Blood cell antigens and antibodies: erythrocytes, platelets and granulocytes

Chapter 21 Blood cell antigens and antibodies

erythrocytes, platelets and granulocytes


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

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.

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.

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).

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

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, Ax) 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.

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.

Lewis System

Rh System

The Rh system, formerly known as the Rhesus system, was so named because the original antibody that was raised by injecting red cells of rhesus monkeys into rabbits and guinea pigs reacted with most human red cells. Although the original antibody (now called anti-LW) was subsequently shown to be different from anti-D, the Rh terminology has been retained for the human blood group system. The clinical importance of this system is that individuals who are D negative are often stimulated to make anti-D if transfused with D-positive blood or, in the case of pregnant women, if exposed to D-positive fetal red cells that have crossed the placenta.

Rh Antigens and Encoding Genes

This is a very complex system. At its simplest, it is convenient to classify individuals as D positive or D negative, depending on the presence of the D antigen. This is largely a preventive measure, to avoid transfusing a D-negative recipient with the cells expressing the D antigen, which is the most immunogenic red cell antigen after A and B. At a more comprehensive level, it is convenient to consider the Rh system as a gene complex that gives rise to various combinations of three alternative antigens – C or c, D or d and E or e – as originally suggested by Fisher. The d gene was thought to be amorphic without any corresponding antigen on the red cell. Subsequently it was confirmed that the RH locus is on chromosome 1 and comprises two highly homologous, very closely linked genes, RHD and RHCE, each with 10 exons. Each gene codes for a separate transmembrane protein with 417 residues and 12 putative transmembrane domains. The D and CE proteins differ at 35 residues. The RHCE gene has four main alleles; CE, Ce, ce and cE. Positions 103 and 226 on the CE polypeptide, situated in the external loops, determine the C/c (serine/proline) and E/e (proline/alanine) polymorphisms, respectively. This concept of D and CcEe genes linked closely and transmitted together is consistent with the Fisher nomenclature.

In Caucasian, D-negative individuals, the RHD gene is deleted, whereas in Black races and other populations, single-point mutations, partial deletions or recombinations have been described. In individuals with a weak D antigen (Du), there is a quantitative reduction in D antigen sites, believed to arise from an uncharacterized transcriptional defect. These individuals do not make anti-D antibodies following a D antigen challenge. Partial D individuals lack one or more epitopes of the D antigen, defined using panels of monoclonal reagents. Dvi is perhaps the most important partial D phenotype because such individuals not infrequently make anti-D. Partial D phenotypes arise from DNA exchanges between RHD and RHCE genes and from other rearrangements. Comprehensive reviews of this system have been provided by Avent and Reid11 and Daniels et al.12

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 Ro (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
CwDe R1w 1
cdE r″ 1
Cde r′ 1
CdE ry Rare


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-CW. 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

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, Kpa, Kpb, Jsa, Jsb
Kidd Jka Jka, Jkb, Jk3
Duffy   Fya, Fyb
MNS   M, S, s, U
Lutheran   Lub
Lewis Lea  
Cartwright   Yta
Colton   Coa, Cob
Dombrock   Doa, Dob

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-Fya 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.

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-Lua and anti-Lub. 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).

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-Kna 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

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:

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:

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:

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:

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 Directive24 (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.

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Jun 12, 2016 | Posted by in HEMATOLOGY | Comments Off on Blood cell antigens and antibodies: erythrocytes, platelets and granulocytes
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