Contents

Abbreviations

CHAPTER 1 An introduction to blood groups

What is a blood group?

Blood group antibodies

Clinical importance of blood groups

Biological importance of blood groups

Blood group systems

Blood group terminology and classification

CHAPTER 2 Techniques used in blood grouping

Factors affecting antigen–antibody reactions

Stages of haemagglutination reactions

Direct agglutination

Indirect agglutination

Elution techniques

Automation of test procedures

Flow cytometry

Molecular blood group genotyping

CHAPTER 3 The ABO blood groups

Introduction

ABO antigens, antibodies, and inheritance

A₁ and A₂

Antigen, phenotype, and gene frequencies

ABO antibodies

Importance of the ABO system to transfusion and transplantation medicine

Biochemical nature of the ABO antigens

Biosynthesis of the ABO antigens and ABO molecular genetics

H, the precursor of A and B

ABH secretion

H-deficient red cells

Further complexities

Acquired changes

Associations with disease and functional aspects

CHAPTER 4 The Rh blood group system

Introduction – Rh, not rhesus

Haplotypes, genotypes, and phenotypes

Biochemistry and molecular genetics

D antigen (RH1)

C, c, E, and e antigens (RH2, RH4, RH3, RH5)

Other Rh antigens

Rh-deficient phenotypes – Rhnull and Rhmod

Putative function of the Rh proteins and RhAG

CHAPTER 5 Other blood groups

The Kell system

The Duffy system

The Kidd system

The MNS system

The Diego system

The Lewis System

Some other blood group systems

Antigens that do not belong to a blood group system

CHAPTER 6 Clinical significance of blood group antibodies

Antibody production and structure

Factors affecting the clinical significance of antibodies

Haemolytic transfusion reactions (HTR)

Haemolytic disease of the fetus and newborn (HDFN)

Autoantibodies

Tests to assess the potential significance of an antibody

Decision-making for transfusion

CHAPTER 7 Blood grouping from DNA

Fetal blood grouping

Blood group typing of patients and donors

Next generation sequencing

The future of blood group serology

CHAPTER 8 Quality assurance in immunohaematology

Achieving total quality

Frequency and specificity of control material

Quality requirements for safe transfusion practice

Checklist of critical control points

Laboratory errors, root cause analysis (RCA), and corrective and preventive action (CAPA)

CHAPTER 9 Trouble-shooting andproblem-solving in thereference laboratory

ABO grouping

Rh grouping

Problems in antibody screening, identification, and crossmatching

CHAPTER 10 Frequently asked questions

What is the difference between sensitivity and specificity and how can these be determined?

Why is anti-A,B no longer obligatory in ABO typing?

Why are two anti-D reagents often recommended for RhD typing?

What is the importance of detecting D variant (weak D and partial D) phenotypes?

How do I control the results for antiglobulin testing?

Why should RhD positive women be tested more than once during pregnancy?

How often should transfusion recipients be tested for the presence of antibodies?

How can passive anti-D be differentiated from anti-D due to alloimmunisation?

Why do we need to perform antibody screening? Isn’t a crossmatch by IAT at 37°C enough to detect incompatible blood?

What is the incidence of alloimmunisation post-transfusion?

How do I determine and identify antibodies present in a sample?

What is a compound antibody?

How can the incidence of compatible donors for a recipient with multiple antibodies be calculated?

Why can’t the droppers in bottles of reagents be used instead of a volumetric pipette?

What cells should be used when performing an antibody titration?

How are the results of titrations reported?

What is a Major Obstetric Haemorrhage?

What is ‘Massive Transfusion’?

When group-specific blood is in short supply, how do I select the ‘next best’ for transfusion?

How are high-titre haemagglutinins classified?

What is an ‘immediate spin’ crossmatch?

What is an ‘electronic crossmatch’?

Which patients are not eligible for electronic issue of blood?

What is ‘bed-side’ testing?

What are signs and symptoms of a suspected transfusion reaction?

What action should be taken in the event of a suspected transfusion reaction?

In haemovigilance, how should ‘near-miss’ events be characterised?

Recommended reading and web sites

Index

This edition first published 2014, © 2014 by John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data

Daniels, Geoff, author.
Essential guide to blood groups / Geoff Daniels, Imelda Bromilow. – Third edition.
p. ; cm.
Includes bibliographical references and index.

ISBN 978-1-118-68892-2 (pbk.)
I. Bromilow, Imelda, author. II. Title.
[DNLM: 1. Blood Group Antigens–Handbooks. WH 39]
QP91
612.1′1825–dc23

2013019571

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: Bubaone

Abbreviations

CHAPTER 1

An introduction to blood groups

What is a blood group?

In 1900, Landsteiner showed that people could be divided into three groups (now called A, B, and O) on the basis of whether their red cells clumped when mixed with separated sera from other people. A fourth group (AB) was soon found. This is the origin of the term ‘blood group’.

A blood group could be defined as, ‘An inherited character of the red cell surface, detected by a specific alloantibody’. Do blood groups have to be present on red cells? This is the usual meaning, though platelet- and neutrophil-specific antigens might also be called blood groups. In this book only red cell surface antigens are considered. Blood groups do not have to be red-cell specific, or even blood-cell specific, and most are also detected on other cell types. Blood groups do have to be detected by a specific antibody: polymorphisms suspected of being present on the red cell surface, but only detected by other means, such as DNA sequencing, are not blood groups. Furthermore, the antibodies must be alloantibodies, implying that some individuals lack the blood group.

Blood group antigens may be:

• proteins;
• glycoproteins, with the antibody recognising primarily the polypeptide backbone;
• glycoproteins, with the antibody recognising the carbohydrate moiety;
• glycolipids, with the antibody recognising the carbohydrate portion.

Blood group polymorphisms may be as fundamental as representing the presence or absence of the whole macromolecule (e.g. RhD), or as minor as a single amino acid change (e.g. Fy^a and Fy^b) or a single monosaccharide difference (e.g. A and B).

Fig. 1.1 Diagram of different types of blood group active proteins and glycoproteins based on their integration into the red cell surface membrane. Listed are examples of blood group antigens for each type. (Type 4 proteins are cytoplasmic and not present in red cells.)

Blood group proteins and glycoproteins are integral structures of the red cell membrane. Diagrammatic representations of some blood group proteins and glycoproteins in the membrane are shown in Fig. 1.1. Some pass through the membrane once. These generally have an external N-terminal domain and a cytoplasmic C-terminal domain (Type 1), though in one case (the Kell glycoprotein) the C-terminus is external and the N-terminus internal (Type 2). Some are polytopic (Type 3); that is, they cross the membrane several times. Usually both termini are cytoplasmic, but the Duffy glycoprotein has an odd number of membrane-spanning domains and an extracellular N-terminal domain. Finally, some have no membrane-spanning domain, but are anchored to the membrane by a lipid tail (called a glycosylphosphatidylinositol or GPI anchor), which is attached to the C-terminus of the protein through carbohydrate (Type 5). There are no Type 4 glycoproteins, which have no external domain, in the red cell membrane.

Most red cell surface proteins are glycosylated, the only exceptions being the Rh and Kx proteins. This glycosylation may be (1) N-glycosylation, large, branched sugars attached to asparagine residues of the amino acid backbone, or (2) O-glycosylation, smaller glycans (usually tetrasaccharides) attached to serine or threonine residues.

Blood group antibodies

Blood groups are antigens and, by definition, a molecule cannot be an antigen unless it is recognised by an antibody (or T cell receptor); therefore, all blood group specificities are defined by antibodies. Most adults have antibodies to the A or B antigens, or to both; that is, they have ‘naturally occurring’ antibodies to those ABO antigens they lack. For most other blood groups, corresponding antibodies are not ‘naturally occurring’, but are only formed as a result of immunisation by transfused red cells or by fetal red cells leaking into the maternal circulation during pregnancy or childbirth.

Blood group antibodies are usually IgM or IgG, although some may be IgA (Chapter 6). ‘Naturally occurring’ antibodies are usually predominantly IgM, whereas ‘immune’ antibodies are predominantly IgG. As a general rule, IgM antibodies will directly agglutinate antigen-positive red cells in a saline medium, whereas most IgG antibodies require potentiators or anti-human globulin to effect agglutination (Chapter 2).

Clinical importance of blood groups

Blood groups are of great clinical importance in blood transfusion and in transplantation. In fact, the discovery of the ABO system was one of the most important factors in making the practice of blood transfusion possible. Many blood group antibodies have the potential to cause rapid destruction of transfused red cells bearing the corresponding antigen, giving rise to a haemolytic transfusion reaction (HTR), either immediately or several days after the transfusion. At their worst, HTRs give rise to disseminated intravascular coagulation, renal failure, and death. At their mildest, they reduce the efficacy of the transfusion (Chapter 6).

IgG blood group antibodies can cross the placenta during pregnancy and haemolyse fetal red cells expressing the corresponding antigen. This may cause alloimmune fetal haemolytic anaemia, more commonly known as haemolytic disease of the fetus and newborn (HDFN). Many blood group antibodies have the potential to cause HDFN, but the most common culprits are D and c of the Rh system and K of the Kell system.

Biological importance of blood groups

The biological importance of many blood group antigens is either known or can be surmised from their structure. The following functions have been attributed to blood group antigens: transporters of biologically important molecules across the red cell membrane; receptors of external stimuli and cell adhesion; regulators of autologous complement to prevent red cell destruction; enzymes; anchors of the red cell membrane to the cytoskeleton; and providers of an extracellular carbohydrate matrix to protect the cell from mechanical damage and microbial attack. Very little is known, however, about the functions of the blood group polymorphisms, but it is likely that they arose from selection pressures created by pathogens exploiting blood group molecules for attachment to the cells and subsequent invasion.

Blood group systems

The International Society of Blood Transfusion (ISBT) recognises 339 blood group antigens; 297 of these are classified into 1 of 33 blood group systems (Table 1.1 and see the Red Cell Immunogenetics and Blood Group Terminology Working Party area of the ISBT website: www.isbtweb.org). Each blood group system represents either a single gene or a cluster of two or three closely linked genes of related sequence and with little or no recognised recombination occurring between them. Consequently, each blood group system is a genetically discrete entity. The MNS system comprises three genes, Rh, Xg, and Chido/Rodgers, two genes each, and each of the remainder represents a single gene. Rh and MNS are the most complex systems, with 54 and 46 antigens, respectively; nine systems consist of just a single antigen.

Blood group terminology and classification

Since the discovery of the ABO system in 1900, a multitude of blood group antigens have been identified and many different styles of terminology have been used. These include the following to represent alleles: upper case letters (e.g. A, B; M, N); upper and lower case letters to represent antithetical antigens, the products of alleles (S, s; K, k); superscript letters (Fy^a, Fy^b), and numbers (Lu6, Lu9). A variety of different styles of terminology have been used even within one system (e.g. Kell system: K, k; Kp^a, Kp^b, Kp^c; K12, K13).

In 1980, the ISBT established a Working Party to devise a genetically based numerical terminology for blood groups. This terminology is based on the blood group systems (Table 1.1). Each system has a three-digit number plus a 3–5 upper case letter symbol. For example, the Kell system is 006 or KEL (Table 1.2). Each antigen within that system has a three-digit number. K is 001 and Kp^a is 003, and so become 006001 or KEL1 and 006003 or KEL3, respectively. The full numerical symbol is seldom used and the alphanumerical symbols, with the redundant zeros removed, are more commonly employed. Phenotypes consist of the system symbol, followed by a colon, followed by a list of antigens shown to be present. Absent antigens are also listed, preceded by a minus symbol (Table 1.2). Gene terminology is more complex and still being developed, but basically alleles have the system symbol followed by an asterisk, followed by the number of the antigen encoded (see ISBT website). In some cases, the number may be replaced by a letter (e.g. JK*01 or JK*A).

Table 1.1 The blood group systems.

Table 1.2 Some examples of blood group terminology.

For an antigen to join an existing blood group system, there must be substantial evidence that it is encoded by the gene (or cluster of genes) producing the other antigens in the system. For one or more antigens to form a new system, the gene must have been identified and must be discrete from all other blood group genes.

Because the ISBT terminology is based on the blood groups systems, it functions as a blood group classification. It is not essential to use the numerical terminology. Indeed, it is not generally used in this book. It is, however, important to understand it, so as to understand the classification of blood groups.

Some blood group antigens have not been allocated to systems, owing to insufficient genetical evidence. If they are of low frequency, they are placed in the 700 Series of antigens and if of high frequency, in the 901 Series. If two or more antigens are categorised together on the basis of genetical, serological, or biochemical information, but, due to lack of appropriate evidence, cannot be allocated to a system or form a new system, then they can form a blood group collection. The series and collections are described in Chapter 5.

CHAPTER 2

Techniques used in blood grouping