Chapter 136: Erythrocyte Antigens and Antibodies

A blood group system consists of a group of antigens encoded by alleles at a single gene locus or at gene loci so closely linked that crossing over does not occur or is very rare. An antigen collection consists of antigens that are phenotypically, biochemically, or genetically related, but the genes encoding them have not been identified.¹ Placement of a blood group antigen into a system or collection begins with the discovery of an antibody, usually in the serum of a multiparous woman or a multiply transfused recipient, with a unique pattern of reactivity. The antibody can be used to study basic biochemical properties of the corresponding antigen, to enable recognition of the pattern of inheritance of the antigen in families and in populations, to identify red blood cells (RBCs) that lack the antigen, and to search for an antithetical antigen. Identified characteristics, such as prevalence of positive reactions or sensitivity or resistance to specific enzymes, are compared to antigens in known systems and collections. A newly recognized antigen is also evaluated using biochemical and molecular genetic methods. Orphan antigens of low or high prevalence are placed in “holding tanks” until the gene that encodes them is established.

The majority of genes encoding blood group antigens have been cloned and sequenced,² and the molecular bases of most blood group antigens have been determined.^3,4,5,6 Details on the alleles associated with blood group antigens and phenotypes can be obtained from the National Center for Biotechnology Information (NCBI) “dbRBC” website: http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home and from the International Society of Blood transfusion (ISBT) website: www.isbt-web.org.

RBC antigens are inherited carbohydrate or protein structures located on the outside surface of the RBC membrane (Fig. 136–1). Although most of the protein blood group antigens are carried on integral transmembrane proteins (either single-pass type I or type II, or multipass; Fig. 136–1), a few are carried on glycosylphosphatidylinositol (GPI)-linked proteins or adsorbed from plasma. Some carbohydrate antigens are attached to proteins or lipids and some require a combination of a specific portion of protein and carbohydrate. Blood group antigens have revealed that certain transmembrane proteins interact with other transmembrane proteins (e.g., band 3 and glycophorin A [GPA]; Kell and Kx; Rh and RhAG), with lipids (e.g., Rh), or with proteins in the membrane skeleton (e.g., band 3 and ankyrin, glycophorin C [GPC], and protein 4.1 and p55). Many of the proteins carrying blood group antigens reside in the erythrocyte membrane as complexes.^7,8,9,10 Many components carrying blood group antigens have been assigned cluster of differentiation (CD) numbers (Table 136–1). In human blood grouping, agglutination of RBCs usually serves as the detectable end point, but it can also be hemolysis.¹¹ Our ability to detect and identify blood group antigens and antibodies has contributed significantly to current safe blood transfusion practice, reducing death from hemolytic disease of the fetus and newborn (HDFN) from 40 percent to less than 2 percent, and supporting patients receiving chemotherapy or organ transplantation.

The naming of blood group antigens usually does not follow the classic convention wherein dominant traits are given capital letters and recessive traits are designated with lowercase letters. For example, in the ABO blood group system, A and B are codominant and the recessive O phenotype is encoded by a gene designated O, whereas in the MNS system the genes S and s are codominant. To standardize terminology used to describe RBC blood groups, the ISBT Working Party for Terminology for Red Cell Surface Antigens recommended using the traditional name for an antigen for verbal communication and a numerical system in computer databases (see Blood Group Terminology website at www.isbt-web.org). The working party has placed blood group antigens into four categories: (1) genetically discrete blood group systems; (2) serologically, biochemically, or genetically related antigens in blood group collections; (3) series of low-incidence antigens; and (4) series of high-incidence antigens. Each system and collection has been given a number and letter designation, and each antigen within the system is numbered sequentially in order of discovery. At the time of going to press (2015), 35 blood group systems and 6 antigen collections are defined (see Table 136–1; www.isbt-web.org).^{1,5,6,12,13,14} Over time, notations devised to describe blood group antigens have changed. A single letter (e.g., A, D, K), a symbol with a superscript (e.g., Fy^a, Jk^b, Lu^a), a symbol with a number (e.g., Fy3, Lu4, K12), and three to four letters (e.g., Vel, JMH, ELO, FPPT) are all used, sometimes within the same blood group system. The ISBT Working Party name has changed to ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology, which reflects that DNA testing is now often used to predict a blood group.

Tables 136–1 and 136–2 summarize the characteristics of common blood group antigens. The following sources provide more detail: Issitt and Anstee,⁵ Reid, Lomas-Francis and Olsson,⁶ Reid and Lomas-Francis,¹⁵ Mollison and colleagues,¹⁶ Daniels,⁴ and Fung and associates.¹¹ In the interest of space, reviews or books are referenced in place of original reports.

ABO BLOOD GROUP SYSTEM

The ABO blood group system was the first system described and remains the most significant in transfusion medicine. A mismatch of ABO may be fatal, whereas a mismatch of other blood groups initially mostly is harmless. This situation occurs because anti-A and anti-B antibodies usually are present in the blood of adults lacking the corresponding antigen. These antibodies are stimulated by the ubiquitous distribution of the antigen that forms part of the membrane structure of many bacteria, plants, and animals. For this reason, all donor blood for transfusion is tested and labeled with the ABO group. The four main phenotypes are A, B, AB, and O, the latter indicating a lack of A and B antigens. The sugars defining A and B antigens are added to carbohydrate chains carrying the H antigen (fucose), which is “hidden” by the A (GalNAc) or B (Gal) sugar. Thus, group A or B erythrocytes appear to have less H antigen than group O cells. Nonetheless, H is found on all human erythrocytes except those from rare individuals of the O_h (Bombay) phenotype.

Anti-A or anti-B immunoglobulins can cause intravascular hemolysis when ABO-incompatible RBCs are transfused. Because A and B antigens also are expressed on most tissue cells, ABO compatibility is a significant consideration in solid-organ transplantation. However, ABO incompatibility only rarely causes severe HDFN because antibodies directed against A and B antigens are predominantly immunoglobulin (Ig) M, which do not cross the placenta, in addition, A and B antigens are not fully developed on RBCs from a fetus (Chap. 55).

Although the ABO blood group system has only four phenotypes, hundreds of alleles have been identified by DNA analyses. The ABO gene was cloned in 1990 following purification of A transferase.^17,18 A and B transferases have only four amino acid differences in the catalytic domain, two of which (Leu266Met and Gly268Ala) are primarily responsible for substrate specificity.¹⁹ The group O phenotype results from nucleotide changes in A and/or B alleles that cause loss of glycosyltransferase activity. The most common group O (O₁) results from a single nucleotide deletion near the 5′ end of the gene that causes a frameshift and early termination with no active enzyme production.²⁰ The ABO gene has seven exons, and A or B subgroups (with only few exceptions) result from a variety of nucleotide changes in exon 7 that cause alterations in the catalytic domain of the glycosyltransferase (reviewed by Chester and Olsson²¹). The rare B(A), A(B), and cis-AB phenotypes expressing both A and B antigens result from variant glycosyltransferases that have a combination of A- and B-specific residues.²¹ Numerous common and rare ABO alleles have been reported, and current information is available on websites: http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home and www.isbt-web.org. In addition to nucleotide changes, recombinations and gene rearrangements can result in hybrid alleles that encode for unexpected transferase activity. This situation makes typing of ABO by DNA analysis difficult to interpret.²² The function of the ABO system is not known, although several disease associations are well established.²³

Rh BLOOD GROUP SYSTEM

The Rh (not Rhesus) system is the second most important blood group system in transfusion medicine because antigen-positive RBCs frequently immunize antigen-negative individuals through transfusion and pregnancy.

Inheritance of Rh antigens is determined by a complex of two closely linked genes: one encodes the protein carrying D antigen (RhD); the other encodes the protein carrying C or c and E or e antigens (RhCE). RBCs from Rh-positive people have both RhD and RhCE, whereas Rh-negative RBCs have only RhCE. In the Rh system, eight common antigen combinations or haplotypes are possible: Dce (R₀, Rh₀), DCe (R₁, Rh₁), DcE (R₂, Rh₂), DCE (R_Z, Rh_z), ce (r, rh), Ce (r′, rh′), cE (r′′, rh′′), and CE (r^y, rh^y). The letter “d” is commonly used to designate the lack of D, but there is no d antigen or anti-d.

Several nomenclatures can be used to describe Rh genes and antigens. The Fisher-Race nomenclature, which uses CDE terminology, is more commonly used for antigens; the Wiener nomenclature, which uses Rh/rh (or R/r) designations, is favored for haplotypes and gene complexes; and the Rosenfield and Rubinstein nomenclature, which uses numerical designations, was introduced to allow interpretation without bias.²⁴

The Rh blood group system has over 50 antigens (the ABO system has 4). By far the most important and immunogenic antigen is D (Rh₀ in Wiener terminology, referring to Wiener’s discovery that a rhesus monkey injected with human RBCs would produce antibody that agglutinated the RBCs of 85 percent of white New Yorkers). For most clinical purposes, testing individuals for the D antigen and classifying them as D+ (or Rh-positive), or D– (or Rh-negative) is sufficient. Approximately 85 percent of the white population is Rh-positive, and 15 percent is Rh-negative. Most Rh-negative recipients produce anti-D if they receive Rh-positive blood. Anti-D can cause hemolysis in adults following an Rh-mismatched transfusion and in the newborn (HDFN) if antibodies were made by the mother from a prior transfusion or pregnancy. Thus, donors and recipients are routinely typed and matched for D. The risk of anti-D sensitization by transfusion is essentially eliminated by matching. The risk of anti-D sensitization in pregnancy is minimized by passive immunization of mothers at risk against D.

The antigens C, c, E, and e are less immunogenic and become important in patient care only after the corresponding antibody develops or when the basic Rh haplotype must be determined. The remaining 45+ antigens are other Rh protein epitopes whose corresponding antibodies are seldom encountered. Some are encoded by variant Rh alleles and appear as antithetical antigens to C, c, E, or e, or as related “extra” antigens. Others are referred to as compound antigens or cis gene products. For example, the protein produced by RHCE^*ce encodes c and e antigens, and the compound f (or ce) antigen. Other compound antigens include Ce (rh_i), cE and CE. Still other Rh antigens are related to the complex “mosaic” nature of D and e, and less commonly C, c and E antigens. If immunized, individuals who lack a part of an antigen and who make antibody to the portion they lack, can present with a challenging serologic picture. For example, the D+ person who lacks part of the D antigen and makes an antibody to the missing portion appears to make alloanti-D because normal D+ RBCs carry all D epitopes.²⁵

Some, but not all, individuals who lack part of the D antigen (partial D) have weak expression of D on their red cells that is detected only by the antiglobulin test. Having a RHCE^*C gene in trans position to a RHD gene (e.g., Dce/Ce or DCe/Ce genotypes) also can weaken expression of D in some individuals. A third type of weak D expression results from inheriting a RHD gene that encodes all epitopes of D, but in less-than-normal quantity.

DNA analyses have revealed the molecular basis underlying antigens and phenotypes. A list of the alleles that have been described to date is available at: http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home and www.isbt-web.org. Rh blood group orthologs are present in nonhuman primates and other species on the evolutionary tree.²⁶ The RhD and RhCE proteins complex with the Rh-associated glycoprotein (RhAG) in the membrane. RhAG acts as a transporter of gases, most likely for ammonium, nitrous oxide, CO₂ and/or O₂ but confirmation is needed as to which it is.

OTHER BLOOD GROUP SYSTEMS

In terms of transfusion and HDFN, the other blood group systems and their antigens become clinically relevant only when antibody develops. Transfusion service laboratories identify (antibody identification) the specificity and characterize the reactivity of antibodies detected in routine testing (antibody screening). Once this information is known, the blood bank assesses the clinical significance of the antibody and selects the most appropriate blood for transfusion. Tables 136–1 and 136–2 summarize the number of antigens in each blood group system and other relevant information. A detailed description of all the blood group antigens is beyond the scope of this chapter. Because the molecular bases of most blood group antigens and phenotypes are known,⁶ DNA analysis can be used to predict the type of transfused patients and to identify the fetus at risk for HDFN.²⁷

An antigen is a substance that can evoke an immune response when introduced into an immunocompetent host and react with the antibody produced from that immune response. An antigen can have several epitopes, which together are called an antigenic determinant, each of which is capable of eliciting an antibody response.

The ability of an antigen to stimulate an immune response is called immunogenicity, and its ability to react with an antibody is called antigenicity. These primary characteristics are affected by antigen size, shape, rigidity, and the number and location of the determinants on the red cell membrane.

IMMUNOGENICITY

Immunogenicity depends on many antigen characteristics, not just the number of antigen sites. Relative immunogenicity is estimated by comparing the actual observed incidence of an antibody to the calculated likelihood of a possible immunizing event. After A and B, the D antigen is most immunogenic (early work suggested that approximately 80 percent of Rh-negative individuals produce anti-D after receiving a single Rh-positive RBC component but more recent studies indicate it is more like 20 to 30 percent⁴), followed by K, which stimulates anti-K in approximately 10 percent of cases.¹⁶ The antigens c and E are one-third as immunogenic; Fy^a is one-twenty-fifth as potent; and Jk^a is one-fiftieth to one-one hundredth times as potent as K.²⁸ It should be noted that immunogenicity does not always correlate with the hemolytic potential of an antibody specificity; for example, K is more immunogenic than Jk^a but anti-Jk^a is more likely to cause hemolysis.

NUMBER OF ANTIGEN SITES

The number of antigen sites per RBC has been estimated by measuring the uptake of ¹²⁵I-labeled antibody or of ferritin-conjugated anti-IgG. Numbers vary widely among blood group systems from a few hundred to over a million (see Table 136–2). Also, estimates for any given antigen can vary greatly.

ANTIGEN DEVELOPMENT ON FETAL ERYTHROCYTES

Most RBC antigens can be detected early in fetal development (A, B, and H antigens can be detected at 5 to 6 weeks’ gestation), but not all are fully developed at birth. A, B, H, I, P1, Lu^a, Lu^b, Yt^a, Xg^a, Vel, Bg, Do^a, Do^b, and Kn^a antigen expression is considerably weaker on cord RBCs than on RBCs from adults. Le^a, sometimes Le^b, Ch/Rg, AnWj, and Sd^a, are not readily detectable, although 50 percent of cord samples type Le(a+) with more sensitive test methods. Full expression of A, B, H, I, and Lewis antigens usually is present by age 3 years, whereas full expression of P1 and Lutheran antigens may not occur until age 7 years.

VARIATION IN ANTIGEN EXPRESSION

RBCs from individuals who are homozygous for an allele typically have a greater number of antigen sites than do RBCs from individuals who are heterozygous. Consequently, their RBCs can react more strongly with antibody. This difference in expression and antigen–antibody reactivity because of zygosity is known as dosage. For example, RBCs from a homozygous MM individual carry a double dose of M antigen and react more strongly with anti-M than do RBCs from a MN heterozygous individual carrying only a single dose of M. Antithetical antigens C/c, E/e, M/N, S/s, and Jk^a/Jk^b commonly show dosage effect. Dosage is less obvious with D, K/k, and Lu^a/Lu^b antigens. It typically is more apparent within a family than between families. Dosage within the Duffy system also may not be serologically obvious because Fy(a+b–) or Fy(a–b+) phenotypes are seen in either homozygous (Fy^aFy^a or Fy^bFy^b) or hemizygous (Fy^aFy or Fy^bFy) individuals.

Some blood group antigens are inherited as closely linked genes or haplotypes. Haplotype pairings and gene interaction (either cis or trans) also can affect phenotypic expression. For example, the pairing of RHCE^*C in trans position to RHD can result in weak expression of D (see “Rh Blood Group System” above), whereas RHCE^*E in cis position with RHD is associated with strong expression of D. Among the common phenotypes, R₂R₂ RBCs carry the strongest expression of D. In the Kell system, Kp^a is associated with weakened expression of in cis k and Js^b antigens.

Still other antigens are affected by regulator genes.²⁹ The dominant type of the Lu(a–b–) phenotype [In(Lu)] results from heterozygosity for an allele of the KLF1 gene, the gene that encodes erythroid Krüppel-like factor (EKLF). The dominant inhibitor gene KLF1 suppresses expression of Lutheran, P1, i, and many other antigens.³⁰ The dominant inhibitor In(Jk) suppresses expression of Jk^a and Jk^b antigens.³¹ Rare variants of the RHAG gene depress or prevent expression of the Rh antigens (see “Rhnull Syndrome” below).

An antibody typically recognizes an epitope consisting of four to five amino acids on linear proteins or one to seven sugars. Alternatively, the antibody-binding site may encompass a more complex three-dimensional structure with branches or folds, and recognition may depend on both amino acids and sugars. Tables 136–2 and 136–3 and Fig. 136–1 summarize blood group biochemistry and antigen structure.^4,6,16

CARBOHYDRATE ANTIGENS

Polysaccharides with blood group activity are made by sequential addition of specific sugars (or sugar derivatives) to specific precursors in specific linkages by specific transferases. Sugars commonly involved are galactose (Gal), N-acetyl-D-galactosamine (GalNAc), N-acetylglucosamine (GlcNAc), fucose (Fuc), and N-acetylneuraminic acid (NeuAc).

Antigens in ABO, LE, P1PK, and GLOB blood group systems depend on an immunodominant sugar, usually terminally located, the polysaccharide to which the sugar is attached, and the type of linkage involved. I/i specificity is defined by a series of sugars on the inner portion of ABH saccharide chains. The presence of at least two repeating Gal(β1–4)GlcNAc(β1–3)Gal units in a linear structure defines i activity. I activity involves these same sugars in branched form (see Table 136–3). The gene for I (GCNT2) encodes the transferase responsible for branching (β(1–6) glucosaminyltransferase). During the first years of a child’s life, linear chains are modified into branched chains, resulting in the appearance of I antigens.³² The I antigen is reduced on RBCs from fetuses and infants. A rare i phenotype occurs in adults (see “I-Negative Phenotype [i Adult]” below).

Polysaccharide chains are attached to glycoproteins in secretions (on type 2 chains), to glycolipids in plasma (on type 1 chains), and to both on the RBC membrane. Approximately 70 percent of A, B, H, and I antigens on the RBC membrane are carried on glycoproteins, primarily on the anion transporter, but also on the glucose transporter, the RhAG, and others. Approximately 10 percent of these antigens are on NeuAc-rich glycoproteins, 5 percent on simple glycolipids, and the remainder on polyglycosylceramide.¹⁶ P1, P^k, and P antigens are found on glycolipids both on the membrane and in plasma.³³

Lewis antigens are unique because they occur only on type 1 polysaccharide chains, which are found in plasma and secretions but not made by RBCs. Hence, they exist on RBCs only by adsorption of Lewis substance from plasma. The Le (or FUT3) gene encodes an α(1–4)fucosyltransferase. Whether the resulting antigen is Le^a or Le^b depends on the secretor gene Se (or FUT2), which encodes an α(1–2)fucosyltransferase.

PROTEIN ANTIGENS

Protein structures that carry blood group antigens can be grouped into three categories: (1) those that make a single pass through the erythrocyte membrane, (2) those that make multiple passes through the membrane, and (3) those that are attached to the membrane through a covalent linkage to lipid (GPI-linked; see Fig. 136–1).

Single-pass proteins include GPA with M and N antigens, glycophorin B (GPB) with S, s, and U antigens, GPC and glycophorin D (GPD) with Gerbich antigens, and the Lutheran, LW, Indian, Knops, Xg, Ok, and Scianna proteins (see Fig. 136–1). These proteins have an extracellular amino-terminus and an intracellular carboxyl-terminus (referred to as type I). In contrast, the Kell glycoprotein has an extracellular carboxyl-terminus and an intracellular amino-terminus (referred to as type II).

Most proteins that carry blood group antigens and make multiple passes through the erythrocyte membrane have both carboxyl- and amino-terminal ends that are intracellular, are hydrophobic, and have a transport function. Rh, RhAG, Diego, Colton, Kidd, Kx, GIL, and Raph proteins are included in this category. Duffy and Lan are multipass proteins, but they have an extracellular amino-terminus. Duffy has homology with a family of cytokine receptors and the Lan protein belongs to the family of ATP-binding cassettes.^4,6,34

Lipid-linked proteins have their carboxyl-terminus attached to the lipid GPI and are said to be GPI-linked or anchored. Cromer, Yt, Dombrock, and JMH proteins belong to this category. GPI-linked proteins are of special interest to hematologists because defective synthesis of the GPI anchor is responsible for paroxysmal nocturnal hemoglobinuria (PNH).³⁵ Thus, PNH-III RBCs lack all proteins attached by a GPI anchor, including those carrying blood groups (Chap. 40).

Expression of an RBC antigen is determined by its exposure as a result of its position on the cell surface and its biochemical structure. Expression can be modified with treatment of RBCs by enzymes and other chemicals. These reagents are used to help identify complex mixtures of antibodies and to help characterize antibody specificity when identity is not readily apparent.

Proteolytic enzymes, such as ficin, papain, bromelin, trypsin, and α-chymotrypsin, cleave proteins from the erythrocyte membrane at specific amino acids. Enzyme treatment of RBCs cleaves certain protein antigens and allows carbohydrate and other resistant protein antigens to react more strongly with their antibody. The reactivity of antibodies with antigens in ABO, I, P1PK, LE, RH, and JK systems is enhanced after enzyme treatment of the RBCs, whereas reactivity of antibodies to M, N, Fy^a, Fy^b, and many minor antigens (Xg^a, Ch, Rg, JMH, In^b, Ge2, Ge4, Pr, Tn, and some examples of Yt^a) is reduced or eliminated. S and s are variably affected by enzyme treatment, and Kell and Scianna antigens are relatively unaffected.^4,5,6

Reagents that reduce disulfide bonds, such as 2-mercaptoethanol (2-ME), dithiothreitol (DTT), and 2-aminoethylisothiouronium bromide (AET), denature Kell blood group antigens but enhance Kx. Reducing reagents also denature antigens in LW, SC, IN, JMH, and YT systems and weaken antigens in LU, DO, CROM, KN, and RAPH systems and the AnWj antigen.^4,5,6

Acid treatment of RBCs (ethylenediaminetetraacetic acid [EDTA]/glycine/acid reagent), which is frequently used to remove IgG from RBCs, can weaken or completely denature antigens in the KEL blood group system. Chloroquine treatment of erythrocytes (also sometimes used to remove IgG from RBCs) at room temperature has little effect on most antigens. However, treatment for 30 minutes at 37°C can weaken expression of many antigens, including Fy^b, Lu^b, Yt^a, JMH, and those in the RH, DO, and KN systems.

Protein antigens are direct gene products: The gene encodes a protein that expresses one or more antigens. Carbohydrate antigens, made by transferase action, are indirect gene products. Most blood group genes are located on autosomes; only two, Xg and XK, are located on the X chromosome (see Table 136–1 for locations of genes on chromosomes).

Most genes that encode blood groups have two or more alleles. Individuals who inherit two identical alleles are homozygous and make a double dose of a single gene product, whereas those who inherit two different alleles are heterozygous and make single dose of each of two gene products. Males are hemizygous for the genes located on their single X chromosome and make a single gene product. In contrast, females produce a double dose of the Xg and XK gene products, as X-chromosome inactivation does not involve Xg^a or Kx antigens.³⁶

ALLELES

Alleles encoding blood group antigens commonly arise from only a single or a few nucleotide changes. For example, A and B alleles differ by only seven DNA base substitutions, which result in four amino acid substitutions in their respective transferases.^4,5,6 The common O allele is similar to A except for a single base deletion at nucleotide 261 that shifts the reading frame during RNA translation. The resulting protein is truncated and has no transferase activity. Another variant O allele encodes a transferase identical to that of B except it has arginine instead of alanine at amino acid position 268, which blocks the enzyme activity. A comprehensive listing of blood group alleles is available at the following websites:http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home and www.isbt-web.org.

GENE COMPLEXES

Some blood group genes are complexes of several closely linked genes or loci that evolved through duplication of an ancestral gene. The antigens they encode are inherited as a haplotype with no or few crossovers. Blood group examples include the Rh system with genes RHD and RHCE, and the MNS system with genes GYPA, GYPB, and GYPE.

RHD and RHCE show remarkable homology between them and with RHAG, which encodes the RhAG. GYPA and GYPB probably arose by duplication of an ancestral GYPA gene encoding the N antigen.³⁷ The most common MNS complex is Ns, followed by Ms, MS, and NS.

In both RH and MNS systems, other antigens arose by further nucleotide changes, deletions, or rearrangements within the gene complex. Unequal pairing of GYPA and GYPB during meiosis, with subsequent recombination, resulted in several hybrids, such as GYP(A-B) (called Lepore type, by analogy with a similar hemoglobin hybrid), which encodes a protein with the amino-terminal end of GPA but the carboxyl-terminal end of GPB. Anti–Lepore-type hybrids, GYP(B-A) (amino-terminal end of GPB and carboxyl-terminal end of GPA), and other rearrangements (e.g., GYP[B-A-B] and GYP[A-B-A]) are known. Within the Rh complex, numerous hybrids of RH(D-CE-D) and RH(CE-D-CE) have been identified. Such hybrids can result in altered antigen expression and new antigens.^4,5,6

Kell and Lutheran proteins are single-gene products that carry multiple antigens. The most common alleles in humans are kKp^bJs^bK¹¹ and Lu^bLu⁶Lu⁸Au^a. Antigens of lower prevalence (K, Kp^a/Kp^c, or Js^a, and Lu^a, Lu9, Lu14, or Au^b) arise from separate nucleotide changes.

SILENT ALLELES

Some blood group alleles are amorphs, or silent; that is, they do not produce a recognizable antigen, although they may encode a product that is simply not detected with standard test methods. As discussed with regard to the ABO system, A and B genes produce transferases that add GalNAc or Gal, respectively, to the same precursors, but O produces no active enzyme. AB individuals express both A and B antigen, but AA and AO individuals express A, and BB and BO individuals express B. Amorphic alleles are recognized only in a homozygous state, and the result is a “null” phenotype. Null phenotypes exist in most blood group systems (see Table 136–1). Group O is the most common, followed by Fy(a–b–) and Le(a–b–) in Africans. Other null phenotypes are rare.

The Fy(a–b–) phenotype is especially interesting. Fy(a–b–) Africans have Fy^b genes that express normal Fy^b glycoprotein on tissue cells but not on RBCs. A nucleotide change that disrupts the GATA-1 binding site for RBC transcription is present in these individuals,³⁸ which helps explain why many Fy(a–b–) Africans do not make anti-Fy^b despite exposure to antigen-positive RBCs from transfusion.

GENE FREQUENCIES

Gene and phenotype frequencies vary widely with race and geographical boundaries.^6,11,16,39 This information is useful when estimating the availability of compatible blood and the probability of HDFN.

EXPRESSION OF RED CELL ANTIGENS IN OTHER BODY TISSUES AND FLUIDS

Antigens in the RH and JK blood group systems are present only on RBCs and have not been detected on platelets, lymphocytes, or granulocytes or in plasma, other body tissues, or secretions (saliva, milk, amniotic fluid).^4,5,6,16 Antigens in MNS, LU, KEL, and FY systems are found on RBCs and other body tissues (see Table 136–2).

ABH antigens have broad tissue distribution. In embryos, A, B, and H antigens are detectable on all endothelial cells and all epithelial cells except those of the central nervous system. Antigens in ABO, P1PK, LE, H, and I systems are in plasma and on platelets and lymphocytes. Granulocytes carry I antigen but no ABH. ABH on platelets and lymphocytes may be acquired at least in part by adsorption from plasma. Lewis antigen is acquired by RBCs by adsorption. Secretions (saliva, milk, sweat, semen, and urine, but not cerebral spinal fluid) contain soluble A, B, H, I, and Le^a and Le^b antigens but no P1PK or GLOB system antigens. Sd^a antigen is found in most body secretions, with the greatest concentration in urine.^5,16

ANTIGENS ASSOCIATED WITH POSSIBLE SUSCEPTIBILITY TO DISEASE

Some blood groups are statistically associated with medical conditions or disease (Table 136–4).^4,5,6,16 For example, blood group A is more common in persons with cancer of the salivary glands, stomach, colon, or ovary and with thrombosis (because of higher levels of coagulation factors VIII, V, and IX). Blood group O is more common in patients with duodenal and gastric ulcers, rheumatoid arthritis, and von Willebrand disease.

Associations with infection arise when microorganisms carry structures homologous with blood group activity. The presence of blood group antibody and/or soluble blood group antigen in secretions may help confer protection. Having anti-B may offer protection against Salmonella, Shigella, Neisseria gonorrhoeae, and some Escherichia coli infections. An association exists between nonsecretion of ABH antigen and susceptibility to Candida albicans, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae.⁶

A number of disease associations with globoside have been identified. Streptococcus suis, which can cause meningitis and septicemia in humans, binds exclusively to P^k antigen. A class of toxins secreted by Shigella dysenteriae, Vibrio cholerae, and Vibrio parahaemolyticus have binding specificity for Gal(α1–4)-Gal(β1–4). In addition, globoside is the receptor of human parvovirus B19. Some strains of E. coli use the disaccharide receptor Gal(α1–4)-Galβ on uroepithelial cells to gain entry to the urinary tract receptors associated with P1, P^k, and P antigens.^5,33 People with the rare p phenotype lack this disaccharide and are not susceptible to acute pyelonephritis from such E. coli strains nor to infection by human parvovirus B19.

PHENOTYPES ASSOCIATED WITH DISEASE RESISTANCE

Erythrocytes lacking Fy^a and Fy^b antigens are not infected by the malarial parasite Plasmodium vivax or by the simian malarial parasite Plasmodium knowlesi. These parasites attach to the Fy(a–b–) RBC membrane, but penetration does not take place. The Fy6 antigen is the critical receptor for P. vivax attachment.⁵ Plasmodium falciparum attaches to RBC glycophorins and their O-linked oligosaccharides (carrying NeuAc). RBCs with the following phenotypes have a decreased rate of infection: M–N– (GPA-deficient), S–s–U– (GPB-deficient), Ge– (Leach type or GPC/GPD-deficient), and Cad-positive and Tn-positive RBCs (which have abnormal O-linked sugars).

DISEASES ASSOCIATED WITH ALTERED ANTIGEN EXPRESSION

Antigen expression can be altered with inherited or acquired disease. Inherited changes are fixed and consistent; acquired changes can disappear with remission or recovery. In some diseases, antigen expression weakens; in others, antigen expression increases or new antigens appear.

Weakened ABH expression on RBCs has been noted in acute myeloid leukemias and may result from reduced transferase activity.^5,16 Normal antigen expression returns with disease remission. Transient weakened expression of target antigen also occurs in some cases of autoimmune hemolytic anemia. Weak Rh, Kell, Kidd, LW and AnWj blood group activity has been reported with concurrent autoantibody.^5,16,40

Increased expression of i on RBCs is associated with inherited disorders, such as thalassemia, sickle cell disease, Diamond-Blackfan syndrome, and hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS). Increased i expression also is noted with acquired conditions that decrease the red cell maturation time in the marrow, such as myeloblastic or sideroblastic myeloblastic erythropoiesis, refractory anemia, and excessive phlebotomy.^16,23 Expression of the de novo antigen Tn is caused by a galactosyltransferase deficiency acquired by somatic mutation in a population of stem cells. The antigen is present on RBCs, platelets, and granulocytes arising from these stem cells. This condition (seen as persistent mixed-field agglutination because of the presence of both normal and abnormal cells) causes other RBC changes, such as depressed MN expression, enhanced H, and reduced NeuAc content. Tn antigen exposure is associated with myelodysplastic syndrome and acute myelomonocytic leukemia.¹⁶ Other antigens (T, Tk) occur as a result of infection when microbes produce enzymes that remove some sugars (NeuAc) and expose new ones. Group A individuals can appear to acquire a B antigen when bacterial deacetylase removes the acetyl group on GalNAc.^4,16 This phenomenon is associated with severe infection, gastrointestinal lesions, and malignancies.

RBCs may acquire blood group activity when they adsorb material from certain microorganisms. Group B activity has been associated with E. coli⁸⁶ and Proteus vulgaris infection, and K antigen with Enterococcus faecium. Acquired Jk^b-like activity has been associated with E. faecium and Micrococcus infections, although the mechanism is not clear.⁴¹

DISEASES ASSOCIATED WITH ABSENT ANTIGENS OR NULL PHENOTYPES

Rh_null Syndrome

The Rh_null phenotype is associated with hereditary stomatocytosis, hemolytic anemia (usually mild and well compensated), and a lack of proteins carrying Rh antigens. The Rh protein resides in the RBC membrane, interacts with other membrane proteins and possibly the membrane skeleton, and may help regulate or organize the lipids within the red cell membrane bilayer.^9,10 Hence, it is an important determinant of membrane shape and expression of other antigens. Rh_null cells have depressed expression or absence of S, s, U, LW, and Fy5 antigens.

Most Rh_null red cells are stomatocytes or occasionally spherocytes and demonstrate increased osmotic fragility, increased potassium permeability, and higher potassium pump activity. They have reduced cation and water content and a relative deficiency of membrane cholesterol. Although these abnormalities are assumed to contribute to shortened in vivo survival, Rh_null RBCs survive normally in splenectomized patients, suggesting their removal is related more to splenic clearance because of shape rather than some other intrinsic factor.

Two genetic mechanisms account for the Rh_null phenotype. Persons with the amorphic type are homozygous for the silent RHCE gene on a deleted RHD background. Individuals with the more common regulator type of Rh_null have normal RH genes but an altered (silenced) RHAG gene. RhAG is required for expression of Rh antigens. Individuals with the Rh_mod phenotype have similar membrane and clinical anomalies associated with Rh_null syndrome but demonstrate some Rh antigen expression. The reduced expression of Rh antigens results from the presence of an altered form of RhAG.^24,26,42

McLeod Phenotype

Numerous males (but no females) with the McLeod phenotype have been identified. These individuals have acanthocytosis, decreased RBC survival, very weak expression of Kell blood group antigens, lack of Kx antigen on RBCs, and a well-compensated hemolytic anemia.⁴³

Kx antigen is carried on the Xk protein encoded by the XK gene on the X chromosome, which interacts with the RBC membrane skeleton and helps stabilize the membrane. The absence of Kx is associated with a lipid deficiency in the membrane bilayer that may be critical to the Kell glycoprotein and general RBC discoid shape. RBCs with the McLeod phenotype show a defect in water transport, increased mobility of phosphatidylcholine across the membrane, and increased phosphorylation of protein band 3 and β-spectrin.⁴³

After age 40 years, patients with the McLeod phenotype develop a slowly progressive form of muscular dystrophy that is associated with areflexia, choreiform movements, and cardiomegaly, leading to cardiomyopathy. They have elevated levels of serum creatine kinase and carbonic anhydrase III. Some patients with the McLeod phenotype and X-linked chronic granulomatous disease (CGD) have a deletion of both the XK and Phox-91 genes (Chap. 66). The McLeod phenotype results from deletions or nucleotide changes in the XK gene.⁴⁴

Gerbich-Negative Phenotype

The GYPC on chromosome 2 encodes two proteins: GPC, with antigens Ge3 and Ge4 (the Ge2 portion is “hidden” by the Ge4-bearing terminal end), and its shorter partner GPD, with antigens Ge2 (now exposed) and Ge3. GPC and GPD interact with membrane skeleton proteins 4.1 and p55, which are involved in cell deformability and membrane stability. Gerbich-negative RBCs of the Leach type (Ge:–2, –3, –4) lack both GPC and GPD, have reduced protein 4.1, and elliptocytosis but exhibit normal survival in vivo.^6,10

Bombay (O_h) Phenotype

Rare people lack A, B, and H antigens and have naturally occurring anti-A, anti-B, and anti-H in their plasma. Such people are said to have the Bombay (O_h) phenotype. In rare people with the Le(a–b–) Bombay phenotype, the gene that encodes the Fuc transporter is silenced. As a consequence, all cells lack Fuc. Without Fuc, neutrophils lack sialyl Le^X and thus cannot roll and ingest bacteria. These patients have a high white blood cell count and severe recurrent infections. The condition is called leukocyte adhesion deficiency II (LADII) or congenital disorder of glycosylation II (CDG II).^45,46

I-Negative Phenotype (i Adult)

The gene encoding the I-branching β-1,6-N-acetylglucosaminyltransferase (GCNT2) has three alternative forms of exon 1, with common exons 2 and 3. Mutations in exon 2 or 3 silence GCNT2 and give rise to the form of I-negative phenotype associated with congenital cataracts in Asians.^47,48 Mutations in exon 1C (IGnTC or IGnT3) silence the gene in RBCs but not in other tissues, and lead to the I-negative phenotype (i adult) without cataracts.⁴⁹

CO_null Phenotype

Antigens of the Colton blood group system are carried on the water transporter (aquaporin [AQP1]). Although an absence of this protein from the RBC membrane was thought to be incompatible with life, in reality these rare individuals have been shown only to be unable to maximally concentrate urine.⁵⁰

RAPH_null Phenotype

The MER2 antigen in the RAPH blood group system is carried on CD151. Rare individuals who lack CD151 have chronic renal failure, skin ulcers, and deafness.⁵¹

Other Null Phenotypes

Patients with null phenotypes can develop RBC antibodies that make it difficult to find compatible blood to avoid the otherwise serious hemolytic transfusion reactions. For example, people with the Bombay phenotype (O_h or H_null) demonstrate no red cell abnormality but make potent hemolytic anti-H as well as anti-A and anti-B. These antibodies are incompatible with all RBCs except those from other persons with the Bombay phenotype. Likewise, p individuals (PP1P^k-negative) or P^k individuals (P-negative) can make hemolytic antibodies to the antigens they lack. Anti-PP1P^k and anti-P also are associated with spontaneous abortions in the first trimester.¹⁶ Women with such antibodies (notably IgG anti-P), even those with a history of spontaneous abortions, have delivered viable infants after plasmapheresis.⁵²

Null phenotypes in the MNSs and Lutheran systems are interesting because several types of null phenotypes are known. Within the MNSs blood group system, people may lack GPA (En[a–] or MN-negative), GPB (SsU-negative), or both (M^kM^k phenotype). The rare Lu(a–b–) phenotype is caused by a dominant inhibitor called In(Lu), by homozygous pairing of the silent allele Lu, or by a recessive sex-linked inhibitor XS2.^5,16 Only the LuLu-type null (recessive Lu[a–b–]) is associated with antibody production because the inhibitor type nulls produce small amounts of Lutheran antigen. In(Lu) type, Lu(a–b–) RBCs have low expression of CD44 and AnWj, and have varying degrees of poikilocytosis and acanthocytosis. RBCs of this type tend to hemolyze more quickly during storage, even though they demonstrate normal osmotic fragility.⁵³ Inactivating nucleotide changes in KLF1, which encodes an altered transcription factor, cause the InLu phenotype.³⁰

The Jk(a–b–) phenotype is caused by the silent alleles JkJk or the dominant inhibitor In(Jk). RBCs having the Jk(a–b–) phenotype resist lysis in 2M urea,⁵⁴ a reagent commonly used in automated platelet counting systems; resulting in erroneously high platelet counts. No significant clinical abnormalities have been identified to date, although Jk(a–b–) individuals have reduced ability to concentrate urine.⁵⁵

The following diagnoses are made easily by simply typing the RBCs with appropriate antisera: Rh syndrome, McLeod syndrome, and LAD II.

IMMUNOLOGY OF RED CELL ANTIBODIES

Blood group antibodies are classified as alloantibodies if they only react with antigens present on the RBCs of other people and as autoantibodies if they react with self-antigens present on the patient’s own RBC. Alloantibodies also can be classified according to their mode of sensitization as naturally occurring (no apparent sensitization) or immune (following sensitization). Table 136–5 summarizes the common anti-erythrocyte antibodies.^4,6,15,16

IMMUNOGLOBULIN CLASSES ASSOCIATED WITH BLOOD GROUP ACTIVITY

Immunoglobulin G

IgG is the predominant antibody made in an immune response and constitutes approximately 80 percent of total serum Ig (Chap. 75). These antibodies, when specific for RBC antigens, can attach to or induce hemolysis of transfused antigen-positive RBCs. Receptors on macrophages in the liver and spleen allow the macrophages to remove IgG-coated RBCs from the circulation. IgG blood group antibodies also are capable of fixing complement, although some subclasses do so less efficiently than others: IgG₃ > IgG₁ > IgG₂ > IgG₄. How well an IgG erythrocyte antibody binds complement, depends on the surface density and location of the recognized antigen. This situation occurs because C1q, the initiator of the classic complement cascade, requires binding of at least two IgG molecules to the RBC within a span of 20 to 30 nm to initiate the complement cascade.¹⁶ For example, IgG anti-D rarely binds complement, presumably because most D sites are spaced too far apart.¹⁶ Most IgG blood group antibodies do not agglutinate saline-suspended RBCs, presumably because the IgG molecule is too small to span the distance between RBCs, although some exceptions are known (i.e., potent IgG examples of anti-A, anti-B, anti-M, and anti-K). Some IgG anti-D can directly agglutinate RBCs with the D-phenotype. Most IgG antibodies sensitize RBCs at 37°C and are detected with an antiglobulin reagent.¹¹

Immunoglobulin M

IgM is a pentamer of five basic units (having μ heavy chains plus a short J, or joining, chain) and makes up only approximately 4 percent of total serum Ig (Chap. 75). IgM is the first class of Ig produced by the fetus and is the predominant antibody in a primary immune response, but it does not cross the placenta. Because of their pentameric structure, even low-affinity IgM blood group antibodies can agglutinate RBCs and activate complement. Both hemolyzing and agglutinating abilities of IgM molecules are destroyed by reducing reagents, such as 2-ME and DTT. IgM antibodies of low affinity may agglutinate RBCs only at temperatures below 37°C. Such antibodies still may fix complement onto the RBC membrane in vivo, at the lower temperatures in the extremities, and activate the complement cascade in the core of the body. Because such IgM antibodies dissociate from RBCs at higher temperatures, their reactivity may be detected in routine antiglobulin tests (using polyspecific antiglobulin) by virtue of the complement components that remain bound to the red cell membrane.^11,16

Immunoglobulin A

IgA is the primary Ig in body secretions, where it exists predominantly as a dimer with a secretory component (Chap. 75). IgA does not cross the placenta or fix complement, but aggregated IgA can activate the alternative pathway of complement, and IgA can trigger cell-mediated events. Multimeric IgA antibodies in serum are seen as hemagglutinins in blood bank tests and most often are associated with anti-A or anti-B.

IMMUNOGLOBULIN IN THE FETUS AND NEWBORN

Initially, the fetus acquires low levels of maternal IgG, probably by diffusion across the placenta. These levels rise significantly between 20 and 33 weeks’ gestation as a selective transport system matures and maternal IgG is actively transported across the placenta. Thus, almost all blood group antibodies detected in the fetus and newborn originate from the mother and disappear within the first few months of life.

Actual fetal antibody production begins shortly before birth with low levels of IgM, followed by IgG and IgA several weeks after birth. Anti-A and anti-B usually are readily detected by age 2 to 6 months.

Because of this late immune response in the newborn and because maternal antibody is so predominant at birth, blood bank standards permit abbreviated testing on neonates younger than 4 months.⁵⁶ If available, the mother’s serum is used (and preferred) for identifying antibodies in a newborn and for crossmatching RBC components.

NATURALLY OCCURRING ANTIBODIES

Naturally Occurring Antibodies in Development

An antibody is said to be naturally occurring when it is found in the serum of an individual who has not been exposed to the antigen through transfusion or pregnancy. These antibodies most likely are heteroagglutinins produced in response to substances in the environment that are similar to those on RBC antigens.

Evidence supporting this concept has come from studies on the formation of anti-B in chickens.⁵⁷ Chicks raised in a normal environment made anti-B within the first 30 days of life, whereas chicks raised in a germ-free environment did not make anti-B by day 60. Naturally occurring anti-A and anti-B in humans, also called isoagglutinins, can increase in titer following ingestion or inhalation of suitable bacteria.⁵⁸

However, a great many antigens that likely are not present in the environment have been associated with naturally occurring antibodies, so the stimulus for naturally occurring antibodies is not clearly known.

Blood Group Associations and Presence of Naturally Occurring Antibodies

Naturally occurring alloantibodies are commonly associated with the carbohydrate antigens of the ABO, LE, and P1PK blood group systems. Anti-A and anti-B are expected in people who lack the corresponding antigens, as are antibodies specific for H, PP1P^k, or P antigens. Naturally occurring antibodies reactive with A1, Le^a, Le^b, or P1 determinants also are seen frequently. Carbohydrate antigens, especially those with repetitive epitopes, can stimulate B cells to make specific antibody without the aid of helper T cells. Such thymus-independent immune responses typically result in antigen-specific antibodies of the IgM class.

Within other systems,¹⁶ anti-Sd^a, anti-Vw, and anti-Wr^a are found in up to 2 percent of normal people. Other, less-common antibody specificities in approximate order of descending occurrence are anti-M, -S, -N, -Ge, -K, -Lu^a, -Di^a, and -Xg^a. Rh antigens are thought to reside only on RBCs, but apparent naturally occurring anti-D has been reported in 0.15 percent of Rh-negative donors and anti-E in more than 0.1 percent of Rh-positive donors when more sensitive enzyme detection methods are used. Examples of naturally occurring anti-C, anti-C^W, and anti-C^X also have been described.^4,5,6

Some naturally occurring antibodies exist as autoagglutinins (e.g., anti-H and anti-I). Patients with autoimmune hemolytic anemia can produce many antibodies to low-prevalence antigens with no specific stimulus, in addition to autoantibody.^5,6,16,40

Characteristics of Naturally Occurring Alloantibodies

Most naturally occurring antibodies are IgM, but some have an IgG component and a few are predominantly IgG. Some anti-A or anti-B may even be of the IgA class. Antibodies that cause direct agglutination of saline-suspended RBCs most commonly are of the IgM class. However, even IgG antibodies may cause agglutination of RBCs when they bind antigens that are present at high density on the RBC membrane, such as the ABO or MN antigens. With the exception of anti-A and anti-B, most common naturally occurring antibodies do not react at body temperature and are considered clinically insignificant. However, if they are found to react at 37°C, providing crossmatch-compatible blood for transfusion is prudent.

ANTIBODIES GENERATED IN RESPONSE TO IMMUNIZATION: IMMUNE ANTIBODIES

Blood Group Associations and Occurrence of Immune Antibodies

Immune antibodies are produced following exposure to foreign RBC antigens through pregnancy or transfusion. The primary immune response is seen several weeks to several months after the first exposure to antigen. IgM usually is associated with early primary responses, but whether it is always the first antibody class made is unclear. In most individuals, IgG soon predominates. This process is characteristic of a thymus-dependent immune response, where T cells help induce B cells to undergo isotype switching from IgM to IgG.

In a secondary or anamnestic response, antibody concentration starts to increase several days to several weeks following exposure, and IgG may rise to very high levels. Some IgG antibodies remain detectable for decades after a stimulus. Others, especially Kidd antibodies, can disappear after several months and are more commonly associated with delayed hemolytic transfusion reactions.^5,6,16

Immune antibodies are found more commonly in individuals who have been multiply transfused than in multiparous women. This situation occurs because in pregnancy the immunizing dose of red cells often is too small to elicit a primary response and the foreign antigens are limited to those of the father.¹⁶

Anti-D used to be the most common immune antibody, but with the advent of Rh matching of donors and recipients in the late 1940s and use of RhIg prophylaxis since the 1970s, its incidence has sharply decreased. Anti-D is present in 0.27 to 0.56 percent of transfusion recipients, 0.10 to 0.20 percent of pregnant women, and 0.16 to 0.25 percent of healthy blood donors.¹⁶

In contrast, the occurrence of immune antibodies other than anti-D has increased. Specificities other than anti-D have been reported in approximately 0.6 percent of transfusion recipients, 0.14 percent of pregnant women, and 0.19 percent of healthy blood donors. Pooled data from three 5-year periods and approximately 300,000 patients suggest the absolute occurrence of Rh antibodies other than anti-D is 0.22 percent, other than anti-K is 0.19 percent, other than anti-Fy^a is 0.05 percent, and other than anti-Jk^a is 0.04 percent.¹⁶ The rate of alloimmunization in patients with sickle cell anemia was 18.6 percent in one survey, and 55 percent of the immunized patients made more than one antibody. The most common specificities were anti-C, anti-E, and anti-K.¹⁶

Characteristics of Immune Antibodies

Immune antibodies most often are IgG but may be IgM and sometimes are IgA. Most immune antibodies react at body temperature and are considered clinically significant, except those directed against Bg, Knops, Cs^a, JMH, and sometimes Yt^a and Lutheran antigens.

Information about the clinical significance of alloantibodies is available at www.nybloodcenter.org.^59,60

HEMOLYTIC TRANSFUSION REACTIONS

Clinically significant antibodies are capable of destroying transfused RBCs. The severity of the reaction varies with antigen density and antibody characteristics.

Antibodies commonly associated with intravascular hemolysis include anti-A, anti-B, anti-Jk^a, and anti-Jk^b. ABO incompatibility is the most potent cause of immediate hemolytic reactions because A and B antigens are strongly expressed on RBCs and the antibodies so efficiently bind complement. Kidd antibodies are associated more often with delayed hemolytic reactions because they typically are difficult to detect and can disappear quickly from the circulation. IgG anti-Jk^a appears to bind complement only when traces of IgM anti-Jk^a are present.¹⁶ Anti-PP1P^k, anti-Vel, and anti-Le^a have been associated with hemolysis, but such examples are rare.

Extravascular hemolysis occurs with IgG₁ and IgG₃ antibodies that react at body temperature; that is, immune antibodies reactive with Rh, Kidd, Kell, Duffy, or Ss antigens. These antibodies make up the bulk of clinically significant antibodies. Antibodies not expected to cause RBC destruction are those that react only at temperatures below 37°C and IgG antibodies of the IgG₂ or IgG₄ subclass.¹⁶

HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN

HDFN is caused by blood group incompatibility between a sensitized mother and her antigen-positive fetus (Chap. 55). The antibodies most significant in HDFN are those that cross the placenta (IgG₁ and IgG₃), react at body temperature to cause red cell destruction, and are directed against well-developed RBC antigens. ABO incompatibility most commonly is seen, but ABO HDFN is clinically mild, presumably because the antigens are not fully expressed at birth. Antibodies directed against the D antigen can cause severe HDFN, and fetal health should be carefully monitored when anti-D titers are greater than 16. The severity of HDFN is less predictable with other blood group antibodies and can vary from mild to severe. For example, anti-K and anti-Ge3 not only causes red cell hemolysis but also may suppress erythropoiesis.^4,6

AUTOIMMUNE HEMOLYTIC ANEMIA

Autoimmune hemolytic anemia is caused by the production of “warm-” or “cold-” reactive autoantibodies directed against RBC antigens (Chap. 54).⁴⁰ Production can be triggered by disease, viral infection, or drugs; from breakdown in immune system tolerance to self-antigens; or from exposure to foreign antigens that induce antibodies that crossreact with self-RBC antigens. Autologous specificity is not always obvious because antigen expression can be depressed when autoantibody is present.⁴⁰

Warm autoantibodies react best at 37°C and are primarily IgG (rarely IgM or IgA). Most are directed against the Rh protein, but Wr^b, Kell, Kidd, and U blood group specificities have been reported.⁴⁰

Cold-reactive autoantibodies are primarily IgM. They react best at temperatures below 25°C but can agglutinate RBCs or activate complement at or near 37°C, causing hemolysis or vascular occlusion upon exposure to cold.¹⁶ Patients with cold agglutinin disease often have C3d on their RBCs, which can provide some protection from hemolysis. Most cold-reactive autoantibodies have anti-I activity. Reactivity with i, H, Pr, P, or other antigenic specificities is much less common.

The biphasic cold-reactive IgG antibody associated with paroxysmal cold hemoglobinuria (“Donath-Landsteiner” antibody) typically reacts with the high-prevalence antigen P (GLOB). It attaches to RBCs in the cold and very efficiently activates complement before it dissociates at warmer temperatures.

DISEASES ASSOCIATED WITH ANTIBODY PRODUCTION

Table 136–4 lists diseases associated with specific antibody production. These antibodies cause autoimmune hemolytic anemia only if the patient carries the corresponding antigen.

ABO

ABO grouping is the single most important test performed in the transfusion service because it is the fundamental basis for determining blood compatibility. ABO grouping is determined by testing RBCs with licensed antisera to identify the A or B antigens they carry (forward, or cell, grouping) and by testing the corresponding serum or plasma with known A and B cells to identify the antibodies present (reverse, or serum, grouping). Positive reactions are seen as hemagglutination or hemolysis, and the results of one test should confirm the results of the other.

If results are discrepant or reactions are weaker than expected, the cause must be investigated before the ABO group can be interpreted with confidence. Discrepancies can be related to RBC anomalies, serum anomalies, or both, and they may be associated with disease.^5,11,16 Table 136–6 lists common causes, excluding clerical and technical error. If the ABO group of a patient cannot be determined, group O blood can be used for transfusion.

Rh

The D type is the next most important test performed for blood compatibility. Individuals whose RBCs type D+ are called Rh-positive, and those who type D– are called Rh-negative, provided controls are acceptable. Blood donors who type D– using standard typing sera are tested further for weak D expression using more sensitive methods, such as an indirect antiglobulin test. Donors with weak D antigen are considered Rh-positive. Testing for weak D is optional for transfusion recipients and pregnant women.⁵⁶

EXTENDED ANTIGEN PHENOTYPING

Reagent antisera to detect other common antigens (e.g., CcEe, MNSs, Kk, Fy^aFy^b, Jk^aJk^b) are available and used when identification of the red cell phenotype is essential to antibody identification, blood compatibility, determination of zygosity, or paternity or forensic issues. Extended phenotyping is especially important to patients who are at high risk for alloimmunization from chronic blood transfusion, for example, those with sickle cell anemia or thalassemia. Ideally, an extended RBC phenotype of patients who are likely to be chronically transfused should be determined prior to initiation of transfusion therapy. Prediction of a blood group antigen can be made by testing DNA of a patient, even in the presence of transfused RBCs.²⁷

ANTIBODY SCREEN

The antibody screen, or indirect antiglobulin test, detects “atypical” or “unexpected” antibodies in the serum (i.e., other than anti-A and anti-B) using group O reagent red cells that are known to carry various combinations of antigens. The methods used must be able to detect clinically significant antibodies. Typically, serum or plasma and screening cells are incubated at 37°C with an additive to potentiate antibody–antigen reactions, then an indirect antiglobulin test is performed. Hemagglutination or hemolysis at any point is a positive reaction, indicating the presence of naturally occurring or immune alloantibody or autoantibody. The antibody screen will not detect all atypical antibodies in serum, such as antibodies to low-prevalence antigens not present on screening cells and antibodies that are not apparent at 37°C and in the antiglobulin phase.

DIRECT ANTIGLOBULIN TEST

The direct antiglobulin test (often referred to as the direct Coombs test, a term discouraged by Robin Coombs because he said that Race and Mourant were also key to the description of the test) detects antibody or complement bound to RBCs in vivo. Red cells are washed free of serum and then mixed with an antiglobulin reagent that agglutinates RBCs coated with IgG or the C3 component of complement.

Positive direct antiglobulin test results are associated with the following: (1) transfusion reactions, in which recipient alloantibody coats transfused donor RBCs or transfused donor antibody coats recipient RBCs; (2) HDFN, in which maternal antibody crosses the placenta and coats fetal RBCs; (3) autoimmune hemolytic anemias, in which autoantibody coats the patient’s own RBCs; (4) drug or drug–antibody complex interactions with RBCs that sometimes lead to hemolysis; (5) passenger lymphocyte syndrome, in which transient antibody produced by passenger lymphocytes from a transplanted organ coats recipient RBCs; and (6) hypergammaglobulinemia, in which Ig nonspecifically adsorb onto circulating RBCs.

A positive direct antiglobulin test result does not always indicate decreased red cell survival. As many as 10 percent of hospital patients and 0.1 percent of blood donors have a positive direct antiglobulin test result with no clinical indication of hemolysis.¹¹

COMPATIBILITY TESTING

Compatibility testing refers to a set of donor and recipient tests that are performed prior to red cell transfusion. The collecting facility tests donors for ABO, Rh, and unexpected antibody. However, transfusing hospitals retest the ABO (and D on Rh-negative units) to verify the accuracy of the blood label.⁵⁶ Routine recipient testing includes an ABO, D, and antibody screening on a blood sample collected within 3 days of the intended transfusion. Results are checked against historical records to verify ABO, D, and antibody status.⁵⁶

If the recipient has a negative antibody screening test result and no history of clinically significant antibodies, a serologic immediate spin crossmatch between recipient serum and donor red cells or a “computer crossmatch” (wherein computer software compares the ABO test results of both donor and recipient) is required to confirm ABO compatibility.¹¹

If clinically significant antibodies are detected in a recipient’s serum or previously were identified, red cell components should test negative for the corresponding antigens and be crossmatch compatible at 37°C by the antiglobulin test. The chance of finding compatible units usually reflects the antigen prevalence in the population, that is, 91 percent of units should be compatible with a patient making anti-K because 9 percent of the population is K+. This reasoning will not be valid if the local donor population varies significantly from the general population. When more than one antibody is present, the probability of finding compatible blood is the product of the prevalence (probability) of each independent antigen tested. For example, only 21 percent of units will be compatible for the recipient having both anti-K and anti-Jk^a: (0.91 for K–) × (0.23 for Jk[a–]) = 0.21.

When multiple clinically significant antibodies or an antibody directed against a high-prevalence antigen are present, finding compatible RBC components can be extremely difficult. Such antibody producers should be encouraged to give autologous donations prior to their elective blood needs. If the patient is not a candidate for autologous donation, compatible units may be found by testing the patient’s siblings or by asking regional blood suppliers to check their rare donor inventories and files. Such procurement requires additional time.

Repeat donor testing and crossmatching are not performed for plasma and platelet components, but the recipient’s ABO and Rh phenotypes must be known for appropriate selection of components. Table 136–7 gives general ABO-D compatibility guidelines.

ANTIBODY IDENTIFICATION

All unexpected antibodies should be investigated. Those detected in serum or plasma as an ABO discrepancy, a positive antibody screening result, or an incompatible crossmatch are identified using a panel of eight to 16 different group O red cells that have been typed for antigens corresponding to clinically significant antibodies. Serum reactions with these RBCs are compared to their antigen typing to determine specificity.¹¹ For example, an antibody that reacts with all K+ RBCs but not with K– cells most likely is anti-K.

A control of autologous RBCs and serum is tested concurrently with panel RBCs. Absence of reactivity with autologous cells implies the antibody is an alloantibody, whereas a positive result suggests autoantibody or a positive direct antiglobulin test result. Once antibody specificity is identified, the patient’s RBCs are tested for the corresponding antigen. If the alloantibody is anti-K, the cells should type K–. Such antigen typing helps to confirm serum findings.

When antibody is detected both on red cells (a positive direct antiglobulin test result) and in serum, only the antibody in serum is identified unless a review of the medical, pregnancy and transfusion history offers evidence that the antibodies might be different. When antibody is detected only on RBCs and in vivo hemolysis is suspected, the antibody can be eluted from the patient’s RBCs and tested against panel RBCs to identify the specificity.