Blood cell antigens and antibodies: erythrocytes, platelets and granulocytes

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Chapter 21 Blood cell antigens and antibodies

erythrocytes, platelets and granulocytes

Fiona Regan

Erythrocytes

Red Cell Antigens

Since Landsteiner’s discovery in 1901, that human blood groups existed, a vast body of serological, genetic and biochemical data on red cell (blood group) antigens has been accumulated. More recently, the biological functions of some of these antigens have been appreciated.

A total of 30 blood group systems have been described (Table 21.1). Each system is a series of red cell antigens, determined either by a single genetic locus or very closely linked loci. In addition to the blood group systems, there are six ‘collections’ of antigens (e.g. Cost), which bring together other genetically, biochemically or serologically related sets of antigens and a separate series of low-frequency (e.g. Rd) and high-frequency (e.g. Vel) antigens, which do not fit into any system or collection. A numeric catalogue of red cell antigens is being maintained by an International Society of Blood Transfusion (ISBT) Working Party.¹

Table 21.1 Blood group systems recognized by the ISBT Working Party

Apart from those of the ABO system, most of these antigens were detected by antibodies stimulated by transfusion or pregnancy.

Alternative forms of a gene coding for red cell antigens at a particular locus are called alleles and individuals may inherit identical or non-identical alleles. Most blood group genes have been assigned to specific chromosomes (e.g. ABO system on chromosome 9, Rh system on chromosome 1). The term genotype is used for the sum of the inherited alleles of a particular gene (e.g. AA, AO) and most red cell genes are expressed as codominant antigens (i.e. both genes are expressed in the heterozygote). The phenotype refers to the recognizable product of the alleles and there are many racial differences in the frequencies of red cell phenotypes, as shown in Table 21.2.

Table 21.2 Frequencies of red cell phenotypes in US Black and White populations

Red cell antigens are determined either by carbohydrate structures or protein structures. Carbohydrate-defined antigens are indirect gene products (e.g. ABO, Lewis, P). The genes code for an intermediate product, usually an enzyme that creates the antigenic specificity by transferring sugar molecules onto the protein or lipid. Protein-defined antigens are direct gene products and the specificity is determined by the inherited amino acid sequence and/or the conformation of the protein. Proteins carrying red cell antigens are inserted into the membrane in one of three ways: single pass, multipass or linked to phosphatidylinositol (GPI-linked). Only a few red cell antigens are erythroid-specific (Rh, LW, Kell and MNSs), the remainder being expressed in many other tissues. The structure and functions of the membrane proteins and glycoproteins carrying blood group antigens have been reviewed by Daniels.² An illustration of the putative functions of molecules containing blood group antigens is provided in Table 21.3.

Table 21.3 Putative functions of molecules containing blood group antigens

However, the main clinical importance of a blood group system depends on the capacity of alloantibodies (directed against the antigens not possessed by the individual) to cause destruction of transfused red cells or to cross the placenta and give rise to haemolytic disease in the fetus or newborn. This in turn depends on the frequency of the antigens and the alloantibodies and the characteristics of the latter: thermal range, immunoglobulin class and ability to fix complement. On these criteria, the ABO and Rh systems are of major clinical importance. Anti-A and anti-B are naturally occurring and are capable of causing severe intravascular haemolysis after an incompatible transfusion. The RhD antigen is the most immunogenic red cell antigen after A and B, being capable of stimulating anti-D production after transfusion or pregnancy in the majority of RhD-negative individuals.

ABO System

Discovery of the ABO system by Landsteiner marked the beginning of safe blood transfusion. The ABO antigens, although most important in relation to transfusion, are also expressed on most endothelial and epithelial membranes and are important histocompatibility antigens.³ Transplantation of ABO-incompatible solid organs increases the potential for hyperacute graft rejection, although ABO-incompatible renal transplantation can be successfully carried out with plasmapheresis in addition to immunosuppression of the recipient.⁴ Major ABO-incompatible stem cell transplants (e.g. group A stem cells into a group O recipient) will provoke haemolysis, unless the donation is depleted of red cells.

ABO Antigens and Encoding Genes

There are four main blood groups: A, B, AB and O (Table 21.4). In the British Caucasian population, the frequency of group A is 42%, B 9%, AB 3% and O 46%, but there is racial variation in these frequencies.⁵ The epitopes of ABO antigens are determined by carbohydrates (sugars), which are linked either to polypeptides (forming glycoproteins) or to lipids (glycolipids).

Table 21.4 ABO blood group system

The expression of ABO antigens is controlled by three separate genetic loci: ABO located on chromosome 9 and FUT1 (H) and FUT2 (Se), both of which are located on chromosome 19. The genes from each locus are inherited in pairs as Mendelian dominants. Each gene codes for a different enzyme (glycosyltransferase), which attaches specific monosaccharides onto precursor disaccharide chains (Table 21.5). There are four types of disaccharide chains known to occur on red cells, on other tissues and in secretions. The Type 1 disaccharide chain is found in plasma and secretions and is the substrate for the FUT2 (Se) gene, whereas Types 2, 3 and 4 chains are only found on red cells and are the substrate for the FUT1 (H) gene. It is likely that the O and B genes arose by mutation of the A gene. The O gene does not encode for the production of a functional enzyme; group O individuals commonly have a deletion at nucleotide 261 (the O1 allele), which results in a frame-shift and premature termination of the translated polypeptide and the production of an enzyme with no catalytic activity. The B gene differs from A by consistent nucleotide substitutions.⁶ The expression of A and B antigens is dependent on the H and Se genes, which both give rise to glycosyltransferases that add L-fucose, producing the H antigen. The presence of an A or B gene (or both) results in the production of further glycosyltransferases, which convert H substance into A and B antigens by the terminal addition of N-acetyl-D-galactosamine and D-galactose, respectively (Fig. 21.1). Because the O gene produces an inactive transferase, H substance persists unchanged as group O. In the extremely rare Oh Bombay phenotype, the individual is homozygous for the h allele of FUT1 and hence cannot form the H precursor of the A and B antigen. Their red cells type as group O, but their plasma contains anti-H, in addition to anti-A, anti-B and anti-A,B, which are all active at 37°C. As a consequence, individuals with an Oh Bombay phenotype can only be safely transfused with other Oh red cells.

Table 21.5 Glycosyltransferases produced by genes encoding antigens within the ABO, H and Lewis blood group systems

Gene	Allele	Transferase
FUT1	H	α-2-L-fucosyltransferase
	h	None
A	A	α-3-N-acetyl-D-galactosaminyltransferase
B	B	α-3-D-galactosyltransferase
O	O	None
FUT2	Se	α-2-L-fucosyltransferase
	se	None
FUT3	Le	α-3/4-L-fucosyltransferase
	le	None

Figure 21.1 Pathways from HAB blood group genes to antigens. *Glycosyltransferase H transfers L-fucose; A transfers N-acetyl-D-galactosamine; B transfers D-galactose; O is inactive.

Serologists have defined two common subgroups of the A antigen. Approximately 20% of group A and group AB individuals belong to group A₂ and group A₂B, respectively, the remainder belonging to group A₁ and group A₁B. These subgroups arise as a result of inheritance of either the A¹ or A² alleles. The A₂ transferase is less efficient in transferring N-acetyl-D-galactosamine to available H antigen sites and cannot utilize Types 3 and 4 disaccharide chains. As a consequence, A₂ red cells have fewer A antigen sites than A₁ cells and the plasma of group A₂ and group A₂B individuals may also contain anti-A₁. The distinction between these subgroups can be made using the lectin Dolichos biflorus, which only reacts with A₁ cells. The H antigen content of red cells depends on the ABO group and, when assessed by agglutination reactions with anti-H, the strength of reaction tends to be graded O > A₂ > A₂B > B > A₁ > A₁B. Other subgroups of A are occasionally found (e.g. A₃, A_x) that result from mutant forms of the glycosyltransferases produced by the A gene and are less efficient at transferring N-acetyl-D-galactosamine onto H substance.⁶

The A, B and H antigens are detectable early in fetal life but are not fully developed on the red cells at birth. The number of antigen sites reaches ‘adult’ level at around 1 year of age and remains constant until old age, when a slight reduction may occur.

Secretors and Non-Secretors

The ability to secrete A, B and H substances in water-soluble form is controlled by FUT2 (dominant allele Se). In a Caucasian population, about 80% are secretors (genotype SeSe or Sese) and 20% are non-secretors (genotype sese) (Table 21.6). Secretors have H substance in the saliva and other body fluids together with A substances, B substances or both, depending on their blood group. Only traces of these substances are present in the secretions of non-secretors, although the antigens are expressed normally on their red cells and other tissues.

Table 21.6 Secretor status in the Caucasian population

An individual’s secretor status can be determined by testing for ABH substance in saliva (see p. 504).

ABO Antigens and Disease

Group A individuals rarely may acquire a B antigen from a bacterial infection that results in the release of a deacetylase enzyme. This converts N-acetyl-D-galactosamine into α-galactosamine, which is similar to galactose, the immunodominant sugar of group B, thereby sometimes causing the red cells to appear to be group AB. In the original reported cases, five out of seven of the patients had carcinoma of the gastrointestinal tract. Case reports attest to the danger of individuals with an acquired B antigen being transfused with AB red cells, resulting in a fatal haemolytic transfusion reaction following the production of hyperimmune anti-B.⁷

The inheritance of ABH antigens is also known to be weakly associated with predisposition to certain diseases. Group A individuals have 1.2 times the risk of developing carcinoma of the stomach than group O or B; group O individuals have 1.4 times more risk of developing peptic ulcer than non-group O individuals; and non-secretors of ABH have 1.5 times the risk of developing peptic ulcer than secretors.⁸ The ABO group also affects plasma von Willebrand factor (VWF) and factor VIII levels; group O healthy individuals have levels around 25% lower than those of other ABO groups.⁹ ABO blood group appears to mediate its effect by accelerating clearance of VWF but the mechanism is not yet clear¹⁰ ABH antigens are also frequently more weakly expressed on the red cells of persons with leukaemia.

ABO Antibodies

Anti-A and anti-B

ABO antibodies, in the absence of the corresponding antigens, appear during the first few months after birth, probably as a result of exposure to ABH antigen-like substances in the diet or the environment (i.e. they are ‘naturally occurring’) (Table 21.4). This allows for reverse (serum/plasma) grouping as a means of confirming the red cell phenotype. The antibodies are a potential cause of dangerous haemolytic transfusion reactions if transfusions are given without regard to ABO compatibility. Anti-A and anti-B are always, to some extent, immunoglobulin M (IgM). Although they react best at low temperatures, they are nevertheless potentially lytic at 37°C. Hyperimmune anti-A and anti-B occur less frequently, usually in response to transfusion or pregnancy, but they may also be formed following the injection of some toxoids and vaccines. They are predominantly of IgG class and are usually produced by group O and sometimes by group A₂ individuals. Hyperimmune IgG anti-A and/or anti-B from group O or group A₂ mothers may cross the placenta and cause haemolytic disease of the newborn (HDN). These antibodies react over a wide thermal range and are more effective haemolysins than the naturally occurring antibodies. Group O donors should always be screened for high-titre anti-A and anti-B antibodies, which may cause haemolysis when group O platelets or plasma are transfused to recipients with A and B phenotypes.

Plasma-containing blood components from such high-titre universal donors should be reserved for group O recipients.

Anti-A₁ and anti-H

Anti-A₁ reacts only with A₁ and A₁B cells and is occasionally found in the serum of group A₂ individuals (1–8%) and not uncommonly in the serum of group A₂B subjects (25–50%). However, anti-A₁ normally acts as a cold agglutinin and is very rarely reactive at 37°C, when it is only capable of limited red cell destruction. There have been a few reports of red cell haemolysis ascribed to anti-A₁, which some authors have questioned because, although the antibodies reacted only with A₁ red cells, no attempts were made to absorb them with A₂ cells, which would have revealed their anti-A specificity.

Anti-H reacts most strongly with group O and A₂ red cells and also normally acts as a cold agglutinin. A notable, but rare, exception is the anti-H that occurs in the Oh Bombay phenotype, which is an IgM antibody and causes lysis at 37°C (Table 21.4) so that Oh Bombay phenotype blood would be required for transfusion.

Lewis System

Lewis Antigens and Encoding Genes

The Lewis antigens (Le^a and Le^b) are located on soluble glycosphingolipids found in saliva and plasma and are secondarily absorbed into the red cell membranes from the plasma.

The Le gene at the FUT3 (LE) locus is located on chromosome 19 and codes for a fucosyltransferase, which acts on an adjacent sugar molecule to that acted on by the Se gene. Where Se and Le are present, the Le^b antigen is produced; where Le but not Se is present, Le^a is produced; and where Le is not present, neither Le^a nor Le^b is produced. After transfusion of red cells, donor red cells convert to the Lewis type of the recipient owing to the continuous exchange of glycosphingolipids between the plasma and red cell membrane.

Neonates have the phenotype Le(a−b−) because low levels of the fucosyltransferase are produced in the first 2 months of life.

Lewis Antibodies

Lewis antibodies are naturally occurring and are usually IgM and complement binding. In vitro, their reactivity is enhanced with the use of enzyme-treated red cells, when lysis may occur. However, only rare examples of anti-Le^a that are strictly reactive at 37°C have given rise to haemolytic transfusion reactions and there is no good evidence that anti-Le^b has ever caused a haemolytic episode. Explanations for the relative lack of clinical significance include their thermal range, neutralization by Lewis antigens in the plasma of transfused blood and the gradual elution of Lewis antigens from the donor red cells. Consequently, it is acceptable to provide red cells for transfusion that have not been typed as negative for the relevant Lewis antigen but are compatible with the recipient plasma when the compatibility test is performed strictly at 37°C.

Lewis antibodies have not been implicated in haemolytic disease of the fetus or newborn. The role of Lewis in influencing the outcome of renal transplants is unclear.

The P System and Globoside Collection

The Rh haplotypes are named either by the component antigens (e.g. CDe, cde) or by a single shorthand symbol (e.g. R¹ = CDe, r = cde). Thus, a person may inherit CDe (R¹) from one parent and cde (r) from the other and have the genotype CDe/cde (R¹r). The haplotypes in order of frequency and the corresponding shorthand notation are given in Table 21.7. Although two other nomenclatures are also used to describe the Rh system, namely, Wiener’s Rh-Hr terminology and Rosenfield’s numeric notation, the CDE nomenclature, derived from Fisher’s original theory, is recommended by a World Health Organization Expert Committee¹³ in the interest of simplicity and uniformity. The Rh antigens are defined by corresponding antisera, with the exception of ‘anti-d’, which does not exist. Consequently, the distinction between homozygous DD and the heterozygous Dd cannot be made by direct serological testing but may be resolved by informative family studies. It is still routine practice to predict the genotype from the phenotype on the basis of probability tables for the various Rh genotypes in the population (Table 21.7). However, in women with immune anti-D and a history of an infant affected by HDN, RH DNA typing is used in prenatal testing for the fetal D status to decide on the clinical management of the pregnancy, e.g. the need for monitoring for fetal anaemia using middle cerebral artery Doppler ultrasound. Suitable sources include amniotic fluid (amniocytes) and trophoblastic cells (chorionic villi) or after 15 weeks’ gestation, maternal blood can be used because it contains fetal DNA.^14,¹⁵ In practice, multiplex polymerase chain reaction (PCR) is used, with more than two primer sets, to detect the different molecular bases for D-negative phenotypes in non-Caucasians. RH DNA typing also has applications in paternity testing and forensic medicine. There are racial differences in the distribution of Rh antigens, e.g. D negativity is more common in Caucasians (approximately 15%), whereas R^o (cDe) is found in approximately 48% of Black Americans but is uncommon (approximately 2%) in Caucasians. The Rh antigens are present only on red cells and are a structural part of the cell membrane. Complete absence of Rh antigens (Rh-null phenotype) may be associated with a congenital haemolytic anaemia with spherocytes and stomatocytes in the blood film, increased osmotic fragility and increased cation transport. This phenotype arises either as a result of homozygosity for silent alleles at the RH locus (the amorph type) or more commonly by homozygosity for an autosomal suppressor gene (X), genetically independent of the RH locus (the regulator type). Rh antigens are well-developed before birth and can be demonstrated on the red cells of very early fetuses.

Table 21.7 The Rh haplotypes in order of frequency (Fisher nomenclature) in Caucasians and the corresponding short notations

Fisher	Short Notations	Approximate Frequency (%)
CDe	R¹	41
cde	r	39
cDE	R²	14
cDe	R⁰	3
C^wDe	R^1w	1
cdE	r″	1
Cde	r′	1
CDE	R^Z	Rare
CdE	r^y	Rare

Antibodies

Fisher’s nomenclature is convenient when applied to Rh antibodies, and antibodies directed against all Rh antigens, except d, have been described: anti-D, anti-C, anti-c, anti-E and anti-e. Rh antigens are restricted to red cells and Rh antibodies result from previous alloimmunization by previous pregnancy or transfusion, except for some naturally occurring forms of anti-E and anti-C^W. Immune Rh antibodies are predominantly IgG (IgG1 and/or IgG3), but may have an IgM component. They react optimally at 37°C, they do not bind complement and their detection is often enhanced by the use of enzyme-treated red cells. Haemolysis, when it occurs, is therefore extravascular and predominantly in the spleen.

Anti-D is clinically the most important antibody; it may cause haemolytic transfusion reactions and was a common cause of fetal death resulting from haemolytic disease of the newborn before the introduction of anti-D prophylaxis. Anti-D is accompanied by anti-C in 30% of cases and anti-E in 2% cases. Primary immunization following a transfusion of D-positive cells becomes apparent within 2–5 months, but it may not be detectable following exposure to a small dose of D-positive cells in pregnancy. However, a second exposure to D-positive cells in a subsequent pregnancy will provoke a prompt anamnestic or secondary immune response.

Of the non-D Rh antibodies, anti-c is most commonly found and can also give rise to severe haemolytic disease of the fetus and newborn. Anti-E is less common, whereas anti-C is rare in the absence of anti-D.

Kell and Kx Systems

Antigens and Encoding Genes

A total of 34 antigens have been identified (K1–K34), but three very closely linked sets of alleles are clinically important: K (KEL1) and k (KEL2); Kp^a (KEL3), Kp^b (KEL4) and Kp^c (KEL21); and Js^a (KEL6) and Js^b (KEL7). These antigens are encoded by alleles at the KEL locus on chromosome 7, but their production also depends on genes at the KX locus on the X chromosome. The K antigen is present in 9% of the English population. The Kp^b antigen has a high frequency in Caucasians; the Js^b antigen is universal in Caucasians and almost universal in black races.

The Kell protein is a single-pass glycoprotein and is believed to be complexed by a disulphide bridge to the Kx protein, which is multipass with 10 putative transmembrane domains. It has considerable sequence homology to other neutral endopeptidases.

In the McLeod phenotype, red cells lack Kx and there is a marked decrease in all Kell antigens, an acanthocytic morphology and compensated haemolysis. The McLeod syndrome is X-linked with slow progression to cardiomyopathy, skeletal muscle wasting and neurological defects.

Kell Antibodies

Immune anti-K is the most common antibody found outside the ABO and Rh systems. It is commonly IgG1 and occasionally complement binding. Other immune antibodies directed against Kell antigens are less common. The presence of some of these antibodies, such as anti-k, anti-Kp^b and anti-Js^b, may cause extensive difficulties in the selection of antigen-negative units for transfusion.

Duffy System

Duffy antigens and encoding genes

The Duffy (Fy) locus is on chromosome 1 and encodes a multipass protein with seven or nine putative transmembrane domains.

The locus has the following alleles: Fy^a, Fy^b, which code for the co-dominant Fy^a and Fy^b antigens, respectively; Fy^x, which is responsible for a weak Fy^b antigen; and Fy, which is responsible when homozygous for the Fy(a−b−) phenotype in black races. This Fy gene is identical to the Fy^b gene in its structural region but has a mutation in the promoter region, resulting in the lack of production of red cell Duffy glycoprotein.

The Fy glycoprotein (also known as Duffy antigen receptor for chemokines, DARC) is a receptor for the CC and CXC classes of proinflammatory chemokines and is expressed on vascular endothelial cells and Purkinje cells in the cerebellum, but its precise role as a potential scavenger of excess chemokines is unknown. The Fy glycoprotein is also a receptor for Plasmodium vivax.

Duffy antibodies

Anti-Fy^a is much more common than anti-Fy^b and all other Duffy antibodies are rare apart from anti-Fy³ (to both Fy^a and Fy^b), which occurs in some African/Afro-Caribbean patients, in whom Fy(a−b−) antigen status is common. They are predominantly IgG1 and are sometimes complement binding.

Kidd (JK) System

Kidd antigens and encoding genes

Genes at the HUT 11(JK) locus on chromosome 18 encode for a multipass protein, which carries both the Kidd antigens and the human erythroid urea transporter. The codominant alleles, Jk^a and Jk^b, represent a polymorphism on HUT 11, which differs by a single amino acid substitution at position 280 (Asp/Asn).

The Jk(a−b−) phenotype is very rare and is caused by homozygous inheritance of the silent allele, Jk, at the JK locus or by inheritance of the dominant inhibitor gene In (Jk) unlinked to the JK locus. These Jk(a−b−) cells are resistant to lysis by solutions of urea and have a selective defect in urea transport.

Kidd antibodies

Anti-Jk^a is more common than anti-Jk^b; both are usually IgG. Kidd antibodies are usually complement binding, which is thought to be because most of them contain an IgG3 fraction. Anti-Jk3 is produced by individuals of the rare Jk(a−b−) phenotype.

Kidd antibodies can be difficult to detect because they often show dosage (may only react with cells showing homozygous expressions of Jk^a or Jk^b), they fall to undetectable levels in plasma and they are often present in mixtures of alloantibodies. A previous history of antibodies is therefore important, to avoid a post-transfusion haemolytic reaction, due to an anamnestic response by an antibody that was below the level of detection before transfusion.

MNSs System

MNSs antigens and encoding genes

GYPA and GYPB are closely linked genes on chromosome 4 and encode glycophorin A (GPA) and glycophorin B (GPB), respectively. Both GPA and GPB are single-pass membrane sialoglycoproteins. M and N are alleles of GYPA (encoding the M and N antigens on GPA) and S and s are alleles of GPYB (encoding the S and s antigens on GPB). Many rare variants have been described owing to gene deletions, mutations and segmental exchanges.

The U antigen is found on the red cells of Caucasians and 99% of black races. U-negative individuals are, with rare exceptions, S−s− and lack GPB or have an altered form of GPB.

MNSs antibodies

Anti-M is a relatively common antibody that may be IgM or IgG. Rare examples are reactive at 37°C when they can give rise to haemolytic transfusion reactions. Anti-M very rarely gives rise to HDN.

Anti-N is uncommon and of no clinical significance.

Anti-S and anti-s are usually IgG; both rarely have been implicated in haemolytic transfusion reactions and HDN.

Anti-U is a rare immune antibody, usually containing an IgG1 component. It has been known to cause fatal haemolytic transfusion reactions and occasional severe HDN.

Other Blood Group Systems

Lutheran system

The antigens in the Lutheran system are not well-developed at birth and as a consequence there are no documented cases of clinically significant haemolytic disease owing to Lutheran antibodies.

Anti-Lu^a is uncommon and rarely of clinical significance. Anti-Lu^b has caused extravascular haemolysis.

Yt (Cartwright) system

The antigens Yt^a and Yt^b are found on GPI-linked acetylcholinesterase. Some examples of anti-Yt^a have caused accelerated red cell destruction.

Colton system

The antigens in the Colton system, Co^a and Co^b, are carried on the water-transport protein, channel-forming integral protein (CHIP-1). Anti-Co^a and the rarer anti-Co^b are both sometimes clinically significant.

Dombrock system

The antigens in the Dombrock system include Do^a and Do^b and also include the high-incidence antigens Gy^a, Hy and Jo^a. Antibodies of this system are usually weak, but all should be considered as potentially significant.

Clinical Significance of Red Cell Alloantibodies

The significance of the alloantibodies described, with respect to the nature of the haemolytic transfusion reaction they produce, is provided in Table 21.8. The majority of haemolytic transfusion reactions, however, are the result of ABO incompatibility.¹⁶

Table 21.8 Antibody specificities related to the mechanism of immune haemolytic destruction

Blood Group System	Intravascular Haemolysis	Extravascular Haemolysis
ABO,H	A, B, H
Rh		All
Kell	K	K, k, Kp^a, Kp^b, Js^a, Js^b
Kidd	Jk^a	Jk^a, Jk^b, Jk³
Duffy		Fy^a, Fy^b
MNS		M, S, s, U
Lutheran		Lu^b
Lewis	Le^a
Cartwright		Yt^a
Colton		Co^a, Co^b
Dombrock		Do^a, Do^b

Mollison et al.¹⁷ analysed the significance of blood group antigens other than those of the ABO system and D by looking at the prevalence of transfusion-induced red cell alloantibodies, excluding anti-D, -CD and -DE (Table 21.9). Rh antibodies, mainly anti-c or anti-E, accounted for 53% of the total and anti-K and anti-Fy^a accounted for a further 38%, leaving only about 9% for all other specificities. A similar distribution of the different red cell antibodies was found in a smaller group of patients who experienced immediate haemolytic transfusion reactions (HTR). However, the figures for delayed HTR showed a striking increase in the relative frequency of Jk antibodies, which reflects the outlined characteristics of Jk antibodies.

Table 21.9 Relative frequency of immune red cell alloantibodies*

Haemolytic disease of the fetus and newborn has not been associated with antibodies directed against Lewis antigens and only very mild disease is produced by anti-Lu^a and anti-Lu^b. With these exceptions, all other IgG antibodies directed against antigens in the systems mentioned should be considered capable of causing haemolysis in this setting.

The significance of the many other blood group antigens not referred to in the text is summarized in Table 21.10. However, it should be noted that the antibodies listed are usually wholly or predominantly IgG and would be detectable in routine pretransfusion testing using the indirect anti-globulin test (IAT).

Table 21.10 ‘Minor’ blood group antigens

It is difficult to find suitable blood for transfusion to a patient whose plasma contains an antibody, such as anti-Vel, which has a specificity for a high-frequency antigen and which can cause severe haemolytic transfusion reactions. In addition to using blood from a frozen blood bank and calling up rare phenotype donors, autologous blood could be considered for planned elective procedures and if necessary, the compatibility of red cells from close relatives (particularly siblings) can be investigated. Antibodies such as anti-Kn^a are commonly found and not clinically important, but their presence may cause delay in the provision of blood until their specificity has been determined.

Mechanisms of Immune Destruction of Red Cells

Immune-mediated haemolysis of red cells depends on the following:¹⁸

1. The immunoglobulin class of the antibody (for all practical purposes, antibodies directed against red cell antigens are either IgM or IgG or both)

2. The ability of the antibody to bind complement.

3. Interaction with the reticuloendothelial system (mononuclear phagocytic system). The most important phagocyte participating in immune haemolysis is the macrophage, predominantly in the spleen.

The mechanism of immune haemolysis also determines the site of haemolysis:

a. Intravascular haemolysis owing to sequential binding of complement components (C1 to C9) cascade and the formation of the membrane attack complex (MAC; C5b678(9)_n). This is characteristic of IgM antibodies, but some IgG antibodies can also act as haemolysins. Red cells are usually destroyed by intravascular complement lysis in ABO incompatible transfusion reactions (see p. 543). Most other alloimmune red cell destruction is extravascular and mediated by the mononuclear-phagocytic system.

Red cell autoantibodies may also cause intravascular lysis, especially the IgG autoantibody of PCH (see p. 277) and some autoantibodies of the cold haemagglutinin disease (CHAD) (see p. 277). Complement-mediated intravascular lysis may also occur in drug-induced immune haemolysis (see p. 289).

b. Extravascular haemolysis by the mononuclear phagocytic system is characteristic of IgG antibodies and occurs predominantly in the spleen. This is caused by non-complement-binding IgG antibodies or those that bind sublytic amounts of complement. Macrophages have Fcγ receptors for cell-bound IgG and sensitized red cells may be wholly phagocytosed or lose part of the membrane and return to the circulation as microspherocytes Spherocytes are less deformable and more readily trapped in the spleen than normal red cells; this shortens their lifespan. In addition to Fc receptor-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC) may also contribute to cell damage during the close contact with splenic macrophages. Red cells are destroyed external to the monocyte membrane by lysosomal enzymes secreted by the monocyte.¹⁹

Complement components may enhance red cell destruction. Complement activation by some IgM and most IgG antibodies is not always complete and the red cell escapes intravascular lysis. The activation of complement stops at the C3 stage and, in these circumstances, complement can be detected on the red cell by the antiglobulin test using appropriate anticomplement reagents. The first activation product of C3 is membrane-bound C3b, which is constantly being broken down to C3bi. Red cells with these components on their surface adhere to phagocytes (monocytes, macrophages and neutrophils), which have complement receptors, CR1 (CD35) and CR3 (CD 11b/CD 18). These sensitized cells are rapidly sequestered in the liver because of its bulk of phagocytic cells (Küpffer cells) and large blood flow, but no engulfment occurs. When C3bi is cleaved, leaving only C3dg on the cell surface, the cells tagged with ‘inactive’ C3dg return to the circulation, as in chronic cold haemagglutinin disease. However, when IgG is also present on the cell surface, C3b enhances phagocytosis and under these circumstances both liver and spleen are important sites of extravascular haemolysis. Hence, C3b and C3bi augment macrophage-mediated clearance of IgG-coated cells and antibodies binding sublytic amounts of complement (e.g. Duffy and Kidd antibodies) often cause more rapid destruction and more marked symptoms than non-complement binding antibodies (e.g. Rh antibodies).

Macrophage activity is an important component of cell destruction and further study of cellular interactions at this stage of immune haemolysis may provide an explanation for the differing severity of haemolysis in patients with apparently similar antibodies. In vitro macrophage (monocyte) assays have been used sometimes to supplement conventional serological techniques to assess this aspect of immune haemolysis.²⁰

Factors that may affect the interaction between sensitized cells and macrophages include the following:

1. IgG subclass. IgG1 and IgG3 antibodies have a higher binding affinity to mononuclear Fcγ receptors than IgG2 and IgG4 antibodies.

2. Antigen density. This affects the number of antibody molecules bound to the cell surface.

3. Fluid-phase IgG. Serum IgG concentration is a determinant of Fc-dependent mononuclear-phagocytic function. Normal levels of IgG block the adherence of sensitized red cells to monocyte Fc receptors (particularly FcγR1) in vitro. Haemoconcentration within the splenic sinusoids is probably a major factor in minimizing this effect in vivo, which may explain why the spleen is about 100 times more efficient at removing IgG-sensitized cells than the liver despite the greater macrophage mass and higher blood flow of the latter organ.

The initial effect of high-dose intravenous IgG is to cause blockade of macrophage FcγR. This reduces the immune clearance of antibody-coated cells and has particular application in the management of autoimmune thrombocytopenia and post-transfusion purpura.

4. Regulation of macrophage activity. Cytokines are known to be important in the upregulation of macrophage receptors. Interferon gamma enhances macrophage phagocytic activity by increasing the expression of FcγRI in vitro and in vivo and also activates FcγII without increasing the number of these receptors.²¹

Interleukin-6 also enhances FcγRII activation and increased activity of the CR1 receptor occurs through the action of T-cell cytokines and through chemotactic agents released in the inflammatory response.²² The increased levels of proinflammatory cytokines and other biological mediators and their effects on the activity of the monocyte phagocytic system have been monitored in patients with systemic inflammatory response syndrome.²³ It is therefore possible that release of cytokines during viral and bacterial infections could, at least in part, trigger some episodes of autoimmune cell destruction.

The rate of immune destruction is therefore determined by antigen and antibody characteristics and the level of activation of the monocyte phagocytic system.

Antigen–Antibody Reactions

The red cell is a convenient marker for serological reactions. Agglutination or lysis (owing to complement action) is a visible indication (endpoint) of an antigen–antibody reaction. The reaction occurs in two stages: in the first stage the antibody binds to the red cell antigen (sensitization) and the second stage involves agglutination (or lysis) of the sensitized cells.

The first stage (i.e. association of antibody with antigen – sensitization) is reversible and the strength of binding (equilibrium constant) depends on the ‘exactness of fit’ between antigen and antibody. This is influenced by the following:

1. Temperature. Cold antibodies (usually IgM) generally bind best to the red cell at a low temperature (e.g. 4°C), whereas warm antibodies (usually IgG) bind most efficiently at body temperature (i.e. 37°C).

2. pH. There is relatively little change in antibody binding over the pH range 5.5–8.5, but to ensure comparable results, it is preferable to buffer the saline in which serum or cells are diluted to a fixed pH, usually 7.0. Some antibody elution techniques depend on altering the pH to <4 or >10.

3. Ionic strength of the medium. Low ionic strength increases the rate of antibody binding. This is the basis of antibody detection tests using low ionic strength saline (LISS).

The second stage depends on various laboratory manipulations to promote agglutination or lysis of sensitized cells. The cell surface is negatively charged (mainly owing to sialic acid residues), which keeps individual cells apart; the minimum distance between red cells suspended in saline is about 18 nm. Agglutination is brought about by antibody crosslinking between cells. The span between antigen-binding sites on IgM molecules (30 nm) is sufficient to allow IgM antibodies to bridge between saline-suspended red cells (after settling) and so cause agglutination. IgG molecules have a shorter span (15 nm) and are usually unable to agglutinate sensitized red cells suspended in saline; notwithstanding this, heavy IgG sensitization owing to high-antigen density lowers intercellular repulsive forces and is able to promote agglutination in saline (e.g. IgG anti-A, anti-B). The agglutination of red cells coated by either IgM or IgG antibodies is enhanced by centrifugation. However, it is standard procedure to promote agglutination of IgG-sensitized red cells by the following:

1. Reducing intercellular distance by pretreatment of red cells with protease enzymes (e.g. papain or bromelin), which reduce the surface charge of red cells (see p. 498)

2. Adding polymers (e.g. albumin), although the mechanism by which albumin or other water-soluble polymers enhance agglutination is uncertain

3. Bridging between sensitized cells with an anti-globulin reagent in the antiglobulin test (see p. 500).

Some complement-binding antibodies (especially IgM) may cause lysis in vitro (without noticeable agglutination), which can be enhanced by the addition of fresh serum as a source of complement. However, complement activation may only proceed to the C3 stage; in these circumstances cell-bound C3 can be detected by the antiglobulin test using an appropriate anticomplement reagent (see p. 500).

Quality Assurance within the Laboratory

It has long been appreciated that the test systems used for routine pre-transfusion testing are of the utmost importance because errors can and do lead to patient morbidity and mortality. It is therefore of little surprise that within the European Union all reagents, calibrators and control materials for red cell typing and for determining the presence of ‘irregular anti-erythrocytic antibodies’ have been included under the In-vitro Diagnostics (IVD) Medical Devices Directive ²⁴ (see p. 588). This means that all reagents sold within the European Union must display the CE mark to show that they conform to the agreed Common Technical Specifications (CTS). In each European country, a Competent Authority will be able to withdraw or suspend certification of any reagent, depending on the information received from its Notified Body, which will perform batch release approval and monitor the performance of the manufacturer and the product.

The arrival of this Directive further reinforces the potential liabilities of an individual laboratory, which takes on the product liability of a manufacturer if reagents are made ‘in-house’ or if the manufacturer’s recommended method is not strictly adhered to.

The majority of the following points are taken from the British Committee for Standards in Haematology (BCSH) guidelines ²⁵ for pre-transfusion compatibility testing:

1. General aspects

a. The laboratory should document its Quality System, appropriate to its requirements.

b. Attention should be given to the sensible inclusion of internal controls in all the tests undertaken.

c. The laboratory should participate in External Quality Assessment exercises.

d. The laboratory should only make use of systems that have been validated against its documented requirements.

e. The laboratory should ensure that they have procedures to cover the failure of automated equipment and computer(s). The laboratory should develop procedures to build in checks for all critical points in transfusion testing (e.g. preserving the identity of patient samples, transcribing results).

2. Reagents

a. The head of the laboratory should refer to available specifications for reagents given by, for example, the International Society of Blood Transfusion (ISBT), the American Association of Blood Banks (AABB) or the Guidelines for the Blood Transfusion Services.^26,²⁷

b. All reagents or systems should be used in accordance with the manufacturer’s instructions. Where this is not possible, the procedure should be validated in accordance with the BCSH Guidelines on evaluation, validation and implementation of new techniques for blood grouping, antibody screening and crossmatching.²⁸

c. There should be a record of all batch numbers and expiry dates of all reagents used in the laboratory.

3. Techniques

a. All procedures used should be in accordance with recommended practice as outlined here.

b. It is essential that the antiglobulin technique chosen has been validated against the documented requirements of the laboratory and has been subjected to a thorough field trial before being introduced into the laboratory.²⁹

c. All changes in techniques must be thoroughly validated in accordance with the BCSH Guidelines on evaluation, validation and implementation of new techniques before being introduced into routine use.

d. Written authorized standard operating procedures (SOPs) which cover all aspects of the laboratory work must be available and reviewed regularly.

e. The regular checking and maintenance of all laboratory equipment must be documented. In particular, there should be a documented quality-assurance procedure for cell washers (e.g. using the National Institute of Biological Standards and Control, NIBSC anti-D standard)³⁰.

4. Staff training and proficiency

a. There should be a documented programme for training laboratory staff, which covers all SOPs in use and which fulfils the documented requirements of the laboratory.

b. Laboratory tasks should only be undertaken by appropriately trained staff.

c. There must be a documented programme for assessing staff proficiency (e.g. replicate testing for the IAT), which should include details of the action limits for retraining.³¹

5. Auditing and reviewing practice

a. There should be a system in place for documenting and reviewing all incidents of non-compliance with procedures.

b. The systems should enable a full audit trail of laboratory steps, including the original results, interpretations, authorizations and all staff responsible for conducting each step.

c. A programme of independent audits should be conducted to assess compliance with documented ‘in-house’ procedures.

6. Health and safety

When appropriate, reagents should have been screened for human immunodeficiency virus and hepatitis B and C virus. All high-risk samples must be handled in accordance with the laboratory safety code (see Chapter 25).

General Points of Serological Technique

Serum versus Plasma

Serum is preferred to plasma for the detection of red cell alloantibodies. Nevertheless, plasma is being used increasingly for convenience in microplate technology and in automated systems.

When plasma is used, complement is inactivated by the ethylenediaminetetra-acetic acid (EDTA) anticoagulant. This is relevant for the detection of some complement-binding antibodies (e.g. of Kidd specificity) that may be missed or give only weak reactions with anti-IgG in the routine antiglobulin test but can be readily detected by anticomplement (see p. 500). It is therefore essential, before using plasma, to optimize the sensitivity of techniques for detecting weak IgG antibodies and to validate the procedure (see p. 500). For example, in antibody screening, increased sensitivity can be achieved by using panel cells with homozygous expression of selected antigens (see p. 528).

Collection and Storage of Blood Samples

Positive identification of the patient and careful labelling of blood samples are essential to avoid misidentification errors. Venous blood is desirable for blood-grouping purposes and 5–10 ml of blood should be taken and either allowed to clot at room temperature or anticoagulated with EDTA in a sterile plastic tube. This will provide serum or plasma and red cells. If serum is required urgently, the specimen may be placed in a 37°C waterbath and centrifuged as soon as the clot can be seen to have started to retract.

Storage of Sera or Plasma

Great care must be taken to identify and label correctly any serum or plasma separated from the patient’s original sample.

Whole blood samples will deteriorate over time. Problems associated with storage include red cell lysis; loss of complement in the serum; decrease in potency of red cell antibodies, particularly IgM antibodies; and bacterial contamination. However, in the absence of evidence, it has been suggested that whole blood can be stored at room temperature for up to 48 h and up to 7 days at 4°C. It has also been recommended that laboratories evaluate the stability of weak antibodies before making local decisions for storage conditions. Patient’s serum or plasma is best stored frozen at −20°C or below in 1–2 ml volumes in plastic vials. Repeated thawing of a sample is harmful. If the sera are stored at −20°C or below, no precautions are necessary with respect to sterility. Complement deteriorates rapidly on storage, but sera separated from blood as quickly as possible and stored at −20°C retain most of their complement activity for 1–2 weeks. For compatibility tests, samples of serum should be separated from the red cells as soon as possible and stored at −20°C until used because the content of complement may be important for the detection of some antibodies.

Red Cell Suspensions

Normal ionic strength saline

A 2–3% suspension of washed red cells in phosphate buffered saline (PBS), pH 7.0, is generally recommended. Cells suspended in normal ionic strength saline (NISS) are routinely used for antibody titrations, but their use in routine pretransfusion testing has declined over the last decade as observations from external quality assessment exercises have demonstrated that laboratories using NISS have a significantly lower detection rate of antibodies than those using other technologies.³²

Low ionic strength saline

It is known that the rate of association of antibodies with red cell antigens is enhanced by lowering the ionic strength of the medium in which the reactions take place. Hence, a major advantage of low ionic strength saline (LISS) is that the incubation period in the IAT (see p. 529) can be shortened while maintaining or increasing sensitivity to the majority of red cell antibodies. The LISS solution can be made up in the laboratory (see p. 620) or purchased commercially.

There was historical reluctance to use low ionic strength media in routine laboratory work for two reasons: first, nonspecific agglutination may occur when NaCl concentrations <2 g/l (0.03 mol/l) are used and second, complement components are bound to the red cells at low ionic strengths.

To avoid false-positive results, the following rules should be followed:

a. Red cells resuspended in LISS and serum or plasma should be incubated together in equal volumes: 2 volumes of cells to 2 volumes of serum are recommended to ensure the optimal molarity in the test of the order of 0.09 mol. Doubling the serum to cell ratio (by halving the cell concentration from 3% to 1.5%) will enhance the detection of some antibodies (e.g. anti-K) that might otherwise be missed.³³

b. The red cells should be washed in saline twice and then once in LISS before suspending in LISS at 1.5–2% cell suspension.

c. The working solution of LISS should be freshly made and kept at room temperature.

d. Centrifugation force and time should be optimal to give maximum sensitivity with freedom from false-positive or false-negative reactions (see p. 501).

False-positive reactions may still infrequently occur with some sera/plasma. If plasma is used, subsequent serological work may be performed using NISS; if serum is used, anti-IgG should replace the polyspecific antiglobulin reagent.

Reagent Red Cells

Red cells of selected phenotypes are required for ABO and RhD grouping, Rh phenotyping and antibody screening and identification (see Chapter 20). Such cells are available commercially or from blood transfusion centres.

Use of Enzyme-Treated Cells

Enzyme-treated red cells are useful reagents in the detection and investigation of autoantibodies and alloantibodies. Papain and bromelin are currently used for this purpose. Enzyme treatment is known to increase the avidity of both IgM and IgG antibodies. The receptors of some red cell antigens, however, may be inactivated by enzyme treatment (e.g. M, N, S, Fy^b).

The most sensitive techniques are those using washed enzyme-pretreated red cells (two-stage), which should match the performance of the spin-tube LISS antiglobulin test (see p. 501) One-stage mixtures and papain inhibitor techniques are relatively insensitive and are not recommended. An ISBT/International Council for Standardization in Haematology (ICSH) protease enzyme standard and an agreed method for its use are available.

Methods for the preparation of papain solution (Low’s method and the two-stage method) and for preparation of bromelin solution have been described.³⁴^–³⁶

Agglutination of Red Cells by Antibody: A Basic Method

Agglutination tests are usually carried out in tubes, microtitre plates or column agglutination (gel) technology, using centrifugation or sedimentation. Slide tests are rarely used for emergency ABO and D grouping (see p. 524). For microplate tests, see p. 525.

Tube Tests

Add 1 volume of a 2% red cell suspension to 2–3 volumes of plasma in a disposable plastic tube. Mix well and leave undisturbed for the appropriate time (see later).

Tubes

For agglutination tests, use medium-sized (75 × 10 or 12 mm) disposable plastic tubes. Similar tubes should be used for lysis tests when it is essential to have a relatively deep layer of serum to look through, if small amounts of lysis are to be detected. The level of the fluid must rise much higher than the concave bottom of the tubes.

Glass tubes should always be used if the contents are to be heated to 50°C or higher or if organic solvents are being used. Glass tubes, however, are difficult to clean satisfactorily, particularly small-bore tubes and cleaning methods such as those given on p. 623 should be followed carefully.

Temperature and Time of Exposure of Red Cells to Antibody

In blood group serology, tube tests are generally done at 37°C, room temperature or both. There is some advantage in using a 20°C waterbath rather than relying on ‘room temperature’, which in different countries and seasons may vary from 15°C (or less) to 30°C (or more).

Sedimentation tube tests are usually read after 1–2 h have elapsed. Strong agglutination will, however, be obvious much sooner than this. In spin-tube tests, agglutination can be read after only 5–10 min incubation if the cell–plasma mixture is centrifuged.

Slide Tests

These are used rarely in a few parts of the world. Because of evaporation, slide tests must be read within about 5 min. Reagents that produce strong agglutination within 1–2 min are normally used for rapid ABO and RhD grouping. Because the results are read macroscopically, strong cell suspensions should be used (35–45% cells in their own serum or plasma)

Reading Results of Tube Tests

Only the strongest complete (C) grade of agglutination seems to be able to withstand a shake procedure without some degree of disruption, which may downgrade the strength of reaction. The BCSH Blood Transfusion Task Force has therefore recommended the following reading procedure.³⁷

Microscopic reading

It is essential that a careful and standardized technique be followed. Lift the tube carefully from its rack without disturbing the button of sedimented cells. Holding the tube vertically, introduce a straight-tipped Pasteur pipette. Carefully draw up a column of supernatant about 1 cm in length and then, without introducing an air bubble, draw up a 1–2 mm column of red cells by placing the tip of the pipette in the button of red cells. Gently expel the supernatant and cells onto a slide over an area of about 2 × 1 cm. It is important not to overload the suspension with cells and the method described earlier achieves this.

A scheme of scoring the results is given in Table 21.11.

Table 21.11 Scoring of results in red cell agglutination tests

Symbol	Agglutination Score*	Description
4+ or C (complete)	12	Cell button remains in one clump, macroscopically visible
3+	10	Cell button dislodges into several large clumps, macroscopically visible
2+	8	Cell button dislodges into many small clumps, macroscopically visible
1+	5	Cell button dislodges into finely granular clumps, macroscopically just visible
(+) or w (weak)	3	Cell button dislodges into fine granules, only visible microscopically^†
−	0	Negative result – all cells free and evenly distributed

* Titration scores are the summation of the agglutination scores at each dilution.

† May be further classified depending on the number of cells in the clumps (e.g. clumps of 12–20 cells [score 3]; 8–10 cells [score 2]; 4–6 cells [score 1]. This is the minimum agglutination that should be considered positive.

Macroscopic reading

A gentle agitation tip-and-roll ‘macroscopic’ method is recommended. It is possible to read agglutination tests macroscopically with the aid of a hand reading glass or concave mirror, but it is then difficult to distinguish reactions weaker than + (microscopic reading) from the normal slight granular appearance of unagglutinated red cells in suspension. Macroscopic reading thus gives lower titration values than does microscopic reading, but the former is recommended. Follow the system of scoring in Table 21.11.

A good idea of the presence or absence of agglutination can often be obtained by inspection of the deposit of sedimented cells: a perfectly smooth round button suggests no agglutination, whereas agglutination is shown by varying degrees of irregularity, ‘graininess’, or dispersion of the deposit (Fig. 21.2).

Figure 21.2 Macroscopic appearances of agglutination in round-bottom tubes or hollow tiles. Agglutination is shown by various degrees of ‘graininess’; in the absence of agglutination, the sedimented cells appear as a smooth round button, as on the extreme right.

Demonstration of Lysis

Many blood-group antibodies lyse red cells under suitable conditions in the presence of complement. This is particularly true of anti-A and anti-B, anti-P, anti-Le^a and Le^b, anti-PP₁P^k (anti-Tj^a) and certain autoantibodies (see p. 284). If it is necessary to add fresh complement, this should be mixed with the serum being tested before the addition of red cells. Otherwise, agglutination occurs and could block complement access. Lysis should be looked for at the end of the incubation period before the tubes are centrifuged, if the cells have sedimented sufficiently; lysis may be scored semiquantitatively after centrifuging the suspensions and comparing the colour of the supernatant with that of the control.

If the occurrence of lysis is of interest, then the final volume of the cell–serum suspension has to be greater than is required for the reading of agglutination. Tubes (75 × 10 or 12 mm) should be used and the level of the cell–serum suspension must rise much higher than the concave bottom of the tubes.

In testing for lytic activity, a high concentration of complement may be required. Therefore, in contrast to tests for agglutination, it is advantageous to use a stronger red cell suspension (c5%).

Lysis tests are usually carried out at 37°C, but with cold antibodies a lower temperature (e.g. 20°C or 30°C) would be appropriate, depending on the upper thermal range of activity of the antibody or, in the case of the Donath–Landsteiner antibody, 0°C followed by 37°C (see p. 277).

With certain antibodies the pH of the cell–serum suspension affects the occurrence of lysis. In these, optimal pH is 6.5–6.8.

Controls

It is necessary to be sure that any lysis observed is not artefactual (i.e. that lysis is brought about by the serum under test and not by the serum added as complement) and that the added complement is potent. A complement control (no test serum) is thus necessary, as is a control using a serum known to contain a lytic antibody.

In lysis tests, great care should be taken to deliver the cell suspension directly into the serum. If the cell suspension comes into contact with the side of the tube and starts to dry, this in itself will lead to lysis.

Antiglobulin Test

The antiglobulin test (Coombs test) was introduced by Coombs and colleagues in 1945 ³⁸ as a method for detecting ‘incomplete’ Rh antibodies (i.e. IgG antibodies capable of sensitizing red cells but incapable of causing agglutination of the same cells suspended in saline), as opposed to ‘complete’ IgM antibodies, which do agglutinate saline-suspended red cells.

Direct and indirect antiglobulin tests can be carried out. In the direct antiglobulin test (DAT), the patient’s cells, after careful washing, are tested for sensitization that has occurred in vivo; in the indirect antiglobulin test (IAT), normal red cells are incubated with a serum suspected of containing an antibody and subsequently tested, after washing, for in vitro-bound antibody.

The antiglobulin test is probably the most important test in the serologist’s repertoire. The DAT is used to demonstrate in vivo attachment of antibodies to red cells, as in autoimmune haemolytic anaemia (see p. 275), alloimmune HDN (see p. 535) and alloimmune haemolysis following an incompatible transfusion (see p. 542). The IAT has wide application in blood transfusion serology, including antibody screening and identification and crossmatching.

Antiglobulin Reagents

Polyspecific (broad-spectrum) reagents

The majority of red cell antibodies are non-complement-binding IgG; anti-IgG is therefore an essential component of any polyspecific reagent. Anti-IgA is not required because IgG antibodies of the same specificity almost always occur in the presence of IgA antibodies. Anti-IgM is also not required because clinically significant IgM alloantibodies that do not cause agglutination in saline are much more easily detected by the complement they bind.

Anticomplement has also traditionally been considered essential – namely, anti-C3c and anti-C3d. However, if plasma is used, only anti-IgG is necessary because EDTA prevents complement activation. In addition, it seems that most, if not all, antibodies detected by the C3-anti-C3 reaction in normal ionic strength tests can be detected with anti-IgG in polybrene, polyethylene glycol (PEG) and LISS. Laboratories using techniques other than NISS have adopted the use of anti-IgG alone, supported by changes to guidelines from the AABB in 1990 and from the BCSH in 1996. Nevertheless, the BCSH guidelines stress the importance of having screening cells with homozygous expression of Jk^a before deciding to use anti-IgG rather than a polyspecific antiglobulin reagent. Anti-C3 will certainly be required for DATs for the diagnosis of autoimmune haemolytic anaemia.

Monospecific reagents

Monospecific reagents can be prepared against the heavy chains of IgG, IgM and IgA and are referred to as anti-γ, anti-μ and anti-α; antibodies against IgG subclasses are also available. Specific antibodies against the complement components C4 and C3 and C3 breakdown products can be prepared as mentioned earlier.

The main clinical application of these monospecific reagents is to define the immunochemical characteristics of antibodies. This is relevant to the mechanisms of in vivo cell destruction and, in the case of IgG, the subclasses have different biological properties (see p. 494).

Quality Control of Antiglobulin Reagents

This is not commonly done in UK hospital laboratories, as they use commercial antiglobulin reagents. The quality control of antiglobulin reagents must always be carried out by the exact technique by which they are to be used. All reagents should be used according to the manufacturer’s instructions, unless appropriately standardized for other methods.

An ISBT/ICSH freeze-dried reference reagent is available for evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components.²⁶ The validation of a new antiglobulin reagent should assess the following qualities of the reagent:

1. Specificity. The reagent should only agglutinate red cells sensitized with antibodies and/or coated with significant levels of complement components

2. Potency of anti-IgG by serological titration

3. Specificity and potency of anticomplement antibodies. A polyspecific reagent should contain anti-C3c and anti-C3d at controlled levels to avoid false-positive reactions or a suitable potent monoclonal anti-C3d (e.g. BRIC-8). It should contain little or no anti-C4. The assessment of these qualities requires red cells specifically coated with C3b, Cb3i, C3d and C4. Details of the procedures recommended for the preparation of such cells have been published by an ISBT/ICSH Working Party.³⁹

It is appreciated that some hospital blood banks worldwide will be unable to evaluate an antiglobulin reagent as comprehensively as outlined earlier. They should, however, carry out the following minimal assessment of all new antiglobulin reagents:

1. Test the antiglobulin reagent for freedom from false-positive results by simulated crossmatch tests:

a. Test for excess anti-C3d by incubating fresh serum at 37°C by NISS or LISS tests with 6 ABO-compatible cells from CPD-A1 donor unit segments (10–30 days old). This is a critical test for false-positive results owing to C3d uptake by stored blood, which is further augmented by incubation with fresh serum

b. Tests for contaminating red cell antibodies (against washed A₁, B and O cells) must be negative.

Only proceed further if the antiglobulin reagent passes the previously listed tests.

2. Compare the antiglobulin reagent with the current reagent using a selection of weak antibodies. These antibodies may be selected from those encountered in routine work or can be obtained from a transfusion centre or reference laboratory. Store such antibodies in small volumes at 4°C for repeated tests.

3. Dilute a weak IgG anti-D (0.8 iu/ml), as used for routine antiglobulin test controls, from undiluted (neat) to 1 in 16 and sensitize R₁r red cells with each dilution of anti-D. These sensitized cells (washed four times) should then be tested with neat to 1 in 8 dilutions of the antiglobulin reagents. The antiglobulin reagent should not show prozones by immediate spin tests using 2 volumes of antiglobulin per test. The potency of the test antiglobulin should at least match the current antiglobulin reagent.

The ISBT/ICSH antiglobulin reference reagent can be used to calibrate an ‘in-house’ antiglobulin reagent for use as a routine standard.

The quality control of Ig class and subclass specific antiglobulin reagents, although following the previously listed general principles, is more complex. Details of the appropriate techniques are beyond the scope of this chapter; the reader should consult the review by Engelfriet et al.⁴⁰

Recommended Antiglobulin Test Procedure

A spin-tube technique is recommended for the routine antiglobulin test; the procedure described here is based on BCSH Guidelines for Compatibility Testing in Hospital Blood Banks.^27,³⁷ Reliable performance depends on the correct procedure at each stage of the test and appropriate quality-control measures.

The test is preferably carried out in glass tubes (75 × 10 or 12 mm), as plastic tubes may adsorb IgG, which could neutralize anti-IgG of the antiglobulin reagent.

1. Sensitize red cells (not relevant to the direct test) by using the following serum:cell ratios:

a. For NISS, use at least 2 volumes of serum (preferably 4) and 1 volume of a 3% suspension of red cells washed (3 times) and suspended in PBS or 0.15 mol/l NaCl (see p. 621).

b. For LISS, use 2 volumes of serum and 2 volumes of a 1.5% suspension of red cells washed twice in PBS or 0.15 mol/l NaCl and washed once in LISS and then suspended in LISS (see p. 621).

c. For commercial low ionic strength additive solutions, the manufacturer’s instructions must be followed.

Because the volume of ‘a drop’ varies according to the type of pipette or dropper bottle, a measured or known drop volume should be used to ensure that appropriate serum:cell ratios are maintained.

Mix the reactants by shaking, then incubate at 37°C, preferably in a waterbath, for a minimum period of 15 min for LISS tests and 45 min for NISS tests.

2. Wash the test cells four times with a minimum of 3 ml of saline per wash. Vigorous injection of saline is necessary to resuspend the cells and achieve adequate mixing. As much of the supernatant as possible should be removed after each wash to achieve maximum dilution of residual serum.

3. Add 2 volumes of a suitable antiglobulin reagent to each test tube and centrifuge without delay after thorough mixing. The combinations of centrifugal force (RCF) and time for spin-tube tests are as follows:

4. Read agglutination as previously described (see p. 498).

5. Quality control of the test should be monitored by the following:

a. An IgG anti-D diluted to give 1+ or 2+ reactions with RhD-positive (R₁r) cells as a positive control.

b. An inert group AB serum with the same RhD-positive cells as a negative control; this is not essential because most tests are negative.

c. The addition of sensitized cells to all negative tests. This is widely used to detect neutralization of the antiglobulin reagent owing to incomplete removal of serum by the wash step. The value of this test as a control depends on the strength of reaction of the sensitized cells. Appropriate control cells sensitized with IgG anti-D should give a 3+ reaction when tested directly with the antiglobulin reagent and should still be positive (if the reagent is potent) when added to negative tests but downgraded (1+ or 2+) owing to the ‘pooled-cell’ effect of the non-sensitized cells. The reaction will, of course, be negative if the antiglobulin has been neutralized by residual serum.

The production of satisfactory antiglobulin control cells can be achieved by limiting the level of anti-D sensitization to that which gives a negative test in the presence of 1 in 1000 parts serum in saline.³⁷

The suitability of the antiglobulin control cells can be checked as follows:

i. Prepare two tubes (10 × 75 mm) with 1 volume of 3% unsensitized cells; wash four times.

ii. Add 2 volumes of antiglobulin to each of the tubes, mix well, spin and read the tubes to confirm the tests are negative.

iii. Add 1 volume of 1 in 1000 serum in saline to one tube and 1 volume of saline as a control to the other tube. Mix and incubate for 1 min at room temperature.

iv. Add 1 volume of control cells to each tube, mix, spin and read the tests.

The test containing 1 in 1000 serum in saline should be negative and the control tube should give at least 2+ reaction. A negative reaction with the control tube suggests a washing deficiency and demands corrective action. If an automated cell-washing centrifuge is used, the washing efficiency should be checked.^30,³⁷

Alternative Technology for Antibody Detection by the Antiglobulin Test

Alternative techniques, now commonly in use, have a simpler reading phase than the manually read spin-tube IAT. These are of two main types: solid-phase red cell adherence methods ⁴¹ and column agglutination techniques. A well-performed spin-tube IAT, as described earlier, is the standard against which any new system should be compared.

Solid-phase red cell adherence methods involve systems in which known red cells, which may also be sensitized, are immobilized on a solid matrix. In the method referenced, ABO and D typing plates are prepared by immobilizing A₁-, B- and D-positive red cells to chemically modified U-bottom strips. The cells are then exposed to the appropriate antibody and the sensitized red cell monolayers are then dried. The unknown test cells are added and the plates are centrifuged after incubation. In a positive reaction, the cells spread over the surface of the well because they have adhered to the bound antibody. In a negative reaction, there is no adherence and the cells form a small button in the centre of the well when the plates are centrifuged.

For reverse typing and antibody screening, A₁, B and O screening cell monolayers are prepared and dried. The test serum is added and, if antibodies to any of the immobilized antigens are present, they attach to the monolayer. The tests are read by the addition of A₁B cells that are coated with anti-IgG.

Solid-phase methods are highly suited for automated reading by passing a light beam through the well at a point at which it will not be interrupted by the button of cells in a negative test but will be dispersed by the layer of red cells spread across the well in a positive test.

With column agglutination techniques very small volumes of serum and cells are mixed in a reservoir at the top of a narrow column that contains either a Dextran gel (DiaMed, AG, Switzerland) or glass beads (Bio Vue, Ortho-Clinical Diagnosis, NJ).⁴² The columns with the integral reservoirs are supplied in card or cassette form, respectively. After a suitable incubation period, the cards/cassettes containing the tests are spun in a centrifuge in which the axis of the column is strictly in line with the centrifugal force. The red cells, but not the medium in which they are suspended, enter the column. Agglutinated red cells are trapped at the top of the column and unagglutinated red cells form a pellet at the bottom of the column (see Fig. 22.6, see p. 526).

The columns can also contain an antiglobulin reagent for performing DATs or IATs. Because, during centrifugation, the red cells but not the suspending fluid pass through the gel, the red cells do not have to be washed before coming into contact with the antiglobulin reagent. The columns can also include an antibody (e.g. anti-D) for cell typing. Antigen positive cells are agglutinated and trapped in the upper portion of the column.

The advantages of column agglutination technology are as follows:

1. Ease of use and reading and can theoretically be performed by relatively unskilled staff.

2. There is less chance of aerosol contamination from infected samples because of no cell washing before IATs.

3. The cards can be kept for up to 24 h, enabling the results to be reviewed by experienced staff.

4. Ease of automation and positive sample identification.

However, the technology is relatively expensive and its performance does not always compare favourably with the standard LISS-IAT in experienced hands.

Assessment of Individual Worker Performance

The procedure is as follows:

1. A low-titre (8–16) IgG anti-D, as used for the control of the antiglobulin test, should be titrated against OR₁r or pooled O RhD-positive cells to find the dilution of anti-D that gives 1+ or 2+ sensitized cells (most workers use around 0.3 iu/ml). A standard BCSH-NIBSC anti-D reference reagent ⁴³ is available for this purpose (available from NIBSC).

2. A batch of sensitized cells is prepared (e.g. by incubating 16 ml of the selected anti-D dilution with 8 ml of 3% washed OR ₁ red cells at 37°C for 45 min).

3. Twelve tubes are labelled for blind tests by another person. One volume of 3% 1+ or 2+ sensitized cells and 2 volumes of group AB inert serum (to simulate the volumes of serum used in routine tests) are placed in 9 random tubes and then 1 volume of unsensitized cells + 2 volumes of group-AB inert serum are placed in the remaining tubes. The position of the various tests is recorded.

4. The cells are washed thoroughly four times, anti-globulin is added and the tubes are spun and read.

5. The number of false-negative (and false-positive) results are recorded for each worker and analysed in relation to reading and/or washing technique. It is advisable to give immediate tuition to any workers with washing or reading test faults, followed by further blind replicate trials to demonstrate improvement in procedure and to restore confidence.

Titration of Antibodies

A method for preparing primary dilutions of serum and subsequent antibody titration is illustrated in Figure 21.3.

Figure 21.3 Diagram illustrating method of preparing four sets of four-fold dilutions of a serum. The large circles at the top represent the large tubes in which the primary dilutions are made; the smaller circles represent the tubes in which the titrations are carried out. The figures represent drops or volumes. The patient’s neat serum is indicated by the bold type.

External quality assessment exercises have demonstrated the wide range of titres reported for a single sample, reflecting the differing sensitivities of technologies in use and have also highlighted the lack of reproducibility.⁴⁴ The following points are taken from an addendum to the BCSH guidelines.⁴⁵

Preparation of serial dilutions of patient’s or other sera

1. All dilutions and titrations should be made using calibrated pipettes and a separate tip for each step.

2. The diluent should be buffered saline, pH 7.0, for agglutination tests; for lysis tests undiluted ABO-compatible fresh normal human serum should be acidified so that the pH of the cell–serum mixture is c 6.8. The normal serum serves as a source of complement.

3. Tube sizes and assay volumes should be chosen to permit thorough mixing of the dilutions.

4. When assaying high-titre samples, an initial dilution should be made to reduce the number of doubling serial transfers to less than 10. A sufficient range of dilutions should be chosen to ensure that two negative results can be observed.

5. The endpoint should be macroscopic and well-defined. The use of visual comparator aids should be considered where possible.

6. Wherever possible, each sample should be tested in parallel with the previous sample.

7. Titrations should be repeated if there is more than a one-tube difference in the titres obtained from sequential samples.

Addition of red cell suspensions to dilutions of serum

It is conventional to add 1 volume of red cell suspension to 1 volume of serum or serum dilution. This means that each antibody dilution, and hence the ‘final’ titre, will be twice that of the original serum dilution. Because red cell antigen expression varies with the source and age of the sample, wherever possible, the same cell sample should be used.

Test for ABH Substance Secretion

In the majority of the population, substances with the appropriate A, B and H antigenic activity are distributed widely in saliva and all body fluids, controlled by a regulator secretor gene (Se), which is inherited independently of ABH genes. Only about 20% of people are non-secretors. Although rarely used, an individual’s secretor status can be determined by testing saliva.

Method

Dilute an anti-A or anti-B serum so that it gives good visible agglutination with A₂ or B cells at the end of 1 h at room temperature (e.g. if the titre of the serum is 128, use it at a dilution of 1 in 16).

Collect several millilitres of saliva in a centrifuge tube. Place the tube in boiling water for 10 min and then centrifuge. Serially dilute the clear supernatant in saline so as to give dilutions ranging from 1 in 2 to 1 in 32. Use a tube containing saliva alone as a control. Add an equal volume of the diluted anti-A (or anti-B) serum to each tube and, after shaking the rack of tubes, allow them to stand at room temperature for 10–15 min. Then add an equal volume of a 2% suspension of A₂ (or B) red cells in saline to each tube. Mix the contents and allow to stand at room temperature for 1–2 h; then inspect for agglutination. If the saliva contains A or B substances, agglutination is usually inhibited in all the tubes except the saline control tube.

H substance can be demonstrated in a similar way using an extract of Ulex, eel serum or the naturally occurring ‘incomplete’ cold antibody as the source of anti-H.

Red Cell Genotyping

Red cell genotyping is not currently in widespread use, but is emerging as a potential alternative to serological provision of compatible blood for patients in future and is currently used occasionally in situations when serological techniques prove difficult, e.g. phenotyping of a recently transfused patient or in a patient with autoimmune haemolytic anaemia.⁴⁶

Platelet and Neutrophils

Platelet and Neutrophil Alloantigen Systems

Platelet and neutrophil alloantigens may be exclusive to each cell type (cell-specific) or shared with other cells. The currently recognized human platelet antigens (HPA) and human neutrophil antigens (HNA) are shown in Tables 21.12–21.14.⁴⁷^–⁴⁹ The historical nomenclature for granulocyte antigens used the letter N to indicate neutrophil specificity and this has been retained, although it is recognized that many studies used granulocytes rather than pure neutrophils and many ‘neutrophil-specific’ antibodies can also target granulocyte precursors. In the HPA nomenclature, HPA-1, -2, -3, -4 and -5 were designated as separate diallelic alloantigen systems. The high-frequency allele of a system was designated with the letter ‘a’ and the low-frequency allele was designated with the letter ‘b’. However, this system is difficult to reconcile with recent molecular genetic knowledge, which suggests that each new base exchange does not constitute a new diallelic alloantigen system but rather defines a single allele that expresses a single new epitope. Currently, nine different GpIIIa alleles have been found in the human gene pool,⁵⁰ four allelic variants have been described for GpIa and GpIIb and two allelic variants have been found for the GpIbα and GpIbβ subunits⁵⁰ (a database of human platelet antigens is available at: www.ebi.ac.uk/ipd/hpa).⁵¹ Of the shared antigens, the HLA system is the most important clinically; only class 1 antigens (HLA-A, -B and to a lesser extent -C) are expressed on platelets and granulocytes. ABH antigens are also expressed on platelets (in part absorbed from the plasma) but cannot be demonstrated on granulocytes.

Table 21.12 Molecular genetics of human platelet antigens (HPA)

Table 21.13 Human platelet antigen frequencies (%) in different populations

Table 21.14 The human neutrophil antigen systems

Clinical Significance of Platelet and Neutrophil Antibodies

Platelet and neutrophil antibodies may be classified on the basis of the antigenic stimulus (e.g. allo-, iso-, auto- and drug-induced antibodies).

Alloantibodies

Alloimmunization to platelet and neutrophil antigens is most commonly a result of transfusion or pregnancy. The associated clinical problems depend on the specificity of the antibody, which determines the target cell involved. Cell-specific alloantibodies are associated with well-defined clinical conditions, which are summarized in Tables 21.15 and 21.16.

Table 21.15 Clinical significance of platelet-specific alloantibodies ⁵²

1.	Neonatal alloimmune thrombocytopenia
2.	Post-transfusion purpura
3.	Refractoriness to platelet transfusion Usually as a result of human leucocyte antigen antibodies.

Table 21.16 Clinical significance of neutrophil-specific alloantibodies ⁵³^–⁵⁵

1.	Neonatal alloimmune neutropenia
2.	Febrile reactions following transfusion (HLA antibodies also involved)
3.	Transfusion-related acute lung injury (TRALI) (transfusion of high-titre antibody)
4.	Poor survival and function of transfused neutrophils (HLA antibodies also involved)
5.	Autoimmune neutropenia – some autoantibodies have allospecificity for HNA system antigens.

HLA, human leucocyte antigen; HNA, human neutrophil antigen.

Alloimmune fetal and neonatal thrombocytopenia are commonly caused by anti-HPA-1a and less frequently by anti-HPA-5b. The chance of HPA-1a alloimmunization is strongly associated with maternal HLA class-II DRB3*0101 (DR52a) type.⁵⁶ Partners should be offered HPA genotyping and, if heterozygous, with a severely affected previous child, fetal HPA grouping should be considered in the first trimester of the next pregnancy using amniocyte DNA. Potential strategies for routine antenatal screening and the acceptability and cost-effectiveness of such a programme are discussed in several publications.^57,⁵⁸

Post-transfusion purpura is most commonly caused by anti-HPA-1a but can be associated with HPA antibodies with other specificities against HPA-1b, HPA-2b, HPA-3a, HPA-3b, HPA-4a and HPA-5b.⁵⁹

Immunological refractoriness to platelet transfusions is usually the result of anti-HLA antibodies. However, in multitransfused patients with HLA immunization, up to 25% may also have anti-HPA antibodies.^60,⁶¹

Isoantibodies

Rarely, after blood transfusion or pregnancy, patients with type I Glanzmann’s disease make antibodies that react with platelet glycoprotein (Gp) IIb/IIIa not present on their own platelets but present on normal platelets (i.e. isotypic determinants).⁶²^–⁶⁵ Similarly, patients with Bernard–Soulier syndrome may make antibodies against isotypic determinants on GpIbVIX not present on their own platelets.⁶⁶ This may present a serious clinical problem because no functional compatible donor platelets can be found to treat severe bleeding episodes.

Autoantibodies

Autoimmune thrombocytopenia may be idiopathic or secondary in association with other conditions. Demonstration of a platelet autoantibody is not required; even with the most suitable techniques now available, platelet autoantibodies remain elusive in a variable proportion (10–20%) of patients. The autoreactive antibodies target epitopes on certain glycoproteins. In 30–40% of patients these are directed against epitopes on the αIIbβ3 integrin heterodimer, platelet glycoprotein GpIIbIIIa (CD41) and in 30–40% against the von Willebrand receptor or complex GpIbα/GPIbβ/IX (CD42).⁶⁷^–⁷⁰

In the diagnosis of autoimmune thrombocytopenia it is important to consider and exclude three other immunological conditions:

1. Post-transfusion purpura (PTP). A blood transfusion within 2 weeks will suggest this possibility, although it is very rare in the UK since leucodepletion of blood components.^59,¹⁶

2. Drug-induced immune thrombocytopenia. A drug history is essential. Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced thrombocytopenia and can be confirmed by the demonstration of antibodies to the heparin/platelet factor 4 (PF4) complex by ELISA (enzyme-linked immunosorbent assay).⁷¹

3. Pseudothrombocytopenia. The patient has an EDTA-dependent platelet antibody that is active only in vitro. The antibody (IgG and/or IgM) reacts with hidden (cryptic) antigens on platelet GpIIbIIIa, which are exposed owing to confirmational changes in the complex caused by the removal of Ca²⁺ by EDTA.⁷² The antibody causes platelet agglutination in the EDTA blood sample associated with large platelet clumps on the blood film or platelet satellitism around neutrophils, both of which lead to a falsely low platelet count. To overcome this, blood should be taken into a tube containing citrate instead.

Autoimmune neutropenia may be idiopathic or secondary. Idiopathic autoimmune neutropenia is more common in infants than in adults, in whom it is usually associated with other disorders that have in common a postulated imbalance of the immune system.⁷³ However, it is the least well-studied of the autoimmune cytopenias because it is rare and performing granulocyte assays is difficult, lengthy, labour-intensive and expensive.

Neutrophil autoantibodies (which are usually IgG) are unusual in that they often have well-defined specificity for alloantigens, especially NA1 or NA2.⁷⁴ These autoantibodies may suppress granulocyte precursors in the bone marrow and cause more severe neutropenia. The investigation of suspected autoimmune neutropenia should, when possible, include granulocyte immunology and studies of colony growth (e.g. CFU-GM) to identify any suppression of bone marrow precursors, as a result of interaction with autoantibodies.

Drug-Induced Antibodies

Drug-induced antibodies may cause selective haemolytic anaemia (see p. 289), thrombocytopenia or neutropenia or various combinations of these in the same patient.^75,⁷⁶

A drug may cause an immune cytopenia by stimulating production of either an autoantibody (which reacts directly with the target cell independently of the drug itself) or a drug-dependent antibody (which destroys the target cell by reacting with a drug–membrane complex on the target cell).⁷⁷ Laboratory tests may demonstrate both types of antibody in some patients.⁷⁸

Demonstration of Platelet and Neutrophil Antibodies

No single method will detect all types of platelet and neutrophil antibodies equally well. In practice, it is useful to have a basic screening method that will detect most commonly occurring antibodies, both cell-bound (direct test) and in serum (indirect test), and to supplement this with other selected methods for demonstrating particular properties of an antibody and for measuring the amount of cell-bound antibody.

Alloantibodies

Reports of national and international workshops make it possible to formulate guidelines for platelet immunological tests. The basic procedure for demonstrating platelet alloantibodies should include the following:

1. A platelet test for platelet-reactive antibodies. The ISBT/ICSH Working Party on Platelet Serology ⁷⁹ recommended the platelet suspension immunofluorescence test⁸⁰ as the standard for assessment of other platelet antibody techniques.

It is important to combine a sensitive binding assay, such as the platelet immunofluorescence test (PIFT), with an antigen-capture method, such as the monoclonal antibody immobilization of platelet antigens (MAIPA),⁸¹ to increase the chance of detecting weak antibodies or those that react with relatively few antigen sites.

2. A lymphocyte test for detecting HLA antibodies. Because HLA antibodies also react with platelets, a lymphocyte cytotoxicity and/or ELISA assay should be included in the basic antibody screening procedure.

3. Tests to differentiate platelet-specific from HLA antibodies. The MAIPA technique using appropriate monoclonal antibodies is particularly useful for resolving mixtures of platelet-reactive antibodies (see p. 512). The chloroquine-‘stripping’ technique to inactivate HLA Class I molecules on platelets⁵² is also helpful in this respect (see p. 511). Conventional serological techniques (e.g. differential reactions with a panel of normal lymphocytes and platelets; differential absorption of HLA antibodies) can also be used to differentiate cell-specific and HLA antibodies, but these are less suitable for rapid screening than the chloroquine-‘stripping’ technique.

Further characterization of platelet-specific antibodies will require referral to a reference laboratory. Identification of allospecificity should be carried out as for red cell antibodies by reaction with a selected genotyped panel of group O platelets, preferably with reference to the patient’s platelet genotype.

An important consideration in platelet serology is the occasional occurrence of antibodies against hidden (cryptic) antigens of the GpIIbIIIa complex, which are exposed by EDTA and paraformaldehyde (PFA) fixation.⁸² These antibodies, which are only active in vitro, are unpredictable but when suspected can be avoided by using unfixed test platelets from citrated blood.

The detection and identification of granulocyte alloantibodies should be left to experienced reference laboratories, but should follow a similar schedule with the use of monoclonal antibody immobilization of granulocyte antigens (MAIGA)^83,⁸⁴ or adsorption of the sera with pooled platelets to differentiate between granulocyte-specific and HLA antibodies.

Autoantibodies

The detection of autoantibodies and drug-induced antibodies requires special consideration.

It can be misleading, when looking for platelet (or granulocyte) autoantibodies, only to test the patient’s serum against normal platelets (granulocytes) because positive reactions may result from the presence of alloantibodies (e.g. HLA or cell-specific) induced by previous transfusion or pregnancy. It is important to show that an autoantibody in the patient’s serum reacts with the patient’s own cells. Ideally a DAT (e.g. PIFT) should be performed, before treatment is given, to detect antibody bound in vivo. Where a severe cytopenia exists, it may not be possible to harvest enough cells for the test; nevertheless, serum samples should be stored at −20°C and tested retrospectively against the patient’s cells when the peripheral platelet (or neutrophil) count has increased in response to treatment.

A major interest in platelet autoimmunity has been the quantitative measurement of platelet-associated immunoglobulins as an indication of in vivo sensitization. A criticism of these quantitative methods is that they detect not only platelet autoantibody but also Ig nonspecifically trapped or bound to platelets and platelet fragments.⁸⁵ and are therefore generally nonspecific in the diagnosis of autoimmune thrombocytopenia.⁸⁶ It is now customary to use the direct PIFT,^87,⁸⁸ using flow cytometry. The patient’s platelets are incubated with isotype-specific fluorescein-isothiocyanate (FITC)-labelled conjugates (anti-IgG, anti-IgM and anti-IgA) and the test is reported as positive when the fluorescence intensity is >mean + 2SD when compared with the results obtained with pooled (10 or more) normal donor platelet suspensions. In a study of 75 patients with idiopathic thrombocytopenic purpura, using microscopy rather than flow cytometry, von dem Borne and colleagues⁸⁹ found a weak positive (± to +) direct PIFT in 60% of patients and strong reactions (++ to ++++) in only 26% of patients. In the same study, the indirect PIFT was positive with the patient’s serum in 66% of cases who had a positive direct PIFT and it was positive with an ether eluate of the patient’s platelets in 94% of the same cases. Although these results may be a reflection of the relative insensitivity of the method, they may result from a low-affinity antibody that is easily eluted during the assay procedure⁸⁵ or indicate an alternative immune mechanism for thrombocytopenia in some cases.

The Ig class of platelet autoantibodies is similar in idiopathic and secondary autoimmune thrombocytopenia; mostly it is IgG (92%), but often (also) it is IgM (42%) and sometimes (also) IgA (9%).⁸⁹ All IgG subclasses occur, but IgG1 and/or IgG3 are the most frequent.

A combination of the granulocyte immunofluorescence test (GIFT)⁹⁰ and the granulocyte agglutination test (GAT)⁹¹ provides the most effective means of granulocyte antibody detection. However, immune complexes and aggregates in a patient’s serum can still cause false-positive results. This can cause a problem for sera from adult patients with secondary autoimmune neutropenia, which should also be investigated for immune complexes (e.g. Clq-enzyme-linked immunosorbent assay). The granulocyte chemiluminescence test (GCLT)⁹² is relatively insensitive to the presence of immune complexes when inactivated serum is used, but it is unable to detect antibodies of the IgM. Several reviews provide an appraisal of the techniques available for detecting granulocyte-specific antibodies and antigens,^93,⁹⁴ including a recent review of investigations for transfusion-related acute lung injury (TRALI).⁹⁵

Drug-Induced Antibodies

The serological investigation of drug-induced immune thrombocytopenia (neutropenia) follows the same pattern as for haemolytic anaemia (see p. 289), with the exception that it is not always possible to collect enough cells to test at the nadir of thrombocytopenia or neutropenia. The following blood samples are therefore necessary:

1. Acute-phase blood sample when the cell count is at the nadir. If there are too few cells to test for cell-bound antibody and complement at this time, it is necessary to test the acute-phase serum against the patient’s cells during remission. These tests will demonstrate the immune basis of the cytopenia.

If the patient’s acute-phase serum is tested against normal donor cells, it is essential to take account of positive reactions owing to HLA or cell-specific alloantibody in the patient’s serum. Furthermore, negative results with normal donor cells may be the result of absence of the antigen for the particular drug-dependent antibody (e.g. owing to genetic restriction of the antigen concerned).⁹⁶

2. Subsequent samples after stopping the drug. Ideally, sampling should be done when the drug has been eliminated and the antibody is still detectable. Tests using this sample with and without the drug in the assay system are necessary to demonstrate the part played by the drug in causing the immune cytopenia. The drug may be added directly to the assay system (and included in the wash solution) or the cells may be pretreated with the drug. For some drugs, a metabolite and not the native drug is the appropriate antigen for testing; in these cases an ‘ex vivo’ drug antigen from urine or plasma may be used.⁹⁷

Methods of Demonstrating Antibodies

The basic immunofluorescent antiglobulin method and the MAIPA assay will be described in detail. Only brief mention will be made of other methods.

The Immunofluorescent Antiglobulin Methods

The immunofluorescent antiglobulin methods are based on the conventional antiglobulin technique (see p. 500) and are suitable for platelet,⁸⁰ granulocyte⁹⁰ and lymphocyte⁹⁸ serology. The PIFT and GIFT (Fison Ltd, Loughborough, UK) are described in detail in this chapter.

These tests can either be read by direct examination of a cell suspension using fluorescence microscopy or by flow cytometry. These tests can detect allo-, auto- and drug-induced antibodies and, by using appropriate monospecific antiglobulin reagents, can determine the Ig class and subclass of the antibody and cell-bound complement components. Both tests can be used with chloroquine-treated cells to differentiate cell-specific from HLA antibodies.⁹⁹

Patient’s and Screening Panel Cells

Platelets and granulocytes are prepared from venous blood taken into 5% (w/v) Na₂EDTA in water (9 volumes blood:1 volume anticoagulant).

Screening panel cells should be obtained from group O donors for platelet serology to avoid positive reactions owing to anti-A and anti-B, but this is not necessary for granulocyte serology because A and B antigens cannot be demonstrated on granulocytes. If a patient’s serum must be tested with ABO-incompatible platelets, anti-A and/or anti-B can be absorbed with corresponding red cells or A or B substance.

The best results are obtained with the freshest cell preparations, but some delay is tolerable (see later). Neutrophils are more susceptible to storage damage than platelets; cells should be fixed (see later) on the day of collection, but serology may be delayed to the following day. Platelets are more resilient and an anticoagulated blood sample may be satisfactory for testing for up to 2 days at ambient temperature (c20°C). Once fixed, platelets may be kept for 3–4 days at 4°C before serological testing. For longer storage, platelet-rich plasma may be kept at −40°C for at least 2 months; however, there is some membrane damage after recovery of frozen platelets, which causes increased background fluorescence that may limit the sensitivity of the test.^100,¹⁰¹ For longer-term storage a cryoprotectant (e.g. DMSO) may be used.¹⁰¹

Patient’s Serum

Serum from clotted venous blood should be heated at 56°C for 30 min to inactivate complement and stored in 1–2 ml volumes at −40°C (to avoid repeated thawing of a stock).

Control Sera

Negative control serum is prepared from a pool of 10 sera from normal group AB male donors who have never been transfused. Positive control sera containing platelet-specific antibodies (e.g. anti-HPA-1a), granulocyte-specific antibodies or multispecific HLA antibodies should be obtained from reference centres.

Eluate from Patient’s Sensitized Cells

Elution is important to confirm the antibody nature of cell-bound immunoglobulin and to determine the specificity of antibodies. This applies especially when no antibody is demonstrable in the patient’s serum, which often occurs in patients with autoimmune thrombocytopenia and neutropenia.

Elution by lowering the pH of the medium, by ether (or DMSO) and by heating to 56°C, has been used.¹⁰² For routine platelet serology, ether elution for platelet autoantibodies or heating to 56°C for platelet-specific alloantibodies could be used.

Heat Eluate

Incubate platelets or granulocytes suspended in 0.5 ml of 0.2% bovine serum albumin (BSA) in PBS for 60 min at 56°C. Centrifuge and remove the supernatant that contains the eluted antibody.

Platelet Preparation

1. Prepare platelet-rich plasma (PRP) by centrifugation of anticoagulated blood (200 g, 10 min).

2. Wash the platelets three times (2500 g, 5 min) in PBS/EDTA buffer (8.37 g of Na₂ EDTA dissolved in 2.5 1 of PBS, pH 7.2); resuspend the platelets thoroughly each time.

3. Fix the platelets in 3 ml of 1% paraformaldehyde solution for 5 min at room temperature. A stock solution of PFA is prepared by dissolving 4 g of PFA (BDH) in 100 ml of PBS by heating to 70°C with occasional mixing. Add 1 mol/l NaOH dropwise with continuous mixing until the solution clears. This 4% stock solution may be stored at 4°C protected from light for several months. Prepare a 1% PFA working solution by adding 1 volume of the 4% PFA stock solution to 3 volumes of PBS and by correcting the pH if necessary to 7.2–7.4 with 1 mol/l HCI.

Wash the platelets twice as before and resuspend in PBS/EDTA buffer at a concentration of 250–500 × 10⁹/l for use in the PIFT.

Granulocyte Preparation

1. Mix anticoagulated blood or blood retained from platelet preparation after removal of PRP (and made up to its original volume with PBS) with 2 ml of Dextran solution per 10 ml of blood (Dextran 150 injection BP in 5% dextrose). Incubate this mixture at 37°C for 30 min at an angle of about 45° to accelerate red cell sedimentation and then remove the leucocyte-rich supernatant (LRS).

2. Granulocytes can be separated by double-density sedimentation (Fig. 21.4). The LRS is underlayered with 2 ml of lymphocyte separating medium (LSM) (LSM = Ficoll-Hypaque sp gr 1.077), which is then underlayered with 2 ml of mono-poly resolving medium (MPRM) (MPRM = Ficoll-Hypaque sp gr 1.114) (LSM and MPRM supplied by Flow Labs Ltd). The density gradient tube is then centrifuged at 2500 g for 5 min. Granulocytes form an opaque layer at the LSM/MPRM interface from which they are harvested by careful pipetting (microscopic examination shows that the cells from this layer are predominantly neutrophil polymorphs). Lymphocytes can similarly be harvested from the plasma/lSM interface (e.g. for use in the lymphocyte immunofluorescence test or LIFT).⁹⁸

3. Wash the granulocytes three times at 400 g for 5 min) in PBS/BSA buffer (PBS pH 7.2 with 0.2% BSA).

4. Fix the granulocytes in 3 ml of 1% PFA for 5 min at room temperature.

5. Wash the granulocytes twice as before and resuspend in PBS/BSA buffer at a concentration of about 10 × 10⁹/l for use in the GIFT.

Figure 21.4 Double-density separation of lymphocytes and granulocytes. A leucocyte-rich supernatant is underlayered with Ficoll–Hypaque with a specific gravity of 1.077 (Solution I) and 1.114 (Solution II) and then centrifuged at 2500 g for 5 min. Lymphocytes concentrate in layer 1; granulocytes concentrate in layer 2.

Platelet and Granulocyte Immunofluorescence Tests

The serological methods for testing platelets and granulocytes in the suspension immunofluorescence test are similar, except that platelets are washed throughout in PBS/EDTA buffer and granulocytes are washed in PBS/BSA buffer. A flow diagram of the PIFT is shown in Figure 21.5.

Figure 21.5 Platelet immunofluorescence test. PRP, platelet-rich plasma; PBS, phosphate buffered saline; PFA, paraformaldehyde; RBC, red blood cells.

FITC-labelled antiglobulin reagents are used as follows: anti-Ig (polyspecific), anti-IgG, anti-IgM and anti-C3. F(ab)₂ fragments of these reagents should be used to minimize nonspecific membrane fluorescence owing to Fc receptor binding, which is a particular problem with granulocytes. The optimal dilution for each reagent should be determined by chequer-board titration. Centrifuge the FITC conjugates at 2500 g for 10 min before use to remove fluorescent debris and reduce background fluorescence.

Positive and negative controls (as described earlier) should be included with each batch of tests.

Indirect Test

1. In plastic precipitin tubes (50 × 7 mm), mix 0.1 ml of serum and 0.1 ml of the appropriate cell suspension, as prepared earlier. (The method can also be adapted for use with microtitre plates, which has the advantage of using smaller volumes.)

2. Incubate for 30 min at 37°C (for IgG and C3 tests) and at room temperature (for IgM tests). For C3 tests only, sediment cells (1000 g, 5 min), remove the supernatant and resuspend the cell button in 0.1 ml of freshly thawed human serum as a source of complement. Incubate for 30 min at 37°C.

3. Wash the cells three times at 1000 g, for 5 min with appropriate buffer – PBS/EDTA for platelets, PBS/BSA for granulocytes; decant the final supernatant. This and subsequent steps are common for both the indirect test (i.e. patient’s serum with donor cells) and the direct test (i.e. patient’s own cells to detect in vivo sensitization).

4. Add the fluorescent antiglobulin reagent (0.1 ml of the appropriate dilution determined by chequer-board titration), mix with the cell button and leave at room temperature for 30 min in the dark.

5. Wash twice as before and remove the supernatant.

6. Mix 0.5 ml of glycerol – PBS (3 volumes glycerol:1 volume PBS) with the cell button and mount on a glass slide under a coverslip.

7. Examine microscopically using ×40 objective and epifluorescent ultraviolet illumination.

Scoring Results

Reactions in the PIFT and GIFT may be scored on a scale from negative (−) through graded positives + to + + + +. Although subjective, this method of scoring in experienced hands can produce semiquantitative results in the PIFT.¹⁰³

In general, normal platelets and granulocytes incubated with AB serum do not fluoresce after incubation with an appropriately diluted FITC antiglobulin reagent. Sometimes the negative control may show weak fluorescence (up to two fluorescing points on some cells); in these cases, the test result is classified as positive only if it is clearly stronger than the negative control (AB serum). Stronger fluorescence in the negative control should raise doubts about the performance of the test.

Use of Flow Cytometry

With simplification of flow cytometers and improved software, more platelet reference laboratories are using them for primary analysis in PIFT because sensitivity is improved. Nevertheless, platelets are more difficult to work with flow cytometrically than other cells and particular attention has to be paid to prevent aggregation and to ensure single cell suspensions. Presence of platelet particles and debris may also cause confusion. The technical considerations of applying flow cytometry to platelet work are the subject of several reviews.^87,⁸⁸

Chloroquine Treatment of Platelets and Granulocytes

Platelets for chloroquine treatment should be prepared from fresh blood or blood stored overnight at 4°C; granulocytes are suitable only if freshly prepared.^52,¹⁰⁴ An important consideration is the extent of chloroquine-induced cell membrane damage, which is minimal with fresh cells.

1. Cells are prepared as already described. Two-thirds of the cells are treated with chloroquine; the remaining one-third are not treated. After washing and before PFA fixation, the cell button is incubated with 4–5 ml of chloroquine diphosphate in PBS (200 mg/ml, pH adjusted to 5.0 with 1 mol/l NaOH) for 2 h at room temperature with occasional mixing or overnight at 4°C without mixing, if this is more convenient for the laboratory routine.

2. Wash three times in the appropriate buffer and fix in 1% PFA as previously described. Cell clumping during washing may be a problem after chloroquine treatment, especially with granulocytes; cell clumps should be dispersed by repeated gentle aspiration with a Pasteur pipette. The final cell suspension for serological testing should be prepared as previously described.

When reading the test by fluorescence microscopy, it is important to recognize and allow for any fluorescence owing to chloroquine-induced cell damage, which is more likely to occur with granulocytes than platelets. Damaged cells are easily recognized by bright homogeneous fluorescence. Such cells should be excluded from assessment; only cells showing obvious punctuate fluorescence should be considered positive.

Chloroquine-treated cells were tested initially in the fluorescent antiglobulin method, but they may also be used in enzyme and radionuclide-labelled antigen methods.

Interpretation of Results with Chloroquine-Treated Cells

Typical results with HLA- and cell-specific antibodies are shown in Table 21.17. If a serum that has been shown to contain HLA antibodies by LCT and/or LIFT gives equal or stronger reactions with chloroquine-treated cells than with untreated cells, then a cell-specific antibody is also present. The Second Canadian Workshop on Platelet Serology¹⁰⁰ concluded that a weaker reaction with chloroquine-treated platelets should be interpreted with caution; this could indicate residual HLA reactivity, especially in the presence of high-titre multispecific HLA antibodies. If a platelet-specific antibody is nevertheless still suspected, other methods should be used to confirm this (e.g. MAIPA using appropriate monoclonal antibodies for capture; see later).

Table 21.17 Platelet and granulocyte antibody reactions using cells prepared with and without treatment with chloroquine

MAIPA Assay

The principle of the MAIPA assay is shown in Figure 21.6. The test is based on the use of monoclonal antibodies, such as anti-IIbIIIa, anti-IbIX, anti-IaIIa and anti-HLA class I, to ‘capture’ specific platelet membrane glycoproteins. The availability of appropriate monoclonal antibodies has led to the wider clinical application of this method.⁸¹ The same principle can be used with granulocytes, depending on the availability of appropriate monoclonal antibodies.^81,¹⁰⁵