease, but there are Factor VIII
edema with FFP administration. The need
concentrates that contain vWF and are
for treatment of isolated deficiency of
more appropriate if available (see Table
Factors II, V, VII, X, or XI is uncommon;
21-4). CRYO can be used in isolated Fac-
guidelines for initial therapy are given in
tor XIII deficiency and to ameliorate the
Table 21-3 (however, for a more complete
platelet dysfunction associated with ure-
treatment, refer to one of the standard he-
mia. It is also used topically as a fibrin
matology or coagulation texts).
sealant, although a commercial prepara-
Plasma can also be used to replace pro-
tion is available. CRYO is seldom used for
teins C and S, and antithrombin; these are
patients with hemophilia because Factor
discussed separately below.
VIII concentrates are available commer-
cially and have been processed to reduce
Misuse of Fresh Frozen Plasma or eliminate the risk of blood-borne viral
Plasma should not be used as a volume infection; CRYO is used as a last resort for
expander, as a nutritional source, or to this indication. Because CRYO contains

ABO antibodies, consideration should be platelet plug formation and partial defi-
given to ABO compatibility when the in- ciency of Factor VIII. The former may
fused volume will be large relative to the manifest as a prolonged bleeding time
recipient’s red cell mass. and the latter as a prolonged aPTT, but
these abnormalities vary between syn-
Calculating the CRYO Dose for Fibrinogen dromes. vWF exists in the plasma as a
Replacement family of multimeric molecules with a
On average, one unit of CRYO contains wide range of molecular weights. The
approximately 250 mg of fibrinogen; the high-molecular-weight species of vWF are
minimum required by AABB Standards is the most hemostatically effective. Labora-
150 mg.59(p30) The amount of transfused tory evaluation demonstrates a specific de-
CRYO required to correct the fibrinogen ficiency in the level of vWF, often mea-
level depends upon the nature of the sured as ristocetin cofactor activity because
bleeding episode and the severity of the vWF is required for the platelet-agglutinat-
initial deficiency and can be calculated as ing effect of ristocetin in vitro.
follows: Mild cases of von Willebrand syndrome
1. Weight (kg) × 70 mL/kg = blood vol- can often be treated with DDAVP, which
ume (mL). causes a release of endogenous stores of
2. Blood volume (mL) × (1.0 – hemato- Factor VIII and vWF. Many Factor VIII con-
crit) = plasma volume (mL). centrates do not contain therapeutic levels
3. Mg of fibrinogen required = (Desired of vWF, but several with satisfactory levels
fibrinogen level in mg/dL – initial are commercially available and one is li-
fibrinogen level in mg/dL) × plasma censed for this indication (see Table 21-4).
volume (mL) ÷ 100 mL/dL. In the absence of a suitably therapeutic vi-
4. Bags of CRYO required = mg of fibrino- rus-inactivated concentrate, severe von
gen required ÷ 250 mg fibrinogen/ Willebrand syndrome can be treated with
bag of CRYO CRYO. The quantity of CRYO required to
This calculation assumes that 100% of treat bleeding episodes or to prepare for
administered fibrinogen is recovered as major surgery varies greatly among patients
measurable intravascular fibrinogen, but, with von Willebrand syndromes. In addi-
because the content of CRYO is variable, tion to the clinical response of the patient,
further refinements are unproductive. the template bleeding time, the level of Fac-
tor VIII, or the ristocetin cofactor activity
may help to guide therapy.
von Willebrand Syndromes
von Willebrand syndromes are the most
Fibrinogen Abnormalities
common major inherited coagulation ab-
normalities.78 These conditions are usu- Hypofibrinogenemia may occur as a rare
ally autosomal dominant and represent a isolated congenital deficiency, may be ac-
collection of quantitative and qualitative quired as part of the DIC syndrome, or may
abnormalities of vWF, the most important be due to obstetric complications such as
protein mediating platelet adhesion to abruptio placentae. Dysfibrinogenemias
damaged endothelial surfaces. The pro- may be congenital or acquired and repre-
tein also transports Factor VIII. As a resent conditions in which fibrinogen is
sult, patients with von Willebrand syn- present as measured by immunoassays
dromes have varying degrees of abnormal but functionally defective as measured by

the thrombin time. Patients with severe in the setting of hemorrhage that results
liver disease frequently exhibit a dys- from DIC once the fibrinogen is above 100
fibrinogenemia. mg/dL.
Disseminated Intravascular Coagulation Topical Use

The fibrinogen in CRYO has been used
DIC occurs when circulating thrombin in-
during surgery as a topical hemostatic
duces widespread fibrin formation in the
preparation, so-called fibrin sealant or fi-
microcirculation and consumption of pla-
brin glue, made from one or two units of
telets and coagulation factors, particularly
CRYO, which may be of autologous origin.
fibrinogen, prothrombin, Factor V, and
The fibrinogen is converted to fibrin by
Factor VIII. Fibrin strands in the micro-
the action of bovine thrombin at the site
circulation may cause mechanical damage
that is bleeding or to be “glued.” Commer-
to red cells, a condition called microangio-
cial preparations of fibrin sealant are
pathic hemolysis, manifest as schistocytes
available that have a higher fibrinogen
(fragmented red cells) in the circulation.
concentration than that of CRYO and in-
Widespread microvascular thrombi pro-
clude human thrombin and bovine apro-
mote tissue ischemia and release of tissue
tinin (see below) to decrease the lysis of
factor, which further activates thrombin.
the resulting fibrin. These pooled, virus-
Lysis of microvascular fibrin causes in-
inactivated products have been licensed
creased quantities of fibrin degradation
for the reduction of bleeding in cardiovas-
products to enter the bloodstream.
cular surgery, 7 9 for repairing splenic
Several clinical conditions can initiate
trauma, and for colostomy closure. Fibrin
DIC, including shock, tissue ischemia, sep-
sealants have also been used for a variety
sis, hemolytic transfusion reactions, dis-
of indications in which it is desired to
seminated cancer (particularly adeno-
bind two tissue surfaces together, includ-
carcinoma), acute promyelocytic leukemia,
ing repair of the dura mater or eardrum.
tumor lysis syndrome, and obstetric com-
Use of bovine thrombin can stimulate
plications such as amniotic fluid embolism.
the formation of antibodies against thrombin
The common precipitating event is a pro-
and other contaminant proteins including
coagulant signal for thrombin production that
Factor V.80 These antibodies can cross-react
exceeds the normal physiologic defenses
with human thrombin and Factor V, caus-
against disseminated thrombin activity.
ing abnormal clotting times and, in some
Treatment of DIC depends on correcting
cases, bleeding. For this reason, it has been
the underlying problem and preventing
suggested that use of “homemade” fibrin
further hypotension and tissue ischemia.
sealants be replaced by use of the commer-
Replacement therapy focuses on the build-
cial product.80
ing blocks of the thrombus (platelets and
fibrinogen), and secondarily on other coag-
ulation factors, including Factors VIII, XIII, Hemophilia A
V, and II. Thus, in bleeding patients, platelet Each unit of CRYO prepared from a single
transfusion is indicated when the platelet blood donation should contain a minimum
count falls below 50,000/µL, and cryopre- of 80 international units (IU) of Factor
cipitate is supplemented if the fibrinogen VIII.59(p30) Although no longer the compo-
level is below 100 mg/dL. FFP is indicated nent of choice, CRYO can serve as replace-

ment therapy for patients with hemo- storage sites. However, DDAVP is ineffective
philia A if virus-inactivated Factor VIII in patients with severe hemophilia A; in
39
concentrates are unavailable. If CRYO is such cases, Factor VIII replacement is re-
used, the amount required to provide a quired. The amount of Factor VIII infused
therapeutic dose of Factor VIII is based on depends upon whether therapy is intended
calculations similar to those used for AHF. to prevent bleeding or, if bleeding has oc-
Hemophilia A is a sex-linked recessive curred, the nature of the bleeding episode
trait (ie, affected males, carrier females) and the severity of the initial deficiency (see
causing deficiency of Factor VIII (anti- Table 21-6). For example, treatment for
hemophilic factor, AHF).81 The responsible hemarthrosis ordinarily requires more Fac-
genes usually produce a protein with re- tor VIII than epistaxis.
duced activity, so immunologic measure-
ment of Factor VIII antigen gives normal re-
Calculating the Dose of Factor VIII
sults despite deficient Factor VIII coagulant
activity. In contrast, antigen is typically de- When the desired result is determined
pressed in von Willebrand disease. Charac- (see Table 21-6), the amount of Factor VIII
teristic laboratory findings include a pro- required for transfusion can be calculated
longed aPTT, normal PT and template by one of the following formulas:
bleeding time, and decreased Factor VIII
activity. Factor VIII dose (IU/kg) =
The severity of hemophilia A depends on Desired factor increase (%) × 0.5 (1
the patient’s level of Factor VIII activity, and IU/kg typically raises the Factor VIII
this varies; patients with severe hemophilia level by 2%)
have Factor VIII levels below 1%, whereas
those with moderate disease typically have Total dose =
1% to 5% activity, and severity is mild with (Patient mass × 70mL/kg) × (1 – Hct) ×
levels of 6% to 30%.81 One unit of Factor VIII (Desired activity – current activity)
activity is defined as the Factor VIII content
of 1 mL of fresh, citrated, pooled normal Example: A 70-kg patient with severe he-
plasma. The measured level of Factor VIII mophilia has an initial Factor VIII level of
can be expressed as a concentration, as 2% (0.02 unit/mL) and a hematocrit of 40%.
percent activity, or as a decimal fraction. How many units of Factor VIII concentrate
For example, a patient with mild hemo- should be given to raise his Factor VIII level
philia with one-tenth the normal activity of to 50% (0.5 unit/mL)?
Factor VIII can be said to have a Factor VIII
level of 10 units/dL or 0.1 unit/mL or 10% (70 × 70) × (1 – 0.4) × (0.5 – 0.02) =
activity. 1411 units
Patients with mild-to-moderate hemo-
philia can often be managed without re- The therapy of choice for severe hemo-
placement therapy.81 Careful attention to lo- philia A is a Factor VIII concentrate (see Ta-
cal hemostasis and the use of topical ble 21-4). CRYO could be used to supply
antifibrinolytics may prevent the need for 1411 units of Factor VIII, but, at 80 IU per
further replacement. Systemic levels of Fac- bag, this would require at least 18 bags (and
tor VIII can be raised in mild hemophilia 18 allogeneic donor exposures). The half-
with the use of DDAVP, which stimulates life of Factor VIII is about 12 hours, so infu-
the release of endogenous Factor VIII from sions are repeated at 8- to 12-hour intervals

Table 21-6. General Factor Replacement Guidelines for the Treatment of Bleeding
in Hemophilia
Initial
Minimum
Desired Factor VIII Factor IX
Factor Level Dose* Dose* Duration
Indication (%) (IU/kg) (IU/kg) (days)
Severe epistaxis, oral 20-30 10-15 20-30 1-2
mucosal bleeding†
Hemarthrosis, hematoma, 30-50 15-25 30-50 1-3
persistent hematuria,‡
gastrointestinal tract
bleeding, retroperitoneal
bleeding
Trauma without signs of 40-50 20-25 40-50 2-4
bleeding, tongue/
retropharyngeal
bleeding†
Trauma with bleeding, 100 50 100 10-14
surgery,§ intracranial
bleeding§
69
Data from USP.
*Dosing intervals are based on a half-life for Factor VIII of 8 to 12 hours (2 to 3 doses/day) and a half-life for Factor IX
of 18 to 24 hours (1 to 2 doses/day). Maintenance doses of one half the initial dose may be given at these intervals. The
frequency depends on the severity of bleeding, with more frequent dosing for serious bleeding.
†
In addition to antifibrinolytics.
‡
Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to
maintain renal output.
§
Continuous factor infusion may be administered. Following the initial loading dose, a continuous infusion at a dose of 3
IU/kg per hour is given. Subsequent doses are adjusted according to the plasma factor levels.
to maintain hemostatic levels. The duration ease or genetic defects involving large
of treatment with Factor VIII infusions de- portions of the molecule, develop a de-
68,81
pends upon the type and location of the tectable inhibitor to human Factor VIII.
hemorrhage or the reason for prophylaxis, These antibodies are directed against the
and the clinical response of the patient (see active site of Factor VIII, rendering the pa-
Table 21-6). After major surgery, the Factor tient relatively unresponsive to infusion.
VIII level should be maintained above 40% Patients having an elective invasive pro-
to 50% for at least 10 days. When elective cedure should be screened for such inhi-
surgery is planned, Factor VIII assays bitors. Management is difficult; ap-
should be made available to serve as a proaches have included attempts to
guide to therapy. overwhelm the inhibitor with very large
doses of human Factor VIII; use of por-
Treatment of Inhibitors of Factor VIII cine Factor VIII, which has low cross-reac-
About 10% to 35% of patients with hemo- tivity with human Factor VIII antibody81;
philia A, typically, those with severe dis- use of Factor VIII bypassing agents in-

cluding Factor IX complex, activated Fac- administration contain aggregates that

tor IX complex, and activated Factor VII may activate the complement and kinin
(see Table 21-4); and desensitization ther- systems and produce hypotensive and/or
apy. The latter includes large daily doses anaphylactoid reactions if administered
of AHF in conjunction with cortico- intravenously, but the intravenous prod-
steroids or Immunoglobulin Intravenous uct contains almost exclusively mono-
(IGIV) and cyclophosphamide. Success rates meric IgG molecules.
83
of 50% to 80% are reported. If hemorrhage The indications for the use of IGIV are
is life-threatening, intensive plasma- 83,84
evolving. Some conditions in which IGIV
pheresis to remove the inhibiting anti- is used are listed in Table 21-7. Infusion of
body, coupled with immunosuppression, IGIV can induce such reactions as head-
as well as infusions of Factor VIII and pos- ache, vomiting, volume overload, allergic
sibly antifibrinolytic therapy (see below), reactions, renal failure, and pulmonary re-
can be employed. actions, but they can usually be prevented
by infusing slowly and pretreating with
Hemophilia B diphenhydramine and/or hydrocortisone.
Passively transferred blood group allo- and
Factor IX deficiency (hemophilia B, Christ-
autoantibodies and/or therapy-induced
mas disease) is clinically indistinguish-
hypergammaglobulinemia may cause a
able from Factor VIII deficiency in that
positive DAT result in recipients, but signif-
both are sex-linked disorders that cause a
icant hemolysis is rarely noted.
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- Antiprotease Concentrates
centrate has been used for treatment of Antithrombin, also known as heparin co-
hemophilia B for the past 2 decades, but factor, is a serine protease inhibitor syn-
the new, more pure forms of Factor IX 85
thesized in the liver. It circulates in nor-
concentrate (see Table 21-4) are preferred mal plasma at a concentration of 15 mg/
because they carry much less risk of in- dL but is typically measured as % activity,
82
ducing thrombosis. with a normal range of 84% to 116%. Anti-
The formula for calculating of Factor IX thrombin inactivates serine proteases in-
dosage is similar to that for Factor VIII, but cluding thrombin, and Factors IXa, Xa,
the units to be given should be doubled be-
XIa, and XIIa by covalently bonding at the
cause only half of the infused Factor IX
serine site, followed by a conformation
dose is recovered in the vascular space. The 86
change. This activity is greatly acceler-
biologic half-life is 18 to 24 hours, so doses
ated by heparin, which induces a confor-
are given 1 or 2 times/day. As with hemo-
mation change in antithrombin and helps
philia A, recommended dose and treatment
approximate thrombin and antithrombin
schedules vary with the severity and type of
as well.
bleeding (see Table 21-6).
Patients who are deficient in anti-
thrombin are prone to thromboembolic
Immunoglobulin, Intravenous complications.87 Such deficiency can be
IGIV is prepared from modified Cohn frac- congenital or acquired. Acquired deficiency
tion II and subjected to virus inactivation. occurs in a wide variety of disease states,
Preparations intended for intramuscular including decreased synthesis due to liver

Table 21-7. Potential Indications and Clinical Uses for Intravenous Immunoglobulin
Preparations
Congenital immune deficiencies
Hypogammaglobulinemia and agammaglobulinemia
Selective antibody deficiency
IgG subclass deficiency and recurrent infection
Premature newborns
Acquired antibody deficiency
Malignancies with antibody deficiency and recurrent infection: multiple myeloma,
chronic lymphocytic leukemia
Protein-losing enteropathy
Drug- or radiation-induced humoral immunodeficiency
Prophylaxis or treatment of bacterial and viral diseases

Pediatric HIV infection for prevention of bacterial and secondary viral infections
Cytomegalovirus infection in transplant recipients
Neonatal sepsis
Other
HIV-related immune thrombocytopenic purpura
Immune cytopenias (ITP, NAIT, PTP, WAIHA)
Kawasaki syndrome
Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy
Acquired Factor VIII inhibitors
Myasthenia gravis
Multiple sclerosis
disease or malnutrition; losses due to thromboembolic episodes and for prophy-

nephrotic syndrome and gastrointestinal lactic use in perioperative, postoperative,
states; accelerated consumption as in DIC, and peripartum settings. Several off-label
surgery or trauma, and preeclampsia; and uses of antithrombin exist, eg, patients with
associated with pharmacotherapy includ- low antithrombin levels and DIC, neonates
ing heparin, L-asparaginase, and oral born to mothers with antithrombin defi-
contraceptives. ciency, and liver transplant recipients.
Antithrombin is stable in plasma so defi- The half-life of purified antithrombin is
ciency can be treated with FFP or with Liq- long, approximately 60 to 90 hours,88 but is
uid or Thawed Plasma. A heat-treated con- abbreviated when replacement is for con-
centrate of antithrombin is also available; sumptive states. The dose is calculated on
recombinant and transgenic sources are the basis of an expected increment of 1.4%
under investigation. per U per kg, with an initial target of 120%.
Clinical uses of antithrombin have re- Other available concentrates of antipro-
87
cently been reviewed. Antithrombin is ap- teases include alpha-1-proteinase inhibitor
proved for use in hereditary antithrombin (alpha-1-antitrypsin). C1-esterase inhibitor
deficiency as part of the treatment for is not available in the United States.

Protein C and Protein S Daily albumin synthesis in a normal adult

Protein C and protein S are vitamin-K-de- approximates 16 g. Albumin has complex
pendent proteins with anticoagulant ef- roles in normal physiology and disease in
fects.86 Protein S is a cofactor for activated addition to its obvious one of mainte-
protein C, which, in turn, inactivates Fac- nance of intravascular volume.90 Hypo-
tor Va and Factor VIIIa. Patients with defi- albuminemia resulting from decreased
ciencies of protein C or protein S have a synthesis, increased catabolism or losses,
predisposition to thrombotic complications and shifts between different fluid com-
and are often treated with anticoagulants.
85 partments is common in acute and chronic
Warfarin treatment, however, can cause illness.
these vitamin-K-dependent proteins to
decrease to dangerously low levels, lead- Replacement
ing to skin necrosis and exacerbating
Albumin solutions are effective volume
thrombosis. Transfusion of FFP can serve
expanders, with the promise of better
as an immediate source of supplemental
intravascular fluid retention than simpler
protein C or protein S for patients with se-
and less expensive crystalloids (eg, nor-
vere deficiencies, and a human protein C
mal saline or lactated Ringer’s solution).
concentrate is under development.
Because hypoalbuminemia is involved in
Patients with heterozygous protein C de-
the pathogenesis of many disease states
ficiency have plasma levels 40% to 60% of
and may correlate with their prognosis, it
normal and characteristically have minimal
symptoms, rarely requiring treatment with has been tempting to try to alter their
protein C supplementation.85 If treatment is course by exogenous supplementation,
needed for a thrombotic episode, anticoag- particularly in view of the perceived low
ulants suffice. Homozygous protein C defi- risk status of albumin solutions. However,
ciency causes neonatal purpura fulminans, this low-risk is in question.
which requires immediate administration of Indications for albumin approved by a
protein C, along with complex regulation of consensus panel91 include:
the rest of the coagulation cascade. The 1. Volume replacement in nonhemor-
half-life of infused protein C is 6 to 16 hours. rhagic shock unresponsive to crystal-
loid or in the presence of capillary
Colloid Solutions leak syndromes.
2. Volume replacement after the first
Human albumin (5% and 25%) and plasma day in patients with extensive burns
protein fraction (PPF) provide volume ex- (>50%) unresponsive to crystalloid.
pansion and colloid replacement without 3. Replacement after removal of large
risk of transfusion-transmitted viruses.89 volumes (>4 L) of ascitic fluid in pa-
PPF has a greater concentration of non- tients unresponsive to crystalloid.
albumin plasma proteins than 5% albumin. 4. Replacement of ascitic fluid or post-
Pharmacologic agents such as hydroxyethyl operative treatment of ascites and
starch or dextran are also commonly used peripheral edema in hypoalbumine-
for volume expansion. mic liver transplant recipients.
5. Replacement during large-volume
Physiology of Albumin plasma exchange.
The total body albumin mass is about 300 6. Volume replacement in patients with
g, of which 40% (120 g) is in the plasma. severe necrotizing pancreatitis.

7. Diarrhea (>2 L/day) in hypoalbum- poietic transplantation, but because the

inemic (<2.0 g/dL) patients on enteral anemia can be controlled with red cell
feedings, unresponsive to short chain transfusions and concurrent iron chela-
peptide supplementation. tion therapy, the use of this expensive and
Nonalbumin colloid solutions were con- potentially hazardous therapy is contro-
sidered less expensive first alternatives in versial. Transfusion of thalassemia pa-
several of these situations. tients is discussed in Chapter 24.
In spite of the conceptual attractiveness,
the long history of albumin replacement, Sickle Cell Disease
and the consensus on its use, a number of
prospective randomized trials have sug- Sickle cell disease (SCD) results from a
gested that albumin use was ineffective or single base substitution in the gene for
increased mortality. A meta-analysis of 30 the beta chain of hemoglobin. The hemo-
randomized trials including 1419 patients globin of individuals homozygous for this
grouped according to indication (namely, abnormality can irreversibly polymerize
hypovolemia, burns, and hypoproteinemia) and cause red cells to deform or “sickle.”
demonstrated higher mortality with albu- Such cells may initiate blockage of the
min treatment for each of the groups.92 In microvasculature directly or in associa-
response to this outcome, the authors of the tion with endothelial damage and throm-
meta-analysis called for an immediate re- bosis. They also have a decreased life
view of albumin use in critically ill patients. 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-
Special Transfusion plications or “crises,” including pain crisis
Situations due to musculoskeletal or other tissue
ischemia, splenic or pulmonary seques-
Thalassemia tration crisis (chest syndrome), aplastic
Thalassemia and sickle cell disease are in- crisis due to transient marrow suppression
herited syndromes characterized by defi- by viruses (particularly parvoviruses), leg
cient or abnormal hemoglobin structures ulcers, priapism, tissue infarction, and
and anemia. Thalassemia is caused by a stroke.
deficiency in alpha or beta chain produc- Most patients with sickle cell disease are
tion that ranges from mild to severe. Total asymptomatic most of the time and do not
absence of synthesis of one of the alpha require routine transfusion. Although
chains is lethal in utero; absence of beta sickling can be prevented or reversed by
chain synthesis (thalassemia major) re- maintaining the level of normal hemoglo-
sults in a progressive anemia in the new- bin above 50% to 70%, the risks from allo-
born period. In an attempt to compensate immunization, iron overload, and disease
for significant degrees of anemia, hemato- transmission outweigh the benefits of pro-
poietic tissue expands, causing character- phylactic transfusion in most patients.
istic bone abnormalities and enlargement Moreover, uncomplicated pain crises do not
of the liver and spleen. Tissue iron accu- respond well to simple transfusion. Simple
mulates as a result of increased adsorp- transfusion is indicated for symptomatic
tion and transfusion (see Chapter 27). The anemia, aplastic crises, and blood loss.
only current cure for thalassemia is hemato- Sometimes, patients with a history of stroke

or pulmonary or cardiac disease are some- cells. Other situations in which all units
times treated with a hypertransfusion proto- appear incompatible include the presence
col or chronic red cell exchange program to of alloantibodies to high-incidence anti-
maintain their hematocrit at 25% to 30% gens and multiple antibody specificities.
and the proportion of hemoglobin S below If serologic testing fails to resolve the
about 30%. Care should be taken to avoid problem, or if the problem is identified but
raising the hematocrit above 35% because time is not sufficient for acquisition of
of the risks of hyperviscosity. Red cell ex- compatible units, the physician must weigh
change is used to manage and/or prevent the risks and benefits of transfusion and
life- or organ-threatening complications, consider what alternative therapies are
particularly stroke and pulmonary crisis. suitable. If the need is sufficiently urgent,
Red cell exchange has also been used to incompatible red cells of the patient’s ABO
prepare patients for surgery, but a random- and Rh type may have to be given. Depend-
ized controlled trial did not support this ing on the alloantibody, incompatible
measure over simple transfusion to a he- transfusion does not always result in imme-
moglobin of 10 g/dL.93 diate hemolysis, and the incompatible cells
The clinical management of sickle cell may remain in the circulation long enough
disease is complex and the reader is re- to provide therapeutic benefit.96
ferred to recent reviews for more details.94,95 If time permits and if equipment is avail-
Patients with thalassemia and sickle cell able, the survival of a radiolabeled aliquot
disease can receive standard red cell com- of the incompatible cells can be deter-
ponents. However, leukocyte reduction is mined. Alternatively, an “in-vivo cross-
generally offered to avoid febrile, non- match” can be performed by cautiously
hemolytic transfusion reactions. Pheno- transfusing 25 to 50 mL of the incompatible
typing the patient’s red cells and providing cells, watching the patient’s clinical re-
antigen-matched units for transfusion sponse, and checking a 30-minute post-
helps reduce alloimmunization to red cell transfusion specimen for hemoglobin-
antigens and delayed hemolytic transfusion tinged serum. Such assessment does not
reactions, although the cost and logistics of guarantee normal survival, but it can indi-
such a program may be impractical for cate whether an acute reaction will occur. If
many institutions. A frequent compromise no adverse symptoms or hemolysis are ob-
is to match for Rh system and K antigens. served, the remainder of the unit can be
Patients with SCD should be given hemo- transfused slowly with careful clinical mon-
globin-S-negative RBC units. For more itoring. If the transfusion need is life-
details, see Chapter 24. threatening, RBC units may sometimes be
given without special testing, but clinical
staff should be prepared to treat any
Transfusing Known Incompatible Blood
reaction that may result.
Clinicians must occasionally transfuse a
patient for whom no serologically com-
Transfusing Patients with Autoimmune
patible RBC units are available. This most
Hemolytic Anemia
often occurs in patients with autoanti-
bodies, which typically react with all red Because of the serologic difficulties that
cells; however, once alloantibodies are accompany autoimmune hemolytic ane-
ruled out, the transfused cells are ex- mia and the expected short red cell sur-
pected to survive as long as autologous vival, a conservative approach to trans-

fusion is recommended. The presence of group alloantibodies, Rh-positive RBCs can

underlying alloantibodies should be in- be used with similar safety.
vestigated before beginning transfusions, The use of so-called “universal donor”
time permitting. It is very helpful to es- RBCs, as discussed above, has several draw-
tablish the patient’s phenotype before backs. Group O RBCs are typically in short
transfusion in order to simplify subse- supply because of demographic differences
quent investigation for the presence of between the donor and recipient popula-
possible alloantibodies. Chapter 20 con- tions in the United States, and such univer-
tains a more complete discussion. sal donor practices accentuate this prob-
lem. ABO typing can be performed very
Massive Transfusion quickly, and, in most emergencies, there is
time for ABO typing of the recipient and
Massive transfusion is defined as replace- provision of ABO-specific components.
ment approximating or exceeding the pa- Second, transfusion of large quantities of
tient’s blood volume within a 24-hour in- group O RBCs before a recipient blood
terval. The most important factor in sample is obtained may obscure subse-
supporting tissue oxygenation is mainte- quent immunohematologic testing. There-
nance of adequate blood flow and blood fore, even if universal donor RBCs are to be
pressure by infusing a sufficient volume used, a blood sample should be obtained
of crystalloid or blood components to before transfusion; this is possible in all but
correct or prevent hypovolemic shock. the most dire cases of exsanguinating hem-
orrhage. Finally, unexpected blood group
Emergency Issue antibodies can cause fatal transfusion reac-
tions, particularly in multitransfused pa-
The transfusion service should establish a tients such as those with SCD or liver dis-
standard operating procedure for emer- ease who present to an emergency room
gency provision of blood. Immunohema- with severe anemia or bleeding. In such
tologic testing is relatively time-consum- situations, a call to another hospital trans-
ing, so it may have to be abbreviated in fusion service at which the patient was pre-
trauma cases or other hemorrhagic emer- viously transfused can be life-saving. Indi-
gencies. Because ABO-compatible com- viduals caring for such patients must
ponents are entirely compatible in the understand the above principle concerning
vast majority of cases, particularly if the the priority of volume deficits over anemia.
patient has never been transfused before, Emergency situations should not be
group O RBCs are often used for emer- construed as justification for exceptions to
gency transfusion before completion of strict identification of recipient blood sam-
any compatibility tests. In this situation, ples. On the contrary, increased attention to
Rh-negative RBCs should be used for fe- patient identification is warranted, and one
males of childbearing potential because indication for use of universal donor RBCs is
of the concern for immunizing such indi- in situations when multiple trauma victims
viduals and possibly causing hemolytic arrive concurrently, when there is a high
disease of the newborn in the future. For risk of recipient misidentification.
women beyond their childbearing years or AABB Standards requires physicians who
men, only the current presence of anti-D order transfusion before completion of
is a concern. Because this antibody is no standard compatibility tests to document the
more common than certain other blood need by signing some type of “emergency

59(p46)
release form.” This documentation should which hemostasis was previously obtained.
be required only after the emergency is These situations have been attributed to
over, typically within 24 hours, and signa- the dilution of platelets or coagulation
tures on such forms should not be con- factors, but consumptive coagulopathy
strued as a release of the transfusion ser- also plays a role (see Chapter 27). Inade-
vice’s responsibility. The transfusion service quate volume resuscitation and poor tis-
must insist on strict identification of sam- sue perfusion not only promote the re-
ples, documentation of unit disposition, lease of tissue procoagulant material
and documentation of the emergency sta- leading to DIC but also result in lactic aci-
tus of the transfusion. However, it also has dosis, acidemia, and poor myocardial
the responsibility to perform testing on a performance. If MVB occurs, the results of
STAT basis, provide consultation, and avoid platelet counts, fibrinogen level, PT, and
unnecessarily restrictive practices. aPTT ideally should guide the need for
hemostatic components. Empiric therapy
Changing Blood Types with platelets and/or plasma may be initi-
ated immediately after specimens are ob-
The transfusion service should establish
tained. Additional tests may be indicated
guidelines for switching blood types dur-
to evaluate the possibility of DIC. In this
ing massive transfusion. An alternative to
situation, a platelet count less than
ABO-identical RBCs is the use of ABO-
50,000/µL and a fibrinogen level less than
compatible units (see Table 21-1). The age
100 mg/dL are better predictors of hem-
and sex of the patient are important con-
siderations. For example, when transfus- orrhage than the PT and aPTT.97 PT results
ing a young group B, Rh-negative woman, below 1.5 times normal are usually associ-
it is preferable to switch to group O, Rh- ated with adequate hemostasis during
negative RBCs before switching to group surgery.3 In most adult patients, these lev-
B, Rh-positive cells. The clinical situation els are encountered only after transfusion
should be evaluated by the transfusion of 15 to 20 RBC units (1.5 to 2 red cell vol-
service’s physician. If the continuing umes). FFP or platelets should not be ad-
transfusion requirement is expected to ministered in a fixed ratio to the number
exceed the available supply of Rh-nega- of RBC units given.
tive blood, evaluation of the change to
Rh-positive blood should be made early, Hypothermia, Tissue Oxygenation, and
to conserve blood for other recipients. Once 2,3-DPG
the patient receives one or more Rh-posi-
Hypothermia as a complication of trans-
tive units, there may be little advantage in
fusion is discussed in Chapter 27. In hypo-
returning to Rh-negative blood.
volemic shock, the underlying patho-
physiologic defect is inadequate tissue
Coagulation Support During Massive oxygenation. Oxygen supply to the tissues
Transfusion is determined by many factors, the most
Massive transfusion is often associated with important of which are blood flow (perfu-
coagulation abnormalities that may man- sion) and hemoglobin concentration. The
ifest as microvascular bleeding (MVB) in level of 2,3-DPG decreases in stored RBCs,
the form of oozing from multiple IV sites, and this decrease has been suggested as a
failure of blood shed into body cavities to potential cause of poor tissue oxygena-
clot, and bleeding from tissue surfaces on tion after massive transfusion. Low 2,3-DPG

levels have not been shown to be detri- need for transfusion in patients with end-
mental to massively transfused patients, stage renal disease and is indicated for
although for infants undergoing exchange the treatment of anemia in patients in-
transfusion, blood with near-normal fected with human immunodeficiency vi-
2,3-DPG levels is frequently requested. rus. Intensive EPO treatment (40,000 units
Within 3 to 8 hours after transfusion, pre- weekly) reduced the transfusion require-
viously stored red cells regenerate 50% of ment of ICU patients.101 It may also have a
normal 2,3-DPG levels.98 role in treating anemia due to chronic dis-
ease or to receipt of other medications
that suppress the marrow.
Pharmacologic Alternatives
Other Blood Cell Growth Factors
to Transfusion Granulocyte-macrophage colony-stimu-
Concern over the risks and limitations of lating factor (GM-CSF) and G-CSF stimu-
transfusion has led to examination of late marrow production of granulocytes.100
pharmacologic alternatives. Such alterna- G-CSF is approved for the treatment of
tives might: 1) stimulate increased pro- chemotherapy-induced neutropenia, for
duction or release of blood elements that patients undergoing peripheral blood
otherwise would require replacement (eg, progenitor cell collection and therapy,
erythropoietin); 2) substitute for a blood and for patients with chronic neutropenia.
component (eg, colloid solutions or oxy- The use of these stimulants decreases the
gen-carrying solutions including chemi- duration of neutropenia, increases toler-
cally modified hemoglobins); or 3) alter ance to cytotoxic drugs, and decreases the
physiologic mechanisms to reduce the need for granulocyte transfusions. GM-
need for replacement (eg, fibrinolytic in- CSF is approved for use in patients under-
hibitors).99 going autologous marrow transplanta-
tion. Another potential use for GM-CSF
Recombinant Growth Factors and G-CSF is support of patients under-
Growth factors are low-molecular-weight going allogeneic marrow transplantation
protein hormones that regulate hemato- or patients receiving antiviral agents that
poiesis by specific interaction with recep- suppress the marrow. Recombinant inter-
tors found on progenitor cells. The use of leukin-11 is licensed for cancer patients
growth factors to stimulate endogenous with thrombocytopenia, but other activa-
blood cell production is an important al- tors for thrombopoietin receptors have
ternative to the use of blood.100 been disappointing and none are avail-
able at this time.
Erythropoietin
Recombinant erythropoietin (EPO) is a Oxygen Therapeutics (Carriers)
growth factor that stimulates RBC pro- Stroma-free hemoglobin solution, in which
duction.100 It has been approved for pre- free hemoglobin has been separated from
surgical administration to increase preop- cell membranes, has several characteris-
erative hemoglobin and hematocrit levels; tics that render it unsuitable as a blood
typical dose regimens range from 300 to substitute, including a low p50, short cir-
600 U/kg by subcutaneous injection weekly. culation time, high oncotic pressure, and
The use of EPO has markedly reduced the vasopressor/nephrotoxic properties.102,103

However, chemical modifications of he- usually given as a single injection (0.3 to 0.4
moglobin solutions may successfully µg/kg) to treat bleeding or for prophylaxis
overcome these disadvantages and such before a procedure. Doses are not usually
products are in Phase III clinical trials at repeated within a 24- to 48-hour period be-
the time of this writing. Patients in these cause of tachyphylaxis (the loss of biologic
trials have survived very severe anemia effect with repeated administration of an
(residual cellular hemoglobin as low as 1 agent) and the induction of water retention
g/dL) without significant toxicity when and hyponatremia. Some patients experi-
supported by these agents.104 Bovine he- ence facial flushing or mild hypotension,
moglobin is approved for veterinary use. but side effects are rare. Its effect on vWF
Hemoglobin produced by recombinant occurs within 30 minutes and lasts 4 to 6
DNA techniques has also been investi- hours.105
gated. Finally, fluorocarbon products that
bind oxygen have been extensively inves-
tigated.103 One of the latter, Fluosol, was
Fibrinolytic Inhibitors
approved by the FDA for use during per-
cutaneous transluminal angioplasty, but Epsilon aminocaproic acid (EACA) and
it is no longer available in the United tranexamic acid—synthetic analogues of
States. lysine—competitively inhibit fibrinolysis
by saturating the lysine binding sites at
which plasminogen and plasmin bind to
DDAVP
fibrinogen and fibrin. The drugs can be
DDAVP is a synthetic analogue of the hor- used locally or systemically and can be
mone vasopressin that lacks significant given orally. Aprotinin is a polypeptide
pressor activity.99,105 First used in the treat- prepared from bovine lung that inhibits
ment of diabetes insipidus, DDAVP is also proteinases including plasmin, kallikrein,
useful in promoting hemostasis because trypsin, and, to some extent, urokinase.
of its ability to cause the release of endog- Thus, it has an antifibrinolytic action but
enous stores of high-molecular-weight may also inhibit coagulation because
vWF from the vascular subendothelium kallikrein activates Factor XII. Aprotinin is
and the concomitant increase in Factor used intravenously. Because it is a poly-
VIII. Because of its effect on Factor VIII peptide, hypersensitivity reactions can
and vWF, DDAVP is used as a hemostatic occur.99,107
agent in patients with mild-to-moderate Antifibrinolytic agents have been used
hemophilia A and in patients with some successfully in cardiac surgery, prostatec-
von Willebrand syndromes. Because tomy, and liver transplantation. EACA and
platelet adhesion and the subsequent for- tranexamic acid can also be used locally at
mation of a platelet plug depend upon sites where fibrinolysis contributes to
vWF, DDAVP may also be beneficial in a bleeding, as from mucosal lesions of the
wide variety of platelet function disor- mouth and gastrointestinal tract, and are of
ders, including uremia, cirrhosis, drug-in- benefit in the control of hemorrhage fol-
duced platelet dysfunction (including as- lowing dental extractions in patients with
pirin), primary platelet disorders, and hemophilia and in the control of gastroin-
myelodysplastic syndromes.99,105,106 testinal bleeding. EACA and tranexamic
DDAVP can be administered intrave- acid may be helpful in controlling bleeding
nously, subcutaneously, or intranasally. It is due to severe thrombocytopenia. Systemic

administration of fibrinolytic inhibitors has and scores the institution on the appro-
been associated with serious thrombotic priateness of the selected performance
complications, including ureteral obstruc- measures and the size of the data sam-
tion due to clot formation and thrombosis ple.110 Moreover, the JCAHO standards re-
of large arteries and veins. When used in quire that the medical staff take the lead-
excessive doses, fibrinolytic inhibitors can ership role in measurement, assessment,
prolong the bleeding time. These drugs and improvement of clinical processes re-
should be employed by physicians with lated to the use of blood and blood com-
experience in their use. ponents. This assessment process must
All three of these antifibrinolytic agents, include peer review, and its findings must
as well as DDAVP, have been used in an at- be communicated to involved staff mem-
tempt to decrease blood use in cardiac sur- bers as well as being a part of the renewal
gery, and meta-analyses have shown a of their clinical privileges.
decrease in the proportion of patients re- Typically, this function has been dele-
ceiving allogeneic RBCs, the number of units gated to a medical staff committee, often a
transfused,108,109 estimated blood losses,108 dedicated “Transfusion Committee.” The
and the number of patients requiring re- medical director of the transfusion service
108,109
operation for bleeding. Of the three should be a member of the committee. This
agents, a much larger data base exists for committee should review blood bank activ-
aprotinin, which is the most frequently ities and statistics, blood ordering, and
used. DDAVP was not effective overall but transfusion practices and should have a
may be useful in patients taking aspirin.108 process to review records of patients trans-
Of concern has been a trend toward infused with blood or components. The com-
creased thrombotic complications (myo- mittee should monitor significant develop-
cardial infarction and graft thrombosis) ments in transfusion medicine that would
with aprotinin, but they do not appear to affect patients in the health-care institution
be statistically significant.107,108 Unfortu- and take appropriate action regarding these
nately, very large studies would be required developments.
to demonstrate whether the risk profile of The College of American Pathologists
these agents is superior to that of blood, (CAP) laboratory accreditation program
particularly in view of the decreasing risks also requires transfusion oversight. This is
of transfusion. 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-
Oversight of Transfusion put in a multidisciplinary fashion.111 More-
Practice over, the CAP accreditation checklist for
Of the various institutions that regulate or blood banks seeks documentation that “. . .
accredit aspects of transfusion, the Joint the transfusion service medical director ac-
Commission for Accreditation of Health- tively participates in establishing criteria
care Organizations (JCAHO) has histori- and in reviewing cases not meeting transfu-
112
cally emphasized oversight of transfusion sion audit criteria.”
practice as a requirement for accredita- Finally, the AABB Standards requires that
tion. As part of its performance improve- there be a peer-review program that moni-
ment standards, the JCAHO requires col- tors appropriateness of use of blood com-
lection of data regarding the use of blood ponents.59(p86)

after elective operations for myocardial

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ated graft-versus-host disease not prevented patients undergoing resternotomy or re-
by white cell-reduction filters. Transfusion operation after cardiac operations. J Thorac
1992; 32:169-72. Cardiovasc Surg 1989;97:194-203.
67. Roberts HR, Monroe DM III, Hoffman M. 80. Streiff MB, Ness PM. Acquired FV inhibitors:
Molecular biology and biochemistry of the A needless iatrogenic complication of bovine
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tasis. In: Beutler E, Lichtman MA, Coller BS, 18-26.
et al. Williams’ hematology. 6th ed. New 81. Roberts HR, Hoffman M. Hemophilia A and
York: McGraw-Hill, 2001:1409-34. hemophilia B. In: Beutler E, Lichtman MA,
68. van Aken WG. Preparation of plasma deriva- Coller BS, et al, eds. Williams’ hematology.
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82. Smith KJ. Factor IX concentrates: The new ment of sickle cell disease. 4th ed. Bethesda,
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32. 97. Ciavarella D, Reed RL, Counts RB, et al. Clot-
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stone, 1995:985-1010. generation of red cell 2,3-diphosphoglycerate
85. G o o d n i g h t S W, G r i f f i n J H . He r e d i t a r y following transfusion of DPG-depleted AS-1,
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6th ed. New York: McGraw-Hill, 2001:1697- 99. Mannucci PM. Drug therapy: Hemostatic
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86. Griffin JH. Control of coagulation reactions.
100. Kruskall MS. Biologic response modifiers—
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antithrombin III concentrate in congenital
101. Corwin HL, Gettinger A, Pearl RG, et al. Effi-
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92. Cochrane Injuries Group Albumin Reviewers. qualitative platelet disorders due to diseases,
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93. Vichinsky EP, Haberkern CM, Newmayer L, et 602.
al. A comparison of conservative and aggres- 107. Bachman F. Disorders of fibrinolysis and use
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Bethesda, MD: AABB Press, 1998. perioperative blood loss in cardiac surgery:
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Healthcare Organizations. 2004 Comprehen- 112. CAP Transfusion Medicine Checklist. Revi-
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standard PI.3.1.1 and MS.8.1.3 and MS.8.3. of American Pathologists, 2003.

Chapter 22: Administration of Blood and Components
Chapter 22
Administration of Blood and

Components
G
OOD TRANSFUSION PRACTICE adult but also for any child who has the
requires that comprehensive poli- ability to understand the process. In the
cies and procedures for blood ad- latter situation, it is appropriate to edu-
ministration be designed to prevent errors. cate the parents, so that they are better
The development of these policies should prepared to support their child through-
be a collaborative effort between the medi- out the transfusion. The transfusionist
cal director of the transfusion service, the should explain how the transfusion will be
directors of the clinical services, both nurs- given, how long it will take, what the ex-
22
ing and medical, and all personnel involved
pected outcome is, what symptoms to re-
in blood administration. Policies and pro-
port, and that vital signs will be taken. The
cedures must be accessible, periodically
physician has a responsibility to explain
reviewed for appropriateness, and moni-
the benefits and risks of transfusion ther-
tored for compliance. In addition to blood
apy as well as the alternatives in a fashion
administration policies and procedures,
this chapter discusses pretransfusion pre- that the patient can comprehend. Other
paration, issuing of blood components, than in emergency situations, the patient
the equipment used in blood administra- should be given an opportunity to ask
tion, and compatible intravenous solutions. 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.
Pre-Issue Events Some states have specific requirements
Patient Education and Consent for blood transfusion consent. Institu-
Patients who are aware of the steps in- tions should be careful to ensure that
volved in a transfusion will experience less their individual processes and procedures
anxiety. This is important not only for an comply with applicable laws.
521

Individual institutions have different re- vice must strive to make every effort to
quirements for obtaining and documenting ensure that the component is ready when
this interaction, as well as different policies needed, but not so early that it expires be-
about how often it is necessary. Some facili- fore administration.
ties require the use of a formal consent Medical and nursing staffs need to be
form, which provides information in unaware of special requirements for prepara-
derstandable language, signed by the pation and to understand that these times
tient. Others expect the physicians to make cannot be significantly shortened, even in
a note in the medical record stating that the urgent situations. Close communication is
risks of, and alternatives to, blood transfu- required. Some examples of pretransfusion
sions were explained and that the patient processing procedures are given in Table
consented. If a patient is unable to give 22-1.
consent, a responsible family member
should be asked. If no family member is Patient Considerations
available or if the emergency need for Patients with a history of recurrent aller-
transfusion leaves no time for consent, it is gic transfusion reactions may benefit from
prudent to note this in the medical record.1-3 premedication with antihistamines or by
slowing the rate of transfusion. Routine
premedication with antipyretics should
Prescription and Special Instructions be discouraged because delaying a rise in
There must be documentation of the or- temperature may mask one sign of a
der by the physician for the blood compo- hemolytic reaction and partly because
nent(s).4 Although a telephone order may they may be ineffective.5-7
be acceptable during urgent situations, this Antipyretics typically do not mask other
must be followed by a written request. clinical features of hemolysis, such as
Special instructions should be indicated changes in blood pressure, pulse, or respi-
regarding the transfusion relating to the: ration. Premedication orders should be
■ Component—eg, washed, irradiated, carefully timed with the anticipated admin-
leukocyte-reduced, cytomegalovirus istration of the unit. Medication ordered in-
negative travenously may be given immediately be-
■ Patient—eg, premedication, timing fore the start of the transfusion, but orally
■ Process—flow rates, rate of infusion, administered drugs need to be given 30 to
use of a blood warmer or electrome- 60 minutes before the start of the transfu-
chanical pump sion.
■ Need for emergency release
Process Considerations
Blood warmers and electromechanical
Component Considerations pumps need to be available if required for
Some components require special prepa- the transfusion.
ration before release for transfusion. Be-
cause these steps are time-consuming and Emergency Release
may significantly shorten the shelf life of Blood may be released without complet-
the component, preparation should be ing pretransfusion testing if it is urgently
carefully coordinated with the anticipated needed for a patient’s survival, provided
time of transfusion. The transfusion ser- that: 1) the records properly document

Chapter 22: Administration of Blood and Components 523
Table 22-1. Component Preparation Times
Component Minimum Time* Shelf Life
RBCs: saline-washed 45 minutes 24 hours

RBCs: thawed-deglycerolized 75 minutes 24 hours or 2 weeks†
Fresh Frozen Plasma: thawed 30 minutes 24 hours
Thawed Plasma — 5 days
Platelets: pooled 15 minutes 4 hours
CRYO: thawed (single unit) 15 minutes 6 hours
CRYO: pooled 15 minutes 4 hours
*Will vary with institutional procedures.
†
Depends on the method used.
RBCs = Red Blood Cells; CRYO = Cryoprecipitated AHF.
the emergency request and 2) the issued without damaging the vein. There are no
units are of an ABO group unlikely to strict guidelines limiting the size of the
cause immediate harm to the recipient. 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
Venous Access
small veins require much smaller catheters.
To avoid any delay in transfusion and po- High-pressure flow through needles or
tential wastage of blood components, ve- catheters with a small lumen may damage
nous access should be established before red cells8-10 unless the transfusion compo-
10
the component is issued. If a pre-existing nent is sufficiently diluted. Undiluted
line is to be used, it should be checked for preparations of red cells flow very slowly
patency; signs of infiltration, inflamma- through a 23-gauge needle, but dilution
tion, or infection; and the compatibility of with saline to increase the flow rate may
any intravenous solutions (see below). Many cause unwanted volume expansion. Even
venous access devices can be used for in patients with cardiac disease or volume
blood component transfusion. Selection expansion, transfusions should be able to
depends on the location, size, and integ- be given safely within 4 hours. For rare pa-
rity of the patient’s veins; the type of med- tients unable to tolerate a transfusion with-
ication or solution to be infused; the type in 4 hours, local policy should be developed
of component to be transfused; the vol- regarding whether to split units or to dis-
ume and timing of the administration; the card the unused portion. Specific models of
possibility of interactions among paren- infusion pumps have been approved for
teral solutions; and expected duration of use in blood transfusion. These pumps
intravenous therapy. maintain a constant delivery of blood, and
The lumen of needles or catheters used studies have indicated no significant evi-
for blood transfusion should be large dence of hemolysis as the needle size var-
enough to allow appropriate flow rates ies.11

Central venous catheters are used for completed, the patient is properly pre-
medium- and long-term therapy or for the pared, and the transfusionist is ready to
administration of solutions potentially toxic begin the procedure. There must be a
to a peripheral vein to allow the dilution mechanism to identify the intended re-
achieved with high-volume blood flow. cipient and the requested component at
Some catheters are placed via special intro- the time of issue. It is optimal to identify
ducing needles and guide-wires; others are the transporter.
surgically implanted. Catheters with a Transfusion service personnel will re-
multilumen design have separate infusion view identifying information, inspect the
ports for each lumen, permitting the simul- appearance of the component before re-
taneous infusion of fluids without intermix- lease, and ensure there is a system to main-
ture in the infusion line, thereby avoiding tain proper storage temperature during
the potential for hemolysis from incompat- transport. The safest practice is to issue one
ible fluids. unit to one patient at a time. For patients
who are rapidly bleeding or who have mul-
Need for Compatibility Testing tiple venous access sites, multiple units
The transfusion service personnel deter- may be issued to a single patient. It is not
mine what pretransfusion testing is re- recommended that blood for two or more
quired. Compatibility testing must be per- patients be issued simultaneously to one
formed for transfusion of Whole Blood, transporter because this could increase the
components with a clinically significant chance of transfusion error. However, logis-
red cell content, and all red cell compo- tical considerations may make this imprac-
nents. For test and specimen require- tical. The transporter should transport the
ments, refer to Chapter 18. Compatibility blood to the intended site of transfusion
testing other than ABO and Rh typing is without delay—preferably to the transfu-
not required for platelet and plasma com- sionist. It is preferable to place the unit of
ponents, but most facilities require that blood in a protective container that would
the recipient’s ABO and D types be known contain any spillage in the event of inadver-
(on file) before such components are se- tent breakage during transport.
lected for issue. Whenever possible, plasma- The responsibility for accurately identi-
containing components should be com- fying a transfusion component rests with both
patible with the patient’s red cells (see the transfusion service personnel who issue
Chapter 21). the blood and the transfusionist who re-
ceives it. Before a unit of blood is issued,
transfusion service personnel complete the
following steps:
Blood Issue and 1. The records that identify the in-
Transportation tended recipient and the requested
component are reviewed.
Delivering Blood to the Patient Area 2. The identifiers (Standards requires
Institutions should define blood pick-up at least two) of the intended recipi-
and delivery policies and appropriate ent, the ABO and D type of the recip-
training programs for the staff assigned to ient, the component unit number,
these functions. Blood is not routinely the ABO and D type of the donor
dispensed from the controlled environ- unit, and the interpretation of com-
ment of the blood bank until all testing is patibility tests (if performed) are re-

corded on a transfusion form for turned to the blood bank after a period
each unit. This form, or a copy, be- outside monitored refrigeration will be
comes a part of the patient’s perma- unsuitable for reissue if the sterility of the
nent medical record after comple- container is compromised or if the tem-
tion of the transfusion. In some perature has risen to 10 C or above, which
institutions, the transfusion form is is generally considered to happen in less
attached to the unit and, therefore, than 30 minutes. If the units have been
serves as the tie tag that is required kept in suitable conditions, such as iced
and described below. This form typi- coolers that have been validated for sev-
cally will have fields to identify the eral hours of storage, longer periods are
transfusionist and co-identifier (if re- acceptable. Requirements for blood col-
quired) and other information, such lected intraoperatively differ. See Table
as pre- and posttransfusion vital signs, 5-8. Units that have been entered (punc-
amount of blood given, whether a re- tured) after release from the blood bank
action occurred, etc. cannot be accepted into general inven-
3. A tie tag or label with the name and tory for later reissue.
identification number of the in-
tended recipient, the component unit
number, and the interpretation of Pre-Administration Events
compatibility tests (if performed) must
Identifying the Recipient and Donor Unit
be securely attached to the blood
container. Accurate identification of the transfusion
4. The appearance of the unit is checked component and the intended recipient
before issue and a record is made of may be the single most important step
this inspection. in ensuring transfusion safety.12-15 Most
5. The expiration date (and time if ap- hemolytic transfusion reactions and
plicable) is checked to ensure that the deaths occur because of inadvertent ad-
unit is suitable for transfusion. ministration of ABO-incompatible red
6. The name of the person issuing the cells.14-15 Plasma and platelets are also ca-
blood and the date and time of issue pable of causing serious transfusion reac-
are recorded. Recording the name of tions.16 Identification and labeling of do-
the transporter to whom the blood is nor blood are discussed in Chapter 7;
issued is optimal. procedures to identify the patient’s speci-
men used for compatibility testing are
discussed in Chapter 18. The most impor-
Delay in Starting the Transfusion tant steps in safe transfusion administra-
Ideally, blood should be requested from tion are clerical and occur when the
the blood bank only at the time when it is transfusion service issues blood for a spe-
intended to be administered. If the trans- cific patient and when the blood is ad-
fusion cannot be initiated promptly, the ministered.
blood should be returned to the blood The transfusionist who administers the
bank for storage, unless the transfusion to blood represents the last point at which
the originally intended recipient can be identification errors can be detected before
completed within 4 hours. It should not transfusion of the component is initiated.
be left at room temperature or stored in The transfusionist must check all identify-
an unmonitored refrigerator. Units re- ing information immediately before begin-

ning the transfusion and record that this in- ponent may not be identical (see
formation has been checked and found to Chapter 21), but the information on
be correct, typically, on the transfusion the transfusion form and that on the
form. Any discrepancy must be resolved container label must be the same.
before the transfusion is started. It is com- 5. Expiration. The expiration date and
mon practice in many institutions that a time of the component should be
second person (co-identifier) confirms the verified as acceptable.
identity of the blood unit and of the patient. 6. Compatibility. The interpretation of
Some institutions may require that the compatibility testing (if performed)
transfusionist check for documentation of must be recorded on the transfusion
patient consent before blood is given. It is form and on the tag attached to the
also common practice to check the ABO unit (if not the same). If blood was
and D type as written in the transfusion issued before compatibility tests were
form with a record of ABO and D type in completed, this must be conspicu-
the patients’ chart. The following informa- ously indicated.
tion, however, must be reviewed and found In certain clinical situations, such as the
to be correct: operating room (OR), emergency room, or
1. Physician’s order. The nature of the in the outpatient setting, an identifying
blood or component should be band may not be attached to all patients at
checked against the physician’s writ- the time of transfusion. Wristbands are fre-
ten order to verify that the correct quently removed in the OR for arterial line
component and dose (number of insertions and identity confirmation may
units) are being given. All identifica- not be possible in this manner. Further-
tion attached to the container must more, a co-identifier confirming identity,
remain attached until the transfu- although always desirable, may not func-
sion has been completed. tion for all circumstances in this setting.
2. Recipient identification. The patient’s Institutions should address such clinical
identifiers on the patient’s identificat- situations, and site-specific transfusion
ion band must be identical with the protocols should be developed.18
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-
Starting the Transfusion
cause the information on the identifi-
cation band may be in error.17 After checking all the identifying informa-
3. Unit identification. The unit identifi- tion, the transfusionist (and co-identifier)
cation number on the blood con- must indicate in the medical record that
tainer, the transfusion form, and the the identification was correct (such as by
tie tag attached to the unit (if not the signing the transfusion form) to docu-
same as the latter) must agree. ment who started the transfusion and to
4. ABO and D. The ABO and D type on record the date and time. Vital signs
the primary label of the donor unit should be taken and recorded, if not done
must agree with those recorded on previously. A record of the date and time
the transfusion form. The recipient’s of transfusion, the name and volume of
ABO and D type must be recorded the component, and vital signs may be re-
on the transfusion form. The pa- quired on other parts of the medical re-
tient’s type and the type of the com- cord, such as intake/output records, anes-

thesia records, or intensive care flow Compliance with institutional blood ad-
sheets, depending on the institution’s pol- ministration policies requires a Quality
icy. This may suffice for documentation Assurance/Continuous Improvement pro-
purposes. gram in which continued monitoring and
Several reports have documented the oc- re-education of staff occur when variance
currence of errors at the point of blood ad- with procedures is observed. Such an ap-
ministration.19-22 In particular, Baele and proach has been initiated in some institu-
colleagues19 studied the charts and records tions, resulting in improvement in transfu-
of 808 patients who received 3485 units of sion practice. 2 6 Direct observation of
27 28
blood over a period of 15 months, to deter- administration or educational videos
mine if there had been errors in blood ad- may also be useful.
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 Administration
patient misidentification that resulted in Infusion Sets
blood being given to patients for whom it
Any blood component must be adminis-
was not intended, constituting 0.74% of pa-
tered through a filter designed to retain
tients and 0.2% of units. One error resulted
blood clots and particles potentially harm-
in an ABO-incompatible hemolytic reaction
ful to the recipient.4,29(p48) All filters and in-
that was not reported to the blood bank.
fusion devices must be used according to
Eight other major errors occurred in four
the manufacturer’s directions.
patients (0.5%), including the administra-
tion of five allogeneic units to a patient for
whom autologous blood was available, and Standard Sets
the transfusion of one anemic patient Standard blood infusion sets have inline
whose doctor had ordered only a cross- filters (pore size: 170 to 260 microns), drip
match. The remaining 150 errors included chambers, and tubing in a variety of con-
misrecording (n = 61), mislabeling (n = 6), figurations. Sets should be primed ac-
and failure to adequately document the cording to the manufacturer’s directions,
transfusion (n = 83). using either the component itself or a so-
Both mechanical barrier systems23 and lution compatible with blood (see the sec-
electronic means of patient and product tion on Compatible IV Solutions). For op-
identifications are marketed to supplement timal flow rates and performance, filters
(but not substitute for) the paper identifica- should be fully wetted. Drip chambers
tion process. In electronic systems, a bar should be half-filled to allow observation
code or radio frequency identifiers are at- of blood flow.
tached to the blood component (and trans- Many institutions have a policy of chang-
fusion form) that, when scanned/read, will ing sets after every transfusion or of limit-
match the patient’s information on the ing their use to several units or several
identification band.24 It is likely that these hours in order to reduce the risks of bacte-
electronic means will find more widespread rial contamination. A reasonable time limit
use in the future. Empiric experience with is 4 hours; this is consistent with the 4-hour
medication administration, however, sug- outdate that the Food and Drug Adminis-
gests that unanticipated side effects of such tration (FDA) places on blood in an open
procedures may need consideration. 25 system held at room temperature. Most

standard filters are designed to filter 2 to 4 ever, the large volume required for priming
units of blood, but if the first unit required 4 causes a significant portion of these com-
hours for infusion, the filter should not be ponents to be lost if the set is not flushed
reused. The filter traps cell aggregates, cel- with saline afterward. Depth-type micro-
lular debris, and coagulated proteins, re- aggregate filters, or any filters capable of re-
sulting in a high protein concentration at moving leukocytes, must not be used for
the filter surface. The high protein milieu the transfusion of granulocyte concen-
and room temperature conditions promote trates.29(p48) Hemolysis of red cells has been
30
growth of any bacteria that might be pres- reported with microaggregate filters.
ent. Accumulated material also slows the
30
rate of flow.
Leukocyte Reduction Filters
Special “third-generation” blood filters
Special Sets can reduce the number of leukocytes in
red cell or platelet components to less
High flow sets for rapid transfusion have
than 5 × 106, a level that reduces the risk of
large filter surface areas, large-bore tub-
HLA alloimmunization and the transmis-
ing, and may have an inline hand pump.
sion of cytomegalovirus as well as the in-
Sets designed for rapid infusion devices
cidence of febrile nonhemolytic transfu-
also may have “prefilters” to retain parti-
sion reactions31-35 (see Chapters 8, 21, and
cles over 300 microns in diameter and to
27). These filters contain multiple layers
extend the life of standard blood filters
of synthetic nonwoven fibers that selec-
“downstream.” Gravity-drip sets for the
tively retain leukocytes but allow red cells
administration of platelets and cryopreci-
or platelets to pass, depending on the fil-
pitate have small drip chamber/filter ar-
ter type. Selectivity is based on cell size,
eas, shorter tubing, and smaller priming
surface tension characteristics, the differ-
volumes. Syringe-push sets for compo-
ences in surface charge, density of the
nent administration have the smallest
blood cells, and, possibly, cell-to-cell in-
priming volumes and an inline blood fil-
teractions and cell activation/adhesion
ter that may be inconspicuous.
properties.36 Because filters for red cells
and filters for platelets do not use the
same technology for leukocyte removal
Microaggregate Filters and may have strict priming and flow rate
Microaggregate filters are designed for the requirements, they must be used only
transfusion of red cells. Microaggregates with their intended component and only
(smaller than 170 microns in size) pass according to the manufacturer’s direc-
through standard blood filters. Screen- or tions.37
depth-type filters have an effective pore The use of these filters at the bedside is
size of 20 to 40 microns and trap the more complex than the use of standard in-
microaggregates composed of degenerat- fusion sets. The filters are expensive and
ing platelets, leukocytes, and fibrin strands can be ineffective or may clog, if improp-
38-40
that form in blood after 5 or more days of erly used. Those designed only for grav-
refrigerated storage. ity infusion should not be used with infu-
Microaggregate filters may be used for sion pumps or applied pressure. A quality
other components if this use is mentioned control program that measures the effec-
in the manufacturer’s instructions; how- tiveness of leukocyte reduction is impor-

tant, but impractical, at the bedside; there- Devices should have a visible thermom-
fore, adherence to proper protocol is very eter and, ideally, an audible alarm that
important. sounds before the manufacturer’s desig-
nated temperature limit is exceeded. The
standard operating procedure for warming
Blood Warmers blood should include guidelines on per-
forming temperature and alarm checks,
It is desirable for the medical staff of the and instructions on what action to take
transfusion service to participate in the when warmers are out of range or the alarm
assessment and selection of transfusion activates.44 Conventional microwave ovens
equipment and ensure that such items and microwave devices for thawing plasma
are included in the facility’s quality assur- are not designed for warming other blood
ance program. The performance of de- components and can damage red cells.
vices such as blood warmers or infusion
pumps must be validated before the
equipment is used and must be moni-
Electromechanical Infusion Devices
tored regularly throughout the facility to
identify malfunctions and ensure appro- Mechanical pumps that deliver infusions
priate use. This characteristically requires at a controlled rate are useful, especially
cooperation among personnel of several for very slow rates of transfusion used for
hospital departments, including transfu- pediatric, neonatal, and selected adult
sion medicine, nursing, anesthesiology, patients. Some pumps use a mechanical
quality assurance, and clinical engineer- screw drive to advance the plunger of a
ing. syringe filled with blood; others use roller
Patients who receive blood or plasma at pumps or other forms of pressure applied
rates faster than 100 mL/minute for 30 to the infusion tubing. Although some can
minutes are at increased risk for cardiac ar- be used with standard blood administra-
rest unless the blood is warmed.41 Rapid in- tion sets, many require special plastic
fusion of large volumes of cold blood can disposables or tubing supplied by the
lower the temperature of the sinoatrial manufacturer. Blood filters can be added
node to below 30 C, at which point an ar- to the required setups upstream of the
rhythmia can occur. pumps.
Transfusions at such rapid rates gener- The manufacturer should be consulted
ally occur only in the OR or trauma settings. before blood is administered with an infu-
There is no evidence that patients receiving sion pump designed for crystalloid or
1 to 3 units of blood over several hours have colloid solutions. Many induce hemolysis,
a comparable risk for arrhythmias; there- but of a magnitude that does not adversely
fore, routine warming of blood is not rec- affect the patient.43 Red cells in components
ommended. 4 2 Several types of blood with high hematocrit and high viscosity are
warmers are available: thermostatically more likely to be hemolyzed when infused
controlled waterbaths; dry heat devices under pressure than red cells in Whole
with electric warming plates; and high-vol- Blood or red cell components prepared in a
ume countercurrent heat exchange with wa- manner that reduces viscosity, such as ad-
ter jackets.43 Blood warming devices must ditive solutions.45 Platelets and granulocytes
not raise the temperature of blood to a level appear to sustain no adverse effects when
that causes hemolysis.29(p6) infused with a pumping device.46,47 Proper

training of personnel and appropriate poli- additive solution (AS) ordinarily do not
cies for maintenance and quality control require dilution. These red cell compo-
should reduce the chances of damage to nents have a hematocrit of approximately
transfused components. 60%. Other solutions intended for intrave-
nous use may be added to blood or com-
ponents or may come into contact with
Pressure Devices blood in an administration set only if they
have been approved for this use by the
Urgent transfusion situations may require
FDA or if there is documentation to show
flow rates faster than gravity can provide.
that their addition to blood is safe and ef-
The simplest method to speed infusion is 29(p6),48
ficacious. Calcium-free, isotonic
to use an administration set with an inline
electrolyte solutions that meet the above
pump that the transfusionist squeezes by
hand. Pressure bags specially designed as requirements also may be used, but they
compression devices are also available. usually are more expensive than saline
These devices operate much like blood and offer little benefit in routine transfu-
pressure cuffs except that they completely sion. Lactated Ringer’s solution, 5% dex-
encase the blood bag and apply pressure trose, and hypotonic sodium chloride so-
more evenly to the bag’s surface. Such de- lutions should not be added to blood.
vices should be carefully monitored dur- Dextrose solution may cause red cells to
ing use because pressures greater than clump in the tubing and, more important,
300 mm Hg may cause the seams of the to swell and hemolyze as dextrose and as-
blood bag to rupture or leak and air em- sociated water diffuse from the medium
bolism is a concern. Large-bore needles into the cells. Lactated Ringer’s solution
are traditionally recommended for venous contains enough ionized calcium (3
access when the use of external pressure mEq/L) to overcome the chelating agents
is anticipated, but recent data question in anticoagulant-preservative or additive
this practice.11 Manually forcing red cells solutions, which results in clot develop-
49-51
through a small-gauge line has been ment.
shown to cause hemolysis.45
Devices for intraoperative and postoper-
ative blood collection are discussed in Patient Care During Transfusion
Chapter 5.
The transfusionist should either remain
with, or be in a position to closely observe,
Compatible IV Solutions the patient for at least the first 15 minutes
AABB Standards for Blood Banks and of the infusion. The transfusion should be
29(p48)
Transfusion Services and the Circular started slowly at a rate of approximately 2
of Information for the Use of Human mL/minute except during urgent restora-
Blood and Blood Components48 are explicit tion of blood volume. Catastrophic reac-
in stating that medications must not be tions from acute hemolysis, anaphylaxis,
added to blood or components. If red cells transfusion-related acute lung injury, or
require dilution to reduce their viscosity bacterial contamination can become ap-
or if a component needs to be rinsed from parent after a very small volume enters
the blood bag or tubing, normal saline the patient’s circulation (see Chapter 27).
(0.9% sodium chloride injection, USP) After the first 15 minutes, some institu-
can be used. Red cells prepared with an tions record vital signs, but this is unneces-

sary if the patient’s condition is satisfactory. cells, if there is an order permitting

The rate of infusion can be increased to such addition.
that specified in the order or to be consis- Clinical personnel should continue to
tent with institutional practice (approxi- observe the patient periodically throughout
mately 4 mL/minute). The desirable rate of the transfusion (eg, every 30 minutes) and
infusion depends upon the patient’s blood up to an hour after completion.
volume, cardiac status, and hemodynamic
condition. No experimental or clinical data Action for Suspected Reactions
exist to support a specific time restriction; Most transfusions proceed without com-
however, the Circular of Information48 gives plication, but when adverse reactions do
4 hours as the maximum duration for an occur, medical and nursing staff must be
infusion. Maximum time should not be prepared to deal with them immediately.
confused with recommended time. Most Different types of reactions, their etiology,
RBC units are transfused within 1 to 2 symptoms, treatment, and prevention are
hours, whereas platelet or plasma transfu- discussed in Chapter 27. Because severity
sions are commonly administered over a can vary significantly and symptoms are
shorter period (30 to 60 minutes). However, not specific, all transfusions must be care-
there is no physiologic reason to administer fully monitored and stopped as soon as a
compatible red cells more slowly than reaction is suspected. It may be helpful to
plasma or platelets and rapid infusion of summarize common symptoms and the
these products may increase the risk of ad- immediate steps to take on the transfu-
verse events. If rapid transfusion is needed, sion form that accompanies the unit. This
blood can be infused as rapidly as the pa- eliminates the need to search for instruc-
tient’s circulatory system will tolerate and tions and helps standardize patient care
the type of vascular access will allow. in an urgent situation.
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 Post-Administration Events
clinical situation. Administration rates are After each unit of blood has been infused,
calculated by counting the drops per min- personnel should measure vital signs; re-
ute in the drip chamber and dividing this cord the time, the volume, and the com-
number by the “drop/mL” rating of the in- ponent given; record the patient’s condi-
fusion system. Blood may flow more slowly tion; and record the identity of the person
than desired as a result of obstruction of who stopped the transfusion (if not the
the filter or when there is excessive viscos- transfusionist), made the observations,
ity of the component. Steps to investigate and measured and recorded vital signs.
and correct the problem include the follow- Many transfusion services require that a
ing: copy of the completed transfusion form
■ Elevate the blood container to in- be returned to the laboratory to docu-
crease hydrostatic pressure. ment the unit’s disposition for laboratory
■ Check the patency of the needle. records. The empty blood bag need not be
■ Examine the filter of the administra- returned after uncomplicated transfu-
tion set for excessive debris. sions, but bags, tubing, and attached so-
■ Consider the addition of 50 to 100 lutions should be returned to the transfu-
mL of saline to a preparation of red sion service if a complication occurs.

Some transfusion services choose to have

all bags returned following all transfu-
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cipient, and selection and proper use of
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13. McClelland DBL, Phillips P. Errors in blood
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ated deaths: 1976-1985. Transfusion 1990:30:
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583-90.
analysis because such errors are often in- 16. McManigal S, Simms KL. Intravascular
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202-6. transfusion medicine. 3rd ed. Baltimore, MD:
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band identification error reporting in 712 32. Novotny VM, van Doorn R, Witvliet MD, et al.
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43. 33. Brand A, Claas FH, Voogt PJ, et al. Alloim-
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21. Sang 1998;54:160-6.
20. Murphy MF, Atterbury CLJ, Chapman JF, et al. 34. Bowden RA, Slichter SJ, Sayers M, et al. A
The administration of blood and blood com- c o m p a r i s o n o f l e u k o c y t e - red u c e d a n d
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strategy for improving transfusion safety. vitro storage time of platelet concentrates on
Transfusion 1994;34:11-15. clogging of white cell reduction filters. Trans-
27. Whitsett CF, Robichaux MG. Assessment of fusion 1994;34:740-1.
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29. Silva MA, ed. Standards for blood banks and 43. Iserson KV, Huestis DW. Blood warming: Cur-
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30. Schmidt WF, Kim HC, Tomassini N, Schwartz 44. Uhl L, Pacini D, Kruskall MS. A comparative
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46. Snyder EL, Ferri PM, Smith EO, Ezekowitz MD. 49. Ryden SE, Oberman HA. Compatibility of
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sion 1984;24:524-7. 50. Dickson DN, Gregory MA. Compatibility of
47. Snyder EL, Malech HL, Ferri PM, et al. In vitro blood with solutions containing calcium. S
function of granulocyte concentrates follow- Afr Med J 1980;57:785-7.
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cular of information for the use of human

Chapter 23: Perinatal Issues in Transfusion Practice
Chapter 23
Perinatal Issues in
P
REGNANCY PRESENTS SPECIAL directed against a paternally inherited an-
immunohematologic problems for tigen present on the fetal cells that is
the transfusion service. The mother absent from maternal cells. The IgG-
may exhibit alloimmunization to antigens coated cells may undergo accelerated de-
on fetal cells, and the fetus may be affected struction both before and after birth, but
by maternal antibodies provoked by pre- the severity of the disease can vary from
vious pregnancies, by previous or present serologic abnormalities detected in an
transfusions, or by the ongoing pregnancy. asymptomatic infant to intrauterine death.
This chapter discusses hemolytic disease
of the fetus and newborn (HDFN) and neona- Pathophysiology
tal alloimmune thrombocytopenia (NAIT)—
the two primary immunohematologic Accelerated red cell destruction stimulates
concerns during the perinatal period. Also increased production of red cells, many of
included is a brief discussion of neonatal which enter the circulation prematurely as 23
thrombocytopenia secondary to maternal nucleated cells, hence the term “erythro-
idiopathic thrombocytopenic purpura. blastosis fetalis.” Severely affected fetuses
may develop generalized edema, called
“hydrops fetalis.” In HDFN resulting from
anti-D, erythropoiesis in the fetal liver
Hemolytic Disease of the may be so extensive that portal circulation
is disrupted and albumin synthesis im-
Fetus and Newborn paired, thereby reducing plasma colloid
In HDFN, fetal red cells become coated osmotic pressure. The severe anemia may
with IgG alloantibody of maternal origin, cause cardiovascular failure, tissue hypo-
535

xia, and death in utero. Intrauterine trans- group is discussed in greater detail
fusion may be lifesaving in these circum- in Chapter 14.)
stances. If live-born, the severely affected 2. “Other” hemolytic disease caused by
infant exhibits profound anemia and antibodies against other antigens in
heart failure.1 Less severely affected in- the Rh system or against antigens in
fants continue to experience accelerated other systems; anti-c and anti-K1 are
red cell destruction, which generates most often implicated.5
large quantities of bilirubin. Unlike HDFN 3. ABO HDFN caused by anti-A,B in a
due to anti-D, HDFN due to anti-K1 re- group O woman or by isolated anti-A
sults from suppression of fetal erythropo- or anti-B.
iesis in addition to causing peripheral red In all but ABO HDFN, maternal antibod-
2
cell destruction. ies reflect alloimmunization by pregnancy
Before birth severs the communication or transfusion. Rising titers of antibody can
between maternal and fetal circulation, fe- be documented, at least in the first affected
tal bilirubin is processed by the mother’s pregnancy, and the infant may be symp-
liver. At birth, the infant’s immature liver is tomatic at birth as a result of effects on the
incapable of conjugating the amount of bil- fetus in utero. In contrast, ABO fetomater-
irubin that results from destruction of anti- nal incompatibility cannot be diagnosed
body-coated red cells. Unconjugated biliru- during pregnancy and the infant is rarely
bin is toxic to the developing central symptomatic at birth.
nervous system (CNS), causing brain dam-
age referred to as “kernicterus.” For the
live-born infant with HDFN and rising lev- Pregnancy as the Immunizing Stimulus
els of unconjugated bilirubin, kernicterus Pregnancy causes immunization when fe-
may pose a greater clinical danger than the tal red cells, possessing a paternal antigen
consequences of anemia.3 Prematurity, aci- foreign to the mother, enter the maternal
dosis, hypoxia, and hypoalbuminemia in- circulation as a result of fetomaternal he-
crease the risk of CNS damage. Decisions morrhage (FMH). FMH occurs in the vast
about undertaking exchange transfusion majority of pregnancies, usually during the
are based primarily on the bilirubin level, third trimester and during delivery.6 De-
the rate of bilirubin accumulation, and, to a livery is the most common immunizing
lesser degree, on the severity of the anemia. event, but fetal red cells can also enter the
Recently, the American Academy of Pediat- mother’s circulation after amniocentesis,
rics has published guidelines aimed toward spontaneous or induced abortion, chori-
preventing and managing hyperbiliru- onic villus sampling, cordocentesis, rup-
binemia in infants ≥35 weeks of gestation.4 ture of an ectopic pregnancy, and blunt
trauma to the abdomen.
Immunogenic Specificities. The antigen
Mechanisms of Maternal Immunization
that most frequently induces immunization
HDFN is often classified into three cate- is D, but, in theory, any red cell antigen
gories, on the basis of the specificity of the present on fetal cells and absent from the
causative IgG antibody. In descending or- mother can stimulate antibody production.
der of potential severity, they are: One retrospective study determined that
1. D hemolytic disease caused by anti-D there was a 0.24% prevalence of production
alone or, less often, in combination of clinically significant antibodies other
with anti-C or anti-E. (The Rh blood than anti-D during pregnancy. Because

Chapter 23: Perinatal Issues in Transfusion Practice 537
other red cell antigens are less immuno- tern of clinical disease in subsequent preg-
genic than D, sensitization is more likely to nancies.
result from exposure to a large volume of Effect of ABO Incompatibility. Rh im-
red cells, such as during blood transfusion. munization of untreated D-negative women
Immunization to D, on the other hand, can occurs less frequently after delivery of an
occur with volumes of fetal blood less than ABO-incompatible D-positive infant than
0.1 mL.7 when the fetal cells are ABO-compatible
Frequency of Immunization. The proba- with the mother. ABO incompatibility be-
bility of immunization to D correlates with tween mother and fetus has a substantial
the volume of D-positive red cells entering but not absolute protective effect against
the D-negative mother’s circulation.6 The maternal immunization by virtue of the in-
overall incidence of D sensitization in un- creased rate of red cell destruction by anti-A
treated D-negative mothers of D-positive or anti-B. The rate of immunization is de-
infants is about 16%; 1.5% to 2% become creased from 16% to between 1.5% and 2%.7
sensitized at the time of their first delivery,
an additional 7% become sensitized within
6 months of the delivery, and the final 7% Transfusion as the Immunizing Stimulus
become sensitized during the second af- It is extremely important to avoid trans-
fected pregnancy.8 The sensitization during fusing D-positive whole blood or red cells
the second affected pregnancy probably re- to D-negative females of childbearing
flects primary immunization during the potential because anti-D stimulated by
first D-positive pregnancy and delivery that transfusion characteristically causes severe
happened without production of detectable HDFN in subsequent pregnancies with a
levels of antibody. The small numbers of D-positive fetus. Red cells present in pla-
D-positive fetal red cells entering the ma- telet or granulocyte concentrates can con-
ternal circulation early during the second stitute an immunizing stimulus; if com-
affected pregnancy constitute a secondary ponents from D-positive donors are
stimulus sufficient to elicit overt production necessary for young D-negative female
of IgG anti-D. In susceptible women not recipients, Rh immunoprophylaxis should
immunized after two D-positive pregnan- be considered. This is discussed further in
cies, later pregnancies may be affected but Chapter 21.
with diminished frequency. The incidence The risk of immunization to a red cell
of the more common genotypes in D-posi- antigen other than D after an allogeneic red
tive individuals can be found in Table 14-4. cell transfusion has been estimated to be
This can be used to get a general idea of the 1% to 2.5% in the general hospital popula-
likelihood of how often an infant with a tion.7 This will endanger the fetus only if the
D-positive father and D-negative mother antibody is IgG and directed against an an-
will express the D antigen. tigen that is also present on the fetal red
Once immunization has occurred, suc- cells. For a couple planning to have chil-
cessive D-positive pregnancies often mani- dren, the woman should not be transfused
fest HDFN of increasing severity, particu- with red cells from her sexual partner or his
larly between the first and second affected blood relatives. This form of directed dona-
pregnancies. After the second affected tion increases the risk that the mother will
pregnancy, the history is predictive of out- be immunized to paternal red cell, leuko-
come, although, in rare instances, some cyte, and/or platelet antigens, which could
women have a stable or diminishing pat- cause alloimmune cytopenias in future

children who share the same paternal anti- studies should be performed on all preg-
gens. Programs using parents as directed nant women as early in pregnancy as
donors for their sick newborns deserve spe- possible; they should include tests for
cial consideration because of these unique ABO and D, and a screen for unexpected
issues in the face of strong parental desires.9,10 red cell antibodies.12 If a woman’s red cells
are not directly agglutinated by anti-D,
ABO Antibodies testing for weak D is not required. When
testing for weak D is not performed,
The IgG antibodies that cause ABO HDFN
women with weak D will be labeled as D
nearly always occur in the mother’s circu-
negative, although they are in fact D posi-
lation without a history of prior exposure
tive; the only potential negative outcome
to human red cells. ABO HDFN can occur
is that these women will receive unneces-
in any pregnancy, including the first. It is
sary Rh Immune Globulin (RhIG). Women
restricted almost entirely to group A or B
with some partial D phenotypes, such as
infants born to group O mothers because
DVI, will also most likely type as D negative
group O individuals make the IgG anti-
in direct tests, and, in the absence of weak
body, anti-A,B. Group A or B mothers with
D testing, these women are also candi-
an A- or B-incompatible fetus predomi-
dates for RhIG antenatal prophylaxis.
nantly produce IgM antibody, with only
With the application of molecular tech-
small amounts of IgG antibody capable of
niques, knowledge of the RHD gene is
crossing the placenta.
evolving. Not all weak D red cells are the re-
sult of a decreased expression of the D anti-
Prenatal Evaluation gen, but, rather, some have altered RhD
Maternal History proteins. Consequently, these patients are
at risk for immunization when exposed to
Information about previous pregnancies or
the D antigen, explaining formation of
blood transfusions is essential in evaluat-
anti-D in some patients classified as weak
ing fetal risk. Invasive tests, which carry
D. As more is learned about the D antigen,
risk to the fetus, should be performed
the distinction between partial D and weak
only for pregnancies in which the fetus is
D is blurring.13,14
at risk for HDFN, by history and/or sero-
Whether or not prophylaxis would be
logic testing. For a woman with a history
successful in the setting of partial D remains
of an infant with hydrops fetalis due to
unknown. The appropriate dose is also sub-
anti-D, there is a 90% or more chance of a
ject to speculation. As a result, some practi-
subsequent fetus being similarly affected.5
tioners administer RhIG to these women,
In contrast, during the first sensitized
whereas others consider it unnecessary.
pregnancy, the risk of a hydropic fetus is
Very rarely, a mother with partial D antigen
8% to 10%. Experience with other allo-
produces anti-D as a result of pregnancy.
antibodies has not been as extensive as with
If weak D testing is performed and the
anti-D; in some series, anti-c and anti-K1
test is clearly positive, the woman should
were by far the most common causes of
be regarded as D positive. If testing for
severe HDFN, other than anti-D.5,11
weak D is not performed, women whose
red cells do not react in direct tests with
Serologic Studies anti-D can be considered candidates for
Alloantibodies capable of causing HDFN RhIG prophylaxis. Some laboratories con-
can be detected during pregnancy. Initial tinue to do weak D testing to avoid confu-

sion in the interpretation of the FMH screen sampling is discouraged because it causes
during postpartum testing.12 Chapter 14 FMH and has been associated with more
contains a more complete discussion of the severe HDFN. Amniotic fluid samples are
Rh blood group. recommended over cordocentesis because
A woman should be classified as D posi- cordocentesis has a fourfold or higher rate
tive if the test for either D or weak D is posi- of perinatal loss over amniocentesis.18 The
tive. If a D-negative woman has a negative use of molecular techniques can help de-
initial antibody screen, the test can be re- tect variations in the RHD gene that might
peated at 28 weeks’ gestation before ad- go undetected using serology alone. It is
ministration of RhIG to detect immuniza- preferable that both paternal and maternal
tion that might have occurred before 28 weeks, blood samples accompany the fetal sam-
in accordance with AABB recommendations. ples.19 Fetal DNA typing is also available for
a b 20 21 22 23
Because the incidence of immunization Jk /Jk , K1/K2, c, and E/e antigens. A
during this period of pregnancy is extremely more recent development in fetal RhD typ-
low, the American College of Obstetricians ing involves the isolation of free fetal DNA
and Gynecologists (ACOG) points out that in the maternal serum. Although not rou-
no data exist that support the cost-effective- tinely available in the United States at this
15
ness of this practice. Repeat antibody time, this will likely replace amniocentesis
screening of D-positive women may be rec- for fetal genotyping in the near future.24
ommended if there is a history of clinically
significant red cell antibodies associated
with HDFN, previous blood transfusion, or Maternal Antibody Titer
trauma to the abdomen. Antibody titrations can help in decisions
Antibody Specificity. All positive screens about the performance and the timing of
for red cell antibodies require identification invasive procedures, especially if the anti-
of the antibody.12 The mere presence of an body is anti-D. The antibody titer should
antibody, however, does not indicate that be established in the first trimester to
HDFN will occur. Non-red-cell-stimulated serve as a baseline, and the specimen
IgM antibodies, notably anti-Lea and anti-I, should be frozen for future comparisons
are relatively common during pregnancy but (see Method 5.3).7 Because invasive tests
do not cross the placenta. In addition, the will not be undertaken before 16 to 18
fetal red cells may lack the antigen corre- weeks’ gestation, no further titration is in-
sponding to the mother’s antibody; the like- dicated until this time. The true signifi-
lihood of fetal involvement can often be cance of an antibody titer in maternal se-
predicted by typing the father’s red cell an- rum is controversial because some studies
tigens.16 The laboratory report on prenatal have shown poor correlation between the
antibody studies should include sufficient level of the titer and effects on the fetus.
information to aid the clinician in deter- For antibodies other than anti-D, critical
mining the clinical significance of the iden- titers have not been identified, although a
tified antibody. critical titer similar to that used in cases
Typing the Fetus. The fetal D type can be of anti-D alloimmunization is often uti-
established by using the polymerase chain lized.25 These techniques continue to be
reaction (PCR) to amplify DNA obtained performed because they represent a non-
from amniotic fluid, chorionic villus sam- invasive way to try to assess the presence
ples, or by serologic typing of fetal blood and severity of alloimmunization. When
17
obtained by cordocentesis. Chorionic villus performed, it is important that successive

titrations use the same methods and test affected pregnancies or have an antibody
cells of the same red cell phenotype. Test- titer at or above the critical titer.28 Because
ing previously frozen serum samples in fetal anemia secondary to K1 alloimmuni-
parallel with a current specimen mini- zation is not always associated with ele-
mizes the possibility that changes in the vated levels of bilirubin in amniotic fluid,
titer result from differences in technique. it has been recommended that fetal blood
The critical titer for anti-D (the level be- sampling be used instead of serial amnio-
low which HDFN and hydrops fetalis are centesis when anti-K1 is detected in a
considered so unlikely that no further in- pregnant woman.2
vasive procedures will be undertaken) Amniotic fluid is obtained by inserting a
should be selected at each facility and is long needle through the mother’s abdomi-
usually 16 or 32 in the antihuman globu- nal wall and uterus into the uterine cavity
lin phase.26,27 Follow-up testing is recom- under continuous ultrasound guidance.
26
mended for any titer greater than 8. The The aspirated fluid is scanned spectropho-
critical titer for anti-K1 may be lower than tometrically at wavelengths of 350 to 700
anti-D, typically a value of 8.25,28 Currently, nm. Peak absorbance of bilirubin is at 450
it is not recommended that gel technol- nm. An increase in optical density from the
ogy be used for prenatal antibody titra- projected baseline at 450 nm (∆OD450) is a
tion because of the lack of data showing a measure of the concentration of bile pig-
correlation between gel and tube aggluti- ments.30,31 The ∆OD450 value is plotted on a
nation titers.12 graph against the estimated length of gesta-
tion, because bile pigment concentration
Other Measures of HDFN Severity has different clinical significance at differ-
ent gestational ages. Liley’s system31 (Fig
Numerous laboratory procedures have been
23-1) of predicting the severity of fetal dis-
investigated to improve the accuracy of
ease based on the ∆OD450 has been used for
predicting the severity of hemolysis.29 The
decades. It delineates three zones to esti-
antibody titer discussed above is not al-
mate severity of disease: a top zone (zone 3)
ways reliable, nor is the serial change in
indicates severe disease, the bottom zone
titer. Functional assays, including measures
(zone 1) indicates mild or no disease, and
of adherence, phagocytosis, antibody-de-
mid-zone (zone 2) values require repeat de-
pendent cytotoxicity, and chemilumines-
termination to establish a trend. This
cence have been investigated, but their use
method is applicable to pregnancies from
has been limited and remains controver-
27 weeks through term. Queenan et al32
sial. These procedures are usually per-
have proposed a system for managing
formed in referral centers and may be use-
D-immunized pregnancies based on the
ful when additional information is required
∆OD450 from as early as 14 weeks’ gestation.
to manage difficult and complex cases.
They identified four zones (Fig 23-2), with
early invasive intervention recommended if
Amniotic Fluid Analysis ∆OD450 values fall in the highest zone. With
A good index of intrauterine hemolysis both systems, the severity of HDFN is more
and fetal well-being is the level of biliru- accurately predicted with serial ∆OD450
bin pigment found in amniotic fluid ob- measurements than with a single observa-
tained by amniocentesis. Amniocentesis tion, to evaluate whether readings are fall-
is usually performed in alloimmunized ing, rising, or stable. In general, the higher
women who have a history of previously the pigment concentration, the more se-

Figure 23-1. Liley graph for collecting data from amniotic fluid studies. Intrauterine transfusion should
be done if the ∆OD 450 value is in the top zone before 32 weeks’ gestation. After 34 weeks, top zone val-
ues indicate immediate delivery. Either intrauterine transfusion or immediate delivery may be indi-
cated for top zone ∆OD 450 between 32 and 34 weeks, depending on studies of fetal maturity. Modified
from Liley.31
vere the intrauterine hemolysis. It is impor- Percutaneous Umbilical Blood Sampling

tant to perform an ultrasound to establish
the correct gestational age so that the test In the early 1980s, the use of sophisticated
can be interpreted and the course of ther- ultrasound equipment made it feasible to
apy for a particular ∆OD450 will be appro- direct a needle into an umbilical blood
priate. Alternatively, a ∆OD450 value in the
18
vessel, preferably the vein at its insertion
upper mid-zone of the Liley curve indicates into the placenta, and obtain a fetal blood
the need for fetal blood sampling. sample. Percutaneous umbilical blood
Amniocentesis, particularly if the needle sampling (PUBS, or cordocentesis) allows
goes through the placenta, or cordocentesis direct measurement of hematologic and
may cause FMH, which can boost the titer biochemical variables. Determination of the
of existing red cell alloantibody, thereby in- fetal hematocrit provides an accurate as-
creasing the severity of HDFN, or inducing sessment of the severity of fetal hemolytic
18 18
immunization to additional antigens. disease. It is important to verify that the
Therefore, when amniocentesis or cordo- sample has been obtained from the fetus.
centesis is performed for any reason on a Fetal and maternal red cells can be distin-
D-negative woman who does not have guished because of differences in red cell
anti-D, Rh immunoprophylaxis should be size and red cell phenotyping, as well as
33
given. by the presence of fetal hemoglobin.

Figure 23-2. Amniotic fluid OD 450 management zones. (Reproduced with permission from Queenan et
al.32 )
The fetal mortality of intrauterine fetal ies have found good correlation between
blood sampling has been reported to be 1% middle cerebral artery (MCA) peak veloc-
to 2%,34 and the procedure carries a high ity, fetal hemoglobin, and ∆OD450 read-
36
risk of FMH. Its use is recommended only ings. Many centers routinely use an MCA
for certain circumstances, such as when se- peak systolic velocity value of greater than
rial amniotic fluid determinations indicate 1.5 multiples of the median to proceed
severe HDFN, when hydrops is present, with cordocentesis to determine if the fe-
when the D titer is high or rising, or when tus is anemic. In such centers, amniocen-
HDFN occurred in previous pregnancies. In tesis is performed only after 35 weeks’
addition to diagnosis, PUBS allows treat- gestation when MCA Doppler is associ-
ment of the affected fetus. ated with a high false-positive rate for the
diagnosis of fetal anemia (Moise K, per-
Doppler Flow Studies sonal communication).
Because fetal anemia results in increased
cardiac output, several investigators have Suppression of Maternal
measured various blood velocities in fetal Alloimmunization
vessels using Doppler ultrasonography to Several approaches to suppress maternal
determine the clinical status of the fetus alloimmunization have been attempted,
in a noninvasive manner.18,35 Recent stud- two of which have limited clinical benefit

in reducing maternal antibody levels: informed, intrauterine transfusion remains

tensive plasma exchange and the admin- the mainstay of standard therapy.
istration of immunoglobulin (intravenous)
(IGIV).5,18 Plasma exchange can reduce an- Intrauterine Transfusion
tibody levels by as much as 75%. Unfortu- Intrauterine transfusion can be performed
nately, rebound usually follows because by the intraperitoneal route (IPT) or the
the IgG antibody is mostly extravascular direct intravascular approach (IVT) by the
and antigen exposure may be ongoing. umbilical vein. In many instances, IVT is
Plasma exchange has been proposed as a the procedure of choice, but there may be
way to delay the need for fetal interven- problems of access that make IPT prefera-
tion, particularly when there is a previous ble; a combination may also be used to
pregnancy complicated by early hydrops.37 minimize peaks and troughs of fetal hema-
In this setting, plasma exchange can delay tocrit between procedures. Intrauterine
the need for more invasive procedures un- transfusion is seldom feasible before the
til the second trimester. The AABB and 20th week of gestation; once initiated,
the American Society for Apheresis (ASFA) transfusions are usually administered pe-
categorize plasma exchange as treatment riodically until delivery. It is important
Category III because its efficacy and safety that blood is available and ready at the
have not been proven for this indication time of the first diagnostic, and subse-
(see Chapter 6). With the increasing safety quent, cordocenteses. If fetal anemia is
of intrauterine transfusion through ultra- detected, an intrauterine transfusion can
be performed at once, minimizing fetal
sound guidance and the decreasing inci-
risks. The interval between transfusions
dence of HDFN due to anti-D, the use of
depends on the presence or absence of
plasma exchange as a treatment modality
hydrops, the gestational age, and the
has declined.
amount of blood infused. Good outcomes
IGIV infusion has also been shown to
are achieved over 80% of the time and
stabilize anti-D titers, with best results ob-
94% of nonhydropic fetuses survive.40 Be-
tained when started before 28 weeks’ gesta-
cause intrauterine transfusion carries a 1%
tion and when the fetus is not hydropic.38 In
to 2% risk of perinatal loss, it should be
a small study assessing the efficacy of IGIV,
performed only after careful clinical eval-
it was found to be well tolerated and there
uation.18,34,41,42 Other perinatal conditions
was a decrease in hemolysis.39 The mecha-
that have been treated with intrauterine
nism of IGIV effect is not clear, although it
transfusion include parvovirus infection,
may work by saturating placental Fc recep-
large FMH, and alpha thalassemia.43
tors and inhibiting the transplacental trans-
fer of maternal antibody or by suppressing
ingestion of IgG-coated red cells by the fetal Techniques
reticuloendothelial system. An alternative IPT is performed through a needle passed,
explanation is that the introduction of with ultrasonographic monitoring, through
anti-idiotype antibodies modifies maternal the mother’s abdominal wall into the
antibody production. IGIV has also been abdominal cavity of the fetus. Transfused
used with plasma exchange to reduce the red cells enter the fetal circulation through
antibody rebound that has been seen fol- lymphatic channels that drain the peri-
lowing plasma exchange.18 Until larger stud- toneal cavity. In IVT, the umbilical vein is
ies assessing safety and efficacy can be per- penetrated under ultrasound guidance,

and a blood sample is taken to verify posi- the neonatal period should be irradiated to
tioning in the fetal vasculature. Injection prevent transfusion-associated graft-vs-host
of saline can also confirm correct place- disease because the fetus is considered im-
45,46
ment because it can be visualized by ul- munologically naïve and tolerant.
trasound. Blood is infused directly, as ei-
ther a simple transfusion or as a partial Volume Administered
exchange transfusion. IVT can be particu-
The volume transfused varies with the
larly valuable for very severe cases of
technique used as well as the fetal size,
HDFN associated with hydrops fetalis. In
initial hematocrit, and gestational age.
hydropic infants, red cells administered For IPT, a volume calculated by the for-
by IPT are not efficiently absorbed. mula V = (gestation in weeks – 20) × 10 mL
appears to be well tolerated by the fetus.
Selection of Red Cells The volume of red cells transfused by IVT
can be calculated by the following for-
The red cells used should be group O,
mula.47
D-negative, or negative for the antigen
corresponding to the mother’s antibody if
Fetoplacental volume (mL) =
the specificity is not anti-D. Blood for
ultrasound estimated fetal weight
intrauterine transfusion should be irradi-
ated (see Chapter 27), and should be cyto- (g) × 0.14
megalovirus (CMV)-reduced-risk. It may
Volume to transfuse (mL) =
also be desirable to transfuse blood that is
known to lack hemoglobin S in order to Fetoplacental volume ×
transfuse red cells with maximal oxygen- (Hct after IVT – Hct before IVT)
transporting capacity, in the setting of low Hct of donor cells
oxygen tension. For optimal survival of the
transfused cells, blood used for intraute- where Hct = hematocrit
rine transfusion should be drawn as re- Transfusion is repeated on the basis of
an estimated decline in fetal hematocrit of
cently as possible, generally less than 7
approximately 1% per day in an effort to
days old.
maintain the fetal hematocrit in the range
The hematocrit of the RBCs prepared for
of 27% to 30%.48
exchange transfusion is usually high to
minimize the chance of volume overload in
the fetus. Washed, irradiated maternal Postpartum Evaluation
blood has also been used for intrauterine It may be desirable to collect a sample of
44
transfusion. To remove the offending anti- cord blood, preferably by cannulation of
body, the red cells are washed and resus- an umbilical vessel at delivery, from new-
pended in saline to a final hematocrit borns where there is a risk of HDFN (eg,
between 75% and 85%. Washed or degly- Rh-positive infants born to Rh-negative
cerolized preparations have been used as a mothers, type A and B infants born to
means to remove plasma, anticoagulant/ type O mothers). This sample should be
preservative solutions, and excess electro- identified as cord blood and labeled in
lytes that might accumulate during pro- the delivery suite with the mother’s name,
longed storage. Blood for intrauterine the date, and two unique forms of identi-
transfusions and all blood and cellular fication for the infant (eg, name and med-
components subsequently transfused in ical record number).

In cases of suspected HDFN, samples of even negative in HDFN resulting from

both cord and maternal blood should be ABO antibodies. However, the strength of
tested. When the mother is known to have the DAT does not correlate with the sever-
antibodies capable of causing HDFN, the ity of hemolysis, especially in ABO HDFN.
hemoglobin or hematocrit and the biliru- Infants who have received an intrauterine
bin level of cord blood should also be de- transfusion may have a weakly positive
termined. If the mother is D negative and DAT with a mixed-field pattern of aggluti-
the infant D positive, the mother’s blood nation. If the DAT on cord cells is positive,
should be tested to determine the volume the antibody can be eluted from the red
of FMH, as discussed later. Testing cord cells and tested for specificity. It is not
blood may present some special problems, necessary to make and test an eluate if
which are described below. the maternal serum has been shown to
contain a single red cell antibody. All clin-
ically significant red cell antibodies in the
ABO Testing maternal serum must be respected. (See
ABO testing on newborns relies entirely on the section on Selection of Blood, later in
red cell typing because ABO antibodies in this chapter.) If the DAT is positive and
cord serum are nearly always of maternal the maternal antibody screen is negative,
origin and are IgG. However, in the inves- investigation should turn toward ABO an-
tigation of possible HDFN due to ABO in- tibodies or HDFN caused by an antibody
compatibility, cord serum should be tested directed against a low-incidence antigen
for antiglobulin-reactive ABO antibodies. not present on reagent red cells.
Also, if the infant will receive non-group-O Evaluation of ABO Antibodies. ABO
red cells, testing must be taken to the HDFN may be suspected on clinical
antiglobulin phase.49(p42) grounds even though the DAT is negative.
Testing the eluate from the cord cells
against A1 and B red cells should establish
the diagnosis of ABO HDFN. Cord blood or
D Testing
peripheral blood serum should be tested by
Newborns who have had successful intra- an indirect antiglobulin technique against
uterine transfusions often type at birth as A1, B, and O red cells. The presence of anti-A,
D negative or very weakly positive be- anti-B, or anti-A,B confirms the potential
cause over 90% of their circulating red for ABO HDFN. It is often possible to elute
cells may be those of the donors. The ABO anti-A and/or anti-B from the infant’s red
and direct antiglobulin tests may also give cells despite a negative DAT, but this step is
misleading results. If the infant’s red cells not necessary for the presumptive diagno-
are heavily coated with IgG antibodies, tests sis. In the rare cases of ABO HDFN that re-
with anti-D may give either false-positive quire transfusion, D-compatible group O
or false-negative results (see Chapter 14). blood should be transfused, whether or not
the diagnosis has been serologically con-
firmed. Nonimmune causes of hyperbiliru-
Antiglobulin Testing binemia and hemolysis should still be con-
The direct antiglobulin test (DAT) is usu- sidered in an infant with a negative DAT
ally strongly positive in HDFN resulting before concluding that it is due to ABO
from anti-D or antibodies to other blood HDFN because other hematologic disor-
groups; reactions are much weaker or ders might be present.50

Antibodies to Low-Incidence Antigens. Selection of Blood

If ABO HDFN is ruled out, antibody against
a low-incidence red cell antigen should be In most cases, the mother’s serum is used
suspected. Testing an eluate or maternal for crossmatching and the red cells se-
serum against the father’s red cells with an lected for transfusion are compatible with
antiglobulin technique may provide an an- her ABO antibodies as well as any addi-
swer. Maternal serum must be ABO com- tional antibody(ies) responsible for the
patible, if it is used. If either or both of these hemolytic process. Group O red cells re-
tests are positive, it indicates that the infant suspended in AB plasma are commonly
has an antigen of paternal origin that the used. In ABO HDFN, the red cells used for
mother lacks, causing her to make an IgG exchange must be group O. If the anti-
antibody directed against this antigen. Un- body is anti-D, the red cells must be D
less the mother has been exposed to red cells negative, but not every exchange transfu-
from the father or his blood relations, trans- sion requires group O RBCs. If mother
fusion would be an unlikely immunizing and infant are ABO-identical, group-spe-
event for a low-incidence antigen. Because cific red cells or whole blood can be used.
there should be no difficulty in obtaining If the implicated antibody is not anti-D,
compatible blood, diagnostic studies can D-positive red cells may be given to a
be performed after initial clinical concerns D-positive infant.
have been resolved. If the DAT is positive Maternal serum or plasma is the speci-
and all attempts to characterize a coating men of choice for crossmatching in ex-
red cell antibody are consistently negative, change transfusion; it is available in large
causes of a false-positive DAT should be quantities, decreases the volume of blood
considered (see Chapter 20). taken from the infant, has the red cell anti-
body present in high concentration, and
can be analyzed accurately and completely
Exchange Transfusion
before delivery. On the other hand, use of
Exchange transfusion for HDFN achieves maternal serum may be problematic if it
several desired effects, including: contains antibodies directed against anti-
1. Removal of antibody-coated fetal red gens not present on the infant’s red cells
cells. because of other sources of sensitization, or
2. Removal of maternal antibody. if it contains IgM antibodies that have not
3. Removal of bilirubin. crossed the placenta. These additional
4. Replacement of red cells, thereby antibodies could complicate the serologic
treating anemia. picture.
The red cells used for replacement must If maternal blood is not available or is
be compatible with the causative antibody. unsuitable for crossmatching, the infant’s
Fresh Frozen Plasma is frequently used to serum and/or, preferably, an eluate from
reconstitute whole blood because it pro- the infant’s red cells can be used for cross-
vides coagulation factors. Platelet values matching. The concentration of antibody in
should also be monitored and transfusion the infant’s serum may be low, especially if
used as necessary. Plasma frozen within 24 most of the molecules are bound to the red
hours and thawed plasma can be used too, cells. Use of the eluate or serum, or of both
with the understanding that there might be together, may be indicated if attempts to
decreases in the activity of the labile obtain a maternal specimen would delay
clotting Factors V and VIII. therapy. Blood used for exchange transfu-

sion should be irradiated. Typically, a vol- line washing, and resuspend the red
ume of twice the infant’s blood volume is cells in compatible plasma to the de-
used for exchange.51 sired hematocrit.
2. If time permits, test the mother’s sib-
Subsequent Transfusion lings or other close relatives for
compatibility and eligibility.
Bilirubin may reaccumulate rapidly after
3. Use incompatible donor blood for
a successful exchange transfusion despite
the exchange transfusion if the clini-
appropriate phototherapy. This occurs
cal situation is sufficiently urgent.
because most bilirubin in extravascular
The exchange will reduce the biliru-
fluid will reequilibrate by entering the
bin load, the most heavily anti-
intravascular space and also because re-
body-coated cells, and the number
sidual antibody-coated cells continue to
of unbound antibody molecules.
hemolyze. If rising bilirubin levels make a
However, residual antibody will at-
second or third exchange transfusion nec-
tach to the transfused cells, and one
essary, the same considerations of red cell
or more additional exchanges will
selection and crossmatching apply.
probably be needed as bilirubin ac-
Infants who have undergone intraute-
cumulates.
rine transfusion need to be followed closely
after birth because intrauterine transfusion
suppresses fetal erythropoiesis. Weekly Rh Immune Globulin
hematocrit and reticulocyte counts should
RhIG is a concentrate of predominantly
be performed on the neonate for a 1- to
IgG anti-D derived from pools of human
3-month period.18 Many of these infants
plasma. A full dose of anti-D (300 µg, 1500
will subsequently develop anemia and
IU, or the actual content of a “dose” as in-
need to be supported with red cell transfu-
dicated by the individual manufacturer53)
sion until their own production begins, as
is sufficient to counteract the immunizing
evidenced by reticulocytosis and age-ap-
effects of 15 mL of D-positive red cells;
propriate hemoglobin levels.18,52
this corresponds to approximately 30 mL
of fetal whole blood. RhIG is available in a
Antibody Against a High-Incidence Antigen reduced dose, approximately 50 µg, which
Rarely, the mother’s antibody reacts with is protective for up to 2.5 mL of D-positive
a high-incidence antigen and no compati- fetal red cells. This dose can be used for
ble blood is available. If this problem is first-trimester abortion or miscarriage, when
recognized and identified before delivery, the total blood volume of the fetus is less
the mother’s siblings can be evaluated for than 2.5 mL. However, because of fears of
compatibility and suitability, or compati- miscalculating the length of pregnancy
ble donors can be sought through a rare and concerns of inadvertently mixing up
donor file. Maternal red cells can also be inventory resulting in undertreatment, a
collected and frozen. Any products collected full dose is usually administered and
from blood relatives must be irradiated. If these low doses are frequently not stocked.
this very rare event is not recognized until The protective effect of RhIG on D-nega-
after delivery, three choices are available: tive individuals exposed to D-positive
1. Collect blood from the mother, if the cells probably results from interference
obstetrician agrees. Remove as much with antigen recognition in the induction
plasma as possible, preferably by sa- phase of primary immunization.54

RhIG is available in two formulations: 1) Postpartum Administration

for intramuscular (IM) injection only and 2)
for either IM or intravenous (IV) adminis- Cord blood from infants born to D-nega-
tration. The dose of the intravenous prepa- tive mothers should be tested for the D
ration is expressed in international units (5 antigen. A D-negative woman with a
IU is equivalent to 1 µg), with 1500 IU (300 D-positive infant should receive one full
µg) neutralizing 17 mL of D-positive red dose of RhIG within 72 hours of delivery,
cells, according to the package insert. unless she is known to be alloimmunized
to D previously. The presence of residual
anti-D from antepartum RhIG does not
indicate ongoing protection.
Antepartum Administration Active vs Passive Antibody. In-vitro clues
Widespread postpartum use of Rh immuno- can help distinguish passively administered
prophylaxis has reduced pregnancy-asso- RhIG from the anti-D formed as a result of
ciated immunization to the D antigen to active alloimmunization. Passively acquired
1% to 2%.8 This risk is further decreased to anti-D is entirely IgG; if a woman’s anti-D is
0.1% if RhIG is also given antepartum at saline-reactive or can be completely or par-
28 weeks of gestation.7 The ACOG recom- tially inactivated by treating the serum with
mends antepartum RhIG prophylaxis at 2-mercaptoethanol or dithiothreitol, it has
28 weeks of gestation, based on the obser- an IgM component and probably repre-
vation that, of women who develop anti-D sents active immunization. Passively ac-
during pregnancy, 92% do so at or after 28 quired anti-D rarely achieves an anti-
weeks.7,15 globulin titer above 4, so a high-titered
Blood obtained before injection of RhIG antibody or rising antibody titer is likely to
should be tested for ABO and D. A D-nega- indicate active immunization. It is desirable
tive woman who has antibodies other than to obtain confirmation from the physician’s
anti-D (eg, anti-G) is still a candidate for records, but RhIG should always be given
anti-D immunoprophylaxis. When the when doubt cannot easily be resolved. It
mother receives RhIG during pregnancy, should also be given if there is any problem
the infant may be born with a positive DAT, determining the Rh type.
but without hemolysis. The mother’s serum Postpartum Evaluation. A sample of the
will often exhibit anti-D reactivity. There mother’s blood should be drawn, preferably
must be good communication between the within 1 hour after delivery, and evaluated
patient’s physician and the blood bank staff for FMH of a quantity greater than that for
at the institution where delivery takes which 300 µg RhIG is immunosuppressive.
place, to ensure correct interpretation of If the screening test demonstrates the pres-
laboratory tests made at the time of deliv- ence of fetal cells, the extent of FMH must
ery. The half-life of an injected dose of RhIG, be determined so that an appropriate dose
49(pp48,49),55
in the absence of significant FMH, is ap- of RhIG can be administered.
proximately 21 days. Therefore, of 300 µg of Postpartum RhIG should be given within
anti-D given at 28 weeks, 20-30 µg could re- 72 hours of delivery. If prophylaxis is de-
main at the time of delivery 12 weeks later. layed, the likelihood that alloimmunization
Some practitioners will administer another will be prevented decreases. Despite the de-
dose of RhIG if delivery is delayed beyond crease seen, the ACOG recommends that
40 weeks.18 Anti-D can be detected in the treatment still be administered because
maternal circulation for as long as 6 months. some studies have found partial protection

has occurred as late as 13 days after expo- receive a full dose of RhIG at that time, a
sure and, possibly, as late as 28 days.15 second full dose at 28 weeks of gestation,
The following women are not candidates and the usual postpartum dose if the in-
for RhIG: fant is D positive. If a nonimmunized
1. The D-negative woman whose infant D-negative woman undergoes amniocen-
is D-negative. tesis for any reason in the second or third
2. Any D-positive woman. Very rare cases trimester, a full dose of RhIG is indicated.
of HDFN have been reported in in- If the procedure is repeated more than 21
fants whose mothers had a weak/par- days later, an additional full dose should
18
tial D phenotype, but routine RhIG be given. If amniocentesis is performed
prophylaxis is not routinely recom- to assess fetal maturity, and if delivery is
mended for women of the weak/ expected within 48 hours of the proce-
partial D phenotype.12 dure, RhIG can be withheld until the in-
3. A D-negative woman known to be fant is born and confirmed to be D posi-
immunized to D. tive. If more than 48 hours will elapse,
RhIG should be given following amnio-
Other Indications for RhIG centesis. If delivery occurs within 21 days
thereafter and there is no evidence of a
RhIG should be given to a D-negative woman 15
massive FMH, additional RhIG may not
after any obstetric event that might allow
be essential, but prudent management
fetal cells to enter the mother’s circula-
suggests repeat RhIG administration at
tion: spontaneous or therapeutic abor-
tion, ectopic pregnancy, amniocentesis, delivery.
chorionic villus sampling, molar preg-
nancy, cordocentesis, antepartum hemor-
rhage, blunt abdominal trauma, or fetal Screening for Large-Volume FMH
death.55 As mentioned earlier, at 12 weeks
Postpartum administration of RhIG may
of gestation or earlier, a 50 µg dose of RhIG
not prevent immunization if the quantity
would be adequate to protect against the
of D-positive fetal red cells entering the
small fetal blood volume during the first
mother’s circulation exceeds the immuno-
trimester. From 13 weeks’ gestation until
suppressive capacity of RhIG. One 300-µg
term, a full dose of RhIG should be given.
dose protects against 15 mL of D-positive
At <20 weeks, the fetal blood volume is
red cells or 30 mL of fetal blood. Only 0.3%
rarely more than 30mL,56 small enough
of pregnancies are estimated to sustain
that a single dose of 300 µg Rh immune
FMH greater than 30 mL, but large FMH
globulin will be sufficient for prophylaxis
is an important and preventable cause of
for any FMH. Therefore, it is not neces- 7
failed immunoprophylaxis. The ACOG
sary to quantitate fetal red cells in the ma-
recommends postpartum testing for large
ternal circulation before 20 weeks of ges- 15
FMH only for high-risk pregnancies, but
tation.57 58
Ness and colleagues showed that testing
based only on the ACOG criteria would
Amniocentesis miss 50% of mothers exposed to large-vol-
Amniocentesis can cause FMH and con- ume FMH. AABB Standards for Blood
sequent Rh immunization. The D-negative Banks and Transfusion Services requires
woman who has amniocentesis at 16 to 18 examination of a postpartum specimen
weeks’ gestation for genetic analysis should from all D-negative women at risk of im-

munization, to detect the presence of FMH enzyme-linked antiglobulin test (ELAT,

49(p49)
that requires more than one dose of RhIG. which is in limited use). Each of these
In rare cases, a massive FMH can cause methods has various advantages and disad-
fetal death and infuse enough Rh-positive vantages that must be evaluated by each in-
cells into the maternal circulation to simu- stitution. The use of nucleic acid amplifica-
late a weak D phenotype in an Rh-nega- tion techniques, designed to detect very
tive patient. 15,59 Unless recognized and small amounts of fetal cells, remains a re-
61-63
treated with an adequate dose of RhIG, this search endeavor.
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 Quantifying FMH
microscopically (“microscopic weak D Historically, quantification of FMH has
test”); mixed-field reactivity indicated a been achieved by the Kleihauer-Betke
substantial admixture with D-positive cells. acid-elution test, which relies on the dif-
This procedure should not be used to iden- ferences between fetal and adult hemo-
tify large FMH, however, because of its lack globin in resistance to acid elution (see
of reliability.60 Method 5.2). Results are reported as a per-
The Rosette Test. The rosette test dem- centage of fetal cells, but the precision
onstrates small numbers of D-positive cells and accuracy of the procedure may be
in a D-negative suspension. The suspen- poor. Because 300 µg of RhIG will protect
sion is incubated with an anti-D reagent of against FMH of 30 mL of D-positive fetal
human origin, and antibody molecules at- blood, the number of doses of RhIG re-
tach to sites on D-positive cells in the sus- quired is determined by dividing the esti-
pension. Indicator D-positive cells are mated volume of fetal blood present by 30.
added, which react with antibody mole- For example:
cules bound to the surface of the al- 1. Kleihauer-Betke test results reported
ready-present D-positive cells and form vis- as 1.3%
ible agglutinates (rosettes) around them 2. (1.3/100) × 5000 mL* = 65 mL of fetal
(see Method 5.1). This method will detect blood
FMHs of approximately 10 mL,60 a sensitiv- 3. 65/(30 mL per dose) = 2.2 doses of
ity that provides a desirable margin of RhIG required
safety for a screening test. Weak D-positive * = mother’s arbitrarily assigned blood volume
cells do not react as strongly in the rosette
procedure as normal D-positive cells. If the Because quantification by this procedure
newborn has a weak D phenotype, FMH is inherently inaccurate and because the
can be evaluated by the Kleihauer-Betke consequences of undertreatment can be
acid-elution test (see below), which identi- serious, it is desirable to provide a safety
fies fetal hemoglobin, not a surface antigen. margin in calculating RhIG dosage. One
In all cases, the rosette test gives only quali- approach is as follows:
tative results and a positive result must be 1. When the number to the right of the
followed by a quantitative test, such as an decimal point is less than 5, round
acid-elution procedure. Other tests that can down and add one dose of RhIG (ex-
be used to detect and/or quantify FMH are ample: If the calculation comes to
flow cytometry, gel agglutination, and the 2.2 doses, give 3 doses).

2. When the number to the right of the Neonatal Alloimmune Thrombocytopenia

decimal point is 5 or greater, round
up to the next number and add one The mechanism of NAIT is similar to that
dose of RhIG (example: If the calcu- of HDFN. Fetal platelets, expressing a pa-
lation comes to 2.8 doses, give 4 ternal antigen absent from the mother’s
doses). (See Table 23-1). cells, may enter the mother’s circulation
Not more than five doses of RhIG should during gestation or delivery. If she becomes
be injected intramuscularly at one time. For immunized, the maternal IgG antibody
larger quantities, injections can be spaced crosses the placenta and causes fetal and
over a 72-hour period for the patient’s com- neonatal thrombocytopenia. The maternal
fort; an optimal time sequence has not platelet count remains normal. The inci-
been established. The intravenous prepara- dence of NAIT is approximately 1 in 1500
tion of RhIG can be used when higher to 2000 live births.64,65 NAIT is the cause of
doses are required. According to the pack- the majority of cases of intracranial hem-
age insert, a maximum dose of 300 IU orrhage due to thrombocytopenia, greater
should be given at each injection, every 8 than all other etiologies of thrombocyto-
hours, until the total calculated dose has penia combined.66
been administered. 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
Neonatal Immune resolving in 2 to 3 weeks. NAIT varies in se-
Thrombocytopenia verity from mild thrombocytopenia with no
Maternal IgG antibodies to platelets can clinical signs to overt clinical bleeding. The
cross the placenta and cause severe ante- incidence of intracranial hemorrhage has
natal and neonatal thrombocytopenia. Two been reported as 10% to 30%, with approxi-
categories of immune thrombocytopenia mately half occurring in utero.65,67 Recur-
are recognized, and the distinction be- rence in subsequent pregnancies is fre-
tween them is therapeutically important. quent, with equal or increasing severity, so
Table 23-1. RhIG Dosage for Massive Fetomaternal Hemorrhage, Based on the Acid
Elution Test
Dose
% Fetal Cells Vials of RhIG to Inject In g In IU
0.3 - 0.8 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 about 33% will have neonates with clini-
receive skilled antenatal attention. cally important thrombocytopenia, this has
not been widely adopted. In addition, the
cost and logistics of performing platelet an-
Serologic Testing tigen typing are impediments to broad im-
Serologic diagnosis should be sought in a plementation.65,71
woman whose infant has had NAIT if fur-
ther pregnancies are planned. Several
platelet-specific antigen systems have Prenatal Considerations
been associated with NAIT, with HPA-1a With knowledge of antibody specificity
antigen (PlA1) accounting for the vast ma- and gene frequencies, the likelihood of
jority of cases in Caucasians.64 Pregnancy, subsequent offspring being affected can
rather than transfusion, is the usual im- be predicted (see Table 16-1). The recog-
munizing event. Approximately 2% of the nized platelet-specific antigens occur in
population is HPA-1a negative; approxi- diallelic systems, so typing the father’s
mately 10% of HPA-1a-negative women platelets indicates zygosity. If the father is
with HPA-1a-positive infants become im- homozygous for the expression of the an-
68
munized. Some studies have shown an tigen, there is no need to determine the
association between developing anti-HPA- fetal antigen status because all offspring
1a and possessing the HLA phenotype will be affected. If the father is heterozy-
DRw52a.66,68 Chapter 16 contains more in- gous for the expression of the antigen,
formation about platelet antigens. Al- then there is a 50% chance that subse-
though antibodies to HLA Class I antigens quent offspring will have the offending
are frequently encountered in pregnancy, antigen. In an at-risk pregnancy, the ge-
and platelets express Class I antigens, this notype of the fetus (and by inference the
is a rare cause of NAIT, and the true role of platelet phenotype) can be determined by
HLA antibodies in this setting remains DNA typing on fetal cells obtained by am-
controversial.69,70 niocentesis.
Any family with a history of an infant When the risk of NAIT is high, a fetal
born with a platelet count of <50,000/µL blood sample for platelet count determina-
should be evaluated. Ideally, serologic test- tion can be obtained by cordocentesis as
ing uses maternal serum and maternal and early as 20 weeks’ gestation. Because
paternal whole blood from which platelets cordocentesis carries a risk of serious
are isolated. Maternal serum is screened for bleeding in a thrombocytopenic fetus,
both platelet-nonspecific and platelet-spe- compatible platelets must be available at
cific antibodies against paternal cells, as the time of the procedure and are often in-
well as panels of phenotyped platelets. Ma- fused if the platelet count is low. Some in-
ternal and paternal platelet typing can be stitutions will infuse platelets during the
performed using serologic and/or molecu- procedure, before knowing the platelet
lar typing methods. Some clinicians have count, because of the risk of bleeding dur-
proposed screening pregnant women for ing the cannulation itself. When the fetus
HPA-1a antigen because it is the most com- is found to be thrombocytopenic, the
monly implicated antigen causing incom- mother is often given infusions of IGIV in
patibility in Caucasians. Because only 10% weekly doses of 1 g/kg, with or without ste-
of HPA-1a-negative women are truly at risk roids, until delivery.64-67,72 Alternatively, some
for forming antibody, and, of those, only would recommend empiric treatment with

IGIV in cases of a homozygous paternal ge- peated cordocentesis are reserved for pa-
notype for the specific platelet antigen or tients when noninvasive forms of therapy
in situations when PCR performed on are not effective. Another approach is ad-
amniotic fluid reveals that the fetus carries ministration of a single platelet transfusion
that platelet antigen. Many centers will pro- just before delivery if cordocentesis reveals
ceed with elective cesarean section instead severe thrombocytopenia. This approach is
of cordocentesis near term to determine usually reserved for pregnancies at extreme
the fetal platelet count. risk for intracranial hemorrhage.
Sources of Platelets. Maternal platelets
are often prepared for use at cordocentesis Management After Delivery
or delivery. The mother will undergo re-
Platelet counts can continue to decrease
quired testing for infectious disease mark-
after birth and should be monitored. For
ers. Prior administration of high-dose IGIV
patients at increased risk of bleeding due
may cause false-positive immunoassays;
to severe thrombocytopenia, compatible
therefore, it is desirable to test the mother
platelets should be given prophylactically.
before initiating IGIV therapy. Those with
If compatible platelets are not available,
confirmed positive results (eg, hepatitis C
the use of high-dose IGIV should be con-
virus, which is transmitted more efficiently
sidered, but response to this treatment is
by transfusion than perinatally) should not
variable. In patients who do respond, pla-
be used as a source of platelets because
telet counts usually start increasing with-
these results are more likely to represent
in 24 to 48 hours, although it may take
maternal infection. Of note, pregnancy it-
longer in some patients. 64 Because re-
self can also cause false-positive results on
sponse is slow, the neonate with an urgent
serologic infectious disease tests.
need for transfusion and no available
Platelets can be collected either from the
compatible platelets can receive platelets
mother or from another donor whose
from random donors, frequently resulting
platelets lack the corresponding antigen
in an adequate response. For patients re-
and whose plasma is compatible with the
quiring platelet transfusion, giving con-
fetal red cells. If maternal platelets are used,
current IGIV can accelerate the recovery
the antibody-containing plasma should be
of the patient’s own platelets and shorten
removed or reduced and the platelets re-
the period of transfusion dependency. If
suspended in compatible plasma or saline
the patient has mild thrombocytopenia
with reduced volume (see Method 6.15). All
without bleeding, it can be managed
components for intrauterine transfusion must
without specific therapeutic intervention.
be irradiated (see Chapter 27) and should
be CMV-reduced risk.73
Scheduling Therapy. Various strategies Thrombocytopenia Secondary to
have been used in the management of fetal Maternal ITP
thrombocytopenia. Although weekly plate- Infants born to mothers with active idio-
let transfusions have been used in the past, pathic (immune) thrombocytopenic purpura
the inherent risk of repeated cordocentesis (ITP) are often not profoundly thrombo-
makes the administration of IGIV to the cytopenic and have a smaller risk of hem-
69,74
mother the preferred treatment in the orrhage than infants with NAIT. The
United States. Practice is different in Eu- antibody in ITP is usually IgG, which
rope, where weekly platelet transfusions are readily crosses the placenta. Occasionally,
still performed. Platelet transfusion and re- delivery of a severely thrombocytopenic

infant has led to the diagnosis of previ- 3. Dennery PA, Seidman DS, Stevenson DK.
Neonatal hyperbilirubinemia. N Engl J Med
ously unsuspected ITP in a moderately af-
2001;344:581-90.
fected mother (postpartum platelet count 4. American Academy of Pediatrics Subcommit-
75,000-100,000/µL). Such cases of mild ITP tee on Hyperbilirubinemia. Management of
should be distinguished from gestational hyperbilirubinemia in the newborn infant 35
or more weeks of gestation. Clinical practice
thrombocytopenia, in which a mother guideline. Pediatrics 2004;114:297-316.
with no history of autoimmune thrombo- 5. Bowman JM. Intrauterine and neonatal
cytopenia has a platelet count less than transfusion. In: Anderson KC, Ness PM, eds.
Scientific basis of transfusion medicine: Im-
150,000/µL. In cases of maternal ITP, the plications for clinical practice. Philadelphia:
risk of severe fetal thrombocytopenia WB Saunders, 2000:307-20.
(usually defined as a platelet count less 6. Bowman JM. Treatment options for the fetus
than 50,000/µL) is 7% to 10%.74,75 The risk with alloimmune hemolytic disease. Transfus
Med Rev 1990;4:191-207.
of intracranial hemorrhage in infants 7. Bowman JM. The prevention of Rh immuni-
born to mothers with ITP is low (≤1%), zation. Transfus Med Rev 1988;2:129-50.
with only a few cases reported in the liter- 8. Bowman JM. Controversies in Rh prophy-
laxis. Who needs Rh immune globulin and
ature. This is lower than the rate in NAIT
when should it be given? Am J Obstet Gynecol
because, infants born to mothers with ITP 1985;151:289-94.
are generally born with higher platelet 9. Strauss RG, Burmeister LE, Johnson K, et al.
counts and their platelet function is not Randomized trial assessing the feasibility and
safety of biologic parents as RBC donors for
impaired, as it seems to be in NAIT. Rou- their preterm infants. Transfusion 2000;40:450-6.
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ommended and cesarean section is re- mothers may be dangerous blood donors for
74,76 their neonates. Acta Hematol 1991;85:189-91.
served for obstetric indications only.
11. Geifman-Holtzman O, Wojtowycz M, Kosmas
The antibody in ITP has broad reactivity K, Artal R. Female alloimmunization with an-
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12. Judd WJ. Practice guidelines for prenatal and
uniformly short survival of platelets from
perinatal immunohematology, revisited.
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times occur, and, in the presence of hemor- quencing: A new tool for decision making on
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Practice Bulletin Number 4. Washington, DC:
American College of Obstetricians and Gyne-
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16. Kanter MH. Derivation of new mathematic
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57. Vengelen-Tyler V, Telen MJ. Collected ques- doing it? Vox Sang 2002;83(Suppl 1):409-16.
tions and answers. 5th ed. Bethesda, MD: 72. Gaddipati S, Berkowitz RL, Lembet AA, et al.
AABB, 1997:52-3. Initial fetal platelet counts predict the re-
58. Ness PM, Baldwin ML, Niebyl JR. Clinical sponse to intravenous gammaglobulin ther-
high risk designation does not predict excess apy in fetuses that are affected by PLA1 in-
fetal maternal hemorrhage. Am J Obstet compatibility. Obstet Gynecol 2001;185:976-80.
Gynecol 1987;156:154-8. 73. Leukocyte reduction. Association Bulletin
59. Owen J, Stedman CH, Tucker TL. Comparison 99-7. Bethesda, MD: AABB, 1999.
of predelivery versus postdelivery Kleihauer- 74. Bussel JB. Immune thrombocytopenia in
Betke stains in cases of fetal death. Am J pregnancy: Autoimmune and alloimmune. J
Obstet Gynecol 1989;161:663-6. Reprod Immunol 1997;37:35-61.
60. Sebring ES. Fetomaternal hemorrhage—inci- 75. Payne SD, Resnik R, Moore TR, et al. Maternal
dence and methods of detection and quan- characteristics and risk of severe neonatal
titation. In: Garratty G, ed. Hemolytic disease thrombocytopenia and intracranial hemorrhage
of the newborn. Arlington, VA: AABB, 1984:87-117. in pregnancies complicated by autoimmune
61. Duguid JKM, Bromilow IM. Laboratory mea- thrombocytopenia. Am J Obstet Gynecol
surement of fetomaternal hemorrhage and 1997;177:149-55.
its clinical relevance. Transfus Med Rev 1999; 76. Blanchette VS, Kirby MA, Turner C. Role of in-
13:43-8. travenous immunoglobulin G in autoim-
62. Riley JZ, Ness PM, Taddie SJ, et al. The detec- mune hematologic disorders. Semin Hematol
tion and quantitation of fetal-maternal hem- 1992;29:72-82.

Chapter 24: Neonatal and Pediatric Transfusion Practice
Chapter 24
Neonatal and Pediatric

M
ANY PHYSIOLOGIC CHANGES patients, whose small blood volumes and
accompany the transitions from impaired or immature organ functions pro-
fetus to neonate, neonate to vide little margin for safety. Ill neonates are
infant, and throughout childhood. Hema- more likely than hospitalized patients of
tologic values, blood volume, and physio- any other age group to receive red cell
logic responses to stresses such as hypo- transfusions.1 Advances in critical-care neo-
volemia and hypoxia vary widely. The natology, such as surfactant therapy, nitric
most rapid changes occur during early in- oxide therapy, use of high-frequency venti-
fancy. Consequently, discussions of pedi- lators, and adherence to transfusion prac-
atric transfusion are usually divided into tice guidelines, have diminished the num-
two periods: neonates from birth through ber of blood transfusions given; most are
4 months, and older infants (>4 months) now given to infants with birthweights less
and children. Some concerns in neonatal than 1000 g.1 The doses of various compo-
transfusion practice overlap with those of nents used for simple, small-volume trans-
the perinatal period and are discussed in fusions are given in Table 24-1.2
Chapter 23.
Advances in medical care now permit
the survival of extremely premature neo-
nates. Blood providers must be capable of
Fetal and Neonatal
furnishing blood components that are tai- Erythropoiesis
lored to satisfy the specific needs of very The predominant sites of hematopoiesis 24
low birthweight (VLBW <1500 g) and ex- in the developing embryo shift from the
tremely low birthweight (ELBW <1000 g) wall of the yolk sac to the liver to the mar-
557

Table 24-1. Volumes for Simple, Small-Volume Transfusions of Neonates

Component Volume Estimated Change
RBC 10-15 mL/kg Hemoglobin ↑ 2-3 g/dL

Platelet 5-10 mL/kg Platelet ↑ 50,000-100,000/µL
Granulocyte ≥1 × 10 neutrophils/kg in
9
Repeat until clinical response
volume of 10-15 mL/kg
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)
2
Adapted with permission from Roseff.
3
row in the first 24 weeks. Hematopoiesis tent of 2,3-diphosphoglycerate (2,3-DPG)
is regulated by gradually increasing eryth- and hemoglobin A, which enhance the re-
ropoietin (EPO) levels stimulated by low lease of oxygen to the tissues. As tissue oxy-
oxygen tensions during intrauterine life. genation improves, levels of EPO decline
Fetal red cells, rich in hemoglobin F, are and erythropoiesis diminishes. This, along
well adapted to low intrauterine oxygen with decreased survival of fetal red cells
tensions. The high oxygen affinity of fetal and expansion of the blood volume due to
hemoglobin enhances transfer of oxygen rapid growth, causes the hemoglobin con-
from maternal erythrocytes to fetal eryth- centration to decline. The rate of decline is
rocytes throughout pregnancy. dependent on gestational age at birth; he-
The “switch” from fetal to adult hemo- moglobin may drop to as low as 8.0 g/dL at
globin begins at about 32 weeks’ gestation; 4 to 8 weeks of age in preterm infants with
at birth, 60% to 80% of the total hemoglo- birthweights of 1000 to 1500 g, and 7.0 g/dL
bin is hemoglobin F. Preterm neonates, in neonates with birthweights less than
therefore, are born with higher levels of fe- 1000 g.3
tal hemoglobin than those born at term. Despite hemoglobin levels that would
The mean cord hemoglobin of healthy term indicate anemia in older children and
neonates is 16.9 ± 1.6 g/dL, and that of adults, the normally developing infant usu-
preterm neonates is 15.9 ± 2.4 g/dL.4 He- ally maintains adequate tissue oxygenation.
moglobin concentration gradually falls in Physiologic anemia requires treatment only
the first few weeks of life. This has been if the degree or timing of the anemia causes
called “physiologic anemia of infancy” in symptoms in the patient.
term newborns and “physiologic anemia of
prematurity” in preterm newborns. The
anemia is considered physiologic because
it is self-limited, is usually well tolerated, Unique Aspects of Neonatal
and is not associated with any deleterious
effects to the infant. Erythropoietic activity
Physiology
diminishes secondary to an increase in pul- Infant Size and Blood Volume
monary blood flow and a rise in arterial Full-term newborns have a blood volume
pO2, as well as the increase in red cell con- of approximately 85 mL/kg; preterm low

Chapter 24: Neonatal and Pediatric Transfusion Practice 559
birthweight newborns have an average lated based on the time of conception, not
blood volume of 100 mL/kg. As survival birth, and possibly not beginning until
rates continue to improve for infants term. The most premature infants produce
weighing 1000 g or less at birth, blood the least amount of EPO for any degree of
banks are being asked to provide blood anemia; this may reflect the absence of the
components for patients whose total developmental shift of erythropoietin pro-
blood volume is less than 100 mL on a duction from the liver to the kidneys.6 Sick
more frequent basis. The need for fre- preterm neonates who receive many trans-
quent laboratory tests has made replace- fusions shortly after birth have reduced cir-
ment of iatrogenic blood loss the most culating levels of fetal hemoglobin. Circu-
common indication for transfusion of low lating EPO levels are lower, for a given
birthweight preterm neonates. However, hematocrit, in preterm neonates with
the previous practice of replacing blood higher proportions of hemoglobin A rela-
mL for mL is giving way to replacement as tive to hemoglobin F, which favors release
6
needed in order to maintain a target hema- of oxygen to the tissues. Erythroid progeni-
tocrit in certain clinical situations.1 tor cells in the hypoproliferative marrow of
Newborns do not compensate for hypo- these preterm infants show normal intrin-
volemia as well as adults. After 10% volume sic sensitivity to EPO. Clinical trials of re-
depletion in a newborn, left ventricular combinant human erythropoietin (rHuEPO)
stroke volume is diminished without in- in premature neonates show that the num-
creasing heart rate. To maintain systemic ber of transfusions and severity of anemia
7
blood pressure, peripheral vascular resis- can be lessened. Adverse effects of rHuEPO
tance increases, and this, combined with a in this age group are also different from
decreased cardiac output, results in poor those seen in older children and adults and
tissue perfusion, low tissue oxygenation, include transient, reversible neutropenia.
and metabolic acidosis.5 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-
Erythropoietin Response
cent multicenter study in Europe showed
Erythropoietin response in newborns dif- decreased need for transfusion when ad-
fers from that in adults and older chil- ministering EPO to ELBW infants within 3
dren. In older children and adults, oxygen to 5 days of life (early EPO administration)
sensors in the kidney recognize dimin- and continuing for 9 weeks.8 The mean
ished oxygen delivery and release EPO number of transfusions dropped from 2.66
into the circulation. In the fetus, the oxy- to 1.86, with a reduction in donor expo-
gen sensor that stimulates EPO produc- sures from 2 to 1. Questions regarding opti-
tion is believed to be the liver, which ap- mal dosing, route of administration, and
pears to be programmed for the hypoxic use of supplemental iron remain to be an-
intrauterine environment. swered.1,7-10 In any event, with the tremen-
This hyporesponsiveness to hypoxia pro- dous strides made in decreasing donor ex-
tects the fetus from becoming poly- posure in transfused newborns by using
cythemic in utero. Eventually, EPO produc- restrictive transfusion practices alone, the
tion shifts from the liver to the kidneys, a role of EPO for this purpose may no longer
developmental change thought to be regu- be relevant.

Cold Stress the membrane of placental cells. IgG1, the

predominant subclass in maternal blood,
Hypothermia in the newborn causes ex-
crosses the placenta first and is transported
aggerated effects, including increased
in greatest quantity. Cord blood has higher
metabolic rate, hypoglycemia, metabolic
antibody concentrations than maternal
acidosis, and a tendency toward apneic
blood. Catabolism of IgG occurs more
episodes that may lead to hypoxia, hypo-
slowly in the fetus than in the mother, so
tension, and cardiac arrest. Blood used for
that transplacental maternal antibody is
exchange transfusion should be warmed
conserved during the neonatal period.
because blood at room temperature may
A fetus exposed to an infectious process
decrease a newborn’s core temperature by
in utero or an infant exposed shortly after
0.7 to 2.5 C. The usual method is to use an
birth may produce small amounts of IgM
inline warmer. Blood, either large or small
detectable by sensitive techniques, but un-
volumes, should not be warmed under
expected red cell alloantibodies of either
a radiant heater because of the risk of
IgG or IgM class are rarely formed during
hemolysis in an unmonitored apparatus.
the neonatal period. The mechanisms
When transfusions are given to infants
responsible for the lack of alloantibody pro-
undergoing phototherapy, the tubing
duction in the neonate are not clearly un-
should be positioned to minimize expo-
derstood and are most likely multifactorial,
sure to the phototherapy light in order to
including deficient T helper function, en-
prevent hemolysis.11
hanced T suppressor activity, and poor an-
tigen-presenting cell function.13
Immunologic Status The cellular immune response is critical
Infants have an immature and inexperi- to the occurrence of transfusion-associated
enced cellular and humoral immune system. graft-vs-host disease (TA-GVHD). In the
Antibodies present derive almost entirely newborn, TA-GVHD has been reported
from the maternal circulation. Trans- most often in the clinical setting of con-
placental transfer of immunoglobulin and firmed or suspected congenital immunode-
other proteins is independent of molecu- ficiency. It is recommended that infants
lar size; IgG (150 kD) is transferred much with suspected and/or documented T-cell
more readily than albumin (64 kD). In hu- immunodeficiency receive irradiated blood
mans, maternal IgM does not reach the components. The majority of TA-GVHD cases
fetus and IgA is not readily transferred, al- reported in nonimmunocompromised hosts
though low levels have been found in the have occurred in infants who received intra-
newborn. uterine transfusion followed by postnatal
All four subclasses of IgG are transported exchange transfusion.14 A proposed expla-
across the placenta, but the rate varies be- nation is that lymphocytes given during
tween individual mother-fetus pairs. Early intrauterine transfusion could induce host
in pregnancy (approximately 12 weeks), IgG tolerance, impairing rejection of lymphocytes
probably passes from mother to fetus by given in the subsequent exchange transfu-
diffusion, and concentration in fetal serum sions. There have also been rare cases of
is low for all subgroups.12 Between 20 and TA-GVHD reported in association with ex-
33 weeks of gestation, fetal IgG levels rise treme prematurity, neonatal alloimmune
markedly, apparently because of matura- thrombocytopenia, and the use of extracor-
tion of a selective transport system that in- poreal membrane oxygenation (ECMO).14,15
volves, in part, specific protein receptors on Neonates present with TA-GVHD after a

longer latent period than adults, with fever days in extended storage medium would
occurring at an average of 28 days after deliver 2 mL of plasma containing only
exposure, rather than 10 days for immuno- 0.1 mmol/L of potassium. This is much
competent adults. There may be several less than the daily potassium requirement
risk factors other than the immune status of of 2 to 3 mmol/L for a 1-kg patient.17 How-
the recipient that predispose to TA-GVHD, ever, serum potassium may, rise rapidly
such as the number and viability of lym- after infusion of large volumes of red cells
phocytes in the transfused component and in such circumstances as surgery, ex-
donor-recipient HLA compatibility. The change transfusion, or extracorporeal cir-
true incidence of TA-GVHD in the neonatal culation, depending upon the plasma
setting is not known. However, on the basis potassium levels in the blood and manip-
of data from Japan, it appears that the inci- ulation of the blood component. 18,19 Of
dence of reported TA-GVHD is far lower interest, a unit of Red Blood Cells (RBCs)
than would be expected.15 The postulated preserved in AS-1 will deliver less extra-
mechanism for this apparent decreased cellular potassium compared to the
susceptibility of newborns to TA-GVHD is amount in RBCs stored in CPDA-1.16,20 In
thought to be extrathymic and/or thymic stored irradiated blood, the problem of
semitolerance of allogeneic donor T lym- potassium leak is potentiated; for selected
phocytes. As for all patients, directed-donor patients, it may be desirable to wash irra-
units from biologic relatives must be irradi- diated cells, if they have subsequently
ated.15,16 There are no data to support the been stored >24 hours. 19 There are in-
practice of universal irradiation of blood creasing anecdotal reports of infants who
transfused to all infants or children. receive either older RBC units or units ir-
radiated more than 1 day before transfu-
sion having severe adverse effects (eg,
Metabolic Problems cardiac arrest, death) after transfusion of
Acidosis or hypocalcemia may occur after these products into central lines or intra-
large-volume whole blood or plasma cardiac lines.21,22
transfusion because the immature liver of The untoward consequences of washing
the newborn metabolizes citrate ineffi- blood, such as reducing its shelf life and the
ciently. Immature kidneys have reduced possible introduction of bacteria, must be
glomerular filtration rate and concentrat- considered. It is preferable to perform irra-
ing ability, and newborns may have diffi- diation as close to the time of administra-
culty excreting excess potassium, acid, tion as possible, obviating the concern for
and/or calcium. high levels of potassium in the transfused
product.
Potassium
Although potassium levels increase rapidly 2,3-Diphosphoglycerate
in the plasma of stored red cells, small- Neonates with respiratory distress syn-
volume, simple transfusions administered drome or septic shock have decreased
slowly have little effect on serum potas- levels of intracellular red cell 2,3-DPG.
sium concentration in newborns. It has Alkalosis and hypothermia may further
been calculated that transfusion of 10 increase the oxygen affinity of hemoglo-
mL/kg of red cells (hematocrit 80%) ob- bin, shifting the dissociation curve to the
tained from a unit of blood stored for 42 left and making oxygen even less available

to the tissues. Arterial oxygenation may children born to seropositive moth-

be further compromised by respiratory ers.25
distress syndrome or other pulmonary 3. The risk of symptomatic posttrans-
disease. Mechanisms that compensate for fusion infection is high in multi-
hypoxia in adults, such as increased heart transfused preterm infants weighing
rate, are limited in newborns. If a large less than 1200 g who are born to
proportion of an infant’s blood volume has seronegative mothers.17,26
come from transfusion of 2,3-DPG-de- 4. The risk of acquiring CMV infection
pleted blood, this may cause problems is directly proportional to the cumu-
that would not affect older children or lative number of donor exposures
adults. Because 2,3-DPG levels decline incurred via transfusion.
rapidly after the first week of storage, the 5. Cytomegalovirus in blood is associ-
freshest blood conveniently available (up ated with leukocytes. The risk of vi-
to 14 days) should be used for exchange rus transmission can be reduced by
transfusion in newborns. For small-volume transfusing CMV-reduced-risk blood
transfusions, the medical necessity for from seronegative donors or by us-
fresh blood has never been demonstrated ing leukocyte-reduced components.
and arguments have been raised to sug- Although deglycerolized and washed
gest it is unnecessary.1,16,19 red cells also have a reduced risk of
CMV infection, leukocyte reduction
by filtration is the technique of
choice.16,27-30
Cytomegalovirus Infection
Perinatal infection with cytomegalovirus
(CMV) may occur, acquired either in utero
or during the birth process. Neonates can
Red Cell Transfusions in
be infected during breast-feeding or by Infants Less than 4 Months
close contact with mothers or nursery of Age
personnel. CMV can also be transmitted
by transfusion, although the risk from the RBCs are the component most often
current blood supply is small.23,24 transfused during the neonatal period.
CMV infection in newborns has ex- Many of the physiologic considerations
tremely variable manifestations, ranging mentioned above directly affect decisions
from asymptomatic seroconversion to death. regarding indications for transfusion, se-
Studies of CMV in neonatal transfusion re- lection, and administration of red cell
cipients reveal the following observations: components, as well as the requirements
1. The overall risk of symptomatic post- for compatibility testing.
transfusion CMV infection seems to
be inversely related to the seropo- Compatibility Testing
sitivity rate in the community. Al- Because the neonate and young infant are
though many adults are positive for immunologically immature, alloimmuni-
CMV antibodies, the rate of symp- zation to red cell antigens is rare during
tomatic CMV infection in newborns the neonatal period. A study of 90 neo-
is low. nates who received 1269 transfusions from
2. Symptomatic CMV infection during different donors found no instances of
the neonatal period is uncommon in antibody production even with use of

31
very sensitive detection techniques. Other and/or provision of blood lacking the target
investigators confirm the relative infre- antigen are unnecessary. It is important to
quency of alloantibodies directed against avoid transfusion of any component that
13,28
red cell, as well as HLA, antigens. may transfer unexpected antibody or ABO-
Because alloimmunization is extremely incompatible antibodies to the infant.
rare and repeated testing increases iatro-
genic blood loss, AABB Standards for Blood
Indications for Red Cell Transfusion
Banks and Transfusion Services32(p42) requires
only limited pretransfusion serologic test- Certain events in the perinatal period cause
ing for infants under 4 months old. Initial anemia, for which the benefits of red cell
testing must include ABO and D typing of transfusion are unquestioned. These in-
red cells and a screen for red cell antibod- clude spontaneous fetomaternal or feto-
ies, using either serum or plasma, from the placental hemorrhage, twin-twin transfu-
mother or the infant. sion, obstetric accidents, and internal
During any one hospitalization, compat- hemorrhage. A venous hemoglobin of less
ibility testing and repeat ABO and D typing than 13 g/dL in the first 24 hours of life in-
may be omitted, provided that the screen dicates significant anemia.33 For severely
for red cell antibodies is negative; that all anemic neonates with congestive heart
red cells transfused are group O or ABO- failure, it may be necessary to remove ali-
identical or ABO-compatible; and that red quots of their dilute blood and transfuse
cells are either D negative or the same D concentrated red cells. This “partial ex-
type as the patient. It is unnecessary to test change” transfusion will prevent intravas-
the infant’s serum for anti-A and/or anti-B cular volume overload. Most red cell
as a component of blood typing. Before giv- transfusions in the neonatal period, how-
ing non-group-O red cells, the neonate’s se- ever, are given either to replace iatrogenic
rum must be checked for passively ac- blood loss or to treat the physiologic de-
quired maternal anti-A or anti-B and must cline in hemoglobin (anemia of pre-
include the antiglobulin phase. If the anti- maturity) when it complicates clinical
body is present, ABO-compatible red cells problems.
lacking the corresponding A or B antigen Because tissue demand for oxygen can-
must be used until the antibody is no lon- not be measured directly and because so
ger detected. In this setting, it is not neces- many variables determine oxygen availabil-
sary to perform crossmatches. If an unex- ity, no universally accepted criteria exist for
pected non-ABO red cell antibody is transfusion of preterm or term neonates.
detected in the infant’s specimen or the Despite the widespread use of micro-
mother’s serum contains a clinically signifi- methods for laboratory tests and growing
cant red cell antibody, the infant should be use of bedside or noninvasive monitoring
given either RBC units tested and found to devices, infants still sustain significant cu-
lack the corresponding antigen(s) or units mulative blood loss from laboratory sam-
compatible by antiglobulin crossmatch. pling. In a sick neonate, red cell replacement
This practice should continue for as long as is usually considered when approximately
maternal antibody persists in the infant’s 10% of the blood volume has been re-
blood. The institution’s policy will deter- moved. The decision to transfuse a new-
mine how frequently to recheck the screen born for anemia should include evaluation
for red cell antibodies; once a negative re- of the hemoglobin levels expected for the
sult is obtained, subsequent crossmatches patient’s age and clinical status, as well as

the amount of blood loss over time. Trans- transfusions an infant can receive from
fusion may be more aggressive in the infant one donor. Because the original seal re-
in respiratory distress who is hypoxic and mains intact, each container has the expi-
more vulnerable to cerebral hemorrhage. ration date of the original unit. With use
Considerable controversy surrounds the of a sterile connecting device, multiple
correlation of the “signs of anemia” in the bags called pedi-packs or specially de-
preterm infant (tachycardia, tachypnea, signed syringe systems can be integrally
bradycardia, recurrent apnea, and poor attached to a unit of RBCs after compo-
weight gain) with response to red cell trans- nent preparation. This maintains a closed
fusions.1 When red cells are transfused, they system and further increases the number
are usually given in small volumes of 10 to of small-volume transfusions obtained
15 mL/kg (or less if the infant cannot toler- from a single donor. Sterile connecting
ate this volume). The hematocrit of the red devices can be used to prepare small
cell component transfused will depend on aliquots for transfusion in the blood bank.
the anticoagulant/preservative used and If aliquots are prepared by entering the
how the original unit is processed to pro- bag through a port, the unit and the
vide small component transfusions for neo- aliquot are assigned a 24-hour shelf life, if
nates. A transfusion of 10 mL/kg of red cells refrigerated.
adjusted to a hematocrit greater than 80% Each aliquot must be fully labeled as it is
just before release for transfusion should prepared, including the time it outdates.
raise the hemoglobin concentration by ap- The origin and disposition of each aliquot
proximately 3 g/dL. A transfusion of 10 must be recorded. Using these techniques,
mL/kg of red cells in additive solution, a recipient can receive multiple small-vol-
which have a hematocrit of approximately ume transfusions from a single donation
65%, will result in a posttransfusion hemo- until the expiration of the original unit,
globin increment less than 3 g/dL. thereby reducing donor exposure.34,36 The
disadvantage of any method that creates
Red Cell Components Used for Neonatal aliquots from a single donation is that any
Transfusion undetected, transmissible pathogen that
The small-volume requirements of trans- might be present in the primary unit can be
fusion to neonatal recipients make it pos- disseminated to multiple recipients.
sible to prepare several aliquots from a In order to prevent waste, many transfu-
single donor unit, thus limiting donor ex- sion services assign a unit of RBCs to one or
posure and decreasing donor-related risks. more infants based on their weight. As an
Several technical approaches are avail- example, 1 or 2 lower birthweight infants
able to realize this advantage and to mini- may be assigned to one unit because they
mize wastage.34 will most likely require the greatest number
of transfusions. On the other hand, four
larger infants may be assigned to one unit
Aliquoting for Small-Volume Transfusion
because their transfusion needs will not be
A multiple-pack system is a common tech- as great.37,38
nique for providing small-volume red cell
transfusions.34,35 Quad packs, where a sin-
gle unit of Whole Blood is collected into a Red Cells with Additive Solution
bag with four integrally attached contain- RBCs used for pediatric transfusions were
ers, can be used to increase the number of traditionally stored in CPDA-1.35 Additive

solutions (AS) used as anticoagulants/ fusion, has not been studied. Concern still
preservatives contain additional adenine remains regarding the use of blood stored
and dextrose and some contain mannitol. in additive solutions being used outside the
There has been concern about the poten- setting of simple, small-volume transfu-
tial side effects of these additives because sion.40,41 However, with extensive anecdotal
large amounts of adenine and mannitol use in large transfusion services, there have
have been associated with renal toxicity. not been reports of deleterious effects from
Mannitol is also a potent diuretic and, be- the infusion of additive solutions.42,43
cause of its effect on the fluid dynamics in Whether or not placental/umbilical cord
preterm infants, may cause unacceptable blood will become an acceptable form of
fluctuations in cerebral blood flow. The neonatal transfusion remains to be seen.
different constituents of these solutions Although use of this type of “autologous”
are found in Chapter 8. However, when blood would eliminate some infectious dis-
the dose of transfused red cells is small (5 ease risks, questions regarding quantity,
to 15 mL/kg), the recipient is exposed to quality, and sterility raise concerns about
relatively small amounts of the preserva- its safety and efficacy.18
tive solutions. Clinical studies comparing
red cells stored in AS-1 and AS-3 solutions
Transfusion Administration
have shown no apparent detrimental ef-
fects in neonates receiving simple trans- Vascular access is often difficult in the tiny
fusions, and, after adjustment for the newborn and in any infant requiring
lower hematocrit of the component, they long-term or repeated intravenous infu-
are as effective as CPDA-1 cells in increas- sions. Within a short time after birth, the
ing hemoglobin. Furthermore, the addi- umbilical artery may be cannulated. Trans-
tional sugars present have been shown to fusion through a needle as small as 25-
benefit glucose homeostasis in compari- gauge or a vascular catheter as small as
son with CPDA-1.39 Studies on the safety 24-gauge has been shown to cause little
of AS-3-preserved red cells in neonates have hemolysis and to be safe when constant
been published.38,40 Because the compo- flow rates are used. Transfusion through
nents of AS-5 are the same as those found smaller gauge catheters has not been
in the other additive solutions, it is con- thoroughly evaluated.
sidered acceptable for use in neonates. It is not usually necessary to warm small-
Using theoretical calculations in a vari- volume transfusions that are given slowly,
41
ety of clinical situations, Luban et al dem- but it is important to be able to control the
onstrated that red cells preserved in volume and rate of infusion. Constant-rate
extended-storage media present no sub- electromechanical syringe delivery pumps
stantive risks when used for small-volume provide this control and cause minimal
transfusions. For preterm infants with se- hemolysis, even when used with inline leu-
vere hepatic or renal insufficiency, however, kocyte reduction filters.44,45
removing the additive-containing plasma The length of the plastic tubing used can
may be beneficial. This is particularly im- add significantly to the volume required for
portant if there will be multiple transfu- transfusion. Infusion sets identified as suit-
sions that could have a cumulative effect. able for platelets or components have less
The safety of red cells stored in additive so- dead space than standard sets because they
lutions and used for massive transfusions, have short tubing and a small 170-micron
such as cardiac surgery or exchange trans- filter. Pediatric microaggregate filters (20-

or 40-micron) are often used for their small therapy with fluorescent blue lights is the
priming volume, not for the removal of most common treatment for hyperbiliru-
microaggregates. Hemolysis may occur binemia; exchange transfusion is reserved
when stored blood is given by negative for phototherapy failures. The most com-
pressure filtration through these filters.46 mon reason, however, for an exchange to
Administration rates for RBCs have not be performed in a neonate is to correct
been extensively studied, nor are there hyperbilirubinemia.
standard practices; rates of transfusion as Pathologic processes that may result in
well as devices used vary with institutions. excessively high unconjugated bilirubin
The rate of administration of blood and levels in neonates include immune-medi-
blood components in neonates and infants ated hemolysis, nonimmune hemolysis,
should be individualized on the basis of the bile excretion defects and impaired albu-
patient’s clinical needs. Theoretical con- min binding. Exchange transfusion re-
cerns regarding rapid changes in intra- moves unconjugated bilirubin and provides
vascular volume and electrolyte changes in additional albumin to bind residual biliru-
these small, labile patients have focused on bin. If hyperbilirubinemia is due to anti-
an increased risk for intracranial hemor- body-mediated hemolysis, exchange trans-
rhage. This has not been clearly demon- fusion is of additional benefit by removing
strated. For simple RBC transfusions, trans- free antibody and antibody-coated red cells
fusing products over 2 to 4 hours is usually while providing antigen-negative red cells
adequate. When there is an urgent need be- that will survive normally.
cause of shock or severe bleeding, infusion Exchange transfusion should be per-
should be as rapid as possible. Products formed before bilirubin rises to levels at
can be transfused safely using a variety of which CNS damage occurs. Several factors
devices. It is important that mechanical affect the threshold for toxicity. CNS dam-
systems be tested and validated for use age occurs at lower levels if there is pre-
with blood and blood components. maturity, decreased albumin binding capac-
ity, or the presence of such complicating
conditions as sepsis, hypoxia, acidosis, hy-
Exchange Transfusion for
pothermia, or hypoglycemia. In full-term
Hyperbilirubinemia
infants, kernicterus rarely develops at indi-
The fetal liver has limited capacity to con- rect bilirubin levels less than 25 mg/dL, but,
jugate bilirubin. In utero, unconjugated in sick VLBW infants, kernicterus has oc-
47
bilirubin crosses the placenta for excre- curred at levels as low as 8 to 12 mg/dL.
tion through the mother’s hepatobiliary The rate at which bilirubin rises is more
system. After birth, transient mild hyper- predictive of imminent need for exchange
bilirubinemia normally occurs during the transfusion than the absolute level attained.
first week of life and is referred to as “phy- Neonates with severe anemia and a rapid
siologic jaundice.” Liver function is less ma- rise in bilibrubin, despite phototherapy, re-
ture, and jaundice worsens in premature quire exchange transfusion. A two-volume
neonates. When the level of unconjugated exchange transfusion removes approxi-
bilirubin is excessive, bilirubin may cross mately 70% to 90% of circulating erythro-
the blood-brain barrier and concentrate cytes and about 25% of the total bilirubin.
in the basal ganglia and cerebellum; the Because of reequilibration between the
resulting damage to the central nervous extravascular tissue and plasma bilirubin,
system (CNS) is called kernicterus. Photo- levels may again rise, resulting in the need

48
for a second exchange transfusion. Indica- used, depending on the clinical situation,
tions for repeat exchange are similar to some institutions would choose to remove
those for the initial exchange. the additive-containing plasma, to reduce
The American Academy of Pediatrics re- the volume transfused. As discussed ear-
cently released new guidelines for the man- lier, washing components may not be
agement of newborn infants born ≥35 necessary or desirable. Many transfusion
weeks of gestation with hyperbilirubine- services use red cells that have been
mia. It is hoped that raising awareness screened and found to lack hemoglobin S
about the potential for hyperbilirubinemia for exchange transfusion, to avoid the
in this patient group will reduce its fre- possibility of intravascular sickling.
quency and provide a framework for opti- The glucose load administered during
mal treatment, to include the use of photo- exchange transfusion can be extremely
therapy, exchange transfusion, and Immune high. This stimulates the infant to secrete
49
Globulin, Intravenous (IGIV). insulin, which may lead to rebound hypo-
glycemia. It is important to monitor blood
Exchange Transfusion for Other Causes glucose levels for the first few hours after
The safety and efficacy of exchange trans- the procedure.
fusion in the neonatal period for other in- Because unconjugated bilirubin binds to
dications should be evaluated on a case- albumin, albumin is frequently used to in-
by-case basis, using guidelines in pub- crease intravascular binding. With addi-
lished literature. Treatment categories for tional albumin in the circulation, bilirubin
disorders based on proven vs theoretical from the extravascular space diffuses out to
benefits have been recommended (see the intravascular space. This, in turn, in-
Table 6-1). The treatment of disseminated creases the total quantity of bilirubin re-
intravascular coagulation (DIC) using ex- moved during the exchange. There have
change transfusion has yielded variable been conflicting results, however, about the
results, perhaps because only the sickest efficacy of administering albumin either
infants have been selected to receive this before or during exchange to enhance bili-
therapy. The most important aspect of rubin removal. A study that compared 15
therapy for neonatal DIC is to treat the hyperbilirubinemic neonates given albu-
underlying disease. min with 27 who received none found simi-
Exchange transfusion is occasionally lar efficiency of bilirubin removal in both
used to remove other toxins, such as drugs groups.52 Infusing albumin raises the colloid
or chemicals given to the mother near the osmotic pressure and increases intravas-
time of delivery, drugs given in toxic doses cular volume. Therefore, it should be given
to the neonate/infant, or substances such cautiously, if at all, to neonates or infants
as ammonia that accumulate in the new- who are severely anemic, have increased
born because of prematurity or inherited central venous pressure, or are in renal or
metabolic diseases.50,51 congestive heart failure.
Exchange transfusion may cause dilu-
Technique of Exchange Transfusion tional thrombocytopenia and/or coagulo-
pathy that require transfusion of platelets
Choice of Components and/or other components containing coag-
Red cells are resuspended in compatible ulation factors. Platelet counts and coagu-
thawed Fresh Frozen Plasma (FFP) for ex- lation parameters should be monitored af-
change transfusion. If AS-RBC units are ter exchange transfusion.

Volume and Hematocrit drawal and the umbilical vein for infu-
sion.
An exchange transfusion equal to twice the
The manual push-pull technique can be
patient’s blood volume is typically recom-
accomplished through a single vascular ac-
mended for newborns; rarely is more than
cess. A three-way stopcock joins the unit of
one full unit of donor blood required. In
blood, the patient, and an extension tube
practice, the volume calculated for ex-
that leads to the graduated discard con-
change is an estimate. The final hemato-
tainer. An inline blood warmer and a stan-
crit of transfused blood should be approx-
dard blood filter should be incorporated in
imately 40% to 50%, with sufficient plasma
the administration set. The maximum vol-
to provide clotting factors, if needed. In
ume of each withdrawal and infusion will
the unusual event that the infant’s condi-
depend on the infant’s size and hemo-
tion demands a high postexchange hemato-
dynamic status. The rate at which exchange
crit, a small-volume transfusion of red cells
transfusion occurs may alter the infant’s
can be given after the exchange, or units
hemodynamic status. It is important to
with a higher hematocrit used for ex-
maintain careful records during an ex-
change. It is important to keep the blood
change transfusion. The procedure should
mixed during the exchange; if it settles in
take place over 1 to 1.5 hours.
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.
Transfusion of Other
Components
Vascular Access Although the percentage of VLBW and
Exchange transfusions in the newborn ELBW infants being transfused has de-
period are usually accomplished via cath- creased significantly since the 1980s, be-
eters in the umbilical vessels. Catheteriza- tween 61% and 94% of these neonatal pa-
tion is easiest within hours of birth, but it tients can be expected to receive multiple
may be possible to achieve vascular ac- red cell transfusions. The smallest pa-
cess at this site for several days. The cath- tients will receive the greatest number of
eters should be radio-opaque to facilitate transfusions. It is estimated that a much
radiographic monitoring during and after lower percentage of infants receive other
placement. If umbilical catheters are not components.1,9,53,54
available for exchange transfusion, small
central venous or saphenous catheters Platelet Transfusion
may be used. The normal platelet count of newborns is
similar to that of adults. A platelet count
Methods Used less than 150,000/µL in a full-term or pre-
Two methods of exchange transfusion are mature infant is abnormal. Approximately
in common use. In the isovolumetric 20% of infants in neonatal intensive care
method, there is vascular access through units have mild-to-moderate thrombo-
two catheters of identical size. Withdrawal cytopenia, which is the most common
and infusion occur simultaneously, regu- hemostatic abnormality in the sick in-
55
lated by a single peristaltic pump. The fant. Neonatal thrombocytopenia may
umbilical artery is usually used for with- result from impaired production or in-

creased destruction of platelets, abnormal dict hemostatic efficacy. Repeated platelet

distribution, or a dilutional effect second- transfusions, without an appropriate rise,
ary to massive transfusion such as ex- may not be beneficial.
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 Platelet Components
thrombocytopenia is discussed in Chap-
A platelet dose of 5 to 10 mL/kg body
ter 23.
weight should raise the platelet count of
an average full-term newborn by 50,000
to 100,000/µL, depending on the platelet
Indications concentration in the component used.17,57
Platelet transfusion is indicated in neo- The platelet component should be group
nates and young infants with platelet specific, if possible, and should not con-
counts below 50,000/µL who are experi- tain clinically significant unexpected red
55
encing bleeding. The use of prophylactic cell antibodies. Transfusion of ABO-in-
platelet transfusions in the newborn re- compatible plasma is more dangerous in
mains controversial. In thrombocyto- infants than in adults because of their
penic adults, the risk of severe bleeding is very small blood volumes. If it is neces-
rare unless the platelet count is less than sary to give a platelet unit that contains
56
10,000/µL. Conversely, preterm neonates incompatible plasma (due to antibodies
and infants with other complicating ill- in the ABO or other blood groups), plasma
nesses may bleed at higher platelet counts. can be removed (see Method 6.15) and
Factors contributing to this increased risk the platelets resuspended in saline. FFP
of bleeding include a quantitatively lower can be substituted as the resuspending
concentration of plasma coagulation fac- medium if the patient also requires clot-
tors; circulation of an anticoagulant that ting factors, but it carries the risk of infec-
enhances inhibition of thrombin; intrin- tious disease transmission. Routine cen-
sic or extrinsic platelet dysfunction; and trifugation of platelets to reduce the
57
increased vascular fragility. Of major volume of transfusion is not necessary.53,55
concern is intraventricular hemorrhage, If platelets have been volume reduced
which occurs in up to 40% of preterm ne- and placed in a syringe, the pH declines
rapidly, a potential problem for an already
onates in the first 72 hours. Although pro-
ill, acidotic patient.58 Therefore, if there is
phylactic platelet transfusions increase
a need to reduce the volume of platelets,
platelet counts and shorten the bleeding
it should be done just before transfusion
time in these infants, the incidence or ex-
and the component must be infused
tent of intraventricular hemorrhage is not
57 within 4 hours, if done in an open system.
reduced. Because of the apparent lack of
clinical benefit, controversy exists about
the use of platelet transfusion in this set- Granulocyte Transfusion
ting, as well as the selection of an optimal Neonates are more susceptible to severe
dose. After a platelet transfusion, a post- bacterial infection than older children be-
transfusion platelet count soon after cause of both the quantitative and quali-
transfusion can be used to evaluate sur- tative defects of neutrophil (polymorpho-
vival in the circulation but may not pre- nuclear cell or PMN) function and, in the

absence of pathogen-specific maternal granulocyte transfusion may be consid-

antibody, to deficiency of humoral immu- ered as an adjunct to antibiotic therapy.
nity. Group B streptococcus is the most Candidates for possible granulocyte
frequent cause of early-onset neonatal transfusion are infants with strong evi-
sepsis, and, despite improvement in dence of bacterial septicemia, an absolute
antimicrobial therapy and intensive care, neutrophil count below 3000/µL, and a
it is still associated with a high mortality diminished marrow storage pool, such
rate. Controversy surrounds several issues that less than 7% of their nucleated cells
in granulocyte transfusions for neonates, in the marrow are granulocytes at the
including dose, neutrophil level at which stage of metamyelocytes or more mature
to transfuse, and efficacy as compared to forms.18,69
59
other forms of therapy. Because it ap-
pears that efficacy is related to dose, the Granulocyte Components
smaller blood volume of infants and
Granulocytes are harvested by standard
young children may result in these pa-
apheresis techniques. For infants, a dose
tients having a better response to this
of 10 to 15 mL/kg is recommended, which
form of therapy. A meta-analysis pub-
is about 1 × 109 to 2 × 109 PMN/kg.17,60 Be-
lished in 1996 concluded that granulocyte
cause neonatal neutrophil function is of-
doses greater than 1 × 10 PMN/kg re-
9
60 ten abnormal, the use of granulocyte

sulted in a better clinical response.
transfusions in this age group may be
Granulocyte concentrates prepared by
beneficial.17,60,67,70-72 Administration should
apheresis are more desirable than those
be continued daily until an adequate
prepared from buffy coats because they
17,60
white cell mass is achieved or the patient
produce a higher yield. Products col-
has clinically improved. Based on the fact
lected from donors on a regimen of
that granulocyte concentrates contain
granulocyte colony-stimulating factor
large numbers of lymphocytes, there is
(G-CSF) and steroid mobilization can
general consensus that all granulocyte
yield higher numbers of granulocytes
transfusions should be irradiated to pre-
than those collected from an unstimulat-
vent graft-vs-host disease. For granulo-
ed donor. There have been some encour- cyte transfusions to neonates, donors are
aging observations on the use of IGIV in usually selected to be CMV seronegative
the treatment of early neonatal sepsis, al- and must be ABO compatible with the in-
though conflicting data result in a lack of fant, in accordance with Standards, be-
59,61-64
consensus on its use. Preliminary stud- cause there is a significant volume of red
ies of hematopoietic growth factors (eg, cells in these products.32(p40) Many institu-
G-CSF) show promise in the treatment of tions also provide products that are
overwhelming bacterial infection in the D-compatible (see Chapter 21).
59,61,65,66
newborn. For adults and larger chil-
dren, a minimum dose of 1 × 10 PMN/kg
10
60,67,68 Transfusion to Enhance Hemostasis

is recommended.
The elements of the hemostatic system of
the newborn are similar to those of older
Indications children and adults, but the concentration
Although the precise role of granulocyte of many plasma proteins is decreased.
transfusion for neonatal sepsis is unclear, Coagulation factors do not cross the pla-
certain clinical situations exist in which centa, but are independently synthesized

by the fetus. Plasma levels of coagulation rience complications in the absence of an-
proteins increase progressively with ges- other pathologic insult. However, the ho-
tational age. At birth, the infant’s pro- mozygous form of protein C deficiency has
thrombin time and partial thrombo- caused life-threatening thrombotic compli-
plastin time are prolonged, compared to cations in the newborn period. In countries
older children and adults, primarily the where it is available, protein C concentrates
result of physiologically low levels of the prepared from human plasma should be
vitamin-K-dependent factors (II, VII, IX, used as the initial treatment for neonates
and X) and contact factors (XI, XII, pre- with homozygous protein C deficiency pre-
kallikrein, and high-molecular-weight senting with purpura fulminans. Protein C
kininogen).73 Proteins C and S and anti- concentrates may be available on a com-
thrombin inhibitors of coagulation are passionate use basis in the United States.
also at low levels. These two systems usu- Otherwise, plasma infusion is used as the
ally balance each other, so that spontane- initial treatment during the acute event,
ous bleeding and thrombosis in the healthy with subsequent anticoagulant therapy for
newborn are rare, but very little reserve long-term management.74,75
capacity exists for response to pathologic
insults. Therefore, serious bleeding may
occur in the first week of life in the sick Fresh Frozen Plasma
premature infant as a result of hemostatic Fresh frozen plasma may be used to re-
immaturity coupled with an acquired displace coagulation factors in newborns,
order of hemostasis. particularly if multiple factors are in-
In addition to having physiologically low volved, such as in vitamin K deficiency.
levels of the vitamin-K-dependent factors, The usual dose is 10 to 15 mL/kg, which
neonates may also become vitamin-K-defi- should increase factor activity by 15% to
cient during the first 2 to 5 days of life, plac- 20% unless there is marked consumptive
ing them at risk of bleeding. This “hemor- coagulopathy.74 As with red cell transfu-
rhagic disease of the newborn” is rare in sions, there are several methods to pro-
developed countries because intramuscular vide small-volume FFP infusions while
vitamin K is routinely given at birth. If vita- limiting donor exposure and wastage of
min K therapy is omitted, especially if the components. Blood can be collected into
neonate is breast fed, life-threatening hem- a system with multiple integrally attached
orrhage may occur. This should be treated bags, creating aliquots that can be pre-
with FFP.2,74 pared for freezing.34 Once thawed, these
Although hereditary deficiencies of coag- aliquots can be further divided and used
ulation factors may be apparent in the for several patients within a 24-hour pe-
newborn, significant bleeding is rare. riod. If not used within 24 hours as aliquots,
Coagulopathy more often results from an the thawed plasma (stored at 1 to 6 C) can
acquired defect such as liver disease or still be used as a means to decrease donor
DIC.74 Although component therapy re- exposure. As with all patients, newborns
placement may temporarily correct the must receive FFP that is ABO compatible
hemostatic problem, treatment of the un- and free of clinically significant unex-
derlying disease will ultimately reduce the pected antibodies. Group AB FFP is often
need to treat the acquired defect. used because a single unit provides com-
Newborns who are heterozygous for de- patible small aliquots for several neonates
ficiencies of inhibitory proteins rarely expe- requiring FFP simultaneously.

Cryoprecipitate ability to increase cardiac output to com-

pensate for hyperviscosity and may
Cryoprecipitate is rich in fibrinogen, co-
develop congestive heart failure. Impair-
agulation Factors VIII and XIII, and von
ment of blood flow can cause CNS abnor-
Willebrand factor. This component is of-
malities, pulmonary and renal failure, and
ten used in conjunction with platelet
necrotizing enterocolitis. Phlebotomy can
transfusions to treat DIC in the newborn.
be used to normalize the hematocrit to
In DIC, fibrinogen and platelets are the el-
55% to 60% and improve tissue perfusion,
ements most often severely depleted. The
while maintaining the blood volume.
plasma in which the platelets are sus-
To treat polycythemia, whole blood is re-
pended is a source of stable coagulation
moved and the volume replaced with
factors. Cryoprecipitate provides concen-
crystalloid (such as normal saline), the
trated levels of additional fibrinogen and
choice being based on the quantity needed
storage-labile Factor VIII. For an infant, one
and on the infant’s clinical condition.
bag is sufficient to achieve hemostatic
Plasma is not recommended because the
levels. As with FFP and platelets, the
volume administered will be insufficient to
cryoprecipitate should be ABO compati-
correct any coagulopathy and because
ble with the neonatal recipient. Directed
necrotizing enterocolitis has been reported
donor cryoprecipitate is not recommended
when it is used in this procedure.78 A for-
as first-line therapy in the newborn with
mula to approximate the volume of colloid
hemophilia A because safer alternatives are
replacement required (and the volume of
available. Recombinant Factor VIII prod-
blood to be drawn) for the exchange is47:
ucts or virus-inactivated, monoclonal-an-
tibody-purified, plasma-derived products
Volume of replacement fluid =
are the standard treatment.76 Cryoprecipi-
blood volume ×
tate should be used to treat von Wille-
(observed hematocrit – desired hematocrit)
brand disease only as a last resort, when
observed hematocrit
safer products are not readily available.
More information on the use of cryopreci-
pitate can be found in Chapter 21.
Extracorporeal Membrane
Oxygenation
Neonatal Polycythemia ECMO is a modified cardiopulmonary by-
A venous hematocrit greater than 65% or pass technique that has been used for
hemoglobin in excess of 22 g/dL any time short-term support for cardiac or respira-
in the first week of life defines polycythemia, tory failure. It is performed in specialized
a condition that occurs in approximately centers and only for patients in whom
5% of all newborns. Small-for-gestational- conventional medical therapy has failed
age infants and infants of diabetic moth- and anticipated survival with such ther-
ers are at increased risk for developing apy is limited (see Table 24-2). The use of
polycythemia. As the hematocrit rises ECMO in patients other than neonates is
above 65%, the viscosity of blood in- not widespread. The therapy is more suc-
creases exponentially and oxygen trans- cessful in infants, whose small blood vol-
port decreases. For neonates, the expo- ume allows for total cardiorespiratory
nential rise may occur at a hematocrit support and whose primary respiratory
closer to 40%. 77 Infants have a limited problem often resolves after 1 to 2 weeks

47,79
Table 24-2. Disorders Treated by ECMO and supplement them as needed. (See
Tables 24-3 and 24-4.)
■ Meconium aspiration
■ Diaphragmatic hernia
■ Persistent pulmonary hypertension of the
newborn Leukocyte Reduction
■ Severe group B streptococcal sepsis 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
of support. ECMO provides gas exchange
study. In addition, infants are rarely alloi-
independent of the patient’s lungs, allow-
mmunized because of the immaturity of
ing them time to improve or heal without
their immune system during this period
exposure to aggressive ventilator support
of development. The reduction of risk of
and the secondary lung damage this may
79 CMV transmission to infants is the only
cause.
benefit of leukocyte reduction that has
Individual ECMO centers establish their
been well documented, as discussed ear-
own specific criteria for transfusion and
lier.16,30,80
blood component selection, and standard
Of interest, a recent study in Canada
transfusion practices are lacking. Because
compared the clinical outcomes of prema-
of the combination of factors present (in-
ture infants weighing <1250 g before and
cluding systemic heparinization, platelet
after implementation of universal leukocyte
dysfunction, thrombocytopenia, and other
reduction. Although neither mortality nor
coagulation defects) as well as the ECMO
bacteremia were reduced in the setting of
circuitry itself, bleeding complications are
universal leukocyte reduction, other sec-
frequent. The ECMO team should be in
ondary clinical outcomes, such as retino-
close communication with the blood bank
pathy of prematurity and bronchopul-
or transfusion service staff, and there
monary dysplasia, were improved. Length
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- Table 24-3. Risks of ECMO
sions. The amount of red cell, platelet, and ■ Bleeding
FFP support required to maintain hemato- ■ Thrombosis
logic and hemodynamic equilibrium will ■ Thrombocytopenia
vary depending on the clinical situation ■ Neutropenia
and in accordance with the institutions’ ■ Platelet dysfunction
practices.68 When platelet transfusion is re- ■ Stroke
quired, some practitioners think it is im- ■ Seizure
■ Air embolism
portant to transfuse through peripheral ac-
■ Hemolysis
cess to avoid platelet damage, whereas
■ Systemic hypertension
others will transfuse directly into the ECMO
■ Cannulization of carotid artery
circuitry because all blood will eventually ■ Infectious complications of blood
flow through the equipment. It is also im- transfusion
portant to monitor ionized calcium levels

Table 24-4. Contraindications for ECMO required, many of the methods described
to provide small-volume transfusions to ne-
■ High risk for intraventricular hemorrhage onates could be applied. All pediatric pa-
■ Irreversible lung disease tients over 4 months of age, however, must
■ Systemic bleeding
be tested for ABO and D type as well as for
■ Severe asphyxia
the presence of clinically significant antibod-
■ Presence of lethal malformations
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
of stay was decreased with universal leuko-
81 does decrease the occurrence of febrile non-
cyte reduction.
hemolytic transfusion reactions in pediatric
hematology/oncology patients.82
Transfusion Practices in Red Cell Support for Children with

Older Infants and Children Hemoglobinopathies
The indications for transfusion of red cells In certain childhood conditions, chronic
and other components in older infants red cell transfusions are given not only to
(>4 months) and children are similar to treat tissue hypoxia but also to suppress
those for adults but must take into ac- endogenous hemoglobin production. Ap-
count differences in blood volume, ability proximately 6% to 10% of children with
to tolerate blood loss, and age-appropri- sickle cell disease suffer a stroke, with
ate hemoglobin and hematocrit levels. two-thirds experiencing a recurrence.83
The most common indication for red cell The goal of transfusion for these patients
transfusion in children is to reverse or is to reduce the risk of stroke by decreas-
prevent tissue hypoxia resulting from de- ing the percentage of circulating red cells
creased red cell mass associated with sur- capable of sickling, while simultaneously
gical procedures or in response to anemia avoiding an increase in blood viscosity. It
of chronic diseases or hematologic malig- is important to remember that raising the
nancies. It is important to remember that hematocrit, without significantly reduc-
normal hemoglobin and hematocrit levels ing the percent of sickle cells, could in-
are lower in children than adults. Pediat- crease viscosity and negate any beneficial
ric patients may remain asymptomatic effects of the transfusion.83,84 The rate of
despite extremely low levels of hemoglo- recurrent stroke can be reduced to less
bin, particularly if the anemia has devel- than 10% by maintaining a hemoglobin
oped slowly. level of 8 to 9 g/dL, with a hemoglobin S
The decision to transfuse should be level less than 30%, in children who have
based not only on the hemoglobin level but had a cerebrovascular accident. This can
also on the presence or absence of symp- usually be achieved with a simple or par-
toms, the functional capacity of the child, tial exchange transfusion every 3 to 4 weeks.
the etiology of the anemia, the possibility of Therapy is continued indefinitely, as ces-
using alternative therapies, and the pres- sation can lead to subsequent stroke.83,84
ence or absence of additional clinical con- Because of concern about iron overload,
ditions that increase the risk for developing some workers follow several uneventful
hypoxia. If small-volume transfusions are years of transfusions to keep hemoglobin

S below 30% with a less aggressive proto- proach to decrease hemoglobin S levels to
col that maintains hemoglobin S between below 30%.90
40% and 50%.84 Erythrocytapheresis has Patients with sickle cell disease might
been shown to improve iron balance in a also be at risk for severe delayed hemolytic
85
small cohort of patients. Adams et al transfusion reactions that could be life-
showed that transfusion to maintain a he- threatening because of the coincident sup-
moglobin S level less than 30% in children pression of erythropoiesis. When a patient’s
who have abnormal results on trans- hemoglobin level decreases after transfu-
cranial Doppler ultrasound reduced the sion, this “hyperhemolytic” syndrome—
86
risk of a first stroke. The benefits of this wherein it appears that autologous red cells
transfusion therapy, however, must be are destroyed through an innocent by-
weighed against the complications of stander mechanism—should be suspected.
transfusion, such as iron overload and In these circumstances, transfusion should
alloimmunization, as well as the risks of be stopped and corticosteroid therapy, or a
increased donor exposure during erythro- combination of corticosteroid therapy
cytapheresis. Although simple transfusion and IGIV, considered, based on reports of
increases blood viscosity, exchange trans- efficacy in case studies.91,92 Autoantibody
fusion does not. Blood for transfusion to a formation also occurs in these patients af-
patient with sickle cell disease should ide- ter transfusion.93 In the hope of decreasing
ally be screened for hemoglobin S. In ad- the need for transfusion, medical interven-
dition, most centers now provide leuko- tions are being explored. One therapy uses
cyte-reduced blood for patients with sickle hydroxyurea to increase the percentage of
cell disease to prevent alloimmunization hemoglobin F. By having a higher percent-
to platelets, which could complicate trans- age of cells that are hemoglobin F, the for-
87
plantation. mation of hemoglobin S polymers is re-
Red cell transfusions are also used to duced. In addition, the concomitant decrease
treat acute complications associated with in neutrophil counts that accompanies
sickle cell disease, such as splenic seques- hydroxyurea administration was found to
tration and aplastic crisis.88 Acute chest syn- be independently associated with a reduc-
drome, a new pulmonary infiltrate in a pation in the rate of crisis.94 Marrow trans-
tient with sickle cell disease, other than plantation has also been used in some pa-
atelectasis, with additional respiratory tients and may have a role in the future
symptoms and fever, carries a poor progno- treatment of patients with sickle cell dis-
sis if untreated. Simple transfusion can be ease.95,96
used as a first-line therapy to improve oxy- For children with thalassemia and severe
genation. For those patients who continue anemia, transfusion not only improves tis-
to deteriorate or who do not improve, red sue oxygenation but also suppresses
cell exchange transfusion should be per- erythropoiesis. By suppressing ineffective
formed. There are no randomized con- erythropoiesis, many of the complications
trolled trials comparing exchange and sim- associated with the disease are amelio-
ple transfusion in this setting.89 A study rated. So-called hypertransfusion, in which
evaluating preoperative transfusion proto- the pretransfusion hemoglobin is kept be-
cols found that a conservative protocol, in tween 8 and 9 g/dL, allows normal growth
which the hemoglobin was raised to 10 g/ and development, as well as normal levels
dL, was as effective in preventing perio- of activity for the child’s age. Supertransfu-
perative complications as an aggressive ap- sion programs aim to maintain a pre-

transfusion hemoglobin concentration be- typically matched units may be a reason-

tween 11 and 12 g/dL, in order to decrease able approach to prevent further allo-
iron absorption from the gastrointestinal immunization.102 Method 2.16 can be used
tract. The results of maintaining near-nor- to perform red cell phenotyping on auto-
mal hemoglobin levels are still controver- logous red cells of recently transfused pa-
sial. Iron overload is a complication of tients.
treatment, requiring chelation therapy be- African Americans with sickle cell dis-
97
ginning early in childhood. ease frequently become immunized be-
cause the majority of red cell transfusions
are obtained from Caucasian donors, with
Antibody Production in Sickle Cell and
major differences in antigen exposure. In
Thalassemia Patients
some areas, blood collectors have devel-
The frequency of red cell alloimmuni- oped programs to specifically recruit Afri-
zation in chronically transfused children can American donors to supply blood for
varies with the disease, the age of first patients with sickle cell disease, in order to
101
transfusion, the number of transfusions reduce rates of alloimmunization. Leuko-
given, and the ethnic background of do- cyte-reduced blood components may be of
nors and recipients, although patients particular value for these chronically trans-
with sickle cell disease have the highest fused patients. One benefit would be to di-
rates of alloimmunization of any patient minish the development of alloimmuniza-
group.98-100 Antibodies to the common an- tion to HLA antigens, in light of the prospect
tigens of the Rh, Kell, Duffy, and Kidd of future marrow transplantation.87 There
systems are often identified. It may be are conflicting data whether leukocyte re-
prudent, therefore, to phenotype the pa- duction can prevent alloimmunization to
tient’s red cell antigens as completely as red cell transfusion.104,105 Preventing febrile
possible before beginning transfusion transfusion reactions is also important for
therapy and to maintain a permanent re- patients who may be receiving a unit of
cord of the results. This can be helpful in phenotypically matched blood. Discarding
selecting compatible blood if alloim- such a unit would be both wasteful and
munization occurs. There is evidence that may have an impact on the ability to ade-
transfusing K, C, and E antigen-negative quately transfuse the patient.106
red cells can significantly reduce the rate
of alloimmunization. 1 0 1 However, the
Platelets and Plasma
practice of transfusing only phenotypi-
cally matched units is controversial, espe- The indications for FFP and platelet
cially in patients who have not yet devel- transfusions in older infants and children
oped the corresponding antibody, because parallel those for adults. Platelet transfu-
many of these units are difficult to ob- sions are most often given as prophylaxis
83,102
tain. A recent survey of 50 academic to children receiving chemotherapy. Pro-
medical centers in the United States and phylactic platelet transfusions are seldom
Canada found that the most common given when platelet counts are above
practice is to perform pretransfusion 10,000 to 20,000/µL, but, as with red cell
phenotypic matching for C, E, and K.103 In transfusion and hemoglobin, the indica-
patients who have already become immu- tion for platelet transfusion should not be
nized and who are at high risk of develop- based solely on the platelet count. When
ing additional antibodies, use of pheno- additive risk factors such as fever, sepsis,

DIC, or clotting abnormalities are present, 12. Simister NE. Placental transport of immuno-
globulin G. Vaccine 2003;21:3365-9.
the platelet count may need to be higher
13. DePalma L. Red cell alloantibody formation
to prevent spontaneous hemorrhage. In the in the neonate and infant: Considerations
absence of such factors, a much lower level for current immunohematologic practice.
may be safe.56 In recent studies, the use of Immunohematology 1992;8:33-7.
14. Sanders MR, Graeber JE. Posttransfusion graft-
ABO-compatible platelets has been asso- versus-host disease in infancy. J Pediatr 1990;
ciated with better clinical outcomes.107-109 117:159-63.
15. Ohto H, Anderson KC. Posttransfusion graft-
versus-host disease in Japanese newborns.
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Pediatr Hematol Oncol 2002;24:512-4. cardiac surgery. Transfusion 2001;41:790-3.

Chapter 25: Cell Therapy and Cellular Product Transplantation
Chapter 25
25
Cell Therapy and Cellular

Product Transplantation
T
HERAPEUTIC CELLS INCLUDE cell sufficient numbers. This chapter primarily
populations collected and proces- discusses the hematopoietic repopulation
sed to provide a special therapeu- cells. Cell preparations for HPC transplan-
tic effect. The classic example of cell tation are thought to contain both hemato-
therapy is the use of pluripotent stem poietic stem cells (HSCs) capable of
cells, which are capable of self-renewal self-renewal and HPCs committed to a
and differentiation into all blood-cell lin- blood-cell lineage. However, committed
eages. These stem cells—transplanted in progenitor cells lack capacity for sus-
preparations of marrow, stimulated pe- tained self-renewal or the ability to differ-
ripheral blood mononuclear preparations, entiate into other blood-cell lineages. The
or cord blood—also give rise to other re- committed cells are important for the
generative tissues such as hepatocytes, speed of engraftment.2 Current measure-
endothelial cells, and other tissue under ment methods cannot easily separate the
the proper microenvironmental circum- earlier and more committed cell popula-
1
stances. Other cell populations may serve tions. Both cell populations are referred to
therapeutic purposes such as immune as HPCs in this chapter. HPCs collected
modulation in posttransplant donor lym- from peripheral blood via apheresis are
phocyte infusion (DLI). The hemato- referred to as HPC-A. Those collected by
poietic progenitor cells (HPCs), which are harvesting marrow are termed HPC-M and
committed to a blood-cell lineage, were those from cord blood are called HPC-C.
first studied in the laboratory for their HPC transplantation has advanced from
power to give rise to complete, sustained a research procedure performed in a few
hematopoietic engraftment when given in centers to a common medical procedure
581

performed in many tertiary care centers. has been such a successful treatment for
HPC transplantation can be classified ac- this disease that the number of CML trans-
cording to the source of the HPCs used for plants has dropped sharply since licensure
engraftment as autologous, allogeneic, of this drug.3
syngeneic, and xenogeneic. Another advance in marrow transplanta-
Autologous HPC transplantation is tech- tion has been the recognition and exploita-
nically not a transplant but, rather, the “res- tion of the effects of transplanted allogeneic
cue” of a patient with the patient’s own immune cells on the malignancy of the pa-
HPCs, which are removed and stored frozen tient. Because the transplanted cells are the
or in nonfrozen conditions for later infusion treatment modality for the patient, the
as protection from the lethal effects of ther- preparation for transplantation needs only
apeutic or ablative irradiation or chemo- to modify the patient’s immune system to
therapy. The autologous graft may be ma- allow the new cells to engraft. This non-
nipulated to retain HPCs and leave behind myeloablative transplantation expands the
or exclude tumor cells or damaging im- number of patients eligible for transplant
mune cells. Autologous HPCs are reinfused by accepting patients of more advanced age
to repopulate the patient’s marrow after an and with more health problems. These pa-
otherwise lethal or near-lethal dose of radi- tients would not be candidates to enter
ation or chemotherapy given to treat malig- more toxic myeloablative regimens because
nancies of the marrow and metastatic or re- the risk of the treatment would be so great.
current solid tumors. In the special case of In this setting, chemotherapy and/or low-
an identical twin donor and recipient, such dose total body irradiation targets the re-
transplants are referred to as syngeneic. cipient’s T lymphocytes to allow tolerance
Allogeneic HPC transplantation involves for the healthy, allogeneic graft.
the infusion of HPCs from another human In successful marrow transplants, the
(an HLA-matched related or unrelated cells gradually produce full chimerism in
donor) in order to establish donor cell the patient’s marrow and attack and elimi-
chimerism to rescue the patient from dose- nate the tumor cells immunologically.4-6 In
intense therapy and/or as an active immuno- patients where healthy cell replacement is
therapy against a disease after a potentially the intended result of treatment, such as in
lethal dose of radiation or chemotherapy. children with immune deficiencies, this
Such transplants are preferred in patients method allows replacement cells to engraft
who have acute myelogenous leukemia without putting the child through the dan-
(AML), acute lymphocytic leukemia (ALL), ger of full myeloablation. The patient does
severe immunodeficiency, aplastic anemia, not have to undergo extensive periods of
marrow involvement with their malignancy, cytopenia with exposure to infection and
or those who are incapable of supplying bleeding risk. The engrafted cells become
their own autologous “normal” HPCs, as the treatment agent, allowing the chemo-
with hemoglobinopathies (thalassemias or therapy and/or radiation to be low-dose
sickle cell disease). As new indications for and relatively low toxicity. This approach
transplantation are developed, some others uses the immune reconstitution as the tool
are drastically changed by new discoveries. to control disease, but it is not without risk
For instance, patients with chronic myelo- because the immune cells can also attack
genous leukemia (CML) used to receive the the healthy tissues of the patient, causing
largest number of adult hematopoietic graft-vs-host disease (GVHD).7 The number
transplants; however, imatinib mesylate of nonmyeloablative transplants has in-

Chapter 25: Cell Therapy and Cellular Product Transplantation 583
creased from 20 in 1997 to 870 in 2001, ac- lignant diseases have also been treated with
cording to the Center for International life-saving transplantation, as a group,
Blood and Marrow Transplant Research they represent less than 15% of all trans-
(CIBMTR) reporting system.3 3,9
plants. The success rate of HPC transplan-
Xenogeneic HPC transplantation would tation depends on the condition and age
involve HPC transplants derived from a of the patient, type and stage of the dis-
nonhuman species. However, because of ease being treated, the cell dose, and de-
currently insurmountable immunologic gree of HLA matching between the donor
barriers and disease concerns, these trans- and the patient. Overall, long-term sur-
plants are not now clinically feasible. vival rates are generally 30% to 60% for
Sources of progenitor cells include mar- otherwise fatal diseases. Table 25-1 de-
row, peripheral blood, umbilical cord scribes general outcomes.10
blood, and fetal liver (although this source
is experimental and not in routine clinical
use).1 Once collected, the HPCs may be
subjected to ex-vivo processing, eg, re- Sources of Hematopoietic
moval of incompatible red cells or plasma Progenitor Cells
and/or cell selection (purging) before the Historically, marrow was the primary source
transplantation procedure. In some cases, of hematopoietic cells for transplantation.
certain cell populations are “positively” se- However, HPC-A transplants constituted
lected (selectively isolated) for their special approximately 90% of adult autologous
therapeutic effects. T lymphocytes may be and about half of allogeneic transplanta-
isolated and later infused in measured tion procedures in the 1998-2000 Interna-
doses for antitumor effects, thus minimiz- tional Bone Marrow Transplant Registry
ing GVHD. Separation of cell populations report. 3 Data on umbilical cord blood
can be accomplished using a cell separa- transplants have shown promising results
tion device approved by the Food and Drug in pediatric patients for whom a matched
Administration (FDA), such as the Isolex unrelated allogeneic HPC-M or HPC-A do-
system (Baxter Healthcare, Deerfield, IL). nor is unavailable. Clinical studies are in
Alternatively, some cell populations may be progress to determine 1) the safety and ef-
“negatively selected” (culled out or de- ficacy of cord blood transplantation, 2)
stroyed), as by antibody-mediated lysis of whether adults can be successfully trans-
malignant cells or by flow cytometry sepa- planted, and 3) the extent of HLA mis-
ration of cell populations in clinical trial sit- match that can safely and effectively pro-
uations. vide durable engraftment.
HPCs from Marrow (Autologous)

Diseases Treated with Currently, only about 5% of autologous
transplants are performed with HPC-M.
Hematopoietic Cell This source has been largely replaced by
Transplantation mobilized autologous HPC-A.
Many diseases have been treated with
8
HPC transplantation. Malignant diseases HPCs from Marrow (Allogeneic)
are the most frequent indications for HPC Allogeneic transplants address the prob-
transplantation. Although many nonma- lem of the inability to obtain a tumor-free

Table 25-1. Examples of Diseases Responsive to Hematopoietic Stem Cell

10
Transplantation
Disease Type of Transplant Timing Clinical Results
AML Allogeneic First CR OS 40%-50%

ALL (children) Allogeneic Second CR OS 40%-65%
ALL (high risk) Allogeneic First CR OS 50%
Chronic phase CML Allogeneic Chronic phase (CP) OS 50%-80%
Accelerated phase CML Allogeneic Individualized OS 30%-40%
Blast phase CML Allogeneic Second CP OS 15%-25%
Myelodysplastic Allogeneic Age <60 OS 40%
syndrome
Aplastic anemia Allogeneic Individualized OS 70%-90%
CLL Allogeneic or Participation in Small series of patients
autologous clinical trial with durable CR;
nonablative transplants
under investigation
Intermediate-grade NHL Autologous Chemosensitive OS 40%-50%
relapse
High-risk NHL Autologous First CR OS 50%-60%
Low-grade NHL Allogeneic or autologous Chemosensitive DFS 25%-50% at 5 years
relapse
Mantle cell lymphoma Allogeneic or autologous First CR Small series with durable
clinical trial CR rates of 25%-50%
Lymphoblastic lymphoma Allogeneic or autologous Chemosensitive Small series with durable
clinical trial relapse or first CR CR
NHL or Hodgkin's disease Allogeneic clinical trial Advanced refractory DFS 15%-25%
disease
Multiple myeloma Autologous Chemosensitive re- OS 50% at 5 years; DFS
lapse or first CR 20%
High-risk breast, Autologous clinical trial Chemosensitive Improved survival over
testicular, or ovarian disease historical controls not
cancer confirmed in
randomized trials
Renal cell carcinoma Nonablative allogeneic Clinical trial Small series with durable
CR
Thalassemia Allogeneic Clinical trial OS 75% for patients
without cirrhosis
Sickle cell anemia Allogeneic Clinical trial OS 75%
Autoimmune disorders Allogeneic Clinical trial Small series of remissions
CR = complete response; DFS = disease-free survival; OS = overall survival; AML = acute myelogenous leukemia; CML =
chronic myelogenous leukemia; ALL = acute lymphocytic leukemia; CP = chronic phase; NHL = non-Hodgkin's lymphoma.
Netter examples used with permission from Icon Learning Systems, a division of MediMedia USA, Inc. All rights reserved.

11
graft with an autologous transplant for a filgrastim). The donors’ physical symp-
patient with malignant disease and pro- toms as well as their attitudes and feelings
vide possible immune help after trans- about the process are being monitored for
plantation from graft-vs-tumor response. the trial. The use of HPC-A offers the poten-
For other patients, such as those with tial advantage of improved engraftment ki-
marrow failure, immunodeficiency, in- netics and enhanced graft-vs-leukemia
born errors of metabolism, or hemoglob- (GVL) effect. In allogeneic, related trans-
inopathy, an allogeneic transplant is the plant settings clinical trials comparing
only appropriate type of graft. Sources of HPC-M to HPC-A, the latter showed im-
allogeneic HPC-M may be from matched proved survival. Although chronic GVHD
or partially matched related or unrelated was a problem in the HPC-A study arm, it
donors. was manageable in a sufficient number of
cases to give those patients a survival ad-
vantage.12
Matched, Unrelated Donor Transplantation Initially, the most common diagnosis in
Matched, unrelated donor searches can patients undergoing matched, unrelated
be initiated for the 60% to 70% of candi- donor transplantation was CML. The dis-
dates without an HLA-identical related covery of imatinib mesylate has revolution-
(usually sibling) donor. Several marrow ized the treatment of CML such that the
donor databases are available worldwide. most common indication for allogeneic
The largest is the National Marrow Donor transplantation is acute leukemia followed
Program (NMDP) database. Since its by non-Hodgkin’s lymphomas, multiple
founding in 1986, the NMDP has facili- myeloma, and other diseases of marrow
tated approximately 12,000 transplant failure such as aplastic anemia.13
procedures with a total of over 2000 trans- Traditionally, HLA typing for Class I
plants per year. A directory of transplant (HLA-A and HLA-B) has been dependent
centers, outcome results, and charges is on serologic techniques. However, it is
available from the NMDP. (See Appendix likely that the posttransplant complications
11 at the end of the book). of GVHD and failure to engraft result from
Upon initial search, 80% of transplanta- use of phenotypically matched, unrelated
tion candidates usually find an HLA pheno- donors with significant disparities in allo-
typic match. However, patients from racial antigens that were not identified through
or ethnic minorities have a lower chance of serologic matching techniques.14 HLA-A
success in such a donor search (Caucasian, and HLA-B molecular Class I typing, inter-
81%; Hispanic, 64%; Asian/Pacific Islander, mediate resolution, and high-resolution
55%; African American, 47%; and American typing of Class II alleles are the current
Indian/Alaska Native, 50%; according to the standard of care, particularly in unrelated
NMDP). The median time from initiating a transplants.
search to receiving a transplant has been The risk of GVHD is greater with HLA
120 days, but a new expedited search and Class II disparity than with Class I dispar-
15
donor preparation program is being imple- ity. HLA typing for HLA-DR and HLA-DQ
mented to shorten this time. is routinely performed by DNA-based tech-
The NMDP is conducting a clinical trial niques. Molecular technology provides
of HPC-A collections from unrelated, HLA- greater resolution, including subtypes of al-
matched volunteers stimulated with granu- leles identified as cross-reactive groups us-
locyte colony-stimulating factor (G-CSF or ing conventional serologic techniques. Mis-

matching for a single Class I or Class II tion or after acute GVHD (generally after
antigen has no effect on survival, but mor- posttransplant day 50) and may severely af-
tality increases with more than one Class I fect the patient’s quality of life. In addition
mismatch or simultaneous mismatches in to the symptoms found in the acute form,
Class I and Class II antigens.15 Recent stud- chronic autoimmune-type disorders such
ies demonstrated the importance of recipi- as biliary cirrhosis, Sjogren’s syndrome, and
ent HLA-DRB1 and HLA-DQB1 allele dis- systemic sclerosis may develop as the
16,17
parity in the development of GVHD. transplanted immune cells attack the secre-
With further experience in molecular typ- tory epithelial cells in the saliva glands, the
ing and transplant outcomes, the extent to biliary tree, or the patient’s connective tis-
which successfully transplanted cells can sue. Chronic GVHD was reported in 55% to
tolerate disparities in specific alleles will be 65% of allogeneic transplant patients who
elucidated. Although the importance of survived beyond day 100 in a large study.2
HLA-A, -B, and -DR disparities is well Both forms of GVHD impair the patient’s
known, the significance of HLA-C disparity immune response and predispose the pa-
is being investigated. HLA-C typing has been tient to infections. To decrease or eliminate
hampered by poor serologic identification, GVHD in these transplants, HPCs can
and its significance has been thought, until undergo procedures for T-cell reduction
recently, to play a minor role in the T-cell (depletion) and the patient can be treated
immune response because of its reduced prophylactically with a variety of immuno-
polymorphism and low level of cell surface suppressive drug therapies.
expression. Early studies showed that GVHD has been associated with both a
HLA-C antigens can be recognized by decreased disease relapse and an improved
alloreactive cytotoxic T lymphocytes and overall survival in leukemia patients if the
natural killer cells, which may be associated GVHD is relatively mild. Such a GVL effect
with an increased risk of graft failure.18 is believed to be secondary to the graft at-
tacking residual malignant cells. A major
clinical challenge is to maximize the GVL
Graft-vs-Host Disease effect while minimizing the adverse se-
The negative outcomes of HLA mismatch- quelae of GVHD. The mechanism of the
ing are graft rejection, host-vs-graft reac- GVL effect is incompletely understood, but
tions, and graft-vs-host reactions. In acute donor-derived cytotoxic T lymphocytes
GVHD, the transplanted cells may attack specific for the patient’s minor histocom-
the tissues of the recipient early in the patibility antigens may contribute to the ef-
engraftment—within 100 days after the fect. Donor lymphocyte infusion of thera-
initial engraftment-associated events. The peutic T cells in patients with leukemic
skin, the gastrointestinal tract, and the relapse after allogeneic transplantation has
liver are most commonly involved, al- been attempted to induce a GVL effect.20
though usually not concurrently. The site Conflicting data exist about whether a GVL
and severity determine the clinical grade effect can occur independently of GVHD. A
of acute GVHD. The risk of GVHD is strategy to maximize the GVL effect and
greater with HLA-mismatched, unrelated minimize GVHD has been to titrate the
and related transplants than with HLA- number of donor lymphocytes until GVHD
identical transplants.19 Grade II-III occurs in order to create a GVL
Chronic GVHD characteristically occurs effect without severe GVHD. However, the
spontaneously months after transplanta- optimal number of donor lymphocytes ca-

pable of inducing a GVL effect without sig- with or without treatment with chemo-
nificant GVHD is not known. Immunosup- therapy before collection. Once in the
pression, number of T cells transfused, circulation, the HPCs are collected by
T-cell phenotype, myelosuppression, and leukapheresis. HPC-A collection carries
the timing of the DLI of therapeutic T cells no anesthesia risk, is less invasive, and
all play a role in the balance between the contains fewer tumor cells than marrow
GVL effect and GVHD. The GVL effect is best harvests.
documented in CML and less well shown in
AML, ALL, and other lymphoid malignancies. HPCs by Apheresis (Allogeneic)
However, because it is difficult to identify
a good HLA match, allogeneic transplanta- Related Transplantation
tion is associated with a major risk that For adult allogeneic transplantation, the
immunocompetent donor T cells reacting best clinical results are obtained with a
against recipient tissues will cause GVHD. completely HLA-matched, related donor.
Even in HLA-identical (six-antigen match) The best chance of finding a six-antigen
donor/recipient pairs, up to 6% of the grafts HLA match is among the patient’s genetic
will fail and GVHD will occur in 20% to 60% sisters and brothers. Parents and children
of cases as a result of nucleated cells exhib- will be at least a haplotype match.
iting minor histocompatibility antigens that Genetically, there is a 25% chance of a
are not linked to the major histocompati- sibling being a complete match, a 50%
bility complex antigens.21 Improved HLA chance of a haplotype match, and a 25%
typing techniques now employ molecular chance of a complete mismatch. Pediatric
techniques that give a fuller picture of the patients are more tolerant of partially mis-
antigen mapping and match/mismatch matched grafts and, therefore, have a larger
picture, but serologic terms of estimation of available donor pool.24 In the rare instance a
number of matches are still frequently used recipient has an identical twin, a syngeneic
in clinical descriptions for reference. This transplant may be optimal because the do-
occurs despite immunosuppressive therapy nor and recipient cells are genotypically
administered for several months after the identical and the risk of GVHD is reduced.
procedure.22 New and emerging immune However, syngeneic grafts do not provide
cell manipulations are being explored to the graft-vs-tumor effect found in allo-
exploit the cancer-controlling effects of geneic transplants.
these cells while seeking to avoid the un- HPC-A has largely replaced HPC-M for
wanted consequences of GVHD. Extracor- related transplantation.25 The use of G-CSF
poreal photopheresis is being used to treat in healthy donors has been shown to mobi-
patients with GVHD and to reduce the dose lize sufficient HSCs for allogeneic trans-
of immunosuppressive medications re- plantation,26 thus avoiding the need for an-
23
quired. esthesia and a marrow harvest. Clinical
data comparing allogeneic HPC-A vs allo-
geneic HPC-M from HLA-identical siblings
HPCs by Apheresis (Autologous)
have shown that HPC-A recipients have
Autologous HPC-A collection involves faster engraftment, improved immune re-
mobilizing the hematopoietic cells from constitution, lower transplant-related mor-
the patient’s marrow compartment into bidity, and a similar incidence of acute
2,27
the peripheral blood with hematopoietic GVHD. Retrospective analyses reported a
growth factors, most commonly filgrastim, higher incidence of chronic GVHD in re-

28
lated allogeneic HPC-A recipients. How- techniques, but the preferred system uses a
ever, a recent prospective randomized small collection bag (150 to 250 mL) with
study found no difference in the risk of appropriate anticoagulant. In-vitro data
chronic GVHD.12 have suggested that placental blood has an
increased capacity for proliferation.32 The
volume of units retained for processing is
HPCs from Cord Blood
typically 80 to 100 mL (range, 40-240 mL),
Despite the fact that over 3 million indi- with a median nucleated cell count of 1.2 ×
viduals are registered with the NMDP, pa- 109 in the units chosen for transplantation
tients in need of an allogeneic HPC trans- and a median CD34+ cell count of 2.7 ×
plant have ≤85% chance of finding a 105/kg.33 Clinical studies have reported suc-
matched donor. Finding and qualifying a cessful engraftment in children.34,35 The me-
willing donor for HPC-A or HPC-M collec- dian time to neutrophil engraftment (500/
tion typically takes weeks. Because of the µL) is 30 days; median time to platelet
time and availability constraints, atten- engraftment (50,000/µL) is 56 days.36 Com-
tion has turned to the third available pared with engraftment observed after allo-
source—HPC-C. Umbilical cord blood, geneic marrow transplantation, neutrophil
which in clinical practice is routinely dis- and platelet engraftment appear to be de-
carded, is being banked as an alternative layed. Clinical studies have also suggested
source of HPCs, especially for children, that unrelated HPC-C transplants are asso-
for whom smaller collection numbers of ciated with a lower risk of GVHD compared
HPCs are adequate for successful engraft- with unrelated HPC-M transplants in chil-
ment. The advantages of cord blood as a dren, even considering the lower risk sus-
31,34,36,37
source of HPCs for transplantation in- ceptibility of pediatric patients to GVHD.
clude: no risk to the donor, potential Two studies have compared engraftment
availability of cord blood from donor pop- and outcomes of transplantation of adults
ulations underrepresented in the NMDP, with one- or two-antigen mismatched
more rapid availability, and possibly HPC-C, matched HPC-M, and one-antigen
lower risk of viral infection and GVHD. Ar- mismatched HPC-M. The findings in the
eas of concern regarding HPC-C include two studies were very similar: HPC-M
ethical and informed consent issues, speed transplant recipients recovered neutrophils
of engraftment, higher mortality in the on day 18 to 19, and HPC-C recipients re-
posttransplant period, and ability to covered neutrophils on day 26 to 27.38,39
achieve engraftment in adults as a result Platelet engraftment in these studies re-
of the limited number of nucleated cells peated the finding that HPC-M transplan-
in cord blood. tation provided more rapid recovery at 29
Cord blood obtained from a delivered days compared with 60 days for HPC-C.
placenta is known to be rich in early and Both of these studies found that the HPC-C
committed progenitor cells.29 Since the first transplantation compares favorably with
cord blood transplant was reported in 1989 one-antigen mismatched HPC-M trans-
for Fanconi anemia,30 more than 2500 pa- plantation, with equal advantage for sur-
tients have received cord blood for a variety vival for each group. A matched HPC-M
of hematologic malignant and nonmalig- transplant was superior to HPC-C or mis-
nant conditions.31 matched HPC-M, but the advantage was
Cord blood is collected from the placenta small. This is encouraging news for adults
at the time of delivery using a variety of who need an HPC-M transplant but have

difficulty finding a suitably matched donor. ucts in 1992. The program has stored in
Current clinical trials are testing whether excess of 14,000 cord blood units and has
multiple HPC-C units received by adults provided cells for more than 1500 trans-
will facilitate faster engraftment. A study of plant procedures. The outcomes of the
23 such recipients with advanced-stage dis- first 562 procedures have been reported
ease showed a mean neutrophil engraft- in detail.34 The NMDP has developed a
ment of 23 days and a 57% chance of dis- registry of participating cord blood banks.
ease-free survival after 1 year. In these The National Cord Blood Bank of the New
two-unit transplants, the cells of one of the York Blood Center, NetCord, Bone Marrow
units prevailed and provided the lasting Donors Worldwide, and the Caitlin Ray-
40
engraftment population. mond Registry are additional search sites
available today.
Related Cord Blood Donors
Sibling-derived cord blood has been used
as a source of hematopoietic engraftment Donor Eligibility
in more than 250 allogeneic transplants in
Europe and North America, representing Donor Evaluation: Autologous Setting
15% of the allogeneic cord blood trans- In the autologous setting, the major con-
plants.31,35 The kinetics of hematopoietic cern regarding eligibility for transplanta-
recovery are similar to those observed with tion arises from the condition of the pa-
unrelated cord blood recipients.31 Times to tient. For HPC-M, the patient’s marrow
both platelet and neutrophil engraftment first should be assessed for residual ma-
are slower than those observed in HLA- lignancy and marrow cellularity. Patients
matched sibling marrow transplants. How- scheduled for an autologous transplant
ever, recipients of cord blood from HLA- should undergo an extensive history and
identical siblings have a lower incidence physical examination to identify any risks
of acute and chronic GVHD compared with from the marrow harvest and/or aphere-
marrow from HLA-identical siblings.31 sis procedures. For HPC-A, the patient/
donor is evaluated for the likelihood that
Autologous Cord Blood he or she can undergo successful mobili-
Companies are marketing the freezing and zation and collection.
long-term storage of an infant’s cord blood
cells to parents, in case the child may need Donor Evaluation: Allogeneic Setting
them. 41,42 The chance of an individual Allogeneic donors must be selected ac-
needing a cord blood transplant by age 18 cording to their degree of HLA match;
is estimated to be 1 in 200,000.42 The first qualifications according to the FDA regu-
successful autologous cord blood transplan- lations; standards of the AABB, NMDP,
tation procedure was reported in a 14- and Foundation for the Accreditation of
month-old patient with neuroblastoma.43 Cellular Therapy (FACT); and physical
ability and willingness of the donor to un-
Cord Blood Banks dergo the collection procedure. Ideally, a
The first large-scale community cord full six-antigen match should be found;
blood program was the Placental Blood however, transplant procedures have been
Program at the New York Blood Center, performed successfully using one-antigen
which began collecting placental prod- mismatched and haploidentical donors.

Cytomegalovirus (CMV) status of the doing of the donor to the level of risk to
nor is also a deciding factor in the selec- which the recipient may be subjected.
tion process in the case of CMV-negative Thus, unrelated allogeneic donors repre-
recipients. The use of parous females or sent the highest level of risk, whereas
gender-mismatched individuals as donors family members or regular sexual partners
is associated with an increased risk of are considered a lower risk source of new
GVHD, as is a history of transfusion in the exposure to patients.
donor.44-46 Therefore, the preferred HLA- The donor must be screened in order to
identical donor would be CMV negative minimize the risk of disease transmission
(in the case of CMV-negative recipients); to an already immunocompromised recipi-
of the same gender as the recipient; if fe- ent. FDA regulations specify that the HPC
male, nonparous; and untransfused. donor sample should normally be tested up
to 7 days before collection, or at the time of
Infectious Disease Testing collection but before release of the product.
HPC-A donor samples may be obtained up
Autologous and tested allogeneic donors to 30 days before collection in order to have
must be screened and tested for certain infectious disease testing completed before
infectious diseases.47 In the Code of Fed- the patient begins myeloablative chemo-
eral Regulations (CFR) Title 21, sections therapy [21 CFR 1271.80(b)]. For purposes
of the regulations relevant to cell therapy of optimal donor selection, it may be advis-
donors are Donor Screening (1271.75), able to test the donor earlier in the trans-
Donor Testing: General (1271.80), and Do- plantation workup period as well.48,49
nor Testing: Specific Requirements The use of approved nucleic acid tests
(1271.85). The rules are described by the for HIV and HCV has considerably short-
FDA as “comprehensive, but flexible.” The ened the possible “window period” in do-
donor screening rules are consistent with nors who may have contracted either of
the 1994 Centers for Disease Control and these infections but who do not yet test
Prevention (CDC) guidelines to prevent positive on antibody tests. Donors being
transmission of human immunodeficiency stimulated with filgrastim develop higher
virus (HIV), and 1993 FDA guidelines for sample-to-cutoff ratios. This makes false-
prevention of transmission of hepatitis B positive reactions more likely in the
virus (HBV) and hepatitis C virus (HCV). immunoassays currently used for infectious
Testing for syphilis is required, and tissue disease. False-positive test results for hepa-
rich in leukocytes must be tested for hu- titis B surface antigen and HCV antibody
man T-cell lymphotropic virus (HTLV ) have been associated with G-CSF adminis-
and CMV. They also include requirements tration in normal donors.50,51
for questioning donors about risk factors Donors who are confirmed positive for
for variant Creutzfeldt-Jakob disease and HIV should not be used as a source for the
new or emerging pathogens such as West transplant. Other positive disease markers
Nile virus and severe acute respiratory do not necessarily prohibit use of collec-
syndrome, and requirements for tests for tions from a particular donor (eg, anti-HBc
carriers of these illnesses as soon as they positive), and special evaluation of donors
are practically available. The regulations with this marker is under way by the NMDP
and guidance are extensive and fit the at this time. The allograft material from
FDA’s stated “layered approach” that in- such donors may be used when the pa-
tensifies the amount of screening and test- tient’s transplantation physician informs

him or her of the status of the donor and diluted with an anticoagulant (usually
documents consent of the patient. Segre- preservative-free heparin and/or ACD).
gated methods of storage must be used for The marrow aliquots are pooled into a
the biohazardous material collected until it sterile vessel or a harvest collection bag
is infused so that cross-contamination of equipped with filters of graduated pore
other stored products is prevented.48(p78),49,52 size. The marrow is then transferred to a
In cases of allogeneic HPC-C transplants, sterile blood bag and transported to the
a sample for infectious disease studies processing laboratory, where samples are
should be obtained from the donor’s removed for graft evaluation, quality as-
mother within 48 hours of collection of the surance testing, possible manipulation of
cord blood and tested. Any positive results the product, final labeling, and/or cryo-
should be reported to the mother and the preservation.
mother’s physician. The HPC-C donation
involving a mother or a child who has a Collection Targets
positive infectious disease history or results
The recipient’s body weight and type of
should either be discarded or the patient
manipulation of the collection, if any, will
must go through a special notification and
determine the volume of marrow to be
consent process.52 In cases of allogeneic
collected. A frequently used minimum
transplantation, CMV status of the donor
target (after processing), for both autologous
and the recipient should be carefully con-
and allogeneic transplants, is 2.0 × 108 nu-
sidered.
cleated cells per kilogram of recipient
A recipient may develop primary CMV
body weight. Marrow harvests in autolo-
infection if he or she is CMV negative and
gous patients who have received alkylat-
receives a CMV-positive graft but may have
ing agents as therapy may yield fewer pro-
some protection from the immunity in the
genitor cells relative to the total nucleated
graft. CMV-positive individuals receiving a
cells collected. In these cases, extra mar-
CMV-negative graft may have a severe pri-
row should be obtained if possible.53 If the
mary infection of the graft from virus resi-
harvested product is to be processed (ie,
dent in the recipient’s tissue.46
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
Collection of Products its donors to a maximum of 1500 mL. Many
HPC-M Collection centers use the cell number requested for
transplant and the cell count on the prod-
A marrow harvest is the same for an allo-
uct during collection to determine the fi-
geneic donor as for an autologous patient.
nal collection volume.
The procedure is performed under sterile
conditions in the operating room. The
posterior iliac crest provides the richest Clinical Considerations
site of marrow. In the autologous patient, The age of the donor and, in the autolo-
prior radiation therapy to an aspiration gous setting, the underlying disease and
site may result in hypocellular yields. In previous treatment regimen influence the
general, these sites are unsuitable for har- HPC yield (Table 25-2).54 Autologous or allo-
vest and should be avoided. Once aspi- geneic donors may require RBC transfu-
rated, the marrow should be mixed and sions to replace blood taken with marrow

collection. Frequently, allogeneic marrow well as the pluripotent stem cells. CD34+
donors will have an autologous RBC unit cells purified from marrow are capable of
55
collected 2 to 3 weeks before the harvest. trilineage reconstitution in humans. One
Most RBC transfusions occur after the har- to three percent of normal marrow cells
vest to avoid marrow dilution. Allogeneic express this antigen. In normal peripheral
blood components must be irradiated if blood, CD34+ cells circulate in low num-
given before or during the procedure, to bers (0.01-0.1%).56 Monoclonal antibodies
incapacitate donor lymphocytes from re- (MoAbs) to CD34 have been developed
plication and attack of the transplant re- and are used in flow cytometric assays to
cipient, possibly causing GVHD. In trans- measure the stem/progenitor content of
plants that require marrow manipulation, HPC collections and CD34+ cell concen-
such as red cell depletion, the recovered tration in donor peripheral blood.57
red cells may be returned to the donor.
HPC-A Collection HPC-A Collection (Autologous)

HPCs can be mobilized into the periph- In the autologous setting, HPC-A donors
eral blood with the use of recombinant are routinely mobilized with hematopo-
colony-stimulating factors and collected ietic growth factors (with or without che-
in sufficient numbers to achieve long- motherapy), which include G-CSF and
term hematopoietic engraftment in a trans- granulocyte-macrophage colony-stimu-
plant recipient. lating factor (GM-CSF) or a combination
of the two. Mobilization with chemother-
apy or growth factors alone results in a
CD34 10- to 30-fold increase in the concentra-
CD34 is the cluster designation given to a tion of HPCs over the steady state.8 Com-
transmembrane glycoprotein present on bined mobilization with growth factors
immature hematopoietic cells, some ma- and chemotherapy enriches HPC concen-
ture endothelial cells, and stromal cells.55 trations 50- to 200-fold.58 Transplantation
The antigen has an approximate molecu- of autologous HPC-A results in a reduced
lar weight of 110 kD and carries a negative time to hematopoietic recovery compared
charge. Cells expressing the CD34 antigen with autologous HPC-M.59 Platelet recon-
encompass the lineage-committed cells as stitution is most striking and patient hos-
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. In most autologous patients, venous ac-
60
Schwella and colleagues demonstrated cess is obtained through a dual- or triple-
that the median time to an unsupported lumen central venous apheresis catheter.
platelet count of ≥20,000/µL was 10 days Operators of blood cell separators generally
in the group receiving chemotherapy plus process two to three blood volumes per
G-CSF-mobilized HPCs vs 17 days in the procedure. In the pediatric setting, it may
group receiving autologous HPC-M. Ef- be necessary to prime the cell separator
fective mobilization in the autologous pa- with compatible, irradiated red cells. The
tient is influenced by previous chemo- donor will undergo daily procedures of ap-
therapy or radiation. proximately 2 to 5 hours each. Large-vol-
There is a linear relation between the ume leukapheresis procedures (processing
precollection peripheral blood CD34 count at least three blood volumes or 15-20 liters)
and the volume of blood that needs to be are performed at many centers to reduce
processed to achieve a target dose. Graphs the overall number of collections. Investiga-
and predictive models would be expected tional studies have suggested that there is
to vary based on the efficiency of CD34+ equilibration of noncirculating HPCs into the
collection related to the specific equipment peripheral circulation with large-volume
and software employed. It would also be collections.70-72 Hillyer73 reported a 2.5-fold
expected to be least predictive at low pe- increase in colony-forming units–granulo-
ripheral blood CD34+ concentrations, where cyte-macrophage (CFU-GM) per mL pro-
the enumeration is least accurate. White cessed in collection when the collection
cell counts, the number of mononuclear volume was 15 liters compared to the yield
cells, and the number of circulating CD34+ in the first blood volume of the collection.
cells have all been used as surrogate mark- Collection Targets. The adequacy of
ers for the timing of the HPC-A collec- autologous HPC collection is gauged by the
tion.59,61-63 It has been suggested that the op- CD34+ dose (the number of CD34+ cells per
timal time to begin HPC collection in pa- kilogram of recipient body weight). The re-
tients who have been primed with chemo- ported minimum threshold of CD34+ cells
therapy is when the white cell count first necessary for neutrophil and platelet engraft-
exceeds 5 × 109/L.59 However, the white cell ment in the autologous patient has ranged
concentration does not correlate with the from 0.75 to 1.0 × 106/kg.8,64 This minimum
number of HPCs in the peripheral blood. target is a broad guideline and higher doses
Phenotypic analysis of CD34+ cells by flow have been associated with accelerated
cytometry provides a more real-time mea- platelet engraftment,74 reduced febrile com-
surement of CD34+ cell content in an HPC plications, and use of antibiotic therapy af-
75
collection or hematopoietic graft. The tech- ter transplantation. Recent data support
nique can be used to indicate the timing of an economic benefit associated with greater
the first collection and the total number of CD34+ cell collections (greater or equal to
CD34+ cells/kg finally collected (as a func- 5.0 × 106/kg) compared to the minimum ac-
56,57,64-66
tion of the volume of blood processed). ceptable collections required for engraft-
ment (1.0 × 10 /kg). The type of processing
6 76
The length of time for engraftment corre-
lates with the number of CD34+ cells in the will also influence the volume necessary for
collection.65,67-69 In general, a peripheral blood collection.
CD34+ cell concentration of 10/µL can be Poor Autologous HPC Mobilization. Pa-
expected to result in a yield of at least tients who have been heavily pretreated
1.0 × 106 CD34+ cells/kg.60,61,64 with chemotherapy and/or radiation ther-

apy may fail to mobilize enough HPCs after crease from 25% to 40% because the HPCs
stimulation with growth factors/chemo- lie close to the platelet layer in the buffy
therapy. The best way to obtain an ade- coat. Collection can be performed via pe-
quate collection from poorly mobilized pa- ripheral or central venous access. Patients
tients is unknown. Collection of HPC-M in who have received prior chemotherapy are
combination with, or in place of, HPC-A is more likely to need a central venous cathe-
frequently ineffective in improving engraft- ter because of poor peripheral access. Cath-
ment.77 Second attempts at mobilization eter-associated thrombosis (either in the
with G-CSF alone have been successful in catheter or in the vein surrounding it) is the
obtaining adequate CD34+ cell counts and most common complication associated
can be as effective as G-CSF and chemo- with HPC-A collection.82
therapy.78 Increasing the dose of G-CSF or
combining with GM-CSF has also success-
fully mobilized some autologous donors af- HPC-A Collection (Allogeneic)
ter prior failed attempts.79 Collection of allogeneic HPCs from HLA-
Mobilization can be very difficult in some matched relatives is primarily performed
patients, particularly if they have been using G-CSF mobilization. Typically, do-
heavily pretreated or are older. Another ap- nors are given 10 to 16 µg/kg as a subcu-
proach to increase production of stem/pro- taneous injection once or twice daily. The
genitor cells by growth factors is manipula- concentration of CD34+ cells in the pe-
tion of the chemokines that attach the cells ripheral blood begins to rise after 3 days
to their microenvironment. The interaction of G-CSF administration and peaks after 5
of stromal-derived growth factor-1 (SDF-1) to 6 days. Standard volume leukapheresis
and CXCR4, its ligand, controls mobiliza- (processing 8-9 L) results in a component
tion. The molecule AMD 3100 was devel- with the following median values: white
oped originally as a potent and selective cells, 32.4 × 109; mononuclear cell count,
31.4 × 10 ; CD34+ cells, 330 × 10 ; plate-
9 6
inhibitor of CXCR4 in order to control repli-
lets, 470 × 10 ; and RBCs, 7.6 mL. Clinical
9 83
cation of HIV-containing cells. An observed
side effect of the drug was a rapid increase trials have suggested a minimum dose of
in white cells. Broxmeyer and colleagues 2.0 × 106/kg of CD34+ cells to be adequate
84
measured a 40-fold increase in HPCs after for allogeneic HPC-A transplants.
injection of AMD 3100 into human volun- Allogeneic donors with poor venous ac-
teers.80 Phase I and II clinical trials are un- cess may require central venous catheters.
der way in conjunction with G-CSF to en- In the NMDP report on normal donor col-
hance mobilization and HPC-A yields in lections of HPCs, 5% of men and 20% of
81
difficult-to-mobilize patients. women require central venous access for
85
Clinical Considerations for Autologous collection. There is a 1% risk of complica-
HPC-A Collection. The frequency and length tions associated with these catheters includ-
of HPC-A collections may result in donor ing infection, hemorrhage, and pneumo-
discomfort and side effects. Complete blood thorax. Thrombocytopenia has been reported
counts are performed before and after each as a complication of G-CSF administration
86,87
apheresis procedure to monitor the hemato- and HPC-A collection in healthy donors.
crit and platelet values. Red cell and/or There is also a risk in exposing normal
platelet transfusions may be required. Red donors to G-CSF. Ninety percent of donors
cell loss should be minimal in HPC-A col- experience side effects.88 The most com-
lection, but platelet counts typically demon complaint is bone pain followed by

headaches, body aches, fatigue, nausea, and have a neutral pH. However, other
and/or vomiting. Bone pain, headaches, closed system arrangements using ACD or
and body aches can be successfully man- heparin are in use in some centers. In-
aged with nonsteroidal analgesics such as formed consent from the biologic mother
acetaminophen. As mentioned earlier, or legal representative of the child must
false-positive infectious disease serologies be obtained, preferably before delivery.
have been reported.51 Serious adverse ef- A personal and family medical history of
fects are rare but include reports of splenic the biologic mother (and, if available, of the
rupture,89,90 neutrophilic infiltration of the infant donor) must be obtained and docu-
91
skin (Sweet’s syndrome), arterial thrombo- mented before, or within 48 hours of, the
92 93
sis, and an anaphylactoid reaction. Do- collection. If available, his medical history
nors with complex hemoglobinopathies should be obtained from the biologic fa-
(eg, sickle trait and β+ thalassemia) have ther. HPC-C collections are not acceptable
been observed to have complications with for allogeneic use if there is a family history
G-CSF stimulation for the purpose of be- (biologic mother, father, or sibling) of ge-
94,95
coming an HPC donor. 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.
HPC-C Collection
Red cells from the HPC-C collection or
Umbilical cord blood can be collected from from the infant donor must be typed for
either a delivered or an undelivered pla- ABO/Rh and a screen for unexpected red
centa.96 Cord blood collection should not cell antibodies must be performed using ei-
interfere with obstetric care of the mother ther the mother’s serum or plasma or a
or the infant. If the birth provider intends sample from either the infant donor or the
52,56
to collect the cord blood, plans should be cord blood collection. White cells from the
made in advance to abandon the collec- cord blood must be typed for HLA (HLA-A
tion if care of the mother or the infant re- and -B antigen testing, and DNA-based
quires the care provider’s attention in- Class II typing) by a laboratory accredited
stead. Cord blood collected after delivery by the American Society of Histocompati-
of the placenta is preferably initiated bility and Immunogenetics (ASHI) or an
within 15 minutes of parturition, if ade- equivalent accrediting organization.49,52 In
quate volumes are to be obtained.97 To order for HPC-C to be a feasible alternative
minimize the risk of bacterial contamina- to allogeneic HPC-M or HPC-A transplants,
tion, the surface of the cord should be it is essential to have a frozen inventory of
cleaned with alcohol, then disinfected, in ready-to-use HLA-typed products.
a similar manner to the preparation of skin Samples, preferably from an attached
for a blood donation. A large-bore needle segment, should be frozen for pretrans-
connected to a blood collection bag con- plant confirmation of HLA type by the
taining CPDA, CPD, or ACD anticoagulant transplant facility. Many centers freeze a
is inserted into the umbilical vein so that sample of the mother’s serum to allow new
the placental blood drains by gravity into infectious disease tests to be done if they
the bag. Alternatively, a delivered pla- become available after the HPC-C has been
centa can be suspended above the collec- collected and are thought to be important
tion bag. CPDA or CPD are the preferred at the time the transplant is planned. The
anticoagulants because they are isotonic emergence of West Nile virus as a threat to

mothers, infants, and transplant recipients mor or T-cell reduction is under investiga-
is an example. A sample of the mother’s tion.102
blood containing cells or DNA (a dried filter Immunomagnetic Separation. Various
paper sample is adequate) may be useful if immunomagnetic separation techniques,
questions concerning the child’s HLA type direct or indirect, are available. Typically, a
arise. These samples from the mother were CD34 antibody is coupled to a magnetic
more useful when HLA typing was done by bead. This complex is incubated with mono-
serologic methods to investigate ambigu- nuclear cells, and the cells expressing the
ous types in the infant. CD34+ cell enumer- CD34 antigen bind to the antibody-coated
ation at the time of thawing may also be beads, forming rosettes. A magnet is ap-
performed to estimate the stem cell con- plied to separate the rosetting CD34+ cells
tent of the HPC-C graft but is useful only if from the nonrosetting cells. Bead detach-
a viability marker is employed so that only ment, which varies among methods, may
viable cells are counted. be accomplished through anti-Fab frag-
The desire to increase the dose of stem ments or enzymatic treatment (eg, chymo-
103
cells in HPC-C has fueled interest in ex-vivo papain). The Isolex 300 system (Baxter
expansion of the cells in a laboratory set- Healthcare Immunotherapy Division, Irvine,
ting.98 Early clinical trials with expanded CA), a magnetic cell separator, is a semi-
cord blood progenitors are promising, hav- automated instrument for clinical-scale
ing shown successful engraftment; how- CD34+ selection applications. This method
ever, no improvement in patient survival uses antibody-coated Dynal paramagnetic
has been reported.99,100 Ex-vivo expansion of beads to rosette the CD34+ cells. A fully au-
cord blood is an area of active experimental tomated device (Isolex 300i, Baxter) and a
investigation. 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-
Processing of Hematopoietic face.105 One such method, magnetic cell
Progenitor Cells sorting (MACS, Miltenyi Biotec, Bergisch
Techniques for Cell Selection and/or Gladbach, Germany), was first introduced
Purging of Hematopoietic Progenitor Cells on a small scale by Miltenyi. 1 0 6 The
CliniMACS (Miltenyi Biotec) is a clinical-
Selection of the CD34+ Cells scale version of the MACS system and is
As an HPC undergoes differentiation and available in the United States for investiga-
maturation, CD34 antigen levels decrease. tional use only. This system employs anti-
This property, coupled with the use of MoAbs body-conjugated iron-dextran microbeads.
specific for the different epitopes of the The magnetically stained cells are sepa-
CD34 molecule, permits physical separa- rated over a high gradient magnetic column
tion procedures. There are several meth- and a microprocessor controls the elution
ods of immunoselection available, such as of the CD34+ cells.
fluorescence-activated cell sorting (FACS) Counterflow Centrifugal Elutriation.
101
and immunomagnetic beads. Selection Counterflow centrifugal elutriation (CCE) is
of CD34+ HPCs may be associated with a a method of separating cells based on their
reduction of tumor cells (autologous grafts) size and density. A continuous-flow centri-
or T cells (allogeneic grafts). The clinical fuge (Beckman Instruments, Palo Alto, CA,
utility of CD34 and AC133 selection for tu- and Gambro, Golden, CO) and unique

111
chamber design allow for the basic separa- with tumor purging. Numerous tech-
tion principle: two opposing forces (centrif- n i q u e s f o r a u t o l o g o u s p u r g i n g a re
ugal force and counter media flow) acting available. The goal of all purging meth-
upon cells at the same time. As cells are ods— whether physical, immunologic, or
pumped into the chamber (centripetal di- pharmacologic—is the destruction or re-
rection), they align according to their sedi- moval of the malignant clone while main-
mentation properties. With adjustment of taining the efficacy of the HPCs necessary
the counterflow rate, the centrifugal and for engraftment.112
counterflow forces are balanced, and a gra- Pharmacologic Techniques. In-vivo
dient of flow rates exists across the cham- antineoplastic therapy produces greater tu-
ber. By gradually increasing the flow rate of mor cell kill because of the differential sen-
the medium or decreasing the speed of the sitivity of malignant cells over normal cells.
rotor, cells can be eluted out of the cham- In-vitro pharmacologic purging is an effort
ber and collected. Smaller, slower sedi- to expand this therapeutic ratio. In-vitro
menting cells elute first. Although this tech- purging allows for dose and exposure in-
nique is used primarily for T-cell depletion, tensification without concern for organ tox-
CCE has been applied to CD34+ cell selec- icity because higher drug concentrations
tion. CD34+ cells are heterogeneous and can be used on isolated hematopoietic
will elute in subset fractions that are useful grafts ex vivo, which then can be adminis-
for repopulation experiments and ex-vivo tered in vivo. However, the drug concentra-
107
expansion trials. tion must be at nontoxic levels before re-
infusion. Activated oxazaphosphorines
(4-hydroperoxycyclophosphamide, mafos-
Autologous Tumor Purging famide) were the most frequently used
Purging or negative selection refers to the compounds but are generally no longer in
removal of tumor cells that may contami- use in the United States. Other chemo-
nate the autologous graft. In patients with therapeutic agents have been investigated
hematopoietic disease or malignancies in preclinical models but are not in clinical
that frequently involve the marrow (eg, use. Results from purged autologous trans-
lymphomas), minimal residual disease plants for AML with either of these drugs
contributes to relapse.108 Although autolo- showed prolonged aplasia with less than
gous HPC-A collections have a lower prob- 1% CFU-GM survival.112
ability than HPC-M of tumor contamina- Physical Techniques. Separation meth-
tion and fewer tumor cells/mL, they still ods based on cell size and density through
may contain large numbers of viable tu- gradient-generating reagents or CCE do not
mor cells. Studies have demonstrated that achieve adequate tumor cell depletion.111
tumor cells may be mobilized from the However, the combined use of physical and
marrow into the peripheral circula- immunologic techniques requires further
tion. 1 0 9 , 1 1 0 Whether tumor purging de- study.
creases the likelihood of relapse is contro- Immunologic Techniques. The develop-
versial. Because some purging methods ment of MoAbs coupled with the discovery
may damage HPCs, the probability of re- of tumor-associated antigens opened the
sidual disease or relapse should be care- field for immunologic purging.113 MoAbs
fully balanced against the higher graft may be directed at tumor-specific antigens
failure rate or increased mortality from or cell-differentiation antigens.111 The im-
prolonged aplasia that may be associated munologic techniques differ primarily by

the method of target cell removal. MoAbs Table 25-3. Methods of T-Cell Depletion
are used in conjunction with complement,
bound to toxins, or coupled to magnetic Nonimmunologic
beads. The choice of MoAb and the hetero- Counterflow centrifugal elutriation
geneity of antigen expression on the target Pharmacologic/cytotoxic drugs
cell affect the success of the purge or the Immunologic
Monoclonal antibodies with or without
level of depletion. Many investigators em-
complement
ploy a cocktail of MoAbs in an effort to en-
Immunotoxins
hance the purging efficiency.114,115 Comple- Immunomagnetic beads
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 Research and developmental efforts have
by direct or indirect method, employs an focused on determining the optimal level of
antibody-coated magnetic bead to target T-cell depletion. Dreger et al115 reported a
the antigen or antigens of interest. A recent comparison of T-cell depletion methods
study described a 5-log tumor cell deple- (CAMPATH-1 plus complement, immuno-
tion with two cycles using the indirect magnetic CD34+ selection, and biotin-
method for B-cell lymphoma. Colony assays avidin-mediated CD34+ selection). The
showed only a 20% reduction in CFU-GM immunomagnetic method provided a 4-log
and multipotential CFU (CFU-GEMM).114 reduction in T cells vs a 3.1-log reduction
with the biotin-avidin method. MoAb treat-
ment with autologous complement yielded
T-Cell Depletion a 2.1-log reduction. The challenge of mini-
GVHD is a significant complication in mizing the severity of GVHD and maintain-
allogeneic HPC transplantation. Because ing the GVL effect is ongoing. Additional
the disease process is mediated by donor studies are examining the role of sub-
T cells that are reactive against the host, populations of T cells and/or cytokines in
depletion strategies are used to target and potentiating the GVL effect.117
remove these cells in an effort to decrease
the incidence or at least lessen the sever- ABO Incompatibility
ity of GVHD. Many of the techniques out- Although HLA compatibility is crucial in
lined for positive selection and tumor purg- the successful engraftment of myelosup-
ing are applicable to T-cell depletion (Table pressed or ablated patients, ABO compat-
25-3).17 ibility is not. Pluripotent and very early
In T-cell-depleted grafts, two major areas committed HPCs do not possess ABH anti-
of concern are graft failure and recurrent gens, allowing engraftment to occur suc-
leukemia. Most instances of graft failure cessfully regardless of the ABO compati-
(initial or late) are caused by immunologic bility between the recipient and donor.
rejection. Early transplants involving de- ABO incompatibility does not affect neutro-
pleted grafts in patients with CML showed phil or platelet engraftment, graft failure,
a 50% or greater relapse rate. In contrast, or rejection. However, delayed red cell
patients who developed GVHD had a lower engraftment may occur after a major
relapse rate. Subsequent clinical and inves- ABO-incompatible transplant and delayed
tigational data provided evidence of an im- hemolysis may occur after a minor ABO-
mune-mediated GVL effect.116 incompatible transplant where the donor

has antibodies to the recipient’s blood cyte and platelet production are not
cells (Table 25-4).118 affected.119 Red cell engraftment may be
delayed to ≥40 days after the transplanta-
Major ABO Incompatibility tion.120 Red cells used for transfusion of the
Major incompatibility occurs when the recipient must be compatible with both the
recipient has ABO antibodies or other red donor and the recipient. In some centers,
cell antibodies, such as anti-D or anti- group O red cells are given to all major
Kell, against the donor red cell antigens. ABO-incompatible transplant recipients
The HPC preparation can be processed to in order to avoid confusion. Recommen-
remove mature red cells. The group O re- dations for optimal blood component se-
cipient who receives a group A graft may lection are shown in Table 25-5.
continue to produce anti-A and anti-B for
3 to 4 months or longer in rare instances, Minor ABO Incompatibility
and the presence of anti-A will delay Minor ABO incompatibility occurs when
erythropoiesis by the group A graft; group the donor has antibodies against the re-
A red cells appear in the circulation when cipient red cell antigens (such as a group
the recipient’s anti-A disappears. Granulo- O donor to a non-group O recipient). Be-
Table 25-4. Potential Problems with ABO- and Rh-Incompatible HPC

Transplantation
Example
Incompatibility Donor Patient Potential Problems
ABO (major) Group A Group O 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 Hemolysis of donor RBCs from newly
(with engrafted HPCs (rare), or delayed
anti-D) RBC recovery
Other RBC Negative Positive Hemolysis of patient RBCs by donor an-
antigens tibodies in plasma produced after
engraftment
Positive Negative Hemolysis of donor RBCs from newly
(with engrafted HPCs (rare), or delayed
antibody) RBC recovery

600
Table 25-5. Transfusion Support for Patients Undergoing ABO-Mismatched Allogeneic HPC Transplantation

Phase I Phase II Phase III
First
Mismatch All Choice Next Choice All
Recipient Donor Type Components RBCs Platelets Platelets* FFP 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
121
*Platelet concentrates should be selected in the order presented. Modified from Friedberg et al.
Phase I = the time when the patient/recipient is prepared for HPC transplantation.
Phase II = from the initiation of myeloablative therapy until:
For RBC—DAT is negative and antidonor isohemagglutinins are no longer detectable (ie, the reverse typing is donor type).
For FFPs—recipient’s erythrocytes are no longer detectable (ie, the forward typing is consistent with donor’s ABO group).
Phase III = after the forward and reverse type of the patient are consistent with donor’s ABO group.
Beginning from Phase I, all cellular components should be irradiated and leukocyte reduced.
124
fore infusion of a graft from a plasma-in- nologic tolerance. This tolerance devel-
compatible donor, the plasma may be re- ops in successful transplants and allows
moved to avoid infusion of preformed normal immune reconstitution. Full chim-
anti-A and/or anti-B. Minor ABO-incom- erism (replacement of host marrow cells
patible transplants can be characterized by entirely by donor cells) usually occurs in
rather abrupt onset of immune hemolysis, successful HPC-M transplantation.
which begins about 7 to 10 days after trans-
plantation and may last for 2 weeks. The
Processing in the Presence of ABO
direct antiglobulin test (DAT) is positive;
Incompatibilities
anti-A and/or anti-B can be recovered in
the eluate and hemoglobinemia and/or Major ABO Incompatibility. Two ap-
hemoglobinuria may occur. An additional proaches have been employed with major
30% of such transplant recipients develop ABO incompatibilities: 1) removal of or
a positive DAT without experiencing gross decrease in the isoagglutinin level in the
hemolysis. This phenomenon is the result recipient or 2) removal of the red cells in
of red cell antibodies produced by pas- the HPC-M collection. Processing is not
senger B lymphocytes in the stem cell generally required with HPC-A because
121
graft. Non-ABO antibodies from passen- such a small volume of red cells is usually
ger lymphocytes in HPC transplants have included.
122
also been reported. Although transient, Attempts to remove or decrease the iso-
the hemolysis may persist for up to 2 weeks agglutinin titer in recipients involve the use
and may require transfusion with group O of one or more large-volume plasma ex-
red cells. Minor ABO-mismatched HPC changes with or without the subsequent in-
transplants may be associated with a fusion of donor-type red cells as a secondary
higher risk of hemolysis, possibly caused effort to absorb any additional isoaggluti-
123
by greater B-cell content. In all cases, nins.125,126
plasma used for transfusion should be Other approaches to ABO-incompatible
compatible with both the donor and the products include red cell depletion. The
recipient. In some centers, group AB plasma most prevalent method in use is red cell
products are given to all minor ABO-in- sedimentation.
compatible transplant recipients in order Some institutions accomplish red cell
to avoid confusion. RBC transfusions, be- depletion using density gradient separation
ginning with the conditioning regimen, and cell washers (Gambro BCT, Lakewood,
should be of donor type. Recommenda- CO); others use continuous-flow apheresis
tions for optimal blood component selec- machines such as the Spectra (Gambro) and
tion are shown in Table 25-5. the Fenwal CS-3000 (Baxter Healthcare),
with mononuclear cell recoveries of 94%
and 87% and red cell depletion of 99% and
Chimerism 98%, respectively.
In spite of intensive pretransplant chemo- Minor ABO Incompatibility. Theoreti-
therapy and irradiation, some of the host’s cally, the level of hemolysis is dependent on
hematopoietic cells may survive and sub- the volume of HPC-M infused relative to
sequently coexist with cells produced by the recipient’s plasma volume and the IgM
the transplanted HPCs. This dual cell pop- titer of the donor. Incompatible plasma can
ulation, called partial hematopoietic be easily removed by centrifugation.
chimerism, may have an effect on immu- Plasma removal using this method removes

approximately 75% of the plasma volume This is particularly important with HPC-M,
while recovering >70% of the initial nucle- which is frequently collected in an open
ated cell count. Plasma removal is not gen- system, and with products that require
129-134
erally required in HPC-A because the vol- multiple manipulations (Table 25-6).
ume of incompatible plasma is usually small. Culture growth can result from contami-
nation during collection or product pro-
Umbilical Cord Blood Processing cessing, or as a result of an infected cathe-
Advances in the processing of umbilical ter or the patient’s sepsis. Skin commensals
cord blood cells have solved the early are the predominant isolates from these
problems of unacceptably high progeni- cultures. In all cases it is important to
tor cell losses with manipulation.127 Unlike identify the source, the degree of contam-
cryopreservation laboratories that store ination, and the causative organism,
relatively few autologous or allogeneic given the fact that this product is in-
HPC-M or HPC-A products (typically, less tended for transplantation in an immuno-
than a few hundred products at any one compromised recipient. Each product
time), a successful cord bank would be must be tested for microbial contamina-
expected to store thousands of products tion at least once during the course of
for prolonged periods. Therefore, attempts processing, usually just before freezing or
to reduce umbilical cord blood bulk (and, infusion with allogeneic transplants.48 A
therefore, storage space, liquid nitrogen positive culture does not necessitate im-
requirements, and cost) have been ac- mediate discard of the product because
tively pursued. Currently, many centers these are frequently irreplaceable cells.135
are following the approach used by the Thus, positive results need to be reviewed
New York Blood Center, which involves by the appropriate physician on a case-
sedimentation and volume reduction be- by-case basis and sensitivity tests ordered
fore cryopreservation.128 to better define the organism and to allow
for appropriate clinical management at
Cultures for Microbial Contamination the time of transplant. This should also
Sterility is essential in blood products for serve as the impetus to review all proce-
infusion to immunosuppressed patients. dures and techniques to ensure that they
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- proach to CD34 analysis have prompted

tion. several collaborative groups such as the In-
ternational Society for Cellular Therapy
(ISCT) to propose guidelines for CD34+ cell
Stem Cell Enumeration: CD34 Analysis,
determination by flow cytometry.56,141 Alter-
Aldehyde Dehydrogenase Activity, and Others
nate approaches are available and will con-
As stem cells differentiate into hemato- tinue to be developed and investigated as
poietic cells, CD34 surface markers ap- more is discovered about the CD34 antigen
pear. As the cells continue to differentiate and other markers for the pluripotent stem
142
and acquire other surface markers, CD34 cell.
expression diminishes. Stages of differen- A second method for enumeration of
tiation may be studied by the expression early cells has gained FDA clearance. The
or co-expression of other antigens or cytosolic expression of aldehyde dehydro-
properties. There are currently two assays genase (ALDH) in stem cells was found to
for CD34 that are FDA-cleared for in-vitro be the protective property of HSCs in cyclo-
diagnostic use. These CD34+ measure- phosphamide treatment of patients and in
ment methods are the most widely used preservation of stem cells in graft purging
for the evaluation of grafts, have the lon- with 4HC. Flow cytometric analysis of
gest history of use, and show the most ALDH-positive cells with the enzymatic ex-
clinical correlations in the marrow trans- citation and trapping inside the stem cells
plantation literature. of a fluorescent substrate (because the
The principle of gain and later loss of fluorophore becomes charged and does not
CD34 expression has been applied to evalu- diffuse freely) allows measurement of a via-
ate the quality of HPC grafts. The current ble population enriched in colony-forming
approach is to use a method based on progenitor cells. Both long-term repopulat-
multiparameter flow cytometry to deter- ing and committed progenitor cells are
mine the number of CD34+ cells and to cal- enriched and the ALDHbr contains the pop-
culate a subsequent CD34+ dose based on ulation responsible for hematopoietic engraft-
the recipient’s weight. Numerous flow ment of NOD-SCID mice.143,144 This method
br lo
cytometric analysis methods for CD34 enu- to enumerate ALDH SSc cells identifies
meration exist.56,136,137 Because these meth- only cells with intact membranes and is
ods differ, correlation of CD34 dose values well correlated with engraftment in post-
from site to site is often unreliable. Multiple thaw HPC-A grafts.145 Another measure-
studies have documented the variability of ment technique involving the use of Flk-2
CD34 analysis methods and results and and Thy 1.1lo showed that Flk-2–, Thy 1.1lo
have examined possible causes of the vari- cells represented long-term repopulating
lo
ability.56,57,138,139 Because of the “rare event” cells, whereas Flk-2+, Thy 1.1 cells were
nature of CD34 enumeration, several pro- short-term engraftment-enhancing cells.
cedural components play a critical role in These tools may be developed for use in fu-
the assay. Selection of the CD34 antibody ture graft engineering approaches to finely
146
clone, fluorescent conjugate, lysing solu- separate early cell populations.
tion, lyse-wash format, gating strategy, and
the number of events analyzed are some of
Colony-Forming Cell Assays
the factors that influence the end result.140
Growing awareness of, and concern Culture systems are available that can
about, the need for a standardized ap- demonstrate in-vitro proliferative capacity

of a hematopoietic sample. It is thought HPC-A, HPC-M, and HPC-C are frozen

that short-term (generally within 2 weeks) and stored using the same techniques. The
repopulating potential is produced from general parameters include cryopreservation
the committed HPCs. Long-term repopu- in dimethylsulfoxide (DMSO) and a source
lating ability is thought to be a result of of plasma protein with or without hydrox-
the pluripotential HPCs that are princi- yethyl starch (HES), cooling at 1 to 3 C/
pally necessary for a complete and sus- minute, and storage at –80 C or colder. Vari-
tained engraftment. Cultures may be use- ations on this technique include the con-
ful to assess engraftment potential; however, centration at which the cells are frozen, the
because media, culture techniques, and amount and source of the plasma protein,
148
colony identification are quite variable, and the cooling techniques used. Most of
interinstitutional comparisons are diffi- these variations probably have little effect
cult. Reported CFU-GM doses below on the survival of the HPCs as shown by the
which engraftment may be delayed range consistent engraftment of cryopreserved
from 1.5 × 105 to 5.0 × 105/kg.147 components. However, cryopreservation
Long-term cultures are not routinely used results in the loss of an undefined but po-
clinically because of the 5- to 8-week incu- tentially substantial proportion of HPCs, and
bation requirements. However, they may be delay in engraftment can occur if the com-
useful in evaluating developmental proce- ponent being frozen has borderline quanti-
dures involving product manipulation. 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-
Freezing and Storage tion techniques.
Although easily performed by many trans-
plant centers, HPC cryopreservation and
Allogeneic Products
reinfusion are not without risk of loss of
HPCs. The infusion of cryopreserved cells The collection of allogeneic marrow is
also results in risks to the recipient, many generally timed to coincide with the com-
of which are life-threatening, and some of pletion of the recipient’s preparative regi-
which are not well understood. The loss of men. Because of the brief storage period
HPCs from cryopreservation and the ef- required, the product is maintained in the
fect of this loss on engraftment speed have liquid state. Unseparated HPC-M can be
not been quantified. There are multiple stored in the liquid state for up to 3 days
aspects to successful cryopreservation of at either 4 C or 22 C without any signifi-
HPCs. Each of these variables affects the cant loss in viability of either uncommit-
recovery of HPCs during the cryopreser- ted or committed progenitors. The ability
vation steps and, as with any manufacturing to store unmanipulated marrow under
process, must be rigidly controlled for re- these conditions is vital in the context of
producible results. The question that still the NMDP or other transplant registries
faces cryopreservation facilities is whether where unrelated HPC-M units are col-
other solutions or processing techniques lected and then transported great dis-
will provide better cryosurvival of cells, at tances for transplantation. Similarly,
less cost, with greater simplicity, or with HPC-A units may be stored in the liquid
less toxicity to either the recipient or to state under the same conditions as HPC-M
149,150
the cell type being frozen. for up to 3 days at 4 C. The cell con-

centration is important in determining dehydration can be minimized by a slow

the storage time and temperature that the cooling rate and the addition of a cryo-
cells will tolerate without damage. High protective agent such as DMSO. Cryo-
concentrations of white cells in liquid protectants such as DMSO prevent the for-
storage at room temperature quickly ex- mation of large ice crystals within the cells
pend the supplies of oxygen and glucose by penetrating the cell and providing some
and cause a decrease in pH in the storage balance of the external hyperosmolar sol-
medium that is toxic to the cells. A lower ute conditions in the freezing medium sur-
concentration of cells (higher volume of rounding the cell. 153 A commonly used
suspending medium, often autologous cryoprotectant consists of 20% DMSO and
plasma) and refrigerated storage are pro- 20% plasma or albumin prepared in a buf-
tective. fered electrolyte solution or tissue culture
media. The plasma or albumin provides a
Autologous Products protein source, which aids in preventing cell
damage during freezing and thawing. The
Because of the length of time required for
cryoprotectant is combined with an equal
treatment and/or HPC product collection,
volume of product immediately before
autologous products are usually cryopre-
freezing, resulting in a final cell suspension
served and stored until the time of infu-
containing 10% DMSO and 10% plasma.
sion. Cryopreservation also allows HPCs to
Phase transition time is a physical phe-
be collected while the patient is in remis-
nomenon of freezing water where forming
sion and stored for use in case a relapse
ice crystals release heat energy into the cell
occurs (prophylactic storage). At present,
solution that needs to be offset by contin-
no expiration date has been defined for
ued cooling to prevent crystallization of ice,
these products; however, HPC-M stored
thawing, and recrystallization during this
for 11 years has been used for transplanta-
energy exchange, with damage to the cell
tion, with sustained engraftment.151
membrane.152 This method of freezing re-
quires physical conditions that support this
Computer-Controlled Cryopreservation process. This may be accomplished by a
The purpose of cryopreservation is to freeze computerized programmable freezing
cells in such a way as to allow for their chamber that adds liquid nitrogen in suffi-
long-term storage with a minimal loss of cient amounts to push the freezing process
cell viability or reconstitutive ability upon through the phase change smoothly. The
thaw. The main obstacle to maintaining freezer is programmed to cool cells at an
viability during cryopreservation and optimal rate of 1 to 3 C/minute until a tem-
storage is the formation of intracellular perature of –90 to –100 C is reached.154-156 With
ice crystals along with an increase in ex- the combination of a cryoprotectant and a
ternal osmolarity, causing exit of water from programmed rate of freezing, cryopreserva-
the cell, both resulting in cell lysis. Ther- tion and long-term storage of HPCs are
mal shock, the time required for phase possible, with minimal damage to the cells.
change (liquid state to solid state), and
the posttransition freezing rate also pres-
Passive Controlled-Rate Freezing
ent specific conditions to be managed for
cryopreservation.152 Mechanical, methanol bath immersion,
The adverse effects caused by the forma- or “passive controlled-rate” freezing is ad-
tion of intracellular ice crystals or by cell vocated by some investigators as a simple,

reliable, cost-effective, and validated than with liquid phase. The advantage of
method of managing the physical condi- vapor phase storage is the possibly de-
tions of cooling as an alternative to a con- creased risk of cross-contamination that
trolled-rate device.157-159 The principle is has become an issue with liquid phase
that products can be cooled without the storage.161
aid of a programmable freezer or liquid
nitrogen if the conditions of freezing are Storage of Untested or Infectious Products
predetermined, measured, and performed Regulations [21 CFR 1271.60(a) and
according to standard operating proce- 1271.65(a)] and standards48(p35),49 require al-
dures. The products are stored in a –80 C ternative storage for products that are un-
mechanical freezer or colder after the ini- tested or have a positive disease marker
tial freeze. HPCs stored in this fashion test result. Some institutions comply by
have successfully engrafted after as long storing all products in vapor phase and
as 2 years of storage.159 In some cases, the physical separation by category of prod-
combination of the metal canisters the uct. Other institutions use an overwrap,
bags are placed in, the bag itself, and the placing the component in an outer plastic
volume of product frozen produces a bag that is sealed before storage. A third
freezing rate of approximately 3 C/min- alternative is a separate storage compart-
ute, which falls within the optimal range ment for units that are untested or posi-
previously discussed.160 The major deter- tive for infectious disease markers.
rent to use of mechanical freezers is fear
of mechanical failure and the lack of data Final Suitability Criteria
on long-term storage and engraftment.
Before each autologous or allogeneic unit
is released, it must undergo review and
Frozen Storage meet the predetermined release criteria
described in the facility’s quality plan.
Although products frozen in a mechanical
The pertinent facts describing donor se-
freezer are stored at –80 to –150 C, prod-
lection, product collection, processing,
ucts cryopreserved using a programmable
and storage must be reviewed. The con-
freezer generally are stored in a liquid ni-
tainer label and associated information
trogen freezer. The storage temperature
must accurately reflect the product classi-
achieved with vapor phase, although not
fication, storage/preservative medium in
as cold as liquid phase, averages –140 C, a
the final container, the product content
temperature that has been shown to allow
(usually cell content), and results of re-
for viable long-term marrow storage.151
lease testing including infectious disease
The major drawbacks to vapor phase stor-
tests. If exceptions to standard practice
age include the potential for large fluctua-
were made, they must be explained either
tion in temperature when the freezer is
on the label or in the accompanying re-
entered as well as the variation in storage
lease material.
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 Transportation and Shipping
temperatures of the stored products in In some cases, a hematopoietic component
cases of electrical or liquid nitrogen sup- must be transported from one center to
ply emergencies is significantly shorter another. The product must be positively

identified upon its removal from inven- ing; others immerse the bag (all but the
tory in preparation for shipping. In all access ports) directly into sterile water or
cases, precautions should be taken to pro- saline at 37 to 40 C.152 The bag is kneaded
tect the component from rough handling, gently until all solid clumps have thawed.
out-of-range temperatures, X-ray exami- The cells are then infused (usually 10-15
nation, breakage, and spillage. The ship- mL per minute). Products may also be
ping container must undergo quality con- washed and resuspended in the laboratory
trol to ensure that it is capable of holding before infusion to prevent cell aggregation.152
the expected temperature during ship- Side effects associated with infusions in-
ping. In the case of cryopreserved compo- clude nausea, diarrhea, flushing, brady-
nents, the use of a liquid nitrogen “dry cardia, hypertension, and abdominal pain.
shipper” is desirable. Such “dry shippers” In general, such side effects may justify
have liquid nitrogen absorbent material slowing, but not halting, the infusion until
between the walls of the container that al- the symptoms pass. A limit of 1 gram of
lows the inside of the container to main- DMSO per kilogram of body weight of the
tain temperatures in the range of –180 C patient at one infusion is recommended to
for up to 10 to 14 days if they are properly allow the patient to tolerate both the DMSO
filled with liquid nitrogen and shipped in and the volume infusion effects of the ad-
the upright position. Tipping or inversion ministration. Sudden and severe hypoten-
of the container during shipping permits sion can occur in the absence of adequate
the liquid nitrogen to drain out, allowing antihistamine premedication. The patient
the container to warm toward ambient should receive fluids and treatment to en-
temperature. 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-
Thawing and Infusion tions. If the total infusion volume exceeds
For all components, final identification is 10 mL/kg of recipient body weight, many
done by the nurse or physician perform- centers divide the volume over a morning
ing the infusion. Flow through the central and an afternoon infusion or over 2 consec-
venous catheter is confirmed and the cells utive days.
are infused by gravity drip, calibrated It is best to collect postthaw laboratory
pump, or manual push with or without an samples directly from the patient’s infusion
in-line filter (a standard 170-micron red bags instead of freezing separate individual
cell infusion filter is acceptable). Although specimens because these samples are iden-
DMSO was thought to be toxic to HPCs, it tical to the infused product.163
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 Evaluation and Quality Control
DMSO ex vivo at 22 to 37 C may be harm- of Hematopoietic Products
ful to HPCs. To minimize the exposure of
thawed cells to DMSO, many centers rap- Cell Counts
idly thaw one bag at a time near the bed- Each hematopoietic product is analyzed
side. Some centers place product bags in to determine the total cell concentration
secondary containment bags before thaw- and the mononuclear cell concentration,

which are used to calculate the number of ence of tumor cells may be associated
167
cells per kilogram (of the recipient) or cell with a reduced disease-free survival.
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, Regulations
they are used to calculate the percent re-
covery, providing a quality control mea- In 1997, the FDA announced a new com-
sure for processing procedures and prehensive approach to the regulation of
equipment.164 cellular and tissue-based products. 1 6 8
In general, automated cell counts provide HPCs from placental/umbilical cord
the most rapid and accurate value. How- blood and peripheral blood were covered
ever, platelet/cellular aggregates in HPC-A by this proposal. Many of the policies,
or fat globules in HPC-M specimens can proposed regulations, and guidance doc-
falsely decrease or increase cell counts.135,165 uments needed to implement this ap-
47,169-173
In such cases, manual cell counts may be proach have been published.
preferable. It is important that cell counts These regulations require establishment
are not overestimated because this may re- registration and listing of facilities collect-
sult in inaccurate estimation of time to ing, processing, or distributing tissue or cell
engraftment or graft failure. 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-
Engraftment Data
ation. The cGTP regulations (found in Title
Ultimately, engraftment of neutrophils, 21 CFR 1271.145 to 320) are specific in-
platelets, and red cells is the primary de- structions intended to ensure that facilities
terminant of graft quality. Monitoring and establish and maintain a quality program
documenting days to engraftment for that documents that personnel, proce-
neutrophil and platelet lineage are re- dures, facilities, equipment, supplies, and
quired by FACT49 and AABB48(p41) for ac- reagents are set up and maintained in an
creditation. acceptable and a standard manner. Process
control is required: there must be written
procedures and documented validation.
Products are required to be collected,
Tumor Cell Detection
tested, labeled, and stored in ways that pre-
Tumor cell detection techniques have been serve their identity and prevent contamina-
developed to screen products suspected tion and cross-contamination. It may be
of tumor cell contamination and to evalu- necessary to demonstrate regulatory com-
ate purged products. The majority of these pliance during FDA inspection visits. Re-
assays use MoAbs that specifically bind porting of adverse events (when applicable)
tumor antigens. Detection and quantita- is also required within 15 days of receipt of
tion can be done by flow cytometry, immuno- the information if the event is serious as de-
fluorescence, or immunohistochemical fined in 21 CFR 1271.350. This body of reg-
staining. Sensitivity varies with technique ulations represents a major effort on the part
from 0.1% to 0.0004% of cells examined.166 of the FDA to ensure that tissue and cell
Preliminary studies indicate that the pres- therapy products are safe for the recipient.

ing Committee and the European Group for

Standards Blood and Marrow Transplantation (EBMT).
Blood 2000;95:3702-9.
The AABB and FACT have published sepa-
3. Report on state of the art in blood and mar-
rate, but substantially similar, standards row transplantation—the IBMTR/ABMTR
for HPCs.48,49 The AABB Standards for Cel- summary slides with guide (2002). IBMTR/
lular Therapy Product Services addresses ABMTR Newsletter 2002;9(1):4-11. [Available
at http://www.ibmtr.org/newsletter/pdf/
the collection, processing, storage, and 2002Feb.pdf.]
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such as the NMDP also publish voluntary myeloablative preparative regimens for allo-
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and current indications. Oncology (Hunt-
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78-81.
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Chapter 26: Tissue and Organ Transplantation
Chapter 26
26
Tissue and Organ
Transplantation
I
N RECENT YEARS, the numbers of Service Guidelines, and the standards,
cornea, bone, skin, heart valve, and other guidelines, and technical manuals of other
1-3 7-14
tissue donations and transplants have national or local organizations.
4
exceeded those of solid organs, such as
kidneys, livers, hearts, and lungs. The
hospital blood bank, transfusion service,
community blood center, and regional Transplant-Transmitted
blood center are uniquely qualified to Diseases and Preventive
provide essential support for organ and
tissue transplantation and to serve as tis- Measures
5
sue banks (Table 26-1). It is common for The widened availability of tissue and or-
hospital blood banks to provide transfu- gan grafts has encouraged new clinical
sion support for organ and tissue recipi- uses and highlighted not only their effec-
ents and, in some cases, to store, keep records tiveness and advantages but also their
of, and dispense tissue for allografts. AABB drawbacks, side effects, and complications.
Standards for Blood Banks and Transfu- Organs and tissues can transmit bacterial,
6(pp8,14,15,60,79)
sion Services addresses the re- fungal, viral, and prion diseases from the
ceipt, storage, transportation, and records donor to the recipient3,15-26 (Table 26-2),
of tissue allografts. Additional guidance for but careful donor screening and testing,
the collection and preparation of tissue along with disinfection and sterilization
and organ allografts is available from fed- steps for specific tissues, can markedly re-
eral and state regulations, Public Health duce the risk.
617

Table 26-1. Skills and Experience Appropriate for Institutions Undertaking

Tissue Banking
■ Community support
■ Public accountability
■ Public education with a broad-based public information system
■ Donor recruitment
■ Counseling
■ Medical overview
■ Donor selection
■ Donor testing including automated virology testing to avoid transcription errors
■ Cellular cryopreservation
■ Temperature-controlled and monitored storage
■ Regulatory compliance
■ Transportation infrastructure
■ Financial relations with hospitals
■ Computerized inventory control
■ Record-keeping
■ Logistics management
■ Investigation of adverse reactions
■ Peer review of medical, scientific, and operational practice
■ Recipient matching
■ Concern over the balance of the adequacy and safety of supply
■ Reputation for dependable service
■ Commitment to research and development
■ 24-hours-per-day, 7-days-per-week operation
Reproduced with permission from Warwick et al.5
Risk Reduction for Tissues care provider, and review of medical

Crucial to the safety of transplanted tissue records. Review includes an evalua-
is an evaluation of the potential donor’s tion (although not necessarily rejec-
eligibility. Listed below are the questions, tion for asterisked items*) of the po-
examinations, and tests undertaken to en- tential donor for:
sure that material from the potential donor a. History of infection,* malignant
poses a low threat of disease transmission. disease,* or neurodegenerative
Additional measures apply to reproductive disease
10,11
tissue donors. b. History of autoimmune processes*
1. Review of health history, through in- c. History of exposure to hormone
terviews with next of kin and/or sig- derived from human pituitary
nificant other, and possibly health- gland or dura mater transplant

Chapter 26: Tissue and Organ Transplantation 619
Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and
15-26
Tissue Allografts
Allograft Infectious Disease/Disease Agent
Bone HIV-1
Hepatitis C
Hepatitis, unspecified type
Bacteria
Tuberculosis
Cornea Hepatitis B
Creutzfeldt-Jakob disease
Rabies
Cytomegalovirus (?)
Bacteria
Fungus
Dura mater Creutzfeldt-Jakob disease
Heart valve Hepatitis B

Tuberculosis
Skin Bacteria
Cytomegalovirus (?)
HIV-1 (?)
Pericardium Creutzfeldt-Jakob disease

Bacteria
Solid organ (eg, kidney, liver, HIV-1

heart) 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)

Table 26-2. Infectious Diseases Reported to Have Been Transmitted by Organ and
15-26
Tissue Allografts (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
2. Review for evidence of high-risk be- 4. Physical examination to detect:

havior (exclusion for any of the do- a. Evidence of intravenous drug use
nor-deferral criteria included in items b. Jaundice
9c, 9e, and 10 of Standard 5.4.1A of c. External signs of infection
Standards for Blood Banks and Trans- d. Signs of AIDS
fusion Services.
6(pp62-65)
5. Review of results of autopsy exami-
3. Serologic testing on suitable blood nation, if performed.
10
specimens (see below for more de-
tail).
Consent and Donor Eligibility
a. Hepatitis B surface antigen
(HBsAg) Written consent for clinical use of any tis-
b. Antibodies to human immuno- sue or organ must be obtained from a liv-
deficiency viruses 1 and 2 (anti- ing donor or from the next of kin of a de-
HIV-1 and -2) ceased donor (formerly referred to as a
c. Antibody to hepatitis C virus cadaveric donor), except when corneas are
(anti-HCV) procured under statutory consent. State
d. Antibody to human T-cell lym- and federal referral statutes and regula-
photropic virus types I and II tions exist, mandating that hospitals 1)
(anti-HTLV-I and -II) maintain policies for notifying organ pro-
e. Serologic test for syphilis curement organizations and appropriate

tissue banks, including eye banks, when Donation of organs and tissues does not or-
death of a patient has occurred or is im- dinarily cause delays in funerals or prevent
minent and 2) designate requestors to ap- family viewing of the body.
proach families about organ and tissue
donation. All applicable federal, state, and
Serologic Testing
local laws concerning the consent of next
of kin must be obeyed. When the next of Federal regulations require that donors of
kin signs consent for tissue donation, he tissues be tested for HBsAg, anti-HIV-1/2,
or she should specify which tissues may and anti-HCV, and given a syphilis screen-
be donated and whether the permission ing test, with tests licensed by the Food
includes tissue to be used for research or and Drug Administration (FDA). Testing
other specific uses. must be performed by a laboratory that is
Living donors and the families of de- certified under the provisions of the Clini-
ceased donors are not responsible for ex- cal Laboratory Improvement Amendments
penses involved in recovering and process- of 19887,8 or that has met equivalent re-
ing donated tissues and organs. Donors do quirements as determined by the Centers
not receive compensation for the donation, for Medicare and Medicaid Services. A
although donors of reproductive tissue are screening test approved for cadaveric
27
often compensated for their time, risk, and specimens must be used when available.
inconvenience, and donor families may re- National standard-setting organizations,
ceive incentives, such as a contribution to- such as the American Association of Tis-
ward funeral expenses, for donation from sue Banks (AATB),10 Eye Bank Association
12
deceased donors. Tissues can be recovered of America (EBAA), and the United Net-
13
up to 24 hours after death if the body is re- work for Organ Sharing (UNOS), may re-
frigerated within 12 hours of death. Tissues quire additional tests for infectious dis-
can be recovered up to 15 hours after death ease markers. The American Society for
if not refrigerated. Organs must be recov- Reproductive Medicine also has issued
ered from a donor whose neurologic death recommendations for gamete and embryo
has been declared and whose circulation donation. 11 For anonymous semen do-
has been maintained (see Table 26-3). Eyes nors, tests for anti-HIV-1/2 and anti-HCV
as a source of corneal allografts should be must be repeated on a sample obtained 6
removed as soon as possible after death to months after donation and the results
ensure viability of endothelial cells. found negative before semen can be re-
Each prospective deceased donor must leased for use. 9,10 Testing for anti-HBc
be evaluated against eligibility criteria for must also be performed on this sample to
the specific tissue(s) and organ(s) to be col- rule out hepatitis B infection at the time
lected; eg, deceased newborns are not suit- of collection. ABO typing is required for
able for bone donation because of their organ grafts. HLA typing is essential for
cartilaginous skeletal structure but may be organ grafts and hematopoietic progeni-
13 28
candidates for heart valve donation. Al- tor cells but not for tissues. Other tests,
though exceptions may be made in specific such as antibody to cytomegalovirus (anti-
cases, the medical eligibility of a donor is CMV), are also generally performed on or-
determined on the basis of absence of in- gan donors. For deceased donors, tests are
fection and malignancy as revealed by the optimally performed on a blood sample
medical history, physical examination, lab- obtained before administration of transfu-
oratory tests, and autopsy, if performed. sions or fluids; these patients may have re-

Table 26-3. Kinds of Donors Providing Organs and Tissue for Transplantation
Deceased Donor
Deceased Donor (Cardiorespiratory and
Living Donor (Neurologic Death) Neurologic Death)
Amniotic membrane Heart Bone

Bone Kidney Cartilage
Fetal tissues (mother is the Liver Cornea
donor) Lung Dura mater
Foreskin Pancreas (with or without Fascia lata
Kidney small intestine) Heart valve
Liver Marrow
Lung Pericardium
Marrow Skin
Milk Tendon
Pancreas Vascular tissue
Parathyroid (frequently
autologous)
Peripheral blood progenitor
cells
Reproductive tissue
Umbilical cord blood
Umbilical vein
ceived large volumes of replacement 1. The total volume of colloid (plasma,

fluids shortly before death, and the conse- dextran, platelets, or hetastarch) trans-
quent plasma dilution may cause false- fused in 48 hours plus the total vol-
negative results.19,20 ume of crystalloid infused in the
If donor serum collected before blood hour before the sample is obtained
transfusion or intravenous fluid adminis- exceed the patient’s plasma volume.
tration is not available from other sources, a 2. The sum of the volume of blood
pretransfusion sample is often available transfused (RBCs, whole blood) and
from the blood bank because blood banks colloid transfused in 48 hours plus
hold specimens collected for compatibility the total volume of crystalloid infused
for at least 7 days. For samples obtained af- in the hour before the sample is ob-
ter transfusion or infusion of intravenous tained exceed the patient’s blood
fluids, algorithms for determining the suit- volume.
ability of a donor sample are available.7,8 For Testing of cadaveric blood specimens can
example, the sample is not suitable for in- be complicated by postmortem hemolysis,
fectious disease marker testing if either one which can cause misleading test results (eg,
15,29
of the following situations exists: false-positive HBsAg). Many tests li-

censed for use on blood donor specimens freeze-dried to a low residual moisture con-
are not licensed for use with postmortem tent (6% or less by gravimetric analysis or
specimens, but licensed assays for post- 8% by nuclear magnetic resonance spec-
mortem specimens are available for anti- trometry), at room temperature for 5 years
HIV-1/2, HBsAg, and HIV-1/HCV nucleic (Table 26-4). Frozen bone is processed
acid.27 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-
Bone Banking grafts underscores the importance of recov-
Except for blood cells, bone is the most ery cultures of donated tissue.24 Freeze-
1
commonly transplanted tissue or organ. dried tissue has undergone extensive pro-
When bone grafting is needed, fresh auto- cessing to remove blood and marrow and
logous bone, usually removed from the may have been exposed to alcohol. Thus,
iliac crest during surgery, is generally con- the risk of disease transmission is reduced.
sidered the most effective graft material. Most bone allografts used in the United
As with blood, the use of autologous bone States are freeze-dried to simplify storage.
for graft material is not risk free and there Some grafts, whether frozen or freeze-dried,
may be morbidity and infectious compli- may be treated with gamma irradiation or
cations. The quantity of bone graft needed ethylene oxide to reduce the risk of infec-
for some surgical procedures may make tious disease transmission. Demineraliza-
the use of autologous bone impractical. tion of bone is believed to make its proteins
Allografts are used for these patients and and growth factors more readily available,
for patients in whom the prolongation of thereby enhancing its capacity to promote
surgery, extra bleeding, and potential healing and bone formation.
complications of autograft collection are The implantation of frozen bone allo-
considered undesirable. Bone allografts grafts has stimulated blood group antibod-
have achieved widespread clinical appli- ies that have been implicated in hemolytic
cation for acetabular and proximal femur disease of the newborn. Frozen, unpro-
support in revisions of failed hip prosthe- cessed bone allografts contain sufficient red
ses; packing of benign bone cysts; spinal cells to stimulate production of Rh and
fusion to treat disc disease or scoliosis; re- other red cell antibodies. When indicated,
construction of maxillo-facial defects; and the risk of blood group sensitization can be
correction of healed fractures. Demineral- avoided by using processed frozen or freeze-
ized bone powder is commonly used by dried bone allograft, both of which are de-
periodontal surgeons to restore alveolar void of blood cells and marrow. With these
bone in periodontal pockets. grafts, matching blood groups of donors
The surgeon today has access to a wide and recipients is not necessary. When using
choice of processed bone allografts: freeze- unprocessed frozen bone in Rh-negative
dried or frozen, cancellous or cortical, with females with childbearing potential, it is
or without treatment with sterilants. Com- advisable to use bone from Rh-negative do-
mon preparations include frozen or freeze- nors to prevent alloimmunization or ad-
dried cancellous cubes or chips, cortical minister Rh immune globulin prophylacti-
struts, and cortical-cancellous blocks and cally. There is no evidence to date that ABO
dowels. Bone can be stored frozen or, if or Rh incompatibility between the bone

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- 5 years*
ture
Tendon –40 C 5 years*
Fascia lata Lyophilized, room tempera- 5 years*
ture
–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- Not defined
ture
Cornea 2-6 C 14 days
Hematopoietic progenitor Liquid nitrogen, immersed Not defined
cells
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- Not defined
ture
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 and structural integrity in the frozen state
on the success of the bone graft.30 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
Skin Banking substitutes, such as cultured keratinocyte
A human skin allograft is the dressing of allografts and autografts, are available for
choice for deep burn wounds if sufficient treatment of acute and chronic wounds.
amounts of skin for autografting are un- Keratinocytes extracted from foreskin can
available. A skin allograft provides tempo- be seeded onto biodegradable platforms to
rary coverage; speeds reepithelialization; create bioengineered tissue products used
acts as a metabolic barrier against loss of in wound healing or as cadaveric skin sub-
water, electrolytes, protein, and heat; and stitutes for burns.32
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 Heart Valves
used for donor sites for pedicle flaps and Human heart valve allografts provide long-
skin autografts, and for traumatically de- term function for valve replacement—su-
nuded areas or unhealed areas of chronic perior to that of mechanical or porcine
injury, such as decubitus ulcers. valves. Recipients of human heart valve
Following preparation, skin donation in- allografts do not require anticoagulation
volves removing a layer of skin approxi- and the incidence of thromboembolism is
mately 0.015 inch thick. After collection, re- low. These allografts may rarely transmit
frigerated skin can be stored at 1 to 10 C for bacterial or fungal infection. They are the
up to 14 days. For refrigerated storage, stan- graft of choice for children, to avoid long-
dard tissue-culture nutrient media are term anticoagulation; for pregnant women,
used, with added antibiotics. Skin can be to avoid teratogenic risks of anticoagulants;
frozen soon after collection, usually with and for patients with infection at the aor-
10% to 15% glycerol, although dimethyl tic root. Despite their advantages, wide-
sulfoxide (DMSO) is an acceptable alterna- spread use of human valve allografts has
tive.31 Skin is often cryopreserved on fine- been slow because implantation is techni-
mesh gauze in flat cryopreservation bags. cally difficult and appropriate size valve
Cryogenic damage is minimized by con- allografts are not always readily available.
trolled-rate freezing at about 1 C per min- To obtain allograft valves, hearts are
ute, or by freezing, using a validated heat aseptically collected in an operating room,
sinking method, followed by storage in liq- autopsy room, or morgue. Subsequently, in
uid nitrogen or in a mechanical freezer at a the tissue bank, the pulmonic and aortic
temperature colder than –40 C. Alternatively, valves are dissected out, cryopreserved with
skin placed in aluminum plates inside in- DMSO, and stored in liquid nitrogen. Com-
sulated boxes can be placed directly into a pared with valves stored at 5 C in antibiot-
–40 C mechanical freezer. This simple pro- ics and culture medium, cryopreserved heart
cess also provides a slow, predictable freez- valves are associated with increased cell vi-
ing rate and maintains cellular viability. The ability; reduced incidence of valve degener-
optimal freezing procedure and the maxi- ation, rupture, and leaflet perforation; and
mal storage period that maintain viability reduced occurrence of valve-related death.33

lished rules for determining eligibility for

Records of Stored Tissue donation of human cells, tissues, and cel-
Allografts lular and tissue-based products (HCT/
In two cases in which HIV and HCV were Ps)36 that became effective May 25, 2005.
transmitted (through unprocessed frozen Federal rules also require all facilities that
bone or organs and tendon allografts, re- recover, process, store, or distribute HCT/Ps
spectively) from two deceased donors to or screen or test the donor to register with
multiple recipients,22,23 investigations re- the agency and list their products by mail-
37
vealed that several hospitals had insuffi- ing or faxing Form FDA 3356. Informa-
cient records to identify recipients of other tion may be submitted electronically at
tissue from the same infected donors. Vol- the FDA website www.fda.gov/cber/tis-
untary standards of national professional sue/tisreg.htm. Products not covered by
associations and government regulations these regulations include xenogeneic tis-
require tissue banks to have a record- sue, vascularized organs, transfusable
keeping system that identifies the donor blood products, products used in the
and allows tracking of any tissue from the propagation of cells or tissues, and prod-
donor (or supplier source) to the con- ucts that are secreted or extracted from
signee.6(pp12,47,79),10,14,34 Records must show the cells or tissues. Minimally manipulated
source facility, the identification number marrow, such as marrow that undergoes
of the donor or lot, storage temperatures, cell separation, the relevant characteris-
and final disposition of each tissue. These tics of which are not altered by the pro-
records must be retained at least 10 years cessing, is not covered.
beyond the distribution date, transplanta- Under the federal regulations, infectious
tion date, disposition date, or expiration disease testing is required for allogeneic tis-
date, whichever is latest. Donor eligibility sues, except reproductive tissue from sexu-
records for dura mater must be retained ally intimate partners. Current good tissue
indefinitely. Hospitals should also have practice rules to address concerns about
records that identify recipients who re- proper handling, storage and processing of
34
ceived tissue from a specific donor or tis- tissue have been finalized. Until such time
sue lot. Hospitals should have procedures that the comprehensive regulatory frame-
in place to recognize adverse outcomes of work for human cells, tissues, and cellular
tissue use and to report them to the tissue and tissue-based products is effective, in-
bank supplying the tissue. 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-
FDA Regulation of Tissue quirements under the Federal Food, Drug,
38
The FDA regulates human tissue collected and Cosmetic Act. Federal regulation of
for transplantation.7,8 There are require- tissue banks, formerly under the purview of
ments for infectious disease testing, do- the FDA’s Center for Biologics Research and
nor screening, and record-keeping. A tis- Review, Office of Blood Research and Re-
sue establishment must have and follow view, is now the responsibility of the Center
written, validated procedures to prevent for Biologics Evaluation and Research, Of-
contamination and cross-contamination fice of Cellular, Tissue, and Gene Therapies’
during processing.7,35 The FDA has pub- Division of Human Tissues.

pretransplant blood transfusions. The as-

The Importance of ABO sociation between fewer transfusions and
Compatibility declining renal allograft survival led
The ABO antigens are important in trans- transplant centers to initiate deliberate
plantation practice because they constitute pretransplant transfusion protocols in the
very strong histocompatibility antigens that mid-1970s. Subsequent studies have sup-
are expressed on vascular endothelium. ported the theory that pretransplant blood
Major ABO mismatching can cause rapid transfusions enhance renal allograft sur-
graft rejection due to endothelial damage vival through mechanisms of inducing
by ABO antibodies and subsequent wide- tolerance that remain imperfectly under-
45,46
spread thrombosis within the graft. ABO stood. However, with the availability of
matching is important to the success of cyclosporine and other immunosuppres-
vascularized grafts (ie, kidney, 39 heart, sive agents, interest in this approach has
liver, and pancreas), but ABO matching is waned.47 If the controversial practice of
not important in tissue grafts (ie, fascia, transfusion to induce tolerance in patients
bone, heart valves, skin, and cornea).40 before transplantation is begun, nonleuko-
The definition of an ABO-compatible cyte-reduced Red Blood Cells (RBCs) should
graft is the same as that for a red cell trans- be used.48,49 The introduction of erythro-
fusion. A group O donor of tissue or organ poietin has reduced the need for red cell
is a universal donor whose graft can be transfusions in patients awaiting a renal
transplanted into recipients of all blood transplant.
groups. Case reports document rare suc-
cessful organ transplants with major ABO
incompatibility, but these are few in num-
ber.41 In some cases, A2 donor kidneys can
Liver Transplants
be successfully transplanted into group O A liver transplant program presents one of
recipients with survival comparable to that the greatest challenges to the donor cen-
of group O donor kidneys.42 ABO-incompat- ter and hospital transfusion service, de-
ible transplants have occurred, often with manding maximal support in terms of
fatal results, due to errors of record-keeping preparedness, supply, and responsive-
or labeling. It has been estimated that inad- ness. Massive blood loss and hypocoagu-
vertent ABO-incompatible heart or kidney lability due to preexisting liver disease
transplants occur with a frequency of 1 per and/or the anhepatic interval during the
1000.43 This underscores the importance of procedure create complex problems for the
a final ABO check of donor and recipient transfusion service. The liver is the major
blood at the transplant facility to reduce site for synthesis of clotting factors and
this risk. 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,
The Role of Transfusion in anhepatic interval, and biliary reconstruc-
Kidney Transplants tion—seriously derange these functions.
44
In 1973, Opelz et al noted decreasing kid-
ney allograft survival when hemodialysis Support Required
staff attempted to avoid priming the dial- To achieve a successful liver transplant
ysis equipment with blood and to limit program, a major commitment to this sup-

port is required. A successful program re- Considerations of ABO and Rh

quires cooperation and communication
among the hospital administration; oper- Except in emergencies, donor livers
ating room and intensive care unit; respi- should be ABO-compatible with the re-
ratory therapy, radiology, gastroentero- cipient. ABO-identical RBCs and Fresh
logy, and anesthesiology services; the Frozen Plasma (FFP) are generally used
coagulation and transfusion laboratories; for transfusion support of group O and
and the regional donor center. The insti- group A recipients. Group B recipients
tutional commitment must extend 24 who need large quantities of red cells can
be switched to group O RBCs. Group AB
hours a day, 365 days a year, because there
recipients needing massive transfusion are
may be no more than a few hours’ ad-
often switched to group A RBCs to con-
vance notice of a liver transplant. Consis-
serve group O RBCs for other patients. If
tent availability of blood bank staff on
the supply of AB FFP is insufficient, early
short notice is essential to meet the trans-
use of group A RBCs followed by a switch
fusion requirements. The surgical proce- to group A FFP is appropriate. A general
dure frequently takes place at night or on rule for massive transfusions is to switch
weekends because of the availability of the red cells first, then switch plasma, and re-
donor organ and in order to avoid dis- verse the order when returning to the pa-
rupting the operating room schedule. The tient’s original blood group.46
surgical procedure takes an average of 6 Special considerations apply to the re-
to 8 hours but may take up to 24 hours cipient of an out-of-group but ABO-com-
and involve massive blood use and sev- patible liver transplant. In a group A patient
eral surgical teams. receiving a group O liver, lymphocytes of
The blood bank should be notified as donor origin may produce ABO antibodies
soon as the donor organ becomes available that cause hemolysis that begins several
and the decision for transplantation is days after the procedure and may continue
made. The blood bank obtains a generous for 2 weeks or longer.50 Although passenger
blood sample from the recipient for cross- lymphocytes may produce antibodies in
matching, but there may be more than one any out-of-group but compatible combina-
patient waiting for a liver and the surgeons tion, significant hemolysis is seen most
may be undecided about the specific recip- often in the recipient of a group O liver.
ient. Therefore, the blood bank may have to Transfusion support of Rh-negative pa-
perform numerous crossmatches for patients not immunized to the D antigen is
tients who may have different ABO groups not standardized. 46 Because successful
and/or Rh types. Liver transplant programs pregnancy has occurred after liver trans-
initially used hundreds of units of blood plantation, most programs consider it pref-
and components per patient. Although erable to provide D-negative units to
blood use has steadily declined over the D-negative females with childbearing po-
years, liver transplant procedures fre- tential, if needs are expected to be moder-
quently use a volume of blood components ate. Should massive blood loss occur, the
equal to one whole-body blood volume and patient could be switched intraoperatively
sometimes several blood volumes. Intra- to D-positive blood, if necessary. For pre-
operative blood recovery frequently plays a menopausal females without anti-D, some
major role in the conservation of red cells programs reserve 10 units of D-negative
in such cases. RBCs; if more than 10 units are required,

46
they switch to D-positive blood. Produc-
tion of anti-D occurs less frequently in
Other Organ Transplants
D-negative liver transplant patients ex- Blood bank support for cardiac transplan-
posed to the D antigen than in other tation is very similar to that routinely
D-negative patients.51 In some programs, used for other surgical procedures in
patients without anti-D who are D-negative which cardiopulmonary bypass is em-
postmenopausal females or D-negative ployed. The blood bank may also provide
males are transfused exclusively with ABO testing and assist in the release
D-positive blood. of ABO-compatible organs to prevent
ABO-mismatched organ transplantation.
Pancreatic transplants have compara-
tively low transfusion requirements, but a
Red Cell Alloantibodies specimen from the recipient should rou-
tinely be examined for clinically signifi-
Liver transplant patients with clinically cant unexpected red cell antibodies; in
significant red cell alloantibodies repre- some institutions, the protocol calls for
sent a special challenge to blood banks. crossmatching several units.
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 Transfusion Service Support
at the beginning of surgery, when allo- for Organ Transplantation
antibody is present, and at the end of
The blood bank provides vital support for
massive blood loss, when transfused cells
a clinical transplantation program. Close
are expected to remain in circulation. An-
communication with the surgeons and
tibody screening during the interval of
other professionals involved in the pro-
massive blood loss can help guide use of
gram is essential. Transfusion practices in
antigen-positive units during surgery.
the peritransplant period have a major ef-
fect on morbidity, mortality, and graft sur-
vival rates.
Potential recipients of solid organ trans-
Coagulation Considerations plants are generally available well before
During surgery, hemodilution, platelet the procedure, so there is ample time to ob-
consumption, disordered thrombin regu- tain a history and perform laboratory tests.
lation, and fibrinolysis derange the hemo- It is important for the transfusion service to
static process. The coagulopathy is espe- know if there have been previous pregnan-
cially severe during the anhepatic and cies, transplants, or transfusions.
early reperfusion stage. The following tests Laboratory tests routinely performed in-
are useful: the hematocrit guides the use clude: ABO group and Rh type, the direct
of red cells, colloids, and crystalloids; the antiglobulin test (DAT), a screen for unex-
platelet count guides transfusion of plate- pected red cell antibodies, and determina-
lets; the prothrombin time and activated tion of CMV serostatus. HLA typing and
partial thromboplastin time guide FFP HLA antibody studies are routine for organ
use; and fibrinogen determinations guide recipients.
use of Cryoprecipitated AHF and anti- Passenger lymphocyte hemolysis (typi-
fibrinolytic agents.46,52,53 cally “ABO”-incompatible hemolysis), as

discussed previously in regard to liver 2. 2002 EBAA statistical report. Washington, DC:
Eye Bank Association of America, 2002.
transplantation, can occur with other solid
3. Yap PL. Viral transmission by blood, organs
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kidney. In the case of a recipient receiving 44.
an ABO-compatible but non-group-identi- 4. The Organ Procurement and Transplantation
Network data reports, 2003. Richmond, VA:
cal organ, prophylactic use of mutually
United Network for Organ Sharing, 2003.
ABO-compatible erythrocytes (compatible 5. Warwick RM, Eastlund T, Fehily D. Role of the
for the donor and the recipient) has been blood transfusion service in tissue banking.
suggested for intraoperative and postopera- Vox Sang 1996;71:71-7.
tive infusions, during the first postoperative
month, or at the appearance of an anti- AABB, 2005.
body. At present, there is no consensus on 7. Food and Drug Administration. Human tissue
this issue. It is important to remember that intended for transplantation. Fed Regist 1997;
62:40429-47.
if immediate-spin or computer cross-
8. Food and Drug Administration. Guidance for
matching is routinely performed after an industry: Screening and testing of donors of
ABO-unmatched transplant, ABO incom- human tissue intended for transplantation.
patibility due to these IgG antibodies may ( July 1997) Rockville, MD: CBER Office of
Communication, Training, and Manufactur-
be missed. In such cases, the routine use of ers Assistance, 1997.
a crossmatch with an antihuman globulin 9. Centers for Disease Control and Prevention.
phase or the use of a DAT (which may de- Guidelines for preventing HIV transmission
through organ and tissue transplantation.
tect such cases earlier than a crossmatch) is
MMWR 1994;43(RR-8):1-17.
recommended. If ABO hemolysis is present, 10. Woll J, Kasprisin D, eds. Standards for tissue
the patient should be transfused with group banking. McLean, VA: American Association
O RBCs. of Tissue Banks, 2002.
11. American Society for Reproductive Medicine.
CMV infection, a serious and often fatal
Guidelines for gamete and embryo donation.
complication in transplant recipients, is re- Fertil Steril 2002;77:Suppl 5.
lated to the presence of CMV in the donor 12. Medical standards of EBAA. Washington, DC:
and recipient and the degree to which the Eye Bank Association of America, 2002.
13. Articles of incorporation, by-laws, and poli-
recipient is immunosuppressed. The pri-
cies of UNOS. Richmond, VA: United Network
mary test used to determine CMV status is of Organ Sharing, 2004 (revised at least annu-
the demonstration of circulating antibody. ally).
CMV-seronegative recipients of CMV-sero- 14. New York State Department of Health. Tissue
banks and nontransplant anatomic banks,
negative transplants characteristically re- Part 52. New York State Codes, Rules, and Reg-
ceive transfusion components processed to ulations, Title 10. Albany, NY: New York State
reduce risk of CMV transmission, either by Department of Health, 2004. (Available at
http://www.wadsworth.org/labert/blood_tissue.)
preparation from seronegative donors or by
15. Eastlund T. Infectious disease transmission
leukocyte reduction to 5 × 106 cells/compo- through cell, tissue and organ transplanta-
nent or below. tion. Reducing the risk through donor selec-
tion. Cell Transplant 1995;4:455-77.
16. Eastlund T, Strong DM. Infectious disease trans-
mission through tissue transplantation. In:
Phillips GO, ed. Advances in tissue banking.
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17. CJD and the eye. Monograph 7. London:
1. Strong M, Eastlund T, Mowe J. Tissue bank Royal College of Ophthalmologists, 1998.
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1996;3:15-18. lar tissue transplantation. Cornea 1999;18:2-11.

19. Human immunodeficiency virus transmitted 33. O’Brien MF, Stafford EG, Gardner MAH, et al.
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20. Eastlund T. Hemodilution due to blood loss Springer-Verlag, 1988:311-21.
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21. Linden JV, Critser JK. Therapeutic insemina- inspection and enforcement; final rule. (No-
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22. Simonds RJ, Holmberg SD, Hurwitz RL, et al. Industry. Validation of procedures for pro-
Transmission of human immunodeficiency cessing human tissues intended for trans-
virus type 1 from a seronegative organ and plantation. (March 2002) Rockville, MD:
tissue donor. N Engl J Med 1992;326:726-32. CBER Office of Communications, Training,
and Manufacturers Assistance, 2002.
23. Conrad EU, Gretch D, Obermeyer K, et al. The
transmission of hepatitis C virus by tissue 36. Food and Drug Administration. Eligibility de-
transplantation. J Bone Joint Surg 1995;77A: termination for donors of human cells, tissues,
214-23. and cellular and tissue-based products; final
rule. Fed Regist 2004;69:29786-834.
24. Kainer MA, Linden JV, Whaley DN, et al.
37. Food and Drug Administration. Human cells,
Clostridium infections associated with
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Med 2004;350:2564-71.
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25. Centers for Disease Control and Prevention.
38. Food and Drug Administration. Human cells,
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tissues, and cellular and tissue-based prod-
nor and transplant recipients—Alabama, Ar-
ucts; establishment registration and listing;
kansas, Oklahoma, and Texas, 2004. MMWR
interim final rule; opportunity for public
Morb Mortal Wkly Rep 2004;53:586-9.
comment. Fed Regist 2004;69:3823-6.
39. Alkhunaizi AM, de Mattos AM, Barry JM, et al.
Lymphocytic choriomeningitis virus infec-
Renal transplantation across the ABO barrier
tion in organ transplant recipients—Massa-
using A2 kidneys. Transplantation 1999;67:
chusetts, Rhode Island, 2005. MMWR Morb
1319-24.
Mortal Wkly Rep 2005;54:537-9.
40. Eastlund T. The histo-blood group ABO sys-
27. Food and Drug Administration. Guidance for tem and tissue transplantation. Transfusion
industry: Availability of licensed donor 1998;38:975-88.
screening tests for use with cadaveric blood 41. Alexandre GPJ, Squifflet JP, DeBruyere M, et
specimens. ( June 23, 2000) Rockville, MD: al. ABO-incompatible related and unrelated
CBER Office of Communication, Training, and living donor renal allografts. Transplant Proc
Manufacturers Assistance, 2000. 1986;18:1090-2.
28. Choo SY, Eastlund T. Tissue transplantation 42. Breimer ME, Brynger H, Rydberg L, et al.
and HLA typing. Tissue Cell Rep 1995;2:3-4. Transplantation of blood group A2 kidneys to
29. LeFor WM, McGonigle AF, Wright CE, Shires O recipients. Biochemical and immunologi-
DL. The frequency of false positive HBsAg cal studies of group A antigens in human kid-
screening test results with cadaver tissue do- neys. Transplant Proc 1985;17:2640-3.
nors is dependent upon the assay procedure 43. Terasaki PI. Red-cell crossmatching for heart
used. Tissue Cell Rep 1996;3:6-170. transplants (letter). N Engl J Med 1991;325:
30. Eastlund T. Bone transplantation and bone 1748-9.
banking. In: Lonstein JE, Bradford DS, Winter 44. Opelz G, Sengar DPS, Mickey MR, Terasaki P.
RB, Ogilvie JW, eds. Moe’s textbook of scoliosis Effect of blood transfusion on subsequent
and other spinal deformities. 3rd ed. Phila- kidney transplants. Transplant Proc 1973;5:
delphia: WB Saunders, 1995:581-95. 253-9.
31. Bravo D, Rigley TH, Gibran N, et al. Effect of 45. Blumberg N, Heal JM. Transfusion immuno-
storage and preservation methods on viabil- modulation. In: Anderson KC, Ness PM, eds.
ity of transplantable human skin allografts. Scientific basis of transfusion medicine. 2nd
Burns 2000;26:367-78. ed. Philadelphia:WB Saunders, 2000:427-43.
32. Phillips TJ. New skin for old: Developments 46. Dzik WH. Solid organ transplantation. In:
in biological skin substitutes. Arch Dermatol Petz LD, Swisher SN, Kleinman S, et al, eds.
1998;134:344-9. Clinical practice of transfusion medicine. 3rd

ed. New York: Churchill Livingstone, 1996: 50. Triulzi DJ, Shirey RS, Ness PM, Klein AS. Im-
783-806. munohematologic complications of ABO-
47. Lundgren G, Groth CG, Albrechtsen D, et al. unmatched liver transplants. Transfusion
HLA matching and pretransplant blood trans- 1992;32:829-33.
fusions in cadaveric renal transplantation—a 51. Casanueva M, Valdes MD, Ribera MC. Lack of
changing picture with cyclosporin. Lancet alloimmunization to D antigen in D-negative
1986;ii:66-9. immunosuppressed liver transplant recipi-
48. Iwaki Y, Cecka JM, Terasaki PI. The transfu- ents. Transfusion 1994;34:570-2.
sion effect in cadaver kidney transplants, yes 52. Triulzi DJ, Bontempo FA, Kiss JE, Winkelstein
or no. Transplantation 1990;49:56-9. A. Transfusion support in liver transplanta-
49. Opelz G, Vanrenterghem Y, Kirste G, et al. tion. Transfus Sci 1993;14:345-52.
Prospective evaluation of pretransplant 53. Motschman TL, Taswell HF, Brecher ME, et al.
blood transfusions in cadaver kidney recipi- Blood bank support of a liver transplantation
ents. Transplantation 1997;63:964-7. program. Mayo Clin Proc 1989;64:103-11.

Chapter 27: Noninfectious Complications of Blood Transfusion
Chapter 27
Noninfectious
Complications of Blood
Transfusion
27
T
HIS CHAPTER ADDRESSES four fusion reactions and can aid in their rec-
broad categories of transfusion re- ognition. In general, one should consider
actions: 1) acute immunologic, 2) any adverse manifestation occurring at
acute nonimmunologic, 3) delayed immu- the time of the transfusion to be a transfu-
nologic, and 4) delayed nonimmunologic sion reaction until proven otherwise.
complications, as shown in Table 27-1.1,2 ■ Fever with or without chills [gener-
For each individual type of reaction, the ally defined for surveillance pur-
pathophysiology, treatment, and preven- poses as a 1 C (2 F) increase in body
tion are discussed. More detailed cover- temperature] associated with trans-
age is available elsewhere. 1 Infectious fusion. Fever is the most common
risks of transfusion are discussed in symptom of a hemolytic transfusion
Chapter 28. reaction (HTR),3 but more frequently
it has other causes.
■ Shaking chills (rigors) with or with-
out fever.
Manifestations ■ Pain at the infusion site or in the
All personnel involved in ordering and chest, abdomen, or flanks.
administering transfusions must be able ■ Blood pressure changes, usually acute,
to recognize a transfusion reaction so that either hypertension or hypotension.
appropriate actions can be taken promptly. Circulatory shock in combination
Listed below are signs and symptoms that with fever, severe chills, and high-
are typically associated with acute trans- output cardiac failure suggests acute
633

634
Table 27-1. Categories and Management of Adverse Transfusion Reactions*
Therapeutic/Prophylactic

Type Incidence Etiology Presentation Diagnostic Testing Approach
Acute (<24 hours) Transfusion Reactions–Immunologic

Hemolytic 1:38,000- Red cell incompatibility Chills, fever, ■ Clerical check ■ Keep urine output >100
1:70,000 hemoglobinuria, ■ DAT mL/hr with fluids and IV
hypotension, renal failure ■ Visual inspection (free diuretic (furosemide)
with oliguria, DIC (oozing Hb) ■ Analgesics (may need

from IV sites), back pain, ■ Repeat patient ABO, pre- morphine)
pain along infusion vein, and posttransfusion ■ Pressors for
anxiety sample hypotension (low-dose
■ Further tests as dopamine)
indicated to define ■ Hemostatic components
possible incompatibility (platelets, cryo, FFP) for
■ Further tests as bleeding
indicated to detect
hemolysis (LDH,
bilirubin, etc)
Fever/chill, RBCs: ■ Antibody to donor WBCs Fever, chills/rigors, head- ■ Rule out hemolysis ■ Antipyretic
nonhemoly- 1:200-1:17 ■ Accumulated cytokines ache, vomiting (DAT, inspect for premedication
tic (0.5-6%) in platelet unit hemoglobinemia, repeat (acetaminophen, no
Plts: patient ABO) aspirin)
1:100-1:3 ■ Rule out bacterial ■ Leukocyte-reduced
(1-38%) contamination blood
■ WBC antibody screen
Urticarial 1:100-1:33 Antibody to donor plasma Urticaria, pruritis, flushing ■ Rule out hemolysis ■ Antihistamine, treatment
(1-3%) proteins (DAT, inspect for or premedication (PO or
hemoglobinemia, repeat IV)
patient ABO) ■ May restart unit slowly
after antihistamine if
symptoms resolve
Anaphylactic 1:20,000- Antibody to donor plasma Hypotension, urticaria, ■ Rule out hemolysis ■ Trendelenberg (feet up)
1:50,000 proteins (includes IgA, bronchospasm (respira- (DAT, inspect for position
haptoglobin, C4) tory distress, wheezing), hemoglobinemia, repeat ■ Fluids
local edema, anxiety patient ABO) ■ Epinephrine (adult dose:
■ Anti-IgA 0.3-0.5 mL of 1:1000
■ IgA, quantitative solution SC or IM; in
severe cases, 1:10,000
IV)
■ Antihistamines,
corticosteroids, beta-2

agonists
■ IgA-deficient blood
components
Transfusion- 1:5,000- WBC antibodies in donor Hypoxemia, respiratory ■ Rule out hemolysis ■ Supportive care until
related acute 1:190,000 (occasionally in recipient), failure, hypotension, fever, (DAT, inspect for recovery
lung injury other WBC-activating bilateral pulmonary edema hemoglobinemia, repeat ■ Defer implicated donors
agents in components patient ABO)
■ WBC antibody screen in
donor and recipient. If
positive, antigen typing
may be indicated
■ WBC crossmatch
■ Chest X-ray
Acute (<24 hours) Transfusion Reactions—Nonimmunologic
Transfusion- Varies by Bacterial contamination Fever, chills, hypotension ■ Gram’s stain ■ Broad spectrum
associated component ■ Culture of component antibiotics (until
sepsis (see Chapter ■ Patient culture sensitivities completed)
28) ■ Rule out hemolysis ■ Treat complications (eg,
(DAT, inspect for shock)
hemoglobinemia, repeat
patient ABO)
635
(cont’d)
636
Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)

Hypotension Dependent Inhibited metabolism of Flushing, hypotension ■ Rule out hemolysis ■ Withdraw ACE inhibition
associated with on clinical bradykinin with infusion of (DAT, inspect for ■ Avoid albumin volume
ACE inhibition setting bradykinin (negatively hemoglobinemia, repeat replacement for
charged filters) or activa- patient ABO) plasmapheresis
tors of prekallikrein ■ Avoid bedside leukocyte
filtration
Circulatory <1% Volume overload Dyspnea, orthopnea, ■ Chest X-ray ■ Upright posture
overload cough, tachycardia, hyper- ■ Oxygen
tension, headache ■ IV diuretic (furosemide)
■ Phlebotomy (250-mL
increments)
Nonimmune Rare Physical or chemical de- Hemoglobinuria, ■ Rule out patient ■ Identify and eliminate
hemolysis struction of blood (heat- hemoglobinemia hemolysis (DAT, inspect cause
ing, freezing, hemolytic for hemoglobinemia,
drug or solution added to repeat patient ABO)
blood) ■ Test unit for hemolysis
Air embolus Rare Air infusion via line Sudden shortness of ■ X-ray for intravascular ■ Place patient on left side
breath, acute cyanosis, air with legs elevated above
pain, cough, hypotension, chest and head
cardiac arrythmia
Hypocalcemia Dependent Rapid citrate infusion Paresthesia, tetany, ■ Ionized calcium ■ Slow calcium infusion
(ionized cal- on clinical (massive transfusion of arrhythmia ■ Prolonged Q-T interval while monitoring ionized
cium) setting citrated blood, delayed on electrocardiogram calcium levels in severe
metabolism of citrate, cases
apheresis procedures) ■ PO calcium supplement
for mild symptoms
during apheresis
procedures
Hypothermia Dependent Rapid infusion of cold Cardiac arrhythmia Central body temperature ■ Employ blood warmer
on clinical blood
setting
Delayed (>24 hours) Transfusion Reactions–Immunologic

Alloimmuni- 1:100 (1%) Immune response to Positive blood group ■ Antibody screen ■ Avoid unnecessary
zation, RBC foreign antigens on antibody screening test ■ DAT transfusions
antigens RBCs, or WBCs and ■ Leukocyte-reduced

platelets (HLA) blood

Alloimmuni- 1:10 (10%) Platelet refractoriness, ■ Platelet antibody screen ■ Avoid unnecessary
zation, HLA delayed hemolytic ■ Lymphocytotoxicity test transfusions
antigens reaction, hemolytic dis- ■ Leukocyte-reduced
ease of the newborn blood
Hemolytic 1:11,000- Anamnestic immune Fever, decreasing ■ Antibody screen ■ Identify antibody
1:5000 response to red cell hemoglobin, new ■ DAT ■ Transfuse compatible
antigens positive antibody screen- ■ Tests for hemolysis red cells as needed
ing test, mild jaundice (visual inspection for
hemoglobinemia, LDH,
bilirubin, urinary
hemosiderin as clinically
indicated)
Graft-vs-host Rare Donor lymphocytes en- Erythroderma, ■ Skin biopsy ■ Corticosteroids,
disease graft in recipient and maculopapular rash, an- ■ HLA typing cytotoxic agents
mount attack on host tis- orexia, nausea, vomiting, ■ Irradiation of blood
sues diarrhea, hepatitis, components for patients
pancytopenia, fever at risk (including related
donors and HLA-
selected components)
637
(cont’d)
638
Table 27-1. Categories and Management of Adverse Transfusion Reactions* (cont’d)
Posttransfusion Rare ■ Recipient platelet Thrombocytopenic ■ Platelet antibody screen ■ IGIV

purpura antibodies (apparent purpura, bleeding, 8-10 and identification ■ HPA-1-negative platelets
alloantibody, usually days after transfusion ■ Plasmapheresis
anti-HPA-1) destroy
autologous platelets
Immunomo- Unknown Incompletely understood Increased renal graft sur- ■ None specific ■ Avoid unnecessary
dulation interaction of donor WBC vival, infection rate, transfusions
or plasma factors with re- postresection tumor recur- ■ Autologous transfusion
cipient immune system rence rate (controversial) ■ Leukocyte-reduced red
cells and platelets
Delayed (>24 hours) Transfusion Reactions–Nonimmunologic
Iron overload Typically af- Multiple transfusions with Diabetes, cirrhosis, ■ Serum ferritin ■ Desferioxamine (iron
ter >100 RBC obligate iron load in trans- cardiomyopathy ■ Liver enzymes chelator)
units fusion-dependent patient ■ Endocrine function tests
*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.
Chapter 27: Noninfectious Complications of Blood Transfusion 639
sepsis but may also accompany an hypotension, or diffuse bleeding at the

acute HTR. Circulatory collapse with- surgical site.
out fever and chills may be the most Such severe acute HTRs today are usu-
prominent finding in anaphylaxis. ally caused by ABO incompatibility5 but oc-
■ Respiratory distress, including dyspnea, casionally may be caused by antibodies
tachypnea, wheezing, or hypoxemia. with other specificities. 6 In contrast,
■ Skin changes, including urticaria, hemolysis of an entire unit of blood can oc-
pruritis (itching), flushing, or local- cur in the virtual absence of symptoms7
ized edema (angioedema). and may be a relatively slow process. In
■ Nausea with or without vomiting. such cases, hemolysis is typically extra-
■ Darkened urine or jaundice. Dark urine vascular, without generation of significant
may be the earliest indication of an systemic levels of inflammatory mediators.
acute hemolytic reaction in anesthe- Complement Activation. The binding of
tized patients. antibody to blood group antigens may acti-
■ Bleeding or other manifestations of vate complement, depending on the char-
a consumptive coagulopathy. 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-
Acute Transfusion Reactions phylatoxin C3a (see Chapter 11), and red
cells coated with C3b are removed by
Immune-Mediated Hemolysis phagocytes with complement receptors,
more rapidly than if antibody is present
Pathophysiology and Manifestations alone. If the enzymatic cascade proceeds to
The most severe hemolytic reactions oc- completion and a membrane attack com-
cur when transfused red cells interact plex is assembled, intravascular hemolysis
with preformed antibodies in the recipi- results, with the production of C5a, which
ent. In contrast, the interaction of trans- is 100 times as potent an anaphylatoxin as
3
fused antibodies with the recipient’s red C3a. This sequence is characteristic of ABO
cells rarely causes symptoms. However, incompatibility and causes the cardinal
there may be accelerated red cell destruc- manifestations of hemoglobinemia and, if
tion, and plasma-containing products with the renal threshold for hemoglobin is ex-
high-titer ABO antibodies can cause acute ceeded, hemoglobinuria.8(p182) Anaphylatoxins
hemolysis. The interaction of antibody interact with a wide variety of cells, includ-
with antigen on the red cell membrane ing monocytes/macrophages, granulocytes,
can initiate a sequence of complement platelets, vascular endothelial cells, and
activation (see Chapter 11), cytokine and smooth muscle cells, the latter leading to
coagulation effects, and other elements of hypotension and bronchospasm. Anaphy-
a systemic inflammatory response4 that latoxins also cause the release or produc-
result in the clinical manifestations of a tion of multiple local and systemic media-
severe acute HTR. Severe symptoms can tors, including granule enzymes, histamine
occur after the infusion of as little as 10 to and other vasoactive amines, kinins, oxy-
15 mL of ABO-incompatible red cells. In gen radicals, leukotrienes, nitric oxide, and
3
anesthetized patients who cannot report cytokines. These mechanisms may cause
symptoms, the initial manifestations of manifestations that mimic allergy, such as
an acute HTR may be hemoglobinuria, flushing and rarely urticaria, wheezing and

chest pain or tightness, and abdominal Coagulation Activation. Several mecha-

pain, nausea, and vomiting. nisms, including those listed above, may be
With most non-ABO blood group anti- responsible for abnormalities of coagula-
bodies, complement activation is usually tion in HTRs.4 The antigen-antibody inter-
incomplete; hemoglobinemia is absent or action may activate the “intrinsic” clotting
mild, but the consequences of complement cascade through Hageman factor. In addi-
activation, most notably the release of ana- tion, activated Hageman factor (Factor XIIa)
phylatoxins and opsonization of red cells, acts on the kinin system to generate brady-
may still have adverse effects. kinin; bradykinin increases capillary per-
Cytokines. The role of cytokines in in- meability and dilates arterioles, causing a
flammatory responses (see Chapter 11), in- decrease in systemic arterial pressure. Sev-
cluding acute HTRs, is increasingly recog- eral factors cited above may increase the
nized.9,10 The known activities of inflammatory expression of tissue factor by leukocytes
cytokines, such as tumor necrosis factor α and endothelial cells, including activated
(TNFα), interleukin-1β and -6 (IL-1β, IL-6), complement components, TNFα, and
and chemokines such as IL-8 and mono- IL-1β. Tissue factor activates the “extrinsic”
cyte chemoattractant protein (MCP), sug- coagulation pathway, and its release is as-
gest that they mediate some of the effects of sociated with disseminated intravascular
alloimmune hemolysis. IL-1 and TNF cause coagulation (DIC), which may, in turn, cause:
fever and hypotension (particularly in syn- 1) formation of thrombi within the micro-
ergy), stimulation of endothelial cells to in- vasculature and ischemic damage to tissues
crease expression of adhesion molecules and organs; 2) consumption of fibrinogen,
and procoagulant activity, and recruitment platelets, and other coagulation factors; 3)
and activation of neutrophils and platelets, activation of the fibrinolytic system and
perhaps through the induction of IL-8 and generation of fibrin degradation products.
MCP. Incubation of whole blood with The outcome can be a hemorrhagic diathesis
washed, ABO-incompatible red cells in vi- characterized by generalized oozing or un-
tro has been shown to cause dramatic in- controlled bleeding.
creases in TNF, IL-8, and MCP. This cyto- Shock and Renal Failure. Considering
kine response is complement dependent. A the absolute mass of antigen and anti-
similar model of hemolysis resulting from body—and the list of mediators that may
anti-D (IgG) showed a different pattern of be involved in HTRs, including anaphyla-
cytokine production with “high-level” re- toxins, vasoactive amines, kinins, and cyto-
sponses of IL-8 and MCP and “low-level” kines—it may not be surprising that shock
responses of IL-1β, IL-6, and TNFα.9,10 can occur. Hypotension provokes a com-
The relevance of these in-vitro models to pensatory sympathetic nervous system re-
HTRs in vivo is suggested by a case in sponse that produces vasoconstriction in
which TNFα and neutrophil elastase levels organs and tissues with a vascular bed rich
were found to be elevated when a group O in alpha-adrenergic receptors, notably, the
patient received 100 mL of group A red renal, splanchnic, pulmonary, and cutane-
cells; elevation of neutrophil elastase is ous capillaries, aggravating ischemia in
consistent with IL-8 activity.11 These find- these sites.
ings may lead to new therapeutic options Renal failure is another sequel of an
for patients. However, the complete role of acute HTR. Although free hemoglobin, his-
cytokines in the consequences of immune torically considered the cause of renal fail-
hemolysis remains to be defined. ure, does impair renal function,12 current

thought attributes renal failure largely to Treatment

hypotension, renal vasoconstriction, anti-
gen-antibody complex deposition, and for- The treatment of an acute HTR depends
4
mation of thrombi in the renal vasculature, on its severity. Vigorous treatment of hypo-
all of which compromise renal cortical tension and promotion of adequate renal
blood supply. blood flow are the primary concerns. If
shock can be prevented or adequately
treated, progression to renal failure may
Frequency be avoided. Adequacy of renal perfusion
Clerical and other human errors leading can be monitored by measurement of
to mistaken identity are the most common urine output, with a goal of maintaining
causes of ABO-incompatible transfusion, urine flow rates above 100 mL/hour in
occurring either at pretransfusion sample adults for at least 18 to 24 hours. The
collection, within the transfusion service, usual first support is intravenous normal
or at the time of blood administration. A saline, but underlying cardiac and/or re-
study of reported transfusion errors in nal disease may complicate therapy, and
New York State over a 10-year period (the it is important to avoid overhydration. In-
1990s) estimated the incidence of ABO- vasive monitoring of pulmonary capillary
incompatible red cell transfusions at wedge pressure is recommended in guid-
1:38,000. Correction for the expected rate ing fluid therapy in the face of hemo-
of fortuitously compatible transfusions
dynamic instability. Diuretics help to im-
led to an estimate of the rate of mis-
prove blood flow to the kidneys and
transfusion of 1:14,000.7 A survey of 3601
increase urine output. Intravenous furo-
institutions by the College of American
semide at a dose of 40 to 80 mg for an
Pathologists found 843 acute HTRs re-
adult or 1 to 2 mg/kg for a child not only
ported over a 5-year period, of which 50
has a diuretic effect but also improves
(6%) were fatal.13 The Serious Hazards of
Transfusion (SHOT) initiative in the United blood flow to the renal cortex. This dose
Kingdom and Republic of Ireland re- may be repeated once, and the patient
ported 161 cases of ABO-incompatible should be adequately hydrated. Mannitol,
transfusion, with nine fatal cases (five def- an osmotic diuretic, has been used in the
initely related deaths, one probably re- past, but furosemide is better for main-
lated death, and three possibly related taining renal cortical blood flow. If no di-
deaths) in 5 years.14 Although no precise uretic response occurs within a few hours
denominator is available for these confi- of instituting fluid and diuretic therapy,
dential reports, it is believed that over there is a strong likelihood that acute tu-
90% of the total transfusions were re- bular necrosis has occurred, and further
viewed and approximately 2.5 million fluid administration may be harmful.
RBC units were issued each year, for a rate Treatment of hypotension with pressor
of no less than 1 in 78,000. These values agents that decrease renal blood flow, such
probably underestimate the true fre- as dopamine in higher doses, should be
quency, because even acute HTRs go un- avoided if possible. The use of low-dose do-
recognized or unreported. Estimates of pamine (2-5 µg/kg/minute), as an agent to
mortality rates from acute HTRs are gener- protect renal function, has been recom-
4
ally in the range of 1 in 1,000,000 transfu- mended in the management of acute HTRs.
sions.5,7,14 However, evidence suggests that it is not ef-

fective in this role, and it has many toxici- and particularly emphasize the impor-
ties.15 tance of the bedside check at the time of
Consumptive coagulopathy, with resul- transfusion.14 Ensuring that all clinical staff
tant bleeding or generalized oozing, may be recognize the signs of acute reactions and
a prominent clinical finding in some HTRs stop the transfusion before a critical vol-
and may be the initial presentation in an ume of blood has been administered is es-
anesthetized patient. Heparin has been rec- sential to preventing harm to the patient.
ommended by some, both to forestall DIC Crucial in the prevention of transfusion
when an ABO incompatibility is first dis- mishaps are training and assessment of
covered and to treat the established coa- personnel performing transfusions. Active
gulopathy. Others believe the dangers of participation by physicians and manage-
heparin outweigh its potential benefits, es- ment, as well as by nursing, technical, and
pecially because the immune event that clinical personnel, is essential.
provoked the DIC is self-limited. Adminis-
tration of Platelets, Fresh Frozen Plasma
(FFP), and Cryoprecipitated AHF, a source Nonimmune-Mediated Hemolysis
of fibrinogen and Factor VIII, may be nec-
essary. Red cell exchange may be consid-
Causes
ered in patients with a significant load of
circulating incompatible red cells.
Red cells may undergo in-vitro hemolysis
Acute hemolytic reactions are rare and
if the unit is exposed to improper temper-
few clinicians have first-hand experience
atures during shipping or storage or is
with their treatment. Because medical
mishandled at the time of administration.
management of an acute HTR is often com-
Malfunctioning blood warmers, use of
plicated and may require aggressive inter-
ventions such as hemodialysis, consultation microwave ovens or hot waterbaths, or in-
with physicians experienced in the organ advertent freezing may cause tempera-
systems most damaged or specialists in ture-related damage. Mechanical hemolysis
critical care medicine may be prudent when may be caused by the use of roller pumps
treating a patient with a severe acute HTR. (such as those used in cardiac bypass sur-
gery), pressure infusion pumps, pressure
16
cuffs, or small-bore needles. Osmotic
Prevention hemolysis in the blood bag or infusion set
Because clerical errors cause the majority may result from the addition of drugs or
of acute, immune-mediated HTRs, the hypotonic solutions. Inadequate degly-
best hope for prevention lies in prevent- cerolization of frozen red cells may cause
ing or detecting errors in every phase of the cells to hemolyze after infusion. Finally,
the transfusion process. In each institu- hemolysis may be a sign of bacterial
tion, there should be systems designed to growth in blood units. In a patient with
prevent and detect errors in patient and transfusion-associated hemolysis for which
unit identification at the time of phlebot- both immune and nonimmune causes
omy (sample acquisition), at all steps in have been eliminated, the possibility might
laboratory testing, at the time of issue, be considered that the patient or donor has
and when the transfusions are given. The an intrinsic red cell defect, such as glu-
SHOT reports document multiple errors cose-6-phosphatase dehydrogenase defi-
in a majority of mistransfusion incidents ciency, causing coincidental hemolysis.

Treatment perature increase of >1 C associated with

transfusion and without any other expla-
Treatment depends on the severity of the
nation. Such reactions are often associated
reaction. If the patient develops a severe
with chills or rigors. The 1 C definition is
reaction with hypotension, shock, and re-
arbitrary; the same events might cause
nal dysfunction, intensive clinical man-
smaller temperature increments. Indeed,
agement is required even before the cause
some authors discuss reactions character-
of the mishap is investigated. If the pa-
ized by rigors or other symptoms, in the
tient exhibits only hemoglobinemia and
absence of fever, as FNHTRs because of a
hemoglobinuria, supportive therapy may
presumed common mechanism.2 In one
be sufficient.
study of 108 reactions characterized by
chills, cold, or rigors, only 18 involved a
Prevention rise in temperature. 19 Febrile reactions
There should be written procedures for all complicate 0.5% to 6% of nonleukocyte-
aspects of procuring, processing, and is- reduced red cell transfusions. Previous
suing blood, and administering transfusions. opportunities for alloimmunization, es-
All staff should be trained in the proper pecially pregnancies and multiple trans-
use of equipment, intravenous solutions, fusions, increase the frequency of FNHTRs
and drugs used during the administration to red cells. The rate of such reactions is
of blood and blood components. Equip- much higher after platelet transfusion
ment must be properly maintained and (1-38%). Most FNHTRs are benign, al-
records kept of how and when items are though some may cause significant dis-
used. Intravenous medications shall not comfort and hemodynamic or respiratory
be injected into blood bags, unless ap- changes. The temperature increase may
proved by the Food and Drug Administra- begin early in the transfusion or be de-
tion (FDA) or documented to be safe for layed in onset for hours after completion
that purpose,17(p48) and care must be exer- of the transfusion.
cised in the selection and use of intrave- Many febrile reactions are thought to re-
nous access devices. Chapter 22 discusses sult from an interaction between antibod-
the details of administering transfusions. ies in the recipient’s plasma and antigens
present on transfused lymphocytes, granu-
Transfusion-Associated Sepsis locytes, or platelets, most frequently HLA
Bacterial contamination of transfused blood antigens. There is also evidence that febrile
should be considered if the patient expe- reactions, particularly those due to plate-
riences severe rigors, especially if they are lets, may be caused by the infusion of bio-
accompanied by cardiovascular collapse logic response modifiers, including cyto-
18
and/or fever over 40 C. For a more de- kines, that accumulate in the blood bag
tailed discussion of this potentially life- during storage. Cytokine release in the re-
threatening transfusion complication, see cipient undoubtedly contributes to those
Chapter 28. reactions that begin with recipient antibody
against donor leukocytes.2,20-22 Because fever
Febrile Nonhemolytic Reactions may be an initial manifestation of an acute
HTR or a reaction to transfusion of blood
Pathophysiology and Manifestations contaminated with bacteria, any observa-
A febrile nonhemolytic transfusion reaction of an increase in temperature associ-
tion (FNHTR) is often defined as a tem- ated with transfusion warrants prompt at-

tention. The diagnosis of an FNHTR is Allergy; Urticaria (Hives) to Anaphylaxis

made after excluding other possible expla-
nations for the fever, particularly a Pathophysiology and Manifestations
hemolytic or septic reaction. Guidelines for Allergic reactions to transfusion form a
evaluating a suspected acute transfusion continuum, with the vast majority clus-
reaction are presented later in this chapter. tered at the mild end, in the form of urti-
caria or “hives”—erythematous, sharply
Treatment circumscribed raised lesions, most often
present over the upper trunk and neck,
Traditionally, transfusion was discontin-
23 which may itch and which are not usually
ued when an FNHTR occurred. However,
accompanied by fever or other adverse
some clinicians believe that fever should
findings. At the other end of the spectrum
not routinely cause discontinuation of a
are anaphylactic reactions, in which there
transfusion,2,24 depending on whether the
are systemic symptoms including hypo-
patient has symptoms, signs, or labora-
tension, loss of consciousness, shock,
tory data that suggest hemolysis, transfu-
and, in rare cases, death. The latter may
sion-related acute lung injury (TRALI), or
begin after infusion of only a few millili-
bacterial contamination. The fever of an
ters, but less severe reactions tend to take
FNHTR usually responds to antipyretics.
longer to develop. The term “anaphylac-
Acetaminophen is preferred to the use of
toid” is used in transfusion medicine to
salicylates because the former drug does
denote reactions in between these ex-
not affect platelet function. Meperidine
tremes, but it is also used to denote reac-
injection may be useful in patients with
tions that have clinical similarities to
severe shaking chills. Antihistamines are
anaphylaxis but different mechanisms.
not indicated because most FNHTRs do
Manifestations of these reactions may in-
not involve histamine release.
volve one or several systems, notably, the
skin (urticaria, generalized flushing or
Prevention rash, localized swelling or “angioedema”),
Febrile reactions in an alloimmunized respiratory tract (upper or lower respira-
individual can often be prevented by tory tract obstruction with cough, hoarse-
transfusion of leukocyte-reduced blood ness, stridor, wheezing, chest tightness or
components. Prevention of reactions pain, dyspnea), the gastrointestinal tract
caused by cytokine accumulation during (cramps, nausea, vomiting, diarrhea), or
storage requires that the leukocyte reduc- the circulatory system (tachycardia and
2,19
tion be performed before storage, but other arrhythmias including cardiac ar-
some patients will still react. With non- rest).26 Fever is characteristically absent, a
leukocyte-reduced platelets, cytokine- feature that aids in differentiating these
mediated reactions may be less frequent reactions from hypotension due to a hemoly-
when the component(s) are less than or tic reaction or bacterial contamination,
equal to 3 days old. Plasma removal may and from respiratory compromise caused
also decrease febrile reactions. Aceta- by TRALI (see below). The severity of al-
minophen is commonly given before lergic transfusion reactions may increase
transfusion, but there is no evidence that with successive transfusions.
the premedication lessens the incidence Allergic reactions are attributed to expo-
of FNHTR symptoms due to prestorage sure to a soluble substance in donor plasma
leukocyte-reduced platelets.25 that binds to preformed IgE antibodies on

mast cells, resulting in the activation and Similarly, bradykinin activation by pre-
release of histamine. This presumption is kallikrein activity in plasma protein fraction
based on the facts that reactions tend to re- has also been implicated in hypotensive re-
cur in an affected recipient and that they actions,36 and a similar mechanism is prob-
can be prevented by removal of the plasma ably responsible for the many patients
from cellular components or, in the case of taking ACE inhibitors reported to have
urticaria, by antihistamines. Anaphylactic hypotensive reactions when receiving
and anaphylactoid reactions are sometimes blood components via bedside leukocyte
associated with class, subclass, and allo- reduction filters.37-39 Similar reactions have
type-specific antibodies against IgA, partic- been observed in association with the con-
27
ularly in IgA-deficient patients. IgE anti- tact of plasma with charged dialysis mem-
IgA has been demonstrated in two patients branes, low-density lipoprotein adsorption
with common variable immunodeficiency columns, and staphylococcal protein A
having reactions to immunoglobulin prep- immunoadsorption columns. Other mech-
arations.28 However, most of the IgA anti- anisms that have been proposed include the
bodies to which anaphylactic reactions are infusion of complement-derived anaphyla-
27 26
attributed are of the IgG or IgM class, and toxins and histamine. The differentiation
these antibodies are demonstrable in only a and appropriate classification of these dif-
minority of the anaphylaxis cases referred ferent reactions will require additional re-
for study (17.5% in the series of Sandler et search and refined diagnostic tools.
al27). Moreover, IgA antibodies are common
but anaphylactic reactions are not. There-
fore, demonstration of anti-IgA in an indi- Frequency
vidual who has not been transfused does
Urticaria may complicate as many as 1%
not predict anaphylaxis. Other allergens or
to 3% of transfusions, the observed fre-
other mechanisms are likely.
quency depending on how vigorously it is
Severe allergic reactions have been re-
sought. The incidence of anaphylactic re-
ported in patients with antibodies directed
actions fortunately is low, estimated to be
against C4 determinants,29,30 haptoglobin,31
1 in 20,000 to 50,000 units. The SHOT data
and elements of nonbiologic origin such as
suggest that anaphylaxis is much more
ethylene oxide used for sterilizing tubing
common as a complication of plasma and
sets.32 However, the causative antigens have
platelet transfusions, than of red cells14;
not been identified in the vast majority of
although these reactions may have con-
cases. Reactions caused by passively trans-
tributed to the death of a few severely ill
ferred donor antibody have rarely been
33,34 patients, they were not a primary cause of
documented.
death. The mortality rate reported to the
Hypotensive reactions mimicking ana-
FDA is about 1 per year.26
phylaxis have been observed in patients
taking angiotensin-converting enzyme
(ACE) inhibitors who receive albumin dur-
ing plasma exchange.35 They were thought Treatment
to be due to inhibition of bradykinin catab- If urticaria is the only adverse event noted,
olism by the ACE inhibitors combined with the transfusion may be temporarily inter-
bradykinin activation by low levels of pre- rupted while an antihistamine (eg, di-
kallikrein activator (a Hageman factor frag- phenhydramine, 25-50 mg) is adminis-
ment) in the albumin used for replacement. tered orally or parenterally. If symptoms

are mild and promptly relieved, the trans- Prevention

fusion may be resumed, provided the
interrupted infusion can be completed Recipients who have frequent transfusion-
within the acceptable time (see Chapter associated urticarial reactions may re-
22). If the patient develops severe urticaria, spond well to administration of antihista-
a significant local swelling, respiratory or mine (eg, 25-50 mg of diphenhydramine)
gastrointestinal symptoms, or hypotension, one-half hour before transfusion. However,
the transfusion should be discontinued.
26
diphenhydramine should not be given
The immediate treatment of an anaphy- routinely without a history of previous al-
lactic transfusion reaction should be to stop lergic reactions, particularly to elderly pa-
40
the transfusion and treat hypotension by tients. If antihistamine administration is
placing the patient in the Trendelenberg insufficient, 100 mg of hydrocortisone given
(feet up) position and administering a fluid 1 hour before transfusion may be useful.
challenge. If the blood pressure does not If reactions are recurrent and severe or as-
improve immediately, epinephrine should sociated with other allergic manifestations
be given. In mild to moderate cases, epi- in spite of adequate premedication, trans-
nephrine (1:1000) should be delivered sub- fusion of washed red cell or platelet com-
cutaneously or intramuscularly in a starting ponents, or red cells that have been frozen,
dose of 0.3 to 0.5 mL in adults, or 0.01 thawed, and deglycerolized will usually be
mL/kg in children. This dose may be re- tolerated.
peated a second and third time at 5- to Patients who have had a prior life-threat-
15-minute intervals. In severe reactions (eg, ening anaphylactic reaction and who are
systolic blood pressure below 80 mm Hg, IgA-deficient or have a demonstrated IgA
laryngeal edema with upper airway com- antibody should receive blood components
promise, or respiratory failure), the drug that lack IgA, either by washing or prepara-
should be given intravenously (1:10,000) for tion of components from IgA-deficient
the most rapid effect because drug absorp- blood donors. Severe reactions that are not
tion is unreliable in hypotensive patients. caused by anti-IgA can be prevented only
Aerosolized or intravenous beta-2 agonists by maximal antiallergy immunosuppres-
and theophylline may be required in se- sion or washing. A need for red cells may be
lected patients in whom bronchospasm is met by the use of washed or frozen, thawed,
26
unresponsive to epinephrine treatment, or and deglycerolized units. Washed platelets
in whom epinephrine is ineffective because are generally not readily available and may
of pre-existing beta-blocker therapy. Oxy- result in decreased platelet recovery, func-
41
gen therapy should be administered as re- tion, and survival ; therefore, after the first
quired, with endotracheal intubation if reaction, if there is no evidence that the re-
there is significant upper airway obstruc- action is mediated by IgA, some centers
tion. Continued hemodynamic instability elect to rechallenge the patient under
may require invasive hemodynamic moni- closely controlled circumstances. Preven-
toring. Under no circumstances should the tion of anaphylactoid reactions in patients
transfusion be restarted. Coincidental oc- such as those with thrombotic thrombocy-
currence of myocardial infarction, pulmo- topenic purpura who absolutely require
nary embolism, or other medical catastro- plasma components may be a tremendous
phes could present with hypotension and challenge if IgA-deficient donors will not
respiratory compromise and should be suffice. Pretreatment with antihistamines,
considered.26 corticosteroids (starting with 100 mg of hy-

drocortisone), and ephedrine may help. Fi- include aspiration, pneumonia, toxic inha-
nally, it may be possible to collect and store lation, lung contusion, near drowning,
autologous blood components from patients severe sepsis, shock, multiple trauma, burn
known to have experienced anaphylactic injury, acute pancreatitis, cardiopulmonary
reactions. bypass, and drug overdose. It was noted
that such a definition will not include cases
Transfusion-Related Acute Lung Injury of mild respiratory embarrassment having
a similar pathogenesis, cases of ALI in pa-
Pathophysiology and Manifestations tients with circulatory overload, and cases
TRALI should be considered whenever a in which a transfusion-related process causes
transfusion recipient experiences acute worsening of pre-existing ALI.44
respiratory insufficiency and/or X-ray TRALI may result from multiple mecha-
findings are consistent with bilateral pul- nisms. Donor antibodies directed against
monary edema but has no other evidence recipient HLA class I or II antigens, or
of cardiac failure or a cause for respiratory neutrophil antigens of the recipient, have
failure. The severity of the respiratory dis- been demonstrated45-47 and are thought to
tress is usually disproportionate to the cause a sequence of events that increase the
volume of blood infused. The reaction permeability of the pulmonary microcir-
typically includes fever, chills, and hypo- culation so that high-protein fluid enters
tension, usually occurring during or with- the interstitium and alveolar air spaces. In-
in 1 to 2 after transfusion, often with an frequently, antibodies in the recipient’s cir-
immediate and dramatic onset. Implicated culation against HLA or granulocyte anti-
components always have contained plasma, gens initiate the same events.45,46,48 Although
but the volume may be as small as that of one would expect causative antibodies to
a unit of cryoprecipitate or RBCs in an ad- be far more common in recipients than do-
ditive solution.42,43 nors, the rarity of TRALI due to recipient
Because the manifestations of TRALI are antibody might be due to the fact that the
variable and may overlap with those of the pool of target leukocytes is much smaller in
patient’s underlying medical problems, it is a cellular blood component than in a recip-
useful to define the syndrome, particularly ient’s circulation. Monocyte activation, with
for the purpose of conducting studies of its expression of cytokines including IL-1β,
epidemiology and pathogenesis. A consen- TNFα, and tissue factor, has been demon-
sus conference of the blood services in strated, and these reactions were highly
Canada developed such a definition.44 The specific for cells bearing the target antigens.48
panel defined acute lung injury (ALI) as a Perfusion of neutrophils, complement, and
syndrome of: 1) acute onset; 2) hypoxemia neutrophil-specific antibody into an ex-vivo
(PaO2/FIO2 <300 mm Hg, or O2 saturation rabbit lung preparation causes severe edema,49
<90% on room air, or other clinical evi- and autopsy studies demonstrate neutro-
dence); 3) bilateral lung infiltrates on a phil aggregation in the lungs of patients
chest x-ray; and 4) no evidence of circula- who have died of TRALI.50 These and other
tory overload. TRALI is then defined as: 1) observations suggest that pulmonary edema
new ALI occurring during transfusion or in TRALI is caused by neutrophil-mediated
within 6 hours of completion; and 2) no endothelial damage, initiated by antibodies
other temporally associated ALI risk factors. activating neutrophils directly or via activa-
If the latter are present, the case is consid- tion of monocytes, pulmonary macrophages,
ered “Possible TRALI.” Risk factors for ALI and/or endothelial cells.

As the spectrum of antibodies implicated Treatment

in cases of TRALI broadens, more cases will
If any kind of acute pulmonary reaction is
appear to be antibody-mediated. However,
suspected, the transfusion should be
other mechanisms have been proposed as
stopped immediately and the same unit
causes for transfusion-related respiratory
should not be restarted even if symptoms
failure. Severe pulmonary reactions are re-
abate. Clinical management focuses on
ported after granulocyte transfusions, par-
reversing progressive hypoxemia with ox-
ticularly in patients with known or unap-
ygen therapy and ventilatory assistance, if
parent lung infections or with conditions
necessary. The role of intravenous ste-
likely to promote prompt complement acti-
51 roids is unproved. Unlike other forms of
vation. Other factors may include anaphy-
acute respiratory distress syndrome, most
latoxins C3a and C5a, aggregation of granu-
patients recover adequate pulmonary
locytes into leukoemboli that lodge in the
function within 2 to 4 days,42,45 and the ob-
pulmonary microvasculature, or transfusion
served mortality is less than 6% to 23%
of cytokines that have accumulated in
(Holness L, personal communication).
stored blood components. Recently, reac-
tive lipid products from donor blood cell
membranes have been implicated as po- Prevention
tential granulocyte activators in the patho- If antibody in donor plasma can be shown
genesis of TRALI.52 These substances accu- to have caused an acute pulmonary reac-
mulate during blood bank storage and tion, blood from that donor should not be
prime neutrophils to produce vasoactive used for plasma-containing components.
mediators in response to a second stimulus No special precautions are needed for the
such as infection. One nested case-control patient if the problem was donor-specific
study found that component age and levels and components from other donors are
of bioactive lipids, but not leukocyte anti- available. Current policy in the United
bodies, were associated with TRALI.53 Kingdom is not to prepare plasma from
The incidence rate of TRALI is not known, female donors.
but data from one institution in the 1980s
suggest that this complication may occur as Circulatory Overload
frequently as 1 in 5000 transfusions.45 The
passive surveillance data of 5 years of SHOT Pathophysiology and Manifestations
reports14 include 70 TRALI cases (approxi- Transfusion therapy may cause acute pul-
mately 1 per 250,000 total components), of monary edema due to volume overload,
which 18 cases were fatal (includes six defi- and this can have severe consequences,
nite, two probable, and 10 possible TRALI- including death. Few data are available on
related fatalities). In this series, TRALI was the incidence rate of transfusion-induced
the most common cause of morbidity and circulatory overload in the general popu-
mortality, ahead of transfusion-associated lation, but young children and the elderly
graft-vs-host disease (TA-GVHD), ABO in- are considered most at risk, and incidence
compatibility, and bacterial contamination. rates of up to 1% have been observed in a
The SHOT data suggest that the rate of study of elderly orthopedic patients. 54
TRALI is higher after plasma and platelet Rapid increases in blood volume are es-
transfusion. Conclusions regarding incidence pecially poorly tolerated by patients with
and fatality rates will, of course, depend on compromised cardiac or pulmonary sta-
the definition of TRALI used. tus and/or chronic anemia with expanded

plasma volume. The infusion of 25% albu- tabolism of citrate can occur after mas-
min, which shifts large volumes of extra- sive transfusion but is probably not clini-
vascular fluid into the vascular space, may cally significant. Patients who are losing
also cause circulatory overload. Hyper- blood rapidly may have pre-existing or
volemia must be considered if dyspnea, coexisting hemostatic abnormalities or
cyanosis, orthopnea, severe headache, hy- develop them during resuscitation.
pertension, or congestive heart failure oc- Hemostatic abnormalities may include
cur during or soon after transfusion. Ele- dilutional coagulopathy, DIC, and liver
vated levels of brain natriuretic peptide and platelet dysfunction.
may be seen in cases of circulatory over-
55
load, and this test may be useful in sepa-
rating such cases from cases of TRALI. Citrate Toxicity
Pathophysiology and Manifestations.
Treatment When large volumes of FFP, Whole Blood,
Symptoms usually improve when the in- or Platelets are transfused rapidly, partic-
fusion is stopped, and it should not be re- ularly in the presence of liver disease,
started until volume overload has been plasma citrate levels may rise, binding
addressed. Placing the patient in a sitting ionized calcium and causing symptoms.
position may help. Diuretics and oxygen Citrate is rapidly metabolized, however,
56
are often indicated and, if symptoms are so these manifestations are transient.
not relieved, multiple medical interven- Hypocalcemia is more likely to cause clin-
tions may be required, including phlebot- ical manifestations in patients who are in
omy. shock or are hypothermic. Prolonged
apheresis procedures put patients, and
occasionally blood donors, at some risk.
Prevention
Exchange transfusion, especially in tiny
Except in conditions of ongoing, rapid infants who are already ill, requires care-
blood loss, anemic patients should re- ful attention to all electrolytes.
ceive blood transfusions slowly, with at- A decrease in ionized calcium increases
tention to total fluid input and output. neuronal excitability, leading, in the awake
The administration of diuretics before patient or apheresis donor, to symptoms of
and during the transfusion may be help- perioral and peripheral tingling, shivering,
ful. and lightheadedness, followed by a diffuse
sense of vibration, tetanic symptoms such
Complications of Massive Transfusion as muscle cramps, fasciculations and
Among the numerous complications that spasm, and nausea. In the central nervous
may accompany massive transfusion, system, hypocalcemia is thought to in-
metabolic and hemostatic abnormalities crease the respiratory center’s sensitivity to
are matters of particular concern. Some CO2, causing hyperventilation. Because
or all of the following metabolic derange- myocardial contraction is dependent on
ments can depress left ventricular func- the intracellular movement of ionized cal-
tion: hypothermia from refrigerated blood, cium, hypocalcemia also depresses cardiac
citrate toxicity, and lactic acidosis from function.57
systemic underperfusion and tissue Treatment and Prevention. Massively
ischemia, often complicated by hyper- transfused patients, particularly those with
kalemia. Metabolic alkylosis due to me- severe liver disease or those undergoing

rapid apheresis procedures such as periph- ing destroys red cells and has caused fatali-
eral blood progenitor cell collections, may ties.5
benefit from calcium replacement. It should
be noted, however, that empiric replace-
ment therapy in the era before accurate
Hyperkalemia and Hypokalemia
monitoring of ionized calcium was avail-
able was associated with iatrogenic mortal- Pathophysiology. When red cells are stored
ity.58 Usually, however, unless a patient or at 1 to 6 C, the potassium level in the super-
donor has a predisposing condition that natant plasma or additive solution in-
hinders citrate metabolism, hypocalcemia creases. Although the concentration in the
due to citrate overload requires no treat- plasma/anticoagulant portion of an RBC
ment other than slowing the infusion. Cal- unit may be high (see Chapter 8), because
cium must never be added directly to the of the small volume, the total extracellular
blood container because the blood will clot. 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-
Hypothermia mic problems in the recipient because
Pathophysiology and Manifestations. rapid dilution, redistribution into cells, and
Ventricular arrhythmias may occur in pa- excretion blunt the effect. Hypokalemia is
tients who receive rapid infusions of large probably more often observed63 because
volumes of cold blood, and they can be potassium-depleted red cells reaccumulate
prevented by blood warming.59 The effect this intracellular ion, and citrate metabo-
of cold blood is presumed to be more lism causes movement of potassium into
likely if the blood is administered via cen- the cells in response to the consumption
tral catheters positioned close to the car- of protons. Hyperkalemia may be a prob-
diac conduction system.60 Hypothermia lem in patients with renal failure and in
increases the cardiac toxicity of hypo- premature infants and newborns receiv-
calcemia or hyperkalemia and can result ing relatively large transfusions, such as in
in poor left ventricular performance. cardiac surgery or exchange transfusion;
Other complications of hypothermia in- otherwise, it can be demonstrated only as
clude impaired hemostasis 6 1 and in- a transient effect in very rapid transfusion.
creased susceptibility to wound infec- Treatment and Prevention. No treat-
tions.62 Blood warming is a must during ment or preventive strategy is usually nec-
massive transfusion of cold blood. essary, provided the patient is adequately
Treatment and Prevention. Hypother- resuscitated from whatever condition re-
mia-induced arrhythmias are reduced by quired the massive transfusion.63 For large-
avoiding rapid infusion of cold blood into volume transfusion to sick infants or adults
the cardiac atrium. Generalized effects of at risk, many professionals prefer red cells
hypothermia can be prevented by using that are no more than 5 to 14 days old or
blood warmers. AABB Standards for Blood washed units. However, for infants receiv-
Banks and Transfusion Services mandates ing small-volume transfusions infused
that warmers have a temperature monitor slowly, units may be used safely until their
and a warning system to detect malfunc- expiration date.64 There is no evidence that
tion and prevent hemolysis.17(p6) Attention to routine red cell transfusions require manip-
proper protocol is critical during the use of ulation to lower potassium levels, even in
blood warming devices because overheat- patients with no renal function.

Coagulopathy in Massive Transfusion conclusion was reached by Harke and

Rahman,70 who showed that the degree of
Pathophysiology. Of greater concern is the platelet and clotting abnormalities corre-
occurrence of coagulopathy during mas- lated with the length of time the patient
sive transfusion. Classically, this coagulo- was hypotensive, in groups of patients re-
pathy is ascribed to dilution of platelets ceiving similar transfusion volumes, also
and clotting factors, which occurs as pa- suggesting that the most important cause
tients lose hemostatically active blood. The was DIC due to shock. Taking these data to-
lost blood is initially replaced with red 71
gether, Collins concluded that “...coagulo-
cells and asanguinous fluids. Classic stud- pathy in heavily transfused patients was due
65 66
ies of military and civilian trauma to hypoperfusion, not transfusion.”
patients receiving stored Whole Blood These data may not be generalizable to
demonstrated a progressive increase in the patients undergoing massive transfusion in
incidence of “microvascular bleeding” (MVB) the “clean” setting of the operating room,
characteristic of a coagulopathy with in- where hypotension due to volume loss is
creasing transfusion, typically occurring af- prevented. In this setting, coagulation fac-
ter replacement of two to three blood vol- tor levels may indeed have priority over
umes (20 to 30 Whole Blood units). Although platelet problems.72
platelet counts, coagulation times, and lev- Treatment and Prevention. The dilu-
els of selected clotting factors all correlated tional model of coagulopathy in massive
with volume transfused, contrary to ex- transfusion would suggest that prophylactic
pectations from a simple dilutional model, replacement of hemostatic components
the relationship was marked by tremen- based on the volume of red cells or whole
dous variability. Inspection of laboratory blood transfused would prevent develop-
parameters in the patients developing a ment of a bleeding diathesis. However, pro-
bleeding diathesis, as well as the response spective studies have consistently shown
to various hemostatic components, sug- that such regimens do not work,73 perhaps
gested that platelet deficits were more im- due to patient variability. Instead, replace-
portant in causing the bleeding than were ment of platelets and coagulation factors in
coagulation factor deficiencies. MVB typi- the massively transfused trauma or surgical
cally occurred when the platelet count fell patient should be based on characteriza-
below 50,000 to 60,000/µL. On the other tion of the specific abnormality by use of
hand, no simple relationship could be de- platelet counts, the PT (international nor-
termined between a patient’s coagulation malized rate), aPTT, and fibrinogen levels.
tests and the onset of bleeding. Thromboelastography may also be useful.
Subsequent studies have refined these It is imperative that the laboratory rapidly
observations. Significant platelet dysfunc- complete testing. Empiric therapy with
tion has been demonstrated in massively platelets and/or plasma may be initiated
67,68
transfused trauma patients. In the stud- immediately after specimens are obtained.
66,69
ies of Counts and coworkers, low fibrino-
gen and platelet levels were better pre-
Air Embolism
dictors of hemostatic failure than elevations
of prothrombin time (PT ) and partial Air embolism can occur if blood in an open
thromboplastin time (PTT), suggesting that system is infused under pressure or if air
consumption coagulopathy was an impor- enters a central catheter while containers
tant factor in addition to dilution. A similar or blood administration sets are being

changed. It has been reported in associa- immediately. A responsible physician

tion with intraoperative and perioperative should evaluate the patient to deter-
blood recovery systems that allow air into mine whether a transfusion reaction
the infusion bag.7 The minimum volume is a possibility, what kind it might be,
of air embolism that is potentially fatal for and what immediate actions should
an adult is approximately 100 mL.74 Symp- be undertaken. The possibilities of acute
toms include cough, dyspnea, chest pain, hemolytic reaction, anaphylaxis, trans-
and shock. fusion-induced sepsis, and TRALI
If air embolism is suspected, the patient should be kept in mind because these
should be placed on the left side with the conditions require aggressive medical
head down, to displace the air bubble from management and must be reported
the pulmonic valve. Aspiration of the air is promptly to the laboratory.
sometimes attempted. However, proper use 5. If the observed events are limited to
of infusion pumps, equipment for blood re- urticaria or circulatory overload, the
covery or apheresis, and tubing couplers is transfusion service need not evalu-
still essential to prevent this complication. ate postreaction blood samples. If there
are signs and symptoms other than
urticaria or circulatory overload,
particularly if there is any possibility
Evaluation of a Suspected of acute HTR, anaphylaxis, TRALI,
Acute Transfusion Reaction transfusion-induced sepsis, or other
serious problem, a postreaction blood
The Role of Clinical Personnel Attending sample(s) should be sent to the labo-
the Patient ratory for evaluation. The speci-
Medical personnel attending the patient men(s) must be carefully drawn to
are generally the first to suspect that a avoid mechanical hemolysis and
transfusion reaction has occurred and the must be properly labeled. In addi-
first to take action. The appropriate actions tion, the transfusion container with
should be specified in the institution’s pa- whatever contents remain, the ad-
tient care procedures manual, and trans- ministration set (without the nee-
fusion service personnel should be pre- dle), and the attached intravenous
pared to act as consultants. solutions should be sent to the labo-
1. If a transfusion reaction is suspected, ratory, following standard precau-
the transfusion should be stopped to tions. In some cases, a postreaction
limit the volume of blood infused. urine sample will be useful.
2. All labels, forms, and patient identi-
fication should be checked to deter-
The Role of the Laboratory
mine whether the transfused compo-
nent was intended for the recipient. Whenever hemolysis is a possibility, the
3. An intravenous line should be main- laboratory should perform three steps as
tained with normal saline (0.9% so- soon as possible after receiving notifica-
dium chloride), at least until a medi- tion and the clinical material: check for
cal evaluation of the patient has been clerical errors; perform a visual check for
completed. hemolysis; and check for evidence of blood
4. The transfusion service and the pa- group incompatibility by performing a di-
tient’s physician should be notified rect antiglobulin test (DAT) and a recon-

firmation of the recipient’s ABO type. Some oration. An increase in bilirubin may begin
laboratories do not follow this sequence as early as 1 hour after the reaction, peak
when the only manifestations are urtica- at 5 to 7 hours, and disappear within 24
rial or febrile reactions to ABO-compati- hours if liver function is normal.
ble platelets. In examining a postreaction urine speci-
men, it is important to differentiate among
Check for Identification Errors hematuria (intact red cells in the urine),
hemoglobinuria (free hemoglobin in the
The identification of each patient’s sam-
urine), and myoglobinuria (free myoglobin
ple and the blood component(s) must be
in the urine). In acute HTRs, free hemoglo-
checked for errors. If an error is discov-
bin released from damaged cells can cross
ered, the patient’s physician or other re-
the renal glomeruli and enter the urine, but
sponsible health-care professional must
hematuria and myoglobinuria would not
be notified immediately, and a search of
be expected. Urine examination should be
appropriate records should be initiated to
done on the supernatant fluid after centri-
determine whether misidentification or
fugation of a freshly collected specimen;
incorrect issue of other specimens or
misinterpretation can occur if free hemo-
components has put other patients at risk.
globin is released when previously intact
Once the acute crisis has passed, each step
red cells in a specimen undergo in-vitro
of the transfusion process should be re-
hemolysis during transportation or storage.
viewed to find the source of error.
Visual Check for Hemolysis Serologic Check for Incompatibility

The serum or plasma in a postreaction A DAT must be performed on a postreac-
blood specimen must be inspected for ev- tion specimen, preferably one anticoagu-
idence of hemolysis and compared with a lated with a chelating agent (such as EDTA)
prereaction sample, if available. Pink or to avoid in-vitro coating of red cells by
red discoloration after, but not before, the complement proteins. If the postreaction
reaction suggests destruction of red cells DAT is positive, a DAT should be performed
and release of free hemoglobin. Intravas- on red cells from the pretransfusion spec-
cular hemolysis of as little as 2.5 mL of red imen (unless this was already done as part
cells may produce visible hemoglobinemia.75 of pretransfusion testing) and compared.
Hemolysis resulting from poor collection If transfused incompatible cells have been
technique or other medical interventions coated with antibody but not immediately
can cause hemoglobinemia; if faulty sam- destroyed, the postreaction specimen DAT
pling is suspected, examination of a sec- is likely to be positive, often with a mixed-
ond specimen should resolve the question. field agglutination pattern. If the trans-
Myoglobin, released from injured muscle, fused cells have been rapidly destroyed,
may also cause pink or red plasma and the postreaction DAT may be negative,
might be suspected if a patient has suf- particularly if the specimen is drawn sev-
fered severe trauma or muscle injury.76(p369) eral hours later. If both the pre- and post-
If the sample is not drawn until 5 to 7 reaction DATs are positive, further work-
hours after an episode of acute hemolysis, up is required to rule out incompatibility.
hemoglobin degradation products, espe- Comparison of the graded strength of these
cially bilirubin, may be in the blood- two tests is not a reliable method to rule
stream and cause yellow or brown discol- this out. Nonimmune hemolysis (eg, from

thermal damage or mechanical trauma) previously undetected antibody is

causes hemoglobinemia but not a posi- discovered, it should be identified.
tive DAT. The recipient’s ABO type must Once the antibody has been identi-
also be confirmed on the postreaction fied, retained samples from transfused
specimen. donor units should be tested for the
corresponding antigen. If a previ-
Additional Laboratory Evaluation ously undiscovered antibody is pres-
ent in a postreaction specimen but
If any of the three initial checks and tests not in a prereaction sample, the rea-
(error check, visual inspection for hemo- son may be 1) a sample identifica-
globinemia, DAT and ABO confirmation) tion error, 2) anamnestic antibody
gives positive or suspicious results, the di- production after a recent transfusion,
agnosis of an acute HTR should be vigor- or, less likely, 3) passive transfer of
ously pursued. Even if no error or apparent antibody from a recently transfused
incompatibility is found, the possibility of component. It may be desirable to use
an acute HTR should still be considered if enhancement techniques, such as an
the patient’s clinical presentation is con- increased serum-to-cell ratio, low-
sistent with such a reaction. The tests ionic-strength saline, Polybrene, poly-
listed below help characterize the cause ethylene glycol, or enzyme techni-
of the HTR, if one has occurred, or help ques, when retesting the prereaction
clarify the immunologic and serologic sta- specimen.
tus of patients in whom the diagnosis is 4. Repeat crossmatch tests, with pre-
unclear. Some or all may be performed reaction and postreaction samples in
following a written institutional protocol parallel using the antiglobulin tech-
or at the discretion of the physician in nique. A positive crossmatch in the
charge of the transfusion service. face of a negative antibody screen-
1. If ABO and Rh typing on the prereac- ing test may indicate the presence of
tion and postreaction samples do not an antibody directed against a low-
agree, there has been an error in pa- incidence blood group antigen.
tient or sample identification, or in 5. Perform DAT and antibody detection
testing. If sample mix-up or misla- tests on additional specimens ob-
beling has occurred, another pa- tained at intervals after the transfu-
tient’s specimen may also have been sion reaction. A first postreaction
incorrectly labeled; it is important to sample may have serologically un-
check the records of all specimens detectable levels of a significant allo-
received at approximately the same antibody, especially if all the anti-
time. body molecules have attached to the
2. Perform ABO and Rh testing on blood incompatible transfused cells. In this
from the unit or an attached seg- event, antibody levels would rise
ment. If blood in the bag is not of the rapidly, and antibody detection and
ABO type noted on the bag label, identification would become possi-
there has been an error in unit label- ble within a few days.
ing. 6. Perform frequent checks of the pa-
3. Perform antibody detection tests on tient’s hemoglobin values, to see
the prereaction and postreaction whether the transfused cells produce
samples and on the donor blood. If a the expected therapeutic rise, or

whether a decline occurs after an if the unit had been inappropriately

initial increase. In patients with sickle heated in the container, both the
cell anemia, the survival of trans- blood in the container and in the
fused red cells can be followed by administration tubing would be
evaluation of the levels of hemoglo- hemolyzed. If a faulty infusion de-
bin A. In complex cases, phenotypic vice had been used during blood
differences between autologous and administration, hemolysis might be
transfused cells quantitated by flow present in the administration tubing,
cytometry have been used to follow but not in the container.
survival.77 Additional testing may also be useful for
7. In-vivo red cell survival studies have significant nonhemolytic reactions.
been used to demonstrate the rare 1. If the presentation suggests an ana-
occurrence of an acute HTR in the phylactic reaction, test the patient’s
absence of detectable alloantibody.78 serum for the presence of anti-IgA.
When the patient is phenotyped in Preliminary information can be ob-
preparation for such studies, it is im- tained by quantitation of IgA because
portant that the sample be one that most patients with IgA-related ana-
contains only the patient’s red cells. phylaxis have been IgA deficient.27
This may be difficult if the patient Note, however, that subclass- or allo-
has received transfusions within the type-specific antibodies may de-
previous several weeks. Method 2.15 velop in patients with normal IgA
gives a technique for obtaining auto- levels.26 If additional transfusions are
logous red cells from a patient who required, cellular components can
has been transfused. If an antigen is be washed; if plasma (or platelets;
present on the donor’s red cells and see above) is required, IgA-deficient
absent from those of the patient, its plasma can be used.
presence or absence in postreaction 2. Examine the returned unit for any
samples indicates whether the trans- abnormal appearance, including clots
fused cells have survived and re- or any brownish, opaque, muddy, or
mained in the circulation. purple discoloration. If the clinical
8. Markers of hemolysis, including lac- presentation suggests bacterial sep-
tate dehydrogenase, unconjugated sis, a Gram stain and bacterial cul-
bilirubin, and haptoglobin levels, tures of the contents should be per-
may be useful, particularly if preformed, even if the unit looks normal.
and multiple postreaction measure- Blood cultures should also be per-
ments are available. formed on the patient’s blood. 7 9
9. Examine the blood remaining in the Treatment for suspected bacterial
unit and the administration tubing contamination should be based on
for evidence of hemolysis, especially clinical considerations because a de-
if no immune explanation for hemo- lay in therapy may result in severe
lysis can be demonstrated. Depending morbidity or death. Treatment in-
on how the blood was handled and cludes prompt intravenous adminis-
administered, hemolysis may be tration of broad-spectrum antibiot-
present in the container and the ad- ics after blood and other appropriate
ministration tubing, or only in the cultures have been obtained, combined
administration tubing. For example, with therapy for shock, if present.

3. If the clinical presentation suggests within 7 days of transfusion.84 In two of the

TRALI, test the patient’s pretransfu- 58, only the DAT was positive, but, in all
sion sample and a sample of the do- others, repeat antibody screening would
nor’s plasma for antibodies to HLA and have detected the new antibody. Regard-
neutrophil antigens. Crossmatching less, characterization of an eluate is neces-
recipient lymphocytes or granulocytes sary because alloantibodies may be present
with implicated donor sera can pro- on the red cells that are not in the serum. If
vide supportive evidence for TRALI. 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 Consequences of delayed HTR (DHTR) should be investigated.
Transfusion Delayed Reactions. In most cases, ana-
Alloimmunization to Red Cell Antigens mnestic antibody production does not cause
detectable hemolysis, leading to the desig-
Pathophysiology nation “delayed serologic transfusion reac-
85
Primary alloimmunization, evidenced by tion” (DSTR). However, in some patients,
the appearance of newly formed antibod- clinically apparent hemolysis will result
ies to red cell antigens, becomes apparent from the combination of significant levels
weeks or months after transfusion. It has of antibody with hemolytic potential and
been estimated that alloimmunization large numbers of transfused red cells in the
occurs in unselected immunocompetent circulation; in the study cited above, only
recipients with a risk of 1% to 1.6% per one of the 58 recipients with a new anti-
RBC unit, provided that D-negative recip- body within 7 days of transfusion had clini-
ients receive D-negative cellular compo- cally evident hemolysis.84 This translated
nents.80 Hemolysis has been reported in into a DHTR rate of one per 2082 recipients,
cases of primary immunization, but these or one for every 11,328 units transfused. As
reports are controversial,81 and, even if it would be expected, retrospective studies,
occurs, the phenomenon must be very which would be similar to the routine expe-
rare and usually subclinical. rience of a transfusion service, yield a lower
Serologic Observations. Once allo- rate of DSTRs, but the rate of clinically de-
immunization has occurred, blood group tectable hemolysis may be roughly equiva-
antibodies can become undetectable, espe- lent.85-87 The most common presentation of
cially those of the Kidd system. One investi- a DHTR is a declining hemoglobin and a
gator reported that this occurred to 29% of newly positive antibody screen, but fever,
82
antibodies after a median of 10 months leukocytosis, and mild jaundice may be
and to 41% of antibodies after 5 or more present. Some DHTRs present as the ab-
years.83 If red cells that express the antigen sence of the anticipated increase in hemo-
are subsequently transfused, however, an globin after transfusion. Other clinical
anamnestic response may cause the ap- problems are infrequent; hemoglobinuria is
pearance, within hours or days, of IgG anti- occasionally noted, but acute renal failure
bodies that react with the transfused red is uncommon. However, DHTRs may be
cells. In a prospective study, previously un- particularly problematic in patients with
detected alloantibodies were found in 58 of sickle cell disease. In these patients,
2082 (2.8%) recipients (37% known previ- hemolysis may include autologous red
ously transfused, 36% previously pregnant) cells, a phenomenon termed sickle cell

hemolytic transfusion reaction syndrome mandates permanent preservation of re-

(see Chapter 24).88 cords of clinically significant antibodies,
If a DHTR is suspected, a freshly ob- and review of previous records before red
tained blood sample may be tested for un- cells are issued for transfusion.17(pp39,72) Pro-
expected alloantibodies, both in the serum spective antigen matching may prevent
and, by DAT, on the red cells. Discovery of a DHTRs in selected patients, particularly
new red cell alloantibody in a recently those with sickle cell disease (see Chap-
transfused patient with hemolysis strongly ters 21 and 24).89
suggests a DHTR, and the diagnosis is sup-
ported by demonstration of the corre- Posttransfusion Autoantibody
sponding antigen on the red cells from a Occasionally, transfusion of allogeneic red
retained segment from one or more trans- cells and platelets stimulates production
fused units. Antigen typing of the red cells of autoantibodies; in some of these pa-
circulating in the patient may also suggest tients, hemolytic anemia or thrombocyto-
whether the newly incompatible cells have penia may occur.90 See Chapter 20 for more
been eliminated, or whether some are still details.
circulating. Repeat antibody screening on
the patient’s previous specimen will rule
Alloimmunization to Leukocyte Antigens
out technical errors.
and Refractoriness to Platelet
Transfusions
Treatment
See Chapter 16 and Chapter 17.
Specific treatment is rarely necessary, al-
though it may be prudent to monitor the Transfusion-Associated Graft-vs-Host
patient’s urine output and renal function Disease
and observe for changes in coagulation
Transfusion-associated graft-vs-host dis-
function. If transfusion is still necessary,
ease is a usually fatal immunologic trans-
donor red cells should lack the antigen
fusion complication caused by engraft-
corresponding to the newly discovered
ment and proliferation of donor lympho-
antibody. Passenger lymphocyte hemolysis,
cytes in a susceptible host.91 The engrafted
seen after solid organ transplantation, is a
lymphocytes mount an immunologic at-
variant of DHTR and is covered in Chap-
tack against the recipient tissues, includ-
ter 26.
ing hematopoietic cells, leading to refrac-
tory pancytopenia with bleeding and
infectious complications, which are pri-
Prevention marily responsible for the 90% to 100%
Future transfusions for the patient should mortality rate in afflicted patients. TA-
lack the antigen(s) responsible for the GVHD is rare in US transfusion recipients
anamnestic response, even if the antibody and has been observed almost exclusively
again becomes undetectable. Some facili- in immunocompromised patients. In con-
ties issue a medical alert card with this in- trast, over 200 cases of TA-GVHD have
formation for the patient to carry and been described in Japan,92 with incidence
present at the time of hospitalization or rates reaching 1:660 in patients undergo-
transfusion in a different facility. It is to ing cardiovascular surgery.93 Greater ge-
prevent these problems that Standards for netic homogeneity of the Japanese popu-
Blood Banks and Transfusion Services lation and frequent use of fresh Whole

Blood from related donors are thought to rived lymphocytes in the recipient’s pe-
be the primary reasons for the surpris- ripheral blood or tissues by HLA typing.91
ingly frequent occurrence of TA-GVHD in Factors that determine an individual pa-
that country. The SHOT data14 demon- tient’s risk for TA-GVHD include whether and
strate a significant decline in the inci- to what degree the recipient is immunode-
dence of TA-GVHD since the introduction ficient, the degree of HLA similarity be-
of universal leukocyte reduction, but two tween donor and recipient, and the num-
cases occurred despite leukocyte reduction. ber and type of T lymphocytes transfused
In the first 3 years of this study, the rate of that are capable of multiplication.91 TA-GVHD
TA-GVHD was approximately 1 per 600,000 may occur in an immunologically normal
cellular components transfused and, in recipient if the donor is homozygous for an
the first 5 years, accounted for a greater HLA haplotype for which the recipient is
number of deaths than acute HTRs and heterozygous, a so-called “one-way” HLA
only slightly fewer than did TRALI. match, and if the component contains via-
ble T cells (the fresher the unit, the higher
the risk). Cytokine dysfunction, recruitment
Pathophysiology and Manifestations of host cells into the immune reaction, and
The pathophysiology of TA-GVHD is com- release of biologic mediators, in particular
plex and incompletely understood. The nitric oxide, all play a role in the pathogene-
overall mechanism includes the escape of sis.94 Of interest is the fact that TA-GVHD
donor T lymphocytes present in cellular has not been reported in an AIDS patient.
blood components from immune clearance
in the recipient and subsequent prolifera-
tion of these cells, which then mount an Treatment and Prevention
immune attack on host tissues. Manifes- Treatment of TA-GVHD with immunosup-
tations include fever, enterocolitis, rash, pressive agents has been attempted but
hepatitis, and pancytopenia. The rash rarely succeeds, so prevention is neces-
typically begins as a blanching, macu- sary. Irradiation of cellular blood compo-
lopapular erythema of the upper trunk, nents is the accepted standard method to
neck, palms, soles, and earlobes, which prevent TA-GVHD. The dose mandated by
becomes confluent with additional find- the FDA is a minimum of 25 Gy targeted
ings ranging from edema to widespread to the midline of the container and a min-
blistering. Skin biopsy reveals infiltration imum dose of 15 Gy delivered to all other
of the upper dermis by mononuclear cells parts of the component.95 This renders T
and damage to the basal layer of epithe- lymphocytes incapable of replication
lial cells. Hepatitis manifests as elevations without substantially affecting the function
in alanine and aspartate aminotrans- of red cells, platelets, and granulocytes.
ferases, alkaline phosphatase, and biliru- AABB Standards for Blood Banks and
bin. Enterocolitis causes anorexia, nausea, Transfusion Services requires routine irradi-
and up to 3 to 4 liters per day of secretory ation of cellular components from units
diarrhea. Pancytopenia is associated with collected from the recipient’s blood rela-
a hypocellular marrow. Symptoms typi- tives, and donors selected for HLA compat-
cally appear within 8 to 10 days of the ibility by typing or crossmatching.17(p43) Poli-
transfusion but may occur as early as 3 cies should be in place to define the other
days and as late as 30 days. The diagnosis groups of patients who should receive irra-
is proven by demonstration of donor-de- diated cellular components, and there must

be a process for ensuring that once a pa- cranial hemorrhage can occur. The ratio
tient has been determined to be at risk for of affected patients is five women to one
TA-GVHD, all cellular components will be irra- man, and the median age is 51 years
diated as long as clinically indicated. (range, 16-83). Most cases (68%) involve
Published guidelines96 additionally rec- patients whose platelets lack the HPA-1a
ommend component irradiation for: 1) (PlA1) antigen (<2% of the population) and
hematopoietic progenitor cell (HPC) trans- who form the corresponding antibody.
plant recipients (this includes allogeneic However, immunization to HPA-1b is re-
and autologous HPC transplants), 2) pa- ported in 10%, and other platelet antibod-
tients with hematologic disorders who will ies, including HLA antibodies, have been
be undergoing allogeneic HPC transplanta- associated with the syndrome as well. PTP
tion imminently, 3) intrauterine transfu- is usually self-limited, with full recovery
sions, 4) neonates undergoing exchange within 21 days. Historically, 10% to 15% of
transfusion or use of extracorporeal mem- patients have been reported to die from
brane oxygenation, 5) patients with Hodg- PTP, typically from intracranial bleeding,
kin’s disease, and 6) patients with congeni- so treatment is desirable.
tal cellular immunodeficiencies. TA-GVHD The reason for destruction of the pa-
has also been reported in patients with tient’s own platelets by what appears to be
acute lymphoid and myeloid leukemias, a platelet alloantibody is controversial.
chronic lymphocytic leukemia particularly Three mechanisms have been proposed,
in patients receiving fludarabine phos- including: 1) formation of immune com-
91
phate, patients with B-cell malignancies plexes of patient antibody and soluble do-
including non-Hodgkin’s lymphoma, nor antigen that bind to Fc receptors on the
myeloma, and Waldenstrom’s macroglobu- patient’s platelets and mediate their de-
linemia,14 premature or low-birthweight instruction, 2) conversion of antigen-negative
fants without specific immunodeficiency autologous platelets to antibody targets by
disorders, and children being treated for soluble antigen in the transfused compo-
neuroblastoma and rhabdomyosarcomas.91 nent, and 3) cross-reactivity of the patient’s
antibodies with autologous platelets (ie, the
Posttransfusion Purpura presence of an autoantibody component).
The last of these theories has received the
Pathophysiology and Manifestations
most support.
Posttransfusion purpura (PTP) is an un-
common event, although over 200 cases
have been published. It is characterized by
Treatment
the abrupt onset of severe thrombocyto- Because PTP remits spontaneously, treat-
penia (platelet count usually <10,000/µL) ment may appear falsely efficacious. Ste-
an average of 9 days after transfusion roids are frequently given but their role is
(range, 1-24 days).97 Components provok- controversial. Plasma exchange can achieve
ing the reaction have usually been RBCs platelet counts of 20,000/µL in 1 to 2 days,98
or Whole Blood, but PTP has also been but the use of high-dose Immune Globu-
reported after platelet and plasma trans- lin Intravenous (IGIV) is now supplanting
fusion, and after transfusion of frozen this therapy.98,99 With the use of IGIV, re-
deglycerolized RBCs. Most patients have covery to platelet counts of 100,000/µL is
previously been pregnant or transfused. typically achieved within 3 to 5 days. As it
“Wet purpura” is common, and fatal intra- does in other disorders such as immune

thrombocytopenic purpura, IGIV appears curs initially in reticuloendothelial sites,

to block antibody-mediated clearance of but when they are saturated, there is de-
the target cells, although the mechanism position in parenchymal cells. The
of action has not been established. If ran- threshold for clinical damage is lifetime
domly selected platelets are transfused, exposure to greater than 50 to 100 RBC
patients may experience a febrile transfu- units in a nonbleeding person.106 Iron de-
sion reaction, and, in the vast majority of position interferes with function of the
cases, such transfusions have not been ef- heart, liver, and endocrine glands (eg,
ficacious. Antigen-negative platelets can pancreatic islets, pituitary); hepatic fail-
be of benefit in PTP, and, in conjunction ure and cancer, diabetes mellitus, and car-
with IGIV, reversal of this disorder in 1 day diac toxicity cause most of the morbidity
is now possible.100,101 Unfortunately, the and mortality. Elevated ferritin levels
time necessary to procure such platelets demonstrate increased iron stores, and tis-
often limits their usefulness. After recov- sue damage can be shown with organ-spe-
ery, some authors have suggested that fu- cific assays such as liver enzyme levels or
ture transfusions should be from donors endocrine function tests (eg, glucose, thy-
lacking the offending antigen, or, if they roid-stimulating hormone).
97
are not available, washed platelet units. Treatment is directed at removing iron
without reducing the patient’s circulating
Immunomodulatory Effects of Transfusion hemoglobin. Metered subcutaneous infu-
sion of desferoxamine, an iron-chelating
Transfusion has been known to modulate
agent, can reduce body iron stores in such
immune responses since the 1973 obser-
patients, but the regimen of nightly subcu-
vation by Opelz and coworkers102 of im-
taneous infusion by pump is arduous and
proved renal allograft survival in trans-
expensive, and compliance is often poor. In
fused patients. This beneficial tolerance-
transfusion-dependent patients with hemo-
inducing effect of transfusion raised con-
globinopathies, red cell exchange can mini-
cerns that transfusion may have other ad-
mize additional iron loads and can reduce
verse effects in different clinical settings,
the total iron burden.107 An oral iron che-
including increased rates of postoperative
lator has been studied but is not yet avail-
solid tumor recurrence and bacterial in-
able in the United States.
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- Records of Transfusion
kocyte reduction of transfused components, Complications
remain controversial.104,105
Each transfusion service must maintain
indefinitely the records of patients who
Iron Overload have had transfusion complications or ev-
Every RBC unit contains approximately idence of alloimmunization. Possible cases
200 mg of iron. Chronically transfused of contaminated blood must be reported
patients, especially those with hemoglo- to the institution where the blood was drawn.
binopathies, have progressive and contin- Records must be kept, and consulted, to
uous accumulation of iron and no phy- prevent patients who have had a transfu-
siologic means of excreting it. Storage oc- sion reaction from having a recurrence with

subsequent transfusions. For example, pa- In the absence of such errors as adminis-
tients with a history of IgA-related anaphy- tration of ABO-incompatible blood or of
lactic reactions should be transfused with physiologic events clearly attributable to
plasma products that lack IgA. A history of acute hemolysis, anaphylaxis, TRALI, or
repeated or severe FNHTRs might prompt sepsis, transfusion is highly unlikely to be
the use of leukocyte-reduced cellular blood acutely responsible for death. The review
components. Red cell alloantibodies may should include all available medical and
become undetectable over time as dis- laboratory records and the results of an au-
cussed above, 82,83 so records should be topsy, if performed. On the other hand, if
checked and compatible blood issued in an investigation does reveal evidence or the
order to prevent a DHTR. Routine checking possibility of hemolysis, anaphylactic or
of previous results of ABO and Rh testing pulmonary events, unexplained sepsis, or
may disclose an error in testing or in the ambiguous identification records, the case
identification of a current sample. may warrant more extensive inquiry.
Records of Patients with Special Needs

In addition to records of transfusion reac- References
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records of patients who need specially ed. Bethesda, MD: AABB Press, 2001.
prepared or manipulated components. 2. Heddle NM, Kelton JG. Febrile nonhemolytic
transfusion reactions. In: Popovsky MA, ed.
This is especially important in institu- Transfusion reactions. 2nd ed. Bethesda, MD:
tions where physicians rotate frequently, AABB Press, 2001:45-82.
and the need for irradiated, leukocyte-re- 3. Davenport RD. Hemolytic transfusion reac-
duced, or IgA-deficient components may tions. In: Popovsky MA, ed. Transfusion reac-
tions. 2nd ed. Bethesda, MD: AABB Press, 2001:
not be known to a particular physician 1-44.
writing an individual order. 4. Capon SM, Goldfinger D. Acute hemolytic
transfusion reaction, a paradigm of the sys-
temic inflammatory response: New insights
Reporting Transfusion Fatalities into pathophysiology and treatment. Trans-
fusion 1995;35:513-20 [Erratum in Transfusion
When a complication of blood transfusion
1995;35:794].
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reported to the Director, Office of Compli- ated deaths: 1976-1985. Transfusion 1990;30:
ance, Center for Biologics Evaluation and 583-90.
6. Del Greco F, Kurtides ES. Kell incompatibility
Research, FDA, as soon as possible, with a with acute renal failure. Arch Intern Med 1963;
written report within 7 days (see Chapter 112:727-30.
28 for reporting information). Patients 7. Linden JV, Wagner K, Voytovich AE, Sheehan
who are critically ill and near death often
analysis of 10 years’ experience. Transfusion
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of cause and effect may occasionally be 2nd ed. Boston: Little, Brown & Co, 1996.
9. Davenport RD, Kunkel SL. Cytokine roles in
raised. The overwhelming majority of hemolytic and non-hemolytic transfusion
such deaths are unrelated to transfusion, reactions. Transfus Med Rev 1994;8:157-68.
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hemolytic transfusion reactions. In: Daven-
sion might have contributed to death, it
port RD, Snyder EL, eds. Cytokines in trans-
may be prudent to pursue an investiga- fusion medicine: A primer. Bethesda, MD:
tion. AABB Press, 1996:85-97.

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cidence, long-term serologic findings, and PlA1-negative platelets. Transfusion 1990;30:
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32:814-7. Effect of blood transfusions on subsequent

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371-9. 2002:463-82.
104. Blajchman MA. Allogeneic blood transfusions, 107. Adams DM, Schultz WH, Ware RE, Kinney
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Bethesda, MD: AABB Press, 1999.

Chapter 28: Transfusion-Transmitted Diseases
Chapter 28
Transfusion-Transmitted
Diseases
M
ANY ADVANCES HAVE been newly described putative hepatitis viruses
made in the testing of blood do- (such as TTV and SEN-V). Infectious agents
nations for infectious diseases. pose a serious threat to transfusion recipi-
However, the risk of transmitting viral, ents if they persist in the circulation of
bacterial, and parasitic diseases via trans- asymptomatic blood donors and can 28
fusion still exists, and new agents may cause clinically significant acute or chronic
appear at any time. Thus, infectious com- disease manifestations in recipients.
plications of transfusion remain an im- The vast majority of posttransfusion
portant area of concern in transfusion hepatitis in the past was attributable to
medicine. 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
Hepatitis 1,2
and mortality. These viruses are consid-
Hepatitis is inflammation of the liver that ered in detail below.
can be caused by many different toxins, HAV and HEV, which are enterically trans-
immunologic processes, or infectious agents. mitted viruses, circulate only transiently
Hepatitis linked to transfusion is almost during the acute phase of infection. Be-
exclusively caused by viruses. These viruses cause the viremic individual is usually clini-
include hepatitis viruses A-E (HAV, HBV, cally ill and not a candidate for donation,
HCV, HDV, HEV), cytomegalovirus (CMV), HAV and HEV are not serious threats to trans-
Epstein-Barr virus (EBV ), and possibly fusion recipients. However, HAV viremia
667

may be present for up to 28 days before not currently recommended because disease
symptoms develop, and isolated cases have associations have not been established.
been reported associated with transfusion
of cellular components3 and outbreaks with Clinical Manifestations of Hepatitis
4
Factor VIII concentrate. Because HAV lacks
Most individuals who acquire HBV or
a lipid envelope, it is not inactivated by sol-
HCV infection have a subclinical primary
vent/detergent treatment; additional inac-
infection without obvious symptoms or
tivation methods are under development to
physical evidence of disease. Some de-
prevent recurrence of such outbreaks. HEV
velop overt hepatitis with jaundice, nau-
is rare in the United States, and there have
sea, vomiting, abdominal discomfort, fa-
been no documented cases of transfusion
tigue, dark urine, and elevation of liver
transmission in this country.
enzymes. Signs and symptoms usually re-
HDV, formerly called the delta agent, can
solve spontaneously. Acute hepatitis C
cause infection and serious hepatitis after
tends to be milder than hepatitis B. Un-
transfusion or other parenteral exposure.
commonly, the clinical course of HBV and,
However, because HDV is a defective virus
rarely, HCV infections may be compli-
found only in HBV carriers, screening do-
cated by fulminant hepatitis. Of greater
nors for HBV infection simultaneously
concern is a propensity of hepatitis C to
eliminates the risk of HDV.5 HGV, also called
evolve to chronic hepatitis (75% to 85% of
GBV-C, is distantly related to HCV and has
affected individuals), with a significant
a high prevalence rate (>1%) among asymp-
number demonstrating long-term pro-
tomatic donors. Although HGV is unequiv-
gression to cirrhosis, liver failure, or hepa-
ocally transfusion-transmissible,6 a causal
tocellular carcinoma. Hepatitis A tends to
relationship has not been established be-
be clinically mild in otherwise healthy
tween HGV infection and hepatitis or any
hosts and is not known to progress to
other disease manifestation, despite inten-
chronic hepatitis or a chronic carrier
sive study.
state.9 HEV infection may lead to severe
TTV appears similar to HGV with respect 10
disease in pregnant women. Vaccination
to prevalence, transmissibility, and the lack
is available for hepatitis A and for hepati-
of clinical disease significance. Thus,
tis B, and hepatitis B immune globulin
screening blood donors for HGV or TTV is
(HBIG) has proven useful for post-expo-
not currently recommended. Hepatitis as-
sure prophylaxis for hepatitis B and im-
sociated with CMV or EBV is generally mild
mune serum globulin (ISG) for hepatitis
in the absence of severe immunosuppres-
A.
sion. The frequency and severity of such
hepatitis cases do not justify routine
screening measures.7 SEN-V has been asso- Chronic Carriers of HBV
ciated with transfusion-associated non-A After initial HBV infection, a proportion of
through non-E hepatitis in one study,8 but a patients fail to clear infectious virus from
causal association has not been estab- the bloodstream and become chronic car-
lished, nor has SEN-V been significantly as- riers for years or life. HBV carriers produce,
sociated with chronic non-A through non-E in addition to the infectious viral particle,
hepatitis. SEN-V appears to be distantly re- large amounts of noninfectious envelope
lated to TTV and to be a member of a fam- protein detected by the assay for hepatitis
ily of small, circular DNA viruses called B surface antigen (HBsAg). The risk of be-
Circoviridae. Screening for these agents is coming an HBsAg carrier is strongly age-

Chapter 28: Transfusion-Transmitted Diseases 669
dependent; ≤5% of those infected with and to be a mild infection at almost 20

HBV as adults become chronic HBsAg years of follow-up.14
carriers, whereas ≥95% recover completely Recommendations for clinical manage-
and develop protective antibody against ment of persons with chronic HCV infec-
HBsAg (anti-HBs). In contrast, before rou- tion were developed by a National Insti-
tine prophylactic immune globulin infu- tutes of Health consensus development
sion and immunization, 90% or more of conference15 and have been updated and
16
infants infected perinatally became carri- expanded by others.
ers and became at risk for progressing to
cirrhosis and hepatocellular carcinoma.
Markers of Viral Infection
According to World Health Organization
estimates, the number of HBsAg carriers is Laboratory tests can identify markers of
approximately 400 million worldwide,11 previous exposure and probable current
with a prevalence of up to 10% in some infectivity for HBV and HCV, which are
Asian countries, 0.1% to 0.5% in the gen- useful for screening and diagnostic appli-
eral US population, and 0.02% to 0.04% in cations. Table 28-1 lists the molecular and
US blood donors. Small proportions (<10%) serologic markers commonly used in the
of HBsAg carriers develop clinical mani- diagnosis of hepatitis. Figure 28-1 illus-
festations, such as hepatic insufficiency, trates the sequence of test results typical
cirrhosis, or hepatocellular carcinoma. of individuals with acute HBV infection
that completely resolves.
The period between exposure to HBV
and emergence of circulating markers of in-
Chronic Carriers of HCV
fection (HBV DNA or HBsAg) is usually
3,9
Most people who are initially infected with about 6 weeks. HBV DNA, detectable by
HCV become chronic HCV carriers, with pooled nucleic acid amplification testing
75% to 85% having persistent HCV RNA in (NAT) techniques, is the first marker to ap-
the serum and liver for years to decades. pear, followed by detectable HBsAg. Indi-
At least 50% of such HCV carriers have vidual donor NAT (ID-NAT) is able to detect
biochemical and histologic evidence of HBV DNA approximately 19 days before
chronic liver inflammation.12 Despite this minipooled NAT (MP-NAT).17 Antibody to
chronic inflammatory process, most HCV- the HBV core protein (anti-HBc) usually ap-
infected individuals remain asymptom- pears several weeks later, first as IgM and
atic. During the first 20 years after infec- then as IgG. The clearance of HBsAg and
tion, HCV is usually indolent and associ- appearance of anti-HBs signal resolution of
ated with low mortality and morbidity. infection. However, there has been a report
Those with clinical liver disease repre- of a fatality, after reactivation of HBV in a
sented about 10% of the entire infected patient treated with rituximab, who was
13
cohort in one study. The risk that and the previously anti-HBs-reactive.18 The specific
rate at which chronic hepatitis by itself virus showed multiple mutations in major
will progress to cirrhosis is unknown. It is antigenic sites and was thought to escape
thought that alcohol may play a synergis- the patient’s endogenous immunity.
tic role in exacerbating chronic hepatitis Two additional HBV markers, HBeAg or
C. Posttransfusion HCV in children in- its antibody (anti-HBe), are useful diagnos-
fected after cardiac surgery appears to retic and prognostic markers but are not em-
solve more frequently than that in adults ployed in donor screening. An asymptom-

670
Table 28-1. Molecular and Serologic Tests in the Diagnosis of Viral Hepatitis
Virus Test Reactivity Interpretation

HBV DNA HBsAg Anti-HBc IgM Anti-HBs HBeAg Anti-HBe
Total
+ – – – – – – 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 Recombinant Antigens (RIBA)

(Screening EIA)
5-1-1 c100-3 c33c c22-3
+/– + Not available Probable acute or chronic HCV infection (if RNA
is positive)
– + – – – – False positive
+/– + + + – – 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
+ + + Acute HAV
Chapter 28: Transfusion-Transmitted Diseases

– + – Recovered HAV/vaccinated
HEV RNA Anti-HEV

Total IgM
+ + + Acute HEV
– + – Recovered HEV
*Those with HBeAg are more infectious and likely to transmit vertically.
†
Anti-5-1-1 and anti-c100-3 generally appear later than anti-c22-3 and anti-c33c during seroconversion and may disappear spontaneously, during immunosuppression or after
successful antiviral therapy.
HBsAg = hepatitis B surface antigen; anti-HBc = antibody to hepatitis B core antigen; anti-HBs = antibody to HBsAg; HBeAg = hepatitis B e antigen; anti-delta = antibody to delta
antigen; anti-HAV = antibody to hepatitis A virus; anti-HCV = antibody to hepatitis C virus; anti-HEV = antibody to hepatitis E virus.
671
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.
atic HBsAg-positive individual may either effective detection of seronegative HBV in-
be in the early phase of acute HBV infection fected donors by NAT will require testing at
(without anti-HBc or with IgM anti-HBc) or the single donation level.19 HBV vaccines
a chronic HBV carrier (with IgG anti-HBc). contain noninfectious HBsAg protein, which
HBsAg particles are produced in excess may result in false-positive HBsAg screen-
during acute and chronic infection; blood ing test results for a few days after the inoc-
from individuals with circulating HBsAg ulation. Resulting protective antibodies are
can infect others. Current screening im- directed against HBsAg; vaccination does
munoassays detect approximately 0.2 to 0.7 not produce anti-HBc.
ng/mL HBsAg or ≥3 × 107 particles.3The Tests for antibodies to HCV are enzyme
number of HBsAg particles in most acute immunoassays (EIAs) using recombinant
and chronic infections exceeds this level, antigens of HCV coated on a solid phase as
but transmission of HBV from HBsAg the capture reagent. Current assays detect
seronegative donors has been described. antibodies to c200 (including c33c and
NAT testing allows the detection of as few c100-3), c22-3, and NS-5. Anti-HCV is de-
as 10 genomic copies of HBV DNA.3 The tectable by third-generation EIAs approxi-
value of NAT for the detection of sero- mately 10 weeks after infection. HCV RNA
negative donors infected with HBV is under is present at high concentrations in plasma
study but may be reduced by high sensitiv- during most of the period from exposure to
ity of current assays for HBsAg and the rela- antibody seroconversion. Anti-HCV is de-
tively slow rise in HBV DNA levels con- tected in 40% to 50% of samples from pa-
trasted with HIV and HCV. It is possible that tients at initial diagnosis of acute hepatitis,

either transfusion-transmitted or community- Surrogate Markers

acquired.20
Before HCV was identified and anti-HCV
The clinical significance of a positive
testing became feasible, two nonspecific
screening test for anti-HCV in healthy blood
or “surrogate” tests on donor blood were
donors is unclear without supplemental
introduced to reduce the risk of non-A, non-B
testing. Approximately 0.21% of US blood
(NANB) hepatitis after transfusion. In 1986
donors have repeatedly reactive EIA re-
and 1987, the AABB called for testing of
sults.21 Several generations of recombinant
whole blood donations for alanine amino-
immunoblot assays (RIBA) have been li-
transferase (ALT) and anti-HBc as surro-
censed by the Food and Drug Administra-
gates for the direct detection of the NANB
tion (FDA) for further elucidation of repeat-
agent. Current very sensitive tests for anti-
edly reactive EIA results. An individual who
HCV have essentially eliminated the value
is positive by RIBA is considered to have true
of surrogate tests in preventing hepatitis,25
HCV antibody; in 70% to 90% of these
but anti-HBc testing continues as recom-
cases, HCV nucleic acid is detectable by
mended by FDA to prevent HBV transmis-
NAT methods. The infectivity of units that
sion. HBV from liver transplant donors with
are positive for HCV RNA approaches
22 reactive anti-HBc and negative HBsAg test
100%. In contrast, EIA repeatedly reactive
results has been transmitted to their re-
donors with negative or indeterminate
cipients, and reports in the literature
RIBA 3.0 results, representing 37% of EIA
show that transfusions of blood reactive
repeatedly reactive donors, are rarely in-
for anti-HBc and negative for HBsAg have
fected or infectious. Regardless of RIBA re-
been associated with development of
sults, a donation with a repeatedly reactive
hepatitis B in some recipients. There may
EIA result cannot be used for transfusion.
also be a small number of potential do-
Donors with negative RIBA results may be
nors infected with HBsAg mutants of HBV
considered for reentry (Table 28-2).
that may not be optimally detected by
In 1999, NAT for HCV RNA was imple-
currently licensed HBsAg tests.26 Donors
mented as a donor screening assay under
who test repeatedly reactive for anti-HBc
FDA-sanctioned investigational new drug
on two occasions or who test repeatedly
(IND) protocols. The testing was performed
reactive on tests from two different manu-
in minipools of samples from 16 to 24 whole
facturers should be deferred.
blood donations. The rapid increase in
viremia and high viral load of seronegative,
acutely infected donors allows sensitive de- Current Risk of Posttransfusion Hepatitis
tection of HCV RNA even in these diluted The risk of posttransfusion HBV or HCV
pools. The window period for HCV detec- infection decreased dramatically, to an es-
tion with pooled NAT is reduced to 10 to 30 timated 1 in 60,000 to 1 in 100,000 risk be-
days.17 After testing over 39 million donors, fore implementation of HCV NAT.27 Not a
approximately 1:270,000 donors had been single new case of transfusion-associated
identified as being in the seronegative win- HCV has been detected by the CDC Senti-
dow with a positive NAT result.23 nel Counties Viral Hepatitis Surveillance
NAT results can be used in lieu of sup- System since 1994 (M. Alter, personal com-
plemental testing in specific circumstances. munication, 3/04).1 The development of
An FDA variance is required.24 It is antici- progressively improved HCV antibody
pated that NAT HCV testing will be used in tests and stringent selection measures for
the future for reentry. donors have contributed to this remark-

Table 28-2. Reentry of Donors with Repeatedly Reactive Screening Tests
Reentry
Repeatedly Reactive for Status
Anti-HIV-1
or -1/2 Anti-HIV-2 HIV-1-Ag HBsAg Anti-HCV
Initial sample
Licensed West- Different HIV-2 Confirmed by Confirmed by RIBA indeter- Not eligible
ern blot posi- EIA RR neutraliza- neutraliza- minate or for reentry
tive or inde- tion tion or positive
terminate or anti-HBc
IFA reactive RR
Licensed West- Different HIV-2 Not confirmed HBsAg speci- RIBA negative Evaluate for
ern blot or IFA EIA NR and li- by neutral- ficity not reentry
NR censed West- ization confirmed
ern blot or IFA by neutral-
NR ization and
anti-HBc
NR
Follow-up sample
(Drawn 6 (Drawn 6 (Drawn 8 (Drawn 8 (Drawn 6
months months weeks weeks months
later) later) later) later) later)
EIA RR or West- RR HIV-1 or dif- HIV-1-Ag RR, HBsAg RR or EIA RR or Not eligible
ern blot posi- ferent HIV-2 neutraliza- anti-HBc RIBA inde- for reentry
tive or inde- EIA RR or a li- tion con- RR terminate
terminate or censed West- firmed or or positive
IFA reactive ern blot or IFA not con-
reactive or in- firmed
determinate
Original EIA Screening test HIV-1-Ag and HBsAg NR Licensed Eligible for
method NR and a differ- anti-HIV-1 and multiantigen reentry
and whole vi- ent HIV-2 EIA EIA NR or anti-HBc EIA
rus lysate NR and li- HIV-1-Ag NR method NR
anti-HIV-1 EIA censed West- RR, not and RIBA
NR and li- ern blot or confirmed negative
censed West- IFA NR (temporary
ern blot or IFA deferral for
NR additional
8 weeks)
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 ents notified as a result of the HCV look-back
considered a major risk factor for HCV should be counseled regarding the nature
transmission.28 Nucleic acid screening as- of the subsequent donor test results and of-
says for HCV were implemented by blood fered appropriate testing. If they test posi-
centers in 1999. In 3 years of NAT testing, tive, life style changes (eg, abstinence from
170 HCV NAT-positive, seronegative do- alcohol consumption) and evaluation for
nations were identified in the United chronic liver disease justifying antiviral
States among 39.7 million screened dona- therapy to reduce the likelihood of disease
tions.23 NAT has probably reduced the re- progression may be warranted. Earlier ex-
sidual risk for HCV transmission to ≤1 in periences from Canada and several Euro-
2,000,000 components transfused.29,30 pean countries indicate that the number of
transfusion recipients who ultimately bene-
33
Quarantine and Recipient Tracing fit from look-back efforts is small. A survey
of US blood collection facilities and hospi-
Donations with repeatedly reactive screen- tal transfusion services after implementa-
ing test results (HBsAg, anti-HBc, and/or tion of targeted HCV look-back resulted in
anti-HCV) cannot be used for transfusion. an estimate that notification of the recipi-
In addition, in-date components from ents of 98,484 components would result in
collections preceding the current unsuit- the identification of 1520 infected persons
able donation may need to be quaran- who were previously unaware of their in-
tined as follows, and consignee notifica- 34
fection. This would represent less than 1%
tion for the purpose of recipient tracing of the 300,000 still-living recipients who
(ie, look-back) may be required31,32: may have acquired infection by blood
For HCV: transfusion. More recently, 0.9% to 5.0% of
■ Extending back indefinitely, to the patients tested for hepatitis C were found to
extent that computerized electronic be positive in a review of look-back studies
records exist for anti-HCV repeat- in Canada that notified recipients of any
edly reactive and confirmed dona- previous transfusions of the risk of HCV
tions or repeatedly reactive donations and then provided testing; 42% to 58% of
for which supplemental tests were the cases were newly identified.35
not performed.
■ Extending back to January 1, 1988 if
computerized electronic records are
not available.
Human Immunodeficiency
For HBsAg and anti-HBc: Viruses
■ In-date components, extending back The human immunodeficiency viruses type
5 years, or 12 months from the most 1 (HIV-1) and type 2 (HIV-2) are the
recent negative test result for units etiologic agents of AIDS. The AIDS syn-
that were repeatedly reactive or con- drome was recognized in 1981, well be-
firmed, or for which confirmatory fore the discovery of the causative virus in
testing was not performed. 1984. Wider implications of the immune
Depending on the results of licensed disorder were noted when, in 1982, AIDS
supplemental tests and prior screening was reported in three patients with hemo-
36
tests, the quarantined units may be re- philia, and in a 17-month-old infant
leased for transfusion or further manufac- whose multiple transfusions at birth in-
ture, or may have to be destroyed. Recipi- cluded a unit of platelets from a donor

37
who subsequently developed AIDS. With- tectable in the plasma. During this time,
in a few years, studies established that well about 60% of acutely infected persons de-
over 50% of patients with hemophilia who velop an acute retroviral syndrome, char-
received clotting factor concentrates in acterized by a flu-like illness with fever,
the early 1980s developed HIV-1 infec- enlarged lymph nodes, sore throat, rash,
tion.38 In some regions of the United States, joint and muscle pain—with or without
up to 1% of single-donor unit transfusions headache, diarrhea, and vomiting. As HIV-1
were infected with HIV in the early 1980s.39 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-
Clinical Manifestations of HIV Infection
ted by blood or genital secretions (Fig
HIV is a cytopathic retrovirus that prefer- 28-2).
entially infects CD4-positive T lympho- Persistent infection with an asymptom-
cytes (helper T cells) in lymph nodes and atic clinical status has been estimated to
40
other lymphoid tissue. After primary in- last a median of 10 to 12 years in the ab-
fection, HIV replicates and disseminates sence of treatment.41 After years of asymp-
initially as cell-free virions, and 10 days to tomatic infection, both plasma viremia and
3 weeks after infection, viremia is first de- the percentage of infected T lymphocytes
Figure 28-2. Virologic events during primary HIV infection. After initial infection and propagation of
HIV in lymph nodes, a blood donor becomes infectious (defined as day 0), with HIV RNA being 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 ser extent, recipients of blood transfusions.
served by helper T cells impairs immune By 1989, the rate of infection in each
reactivity, and there may be inappropriate group was no longer increasing exponen-
immune activation and cytokine secretion. tially and appeared to have reached a pla-
Eventually, there is a sharp decline in the teau in the populations most at risk.43 HIV
number of CD4+ T lymphocytes, and the seroprevalence had stabilized in most US
vast majority of infected individuals suc- cities. Heterosexual transmission of HIV
cumb to opportunistic illnesses fostered by represented a progressively larger propor-
profound immunosuppression. tion of US HIV infections and AIDS cases
Enumeration of viral load and CD4+ cells reported in the 1990s.44 This is of impor-
is used to guide clinical and therapeutic tance in transfusion medicine because
management of HIV-infected persons. The screening for heterosexual high-risk be-
AIDS classification system devised by the havior is more problematic than screen-
Centers for Disease Control and Prevention ing for male-to-male sex and parenteral
45
(CDC) is based on the number of CD4+ T drug use.
cells (<200/µL defines AIDS), the presence
or absence of systemic symptoms, and ex-
HIV-2 and HIV-1, Group O
istence of any of the 26 clinical conditions
considered to be AIDS-defining illnesses.42 First discovered in 1985, HIV-2 causes en-
Among these conditions are otherwise un- demic infection in many countries in
usual malignancies, such as Kaposi’s sar- West Africa. Although HIV-2 was initially
coma, central nervous system lymphoma, restricted to West Africa, recent studies in
and a wide array of devastating, potentially European countries such as Great Britain
lethal opportunistic infections with fungi and France (which have significant immi-
and parasites, the most common being gration from West Africa) have observed
Pneumocystis carinii pneumonia. increasing rates of HIV-2 and other HIV
Advances in treatment of HIV and op- subtypes.46,47
portunistic infections have dramatically en- The first case of HIV-2 infection in the
hanced the survival of infected persons. United States was reported in March 1988
Unfortunately, worldwide, the disease is in a young West African who had recently
still spreading rapidly and, for the majority immigrated to the United States.48 The
of HIV-infected individuals in developing spectrum of disease attributable to HIV-2 is
countries, effective therapy is either not similar to that caused by HIV-1; however,
available or not affordable. there appears to be a longer incubation pe-
riod and lower incidence of progression to
AIDS. HIV-2 is spread both sexually and
Risk Factors for HIV Infection
from mother to child, but transmission is
HIV can be transmitted through sexual less efficient than for HIV-1.
contact, childbirth, breast-feeding, and Tests in the United States on parenteral
parenteral exposure to blood. Those iden- drug users, persons with sexually transmit-
tified early as being at highest risk were ted diseases, newborn infants, and homo-
men who had sex with other men; com- sexual men confirm the very limited preva-
mercial sex workers and their contacts; lence and transmission of the agent. HIV-1/
needle-sharing drug users; patients with HIV-2 combination tests were implemented
hemophilia who received human-derived in the United States in 1992. Since then,
clotting factor concentrates; and, to a les- three HIV-2-infected donors have been

identified; none appeared to have been in- over a period spanning two decades, only
fected in the United States.49 three non-B subtypes were identified. Two
50
To date, three groups of HIV-1 viruses of these three donors were born in Africa.
have been identified: group M (major
group); group O (outlier group); and, most Transfusion Considerations
recently, group N. Further, there are 10 sub-
Transfusion-Transmitted HIV-1
types (A-J) of group M. None of 97 donors
retrospectively identified as being HIV-1 in- All blood components can transmit HIV-1.
fected in 1985 and three (1%) of 383 donors Although approximately 1% of all AIDS
prospectively identified between 1993 and exposures have resulted from transfusion
1996 were found to have non-B subtypes or organ or tissue transplantation, the in-
(two subtype As and one subtype C).50 Of troduction of MP-NAT in 1999 virtually
note, this study did find an increase in env eliminated the risk of transfusion-trans-
gene diversity among HIV-1 group B strains mitted HIV.54 The few cases of HIV infec-
over time and called for continued surveil- tion that have been documented since
lance for emergence of non-B subtypes and 1999 were attributed to low-level viremic
development of test systems for their de- units that likely would have been detected
tection. A follow-up study from these same by ID-NAT.55-57
investigators documented characterized Most but not all recipients of HIV-in-
HIV subtypes in 291 infected US donors fected blood transfusions become infected.
identified from 1997 through 2000 and In one large study, HIV infection developed
identified that six (2%) were non-B sub- in 89.5% of recipients who received blood
types of HIV-1 and one was HIV-2. In
51 from anti-HIV-positive donors.58 Transmis-
Cameroon and surrounding West African sion rates correlated with component type
countries, an estimated 1% to 2% of HIV in- and viral load in the donation. With the ex-
fections are caused by group O viral strains.52 ception of coagulation factor concentrates,
As with HIV-2, group O isolates have rarely plasma derivatives (such as albumin and
been seen outside this geographic area. immune globulins) have not been reported
Concern arose when studies demonstrated to transmit HIV infection. No transmission
that some group O viral isolates were not of HIV attributable to coagulation factors has
reliably detected by several EIA tests used been documented in the United States
for blood donor screening. Of the two since implementation of full donor screening
FDA-licensed tests for NAT, both were eval- and virus inactivation techniques in 1987.59
uated by the manufacturer for the detection
of non-B subtypes including group O and Transfusion-Transmitted HIV-2 and HIV-1,
N, using a limited number of specimens. Group O
(although neither test detects HIV-2). How- There have been two reports of possible
ever, until reliable detection of group O in- HIV-2 transmission through blood com-
fections is established, the FDA recom- ponent use, both in Europe. Two women
mends indefinite deferral of blood and were infected by Whole Blood obtained from
plasma donors who were born, resided, or a donor who developed AIDS at least 16
traveled in West Africa since 1977, or had years after becoming infected with HIV-2;
sexual contact with someone identified by both women were asymptomatic 14 years
these criteria.53 The risk of group O infec- after transfusion.60 Two hemophilia pa-
tion in the United States is very low. In a tients who received clotting factors were
survey of HIV subtypes in US blood donors also infected. Because of their extremely

low prevalence, no HIV-2 or HIV-1 group tectable antibody develops 2 to 4 weeks

O transmissions have been reported in after exposure,58 days to a week after the on-
the United States by blood transfusion or set of symptoms in those who have any rec-
any other transmission route. ognized acute illness,64 and about 12 days
after detectable viremia by ID-NAT or 9 to
Current Risk of Posttransfusion HIV 10 days by MP-NAT.17,65
A few days later, HIV-1 antibodies become
With screening tests available before 1992,
detectable by the HIV-1 Western immuno-
the seronegative interval (“window pe-
blot. With very rare exceptions, all persons
riod”) averaged 45 days. More sensitive
infected with HIV develop anti-HIV reactiv-
screening tests for HIV antibody closed
ity detectable by EIA and Western blot that
the antibody-negative window to approxi-
persists for life.
mately 22 days.17 Introduced in 1996, p24
More sensitive tests using NAT technol-
antigen screening further reduced the po-
ogies have been shown to detect additional
tentially infectious window by an esti-
potentially infectious donors. In February
mated 6 days,61 although it appears that
2002, the FDA approved the first NAT sys-
fewer HIV-infected units were intercepted
tem for screening whole blood donors and
by the introduction of this test than had
issued draft guidance for blood establish-
been expected based on the calculated re-
ments; the final guidance was issued in
duction of the infectious window period. 66,67
October 2004. It has been estimated that
Risk from seronegative donations will
HIV NAT has reduced the window period
vary in proportion to the incidence of HIV
for HIV from 16 days to 10 days.17 The use
infection in the donor community. Overall
of NAT for donor testing has not only in-
estimates of posttransfusion HIV risk in the
creased sensitivity, but has also decreased
United States since the implementation of
the number of false-positive tests, increas-
HIV NAT are approximately 1 in 2 million
ing specificity. During the 3 years of inves-
screened donations.23,30
tigational HIV NAT, 12 confirmed HIV-1
RNA-positive antibody-negative donors
HIV Testing of Blood Donors were detected in 37 million donations
screened, or 1 in 3.1 million, of which only
AABB Standards for Blood Banks and Trans- two were detected by HIV-1 p24 antigen.23
fusion Services62(pp33,34) and FDA regulations63
require that all units of blood and compo-
nents be nonreactive for anti-HIV-1 and Confirmatory Testing for Antibodies to
anti-HIV-2 before they are issued for HIV-1/2
transfusion. HIV-1-antigen (HIV-1-Ag) If a disease has low prevalence in the
testing is no more required by the FDA or tested population, the likelihood is high
AABB as long as licensed HIV-1 NAT is in that most positive screening test results will
place. Figure 28-3 shows the sequence of be false positive. More specific supplemen-
screening and confirmatory testing for anti- tal tests are then required to confirm the
HIV-1/2. screening test results. The most commonly
Because the consequence of missing even used of these tests for antibodies to HIV-1/2
one true positive is great, screening tests is the Western blot (see Chapter 7).
are designed to have high sensitivity both According to current FDA and CDC cri-
to immunovariant viruses and to low-titer teria, a sample is defined as anti-HIV-posi-
antibody during seroconversion. EIA-de- tive if at least two of the following bands are

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.
68
present: p24, gp41, and/or gp120/160. fection, but they are not currently eligible
Negative Western blot results have no to donate blood. Several groups have iden-
bands present. Western blot results classi- tified Western blot patterns in blood donors
fied as indeterminate have some bands that were identified as false-positive results;
present but do not have the pattern defin- for these, testing (using NAT) is recom-
ing HIV positivity. Individuals infected with mended to resolve the infectious status of
HIV may have indeterminate patterns when the donor.69
initially tested but develop additional bands Approximately 50% of all HIV screening
within 6 weeks. Healthy individuals with EIA repeatedly reactive donors test indeter-
initial indeterminate patterns continue to minate by licensed HIV-1 Western blot as-
have negative or indeterminate results on says. However, when these donations were
repeat samples and are negative on clinical tested by ID-NAT, less than 0.1% were
examination and additional tests, including shown to contain HIV-1 RNA (1:1450 RNA-
viral cultures and NAT. Healthy donors who positive, indeterminate donors). When com-
continue to show the same indeterminate bined with those donations that tested
pattern for more than 3 months can be reas- Western blot negative, the frequency of a
sured that they are unlikely to have HIV in- donor testing HIV repeatedly reactive and

then Western blot indeterminate or nega- Whether a facility elects to offer autolo-
tive and demonstrating RNA was 1:4.27 gous services is an internal decision. Insti-
million (S. Stramer, personal communica- tutions should consider, however, that
tion, 3/17/04). where feasible for a patient, it is generally
The FDA has approved reentry protocols accepted that the patient should have the
to qualify donors with negative confirma- option to use his or her blood. In addition,
tory test results as eligible for subsequent a US Supreme Court decision in the Brag-
donations (see Table 28-2).70 Reentry currently don v Abbott case ruled that HIV-positive
requires retesting at least 6 months later, to individuals are protected under the Ameri-
detect delayed seroconversion; the use of cans with Disabilities Act.73 Whether this
EIA tests based on whole-virus lysate; and would apply to HIV-positive donations that
use of either a licensed Western blot to en- could represent a risk to hospitalized trans-
sure appropriate sensitivity of the methods fused patients remains controversial.
or an FDA-licensed immunofluorescence
assay. 71 The later sample must also be
Recipient Tracing (Look-Back)
nonreactive in an EIA test for anti-HIV-2, if
standard testing does not include HIV-2. Identification of persons who have received
The FDA recently published draft reentry seronegative or untested blood from a do-
guidelines that will allow reinstatement of nor later found to be infected by HIV is
donors with indeterminate Western blot re- referred to as “look-back.” Because the in-
sults, eliminate the need for viral lysate EIA terval between receipt of an infected
testing, and include testing by HIV-1 NAT. transfusion and onset of AIDS can be very
NAT results can be used in lieu of sup- long, recipients are usually unaware of their
plemental testing in specific circumstances. infection and may be infectious to others.
An FDA variance is required.24 It is antici- To identify these individuals, blood cen-
pated that NAT HIV-1 testing will be used in ters must have procedures to notify recip-
the future for reentry. ients of previous donations from any do-
nor later found to have a confirmed
Positive Tests in Autologous Donors positive test for anti-HIV or a confirmed
positive test for HIV using licensed NAT. If
Whether HIV EIA repeatedly reactive or
a patient with AIDS is known to have do-
NAT-positive autologous donations should
nated previously, recipients of blood or
be withheld from transfusion is contro-
blood components from these donations
versial.72 These units may be supplied for
should be traced and notified. Recipient
autologous use if the following conditions
are met: 1) there is a written, signed, and tracing and testing are usually accom-
dated request from the patient’s physician plished through the patient’s physician, not
authorizing this shipment, 2) there is a through direct contact with the patient. In
written statement from the transfusion companion rules, the FDA and the Cen-
service indicating willingness to receive this ters for Medicare and Medicaid Services
product, and 3) the transfusion service established timelines and standards de-
74-77
takes responsibility for ensuring that there fining look-back. If recipients of units
is documented verification of the accurate that were donated at least 12 months be-
identity of the transfusion recipient. These fore the last known negative test are tested
units must be labeled “BIOHAZARD” and and found negative, earlier recipients are
“FOR AUTOLOGOUS USE ONLY.” probably not at risk because infectivity

earlier than 12 months before a negative noted among some Native American pop-
screening test is extremely unlikely. 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
Human T-Cell Lymphotropic occurrence seems to be somewhat less
Viruses frequent than with HTLV-I.58
Epidemiologic data suggest that there is
HTLV, Type I
an excess of infectious syndromes (eg,
Human T-cell lymphotropic virus, type I bronchitis, urinary infections, and pneu-
(HTLV-I) was the first human retrovirus monia) among blood donors infected with
isolated and the first to be causally associ- HTLV-I or -II.
78-81
ated with a malignant disease of humans,

adult T-cell lymphoma-leukemia (ATL).58
Clinical Observations
HTLV-I is also associated with the neuro-
logic condition HTLV-associated myelo- For both HTLV-I and -II, infection persists
pathy (HAM), often called tropical spastic lifelong, as does the presence of antibody.
paraparesis (TSP). ATL was described be- Studies of prevalence and transmission
fore HAM. Both these conditions occur in use seroconversion as the endpoint for di-
a small minority (no more than 2-4%) of agnosis. Infection does not cause any rec-
persons harboring the virus. Infection ognizable acute events, and with the ex-
during childhood is an important aspect ception of those developing ATL or HAM,
of, and possibly a requirement for, devel- infected individuals experience few, if
oping ATL many years later, whereas any, health consequences. Most carriers
childhood or adult infection can cause are asymptomatic and completely un-
HAM, with a variable latent period. aware of the infection.
Prevalence of HTLV-I infection shows
striking geographic clustering, with pockets
of high endemicity in parts of southern Transmission
Japan and certain Pacific Islands; sub-Saha- Both viruses are very strongly cell-associ-
ran Africa; and the Caribbean basin, Cen- ated. Contact with infected viable lym-
tral America, and South America. Transmis- phocytes can cause infection, but plasma
sion is by mother to child through breast appears to be not, or much less, infective.
milk, by sexual contact (predominantly Cellular components from infected donors
male-to-female), and by exposure to blood. cause seroconversion in at least 50% of
recipients in Japan, but apparently in a
much smaller proportion of US recipi-
HTLV, Type II ents. 58 After refrigerated storage for 10
Human T-cell lymphotropic virus, type II days or more, red cells transfused from an
(HTLV-II) was described several years af- infected donor are far less likely to result
ter HTLV-I. There is at least 60% similarity in seroconversion, presumably due to de-
of genetic sequences to those of HTLV-I; gradation of lymphocytes that transmit
antibodies to either show strong cross-re- the virus. 8 2 , 8 3 Transfusion-transmitted
activity in tests with viral lysates. HTLV-II HTLV-I infection has been associated with
also shows clustering, but in different HAM of rather rapid onset and at least
populations. High prevalence has been one case of ATL.

Donor Tests ries of tests in an algorithm if the donation

tests repeatedly reactive by both the test-
Donor screening for anti-HTLV-I began in of-record and second HTLV-I/II EIAs; if
the United States in late 1988; at that time, supplemental tests are positive, the donor
the rate of confirmed positive tests was is indefinitely deferred (see Table 28-3). Re-
approximately 0.02%, or 1 in 5000 units gardless of the results of these investiga-
collected, a figure that has since declined tional supplemental tests, a donor meeting
at least 10-fold as seropositive persons the EIA criteria for indefinite deferral de-
have been removed from the donor pool.84 scribed above must still be indefinitely de-
The combined risk of transfusion-transferred.
mitted HTLV-I/II infection has been esti-
84
mated as about 1 in 641,000 units. Fur-
ther risk reduction can be expected from Quarantine and Look-Back
the implementation of combination
In-date prior collections of blood or com-
HTLV-I/II EIA tests in blood donor screen-
ponents from donors who subsequently
ing. The first such test was licensed in
are found repeatedly reactive for anti-
1998 in the United States. By using viral HTLV-I/II need to be quarantined. Be-
lysates from both HTLV-I and HTLV-II vi- cause recipients of units from seroposi-
ruses, the test offers sensitive detection of tive donors do not consistently sero-
both anti-HTLV-I and anti-HTLV-II. The convert and because many seropositive
originally licensed anti-HTLV-I EIA donors have lifelong infection, the time
screening tests might have missed up to frame for look-back is not self-evident. No
50% of HTLV-II infections.85 requirement for recipient tracing and no-
A donation that is repeatedly reactive on tification has been established.85 Screen-
EIA may not be used for transfusion. If a ing, in place since 1988, has probably re-
donor tests repeatedly reactive by the EIA moved from the active donor pool most
screening assay on two or more occasions, donors with lifelong infection.
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 West Nile Virus
indefinitely deferred on the basis of that West Nile virus (WNV) is a flavivirus pri-
single donation. Further testing of serum marily transmitted in birds through mos-
that is repeatedly reactive for anti-HTLV-I/II quito bites; humans are incidental hosts.
against antigen preparations specific for In humans, symptoms range from a mild
the two agents (HTLV-I or HTLV-II), or by febrile illness to encephalitis, coma, and
NAT on material from peripheral blood death, although about 80% of infected in-
mononuclear cells, can characterize the individuals remain asymptomatic. WNV
fecting agent. Half or more of US blood do- outbreaks have been reported in Europe,
nors confirmed infected after EIA screening the Middle East, and Russia over the past
prove to have HTLV-II infections. Although decade and have been associated with
there is no FDA requirement to perform ad- human encephalitis and meningitis (in
ditional testing (no confirmatory test for <1%), although no transfusion-associated
HTLV is licensed), most centers do so using cases were reported. WNV was observed
an investigational supplemental test or se- in the United States, in the metropolitan

684
Table 28-3. Recommended Actions for HTLV-I/II Testing
First Donation to Be Tested for HTLV-I/II Antibodies Subsequent Donation(s)
Second
EIA WB/RIPA Donation Donor EIA Manufacturer WB/RIPA Donation Donor
Repeatedly Positive Destroy all Defer and Not applicable/Donor deferred

reactive compo- counsel
nents*
Repeatedly Negative or Destroy all No action Repeatedly Pos Any result Destroy all Defer and
reactive indeterm. compo- reactive compo- counsel
nents* nents*
Nonreactive† Neg Not done All compo- No action
nents ac-
ceptable
*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, sociated WNV cases were reported in 2003;
it dispersed rapidly westward throughout four recipients had WNV encephalitis, one
the country, spread by infected birds. In had West Nile fever, and one critically ill
2001, 66 human cases occurred in 10 patient did not have discernible WNV-com-
states. In 2002, 4161 cases of WNV illness patible illness despite confirmed WNV in-
91
were reported in 39 states, including 284 fection. Some of the donors were identi-
deaths. In 2002, 23 cases were associated fied through retrospective testing of
with transfusion (with an infected donor individual samples, and it appears that all
identified); Red Blood Cells (RBCs), Fresh were related to specimens with very low vi-
Frozen Plasma, and Platelets were impli- ral titers. The number of transfusion-asso-
87
cated. The implicated donations oc- ciated cases undoubtedly would have been
curred between July 22 and October 6, much higher had widespread testing under
2002. Statistical resampling of data avail- IND protocols not been initiated. There
able regarding case onset dates during the were 9862 WNV cases and 264 deaths in the
2002 epidemic was used to generate esti- United States overall during this time.
mates of the mean risk of transfusion-as- The persistent low-level transmission of
sociated WNV transmission (per 10,000 WNV by transfusion in 2003 led to the im-
donations) for six states and six selected plementation of targeted ID-NAT in high
metropolitan areas, with results ranging epidemic regions in 2004. This effort suc-
from a mean of 2.12 to 4.76 and 1.46 to cessfully interdicted low-level viremic units
12.33, respectively.88 that would have been missed by MP-
An FDA guidance document in May NAT.92,93
2003 recommended deferral of donors with In 2004, the virus appeared predomi-
a diagnosis of WNV infection for 28 days af- nantly in western states, with California ac-
ter onset of symptoms or 14 days after reso- counting for 31% of cases.94 Only three
lution, whichever is later, and recom- states (Hawaii, Alaska, and Washington) re-
mended inquiring whether donors had mained free of reports of infection in either
experienced a fever with headache in the humans or animals (mammalian and
week before donation.89 However, because avian). A total of 2470 cases of human WNV
most infected persons are asymptomatic, infection were reported in 40 states plus the
the yield of such measures would be ex- District of Columbia, including 88 fatal
pected to be modest, and testing was cases—far fewer than the previous year. A
sought as the best method to identify in- total of 192 WNV-positive donors were
fected donors. During the summer and fall identified, almost all from states west of the
of 2003, almost 5 million donations consti- Mississippi River.94 Of these donors, three
tuting over 95% of collections in the United subsequently reported neuroinvasive WNV
States were tested for WNV by NAT in illness and 55 subsequently developed
minipools of six or 16 samples under one of WNV fever.94 One probable transfusion-as-
95
two IND protocols—one using a polymer- sociated case was reported.
ase chain reaction method and one using a The experience to date indicates that
transcription-mediated amplification blood screening for WNV has improved
method. During 2003, approximately 1000 blood safety. However, a small risk of WNV
donors were confirmed as viremic for WNV transfusion-associated transmission re-
and approximately 1500 likely infected mains. If the number of WNV cases contin-
components were interdicted.90 However, ues to dramatically decline, the need for
six probable or confirmed transfusion-as- WNV testing will be reassessed. The FDA

has agreed that asking the question con- CMV infection can progress to CMV dis-
cerning fever with headache in the week ease and cause serious morbidity and mor-
before donation may be discontinued (K tality in premature infants, recipients of
Gregor y, personal communication, organ, marrow, or peripheral blood progen-
3/30/05). 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
Herpesviruses and Parvovirus blood transfusions. Other sources of infec-
Cytomegalovirus tion, however, such as organ transplants
from CMV-positive donors or reactivation
CMV, a member of the human herpes-
of latent virus, may be as much or more of a
virus family, is a ubiquitous DNA virus
risk than transfusion.
that causes widespread infection; trans-
mission can occur through infectious
body secretions, including urine, oro-
pharyngeal secretions, breast milk, blood, Transfusion-Transmitted CMV
semen, and cervical secretions. About 1%
Infection with CMV varies greatly accord-
of newborns are infected, transplacentally
ing to socioeconomic status and geo-
or through exposure to infected cervical
graphic region. Although approximately
secretions at delivery or by breast milk. In
50% of blood donors can be expected to
early childhood, CMV is often acquired
be CMV seropositive, it has been esti-
through close contact, especially in day-
mated that, currently, less than 1% of se-
care settings; in adulthood, through sex-
ropositive cellular blood components are
ual intercourse. The prevalence of CMV
able to transmit the virus.96,97 Rarely, post-
antibodies ranges from 50% to 80% in the
transfusion hepatitis may be due to CMV.
general population.96 The rate increases
The postperfusion mononucleosis syn-
with age and is generally higher in lower
drome that first focused attention on
socioeconomic groups, in urban areas,
CMV in transfused components in the
and in developing countries.
early 1960s is now rarely seen. Posttrans-
fusion CMV infection is generally of no
Clinical Observations clinical consequence in immunocompe-
In persons with an intact immune system, tent recipients, and intentional selection
CMV infection may be asymptomatic and of CMV-reduced-risk blood (see below) is
remain latent in tissues and leukocytes not warranted.
for many years. Infection, either primary In light of the potential for severe CMV
or reactivation of latent infection, can be disease in immunocompromised patients,
associated with a mononucleosis-like several categories of recipients have been
syndrome of sore throat, enlarged lymph identified who should be protected from
nodes, lymphocytosis, fever, viremia, transfusion-transmitted CMV.7 These in-
viruria, and hepatitis. Intrauterine infec- clude low-birthweight premature infants
tion may cause jaundice, thrombocyto- born to seronegative mothers; seronegative
penia, cerebral calcifications, and motor recipients of hematopoietic progenitor cells
disabilities; the syndrome of congenital from CMV-negative donors; seronegative
infection causes mental retardation and pregnant women, because the fetus is at
deafness and may be fatal. risk of transplacental infection; and recipi-

ents of intrauterine transfusions. In some by the time they reach adulthood; al-
cases, seronegative recipients of organ though usually asymptomatic, infection
transplants from a seronegative donor; persists. Infection is spread by contact
seronegative individuals who are candi- with infected saliva. Primary infection in
dates for autologous or allogeneic hemato- children is either asymptomatic or is
poietic progenitor cell transplants; and characterized by a sore throat and en-
those few patients with AIDS who are free larged lymph nodes. Primary infection in
of CMV infection are also included.
older, immunologically mature persons
usually causes a systemic syndrome, infec-
tious mononucleosis, with fever; tonsillar
Preventive Measures infection, sometimes with necrotic ulcers;
Blood from donors who test negative for enlarged lymph nodes; hematologic and
CMV antibody has very little risk of trans- immunologic abnormalities; and some-
mitting CMV, but the supply of seronega- times hepatitis or other organ involvement.
tive blood is limited.96,97 Another approach EBV infection targets B lymphocytes,
to reduce risk is to remove leukocytes which undergo polyclonal proliferation and
from donated blood (because leukocytes
then induce a T-lymphocyte response, ob-
are the principal reservoir for CMV).98-100
served as “atypical lymphocytes.”
Although the precise leukocyte population
Transfusion-transmitted EBV infection is
that harbors the virus has not been de-
usually asymptomatic, but it has been a
fined, leukocyte removal with high-effi-
rare cause of the postperfusion syndrome
ciency filters, to 5 × 106 leukocytes per
that follows massive transfusion of freshly
component or fewer, can significantly re-
drawn blood during cardiac surgery and is
duce, if not prevent, posttransfusion CMV
a rare cause of posttransfusion hepatitis.101
in high-risk neonates and transplant re-
EBV plays a role in the development of na-
cipients. Effectively leukocyte-reduced
sopharyngeal carcinoma and at least one
cellular components are considered equiv-
form of Burkitt’s lymphoma and has the
alent to serologically screened compo-
in-vitro capacity to immortalize B lympho-
nents by many experts, although this is
cytes. EBV contributes to the development
controversial.98-100 The incremental benefit
of lymphoproliferative disorders in im-
of serologic testing when added to leuko-
munosuppressed recipients of hemato-
cyte reduction has not been established.
poietic and organ transplants. Given a 90%
Prophylactic therapy with CMV immune
seropositivity rate for EBV among blood do-
globulin and prophylactic use of antiviral
nors and essentially no risk for clinical dis-
agents are being investigated as options
ease from transfusion-transmitted EBV in
for high-risk immunosuppressed organ
immunocompetent recipients, serologic
transplant recipients.96,97
screening for this virus has not been con-
sidered helpful. As is the case for CMV, leu-
kocyte reduction of cellular blood compo-
Epstein-Barr Virus nents would be expected to reduce the risk
EBV causes most cases of infectious mono- of EBV infection in severely immunosup-
nucleosis and is closely associated with pressed seronegative patients who may be
the endemic form of Burkitt’s lymphoma at risk for clinical disease. However, there
in Africa and with nasopharyngeal carci- have been no studies to verify such a
noma. Most persons have been infected reduced risk.

Human Herpesviruses 6 and 8 febrile illness of early childhood. Infections

in adults may be associated with arthritis
As with CMV and EBV, human herpesvirus
but are generally benign. More ominou-
6 (HHV-6) is a cell-associated virus that
sly, parvovirus B19 can infect and lyse red
integrates with the genome of lympho- 109
cell precursors in the marrow. This may
cytes. The seroprevalence in some adult
result in sudden and severe anemia in pa-
populations approaches 100%. Primary
tients with underlying chronic hemolytic
infection in immunocompetent children
disorders who depend on active erythro-
is recognized as exanthem subitum, a fe-
poiesis to compensate for shortened red
brile illness characterized by a rash; it is
cell survival. Patients with cellular immu-
rarely complicated by involvement of other
nodeficiency, including those infected with
organ systems. Immunocompromised pa-
HIV, are at risk for chronic viremia and as-
tients (eg, those with transplants or AIDS)
sociated hypoplastic anemia. Infection dur-
may experience manifestations of reacti-
ing pregnancy predisposes to spontaneous
vation of HHV-6 infection in multiple or-
abortion, fetal malformation, and hydrops
gan systems. Studies have sought an asso- 110
from severe anemia and circulatory failure.
ciation between multiple sclerosis and
The red cell P antigen is the cellular re-
HHV-6 infection, but this remains contro-
ceptor for parvovirus B19, and people who
versial. With the ubiquity of antibodies to
do not have the P antigen are naturally re-
HHV-6 and absence of disease associations
sistant to infection.111 About 30% to 60% of
after transfusion transmission, no recom-
normal blood donors have antibodies to par-
mendations have been made for protec-
vovirus B19, which indicates immunity
tion of seronegative blood recipients from
rather than chronic persistent infection.109
transmission by blood components.102
Viremia occurs only in the early phases of
HHV-8 (also known as Kaposi’s sarcoma-
infection and there is no evidence for a car-
associated herpesvirus or KSHV) is causally
rier state; the incidence of viremia in blood
associated with both Kaposi’s sarcoma and
donors has been estimated to range from 1
body cavity-based lymphomas. It has been
in 3300 to 1 in 40,000.110 Parvovirus B19 lacks
found in apparently healthy blood donors,
a lipid envelope and is therefore not inacti-
but spread appears to be primarily by the
vated by solvent/detergent treatment or heat
venereal route. Low titers of HHV-8 antibodies
inactivation using temperatures below 100
were found in 11% of 91 healthy US blood
C.109 The virus has been found regularly in
donors.103 Among HHV-8-seropositive women,
clotting factor concentrates and has been
injection drug use and indices of sexual ac-
transmitted to persons with hemophilia. Rare
tivity were independent risk factors for
transmission through cellular blood compo-
HHV-8 infection.104 In this study, the associ-
nents and plasma, but not intravenous im-
ation with injection drug use suggests that
munoglobulin and albumin, has been re-
transmission by infected blood is possible.
ported.110
Transmission by organ donation has been
After the description of parvovirus B19
documented.105-107 Epidemiologic studies
seroconversion (without clinical illness) in
suggest that blood transfusion is associated
volunteers during Phase IV clinical evalua-
with a small risk of HHV-8 transmission.108
tion of solvent/detergent-treated plasma,
derivative manufacturers (with the concur-
Parvovirus rence of FDA) began development and im-
Parvovirus B19 is the cause of erythema plementation of NAT screening for high-ti-
infectiosum or “fifth disease,” a contagious ter parvovirus B19 viremic donations in

minipools. The rationale, supported by ob- tations in the prion gene that, in its non-
servations from the Phase IV evaluation, is mutated form, encodes for a normal cel-
that high-titer donations overwhelm neu- lular protein. Worldwide, there is about
tralizing antibody in plasma pools, allowing one case of CJD per million people per
transmission of this highly resistant virus year, nearly all in older individuals. In
by some derivatives. The FDA has classified sporadic CJD, the vast majority of cases,
this MP-NAT as an in-process manufactur- the mode of acquisition is unknown. The
ing control rather than a donor screening agent causing CJD is resistant to commonly
test. Screening of whole blood donations has used disinfectants and sterilants. Iatro-
not been a high priority because of the be- genic CJD has been transmitted by ad-
nign and/or transient nature of most par- ministration of growth hormone and go-
vovirus disease, the availability of effective nadotropic hormone derived from pooled
treatment (intravenous immunoglobulin) human pituitary tissue, through allografts
for chronic hematologic sequelae, and the of dura mater, and through reuse of intra-
extreme rarity of reports of parvovirus B19 cerebral electroencephalographic elec-
transmission by individual components.112 trodes from infected patients.
113
Early experimental studies in animals

raised the possibility that CJD could be
transmitted by blood transfusion. Addition-
Transmissible Spongiform ally, iatrogenic transmission from periph-
Encephalopathies eral injection of human pituitary-derived
hormones has been observed. Neverthe-
The transmissible spongiform encephalo-
less, several population-based, case-con-
pathies (TSEs) are degenerative brain dis-
trolled studies have shown no evidence that
orders caused by agents often called prions,
blood transfusion is a risk factor for the de-
postulated to be infectious proteins. They
velopment of CJD.114-116
are characterized by long incubation peri-
Individuals at increased risk for CJD are
ods, measured in years to decades, and by
excluded from donating blood; this group
the extreme resistance of the pathogens
includes persons who have received tissue
to inactivation by physical and chemical
or tissue derivatives known to be a source
methods sufficient for classic pathogens.
of the CJD agent (eg, dura mater allografts,
Two TSEs, Creutzfeldt-Jakob disease (CJD)
pituitary growth hormone of human origin)
and variant Creutzfeldt-Jakob disease
and persons with a family history of
(vCJD), are of particular interest in trans-
CJD.117-119 For the purposes of donor exclu-
fusion medicine.
sion, unit quarantine, and unit destruction,
family history has been defined as having
Classic CJD one blood relative who has had this diagno-
CJD is a degenerative brain disorder that sis. However, a donor with CJD in a family
is rapidly fatal once symptoms of progres- member may be accepted if gene se-
sive dementia and motor disturbances quences have been tested and found to be
develop. Approximately 85% of cases are normal.
sporadic. Symptoms do not develop until
many years to several decades after the
Variant CJD
initial infection. Ten to fifteen percent of
cases are familial, associated with inheri- In 1996, the first cases of an unusual CJD
tance of one of at least 20 described mu- outbreak or cluster were described in the

United Kingdom (UK). These cases were unrelated to vCJD, but a postmortem exam-
later termed variant CJD (vCJD) and ap- ination revealed the presence of the
peared to be caused by the same prion abnormal prion protein in the patient’s
responsible for bovine spongiform en- spleen and in a lymph node.122 Notably, un-
cephalopathy (BSE). This prion is distinct like previous cases of vCJD (by any method
from the prion found in classical CJD. A of transmission), in which all involved peo-
donor infected by dietary exposure to BSE ple were homozygous for methionine (MM)
during the incubation period of vCJD might at codon 129 of the prion protein gene
theoretically infect a transfusion recipi- (PRNP), this individual was heterozygous
ent. Consequently, UK health authorities (methionine valine— MV). In the UK, the
prohibited the use of UK plasma for fur- population distribution of this gene is MM,
ther manufacture, restricted the use of UK MV, or VV in 42%, 47%, and 11%, respec-
plasma for children (born on or after 1 tively.123
January 1996), and implemented univer- In the United States, blood donors who
sal leukocyte reduction of cellular compo- were in the UK or Europe during the years
nents (to reduce the prions known to be of potential exposure to the BSE agent are
present in white cells). deferred based on the duration of residence
Experimental transfusion transmission there. Balancing the theoretical risk against
of BSE to two sheep (and four cases of considerations of the adequacy of the blood
transmission of natural scrapie—a sheep supply, the recommended deferral is for 3
120
prion illness) have been reported. Positive months of cumulative residence in the UK
transmissions occurred with blood taken at between 1980 and 1996 (and current and
preclinical and clinical stages of infection. former US military personnel, civilian mili-
Two cases of transfusion-transmitted BSE tary employees, and their dependents who
in humans have been observed121,122 as a re- were stationed at European bases for 6
sult of surveillance in the UK of 48 individ- months or more during this period) or 5
uals identified as having received a labile years of cumulative residence in Europe.
blood component from a total of 15 donors Potential donors who may have injected
who later developed vCJD. In the first possi- bovine insulin from the UK or received
ble case, the recipient developed symptoms transfusions in the UK during the BSE epi-
of vCJD 6.5 years after receiving a transfu- demic are also excluded.119
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 Bacterial Contamination
past dietary exposure to the BSE agent, the Bacterial contamination remains an im-
age of the patient was well beyond that of portant cause of transfusion morbidity
most vCJD cases, and the chance of observ- and mortality. Bacterial contamination of
ing a case of vCJD in a recipient in the ab- blood components accounted for 29 (16%)
sence of transfusion-transmitted infection of the transfusion fatalities reported to
was estimated to be about 1 in 15,000 to 1 the FDA between 1986 and 1991. How-
in 30,000, making dietary transmission un- ever, in 2002 alone, there were 17 deaths
likely in this case.121 In the second possible reported to the FDA from bacterial con-
case, the person received a blood transfu- tamination of blood components, most
sion in 1999 from a donor who later devel- commonly caused by contaminated
oped vCJD. This patient died of causes apheresis platelets and whole-blood-de-

124,125
rived platelets. Although the hepatitis Because platelets are stored at 20 to 24 C
viruses, HIV, and WNV have been more to retain their viability and function, they
prominently featured in the media and serve as an excellent growth medium for
remain a primary concern of the public, bacteria. Sepsis resulting from transfusion
bacterial contamination is believed to be of contaminated platelets is believed to be
the most common infectious source of both underrecognized and underreported.
morbidity and mortality related to trans- Sepsis occurring after transfusion of con-
fusion. To place the risk of bacterial contaminated platelets is usually not a cata-
tamination into some perspective, in 2002, strophic event, but it can occur several
there were 23 transfusion-transmitted cases hours or longer after transfusion, making it
of WNV identified in the United States.87 more difficult to connect the transfusion to
Of these 23 recipients, seven died, but the sepsis. Because many of the patients in-
only five of these deaths were associated fected by bacteria from a platelet transfu-
with WNV meningoencephalitis. Thus, the sion are immunocompromised by their
deaths from bacterial contamination were underlying condition and treatment (eg,
more than three times more common than chemotherapy), the event is frequently at-
those from WNV. tributed to other causes, such as an in-
No matter how carefully blood is drawn, fected catheter, which often involves the
processed, and stored, complete elimina- same organisms.
tion of microbial agents is impossible. Bac- In the United States, 4 million platelet
teria are most often believed to originate units are transfused annually (1 million
with the donor, either from the venipunc- apheresis platelets and 3 million whole-
ture site or from unsuspected bacteremia.
126
blood-derived platelet concentrates). 130
Bacterial multiplication is more likely in Given that approximately 1:1000 to 1:2000
blood components stored at room temper- platelet units are contaminated with bacte-
ature than in refrigerated components.126 ria (as measured by aerobic cultures done
Organisms that multiply in refrigerated in multiple studies before 2002), it would
blood components are often psychrophilic be expected that 2000 to 4000 bacterially
gram-negative organisms (such as Yersinia contaminated units would be transfused.131
enterocolitica, Serratia liquifaciens, and Estimates of the fraction of such units that
Pseudomonas fluorescens). Gram-positive would result in signs or symptoms have
organisms are more often seen in platelets been as low as 1 in 10 cases. However, in
stored at 20 to 24 C. the only study that has prospectively cul-
For RBCs, the CDC estimates a sympto- tured platelets that were transfused, symp-
matic contamination rate of approximately toms occurred in 3 of 8 (35.8%) patients
1 case per million units, primarily with Y. who received culture-positive but Gram’s-
127 132
enterocolitica, followed by S. liquifaciens. stain-negative platelet pools. Notably, six
In New Zealand, the incidence of symp- Gram’s-stain-positive pools were inter-
tomatic Yersinia contamination of RBC dicted and never transfused. Thus, of con-
units has been reported to be as high as taminated products, perhaps 1/10 to 2/5
one in 65,000 units, with a fatality rate of would be expected to result in clinical sep-
one in 104,000.128 Transfusion of an RBC sis (200 to 1600 cases) if transfused. Data
unit heavily contaminated with a gram- from national passive reporting studies in
negative organism is often a rapid and cata- the United States, Great Britain, and France
strophic event, with a quick onset of sepsis (Table 28-4) suggest that perhaps 1/5 to 1/3
and a greater than 60% mortality rate.129,130 would result in death (40 to 533 deaths per

Table 28-4. Summary of Organisms Identified in the BaCon, SHOT, and BACTHEM
Studies*
United United
127 133 134
Organism States Kingdom France Total
Gram positive
Bacillus cereus 1 4 (1) 2 7 (1)
Coagulase-negative 9 6 (1) 5 20 (1)
Staphylococci
Streptococcus sp. 3 (1) 2 5 (1)
Staphylococcus aureus 4 2 (1) 6 (1)
Propionibacterium 3 3
acnes
Subtotal 17 (1 = 6%)† 14 (3 = 21%) 10 (0 = 0%) 41 (4 = 10%)
Gram negative
Klebsiella sp. 2 (1) 2 (1)
Serratia sp. 2 (2) 1 (1) 3 (3)
Escherichia coli 5 (1) 2 (1) 1 8 (2)
Acinetobacter 1 1
Enterobacter sp. 2 (1) 1 (1) 1 4 (2)
Providencia rettgeri 1 (1) 1 (1)
Yersinia enterocolitica 1 1
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.
127,133,134
year). This translates to a risk of death similarly observed a fatality rate of approxi-
from a transfusion of a platelet unit con- mately 1:48,000 per whole-blood-derived
taminated with bacteria of between 1:7500 platelet concentrate.136 With the implemen-
to 1:100,000. Clinical observations from tation of bacteria detection of platelets (see
university hospitals with heightened aware- below), it is anticipated that this rate will be
ness of platelet-related sepsis confirm such greatly reduced.
estimates. A fatality rate of 1:17,000 has
been reported by Ness et al, from Johns
Clinical Considerations
Hopkins, with pooled whole-blood-derived
platelets and 1:61,000 with apheresis plate- Severe reactions are characterized by fe-
lets.135 University Hospitals of Cleveland ver, shock, and disseminated intravascular

coagulation (DIC). If bacterial contamina- interventions, or of any constitutional

tion is suspected, the transfusion should symptoms. Questions to elicit the possibil-
be stopped immediately and a Gram’s stain ity of bacteremia are especially important
and blood culture should be obtained for autologous donors, who may have un-
from the unit (not an attached segment of dergone recent hospitalization, antibiotic
tubing because the bag may be contami- therapy, or invasive diagnostic or therapeu-
nated but an isolated segment of tubing tic procedures; there have been several re-
may be sterile) and recipient as promptly ports of Yersinia sepsis complications after
as possible after the reaction is observed. the infusion of stored autologous blood.
Bacterial multiplication may cause the Scrupulous attention must be paid to se-
oxygen in an RBC unit to be consumed, lecting and cleansing the donor’s phlebot-
resulting in hemoglobin desaturation and omy site. Skin preparation reduces but does
erythrocyte lysis, both of which contrib- not completely abrogate the contamination
ute to a darkening of the unit compared of components by bacteria. Scarred or dim-
to the color of the blood in the attached pled areas associated with previous derma-
sealed segments. Color change (to dark pur- titis or repeated phlebotomy can harbor
ple or black), clots in the bag, or hemoly- bacteria and should be avoided. Green
sis suggest contamination, but the ap- soap must not be used to prepare the
pearance of the blood in the bag is often phlebotomy site.
unremarkable. The presence of bacteria Discarding the first aliquot of donor blood
on a Gram’s stain of the component is removed (“diversion”) has been proposed
confirmatory, but absence of visible or- as a measure to reduce bacterial contami-
ganisms does not exclude the possibility. nation of blood components. This measure
Gram’s stain has a sensitivity of only 106 to would remove the skin core that may enter
107 CFU/mL. The patient’s blood, the sus- the collection from the hollow bore needle
pect component, and intravenous solu- used in the phlebotomy. Systems have been
tions in all the administration tubing used developed to facilitate the application of
should be cultured. this approach and would be expected to re-
Treatment should not await the results of duce skin contaminants (mostly gram-pos-
these investigations and should include im- itive organisms).
mediate intravenous administration of an- Phagocytosis of contaminating bacteria
tibiotics combined with therapy for shock, by donor white cells in blood components
renal failure, and DIC, if present. may be important for the minimization of
clinical bacterial contamination. Leukocyte
removal, with coincident removal of adher-
Preventive Measures
ent or engulfed bacteria, has been advo-
Prevention of septic reactions depends upon cated as an approach to reducing Yersinia
reducing or preventing contamination of contamination of RBCs.137,138
components with bacteria. Careful selec- Care in the preparation of components
tion of healthy blood donors is the first and handling of materials used in blood ad-
and most important step. ministration is essential. If a waterbath is
The donor’s present appearance and reused, components should be protected by
cent medical history should indicate good overwrapping, outlet ports should be in-
health; additional questioning may be spected for absence of trapped fluid, and
needed if there is a present or recent his- the waterbath should be frequently emp-
tory of antibiotic use, of medical or surgical tied and disinfected.

Worldwide, screening of platelets for logistics, whole-blood-derived platelets are

bacteria is being implemented. Screening is often tested in the United States with less
mandatory in several countries [eg, Bel- sensitive but more rapid detection strate-
gium (Flemish Red Cross), the Netherlands, gies, such as staining or the use of surrogate
Hong Kong (Red Cross), and Wales].139 markers of bacterial metabolism (eg, pH
In the United States, the College of and glucose). Several other more rapid and
American Pathologists (CAP) Commission sensitive detection strategies are under de-
on Laboratory Accreditation has added a velopment or are not readily available. One
question to the Transfusion Medicine possible investigative strategy after detect-
Checklist to assess the presence of a labora- ing a confirmed culture-positive platelet
tory system to detect bacteria in platelet unit is outlined in Fig 28-4.
140
components (TRM.44955). Similarly, the
62(p11)
AABB requires bacteria detection. Cur- Prospect for Extended Storage
rently, two culture techniques are approved
by the FDA for quality control of leuko- The extent of bacterial growth in platelet
cyte-reduced platelets and are available in components correlates with the duration
the United States. Because of expense and of storage. In 1983, in recognition of tech-
Figure 28-4. Possible investigative strategy for a positive culture in a platelet unit. Modified with per-
141
mission.

nically improved storage conditions, the gesting a low probability that the blood of
FDA increased storage limits of platelets donors who have a confirmed positive
at room temperature from 3 to 7 days. syphilis test result is infectious for syphilis.
However, it reduced the limits to a maxi- Nevertheless, a study from the CDC showed
mum of 5 days in 1986, responding to re- that from 1995-2000, 22 primary, 81 sec-
ports of bacterial contamination after ondary, and 413 early latent syphilis cases
142
more than 5 days of storage. The use of were identified through blood or plasma
144
bacteria detection systems has been given donor screening in the United States.
as the rationale for an extension of plate- Thus, screening of blood donors for syphilis
let storage to 7 days in several European may have broader public health implica-
countries and is being implemented in the tions. Currently, performance of a STS is
United States.139 still required.
62(pp33,34)
Syphilis Tick-Borne Infections

Syphilis is caused by the spirochete Tre- Because many tick-borne infectious agents
ponema pallidum and is characteristically circulate in the blood, it is theoretically
spread by sexual contact. The phase of possible that they will be transmitted by
spirochetemia is brief and the organisms transfusion of blood components.
survive only a few days at 4 C. Although
transmission by transfusion is possible, Babesia
its occurrence is exceedingly rare (the last
case reported in the United States oc- Clinical Events
curred in 1965). Syphilis transmission by In the United States, the most frequently
transfusion may not be effectively pre- recognized transfusion-associated tick-borne
vented by subjecting the donor blood to infection is babesiosis. 145 Babesiosis is
standard serologic tests for syphilis (STS) usually transmitted by the bite of an in-
because seroconversion often occurs after fected deer (black-legged) tick and is re-
the phase of spirochetemia. Most positive ported most frequently in the coastal lands
STS results reflect immunologic abnor- and islands of northeastern United States,
malities unrelated to syphilis (biologic including Martha’s Vineyard, Cape Cod, and
false-positives), inadequately treated non- Long Island. Geographic areas of the hosts
infectious syphilis that is more of a threat and the vectors appear to be expanding.146
to the individual being tested than to a Transfusion-associated babesiosis has been
transfusion recipient, or the serologic re- documented in more than 50 cases, caused
sidual of an effectively treated infection. mostly by Babesia microti from the North-
A recent series described T. pallidum east, but also by the recently recognized
DNA and RNA testing of 169 aliquots from WA1-type Babesia parasite, from asymp-
145-147
platelet concentrates that were reactive by tomatic infected blood donors. As hu-
STS and confirmed positive by fluorescent mans continue to encroach on the habitat
treponemal antibody absorption.143 This se- of vectors and natural reservoirs of infec-
ries included 48 donors who were positive tion [eg, deer (and other Cervidae) and
by rapid plasma reagin tests (compatible mice populations in the northeastern
with recent or active disease). No sample United States], the incidence of transfu-
contained T. pallidum DNA or RNA, sug- sion-transmitted babesiosis may increase.

The vector and reservoir of the Babesia Other Agents

more recently found in the northwestern
and western United States remain to be One case of transfusion transmission of
defined. Babesia species survive blood Rocky Mountain spotted fever (Rickettsia
bank storage for up to at least 35 days and rickettsii) and no cases of human mono-
can be transmitted by both RBCs and cytic ehrlichiosis (caused by Erlichia chaf-
platelet concentrates. Babesiosis classi- feensis) have been documented.150 A single
cally causes a febrile illness with hemoly- possible transfusion transmission of the
tic anemia, but infection can also cause unnamed agent of human granulocytic
chronic asymptomatic or mildly symp- erlichiosis has been reported.151 In 1997,
tomatic parasitemia. Studies suggest that Rocky Mountain spotted fever and/ or hu-
untreated persons can harbor B. microti man monocytic erlichiosis developed in
DNA for long periods, despite mild or ab- National Guard trainees at Fort Chaffee,
sent symptoms, and may transmit infec- AR. Ten components donated by infected
148 trainees had been transfused before a re-
tion for months or possibly longer. Sym-
call; however, none of the persons who re-
ptoms are often so mild that the infection
ceived blood from infected donors became
is not recognized, which likely explains 152
clinically ill.
the low rate of reported transfusion-trans-
Lyme disease is the most common tick-
mitted babesiosis. Symptomatic patients
borne infection in the United States.
develop fever 2 to 8 weeks after transfu-
Borrelia burgdorferi, the causative spiro-
sion, sometimes associated with chills,
chete, is transmitted through bites of the
headache, hemolysis, or hemoglobinuria.
deer (black-legged) tick. No transfusion-
Rarely, life-threatening hemolytic anemia, related cases have been reported, but
renal failure, and coagulopathy develop, chronic subclinical infections do occur
particularly in asplenic or severely im- and experimentally inoculated organisms
munocompromised patients.149 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-
Preventive Measures toms that would render a potential donor
ineligible, and in two reported cases where
The Babesia carrier state may be asymp- the donor became ill shortly after dona-
tomatic and may exceed a year in duration, the recipient did not develop infec-
tion. Persons with a history of babesiosis 149
tion. Potential donors who give a history
are indefinitely deferred, because lifelong of Lyme disease should be completely
parasitemia can follow recovery from asymptomatic and should have com-
symptomatic illness. Restrictive policies, pleted a full course of antibiotic therapy
such as not collecting blood in areas before they are permitted to donate. Trans-
where the disease vectors are endemic fusion transmission of tick-borne agents is
during spring and summer months when biologically plausible and, for some agents,
tick bites are more common, are in prac- has been demonstrated. Nevertheless,
tice in some locations but probably are of modifications to current donor screening
limited value. No test is available for mass are not likely to be useful because of their
screening to detect asymptomatic carriers low predictive value and the potential
of Babesia species. for nonspecific questions to defer large

numbers of donors for a small increment in contains red cells can transmit infection,
transfusion safety.153 via the asexual form of the intraerythrocytic
parasite.
Asymptomatic carriers are generally the
source of transfusion-transmitted malaria,
Other Nonviral Infectious although their parasite density is very low.
Complications of Blood Asymptomatic infections rarely persist
Transfusion more than 3 years, but asymptomatic P.
falciparum and P. vivax infections may per-
Malaria sist for 5 years, P. ovale for 7 years, and P.
Malaria is caused by several species of malariae can remain transmissible for the
the intraerythrocytic protozoan genus lifetime of the asymptomatic individual. In
Plasmodium. Transmission usually results extreme cases, transmission of P. vivax, P.
from the bite of an anopheles mosquito, but ovale, P. falciparum, and P. malariae have
infection can follow transfusion of para- been reported at 27, 7, 13, and 53 years, re-
sitemic blood. Although very rare in the spectively.156 There are no practical sero-
United States, malaria is probably the most logic tests to detect transmissible malaria in
commonly recognized parasitic complica- asymptomatic donors. Malaria transmis-
tion of transfusion; the risk in the United sion is prevented by deferral of prospective
States is estimated to be <0.3 case per mil- donors with increased risk of infectivity,
154,155
lion transfusions. From 1963 to 1999, 93 based on their medical and travel history.
cases of transfusion-transmitted malaria (10 The AABB requires that prospective donors
fatal) in the United States were reported to who have had a diagnosis of malaria, or
CDC.155 who have traveled or lived in a malaria-en-
The species involved in transfusion- demic area and have had unexplained
transmitted malaria in the United States are symptoms suggestive of malaria, be de-
P. falciparum (35%), P. malariae (27%), P. ferred for 3 years after becoming asymp-
vivax (27%), and P. ovale (5%).155 Three per- tomatic.62(p65) Individuals who have lived for
cent were mixed infections, and 2% were at least 5 consecutive years in areas in
caused by unidentified species. Fever, which malaria is considered endemic by
chills, headache, and hemolysis occur a the CDC Malarial Branch shall be deferred
week to several months after the infected for 3 years after departure from that area.
transfusion; morbidity varies but can be se- Individuals who have traveled to an area
vere, and deaths have occurred, especially where malaria is endemic shall be deferred
from P. falciparum. Adding to the risk of a for 12 months after departing that area.
fatal outcome may be a lack of immunity in These deferral periods apply irrespective of
the recipient, the patient’s underlying con- the receipt of antimalarial prophylaxis. Up-
dition(s), and delay in the diagnosis be- dated information on malaria risks world-
cause of lack of suspicion and unfamiliarity wide is available from the CDC, including
with the disease in areas where the parasite an on-line resource (http://www.cdc.gov/
is not endemic. travel/yb/outline.htm#2).
Malaria parasites survive for at least a
week in components stored at room tem-
Chagas’ Disease
perature or at 4 C. The parasites can also
survive cryopreservation with glycerol and American trypanosomiasis, or Chagas’
subsequent thawing. Any component that disease, is endemic in South and Central

158
America and is caused by the protozoan States : in New York, Los Angeles, Texas,
parasite Trypanosoma cruzi. The human and Florida. All occurred in immuno-
host sustains infection after the bite of compromised patients. Additionally, two
reduviid bugs (called cone-nosed or “kiss- cases were reported in Manitoba, Canada.
ing” bugs), which usually exist in hollow In one interesting study, postoperative
trees, palm trees, and in thatched-roofed blood specimens from 11,430 cardiac sur-
mud or wooden dwellings. Naturally ac- gery patients were tested by EIA and, if
quired Chagas’ disease in the United States repeatedly reactive, were confirmed by
is exceedingly rare. Five such cases have radioimmunoprecipitation. Six postoper-
been recognized in the United States since ative specimens (0.05%) were confirmed
1955, the most recent in 1998 in Tennes- positive. All six seropositive patients ap-
see.157 parently were infected with T. cruzi before
surgery; however, a diagnosis of Chagas’
Clinical Events disease was not known or even consid-
ered in any of these patients. No evidence
T. cruzi infects humans whose skin or mu-
for transfusion-transmitted T. cruzi was
cosa comes in contact with feces of in-
found.159
fected reduviid bugs, usually as the result
Reasonably sensitive and specific EIA
of a bite. Recent infections are usually ei-
screening tests for antibodies to T. cruzi, as
ther asymptomatic or the very mild signs,
well as confirmatory Western blot and
and symptoms go undetected. Rarely, the
radioimmunoprecipitation assays, have
site of entry evolves into an erythematous
been developed.158 Testing in several US
nodule called a chagoma, which may be
blood centers located in geographic areas
accompanied by lymphadenopathy. Fever
with a large immigrant population from
and enlargement of the spleen and liver
Central or South America found a sero-
may follow. Recently infected young chil-
prevalence of 0.1% to 0.2% among at-risk
dren may experience acute myocarditis or
donors, who were identified by question-
meningoencephalitis. Acute infection
naire.160,161 However, look-back studies iden-
usually resolves without treatment, but
tified no infected recipients; it is also likely
persisting low-level parasitemia is usual
that not all at-risk donors can be identified
and up to one-third of infected individu-
by questionnaire.160 As a consequence, if blood
als develop a chronic form associated with
donor screening were to be implemented,
cardiac or gastrointestinal symptoms
testing of all donors might be necessary.
years or decades later.158
Transfusion Considerations Other Parasites

Blood transfusion has been a major source Toxoplasmosis is caused by the ubiquitous
of infection with T. cruzi in South Ameri- parasite Toxoplasma gondii, and infection
can urban centers that receive large num- has been reported as an unusual transfu-
bers of immigrants from rural areas where sion complication in immunocompromi-
162
the parasite is endemic. However, in many sed patients. The disease has not been
countries, serologic screening has been considered a problem in routine transfu-
effective in reducing the risk of transfusion practice.
sion-transmitted Chagas’ disease. Four There have been occasional reports of
cases of transfusion-transmitted Chagas’ parasitic worm infections transmitted by
disease have been reported in the United transfusion in countries other than the

162
United States. Microfilariasis is a potential cold ethanol precipitation after removal
transfusion risk in tropical zones, acquired of cryoprecipitate. Historically, when anti-
by donors through bites by insects carrying bodies to HCV were present in the plasma,
Wuchereria bancrofti. Transfusion trans- this process concentrated HCV in the Fac-
mission of Leishmania species is a rare risk tor VIII-rich cryoprecipitate and other
in countries where such organisms are en- fractions and left little in the immuno-
demic. Currently, the AABB defers potential globulin fraction. The immunoglobulin
donors who have been to Iraq in the previ- fraction also has a high concentration of
ous 12 months as a result of possible Leish- virus-neutralizing antibodies and the re-
mania exposure.163 sulting product for intramuscular appli-
cation has a remarkably low risk of virus
transmission.168
Preparations of immunoglobulin intended
Reducing the Risk of for intravenous administration (IGIV) were
Infectious Disease expected to be similarly free of disease
Transmission transmission. However, NANB hepatitis
transmission did occur in the 1980s during
Overall, the risk per unit of transfusion-
early clinical trials of IGIV products in the
transmitted disease is remarkably low
United States and with routinely manufac-
(Table 28-5). This low incidence is due to
tured IGIV products in Europe.169 In late
both donor screening and specific disease
1993 and early 1994, a worldwide outbreak
testing. Nevertheless, in pooled compo-
with more than 200 reported HCV infec-
nents, which may contain elements from
tions was traced to a single IGIV prepara-
thousands of donors, the risk of disease
tion licensed in the United States.170,171 In
transmission is increased. Therefore, sev-
this case, transmission apparently occurred
eral strategies have been developed and
because of lack of virus inactivation steps in
implemented to further reduce the risk of
the specific manufacturing process for this
disease transmission in pooled acellular
product172 and absence of complexing and
components and, in some cases, cellular
neutralizing anti-HCV subsequent to anti-
components.
HCV screening of plasma donors, with re-
sultant accumulation of virus particles in
Inactivation/Destruction of Agents in
the immunoglobulin fraction.170 Anti-HCV-
Derivatives or Plasma Products
positive source plasma has been excluded
The first intervention specifically added from the manufacture of IGIV since 1992.
to reduce the risk of hepatitis transmis- The importance of the manufacturing
sion was heating (to 60 C for 10 hours), method is underscored by outbreaks of HCV
which has been used for albumin prod- infection from intravenous anti-D immu-
ucts since at least 1948.167 In those rare in- noglobulin in Germany in the late 1970s
stances when infections occurred with and in Ireland from the late 1970s to the
plasma protein fractions prepared with early 1990s.173,174 Both products were pre-
this step, the processing had been compared by anion exchange chromatography
promised. rather than cold-ethanol (Cohn) fraction-
ation.175 To prevent further HCV outbreaks,
Immunoglobulins the FDA has required, since 1994, virus
The plasma fractionation process used for clearance steps in the manufacturing pro-
most immunoglobulin products employs cess of immunoglobulin or proof of ab-

Table 28-5. Infectious Risks of Blood Transfusion in the United States
Estimated % of
Infected Units that
Infectious Agent Estimated Risk per Transmit or Cause
or Outcome Unit Transfused Clinical Sequelae* Reference
Viruses
HIV-1 and -2 1:1,400,000- 90 23, 29, 30
1:2,400,000
HTLV-I and -II 1:256,000- 30 84
1:2,000,000
HAV 1:1,000,000 90 164
HBV 1:58,000-1:147,000 70 29
HCV 1:872,000-1: 90 23, 29, 30
1,700,000
B19 parvovirus 1:3,300-1:40,000 Low 110
Bacteria
RBCs 1:1000 1:10,000,000 fatal 165
Platelets 1:2000-1:4000 >40% result in clini- 30, 132, 166
(screened with cal sequelae
Gram’s stain, pH,
or glucose con-
centration)
(screened with <1:10,000 Unknown
early aerobic cul-
ture)
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.

sence of HCV from the final product by ports of viral transmission continue to
NAT. In addition, NAT technology is now occur, possibly resulting from accident or
applied to screening of source plasma as an error during the manufacturing process.
additional layer of safety. The combination of heat treatment, sol-
vent/detergent treatment, and purification
steps with monoclonal antibodies provides
Coagulation Factors clotting factor concentrates with a risk of
Until the early 1980s, clotting factor con- transmitting hepatitis and HIV that is lower
centrates frequently transmitted viral in- than the risk associated with use of Cryo-
fections. As the significance of HIV trans- precipitated Antihemophilic Factor derived
mission was recognized, virus inactivation from individual voluntary whole blood do-
steps were applied more rigorously to nations. Documented transmission of HBV,
Factor VIII and other clotting factor con- HCV, or HIV by US-licensed plasma deriva-
centrates, even though these steps were tives is rare since the introduction of effec-
initially introduced in the hope of reduc- tive virus inactivation procedures and im-
ing hepatitis transmission. Unfortunately, proved viral screening. Although absolute
a large proportion (over 50%) of the hemo- safety of products derived from human
philic population receiving concentrates plasma cannot be guaranteed, starting with
before processing was improved became the safest possible donated plasma reduces
infected with HIV. Chronic hepatitis was the viral load and has contributed to the ex-
an additional complication in almost all cellent safety record of the products sub-
patients with hemophilia receiving older jected to virus inactivation/removal.177
clotting factor products.176 Avoiding Human Plasma. Factor VIII
The thermal instability of Factor VIII concentrates produced by recombinant
made it difficult to develop an effective heat DNA technology are licensed for use and
treatment, until a practical approach was have become the preparation of choice for
widely adopted in 1985. Since then, many previously untreated patients with hemo-
virus inactivation steps have been introduced, philia.178 Batches are produced by culture of
and factor concentrates are now, in general, mammalian cells engineered to secrete
very safe products. Each process has its Factor VIII into the supernatant medium,
own set of advantages and disadvantages. which is purified by ion-exchange chroma-
Application of organic solvents and deter- tography and immunoaffinity chromatog-
gents inactivates viruses with a lipid-con- raphy using a mouse monoclonal antibody.
taining envelope (eg, HIV, HBV, HCV, HTLV, Except for the fact that excipient human al-
EBV, CMV, HHV-6, HHV-8) but is ineffective bumin is sometimes added to stabilize Fac-
against nonenveloped agents such as HAV tor VIII, the product is free of human pro-
and parvovirus B19. Virus inactivation steps teins, HIV, hepatitis viruses, and other
have the potential drawback of reducing the unwanted agents, thus avoiding many of the
potency and biologic effectiveness of the risks associated with using human plasma.
product. Another concern is whether virus On the other hand, recombinant products
inactivation steps affect immunogenicity, have a relatively short history of use and
especially the induction of Factor VIII in- there is no guarantee that they are risk-free.
hibitors in patients with hemophilia.
Current Risks of Human Plasma Deriva- Plasma
tives. Many methods are highly effective Virus reduction steps, originally developed
against enveloped viruses, but sporadic re- for purified plasma protein fractions, have

also been applied to plasma intended for mately eliminate such molecules from
transfusion. Alternative approaches being widespread clinical application.
studied include organic solvents and de-
tergents and use of photochemicals. Sol- Reporting Transfusion-Associated
vent/detergent treatment, which is effec- Infections
tive against lipid-enveloped viruses, involves
Unexplained infectious disease reported
addition of 1% Triton X-100 and 1% tri-n-
in a transfusion recipient must be investi-
butyl phosphate (TNBP) to pooled plasma,
gated for the possibility of transfusion-
followed by oil extraction of the TNBP and
transmitted illness.62(pp83,85) Hepatitis is ex-
chromatographic adsorption of the Triton
pected to become apparent within 2 weeks
X-100. After several years of experience
to 6 months if it resulted from transfu-
with this method in Europe, solvent/de-
sion, but, even within this interval, the
tergent-treated plasma was transiently
cause need not necessarily have been
available in the United States. However,
blood-borne infection. Blood centers and
concern with the use of a pooled product,
transfusion services must have a mecha-
expense, poor market penetration, and
nism to encourage recognition and re-
possible thrombotic (or excessive bleed-
porting of possible transfusion-associated
ing) events led to the discontinuation of
infections. HIV infection thought to be a
this product. A psoralen (S59) activated by
result of transfusion should also be re-
ultraviolet A light is undergoing US clini-
ported to the blood supplier, although the
cal trials for pathogen inactivation in
interval between transfusion and the rec-
platelets and plasma and is available in ognition of infection or symptoms may be
some countries in Europe. years.
Infection in a recipient should be re-
ported to the collecting agency so that do-
Processing Cellular Components nors shown or suspected of being infec-
tious can be evaluated and recipients of
Photochemical and chemical pathogen
other components from the implicated or
inactivation methods are theoretically ap-
other donations can be contacted and, if
plicable to cellular blood components;
necessary, tested. A donor who proves to
another organic chemical (S-303) and a
have positive results on tests during the in-
nucleic acid targeting compound (PEN
vestigation must be placed on an appropri-
110) have undergone initial clinical evalu-
ate deferral list.
ation for pathogen inactivation in red cells.
These methods have the potential for reli-
able inactivation of bacteria, viruses, and Reporting Fatalities
179,180
parasites, including intracellular forms. The Code of Federal Regulations [21 CFR
However, both products have been shown 606.170(b)] requires that fatalities attrib-
to result in the formation of antibodies in uted to transfusion complications (eg,
recipients. Pathogen reduction trials in hepatitis, AIDS, and hemolytic reactions)
platelets (eg, with S-59) have been associ- be reported to the Director, Center for
ated with decreased cell recovery and sur- Bi o l o g i c s Eva l u a t i o n a n d Re s e a rc h
vival and the need for increased platelet ( C B E R ) , O f f i c e o f Co m p l i a n c e a n d
transfusions. Such unintended consequences Biologics Quality, Attn: Fatality Program
of pathogen reduction have resulted in Manager (HFM-650), 1401 Rockville Pike,
some trials being halted and may ulti- Suite 200N, HFM-650, Rockville, MD

20852-1448. A report should be made as physician, a blood bank physician or

s o o n a s p o s s i b l e by t e l e p h o n e other trained staff member should pro-
(301-827-6220), fax (301-827-6748), or vide initial counseling and appropriate
email to fatalities2@cber.fda.gov and a medical referral. The notification process
written report should be submitted within and counseling must be done with tact
7 days. Current information can be found and understanding, and the concerns of
on the Internet at http://www.fda.gov/ the donor should be addressed. The do-
cber/gdlns/bldfatal.pdf. nor should be told clearly why he or she is
deferred and, when appropriate, about
the possibility of being infectious to oth-
Management of Posttransfusion Infections ers. Notification should occur promptly
because a delay in notification can delay
Implicated Donors
initiation of treatment or institution of
If documented transfusion-associated measures to prevent the spread of infec-
hepatitis, HIV, or HTLV-I/II occurs in a pation to others.
tient who received only a single unit, that
donor must be permanently excluded
from future donations, and the name
Use of Immunoglobulins
placed in a file of permanently deferred It is not recommended practice to give in-
individuals. If posttransfusion viral infec- tramuscular or intravenous immune serum
tion occurs after exposure to blood from globulin or HBIG prophylactically to pre-
several donors, it is not necessary to ex- vent posttransfusion hepatitis 181 ; these
clude all of the potentially implicated do- agents have not been shown to prevent
nors. If only a few donors are involved, it posttransfusion hepatitis B, and the avail-
may be desirable to recall them to obtain able evidence is conflicting about their ef-
an interim medical history and to perfect on posttransfusion hepatitis C.182,183 If
form additional tests. Donors found to there has been inadvertent transfusion of
have been implicated in more than one known marker-positive blood or needle-
case of transfusion-associated viral infec- stick exposure to infectious material, HBIG
tion should be appropriately investigated may prevent or attenuate HBV infection.184
and possibly deferred permanently ac- Prophylaxis with immunoglobulin is inef-
cording to procedures established by the fective in preventing HCV transmission
collecting agency. following occupational exposures and is
not recommended for this indication.185
Notification
A donor who will be permanently exclu-
ded as a future blood donor because of a
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3:155-63. 127. Kuehnert MJ, Roth VR, Haley NR, et al. Trans-
115. Evatt B, Austin H, Barnhart E, et al. Surveil- fusion-transmitted bacterial infection in the
lance for Creutzfeldt-Jakob disease among United States, 1998 through 2000. Transfu-
persons with hemophilia. Transfusion 1998; sion 2001;41:1493-9.
38:817-20. 128. Theakston EP, Morris AJ, Streat SJ, et al.
116. van Duijn CM, Delasner ie-Laupretre N, Transfusion transmitted Yersinia entero-
Masullo C, et al. Case-control study of risk colitica infection in New Zealand. Aust N Z J
factors of Creutzfeldt-Jakob disease in Eu- Med 1997;27:62-7.
rope during 1993-95. European Union (EU) 129. Cookson ST, Arduino MJ, Aguero SM, Jarvis
Collaborative Study Group of Creutzfeldt- WR, and the Yersinia Study Group. Yersinia
Jakob Disease (CJD). Lancet 1998;351:1081-5. enterocolitica-contaminated Red Blood Cells

(RBCs)—an emerging threat to blood safety 143. Orton SL, Liu H, Dodd RY, Williams AE. Prev-
(abstract). In: Program and abstracts of the alence of circulating Treponema pallidum
36th Interscience Conference on Antimicro- DNA and RNA in blood donors with con-
bial Agents and Chemotherapy, New Or- firmed positive syphilis tests. Transfusion
leans, September 15-18, 1996. Washington, 2002;42:94-9.
DC: American Society for Microbiology, 144. Gardella C, Marfin AA, Kahn RH, et al. Per-
1996:237. sons with early syphilis identified through
130. National Blood Data Resource Center. Com- blood or plasma donation screening in the
prehensive report on blood collection and United States. J Infect Dis 2000;185:545-9.
transfusion in the United States in 2001. 145. Leiby DA. Babesia and other parasites. In:
Bethesda, MD: AABB, 2003. Brecher ME, ed. Bacterial and parasitic con-
131. Brecher ME, Hay SN. Improving platelet tamination of blood components. Bethesda,
safety: Bacterial contamination of platelets. MD: AABB Press, 2003:179-200.
Curr Hematol Rep 2004;3:121-7. 146. Herwaldt BL, Springs FE, Roberts PP, et al.
132. Dykstra A, Hoeltge G, Jacobs M, et al. Platelet Babesiosis in Wisconsin: A potentially fatal
bacterial contamination (PBC) rate is sur- disease. Am J Trop Med Hyg 1995;53:146-51.
veillance method (SM) dependent (abstract). 147. Herwaldt BL, Kjemtrup AM, Conrad PA, et al.
Transfusion 1998;38(Suppl):104S. Transfusion-transmitted babesiosis in Wash-
133. Serious Hazards of Transfusion (SHOT). An- ington State: First reported case caused by a
nual report 2000-2001. Manchester, UK: WA1-type parasite. J Infect Dis 1997;175:
SHOT Office, 2002. [Available at http://www. 1259-62.
shot.demon.co.uk/toc.htm.] 148. Krause PJ, Spielman A, Telford SR 3rd, et al.
134. Perez P, Salmi LR, Follea G, et al. Determi- Persistent parasitemia after acute babesiosis.
nants of transfusion-associated bacterial N Engl J Med 1998;339:160-5.
c o n t a m i n a t i o n : Re s u l t s o f t h e Fre n c h 149. Cable R, Trouern-Trend J. Tick-borne infec-
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2001;41:862-71. safety and surveillance. New York: Marcel
135. Ness PM, Braine HG, King K, et al. Single do- Dekker, 2001:399-422.
nor platelets reduce the risk of septic trans- 150. Wells GM, Woodward TE, Fiset P, Hornick RB.
fusion reactions. Transfusion 2001;41:857-61. Rocky Mountain spotted fever caused by
136. Engelfriet CP, Reesink HW, Blajchman MA, et blood transfusion. JAMA 1978;239:2763-5.
al. Bacterial contamination of blood compo- 151. Eastlund T, Persing D, Mathiesen D, et al.
nents. Vox Sang 2000;78:59-67. Human granulocytic ehrlichiosis after red
137. Kim DM, Brecher ME, Bland LA, et al. Pre- cell transfusion (abstract). Transfusion 1999;
storage removal of Yersinia enterocolitica 39(Suppl):117S.
from red cells with white cell-reduction fil- 152. Arguin PM, Singleton J, Rotz LD, et al. An in-
ters. Transfusion 1992;32:658-62. vestigation into the possibility of transmis-
138. Buchholz DH, AuBuchon JP, Snyder EL, et al. sion of tick-borne pathogens via blood
Removal of Yersinia enterocolitica from AS-1 transfusion. Transfusion-Associated Tick-
red cells. Transfusion 1992;32:667-72. Borne Illness Task Force. Transfusion 1999;
139. Pietersz RNI, Engelfriet CP, Reesink HW, et 39:828-33.
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Vox Sang 2003;85:224-39. sion Transmission of Tick-Borne Diseases.
140. Commission on Laboratory Accreditation. Transmission of tick-borne agents of disease
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Phase I. Northfield, IL: College of American potential risks in the United States. Transfu-
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http://www.cap.org/html/checklist_html/ 154. Mungai M, Tegtmeier G, Chamberland M,
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141. Brumit MC, Hay SN, Brecher ME. Bacteria in the United States from 1963 through 1999.
detection. In: Brecher ME, ed. Bacterial and N Engl J Med 2001;344:1973-8.
parasitic contamination of blood compo- 155. Centers for Disease Control and Prevention.
nents. Bethesda, MD: AABB Press, 2003:57- Probable transfusion-transmitted malaria—
82. Houston, Texas, 2003. MMWR Morb Mortal
142. Anderson KC, Lew MA, Gorgone BC, et al. Wkly Rep 2003;52:1075-6.
Transfusion-related sepsis after prolonged 156. Katz LM. Transfusion-induced malaria. In:
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tamination of blood components. Bethesda, immunoglobulin (letter). Lancet 1988;ii:501.

MD: AABB Press, 2003:127-55. [Erratum in Lancet 1988;ii:584.]
157. Herwaldt BL, Grijalva MJ, Newsome AL, et al. 170. Yu MW, Mason BL, Guo ZP, et al. Hepatitis C
Use of polymerase chain reaction to diagnose transmission associated with intravenous
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158. Shulman IA. Transfusion of T. cruzi infection hepatitis C associated with intravenous im-
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159. Leiby DA, Rentas FJ, Nelson KE, et al. Evi- mission by intravenous immunoglobulin
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diac surgery. Circulation 2000;102:2978-82. of hepatitis C virus to children and husbands
160. Leiby DA, Read EJ, Lenes BA, et al. Seroepi- by women infected with contaminated anti-D
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J Infect Dis 1997;176:1047-52. tis C viraemia in recipients of Irish intrave-
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175. Foster PR, McIntosh RV, Welch AG. Hepatitis
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Methods
The inclusion of methods in this edition There are often many different ways to
of the Technical Manual is a subjective perform the same test procedure. Although
decision of the Technical Manual Pro- some workers may prefer other methods,
gram Unit. Readers are encouraged to re- those given here are reliable, straightfor-
fer to previous editions of the manual for ward, and of proven value. Although the in-
methods not appearing in this edition be- vestigation of unusual serologic problems
cause exclusion from the current edition often requires flexibility in thought and
does not necessarily indicate that their methodology, the adoption of uniform
use is prohibited. However, some proce- methods for routine procedures in the lab-
dures, such as xylene and chloroform elu- oratory is imperative. In order for labora-
tion techniques, were removed because tory personnel to have reproducible and
Methods
the chemicals used in the procedures comparable results in a test procedure, it is
could present a safety risk. Thus, readers essential that everyone in the laboratory
are cautioned when referring to proce- perform the same procedure in the same
dures in previous editions because they manner.
have not been reviewed for content and
safety.
713

Methods Section 1: General Laboratory Methods
Methods Section 1
General Laboratory Methods
Introduction Reagent Preparation

Many procedures include formulas for re-
The methods outlined in the following
agent preparation. Labels for reagents pre-
sections are examples of acceptable pro-
pared in-house must contain the follow-
cedures. Other acceptable procedures
ing:
may be used by facilities if desired. To the
■ Name of solution.
greatest extent possible, the written pro-
■ Date of preparation.
cedures conform to the Guidelines for
■ Expiration date (if known).
Clinical Laboratory Technical Procedure
■ Storage temperature and/or conditions.
Manuals developed by the National Com-
■ Mechanism to identify the person pre-
mittee for Clinical Laboratory Standards.
paring the solution.
As indicated in Title 21 of the Code of
■ Universal hazardous substance label.
Federal Regulations (CFR) Part 606.65(e),
the manufacturer’s instructions (eg, prod-
uct insert) for reagents and supplies li-
Temperatures
censed by the Food and Drug Administra- Whenever specific incubation or storage
tion (FDA) should be followed. Any deviation temperatures are given, the following ranges
should be validated using appropriate are considered satisfactory:
controls and incorporated into a standard
operating procedure before approval by
the medical director. (Note: Deviations Stated Temperature Acceptable Range
Section 1
may also require concurrence from the 4C 2-8 C

FDA.) It is important to use Standard Pre- Room temperature 20-24 C
cautions when appropriate (see Chapter 37 C 36-38 C
2). 56 C 54-58 C
715

Centrifugation Variables and the spread of disease. The CDC also

serves as a Center for Applied Biosafety and
Centrifugation speeds (relative centrifugal
Training for the World Health Organization
force) and times should be standardized
(WHO)8 and for the UN. The Occupational
for each piece of equipment. (See Meth- 9
Safety and Health Administration (OSHA)
ods Section 8.)
regulates worker safety related to handling,
Reference packing, and transport. Both the DOT and
the IATA require active training for anyone
Guidelines for clinical laboratory technical proce-
dure manuals. 3rd ed. (NCCLS Document GP2-A3,
who packages infectious or toxic materials
Vol. 12, No. 10.) Wayne, PA: National Committee for shipment.3,4 DOT and IATA documents
for Clinical Laboratory Standards, 1996. offer general shipping advice for all hazard-
ous materials because the regulations are
similar and most carriers follow the ship-
Method 1.1. Transportation ping guidelines set forth in the IATA Dan-
gerous Goods Regulations4 and the Infec-
and Shipment of Dangerous tious Substances Shipping Guidelines.
7
Goods Facilities should also consult their local car-

Several agencies specify packaging and riers for additional requirements. In gen-
shipping requirements for dangerous or eral, all dangerous goods regulations are
hazardous materials, depending on how based on public risk from the materials and
the material is shipped (mail, ground, or therefore have specific packaging, labeling,
air). For transport by mail of infectious and documentation requirements. It is the
materials, clinical specimens, or biologic responsibility of the shipper of biologic or
products, the United States Postal Service infectious material to properly classify,
(USPS) Dangerous Goods Regulations package, label, and document the sub-
must be followed.1,2 For interstate trans- stance being shipped.
port of infectious materials by ground or
air, the United States Department of
Transportation (DOT) regulations apply.3 Dangerous Goods Classifications
Most air carriers apply the International
Air Transport Association (IATA)4 regula- IATA classifies hazards into nine catego-
tions and the technical instructions of the ries:
International Civil Aviation Organization 1. Explosives.
(ICAO).5 These agencies adopt the recom- 2. Compressed gases.
mendations of the United Nations (UN) 3. Flammable liquids.
Committee of Experts on the Transport of 4. Other flammable hazards.
Dangerous Goods for the international 5. Oxygen-rich material, oxidizers, and
transport of infectious substances and organic peroxides.
clinical specimens. 6. Material affecting health, poisons,
The Centers for Disease Control and Pre- and infectious substances.
6 7
vention (CDC) and the IATA provide pack- 7. Radioactive materials.
ing and labeling requirements for ship- 8. Corrosive material.
ments of infectious materials in order to 9. Miscellaneous hazards.4
protect the public health by minimizing the The infectious category includes:
potential for direct contact with such mate- ■ Infectious substances: microbiologic
rials, contamination of the environment, agents or their toxins that cause, or

Methods Section 1: General Laboratory Methods 717
may cause, disease; also called etio- be shipped without hazard restrictions.
logic agents. Specimens being shipped for initial diag-
■ Biologic products: products that are nosis purposes and with a low probability
prepared and shipped in compli- that infectious agents are present are
ance with the provisions of 9 CFR packaged as diagnostic samples (IATA
part 102 (Licensed Veterinary Bio- packing instruction 650); specimens from
logical Products), 9 CFR part 103 (Bi- individuals known, or thought likely, to
ological Products for Experimental have an infectious disease are shipped as
Treatment of Animals), 9 CFR part infectious substances (IATA packing in-
104 (Imported Biological Products), struction 602). The IATA Dangerous
21 CFR part 312 (Investigational Goods Regulations should be consulted
New Drug Application), 21 CFR parts for the most recent amendments to ship
600-680 (Biologics), 29 CFR Part biologic specimens.
1910.1030 (Occupational Exposure
to Bloodborne Pathogens), 42 CFR Method 1.1.1. Shipping Diagnostic
Part 72 (Interstate Shipment of Etio- Specimens and Infectious Substances
logical Agents), or 49 CFR Parts 171-
178 (Hazardous Materials Regula-
Principle
tions). The safe transport of hazardous materials
■ Clinical or diagnostic specimens: hu- requires that they be packaged in a way to
man or animal material (including protect the materials and those who han-
excreta, secreta, blood and its com- dle them. Prevention from leakage and
ponents, body fluids, tissue) being from being crushed in unexpected acci-
shipped for the purpose of diagno- dents are required. Primary containers
sis. should be leakproof and securely closed.
■ Genetically modified organisms. The primary container should be placed
■ Clinical and medical waste. in a watertight secondary container. Ab-
sorbent material capable of absorbing the
entire liquid contents in the package is
Blood Bank Applications
placed between the primary and second-
Although the USPS regulates some bio- ary containers. The outer packaging should
logic products, such as live poliovirus vac- have adequate strength for its intended
cine, the DOT and IATA take the position use and capacity and be labeled in accor-
that biologic products and diagnostic dance with applicable regulations. Pack-
specimens may be shipped with less aging requirements for clinical specimens
stringent packaging and labeling require- are similar to those for infectious sub-
ments, unless they contain, or are reason- stances except that performance stan-
ably believed to contain, an infectious dards of the packaging materials are less
substance. The CDC has proposed regula- rigorous.
tions to harmonize these requirements
with other federal and international re-
10
quirements. Units of blood that meet Materials
disease testing requirements and un- 1. Leakproof, watertight primary con-
screened blood from healthy donors tainer (eg, test tube), heat sealed,
meeting all FDA donor eligibility criteria crimped, or otherwise reinforced to
including specimens for screening may prevent cap slippage.

2. Leakproof, watertight secondary pack- Procedure

age (eg, sealable plastic bag or con- 1. Place the sealed primary container
tainer with screw cap). into the secondary container.
3. Nonparticulate absorbent material (eg, 2. Add sufficient absorbent material
paper towels, gauze, disposable dia- around the primary container (be-
per). tween the primary and secondary
4. For infectious substances and for to- containers) to cover all sides of the
tal shipment volumes greater than primary container and to absorb the
50 mL (but less than 4000 mL or 4 entire contents of the primary con-
tainer should breakage occur.
kg), shock-absorbent material equal
3. Seal the secondary container securely.
in volume to the nonparticulate-ab-
4. Place the secondary container into
sorbent material. the applicable outer package.
5. Outer packaging. 5. For infectious substances with a
a. For infectious substances with a shipping volume greater than 50 mL,
shipping volume greater than 50 place shock-absorbent materials at
mL (but less than 4000 mL or 4 the top, bottom, and sides between
kg), an outer package of corru- the secondary container and the
gated cardboard, fiberboard, outer packaging. See Fig 1.1.1-1.
wood, metal, or rigid plastic meet- 6. Place any necessary coolant material
ing UN strength requirements. (eg, wet ice or dry ice) between the
b. For biologic and diagnostic secondary container and the appli-
specimens with a shipping vol- cable outer packaging. Provide inte-
ume less than 50 mL, a non- rior support to secure the secondary
certified outer container. packaging in the original position
6. Coolant material (if necessary). because the ice or dry ice melts or
a. Wet ice, enclosed in sealed bags dissipates.
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- Figure 1.1.1-1. Appropriate packaging of clinical
tions. specimen material.

Table 1.1.1-1. Special Labels for Shipping

Clinical (Diagnostic) Specimens
Specimens collected for diagnosis, research, or other purposes must be labeled as biohazardous. Both the
primary container and the outer packaging must contain a biohazard label. The outer packaging label must
appear as follows:
1. The color of the material on which the label is printed must be bright orange, with the biohazard symbol
and printing in black.
2. The label must be a rectangle measuring 2 inches high by 4 inches long.
3. The biohazard symbol, measuring 1.56 inches in diameter, must be centered on a square measuring 2
inches on each side.
4. The size of the letters, printed in Helvetica, must be:
Biohazard 16 pt.
Clinical specimens 14 pt.
Packaged in compliance with 42 CFR part 72 6 pt.
In case of damage or leakage, notify 10 pt.
Shipper and receiver 10 pt.
Infectious Substances (Etiologic Agents)

For substances known to contain infectious agents or reasonably anticipated to contain such agents,
biohazardous labels must be placed on the primary container and outer packaging. The outer packaging
label must appear as follows:
1. The color of the material on which the label is printed must be white, with the biohazard symbol and
printing in black.
2. The label must be a diamond-on-point measuring, at a minimum, 4 inches on each side.
3. The biohazard symbol, measuring 0.81 inches in diameter, must be centered on a square measuring 2
inches on each side.
4. The Class 6 symbol for infectious substances must be centered at the bottom of the label.
5. The size of the letters, printed in Helvetica, must be:
Infectious substance 16 pt.
Packaged in compliance with 42 CFR part 72 5 pt.
In case of damage or leakage, notify 7 pt.
Immediately notify 7 pt.
Public Health Authority 7 pt.
In the United States 5 pt.
Centers for Disease Control and Prevention 5 pt.
Atlanta, GA 5 pt.
1-800-232-0124 5 pt.
6 24 pt.
Dry Ice
Packages with dry ice must be labeled with the following information:
1. The diamond-shaped Class 9 symbol for miscellaneous hazardous materials.
2. The words “Dry Ice” or “Carbon Dioxide, Solid.”
3. “UN 1845,” the United Nations hazardous material category for dry ice (if shipped out of the country).
4. The name of the contents being cooled.
5. The weight of the dry ice in kg (not to exceed 5 lb if transported by air).
Liquid Nitrogen
Packages with liquid nitrogen must be labeled with a green IATA label stating “Contains Cryogenic Liquid.”
Cargo Aircraft Only

If the mode of transport is air and more than 50 mL of infectious substance is enclosed, the package must
have a warning label “Cargo Aircraft Only” to preclude the carrier from transporting the package on a
passenger plane.

7. Enclose the itemized listing of containers must be able to withstand at

tainer contents between the second- least a 1.2-meter drop on a hard un-
ary and outer packaging. yielding surface without release of
8. Seal the outer packaging and label the container’s contents.
with address label and applicable 8. Ensure that all state and local regu-
special labels. lations are followed.
9. Complete applicable shipping forms 9. Some exceptions may apply for
and send them with the package. ground transportation.
10. If unsure about how to package and
ship materials, contact the CDC.
Notes
11. If breakage occurs during shipment,
1. Package specimens within a con- the package should be handled with
tainer such that they will remain in extreme caution (wear personal pro-
an upright position to help prevent tective equipment) and the entire
leakage. package (including containers, con-
2. Closures on primary containers can tents, and packaging materials) should
be reinforced with adhesive tape or be autoclaved before discard. Super-
paraffin. It is not necessary to rein- visory personnel should be notified
force unopened evacuated specimen if the contents are lost in transit or if
collecting tubes. the damage appears related to inade-
3. For infectious substances, the name, quate packaging by the sender.
address, and telephone number of 12. The carrier, the receiver, or anyone
the shipper must appear on both the handling damaged or leaking pack-
secondary and outer shipping con- ages must isolate the package and
tainers. notify the shipper and intended re-
4. Shipments of infectious substances cipient immediately. In addition, for
may contain multiple secondary con- infectious substances, notify the CDC
tainers, but the total volume of the as soon as possible (1-800-232-0124).
shipment may not exceed 4 L or 4 When notifying the CDC, the caller
kg, excluding the packaging and should provide a description of the
coolant weights. condition of the package, the name,
5. Primary containers or secondary address, and telephone number of
packaging must be capable of with- the shipper, and any other pertinent
standing an internal pressure differ- information, so that decontamina-
ential of 95 kPA (0.95 bar, 13.8 lb/in2) tion and disposal procedures can be
between the temperatures of –40 C provided.
and 55 C.
6. Styrofoam, plastic bags, and paper
envelopes are unacceptable for outer
packaging. Additional Considerations with the Use of
7. UN-certified containers must be able Dry Ice
to withstand a 30-foot “drop test,” as 1. Solid carbon dioxide or “dry ice” is
specified in 49 CFR 178.609, without classified as a hazardous material
breaking enclosed tubes. The UN because it can cause burns on con-
certification number must appear tact and gives off carbon dioxide gas
on the container. Noncertified con- as it volatilizes.

2. Insulated gloves must be worn when area) after a shipment is re-

handling dry ice; eye protection must ceived.
be worn when breaking up chunks
of solid ice or when breaking apart
Additional Considerations with the Use of
ice pellets.
Liquid Nitrogen
3. Dry ice should be handled in a well-
ventilated area; its gas can cause light- 1. Liquid nitrogen is classified as a cryo-
headedness and, in extreme cases, as- genic liquid.
phyxiation. 2. Insulated gloves, eye protection, and
4. When kept in a tightly sealed ship- a protective laboratory coat must be
ping container, dry ice could rupture worn when working with liquid ni-
the packaging. If dry ice is used, it trogen.
must be placed outside the second- 3. A watertight material capable of with-
ary packaging or in an overpack with standing cryogenic temperatures must
one or more complete packages. The be used for the primary container.
outer packaging must be leakproof. This container must maintain its con-
Procedures for packing with dry ice tainment integrity at the tempera-
must include instructions for sealing ture of the refrigerant as well as at
the outer container in a manner that the temperature and pressure of air
allows the gas to escape and pre- transport if refrigeration is lost.
vents loosening of secondary con- 4. The secondary packaging must be
tainers as the dry ice dissipates. designed to withstand cryogenic tem-
Consult the Domestic Mail Manual peratures.
(section C023),2 49 CFR 173.217 and 5. The design of the outer packaging
175.10 (a)(13),3 IATA Dangerous Goods must allow for relief of pressure when
4
Regulations, and IATA packing in- the liquid nitrogen evaporates. A
struction 904. loose-fitting, but secured, container
5. HAZMAT training requirements for cover or pressure relief valve may be
dry ice are found in 49 CFR 172.704.3 used.
Training for staff who come in con-
tact with dry ice should include in- References
formation on:
1. Code of Federal Regulations. Title 39 CFR.
■ Potential hazards. Washington, DC: US Government Printing
■ Personal protective equipment Office, 2004 (revised annually).
to use when handling or chip- 2. Etiologic agent preparations, clinical speci-
mens, and biological products. Domestic
ping. Mail Manual (section C023), issue 57, July 10,
■ Procedures for packing, seal- 2003.
ing, labeling, and handling 3. Code of Federal Regulations. Title 49 CFR Part
boxes containing dry ice. 171-180. Washington, DC: US Government
■ Special considerations in oper-
4. Dangerous goods regulations. 45th ed. Mon-
ating a vehicle used to trans- treal, Canada: International Air Transport As-
port boxes containing dry ice. sociation, 2004 (revised annually).
■ Procedures for storing or dis- 5. Technical instructions for the safe transport
of dangerous goods by air. 2002-2004 ed.
posing of dry ice (eg, allowing
Montreal, Canada: International Civil Avia-
the gas to dissipate by natural tion Organization, Documents 9284 and
means in a well-ventilated 9284SU, 2002.

6. Code of Federal Regulations. Title 42 CFR Part Notes

72. Washington, DC: US Government Printing
Office, 2004 (revised annually). Other suitable methods for monitoring
7. Infectious substances and diagnostic speci- shipments are:
mens shipping guidelines. 4th ed. Montreal, 1. Use time/temperature indicators,
Canada: International Air Transport Associa-
tion, 2003. one such indicator per shipping car-
8. World Health Organization. Guidelines for ton. These indicators will change
the safe transport of infectious substances color or show another visible indica-
and diagnostic specimens. Geneva, Switzer- tion if the temperature has exceeded
land: World Health Organization, 1997.
10 C.
9. Code of Federal Regulations. Title 29 CFR
2. Place a “high-low” thermometer in
Printing Office, 2004 (revised annually). the shipping container. This simple,
10. Centers for Disease Control and Prevention. reusable thermometer measures and
Packaging and handling of infectious sub- records the highest and lowest tem-
stances and select agents, notice of proposed
peratures during any period.
rulemaking. Fed Regist 1999;64(208):58022-
31.
Method 1.1.2. Monitoring Temperature

During Shipment of Blood Method 1.2. Treatment of
Principle Incompletely Clotted Specimens
Some form of temperature indication or
monitoring is desirable when shipping Principle
blood. The temperature of the contents of Fibrin generation may continue in serum
a shipping container used for whole blood separated from incompletely clotted blood,
or liquid-stored red cell components can especially during incubation at 37 C. This
be ascertained when the shipment is re- produces strands of protein that entrap
ceived, as follows: red cells and make it difficult to evaluate
agglutination. Blood from patients who
Procedure have recently received heparin may not
clot at all, and blood from patients with
1. Open the shipping container and excessive fibrinolytic activity may relique-
promptly place the sensing end of a fy or may contain protein fragments that
calibrated liquid-in-glass or elec- interfere with examination for agglutina-
tronic thermometer between two tion.
bags of blood or components (labels
facing out) and secure the “sand-
Materials
wich” with two rubber bands.
2. Close the shipping container. 1. Thrombin: dry human/bovine thrombin
3. After approximately 3 to 5 minutes, or thrombin solution (50 units/mL
read the temperature. in saline).
4. If the temperature of red-cell-con- 2. Glass beads.
taining components exceeds 10 C, 3. Protamine sulfate: 10 mg/mL in sa-
quarantine the units until their ap- line.
propriate disposition can be deter- 4. Epsilon aminocaproic acid (EACA):
mined. 0.25 g/mL in saline.

Procedure bodies, test results must be carefully

observed for false-positive reactions.
1. To accelerate clotting: Either of the
Quality control should be performed
following techniques may be used:
on thrombin reagents before or con-
a. Add to the specimen the amount
current with their use to identify
of dry thrombin that adheres to
those with contaminating antibod-
the tip of an applicator stick or
ies.
1 drop of thrombin solution
per mL of whole blood or se-
rum. Allow 10 to 15 minutes for
the clot to form. Use standard Method 1.3. Solution
centrifugation to separate the
clot and serum.
Preparation—Instructions
b. Gently agitate the separated se- Principle
rum with small glass beads, at
The basic definitions, calculations, and
37 C, for several minutes. Then,
instructions given below serve as a review
use low speed centrifugation to
of simple principles necessary for solu-
pellet the glass beads. Transfer
tion preparation.
the serum to another tube.
1. Mole, gram-molecular weight: Weight,
2. To neutralize heparin: Protamine
expressed in grams equal to the
sulfate can be added to the speci-
atomic or molecular weight of the
men to neutralize heparin; however,
substance.
excess protamine promotes rouleaux
2. Molar solution: A one molar (1 M)
formation and, in great excess, will
solution contains one mole of solute
inhibit clotting. Add 1 drop of prota-
in a liter of solvent. The solvent is as-
mine sulfate solution to 4 mL of
sumed to be distilled or deionized
whole blood and wait 30 minutes to
water unless otherwise indicated.
evaluate the effect on clotting. If
3. Gram-equivalent weight: Weight, in
clotting does not occur, add addi-
grams, of a substance that will pro-
tional protamine sparingly. Note:
duce or react with 1 mole of hydro-
protamine sulfate may work more
gen ion.
rapidly when briefly incubated (5-10
4. Normal solution: A one normal (1 N)
minutes) at 37 C.
solution contains one gram-equiva-
3. To inhibit fibrinolytic activity: Add 0.1
lent weight of solute in a liter of
mL of EACA to 4 mL of whole blood.
solution.
5. Percentage solutions: The percent
Notes designation of a solution gives the
1. The use of anticoagulated (eg, ACD weight or volume of solute present
or EDTA) collection tubes may help in 100 units of total solution. Percent
to avoid the problem of incom- can be expressed as:
pletely clotted specimens. The use of a. Weight/weight (w/w), indicating
anticoagulated specimens must be grams of solute in 100 g of solu-
validated in accordance with each tion.
standard operating procedure. b. Volume/volume (v/v), indicating
2. Because preparations of human milliliters of solute present in
thrombin may contain red cell anti- 100 mL of solution.

c. Weight/volume (w/v), indicating 69 g of the monohydrate crystals

grams of solute in 100 mL of so- made up to 1 L.
lution. Unless otherwise speci- 3. Normal solutions:
fied, a solution expressed in 1 N HCl = 36 g of solute made up to 1
percentage can be assumed to L. One mole HCl dissociates into one
+
be w/v. mole H , so gram-equivalent weight
6. Water of crystallization, water of hy- and gram-molecular weight are the
dration: Molecules of water that form same.
an integral part of the crystalline 12 N HCl = (36 × 12) = 432 g of solute
structure of a substance. A given made up to 1 L.
substance may have several crystal- 1 N H2SO4 = (98 ÷ 2) = 49 g of solute
line forms, with different numbers of made up to 1 L. One mole H2SO4 dis-
+
water molecules intrinsic to the en- sociates to give two moles of H , so
tire molecule. The weight of this wa- the gram-equivalent weight is half
ter must be included in calculating the gram-molecular weight.
molecular weight of the hydrated 4. Percent solution:
substance. 0.9% NaCl (w/v) = 0.9 g of solute
7. Anhydrous: The salt form of a sub- made up to 100 mL of solution.
stance with no water of crystallization.
8. Atomic weights (rounded to whole Notes
numbers): H, 1; O, 16; Na, 23; P, 31; S,
Accurate results require accurate prepara-
32; Cl, 35; K, 39.
tion of reagents. It is important to care-
9. Molecular weights:
fully read and follow all instructions and
HCl: 1 + 35 = 36; NaCl: 23 + 35 = 58
labels.
KCl: 39 + 35 = 74
1. Weigh only quantities appropriate
H2O: (2 × 1) + 16 = 18
for the accuracy of the equipment.
NaH2PO4: 23 + (2 × 1) + 31 + (4 × 16) =
The operator’s manual should give
120
these specifications.
NaH2PO4 • H2O: 23 + (2 × 1) + 31 +
2. Prepare the largest volume that is
(4 × 16) + (2 × 1) + 16 = 138
practical. There is greater accuracy
KH2PO4: 39 + (2 × 1) + 31 + (4 × 16) =
in measuring larger volumes than
136
smaller volumes. If a reagent bal-
H2SO4: (2 × 1) + 32 + (4 × 16) = 98
ance is accurate to ±0.01 g, the po-
tential error in weighing 0.05 g (50
mg) will be 20%, whereas the poten-
Examples tial error in weighing 0.25 g (250 mg)
1. Molar solutions: will be only 4%. If the solution re-
1 M KH2PO4 = 136 g of solute made up tains its activity when stored appro-
to 1 L. priately, it is usually preferable to
0.15 M KH2PO4 = (136 × 0.15) = 20.4 g prepare a large volume. If the solu-
of solute made up to 1 L. tion deteriorates rapidly, smaller vol-
0.5 M NaH2PO4 = (120 × 0.5) = 60 g of umes may be preferred to reduce
solute made up to 1 L. waste.
2. Molar solution with hydrated salt: 3. Note whether a substance is in the
0.5 M NaH2PO4 • H2O = (138 × 0.5) = hydrated or anhydrous form. If the

instructions give solute weight for 5. Adjust the pH of the solution before
one form, and the available reagent bringing it to its final volume so that
is in another form, be sure to adjust the addition of water (or other sol-
the measurements appropriately. vent) does not markedly change the
For example, if instructions for 0.5 M molarity. For example, to bring 500
NaH2PO4 call for 60 g, and the re- mL of 0.1 M glycine to a pH of 3:
agent is NaH2PO4 E H2O, find the ra- a. Ad d 3 . 7 5 g o f g l y c i n e
tio between the weights of the (H2 NCH2 COOH: molecular
two forms. The molecular weight of weight, 75) to 400-475 mL of
NaH2PO4 E H2O is 138 and the mo- water in a beaker. Dissolve
lecular weight of NaH 2 PO 4 is 120. completely, using a magnetic
Therefore, the ratio is 138 ÷ 120 = stirrer.
1.15. Multiply the designated weight b. Add a few drops of concentrated
by the ratio (60 g × 1.15 = 69 g) to ob- (12 N) HCl and measure pH af-
tain the final weight needed. ter acid is thoroughly mixed.
4. Dissolve the solute completely be- Continue adding HCl until pH
fore making the solution to the final is 3.0.
volume. This is especially important c. Transfer the solution to a 500-mL
for substances, such as phosphates, volumetric flask. Rinse beaker
that dissolve slowly. For example, to and stirring bar with aliquots of
make 500 mL of 0.15 M KH2PO4: water, adding the rinse water to
a. Weigh 10.2 g of solute in a weigh- the flask. Use the rinses to con-
ing boat or glass [(0.15 × 136) ÷ tribute to the total 500-mL vol-
2] because only 500 mL will be ume.
made. d. Measure the pH of the solution
b. Place 350 mL of water in a at final volume.
500-mL volumetric flask on a
magnetic stirrer. Add the stir- References
ring bar and adjust it to a slow, 1. Remson ST, Ackerman PG. Calculations for
steady stirring speed. the medical laboratory. Boston, MA: Little,
Brown & Co., 1977.
c. Add 10.2 g of salt, then rinse the
2. Henry JB, ed. Clinical diagnosis and manage-
boat with several aliquots of ment by laboratory methods. 18th ed. Phila-
water until no salt remains. Nu- delphia: WB Saunders, 1991.
merous small-volume rinses
remove adherent material more
effectively than a few larger
volumes. Add the rinse water to
Method 1.4. Serum Dilution
the material in the flask and stir Principle
until the salt has completely Serum is sometimes diluted in saline or
dissolved. other diluents to determine its relative
d. If pH measurement is unneces- antibody concentration. It is customary to
sary, add water to the 500-mL express the dilution as 1 part of serum
mark, adjusting the volume for contained in the total number of parts of
the stirring bar, and mix thor- the dilution. For example, to test the se-
oughly. For solutions needing pH rum at one-tenth its original concentra-
adjustment, see the next step. tion, a dilution of 1 part in 10 may be

made by mixing 1 mL of serum with 9 mL quantity of a new higher final

of saline. The final volume is 10, and the dilution is:
dilution is expressed as a 1 in 10 dilution.
The diluted material contains one-tenth reciprocal of present dilution
(1/10 or 0.1) of the unmodified serum. It volume of present dilution needed
is often customary to report the titer of an
antibody as the reciprocal of the highest = reciprocal of final dilution
dilution that retains a 1+ agglutination. total final volume required
Therefore, serum that reacts at a dilution
of 1/32 is considered to have a titer of 32. b. Example: Present serum dilu-
Note: A 1 in 10 dilution is 1 part in 9 parts, tion is one in two, total final
whereas a 1 to 10 or 1:10 is 1 part in 10 parts. volume is 100 mL, and new fi-
nal serum dilution is 1 in 10.
How much serum (diluted one
Procedure in two) will have to be added to
make up a final volume of 100
1. Diluting an existing dilution: mL of a 1 in 10 dilution?
a. A new higher dilution can be
prepared from diluted material 2 = 10
by adding more diluent. The X 100
formula for calculating either
the new higher final dilution or X = 20 or 20 mL of serum (dilu-
the amount of diluent to add to tion of one in two) must be added
obtain a higher final dilution is: to 80 mL of diluent to obtain
100 mL of a 1 in 10 dilution.
reciprocal of present serum dilution
volume of serum dilution used
= reciprocal of new final dilution Method 1.5. Dilution of %

total final volume
Solutions
b. Example: Serum dilution is one
in two and volume of serum di- Procedure
lution is 1.0 mL. If 4.0 mL of sa-
line is added, what will be the 1. Dilutions can be prepared from more
new final dilution? concentrated solutions by use of the
following formula:
2 = X
1 5 (Volume1 × Concentration1) =
(Volume2 × Concentration2)
X = 10 or 1 in 10 dilution V1 × C1 = V2 × C2
2. Diluting a dilution to a specified volume: where V1 and C1 represent original

a. The formula for calculating the volume and concentration, and V2
volume of diluent to add to a and C2 represent final desired vol-
dilution to achieve a certain ume and concentration.

2. Example: 30% albumin is available, 1. Transfer at least 1 mL of whole blood

but 2 mL of 6% albumin is needed. to a 10-mL tube.
How should the albumin be diluted? 2. Wash the red cells in saline or phos-
V1 × 30 = 2 × 6 phate-buffered saline (PBS), centri-
30V1 = 12 fuging for 5 minutes to pellet the
V1 = 12 ÷ 30 = 0.4 cells. Repeat two or three times. The
Therefore, mix 0.4 mL of 30% albu-
final supernate should be clear and
min with 1.6 mL saline to obtain 2.0
should be completely removed by
mL of 6% albumin, or for small-vol-
ume use, mix 4 drops 30% albumin aspiration.
with 16 drops saline to obtain 20 3. Transfer 0.3 mL of the washed red
drops of 6% albumin. 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-
Method 1.6. Preparation of a line by gently inverting the tube sev-
3% Red Cell Suspension eral times.
Principle 5. To compare the color and density of
A 3% red cell suspension is a common re- the suspension by eye, transfer a vol-
agent in many serologic procedures. The ume of the prepared suspension to a
suspension need not be exactly 3%; an 10 × 75 mm tube. Also transfer a sim-
approximation achieves the appropriate ilar volume of a known 3% red cell
serum-to-cell ratio for most test proce- suspension (eg, commercial reagent
dures and an adequate number of red red cell suspension) to another 10 ×
cells to read and grade the reactions. The 75 mm tube. Hold the two tubes in
following steps are intended to help an in-
front of a light source to compare them.
dividual gain confidence in approximat-
6. To compare the size of the cell pellet
ing a 3% red cell suspension visually, both
expected from a 3% red cell suspen-
as a suspension of cells and in the appro-
priate size of the cell pellet achieved after sion, transfer one drop of the pre-
centrifugation. pared suspension to a 10 × 75 mm
tube. Similarly, transfer one drop of
Materials a known 3% commercial reagent red
1. Whole blood sample. cell suspension to another 10 × 75 mm
2. Test tubes. tube. Centrifuge the tubes in a sero-
3. Disposable pipettes (1 mL and 10 mL logic centrifuge, using the spin time
serologic). designated for “saline.” The size of
4. Saline. the two cell pellets should be similar.
5. Centrifuge (3000 rpm or equivalent).
6. Commercially prepared 3% reagent
red cell suspension. Note
Procedure For best results use red cell suspensions

To prepare 10 mL of a 3% red cell suspen- on the day of preparation only, unless sta-
sion: bility for a longer time has been validated.

Method 1.7. Preparation and References

1. Hendry EB. Osmolarity of human serum and
Use of Phosphate Buffer of chemical solutions of biologic importance.
Clin Chem 1961;7:156-64.
Principle 2. Dacie JV, Lewis SM. Practical haematology. 4th
ed. London, England: J and A Churchill, 1968:
Mixtures of acids and bases can be pre- 540-1.
pared at specific pH values and used to
buffer (render) other solutions to that pH.
The following procedure includes a method
for preparing phosphate-buffered saline Method 1.8. Reading and
(PBS), which can be used as a diluent in Grading Tube Agglutination
serologic tests.
Principle
Reagents The purpose of grading reactions is to al-
1. Prepare acidic stock solution (solu- low comparison of reaction strengths.
tion A) by dissolving 22.16 g of This is beneficial in detecting multiple
NaH2PO4 • H2O in 1 L of distilled wa- antibody specificities or antibodies exhib-
ter. This 0.16 M solution of the mono- iting dosage. The grading of agglutination
basic phosphate salt (monohydrate) reactions should be standardized among
has a pH of 5.0. all members of the laboratory staff, in the
2. Prepare alkaline stock solution (so- interest of uniformity and reproducibility
lution B) by dissolving 22.7 g of of test results. Most laboratories define
Na2 HPO4 in 1 L of distilled water. their own version of a grading system,
This 0.16 M solution of the dibasic which is described in a written procedure
phosphate salt (anhydrous) has a pH available to all staff. Some systems use as-
of 9.0. signed numeric values (scores) for the
observed reactions.
Procedure
Materials
1. Prepare working buffer solutions of
1. Centrifuged serologic tests for agglu-
the desired pH by mixing appropri-
tination.
ate volumes of the two solutions. A
2. Agglutination viewer.
few examples are:
pH Solution A Solution B
5.5 94 mL 6 mL Procedure
7.3 16 mL 84 mL 1. Gently shake or tilt the tube and re-
7.7 7 mL 93 mL suspend the red cell button in the
2. Check the pH of the working solu- tube. The tilt technique uses the me-
tion before using it. If necessary, add niscus to gently dislodge the red cell
small volumes of acid solution A or button from the wall of the tube.
alkaline solution B to achieve the de- 2. Observe the way that cells are dis-
sired pH. persed from the red cell button.
3. To prepare PBS of a desired pH, add 3. Record reactivity by comparing the
one volume of phosphate buffer at agglutinates to the descriptions in
that pH to nine volumes of normal Table 1.8-1. The reactivity should be
saline. assessed when the red cells have been

Table 1.8-1. Interpretation of Agglutination Reactions
Macroscopically Observed Findings Designation Score
One solid agglutinate 4+ 12

Several large agglutinates 3+ 10
Medium-size agglutinates, clear background 2+ 8
Small agglutinates, turbid background 1+ 5
Very small agglutinates, turbid background 1w 4
Barely visible agglutination, turbid background w+ or +/– 2
No agglutination 0 0
Mixtures of agglutinated and unagglutinated red cells mf
(mixed field)
Complete hemolysis H
Partial hemolysis, some red cells remain PH
completely resuspended from the hemolyzed and no hemolytic agent

button. was added to the test.
3. The character of the agglutination
Interpretation should be noted and recorded. This
Refer to Table 1.8-1. information provides valuable clues
in the investigation such as the char-
Notes acteristic refractile agglutination of
anti-Sda.
1. An agglutination viewer may facilitate
4. Mixed-field agglutination is ex-
the reading of tube tests. However,
pected when using pooled cells for
the manufacturer’s test recommen-
donor antibody detection and add-
dation must be followed for inter-
ing check cells to negative antiglo-
preting the test results.
bulin tests.
2. Serum overlying the centrifuged cell
button must be inspected for
hemolysis, which is a positive sign of Reference
an antigen-antibody reaction, pro- Race RR, Sanger R. Blood groups in man. 6th ed.
vided the pretest serum was not Oxford: Blackwell Scientific Publications, 1975.

Methods Section 2: Red Cell Typing
Methods Section 2
Section 2
Red Cell Typing
All reagents must be used in accordance

Method 2.1. Slide Test for with the manufacturer’s instructions.
Determination of ABO Type
of Red Cells Procedure
1. Place 1 drop of anti-A on a clean, la-
Principle beled glass slide.
See Chapter 13 for a discussion of the 2. Place 1 drop of anti-B on a separate
principles of testing for ABO groups. clean, labeled glass slide.
3. Place 1 drop of anti-A,B on a third
Specimen slide, if parallel tests are to be per-
formed with this reagent, or on a
The reagent manufacturer’s instructions
single clean, labeled slide if this is
must be consulted before slide tests are
the only test performed.
performed; some manufacturers recom-
4. Add to each drop of reagent on the
mend performing slide tests with whole
slides 1 drop of well-mixed suspen-
blood, whereas others specify the use of
sion (in saline, serum, or plasma) of
red cell suspensions of lighter concentra-
the red cells to be tested. (Consult
tions prepared in saline, serum, or plasma.
the reagent manufacturer’s instruc-
tions to determine the correct cell
Reagents concentration to be used.)
1. Anti-A. 5. Mix the reagents and red cells thor-
2. Anti-B. oughly, using a clean applicator stick
3. Anti-A,B (optional). for each reagent. Spread the mixture
731

over an area approximately 20 mm × Specimen

40 mm.
The reagent manufacturer’s package in-
6. Gently tilt the slide continuously for
sert must be consulted to determine spe-
up to 2 minutes. Do not place the slide
cific specimen requirements. Generally,
over a heated surface, such as an Rh
clotted or anticoagulated blood samples
viewbox, during this period.
may be used for ABO testing. The red cells
7. Read, interpret, and record the re-
may be suspended in autologous serum,
sults of the reactions on all slides.
plasma, or saline, or may be washed and
resuspended in saline.
Interpretation
1. Strong agglutination of red cells in Reagents
the presence of any ABO typing re- 1. Anti-A.
agent constitutes a positive result. 2. Anti-B.
2. A smooth suspension of red cells at 3. Anti-A,B. Note: Use of this reagent is
the end of 2 minutes is a negative re- optional.
sult. 4. A1, A2, and B red cells. They can be
3. Samples that give weak or doubtful obtained commercially or the testing
reactions should be retested using laboratory can prepare a 2% to 5%
Method 2.2. suspension on each day of use. (Note:
The use of A2 cells is optional.)
Notes All reagents must be used in accordance
1. Slide testing imposes a greater risk with the manufacturer’s instructions.
of exposure to infectious samples.
Personnel should follow safety mea-
sures detailed in the facility’s proce- Procedures
dures manual. Testing Red Cells
2. Slide testing is not suitable for detec-
tion of ABO antibodies in serum/ 1. Place 1 drop of anti-A in a clean, la-
beled test tube.
plasma.
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-
Method 2.2. Tube Tests for formed with this reagent.
Determination of ABO Group 4. Add to each tube 1 drop of a 2% to
5% suspension (in saline, serum, or
of Red Cells and Serum plasma) of the red cells to be tested.
Alternatively, the equivalent amount
Principle of red cells can be transferred to each
See Chapter 13 for a discussion of the tube with clean applicator sticks.
principles of testing for ABO groups. The 5. Mix the contents of the tubes gently
following procedure is an acceptable rep- and centrifuge them for the cali-
resentative method, but the manufacturer’s brated spin time.
instructions for the specific reagents must 6. Gently resuspend the cell buttons
be consulted. and examine them for agglutination.

Methods Section 2: Red Cell Typing 733
7. Read, interpret, and record the test re- interpretation is recorded for the
sults. Compare the red cell test re- patient’s or donor’s ABO group (see
sults with those obtained in the se- Chapter 13).
rum/plasma tests (see below).
Note
Testing Serum/Plasma Positive reactions characteristically show
1. Label two clean test tubes as A1 and 3+ to 4+ agglutination by reagent ABO an-
B. (Note: Label an additional tube if tibodies; reactions between test serum
an optional test with A2 red cells is to and reagent red cells are often weaker.
be performed.) The serum tests may be incubated at
2. Add 2 or 3 drops of serum/plasma to room temperature for 5 to 15 minutes to
each tube. enhance weak reactions. See Chapter 13
3. Add 1 drop of A1 reagent cells to the for a discussion of weakly reactive sam-
tube labeled A1. ples.
4. Add 1 drop of B reagent cells to the
tube labeled B.
5. Add A2 cells to the appropriate tube, Method 2.3. Microplate Test
if this optional test is being per-
formed. for Determination of ABO
6. Mix the contents of the tubes gently Group of Red Cells and
and centrifuge them for the cali-
brated spin time.
Serum
7. Examine the serum overlying the cell Principle
buttons for evidence of hemolysis. See Chapter 13 for a discussion of the
Gently resuspend the cell buttons principles of testing for ABO blood group.
and examine them for agglutination. Microplate techniques can be used to test
8. Read, interpret, and record test results. for antigens on red cells and for antibod-
Compare serum test results with ies in serum.
those obtained in testing red cells A microplate can be considered as a ma-
(see above). trix of 96 “short” test tubes; the principles
that apply to hemagglutination in tube tests
Interpretation also apply to tests in microplates.
1. Agglutination of tested red cells and Microplates may be rigid or flexible, with
either hemolysis or agglutination in either U-shaped or V-shaped bottoms.
tests with serum constitute positive U-shaped bottom plates are more widely
test results. used because results can be read either af-
2. A smooth cell suspension after re- ter centrifuging the plate and observing the
suspension of the cell button is a characteristics of resuspended red cells or
negative test result. by observing the streaming pattern of the
3. Interpretation of serum/plasma and cells when the plate is placed at an angle.
cell tests for ABO is given in Table Either reading technique permits estima-
13-1. tion of the strength of agglutination.
4. Any discrepancy between the results
of the tests with serum/plasma and Specimen
cells should be resolved before an Refer to Method 2.2.

Equipment 4. Group A1, A2, and B red cells. They

can be obtained commercially or the
1. Dispensers (optional): Semiautoma-
testing laboratory can prepare a 2%
ted devices are available for dispens-
to 5% suspension on each day of use.
ing equal volumes to a row of wells.
(Note: The use of A2 cells is optional.)
Special plate carriers can be pur-
chased to fit common table-top cen-
trifuges. Procedure
2. Microplate readers (optional): Auto- Testing Red Cells
mated photometric devices are avail-
able that read microplate results by 1. Place 1 drop of anti-A and anti-B in
the light absorbance in U-shaped separate clean wells of a U-bottom
bottom wells to differentiate be- microplate. If tests with anti-A,B are
tween positive and negative tests. to be performed, add this reagent to
The microprocessor component of a third well.
the reader interprets the reactions 2. Add 1 drop of a 2% to 5% saline sus-
and prints the blood testing results. pension of red cells to each well con-
The manufacturer’s instructions for taining blood typing reagent.
the collection and preparation of se- 3. Mix the contents of the wells by
rum/plasma and cell specimens gently tapping the sides of the plate.
must be followed. 4. Centrifuge the plate at the appropri-
3. Centrifuges: Appropriate conditions ate conditions established for the
must be established for each centri- centrifuge.
fuge. The following times and rela- 5. Resuspend the cell buttons by man-
tive centrifugal forces, expressed as ually tapping the plate or with the
g, are suggested. Consult the manu- aid of a mechanical shaker, or place
facturer’s directions for specific in- the plate at an angle for the tilt and
formation. stream method.
For a flexible U-shaped bottom 6. Read, interpret, and record results.
microplate: 700 × g for 5 seconds for Compare red cell test results with those
red cell testing and serum/plasma obtained in testing serum/plasma.
testing.
For a rigid U-shaped bottom micro- Testing Serum/Plasma
plate: 400 × g for 30 seconds for red
1. Add 1 drop of a 2% to 5% suspension
cell testing and serum/plasma testing.
of reagent A1 and B red cells to sepa-
rate clean wells of a U-bottom micro-
Reagents plate. (Note: If an optional test on A2
Many manufacturers supply ABO or Rh cells will be performed, add A2 cells
typing reagents that are licensed by the to a third well.)
Food and Drug Administration (FDA) for 2. Add 1 drop of serum or plasma un-
use as undiluted reagents in microplate der test to each well.
tests. 3. Mix the contents of the wells by gently
1. Anti-A. tapping the sides of the plate.
2. Anti-B. 4. Centrifuge the plate at the appropri-
3. Anti-A,B. Note: Use of this reagent is ate conditions established for the
optional. centrifuge.

5. Resuspend the cell buttons by man- Specimen

ually tapping the plate or with aid of
Refer to Method 2.2.
a mechanical shaker, or place the plate
at an angle for the tilt and stream
method. Reagents
6. Read, interpret, and record results. 1. Human anti-A and/or anti-B. Be-
Compare test results on serum/plasma cause some monoclonal ABO typing
with those obtained in testing red reagents are sensitive to changes in
cells. pH and osmolarity, they may not be
suitable for use in adsorption/elution
Note tests.
2. Eluting agent: See Methods Section
To enhance weak serum/plasma reac-
4.
tions, the plates may be incubated at
room temperature for 5 to 10 minutes,
then the centrifugation, reading, and re- Procedure
cording steps may be repeated. 1. Wash 1 mL of the red cells to be
tested at least three times with saline.
Remove and discard the supernatant
Interpretation saline after the last wash.
2. Add 1 mL of reagent anti-A (if a weak
1. Agglutination in any well of red cell
variant of A is suspected) or 1 mL of
tests and hemolysis or agglutination
anti-B (if a weak variant of B is sus-
in any well of a serum test constitute
pected) to the washed cells.
positive results.
3. Mix the red cells with the reagent
2. A smooth suspension of red cells af-
antibody and incubate them at 4 C
ter resuspension of the cell button is
for 1 hour, mixing occasionally.
a negative test.
4. Centrifuge the mixture to pack the
3. The interpretation of ABO tests is
red cells. Remove all supernatant re-
given in Table 13-1.
agent.
4. Any discrepancy between results on
5. Transfer the red cells to a clean test
cell and serum/plasma tests should
tube.
be resolved before an interpretation
6. Wash the cells at least eight times
is recorded for the patient’s or donor’s
with large volumes (10 mL or more)
ABO group (see Chapter 13).
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
Method 2.4. Confirmation of recovery of ABO antibodies, eg, heat
Weak A or B Subgroup by or Lui freeze-thaw elution techni-
ques can be used to remove anti-
Adsorption and Elution body from the cells (see Methods
Section 4).
Principle 8. Centrifuge to pack the cells and trans-
See Chapter 13 for a discussion of the fer the supernatant eluate to a clean
principles of testing for ABO groups. test tube.

9. Test the eluate and the final wash beginning the elution, if the cells
solution (from step 6), in parallel, were not adequately washed, or if
against two examples of group O there was dissociation of bound an-
cells and two examples of cells ex- tibody during the wash process.
pressing the relevant antigen (A 1 3. A and B cells can be used in the ad-
cells for suspected anti-A, B cells for sorption/elution procedure as posi-
anti-B). Add 2 drops of eluate or tive/negative controls and tested in
wash to 1 drop of cells and examine parallel. Group O cells can also be
them for agglutination after immedi- used as a negative control.
ate centrifugation; if negative, incu-
bate 15 to 30 minutes at room tem- Note
perature. If these phases are both The eluate may be stained by hemoglobin
negative, a 15-minute incubation at and be difficult to read except at the indi-
37 C and the indirect antiglobulin rect antiglobulin phase.
test may also be performed.
Reference
Interpretation Beattie KM. Identifying the causes of weak or
“missing” antigens in ABO grouping tests. In: The
1. The presence of anti-A or anti-B in investigation of typing and compatibility prob-
the eluate, hence the presence of A lems caused by red blood cells. Washington, DC:
or B antigen on the test cells, is con- AABB, 1975:15-37.
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 Method 2.5. Saliva Testing
a b
c) the final wash solution is non- for A, B, H, Le , and Le
reactive with all four cells.
If the eluate does not react with
Principle
the A or B cells, it may indicate that Approximately 78% of all individuals pos-
the test cells do not express the anti- sess the Se gene that governs the secretion
gen and cannot adsorb the relevant of water-soluble ABH antigens into all
antibody; alternatively, it could re- body fluids with the exception of cere-
flect failure to prepare the eluate brospinal fluid. These secreted antigens
correctly. can be demonstrated in saliva by inhibi-
If the eluate reacts with one or tion tests with ABH and Lewis antisera
both of the A or B cells and also with (see Chapter 13).
one or both or all of the O cells, it in-
dicates recovery of some other or Specimen
additional antibody in the adsorp- 1. Collect 5 to 10 mL of saliva in a small
tion/elution process. beaker or wide-mouthed test tube.
2. If the final wash solution reacts with Most people can accumulate this
the A or B cells, tests on the eluate amount in several minutes. To en-
cannot be considered valid. This can courage salivation, the subject may
occur if unbound reagent antibody be asked to chew wax, paraffin, or a
was not adequately removed before clean rubber band, but not gum or

anything else that contains sugar or or nonsecretors, to use as positive

protein. and negative controls.
2. Centrifuge saliva at 900 to 1000 × g
for 8 to 10 minutes. Procedures
3. Transfer supernatant to a clean test
Selection of Blood Grouping Reagent
tube and place it in a boiling water-
Dilution
bath for 8 to 10 minutes to inactivate
salivary enzymes. 1. Prepare doubling dilutions of the ap-
4. Recentrifuge at 900 to 1000 × g for 8 propriate blood typing reagent.
to 10 minutes, remove clear or slightly 2. To 1 drop of each reagent dilution,
add 1 drop of 2% to 5% saline sus-
opalescent supernatant fluid, and
pension of red cells. Use A, B, or O
discard the opaque or semisolid ma-
cells to determine, respectively, A, B,
terial. Dilute the supernatant fluid
or H secretor status. Use Le(a+b–)
with an equal volume of saline.
red cells to determine Lewis secretor
5. Refrigerate, if testing is to be done
status.
within several hours. If testing will
3. Centrifuge each tube and examine
not be done on the day of collection,
macroscopically for agglutination.
freeze the sample and store it at –20
4. Select the highest reagent dilution
C. Frozen samples retain activity for
that gives 2+ agglutination.
several years.
Inhibition Test for Secretor Status

1. Add 1 drop of appropriately diluted
blood grouping reagent to each of
Reagents
four tubes. For ABH studies, the
1. Human (polyclonal) anti-A and anti-B. tubes should be labeled “Secretor,”
Note: Some monoclonal reagents “Nonsecretor,” “Saline,” and “Un-
may not be appropriate for use; known.” For Lewis studies, they will
therefore, appropriate controls are be “Lewis-positive,” “Lewis-nega-
essential. tive,” “Saline,” and “Unknown.”
2. Anti-H lectin from Ulex europaeus 2. Add 1 drop of the appropriate saliva
obtained commercially or prepared to the “Secretor,” “Nonsecretor,” and
by saline extraction of Ulex euro- “Unknown” tubes, and 1 drop of sa-
paeus seeds. line to the tube marked “Saline.”
3. Polyclonal (rabbit/goat/human) 3. Mix the contents of the tubes. Incu-
a
anti-Le . There are no published data bate the tubes for 8 to 10 minutes at
on the suitability of monoclonal room temperature.
Lewis antibodies. 4. Add 1 drop of 2% to 5% saline sus-
4. A1 and B red cells, as used in Method pension of washed indicator cells to
2.2. each tube, group A, B, or O for ABH
5. Group O, Le(a+b–) red cells, as used secretor status, as appropriate, or
for antibody detection or identifica- Le(a+) for Lewis testing.
tion (see Chapter 19). 5. Mix the contents of the tubes. Incu-
6. Specimens, frozen or fresh saliva, bate the tubes for 30 to 60 minutes
from persons known to be secretors at room temperature.

6. Centrifuge each tube and inspect 4. For further interpretation, see Table
each cell button macroscopically for 2.5-1.
agglutination.
Notes
Interpretation 1. Include, as controls, saliva from a
1. Agglutination of indicator cells by known secretor and nonsecretor. For
antibody in tubes containing saliva ABH status, use saliva from previ-
indicates that the saliva does not ously tested Se and sese persons. For
contain the corresponding antigen. Lewis testing, use saliva from a per-
2. The failure of known antibody to ag- son whose red cells are Le(a+b–) or
glutinate indicator cells after incu- Le(a–b+) as the positive control; use
bation with saliva indicates that the saliva from a Le(a–b–) person as the
saliva contains the corresponding negative control. Aliquots of saliva
antigen. from persons of known secretor sta-
3. The failure of antibody in the saline tus may be frozen for later use.
control tube to agglutinate indicator 2. This screening procedure can be
cells invalidates the results of saliva adapted for the semiquantitation of
tests; this usually reflects use of re- blood group activity by testing serial
agents that are too dilute. Redeter- saline dilutions of saliva. The higher
mine the appropriate reagent dilu- the dilution needed to remove inhib-
tion, as described above, and repeat itory activity, the more blood group
the testing. substance is present in the saliva.
Table 2.5-1. Interpretation of Saliva Testing
Testing with Anti-H
Se Saliva Non- Se Saliva

Unknown (H Substance (H Substance Not Saline
Saliva Present) Present) (Dilution Control) Interpretation
2+ 0 2+ 2+ Nonsecretor of H
0 0 2+ 2+ Secretor of H
a
Testing With Anti-Le
Unknown Le-negative Saline

Saliva Le-positive Saliva Saliva (Dilution Control) Interpretation
2+ 0 2+ 2+ Lewis-negative
0 0 2+ 2+ Lewis-positive*
b a
*A Lewis-positive person shown to be a secretor of ABH can be assumed to have Le as well as Le in saliva. A Le(a+)
a
person who is sese and does not secrete ABH substance will have only Le in saliva.

Saliva should be diluted before it is 2. Rh control reagent: The manufac-

incubated with antibody. To detect turer’s instructions will indicate the
or to measure salivary A or B subtype of reagent to use, if needed.
stance in addition to H substance,
the same procedure can be used Procedure
with diluted anti-A and anti-B re-
1. Place 1 drop of anti-D onto a clean,
agents. The appropriate dilution of
labeled slide.
anti-A or anti-B is obtained by titrat-
2. Place 1 drop of the appropriate con-
ing the reagent against A1 or B red
trol reagent, if needed, onto a sec-
cells, respectively.
ond labeled slide.
3. A Lewis-positive person shown to be
3. To each slide, add 2 drops of a well-
a secretor of A, B, and H can be as-
mixed 40% to 50% suspension (in auto-
sumed to have Leb as well as Lea in
logous or group-compatible serum
the saliva. A Le(a+) person who does
or plasma) of the red cells to be tested.
not secrete A, B, or H substances
4. Thoroughly mix the cell suspension
lacks the Se gene and will have only
and reagent. Using a clean applica-
Lea in the saliva.
tor stick for each test, spread (mix)
4. Specimens with a high concentra-
the reaction mixture over an area ap-
tion of soluble antigen may give a
proximately 20 mm × 40 mm.
false-negative result and require di-
5. Place both slides on the viewbox and
lution before testing.
tilt the slides gently and continu-
ously to observe them for agglutina-
tion (see note 1). Most manufactur-
Method 2.6. Slide Test for ers stipulate that the test must be
Determination of Rh Type read within 2 minutes because dry-
ing of the reaction mixture may
Principle
cause the formation of rouleaux,
See Chapter 14 for a discussion of the which may be mistaken for aggluti-
principles of Rh typing. nation.
6. Interpret and record the results of
Specimen the reactions on both slides.
Interpretation
Equipment
1. Agglutination with anti-D and a
View box. smooth suspension on the control
slide constitute a positive test result
Reagents and indicate that the cells being
1. Reagent anti-D: Suitable reagents in- tested are D+.
clude polyclonal high-protein or low- 2. No agglutination with either anti-D
protein (eg, monoclonal) reagents. or the Rh control suggests that the
Follow the instructions from the cells are D–. Testing by the anti-
manufacturer of the anti-D in use globulin procedure (see Method 2.9)
before performing slide tests; the will show weak expression of D on
method presented here is a repre- cells that are not agglutinated on
sentative procedure. slide testing.

3. If there is agglutination on the con- Procedure

trol slide, results of the anti-D test
1. Place 1 drop of anti-D in a clean, la-
must not be interpreted as positive
beled test tube.
without further testing.
2. Place 1 drop of the appropriate con-
4. Drying around the edges of the reac-
trol reagent, if needed, in a second
tion mixture must not be confused
labeled tube.
with agglutination.
3. Add to each tube 1 drop of a 2% to
5% suspension (in saline, serum or
Notes plasma) of the red cells to be tested;
1. Slide testing imposes a much greater alternatively, the equivalent amount
risk of biohazardous exposure. Per- of red cells can be transferred to each
sonnel should follow safety mea- tube with clean applicator sticks.
sures detailed in the facility’s proce- 4. Mix gently and centrifuge for the
dures manual. time and at the speed specified by
2. For slide tests using low-protein the manufacturer.
anti-D, a negative result on slide 5. Gently resuspend the cell button and
testing with either anti-A or anti-B examine it for agglutination. If a
serves as the control reaction. 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.
Method 2.7. Tube Test for 6. Grade reactions and record test and
control results.
Determination of Rh Type
Principle
See Chapter 14 for a discussion of the Interpretation
principles of Rh typing. 1. Agglutination in the anti-D tube,
combined with a smooth suspension
Specimen in the control tube, indicates that
the red cells under investigation are
D+.
2. A smooth suspension of red cells in
Reagents both the anti-D and the control
1. Reagent anti-D: Suitable reagents in- tubes is a negative test result. Speci-
clude polyclonal high-protein or low- mens from patients may be desig-
protein (eg, monoclonal) reagents. nated as D– at this point. Donor
Follow the instructions from the blood must be further tested for the
manufacturer of the anti-D in use presence of weak D antigen. The se-
before performing tube tests. The rum-and-cell mixture used in steps 1
method presented here is a repre- through 5, above, may be used to test
sentative procedure. for weak D, providing the manufac-
2. Rh control reagent: The manufacturer’s directions state that the re-
turer’s instructions will indicate the agent is suitable for the test for weak
type of control to use, if needed. D.

Notes 2. Add 1 drop of a 2% to 5% saline sus-

pension of red cells to each well.
1. Most commercially prepared antisera
3. Mix the contents of the wells by
provide a 2+ or greater agglutination
gently tapping the sides of the plate.
with D+ cells. A facility may choose
4. Centrifuge the plate at the appropri-
to do additional testing on results
ate conditions established for the
with an agglutination of less than 2+.
centrifuge.
Required testing must be defined in
5. Resuspend the cell buttons by man-
the facility’s procedures manual.
ually tapping the plate or with the
2. A negative tube test with anti-A and/
aid of a mechanical shaker, or place
or anti-B serves as a valid control
the plate at an angle for the tilt and
when a low-protein anti-D reagent
stream method.
has been used.
6. Read, interpret, and record the results.
7. Incubate negative tests at 37 C for 15
minutes.
Method 2.8. Microplate Test 8. Centrifuge the plate at the appropri-
ate conditions established for the
for Determination of Rh Type centrifuge.
9. Resuspend the cell buttons by man-
Principle
ually tapping the plate or with the
See Chapter 14 for a discussion of the aid of a mechanical shaker, or place
principles of Rh typing the plate at an angle for the tilt and
stream method.
Specimen 10. Read, interpret, and record the re-
Refer to Method 2.2. Clotted or anticoagu- sults.
lated samples may be used for Rh testing.
Follow the manufacturer’s instructions for Interpretation
specimen preparation when using semi- Agglutination with anti-D reagent after
automated microplate readers. the immediate-spin or 37 C incubation
phase indicates a positive test provided
Reagents there is no agglutination with the control
Use only anti-D approved for use in micro- reagent. See Table 14-3 for determining
plate tests (see the discussion in Method Rh phenotypes from reactions obtained
2.3). with Rh blood typing reagents.
Procedure Note
The following is a representative method; Refer to the manufacturer’s instructions
the manufacturer’s instructions should be for the necessity for weak D testing.
followed for specific reagents and equip-
ment.
1. Place 1 drop of the Rh reagent in a Method 2.9. Test for Weak D
clean well of the microplate. If the
reagent requires the use of an Rh Principle
control, add 1 drop of the control to Some red cells express the D antigen so
a second well. weakly that most anti-D reagents do not

directly agglutinate the cells. Weak D ex- 4. Mix and incubate both tubes accord-
pression can be recognized most reliably ing to the reagent manufacturer’s di-
by an indirect antiglobulin procedure af- rections. Typically, this is 15 to 30
ter incubation of the test red cells with minutes at 37 C.
anti-D. 5. If a reading is desired after the 37 C
incubation phase, centrifuge the tubes
Specimen according to the reagent manufac-
Refer to Method 2.2. turer’s directions.
6. Gently resuspend the cell buttons
Reagents and examine the tubes for agglutina-
tion. If the test red cells are strongly
1. Reagent anti-D: Suitable reagents in-
agglutinated in the anti-D tube but
clude polyclonal high-protein or low-
not in the control tube, record the test
protein (eg, monoclonal blend) re-
sample as D+ and do not proceed with
agents, but the manufacturer’s pack-
the antiglobulin phase of the test.
age insert should be consulted be-
7. If the test cells are not agglutinated
fore any anti-D reagent is used for
or the results are doubtful, wash the
this purpose.
cells three or four times with large
2. Antihuman globulin reagent, either
volumes of saline.
anti-IgG or polyspecific.
8. Add antiglobulin reagent, according
3. IgG-coated red cells.
to the manufacturer’s directions.
9. Mix gently and centrifuge according
to the calibrated spin times.
Procedure
10. Gently resuspend each cell button,
If the original, direct test with anti-D was examine the tubes for agglutination,
performed by tube testing, the same tube and grade and record the test result.
may be used for the weak D test, provid- 11. If the test result is negative, add IgG-
ing the manufacturer’s directions so state. coated red cells.
In this case, proceed directly to step 4, af-
ter recording the original anti-D tube test
Interpretation
as negative.
1. Place 1 drop of anti-D in a clean, la- 1. Either a diluent control or a direct
beled test tube. antiglobulin test (DAT) must accom-
2. Place 1 drop of the appropriate con- pany the test for weak D. Agglutina-
trol reagent in a second labeled test tion in the anti-D tube and none in
tube. the control tube constitutes a posi-
3. To each tube, add 1 drop of a 2% to tive test result. If the facility chooses
5% suspension in saline of the red to perform the test for weak D, and
cells to be tested. It is permissible to the result is clearly positive, the
use a direct antiglobulin test (DAT) blood should be classified as D+. It is
on the test cells as a control, but an incorrect to report such red cells as
u ”
indirect antiglobulin procedure with being “D–, weak D” or “D–, D .
an Rh control reagent is preferable 2. Absence of agglutination in the tube
because this ensures that all reagent with anti-D is a negative result, indi-
components that might cause a cating that the cells do not express D
false-positive result are represented. and should be classified as D–.

3. If there is agglutination at any phase polyagglutinable red cells are shown in

in the control tube, no valid inter- Table 2.10-1. If commercially made lectins
pretation of the weak D test can be are used, follow the manufacturer’s in-
made. If the specimen is from a po- structions.
tential transfusion recipient, Rh-
negative blood should be given until Reagents
the D type can be resolved. If the Seeds may be obtained from health-food
specimen is from a donor, the unit stores, pharmacies, or commercial seed
should not be used for transfusion. companies. The seeds should be raw.
Note Procedure
Some facilities may elect to do an addi- 1. Grind the seeds in a food processor
tional reading after the 37 C incubation or blender until the particles look
and before completing the antiglobulin like coarse sand. A mortar and pestle
phase of testing. Refer to the manufac- may be used, or seeds can be used
turer’s instructions. If this optional read- whole.
ing is performed, the facility’s procedures 2. In a large test tube or small beaker,
manual should indicate its policy on the place ground seeds and three to four
interpretation of this result and on the ad- times their volume of saline. (Seeds
ditional testing requirements. vary in the quantity of saline they
absorb.)
3. Incubate at room temperature for 4
Method 2.10. Preparation to 12 hours, stirring or inverting oc-
casionally.
and Use of Lectins 4. Transfer supernatant fluid to a cen-
Principle trifuge tube and centrifuge it for 5
Saline extracts of seeds react with specific minutes, to obtain clear superna-
carbohydrates on red cell membranes and
make useful typing reagents that are
highly specific at appropriate dilutions.
Diluted extract of Dolichos biflorus agglu-
tinates A 1 red cells but not A 2 . Ulex Table 2.10-1. Reactions Between Lectins
europaeus extract reacts with the H deter- and Polyagglutinable Red Cells
minant; it agglutinates in a manner pro-
portional to the amount of H present T Th Tk Tn Cad
(O>A2>B>A1>A1B red cells). Other lectins
Arachis hypogaea* + + + 0 0
useful for special purposes include
Dolichos biflorus† 0 0 0 + +
Arachis hypogaea (anti-T), Glycine max
(anti-T, -Tn), Vicia graminea (anti-N), and
Glycine max (soja) + 0 0 + +
the Salvia lectins (S. horminum, anti- Salvia sclarea 0 0 0 + 0
Tn/Cad; S. sclarea, anti-Tn). To investi- Salvia horminum 0 0 0 + +
gate red cell polyagglutination, prepare *T and Th cells give weaker reactions with Arachis after
and test the cells with Arachis, Glycine, protease treatment; Tk reactivity is enhanced after prote-
ase treatment.
Salvia, and Dolichos lectins. The antici- †
A and AB cells may react due to anti-A reactivity of Doli-
pated reactions with various types of chos lectin.

tant. Collect and filter the super- Notes

natant fluid and discard seed residue.
1. To facilitate grinding hard seeds, the
5. Test dilutions of the extract to find
seeds can be covered with saline and
dilution for the desired activity. De-
soaked for several hours before
termine the activity of the extract
grinding. The container used for
with the appropriate red cells, as be-
soaking should not be tightly closed
low.
because some beans release gas dur-
For Dolichos biflorus:
ing the soaking process, which could
a. Add 1 drop of 2% to 5% saline
cause the container to explode.
suspension of known A 1 , A 2 ,
2. The saline extracts may be stored in
A1B, A2B, B, and O red cells to
the refrigerator for several days; they
appropriately labeled tubes.
may be stored indefinitely if frozen.
b. Add 1 drop of the extract to each
3. Tests should include a positive and
tube.
negative control.
c. Centrifuge for calibrated time.
d. Inspect for agglutination and
record results.
e. The lectin should agglutinate Method 2.11. Use of
A1 and A1B cells but not A2, A2B, Sulfhydryl Reagents to
B, or O cells. The native extract
often agglutinates all the cells
Disperse Autoagglutination
tested. To make the product Principle
useful for reagent purposes, add See Chapter 20 for a discussion of auto-
enough saline to the extract so agglutination dispersion.
that there is 3+ or 4+ agglutina-
tion of A1 and A1B cells, but not Specimen
of A2, A2B, B, or O cells.
Immunoglobulin-coated red cells to be
For Ulex europaeus:
evaluated.
a. Add 1 drop of 2% to 5% saline
suspension of known A 1 , A 2 ,
Reagents
A1B, B, and O cells to appropri-
ately labeled tubes. 1. 0.01 M dithiothreitol (DTT): 0.154 g
b. Add 1 drop of extract to each tube. of DTT dissolved in 100 mL of phos-
c. Centrifuge for the calibrated phate-buffered saline (PBS) at pH
time. 7.3, or 0.1 M 2-mercaptoethanol
d. Inspect for agglutination and (2-ME), 0.7 mL of stock solution of
record results. 14 M 2-ME diluted in 100 mL of PBS
e. The strength of the agglutina- at pH 7.3.
tion should be in the order of 2. PBS at pH 7.3.
O>A2>B>A1>A1B.
f. Dilute extract with saline, if Procedure
necessary, to a point that O 1. Dilute red cells to a 50% concentra-
cells show 3+ or 4+ agglutination in PBS.
tion, A2 and B cells show 1+ to 2. Add an equal quantity of 0.01 M DTT
2+ agglutination, and A1 or A1B in PBS, or 0.1 M 2-ME in PBS, to the
cells are not agglutinated. cells.

3. Incubate at 37 C for 10 minutes (2-ME) Specimen

or 15 minutes (DTT).
Test cells with a positive direct antiglo-
4. Wash cells three times in saline and
bulin test (DAT) result.
resuspend them.
5. Dilute the treated red cells to a 2% to
5% concentration in saline for use in Procedure
blood grouping tests. Verify that red 1. Place one volume of washed anti-
cells do not spontaneously aggluti- body-coated red cells and three vol-
nate before typing or use. umes of normal saline in a test tube
of appropriate size. In another tube,
Note place the same volumes of saline
This procedure is normally used only for and washed red cells positive for the
ABO forward cell typing, Rh testing, and antigen under test. This will provide
the direct antiglobulin test. At this con- a check that the elution technique
centration of DTT, some antigens, in par- does not destroy the antigen reactiv-
ticular Jsa and Jsb, may be weakened or de- ity.
stroyed by 0.01M DTT. 2. Incubate the contents of both tubes
at approximately 45 C for 10 to 15
Reference minutes. The tubes should be agi-
tated frequently. The time of incuba-
Reid ME. Autoagglutination dispersal utilizing
sulfhydryl compounds. Transfusion 1978;18:353-5. 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
Method 2.12. Gentle Heat supernatant saline.
Elution for Testing Red Cells 4. Test the person’s cells for degree of
antibody removal by comparing a
with a Positive DAT DAT on the treated cells with the
Principle antiglobulin results on untreated red
Red cells heavily coated with IgG may cells. If the antibody coating is re-
spontaneously agglutinate in high-produced but still present, steps 1
tein reagents and will cause false-positive through 3 can be repeated; the con-
AHG test results. To perform red cell anti- trol cells should be subjected to a
gen typing, it may be necessary to dissoci- similar second treatment.
ate antibody from the cells by elution 5. Test the treated cells for the desired
without damaging membrane integrity or antigen.
altering antigen expression. The gentle
heat elution procedure employed to pre- Notes
pare immunoglobulin-free red cells dif- 1. This procedure may be unnecessary
fers from procedures intended to recover if IgM monoclonal reagents are
active antibody. available; these reagents cause di-
rect agglutination and are not usu-
Reagent ally affected by bound immunoglob-
Antihuman globulin. ulin.

2. As with untreated patient cells, re- phate solution. Similarly treat the
sults of antigen testing in recently control sample.
transfused patients should be inter- 2. Mix and incubate at room tempera-
preted with caution because of the ture for 30 minutes.
potential presence of donor cells. 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.
Method 2.13. Dissociation of 5. If this treatment has rendered the
IgG by Chloroquine for Red cells nonreactive with anti-IgG, wash
Cell Antigen Testing of Red the total volumes of treated test cells
and control cells three times in sa-
Cells with a Positive DAT line and make a 2% to 5% suspen-
Principle sion in saline to use in subsequent
Red cells with a positive direct antiglobulin blood typing tests.
test (DAT) cannot be tested accurately 6. If the treated red cells react with
with blood typing reagents that require an anti-IgG after 30 minutes of incuba-
indirect antiglobulin technique. Under tion with chloroquine diphosphate,
controlled conditions, chloroquine dip- steps 3 and 4 should be repeated at
hosphate dissociates IgG from the red cell 30-minute intervals (for a maximum
membrane with little or no damage to its incubation period of 2 hours), until
integrity. Use of this procedure permits the sample tested is nonreactive
complete phenotyping of red cells coated with anti-IgG. Then proceed as de-
with warm-reactive autoantibody, includ- scribed in step 5.
ing tests with reagents solely reactive by
indirect antiglobulin techniques. Notes
Specimen 1. Chloroquine diphosphate does not

dissociate complement proteins
Red cells with a positive DAT due to IgG from the cell membrane. If red cells
coating.
are coated with both IgG and C3,
only anti-IgG should be used in tests
Reagents performed after chloroquine treat-
1. Chloroquine diphosphate solution ment.
prepared by dissolving 20 g of chloro- 2. Incubation with chloroquine diphos-
quine diphosphate in 100 mL of sa- phate should not be extended be-
line. Adjust to pH 5.1 with 1 N NaOH, yond 2 hours. Prolonged incubation
and store at 2 to 6 C. at room temperature or incubation
2. Control red cells carrying a single-dose at 37 C may cause hemolysis and
expression of antigens for which the loss of red cell antigens.
test samples are to be phenotyped. 3. Some denaturation of Rh antigens
3. Anti-IgG antiglobulin reagent. may occur.
4. Many serologists run chloroquine-
Procedure treated control cells for each antigen
1. To 0.2 mL of washed IgG-coated cells, tested. Select control cells that are
add 0.8 mL of chloroquine diphos- positive for the antigen correspond-

ing to the antisera that will be used Specimen

to type the patient’s cells.
Red cells with a positive direct antiglobulin
5. Chloroquine diphosphate may not
test (DAT).
completely remove antibody from
sensitized red cells. DAT results on
Reagents
red cells from some persons, partic-
ularly those with a strongly positive 1. 10% EDTA prepared by dissolving 2 g
initial test, may only be diminished of disodium ethylenediamine tetra-
in strength. acetic acid (Na2EDTA) in 20 mL of
6. In addition to its use for removal of distilled or deionized water.
autoantibodies, this method can be 2. 0.1 M glycine-HCl buffer (pH 1.5)
used for removal of Bg (HLA)-related prepared by diluting 0.75 g of glycine
antigens from red cells. Appropriate to 100 mL with isotonic (unbuffered)
Bg controls should be used. saline. Adjust the pH to 1.5 using
7. If a commercial kit is used, manufac- concentrated HCl.
turer’s instructions should be followed 3. 1.0 M TRIS-NaCl prepared by dis-
for testing and controls. solving 12.1 g of tris (hydroxymethyl)
aminomethane (TRIS) and 5.25 g of
sodium chloride (NaCl) to 100 mL
References with distilled or deionized water.
1. Edwards JM, Moulds JJ, Judd WJ. Chloro-

quine diphosphate dissociation of antigen-
antibody complexes: A new technique for Procedure
phenotyping rbcs with a positive direct
1. Wash the red cells to be treated six
antiglobulin test. Transfusion 1982;22:59-61.
2. Swanson JL, Sastamoinen R. Chloroquine times with isotonic saline.
stripping of the HLA-A,B antigens from red 2. In a test tube, mix together 20 vol-
cells (letter). Transfusion 1985;25:439-40. 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
Method 2.14. Acid Glycine/ in a clean tube.
4. Add 20 volumes of acid glycine/EDTA.
EDTA Method to Remove 5. Mix the contents of the tube thor-
Antibodies from Red Cells oughly.
6. Incubate the mixture at room tem-
Principle perature for no more than 2 to 3
Acid glycine/EDTA can be used to dissoci- minutes.
ate antibody molecules from red cell 7. Add one volume of 1.0 M TRIS-NaCl
membranes. This procedure is routinely and mix the contents of the tube.
used for blood typing tests or adsorption 8. Centrifuge at 900 to 1000 × g for 1 to
procedures. All common red cell antigens 2 minutes, then aspirate, and dis-
can be detected after treatment with acid card the supernatant fluid.
glycine/EDTA except antigens of the Kell 9. Wash the red cells four times with
system, Bg antigens, and Er antigens. saline.
Thus, cells treated in this manner cannot 10. Test the washed cells with anti-IgG;
be used to determine these phenotypes. if nonreactive with anti-IgG, the cells

are ready for use in blood typing or transfused red cells and may be separated
adsorption procedures. If the DAT is from the transfused population by simple
still positive, one additional treatment centrifugation. Newly formed autologous
can be performed. cells concentrate at the top of the column
of red cells when blood is centrifuged in a
Notes microhematocrit tube, providing a simple
1. Overincubation of red cells with acid method for recovering autologous cells in
glycine/EDTA causes irreversible a blood sample from recently transfused
damage to cell membranes. patients. Note: Red cells from patients
2. Include a parallel control reagent, with hemoglobin S or spherocytic disor-
such as 6% bovine albumin or inert ders are not effectively separated by this
plasma, when typing treated red cells. method (see Method 2.16 for an alterna-
3. Use anti-IgG, not a polyspecific anti- tive procedure).
globulin reagent, in step 10.
4. Many serologists run acid glycine/
EDTA treated control cells for each Specimen
antigen tested. Select control cells that Red cells from whole blood collected into
are positive for the antigen corre- EDTA.
sponding to the antisera that will be
used to type the patient’s cells.
5. If a commercial kit is used, manufac- Materials
turer’s instructions should be fol-
1. Microhematocrit centrifuge.
lowed for testing and controls.
2. Plain (not heparinized) glass or plas-
tic hematocrit tubes.
References 3. Sealant.
1. Louie JE, Jiang AF, Zaroulis CG. Preparation of
intact antibody-free red cells in autoimmune
hemolytic anemia (abstract). Transfusion
1986;26:550. Procedure
2. Champagne K, Spruell P, Chen J, et al. EDTA/
glycine-acid vs. chloroquine diphosphate 1. Wash the red cells three times in sa-
treatment for stripping Bg antigens from red line. For the last wash, centrifuge
blood cells (abstract). Transfusion 1996;36 them at 900 to 1000 g for 5 to 15
(Suppl):21S.
3. Reid ME, Lomas-Francis C. The blood group
minutes. Remove as much of the
antigen factsbook. New York: Academic Press, supernatant fluid as possible with-
2004. out disturbing the buffy coat. Mix
thoroughly.
2. Fill 10 microhematocrit tubes to the
Method 2.15. Separation of 60-mm mark with well-mixed washed
red cells.
Transfused from Autologous 3. Seal the ends of the tubes by heat or
Red Cells by Simple with sealant.
4. Centrifuge all tubes in a microhema-
Centrifugation tocrit centrifuge for 15 minutes.
Principle 5. Cut the microhematocrit tubes 5
Newly formed autologous red cells gener- mm below the top of the column of
ally have a lower specific gravity than red cells. This 5-mm segment con-

tains the least dense, hence youn-

gest, circulating red cells.
Method 2.16. Separation of
6. Place the cut microhematocrit tubes Transfused Red Cells from
into larger test tubes (10 or 12 × 75 Autologous Red Cells in
mm), add saline, and mix well to
flush the red cells from the micro- Patients with Hemoglobin S
hematocrit tubes. Then, either a) Disease
centrifuge them at 1000 × g for 1
minute and remove the empty hema- Principle
tocrit tubes or b) transfer the saline-
suspended red cells to a clean test Red cells from patients with sickle cell
tube. disease, either hemoglobin SS or SC, are
7. Wash the separated red cells three resistant to lysis by hypotonic saline, in
times in saline before resuspending contrast to red cells from normal persons
them to 2% to 5% in saline for test- and those with hemoglobin S trait. This
ing. procedure permits isolation of autologous
red cells from patients with hemoglobin
SS or SC disease who have recently been
Notes transfused.
1. Separation is better if 3 or more days

Specimen
have elapsed since transfusion than
if the sample has been obtained Red cells to be evaluated.
shortly after transfusion.
2. The red cells should be mixed con- Reagents
tinuously while the microhematocrit
1. Hypotonic saline (0.3% w/v NaCl):
tubes are being filled.
NaCl, 3 g; distilled water to 1 L.
3. Separation techniques are only ef-
2. Normal saline (0.9% w/v NaCl):
fective if the patient is producing
NaCl, 9 g; distilled water to 1 L.
normal or above-normal numbers of
reticulocytes. This method will be
ineffective in patients with inade- Procedure
quate reticulocyte production. 1. Place 4 or 5 drops of red cells into a
4. Some red cell antigens may not be as 10 or 12 × 75-mm test tube.
strongly expressed on reticulocytes 2. Wash the cells six times with 0.3%
as on older cells. Particular attention NaCl, or until the supernatant fluid
should be given to determinations of no longer contains grossly visible
the E, e, c, Fya, Jka, and Ge antigens. hemoglobin. For each wash, centri-
fuge at 1000 × g for 1 minute.
References 3. Wash the cells twice with 0.9% NaCl
1. Reid ME, Toy P. Simplified method for recov- to restore tonicity. For each wash,
ery of autologous red blood cells from trans- centrifuge at 200 × g for 2 minutes to
fused patients. Am J Clin Pathol 1983;79:364-6. facilitate removal of residual stroma.
2. Vengelen-Tyler V, Gonzales B. Reticulocyte 4. Resuspend the remaining intact red
rich RBCs will give weak reactions with many
blood typing antisera (abstract). Transfusion cells to a 2% to 5% concentration for
1985;25:476. phenotyping.

Note Reference
Larger volumes, for use in adsorption stud- Brown D. A rapid method for harvesting autolo-
ies, can be processed in a 16 × 100-mm gous red cells from patients with hemoglobin S
disease. Transfusion 1988;28:21-3.
test tube.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing
Methods Section 3
Antibody Detection,
Antibody Identification, and
Section 3
Serologic Compatibility
Testing
the donor red cells in EDTA saline be-

Method 3.1. Immediate-Spin cause high-titered anti-A or -B can
Compatibility Testing to initiate complement coating, which can
Demonstrate ABO cause steric hindrance of agglutina-
tion.2 The use of a patient’s sample
Incompatibility collected in EDTA is an alternative
Principle approach to prevent this phenome-
See Chapter 18 for a discussion of the prin- non.
ciples of compatibility testing.
Specimen Procedure
Patient’s serum or plasma may be used. 1. Label a tube for each donor red cell
The age of the specimen must comply with suspension being tested with the pa-
the pretransfusion specimen requirements tient’s serum.
in AABB Standards for Blood Banks and 2. Add 2 drops of the patient’s serum or
Transfusion Services.1(p38) plasma to each tube.
3. Add 1 drop of the suspension of do-
Reagents nor red cells to the appropriate test
1. Normal saline. tube.
2. Donor red cells, 2% to 5% suspen- 4. Mix the contents of the tube(s) and
sion in normal saline or EDTA saline. centrifuge according to the calibra-
Some serologists prefer to suspend tion of the centrifuge.
751

5. Examine the tube(s) for hemolysis, 2. Bovine albumin (22% or 30%).

gently resuspend the red cell button(s), 3. LISS made as follows:
and examine for agglutination. a. Add 1.75 g of NaCl and 18 g of
6. Read, interpret, and record test results. glycine to a 1-liter volumetric flask.
b. Add 20 mL of phosphate buffer
Interpretation prepared by combining 11.3 mL
1. Agglutination or hemolysis constitutes of 0.15 M KH2PO4 and 8.7 mL of
a positive (incompatible) test result. 0.15 M Na2HPO4.
2. A smooth suspension of red cells af- c. Add distilled water to the 1-liter
ter resuspension of the red cell but- mark.
ton constitutes a negative result and d. Adjust the pH to 6.7 ± 0.1 with
indicates a compatible immediate- NaOH.
spin crossmatch. e. Add 0.5 g of sodium azide as a
preservative.
References Note: LISS may be used as an addi-
tive (Method 3.2.2) or for the suspen-
transfusion services. 23rd ed. Bethesda, MD: sion of test red cells (Method 3.2.3).
AABB, 2005. LISS preparations are also available
2. Judd WJ, Steiner EA, O’Donnell DB, Oberman commercially.
HA. Discrepancies in ABO typing due to
prozone; how safe is the immediate-spin
4. PEG, 20% w/v: To 20 g of 3350 MW
crossmatch? Transfusion 1988;28:334-8. 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.
Method 3.2. Indirect Polyspecific or anti-IgG may be used
Antiglobulin Test (IAT) for unless otherwise indicated.
the Detection of Antibodies 6. Commercially available group O an-
tibody detection cells. Pooled group
to Red Cell Antigens O antibody detection cells may be
Principle used only for donor testing. Testing
of patients’ samples must be per-
For a discussion of the principles of sa-
formed with unpooled cells.
line, albumin, low-ionic-strength saline
(LISS), and polyethylene glycol (PEG) in-
direct antiglobulin testing, see Chapters 12,
18, and 19. Method 3.2.1. Saline Indirect Antiglobulin
Test
Specimen Procedure
Serum or plasma may be used. The age of 1. Add 2 drops of serum or plasma to
the specimen must comply with pretrans- properly labeled tubes.
fusion specimen requirements in AABB 2. Add 1 drop of 2% to 5% saline-sus-
Standards for Blood Banks and Transfusion pended reagent group O cells or do-
Services. nor red cells to each tube and mix.
3. Centrifuge and observe for hemoly-
Reagents sis and agglutination. Grade and re-
1. Normal saline. cord the results.

Methods Section 3: Antibody Detection/Identification and Compatibility Testing 753
4. Incubate at 37 C for 30 to 60 minutes. 3. Add 2 drops of serum to a properly

5. Centrifuge and observe for hemoly- labeled tube.
sis and agglutination. Grade and re- 4. Add 2 drops of LISS-suspended red
cord the results. cells, mix, and incubate at 37 C 10 to
6. Wash the cells three or four times with 15 minutes or follow the manufac-
saline and completely decant the fi- turer’s directions.
nal wash. 5. Centrifuge and observe for hemoly-
7. Add AHG to the dry cell button ac- sis and agglutination by gently re-
cording to the manufacturer’s direc- suspending the cell button. Grade and
tions. Mix well. record results.
8. Centrifuge and observe for aggluti- 6. Perform the test described in Method
nation. Grade and record the results. 3.2.1, steps 6 through 9.
9. Confirm the validity of negative tests
by adding IgG-coated red cells. Method 3.2.4. PEG Indirect Antiglobulin
Test
Method 3.2.2. Albumin or LISS-Additive Procedure
Indirect Antiglobulin Test
1. For each cell sample to be tested, mix
Procedure 2 drops of test serum, 4 drops of 20%
1. Add 2 drops of serum or plasma to PEG in PBS, and 1 drop of a 2% to
properly labeled tubes. 5% suspension of red cells.
2. Add an equivalent volume of 22% or 2. Incubate at 37 C for 15 minutes.
30% bovine albumin or LISS additive 3. DO NOT CENTRIFUGE.
(unless the manufacturer’s directions 4. Wash the cells four times with saline
state otherwise.) and completely decant the final wash.
3. Add 1 drop of a 2% to 5% saline-sus- 5. Perform the AHG test, using anti-IgG,
pended reagent or donor red cells to described in Method 3.2.1, steps 7
each tube and mix. through 9.
4. For albumin, incubate at 37 C for 15 Note: The manufacturer’s instructions
to 30 minutes. For LISS, incubate for should be followed for the proper use
10 to 15 minutes or follow the manu- of commercial PEG solutions.
facturer’s directions.
5. Centrifuge and observe for hemoly- Interpretation (for Antiglobulin Tests,
sis and agglutination. Grade and re- Methods 3.2.1 through 3.2.4)
cord the results. 1. The presence of agglutination/hem-
6. Perform the test described in Method olysis after incubation at 37 C con-
3.2.1, steps 6 through 9. stitutes a positive test.
2. The presence of agglutination after
Method 3.2.3. LISS Indirect Antiglobulin Test addition of AHG constitutes a posi-
tive test.
Procedure 3. Antiglobulin tests are negative when
1. Wash reagent or donor red cells three no agglutination is observed after
times in normal saline and comple- initial centrifugation and the IgG-
tely decant saline. coated red cells added afterward are
2. Resuspend the cells to a 2% to 3% agglutinated. If the IgG-coated red
suspension in LISS. cells are not agglutinated, the nega-

tive result is invalid and the test must

be repeated.
Method 3.3. Prewarming
Technique
Controls
The procedure used for the detection of
Principle
unexpected antibodies in pretransfusion Prewarming may be useful in the detec-
testing should be checked daily with weak tion and identification of red cell antibod-
examples of antibody. Control sera can be ies that bind to antigen only at 37 C. This
prepared from reagent grade typing sera test is particularly useful for testing sera
diluted with 6% bovine albumin to give 2+ of patients with cold-reactive autoantibody
reactions by an IAT. Human sources of IgG activity that may mask the presence of
antibodies are also acceptable. clinically significant antibodies. However,
use of the prewarming technique for this
Notes application has become controversial.1-2 It
1. The incubation times and the volume has been shown to result in decreased
and concentration of red cells indi- reactivity of some potentially significant
cated are those given in the literature. antibodies and weak antibodies can be
Individual laboratories may choose to missed.3 The technique should be used
standardize techniques with somewhat with caution and not used to eliminate
different values. See Chapter 12 for unidentified reactivity.
other limitations when modifying Strong cold-reactive autoantibodies may
procedures. In all cases, the manu- react in prewarmed tests; other techniques
facturer’s package insert should be such as cold allo- or autoadsorption or
consulted before modifying a proce- dithiothreitol treatment of plasma may be
dure. required to detect underlying clinically sig-
2. For the saline procedure, step 3 may nificant antibodies.
be omitted to avoid the detection of
antibodies reactive at room temper- Specimen
ature. Serum or plasma may be used. The age of
3. For the PEG procedure: the specimen must comply with pretrans-
a. Omit centrifugation after 37 C fusion specimen requirements in AABB
incubation because red cells will Standards for Blood Banks and Transfusion
not resuspend readily. Services.4(p38)
b. Use anti-IgG rather than poly-
specific AHG to avoid unwanted
positive reactions due to C3-
Reagents
binding autoantibodies. 1. Normal saline.
4. Steps 6 through 9 of the IAT (Method 2. Anti-IgG.
3.2.1) must be performed without in- 3. Commercially available group O an-
terruption. tibody detection cells. Pooled group
O antibody detection cells may be used
Reference only for donor testing. Testing of pa-
tients’ samples must be done with
Silva MA, ed. Standards for blood banks and trans-
fusion services. 23rd ed. Bethesda, MD: AABB, 2005: unpooled cells.
38. 4. IgG-coated red cells.

Procedure saline instead of 37 C saline is used

in the wash step.2 The use of room-
1. Prewarm a bottle of saline to 37 C.
temperature saline may avoid the
2. Label one tube for each reagent or
elution of clinically significant anti-
donor sample to be tested.
body(ies) from reagent red cells that
3. Add 1 drop of 2% to 5% saline-sus-
can occur with the use of 37 C saline.
pended red cells to each tube.
Some strong cold-reactive autoanti-
4. Place the tubes containing red cells
bodies, however, may still react and
and a tube containing a small volume
therefore require the use of 37 C sa-
of the patient’s serum and a pipette
line to avoid their detection.
at 37 C; incubate for 5 to 10 minutes.
5. Using the prewarmed pipette, trans-
References
fer 2 drops of prewarmed serum to
each tube containing prewarmed red 1. Judd WJ. Controversies in transfusion medi-
cine. Prewarmed tests: Con. Transfusion 1995;
cells. Mix without removing tubes 35:271-7.
from the incubator. 2. Mallory D. Controversies in transfusion med-
6. Incubate at 37 C for 30 to 60 min- icine. Prewarmed tests: Pro—why, when, and
how—not if. Transfusion 1995;35:268-70.
utes. 3. Leger RM, Garratty G. Weakening or loss of
7. Without removing the tubes from the antibody reactivity after prewarm technique.
incubator, fill each tube with pre- Transfusion 2003;43:1611-14.
warmed (37 C) saline. Centrifuge 4. Silva MA, ed. Standards for blood banks and
and wash three or four times with 37 AABB, 2005.
C saline.
8. Add anti-IgG, according to the man-
ufacturer’s directions.
9. Centrifuge and observe for reaction. Method 3.4. Saline
Grade and record the results. Replacement to Demonstrate
by adding IgG-coated red cells.
Alloantibody in the Presence
of Rouleaux
Notes Principle
1. The prewarming procedure described Rouleaux are aggregates of red cells that,
above will not detect alloantibodies characteristically, adhere to one another
that agglutinate at 37 C or lower and on their flat surface, giving a “stack of coins”
are not reactive in the antiglobulin appearance when viewed microscopically.
phase. If detection of these antibod- Rouleaux formation is an in-vitro phe-
ies is desired, testing and centrifuga- nomenon resulting from abnormalities of
tion at 37 C are required. If time per- serum protein concentrations. The pa-
mits, a tube containing a prewarmed tient is often found to have liver disease,
mixture of serum and cells can be multiple myeloma, or another condition
incubated at 37 C for 60 to 120 min- associated with abnormal globulin levels.
utes, and the settled red cells exam- It may be difficult to detect antibody-as-
ined for agglutination by resuspend- sociated agglutination in a test system
ing the button without centrifugation. containing rouleaux-promoting serum. In
2. Cold-reactive antibodies may not be the saline replacement technique, serum
detectable when room-temperature and cells are incubated to allow antibody

attachment, but the serum is removed Reagents

and saline is added as the resuspending
1. Dry enzyme powder, 1 g.
medium.
2. Phosphate-buffered saline (PBS), pH
7.3: see Method 1.7.
Reagents 3. Phosphate buffer, pH 5.4.
Saline.
Procedure
Procedure 1. Place 1 g of powdered ficin in a 100-
After routine incubation and resuspension, mL volumetric flask. Handle the
proceed with the following steps if the ap- ficin carefully; it is harmful if it gets
pearance of the resuspended cells suggests in the eyes or is inhaled. It is desir-
rouleaux formation: able to wear gloves, mask, and apron,
1. Recentrifuge the serum/cell mixture. or to work under a hood.
2. Remove the serum, leaving the cell 2. Add PBS, pH 7.3 to 100 mL, to dis-
button undisturbed. solve the ficin. Agitate vigorously by
3. Replace the serum with an equal inversion, rotate for 15 minutes, or
volume of saline (2 drops). mix with a magnetic stirrer until
4. Resuspend the cell button gently and mostly dissolved. The powder will not
observe for agglutination. Rouleaux dissolve completely.
will disperse when suspended in saline, 3. Collect clear fluid, either by filtration
whereas true agglutination will remain. or centrifugation, and prepare small
aliquots. Store the aliquots at –20 C
Reference or colder. Do not refreeze a thawed
solution.
Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham, NC: Montgomery Scientific Pub-
lications, 1998:1135.
Method 3.5.2. Preparation of Papain
Enzyme Stock, 1% w/v
Principle
Method 3.5. Enzyme The enzyme preparations used in blood
Techniques banking differ from lot to lot; each time a
stock enzyme solution is prepared, its re-
For a discussion of the principles of en-
activity should be tested and incubation
zyme testing, see Chapter 19.
periods standardized for optimal effective-
ness. See Method 3.5.3.
Method 3.5.1. Preparation of Ficin
Enzyme Stock, 1% w/v
Principle Reagents
The enzyme preparations used in blood 1. L-cysteine hydrochloride 0.5 M, 0.88
banking differ from lot to lot; each time a g in 10 mL distilled water.
stock enzyme solution is prepared, its re- 2. Dry enzyme powder, 2 g.
activity should be tested and incubation 3. Phosphate buffer 0.067 M at pH 5.4,
periods standardized for optimal effective- prepared by combining 3.5 mL of
ness. See Method 3.5.3. Na2HPO4 and 96.5 mL of KH2PO4.

Procedure Procedure
1. Add 2 g of powdered papain to 100 1. Prepare 0.1% ficin by diluting one
mL of phosphate buffer (pH 5.4). volume of stock ficin solution with
Handle papain carefully; it is harm- nine volumes of PBS, pH 7.3.
ful to mucous membranes. Use ap- 2. Label three tubes: 5 minutes, 10
propriate protective equipment. minutes, and 15 minutes.
2. Agitate enzyme solution for 15 min- 3. Add equal volumes of washed red cells
utes at room temperature. and 0.1% ficin to each tube.
3. Collect clear fluid by filtration or 4. Mix and incubate at 37 C for the time
centrifugation. designated. Incubation times are
4. Add L-cysteine hydrochloride and easily controlled if the 15-minute
incubate solution at 37 C for 1 hour. tube is prepared first, followed by
5. Add phosphate buffer (pH 5.4) to fi- the 10- and 5 -minute tubes at
nal volume of 200 mL. Store aliquots 5-minute intervals. Incubation will
at –20 C or colder. Do not refreeze be complete for all three tubes at the
aliquots. same time.
5. Immediately wash the red cells three
times with large volumes of saline.
6. Resuspend treated cells to 2% to 5%
Method 3.5.3. Standardization of Enzyme in saline.
Procedures 7. Label four tubes for each serum to
be tested: untreated, 5 minutes, 10
Principle minutes, 15 minutes.
For a two-stage enzyme procedure, the 8. Add 2 drops of the appropriate se-
optimal treatment time must be deter- rum to each of the four tubes.
mined for each new lot of stock solution. 9. Add 1 drop of the appropriate red cell
The technique given below for ficin can suspension to each of the labeled
be modified for use with other enzymes. tubes.
10. Mix and incubate at 37 C for 15 min-
utes.
Reagents 11. Centrifuge and examine for aggluti-
nation by gently resuspending the red
1. 1% stock solution of ficin in PBS, pH
cell button.
7.3.
12. Proceed with the AHG test described
2. Several sera known to lack unex-
in Method 3.2.1, steps 6 through 9.
pected antibodies.
3. Anti-D that agglutinates only en-
zyme-treated D+ red cells and does
not agglutinate untreated D+ cells. Interpretation
4. Anti-Fya of moderate or strong reac- Table 3.5.3-1 shows possible results with
tivity. D+, Fy(a+b–) cells and the sera indicated.
5. D+ and Fy(a+b–) red cell samples. In this case, the optimal incubation time
6. Antihuman globulin (AHG) reagent. would be 10 minutes. Incubation for only
Polyspecific or anti-IgG may be used 5 minutes does not completely abolish Fya
unless otherwise indicated. activity or maximally enhance anti-D re-
7. IgG-coated red cells. activity. Incubation for 15 minutes causes

Table 3.5.3-1. Hypothetical Results with D+, Fy(a+b–) Red Cells

a
Cells and Enzyme Inert Serum Anti-D Anti-Fy
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+
antihuman globulin test 0 2+ 0
antihuman globulin test w+ 2+ w+
false-positive antiglobulin reactivity with Reagents

inert serum.
1. Sera known to contain antibody that
If incubation for 5 minutes overtreats the
will agglutinate enzyme-treated cells.
cells, it is preferable to use a more dilute
2. Sera free of any unexpected antibod-
working solution of enzyme than to reduce
ies.
incubation time because it is difficult to ac-
curately monitor very short incubation
Polyspecific or anti-IgG may be used
times. Additional tests can evaluate a single
unless otherwise indicated.
dilution at different incubation times, or a
single incubation time can be used for
different enzyme dilutions.
Procedure
Method 3.5.4. Evaluating Enzyme-Treated 1. Select an antibody that agglutinates
Red Cells enzyme-treated red cells positive for
the antigen but gives only AHG reac-
Principle
tions with unmodified cells. Many
After optimal incubation conditions have examples of human source anti-D
been determined for a lot of enzyme solu- behave in this way.
tion, treated red cells should be evaluated 2. Add 2 drops of the selected antibody-
before use to demonstrate that they are containing serum to a tube labeled
adequately, but not excessively, modified. “positive.”
Satisfactory treatment produces cells that 3. Add 2 drops of a serum free of unex-
are agglutinated by an antibody that causes pected antibodies to a tube labeled
only indirect antiglobulin test reactivity of “negative.”
unmodified cells but are not agglutinated 4. Add 1 drop of 2% to 5% suspension
or aggregated by inert serum. of enzyme-treated red cells to each
tube.
5. Mix and incubate 15 minutes at 37 C.
Specimen 6. Centrifuge and resuspend the cells by
Enzyme-treated red cells. gentle shaking.

7. Examine macroscopically for the pre- Method 3.5.6. Two-Stage Enzyme

sence of agglutination. Technique
8. Perform the AHG test described in
Method 3.2.1, steps 6 through 9, on
Specimen
the tube labeled “negative.” Serum or plasma to be tested.
Reagent
Interpretation 1. Reagent red cells.
There should be agglutination in the “pos-
Polyspecific or anti-IgG may be used
itive” tube and no agglutination in the
unless otherwise indicated.
“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 Procedure
been inadequate. 1. Prepare a diluted enzyme solution
(papain or ficin) by adding 9 mL of
PBS, pH 7.3, to 1 mL of stock enzyme.
Method 3.5.5. One-Stage Enzyme 2. Add one volume of diluted enzyme
Technique to one volume of packed, washed re-
agent red cells.
Specimen 3. Incubate at 37 C for the time deter-
Serum or plasma to be tested. mined to be optimal for that enzyme
solution.
4. Wash treated cells at least three times
with large volumes of saline and re-
Reagent
suspend the cells to a 2% to 5% con-
1. Reagent red cells. centration in saline.
2. Antihuman globulin (AHG) reagent. 5. Add 2 drops of serum or plasma to
Polyspecific or anti-IgG may be used be tested to an appropriately labeled
unless otherwise indicated. tube.
3. IgG-coated red cells. 6. Add 1 drop of 2% to 5% suspension
of enzyme-treated cells.
Procedure 7. Mix and incubate for 15 minutes at
37 C.
1. Add 2 drops of serum to an appro-
8. Centrifuge; gently resuspend the cells
priately labeled tube.
and observe for agglutination. Grade
2. Add 2 drops of a 2% to 5% saline sus-
and record the results.
pension of reagent red cells.
9. Proceed with the AHG test described
3. Add 2 drops of 0.1% papain solution
in Method 3.2.1, steps 6 through 9.
and mix well.
4. Incubate at 37 C for 15 minutes.
5. Centrifuge; gently resuspend the cells Notes
and observe for agglutination. Grade 1. An alternative method for steps 4 and
and record the results. 5 (Method 3.5.5) or steps 7 and 8
6. Proceed with the AHG test described (Method 3.5.6) is to incubate the se-
in Method 3.2.1, steps 6 through 9. rum and enzyme-treated cells at 37

C for 60 minutes and examine the Procedure

settled cells for agglutination with-
1. Dispense 1 drop of a 2% to 5% sus-
out centrifugation. This can be use-
pension of red cells into each tube.
ful for serum with strong cold-reac-
2. Wash each tube three or four times
tive agglutinins and can sometimes
with saline. Completely decant the fi-
prevent the occurrence of false-posi-
nal wash.
tive results.
3. Immediately add antisera and mix. For
2. Microscopic examination is not rec-
the amount of antisera required, re-
ommended for routine use and is
fer to the manufacturer’s directions.
particularly inappropriate with en-
4. Centrifuge according to the manu-
zyme enhanced tests; false-positive
facturer’s directions.
reactions will often be detected.
5. Examine the cells for agglutination.
3. Either papain or ficin may be used in
Grade and record the reaction.
a two-stage procedure.
6. If using polyspecific AHG or anti-
4. Enzyme preparations are available
C3d, incubate nonreactive tests at
commercially. The manufacturer’s
room temperature for 5 minutes, then
directions should be followed for ap-
centrifuge, and read again.
propriate use and quality control.
by adding IgG-coated red cells to tests
References
containing anti-IgG.
8. Centrifuge according to the manu-
rology, 4th ed. Durham, NC: Montgomery
Scientific, 1998. facturer’s directions.
2. Judd WJ. Methods in immunohematology. 2nd 9. Examine the cells for agglutination and
ed. Durham, NC: Montgomery Scientific, 1994. record the reaction.
Interpretation
Method 3.6. Direct 1. The DAT is positive when agglutina-
Antiglobulin Test (DAT) tion is observed either after im-
Principle mediate centrifugation or after the
centrifugation that followed room-
See Chapter 20 for a discussion of the
temperature incubation. IgG-coated
principles of direct antiglobulin testing.
red cells usually give immediate re-
actions, whereas complement coat-
Specimen
ing may be more easily demonstra-
Red cells from an anticoagulated blood 1,2
ble after incubation. Monospecific
sample. AHG reagents are needed to confirm
which globulins are present.
Reagents 2. The DAT is negative when no agglu-
1. Antihuman globulin (AHG) reagent: tination is observed at either test phase
polyspecific antiglobulin reagent, and the IgG-coated cells added in step
anti-IgG, anti-complement antisera. 7 are agglutinated. If the IgG-coated
2. A control reagent (eg, PBS) is re- cells are not agglutinated, the nega-
quired when all antisera tested give tive DAT result is considered invalid
a positive result. and the test must be repeated. A
3. IgG-coated red cells. negative DAT does not necessarily

mean that the red cells have no at- in a serum sample or to compare the
tached globulin molecules. Poly- strength of antigen expression on differ-
specific and anti-IgG reagents detect ent red cell samples. The usual applica-
as few as 200 to 500 molecules of IgG tions of titration studies are: 1) estimating
per cell,1 but patients may experi- antibody activity in alloimmunized preg-
ence autoimmune hemolytic ane- nant women to determine whether and
mia when IgG coating is below this when to perform more complex invasive
2
level. investigation of the fetal condition (see
3. No interpretation can be made if the Chapter 23); 2) elucidating autoantibody
results with all antisera used to per- specificity (see Chapter 20); 3) character-
form a DAT and the control are reac- izing antibodies as high-titer, low-avidity,
tive. This indicates spontaneous ag- traits common in antibodies to antigens
glutination, which must be resolved of the Knops and Chido/ Rodgers sys-
a
before further testing is performed. tems, Cs , and JMH (see Chapter 15); and
4) observing the effect of sulfhydryl re-
Notes agents on antibody behavior, to deter-
1. Steps 2 through 7 must be performed mine immunoglobulin class (IgG or IgM).
without interruption. See Method 5.3 for titration studies spe-
2. Initial testing may be performed with cifically to assist in monitoring clinically
polyspecific reagent only. If the DAT significant antibodies in the pregnant
is negative with polyspecific reagent, woman.
no further testing is necessary. If the
DAT is positive with polyspecific re-
agent, perform the DAT test with Specimen
monospecific reagents, anti-IgG, and Serum or plasma antibody to be titrated.
anticomplement, to determine which
globulins are present. Reagents
3. Verification of negative results with
1. Red cells that express the antigen(s)
anti-C3d is recommended. Refer to
corresponding to the antibody spec-
the manufacturer’s instructions to
ificity (ies), in a 2% to 5% saline sus-
determine appropriate controls.
pension. Uniformity of cell suspen-
sions is very important to ensure
References comparability of results.
1. Mollison PL, Engelfriet CP, Contreras M, eds. 2. Saline. (Note: Dilutions may be made
Blood transfusion in clinical medicine. 10th
ed. Oxford, England: Blackwell Scientific Pub- with albumin if desired.)
lications, 1997.
2. Petz LD, Garratty G. Immune hemolytic ane-
mia. Philadelphia: Churchill-Livingstone, 2004.
Procedure
The master dilution technique for titration
studies is as follows:
1. Label 10 test tubes according to the
Method 3.7. Antibody Titration serum dilution (eg, 1 in 1, 1 in 2, etc).
Principle A 1 in 1 dilution means one volume
Titration is a semiquantitative method used of serum undiluted; a 1 in 2 dilution
to determine the concentration of antibody means one volume of serum in a fi-

nal volume of two, or a 50% solution Interpretation

of serum in the diluent. See Methods
1.4 and 1.5. 1. Observe the highest dilution that
2. Deliver one volume of saline to all test produces 1+ macroscopic agglutina-
tubes except the first (undiluted 1 in tion. The titer is reported as the re-
1) tube. ciprocal of the dilution level, eg,
3. Add an equal volume of serum to 32—not 1 in 32 or 1:32 (see Table
each of the first two tubes (undiluted
3.7-1). If there is agglutination in the
and 1 in 2).
tube containing the most dilute se-
4. Using a clean pipette, mix the con-
rum, the endpoint has not been
tents of the 1 in 2 dilution several
reached, and additional dilutions
times and transfer one volume into
the next tube (the 1 in 4 dilution). should be prepared and tested.
5. Continue the same process for all di- 2. In comparative studies, a significant
lutions, using a clean pipette to mix difference in titer is three or more di-
and transfer each dilution. Remove lutions. Variations in technique and
one volume of diluted serum from inherent biologic variability can cause
the final tube and save it for use if duplicate tests to give results that
further dilutions are required. differ by one dilution in either direc-
6. Label 10 tubes for the appropriate tion. Serum containing antibody at a
dilutions. true titer of 32 may show, on repli-
7. Using separate pipettes for each dicate tests, the endpoint in the 1:32
lution, transfer 2 drops of each di- tube, the 1:64 tube, or the 1:16 tube.
luted serum into the appropriately 3. Titer values alone can be misleading
labeled tubes and add 2 drops of a without also evaluating the strength
2% red cell suspension. Alterna- of agglutination. The observed strength
tively, for convenience, add 1 drop of of agglutination can be assigned a
a 3%-4% suspension of red cells as
number and the sum of these num-
supplied by the reagent manufac-
bers for all tubes in a titration study
turer, although this method is less
represents the score, another semi-
precise.
quantitative measurement of antibody
8. Mix well and test by a serologic tech-
reactivity. The arbitrarily assigned
nique appropriate to the antibody
(see Chapter 19). threshold for significance in com-
9. Examine test results macroscopically; paring scores is a difference of 10 or
grade and record the reactions. The more between different test samples.
prozone phenomenon (see Chapter See Table 3.7-1.
12) may cause reactions to be weaker 4. Antibodies with high-titer, low-avid-
in the more concentrated serum pre- ity characteristics generally have a ti-
parations than in higher dilutions. ter greater than 64, with most tubes
To avoid misinterpretation of results, showing consistently weak reactivity.
it may be preferable to examine first Table 3.7-1 shows the results obtained
the tube containing the most dilute with three sera, each of which shows no
serum and proceed through the more more agglutination after 1:256. The differ-
concentrated samples to the undiluted ences in score, however, indicate consider-
specimen. able variation in strength of reactivity.

Table 3.7-1. Examples of Antibody Titers, Endpoints, and Scores
Reciprocal of Serum Dilution
1 2 4 8 16 32 64 128 256 512 Titer* Score
Strength: 3+ 3+ 3+ 2+ 2+ 2+ 1+ ± ± 0 64(256)
Sample 1
Score: 10 10 10 8 8 8 5 3 2 0 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: 5 5 5 5 3 3 3 2 2 0 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.
Notes from donors of the same phenotype.

Titration is a semiquantitative technique. When a single serum is to be tested
Technical variables greatly affect the re- against different red cell samples, all
sults and care should be taken to achieve samples should be collected and pre-
the most uniform possible practices. served in the same manner and di-
1. Careful pipetting is essential. Pi- luted to the same concentration be-
pettes with disposable tips that can fore use.
be changed after each dilution are 4. Completely reproducible results are
recommended. virtually impossible to achieve. Com-
2. Optimal time and temperature of in- parisons are valid only when speci-
cubation and time and force of centri- mens are tested concurrently. In tests
fugation must be used consistently. with a single serum against different
3. The age, phenotype, and concentra- red cell samples, material from the
tion of the test red cells will influence master dilution must be used for all
the results. When the titers of several the tests.
antibody-containing sera are to be 5. Measurements are more accurate with
compared, all of them should be large volumes than with small vol-
tested against red cells (preferably umes; a master dilution technique
freshly collected) from the same do- (see above) gives more reliable re-
nor. If this is not possible, the tests sults than individual dilutions for a
should use a pool of reagent red cells single set of tests. The volume needed
for all planned tests should be calcu-

lated and an adequate quantity of 2. 0.01 M dithiothreitol (DTT), prepared

each dilution prepared. by dissolving 0.154 g of DTT in 100
6. When performing a titration for mL of pH 7.3 PBS. Store at –18 C or
anti-D for HDFN, see Method 5.3. lower.
Procedure
Method 3.8. Use of Sulfhydryl 1. Dispense 1 mL of serum or plasma
into each of two test tubes.
Reagents to Distinguish IgM 2. To one tube, labeled dilution control,
from IgG Antibodies add 1 mL of pH 7.3 PBS.
3. To the other tube, labeled test, add 1
Principle mL of 0.01 M DTT.
Treating IgM antibodies with sulfhydryl 4. Mix and incubate at 37 C for 30 to 60
reagents abolishes both agglutinating and minutes.
complement-binding activities. Observa- 5. Use the DTT-treated and dilution con-
tions of antibody activity before and after trol samples in standard procedures.
sulfhydryl treatment are useful in deter-
mining immunoglobulin class. Sulfhydryl
treatment can also be used to abolish IgM Interpretation
antibody activity to permit detection of co- 1. Reactivity in the dilution control
existing IgG antibodies. For a discussion of serum and no reactivity in the DTT-
IgM and IgG structures, see Chapter 11. treated serum indicates an IgM anti-
body.
Specimen 2. Reactivity in the dilution control se-
2 mL of serum or plasma to be treated. rum and the DTT-treated serum in-
dicates an IgG antibody or an IgG
Reagents and IgM mixture. Titration studies
1. Phosphate-buffered saline (PBS) at may be necessary to distinguish be-
pH 7.3. tween them. See Table 3.8-1.
Table 3.8-1. Effect of Dithiothreitol on Blood Group Antibodies
Dilution
Test Sample 1/2 1/4 1/8 1/16 1/32 Interpretation
Serum + DTT 3+ 2+ 2+ 1+ 0
IgG
Serum + PBS 3+ 2+ 2+ 1+ 0
Serum + DTT 0 0 0 0 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-
Method 3.9. Plasma Inhibition
tibody reactivity and an invalid test. to Distinguish Anti-Ch and
-Rg from Other Antibodies
with HTLA Characteristics
Control Principle
A serum or plasma sample known to con- For a discussion of the principles of plasma
tain an IgM antibody should be treated and inhibition of anti-Ch and -Rg, see Chapter 19.
tested in parallel.
Specimen
Serum or plasma to be tested.
Notes Reagents
1. 2-mercaptoethanol can also be used 1. Reactive red cell samples.
for this purpose. See Method 2.11 for 2. A pool of six or more normal plasma
preparation. samples.
2. Sulfhydryl reagents used at low con- 3. 6% bovine albumin, see Method 1.5.
centration may weaken antigens of 4. Anti-IgG.
the Kell system. For investigation of 5. IgG-coated red cells.
antibodies in the Kell system, it may
be necessary to use other methods.
3. Gelling of a serum or plasma sample Procedure
may be observed during treatment with
1. Prepare serial twofold dilutions of test
DTT. This can occur if the DTT has
serum in saline. The dilution range
been prepared incorrectly and has a
should be from 1 in 2 to 1 in 512, or
concentration above 0.01 M. Gelling
to one tube beyond the known titer
may also occur if serum and DTT are
as determined above (Method 3.7). The
incubated too long. An aliquot of the
volume prepared should be not less
sample undergoing treatment can be
than 0.3 mL for each red cell sample
tested after 30 minutes of incubation;
to be tested.
if the activity thought to be due to IgM
2. For each red cell sample to be tested,
has disappeared, there is no need to place 2 drops of each serum dilution
incubate further. Gelled samples can- into each of two sets of appropriately
not be tested for antibody activity be- labeled 10 or 12 × 75-mm test tubes.
cause overtreatment with DTT causes 3. To one set, add 2 drops of pooled
the denaturation of all serum proteins. 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
Reference tube and incubate the tubes at room
temperature for at least 30 minutes.
Mollison PL, Engelfriet CP, Contreras M, eds. Blood
transfusion in clinical medicine. 10th ed. Oxford, 6. Add 1 drop of a 2% to 5% suspension
England: Blackwell Scientific Publications, 1997. of red cells to each tube.

7. Gently agitate the contents of each 2. Ellisor SS, Shoemaker MM, Reid ME. Adsorp-
tion of anti-Chido from serum using autolo-
tube and incubate the tubes at 37 C
gous red blood cells coated with homologous
for 1 hour. C4. Transfusion 1982;22:243-5.
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- Method 3.10. Dithiothreitol
amine for agglutination; confirm all
nonreactive tests microscopically.
(DTT) Treatment of Red Cells
Grade and record the results.
Principle
by adding IgG-coated red cells. DTT is an efficient reducing agent that
can disrupt the tertiary structure of pro-
Interpretation teins by irreversibly reducing disulfide
bonds to free sulfhydryl groups. Without
1. Inhibition of antibody activity in the
tertiary structure, protein-containing an-
tubes to which plasma has been added
tigens can no longer bind antibodies that
suggests anti-Ch or anti-Rg specific-
are specific for them. Red cells treated
ity; this inhibition is often complete.
with DTT will not react with antibodies in
2. The presence of partial inhibition
the Kell blood group system, most anti-
suggests the possibility of additional
bodies in the Knops system, or most ex-
alloantibodies. This can be tested by
amples of anti-LWa, -Yta, -Ytb, -Doa, -Dob,
preparing a large volume of inhibited a a
-Gy , -Hy, and -Jo . This inhibition tech-
serum and testing it against a re-
nique may be helpful in identifying some
agent red cell panel to see if the non-
of these antibodies or in determining if a
neutralizable activity displays anti-
serum contains additional underlying allo-
genic specificity.
antibodies.
3. Lack of reactivity in the control (6%
albumin) indicates dilution of weakly
reactive antibody and an invalid test.
Specimen
Red cells to be tested.
Notes Reagents
1. Antibodies to other plasma antigens 1. Prepare 0.2 M DTT by dissolving 1 g
may also be partially inhibited by of DTT powder in 32 mL of phos-
plasma.1 phate-buffered saline (PBS), pH 8.0.
2. Adsorption with C4-coated red cells Divide it into 1-mL volumes and
is an alternative procedure that may freeze aliquots at –18 C or colder.
be used for identifying anti-Ch or 2. PBS at pH 7.3, see Method 1.7.
anti-Rg and for detecting underlying 3. Red cells known to be positive for the
alloantibodies.2 antigen in question and, as a control,
red cells known to be positive for K,
References which is consistently disrupted by
DTT.
1. Reid ME, Lomas-Francis C. The blood group
antigen factsbook. 2nd ed. New York: Aca- 4. Anti-K, either in reagent form or
demic Press, 2004. strongly reactive in a serum specimen.

Procedure Reference
1. Wash one volume of the test cells and Branch DR, Muensch HA, Sy Siok Hian S, Petz LD.
Disulfide bonds are a requirement for Kell and
the control cells with PBS. After de- Cartwright (Yt a) blood group antigen integrity. Br J
canting, add four volumes of 0.2 M Haematol 1983;54:573-8.
DTT, pH 8.0.
2. Incubate at 37 C for 30 to 45 min-
utes.
3. Wash four times with PBS. Slight
Method 3.11. Urine
a
hemolysis may occur; if hemolysis is Neutralization of Anti-Sd
excessive, repeat the procedure us- Principle
ing fresh red cells and a smaller vol- a
For a discussion of anti-Sd neutralization
ume of DTT, eg, two or three volumes.
by urine, see Chapter 15.
4. Resuspend the cells to a 2% to 5%
suspension in PBS.
Specimen
5. Test DTT-treated cells with serum
containing the antibody in question. Serum or plasma suspected of containing
a
Test K+ red cells with anti-K. anti-Sd .
Reagents
Interpretation 1. Urine from a known Sd(a+) individ-
ual, or from a pool of at least six in-
1. The control K+ red cells should give
dividuals of unknown Sda type, pre-
negative reactions when tested with
pared as follows: Collect urine and
anti-K; if not, the DTT treatment has
immediately boil it for 10 minutes.
been inadequate. Other antigens in
Dialyze it against phosphate-buf-
the Kell system can also serve as the
fered saline (PBS), pH 7.3, at 4 C for
control. 48 hours. Change PBS several times.
2. If reactivity of the test serum is elim- Centrifuge. Dispense supernatant into
inated, the suspected antibody spec- aliquots, which can be stored at –20
ificity may be confirmed. Enough red C until thawed for use.
cell samples should be tested to ex- 2. PBS, pH 7.3. See Method 1.7.
clude most other clinically signifi-
cant alloantibodies. Procedure
1. Mix equal volumes of thawed urine
Note and test serum.
Treatment of red cells with 0.2 M DTT, pH 2. Prepare a dilution control tube con-
8.0, is optimal for denaturation of all anti- taining equal volumes of serum and
gens of the Kell, Cartwright, LW, and PBS.
Dombrock systems, and most antigens of 3. Prepare a urine control tube by mix-
the Knops system. Lower concentrations ing equal volumes of thawed urine
of DTT may selectively denature particu- and PBS.
lar blood group antigens (ie, 0.002 M DTT 4. Incubate all tubes at room tempera-
will denature only Jsa and Jsb antigens). This ture for 30 minutes.
property may aid in certain antibody in- 5. Mix 1 drop of each test red cell sam-
vestigations. ple with 4 drops from each of the

tubes: neutralized serum, serum with

PBS, and urine with PBS. Test each
Method 3.12. Adsorption
one using standard procedures. Procedure
Principle
See Chapter 19.
Interpretation
Specimen
1. Persistent agglutination in the serum
Serum or plasma containing antibody to
sample incubated with urine means
be adsorbed.
either that partial or no neutraliza-
tion was achieved or that underlying
Reagents
antibodies are present. Microscopic
examination may be helpful; aggluti- Red cells (eg, autologous or allogeneic) that
a
nation due to anti-Sd has a refractile, carry the antigen corresponding to the an-
mixed-field appearance on micro- tibody specificity to be adsorbed.
scopic examination.
2. No agglutination in the neutralized Procedure
tube with persistent agglutination in 1. Wash the selected red cells at least
the dilution control tube and absence three times with saline.
of hemolysis and agglutination in 2. After the last wash, centrifuge the red
the urine control tube indicate that cells at 800 to 1000 × g for at least 5
the antibody has been neutralized minutes and remove as much of the
a
and is quite probably anti-Sd . supernatant saline as possible. Addi-
3. The absence of agglutination in the tional saline may be removed by
dilution control tube means that the touching the red cell mass with a
dilution in the neutralization step narrow piece of filter paper.
was too great for the antibody pres- 3. Mix appropriate volumes of the pack-
ent and the results of the test are ined red cells and serum and incubate
valid. The urine control tube provides at the desired temperature for 30 to
assurance that no substances in the 60 minutes.
urine are agglutinating or damaging 4. Mix the serum/cell mixture periodi-
the red cells. cally throughout the incubation
phase.
5. Centrifuge the red cells at 800 to
Note 1000 × g for 5 minutes to pack cells
tightly. Centrifuge at the incubation
Urine may also contain ABO and Lewis temperature, if possible, to avoid
blood group substances, depending upon dissociation of antibody from the red
the ABO, Lewis, and secretor status of the cell membranes.
donor. 6. Transfer the supernatant fluid, which
is the adsorbed serum, to a clean test
Reference tube. If an eluate is to be prepared,
save the red cells.
Judd WJ. Methods in immunohematology. 2nd ed.
Durham, NC: Montgomery Scientific Publications, 7. Test an aliquot of the adsorbed se-
1994. rum, preferably against an addi-

tional aliquot of the cells used for The ARDP maintains a database of rare
adsorption, to see if all antibody has donors submitted by immunohematology
been removed. reference laboratories that are accredited
by the AABB or the American Red Cross
Interpretation (ARC). Donors are considered rare due to
If reactivity remains, the antibody has not the absence of a high-incidence antigen,
been completely removed. No reactivity absence of multiple common antigens, or
signifies that antibody has been com- IgA deficiency.
pletely adsorbed. All requests to the ARDP must originate
from an AABB- or ARC-accredited im-
Notes munohematology reference laboratory to
ensure that the patient in question has
1. Adsorption is more effective if the
been accurately evaluated and reported. All
area of contact between the red cells
shipping and rare unit fees are established
and serum is large; use of a large-
by the shipping institution.
bore test tube (13 mm or larger) is
recommended.
2. Multiple adsorptions may be neces- Procedure
sary to completely remove an anti-
1. A hospital blood bank, transfusion
body, but each successive adsorp-
service, or blood center identifies a
tion increases the likelihood that the
patient who needs rare blood.
s e r u m wi l l b e d i l u te d a n d u n-
2. The institution contacts the nearest
adsorbed antibodies weakened.
AABB- or ARC-accredited immuno-
3. Repeat adsorptions should use a
hematology reference laboratory to
fresh aliquot of cells and not the
supply the needed blood.
cells from the prior adsorption.
3. If the laboratory cannot supply the
4. Enzyme pretreatment of adsorbing
blood, it contacts the ARDP. All re-
cells can be performed to increase an-
quests to the ARDP must come from
tibody uptake for enzyme-resistant
an AABB- or ARC-accredited labora-
antigens.
tory (or another rare donor program).
Requests received directly from a non-
Reference accredited facility will be referred to
Judd WJ. Methods in immunohematology. 2nd ed. the nearest accredited institution.
Durham, NC: Montgomery Scientific Publications,
1994.
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
Method 3.13. Using the by another institution.
American Rare Donor 5. ARDP staff search their database for
Program centers that have identified donors
with the needed phenotype and con-
Principle tact the centers for availability of
The American Rare Donor Program (ARDP) units. ARDP staff give the name(s) of
helps to locate blood products for pa- the shipping center(s) to the request-
tients requiring rare or unusual blood. ing institution.

6. The requesting and shipping institu- communication to all ARDP partici-

tions should discuss and agree on pating centers alerting them to search
charges and testing requirements their inventories and/or recruit do-
before units are shipped. nors matching the needed pheno-
7. If an initial search does not result in type, 2) contacting other rare donor
a sufficient number of units, the fol- files such as those administered by
lowing mechanisms can be used by the World Health Organization, Jap-
ARDP staff to obtain needed units: 1) anese Red Cross, etc.

Methods Section 4: Investigation of a Positive DAT
Methods Section 4
Investigation of a Positive
Direct Antiglobulin Test
Section 4
method (eg, organic solvent) may be indi-
Elution Techniques cated when a nonreactive eluate is not in
agreement with clinical data. The reader
The objective of all elution techniques is should refer to Chapter 2 for the proper
to interfere with the noncovalent binding handling of hazardous chemicals that are
forces that hold antibody-antigen complexes sometimes used in these techniques. Ac-
together on the red cell surface. The cell cess to a chemical fume hood is desirable
membrane can be physically disrupted by when organic solvents are in use.
heat, ultrasound, freezing and thawing, Very thorough washing of the red cells
detergents, or organic solvents. The bind- before elution is essential to ensure that an-
ing forces of antigen-antibody complexes tibody in the eluate is only red cell-bound
can be interrupted by alterations in pH or and does not represent free antibody, eg,
salt concentration. For a comparison of the from plasma. A control to show that all free
advantages and disadvantages of various antibody has been removed by washing can
elution methods, see Chapter 20. Selected be obtained by saving the saline from the
elution methods follow, including one ex- last wash and testing it in parallel with the
ample of an organic solvent method. The eluate. Additionally, transferring the red
cold-acid elution method (Method 4.1) is cells into a clean test tube just before the
the basis of the commercially available acid elution step eliminates the possibility of
elution kits commonly used in the United dissociating antibody that may have non-
States. Because no single elution method specifically bound to the glass test tube
will result in the identification of all during an adsorption or the initial eluate
antibodies, use of an alternative elution preparation steps.
771

4. Quickly centrifuge the tube at 900 to

Method 4.1. Cold-Acid Elution 1000 × g for 2 to 3 minutes.
Principle 5. Transfer the supernatant eluate into
Dissociation of antibodies from red cells a clean test tube and add 0.1 mL of
enables the identification of auto- or allo- pH 8.2 phosphate buffer for each 1
antibodies. Elution methods used in con- mL of eluate (see note 3).
junction with adsorption techniques are 6. Mix and centrifuge at 900 to 1000 × g
also useful in detecting weak antigen ex- for 2 to 3 minutes.
pression on the adsorbing cells and in 7. Transfer the supernatant eluate into
separating mixtures of antibodies against a clean test tube and test it in paral-
red cell antigens. lel with the supernatant saline from
the final wash.
Specimen
Notes
Red cells positive by the direct antiglobulin
test (DAT) washed six times with large 1. Keep glycine-HCl in an ice bath dur-
volumes of saline (save the last wash). ing use, to maintain the correct pH.
2. Phosphate buffer will crystallize dur-
Reagents ing storage at 4 C. Redissolve it at 37
C before use.
1. Glycine-HCl (0.1 M, pH 3.0), prepared
3. The addition of phosphate buffer re-
by dissolving 3.75 g of glycine and
stores neutrality to the acidic eluate.
2.922 g of sodium chloride in 500 mL
Acidity may cause hemolysis of the
of deionized or distilled water. Adjust
reagent red cells used in testing the
the pH to 3.0 with 12 N HCl. Store at
eluate. The addition of 22% bovine
4 C.
albumin (one part to four parts of
2. Phosphate buffer (0.8 M, pH 8.2),
eluate) may reduce such hemolysis.
prepared by dissolving 109.6 g of
Na2HPO4 and 3.8 g of KH2PO4 in ap-
References
proximately 600 mL of deionized or
distilled water and adjusting the fi- 1. Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Pub-
nal volume to 1 L. Adjust the pH, if lications, 1994.
necessary, with either 1 N NaOH or 1 2. Rekvig OP, Hannestad K. Acid elution of blood
N HCl. Store at 4 C (see note 2). group antibodies from intact erythrocytes.
Vox Sang 1977;33:280-5.
3. NaCl, 0.9%, at 4 C.
4. Supernatant saline from the final
wash of red cells to be tested.
Method 4.2.
Procedure Glycine-HCl/EDTA Elution
1. Place 1 mL of red cells in a 13 ×
Principle
100-mm test tube and chill in an ice
water bath for 5 minutes before add- See Method 4.1.
ing the glycine-HCl.
2. Add 1 mL of chilled saline and 2 mL Specimen
of chilled glycine-HCl to the red cells. Red cells positive by the direct antiglobulin
3. Mix and incubate the tube in an ice test (DAT) washed six times with large
water bath (0 C) for 1 minute. volumes of saline (save the last wash).

Methods Section 4: Investigation of a Positive DAT 773
Reagents denatures Kell system antigens and

Era. Wash the red cells at least three
1. Disodium EDTA (10% w/v): Na2EDTA,
times in saline before use.
10 g; distilled water to 100 mL.
2. Red cells modified with glycine-
2. Glycine-HCl (0.1 M at pH 1.5): 0.75 g
HCl/EDTA may be treated with a
glycine diluted to 100 mL with 0.9%
protease and used in autologous ad-
NaCl; adjust to pH 1.5 with 12 N HCl.
sorption studies.
3. TRIS-NaCl (1 M): Tris(hydroxymethyl)
3. Overincubation with the eluting so-
aminomethane [TRIS] or TRIZMA
lution (step 3) will irreversibly dam-
BASE, 12.1 g; 5.25 g NaCl; distilled
age the red cells.
water to 100 mL.
4. TRIS-NaCl is very alkaline and only a
4. Supernatant saline from the final wash
few drops should be required to at-
of the red cells to be tested.
tain the desired pH (step 5).
5. Aliquots of the reagents can be stored
Procedure
frozen and one tube of each can be
1. In a test tube, mix together 20 vol- thawed just before use. The 10% EDTA
umes (eg, drops) of 0.1 M glycine- may precipitate when stored at 2 to 8 C.
HCl buffer and 5 volumes of 10% 6. Stored eluate (4 C or frozen) may be
EDTA. This is the eluting solution. more stable if albumin is added (1
2. In a 12 × 75-mm tube, place 10 vol- volume of 30% bovine albumin for
umes of packed red cells. every 10 volumes of eluate). If albu-
3. Add 20 volumes of the eluting solu- min is added to the eluate, it should
tion to the red cells, mix well, and in- be added to the last wash.
cubate at room temperature for 2
minutes. Do not overincubate. Reference
4. Add 1 volume of TRIS-NaCl, mix,
Byrne PC. Use of a modified acid/EDTA elution
and immediately centrifuge the tube technique. Immunohematology 1991;7:46-7. [Cor-
at 900 to 1000 × g for 60 seconds. rection note: Immunohematology 1991;7:106.]
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
Method 4.3. Heat Elution
checked with pH paper. Principle
6. Centrifuge at 900 to 1000 × g for 2 to Heat elution uses an increase in tempera-
3 minutes to remove the precipitate. ture to dissociate antibodies from red cells.
7. Transfer the supernatant eluate into This method is best suited for the investi-
a clean test tube and test it in paral- gation of ABO hemolytic disease of the fe-
lel with the supernatant saline from tus and newborn and for the elution of IgM
the final wash. antibodies from red cells. It should not
routinely be used for the investigation of
Notes abnormalities caused by IgG auto- or allo-
1. Once the red cells have been rendered antibodies.
DAT negative, they may be tested for
the presence of blood group anti- Specimen
gens, except those in the Kell system. Red cells positive by the direct antiglobulin
Treatment with glycine-HCl/EDTA test (DAT) washed six times with large

volumes of saline; save the last wash (see of the remaining extracellular fluid, which
note). then extracts water from the red cells. The
red cells shrink, resulting in lysis. As the
Reagents membranes are disrupted, antibody is
1. 6% bovine albumin (see Method 1.5). dissociated. This method is used primar-
2. Supernatant saline from the final ily for the investigation of ABO hemolytic
wash of the red cells to be tested. disease of the fetus and newborn.
Procedure
1. Mix equal volumes of washed packed Specimen
cells and 6% bovine albumin in a
13 × 100-mm test tube. 1. Red cells washed six times with large
2. Place the tube at 56 C for 10 min- volumes of saline.
utes. Agitate the tube periodically 2. Supernatant saline from the final
during this time. wash of the red cells to be tested.
3. Centrifuge the tube at 900 to 1000 ×
g for 2 to 3 minutes, preferably in a
heated centrifuge. Procedure
4. Immediately transfer the supernatant
eluate into a clean test tube and test 1. Mix 0.5 mL of the red cells to be tested
in parallel with the supernatant sa- with 3 drops of saline in a test tube.
line from the final wash. 2. Cap the tube, then rotate the tube to
coat the tube wall with cells.
Note 3. Place the tube in a horizontal posi-
For optimal recovery of cold-reactive anti- tion in a freezer at –6 C to –70 C for
bodies, the red cells should be washed in 10 minutes.
ice-cold saline to prevent dissociation of 4. Remove the tube from the freezer and
bound antibody before elution. thaw it quickly with warm, running
tap water.
References 5. Centrifuge for 2 minutes at 900 to
1000 × g.
1. Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Pub- 6. Transfer the supernatant to a clean
lications, 1994. test tube and test it in parallel with
2. Landsteiner K, Miller CP Jr. Serological stud- the supernatant saline from the final
ies on the blood of primates. II. The blood
wash.
groups in anthropoid apes. J Exp Med 1925;
42:853-62.
References
Method 4.4. Lui
Freeze-Thaw Elution 1. Judd WJ. Methods in immunohematology. 2nd
ed. Durham, NC: Montgomery Scientific Pub-
Principle lications, 1994.
2. Feng CS, Kirkley KC, Eicher CA, et al. The Lui
As red cells freeze, extracellular ice crys-
elution technique: A simple and efficient
tals form that attract water from their sur- method for eluting ABO antibodies. Transfu-
roundings. This increases the osmolarity sion 1985;25:433-4.

Method 4.5. Methylene Reference

Judd WJ. Methods in immunohematology. 2nd ed.
Chloride Elution Durham, NC: Montgomery Scientific Publications,
1994.
Principle
Organic solvents can influence antigen-
antibody dissociation by several mecha- Immune Hemolytic Anemia
nisms, including alteration of the tertiary Serum/Plasma Methods
structure of antibody molecules and dis-
Included in this section are methods used
ruption of the red cell membrane. This
to remove warm or cold autoantibody re-
method is suitable for elution of IgG auto-
activity (eg, adsorptions) so that alloanti-
and alloantibodies.
body detection tests and diagnostic tests
for differentiating the immune hemolytic
Specimen anemias can be performed. See Chapter
DAT-positive red cells washed six times with 20 for a discussion of the immune hemoly-
large volumes of saline (save last wash). tic anemias.
Reagents
1. Methylene chloride (dichloromethane). Method 4.6. Cold
2. Supernatant saline from final wash Autoadsorption
of the red cells to be tested.
Principle
Procedure Although most cold autoantibodies do not
1. Mix 1 mL of red cells, 1 mL of saline, cause a problem in serologic tests, some
and 2 mL of methylene chloride in a potent cold-reactive autoantibodies may
test tube, eg, 13 × 100 mm. mask the concomitant presence of clini-
2. Stopper the tube and mix by gentle cally significant alloantibodies. In these
agitation for 1 minute. cases, adsorbing the serum in the cold
3. Remove the stopper and centrifuge with autologous red cells can remove the
the tube at 1000 × g for 10 minutes. autoantibody, permitting detection of un-
4. Remove the lower layer of methylene derlying alloantibodies. In the case of most
chloride with a transfer pipette and nonpathologic cold autoantibodies, a
discard it. simple quick adsorption of the patient’s
5. Place the tube at 56 C for 10 min- serum with enzyme-treated autologous
utes. Stir the eluate constantly with red cells will remove most cold antibody.
wooden applicator sticks in the first See Method 3.5.6. A more efficient method
several minutes to avoid it boiling of removing immunoglobulins is the use
over; thereafter, stir it periodically. of ZZAP reagent, a combination of pro-
6. Centrifuge at 1000 × g for 10 min- teolytic enzyme and a powerful reducing
utes. agent. ZZAP treatment removes IgM and
7. Transfer the supernatant eluate into complement from autologous red cells
a clean test tube and test it in paral- and uncovers antigen sites that can be
lel with the supernatant saline from used to bind free autoantibody in the se-
the final wash. rum.

Specimen 7. After the final adsorption, test the

serum with reagent red cells for allo-
1. 1 mL of serum or plasma to be ad-
antibody activity.
sorbed.
2. Two 1-mL aliquots of autologous red
Notes
cells.
1. To avoid dilution of the serum and
possible loss of weak alloantibody
Reagents
activity, it is important in step 3 to
1. 1% cysteine-activated papain or 1% remove as much of the residual sa-
ficin (see Methods 3.5.1 and 3.5.2). line as possible.
2. Phosphate-buffered saline (PBS), pH 2. If the reactivity of the autoantibody
7.3 (see Method 1.7). is not diminished, the target auto-
3. 0.2 M dithiothreitol (DTT) prepared antigen may have been destroyed by
by dissolving 1 g of DTT in 32.4 mL either the enzyme or the DTT. The
of pH 7.3 PBS. Dispense into 3-mL adsorption should be repeated against
aliquots and store at –18 C or colder. untreated autologous red cells washed
several times in warm saline.
Procedure References
1. Branch DR. Blood transfusion in autoimmune
1. Prepare ZZAP reagent by mixing 0.5
hemolytic anemias. Lab Med 1984;15:402-8.
mL of 1% cysteine-activated papain 2. Branch DR, Petz LD. A new reagent (ZZAP)
with 2.5 mL of 0.2 M DTT and 2 mL having multiple applications in immuno-
of pH 7.3 PBS. Alternatively, use 1 hematology. Am J Clin Pathol 1982;78:161-7.
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. Method 4.7. Determining the
2. Add 2 mL of ZZAP reagent to 1 mL of
autologous red cells. Mix and incu-
Specificity of Cold-Reactive
bate at 37 C for 30 minutes. Autoagglutinins
3. Wash the cells three times in saline. Principle
Centrifuge the last wash for at least 5
For a discussion of specificity of cold-re-
minutes at 900 to 1000 × g and re-
acting autoantibodies, see Chapter 20.
move as much of the supernatant sa-
line as possible (see note 1).
Specimen
4. To the tube of ZZAP-treated red cells,
add 1 mL of the autologous serum. 1. Serum, separated at 37 C from a blood
Mix and incubate at 4 C for 30 min- sample allowed to clot at 37 C, or
utes. plasma, separated from an anti-
5. Centrifuge at 900 to 1000 × g for 4 to coagulated sample after periodic in-
5 minutes and transfer the serum into version at 37 C for approximately 15
a clean tube. minutes.
6. Steps 2 through 5 may be repeated if 2. Autologous red cells.
the first autoadsorption does not sat-
isfactorily remove the autoantibody Reagents
activity. Test red cells of the following phenotypes:

1. A pool of two examples of adult 3. Dispense 2 drops of each dilution

group O I adult red cells; they can be into the appropriate tubes.
the reagent cells routinely used for 4. Add 1 drop of a 3% to 5% saline sus-
alloantibody detection. pension of each red cell sample to
2. Group O i cord red cells. the appropriate set of tubes.
3. The patient’s own (autologous) red 5. Mix and incubate at room tempera-
cells, washed at least three times ture for 30 to 60 minutes.
with 37 C saline. 6. Centrifuge for 15 to 20 seconds at
4. Red cells of the same ABO group as 900 to 1000 × g. Examine the tubes
the patient, if the patient is not one by one macroscopically for agglu-
group O. If the patient is group A or tination, starting with the set of tubes
AB, use both A1 and A2 cells. at the highest dilution for each cell
5. Saline or phosphate-buffered saline tested (ie, read all the tubes for each
(PBS), pH 7.3 (see Method 1.7). dilution as a set). Grade and record
the results.
Procedure 7. Transfer the tubes to 4 C and incu-
bate them at this temperature for 1
1. Prepare serial twofold dilutions of
to 2 hours.
the serum or plasma in saline or
8. Centrifuge for 15 to 20 seconds at
PBS. The dilution range should be
900 to 1000 × g. Immediately place
from 1 in 2 to 1 in 4096 (12 tubes),
the tubes in a rack in an ice water
and the volumes prepared should be
bath. Examine the tubes as in step 6.
more than the total volume needed
Grade and record the results.
to test all of the desired red cells. See
Method 3.7.
2. Label a set of 12 tubes with the dilu- Interpretation
tion (eg, 2, 4, 8, etc) for each of the Table 4.7-1 summarizes the reactions of
red cells to be tested (eg, adult, cord, the commonly encountered cold-reactive
autologous). autoantibodies. In cold agglutinin syn-
Table 4.7-1. Typical Relative Reactivity Patterns of Cold Autoantibodies
Antibody Specificity
T
Red Cells Anti-I Anti-i Anti-I Anti-IH Anti-Pr
O I adult + 0/↓ 0/↓ + +

O i cord 0/↓ + + ↓ +
O i adult 0/↓ + 0/↓ ↓ +
A1 I 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 ferential reactivity may be more
anti-i may also be encountered. When apparent if incubation times are
cord cells react stronger than adult cells, prolonged and agglutination is eval-
the specificity may be anti-i, but adult i uated after settling, without centrifu-
red cells need to be tested to confirm that gation. Settled readings are more ac-
these reactions are due to anti-i and not curate after a 2-hour incubation.
anti-I T . Some examples of anti-I react 4. This procedure can be used to deter-
more strongly with red cells that have a mine both the titer and the specific-
strong expression of H antigen (eg, O and ity. If incubations are started at 37 C
A2 cells); such antibodies are called anti- (set up prewarmed, and readings are
IH. Rarely, the specificity may be anti-Pr, taken sequentially after incubation
which should be suspected if all the cells at each temperature—eg, 37 C, 30 C,
tested react equally. Anti-Pr can be con- room temperature, 4 C), the specific-
firmed by testing enzyme-treated cells; ity, titer, and thermal amplitude of
anti-Pr does not react with enzyme- the autoantibody can be determined
treated cells, whereas anti-I and anti-i with a single set of serum dilutions.
react better with enzyme-treated cells. 5. If testing will also be performed at
Anti-Pr reacts equally with untreated red 30 C and 37 C, include a test of the
cells of I or i phenotypes. neat (undiluted) serum.
Notes Reference
1. It is important to use separate pi- Petz LD, Garratty G. Immune hemolytic anemias.
2nd ed. Philadelphia: Churchill Livingstone, 2004.
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 Method 4.8. Cold Agglutinin
falsely high titration endpoints. The
difference can convert a true titer of
Titer
4000 to an apparent titer of 100,000,
when the use of separate pipettes is Principle
compared with the use of a single pi- Cold-reactive autoantibodies, if present at
pette. very high titers, may suggest a pathologic
2. Serum dilutions can be prepared cold agglutinin disease. This may result in
more accurately with large volumes overt hemolysis and systemic symptoms
(eg, 0.5 mL) than with small vol- and may indicate underlying B-cell hema-
umes. tologic neoplasia.
3. Potent examples of cold-reactive
autoantibodies generally do not
Specimen
show apparent specificity until titra-
tion studies are performed; this Serum, separated at 37 C from a sample
specificity may not even be apparent allowed to clot at 37 C, or plasma, sepa-
with dilutions at room temperature rated from an anticoagulated sample after
or 4 C. In such circumstances, tests periodic inversion at 37 C for approxima-
can be incubated at 30 to 37 C. Dif- tely 15 minutes.

Reagents Notes
1. A pool of 2 examples of washed group 1. It is important to use separate pi-
O I adult red cells, eg, antibody de- pettes for each tube when preparing
tection cells. serum dilutions because the serum
2. Phosphate-buffered saline (PBS), pH carried from one tube to the next when
7.3 (see Method 1.7). a single pipette is used throughout
may cause falsely high titration end-
points.
Procedure
2. Serum dilutions can be prepared
1. Prepare serial twofold dilutions of the more accurately with large volumes
patient’s serum or plasma in PBS. (eg, 0.5 mL) than with small vol-
The dilution range should be from 1 umes.
in 2 to 1 in 4096 (12 tubes). See
Method 3.7. Reference
2. Mix 2 drops of each dilution with 1 Petz LD, Garratty G. Immune hemolytic anemias.
drop of a 3% to 5% cell suspension of 2nd ed. Philadelphia: Churchill Livingstone, 2004.
red cells.
3. Mix and incubate at 4 C for 1 to 2
hours.
4. Centrifuge the tubes for 15 to 20 sec-
Method 4.9. Autologous
onds at 900 to 1000 × g, then place Adsorption of
the tubes in a rack in an ice water Warm-Reactive
bath. Examine the tubes one by one
macroscopically for agglutination,
Autoantibodies
starting with the tube at the highest
Principle
dilution. Grade and record the re-
Warm-reactive autoantibodies in serum
sults.
may mask the concomitant presence of
clinically significant alloantibodies. Ad-
Interpretation sorption of the serum with autologous red
The titer is the reciprocal of the highest cells can remove autoantibody from the
serum dilution at which macroscopic ag- serum, permitting detection of underly-
glutination is observed. Titers above 64 ing alloantibodies. However, autologous
are considered elevated, but hemolytic red cells in the circulation are coated with
anemia resulting from cold-reactive auto- autoantibody. Autologous adsorption of
agglutinins rarely occurs unless the titer is warm-reactive autoantibodies can be fa-
>1000. Titers below 1000 may be obtained cilitated by dissociating autoantibody from
when the autoantibody has a different the red cell membrane, thereby uncover-
specificity (eg, anti-i), or if the cold agglu- ing antigen sites that can bind free auto-
tinin is of the less common low-titer, antibody to remove it from the serum.
high-thermal-amplitude type. If the pa- Some autoantibody can be dissociated by
tient has a positive direct antiglobulin test a gentle heat elution for 3 to 5 minutes at
(DAT) because of complement only and 56 C. Subsequent treatment of the cells
has clinical signs of hemolytic anemia, with enzymes enhances the adsorption
specificity and thermal amplitude studies process by removing membrane struc-
should be performed (see Method 4.7). tures that otherwise hinder the associa-

tion between antigen and antibody. The and remove as much supernatant
most effective procedure involves the use saline as possible.
of ZZAP reagent, a mixture of a proteolytic 4. Add serum to an equal volume of
enzyme and a sulfhydryl reagent. ZZAP ZZAP-treated red cells, mix, and in-
removes immunoglobulins and comple- cubate at 37 C for approximately 30
ment from the red cells and enhances the to 45 minutes.
adsorption process. Red cells from pa- 5. Centrifuge and carefully remove serum.
tients transfused within the last 3 months 6. If the original serum reactivity was
should not be used for autoadsorption only 1+, proceed to step 7; otherwise,
because transfused red cells present in repeat steps 4 and 5 once more us-
the circulation are likely to adsorb the ing the once-adsorbed patient’s se-
alloantibodies that are being sought (see rum and the second aliquot of ZZAP-
Chapter 20). treated cells.
7. Test the serum against a specimen of
Specimen group O reagent cells. If reactivity
persists, repeat steps 4 and 5.
1. 1 mL of serum or plasma (or eluate)
to be adsorbed.
Interpretation
2. 2 mL of autologous red cells.
One or two adsorptions ordinarily remove
sufficient autoantibody so that alloanti-
Reagents
body reactivity, if present, is readily ap-
1. 1% cysteine-activated papain or 1% parent. If the twice-autoadsorbed serum
ficin (see Methods 3.5.1 and 3.5.2). reacts with defined specificity, as shown
2. Phosphate-buffered saline (PBS), pH by testing against a small antibody identi-
7.3 (see Method 1.7). fication panel, then the defined specificity
3. 0.2 M DTT prepared by dissolving 1 of the antibody is probably an alloantibody.
g of DTT in 32.4 mL of pH 7.3 PBS. If the serum reacts with all cells on the
Dispense into 3-mL aliquots and panel, either additional autoadsorptions
store at –18 C or colder. are necessary, the serum contains anti-
body to a high-incidence antigen, or the
Procedure serum contains an autoantibody (eg,
anti-Kpb) that does not react with ZZAP-
1. Prepare ZZAP reagent by mixing 0.5
treated cells and thus will not be adsorbed
mL of 1% cysteine-activated papain
by this procedure. To check this latter
with 2.5 mL of 0.2 M DTT and 2 mL
possibility, test the reactive autoadsorbed
of pH 7.3 PBS. Alternatively, use 1
serum against reagent cells that have
mL of 1% ficin, 2.5 mL of 0.2 M DTT,
been pretreated with the ZZAP reagent.
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 Notes
mL of red cells, add 2 mL of ZZAP re- 1. ZZAP treatment destroys all Kell sys-
agent. Mix and incubate at 37 C for tem antigens and all other antigens
30 minutes with periodic mixing. that are destroyed by proteases, eg,
3. Wash the red cells three times in sa- M, N, Fya, and Fyb. ZZAP reagent also
line. Centrifuge the last wash for at denatures the antigens of the LW,
least 5 minutes at 900 to 1000 × g Cartwright, Dombrock, and Knops

systems. If the autoantibody is sus- blood group systems. The specificity of the
pected to have specificity in any of antibodies that remain after adsorption
these latter blood groups, an alter- can be confirmed by testing against a
native procedure is to perform auto- panel of reagent red cells. This procedure
adsorption with untreated autologous can be used to detect underlying alloanti-
cells or autologous cells treated only bodies if the patient has been recently
with 1% ficin or 1% cysteine-activated transfused, or if insufficient autologous
papain. red cells are available and the patient’s
2. There is no need to wash packed red phenotype is unknown.
cells before treatment with ZZAP. Treating the adsorbing cells with enzyme
3. Cold autoantibodies reactive at room or ZZAP typically enhances the adsorption
temperature can also be present in process. In addition, the treated red cells will
the serum of about 30% of patients lack the antigens destroyed by dithiothrei-
with warm-reactive autoantibodies. tol (DTT) and/or enzymes (see Chapter 19).
Removal of these cold antibodies can
be facilitated by placing the serum Specimen
and cell mixture at 4 C for about 15 Serum/plasma containing warm-reactive
minutes after incubation at 37 C. autoantibodies or eluate from direct anti-
4. As a guide, when the original serum globulin test (DAT)-positive cells.
reactivity is 1+ in the low-ionic-
strength saline indirect antiglobulin Reagents
test (LISS-IAT), usually only one ad-
sorption would be required. Antibod- 1. 1% cysteine-activated papain or 1%
ies with 2+ to 3+ reactivity will gen- ficin (see Methods 3.5.1 and 3.5.2).
erally be removed in two to three 2. ZZAP reagent (papain or ficin plus
adsorptions. Performing greater 0.2 M DTT). See Method 4.9.
than four adsorptions increases the 3. Phosphate-buffered saline (PBS), pH
risk of diluting alloantibody reactiv- 7.3 (see Method 1.7).
ity. 4. Group O red cells of the phenotypes
R1R1, R2R2, and rr; one of these cells
Reference should be Jk(a–b+) and one should
be Jk(a+b–). Additionally, if the red
Branch DR, Petz LD. A new reagent (ZZAP) having
multiple applications in immunohematology. Am
cells are to be only enzyme-treated,
J Clin Pathol 1982;78:161-7. 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.
Method 4.10. Differential
Warm Adsorption Using Procedure
Enzyme- or ZZAP-Treated 1. Wash 1 mL of each red cell specimen
Allogeneic Red Cells once in a large volume of saline,
centrifuge to pack the cells, and re-
Principle move the supernatant saline.
Adsorption of serum with selected red cells 2. To each volume of washed packed
of known phenotypes will remove auto- cells, add one volume of 1% enzyme
antibody and leave antibodies to most solution or two volumes of working

ZZAP reagent. Invert several times to 3. Agitate the serum/cell mixture dur-
mix them. ing incubation to provide maximum
3. Incubate at 37 C: 15 minutes for en- surface contact.
zyme or 30 minutes for ZZAP. Mix 4. A visible clue to the effectiveness of
periodically throughout incubation. adsorption is clumping of the en-
4. Wash the red cells three times with zyme- or ZZAP-treated cells when they
large volumes of saline. Centrifuge are mixed with the serum, especially
at 900 to 1000 × g for at least 5 min- when strong antibodies are present.
utes and remove the last wash as 5. As a guide, when the original serum
completely as possible to prevent di- reactivity is 1+ in the low-ionic-
lution of the serum. strength saline indirect antiglobulin
5. For each of the three red cell speci- test (LISS-IAT), usually only one ad-
mens, mix one volume of treated sorption would be required. Anti-
cells with an equal volume of the pa- bodies with 2+ to 3+ reactivity will
tient’s serum and incubate at 37 C generally be removed in two to three
for 30 minutes, mixing occasionally. adsorptions. Performing greater than
6. Centrifuge at 900 to 1000 × g for ap- four adsorptions increases the risk of
proximately 5 minutes and harvest diluting alloantibody reactivity.
the supernatant serum. 6. If adsorption with enzyme- or ZZAP-
7. Test the three samples of adsorbed treated cells has no effect on the
serum against the cells (untreated) autoantibody, adsorption with un-
used for adsorption, respectively. If treated red cells may be tried.
reactivity is present, repeat steps 5
through 7 with a fresh aliquot of treat- References
ed red cells until no reactivity re- 1. Branch DR, Petz LD. A new reagent (ZZAP)
mains. The three samples of adsorbed having multiple applications in immuno-
hematology. Am J Clin Pathol 1982;78:161-7.
serum can then be tested against an- 2. Judd WJ. Methods in immunohematology.
tibody detection/panel cells and the 2nd ed. Durham, NC: Montgomery Scientific
results compared for demonstration Publications, 1994.
of persisting and removed alloanti-
body activity. See section on allo-
geneic adsorption in Chapter 20. Method 4.11. One-Cell
Notes
Sample Enzyme or ZZAP
1. If the autoantibody is very strong,
Allogeneic Adsorption
three or more aliquots of adsorbing Principle
cells should be prepared. If the first If the Rh and Kidd phenotypes of a re-
adsorption is unsuccessful, the use cently transfused patient are known or can
of a higher proportion of cells to se- be determined, autoantibody activity can
rum/eluate may enhance effective- be adsorbed from the serum onto a single
ness. allogeneic red cell sample, leaving serum
2. The adsorbing red cells should be that can be evaluated for the presence of
tightly packed to remove residual sa- alloantibodies. The red cells used should
line that might dilute the antibodies have the same Rh and Kidd phenotypes as
remaining in the serum/eluate. the patient; they can be treated with en-

zyme or ZZAP to denature antigens (see 7. Test the adsorbed serum against the
Chapter 19). This method is a simplified cells (untreated) used for adsorption.
version of the previous adsorption proce- If reactivity persists, repeat steps 5
dure, but it should be used only if the pa- through 7 with a fresh aliquot of
tient’s Rh and Kidd phenotypes are known treated cells until the serum is no
(see the note). longer reactive.
Specimen Note
Serum, plasma, or eluate to be tested. The s antigen may not be denatured by a
particular enzyme or ZZAP solution. The s
Reagents antigen status of the adsorbing red cells
1. 1% cysteine-activated papain or 1% may need to be considered.
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. Method 4.12. Polyethylene
3. ABO-compatible red cells of the pa-
tient’s Rh and Kidd phenotypes; they Glycol Adsorption
can be reagent cells or cells from any Principle
blood specimen that will yield suffi- Polyethylene glycol (PEG) enhances the
cient cells. adsorption of antibody by untreated red
cells. Testing the adsorbed aliquot against
a panel of red cells can identify the speci-
Procedure ficity of antibodies that remain after ad-
1. Wash the selected allogeneic red cells sorption. This method can be used for both
once in a large volume of saline and autologous and allogeneic adsorption.
centrifuge to pack them.
2. Add one volume of 1% enzyme solu- Specimen
tion or two volumes of ZZAP reagent Serum or plasma to be tested.
to one volume of these packed cells.
Invert several times to mix them.
Reagents
3. Incubate at 37 C: 15 minutes for en-
zyme or 30 minutes for ZZAP. Mix 1. PEG, 20% (20 g PEG, 3350 MW, in
periodically throughout incubation. 100 mL of PBS, pH 7.3) or commer-
4. Wash the cells three times with sa- cial PEG enhancement reagent.
line. Centrifuge at 900 to 1000 × g for 2. Autologous red cells or ABO-com-
at least 5 minutes and remove the patible allogeneic red cells of known
last wash as completely as possible phenotype.
to prevent dilution of the serum.
5. To one volume of treated cells, add Procedure
an equal volume of the patient’s se- 1. Wash aliquots of red cells in large
rum, mix, and incubate at 37 C for 30 volumes of saline three times and
minutes, mixing occasionally. centrifuge for 5 to 10 minutes at
6. Centrifuge at 900 to 1000 × g for ap- 1000 × g. Remove all residual saline.
proximately 5 minutes and harvest 2. To 1 volume (eg, 1 mL) of red cells,
the supernatant serum. add 1 volume of serum and 1 vol-

ume of PEG. Mix well and incubate tained using a different technique.
at 37 C for 15 minutes. To accommodate this potential
3. Centrifuge the serum/PEG/cell mix- weakening of antibody reactivity,
ture for 5 minutes and harvest the some serologists test 6 drops of the
adsorbed serum/PEG mixture. PEG-adsorbed serum.
4. To test the adsorbed serum, add 4 5. Agglutination of the adsorbing red
drops of serum to 1 drop of test red cells does not occur when PEG is
cells, incubate for 15 minutes at 37 used; therefore, there is no visible
C, and proceed to the antiglobulin clue to the efficiency of the adsorp-
test with anti-IgG. The larger volume tion process. As a guide, when the
of serum tested (4 drops) is required original serum reactivity is 1+ in
to account for the dilution of the se- low-ionic-strength saline indirect
rum by the PEG. See the notes. antiglobulin test (LISS-IAT), usually
5. To check for completeness of ad- only one adsorption would be re-
sorption, test the adsorbed serum quired. Antibodies with 2 to 3+ reac-
against the red cells used for the ad- tivity will generally be removed in
sorption. If positive, repeat the ad- two adsorptions.
sorption by adding the adsorbed se-
rum to a fresh aliquot of red cells but References
do not add additional PEG. If the test 1. Leger RM, Garratty G. Evaluation of methods
was negative, test the adsorbed se- for detecting alloantibodies underlying warm
autoantibodies. Transfusion 1999;39:11-6.
rum with a panel of cells. 2. Leger RM, Ciesielski D, Garratty G. Effect of
storage on antibody reactivity after adsorp-
tion in the presence of polyethylene glycol.
Notes Transfusion 1999;39:1272-3.
1. Red cells for adsorption may be chem-
ically modified (eg, with enzymes or
ZZAP) before adsorption if denatur- Method 4.13. The
ation of antigens is desired.
2. The adsorbing cells should be thor-
Donath-Landsteiner Test
oughly packed to remove any resid- Principle
ual saline that could result in dilu- IgG autoantibodies that cause paroxysmal
tion of the antibodies remaining in cold hemoglobinuria (PCH) act as bi-
the serum. phasic hemolysins in vitro. The IgG auto-
3. Test the adsorbed serum on the day antibodies bind to the red cells at cold
it was adsorbed. Weak antibody re- temperatures, and, as the test is warmed
activity may be lost upon storage of to 37 C, complement is activated and lysis
PEG-adsorbed sera, possibly due to of the red cells occurs. The patient for whom
precipitation of the protein noticeable this procedure should be considered is
after 4 C storage. one with a positive direct antiglobulin test
4. Although many laboratories suc- (DAT) resulting from C3; demonstrable
cessfully use the PEG adsorption hemoglobinemia, hemoglobinuria, or
method, some serologists have re- both; and no evidence of autoantibody
ported a weakening or loss of anti- activity in the serum or the eluate made
body reactivity in some samples from the DAT-positive cells. For a discus-
when compared with results ob- sion of PCH, see Chapter 20.

Specimen melting ice (ie, tubes B1, B2). The A3, B3,
and C3 tubes serve as a control of the nor-
Serum separated from a freshly collected
mal sera complement source and should
blood sample maintained at 37 C.
not manifest hemolysis.
Reagents
Notes
1. Freshly collected pooled normal sera
known to lack unexpected antibod- 1. The biphasic nature of the hemoly-
ies, to use as a source of complement. sin associated with PCH requires
2. 50% suspension of washed group O that serum be incubated with cells at
red cells that express the P antigen, a cold temperature first (eg, melting
eg, antibody detection cells. ice bath) and then at 37 C.
2. Active complement is essential for
Procedure demonstration of the antibody. Be-
cause patients with PCH may have
1. Label three sets of three 10 × 75-mm low levels of serum complement,
test tubes as follows: A1-A2-A3; fresh normal serum should be in-
B1-B2-B3; C1-C2-C3. cluded in the reaction medium as a
2. To tubes 1 and 2 of each set, add 10 source of complement.
volumes (eg, drops) of the patient’s 3. To avoid loss of antibody by cold
serum. autoadsorption before testing, the
3. To tubes 2 and 3 of each set, add 10 patient’s blood should be allowed to
volumes of fresh normal serum. clot at 37 C, and the serum sepa-
4. To all tubes, add one volume of the rated from the clot at this tempera-
50% suspension of washed P-posi- ture.
tive red cells and mix well. 4. If a limited amount of blood is avail-
5. Place the three “A” tubes in a bath of able (eg, from young children), set
melting ice for 30 minutes and then up tubes A-1, A-2, A-3 and C-1, C-2;
at 37 C for 1 hour. if there is only enough serum for two
6. Place the three “B” tubes in a bath of tests (ie, 20 drops), set up tubes A-2,
melting ice and keep them in melt- A-3, and C-2.
ing ice for 90 minutes. 5. To demonstrate the P specificity of
7. Place the three “C” tubes at 37 C and the Donath-Landsteiner antibody,
keep them at 37 C for 90 minutes. ABO-compatible p red cells should
8. Centrifuge all tubes and examine the be tested in a second set of tubes
supernatant fluid for hemolysis. A-1, A-2, and A-3. No lysis should
develop in these tubes, confirming
Interpretation the P specificity of the antibody.
The Donath-Landsteiner test is considered
positive when the patient’s serum, with or References
without added complement, causes
hemolysis in the tubes that were incu- 1. Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
bated first in melting ice and then at 37 C
Publications, 1994.
(ie, tubes A1 and A2), and there is no
2. Dacie JV, Lewis SM. Practical hematology. 7th
hemolysis in any of the tubes maintained ed. New York: Churchill Livingstone, 1991:
throughout at 37 C (ie, tubes C1, C2) or in 500-1.

by adding 1 mL of untreated red

Method 4.14. Detection of cells (without the drug) to 15 mL of
Antibodies to Penicillin or the same buffer. Incubate both tubes
Cephalosporins by Testing for 1 hour at room temperature with
occasional mixing. Wash three times
Drug-Treated Red Cells in saline and store in PBS at 2 to 8 C
for up to 1 week. See note 1.
Principle 2. For cephalosporin-treated cells, dis-
See Chapter 20 for a discussion of the solve 400 mg of the drug in 10 mL of
mechanisms by which drugs cause a posi- PBS, pH 7.3. Add 1 mL of red cells. In
tive direct antiglobulin test (DAT). The a separate tube, prepare control cells
preparations of drugs used should, to the by adding 1 mL of untreated red cells
extent possible, be the same as those (without the drug) to 10 mL of PBS.
given to the patient. For drugs other than Incubate both tubes for 1 hour at 37
penicillin and the cephalosporins, refer to C with occasional mixing. Wash three
published reports for the method used to times in saline and store in PBS for
treat the red cells. up to 1 week at 2 to 8 C. See notes 1
and 2.
Specimen 3. Mix 2 or 3 drops of each specimen
Serum or plasma and eluate (and last (serum, eluate, and last wash) and
wash) to be studied. controls with 1 drop of 5% saline
suspension of drug-treated red cells.
Reagents 4. In parallel, test each specimen and
control with the untreated red cells.
1. 0.1 M sodium barbital-buffer (BB) at
See notes 3 and 4.
pH 9.6 to 9.8, prepared by dissolving
5. Incubate the tests at 37 C for 60 min-
2.06 g of sodium barbital in 80 mL of
utes. Centrifuge and examine for
distilled or deionized H20. Adjust the
hemolysis and agglutination. Record
pH to between 9.6 and 9.8 with 0.1 N
the results.
HCl. Bring total volume to 100 mL.
6. Wash the cells four times in saline
Store at 2 to 8 C.
and test by an indirect antiglobulin
technique using polyspecific anti-
7.3 (see Method 1.7).
human globulin or anti-IgG reagent.
3. Drug, eg, penicillin, cephalosporin.
Centrifuge and examine for aggluti-
4. Washed, packed, group O red cells.
5. Normal sera/plasma. nation. Record the results.
6. IgG-coated red cells. 7. Confirm the validity of negative tests
by adding IgG-coated red cells.
Procedure
1. For penicillin-treated cells, dissolve
Interpretation
600 mg of penicillin in 15 mL of BB. Reactivity (hemolysis, agglutination, and/
This high pH is optimal, but if the or positive indirect antiglobulin test) with
buffer is unavailable, PBS, pH 7.3, drug-treated cells, but not with untreated
can be used. Add 1 mL of red cells. In cells, indicates that drug antibodies are
a separate tube, prepare control cells present (see notes 3 and 4). No hemolysis

will be seen in tests with plasma or the test serum at a 1 in 20 dilution in PBS.
eluate. Antibodies to either penicillin or Normal sera diluted 1 in 20 generally
cephalothin may cross-react with cells do not react nonspecifically. Thus,
treated with the other drug (ie, penicillin reactivity of the diluted serum with
antibodies may attach to cephalothin- the drug-treated cells but not with
treated cells and vice versa). Antibodies to the untreated cells indicates that
other cephalosporins may react with drug antibody is present.
cephalothin-treated cells. It is best to treat 5. When testing cefotetan-treated red
cells with the drug that is suspect. cells, test the serum at a 1 in 100 di-
Negative results without a positive con- lution in PBS to prevent a false-posi-
trol can only be interpreted to mean that tive test result. In addition to the
drug antibodies were not detected. The nonspecific uptake of protein onto
drug may or may not be bound to the test cefotetan-treated red cells, some
red cells. normal sera appear to have a “natu-
rally occurring” anticefotetan,2 a few
of which react weakly at a 1 in 20 di-
lution. Cases of cefotetan-induced
Notes
immune hemolytic anemia are asso-
1. The volume of drug-treated red cells ciated with very high antibody titers
can be scaled down as long as the ra- (eg, mean antiglobulin test titer =
3
tio of the 40 mg/mL drug solution to 16,000).
red cells is constant; eg, 120 mg pen- 6. About 30% of patients with antice-
icillin in 3 mL BB plus 0.2 mL red fotetan also have a drug-independ-
3
cells or 100 mg cephalosporin in 2.5 ent antibody ; in these cases, the
mL PBS plus 0.25 mL red cells.1 serum and/or eluate, when tested
2. Cephalosporins do not require a undilute, may react with both the
high pH for optimal coating of red cefotetan-treated and untreated red
cells. In fact, a lower pH, ie, pH 6 to cells.
7, decreases nonspecific protein ad- 7. The last wash control can some-
sorption seen when a high pH buffer times react with cefotetan-treated
is used. The least amount of nonspe- red cells when antibodies to cefo-
cific protein adsorption by drug- tetan are present, regardless of the
treated red cells will occur if a pH 6.0 wash solution used (commercial
buffer is used, but this leads to a acid eluate kit wash solution, 4 C
slight decrease in coating by the LISS, or 4 C PBS) or the number of
drug. washes performed.3
3. Test normal pooled serum and PBS 8. Prepare drug solutions just before
as negative controls and, when avail- use.
able, a specimen known to contain 9. Drug-treated red cells may be kept
an antibody to the drug being inves- in PBS at 4 C for up to 1 week; how-
tigated as a positive control. ever, there may be some weakening
4. To control for nonspecific protein of drug coating upon storage. Drug-
adsorption and nonspecific aggluti- treated and untreated red cells may
nation of normal sera observed with also be stored frozen.
some cephalosporins (eg, cephalo- 10. When antibodies are not detected
thin), test the normal serum and the with drug-treated red cells, test by

the immune complex method (Method 5. Polyspecific antihuman globulin re-

4.15). Antibodies to some third-gen- agent.
eration cephalosporins (eg, ceftriaxone) 6. IgG-coated red cells.
do not react with drug-treated red cells.
Procedure
References
1. Petz LD, Garratty G. Immune hemolytic 1. Prepare a 1 mg/mL solution of the
anemias. 2nd ed. Philadelphia: Churchill drug in PBS. Centrifuge to remove
Livingstone, 2004.
any particulate matter and adjust
2. Ar ndt P, Garratty G. Is severe immune
hemolytic anemia, following a single dose of the pH of the supernatant fluid to
cefotetan, associated with the presence of approximately 7 with either 1 N
“naturally occurring” anti-cefotetan? (ab-
NaOH or 1 N HCl, as required, if the
stract) Transfusion 2001;41(Suppl):24S.
3. Arndt PA, Leger RM, Garratty G. Serology of pH is below 5 or above 8.
antibodies to second- and third-generation 2. Label two sets of tubes for the fol-
cephalosporins associated with immune
lowing test mixtures:
hemolytic anemia and/or positive direct
antiglobulin tests. Transfusion 1999;39:1239- a. Patient’s serum + drug.
46. b. Patient’s serum + PBS.
c. Patient’s serum + complement
(normal serum) + drug.
d. Patient’s serum + complement
Method 4.15. Demonstration (normal serum) + PBS.
of Immune-Complex e. Normal serum + drug.
Formation Involving Drugs f. Normal serum + PBS.
3. Add 2 volumes (eg, 2 drops) of each
Principle component in the appropriate tubes
For a discussion of the mechanism of (eg, 2 drops of serum + 2 drops of
drug-induced immune-complex forma- drug).
tion, see Chapter 20. 4. Add 1 drop of a 5% saline suspen-
sion of untreated group O reagent
Specimen red cells to one set of tubes. Add 1
The patient’s serum. drop of a 5% saline suspension of
enzyme-treated group O reagent red
Reagents cells to the second set of tubes.
1. The drug under investigation, in the 5. Mix and incubate at 37 C for 1 to 2
same form (powder, tablet, capsules) hours, with periodic gentle mixing.
that the patient is receiving. 6. Centrifuge, examine for hemolysis
2. Phosphate-buffered saline (PBS) at and agglutination, and record the re-
pH 7.3 (see Method 1.7). sults.
3. Fresh, normal serum known to lack 7. Wash the cells four times in saline
unexpected antibodies, as a source and test with a polyspecific antiglo-
of complement. bulin reagent.
4. Pooled group O reagent red cells, 5% 8. Centrifuge, examine for agglutina-
suspension, one aliquot treated with tion, and record the results.
a proteolytic enzyme (see Method 9. Confirm the validity of negative tests
3.5.6) and one untreated. by adding IgG-coated red cells.

Interpretation ment may increase the sensitivity of

the test.
Hemolysis, direct agglutination, or posi- 6. If tests for drug antibodies by the
tive indirect antiglobulin tests can occur immune-complex method and drug
together or separately. Reactivity in any of adsorption method are noninforma-
the tests containing the patient’s serum to tive, consider testing with ex-vivo
which the drug was added, and absence antigen (see Method 4.16).
of reactivity in the corresponding control
tests containing PBS instead of the drug, Reference
indicate that antibody to the drug is pres-
Petz LD, Garratty G. Immune hemolytic anemias.
ent. See note 4. 2nd ed. Philadelphia: Churchill Livingstone, 2004.
Notes
1. The drug may be more easily dis- Method 4.16. Ex-Vivo
solved by incubation at 37 C and vig-
orous shaking of the solution. If the
Demonstration of
drug is in tablet form, crush it with a Drug/Anti-Drug Complexes
mortar and pestle and remove any
visible outer tablet coating material Principle
before adding PBS. Immune drug/anti-drug complexes can
2. Not all drugs will dissolve com- activate complement and cause hemolysis
pletely in PBS. Consult the manufac- in vivo. These immune complexes may be
turer or a reference such as the Merck demonstrable by serologic testing in the
Index for the solubility of the drug in presence of the drug, but with some drugs
question. A previous report of drug- (notably nomifensine), antibodies are di-
induced immune hemolytic anemia rected against metabolites of the drug,
resulting from the drug in question rather than the native drug. Serum and/or
may provide information on the urine from volunteers who have ingested
drug solution preparation. therapeutic levels of the drug can be used
3. When available, a serum/plasma as a source of these metabolites. See note
known to contain antibody with the 1.
drug specificity being evaluated This procedure is used to investigate
should be included as a positive drug-associated immune hemolysis, partic-
control. ularly when use of the preceding methods
4. Tests without the drug added may be has been noninformative.
positive if autoantibodies or circu-
lating drug-antibody immune com- Specimen
plexes are present in the patient’s
Patient’s serum.
sample. Autoantibody reactivity would
be persistent over time, whereas cir-
culating immune complexes are Reagents
transient. 1. Polyspecific antihuman globulin
5. Testing with enzyme-treated red (AHG) reagent.
cells and the addition of fresh nor- 2. Drug metabolites from volunteer
mal serum as a source of comple- drug recipients. See note 2.

a. Volunteer serum (VS) obtained 3. Add 1 drop of a 5% saline suspen-

immediately before (VS0), at 1 sion of the untreated group O re-
hour (VS1), and 6 hours (VS6) agent red cells to one set of tubes.
after drug administration. Di- Add 1 drop of a 5% saline suspen-
vide serum into 1-mL aliquots sion of enzyme-treated reagent red
and store them at 2 to 8 C for a cells to the second set of tubes.
few hours or at –20 C or colder 4. Mix the contents of each tube and
until use. incubate at 37 C for 1 to 2 hours, with
b. Volunteer urine (VU) obtained periodic mixing.
immediately before (VU0), at 1 5. Centrifuge, examine for agglutina-
hour (VU1), 3.5 hours (VU3.5), 7 tion and/or hemolysis, and record
hours ( VU 7 ), and 16 hours the results.
(VU 16 ) after drug administra- 6. Wash the cells four times with saline
tion. Divide into 1-mL aliquots and test with a polyspecific antiglo-
and store them at 2 to 8 C for a bulin reagent.
few hours or at –20 C or colder 7. Centrifuge, examine for agglutina-
until use. tion, and record the results.
3. Fresh normal serum, known to lack 8. Confirm the validity of negative tests
unexpected antibodies, as a source of by adding IgG-coated red cells.
complement.
7.3 (see Method 1.7).
5. Pooled group O reagent red cells Interpretation
washed three times with saline and Hemolysis, direct agglutination, or reac-
resuspended to a 5% concentration tivity with AHG in any of the tubes con-
with PBS. taining test serum and VS or VU, and ab-
6. Pooled, enzyme-treated, group O red sence of reactivity in all the control tubes,
cells, 5% suspension in PBS. indicate antibody against a metabolite of
7. IgG-coated red cells. the drug in question.
Procedure
1. For each volunteer serum and/or Notes
volunteer urine sample collected, la- 1. Approval of the institutional ethics
bel two sets of the following test committee should be obtained for
mixtures: the use of volunteers for obtaining
a. Patient’s serum + VS (or VU). drug metabolites.
b. Patient’s serum + PBS. 2. The urine sample collection times
c. Patient’s serum + complement given are those optimal for antibod-
+ VS (or VU). ies to nomifensine metabolites; dif-
d. Patient’s serum + complement ferent collection times may be re-
+ PBS. quired for other drugs.
e. Complement + VS (or VU). 3. Complement may be omitted from
f. Complement + PBS. step 1 if the VS samples have been
2. Add 0.1 mL of each component to the kept on ice and are used for testing
appropriate tubes. within 8 hours of collection.

4. Testing with enzyme-treated red References

cells and the addition of fresh nor- 1. Judd WJ. Methods in immunohematology.
mal serum as a source of complement 2nd ed. Durham, NC: Montgomery Scientific
may increase the sensitivity of the Publications, 1994.
2. Salama A, Mueller-Eckhardt C. The role of me-
test. tabolite-specific antibodies in nomifensine-
dependent immune hemolytic anemia. N
Engl J Med 1985;313:469-74.

Methods Section 5: Hemolytic Disease of the Fetus and Newborn
Methods Section 5
Hemolytic Disease of the

Fetus and Newborn
number of fetal cells. The acid-elution pro-

Method 5.1. Indicator Cell cedure given below and flow cytometry are
Rosette Test for
Section 5
acceptable choices. If a commercial test is
Fetomaternal Hemorrhage available, the directions in the package in-
sert should be followed.
Principle
This test detects D+ red cells in the blood Specimen
of a D– woman whose fetus or recently
A 2% to 5% saline suspension of washed
delivered infant is D+. When reagent
red cells from a maternal blood sample.
anti-D is added to maternal blood con-
taining D+ fetal cells, fetal red cells be-
come coated with anti-D. When D+ re- Reagents
agent cells are subsequently added, easily Prepared reagents are commercially avail-
visible rosettes are formed, with several able. The steps below can be used for in-
red cells clustered around each anti- house preparation.
body-coated D+ red cell. 1. Negative control: a 2% to 5% saline
Although the number of rosettes is suspension of washed red cells known
roughly proportional to the number of D+ to be D–.
red cells present in the original mixture, 2. Positive control: a 2% to 5% saline
this test provides only qualitative informa- suspension of a mixture containing
tion about fetal-maternal admixture. Speci- approximately 0.6% D+ red cells and
mens giving a positive result should be sub- 99.4% D– red cells. The positive con-
jected to further testing to quantify the trol can be prepared by adding 1 drop
793

of a 2% to 5% suspension of D+ con- Interpretation

trol cells to 15 drops of a 2% to 5%
The absence of rosettes is a negative re-
suspension of washed D– control cells.
sult. With enzyme-treated indicator cells,
Mix well, then add 1 drop of this cell
up to one rosette per three fields may oc-
suspension to 9 drops of the 2% to cur in a negative specimen. With un-
5% suspension of D– red cells. Mix well. treated indicator cells and an enhancing
3. Indicator red cells: a 2% to 5% saline medium, there may be up to six rosettes
suspension of group O, R2R2 red cells. per five fields in a negative test. The pres-
Either enzyme-treated or untreated ence of more rosettes than these allow-
cells in an enhancing medium can be able maxima constitutes a positive result,
used. and the specimen should be examined
4. Chemically modified or high-protein using a test that quantifies the amount of
reagent anti-D serum. Some mono- fetal blood present.
clonal/polyclonal blended reagents The presence of rosettes or agglutination
are unsuitable for use in this method. in the negative control tube indicates inad-
The antisera selected for use should equate washing after incubation, allowing
be evaluated for suitability before in- residual anti-D to agglutinate the D+ indi-
corporation into the test procedure. cator cells. A strongly positive result is seen
with red cells from a woman whose Rh
Procedure phenotype is weak D rather than D–; mas-
1. To each of three test tubes, add 1 drop sive fetomaternal hemorrhage may pro-
(or volume specified in the manufac- duce an appearance difficult to distinguish
from that caused by a weak D phenotype,
turer’s instructions) of reagent anti-D.
and a quantitative test for fetal cells should
2. Add 1 drop of maternal cells, nega-
be performed. If the infant’s cells are shown
tive control cells, and positive control
to be weak D, a negative result on the
cells to the appropriately labeled tubes.
mother’s specimen should be interpreted
3. Incubate at 37 C for 15 to 30 min-
with caution. In this situation, a quantita-
utes, or as specified by the manufac-
tive test that does not rely on D antigen
turer’s instructions.
expression should be performed.
4. Wash cell suspensions at least four
times with large volumes of saline, to
remove all unbound reagent anti-D.
Reference
Decant saline completely after last Sebring ES, Polesky HF. Detection of fetal mater-
nal hemorrhage in Rh immune globulin candi-
wash. dates. Transfusion 1982;22:468-71.
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 Method 5.2. Acid-Elution Stain
at 900 to 1000 × g. (Modified Kleihauer-Betke)
7. Resuspend cell button and examine
the red cell suspension microscopi- Principle
cally at 100 to 150 × magnification. Fetal hemoglobin resists elution from red
8. Examine at least 10 fields and count cells under acid conditions, whereas adult
the number of red cell rosettes in each hemoglobin is eluted. When a thin blood
field. smear is exposed to an acid buffer, hemo-

Methods Section 5: Hemolytic Disease of the Fetus and Newborn 795
globin from adult red cells is leached into 2. Fix the smears in 80% ethyl alcohol
the buffer so that only the stroma remains; for 5 minutes.
fetal cells retain their hemoglobin and 3. Wash the smears with distilled wa-
can be identified by a positive staining ter.
pattern. The approximate volume of feto- 4. Immerse the smears in McIlvaine’s
maternal hemorrhage can be calculated buffer, pH 3.2, for 11 minutes at room
from the percentage of fetal red cells in temperature or 5 minutes at 37 C. This
the maternal blood film. reaction is temperature-sensitive.
5. Wash the smears in distilled water.
Specimen 6. Immerse the smears in erythrosin B
Maternal anticoagulated whole blood for 5 minutes.
sample. 7. Wash the smears completely in dis-
tilled water.
Reagents 8. Immerse the smears in Harris hema-
toxylin for 5 minutes.
Prepared reagents are commercially avail- 9. Wash the smears in running tap wa-
able in kits. The steps below can be used ter for 1 minute.
for in-house preparations. 10. Examine dry using 40× magnifica-
1. Stock solution A (0.1 M of citric acid). tion, count a total of 2000 red cells,
C6H8O7•H2O, 21.0 g, diluted to 1 liter and record the number of fetal cells
with distilled water. Keep it in the re- observed.
frigerator. 11. Calculate the percent of fetal red cells
2. Stock solution B (0.2 M of sodium in the total counted.
phosphate). Na2HPO4•7H2O, 53.6 g,
diluted to 1 liter with distilled water.
Interpretation
Keep it in the refrigerator.
3. McIlvaine’s buffer, pH 3.2. Add 75 1. Fetal cells are bright pink and re-
mL of stock solution A to 21 mL of fractile; normal adult red cells ap-
stock solution B. Prepare fresh mix- pear as very pale ghosts.
ture for each test. This buffer mix- 2. The conversion factor used to indi-
ture should be brought to room tem- cate the volume (as mL of whole
perature or used at 37 C. blood) of fetomaternal hemorrhage
4. Erythrosin B, 0.5% in water. is the percent of fetal red cells ob-
5. Harris hematoxylin (filtered). served times 50.
6. 80% ethyl alcohol.
7. Positive control specimen. Ten parts
of anticoagulated adult blood, mixed Note
with one part of anticoagulated ABO- The accuracy and precision of this proce-
compatible cord blood. dure are poor, and decisions regarding Rh
8. Negative control specimen. Anticoa- Immune Globulin (RhIG) dosage in mas-
gulated adult blood. sive fetomaternal hemorrhage should be
made accordingly. If there is a question
Procedure regarding the need for additional RhIG, it
1. Prepare very thin blood smears, di- is preferable to administer another dose
luting blood with an equal volume of to prevent the risks of undertreatment. (See
saline. Air dry. Table 23-1 for dosage.)

Reference Materials
Sebring ES. Fetomaternal hemorrhage—incidence 1. Antihuman IgG: need not be heavy-
and methods of detection and quantitation. In:
chain-specific.
Garratty G, ed. Hemolytic disease of the newborn.
Arlington, VA: AABB, 1984:87-118. 2. Isotonic saline.
3. Volumetric pipettes, or equivalent:
0.1- to 0.5-mL delivery, with dispos-
able tips.
Method 5.3. Antibody 4. Red cells: group O reagent red cells,
Titration Studies to Assist in 2% suspension. (See note 1 regard-
ing the selection of red cells for test-
Early Detection of Hemolytic ing.) Avoid using Bg+ red cells be-
Disease of the Fetus and cause they may result in falsely high
Newborn values, especially with sera from
multiparous women.
Principle 5. IgG-coated red cells.
Antibody titration is a semiquantitative
method of determining antibody concen- Quality Control
tration. Serial twofold dilutions of serum
1. Test the preceding sample in parallel
are prepared and tested for antibody ac-
with the most recent sample.
tivity. The reciprocal of the highest dilu-
2. Prepare the dilutions using a sepa-
tion of plasma or serum that gives a 1+ re-
rate pipette for each tube. Failure to
action is referred to as the titer (ie, 1 in
do so will result in falsely high titers
128 dilution; titer = 128).
because of carryover.
In pregnancy, antibody titration is per-
3. Confirm all negative reactions with
formed to identify women with significant
levels of antibodies that may lead to IgG-coated red cells (see step 9 be-
hemolytic disease of the fetus and newborn low).
(HDFN) and, for low-titer antibodies, to es-
tablish a baseline for comparison with Procedure
titers found later in pregnancy. Titration of 1. Using 0.5-mL volumes, prepare serial
non-Rh antibodies should be undertaken twofold dilutions of serum in saline.
only after discussion with the obstetrician The initial tube should contain un-
about how the data will be used in the clini- diluted serum and the doubling di-
cal management of the pregnancy. The sig- lution range should be from 1 in 2 to
nificance of titers has been sufficiently es- 1 in 2048 (total of 12 tubes). (See
tablished only for anti-D (using a saline Method 3.7.)
technique). 2. Place 0.1 mL of each dilution into
appropriately labeled test tubes.
Specimen 3. Add 0.1 mL of the 2% suspension of
Serum for titration (containing potentially red cells to each dilution. Alterna-
significant unexpected antibodies to red tively, for convenience, add 1 drop of
cell antigens, 1 mL). If possible, test the a solution of a 3% to 4% suspension
current sample in parallel with the most of red cells as supplied by the re-
recent previously submitted (preceding) agent manufacturer, although this
sample from the current pregnancy. method is less precise.

Methods Section 5: Hemolytic Disease of the Fetus and Newborn 797
4. Gently agitate the contents of each body; save an appropriately labeled

tube; incubate at 37 C for 1 hour. aliquot of the serum (frozen at –20 C
5. Wash the red cells four times with or colder) for comparative studies
saline; completely decant the final with the next submitted sample.
wash supernatant. 3. When the titer (eg, >16) and the anti-
6. To the dry red cell buttons thus ob- body specificity have been associ-
tained, add anti-IgG according to the ated with HDFN, it is recommended
manufacturer’s directions. that repeat titration studies be per-
7. Centrifuge as for hemagglutination formed every 2 to 4 weeks, beginning
tests. at 18 weeks’ gestation; save an ali-
8. Examine the red cells macroscopically; quot of the serum (frozen at –20 C or
grade and record the reactions. colder) for comparative studies with
9. Add IgG-coated red cells to all nega- the next submitted sample.
tive tests; recentrifuge and examine 4. When invasive procedures (eg, am-
the tests for macroscopic agglutina- niocentesis) have demonstrated fetal
tion; repeat the testing if the tests with compromise and are being used to
IgG-coated red cells are nonreactive. monitor the pregnancy, use the opti-
mal method for follow-up of fetal
Interpretation well-being. However, if initial studies
do not show fetal compromise or the
The titer is reported as the reciprocal of
Liley curve result is borderline, addi-
the highest dilution of serum at which 1+
tional titrations may be helpful as a
agglutination is observed. A titer ≥16 (this
means of following the pregnancy in
value may vary according to the labora-
a less invasive manner.
tory) is considered significant and may
5. Each institution should develop a
warrant further monitoring for HDFN.
policy to ensure a degree of unifor-
mity in reporting and interpreting
Notes antibody titers.
1. The selection of the most suitable 6. For antibodies to low-incidence an-
phenotype of red cells to use when tigens, consider using putative pa-
performing titration studies for HDFN ternal red cells, having established
is controversial. Some workers select that they express the antigen in ques-
red cells that have the strongest ex- tion.
pression of antigen, such as R2R2 for 7. Do not use enhancement techniques
anti-D. Others select red cells with the [albumin, polyethylene glycol, low-
phenotype that would be expected ionic-strength saline (LISS)] or en-
in fetal circulation—ie, red cells that zyme-treated red cells because falsely
express a single dose of the antigen, elevated titers may be obtained. Gel
such as R 1 r for testing for anti-D. testing is not recommended.
Whichever viewpoint is followed, it 8. LISS should not be used as a diluent
is important that the laboratory be in titration studies; nonspecific up-
consistent and use red cells of the take of globulins may occur in serum-
same phenotype for future titrations LISS dilutions.
to test the same patient’s serum. 9. Failure to obtain the correct results
2. Titration studies should be performed may be caused by 1) incorrect tech-
upon initial detection of the anti- nique, notably, failure to use sepa-

rate pipette tips for each dilution or mendations for serologic management of the
fetus, newborn infant, and obstetric patient.
2) failure to adequately mix thawed
frozen serum. 3. Judd WJ. Methods in immunohematology.
2nd ed. Durham, NC: Montgomery Scientific
References Publications, 1994.
4. Judd WJ. Practice guidelines for prenatal and
1. Issitt PD, Anstee DJ. Applied blood group se- perinatal immunhematology, revisited. Trans-
rology. 4th ed. Durham, NC: Montgomery fusion 2001;41:1445-52.
Scientific Publications, 1998:1067-9.
2. Judd WJ, Luban NLC, Ness PM, et al. Prenatal
and perinatal immunohematology: Recom-

Methods Section 6: Blood Collection, Storage, and Component Preparation
Methods Section 6
Blood Collection, Storage,

and Component Preparation
are rare; donors whose drop of blood sinks

Method 6.1. Copper Sulfate nearly always have an acceptable hemoglo-
Method for Screening Donors bin level. False-negative reactions occur
for Anemia fairly commonly and can cause inappropri-
ate deferral.1,2 Measuring hemoglobin by
Principle
another method or determining hematocrit
This method estimates the hemoglobin sometimes reveals that the prospective do-
content of blood from its specific gravity. nor is acceptable.
A drop of blood in contact with copper
sulfate solution of specific gravity 1.053
Reagents and Materials
becomes encased in a sac of copper pro-
teinate, which prevents dispersion of the 1. Copper sulfate solution at specific
fluid or any change in specific gravity for gravity 1.053, available commer- Section 6
about 15 seconds. If the specific gravity of cially. Store it in tightly capped con-
the blood is higher than that of the solu- tainers to prevent evaporation. The
tion, the drop will sink within 15 seconds; solution should be kept at room tem-
if not, the drop will hesitate and remain perature or brought to room temper-
suspended or rise to the top of the solu- ature before it is used.
tion. A specific gravity of 1.053 corre- 2. Sterile gauze, antiseptic wipes, and
sponds to a hemoglobin concentration of sterile lancets.
12.5 g/dL. 3. Containers for the disposal of sharps
This is not a quantitative test; it shows and other biohazardous materials.
only whether the prospective donor’s he- 4. Capillary tubes and dropper bulbs or
moglobin is below or above the acceptable a device to collect capillary blood
level of 12.5 g/dL. False-positive reactions without contact.
799

Procedure at an acceptable level for blood do-

nation. If time and equipment permit,
1. Into a labeled, clean, dry tube or
it is desirable to perform a quantita-
bottle, dispense a sufficient amount
tive measurement of hemoglobin or
(at least 30 mL) of copper sulfate so-
hematocrit.
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 Notes
before beginning each day’s deter- 1. A certificate of analysis from the man-
minations. ufacturer should be obtained with
2. Clean the site of the skin puncture each new lot of copper sulfate solu-
thoroughly with antiseptic solution tion.
and wipe it dry with sterile gauze. 2. Used solution should be disposed of
3. Puncture the finger firmly, near the as biohazardous or chemical mate-
end but slightly to the side, with a rial because of the blood in the con-
sterile, disposable lancet or spring- tainer. Refer to local and state laws
loaded, disposable needle system. A regarding disposal.
good free flow of blood is important. 3. Use care to prevent blood from con-
Do not squeeze the puncture site re- taminating work surfaces, the donor’s
peatedly because this may dilute the clothing, or other persons or equip-
drop of blood with tissue fluid and
ment.
lower the specific gravity.
4. Cover the container between uses to
4. Collect the blood in a capillary tube
prevent evaporation.
without allowing air to enter the
tube.
5. Let one drop of blood fall gently
from the tube at a height about 1 cm References
above the surface of the copper sul- 1. Lloyd H, Collins A, Walker W, et al. Volunteer
fate solution. blood donors who fail the copper sulfate
screening test: What does failure mean, and
6. Observe for 15 seconds. what should be done? Transfusion 1988;28:
7. Dispose of lancets and capillary tubes 467-9.
in appropriate biohazard containers. 2. Morris MW, Davey FR. Basic examination of
Dispose of gauze appropriately; blood. In: Henry JB, ed. Clinical diagnosis
and management by laboratory methods.
gauze contaminated with droplets of 20th ed. Philadelphia: WB Saunders, 2001:
blood that subsequently dry such 479-519.
that the item is stained but not
soaked or caked may be considered
nonhazardous.
Method 6.2. Arm Preparation
Interpretation
for Blood Collection
1. If the drop of blood sinks, the do-
nor’s hemoglobin is at an acceptable Detailed instructions are specific to each
level for blood donation. manufacturer and should be followed as
2. If the drop of blood does not sink, indicated. The following procedure is
the donor’s hemoglobin may not be written in general terms as an example.

Methods Section 6: Blood Collection, Storage, and Component Preparation 801
Principle chlorhexidine and 70% isopropyl al-

cohol) should be designated by the
Iodophor compounds, or other sterilizing
blood bank physician. Green soap
compounds, are used to sterilize the veni-
should not be used.
puncture site before blood collection.
2. For donors sensitive to both iodine
and chlorhexidine, a method using
Materials only isopropyl alcohol could be con-
1. Scrub solution: Disposable povidone- sidered. The preferred procedure is
iodine scrub 0.75% or disposable the use of a 30-second up-and-down
povidone-iodine swabstick 10%; avail- scrub, followed by enough time for
able in prepackaged single-use form. the skin to dry. A second scrub is
2. Preparation solution: 10% povidone- then applied. This method may re-
iodine; available in prepackaged sin- quire a variance from the Food and
gle-use form. Drug Administration.
3. Sterile gauze. 3. Arm preparation methods approved
by the Food and Drug Administra-
tion are available at http://www.fda.
Procedure gov/cber/infosheets/armprep.htm.
1. Apply tourniquet or blood pressure
Reference
cuff; identify venipuncture site, then
release tourniquet or cuff. Goldman M, Roy G, Frechette N, et al. Evaluation
of donor skin disinfection methods. Transfusion
2. Scrub area at least 4 cm (1.5 inches) 1997;37:309-12.
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-
Method 6.3. Phlebotomy and
lution of iodophor compound. Excess Collection of Samples for
foam may be removed, but the arm Processing and Compatibility
need not be dry before the next step.
3. Starting at the intended site of veni- Tests
puncture and moving outward in a Principle
concentric spiral, apply “prep” solu- Blood for transfusion and accompanying
tion; let stand for 30 seconds or as samples is obtained from prominent veins
indicated by manufacturer. on the donor’s arm, usually in the area of
4. Cover the area with dry, sterile gauze the antecubital fossa.
until the time of venipuncture. After
the skin has been prepared, it must Materials
not be touched again. Do not repal-
1. Sterile collection bag containing an-
pate the vein at the intended veni-
ticoagulant, with integrally attached
puncture site.
tubing and needle.
2. Metal clips and hand sealers.
Notes 3. Balance system to monitor volume
1. For donors sensitive to iodine (tinc- of blood drawn.
ture or povidone preparations), an- 4. Sterile gauze and clean instruments
other method (eg, ChloraPrep 2% (scissors, hemostats, forceps).

5. Test tubes for sample collection. the skin, palpation of the skin above
6. Device for stripping blood in the the needle stem may be performed
tubing. with a gloved finger, provided the
7. Dielectric sealer (optional). needle is not touched. When the
needle position is acceptable, tape
Procedure the tubing to the donor’s arm to hold
the needle in place and cover the
1. Ask donor to confirm his or her iden- site with sterile gauze.
tification. 8. Release the hemostat. Open the tem-
2. Ensure that all labeling on blood con- porary closure between the interior
tainer, processing tubes, retention of the bag and the tubing.
segment, and donor records is cor- 9. Ask the donor to open and close hand
rect. slowly every 10 to 12 seconds during
3. Prepare donor’s arm as described in collection.
Method 6.2. 10. Keep the donor under observation
4. Inspect bag for any defects and dis- throughout the donation process.
coloration. The anticoagulant and The donor should never be left unat-
additive solutions should be intended during or immediately after
spected for particulate contaminants. donation.
5. Position bag below the level of the 11. Mix blood and anticoagulant gently
donor’s arm. and periodically (approximately ev-
a. If a balance system is used, be ery 45 seconds) during collection.
sure the counterbalance is level Mixing may be done by hand or by
and adjusted for the amount of continuous mechanical mixing.
blood to be drawn. Unless metal 12. Be sure blood flow remains fairly
clips and a hand sealer are used, brisk, so that coagulation activity is
make a very loose overhand knot not triggered. If there is continuous,
in the tubing. Hang the bag and adequate blood flow and constant
route the tubing through the agitation, rigid time limits are not
p i n c h c l a m p. A h e m o s t a t necessary. However, units requiring
should be applied to the tubing more than 15 minutes to draw may
before the needle is uncapped not be suitable for preparation of
to prevent air from entering the Platelets, Fresh Frozen Plasma, or
line. Cryoprecipitated AHF. The time re-
b. If a balance system is not used, quired for collection can be moni-
be sure to monitor the volume tored by indicating the time of phle-
of blood drawn. botomy or the maximal allowable
6. Reapply tourniquet or inflate blood time (start time plus 15 minutes) on
pressure cuff. Ask the donor to open the donor record.
and close hand until previously se- 13. Monitor volume of blood being drawn.
lected vein is again prominent. If a balance is used, the device will
7. Uncover sterile needle and perform interrupt blood flow after the proper
the venipuncture immediately. A amount has been collected. One mL
clean, skillful venipuncture is essen- of blood weighs at least 1.053 g, indi-
tial for collection of a full, clot-free cated by the minimum allowable
unit. Once the bevel has penetrated specific gravity for donors. A conve-

nient figure to use is 1.06 g/mL; a unit a segment of the tubing free of
containing 405 to 550 mL should blood between the knot and
weigh 429 to 583 g plus the weight of the needle (about 1 inch in
the container and anticoagulant. For length). Reapply the hemostat
a 500-mL bag, this is 565 to 671 g. and cut the tubing in the strip-
14. Clamp the tubing near the veni- ped area between the knot and
puncture using a hemostat, metal the hemostat. Fill the required
clip, or other temporary clamp. Re- tube(s) by releasing the hemo-
lease the blood pressure cuff/tourni- stat and then reclamp the tub-
quet to 20 mm Hg or less and fill the ing with the hemostat. Because
tube(s) for blood processing sam- this system is open, Biosafety
ple(s) by a method that prevents Level 2 precautions should be
contamination of the contents of the followed.
bag. This can be done in several ways. 15. Deflate the cuff and remove the
a. If the blood collection bag con- tourniquet. Remove the needle from
tains an inline needle, make an the donor’s arm, if not already re-
additional seal with a hemo- moved. Apply pressure over the gauze
stat, metal clip, hand sealer, or and ask the donor to raise his or her
a tight knot made from previ- arm (elbow straight) and hold the
ously prepared loose knot just gauze firmly over the phlebotomy
distal to the inline needle. Open site with the other hand.
the connector by separating 16. Discard the needle assembly into a
the needles. Insert the proximal biohazard container designed to pre-
needle into a processing test vent accidental injury to, and con-
tube, remove the hemostat, al- tamination of, personnel.
low the tube to fill, and re- 17. Strip donor tubing as completely as
clamp the tubing. The donor possible into the bag, starting at the
needle is now ready for removal. seal. Work quickly, to prevent the
b. If the blood collection bag con- blood from clotting in the tubing. In-
tains an inline processing tube, vert the bag several times to mix the
be certain that the processing contents thoroughly; then allow the
tube, or pouch, is full when the tubing to refill with anticoagulated
collection is complete and the blood from the bag. Repeat this pro-
original clamp is placed near cedure a second time.
the donor needle. The entire 18. Seal the tubing attached to the col-
assembly may now be removed lection bag into segments, leaving a
from the donor. segment number clearly and com-
c. If a straight-tubing assembly pletely readable. Attach a unit iden-
set is used, the following proce- tification number to one segment to
dure should be followed. Place be stored as a retention segment.
a hemostat on the tubing, al- Knots, metal clips, or a dielectric
lowing about four segments be- sealer may be used to make seg-
tween the hemostat and the ments suitable for compatibility
needle. Pull tight the loose testing. It must be possible to sepa-
overhand knot made in step 3. rate segments from the unit without
Release the hemostat and strip breaking sterility of the bag. If a di-

electric sealer is used, the knot or

clip should be removed from the dis-
Method 6.4. Preparation of
tal end of the tubing after creating a Red Blood Cells
hermetic seal.
19. Reinspect the container for defects. Principle
20. Recheck numbers on the container, Red Blood Cells (RBCs) are obtained by
processing tubes, donation record, removal of supernatant plasma from cen-
and retention segment. trifuged Whole Blood. The volume of
21. Place blood at appropriate tempera- plasma removed determines the hema-
ture. Unless platelets are to be re- tocrit of the component. When RBCs are
moved, whole blood should be placed preserved in CPDA-1, maximal viability
at 1 to 6 C immediately after collec- during storage requires an appropriate ra-
tion. If platelets are to be prepared, tio of cells to preservative. A hematocrit of
blood should not be chilled but 80% or lower ensures the presence of ade-
should be stored in a manner in- quate glucose for red cell metabolism for
tended to reach a temperature of 20 up to 35 days of storage.
to 24 C until platelets are separated.
Platelets must be separated within 8
hours after collection of the unit of Materials
Whole Blood.
1. Freshly collected Whole Blood, ob-
tained by phlebotomy as described
in Method 6.3. Collect blood in a col-
Notes lection unit with integrally attached
1. If the needle is withdrawn and veni- transfer container(s).
puncture is attempted again, prepa- 2. Plasma extractor.
ration of the site must be repeated as 3. Metal clips and hand sealer.
in Method 6.2. 4. Clean instruments (scissors, hemo-
2. In addition to routine blood donor stats).
phlebotomy, this procedure may be 5. Dielectric sealer (optional).
adapted for use in therapeutic phle- 6. Refrigerated centrifuge.
botomy. 7. Scale.
References Procedure
1. Silva MA, ed. Standards for blood banks and 1. Centrifuge whole blood using a “heavy”
transfusion services. 23rd ed. Bethesda, MD: spin (see Method 7.4), with a tem-
AABB, 2005. perature setting of 4 C.
2. Smith LG. Blood collection. In: Green TS, 2. Place the primary bag containing
Steckler D, eds. Donor room policies and pro-
cedures. Arlington, VA: AABB, 1985:25-45. centrifuged blood on a plasma ex-
3. Huh YO, Lightiger B, Giacco GG, et al. Effect pressor, and release the spring, al-
of donation time on platelet concentrates lowing the plate of the expressor to
and fresh frozen plasma. Vox Sang 1989;56: contact the bag.
21-4.
3. Temporarily clamp the tubing be-
4. Sataro P. Blood collection. In: Kasprisin CA,
Laird-Fryer B, eds. Blood donor collection tween the primary and satellite bags
practices. Bethesda, MD: AABB, 1993:89-103. with a hemostat or, if a mechanical

sealer will not be used, make a loose

Table 6.4-1. Removing Plasma from
overhand knot in the tubing.
Units of Whole Blood (To Prepare RBCs
4. If two or more satellite bags are at-
in Anticoagulant-Preservative with a
tached, apply the hemostat to allow
Known Hematocrit)
plasma to flow into only one of the
satellite bags. Penetrate the closure
Hematocrit of Volume of Final
of the primary bag. A scale, such as a
Segment from Plasma Hematocrit of
dietary scale, may be used to mea- Whole Blood to Be Red Blood Cell
sure the expressed plasma. Remove Unit Removed Unit
the appropriate amount of plasma
to obtain the desired hematocrit. 40% 150 mL 56%
5. Reapply the hemostat when the de- 39% 150 mL 55%
sired amount of supernatant plasma
38% 160 mL 55%
has entered the satellite bag. Seal the
tubing between the primary bag and 37% 165 mL 54%
the satellite bag in two places. 36% 170 mL 54%
6. Check that the satellite bag has the 35% 180 mL 54%
same donor number as that on the 34% 195 mL 55%
primary bag and cut the tubing be-
33% 200 mL 55%
tween the two seals.
Notes
of the red cells in the anticoagulant-
1. If blood was collected in a single bag, preservative solution will generally
modify the above directions as fol- result in a red cell component with a
lows: after placing the bag on the hematocrit between 70% and 80%.
expressor, apply a hemostat to the 4. See Table 6.4-1 to prepare Red Blood
tubing of a sterile transfer bag, asep- Cells with a specific (desired) hema-
tically insert the cannula of the trans- tocrit.
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. Method 6.5. Preparation of
2. Collection of blood in an additive so- Prestorage Red Blood Cells
lution allows removal of a greater
volume of plasma in step 4. After the
Leukocytes Reduced
plasma has been removed, the addi- Principle
tive solution is allowed to flow from The general principle and materials of
the attached satellite bag into the red Method 6.4 apply, except that the red cells
cells. This will result in a hematocrit are filtered using a special leukocyte re-
of 55% to 65%. Be sure that an ap- duction filter. All red cell leukocyte reduc-
propriate label and dating period are tion filters licensed in the United States
used. remove platelets to some degree. Anti-
3. The removal of 230 to 256 g (225 to coagulated whole blood may be filtered,
250 mL) of plasma and preservation from which only platelet-poor plasma

(leukocyte reduced) and red cells may be Notes

made. Alternatively, the red cells may be
1. If the collection system does not in-
filtered in additive solution, potentially
clude an in-line filter, a sterile con-
allowing the preparation of platelets,
nection device can be used to attach
plasma, and red cells. Nonleukocyte-re-
a leukocyte reduction filter to the
duced red cells may also undergo leuko-
collection system. The filter should
cyte reduction after preparation by at-
be used according to the manufac-
taching a leukocyte reduction filter con-
turer’s directions.
nected to a storage container using a ster-
2. Whole-blood-derived platelets can
ile connection device.
be manufactured only before leuko-
cyte reduction (see Method 6.14).
Procedure
1. Before centrifugation, the antico-
agulated Whole Blood may be fil- Method 6.6. Rejuvenation of
tered by hanging the container up- Red Blood Cells
side down and allowing the blood to
Principle
flow through an in-line filter by grav-
ity into a secondary container. The Rejuvenation is a process to restore de-
steps in Method 6.4 are then fol- pleted metabolites and improve the func-
lowed (see note 2, for addition of ad- tion and posttransfusion survival of stored
ditive solution). red cells. The rejuvenating solution is not
2. The anticoagulated Whole Blood intended for intravenous administration;
may be centrifuged with the in-line after warm incubation with the solution,
filter attached. After centrifugation, the red cells are washed and either gly-
the plasma is expressed. The addi- cerolized for frozen storage or kept at 1 to
tive solution is added, and the red 6 C for transfusion within 24 hours.
cells in the additive solution are fil- The rejuvenating solution approved by
tered by gravity, as in step 1 above. the Food and Drug Administration contains
3. A red cell component prepared us- pyruvate, inosine, phosphate, and adenine.
ing Method 6.4 either in residual Its use is permitted only with RBCs pre-
anticoagulated plasma or in additive pared from Whole Blood collected into
solution (AS-1, AS-3, AS-5) may have CPD, CP2D, or CPDA-1, and it may be
a secondary container with an in- added at any time between 3 days after col-
line filter attached using a sterile lection of the blood and 3 days after the ex-
connection device. Filtration can piration of the unit. However, the use of the
proceed according to the manufac- rejuvenation solution with RBC units be-
turer’s directions using gravity, as in fore 14 days of storage is not routinely ac-
step 1. The timing of this filtration is cepted because the treated cells may develop
often within 24 hours of collection supranormal levels of 2,3-diphospho-
but can be up to 5 days. glycerate, which impairs oxygen uptake.
4. Red cells that are leukocyte reduced
are labeled “Red Blood Cells Leuko- Reagents and Materials
cytes Reduced.” There is no specific 1. RBCs stored at 1 to 6 C and prepared
label for prestorage leukocyte reduc- from Whole Blood collected in CPD
tion. or CPDA-1. After collection, RBCs

suspended in CPD from day 3 to day 6. Be sure that units are appropriately
24 (or in CPDA-1 from day 3 to day labeled and that all applicable re-
38) may be used. The solution is not cords are complete.
approved for use with cells stored in
additive solutions. Reference
2. Red Blood Cell rejuvenation solu- Valeri CR, Zaroules CG. Rejuvenation and freezing
tion, in 50-mL sterile vial (Rejuvesol, of outdated stored human red cells. N Engl J Med
1972;287:1307-13.
Cytosol Laboratories, Braintree, MA);
also called rejuvenating solution.
3. Waterproof plastic bag.
4. Metal clips and hand sealer. Method 6.7. Red Cell
5. Sterile airway. Cryopreservation Using
Procedure
High-Concentration
1. Connect the container of rejuvenat-
Glycerol—Meryman Method
ing solution to the RBCs, using a Principle
transfer set and aseptic technique. Cryoprotective agents make possible the
2. Allow 50 mL of rejuvenating solution long-term (10 or more years) preservation
to flow by gravity into the container of red cells in the frozen state. High-con-
of red cells. Gently agitate the cell/ centration glycerol is particularly suitable
solution mixture during this addition. for this purpose. A practical method for
Note: A sterile airway is required if RBCs collected in a 450-mL bag is de-
the solution is in a bottle. scribed below.
3. Seal the tubing near the blood bag
and incubate the mixture for 1 hour
at 37 C. Either a dry incubator or cir- Materials
culating waterbath can be used. If (See Chapter 8 for additional information
placed in a waterbath, the container on frozen cellular components.)
should be completely immersed; use 1. Donor blood, collected into CPD,
of a waterproof overwrap is essential CD2D, CPDA-1, or AS.
to prevent contamination. a. Complete all blood processing
4. For use within 24 hours, wash the re- on units intended for freezing.
juvenated cells with saline (2 L un- b. RBCs preserved in CPD or CPDA-1
buffered 0.9% NaCl) by the use of an may be stored at 1 to 6 C for up
approved protocol. Storage of the to 6 days before freezing.
washed cells from the start of the c. RBCs preserved in AS-1 and AS-3
wash procedure should be at 1 to 6 C may be stored at 1 to 6 C for up
for no longer than 24 hours. to 42 days before freezing.
5. If the rejuvenated cells are to be cryo- d. RBCs that have undergone re-
preserved, the standard glyceroli- juvenation (see Method 6.6) may
zation protocol adequately removes be processed for freezing up to
the rejuvenation solution from the 3 days after their original expi-
processed cells. Expiration date re- ration.
mains 10 years from the date of col- e. RBCs in any preservative solu-
lection. tion that have been entered for

processing must be frozen with- procedure. If applicable, document

in 24 hours of puncturing the the lot number of the transfer bag.
seal. 4. Warm the red cells and the glycerol
2. Storage containers, either polyvinyl to at least 25 C by placing them in a
chloride or polyolefin bags. dry warming chamber for 10 to 15
3. 6.2 M of glycerol lactate solution minutes or by allowing them to re-
(400 mL). main at room temperature for 1 to 2
4. Cardboard or metal canisters for hours. The temperature must not
freezing. exceed 42 C.
5. Hypertonic (12%) sodium chloride 5. Apply a “Red Blood Cells Frozen” la-
solution. bel to the freezing bag in which the
6. 1.6% NaCl, 1 liter for batch wash. unit will be frozen. The label must
7. Isotonic (0.9%) NaCl with 0.2% dex- also include: name of the facility
trose solution. freezing the unit; Whole Blood num-
8. 37 C waterbath or 37 C dry warmer. ber; ABO group and Rh type; date
9. Equipment for batch or continuous- collected; date frozen; the cryopro-
flow washing, to deglycerolize cells tective agent used; and the expira-
frozen in high-concentration glyc- tion date.
erol.
10. Freezer tape.
11. Freezer (–65 C or colder).
Glycerolization
1. Document the lot numbers of the
glycerol, the freezing bags, and, if
used, the 0.9% NaCl.
Procedure 2. Place the container of red cells on a
shaker and add approximately 100
Preparing RBCs for Glycerolization mL of glycerol as the red cells are
1. Prepare RBCs from Whole Blood gently agitated.
units by removal of supernatant an- 3. Turn off the shaker and allow the
ticoagulant-preservative or additive cells to equilibrate, without agita-
solution. Weigh the RBC unit to be tion, for 5 to 30 minutes.
frozen and obtain the net weight of 4. Allow the partially glycerolized cells
the RBCs. The combined weight of to flow by gravity into the freezing bag.
the cells and the collection bag 5. Add the remaining 300 mL of glyc-
should be between 260 g and 400 g. erol slowly in a stepwise fashion,
2. Underweight units can be adjusted with gentle mixing. Add smaller vol-
to approximately 300 g either by the umes of glycerol for smaller volumes
addition of 0.9% NaCl or by the re- of red cells. The final glycerol con-
moval of less plasma than usual. Re- centration is 40% w/v. Remove any
cord the weight and, if applicable, air from the bag.
document the amount of NaCl added. 6. Allow some glycerolized cells to flow
3. Record the Whole Blood number, back into the tubing so that seg-
ABO group and Rh type, anticoagu- ments can be prepared.
lant, date of collection, date frozen, 7. Maintain the glycerolized cells at
expiration time, and the identifica- temperatures between 25 and 32 C
tion of the person performing the until freezing. The recommended in-

terval between removing the RBC Deglycerolized” label to the transfer

unit from refrigeration and placing pack and be sure that the label in-
the glycerolized cells in the freezer cludes identification of the collect-
should not exceed 4 hours. ing facility, the facility preparing the
deglycerolized cells, the ABO group
Freezing and Storage and Rh type of the cells, the Whole
Blood number, and the expiration
1. Place the glycerolized unit in a card-
date and time.
board or metal canister and place it
5. Dilute the unit with a quantity of
in a freezer at –65 C or colder.
hypertonic (12%) NaCl solution ap-
2. Label the top edge of the canister
propriate for the size of the unit. Al-
with freezer tape marked with the
low it to equilibrate for approxi-
Whole Blood number, ABO group
and Rh type, the date frozen, and the mately 5 minutes.
expiration date. 6. Wash the cells with 1.6% NaCl until
3. Do not bump or handle the frozen deglycerolization is complete. Ap-
cells roughly. proximately 2 liters of wash solution
4. The freezing rate should be less than are required. To check for residual
10 mL/minute. glycerol, see Method 6.8.
5. Store the frozen RBCs at –65 C or 7. Suspend the deglycerolized cells in
colder for up to 10 years. For blood isotonic (0.9%) saline with 0.2% dex-
of rare phenotypes, a facility’s medi- trose.
cal director may wish to extend the 8. Fill the integrally attached tubing
storage period. The unusual nature with an aliquot of cells sealed in
of such units and the reason for re- such a manner that it will be avail-
taining them past the routine 10- able for subsequent compatibility
year storage period must be docu- testing.
mented. 9. Deglycerolized RBCs must be stored
at 1 to 6 C for no longer than 24 hours.
(A closed system has been licensed
Thawing and Deglycerolizing
that allows storage of deglycerolized
1. Put an overwrap on the protective RBCs at 1 to 6 C for 2 weeks.)
canister containing the frozen cells
and place it in either a 37 C water-
Notes
bath or 37 C dry warmer.
2. Agitate it gently to speed thawing. 1. An aliquot of the donor’s serum or
The thawing process takes at least 10 plasma should be frozen and stored
minutes. Thawed cells should be at at –65 C or colder for possible future
37 C. use if new diagnostic tests are imple-
3. After the cells have thawed, use a mented.
commercial instrument for batch or 2. When new diagnostic tests have
continuous-flow washing to degly- been implemented and stored units
cerolize cells. Follow the manufac- do not have aliquots available for
turer’s instructions. testing, the units may have to be is-
4. Record the lot numbers and manu- sued with a label stating that the test
facturer of all the solutions and soft- has not been performed. The reason
ware used. Apply a “Red Blood Cells for distributing an untested compo-

nent should be documented. If a 10. Sterile filtered airway needle (BD

specimen from the donor is obtained 5200), for Fenwal rejuvenation har-
and tested after the unit was stored, ness only.
the date of testing should be noted 11. 500 mL of glycerolyte 57 solution
on the unit when it is issued. (Fenwal 4A7833) or 500 mL of 6.2 M
glycerolization solution (Cytosol
Reference PN5500).
12. Labels—Red Blood Cells Frozen Re-
Meryman HT, Hornblower M. A method for freez-
juvenated.
ing and washing RBCs using a high glycerol con-
centration. Transfusion 1972;12:145-56. 13. Corrugated cardboard storage box
(7" × 5.5" × 2" outside dimensions).
14. Heat sealing device.
15. Plastic bag for overwrapping.
Method 6.8. Red Cell

Cryopreservation Using Procedure
High-Concentration Preparing RBCs for Glycerolization
Glycerol—Valeri Method 1. Collect 450 mL of Whole Blood in
the primary bag. Invert the bag, fold
Principle it about 2 inches from the base, se-
cure the fold with tape, and place
RBCs collected in an 800-mL primary col-
the bag upright in a centrifuge. Cen-
lection bag in CPDA-1 and stored at 1 to 6
trifuge and remove all visible super-
C for 3 to 38 days can be biochemically re-
natant plasma. The hematocrit of
juvenated and frozen with 40% w/v glyc-
the RBC unit must be 75% ± 5%.
erol in the 800-mL primary container. See
2. Store RBCs at 1 to 6 C in the 800-mL
Method 6.6 for additional information.
primary bag, along with the adapter
port on the tubing that connects the
Materials primary bag and transfer pack.
1. Quadruple plastic bag collection 3. Centrifuge the stored cells to remove
system with 800-mL primary bag. all visible plasma before undertaking
2. Hand sealer clips. rejuvenation. The gross and net
3. Empty 600-mL polyethylene cryo- weights of the RBCs should not ex-
genic vials (eg, Corning 25702 or ceed 352 g and 280 g, respectively.
Fisher 033746). 4. Transfer the plasma to the integrally
4. Sterile connection device with wa- connected transfer pack, fold the in-
fers. tegral tubing, and replace the hand
5. Freezer tape. sealer clip (not crimped).
6. 600-mL transfer bag. 5. Attach an empty 600-mL transfer
7. 50 mL of Red Blood Cell Processing pack to the integral tubing of the pri-
Solution (Rejuvesol, Cytosol Labora- mary collection bag, using a sterile
tories, Braintree, MA). connection device.
8. Heat-sealable 8" × 12" plastic bags. 6. Transfer 1 mL of plasma to each of
9. Rejuvenation harness (Fenwal 4C1921 three cryogenic vials to be used for
or Cutter 98052). future testing.

Biochemical Modification of the Cells nected empty transfer pack, and the
coupler of the Y-type harness and
1. Using the Fenwal Rejuvenation Har-
incubate them in a 37 C waterbath
ness: Aseptically insert the needle of
for 1 hour.
the Y-type Fenwal Harness into the
rubber stopper of a 50-mL Red
Blood Cell Processing Solution bot- Glycerolization
tle and the coupler of the set into the 1. Remove the numbered crossmatch
adapter port of the primary collec- segments, leaving the initial seg-
tion bag. Insert the filtered airway ment and number attached to the
needle into the rubber stopper of the collection bag. Weigh the unit.
Red Blood Cell Processing Solution 2. Determine the amount of glycerol to
bottle. be added, based on the gross or net
2. Using the Cutter Rejuvenation Har- weight of the unit, from the values
ness: Aseptically insert the vented shown in Table 6.8-1.
white spike with the drip chamber 3. Aseptically insert the coupler of the
into the rubber stopper of the Red rejuvenation harness into the outlet
Blood Cell Processing Solution bot- port of the rubber stopper on the
tle and the nonvented spike into the glycerol solution bottle. For the
special adapter port on the primary Fenwal harness only, insert a filtered
collection bag. airway needle into the vent portion
3. With gentle manual agitation, allow of the glycerol bottle stopper.
50 mL of Red Blood Cell Processing 4. Place the bag on a shaker. Add the
Solution to flow directly into the red amount of glycerol shown in Table
cells. 6.8-1 for the first volume while the
4. Heat-seal the tubing of the harness bag is shaking at low speed (180 os-
set that connects the Red Blood Cell cillations/minute).
Processing Solution to the adapter 5. Equilibrate the mixture for 5 min-
port. The second tubing of the har- utes without shaking and add the
ness Y-set is used to add glycerol second volume. Equilibrate it for 2
(see below). minutes. Add the third volume of
5. Completely overwrap the 800-mL glycerol, using vigorous manual
primary bag, the integrally con- shaking.
Table 6.8-1. Amount of Glycerol Needed for Different Weights of Red Cell Units
Gross Weight Net Weight Initial Second Third Total

of Unit of Unit Addition of Addition of Addition of Glycerol
(grams)* (grams) Glycerol (mL) Glycerol (mL) Glycerol (mL) 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 Thawing and Deglycerolization

empty bottle of glycerol and the tub-
See Method 6.7. Note, however, that the
ing proximal to the adapter port. En-
supernatant glycerol is removed before
sure that the transfer pack remains
freezing. Therefore, only two salt solu-
integrally attached to the primary
tions (the hypertonic 12% saline and the
collection bag.
0.9% saline-0.2% dextrose solution) are
7. Centrifuge the mixture of red cells
used in the deglycerolization process.
and glycerol and transfer all visible
supernatant glycerol to the transfer
References
pack, resuspend, and mix. Note: This
step differs from Method 6.7. 1. Rejuvesol Package insert. Braintree, MA:
Cytosol Laboratories, 2002.
8. Seal the tubing 4" from the primary
2. Valeri CR, Ragno G, Pivacek LE, et al. A
collection bag, detach the transfer multicenter study of in vitro and in vivo val-
pack containing the supernatant fluid, ues in human RBCs frozen with 40% (wt/vol)
and discard it. glycerol and stored after deglycerolization for
15 days at 4 C in AS-3: Assessment of RBC
9. Affix an overlay blood component processing in the ACP 215. Transfusion 2001;
label, the facility label, and an ABO/ 41:933-9.
Rh label. Record the expiration date
on the label.
10. Weigh the unit just before freezing
and record the weight. Method 6.9. Checking the
11. Fold over the top portion of the pri- Adequacy of
mary bag (approximately 2"). Place
the primary bag into a plastic bag
Deglycerolization of Red
overwrap and heat-seal the outer Blood Cells
bag across the top so that there is as
little air as possible between the bags. Principle
12. Place one vial of plasma and the Glycerolization of red cells for frozen stor-
plastic bag containing the glyceroli- age creates a hyperosmolar intracellular
zed red cells in the cardboard box. fluid, which must be restored to physio-
Store the other two vials, suitably logically compatible levels before the cells
identified, at –65 C or colder for fu- are transfused. Inadequately deglycerolized
ture testing, if needed. red cells will be hemolyzed by contact
13. Affix a “Red Blood Cells Frozen Reju- with normal saline, or with serum or
venated” label, an ABO/Rh label, a plasma if subjected to crossmatching.
facility label, and the original unit During deglycerolization, the last solution
number on the outside of the box. in contact with the cells is normal saline.
Record separately or affix on the The easiest way to determine adequacy of
cardboard box the collection, freez- glycerol removal is to determine the level
ing, and expiration dates. of free hemoglobin (mg/dL) in the final
14. Freeze the unit in a –80 C freezer. No wash. An adequate estimate of hemolysis
more than 4 hours should be al- can be achieved by comparing the color
lowed to elapse between the time of the final wash fluid with the blocks in a
the unit was removed from the 4 C commercially available color comparator.
refrigerator and the time the cells Alternatively, normal saline can be added
are placed in the –80 C freezer. to an aliquot of deglycerolized cells and

the color of the supernatant fluid evalua- time frame required for the anticoagulant
ted against the color comparator. or collection process.
Materials and Equipment Materials

1. Semiautomated instrument for de-
glycerolizing cryopreserved RBCs. 1. Freshly collected Whole Blood, ob-
2. Transparent tubing, as part of dis- tained by phlebotomy as described
posable material used to deglycerol- in Method 6.3, in a collection unit
ize individual unit. with integrally attached transfer con-
3. Color comparator, available com- tainer(s).
mercially. 2. Metal clips and hand sealer.
3. Clean instruments (scissors, hemo-
stats).
4. Dielectric sealer (optional).
Procedure
5. Plasma extractor.
1. Interrupt the last wash cycle at a 6. Freezing apparatus.
point when wash fluid is visible in 7. Refrigerated centrifuge.
the tubing leading to the disposal bag. 8. Scale.
2. Hold the comparator block next to
an accessible segment of tubing,
against a well-lighted white back- Procedure
ground. 1. Centrifuge blood soon after collection,
3. Note coloration of the wash fluid, using a “heavy” spin (see Method 7.4).
which should be no stronger than Use a refrigerated centrifuge at 1 to 6
the block, indicating 3% hemolysis C unless also preparing platelets (see
(3% of the red cells are hemolyzed). Method 6.13).
4. If the level of hemolysis is excessive, 2. Place the primary bag containing
continue the wash process until the centrifuged blood on a plasma ex-
color is within acceptable limits. tractor and place the attached satel-
5. Record observation for the individ- lite bag on a scale adjusted to zero.
ual unit and for the quality assurance Express the plasma into the satellite
program. bag and weigh the plasma.
6. If unacceptable hemolysis occurs re- 3. Seal the transfer tubing with a di-
peatedly, document corrective action. electric sealer or metal clips but do
not obliterate the segment numbers
of the tubing. Place another seal
Method 6.10. Preparation of nearer the transfer bag.
4. Label the transfer bag with the unit
Fresh Frozen Plasma from number before it is separated from
Whole Blood the original container. Record the
volume of plasma on the label.
Principle 5. Cut the tubing between the two seals.
Plasma is separated from cellular blood The tubing may be coiled and taped
elements and frozen to preserve the activ- against the plasma container, leaving
ity of labile coagulation factors. Plasma the segments available for any test-
must be placed in the freezer within the ing desired.

6. Place the plasma at –18 C or colder Procedure

within the time frame required for the
anticoagulant or collection process. 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
Method 6.11. Preparation of (see Method 7.4). Collect at least 200
Cryoprecipitated AHF from mL (205 g) of cell-free plasma for
Whole Blood processing into cryoprecipitate.
3. Promptly place plasma in a freezing
device so that freezing is started
Principle within the time frame required for
Coagulation Factor VIII (antihemophilic the anticoagulant or collection pro-
factor, AHF) can be concentrated from cess. Plasma containers immersed in
freshly collected plasma by cryoprecipita- liquid must be protected with a plas-
tion. Cryoprecipitation is accomplished tic overwrap.
by slow thawing, at 1 to 6 C, plasma that 4. Allow the frozen plasma to thaw at 1
has been prepared for freezing within the to 6 C by placing the bag in a 1 to 6 C
time frame required for the anticoagulant circulating waterbath or in a refrig-
or collection process. erator. If thawed in a waterbath, use
a plastic overwrap (or other means)
to keep container ports dry.
Materials
5. When the plasma has a slushy con-
1. Freshly collected Whole Blood, ob- sistency, separate liquid plasma from
tained by phlebotomy as described the cryoprecipitate by one of the
in Method 6.3, in a collection unit procedures below:
with at least two integrally attached a. Centrifuge the plasma at 1 to 6
transfer containers. C using a “heavy” spin. Hang the
2. Metal clips and hand sealer. bag in an inverted position and
3. Clean instruments (scissors, hemo- allow the separated plasma to
stats). flow rapidly into the transfer
4. Dielectric sealer (optional). bag, leaving the cryoprecipitate
5. Plasma extractor. adhering to the sides of the pri-
6. Refrigerated centrifuge. mary bag. Separate the cryo-
7. Freezing apparatus: suitable freezing precipitate from the plasma
devices include blast freezers or me- promptly, to prevent the cryo-
chanical freezers capable of main- precipitate from dissolving and
taining temperatures of –18 C or flowing out of the bag. Ten to
colder; dry ice; or an ethanol dry ice 15 mL of supernatant plasma
bath. In a bath of 95% ethanol and may be left in the bag for resus-
chipped dry ice, freezing will be pension of the cryoprecipitate
complete in about 15 minutes. after thawing. Refreeze the cryo-
8. 1 to 6 C circulating waterbath or re- precipitate immediately.
frigerator. b. Place the thawing plasma in a
9. Scale. plasma expressor when appro-

ximately one-tenth of the con- are available commercially, as are

tents is still frozen. With the specially designed dry heat devices).
bag in an upright position, al- 2. Medication injection ports.
low the supernatant plasma to 3. Sterile 0.9% sodium chloride for in-
flow slowly into the transfer jection.
bag, using the ice crystals at 4. Syringes and needles.
the top as a filter. The cryo-
precipitate paste will adhere to Procedure
the sides of the bag or to the ice. 1. Cover the container with a plastic
Seal the bag when about 90% of overwrap to prevent contamination
the cryoprecipitate-reduced of the ports with unsterile water, or
plasma has been removed and use a device to keep the containers
refreeze the cryoprecipitate im- upright with the ports above water.
mediately. 2. Resuspend the thawed precipitate
6. The cryoprecipitate should be re- carefully and completely, either by
frozen within 1 hour of thawing. kneading it into the residual 10 to 15
Store at –18 C or colder, preferably mL of plasma or by adding approxi-
–30 C or colder, for up to 12 months mately 10 mL of 0.9% sodium chlo-
from the date of blood collection. ride and gently resuspending.
3. Pool by inserting a medication injec-
Note tion site into a port of each bag. As-
Cryoprecipitated AHF may be prepared pirate contents of one bag into a sy-
from Fresh Frozen Plasma at any time ringe and inject into the next bag. Use
within 12 months of collection. The expi- the ever-increasing volume to flush
ration date of Cryoprecipitated AHF is 12 each subsequent bag of as much dis-
months from the date of phlebotomy, not solved cryoprecipitate as possible,
from the date it was prepared. until all contents are in the final bag.
4. Thawed Cryoprecipitated AHF must
be stored at room temperature. If
pooled, it must be administered
Method 6.12. Thawing and within 4 hours. Thawed single units,
if not entered, must be administered
Pooling Cryoprecipitated within 6 hours of thawing if intend-
AHF ed for replacement of Factor VIII.
Pools of thawed individual units may
Principle not be refrozen.
Cryoprecipitated AHF should be rapidly
thawed at 30 to 37 C but should not re-
main at this temperature once thawing is Method 6.13. Preparation of
complete. The following method permits
rapid thawing and pooling of this product. Platelets from Whole Blood
Principle
Materials Platelet-rich plasma is separated from
1. Circulating waterbath at 37 C (water- Whole Blood by “light-spin” centrifugation
baths designed for thawing plasma and the platelets are concentrated by

“heavy-spin” centrifugation, with subse- 4. Express the platelet-poor plasma

quent removal of supernatant plasma (see into the second transfer bag and seal
Method 7.4). the tubing. Some plasma should re-
main on the platelet button for stor-
Materials age, but no exact volume can be des-
ignated. AABB Standards for Blood
1. Freshly collected Whole Blood, ob-
Banks and Transfusion Services re-
tained by phlebotomy as described
in Method 6.3, in a collection unit quires that sufficient plasma remain
with two integrally attached transfer with the platelet concentrate to main-
containers. The final container must tain the pH at 6.2 or higher for the
be a plastic approved for platelet entire storage period. This usually
storage. Keep blood at room temper- requires a minimum of 35 mL of
ature (20 to 24 C) before separating plasma when storage is at 20 to 24 C,
platelet-rich plasma from the red cells. but 50 to 70 mL is preferable.
This separation must take place 5. The platelet concentrate container
within 8 hours of phlebotomy. should be left stationary, with the la-
2. Metal clips and hand sealer. bel side down, at room temperature
3. Scissors, hemostats. for approximately 1 hour.
4. Plasma extractor. 6. Resuspend the platelets in either of
5. Dielectric sealer (optional). the following ways:
6. Centrifuge, calibrated as in Method a. Manipulate the platelet con-
7.4. tainer gently by hand to achieve
7. Scale. uniform resuspension.
8. Rotator. b. Place the container on a rotator
at room temperature. The slow,
Procedure gentle agitation should achieve
uniform resuspension within 2
1. Do not chill the blood at any time hours.
before or during platelet separation. 7. Maintain the platelet suspensions at
If the temperature of the centrifuge 20 to 24 C with continuous gentle
is 1 to 6 C, set the temperature con- agitation.
trol of the refrigerated centrifuge at 8. Platelets should be inspected before
20 C and allow the temperature to issue to ensure that no platelet ag-
rise to approximately 20 C. Centri-
gregates are visible.
fuge the blood using a “light” spin
(see Method 7.4).
2. Express the platelet-rich plasma into
Notes
the transfer bag intended for platelet The platelet-reduced plasma may be frozen
storage. Seal the tubing twice be- promptly and stored as Fresh Frozen
tween the primary bag and Y con- Plasma (FFP), if the separation and freez-
nector of the two satellite bags and ing are completed within the time frame
cut between the two seals. Place the required for the anticoagulant or collec-
red cells at 1 to 6 C. tion process. The volume of FFP prepared
3. Centrifuge the platelet-rich plasma after platelet preparation will be substan-
at 20 C using a “heavy” spin (see tially less than that prepared directly from
Method 7.4). Whole Blood.

Reference centrifuged and much of the plasma re-

Silva MA, ed. Standards for blood banks and trans-
moved shortly before transfusion, but
fusion services. 23rd ed. Bethesda, MD: AABB, appropriate resuspension is essential.
2005:30. The platelets must remain at room tem-
perature, without agitation, for 20 to 60
minutes before resuspension into the re-
Method 6.14. Preparation of maining plasma. Transfusion must take
place within 4 hours of the time the
Prestorage Platelets platelet bag was entered. Volume reduc-
Leukocytes Reduced from tion can be performed on individual or
Whole Blood pooled units.
No consensus exists regarding the opti-
Principle mal centrifugation rate. One study1 found
Prestorage leukocyte-reduced platelets 35% to 55% platelet loss in several units
may be prepared from whole blood using centrifuged at 500 × g for 6 minutes, com-
in-line filtration of the platelet-rich plasma pared with 5% to 20% loss in units centri-
(PRP). The resulting intermediate product fuged at 5000 × g for 6 minutes or 2000 × g
is a filtered PRP, from which a leukocyte- for 10 minutes. The authors recommend
reduced platelet concentrate and leuko- 2000 × g for 10 minutes, to avoid any risk
cyte-reduced plasma may be manufac- that a higher centrifugal force might inflict
tured. Materials and procedures are the on the plastic container. A study by Moroff
2
same as for Method 6.13, except that the et al found mean platelet loss to be less
PRP is expressed through an in-line filter. than 15% in 42 units centrifuged at 580 × g
for 20 minutes. High g forces are of theoret-
References ical concern because they may damage the
1. Sweeney JD, Holme S, Heaton WAL, Nelson E. platelets when they are forced against the
Leukodepleted platelet concentrates pre- wall of the container and also increase the
pared by in-line filtration of platelet rich
plasma. Transfusion 1995;35:131-6.
possibility of container breakage.
2. Sweeney JD, Kouttab N, Penn LC, et al. A
comparison of prestorage leukoreduced Materials
whole blood derived platelets with bedside
filtered whole blood derived platelets in 1. Platelet concentrate(s), prepared as
autologous stem cell transplant. Transfusion described in Method 6.13.
2000;40:794-800.
2. Metal clips and hand sealer.
3. Scissors, hemostats.
4. Dielectric sealer (optional).
Method 6.15. Removing 5. Centrifuge, calibrated as in Method
Plasma from Platelet 7.4.
6. Plasma extractor.
Concentrates (Volume
Reduction)
Principle Procedure
Although optimal storage of platelets re- 1. Pool platelets, if desired, into a trans-
quires an adequate volume of plasma, a fer pack, using standard technique.
few patients may not tolerate large-vol- Single platelet concentrates may
ume infusion. Stored platelets may be need volume reduction for pediatric

recipients. Apheresis components may pheresis component or individual

be processed directly. platelet concentrate, the unit can be
2. Centrifuge at 20 to 24 C, using one of considered sterile and it is not nec-
the following protocols: essary to impose the 4-hour expira-
a. 580 × g for 20 minutes. tion interval required for entered
b. 2000 × g for 10 minutes. Platelets. However, no data exist to
c. 5000 × g for 6 minutes. support storage of reduced volume
3. Without disturbing the contents, platelet concentrates; therefore, it is
transfer the bag to a plasma extrac- preferable to transfuse them as soon
tor. Remove all but 10 to 15 mL plasma as possible.
from single units, or somewhat more 2. Reduced-volume platelet concentrates
volume, proportionately, from a pool may not be distributed as a licensed
or from a component prepared by product.
apheresis. 3. Platelets that have been pooled must
4. Mark expiration time on bag as 4 be used within 4 hours of entering
hours after the time the unit was en- the units, whether or not they have
tered. been volume-reduced. Pooled plate-
5. Leave bag at 20 to 24 C without agi- lets may not be distributed as a li-
tation for 20 minutes if centrifuged censed product.
at 580 × g, or for 1 hour if centrifuged
at 2000 or 5000 × g. References
6. Resuspend platelets as described in 1. Simon TL, Sierra ER. Concentration of plate-
Method 6.13. let units into small volumes. Transfusion
1984;24:173-5.
Notes 2. Moroff G, Friedman A, Robkin-Kline L, et al.
Reduction of the volume of stored platelet
1. If a sterile connection device is used concentrates for use in neonatal patients.
for removing plasma from a hema- Transfusion 1984;24:144-6.

Methods Section 7: Quality Control
Methods Section 7
Quality Control
Method 7.1. Quality Control Procedure
for Copper Sulfate Solution 1. Obtain several (three to six, if possi-

ble) blood samples with known he-
Either of the two methods presented be- moglobin levels. Samples should in-
low is acceptable for quality control of cop- clude hemoglobin levels slightly
per sulfate solution. above and below 12.5 g/dL.
2. Gently place a drop of each blood
Method 7.1.1. Functional Validation of sample into a vial of copper sulfate
Copper Sulfate Solution solution of stated specific gravity of
1.053.
Principle 3. Drops of all blood samples with he-
Copper sulfate solution can be checked for moglobin at or above 12.5 g/dL must
suitability in donor screening by observing sink and those with hemoglobin lev-
the behavior (sinking or floating) of drops els below 12.5 g/dL must float.
of blood of known hemoglobin concentra- 4. Record the date of testing; the man-
tion. ufacturer, lot number, and expiration
date of the copper sulfate; sample
identity; the results; and the identity
Section 7
Materials of the person performing the test.

1. Copper sulfate—specific gravity 1.053. 5. Document the corrective action taken
2. Capillary tubes. if the results are outside acceptable
3. Worksheet for recording results. limits.
819

Method 7.1.2. Use of Measurement stead of an ellipse. Read at the point

Instruments for Specific Gravity, Density, where the line intersects the scale on
or Refractive Index of Copper Sulfate the instrument.
Solution Refractometer method:
1. Follow manufacturer’s instructions for
Principle operation and maintenance of the in-
The specific gravity, density, or refractive strument.
index of the copper sulfate solution can 2. Add a drop of copper sulfate to the
be measured directly and the result com- refractometer.
pared with the value stated by the manu- 3. Observe refractive index.
facturer. An error in the specific gravity Note: Some refractometers designed
reading of ±0.0001 corresponds to ±0.06 for urine analysis provide both a re-
g/dL hemoglobin in whole blood.1 There- fractive index scale and a specific
fore, a copper sulfate solution with a spe- gravity scale. Do not read specific
cific gravity of 1.053 ± 0.0003 would result gravity directly from this type of
in a corresponding hemoglobin range of refractometer.
12.5 ± 0.18 g/dL. 4. A copper sulfate solution with a spe-
cific gravity of 1.053 will have a re-
Materials fractive index of about 1.3425 at room
1. Copper sulfate—specific gravity 1.053. temperature.2
2. Measurement instruments Both methods:
• For specific gravity—high-preci- 1. Record the results; the date of test-
sion hydrometer, with gradations ing; the manufacturer, lot number,
of 0.0005 or smaller. and expiration date of the copper
• For refractive index—refracto- sulfate solution; the identity of the
meter. person performing the test; and the
3. Pipette. identification of the instrument used
4. For specific gravity method—gradu- for measurement.
ated cylinder or other container suit- 2. Document the corrective action taken
able for use with a hydrometer. if the results are outside the accept-
5. Alcohol for cleaning hydrometer. able limits.
6. Worksheet for recording results.
Procedure Note
Hydrometer method: A liquid densitometer may be used to
1. Wipe the hydrometer clean with al- measure density directly. At 4 C, the den-
cohol and dry it. sity of the solution will be exactly the
2. Gently lower the hydrometer into the same as the specific gravity (specific
solution until it floats on its own. gravity = density of solution/density of
Drops of solution on the stem will water; density of water at 4 C = 1 g/mL).
cause inaccurate readings. When density is measured at room tem-
3. To read, observe a point just below perature, a conversion factor of 0.9970
the plane of the liquid surface and (the density of water at 25 C) is used to
then raise the line of vision until the calculate specific gravity.2 Specific gravity
surface is seen as a straight line in- of copper sulfate solution at 25 C = the

Methods Section 7: Quality Control 821
observed density in g/mL divided by 3. Suitable container, eg, 250-500 mL

0.9970 g/mL. beaker.
4. Water.
References 5. Crushed ice.
1. Phillips RA, Van Slyke DD, Hamilton PB, et al. 6. 37 C waterbath.
Measurement of specific gravities of whole 7. Worksheet for recording results.
blood and plasma by standard copper sulfate
solutions. J Biol Chem 1950;183:305-30.
2. Blood hemoglobin screening (specific gravity Method 7.2.1. Liquid-in-Glass Laboratory
method). Technical Reference Document 12. Thermometers
Arlington, TX: Ricca Chemical Company,
2002. Procedure
1. Before choosing a thermometer for a
particular application, consider all
Method 7.2. Standardization the governing factors; be sure that
the thermometer will be used at its
and Calibration of proper immersion; and follow the
Thermometers manufacturer’s instructions for its
proper use. When using a certified
Principle
thermometer, read and follow the
Thermometers used during laboratory applicable notes. Be sure to include
testing and in the collection (donor suit- any correction factors noted on the
ability), processing, and storage of blood certificate for the NIST-traceable
components and reagents should be cali- thermometer and apply them in
brated and standardized to ensure accu- calculations.
rate indication of temperatures. Calibra- 2. Categorize the thermometers by key
tion should be performed at temperatures factors, such as immersion, incre-
close to the temperature at which the ments, and temperature of intended
thermometers will be used. Over time, use. Test them in groups, comparing
liquid-in-glass thermometers may give a similar thermometers. Do not attempt
different reading at a given temperature to compare dissimilar thermometers
because of permanent changes in the vol- in a single procedure.
ume of the bulb related to relaxation of 3. Number each thermometer being
the glass.1 Each thermometer should be tested (eg, place a numbered piece
calibrated before initial use and periodi- of tape around the top of each ther-
cally thereafter, as well as any time there mometer or use the manufacturer’s
is reason to suspect change or damage. serial number).
Calibration must be verified for all elec- 4. Perform calibration with water at a
tronic thermometers, even those describ- temperature close to that which the
ed as “self-calibrating.” thermometer will monitor.
a. To calibrate at 37 C, place the
Materials thermometers to be tested and
1. National Institute of Standards and the NIST thermometer at a uni-
Technology (NIST)-certified thermo- form depth in a standard 37 C
meter or thermometer with NIST- waterbath, making sure that the
traceable calibration certificate. tips of all devices are at the same
2. Thermometer to be calibrated. level in the liquid.

b. To calibrate at 1 to 6 C, fill a results should be within 1 C of the

suitable container with water. NIST thermometer to be considered
Add crushed ice until the ap- acceptable.
proximate desired temperature 2. Thermometers should be observed
is reached. Place the thermom- routinely for any split in the column
eters to be tested and the NIST because this will cause inaccurate
thermometer at a uniform depth readings. The methods for reuniting
in the water/ice mixture, mak- the separation can be found in NCCLS
ing sure that the tips of all de- Standard I2-A2.2 When this occurs,
vices are at the same level and document corrective action and
are in the liquid, not the upper recalibrate the thermometer.
ice.
5. Stir constantly in a random motion
References
until the temperature equilibrates,
approximately 3 to 5 minutes. 1. Wise JA. A procedure for the effective recali-
bration of liquid-in-glass thermometers.
6. Observe temperatures. Record each NIST Special Publication 819. Gaithersburg,
thermometer’s identification and re- MD: National Institute of Standards and
sults. Acceptance criteria depend on Technology, 1991.
the level of precision required, but, 2. Temperature calibration of water baths, in-
struments, and temperature sensors. 2nd ed;
for most blood banking applications, approved standard I2-A2 Vol. 10 No. 3. Wayne,
agreement within 1 C between the two PA: National Committee for Clinical Labora-
thermometers may be considered tory Standards, 1990.
acceptable. If the reading varies by
more than one degree from the stan- Method 7.2.2. Electronic Oral
dard, the thermometer may be re- Thermometers
turned to the distributor (if newly
purchased), labeled with the correc- Procedure
tion factor (degrees different from 1. Use any of the following methods to
the NIST thermometer) that must be verify calibration:
applied to each reading, or dis- a. Follow manufacturer’s instruc-
carded. tions for verifying calibration.
7. Complete the calibration record b. Use a commercially available
with the date of testing and identity calibration device by following
of the person who performed the the instructions provided by
test. the device’s manufacturer.
c. Calibrate the thermometer by
inserting the probe in a 37 C
Notes waterbath with a NIST-certified
1. If a thermometer is to be used for thermometer.
temperatures over a range greater 2. A result is acceptable if the reading
than a few degrees (eg, 10 degrees), a on the thermometer agrees with the
three-point calibration should be NIST thermometer within 0.1 C. If
performed. Use water of appropriate expected results are not achieved,
temperature. Test at temperatures unsatisfactory thermometers should
just below, just above, and at the be returned to the distributor. Docu-
midway point of intended use. All ment corrective actions.

3. Record the date of testing, thermom- manufacturer or other equipment storage

eter identification numbers, temper- expert. The facility procedures manual
ature readings, and the identity of the must include a detailed description of the
person performing the test. method(s) in local use. (See Appendix 10 for
quality control testing intervals.)
Method 7.3. Testing Blood Method 7.3.1. Testing Refrigerator Alarms
Storage Equipment Alarms Principle

Blood storage refrigerators and freezers Refrigerator temperatures may increase
must be equipped with a system for con- beyond acceptable limits for several rea-
tinuous temperature monitoring and an sons, including:
audible alarm. If a storage unit goes into 1. Improperly closed door.
alarm, it is essential that personnel know 2. Insufficient refrigerant.
the appropriate actions to take. Direc- 3. Compressor failure.
tions for such events should be available 4. Dirty or blocked heat exchanger.
in a conspicuous location, and personnel 5. Loss of electrical power.
should be trained to initiate these actions
if the temperature cannot be corrected Materials
rapidly. The alarm on each storage unit
1. Calibrated thermometer.
should be checked periodically for proper
2. Pan large enough to hold the ther-
functioning. Monthly checks are appro-
mocouple container.
priate until consistent behavior of a par-
3. Water.
ticular storage unit has been demon-
4. Crushed ice.
strated. Thereafter, alarms should be
5. Table salt.
tested regularly and frequently enough to
achieve and maintain personnel compe-
tency as well as to detect malfunctions.
For equipment in good condition, quar- Procedure
terly checks are usually sufficient. Be- 1. Verify that the alarm circuits are op-
cause alarms may be disconnected or si- erating, the alarm is switched on, and
lenced during repairs, it is also prudent to the starting temperature is 1 to 6 C.
verify alarm functioning after repairs. Immerse an easy-to-read calibrated
The high and low temperatures of activa- thermometer in the container with
tion must be checked and the results re- the alarm thermocouple.
corded. AABB Standards for Blood Banks For low activation:
and Transfusion Services1(p5) requires that 2. Place the container with the thermo-
the alarm be set to activate at a tempera- couple and thermometer in a pan
ture that will allow appropriate interven- containing an ice and water slush at
tion before blood or components reach an a temperature of –4 C or colder. To
undesirable temperature. Because of the di- achieve this temperature, add several
versity of equipment available, it is not pos- spoonfuls of table salt to the slush.
sible to give specific instructions for all ap- 3. Close the refrigerator door to avoid
plicable alarm systems. If the equipment changing the temperature of the
user’s manual does not provide suitable di- storage compartment. Keep the con-
rections for testing the alarm, consult the tainer in the pan of cold slush, and

gently agitate it periodically until the change in temperature may give the
alarm sounds. false impression that the alarm does
4. Record this temperature as the low- not sound until an inappropriate
activation temperature. temperature is registered.
For high activation: 4. The low temperature of activation
5. Place the container with thermocou- should be greater than 1 C (eg, 1.5 C);
ple and thermometer in a pan con- the high temperature of activation
taining cool water (eg, 12 to 15 C). should be less than 6 C (eg, 5.5 C).
6. Close the refrigerator door. Allow the 5. Alarms should sound simultaneou-
fluid in the container to warm slowly, sly at the site of the refrigerator and
with occasional agitation. at the location of remote alarms,
7. Record the temperature at which the when employed. If remote alarms
alarm sounds as the high-activation are used, the alarm check should in-
temperature. clude a verification that the alarm
8. Record the date of testing, the refrig- sounded at the remote location.
erator identification, the thermome- 6. The amount of fluid in which the
ter identification, and the identity of thermocouple is immersed must be
the person performing the test. no larger than the volume of the
9. If temperatures of activation are too smallest component stored in that
low or too high, take appropriate refrigerator. The thermocouple may
corrective actions such as those sug- be immersed in a smaller volume,
gested by the manufacturer, record but this means that the alarm will
the nature of the corrections, and re- go off with smaller temperature
peat the alarm check to document changes than those registered in a
that the corrections were effective. larger volume of fluid. Excessive sen-
sitivity may create a nuisance.
7. With the one-time assistance of a
qualified electrician, the required re-
Notes frigerator alarm checks of units with
1. The thermocouple for the alarm should virtually inaccessible temperature
be easily accessible and equipped probes can be performed with an
with a cord long enough so that it electrical modification cited by Wenz
can be manipulated easily. and Owens.2
2. The thermocouple for the continu-
ous temperature monitor need not References
be in the same container as that of
the alarm. If it is in the same con- transfusion services. 23rd ed. Bethesda, MD:
tainer, a notation should be made in AABB, 2005.
the records that explains any out- 2. Wenz B, Owens RT. A simplified method for
monitoring and calibrating refrigerator alarm
of-range temperature registered as a systems. Transfusion 1980;20:75-8.
result of the alarm check.
3. When the temperatures of alarm ac- Method 7.3.2. Testing Freezer Alarms
tivation are checked, the temperature
change should occur slowly enough Principle
so that the measurements and re- Freezer temperatures may rise to unac-
cording are accurate. Too rapid a ceptable levels for a variety of reasons.

Common causes of rising temperatures 5. Return the freezer and the alarm sys-
include: tem to their normal conditions.
1. Improperly closed freezer door or lid. 6. If the alarm sounds at too high a
2. Low level of refrigerant. temperature, take appropriate cor-
3. Compressor failure. rective actions such as those sug-
4. Dirty or blocked heat exchanger. gested by the manufacturer, record
5. Loss of electrical power. the nature of the correction, and re-
peat the alarm check to document
that the corrections were effective.
Materials
1. Protection for the freezer contents,
eg, a blanket. Notes
2. Calibrated thermometer or thermo- 1. Alarms should sound simultaneou-
couple independent from that built sly at the site of the freezer and at
into the system. the location of the remote alarms,
3. Warm water or an oven mitt. when employed. If remote alarms
4. Worksheet for recording results. are used, the alarm check should in-
clude a verification that the alarm
sounded at the remote location.
Procedure 2. Test battery function, electrical cir-
1. Protect frozen components from ex- cuits, and power-off alarms more
posure to elevated temperatures dur- frequently than the activation tem-
ing the test. perature. Record function, freezer
2. Use a thermometer or thermocou- identification, date, and identity of
ple, independent from that built into person performing the testing.
the system, that will accurately indi- 3. For units with the sensor installed in
cate the temperature of alarm acti- the wall or in air, apply local warmth
vation. Compare these readings with to the site or allow the temperature
the temperatures registered on the of the entire compartment to rise to
recorder. the point at which the alarm sounds.
3. Warm the alarm probe and thermo- Remove the frozen contents or pro-
meter slowly (eg, in warm water, by tect them with insulation while the
an oven-mitt-covered hand, expo- temperature rises.
sure to air). The specific temperature 4. For units with the thermocouple lo-
of activation cannot be determined cated in antifreeze solution, pull the
accurately during rapid warming, container and the cables outside the
and the apparent temperature of ac- freezer chest for testing, leaving the
tivation will be too high. door shut and the contents protected.
4. Record the temperature at which the 5. For units with a tracking alarm that
alarm sounds, the date of testing, sounds whenever the temperature
the identity of the person perform- reaches a constant interval above
ing the test, the identity of the the setting on the temperature con-
freezer and calibrating instrument, troller, set the controller to a warmer
and any observations that might setting and note the temperature in-
suggest impaired activity. terval at which the alarm sounds.

6. Liquid nitrogen freezers must have Procedure

alarm systems that activate at an un-
safe level of contained liquid nitro- Preparation of Platelet-Rich Plasma
gen. 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. Calculate and record the number of
Method 7.4. Functional platelets in the Whole Blood unit: pla-
Calibration of Centrifuges for telets/µL × 1000 × mL of whole blood
Platelet Separation = number of platelets in whole blood.
3. Prepare PRP at a selected speed and
time. (See “light spin” in Table 7.4-1
Principle or guidance provided by the centri-
Successful preparation of platelet concen- fuge manufacturer.)
trates requires adequate but not excessive 4. Place a temporary clamp on the tub-
centrifugation; the equipment used must ing so that one satellite bag is closed
perform in a consistent and dependable off. Express the PRP into the other
manner. Each centrifuge used to prepare satellite bag. Seal the tubing close to
platelets should be calibrated upon re- the primary bag, leaving a long sec-
ceipt and after adjustment or repair. tion of tubing, the “tail.” Disconnect
Functional calibration of the centrifuge the two satellite bags from the pri-
for both the preparation of platelet-rich mary bag. Do not remove the tem-
plasma (PRP) from whole blood and sub- porary clamp between the satellite
sequent preparation of platelet concen- bags until the platelets are prepared
trate from PRP can be performed during (see next section).
the same procedure. 5. Strip the tubing and “tail” several
times so that they contain a repre-
sentative sample of PRP.
Materials
6. Seal off a segment of the “tail” and
1. Freshly collected whole blood, ob- disconnect it, so that the bag of PRP
tained by phlebotomy into a bag with remains sterile.
two integrally attached transfer con- 7. Perform a platelet count on the sam-
tainers. ple of PRP in the sealed segment.
2. A specimen of blood from the donor, Calculate and record the number of
anticoagulated with EDTA and col- platelets in the bag of PRP: platelets/
lected in addition to the specimens µL × 1000 × mL of PRP = number of
drawn for routine processing. platelets in PRP.
3. Metal clips and hand sealer or di- 8. Calculate and record percent yield:
electric sealer. (number of platelets in PRP × 100)
4. Clean instruments (scissors, hemo- divided by (number of platelets in
stats, tubing stripper). whole blood) = % yield.
5. Plasma extractor. 9. Repeat the above process three or four
6. Centrifuge suitable for preparation times with different donors, using
of platelet concentrates. different speeds and times of centri-
7. Worksheet for recording results. fugation, and compare the yields

achieved under each set of test con- 3. Allow the platelets to rest for approx-
ditions. imately 1 hour.
10. Select the shortest time/lowest speed 4. Place the platelets on an agitator for
combination that results in the high- at least 1 hour to ensure that they are
est percent of platelet yield without evenly resuspended. Platelet counts
unacceptable levels of red cell con- performed immediately after centri-
tent in the PRP.
fugation will not be accurate.
11. Record the centrifuge identification,
5. Strip the tubing several times, mix-
the calibration settings selected, the
ing tubing contents well with the
date, and the identity of the person
contents of the platelet bag. Seal off
performing the calibration.
a segment of the tubing and discon-
nect it, so that the platelet bag re-
Preparation of Platelets
mains sterile.
1. Centrifuge the PRP (as prepared 6. Perform a platelet count on the con-
above) at a selected time and speed
tents of the segment.
to prepare platelets. (See “heavy spin”
7. Calculate and record the number of
in Table 7.4-1 or guidance provided
platelets in the concentrate: platelets/
by the centrifuge manufacturer.)
µL × 1000 × mL of platelets = number
2. Remove the temporary clamp be-
tween the two satellite bags and ex- of platelets in platelet concentrate.
press the platelet-reduced plasma 8. Calculate and record percent yield.
into the second attached satellite 9. Repeat the above process with PRP
bag, leaving approximately 55 to 60 from different donors, using differ-
mL volume in the platelet bag. Seal ent speeds and times of centrifuga-
the tubing, leaving a long section of tion, and compare the yields achieved
tubing attached to the platelet bag. under each set of test conditions.
Table 7.4-1. Centrifugation for Component Preparation

Heavy Spin
Red cells
Platelets from whole blood }5 5000 × g, 5 minutes*
Cryoprecipitate }5
Cell- free plasma 5000 × g, 7 minutes*
Light Spin
Platelet-rich plasma 2000 × g, 3 minutes*
To calculate relative centrifugal force in g:
rcf (in g) = 28.38 × R† × (rpm/1000)2
*Times include acceleration but not deceleration times. Times given are approximations only. Each individual centrifuge
must be evaluated for the preparation of the various components.
†
R = radius of centrifuge rotor in inches.

10. Select the shortest time/lowest speed 2. McShine R, Das P, Smit Sibinga C, Brozovic B.
Effect of EDTA on platelet parameters in
combination that results in the high-
blood and blood components collected with
est percent of platelet yield in the CPDA-1. Vox Sang 1991;61:84-9.
platelet concentrate.
11. Record the centrifuge identification,
the calibration settings selected, the
date, and the identity of the person Method 7.5. Functional
performing the calibration.
Calibration of a Serologic
Centrifuge
Notes
Principle
1. It is not necessary to perform func-
Each centrifuge should be calibrated
tional recalibration of a centrifuge
upon receipt, after adjustments or repairs,
unless the instrument has under-
and periodically. Calibration evaluates the
gone adjustments or repairs, or
behavior of red cells in solutions of differ-
component quality control indicates
ent viscosities, not the reactivity of differ-
that platelet counts have fallen be-
ent antibodies.
low acceptable levels. However,
timer, speed, and temperature cali-
brations of the centrifuge should oc- For Immediate Agglutination
cur on a regularly scheduled basis Materials
(see Appendix 10).
1. Test tubes, 10 × 75 mm or 12 × 75
2. Each centrifuge used for preparing
mm (whichever size is routinely
platelets must be calibrated individ-
used in the laboratory)
ually. Use the conditions determined
to be optimal for each instrument.
3. For saline-active antibodies:
3. When counting platelet samples on
■ Serum from a group A person
an instrument intended for whole
(anti-B) diluted with 6% albu-
blood, it may be necessary to use a
min to give 1+ macroscopic ag-
correction factor to obtain accurate
glutination (3 mL of 22% bovine
results.
albumin + 8 mL of normal sa-
4. When determining the appropriate
line = 6% bovine albumin). See
time and speed of centrifugation,
Method 1.5.
consideration should also be given to
■ Positive control: Group B red cells
other products that will be prepared
in a 2% to 5% saline suspension.
from the whole blood. Final size and
■ Negative control: Group A red cells
hematocrit of red cell and plasma
in a 2% to 5% saline suspension.
volume made available for further
4. For high-protein antibodies:
processing are important factors to
■ Anti-D diluted with 22% or 30%
consider.
albumin to give 1+ macro-
scopic agglutination.
References
■ Positive control: D+ red cells in
1. Kahn R, Cossette I, Friedman L. Optimum a 2% to 5% saline suspension.
centrifugation conditions for the preparation
of platelet and plasma products. Transfusion ■ Negative control: D– red cells in
1976;16:162-5. a 2% to 5% saline suspension.

Procedure 6. Select the optimal time of centrifu-

gation, which is the shortest time re-
1. For each set of tests (saline and high- quired to fulfill the following criteria:
protein antibodies), label five test a. Agglutination in the positive
tubes for positive reactions and five tubes is as strong as determined
for negative reactions. in preparing reagents.
2. In quantities that correspond to rou- b. There is no agglutination or
tine use, add diluted anti-B to each ambiguity in the negative tubes.
of 10 tubes for the saline test and c. The cell button is clearly delin-
add diluted anti-D to each of 10 tubes eated and the periphery is
for the high-protein test. Add serum sharply defined, not fuzzy.
and reagents in quantities that cor- d. The supernatant fluid is clear.
respond to routine use. e. The cell button is easily resus-
3. Add the appropriate control cell suspended.
pension to one set of tubes (one pos- In the example shown in Ta-
itive and one negative tube for the ble 7.5-1, these criteria are met
saline test, and one positive and one by the 30-second and the 45-
negative tube for the high-protein second spins; the optimal time
for these tests in this centrifuge
antibody test). Centrifuge immedi-
is 30 seconds.
ately for the desired time interval
7. Record centrifuge identification, the
(eg, 10 seconds).
times selected, the date, and the
4. Observe each tube for agglutination
identity of the person performing
and record observations. (See exam-
the calibration.
ple in Table 7.5-1.)
5. Repeat steps 2 and 3 for each time
interval (eg, 15, 20, 30, and 45 sec- For Washing and Antiglobulin Testing
onds). Do not allow cells and sera to Tests in which antihuman globulin (AHG)
incubate before centrifugation. serum is added to red cells may require
Table 7.5-1. Example of Serologic Centrifuge Test Results*
Time in Seconds
Criteria 10 15 20 30 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 pended in the residual fluid. The

those for immediate agglutination. Cen- optimal time for washing is the
trifugation conditions appropriate for shortest time that accomplishes
both washing and AHG reactions can be these goals.
determined in one procedure. Note that 3. Repeat washing process on all pairs
this procedure does not monitor the com- three more times, using time deter-
pleteness of washing; use of IgG-coated cells mined to be optimal.
to control negative AHG reactions provides 4. Decant supernatant saline thoroughly.
this check. The following procedure ad- 5. Add AHG to one positive control test
dresses only the mechanics of centrifu- tube and one negative control test tube.
gation. Centrifuge immediately for the de-
sired interval (eg, 10 seconds).
Materials 6. Observe each tube for agglutination
and record observations.
1. AHG reagent, unmodified.
7. Repeat steps 5 and 6 for each inter-
2. Saline, large volumes.
val (eg, 15, 20, 30, and 45 seconds).
3. Test tubes, 10 × 75 mm or 12 × 75
Do not allow cells and AHG to incu-
mm (whichever size is routinely used
bate before centrifugation.
in the laboratory).
8. Select optimal time as in immediate
agglutination procedure.
5. Positive control: a 2% to 5% saline
9. Record centrifuge identification, the
suspension of D+ red cells incubated
times selected, the date, and the
for 15 minutes at 37 C with anti-D
identity of the person performing
diluted to give 1+ macroscopic ag-
the calibration.
glutination after addition of AHG.
6. Negative control: a 2% to 5% suspen-
Notes
sion of D+ red cells incubated for 15
minutes at 37 C with 6% albumin. Periodic recalibration is performed to ver-
[Note: D– red cells incubated with ify that the timing in use continues to be
diluted anti-D may also be used as a the optimal timing. This may be accom-
negative control.] plished by using a shortened version of
the procedures outlined above. For exam-
ple, use the current timing for a particular
Procedure
centrifuge and each medium and those
1. Prepare five test tubes containing 1 times just above and just below the cur-
drop of positive cells and five tubes rent timing.
containing 1 drop of negative con-
trol cells.
2. Fill tubes with saline and centrifuge
them in pairs, one positive and one Method 7.6. Performance
negative, for different times (eg, 30, Testing of Automatic Cell
45, 60, 90, and 120 seconds). The red
cells should form a clearly delin-
Washers
eated button, with minimal cells Principle
trailing up the side of the tube. After Antihuman globulin (AHG) is inactivated
the saline has been decanted, the readily by unbound immunoglobulin. The
cell button should be easily resus- red cells to which AHG will be added

must be washed free of all proteins and 5. Continue the washing cycle.
suspended in a protein-free medium. A 6. After addition of saline in the third
properly functioning cell washer must add cycle, stop the cell washer and in-
large volumes of saline to each tube, respect tubes as above. Record obser-
suspend the cells, centrifuge them ade- vations.
quately to avoid excessive red cell loss, and 7. Complete the wash cycle.
decant the saline to leave a dry cell button. 8. At the end of the wash cycle, inspect
all tubes to see that saline has been
Materials completely decanted and that each
tube contains a dry cell button. Re-
1. Test tubes routinely used in the lab-
cord observations.
oratory, 10 × 75 mm or 12 × 75 mm.
9. Add AHG according to the manufac-
2. Additive routinely used to potentiate
antigen-antibody reactions. turer’s directions, centrifuge, and ex-
3. Human serum, from patient or do- amine all tubes for agglutination. If
nor. the cell washer is functioning prop-
4. IgG-coated red cells, known to give a erly, the size of the cell button
1 to 2+ reaction in antiglobulin test- should be the same in all tubes. All
ing. tubes should show the same degree
5. Normal saline. of agglutination. Record observa-
6. AHG reagent, anti-IgG or polyspeci- tions.
fic. 10. Record identity of centrifuge, the date
7. Worksheet for recording results. of testing, and the identity of the
person performing the testing.
Procedure
Notes
1. To each of 12 tubes, add potentiator
and human serum in quantities that 1. Further investigation is needed if:
correspond to routine use and 1 drop a. The amount of saline varies
of IgG-coated red cells. significantly from tube to tube
2. Place the tubes in a centrifuge car- or cycle to cycle.
rier, seat the carrier in the cell washer, b. The cell button is not resus-
and start the wash cycle. pended completely after being
3. After addition of saline in the second filled with saline.
cycle, stop the cell washer. Inspect c. Any tube has weak or absent
the contents of all tubes. There agglutination in the antiglobu-
should be an approximately equal lin phase.
volume of saline in all tubes; some d. Any tube has a significant de-
variation is acceptable. Tubes should crease in the size of the cell
be approximately 80% full, to avoid button.
splashing and cross-contamination. 2. Cell washers that automatically add
(Refer to manufacturer’s instructions AHG should also be checked for uni-
for specific requirements.) Record form addition of AHG. In step 9
observations. above, AHG would be added auto-
4. Observe all tubes to see that the red matically, and failure of addition
cells have been completely resus- would be apparent by absence of ag-
pended. Record observations. glutination. The volume of AHG

should be inspected and found to be 3. Seal a 5- to 8-cm (2- to 3-inch) seg-

equal in all tubes. The volume of ment distal to the collection bag.
AHG delivered automatically by cell There should be approximately 2 mL
washers should be checked monthly of fluid in the segment. Double-seal
to ensure that it is as specified in the the end of the tubing next to the
manufacturer’s directions and that component bag and detach the seg-
delivery is uniform in all tubes. ment.
3. Some manufacturers market AHG 4. Empty the contents of the segment
colored with green dye for use in au- into a suitably labeled tube.
tomated cell washers so that it will 5. Determine and record cell counts in
be immediately obvious if no re- cells/mL.
agent has been added. 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,
Method 7.7. Monitoring Cell change values to cells/mL by
Counts of Apheresis dividing by 1000 (or 103).
6. Multiply cells/mL by the volume of
Components the component, in mL, to obtain to-
Principle tal cell count in the component.
When cellular components are prepared 7. Record component’s identity, the date,
by apheresis, it is essential to determine and the identity of the person per-
cell yields without compromising the ste- forming the testing.
rility of the component.
Note
Materials Refer to manufacturer’s directions for any
1. Component collected by apheresis. additional requirements.
2. Metal clips and hand sealer or di-
electric sealer.
3. Tubing stripper. Method 7.8. Manual Method
4. Clean instruments (scissors, hemo-
stats). for Counting Residual White
5. Test tubes. Cells in Leukocyte-Reduced
6. Cell-counting equipment.
Blood and Components
Principle
Procedure The residual white cell content of leuko-
1. Ensure that the contents of the aphere- cyte-reduced whole blood and components
sis component bag are well mixed. can be determined using a large-volume
2. Strip the attached tubing at least four hemocytometer. For red-cell-containing
times, mixing the contents of the components, the red cells in the aliquot to
tubing with the contents of the bag, be counted are first lysed. Crystal violet is
to ensure that the contents of the used to stain the leukocyte nuclei. The
tubing accurately represent the en- Nageotte counting chamber has a volume
tire contents of the bag. 56 times that of the standard hemocyto-

meter. Accuracy of counting is improved no longer coated with in-

by examining a larger volume of mini- tact red cells.
mally diluted specimen, compared with 4) Pipette 360 µL of crystal
standard counting techniques. violet stain into the mix-
ture and mix fluids by
Materials pipetting up and down
several times. The final
1. Hemocytometer chamber with 50 µL
volume is now 500 µL.
counting volume (eg, Nageotte Brite
b. For Platelets
Line Chamber).
1) Place a representative sam-
2. Crystal violet stain: 0.01% w/v crys-
ple of the platelet in a clean
tal violet in 1% v/v acetic acid (eg,
test tube.
Turks solution).
2) Pipette 100 µL of the pla-
3. Red cell lysing agent (eg, Zapoglobin,
Coulter Electronics, Hialeah, FL), for telet sample into a clean
red-cell-containing components test tube.
only. 3) Pipette 400 µL of crystal
4. Pipettor (40 µL and 100 µL) with dis- violet stain into the 100 µL
posable tips. of platelets and mix fluids
5. Talc-free gloves, clean plastic test by pipetting up and down
tubes, plastic petri dish, filter paper. several times. The final
6. Light microscope with 10× ocular volume is now 500 µL.
lens and 20× objective. 2. Fit the hemocytometer with a cover-
7. Worksheet for recording results. slip and, using a pipette, load the
mixture until the counting area is
completely covered but not over-
Procedure
flowing.
1. Dilute and stain leukocyte-reduced 3. Cover the hemocytometer with a
blood and component samples as moist lid to prevent evaporation (a
follows: plastic petri dish into which a piece
a. For Red-Cell-Containing Com- of damp filter paper has been placed
ponents works well) and let it rest undisturbed
1) Pipette 40 µL of lysing for 10 to 15 minutes, to allow the
agent into a clean test tube. white cells to settle in the counting
2) Place a representative area of the chamber.
sample of the component 4. Remove the moist lid, place the
to be tested in a clean test hemocytometer on the microscope
tube. The hematocrit of the and, using a 20× objective, count the
sample to be tested should white cells present in the entire
not exceed 60%. 50-µL volume of the counting cham-
3) Pipette 100 µL of the sam- ber.
ple into the tube contain- 5. Calculate and record results.
ing 40 µL of lysing agent. a. White cell concentration:
Rinse the pipette several
times to mix the two flu- leukocytes/µL = (cells counted/50 µL) × 5
ids, until the pipette tip is = cells counted/10

where 50 µL is the volume 4. The accuracy of the counting method

counted and 5 is the dilution can be validated from a reference
factor resulting from the addi- sample with a high white cell con-
tion of lysing agent and stain. tent that has been quantified by an-
b. Total white cell content of the other means. This reference sample
leukocyte-reduced component: can be used for serial dilutions in
blood or a component that has been
leukocytes/µL × rendered extremely leukocyte re-
leukocytes/
= 1000 µL /mL × duced by two passages through a
component volume in mL
of the component leukocyte reduction filter. Counts
obtained on the serially diluted sam-
6. Record the component’s identity, the ples can be compared with the ex-
date, and the identity of the person pected concentration derived by cal-
performing the testing. culation.
5. This counting technique is not known
Notes to be accurate at concentrations lower
than 1 white cell/µL.
1. White cells deteriorate during refrig-
erated storage; counts on stored blood
References
or red cell components may give in-
1. Lutz P, Dzik WH. Large-volume hemocyto-
accurate results. meter chamber for accurate counting of
2. Use of talc-free gloves is recom- white cells (WBCs) in WBC-reduced platelets;
mended because talc particles that validation and application for quality control
of WBC-reduced platelets prepared by
contaminate the counting chamber apheresis and filtration. Transfusion 1993;33:
can be misread as white cells. 409-12.
3. Experience identifying crystal-violet- 2. Dzik WH, Szuflad P. Method for counting
white cells in white cell-reduced red cell con-
stained white cells can be obtained
centrates (letter). Transfusion 1993;33:272.
by examining samples from compo-
nents that have not been leukocyte
reduced.

Appendices
Appendices
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 A2 0.015-0.035 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
835

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
Thereafter Preterm <34 mmol/L <2 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
3-5 years 701-1157 mg/dL 38-74 mg/dL 66-120 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.

Appendix 4. Coagulation Factor Values in Platelet Concentrates

Factor/ Normal
Protein 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 198-434 217-308 278-313 310 265-323 302 221-299
mg/dL
Ristocetin 50-150 106 124 125 133 116 127
cofactor %
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
1 2
Infant Adult
Term Birth at
Premature 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
4
The adult values should be modified to correct Estimation of Body Surface Area :
for:
Ht(cm) × Wt(kg) Ht(in) × Wt(lb)
1. Below age 18: increase values by 10%. BSA(m 2 ) = or
3600 3131
2. Weight loss:
5
a. Marked loss within 6 months—calcula- Blood Volume (BV) :
tions made at original weight. BV = 2740 mL/m2—males
b. Gradual loss over a longer time—calcu- BV = 2370 mL/m2—females
6
lations made at present weight and Hematocrit :
raised 10% to 15%. Venous hematocrit = Hv (blood obtained by
3. Obese and short: values are reduced by vein or finger puncture)
10%. Whole-body hematocrit = HB
4. Elderly: values are reduced by 10%. HB = (Hv) × (0.91)
5. Pregnancy3:
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.

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) Me (MNS13) Dantu (MNS25) ERIK (MNS37)

MN (MNS/002) N (MNS2) Mta (MNS14) Hop (MNS26) Osa (MNS38)
S (MNS3) Sta (MNS15) Nob (MNS27) ENEP (MNS39)
s (MNS4) Ria (MNS16) Ena (MNS28) ENEH (MNS40)
U (MNS5) Cla (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
Appendix 6. Blood Group Antigens Assigned to Systems (cont’d)

System (ISBT
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)
(cont’d)


System (ISBT
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)

Appendices 843

System (ISBT
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 JMH (JMH1)

Hagen
(JMH/026)
I (I/027) I (I1)
Globoside P (GLOB1)
(GLOB/028)
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 7. Examples of Gene, Antigen, and Phenotype Terms

System Genes Antigens Phenotypes
ABO A A1 A2 B A A1 A2 B A A1 A2 B
Rh DCEce DCEce D+C+E–c+e+
MN MNSs MNSs M+N+S–s+
P P1 P1 P1+ P1–
Lewis Le le Lea Leb Le(a+)Le(a–b+)
Kell K k Kpa Jsa K k Kpa Jsa K–k+Kp(a+)Js(a–)
Kell K1 K2 K3 K1 K2 K3 K:–1,2,–3
Scianna Sc1 Sc2 Sc Sc1 Sc2 Sc:–1,–2,–3
Kidd Jka Jkb Jk3 Jka Jkb Jk3 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(+), Fya+, Fya(+), Duffya+,

Duffya-positive
Phenotype Fy(a+b–) Fya+b–, Fy(a+b–), Fya(+)b(–), Fya(+)b(–)
Antibody Anti-Fya Anti Fya, Anti-Duffy
Antigen 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.

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.
Appendices
845
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
3. Alarm system board (if applicable) Daily
C. Platelet incubators
1. Recorder Daily
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 Monthly
(if applicable)
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
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 Twice yearly
(usually at temperature extremes)
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.

Appendix 11. Directory of Organizations

AABB American Society of Hematology (ASH)
8101 Glenbrook Road 1900 M Street, NW, Suite 200
Bethesda, MD 20814-2749 Washington, DC 20036
(301) 907-6977 (202) 776-0544
FAX: (301) 907-6895 FAX: (202) 776-0545
www.aabb.org www.hematology.org
American Association of Tissue Banks (AATB) America’s Blood Centers (ABC)

1320 Old Chain Bridge Road, Suite 450 725 15th Street, NW
McLean, VA 22101 Suite 700, The Folger Building
(703) 827-9582 Washington, DC 20005
FAX: (703) 356-2198 (202) 393-5725
www.aatb.org FAX: (202) 393-1282
www.americasblood.org
American Medical Association (AMA)
515 N. State Street Armed Services Blood Program Office (ASBPO)
Chicago, IL 60610 5109 Leesburg Pike, Suite 698
(800) 621-8335 Falls Church, VA 22041-3258
www.ama-assn.org (703) 681-8024
FAX: (703) 681-7541
American Red Cross National Headquarters (ARC) www.tricare.osd.mil/asbpo
2025 E Street, NW
Washington, DC 20006 Association of Donor Recruitment Professionals
(202) 303-4498 (ADRP)
Disaster Assistance: (866) 438-4636 P.O. Box 540524
www.redcross.org Grand Prairie, TX 75054-0524
www.adrp.org
American Society for Apheresis (ASFA)
570 West 7th Avenue, Suite 402 College of American Pathologists (CAP)
Vancouver, BC, Canada V5Z 1B3 325 Waukegan Road
(604) 484-2851 Northfield, IL 60093-2750
FAX: (604) 874-4378 (800) 323-4040
www.apheresis.org FAX: (847) 832-8000
www.cap.org
American Society for Clinical Pathology (ASCP)
2100 West Harrison Street Foundation for the Accreditation of Cellular Therapy
Chicago, IL 60612-3798 (FACT)
(312) 738-1336 University of Nebraska Medical Center
Outside Illinois: (800) 621-4142 986065 Nebraska Medical Center
FAX: (312) 738-1619 Omaha, NE 68198-6065
www.ascp.org (402) 559-1950
FAX: (402) 559-1951
American Society for Histocompatibility www.factwebsite.org
and Immunogenetics (ASHI)
15000 Commerce Parkway, Suite C ICCBBA, Inc.
Mount Laurel, NJ 08054 204 St. Charles Way, Unit 179E
(856) 638-0428 York, PA 17402
FAX: (856) 439-0525 (717) 845-4790
www.ashi-hla.org FAX: (717) 845-9727
www.iccbba.com
American Society of Anesthesiologists (ASA)
520 N. Northwest Highway
Park Ridge, IL 60068-2573
(847) 825-5586
FAX: (847) 825-1692
www.asahq.org

Appendix 12. Resources for Safety Information
Centers for Disease Control and Prevention (CDC) International Air Transport Association (IATA)
Office of Health and Safety, Biosafety Branch 1776 K Street, NW, Suite 400
Mail Stop F-05 Washington, DC 20006
1600 Clifton Road (202) 293-9292
Atlanta, GA 30333 FAX: (202) 293-8448
(404) 639-2453 www.iata.org
FAX: (404) 639-2294
www.cdc.gov International Civil Aviation Organization (ICAO)
999 University Street
Clinical and Laboratory Standards Institute (CLSI) Montreal, Quebec
940 West Valley Road, Suite 1400 Canada H3C 5H7
Wayne, PA 19087-1898 (514) 954-8220
(610) 688-0100 FAX: (514) 954-6376
FAX: (610) 688-0700 www.icao.int
www.clsi.org
National Fire Protection Association (NFPA)
Department of Transportation (DOT) 1 Batterymarch Park
Office of Hazardous Materials Standards Quincy, MA 02169-7471
Research and Special Programs Administration (617) 770-3000
DHM-10 FAX: (617) 770-0700
400 7th Street, SW www.nfpa.org
Washington, DC 20590-0001
(202) 366-8553 National Institute for Occupational Safety and
FAX: (202) 366-3012 Health (NIOSH)
www.dot.gov Education and Information Division
4676 Columbia Parkway
Environmental Protection Agency (EPA) Cincinnati, OH 45226-1998
Chemical Emergency Preparedness and Prevention (800) 356-4674
Office (5104A) Outside the US: (513) 533-8328
1200 Pennsylvania Avenue, NW Clinicians’ Post-Exposure Prophylaxis Hotline:
Washington, DC 20460 (888) 448-4911
(800) 424-9346 FAX: (513) 533-8588
In Washington, DC metropolitan area: (703) 412-9810 www.cdc.gov/niosh
www.epa.gov
National Institutes of Health (NIH)
Environmental Protection Agency (EPA) Division of Safety
Office of Solid Waste (5305W) Building 13, Room 3K04
1200 Pennsylvania Avenue, NW Bethesda, MD 20892
Washington, DC 20460 (301) 496-2346
(800) 424-9346 FAX: (301) 402-0313
www.epa.gov/osw www.nih.gov
Food and Drug Administration (FDA) Nuclear Regulatory Commission (NRC)

Center for Biologics Evaluation and Research Office of Public Affairs
Division of Blood Applications, HFM-370 Washington, DC 20555
1401 Rockville Pike (800) 368-5642
Rockville, MD 20852-1448 In Washington, DC metropolitan area: (301) 415-8200
(301) 827-3524 FAX: (301) 415-2234
FAX: (301) 827-3535 www.nrc.gov
www.fda.gov/cber

Appendices 849
Appendix 11. Directory of Organizations (cont'd)
International Society for Cellular Therapy (ISCT) National Marrow Donor Program (NMDP)
570 West 7th Avenue, Suite 402 3001 Broadway Street NE, Suite 500
Vancouver, BC, Canada V5Z 1B3 Minneapolis, MN 55413-1753
(604) 874-4366 (800) 627-7692
FAX: (604) 874-4378 Outside the US: (612) 627-5800
www.celltherapy.org www.marrow.org
International Society of Blood Transfusion (ISBT) Plasma Protein Therapeutics Association (PPTA)
Central Office 147 Old Solomons Island Road, Suite 100
C/O Jan van Goyenkade 11 Annapolis, MD 21401
1075 HP Amsterdam (410) 263-8296
The Netherlands FAX: (410) 263-2298
+ 31 (0) 20 679 3411 www.plasmainfo.org
FAX: + 31 (0) 20 673 7306
www.isbt-web.org United Network for Organ Sharing (UNOS)
700 North 4th Street
Joint Commission on Accreditation of Healthcare P.O. Box 2484
Organizations (JCAHO) Richmond, VA 23218
One Renaissance Boulevard (804) 782-4800
Oakbrook Terrace, IL 60181 FAX: (804) 782-4817
(630) 792-5000 www.unos.org
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
Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publi-
cation.

Appendices 851
Appendix 12. Resources for Safety Information (cont'd)
Occupational Safety and Health Administration (OSHA) US Postal Service (USPS)

Office of Communications Headquarters, Room 9301
Room N-3647 475 L’Enfant Plaza, SW
200 Constitution Avenue, NW Washington, DC 20260
Washington, DC 20210 (800) 275-8777
(202) 693-1999 www.usps.com
Workplace safety and health-related questions:
(800) 321-6742
www.osha.gov
Note: Contact information changes rapidly. Therefore, the data listed above may not be current for the entire life of this publi-
cation.

Index
Index
Index
Page numbers in italics refer to tabular or illustrative material.
A passively acquired, 563

A, B, H substances, 290-291, 298, 301, 445, reactivity, 295
736-739 antigens, 293
ABO compatibility in newborns, 293
of bone grafts, 623-624 nomenclature, 226, 840, 844
of Cryoprecipitated AHF, 411, 500-501 on platelets, 361-362, 397
of Granulocytes Pheresis, 411 weakly expressed, 297-298, 300-301
of HPC transplants, 598-599, 600, 601-602 B(A) phenotype, 298, 301
of kidney transplants, 401 discovery of, 289
of liver transplants, 402, 628 genetics and biochemistry, 229-230, 235,
of plasma products, 411, 496, 498 290-293, 844
of platelet components, 361-362, 411, phenotypes, 844, 845
489-490, 569 soluble substances, 290-291, 298, 301, 445,
of Red Blood Cells, 411, 418, 486 736-739
of solid organ transplants, 402, 627, 629-630 subgroups, 293-294
of Whole Blood, 411, 486 in ABO discrepancies, 302
ABO hemolytic disease of the fetus and new- confirmation of by adsorption/elution,
born, 536, 537, 538, 545 735-736
ABO system (ISBT 001), 289-303 testing for, 296
acquired B phenotype, 298, 301-302 ABO testing
antibodies, 294-296 of autologous blood, 122
age variations, 295, 298, 299 of blood components, 164, 165
anti-A and anti-B, 294-296 in children, 296, 415, 545, 563, 574
anti-A1, 295-296, 302 with cold autoagglutinins, 299, 302-303, 469
anti-A,B, 295, 296 comparison with previous records, 413, 526
in HDFN, 536, 538, 545 confirmation of weak A or B subgroup,
735-736
853

of cord blood, 545, 595 Additives

discrepancies, 296-303, 469 anticoagulants-preservatives for red cells,
absence of expected antibodies, 301 176, 186, 564-565
absence of expected antigens, 300-301 in serologic testing, 276-277, 437-438, 443,
causes, 297 753-754
mixed-field agglutination, 298, 299 Adhesion molecules, 246, 269
resolving, 300-303 Adsol (AS-1), 176, 186, 565
specimen-related problems, 297-299 Adsorption
technical errors, 299-300 allogeneic, 471-472, 781-782
unexpected red cell reactions, 301-302 autologous cold, 438, 466, 472, 775-776
unexpected serum reactions, 302-303 autologous warm, 470-471, 779-781
hemolysis in, 295 differential, 781-782
microplate tests, 733-735 and elution, 300-301, 448, 735-736
for organ transplantation, 621, 629 methods, 768-769, 775-776, 779-784
of Platelets Pheresis, 142 polyethylene glycol, 783-784
in prenatal studies, 538 purposes, 446-447
reagents, 296 with rabbit red cells, 439, 466
of recipients, 122, 410, 411 Adsorption separation, 140
routine, 290, 296 Adult T-cell lymphoma/leukemia (ATL), 682
slide tests, 731-732 Adverse reactions
in transfusion reaction evaluation, 654 to apheresis, 141, 150-153
tube tests, 732-733 to DMSO, 607
ABTI antigen, 355 in donors, 19, 107-109
Accidents, 45-46 to G-CSF, 594-595
ACE (angiotensin-converting enzyme) inhibi- management, 19, 20, 21
tion, 636, 645 to transfusions
Acid-elution stain (Kleihauer-Betke), 550-551, action for, 531
794-796 autologous, 123
Acid glycine/EDTA, 446, 469, 747-748 infectious, 667-703
Acidosis, in neonates, 561 noninfectious, 633-661
Acquired antigens, 298, 301-302 records, 660-661
Activated partial thromboplastin time (aPTT) reporting, 19, 20, 21 , 661, 702-703
in massive transfusion, 511 AET (2-aminoethylisothiouronium bromide),
monitoring hemostasis with, 494, 496 342, 446
normal values, 837 Affinity constant (Ko), 273
in vitamin K deficiency, 498 Age
Active failures, 26 of blood samples, 410, 431, 433
Acute lung injury (ALI), 647 of donor, 98
Acute normovolemic hemodilution (ANH), 117, effect on ABO antigens/antibodies, 293, 295,
126-130 298, 299
clinical studies, 128, 129, 130 Agglutination
physiologic considerations, 127-128 defined, 271
practical considerations, 128, 130 factors affecting, 272-275
selection of patients, 131 antigen-antibody proportions, 274-
Acute transfusion reactions. See Transfusion re- 275
actions chemical bonding, 273
Adaptive (acquired) immunity, 243-244 equilibrium (affinity) constant, 273
ADCC (antibody-dependent cellular incubation time, 274, 444
cytotoxicity), 255, 441 ionic strength, 274

Index 855
pH, 274, 444 clinical significance, 411, 418, 423-424, 439, 441
temperature, 273-274, 336, 443 dosage effect, 225, 227, 327, 345, 425, 431
grading reactions, 412, 728-729 to high-incidence antigens, 356-357,
inhibition, 275-276, 444-445 435-436, 441-442, 547
mixed-field, 298, 299, 348, 357 in liver transplant patients, 629
stages, 272-275 to low-incidence antigens, 340, 358, 436-437,
tests for granulocyte antibodies, 380 546
Agreements, 10, 177 multiple, 236, 434-435, 441-442, 449
AHF. See Cryoprecipitated Antihemophilic Fac- in selection of units, 418, 439-443
tor serologic behavior, 336
AHG test. See Antiglobulin test in sickle cell disease, 576
AIDS (acquired immunodeficiency syndrome), temperature of reaction, 273-274, 336, 443
675-676, 677. See also HIV in thalassemia patients, 576
AIHA. See Autoimmune hemolytic anemia titration, 449, 539-540, 761-764, 796-798
Air embolism, 636, 651-652 Allogeneic adsorption, 471-472, 781-782
AITP (autoimmune thrombocytopenic Allogeneic HPC transplantation
purpura), 158, 373-374, 492, 553-554 ABO incompatibilities, 598-599, 600, 601-602
Alanine aminotransferase (ALT), 164, 673, 835 cell processing, 596-597, 598, 601-604
Alarm systems, 185, 198-199, 823-826, 846 collection of products, 591-596
Albumin defined, 582
antibodies to ingredients in, 437-438 donor evaluation, 589-591
as colloid replacement, 507-508 evaluation and QC of products, 607-608
physiology, 507 freezing and storage of products, 604-606
recombinant, 219 graft-vs-host disease in, 586-587
as replacement fluid in apheresis, 150, 151 of HPC-A (apheresis), 587-588, 592, 594-595
in serologic testing, 276, 427, 753 of HPC-C (cord blood), 588-589, 595-596
use in exchange transfusion, 567 of HPC-M (marrow), 583, 585-587, 591-592
Aldehyde dehydrogenase (ALDH), 603 indications for, 582
ALI (acute lung injury), 647 infectious disease testing, 590-591
Aliquoting components, 193-194, 564, 571 matched, unrelated transplantation,
Allele-specific oligonucleotide (ASO) hybridiza- 585-586, 589
tion. See Sequence-specific oligonucleotide mobilization of HPCs, 587, 594-595
probes nonmyeloablative, 582-583
Alleles, 225, 227. See also specific blood groups positive DAT after, 455
blank, 389 regulations, 608
dosage effect, 225, 227 related transplantation, 587-588, 589
frequencies, 227-229 standards, 609
rare, 233, 234 thawing and infusion of products, 607
Allelic, defined, 241 transfusion support in, 591-592, 599, 600,
Allergic reactions 601
in apheresis, 152 transportation and shipping of products,
to latex, 46-47 606-607
to transfusions, 522, 634, 644-647 Allografts. See Organ donation and transplanta-
Alloantibodies, red cell. See also Antibody detection; Tissue transplantation
tion; Antibody identification; specific blood Alloimmune hemolytic anemia. See Hemolytic
groups disease of the fetus and newborn; Hemolytic
in ABO discrepancies, 299, 303 transfusion reactions
with autoantibodies, 438-439, 470-472 Alloimmunization. See also Alloantibodies, red cell
in bone graft patients, 623 direct/indirect allorecognition, 265

HLA, 265, 266, 362-366, 397-398, 637 Angiotensin-converting enzyme (ACE) inhibi-
in sickle cell disease, 575, 576 tion, 636, 645
as transfusion complication, 265, 637, ANH. See Acute normovolemic hemodilution
656-657 Anhydrous, defined, 724
Allotypic, defined, 269 Ankylosing spondylitis, 403
α-methyldopa, 455, 475-476 Anti-A and anti-B, 294-296
ALT (alanine aminotransferase), 164, 673, 835 Anti-A1, 293, 295-296, 302
AMD 3100, 594 Anti-A,B, 295, 296
American Rare Donor Program (ARDP), 441-442, Anti-C3b, -C3d, 279, 280, 456
769-770 Anti-CMV, 170
American trypanosomiasis (Chagas’ disease), Anti-D
697-698, 700 active vs passive antibody, 548
2-aminoethylisothiouronium bromide (AET), antibody titrations, 539-540
342, 446 in D+ individuals, 327
Amniocentesis, 539, 540, 541, 549 discovery of, 315-316
Amniotic fluid analysis, 540-541, 542 dosage effect, 327
Ana (Gerbich) antigen, 352, 843 in HDFN, 330, 535, 536-537, 539-540
Anaphylactic transfusion reactions, 644-647 in liver transplantation, 628-629
evaluation, 655 partial D, 322-323
frequency, 645 reagents, 328-330
management, 635 and weak D, 323
pathophysiology, 635, 644-645 Anti-Delta, 670
prevention, 646-647 Anti-HAV, 671
symptoms, 639, 644-645 Anti-HBc (antibody to hepatitis B core antigen)
treatment, 645-646 blood component testing, 164, 166, 675
Anaphylactoid transfusion reactions, 644, 645 look-back for, 675
Anaphylatoxins, 262, 639 marker of infection, 669, 670
Anemia. See also Hemolytic disease of the fetus reentry of donors with, 675
and newborn as surrogate marker, 672
alloimmune hemolytic, 459 testing transplant donors for, 621
autoimmune hemolytic, 458-477, 509-510 Anti-HBe (antibody to hepatitis B e antigen),
cold agglutinin syndrome, 459, 460, 464- 669, 670
466 Anti-HBs (antibody to hepatitis B surface anti-
DAT-negative AIHA, 459, 468 gen), 669, 670
drug-induced hemolytic, 459, 460, 472-477, Anti-HCV (antibody to hepatitis C virus)
481-482 blood component testing, 164, 166, 170
IgM warm AIHA, 464 marker of infection, 670-671, 672-673
mixed-type AIHA, 459, 460, 466-467 reentry of donors with, 673, 674, 675
in neonates, 558, 559, 563-564 supplemental testing, 170
oxygen compensation in, 484 testing organ donors for, 620, 621, 623
paroxysmal cold hemoglobinuria, 459, 460, Anti-HEV, 671
467-468 Anti-HIV-1, 2
screening donors for, 102, 799 reentry of donors with positive tests, 674,
sickle-cell disease, 119, 155-156, 508-509, 681
574-576 supplemental testing, 169-170
in thalassemia, 508 testing blood donors for, 164, 166, 169-170,
treatment, 487-488 676, 679-681
warm autoimmune hemolytic anemia, 459, testing organ donors for, 620, 621,
460, 461-464 623

Index 857
Anti-HTLV-I, II Antibody-dependent cellular cytotoxicity

blood component testing, 120, 164, 166, 170, (ADCC), 255, 441
683 Antibody detection
donors for organ transplants, 620 antiglobulin test, 277-281, 282, 283
supplemental testing, 170 with autoantibodies, 438-439, 463-464,
Anti-IgA, 645, 646, 655 470-472
Anti-IgG, 279, 280, 427, 438, 456 in autologous blood, 122
Anti-K1, 536, 538, 540 autologous control, 412, 427, 438-439
Anti-Ku, 343 automated testing, 285
Anti-Pr, 777, 778 in blood components, 165
Antibodies. See also Alloantibodies; Antigen- in children, 415, 563, 574
antibody reactions; Autoantibodies; other column agglutination for, 278, 284-285
Antibody entries; specific blood groups in cord blood, 595
complement-binding, 280 ELISA for, 286
concomitant, 327-328, 441 enhancement, 276-277, 427, 443-444,
defined, 269 753-754
distinguishing IgG and IgM, 764-765 enzyme-linked immunosorbent assay, 286
drug-induced, 374-377, 472-477, 481-482, frequency of testing, 442-443, 463-464
786-791 of granulocyte autoantibodies, 380
equilibrium constant (Ko), 273 of HLA antibodies, 365-366, 397
high-titer, low avidity, 449, 762, 765- immunofluorescence, 285-286
766 indirect antiglobulin test, 277, 278, 752-754
HLA interpreting reactions, 415-416, 417
in antibody identifications, 433 MAIEA assay, 284, 286
and Bg antigens, 358 in organ transplantation, 629
detection, 365-366, 397 of platelet antibodies, 370-373, 374, 375, 377
in platelet refractoriness, 362-365, in Platelets Pheresis, 142
397-398, 491 in positive DAT evaluation, 456
in transfusion reactions, 398-400, 637, practical considerations, 412
647, 656 in prenatal evaluations, 539
monoclonal in pretransfusion testing, 411-413, 417
in ABO testing, 296 prewarming technique, 308, 438, 754-755
anti-D, 328, 329-330 reagents, 278-280, 425, 427, 468
assays utilizing, 284, 373, 380 solid-phase red cell adherence tests, 283-284
in autologous tumor purging, 597-598 specimen requirements, 424
for reagent use, 266-267 in transfusion reaction evaluation, 654
in neonates, 560 Antibody identification, 427-450
platelet ABO type of red cells tested, 434
autoantibodies, 373-374 adsorption, 446-447, 448, 470-472
clinical importance, 370-373 anomalous reactions, 437-438
detecting, 370-373, 374, 375, 377, 552 antibodies to high-incidence antigens,
drug-induced, 374-377 356-357, 435-436, 441-442, 547
HLA, 266, 362-366, 397-398 antibodies to low-incidence antigens, 340,
platelet-specific, 366, 367-368, 370-373, 358, 436-437, 546
552 with antibodies to reagent components,
production, 249 437-438
reagent, 266-267 with autoantibodies, 438-439, 463-464,
to reagent components, 298, 437-438 470-472
temperature of reaction, 273-274, 443 autologous control, 427, 429-430, 438-439

elution, 447-448, 456-458 blood group nomenclature, 238-239,

enhancement techniques, 276-277, 427, 318-319, 840-844
443-444, 753-754 CD, 246, 247-248
flowchart for, 432 collections, 335, 355-356
frequency of testing, 442-443, 463-464 defined, 269
inactivation of antigens, 445-446, 766-767 depressed, 341-342, 468
inhibition tests, 444-445 distribution in population, 335, 337
interpreting results, 428-429 granulocyte/neutrophil, 377-380
medical history in, 424 high-incidence
multiple antibodies, 236, 434-435, 441-442, antibodies to, 356-357, 435-436, 441-442,
449 547
no discernible specificity, 433-434 of Cromer system, 353, 354
phenotyping autologous red cells, 429-430, defined, 335
439 of Gerbich system, 352
with positive DAT, 427, 436, 438-439, 456 GIL, 355
in prenatal evaluations, 539 of Knops system, 354
prewarming technique, 308, 438, 754-755 of Lutheran system, 347
probability values, 429, 430 not assigned to a system or collection,
reagents, 278-280, 425, 426, 427, 468 356-357
and selection of blood, 418, 439-443, 463 with positive DAT, 436
specimen requirements, 424 selecting blood negative for, 441-442
sulfhydryl reagents in, 446, 448-449, 744-745, of Vel collection, 355-356
766-767 HLA, 362, 385, 388-389, 390-391
titration studies, 449, 539-540, 761-764, inactivation, 342, 445-446, 766-767
796-798 low-incidence, 357-358, 436-437, 546
in transfusion reaction evaluation, 654 antibodies to, 340, 358, 436-437, 546
using immunohematology reference labs, of Cromer system, 353, 354
439 defined, 335
variations in antigen expression, 431, 433, of Gerbich system, 352
468 of Lutheran system, 347
Antibody screens. See Antibody detection of MNS system, 227, 228, 337-338, 340
Antibody specificity prediction (ASP) method, not assigned to a system or collection,
365 357
Anticoagulant-preservative solutions, 178-179 of Scianna system, 350
CPD, CP2D, CPDA-1, 176, 178, 186, 564, platelet, 361-363, 366, 367-368, 397, 552
565 public, 392
red cell changes during storage, 185, 186, typing donor units for, 418, 440-441, 657,
187, 431, 433 745-748
shelf life of components, 178, 188, 189-190 variations in expression of, 431, 433, 468
Antifibrinolytic agents, 513-514 Antiglobulin (AHG) test. See also Direct
Antigen-antibody reactions. See also Antibody antiglobulin test
detection; Antibody identification in antibody detection/identification,
agglutination, 271, 272-276 277-281, 282, 283
hemolysis, 271-272 complement in, 280-281, 453
precipitation, 272 in crossmatching, 413, 414
prozone, 272, 275 direct, 278, 454-458, 760-761
Antigen-presenting cells (APCs), 269 indirect, 278, 752-754
Antigens. See also specific blood groups principles, 277-278
acquired, 298, 301-302 reagents, 278-280, 427, 454, 468

Index 859
sources of error, 282, 283 Auto controls. See Autologous controls

use of IgG-coated cells, 281, 412 Autoadsorption. See Adsorption
Antiprotease concentrates, 505-506 Autoantibodies
Antipyretics, 522 cold
Antithrombin, 500, 505-506 ABO discrepancies with, 299, 302-303,
AnWj antigen, 356-357 469
APCs (antigen-presenting cells), 269 adsorption, 438, 466, 472, 775-776
Apheresis with alloantibodies, 438-439, 472
complications, 141, 150-153 anti-I/i, 306, 307-308, 438, 465-466
for component collection, 140-144 anti-IH, 308, 434, 777, 778
donation intervals, 141, 143 in cold agglutinin syndrome, 281, 307,
equipment, 139-140, 152, 847 465-466
HPCs collected by, 581, 587-588, 592-595 and complement, 281
monitoring cell counts, 832 determining specificity, 776-778
records, 142 in mixed AIHA, 466, 467
separation techniques, 139-140 in paroxysmal cold hemoglobinuria,
therapeutic, 144-158 467
indications for, 146-148, 153-158 in Rh testing, 329, 469-470
photopheresis, 158 titration, 458, 465, 778-779
plasma volumes exchanged, 145, 149 use of sulfhydryl reagents with, 469-470,
removal of normal plasma constituents, 744-745
149-150 in warm autoimmune hemolytic anemia,
removal of pathologic substances, 145, 461
148-149 in DAT-negative AIHA, 468
replacement fluids, 150, 151 granulocyte, 380
SPA immunoadsorption, 158 platelet-specific, 373-374
vascular access, 145, 150 posttransfusion, 657
Aprotinin, 502, 513-514 warm
aPTT. See Activated partial thromboplastin time adsorption, 470-472, 779-781
Arachis hypogaea (anti-T), 743 with alloantibodies, 439, 470-472
ARDP (American Rare Donor Program), 441-442, drug-induced, 475-476
769-770 frequency of testing, 463-464
Arrhythmias, 650 mimicking alloantibodies, 472
Arterial transplants, 624 transfusion-stimulated, 462
Articular cartridge transplants, 624 transfusion with, 462-464
AS (additive solutions), 176, 186, 564-565 in warm autoimmune hemolytic anemia,
ASP (antibody specificity prediction) method, 461-462
365 Autoclaving biohazardous waste, 57
Assessments, 22-24 Autoimmune hemolytic anemia (AIHA)
blood utilization, 23, 36-38, 514 classification, 458, 459
competency, 8 cold agglutinin syndrome, 459, 460, 464-466
external, 23-24 complement coating cells, 281, 460, 465
internal, 22 DAT-negative AIHA, 459, 468
proficiency testing, 24 IgM warm AIHA, 464
quality indicators, 22-23, 31 mixed-type AIHA, 459, 460, 466-467
transfusion oversight, 514 paroxysmal cold hemoglobinuria, 459, 460,
Ata antigen, 356 467-468
ATL (adult T-cell lymphoma/leukemia), 682 serologic findings in, 460
Audits, transfusion, 23 transfusion in, 462-464, 467, 468, 509-510

warm autoimmune hemolytic anemia, 459, shipping, 120

460, 461-464 storage, 123
Autoimmune neutropenia, 379-380 supplemental iron use, 121
Autoimmune platelet disorders, 373-374 testing, 119-120, 122
Autoimmune thrombocytopenic purpura timing and red cell regeneration during,
(AITP), 158, 373-374, 492, 553-554 122
Autologous adsorption transfusion of units, 123
cold, 438, 466, 472, 775-776 transfusion trigger, 125
warm, 470-471, 779-781 volume collected, 122
Autologous blood voluntary standards, 119
acute normovolemic hemodilution, 117, weak D in donor, 324
126-130 separation from transfused cells, 748-750
clinical studies, 128, 129, 130 Autologous controls
physiologic considerations, 127-128 in antibody identification, 427, 429-430,
practical considerations, 128, 130 438-439
selection of patients, 131 positive, 417, 438-439
bacterial contamination of, 693 in pretransfusion testing, 412, 417, 454
categories, 117 Autologous HPC transplantation
for HIV-positive donors, 117, 681 autologous tumor purging, 597-598
intraoperative blood collection, 117, 130-133 cell processing, 596-604
clinical studies, 131-132 collection of products, 591-596
controversies in, 132-133 defined, 582
direct reinfusion, 133 donor evaluation, 589, 590-591
equipment for, 132-133 evaluation and QC of products, 607-608
practical considerations, 132 freezing and storage of products, 604,
processing, 132-133 605-606
requirements and recommendations, 133 of HPC-A (apheresis), 587, 592-595
inventory management, 94-95 of HPC-C (cord blood), 589, 595-596
for patients needing rare blood types, 442 of HPC-M (marrow), 583, 591-592
postoperative blood collection, 117, 133-135 infectious disease testing, 590-591
preoperative blood donation, 117, 118-126 mobilization of HPCs, 592-594
advantages/disadvantages, 118 regulations, 608
adverse reactions, 123 standards, 609
aggressive phlebotomy, 125 thawing and infusion of products, 607
collection, 121, 126 transportation and shipping, 606-607
compliance requirements, 119-120 Autologous tumor purging, 597-598
continuous quality improvement, Automated testing platforms, 285
123-124 Autosomal dominant and recessive traits, 233,
contraindications, 119 234
cost-effectiveness, 125, 127
donor deferrals, 120 B
donor screening, 121-122, 124-125
5b antigen, 378
erythropoietin use, 125
B-cell receptor (BCR), 249
establishing program, 120-124
B lymphocytes, 249-250, 252-253
labeling, 120, 122-123
receptors/markers on, 249-253
medical interview, 122
stimulation of, 254-255
in pediatric patients, 118-119
β2-microglobulin, 245
physician responsibility, 120-121
B(A) phenotype, 298, 301
records, 111, 123
Babesiosis, 695-696, 700

Index 861
Bacterial contamination, 690-695 safe work practices, 54-55

clinical considerations, 692-693 shipping dangerous goods, 716-722
fatalities associated with, 690-692 Standard Precautions, 49
of HPCs, 602-603 storage, 54
organisms involved, 691, 692 training, 50
platelet-associated, 690-692, 694-695 waste management, 55-57
preventive measures for, 693-694 Biphasic hemolysin of paroxysmal cold
of reagents, 331, 332 hemoglobinuria, 467
of Red Blood Cells, 691 Blank alleles, 364
testing components for, 195, 655, 693, 694 Bleeding
in transfusion-associated sepsis, 635, 643, indication for transfusion, 486
655, 691-692 microvascular, 511, 651
transfusion risk of, 700 with platelet defects, 490
Bacteriologic studies transfusion-related, 639
of components, 195, 655, 693, 694 Bleeding time, 488, 489, 837
of hematopoietic products, 602-603 Blocking phenomenon, 330
in transfusion reaction evaluation, 655 Blood administration
Bags, blood, 104-105 assessment, 37-38
centrifugation of, 179-180 blood warmers for, 522, 529, 560, 650, 847
for frozen storage, 182 compatibility testing, 524
returning to lab, 531-532 component preparation in, 522, 523
Barcode labels, 171 delays in starting, 525
Basophils, 255 delivering blood to transfusion area, 524-
BCR (B-cell receptor), 249 525
Bennett-Goodspeed (Bg) antigens, 358, 391 electromechanical pumps for, 522, 523,
Bilirubin 529-530, 565
in HDFN, 536, 540-541, 542, 547 emergency release, 522-523
hyperbilirubinemia, exchange transfusion errors, 525-526, 527, 641, 642, 653, 654
for, 566-567 filters for, 528-529, 565-566
total, normal values, 835, 836 identification procedures, 525-526
Bioassays, 65 infusion sets for, 527-528, 565-566
Biohazardous waste IV solutions, 530
defined, 55-56 in neonates, 565-566
disposal, 56-57 patient care during, 530-531
treating, 57 patient consent, 521-522
Biologic product deviations, 19, 21 patient considerations in, 522
Biological products, 717 patient education, 521-522
Biological safety cabinets (BSCs), 50, 51, 52-53 post-administration events, 531-532
Biosafety, 49-57 prescription for, 522-523
biosafety levels and precautions, 49-50, pressure devices for, 530
54-55, 77 quality assurance, 532
Bloodborne Pathogen Standard, 49 reaction evaluation, 531, 652-656
decontamination, 51, 54 special instructions for, 522-523
emergency response plan, 55-57 starting the transfusion, 526-527
engineering controls, 50-51 venous access, 523-524, 565-566
hazard identification and communication, Blood bags, 104-105
50 centrifugation of, 179-180
labeling, 719 for frozen storage, 182
personal protective equipment, 54 returning to lab, 531-532

Blood collection freezing, 180-183, 807-812

adverse donor reactions, 19, 107-109 for hemostasis, 496, 497, 498-507, 570-572
anticoagulants and preservatives, 178-179, identification, 105, 416, 524-526, 527
186 inspection, 93-94, 194-195, 416, 418, 525
blood containers, 104-105 irradiated, 183, 192-193, 493
of blood samples, 105-106, 408-409, 801-804 issuing, 196-197, 416, 418, 524-525
care of donor, 106 labeling, 15, 170-172, 416, 440
of components by apheresis, 140-144 leukocyte reduction, 180, 190, 492-493,
equipment quality control, 847 573-574
fatalities related to, 19 ordering, 36, 91-94, 522-523, 526
identification in, 98, 105 outdating, factors affecting, 90-91
intraoperative, 117, 130-133 pathogen inactivation, 702
phlebotomy, 105-106, 178, 693, 801-804 phenotyping, 440-441
postoperative, 117, 133-135 pooling, 183-184, 193, 815
preoperative, 126 preparation, 173, 179-184, 522, 523, 804-818
preoperative autologous, 118-126 quality control, 197-199, 202
preparing venipuncture site, 105, 800-801 quarantine, 173-174, 194
records, 97-98, 165 records, 165, 172-173, 416, 418
safety, 41, 51 reissuing, 197, 525
volume collected, 101, 122, 178-179 rejuvenation, 194, 806-807
of whole blood, 178-179 returning, 525
Blood component selection storage, 93-94, 184-185, 186, 187
ABO/Rh compatibility in, 411, 418, 486, testing
489-490, 496, 498 ABO and D, 164, 165
after non-group-specific transfusions, alanine aminotransferase, 164
419-420 antibody screening, 165
with alloantibodies, 418, 439-443 cytomegalovirus, 170
for exchange transfusions, 546-547, 567 equipment requirements, 164-165
for intrauterine transfusions, 544 general requirements, 163-164
for massive transfusions, 419, 510-511 hepatitis, 669-673
platelets, 363-365, 489-490, 569 HIV, 164, 166, 169-170, 679-681
rare types, 441-442 HTLV, 120, 164, 166, 170, 683
red cell components, 411, 486 records, 165
in urgent situations, 419, 510-511 syphilis, 165-166, 695
in warm autoimmune hemolytic anemia, viral markers, 166-170
463 thawing, 191-192, 809, 815
Blood components. See also Blood collection; transportation and shipping, 66, 179,
Blood component selection; specific compo- 195-196, 717, 722
nents volume reducing, 193, 490, 569, 817-818
aliquoting, 193-194, 564, 571 washed, 193
appearance, 194, 525, 655 Blood containers, 104-105, 179-180, 182, 564
bacterial contamination, 195, 643, 655, Blood donation. See Blood collection
690-695 Blood exposure, 45, 54-55
for children, 564-572, 574-577 Blood Group Antigen Mutation Database, 236
CMV-negative, 95, 172 Blood group sugars, 445
disposition, 196 Blood group systems. See also specific blood
distribution, handling and dispensing, 36-37 groups
for exchange transfusion, 567 chromosomal assignments, 225, 226,
expiration and shelf life, 178, 188, 189-190 235-236

Index 863
co-dominant traits in, 234-235 Blood utilization

defined, 335 assessments, 23, 36-38
distribution of antigens, 335, 337 blood ordering practices, 36, 91-94, 414
genetics inventory levels, 89-90, 93
basic principles, 223-225 maximum surgical blood order schedules,
and heredity, 225-232 92-93, 414
patterns of inheritance, 232-236 outdating factors, 90-91
population genetics, 236-237 routine vs emergency orders, 93
nomenclature, 238-239, 318-319, 840- of special components, 94-95
844 units processed, transfused and outdated in
serologic behavior of antibodies, 336 2001, 91
Blood inspections Blood volume, 558-559, 839
before release, 194-195, 416, 418, 525 Blood warmers, 522, 529, 560, 650, 847
of inventory, 93-94 Bloodborne Pathogen Standard, 49
Blood loss Bombay (Oh) phenotype, 304
dilutional coagulopathy in, 499-500, 651 Bonds, chemical, 273
due to platelet dysfunction, 490 Bone banking, 623, 625
in preterm neonates, 559 infectious disease transmission in, 619
transfusion in, 486-487 preservation and dating periods for, 624
Blood ordering practices Bone marrow. See Marrow transplantation
assessment, 36 Bovine aprotinin, 502, 513-514
improving, 91-94 Bovine spongiform encephalopathy (BSE), 690
physicians’ orders, 522-523, 526 Burkitt’s lymphoma, 687
for routine vs emergency orders, 93, 414
Blood pressure, 633 C
of donors, 102
C/c antigens and antibodies (Rh system), 316,
hypertension, 633
841
hypotension
cis product antigens, 324
in donors, 107
concomitant antibodies, 327-328, 441
in recipients, 585, 586, 591, 595
dosage in, 317
in therapeutic apheresis, 152
expression of, 320
treatment, 592
gene complexes, 319
Blood pressure cuffs, 847
in HDFN, 536
Blood samples, 409-410
incidence of, 317, 321
age of, 410, 431, 433
phenotypes, 320
appearance, 410
testing for, 330-331, 441
frozen, 184-185
C1-esterase inhibitor, 500
hemolyzed, 410
Calcium, 151-152, 561, 636, 649-650
identification and collection, 105-106,
Calculations
408-409, 801-804
for CRYO dose, 501
incompletely clotted, 409, 722-723
for Factor VIII dose, 503-504
labeling, 409, 719
for multiple alloantibodies, 236, 441
lipemic, 410
Calibration
packaging, 717-718, 720-721
of cell washers, 830-832
requirements for testing, 424, 454
defined, 30
retention and storage, 410
of platelet separation centrifuges, 179,
transportation and shipment, 66, 717-721
826-828
Blood spills, 55, 56
of serologic centrifuges, 828-830
Blood transfusions. See Transfusions
of thermometers, 198, 821-823

CAP (College of American Pathologists), 514 Centromere, 224, 241

Capture-P, 372 Cephalosporins, 476
Cardiovascular system. See Heart and heart valves detection of antibodies to, 786-788
Carriers, defined, 233 in positive DAT, 455, 474, 476, 477
Cartwright (Yt) system (ISBT 011), 226, 336, 348, Cephalothin (Keflin), 476
349, 842 CFR (Code of Federal Regulations), 1, 32
CAS. See Cold agglutinin syndrome CGD (chronic granulomatous disease), 343
Catheters Ch (Chido/Rodgers) antigens, 351-352, 842
for apheresis, 145 Chagas’ disease, 697-698, 700
for HPC collection, 593, 594 Change control, defined, 30
for neonates, 565, 568 Charts, run and control, 23
for transfusions, 523-524 Check cells, 281, 412
Cause-and-effect diagrams, 26 Chemical bonds, 273
CBER (FDA Center for Biologics Evaluation and Chemical Hygiene Plan (CHP), 57-58
Research), 19, 702-703 Chemical safety, 57-63
CCE (counterflow centrifugal elutriation), chemical categories, 82-83
596-597 chemical data sheet, 78-79
CCI (corrected platelet count increment), 362, chemical hygiene officer, 58
363, 489 chemical spills, 61-62, 84-88
CD (cluster of differentiation) markers, 246, chemicals found in blood banks, 80-81
247-248, 250 engineering controls, 61
CD34 general principles, 57-58
analysis/enumeration, 603 hazard communication, 59
collection targets, 593-594 hazardous vapors, 62, 63
defined, 592 health hazards categories, 58, 59
selection of, 596-597 labeling and signs, 59-60
Cefotetan antibodies, 477 material data safety sheets, 59, 60-61, 78-79
Cell adhesion molecules, 246, 269 personal protective equipment, 61
Cell counters, 847 safe work practices, 61, 82-83
Cell counts spills, 61-62, 84-88
in apheresis components, 832 training, 58-59
in HPC products, 607-608 waste disposal, 63
Cell selection techniques, 596-598 Chemiluminescence assay, 380, 441
Cell therapy. See Hematopoietic progenitor cell Chemokines, 594
transplantation Chido/Rodgers system (ISBT 017), 351-352
Cell washers, 830-832, 846 antibodies, 352
Center for Biologics Evaluation and Research antigens, 351-352, 842
(CBER), 19, 702-703 genes, 226
Central venous catheters, 524, 593 plasma inhibition studies, 445, 765-766
Centrifugation Children. See also Hemolytic disease of the fetus
apheresis separation by, 139-140 and newborn
in component preparation, 179-180 ABO antigens/antibodies in, 293, 295, 298,
counterflow elutriation, 596-597 420
in separating autologous cells, 748-749 ABO discrepancies in, 298
standardization of variables, 716 anemia in, 558, 559, 563-564
Centrifuges autologous collection from, 118-119
calibrating, 179, 826-830 blood volume of, 558-559, 839
continuous-flow, 596-597 compatibility testing in, 415, 562-563, 574
QC performance intervals, 846, 847 cytomegalovirus in, 562

Index 865
DIC in, 567, 572 defined, 223-224, 241

erythropoiesis in, 557-558, 559 DNA structure in, 203-204
extracorporeal membrane oxygenation in, locations of blood group genes, 225, 226,
572-573, 574 235-236
graft-vs-host disease in, 560-561 Chronic granulomatous disease (CGD), 343
hematopoietic transplantation in, 587, 593 Chronic inflammatory demyelinating
hypothermia in, 560 polyneuropathy (CIDP), 156
immunologic status, 560-561 Circulatory overload, 636, 648-649
leukocyte reduction for, 573-574, 576 Circulatory shock, 633
Lewis antigens in, 306 Cis product antigens, 319-320, 324, 342
metabolic problems in, 561-562 Citrate toxicity, 151, 636, 649-650
normal laboratory values in, 836-837 CJD (Creutzfeldt-Jakob disease), 689-690
paroxysmal cold hemoglobinuria in, 468 Class I, II, III antigens (HLA), 244-246, 386-394
plasma volume of, 839 biochemistry and structure, 390-391
polycythemia in, 572 biologic function, 393-394
red cell volume of, 839 defined, 269, 385
size of, 558-559 genetics, 386-387
transfusions in nomenclature, 392-393
administration, 565-566 Clinical specimens
aliquoting for small volumes, 193-194, defined, 717
564, 571 labeling, 718, 719
Cryoprecipitated AHF, 572 shipping, 717-718, 720-722
education and consent, 521 Clocks, 847
to enhance hemostasis, 570-572 Clone, defined, 269
exchange, 546-547, 560, 566-568 Cluster of differentiation (CD) markers, 246,
FFP, 571, 576-577 247-248
Granulocytes, 569-570 CMV. See Cytomegalovirus
with hemoglobinopathies, 574-576 Co (Colton) antigens and antibodies, 336, 349,
indications for, 563-564 351
older infants and children, 574-577 Co-dominant traits, 233, 234-235, 241
Platelets, 490, 552-553, 568-569, 576-577 Coagulation
Red Blood Cells, 564-568 in hemolytic transfusion reactions, 640, 642
with sickle cell disease, 574-576 in liver transplants, 629
with thalassemia, 575-576 in massive transfusion, 511, 651
volumes for, 558, 564, 568 monitoring hemostasis, 494, 496
variations in antigen expression, 431 normal test values, 837, 838
Chills with transfusions, 633, 634, 643 physiologic principles, 493-494
Chimerism role of platelets, 488-489
blood group, 237, 299 Coagulation factors
hematopoietic, 298, 601 changes in red cell storage, 187-188
and posttransfusion GVHD, 399 concentrates, 496, 497
transfusion, 298, 399 in Cryoprecipitated AHF, 500-502
Chloroquine diphosphate, 446, 469, 746-747 Factor VIIa concentrates, 497
Chorionic villus sampling, 539 Factor VIII concentrates, 497, 503-504
CHP (chemical hygiene plan), 57-58 Factor IX concentrates, 497, 505
Christmas disease, 505 minimum levels needed for hemostasis, 495
Chromatid, 241 in neonates, 571
Chromatin, 223, 241 normal values, 837
Chromosomes. See also Genes in plasma, 496, 498-500

in platelet concentrates, 838 in warm autoimmune hemolytic anemia,

replacement, 496, 497, 498-508 461
support during massive transfusion, 511 Cold hemagglutinin disease (CHD). See Cold ag-
virus inactivation in concentrates, 701 glutinin syndrome
for vitamin K deficiency, 497, 498-499 Cold reactive alloantibodies, 273-274, 299, 303
for warfarin reversal, 497, 498-499 Cold stress. See Hypothermia
Coagulopathy Collection of blood. See Blood collection
dilutional, 499-500, 567, 651 Collections, antigen, 335, 355-356
in hemolytic transfusion reactions, 640, College of American Pathologists (CAP), 514
642 Colloid solutions, 486, 507-508, 572
in liver disease, 499 Colony-forming cell assays, 603-604
in liver transplantation, 629 Colony-stimulating factors, 264
in massive transfusions, 511, 651 Colton system (ISBT 015), 226, 336, 349, 351, 842
in neonates, 571 Column agglutination technology, 278, 284-285
treatment with heparin, 642 Compatibility testing. See Pretransfusion testing
in vitamin K deficiency, 498-499 Competency assessments, 8
in warfarin reversal, 498-499 Complement, 259-262
Code of Federal Regulations (CFR), 1, 32 in acute transfusion reactions, 639-640
Codons, 206-207 alternative pathway, 260, 261
Cold-acid elutions, 457, 772 in antiglobulin testing, 280-281, 453
Cold agglutinin syndrome (CAS), 464-466 in autoimmune hemolytic anemia, 281, 460,
adsorption procedures, 466 465
classification, 459 C1-esterase inhibitor, 500
complement in, 281 classical pathway, 259-261
pretransfusion testing in, 466 complement receptors, 262, 354
serologic findings in, 460, 465 membrane attack complex, 261-262, 263
specificity of autoantibodies, 307, 465-466 physiologic effects of activation, 262
Cold agglutinin titer, 458, 465, 778-779 and positive DAT, 453
Cold autoadsorption regulation of activation, 262, 263
in cold agglutinin syndrome, 308, 466 Complications. See Adverse reactions
for detection of alloantibodies, 438, 472 Computer crossmatch, 413, 414-415
method, 775-776 Computer systems
in resolving ABO discrepancies, 303 alternative systems, 18-19
Cold autoantibodies backups, 19
ABO discrepancies with, 299, 302-303, 469 for record storage, 17-19
adsorption, 438, 466, 472, 775-776 security, 18
with alloantibodies, 438-439, 472 validation, 13-14, 415
anti-I/i, 306, 307-308, 438, 465-466 Confidential unit exclusion, 101
anti-IH, 308, 434, 777, 778 Confidentiality of records, 18
in cold agglutinin syndrome, 281, 307, Consent
465-466 for apheresis, 140, 142
and complement, 281 for blood donation, 103
determining specificity, 776-778 for transfusion, 521-522
in mixed AIHA, 466, 467 for transplantation, 620-621
in paroxysmal cold hemoglobinuria, 467 Containers
in Rh testing, 329, 469-470 for biological specimens, 717
titration, 458, 465, 778-779 for blood collection, 104-105, 179-180
use of sulfhydryl reagents with, 469-470, damaged or leaking, 720
744-745 freezer storage bags, 182

Index 867
quality control, 847 CP2D (citrate-phosphate-dextrose-dextrose),

for shipments, 195, 607, 717-718 176, 178
Contamination. See Bacterial contamination CPD (citrate-phosphate-dextrose), 176, 178, 186
Continuous-flow centrifuges, 596-597 CPDA-1 (citrate-phosphate-dextrose-adenine),
Continuous Quality Improvement, 123-124 176, 178, 564-565
Contracts, 10 CR (complement receptors), 262, 354
Control charts, 23, 30 Cra (Cromer) antigen/antibody, 353, 354, 843
Controls CREG (cross-reactive groups), 392
autologous, 412, 427, 438-439, 454 Creutzfeldt-Jakob disease (CJD), 689-690
for high-protein reagents, 328-329 Cromer system (ISBT 021), 226, 353, 354, 843
IgG-coated cells, 281, 412 Cross-reactive groups (CREGs), 392
for indirect antiglobulin tests, 754 Crossmatch-to-transfusion (C:T) ratios, 92, 93
for low-protein reagents, 330 Crossmatching
in viral marker testing, 167-169 antiglobulin test in, 413, 414
Convulsions in donors, 108 for children, 415, 546, 563
Copper sulfate computer, 413, 414-415
quality control, 819-821, 847 HLA, 397, 398, 401, 402, 492
in screening donors, 102, 799-800 immediate-spin, 413, 414, 751-752
Cord blood “in-vivo,” 509
antigen expression on, 431, 433 interpretation of results, 415-416, 417
HPC-C transplantation, 588-589 platelets, 364, 365, 398
autologous, 589 in pretransfusion testing, 412, 413-415, 417
collection, 588, 595-596 in transfusion reaction evaluation, 654, 655
colony-forming cell assays, 603-604 Crossover, gene, 208, 209, 230-231, 232, 241, 389
cord blood banks, 589 CRYO. See Cryoprecipitated Antihemophilic Fac-
diseases treated with, 583, 584 tor
engraftment in, 588-589 Cryoglobulinemia, 156
evaluation and quality control, 607- Cryoprecipitate Reduced Plasma, 177, 190, 496
608 Cryoprecipitated Antihemophilic Factor (CRYO)
freezing and storage, 604, 605-606 ABO compatibility, 411, 500-501
infectious disease testing, 591 calculating dose, 501
microbial cultures, 602-603 for coagulation factor replacement, 496
processing, 602 description, 177
regulations, 608 for disseminated intravascular coagulation, 502
related donors, 589 expiration dates, 189
standards, 609 for fibrinogen abnormalities, 501-502
stem cell enumeration, 603 for hemophilia A, 502-503
thawing and infusion, 607 indications for use, 499, 500-503
transportation and shipping, 606-607 inspection, 194
serologic testing, 544-546, 595 inventory management, 94
Cordocentesis, 539, 541-542, 552 for neonates, 558, 572
Corneal transplants, 619, 621, 624 pooled, 183-184, 191, 193, 815
Corrected platelet count increment (CCI), 362, preparation, 814-815
363, 489 quality control, 199, 202
Corrective action, 24-25 room temperature storage, 185
Corticosteroids, 144 thawing, 191, 815
Cost blood group collection, 352, 355 topical use, 502
Counterflow centrifugal elutriation (CCE), transportation and shipping, 196
596-597 for von Willebrand syndromes, 501

Cryopreservation quantitative, 322

agents, 181, 604 in recipients, 323-324, 411
computer-controlled, 605 Dangerous goods, 716-722
effects of, 181 DAT. See Direct antiglobulin test
of hematopoietic progenitor cells, 604-606 DAT-negative autoimmune hemolytic anemia,
passive controlled-rate, 605-606 459, 468
of platelets, 183 DDAVP (desmopressin), 513
of red cells, 181-183, 807-812 as fibrinolytic inhibitor, 514
storage bags, 182 in hemophilia A, 503, 513
Crystalloids, 150, 151, 486, 572 indications for, 513
Cs (Cost) antigens and antibodies, 352, 355 in liver disease, 499
C:T (crossmatch-to-transfusion) ratios, 92, 93 in von Willebrand syndrome, 501, 513
Cultures, bacterial, 195, 602-603, 655, 693, 694 Decay-accelerating factor (DAF), 353
Customer relations, 9-10 Decontamination of biohazardous waste, 51, 54,
CXCR4, 594 57
Cytokines Deferrals, donor
defined, 269 of autologous donors, 120
in immune response, 251, 254, 262-263, 264 for babesiosis, 696
in transfusion reactions, 640, 643 for Creutzfeldt-Jakob disease, 689, 690
Cytomegalovirus (CMV), 686-687 for drugs taken by donor, 100-101, 113
associated with hepatitis, 667, 668 for hepatitis, 674, 703
blood component testing for, 170 for HIV, 674, 680, 681, 703
clinical manifestations, 686 for HTLV, 683, 684, 685, 703
and leukocyte reduction, 687 implicated in posttransfusion infections, 703
in neonates, 562 for Lyme disease, 696-697
prevention, 687 for malaria, 697
transfusion-transmitted, 686-687 records of, 98, 173
in transplantation, 590, 591, 621, 629, 630 Deglycerolized Red Blood Cells
Cytomegalovirus (CMV)-negative products, 95 checking adequacy of deglycerolization,
granulocytes, 492, 570 812-813
for intrauterine transfusion, 544, 553 expiration date, 189
labeling, 172 preparation, 191-192, 809
refreezing, 183
D of sickle cell trait, 192
storage, 192
D antigen. See also Rh system; Rh testing
Delayed hemolytic transfusion reactions
clinical significance, 316
(DHTR), 346, 575, 637, 656-657
discovery of, 315-316
Delayed serologic transfusion reaction (DSTR),
expression of, 319-320
656
in HDFN, 330, 536-537
Delayed transfusion reactions. See also Transfu-
ISBT classification, 841
sion reactions
partial D, 322-323, 538
alloimmunization, 637, 656-657
testing, 165, 328-330, 414
graft-vs-host disease, 399, 560, 637, 657-659
weak D, 322-324
hemolytic, 346, 575, 637, 656-657
in autologous donations, 324
immunomodulation, 638, 660
in donors, 165, 323
iron overload, 638, 660
“microscopic,” 550
Kidd antibodies in, 346, 656
partial D, 322-323
posttransfusion purpura, 366, 370, 638, 659-660
as position effect, 302
Density gradient separation, 601
in pregnancy, 538-539

Index 869
Derivatives. See also specific derivatives in HDFN, 545-546

for coagulation factor replacement, 496 in IgM warm AIHA, 464
virus inactivation, 496, 699, 701 medical history in, 454-456
Desensitization therapy, 505 in mixed-type AIHA, 460, 466
Design output, defined, 4, 30 in paroxysmal cold hemoglobinuria, 460,
Designated donations, 98, 103-104 467
Desmopressin. See DDAVP red cell testing with, 745-747
Deviations, 19, 20, 21 serologic studies, 456
Dextrose solution, 530 in warm autoimmune hemolytic anemia,
Dha (Gerbich) antigen, 352, 843 459, 460, 461
DHTR (delayed hemolytic transfusion reac- in pretransfusion testing, 412, 454
tions), 346, 575, 637, 656-657 principles, 278
Di (Diego) antigens and antibodies, 336, 348, 349 reagents, 278-280 , 427, 454, 468
Diagnostic specimens specimens, 454
defined, 717 in transfusion reaction evaluation, 653-654
labeling, 718, 719 in transplantation, 455, 629
shipping, 717-718, 719, 720-722 vs autologous control, 427
DIC. See Disseminated intravascular coagula- Directed donations, 94-95, 537-538
tion Disaster planning, 67-68
Dichloromethane/methylene chloride elutions, Disinfectants, 51, 54
457, 775 Disposal
Diego system (ISBT 010), 348 of biohazardous waste, 56-57
antigens/antibodies, 336, 348, 842 of blood components, 196
chromosomal location of genes, 226 of chemical waste, 63
and En(a–), 338 of radioactive waste, 66
in HDFN, 348 Disposition of blood components, 196
phenotype frequencies, 349 Disseminated intravascular coagulation (DIC)
in transfusion reactions, 348 in neonates, 567, 572
Differential centrifugation, 179-180 in transfusion reactions, 640, 642
Differential warm adsorption, 781-782 treatment for, 502, 567
Digitonin acid elutions, 457 Dithiothreitol. See DTT
Dilution Diversion, 693
of % solutions, 726-727 DMSO (dimethylsulfoxide), 604, 607
of serum, 725-726 DNA (deoxyribonucleic acid)
Dilutional coagulopathy, 499-500, 567, 651 libraries, 217
Dimethylsulfoxide (DMSO), 604, 607 molecular techniques
2,3-Diphosphoglycerate. See 2,3-DPG cloning, 217, 222
Direct antiglobulin test (DAT) isolation of nucleic acids, 209-211
in classifying AIHA, 458 microarrays, 218
complement in, 280-281 polymerase chain reaction technique,
method, 760-761 211, 212, 213-214, 222, 394-396
positive test profiling (typing or fingerprinting), 216, 222
in antibody identification, 427, 436, recombinant proteins, 218-219, 222
438-439 restriction endonucleases, 214, 215
causes, 453-454 restriction fragment length polymor-
in cold agglutinin syndrome, 460, 465 phism analysis, 214, 215, 222
drug-induced, 455, 472-477, 481-482 sequencing, 217-218, 222, 396
elution with, 456-458, 745-746 structure, 203-204
evaluation, 454-458, 480 transcription, 204, 205

Do (Dombrock) antigens and antibodies, 336, implicated in posttransfusion infection, 703

349, 350-351, 842 information provided to, 98-99, 114
Documents, 14-17. See also Records medical history, 100-101, 110-112, 693
management, 14-15, 17 notification of abnormal tests, 99, 103, 703
quality system, 14, 16 for organ and tissue transplantation, 618,
types, 15 620-623
Dolichos biflorus lectin, 293, 296, 302, 743-744 physical examination of, 100, 101-103, 620
Dombrock system (ISBT 014) for plateletpheresis, 141
antibodies, 336, 350-351 records, 165, 173
antigens, 350, 842 red cell density, 102
genes, 226 reentry with positive screening tests, 673,
phenotype frequencies, 349 674, 675, 681-682
Dominant traits, 232-233, 234, 241 registration, 97-98
Donath-Landsteiner test, 458, 467, 784-785 report of illness after donation, 103
Donation intervals for apheresis, 141, 143 testing
Donation process. See Blood collection ABO, 165, 290, 296, 413
Donor area, 55, 97 alanine aminotransferase, 164
Donor deferrals antibody detection, 165
of autologous donors, 120 for blood group antigens, 418, 440-441,
for babesiosis, 696 745-748
for Creutzfeldt-Jakob disease, 689, 690 cytomegalovirus, 170
for drugs taken by donor, 100-101, 113 equipment requirements, 164-165
for hepatitis, 674, 703 general requirements, 163-164
for HIV, 674, 680, 681, 703 hepatitis, 120, 166-170, 669-673
for HTLV, 683, 684, 685, 703 HIV, 120, 164, 166-170, 679-681
implicated in posttransfusion infections, 703 HTLV, 120, 683
for Lyme disease, 696-697 repeat, 413
for malaria, 697 Rh, 165, 328-332, 413
for positive infectious disease tests, 680, 683, syphilis, 120, 165-166, 695
684, 685 viral markers, 166-170
records, 98, 173 weak D, 165, 323, 328
Donor history questionnaire, 110-112, 114-118 weight, 101, 143
Donors Doppler flow studies, 542
adverse reactions in, 19, 107-109 Dosage effect
autologous, 103, 121-122, 124-125, 131, 135, in antibody identification, 425, 431
693 in Duffy system, 345
care of, after phlebotomy, 106 genetics, 225, 227
confidential unit exclusion, 101 in Rh system, 327
consent, 103, 620-621 Dosimeters, 64-65
designated donations, 98, 103-104 Dot blot, 213, 395
directed donations, 104, 537-538 2,3-DPG
drugs taken by, 100-101, 113, 115 in neonatal transfusions, 561-562
eligibility, 99-100 in red cell storage lesion, 185, 187
family members as, 442 in tissue oxygenation, 484, 511-512
hematocrit, 102 Dra (Cromer) antigen, 353, 354, 843
for hematopoietic transplantation, 589-591 Drug-induced immune hemolytic anemia,
hemoglobin levels, 102, 799-800 472-477
for HLA-matched platelets, 398 autoantibodies, 474, 475-476
hypotension in, 107 classifications, 473-476

Index 871
drug adsorption mechanism, 473, 474, 475 in HDFN, 536

drug-dependent antibodies, 473, 474-475 incidence of, 317, 321
drugs associated with, 476, 481-482 phenotypes, 320
laboratory investigation, 460, 476-477, testing for, 330-331, 441
786-791 E/e antigens and antibodies (Rh system), 316,
metabolites, 477 841
nonimmunologic protein adsorption, 476 EACA (epsilon aminocaproic acid), 513-514
theories of, 472-473 EBV (Epstein-Barr virus), 667, 668, 687-689
Drugs ECMO (extracorporeal membrane oxygenation),
administered for leukapheresis, 143-144 572-573, 574
alteration of pharmacodynamics in Education
apheresis, 150-151 donor, 98-99, 114, 117-118
for autologous tumor purging, 597 patient, 521-522
as cause of thrombocytopenia, 374-377 EIA. See Enzyme-linked immunosorbent assay
for febrile transfusion reactions, 644 ELBW (extremely low birthweight) neonates,
in positive DAT, 455, 472-477, 481-482 557, 568. See also Neonates
to prevent allergic transfusion reactions, 646 Elderly people, 295, 299
taken by donors, 100-101, 113, 115 Electrical safety, 48-49
Dry ice, 196, 719, 720-721 Electromechanical infusion devices, 522,
Dry shippers, 607 529-530, 565
DSTR (delayed serologic transfusion reaction), ELISA. See Enzyme-linked immunosorbent as-
656 say
DTT (dithiothreitol) Elutions
applications for, 448-449 with adsorption, 300-301, 448, 735-736
differentiating IgG from IgM, 764-765 in antibody identification, 447-448
to disperse autoagglutination, 302, 469, 470, in cold agglutinin syndrome, 460, 465
744-745 in IgM warm AIHA, 464
inactivating blood group antigens, 342, 445, methods, 457, 771-775
446, 766-767 cold-acid, 457, 772
Duffy system (ISBT 008), 343-345 dichloromethane/methylene chloride,
antibodies, 336, 345, 844 457, 775
antigens, 343-344, 841 digitonin-acid, 457
biochemistry, 344-345 gentle-heat, 745-746
chromosomal locations of genes, 226 glycine-HCl/EDTA, 772-773
effect of enzymes on, 276, 336, 345 heat, 457, 773-774
in HDFN, 345 Lui freeze thaw, 457, 774
and malaria, 337, 344 in mixed-type AIHA, 460, 467
phenotypes and frequencies, 344, 844 in paroxysmal cold hemoglobinuria, 460, 467
in transfusion reactions, 345 in positive DAT evaluation, 456-458
Dura mater transplants, 619, 624 technical factors in, 447-448
Dysfibrinogenemias, 501-502 uses, 448
in warm antibody AIHA, 460, 461
E Embolism, air, 636, 651-652
Emergency procedures
E/e antigens and antibodies
blood orders, 93
cis product antigens, 324
identification of patients, 408
concomitant antibodies, 327-328, 441
issue of blood, 419, 510-511, 522-523
dosage in, 317
in pretransfusion testing, 419
expression of, 320
Emergency release, 510-511, 522-523
gene complexes, 319

Emergency response plans, 44 evaluating treated red cells, 758-759

for biohazardous waste, 55-57 ficin preparation, 756
for chemicals, 61-62, 63 one-stage technique, 759
for electrical emergency, 49 papain preparation, 756-757
for fires, 48 in resolving ABO discrepancies, 300, 301
for radiation safety, 66 standardization of procedures, 757-758
Emergency showers, 61 two-stage technique, 759-760
Emergency supplies for donor area, 108 Eosinophils, 255
Employees. See Personnel Epistasis, 236
En(a–) red cells, 338 Epitope, defined, 269
Engineering controls EPO. See Erythropoietin
for biosafety, 50-51, 52-53, 54 Epsilon aminocaproic acid (EACA), 513-514
for chemical safety, 61 Epstein-Barr virus (EBV), 667, 668, 687-688
for electrical safety, 48 Equilibrium constant (Ko), 273
for fire prevention, 47 Equipment. See also specific equipment
general guidelines, 43-44, 73-76 for apheresis, 139-140, 152
for radiation safety, 65-66 calibration, 821-823, 826-830
Engraftment data, 608 for component testing, 164-165
Enhancement of antibodies critical, 10
albumin additives, 276, 427, 753 management, 10-11
alteration of pH, 274, 444 personal protective, 43-44, 73-75
in antibody detection/identification, quality control
276-277, 427, 443-444, 753-754 automatic cell washers, 830-832
combined adsorption/elution, 300-301, 448, centrifuge calibration, 826-830
735-736 continuous temperature monitoring sys-
effect of incubation times, 274, 444 tems, 197-198
enzymes, 276, 443, 445 , 446, 756-760 freezer alarms, 198-199, 824-826, 846
increased serum-to-cell ratio, 444 performance intervals, 846-847
LISS and LISS additives, 274, 275, 277, 427, refrigerator alarms, 198-199, 823-824, 846
443, 753, 754 thermometers, 198, 821-823, 847
polyethylene glycol, 276-277, 427, 443, 753 for transfusions, 523-524, 527-530
temperature reduction, 443 validation, 11, 13
Environmental chambers, 185, 197-198, 846 Er blood group collection, 355
Enzyme-linked immunosorbent assay Ergonomics, 41-42
(EIA/ELISA) Errors
in anti-HTLV testing, 683 in ABO typing, 299-300
in antibody detection, 286 in antiglobulin testing, 282, 283
in antigen-antibody detection, 286 identification, 525-526, 527, 641, 642, 653,
for Chagas’ disease testing, 698 654
for heparin-dependent antibodies, 377 Erythroblastosis fetalis. See Hemolytic disease of
in hepatitis testing, 672-673 the fetus and newborn
in HIV testing, 679 Erythropoiesis, 557-558, 559
in viral marker testing, 166 Erythropoietin (EPO)
Enzymes, proteolytic in anemia treatment, 219, 488
in adsorption, 470, 471, 781-782 in autologous donations, 125, 219
in antibody detection/identification, 276, in newborns and infants, 558, 559
443, 445, 446 recombinant, 125, 219, 488, 512, 559
effect on blood group antigens, 276, 336, as transfusion alternative, 512
339-340, 345 Esa (Cromer) antigen, 353, 354, 843

Index 873
Ethnic groups, differences in FACT standards, 609

in ABO system, 845 Factor V, 188
in Cromer system, 353 Factor VIIa, 497, 505
in distribution of antigens, 335, 337 Factor VIII
in Duffy system, 337, 344, 345 calculating dose, 503-504
in HLA system, 585 concentrates, 497, 503-504
in Kell system, 341, 343 deficiency, 503
in Kidd system, 337 for hemophilia A, 502-504
in Lutheran system, 347 inhibitors to, 504-505
in Rh system, 316, 317, 320, 321, 325 in stored blood, 188
Etiologic agents, 719 virus inactivation, 710
Exchange transfusions for von Willebrand syndromes, 501
with antibody against high-incidence anti- Factor IX, 497, 498, 504, 505
gen, 547 Failures, active and latent, 26-27
blood selection for, 546-547, 567 Fainting in donors, 107
blood warming in, 560 False-positive/false-negative results
in DIC, 567 in antiglobulin testing, 282, 283
in HDFN, 546-547 in Rh testing, 329, 330, 331-332
for hyperbilirubinemia, 566-567 Fascia lata transplants, 624
methods used, 568 Fatalities
“partial,” 563 during apheresis, 153
for removal of toxins, 567 due to bacterial contamination, 690-692
in sickle cell disease, 574 due to transfusions, 641, 645, 661, 691,
vascular access, 568 702-703
volume and hematocrit, 568 employees, 46
Executive management, 6 reporting, 19, 46, 661, 702-703
Exons, 206 FDA. See Food and Drug Administration
Expiration dates Febrile nonhemolytic transfusion reactions
checking before issue, 525, 526 (FNHTR), 643-644
of components, 188, 189-190 granulocyte antibodies in, 379
of tissues and organs, 624 HLA antibodies in, 398-399
Exposure control plan, 49, 50 management, 634
External assessments, 23-24 manifestations, 643-644
External controls in viral marker testing, 167-169 pathophysiology, 634, 643-644
Extracorporeal membrane oxygenation (ECMO), preventing, 576, 644
572-573, 574 treatment, 644
Extracorporeal photochemotherapy, 158 Fetal hemoglobin (hemoglobin F), 550, 558, 835
Eye washes, 50, 75 Fetomaternal hemorrhage (FMH)
as immunizing event, 536
F Kleihauer-Betke acid-elution test, 550-551,
794-796
Face shields, 74
microscopic weak D test, 550
Facilities
postpartum evaluation, 548, 549-551
design and workflow, 40
rosette test, 550, 793-794
ergonomics, 41-42
Fetus. See also Hemolytic disease of the fetus
housekeeping, 40-41
and newborn
mobile sites, 41
erythropoiesis in, 557-558, 559
organizational management, 6-7
hematocrit, 541
restricted areas, 41
Rh typing, 539
safety, 27, 39-42

Fever, transfusion-associated, 398-399, 633, 634, quality control, 197-199, 846

643 temperature monitoring systems for,
FFP. See Fresh Frozen Plasma 197-198
Fibrin degradation products, 837 thermometers for, 198
Fibrin sealant, 502 Freezing
Fibrinogen blood samples, 184-185
abnormalities, 501-502 cryoprotective agents, 181, 604
CRYO for replacement of, 500, 501 freeze-thaw damage, 181
in monitoring coagulation, 494, 511 Fresh Frozen Plasma, 180-181, 814
normal values, 837 hematopoietic progenitor cells, 604-606
Fibrinolytic inhibitors, 513-514 Platelets, 183
Ficin, preparation of, 756. See also Enzymes, Red Blood Cells, 181-183, 807-812
proteolytic refreezing deglycerolized cells, 183
Filgrastim. See Granulocyte colony-stimulating skin grafts, 624, 625
factor storage bags for, 182
Filters Fresh Frozen Plasma (FFP)
leukocyte-reduction, 180, 190, 528-529 ABO compatibility, 411, 496, 498
microaggregate, 528, 565-566 coagulation factors in, 496
standard in-line, 527-528 collection by apheresis, 141, 142
Fire safety, 47-48 description, 177
Fish-bone diagrams, 26 expiration dates, 189-190
Fisher/Race Rh terminology, 319 indications for, 496, 498-500
Flow cytometry inspection, 194
in CD34 analysis/enumeration, 603 inventory management, 94
in granuloctye autoantibody testing, 380 misuse, 500
in HLA crossmatch, 397, 401 for neonates, 558, 571
in platelet antibody detection, 372, 375, 376 in older infants and children, 576
to predict clinical significance of antibodies, preparation, 180-181, 813-814
441 small-volume, 571
Flow PRA, 366 thawing, 191
Fluids, replacement, 150, 151 transfusion thresholds, 494, 496
Fluosol, 513 transportation and shipping, 196
FMH. See Fetomaternal hemorrhage virus inactivation, 701-702
FNHTR. See Febrile nonhemolytic transfusion Frozen components. See specific components
reactions Fume hoods, 61
Food and Drug Administration (FDA) Fy (Duffy) antigens and antibodies, 336,
cGMP training, 8 343-344, 345, 841
quality assurance regulations, 1, 2
regulations for autologous blood, 119-120 G
regulations for tissue transplantation, 626
G antigen (Rh system), 324-325, 841
reporting adverse events to, 19, 20, 21, 661,
G-CSF. See Granulocyte colony-stimulating fac-
702-703
tor
Forensic testing, 402
GBV-C virus, 668
Forms, 15, 17. See also Documents; Records
Ge (Gerbich) antigens and antibodies, 352-353,
Foundation for the Accreditation of Cell Therapy
843
(FACT), 609
GEIS antigen, 352, 843
Freeze-thaw elutions, 457, 774
Gel test, 285
Freezers, 185
Gene chips, 218
alarm systems for, 185, 198-199, 824-826
Gene therapy, 217, 219-220

Index 875
Genes Glycine max (anti-T, -Tn), 743

blood group terminology, 844 Glycophorins, 338, 339
chromosomal assignment, 225, 226, 235-236 GM-CSF (granulocyte-macrophage col-
defined, 241 ony-stimulating factor), 264, 512, 592, 594
DNA structure, 203-204 Goggles, safety, 74
frequencies, 227-229, 320-321 Gov antigens, 370
gene conversion, 209, 210 Grading test results, 412, 728-729
major histocompatibility complex, 244-246, Graft failure, 598
386-389 Graft-vs-host disease (GVHD)
modifier, 235-236 acute/chronic, 586
nucleotide insertions and deletions, 208 and graft-vs-leukemia effect, 586-587
nucleotide substitutions in, 207-208, 228 in hematopoietic transplantation, 585-588
obligatory, 237 HLA system in, 399, 400
single crossover, 208, 209, 230-231, 232, 241, 389 in neonates, 560-561
suppressor, 235-236 transfusion-associated, 399, 400, 560-561,
variability, 208-209, 210 637, 657-659
Genetics of blood groups Graft-vs-leukemia (GVL) effect, 586-587
of ABO system, 229-230, 235, 290-293 Gram-equivalent weight, 723
alleles, 225, 227-229 Gram-molecular weight, 723
basic principles, 223-225 Gram’s stain, 693
definitions of terms, 241 Granulocyte colony-stimulating factor (G-CSF)
of H system, 290-293 and false-positive test results, 590
and heredity, 225-232 for HPC mobilization, 587, 592-595
of HLA system, 386-389 in leukapheresis, 144
Lewis system, 305 in neonatal sepsis, 570
of MNS system, 338 production and function, 264
nomenclature, 238-239, 318-319, 840-844 side effects of, 594-595
patterns of inheritance, 232-236 uses for, 218, 222, 512
polymorphism in, 207-208 Granulocyte-macrophage colony-stimulating
population genetics, 236-237 factor (GM-CSF), 264, 512, 592, 594
of Rh system, 316-318 Granulocytes
Genotypes. See also specific blood groups alloantigens, 377-380, 647
defined, 233 antibodies to
frequencies, 227-228 in neonatal alloimmune neutropenia, 379
nomenclature for, 238-239 testing for, 380
Gentle heat elutions, 745-746 in TRALI, 379-380, 647, 656
Gerbich system (ISBT 020), 226, 352-353, 843 in immune system, 255-256
GIL system (ISBT 029), 226, 355, 843 Granulocytes Pheresis
Glanzmann’s thrombasthenia Type I, 366, 369 ABO compatibility, 411
Gleevec (imatinib mesylate), 220 collection, 143-144
Globoside (GLOB) blood group, 226, 308, 843 description, 177-178
Gloves, 46, 55, 73-74, 106 expiration dates, 189
Glycerolization of red cells, 181-182, 808-809, indications/contraindications for use, 492,
810-812 570
Glycine-HCl/EDTA irradiation, 144, 492
to disperse autoagglutination, 469 laboratory testing, 144
to dissociate IgG from red cells, 469, 747-748 for neonates, 558, 569-570
elutions, 772-773 quality control, 202
to inactivate red cell antigens, 446 storage, 144

transfusion, 143, 492, 569-570 transportation and shipping, 66, 716-722

transportation and shipping, 196 waste management, 55-57, 67
validation, 35 HBeAg (hepatitis B e antigen), 669, 670
Group O serum (anti-A,B), 295 HBsAg. See Hepatitis B surface antigen
Growth factors, recombinant HBV. See Hepatitis B virus
for HPC mobilization, 587, 592-595 HCV. See Hepatitis C virus
in leukapheresis, 144 HDFN. See Hemolytic disease of the fetus and
as transfusion alternative, 512, 559 newborn
uses for, 219, 222 HDV (hepatitis D virus), 668, 670
GTI PAKAUTO, 374 Heart and heart valves
Guillain-Barré syndrome, 156 arrhythmias in recipients, 650
GUTI (Cromer) antigen/antibody, 353, 354, 843 cardiac arrest in donors, 108
GVHD. See Graft-vs-host disease cardiac output, 485-486
GVL (graft-vs-leukemia) effect, 586-587 disease of, 119, 487-488
Gya (Dombrock) antigen, 350, 842 infectious disease transmission, 619
preservation, 624
transplantation, 402, 625, 629
H Heat elutions, 457, 745-746, 773-774
Heating blocks, 846
H system (ISBT 018), 303-304
Hemapheresis practitioner (HP), 139
anti-H, 304, 434
Hematocrit
anti-IH, 308, 434, 777, 778
in autologous blood donors, 125, 126
biochemistry and genetics, 290-293
in blood donors, 102
chromosomal locations of genes, 226
of blood in exchange transfusions, 568
H antigen, 290, 303-304, 361, 842
fetal, 541
Oh phenotype (Bombay), 304
in neonates, 572
para-Bombay phenotypes, 304
normal values, 835
saliva testing for substances, 301, 736-739
of red cell components, 805
HAM (HTLV-associated myelopathy), 682
transfusion trigger, 487
Hand-washing, 50, 75
Hematoma, in donors, 108
Haplotypes
Hematopoietic growth factors
ancestral, 389
for HPC mobilization, 587, 592-595
defined, 231
for leukapheresis, 144
of HLA system, 387-388, 389, 400, 402
as transfusion alternative, 512, 559
of Rh system, 318-319
uses for, 218-219, 222
Haptoglobin, 835
Hematopoietic progenitor cell (HPC) transplan-
Hardy-Weinberg law, 227-229
tation, 581-609
HAV (hepatitis A virus), 667-668, 671, 700
ABO incompatibility, 598-599, 600, 601-602
Hazard Communication Standard, 59-60
ABO typing discrepancies in, 299
Hazard identification and communication, 43
allogeneic, 582, 583, 585-588, 589-590,
for biosafety, 50
594-595
for chemical safety, 59-61
autologous, 582, 583, 587, 589, 592-594, 597
for electrical safety, 48
autologous tumor purging, 597-598
for fire prevention, 47
cell processing, 596-604
Hazardous areas of facilities, 41
chimerism in, 298, 601
Hazardous materials
collection of products, 144, 591-596
biohazards, 49-57
colony-forming cell assays, 603-604
chemicals, 57-63
diseases treated with, 583, 584
radioactive, 63-66
donor eligibility, 589-591
safety plan for, 42-47

Index 877
evaluation and QC of products, 607-608 in antibody detection, 412

freezing and storage of cells, 604-606, 624 defined, 271-272
and graft-vs-host disease, 585, 586-587 immune-mediated
HLA testing, 400-401, 585-587 ABO/Rh typing problems with, 469-470
of HPC-A (apheresis), 581, 587-588, 592-595 alloimmune hemolytic anemia, 459
of HPC-C (cord blood), 588-589, 595-596, antibodies associated with, 336
602 antibody detection with, 470-472
of HPC-M (marrow), 583, 585-587, 591-592 classification of anemias, 458, 459
indications for, 582 cold agglutinin syndrome, 459, 460,
infectious disease testing, 590-591 464-466
matched, unrelated donors, 585-586, 589 DAT-negative AIHA, 459, 468
microbial cultures, 602-603 defined, 458-459
mobilization of HPCs, 587, 592-595 drug-induced, 459, 460, 472-477, 481-482
nonmyeloablative, 582-583 in hemolytic transfusion reactions,
positive DAT after, 455 639-642
processing, 596-598, 601-604 IgM warm AIHA, 464
red cell depletion, 601 intravascular/extravascular, 265-266, 267,
regulations, 608 458
related donors, 587-588, 589 mixed-type AIHA, 459, 460, 466-467
selection of CD34+ cells, 596-597 paroxysmal cold hemoglobinuria, 459,
sources of cells for, 583, 584-589 460, 467-468
standards, 609 warm antibody autoimmune hemolytic
stem cell enumeration, 603 anemia, 459, 460, 461-464
suitability criteria, 606 non-immune mediated, 152, 636, 642-643
syngeneic, 582, 587 passenger lymphocyte, 628, 629-630, 657
T-cell depletion, 586, 598 in patient samples, 410
thawing and infusion of products, 607 and positive DAT evaluation, 455
transfusion support, 591-592, 599, 600, 601 in transfusion reaction evaluation, 653, 655
transportation and shipping, 606-607 Hemolytic anemias. See also Hemolytic disease
types, 581-582 of the fetus and newborn
xenogeneic, 582, 583 alloimmune, 459
Hemoglobin classification of, 458, 459
in autologous donors, 125 cold agglutinin syndrome, 459, 460, 464-466
in blood donors, 102 DAT-negative AIHA, 459, 468
bovine, 513 drug-induced, 459, 460, 472-477, 481-482
fetal (F), 550, 558, 835 IgM warm AIHA, 426, 427, 428, 429-431
in neonates, 558, 563, 572, 836 mixed-type AIHA, 459, 460, 466-467
normal values, 835, 836 paroxysmal cold hemoglobinuria, 459, 460,
and oxygen, 185, 187, 483-485 467-468
recombinant, 219, 513 serologic findings in, 460
S, 544, 567, 574, 575 warm autoimmune hemolytic anemia, 459,
in transfusion reaction evaluation, 654-655 460, 461-464
as transfusion trigger, 483, 486-488 Hemolytic disease of the fetus and newborn
Hemoglobin solutions, 512-513 (HDFN), 535-551
Hemoglobinometers, 847 ABO, 536, 537, 538, 545
Hemoglobinopathies, 508-509, 574-576. See also ABO discrepancies in, 302
specific hemoglobinopathies antibodies associated with, 336, 536-537,
Hemolysis 545-546
in ABO testing, 295 elutions in, 456

exchange transfusion in, 546-547 normal test values, 837

intrauterine transfusion in, 541, 543-544 physiologic principles, 488-489 , 493-494
maternal immunization in, 536-538, 542-543 Heparin, 375-377, 505, 642, 723
measuring severity of Heparin co-factor, 505-506
amniotic fluid analysis, 540-541, 542 Heparin-induced thrombocytopenia (HIT),
Doppler flow studies, 542 375-377, 492
maternal antibody titer, 449, 539-540, Hepatitis, 667-675
761-764, 796-798 associated with CMV or EBV, 668
percutaneous umbilical blood sampling, chronic carriers, 668-669
541-542 clinical manifestations, 668-669
pathophysiology, 535-536 donors implicated in, 703
postpartum evaluation, 544-546 markers, 669, 670-671, 672-673, 674
prenatal evaluation, 538-540 non A, non B (NANB), 673, 699
Rh testing in, 330, 538-539, 545 prophylaxis, 45, 668, 672, 703
use of Rh Immune Globulin, 547-551 quarantine and recipient tracing, 675
Hemolytic transfusion reactions (HTR) reentry of donors with, 673, 674, 675
antibodies associated with, 336 reporting cases, 702
coagulation activation in, 640, 642 risk of, 673, 675, 700
complement activation in, 639-640 surrogate markers, 673
cytokines in, 640 vaccination for, 668
delayed, 346, 575, 637, 656-657 viruses, 667-668
due to ABO incompatibility, 639, 640, 641 Hepatitis A virus (HAV), 667-668, 671, 700
elutions in, 456 Hepatitis B core antigen, antibody to (anti-HBc)
frequency, 641 blood component testing, 164, 166, 675
HLA antibodies in, 399-400 look-back for, 675
management, 634 marker of infection, 669, 670
non-immune, 642-643 reentry of donors with, 675
pathophysiology, 639-641 as surrogate marker, 672
prevention, 642 testing transplant donors for, 621
renal failure in, 640-641 Hepatitis B e antigen (HBeAg), 669, 670
shock in, 640 Hepatitis B immune globulin (HBIG), 45, 668,
signs and symptoms of, 633, 639-641 703
treatment, 641-642 Hepatitis B surface antigen (HBsAg)
Hemolytic-uremic syndrome (HUS), 154-155 antibody to (anti-HBs), 669, 670
Hemophilia A, 502-505 blood component testing, 164, 166, 169
calculating Factor VIII dose, 503-504 chronic carriers, 668-669
factor replacement for, 504 look-back for, 675
inheritance of, 233-234 marker of infection, 669, 670, 672
inhibitors of Factor VIII, 504-505 reentry of donors with positive tests, 674, 675
Hemophilia B, 505 testing organ and tissue donors for, 620, 621,
Hemorrhage, intraventricular, 569 623
Hemorrhagic disease of the newborn, 571 Hepatitis B virus (HBV)
Hemostasis chronic carriers, 668-669
in acute normovolemic hemodilution, 128 clinical manifestations, 667, 668-669
coagulation factors needed for, 495 employee exposure to, 45
components for, 496, 497, 498-508, 570-572 HBV DNA, 669, 672
during massive transfusion, 511, 651 look-back for, 675
monitoring, 494, 496 markers of infection, 120, 669, 670, 672, 674
in neonates, 570-571 NAT testing, 669, 672

Index 879
prophylaxis for, 45, 668, 672, 703 of Cromer system, 353, 354
reentry of donors with, 674, 675 defined, 335
testing transplantation donors for, 590 of Gerbich system, 352
transfusion risk of, 672, 679, 700 GIL, 355
Hepatitis C virus, antibody to (anti-HCV) of Knops system, 354
blood component testing, 164, 166, 170 of Lutheran system, 347
marker of infection, 670-671, 672-673 not assigned to a system or collection,
reentry of donors with, 673, 674, 675 356-357
supplemental tests, 170 selecting blood negative for, 441-442
testing organ donors for, 620, 621, 623 of Vel collection, 355-356
Hepatitis C virus (HCV) High-titer, low-avidity antibodies, 449, 762,
autologous blood testing, 120 765-766
blood component testing, 164, 166-167, 170 HIT (heparin-induced thrombocytopenia),
chronic carriers, 669 375-377, 492
clinical manifestations, 667, 668, 669 HIV (human immunodeficiency virus), 675-682
employee exposure to, 45 in AIDS, 675-676
false-positive test results, 590 in autologous donors, 117, 681
infections due to IGIV, 699 clinical manifestations, 99, 676-677
look-back for, 675 confirmatory tests for, 169-170, 679-681
markers of infection, 669, 670-671, 672-673, donors implicated in, 703
674 employee exposure to, 45
NAT testing, 164, 166, 170, 213, 590, 673, 675 false-positive test results, 590
reentry of donors with, 673, 674, 675 HIV-1, 675, 676, 678
testing transplantation donors for, 590, 620, HIV-2, and HIV-1, group O, 675, 677-679
621, 623 information provided to donors about, 99
transfusion risk of, 673, 675, 699, 700, 701 nucleic acid testing, 164, 166-167, 213, 590,
Hepatitis D virus (HDV), 668, 670 678-681
Hepatitis E virus (HEV), 667-668, 671 recipient tracing (look-back), 681-682
Hepatitis G virus (HGV), 668 reentry of donors with positive screens, 674,
Hereditary angioneurotic edema, 500 681
Hereditary hemochromatosis (HH), 387 reporting cases, 702
Heredity, genetics of risk factors for, 677
alleles, 225, 227-229 testing components for, 164, 166-167,
Hardy-Weinberg Law, 227-229 169-170, 213, 679-681
independent assortment, 229-230, 231 testing transplantation donors for, 590, 620,
linkage, 230-231, 232 621, 623
linkage disequilibrium, 231-232, 337, 389 transfusion considerations, 678-679
segregation, 229, 230 transfusion risks of, 679, 700, 701
Herpesviruses, 686-688. See also Hives, 634, 644-647
Cytomegalovirus HLA-B27, 403
HES (hydroxyethyl starch), 143-144 HLA Matchmaker, 365
Heterozygous, defined, 225 HLA system, 385-404
HEV (hepatitis E virus), 667-668, 671 alloimmunization to platelets, 265, 266,
HGV (hepatitis G virus), 668 362-366, 397-398, 637
HH (hereditary hemochromatosis), 387 antibody detection, 365-366, 397
HHV-6/HHV-8 (human herpesviruses), 688 antigens, 362, 385, 388-389, 390-391
High-incidence antigens and Bg antigens, 358
antibodies to, 356-357, 435-436, 441-442, biochemistry, 390-391
547 biologic function, 393-394

and chimerism, 399 testing transplantation donors for, 590, 620

crossmatching, 397, 398, 401, 402, 492 transfusion risk, 700
disease associations, 402-404 transmission, 682
in forensic testing, 402 Type I, II, 682
genetics, 386-389 HTR. See Hemolytic transfusion reactions
in graft-vs-host disease, 399, 400 Human albumin (5% and 25%), 507
major histocompatibility complex, 244-246, Human Genome Project, 217
386-389 Human granulocytic erlichiosis, 696
matching Human herpesviruses, 688
granulocytes, 492 Human immunodeficiency virus. See HIV
in HPC transplantation, 388, 400-401, Human monocytic erlichiosis, 696
585-586 Human resources, 7-9
in organ transplantation, 387-388, Human T-cell lymphotropic virus. See HTLV
401-402 Human thrombin, 502
of platelets, 363-365, 398 Hy (Dombrock) antigen, 350, 842
nomenclature, 391-393 Hydatid cyst fluid, 310, 445
in parentage testing, 402 Hydroxyethyl starch (HES), 143-144
in platelet refractoriness, 362-365, 397-398, Hydroxyurea, 575
491 Hyper-IgM immune deficiency, 255
structure of molecules, 244-246, 390-391 Hyperbilirubinemia, 536, 566-567
tissue distribution, 390-391 Hypercholesterolemia, familial, 140, 157
and transfusion, 397-400 Hyperhemolytic syndrome, 575
in transfusion reactions, 398-400, 637, 643, Hyperkalemia, 650
656 Hyperleukocytosis, 154
in transfusion-related acute lung injury, 399, Hypertension, 633
647 Hypertransfusion programs, 575-576
and transplantation, 388, 400-402 Hyperventilation, in donors, 107
typing, 394-397, 595, 621 Hyperviscosity, serum, 153-154
HMIWI (hospital/medical/infectious waste in- Hypoalbuminemia, 507
cinerators), 57 Hypocalcemia, 561, 636, 649-650
HNA antigens, 378 Hypofibrinogenemia, 500, 501
Homozygous, defined, 225 Hypokalemia, 650
Homozygous type II familial hypercholesterol- Hypotension
emia, 140, 157 associated with ACE inhibition, 636, 645
Housekeeping, 40-41 in donors, 107
HPA antigens, 366, 367-368, 369-370, 552 in recipients, 633, 640, 641, 645
HPCs. See Hematopoietic progenitor cell trans- in therapeutic apheresis, 152
plantation treatment, 646
HTLA (high-titer, low-avidity antibodies), 449, Hypothermia, 511, 560, 637, 650
762, 765-766 Hypovolemia, 141, 152
HTLV-associated myelopathy (HAM), 682
HTLV (human T-cell lymphotropic virus),
682-683 I
autologous blood testing, 120
I/i antigens and antibodies, 306-308
blood component testing, 164, 166, 170, 674,
in ABO discrepancies, 302
683
clinical manifestations, 682
in cold agglutinin syndrome, 307, 465-466
donors implicated in, 703
complex reactivity, 308
quarantine and look-back, 683
ISBT nomenclature, 843

Index 881
in pregnancy, 539 complement activation by, 260

serologic behavior, 307-308, 777, 778 dispersing autoagglutination, 302, 469-470,
IAT. See Indirect antiglobulin test 744-745
ID-NAT (individual donor NAT), 669, 678, 680, 685 distinguishing from IgG, 764-765
Identification normal values, 835, 837
of blood samples, 409 polymers, 257, 258
of components, 105, 416, 524-526, 527 properties, 253
of donors, 98, 105 temperature of reactions, 274
of equipment, 11 IgM warm AIHA, 464
errors in, 525-526, 527, 641, 642, 653, 654 IgSF (immunoglobulin superfamily), 244, 245,
of persons issuing blood, 416, 525 246
of phlebotomists, 409 IL (interleukins), 264, 512, 640
of recipients, 407, 408-409, 418, 524-526, 527 Imatinib mesylate (Gleevec), 220
traceability of components, 173 Immediate-spin crossmatch, 413, 414, 751-752
Idiopathic thrombocytopenic purpura (ITP), Immune complexes
158, 373-374, 492, 553-554 in antigen-antibody reactions, 280, 281
Idiotypes, 256, 269 demonstration of, 788-789
IFC (Cromer) antigen, 353, 354, 843 mechanism of drug-induced antibodies,
IgA, 259 473, 474-475
antibodies to, 645, 646, 655 Immune-mediated hemolysis. See also
normal values, 835, 837 Hemolytic disease of the fetus and newborn
polymers, 258, 259 ABO/Rh typing problems with, 469-470
properties, 253 alloimmune hemolytic anemia, 459
secretory, 258, 259 classification of anemias, 458, 459
IgD, 253, 259, 835 cold agglutinin syndrome, 459, 460, 464-466
IgE, 259 DAT-negative AIHA, 459, 468
in allergic transfusion reactions, 644-645 defined, 458-459
normal values, 835 drug-induced, 459, 460, 472-477, 481-482
properties, 253 in hemolytic transfusion reactions, 639-642
IgG, 258 intravascular/extravascular, 265-266, 267,
anti-A and -B, 295 458
in autoimmune hemolytic anemias, mixed-type AIHA, 459, 460, 466-467
458-459, 460 paroxysmal cold hemoglobinuria, 459, 460,
complement activation by, 260 467-468
dissociation of, by chloroquine, 469, 746-747 warm autoimmune hemolytic anemia, 459,
distinguishing from IgM, 764-765 460, 461-464
in neonates, 560 Immune serum globulin (ISG), 668
normal values, 835, 837 Immune system
in positive DAT, 280, 453, 461 cell adhesion molecules in, 246
properties, 253 cells of, 249-250, 251, 252-256
subclasses, 258, 458-459 cluster of differentiation molecules in, 246,
temperature of reaction, 274 247-248
IgG-coated cells (check cells), 281, 412 complement in, 259-262, 263
IGIV. See Immunoglobulin, Intravenous cytokines in, 262-263, 264
IgM, 258 defined, 269
anti-A and -B, 295 and drug-dependent antibodies, 472-473
in antiglobulin testing, 281 in HLA alloimmunization to platelets, 265,
cold-reactive autoagglutinins, 302-303, 465, 266
466, 467, 469, 776-778 immune response, 243-246, 247-248

immunoglobulin superfamily in, 244, 245 Immunoglobulin superfamily (IgSF), 244, 245,
immunoglobulins in, 253, 256-259 246
innate and adaptive immunity, 243-244 Immunoglobulins, 256-259. See also specific
lymphocytes in, 249-250, 251, 252-255 immunoglobulins
major histocompatibility complex in, classes, 258-259
244-246 Fab and Fc fragments, 256-257
of neonates, 560-561 functions, 256
organs of, 249 interchain bonds, 256
phagocytic cells in, 255-256 J chains, 258
in production of reagent antibodies, 266-267 normal values, 835, 837
receptors/markers, 250 polymers, 257-258
in red cell alloimmunization, 265 properties, 253
in red cell destruction, 265-266, 267 secretory component, 258
signal transduction in, 246 structure, 256, 257
soluble components, 256-263 Immunohematology reference laboratories, 439
Immune thrombocytopenic purpura (ITP), 158, Immunomagnetic cell separation, 596, 598
373-374, 492, 553-554 Immunomodulation, 638, 660
Immune tolerance, 237 In (Indian) antigens and antibodies, 349, 354,
Immunity. See Immune system 843
Immunization, maternal “In-vivo” crossmatch, 441, 509
antibody titers, 449, 539-540, 761-764, Incinerators, hospital/medical/infectious waste
796-798 (HMIWI), 57
mechanisms, 536-538 Incompletely clotted specimens, 409, 722-723
prenatal evaluation, 538-542 Incubation
suppression, 542-543 temperatures, 443, 715
Immunocompromised patients, 298-299 times, 274, 444
Immunofluorescence tests Independent assortment, 229-230, 231
in antigen-antibody detection, 285-286 Indian system (ISBT 023), 226, 349, 354, 843
in confirming HIV tests, 169 Indirect antiglobulin test (IAT)
for granulocyte antibodies, 380 false-positive/false-negative results, 282, 283
for platelet antibody detection, 372 methods, 752-754
Immunogen, defined, 269 principles, 278
Immunoglobulin, Intravenous (IGIV), 505 reagents, 278-280, 425, 427, 468
ABO discrepancies with, 299 role of complement, 280-281
for idiopathic thrombocytopenic purpura, 554 use of additives, 276-277, 443, 753-754
indications for, 505, 506 use of IgG-coated cells, 281, 412
for neonatal immune thrombocytopenia, Infants. See Children; Neonates
552-553 Infectious disease testing
positive DAT with, 456 of autologous blood, 119-120
for posttransfusion purpura, 659-660 of blood components, 166-170
to suppress maternal alloimmunization, 543 external controls in, 167-169
virus inactivation in, 699 of granulocytes, 144
Immunoglobulin products for hepatitis, 669, 670-671, 672-673, 674
hepatitis B immune globulin (HBIG), 45, for HIV, 164, 166-167, 169-170, 213, 679-681
668, 703 of HPC donors, 590-591
immune serum globulin (ISG), 668 for HTLV, 120, 164, 166, 170, 683
immunoglobulin, intravenous (IGIV), 505, invalidation of results, 167-169
506, 543 neutralization, 169
virus inactivation in, 699, 701 notification of abnormal tests, 703

Index 883
in organ and tissue transplantation, 621-623 to Factor VIII, 504-505

of Platelets Pheresis, 142 fibrinolytic, 513-514
records of, 173 Injuries, 45-46, 51
supplemental tests, 169-170 Innate immunity, 243-244
surrogate markers, 673 Inspections
for syphilis, 120, 165-166, 695 of components before release, 194-195, 416,
Infectious diseases. See also Transfusion-trans- 418, 525
mitted diseases external assessments, 23-24
as apheresis complication, 152 of incoming supplies, 10
in blood donors, 99 of inventory, 93-94
safety precautions for, 49-57 Integrins, 246, 369
transfusion-transmitted, 667-699, 700, Interferon γ (IFNγ ), 264
701-703 Interleukins, 264, 512, 640
transmitted by allografts, 617, 619-620 Internal assessments, 22
Infectious mononucleosis, 687 Internal controls, 167
Infectious substances Internal event report, 19, 20
defined, 716-717 International Organization for Standardization
labeling, 718, 719 (ISO), 1-2, 3
shipping, 717-718, 720-722 Intervening sequences, 206
waste, 55-57 Intraoperative blood collection, 117, 130-133
Inflammation, 262 clinical studies, 131-132
Informed consent controversies in, 132-133
for apheresis, 140, 142 direct reinfusion, 133
of blood donors, 103 equipment for, 132-133
for transfusion, 521-522 practical considerations, 132
for transplantation, 620-621 processing, 132-133
Infusions. See also Transfusions requirements and recommendations, 133
of hematopoietic components, 607 Intrauterine transfusions, 541, 543-544
infusion pumps, 522, 523, 529-530, 565 Intraventricular hemorrhage, 569
infusion sets, 527-529, 565-566 Introns, 206
rates for, 531, 566 Inventory
Inheritance patterns counts and inspection, 93-94
autosomal dominant, 233, 234 determining levels, 89-90
autosomal recessive, 233, 234 minimum and ideal levels, 89
blood group co-dominant, 234-235 and outdating, 90-91
chromosomal assignment, 226, 235-236 routine vs emergency orders, 93
dominant and recessive, 232-233 of special products, 94-95
of major histocompatibility complex, Ionic strength, 274
387-389 Iron, supplemental, 121
sex-linked dominant or co-dominant, 233, Iron overload, 638, 660
234 Irradiated blood components, 192-193
sex-linked recessive, 233-234 expiration dates, 183, 189, 192
Inhibition tests Granulocytes, 144, 492
agglutination, 275-276 HPC products, 592
in antibody identification, 444-445 indications for, 183, 193, 493, 658-659
for Chido/Rodgers, 445, 765-766 for intrauterine transfusion, 544
for secretor status, 737-739 inventory management, 95
Inhibitors labeling, 172
of complement activation, 262, 263 platelet products, 144, 189, 192-193, 492, 553

potassium leak in, 561 K

to prevent TA-GVHD, 183, 192, 493, 658-659
K/k (Kell) antigens and antibodies, 336, 340-342,
quality control, 202
343, 841
Red Blood Cells, 189, 192, 193, 493
Kaposi’s sarcoma-associated herpesvirus, 688
to reduce platelet alloimmunization, 365, 398
Kell system (ISBT 006), 340-343
ultraviolet B, 365, 398
anti-Ku, 343
washing, 561
antibodies, 336, 343, 844
Irradiators, blood, 65-66, 192, 846
antigens, 340-342, 468, 841, 844
ISBT (International Society of Blood Transfu-
biochemistry, 342
sion)
genes, 226, 844
128 barcode labeling, 171
in HDFN, 343, 536, 540
nomenclature
inheritance, 229-230
for platelet antigens, 366, 367-368
Kx antigen, 226, 342, 842
for red cell antigens, 226, 238-239,
McLeod phenotype, 342-343
840-843
phenotypes and frequencies, 341, 844
ISG (immune serum globulin), 668
in transfusion reactions, 343
Ishikawa diagrams, 26
Kernicterus, 536, 566
ISO 9001 standards, 1-2, 3
Kidd system (ISBT 009), 345-346
Isotype switching, 252
allele frequencies in, 227-229
Issuing blood
antibodies, 336, 346
delivering blood to patient area, 524-525
antigens, 345-346, 841, 844
identification of recipient and component,
418, 524-526
genes, 844
policies and procedures of, 416, 418, 524-525
in HDFN, 346
reissue, 197, 525
inheritance patterns of, 235
from transfusion service, 196-197
phenotypes and frequencies, 345, 346, 844
in urgent situations, 419, 510-511, 522-523
in transfusion reactions, 346, 656
ITP (idiopathic thrombocytopenic purpura),
Kidney
158, 373-374, 492, 553-554
diseases treated with apheresis, 156-157
IV solutions, 530
failure, 640-641
transplantation, 401-402, 619, 624, 627
J Kinetic PCR, 213
J chains, 258 Kleihauer-Betke acid-elution test, 550-551,
Jaundice, 566, 639 794-796
JCAHO (Joint Commission on Accreditation of Kn (Knops) antigens, 354, 843
Healthcare Organizations), 25, 514 Knops system (ISBT 022), 226, 352, 354, 843
Jk (Kidd) antigens and antibodies, 336, 345-346, Kp (Kell) antigens and antibodies, 336, 341, 343,
841 841
JMH antigen/antibodies, 355, 843 Kx antigen (ISBT 019), 226, 342, 842
Joa (Dombrock) antigen, 350, 842
Job descriptions, 7 L
John Milton Hagen (JMH) system (ISBT 026),
Labeling
226, 355, 843
aliquots, 564
Joint Commission on Accreditation of
for antigen typing, 440
Healthcare Organizations (JCAHO), 25, 514
autologous blood, 120, 122-123
Jra antigen, 356
biohazardous materials, 50, 719
Js (Kell) antigens and antibodies, 336, 341, 343,
blood components, 15, 170-172, 416, 440
841
blood samples, 409
Juran’s Quality Trilogy, 2, 4

Index 885
dry ice, 719 genes, 226, 305

hazardous chemicals, 59-60 Lewis substance, 445
ISBT 128 system, 171 phenotypes, 305
reagents, 715 saliva testing for, 445, 736-739
shipments, 718, 719 transfusion practices, 306
Laboratory coats, 73 Ligands, 269
Lactated Ringer’s solution, 530 Ligase chain reaction (LCR), 213, 214
Lan antigen, 356 Liley graphs, 540, 541
Landsteiner-Wiener blood group (ISBT 016), Linkage, 230-231, 232
226, 327, 351, 842 Linkage disequilibrium, 231-232, 337, 389
Latent failures, 26-27 Lipemic samples, 410
Latex allergies, 46-47 Liquid-in-glass thermometers, 821-822
Law of mass action, 273 Liquid nitrogen
LCR (ligase chain reaction), 213, 214 shipping, 718, 719
LDL apheresis, 140, 157 storage, 185, 189, 606
Le (Lewis) antigens/antibodies, 304-306, 336, Liquid Plasma, 177, 189
844 LISS (low-ionic-strength saline)
Leach phenotype, 352 antibodies to ingredients in, 437-438
Lectins and antigen-antibody proportions, 275, 277
Dolichos biflorus (anti-A1), 293, 296, 302, to enhance antigen-antibody reactions, 277,
743-744 427, 443, 753, 754
preparation and use, 743-744 and incubation time, 274
Ulex europaeus, 304, 743-744 Liver
Leishmania sp., 699 disease, 499
Leukapheresis, 143-144, 154, 587, 593 transplantation, 402, 619, 624, 627-629
Leukocyte-reduction filters, 180, 190, 528-529 LKE (Luke) antigen, 308, 310, 311
Leukocytes-reduced components Locus, defined, 241
for children, 573-574, 576 Long distance PCR, 213, 214
expiration dates, 189 Look-back
indications for, 493 for hepatitis, 675
labeling, 172 for HIV, 681-682
leukocyte content in, 492-493 for HTLV, 683
Platelets, 180, 184, 189, 199, 202, 365, 493, Low-incidence antigens
817 antibodies to, 340, 358, 436-437, 546
Platelets Pheresis, 35, 202, 493 of Cromer system, 353, 354
poststorage, 190 defined, 335
prestorage, 180, 184, 199, 805-806, 817 of Diego system, 348
to prevent bacterial contamination, 693 of Gerbich system, 352
to prevent CMV infection, 687 of Lutheran system, 347
quality control, 202, 832-834 of MNS system, 227, 228, 337-338, 340
Red Blood Cells, 180, 189, 199, 202, 493, not assigned to a system or collection, 357
805-806 of Scianna system, 350
to reduce platelet alloimmunization, 265, Low-ionic-strength saline. See LISS
365 Low-volume units, 101, 122, 179
Whole Blood, 180 Lsa (Gerbich) antigen, 352, 843
Lewis blood group (ISBT 007), 304-306 Lu (Lutheran) antigens and antibodies, 336,
antibodies, 305-306, 336, 434, 539 347-348, 841
antigens, 304-305, 841 Lui freeze-thaw elution, 457, 774
in children, 306 Luke (LKE) antigen, 308, 310, 311

Lung transplants, 402 Manual, safety, 45

Lutheran system (ISBT 005), 347-348 Marrow transplantation
antibodies, 336, 347-348 ABO incompatibilities, 598-599, 600, 601-
antigens, 347, 841 602
biochemistry, 347 ABO typing discrepancies, 299
chromosomal location of genes, 226 allogeneic, 582, 583, 585-587
in HDFN, 348 autologous, 583
phenotypes and frequencies, 347 bacterial contamination, 602-603
LW system (ISBT 016), 226, 327, 351, 842 collection of marrow, 591-592
Lyme disease, 696-697 diseases treated with, 583, 584
Lymphocytes donor databases, 585
B cells, 249-250, 251, 252-253 donor evaluation, 589-591
NK (natural killer) cells, 255 engraftment in, 588-589
passenger, 628, 629-630, 657 evaluation and quality control, 607-608
T cells, 253-254 freezing and storage of product, 604-606
Lymphocytotoxicity assays, 365-366, 395, 396-397 and graft-vs-host disease, 585-587
Lyonization, 224, 241 HLA testing, 400-401, 585-586
infectious disease testing, 590-591
M matched, unrelated donor, 585-586
nonmyeloablative, 582-583
Macrophage chemoattractant protein (MCP),
positive DAT after, 455
640
processing, 596-598, 601-604
Macrophage colony-stimulating factor (M-CSF),
red cell depletion, 601
264
regulations, 608
Macrophages, 250, 255, 256
standards, 609
Magnetic cell separation, 596
thawing and infusion, 607
MAIEA (monoclonal antibody-specific immobi-
transportation and shipping product,
lization of erythrocyte antigens) assay, 284,
606-607
286
MART antigen, 378, 379
MAIGA (monoclonal antibody-specific immobi-
Masks, 75
lization of granulocyte antigens), 380
Massive transfusions
MAIPA (monoclonal antibody-specific immobi-
2,3-DPG levels in, 511-512
lization of platelet antigen), 373
changing blood types, 511, 628-629
Major histocompatibility complex (MHC). See
citrate toxicity in, 649-650
also HLA system
coagulopathy in, 511, 651
Class I, II, III molecules, 244-246, 390-391
emergency issue, 510-511
defined, 269
hyper- and hypokalemia in, 650
organization of regions, 386-387
hypothermia in, 511, 650
patterns of inheritance, 387-389
selection of blood, 419, 510-511
restriction, 393
tissue oxygenation in, 511
Malaria, 697, 700
Material data safety sheets (MSDS), 59, 60-61,
Management
78-79
assessment, 38
Materials, critical, 9
of critical supplies and services, 9-10
Maternal immunization
of documents and records, 14-19
antibody titers, 449, 539-540, 761-764,
of equipment, 10-11
796-798
of facilities, 6-7
mechanisms, 536-538
of organization, 6-7
prenatal evaluation, 538-542
of personnel, 7-9
suppression, 542-543
of safety program, 45

Index 887
Maximum surgical blood order schedules Microplate tests

(MSBOS), 92-93, 414 for ABO group, 733-735
McCoy (McC) antigens, 352, 354 in antigen-antibody detection, 283-284
McLeod phenotype, 342-343 for Rh typing, 328, 741
MCP (macrophage chemoattractant protein), Microscopic weak D test, 550
640 Microvascular bleeding (MVB), 511, 651
2-ME (2-mercaptoethanol) Miltenberger system, 338
applications for, 448-449 Missense responses (mutations), 208, 227
to disperse autoagglutination, 469, 470, Mitosis, 224, 225, 241
744-745 Mixed-field agglutination, 298, 299, 348, 357
inactivation of Kell antigens, 342 Mixed lymphocyte culture (reaction)
Mechanical barrier systems, 527 (MLC/MLR), 397
Mechanical hemolysis, 152, 642 Mixed type AIHA, 466-467
Medical history classification, 459
in allogeneic donor selection, 100-101, serologic findings in, 460, 466-467
110-112 specificity of autoantibodies, 467
in antibody identification, 424 transfusion in, 467
in autologous donations, 122 MLC (mixed lymphocyte culture), 397
in cord blood collection, 595 MLR (mixed lymphocyte reaction), 397
in evaluation of positive DAT, 454-456 MNS system (ISBT 002), 337-340
in prenatal evaluations, 538 antibodies, 303, 336, 340, 444
in tissue and organ transplantation, 618, antigens
620 linkage disequilibrium in, 231, 232, 337
Medical waste, 55, 56, 57 linkage in, 232
Medications. See Drugs low-incidence, 227, 228, 337-338, 340
Meiosis, 224-225, 241 M, N, S, s, U, 337
Membrane attack complex (MAC), 261-262, 263 nomenclature, 840, 844
MER2 (Raph) antigen, 354-355, 843 phenotypes and frequencies, 337, 844
2-mercaptoethanol. See 2-ME biochemistry, 338-339
Meryman method of red cell cryopreservation, effect of enzymes on, 276, 339-340
807-810 genes, 226, 227, 228, 338, 844
Messenger RNA (mRNA) in HDFN, 340
isolation of, 210-211 hybrid molecules, 338
processing, 204-206 in transfusion reactions, 340
translation, 206-207 MoAb. See Monoclonal antibodies
Metabolic abnormalities, in neonates, 561-562 Mobile blood collection, 41, 179
Methemoglobin, 835 Mobilization of hematopoietic progenitor cells,
Methylene chloride (dichloromethane) elutions, 587, 592-595
457, 775 Modifier genes, 235-236
MHC. See Major histocompatibility complex Molar solutions, 723
MHO4 clone, 298, 301 Mole, defined, 723
Microaggregate filters, 528, 565-566 Molecular biology, 203-220
Microangiopathic hemolysis, 502 DNA and mRNA, 203-207
Microarrays, 218 genetic variability, 208-209, 210
Microbead array assay, 395-396 polymorphism, genetic mechanisms in,
Microfilariasis, 699 207-208
Microhematocrit centrifuges, 847 techniques
Microlymphocytotoxicity tests, 365-366, 395, DNA cloning, 217, 222
396-397 DNA microarrays, 218

DNA profiling, 216-217, 222 NAIT. See Neonatal alloimmune

DNA sequencing, 217-218, 222, 396 thrombocytopenia
gene therapy, 219-220 NAN (neonatal alloimmune neutropenia), 379
isolation of nucleic acids, 209-211 Nasopharyngeal carcinoma, 687
phage display/repertoire cloning, 222 NAT. See Nucleic acid amplification test
polymerase chain reaction, 211, 212, National Marrow Donor Program (NMDP), 585,
213-214, 215, 222, 394-396 589
protein and RNA targeted inactivation, 220 Natural killer (NK) cells, 255
recombinant proteins, 218-219, 222 Nausea, 107-108, 639
restriction endonucleases, 214, 216 Needles, 51, 523, 565
restriction fragment length polymor- Negative selection, 597-598
phism analysis, 214, 216, 222 Neonatal alloimmune neutropenia (NAN), 379
Monoclonal antibodies Neonatal alloimmune thrombocytopenia
in ABO testing, 296 (NAIT), 551-553
anti-D, 328, 329-330 management after delivery, 553
assays utilizing, 284, 373, 380 platelet-specific antigens in, 366, 552
in autologous tumor purging, 597-598 prenatal considerations for, 552-553
for reagent use, 266-267 scheduling therapy, 553
Monoclonal antibody-specific immobilization serologic testing in, 552
of granulocyte antigens (MAIGA), 380 sources of platelets for, 553
Monoclonal antibody-specific immobilization Neonatal immune thrombocytopenia (NIT)
of platelet antigens assay (MAIPA), 373 alloimmune (NAIT), 366, 551-553
Monoclonal antibody-specific immobilization secondary to maternal ITP, 553-554
of erythrocyte antigens (MAIEA) assay, 284, Neonatal polycythemia, 572
286 Neonates. See also Hemolytic disease of the fetus
Monocyte monolayer assay, 441 and newborn
Monocytes, 250, 255, 256 ABO antigens/antibodies in, 293, 295, 298
Mononuclear phagocytic system, 255 ABO discrepancies in, 299
Monospecific AHG reagents, 280 ABO/Rh typing in, 415, 545
MP-NAT (minipooled NAT) anemia in, 558, 559, 563-564
for HCV, 669 antigen variations in, 431
for HIV, 678, 679 blood volume, 558-559, 839
for parvovirus, 689 compatibility testing in, 415, 562-563
for West Nile virus, 685 cytomegalovirus infection in, 562
MPHA (mixed passive hemagglutination assay), DIC in, 567, 572
371-372 direct antiglobulin testing in, 545-546
mRNA. See Messenger RNA erythropoiesis in, 557-558, 559
MSBOS (maximum surgical blood order sched- extracorporeal membrane oxygenation in,
ules), 92-93, 414 572-573, 574
MSDS (material data safety sheets), 59, 60-61, graft-vs-host disease in, 560-561
78-79 hypothermia in, 560
Multiplex PCR, 213 immune thrombocytopenia in, 551-554
Muscular spasms, in donors, 108 immunologic status, 560-561
Mutations, genetic, 208, 227, 228 leukocyte reduction for, 573-574
Myasthenia gravis, 156 Lewis antigens in, 306
low birthweight, 557
N metabolic problems in, 561-562
neonatal alloimmune neutropenia, 379
N antigen/antibody, 336, 337, 340, 840
normal laboratory values in, 836-837
NA/NB neutrophil antigens, 378

Index 889
plasma volume in, 839 Nucleic acid amplification test (NAT)

polycythemia in, 572 in HBV testing, 669, 672
red cell volume in, 839 in HCV testing, 164, 166, 213, 590, 672, 673,
size of, 558-559 675, 701
transfusions in in HIV testing, 164, 166-167, 213, 590,
administration, 565-566 678-681
Cryoprecipitated AHF, 558, 572 in HTLV testing, 683
effect of additive/preservative solutions, in parvovirus testing, 688-689
564-565 in viral maker testing, 166-167
to enhance hemostasis, 570-572 in West Nile virus testing, 213
exchange, 546-547, 560, 566-568 Nucleic acids
of Fresh Frozen Plasma, 558, 571 isolation of, 209-211
Granulocytes, 558, 569-570 sequencing, 217-218, 222
indications for, 563-564 Nucleotides
Platelets, 490, 552-553, 558, 568-569 defined, 203
Red Blood Cells, 558, 564-568 insertion and deletion, 208
volumes for, 558, 564, 568, 571 sequences, 204, 205
Neutralization substitution, 207-208, 228
in antibody identification, 445 Nutricel (AS-3), 176, 186, 565
of Sda antigen, 357, 445, 767-768
in viral marker testing, 169 O
Neutropenia, 379-380
Obligatory gene, 237
Neutrophils
Occupational Safety and Health Administration
alloantigens, 377-380, 647
(OSHA), 41
antibodies to
Officers
in neonatal alloimmune neutropenia, 379
chemical hygiene, 58
testing for, 380
safety, 42-43
in TRALI, 379-380, 647, 656
Oh phenotype (Bombay), 304
in immune system, 255
Ok system (ISBT 024), 226, 354, 843
NIT. See Neonatal immune thrombocytopenia
Oligonucleotide probes, sequence-specific
NK (natural killer) cells, 255
(SSOP), 214, 373, 395-396
Nomenclature
OND antigen, 378, 379
of blood group systems, 238-239, 318-319,
“Open” system, 189, 192
840-844
Opsonization, 262
CD (clusters of differentiation), 246, 247-248
Optisol (AS-5), 176, 186, 565
of HLA system, 391-393
Oral thermometers, electronic, 822-823
ISBT, 226, 238-239, 840-843
Ordering policies, 36, 91-94, 414
of platelet antigens, 366, 367-368
Organ donation and transplantation
of Rh system, 315, 317, 318-319
ABO compatibility in, 401, 402, 627, 629-630
Non-A, non-B hepatitis (NANB), 673, 699
consent for, 620-621
Non-immune hemolysis, 636, 642-643
cytomegalovirus in, 629, 630
Nonconforming products or services
disease transmission in, 617, 619-620
management, 19, 20, 21
donor eligibility, 620-621
quarantine, 173-174, 194
of heart, 629
Nonimmunologic protein adsorption, 476
HLA testing, 401-402
Nonsense response (mutations), 208, 227
of kidney, 401-402, 619, 624, 627
Normal solutions, 723
of liver, 402, 627-629
Nuclear Regulatory Commission (NRC), 41, 64,
of pancreas, 401-402, 629
65, 66
positive DAT after, 455

preservation conditions and dating periods, Pain at infusion site, 633

624 Pancreatic transplantation, 401-402, 620, 624,
records, 626 629
recovery of tissue, 621 Panel-reactive antibody (PRA), 362, 397, 401
regulations, 626 Panels, red cell, 425, 426
risk reduction in, 618, 620 Papain, preparation of, 756-757. See also En-
serologic testing for, 621-623, 629 zymes, proteolytic
skills and experience appropriate for, 618 Para-Bombay phenotype, 304
transfusion support, 627, 629-630 Parasitic worms, 698-699
types of donors for, 622 Parentage testing
Organisms genetics, 236-237
in bacterial contamination, 691, 692 HLA testing in, 402
genetically modified, 717 linkage disequilibrium in, 389
Organizational management, 6-7 Paresthesias, 141, 151
Organizations, 848-850. See also specific organi- Pareto analysis, 27
zations Paroxysmal cold hemoglobinuria (PCH),
regulating quality systems, 1-2 467-468
regulating safety, 39-40, 71-72 classification, 459
Organs of immune system, 249 serologic findings in, 460, 467
Orientation program, 8 specificity of autoantibodies, 469
OSHA (Occupational Safety and Health Admin- transfusion in, 467
istration), 41 Paroxysmal nocturnal hemoglobinuria, 378
Outdating, 90-91 Partial D, 322-323
Outpatients, 408-409 Parvovirus B19, 688-689, 700
Oxygen Passenger lymphocyte hemolysis, 628, 629-630,
compensation for anemia, 484 657
delivery of, in ANH, 127-128 Paternity testing. See Parentage testing
dissociation in red cell storage, 185, 187 Pathogen inactivation, 702
in massive transfusion, 511 Patients. See Recipients
measuring adequacy of supply, 484-485 PCH. See Paroxysmal cold hemoglobinuria
supply and demand, 483-484 PCR. See Polymerase chain reaction
treating inadequate supply of, 485 PCR-SSO/PCR-SSOP. See Sequence-specific
Oxygen therapeutics, 512-513 oligonucleotide probes
Pedi packs, 564
P Pediatric patients. See Children; Neonates
Peer review, 23, 514
P blood group (ISBT 003), 308-311
PEG. See Polyethylene glycol
anti-P1, 303, 310-311
Penicillin, 455, 474, 477, 786-788
antibodies, 311, 468
Percentage solutions, 723, 726-727
antigens, 308-309, 310, 311, 840, 844
Percutaneous umbilical blood sampling (PUBS),
association with PCH, 468
541-542
biochemistry and genetics, 309-310, 844
Performance improvement standards, 25
Pericardium transplants, 619
hydatid cyst fluid/P1 substance, 310, 445
Personal protective equipment (PPE), 43-44
phenotypes, 309, 844
for biosafety, 54
P1A1 and P1A2 (HPA-1) antigens, 366, 367, 369,
for chemical safety, 61
552
face shields, 74
p24 antigen, 679
gloves, 46, 55, 73-74, 106
Packaging biological materials, 717-718, 720-721
laboratory coats, 73
PAD. See Preoperative blood donation

Index 891
masks, 75 of donors, 105-106, 800-804

safety goggles, 74 prevention of contamination in, 693
uniforms, 73 Phosphate buffer, 728
Personnel Photochemical inactivation, 702
accidents and injuries in, 45-46 Photopheresis, 158
competency assessment, 8 Physical examination of donors, 100, 101-103, 620
hepatitis prophylaxis for, 45, 703 Physicians’ orders, 522-523, 526
job descriptions for, 7 Physician’s responsibilities, in autologous pro-
latex allergies in, 46-47 gram, 120-121
medical first aid and follow-up, 45 Physiologic anemia of infancy, 558, 559, 563-564
orientation program, 8 Physiologic jaundice, 566
protective equipment for, 43-45, 73-75 Phytanic acid disease, 157-158
records, 19 Pipettes, recalibration, 847
safety monitoring programs, 45 PlA antigens, 366, 552
selection of, 7 Plasma
staffing levels, 8-9 ABO compatibility, 411, 496, 498
training avoiding use, 701
biosafety, 50 coagulation factor replacement with, 496,
chemical safety, 58-59 498-500
computer systems, 32 collection by apheresis, 141, 142-143
electrical safety, 48 components
FDA cGMP, 8 derivatives/substitutes, 496, 503-508, 699,
fire safety, 47 701
general safety, 43, 44 Fresh Frozen Plasma, 142, 177, 496,
new employee, 8 498-500
radiation safety, 65 inventory management, 94
PF4 ELISA, 377 Liquid Plasma, 177, 189
pH, 274, 431, 444 Plasma, 177
pH meters, 846 Plasma, Cryoprecipitate Reduced, 177,
Phage display/repertoire cloning, 222, 267 190, 496, 498
Phagocytic cells, 255-256, 262 Plasma Frozen within 24 Hours after
Phagocytosis, 262, 269 Phlebotomy, 181, 189
Pharmacologic alternatives to transfusion, Pooled Plasma, Solvent/deter-
512-514 gent-treated, 496, 702
Pharmacologic purging, 597 Recovered Plasma (for manufacture), 177
Phenotypes. See also specific blood groups Source Plasma, 142
of autologous red cells, 429-430, 439 Thawed Plasma, 189, 191
calculations for combined phenotypes, 236, constituents removed in apheresis, 149-150
441 description, 177
defined, 233 expiration dates, 189-190
of donor units, 440-441 for pretransfusion testing, 409, 424
frequencies, 236 as replacement fluid in apheresis, 150, 151
nomenclature for, 238-239, 844 use in children, 571
Phlebotomy virus inactivation, 701-702
adverse reactions to, 19, 107-109 volume, normal value, 839
aggressive, in autologous donations, 125 Plasma D-dimers, 837
care of donors after, 106 Plasma exchange. See Therapeutic apheresis
collection of blood samples, 105-106, Plasma inhibition of anti-Ch and-Rg, 445,
408-409, 801-804 765-766

Plasma protein fraction (PPF), 507 Platelet disorders

Plasmapheresis, 142-143, 500 DDAVP in, 513
Plasmids, 217 in liver disease, 499
Platelet chambers, 185, 197-198, 846 thrombocytopenia
Platelet concentrates (Platelets) in blood loss, 499
aliquoting, 193-194 drug-induced, 374-377
bacterial growth in, 690-692, 694-695 heparin-induced, 375-377, 492
biochemical changes during storage, 188 idiopathic thrombocytopenic purpura,
coagulation factors in, 838 158, 373-374, 492, 553-554
description, 176-177, 489 neonatal alloimmune thrombocytopenia,
expiration dates, 188, 189 511-513, 551-553
freezing, 183 posttransfusion purpura, 366, 370, 638,
infectious risks of, 700 659-660
inspection, 194-195 secondary to maternal ITP, 553-554
inventory management, 94, 95 thrombotic thrombocytopenic purpura,
irradiated, 189, 192-193, 553 154-155, 158, 492, 500
leukocyte-reduced, 184, 189, 190, 199, 202, treatment, 158, 490-491
365, 493, 817 Platelet incubators, 185, 197-198, 846
pooled, 184, 189, 193 Platelet-rich plasma (PRP), 176, 177
preparation, 176-177, 815-817, 827-828 Plateletpheresis, 140-142, 154
quality control, 199, 202 Platelets. See also Platelet concentrates; Platelet
storage, 185, 188, 694-695 disorders; Platelets Pheresis
transfusions, 488-492 antigens/antibodies
ABO/Rh matching, 361-362, 489-490, 569 ABH, 361-362, 397
in children, 490, 552-553, 558, 568-569, autoantibodies, 373-374
576-577 clinical importance, 370-373
contraindications to, 491-492 detecting, 370-373, 374, 375, 377, 552
HLA alloimmunization to, 265, 266, drug-induced, 374-377
362-366, 397-398, 637 HLA, 265, 362-366, 397-398
indications for, 490-491, 569 platelet-specific, 366, 367-368, 369-373, 552
physiologic principles, 488-489 assessing function, 488-489
prophylactic, 490-491 corrected count increment, 362, 363, 489
refractoriness to, 362-365, 397-398, 491 life span and kinetics, 489
therapeutic, 490 membrane glycoproteins, 369-370
transportation and shipping, 196 normal values, 835, 836
volume reduction, 193, 490, 569, 817-818 predicted platelet count increment, 362, 363,
washed, 193, 646 489
Platelet counts refractoriness, 362-365
in assessing hemostasis, 488 antibody specificity prediction method
corrected platelet count increment, 362, 363, for, 365
489 causes, 362-363, 364, 397-398, 491
in idiopathic thrombocytopenic purpura, 554 defined, 362
in massive transfusion, 511, 651 finding compatible donors, 398
in NAIT, 552, 553 HLA-matched platelets for, 363-365, 398
in neonates, 568-569 preventing alloimmunization, 365-366
normal values, 835, 836 selecting platelets with, 363-365
predicted platelet count increment, 362, 363, Platelets Pheresis
489 ABO/Rh matching, 361-362, 411, 489-490, 590
transfusion triggers, 490-491, 569, 576 bacterial growth in, 690-692, 694-695

Index 893
collection, 140-141 Polyspecific AHG, 279-280, 427

crossmatching, 364, 365, 398 Pooled components
described, 489 Cryoprecipitated AHF, 183-184, 191, 193, 815
donor selection and monitoring, 141 expiration, 183, 189
HLA matched, 95, 363-365, 398 labeling, 172, 183
infectious risks of, 700 Platelets, 184, 189, 193
inspection, 194-195 preparation, 183
inventory management, 94, 95 Pooled plasma, solvent-detergent-treated, 496, 702
laboratory testing, 142 Population genetics, 236-237
leukocytes reduced, 142, 202, 493 Position effect, 319-320, 324
product validation, 35 Postoperative blood collection, 117, 133-135
quality control, 202 Posttransfusion platelet recovery (PPR), 362,
records, 141 363, 489
storage, 185, 694-695 Posttransfusion purpura (PTP), 366, 370, 638,
transfusions, 488-492 659-660
ABO/Rh matching, 361-362, 411, 489-490 Potassium, 187, 561, 650
contraindications to, 491-492 PPE. See Personal protective equipment
HLA alloimmunization to, 265, 266, PPF (plasma protein fraction), 507
362-366, 397-398, 637 PPR (posttransfusion platelet recovery), 362,
physiologic principles, 488-489 363, 489
prophylactic, 490-491 PRA (panel-reactive antibody), 362, 397, 401
refractoriness to, 362-365, 397-398, 491 Preadmission testing, 409
therapeutic, 490 Precipitation, 272
transportation and shipping, 196 Pregnancy. See also Hemolytic disease of the fe-
Pluripotent hematopoietic stem cell, 249, 251 tus and newborn; Prenatal studies
Policies and procedures, 7, 11, 15 autologous collection in, 119
Polyagglutination, 298, 331, 743 as immunizing stimulus, 536-537, 552
Polycythemia, 572 Lewis antibodies in, 305
Polyethylene glycol (PEG) Premedication in transfusions, 522, 646-647
in antibody detection/identification, Prenatal studies
276-277, 427, 443, 753-754 amniotic fluid analysis, 540-541, 542
effect on incubation times, 274 antibody titration, 449, 539-540, 761-764,
use in adsorption, 783-784 796-798
Polymerase chain reaction (PCR) Doppler flow studies, 542
applications, 213-214, 215, 222 maternal history, 538
in HLA typing, 394-396 in neonatal immune thrombocytopenia, 552
kinetic, 213 percutaneous umbilical blood sampling,
ligase chain reaction, 213, 214 541-542
long distance, 213, 214 serologic studies, 538-539
multiplex, 213 typing the fetus, 539
oligonucleotide probes, 214, 373, 395-396 Preoperative blood donation (PAD), 117,
in platelet typing, 373 118-126
reaction procedure, 211, 212, 213 advantages/disadvantages, 118
reverse transcriptase, 214, 217 adverse reactions, 123
sequence-specific primers, 214, 373, 395, 396 aggressive phlebotomy, 125
in testing fetus for D antigen, 538 collection, 121
variations, 213 compliance requirements, 119-120
Polymorphisms, 207-209 component collection, 126
Polymorphonuclear granulocytes, 255 continuous quality improvement, 123-124

contraindications, 119 transfusion requests, 407-408, 522-523

cost-effectiveness, 125, 127 type and screen, 91-93, 414
donor deferrals, 120 in urgent situations, 419, 510-511
donor screening, 121-122, 124-125 Preventive action, 24-25
erythropoietin use, 125 Prewarming technique, 308, 438, 754-755
establishing program, 120-124 Probability values in antibody identification,
labeling, 120, 122-123 429, 430
medical interview, 122 Problem identification and resolution, 25-27
in pediatric patients, 118-119 Procainamide, 455
physician responsibility, 120-121 Procedures and policies, 7, 11, 15
records, 123 Process capability, defined, 30
shipping, 120 Process control, 30
storage, 123 Process flowcharting, 26, 27
supplemental iron in, 121 Process improvement, 24-27
testing, 119-120, 122 Process management, 11-14
timing and red cell regeneration during, 122 computer system validation, 13-14
transfusion, 123 concepts, 4-5, 12
transfusion trigger, 125 equipment validation, 13
volume collected, 122 process validation, 11-12
voluntary standards, 119 quality control, 14
weak D in donor, 324 validation plan, 12-13
Prescriptions for blood orders, 522-523, 526 Processes, 7, 11-12, 15
Preservatives Production, principles of, 5
anticoagulant-preservative solutions, Products, nonconforming, 19, 20, 21
178-179 Proficiency testing (PT), 24
CPD, CP2D, CPDA-1, 178-179, 186, 564, 565 Promoter sequence, 204, 205
red cell changes during storage, 185, 186, Protein C, 500, 507, 571, 837
187, 431, 432 Protein inactivation, 220
shelf life of components, 178, 188, Protein S, 500, 507, 837
189-190 Protein synthesis, 204-207
reagent, antibodies to, 437 Proteolytic enzymes. See Enzymes, proteolytic
Pressure devices, 530 Prothrombin time (PT)
Pretransfusion testing, 407-420 in liver disease, 499
after non-group-specific transfusions, 419-420 in massive transfusion, 511
with autoantibodies, 469-472 in monitoring hemostasis, 494, 496
of autologous blood, 122 normal value of, 837
blood labeling and release, 416, 418 in vitamin K deficiency, 498
blood samples, 409-410, 424 Prozone, 272, 275
in children, 415, 562-563, 574 PRP (platelet-rich plasma), 176, 177
in cold agglutinin syndrome, 466 Psoralen (S59), 702
comparison with previous records, 413, 526 PT. See Prothrombin time
crossmatching, 413-415, 417 PTP (posttransfusion purpura), 366, 370, 638,
interpretation of results, 415-416, 417 659-660
in massive transfusions, 419, 510-511 Public antigens, 392
patient identification, 407, 408-409 PUBS (percutaneous umbilical blood sampling),
procedures included, 524 541-542
selection of units, 418-420 Pulmonary edema, 648-649
serologic testing, 410-413, 417 Pulse, of donor, 102
surgical blood orders, 91-93, 414 Pumps, infusion, 522, 523, 529-530, 565

Index 895
Q process improvement, 24-27

process management, 11-14
QSE (Quality System Essentials), 3
quality assurance, 2
Quad packs, 564
quality concepts, 2, 4-6
Qualification, defined, 30
quality control, 2
Quality assurance (QA)
quality management, 2
of blood administration, 527, 532
work environment, 27-28
defined, 2, 30
Quarantine
Quality control (QC)
of nonconforming components, 173-174
of blood components, 197-199, 202
of repeatedly reactive units, 673, 674, 675,
apheresis, 832
683
Cryoprecipitated AHF, 199
of unusable products, 93-94
leukocyte-reduced components, 199,
832-834
Platelets, 199, 694 R
Red Blood Cells, 199 Race of donor, 98
Red Blood Cells, Deglycerolized, 812-813 Radiation safety, 63-66
of copper sulfate solution, 819-821, 847 blood irradiators, 65-66
defined, 2, 31 effects of radiation, 63-64
of equipment emergency response plan, 66
automatic cell washers, 830-832 exposure limits, 64
centrifuge calibration, 826-830 monitoring radiation, 64-65
continuous temperature monitoring sys- radiation measurement units, 63
tems, 197-198 regulations, 64
freezer alarms, 198-199, 824-826, 846 safe work practices, 66
frequency of, 14, 846-847 training, 65
refrigerator alarms, 198-199, 823-824, 846 waste management, 66
thermometers, 198, 821-823, 847 Raph system (ISBT 025), 226, 354, 843
of hematopoietic products, 607-608 Rapidly progressive glomerulonephritis (RPGN),
in process control, 14 156-157
records, 14 Rare Donor Program, 441-442, 769-770
unacceptable results for, 14 Rd (Scianna) antigen, 349, 350, 842
Quality improvement, 4 Reagents
Quality indicators, 22-23, 31 in ABO testing, 296
Quality management, 2, 4-6, 31 albumin additives, 276, 427, 753
Quality oversight, 6-7, 8 antibodies to components of, 298, 437-438
Quality System Essentials (QSEs), 3 antiglobulin, 278-280 , 427, 454, 468
Quality systems, 1-38 chloroquine diphosphate, 446, 469, 746-
application of principles, 6-28 747
Code of Federal Regulations references, 32 contamination of, 331, 332
common terms, 30-31 for elutions, 457, 772-773, 775
customer and supplier relations, 9-10 enzymes, 276, 443, 445, 446, 756-760
defined, 1-2 glycine-HCl/EDTA, 446, 469, 747-748
deviations and nonconforming products or labeling, 715
services, 19, 20, 21 LISS and LISS additives, 274, 275, 277, 427,
documents and records, 14-15, 16, 17-19 443, 753, 754
equipment management, 10-11 monoclonal, 266-267, 296, 328, 329-330
human resources, 7-9 for phenotyping, 440-441
monitoring and assessment, 22-24, 36-38 polyethylene glycol (PEG), 276-277, 427, 443,
organizational management, 6-7 753-754

preparation of, 715, 743-744, 756-757 donor, 97-98, 103, 165, 173
quality control intervals, 847 electronic, 17-18
red cells, 425, 426, 438 infectious disease testing, 173
in Rh testing, 328-331 linking personnel to, 19
sulfhydryl, 448-449, 744-745, 764-765, management, 17-19
766-767 of patients with special needs, 661
use of manufacturer’s directions, 11 plateletpheresis, 142
ZZAP, 445, 446, 781-783 pretransfusion testing, 413
Receptors storage, 18
on B cells, 249-253 stored tissue allograft, 626
complement, 262, 354 transfer, 173
defined, 269 transfusion, 416
immunoglobulin superfamily, 244, 245 transfusion complication, 660-661
on macrophages and monocytes, 250, Recovered Plasma, 177
255-256 Red blood cells
for pathogens, P antigens as, 311 abnormalities in Rhnull, 326
on phagocytic cells, 255-256 alloimmunization to, 265, 637, 656-657
on T cells, 245, 254, 393, 394 antigen nomenclature, 238-239, 318-319,
Recessive traits, 233-234, 241 840-844
Recipients changes in storage, 185, 186, 187-188, 431,
ABO and Rh testing in, 122, 410, 411 433
care during transfusions, 530-531 immune-mediated destruction, 265-266
education and consent, 521-522 membrane components of, 290-291
identification, 407, 408-409, 416, 524-526, normal values, 835
527 preparation of 3% suspension, 727
phenotyping, 429-430, 439 reagents, 425, 426, 438
records, 413, 416, 660-661 removal from marrow, 601
tracing (look-back), 675, 681-682, 683 separating autologous and transfused,
weak D in, 323-324, 411 748-750
Recombinant human erythropoietin (rHuEPO), survival studies, 441, 509, 655
219 volume, normal value, 839
in anemia treatment, 488 Red Blood Cells, Deglycerolized
in autologous donations, 125 adequacy of deglycerolization, 812-813
in neonates, 559 expiration date, 189
Recombinant immunoblot assay (RIBA), 170, preparation, 191-192, 809
670-671, 672 refreezing, 183
Recombinant interleukin-11, 512 storage, 192
Recombinant proteins Red Blood Cells, Frozen
for HPC mobilization, 587, 592-595 cryoprotective agents for, 181
in leukapheresis, 144 expiration dates, 184, 189
as transfusion alternative, 512, 559 freeze-thaw damage in, 181
uses for, 218-219, 222 preparation, 181-183, 807-812
Records storage, 184-185, 809
archiving, 17-18 thawing and deglycerolizing, 191-192, 809
autologous donation/transfusion, 123 transportation and shipping, 196
blood component, 165, 172-173, 416, 418 Red Blood Cells, Pheresis, 35, 144
changing, 18 Red Blood Cells (RBCs)
checking before blood release, 416, 526 ABO/Rh compatibility, 411, 418, 486
confidentiality, 18 antigen-matched, 95

Index 897
bacterial contamination, 691 quality-related CFR regulations, 32

collection by apheresis, 35, 143 for radiation safety, 64
description, 176 for safety, 28, 39-40, 64, 71-72
expiration dates, 189 for tissue transplantation, 626
freezing, 181-183, 807-812 for transport and shipping dangerous goods,
infectious risks of, 700 716-721
irradiated, 189, 192, 193, 493 Reissuing blood products, 197, 525
leukocytes-reduced, 189, 190, 199, 202, 493, Rejuvenated RBCs, 189, 194, 806-807
805 Relative risk (RR), 403-404
low volume, 101, 122, 179 Release of blood
open system, 189 identification of recipient and component,
preparation, 176, 804-807 524-526
quality control, 199, 202 policies of, 416, 418
rejuvenated, 189, 194, 806-807 reissue, 197, 525
storage, 185, 186, 187-188 in urgent situations, 419, 510-511, 522-523
substitutes, 512-513 Remedial action, 25
transfusion, 483-488 Renal system
in HPC transplantation, 591-592, 599, diseases treated with apheresis, 156-157
600, 601 failure in transfusion reactions, 640-641
indications for, 485-488, 563-564 kidney transplantation, 401-402, 619, 624,
in neonates, 562-568, 588 627
physiologic principles, 483-485 Reports
selection of components, 411, 486 of deviations, nonconformances and com-
transportation and shipping, 195-196 plications, 19, 20, 21
washed, 189, 193 of fatalities, 19, 46, 661, 702-703
Red cell depletion, 601 of injuries, 45-46
Reentry protocols, 673, 674, 675, 681-682 internal event, 19, 20
Reference laboratories, 439 Requests
Refractoriness to platelets, 362-365 for autologous blood collection, 121
antibody specificity prediction method for, for transfusion, 407-408, 522-523
365 Requirement, defined, 31
causes, 362-363, 364, 397-398, 491 Respiratory distress, 152, 639, 647-648
defined, 362 Restricted work areas, 41
finding compatible donors, 398 Restriction endonucleases, 214, 215
HLA-matched platelets for, 363-365, 398 Restriction fragment length polymorphism
platelet crossmatching for, 365, 398 analysis (RFLP), 214, 215, 222, 373
preventing alloimmunization, 365-366 Reticulocyte counts, normal value, 835
selecting platelets with, 363-365 Reverse line technique, 395
Refrigerators, 184 Reverse transcriptase PCR (RT-PCR), 214, 217
alarm systems for, 198-199, 823-824, 846 RFLP (restriction fragment length polymor-
quality control, 197-199, 846 phism analysis), 214, 215, 222, 373
temperature monitoring systems for, Rg (Chido/Rodgers) antigens/antibodies,
197-198 351-352, 842
thermometers for, 198 Rh Immune Globulin (RhIG), 547-551
Refsum’s disease, 157-158 in amniocentesis, 541, 549
Registration of donors, 97-98 antepartum administration of, 538, 541,
Regulations 548
for autologous donations, 119-120 contraindications for, 549
for hematopoietic transplantation, 608 dosage for, 547, 549, 550-551

for fetomaternal hemorrhage, 549-551 of donors and recipients, 328-332, 410, 411,
in platelet transfusions, 490 413, 441
in positive DAT, 456 false-positive/false-negative results, 329,
postpartum administration of, 548-549 330, 331-332
Rh system (ISBT 004), 315-331 of Granulocytes, 144
antibodies, 327-329 in HDFN, 330, 539, 545
anti-D in D+ individuals, 327 high-protein reagents in, 328-329, 330
concomitant, 327-328, 441 low-protein reagents in, 329-330
directed at cis products, 324 microplate test, 328, 741
dosage effect, 327 of Platelets Pheresis, 142
antigens in prenatal evaluation, 538-539
C, c, E, e, 316, 319, 320, 330-331 slide test, 328, 739-740
cis product, 324 in transfusion reaction evaluation, 654
D antigen, 315-316, 319-320 for transplantation, 629
deleted phenotypes, 326 tube test, 740-741
G antigen, 324-325 of umbilical cord blood, 595
incidence, 317 for weak D, 165, 323, 328, 411, 538-539,
nomenclature, 841, 844 741-744
partial D, 322-323 RIBA (recombinant immunoblot assay), 170,
Rhmod, 326 670-671, 672
Rhnull, 325-326 Rigors, 633, 643
variants, 325 Risks of disease transmission
weak D, 322-324 HIV infection, 677
association with LW, 351 with plasma derivatives, 699, 701
in component selection, 418, 441, 486, 490, posttransfusion hepatitis, 673, 675
511 reducing, 618, 620, 699, 701-703
ethnic differences in, 316, 317, 320, 321, with tissue transplantation, 617-618,
325 619-620, 620
genes, 226, 320-321 with transfusions, 700
genetics and biochemistry, 316-318 RNA interference, 220
genotypes, 321-322, 844 RNA (ribonucleic acid)
in HDFN, 536 isolation of, 209-211
phenotypes and haplotypes, 319, 320, 844 mRNA processing, 204-206
position effect in, 319-320, 324 mRNA translation, 206-207
red cell abnormalities in, 326 splicing, 206
RhAG (Rh-associated glycoprotein), 318 transfer, 207
terminology, 315, 317, 318-319 Rocky Mountain spotted fever, 696
in transplantation, 599, 628-629 Rodgers blood group. See Chido/Rodgers
Rh testing, 328-332 Room temperature antibodies, 302-303
with autoagglutinins, 329, 331, 469-470 Room temperature storage, 185
of autologous blood, 122 Rosette test, 550, 793-794
of blood components, 164, 165 Rouleaux
of C, c, E, e, 320, 330-331 in ABO discrepancies, 298, 299, 303
in children, 330, 415, 545, 563, 574 in antibody detection, 412
comparison with previous records, 413, in Rh typing, 329, 330
526 saline replacement technique, 303, 755-
controls, 328-329, 330 756
of cord blood, 545 RT-PCR. See Reverse transcriptase PCR
for D, 319-320, 328-330 Run charts, 23

Index 899
S SDF-1 (stromal-derived growth factor-1), 594

Secretor gene (Se), 290, 291, 304-305, 737-739
S/s antigens and antibodies, 336, 337, 340, 840
Secretory component, 258
Safe work practices
Segregation, 229, 230
for biosafety, 54-55
Selectins, 246
for electrical safety, 48-49
Semen, 620, 621, 624
for fire prevention, 47-48
SEN-V virus, 668
general guidelines, 44, 73-76
Sensitization, 272-275
for radiation safety, 66
Separation techniques
Safety goggles, 74
in apheresis, 139-140
Safety program
immunomagnetic, 596, 598
accidents and injuries, 45-46
of multiple antibodies, 449
biosafety, 49-57, 77
for transfused and autologous cells, 748-750
chemical safety, 57-63, 78-88
Sepsis
disaster planning, 67-68
neonatal, 569-570
electrical safety, 48-49
and positive DAT, 456
emergency response plans, 44
prevention, 693-694
employee monitoring programs, 45
transfusion-associated, 635, 643, 655,
engineering controls, 43
691-692
fire prevention, 47-48
Sequence-specific oligonucleotide probes
first aid and follow-up, 45
(SSOP), 214, 373, 395-396
general elements, 42-47
Sequence-specific primers (SSPs), 214, 373, 395,
hazard identification and communication,
396
43
SERF (Cromer) antigen, 353, 354, 843
hepatitis prophylaxis, 45, 668, 672, 703
Serious Hazards of Transfusion (SHOT) initia-
latex allergies, 46-47
tive, 641
management controls, 45
Serologic centrifuges, calibration, 828-830
personal protective equipment, 43-44
Serologic testing
policy manual, 45
additives in, 276-277, 443, 753-754
radiation safety, 63-66
of autologous blood, 120, 122
regulations and recommendations, 71-72
of blood components, 165-166
resources for information, 850
in cold agglutinin syndrome, 460, 465
safe work practices, 44
comparison with previous results, 413
safety officer, 42-43
of cord blood, 595
shipping hazardous materials, 66
of Granulocytes, 144
training, 43, 44
in HDFN, 538-539, 544-546
waste management, 67
for HLA antigens, 396-397
in work environment, 27-28
in IgM warm AIHA, 464
Saline replacement technique, 303, 755-756
incubation temperatures, 443, 715
Saliva
in mixed-type AIHA, 460, 466-467
ABH substances in, 290-291, 301, 736-739
in neonatal immune thrombocytopenia, 552
Lewis substance in, 445, 736-739
for organ and tissue transplantation, 620,
Salvia lectins, 743
621-623, 629
Samples. See Blood samples
in paroxysmal cold hemoglobinuria, 460,
SBO (standard blood orders), 92-93
467
Scianna system (ISBT 013), 226, 336, 349-350,
for platelet antibodies, 375
842
of Platelets Pheresis, 142
Sda antigen, 357
with positive DAT, 456
agglutination pattern of antibody, 357, 412
in pretransfusion testing, 410-413, 562-563
neutralization of, 357, 445, 767-768

records of, 165 Signs, safety, 47, 51, 60

for syphilis, 165-166, 695 Silent mutations, 207
in transfusion reaction evaluations, 653-654 Single-donor platelets. See Platelets Pheresis
in warm autoimmune hemolytic anemia, Skin appearance
460, 461 in donors, 102-103
in warm IgM AIHA, 464 in transfusion reactions, 639, 644-646
Serotonin release assay, 377 Skin banking, 619, 624, 625
Serum Sl (Knops) antigens, 354, 843
dilution, 725-726 Slide tests
for pretransfusion testing, 409, 424 for ABO group, 731-732
Serum hyperviscosity syndrome, 153-154 for Rh typing, 328, 739-740
Serum proteins in typing discrepancies, 298, Sodium, 187
299, 303, 331 Software, computer, 17
Serum-to-cell ratio, 444 Solid organ transplantation. See Organ donation
Services and transplantation
critical, 6-7 Solid-phase red cell adherence assay (SPRCA)
nonconforming, 19, 20, 21 in detecting antigen-antibody reactions,
quality principles, 5 283-284
Severe acute respiratory syndrome (SARS), 590 in platelet antibody detection, 371, 372, 375
Sex-linked traits, 233-234, 241 in platelet crossmatching, 364
SH antigen, 378 Soluble antigens
Shelf life of components, 178, 188, 189-190 ABH, 290-291, 298, 301, 445, 736-739
Shipments Chido/Rodgers, 445, 765-766
of autologous units, 120 HLA, 391
of blood components, 66, 179, 195-196 Lewis, 445, 736-739
cargo aircraft only, 719 P1, 310, 445
of clinical specimens, 66, 717-718, 720-722 Sda, 357, 445, 767-768
containers for, 195, 847 testing, 301, 445, 736-739
of dangerous goods, 66, 716-722 Solutions
of hematopoietic components, 606-607 additive, 176, 186, 564-565
labeling, 718, 719 colloid, 486, 507-508, 572
monitoring temperature during, 722 dilution, 726-727
packaging with dry ice, 196, 719, 720-721 hemoglobin, 512-513
packaging with liquid nitrogen, 718, 719 IV, 530
Shock, 633, 640 phosphate buffer, 728
Short supply agreement, 177 preparation, 723-725
SHOT (Serious Hazards of Transfusion) initia- Solvent/detergent-treated pooled plasma, 496,
tive, 641 702
Showers, emergency, 61 Somatic cells, 223, 241
Sickle cell disease (SCD) Source Plasma, 142
alloantibody production in, 576 Southern blotting, 214, 216
autologous donation with, 119 SPA immunoadsorption, 158
in children, 574-576 Specification, defined, 31
delayed transfusion reactions in, 656-657 Spills
separation of transfused from autologous blood, 55, 56
cells, 749-750 chemical, 61-62, 84-88
transfusion in, 508-509, 574-576 radioactive, 66
treatment with apheresis, 155-156 SPRCA. See Solid-phase red cell adherence assay
Signal transduction, 246 SRA (C-serotonin release assay), 377

Index 901
SSOP (sequence-specific oligonucleotide STS (serologic testing for syphilis), 165-166,

probes), 214, 373, 395-396 695
SSP (sequence-specific primers), 214, 373, 395, Subgroups of ABO system, 293-294
396 in ABO discrepancies, 302
Staffing, 8-9 confirmation of by adsorption/elution,
Standard blood orders (SBO), 92-93 735-736
Standard Precautions, 49, 54 testing for, 296
Standards Sulfhydryl reagents, 448-449
for autologous donations, 119 to alter blood group antigens, 342, 445, 446,
for hematopoietic transplantation, 609 766-767
ISO 9001, 1- 2, 3 applications for, 448-449
performance improvement, 25 in dispersing autoagglutination, 469-470,
STAR (Scianna) antigen, 349, 350, 842 744-745
Statistical tables for binomial distribution, to distinguish IgM from IgG, 764-765
33-35 effect on Kell antigens, 342
Stem cells, pluripotent, 249, 251, 581 Supertransfusion programs, 575-576
Sterile connection devices, 847 Supplier relations, 9-10
Sterility testing, 195 Supplies, critical, 9, 10, 11
Storage Suppressor genes, 235-236
acceptable temperatures for, 184, 185, 715 Surgical blood order systems, 91-93, 414
antigen deterioration with, 431, 433 Survey meters, 65
of autologous blood, 94-95, 123 Survival studies of red cells, 441, 509, 655
biochemical changes with, 185, 186, 187 Syncope, in donors, 107
of biohazardous material, 57 Syngeneic HPC transplantation, 582, 587
of blood components, 93-94, 184-185, 186, Syphilis, 695
187-188 testing autologous blood, 120
of blood samples, 184-185, 410 testing blood components, 164, 165-166, 695
of directed donor units, 94-95 testing transplantation donors for, 590, 620,
frozen, 184-185, 606 621
of Granulocytes, 144
of hazardous chemicals, 61 T
of hematopoietic progenitor cells, 604-606
T-cell receptor (TCR), 245, 254, 393, 394
liquid, 185, 186, 187-188
T-cell reduction (depletion), 586, 598
liquid nitrogen, 185, 606
T lymphocytes, 249, 253-255
of organs and tissue, 624
cytotoxic T cells, 249, 253-254
of Platelets, 185, 188, 694-695
depletion methods, 598
of records, 18-19
helper T cells, 249, 253-254
of Red Blood Cells, 185, 186, 187-188
receptors (TCR), 245, 254, 393, 394
of Red Blood Cells, Deglycerolized, 192
recognition by T cytotoxic cells, 254
of Red Blood Cells, Frozen, 184-185, 809
stimulation of B cells, 254-255
refrigerated, 184
subpopulations of, 249, 253
room temperature, 185
TA-GVHD. See Transfusion-associated
of untested or infectious products, 606
graft-vs-host disease
vapor-phase, 606
Tc (Cromer) antigens/antibodies, 353, 354, 843
Storage lesion
TCR (T-cell receptor), 245, 254, 393, 394
of platelets, 188
Temperature
of red cells, 185, 186, 187-188
of donor, 101-102
Stroma-free hemoglobin solution, 512-513
effect on agglutination, 273-274, 336, 443
Stromal-derived growth factor-1 (SDF-1), 594
incubation, 443, 715

monitoring systems, 197-198, 823-826 posttransfusion purpura, 366, 370, 638,

during shipments, 195-196, 722 659-660
storage, 184, 185, 715 secondary to maternal ITP, 553-554
Tendon transplants, 624 thrombotic thrombocytopenic purpura,
Test results, grading, 412, 728-729 154-155, 158, 492, 500
Thalassemia, 508, 575-576 treatment, 158, 490-491
Thawed Plasma, 189, 191 Thrombopoietin, recombinant human, 219
Thawing Thrombotic thrombocytopenic
Cryoprecipitated AHF, 191, 815 purpura/hemolytic-uremic syndrome
devices for, 846 (TTP/HUS), 154-155, 158, 492, 500
effects of, 181 Tick-borne infections, 695-697
Fresh Frozen Plasma, 191 Timers/clocks, 847
hematopoietic components, 607 Tissue antigens. See HLA system
Red Blood Cells, Frozen, 191-192, 809 Tissue oxygenation, 484-485, 511-512
Therapeutic apheresis, 144-158 Tissue transplantation
complications, 150-153 ABO compatibility in, 627
in HDFN, 145, 149 bone banking, 623, 625
indications for, 146-148, 153-158, 500 consent for, 620-621
photopheresis, 158 disease transmission in, 617, 619-620
plasma volumes exchanged, 145, 149 donor eligibility, 620-621
removal of normal plasma constituents, heart valves, 625
149-150 preservation conditions and dating periods,
removal of pathologic substances, 145, 624
148-149 records, 626
replacement fluids in, 150, 151 recovery of tissue, 621
SPA immunoadsorption, 158 regulation, 626
vascular access in, 145, 150 risk reduction in, 618, 620
Therapeutic cells, 581. See also Hematopoietic serologic testing for, 621-623
progenitor cell transplantation skills and experience appropriate for, 618
Therapeutic plasma exchange (TPE), 145. See skin banking, 625
also Therapeutic apheresis types of donors for, 622
Thermal amplitude studies, 441, 458, 465 Titration of antibodies
Thermometers, 198 applications, 449
electronic oral, 822-823 cold agglutinins, 458, 465, 778-779
liquid-in-glass, 821-822 maternal antibodies, 449, 539-540, 730-731,
standardization and calibration, 198, 796-798
821-823, 847 methods for, 761-764, 796-798
Thrombin, 502 TNF (tumor necrosis factor), 264, 640
Thrombin time, normal value, 837 Toxoplasmosis, 698
Thrombocythemia, 154 TPE (therapeutic plasma exchange), 145. See
Thrombocytopenia also Therapeutic apheresis
in blood loss, 499 Training
drug-induced, 374-377 biosafety, 50
and exchange transfusion, 567 chemical safety, 58-59
heparin-induced, 375-377, 492 for dry ice shipments, 721
idiopathic thrombocytopenic purpura, 158, electrical safety, 48
373-374, 492, 553-554 FDA cGMP, 8
neonatal alloimmune thrombocytopenia, fire safety, 47
551-553, 568-569 general safety, 43, 44

Index 903
new employee, 8 posttransfusion purpura, 366, 370, 638,

radiation safety, 65 659-660
Traits, 232-236 management, 634-639
TRALI. See Transfusion-related acute lung injury records, 660-661
Tranexamic acid, 513-514 reporting, 19, 661
Trans effect, 319, 320 Transfusion-related acute lung injury (TRALI),
Transforming growth factor-β (TNF-β ), 264 647-648
Transfusion-associated graft-vs-host disease evaluation, 656
(TA-GVHD), 657-659 and granulocyte transfusions, 648
HLA system in, 399, 400 HLA antibodies in, 399, 647
management, 637 management, 635
manifestations, 658 neutrophil antibodies in, 379-380, 647
in neonates, 560-561 pathophysiology, 635, 647
pathophysiology, 637, 658 prevention, 648
treatment and prevention, 658-659 symptoms, 634, 647-648
Transfusion-associated sepsis, 635, 643, 655, treatment, 648
691-692 Transfusion-transmitted diseases
Transfusion Committee, 514 babesiosis, 695-696
Transfusion reactions bacterial sepsis, 635, 643, 655, 691-692
acute Chagas’ disease, 697-698
air embolism, 636, 651-652 cytomegalovirus, 686-687
allergic (urticarial), 522, 634, 644-647 Epstein-Barr virus, 687
anaphylactic, 635, 644-647 erlichiosis, 696
circulatory overload, 636, 648-649 fatalities from, 702-703
citrate toxicity, 649-650 hepatitis, 667-675
coagulopathy, 651 herpesviruses, 686-689
evaluation, 531, 652-656 human immunodeficiency viruses, 675-682
febrile nonhemolytic, 379, 398-399, 576, human T-cell lymphotropic viruses, 682-683,
634, 643-644 684
hemolytic, 336, 399-400, 634, 639-642 Lyme disease, 696-697
hyper- and hypokalemia, 650 malaria, 697
hypocalcemia, 561, 636, 649-650 management, 703
hypotension associated with ACE inhibi- parasitic worms, 698-699
tion, 636, 645 parvovirus, 688-689
hypothermia, 511, 637, 650 reducing risks of, 699, 700, 701-702
nonimmune hemolytic, 636, 642-643 reporting, 702
sepsis, 635, 643, 655, 691-692 Rocky Mountain spotted fever, 696
signs and symptoms, 633, 639 syphilis, 695
transfusion-related acute lung injury, tick-borne infections, 695-697
379-380, 399, 635, 647-648 toxoplasmosis, 698
classification, 633, 634-638 transmissible spongiform encephalopathies,
delayed 689-690
alloimmunization, 637, 656-657 West Nile virus, 683, 685-686
graft-vs-host disease, 399, 400, 560-561, Transfusion triggers
637, 657-659 for autologous transfusions, 125
hemolytic, 346, 575, 637, 656-657 for platelet transfusions, 490-491, 569, 576
immunomodulation, 638, 660 for red cell transfusions, 483, 486-488
iron overload, 638, 660 Transfusions
Kidd antibodies in, 346, 656 administration procedures, 521-532, 565-566

alternatives to, 512-514 of protein C and protein S, 507

of antiprotease concentrates, 505-506 quality assurance, 514, 527, 532
assessments of, 23, 38, 514 of RBCs, 483-488, 558, 562-568
in autoimmune hemolytic anemias, of recombinant proteins, 512
462-464, 467, 468, 509-510 requests for, 407-408, 522-523
of autologous blood, 123 selection of components, 411, 486
cause of ABO discrepancies, 298 in sickle cell disease, 508-509, 574-576
cause of positive DAT, 455, 462 starting, 526-527
in children, 562-572, 574-577 in thalassemia, 508, 575-576
of coagulation factors, 493-494, 495, 496, in transplantation, 627, 629-630
497, 498-505 in urgent situations, 419, 510-511,
of colloid solutions, 486, 507-508, 572 522-523
consent for, 521-522 of Whole Blood, 485, 486
of Cryoprecipitated AHF, 500-502, 572 Transmissible spongiform encephalopathies
delays in starting, 525 (TSEs), 689-690
equipment for, 523-524, 527-530 Transplantation. See Hematopoietic progenitor
events following, 531-532 cell transplantation; Organ donation and
exchange, 546-547, 560, 566-568 transplantation; Tissue transplantation
fatalities from, 19, 641, 645, 661, 690-691, Transplantation antigens. See HLA system
702-703 Transportation
of Fresh Frozen Plasma, 496, 498-500, 571 of autologous units, 120
of Granulocytes, 144, 492, 569-570 of blood components, 66, 179, 195-196
and HLA system, 397-400 of blood to patient area, 524-525
in HPC transplantation, 591-592, 599, 600, cargo aircraft only, 719
601 of clinical specimens, 66, 717-718, 720-722
identification of recipient and components, containers for, 195, 847
524-526, 527 of dangerous goods, 66, 716-722
of Immune Globulin, Intravenous, of hematopoietic components, 606-607
505, 506 labeling, 718, 719
as immunizing stimulus in HDFN, monitoring temperatures during, 722
537-538 packaging with dry ice, 196, 719, 720-721
of incompatible blood, 509, 641 packaging with liquid nitrogen, 718, 719
indications for, 486-488, 563-564 Trial to Reduce Alloimmunization to Platelets
infusion rates, 531, 566 (TRAP) Study Group, 365
infusion sets for, 527-529, 565-566 Tropical spastic paraparesis (TSP), 682-683
intrauterine, 541, 543-544 Trypanosoma cruzi, 698, 700
of irradiated components, 493 TSEs (transmissible spongiform
IV solutions for, 530 encephalopathies), 689-690
of leukocyte-reduced components, 492-493 TTP/HUS. See Thrombotic thrombocytopenic
massive, 419, 510-512, 628-629, 649-651 purpura/hemolytic-uremic syndrome
in neonates, 562-572 TTV virus, 668
non-group-specific, 419-420, 510-511 Tube tests
in older infants and children, 574-577 for ABO group, 732-733
patient care during, 530 for Rh type, 740-741
perioperative, 486-487 Tumor cell detection, 608
of plasma derivatives and substitutes, 496, Tumor necrosis factor (TNF), 264, 640
503-508 Tumor purging, 597-598
of Platelets, 488-492, 553, 568-569 Twitching, in donors, 108
premedication in, 522 Type and screen (T/S), 91-93, 414

Index 905
U Vil antigen, 354

Viral marker testing. See Infectious disease test-
U antigen/antibody (MNS system), 336, 337,
ing
340, 840
Viruses
Ulex europaeus (anti-H), 304, 743-744
inactivation of, 699, 701-702
Ultraviolet B (UVB) irradiation, 365
transmitted by transfusion
Umbilical cord blood. See Cord blood
cytomegalovirus, 686-687
UMC (Cromer) antigen, 353, 354, 843
Epstein-Barr, 687
Unasyn, 455
hepatitis, 667-675
Uniforms, 73
HIV, 675-682
Urgent release of blood, 419, 510-511, 522-523
HTLV, 682-683, 684
Urine
human herpesviruses, 688
examinations in transfusion reactions, 639,
parvovirus, 688-689
653
transmissible spongiform
neutralization of Sda, 357, 445, 767-768
encephalopathies, 689-690
Urticaria (hives), 634, 644-647
West Nile, 683, 685-686
Utilization of blood. See Blood utilization
Viscosity, relative, 835
UVB (Ultraviolet B) irradiation, 365, 398
Vital signs, 526-527, 531
Vitamin K, 498-499, 571
V VLBW (very low birthweight) neonates, 557, 568.
Vaccines, hepatitis, 45, 668, 672, 703 See also Neonates
Valeri method for red cell cryopreservation, Volume of blood
810-812 administered in intrauterine transfusions,
Validation 544
of computer systems, 13-14, 415 administered in neonatal transfusions, 558,
defined, 31 564, 568, 571
of equipment, 13 collections, 101, 122, 178-179
of processes, 11-12 normal values, 558-559, 839
statistical tables for, 33-35 Volume of marrow collections, 591
validation plans, 12-13 Volume of plasma
variances to, 11 exchanged in TPE, 145, 149
Vapors, hazardous, 62, 63 normal values, 839
Variant Creutzfeldt-Jakob disease (vCJD), 590, Volume of red cells, 839
689-690 Volume reduction of platelets, 193, 490, 569,
Vascular access 817-818
in apheresis, 145, 150 Vomiting, 107-108, 639
in HPC collection, 593, 594 von Willebrand syndromes, 501
in neonates, 565-566, 568
for transfusions, 523-524 W
Vasovagal syndrome, 107, 141
WAIHA. See Warm autoimmune hemolytic ane-
vCJD (variant Creutzfeldt-Jakob disease), 590,
mia
689-690
Warfarin, 376, 498-499
Vel collection, 355-356
Warm autoantibodies
Venipuncture, 105-106, 800-804
adsorption, 470-472, 779-781
Venous access. See Vascular access
alloantibodies with, 439, 470-472
Venous transplants, 624
drug-induced, 475-476
Verification, defined, 31
frequency of testing, 463-464
Vicia graminea (anti-N), 743
mimicking alloantibodies, 472
View boxes, 846
transfusion-stimulated, 462

transfusion with, 462-464 Whole blood

in warm autoimmune hemolytic anemia, ABO/Rh compatibility, 411, 486
461-462 biochemical changes during storage, 185,
Warm autoimmune hemolytic anemia (WAIHA), 186, 187-188
459, 461-464 collection, 178-179
antiglobulin testing in, 281 description, 175
chronic, 462-463 equipment quality control, 847
classification, 459 expiration date, 189
serologic findings in, 459, 460, 461 leukocyte reduction, 180
specificity of autoantibody, 461-462 processing, 179-180
transfusion in, 462-464 transfusion, 485, 486
Warm-reactive alloantibodies, 273-274 transportation and shipping, 179,
Washed blood components, 189, 193, 561, 646 195-196
Wastage of autologous blood, 123-124 Wipe tests, 65
Waste Work environment
biohazardous, 55-57 design and workflow, 40
chemical, 63 housekeeping, 40-41
defined, 717 restricted areas, 41
radioactive, 63 safety, 27-28, 54-55
reduction program, 67 Wr (Diego) antigens/antigens, 348, 842
treating, 57 Wristbands, patient, 408, 409, 526
Water of crystallization, 724
Water of hydration, 724
Waterbaths, 693, 846 X
Wb (Gerbich) antigen, 352, 843
X-linked traits, 233-234, 241
Weak D, 322-324
Xenogeneic HPC transplantation, 582
in autologous donations, 324
Xg system (ISBT 012), 226, 336, 348-349, 350,
in donors, 165, 323
842
“microscopic,” 550
partial D, 322-323
in pregnancy, 538-539
quantitative, 322 Y
in recipients, 323-324, 411 York (Yk) antigens, 352, 354, 843
testing for, 165, 323, 328, 741-743 Yt (Cartwright) system (ISBT 011), 226, 336, 348,
Weight, donor, 101, 143 349, 842
WES (Cromer) antigens/antibodies, 353, 354,
843
West Nile virus (WNV), 683, 685-686 Z
fatalities due to, 691
ZENA (Cromer) antigen, 353, 354, 843
preventive measures, 590, 685
Zygosity, 431. See also Dosage effect
testing for, 166, 213, 595-596, 685
ZZAP
Western immunoblot, 169-170, 679-681, 698
in allogeneic adsorption, 471-472, 781-783
WH (Chido/Rodgers) antigen, 352, 842
to alter antigens, 342, 445, 446
White cells
in autologous adsorption, 469, 470, 775-776,
normal values, 835, 836
779-781
residual, in leukocyte-reduced components,
in dispersing autoagglutination, 469
493, 832-834

Glossary of Abbreviations
AATB American Association of Tissue CCI corrected count increment
Banks CD clusters of differentiation
ACD acid-citrate-dextrose CDC Centers for Disease Control and
ACE angiotensin-converting enzyme Prevention
AChE acetylcholinesterase CFR Code of Federal Regulations
ACOG American College of Obstetricians CFU colony-forming unit
and Gynecologists CGD chronic granulomatous disease
ADCC antibody-dependent cellular cGMP current good manufacturing
cytotoxicity practice
AET 2-aminoethylisothiouronium cGy centiGray
AHF antihemophilic factor CHD cold hemagglutinin disease
AHG antihuman globulin CI continuous improvement or
AHTR acute hemolytic transfusion reaction confidence interval
AICC anti-inhibitor coagulation complex CIDP chronic inflammatory demyelinating
AIDS acquired immune deficiency polyneuropathy
syndrome CJD Creutzfeldt-Jakob disease
AIHA autoimmune hemolytic anemia CLIA Clinical Laboratory Improvement
ALG antilymphocyte globulin Amendments
ALT alanine aminotransferase CLSI Clinical and Laboratory Standards
Institute
ANCA antineutrophil cytoplasmic
antibodies CML cell-mediated lympholysis
ANH acute normovolemic hemodilution CMS Centers for Medicare and Medicaid
Services
APC antigen-presenting cell
CMV cytomegalovirus
aPTT activated partial thromboplastin
time CNS central nervous system
AS additive solution CPD citrate-phosphate-dextrose
ASO allele-specific oligonucleotide CPDA-1 citrate-phosphate-dextrose-adenine-1
ATG antithymocyte globulin CR complement receptor
ATL adult T-cell lymphoma/leukemia CREG cross-reactive group
ATP adenosine triphosphate CRYO cryoprecipitated AHF
BFU burst-forming unit CUE confidential unit exclusion
BPO benzyl-penicilloyl DAF decay-accelerating factor
BSA bovine serum albumin or body DAT direct antiglobulin test
surface area DDAVP 1-deamino-8-d-arginine
BSC biological safety cabinet vasopressin
BUN blood urea nitrogen DDR donor deferral registry
C:T crossmatch-to-transfusion DHTR delayed hemolytic transfusion
reaction
CAD cold antibody (agglutinin) disease
DIC disseminated intravascular
CAP College of American Pathologists
coagulation
CAS cold antibody (agglutinin) syndrome
DMSO dimethylsulfoxide
CBER Center for Biologics Evaluation and
DNA deoxyribonucleic acid
Research
2,3-DPG 2,3-diphosphoglycerate
CCE counterflow centrifugal elutriation
DRG diagnosis-related group

DTT dithiothreitol HBV hepatitis B virus
EACA epsilon aminocaproic acid Hct hematocrit
EBAA Eye Bank Association of America HCV hepatitis C virus
EBV Epstein-Barr virus HDFN hemolytic disease of the fetus and
ECMO extracorporeal membrane newborn
oxygenation HDV hepatitis D virus
EDTA ethylenediaminetetraacetic acid HES hydroxyethyl starch
EIA enzyme immunoassay HEV hepatitis E virus
ELAT enzyme-linked antiglobulin test HIV human immunodeficiency virus
ELBW extremely low birthweight HPC hematopoietic progenitor cell
ELISA enzyme-linked immunosorbent HTLV-I human T-cell lymphotropic virus
assay type I
EPO erythropoietin HTR hemolytic transfusion reaction
ESR erythrocyte sedimentation rate HUS hemolytic uremic syndrome
FACT Foundation for the Accreditation of IAT indirect antiglobulin test
Cellular Therapy Ig immunoglobulin
FDA Food and Drug Administration IGIV Immunoglobulin Intravenous
FFP Fresh Frozen Plasma IHA immune hemolytic anemia
FMH fetomaternal hemorrhage IL-1α interleukin 1 alpha
FNHTR febrile nonhemolytic transfusion IL-1ß interleukin 1 beta
reaction
IL-2 interleukin 2
FTA-ABS fluorescent treponemal antibody
IPT intraperitoneal transfusion
absorption test
IS immediate spin
5-FU 5-fluorouracil
ISBT International Society of Blood
G-CSF granulocyte colony-stimulating
Transfusion
factors
ISCT International Society for Cellular
GalNAc N-acetylgalactosamine
Therapy
GM-CSF granulocyte macrophage
ITP idiopathic thrombocytopenic
colony-stimulating factors
purpura
GMP good manufacturing practice
IUT intrauterine transfusion
Gp glycoprotein
IVT intravascular transfusion
GPA glycophorin A
JCAHO Joint Commission on Accreditation
GPB glycophorin B of Healthcare Organizations
GPC glycophorin C L/S lecithin to sphingomyelin
GPD glycophorin D LDH lactate dehydrogenase
GVHD graft-vs-host disease LDL low-density lipoproteins
Gy Gray LISS low ionic strength saline
HAM HTLV-associated myelopathy LT-CIC long-term culture-initiating cells
HAV hepatitis A virus MAC membrane attack complex
HAZMAT hazardous material 2-ME 2-mercaptoethanol
Hb hemoglobin MF mixed field
HBc hepatitis B core antigen MHC major histocompatibility complex
HBIG hepatitis B immunoglobulin MLC mixed lymphocyte (leukocyte)
HBsAg hepatitis B surface antigen culture
(cont’d)

MLR mixed lymphocyte (leukocyte) RBCs Red Blood Cells (blood donor unit)
reaction RCA regulators of complement activation
MoAb monoclonal antibody RES reticuloendothelial system
mRNA messenger ribonucleic acid RFLP restriction fragment length
MSBOS maximum surgical blood order polymorphism
schedule Rh Rhesus factor
MSDS material safety data sheets RhIG Rh Immune Globulin
NAIT neonatal alloimmune RIBA recombinant immunoblot assay
thrombocytopenia
RNA ribonucleic acid
NAT nucleic acid testing
RPGN rapidly progressive
NIH National Institutes of Health glomerulonephritis
NK natural killer RPR rapid plasma reagin (serologic test
NMDP National Marrow Donor Program for syphilis)
NRC Nuclear Regulatory Commission RR repeatedly reactive or relative risk
NT not tested RT room temperature or reverse
OSHA Occupational Safety and Health transcriptase
Administration SBO standard blood order
p probability SCF stem cell factor
PAD preoperative autologous (blood) SGP sialoglycoprotein
donation SOP standard operating procedure
PBPC peripheral blood progenitor cell SPA staphylococcal protein A
PBS phosphate-buffered saline SSO sequence-specific oligonucleotide
PCH paroxysmal cold hemoglobinuria STS serologic test for syphilis
PCR polymerase chain reaction TA transfusion-associated
PEG polyethylene glycol TCR T-cell receptor
PHA phytohemagglutinin TNF-α tumor necrosis factor alpha
PI paternity index TPE therapeutic plasma exchange
PPE personal protective equipment TRALI transfusion-related acute lung
PPF plasma protein fraction injury
PPTA Plasma Protein Therapeutics tRNA transfer ribonucleic acid
Association TTP thrombotic thrombocytopenic
PRA panel reactive antibody purpura
PT prothrombin time or proficiency test UNOS United Network for Organ Sharing
PTP posttransfusion purpura VLBW very low birthweight
PUBS percutaneous umbilical blood vWD von Willebrand disease
sampling vWF von Willebrand factor
PVC polyvinyl chloride WAIHA warm autoimmune hemolytic
QA quality assessment or quality anemia
assurance WB Whole Blood or Western blot
QC quality control XM crossmatch
QSE quality system essential

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Technical Manual

Copyright © 2005 by the AABB. All rights reserved.

Technical Manual and Standards for Blood Banks and

Transfusion Therapy: Clinical Principles and Practice, 2nd Edition

Transfusion Medicine Self-Assessment and Review

Blood Transfusion Therapy: A Physician’s Handbook, 8th Edition

Practical Guide to Transfusion Medicine

Transfusion Medicine Interactive: A Case Study Approach CD-ROM

Copyright © 2005 by the AABB. All rights reserved.

AABB ISBN No. 1-56395-196-7

Technical manual / editor, Mark E. Brecher. —15th ed.

Copyright © 2005 by the AABB. All rights reserved.

Chair and Editor

Regina M. Leger, MSQA, MT(ASCP)SBB, CQMgr(ASQ)

Martha Rae Combs, MT(ASCP)SBB

Gilliam B. Conley, MA, MT(ASCP)SBB

Copyright © 2005 by the AABB. All rights reserved.

James P. AuBuchon, MD W. John Judd, FIBMS, Arell S. Shapiro, MD

Copyright © 2005 by the AABB. All rights reserved.

Copyright © 2005 by the AABB. All rights reserved.

Copyright © 2005 by the AABB. All rights reserved.

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

Copyright © 2005 by the AABB. All rights reserved.

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

Blood Donation and Collection

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

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

Immunologic and Genetic Principles

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

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

Serologic Principles and Transfusion Medicine

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

Copyright © 2005 by the AABB. All rights reserved.

Appendix 20-1. An Example of an Algorithm for Investigating a Positive DAT

Clinical Considerations in Transfusion Practice

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

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

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

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

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

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

Copyright © 2005 by the AABB. All rights reserved.

3. Antibody Detection, Antibody Identification, and Serologic Compatibility

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

Copyright © 2005 by the AABB. All rights reserved.

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

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