14 Other Blood Groups 14

Transcription

1 14 Other Blood Groups 14 In addition to the antigens discussed in Chapters 12 and 13, over 200 others can be detected on red cells, and some are found on other cells or in body fluids. For example, CD44, CR1, and decay accelerating factor (DAF) are molecules that bear blood group antigens but are also expressed on a broad range of hematopoietic cells. 1 Antigens of the HLA system (see Chapter 15), only occasionally detected on red cells, are present on most other human cells. Certain antigenic determinants exist only on granulocytes or platelets, but they deserve brief mention in this chapter for their potential significance in transfusion therapy. Distribution of Antigens Antigens present in almost all persons are known as high-incidence or public antigens. Others, of very low incidence, are sometimes called private antigens. Antigens that occur as codominant traits, especially pairs such as Jk a and Jk b, have more variable incidence, and this may differ from one ethnic group to another as a result of selective pressure. For example, the Duffy glycoprotein is known to be a receptor for the parasite Plasmodium vivax, the causative agent of malaria. In West Africa, where malaria is endemic, the Fy(a b ) red cell phenotype, very rare in Whites, occurs with an incidence of greater than 80%. Each of the known antigens described in this chapter was initially identified through the detection of its specific antibody in a serum. Tables listing phenotype frequencies among Whites and Blacks in the US population are given throughout this chapter. Frequencies among other racial groups in the population are not given, as data are scanty and wide differences among groups of diverse Asian or Native American origins make generalizations about phenotypes inappropriate. Table 14-1 lists the blood group systems as defined by the International Society of Blood Transfusion (ISBT) working party on blood group terminology, and their gene location. 2-4 Table 14-2 shows the serologic behavior and characteristics of the major blood 277

2 278 AABB Technical Manual Table Membrane Component and Chromosomal Assignment of the Human RBC Blood Group Systems ISBT Number Blood Group RBC Membrane Component Chromosome Location 001 ABO Anion exchanger (AE-1), 9q34.1-q34.2 protein 4.5, lipids 002 MNS M,N: glycophorin A 4q28-31 S,s: glycophorin B 003 P1 Glycolipid 22q11.2-ter 004 Rh Rh proteins 1p36.2-p Lutheran Lutheran glycoprotein 19q12-q Kell Kell protein 7q Lewis Type 2 oligosaccharides 19p Duffy Chemokine receptor 1q22-q Kidd Urea transporter 18q11-q Diego AE-1 17q12-q Yt Acetylcholinesterase 7q Xg Xg glycoprotein Xp Scianna Scianna protein 1p36.2-p Dombrock Dombrock glycoprotein Unknown 015 Colton CHIP 28 (Aquaporin) 7p LW LW glycoprotein 19p13.2-cen 017 Chido/Rodgers C4 6p Hh AE-1, protein 4.5, lipids 19q Kx Kx protein Xp Gerbich Glycophorin C and D 2q14-q Cromer Decay accelerating factor (CD55) 1q Knops CR1 (CD35) 1q Indian CD44 11p13 group antibodies derived from human sources. This chapter briefly reviews all the blood group systems, but the reader is advised to read other texts, such as those in the reference list, for more detail on a particular blood group. The MNS Blood Group System (ISBT 002) The MNS blood group system is a complex system of 37 antigens distributed on

3 Chapter 14: Other Blood Groups 279 Table Serologic Behavior of the Principal Antibodies of Different Blood Group Systems In-Vitro Saline Albumin Papain/Ficin Associated With Antibody Hemolysis 4 C 22 C 37 C AGT 37 C AGT HDN * HTR Anti-M 0 Most Some Few Few 0 0 Few Few Anti-N 0 Most Few Occ. Occ. 0 0 Rare No Anti-S 0 Few Some Some Most See text Yes Yes Anti-s 0 0 Few Few Most See text Yes Yes Anti-U 0 0 Occ. Some Most Most Most Yes Yes Anti-Lu a 0 Some Most Few Few Few Few No No Anti-Lu b 0 Few Few Few Most Few Few Mild Yes Anti-K 0 Few Some Most Some Most Yes Yes Anti-k 0 Few Few Most Some Most Yes Yes Anti-Kp a 0 Some Some Most Some Most Yes Yes Anti-Kp b 0 Few Few Most Some Most Yes Yes Anti-Js a 0 Few Few Most Few Most Yes Yes Anti-Js b Most Few Most Yes Yes Anti-Fy a 0 Rare Rare Most 0 0 Yes Yes Anti-Fy b 0 Rare Rare Most 0 0 Yes Yes Anti-Jk a Some Few Few Most Some Yes Yes Yes Anti-Jk b Some Few Few Most Some Most Yes Yes Anti-Xg a 0 Few Few Most 0 0 No report Anti-Di a 0 Some Some Most Some Some Yes Yes Anti-Di b 0 Most Some Some Yes Yes Anti-Yt a Most 0 Some No No Anti-Yt b 0 All No report Anti-Do a Some Some Most No Yes Anti-Do b 0 All All No report Anti-Co a Some Some Most Yes Yes Anti-Co b Some Some Most No report Anti-Sc1 0 All No report Anti-Sc2 0 Some Some Most Most Most No report *Hemolytic disease of the newborn. Hemolytic transfusion reaction. The reactivity shown in the table is based on the tube methods in common use. If tests are carried out by more sensitive test procedures (such as in capillary tubes, in microtiter plates or by the albumin layering method), direct agglutination (prior to the antiglobulin phase) may be observed more often with some antibodies. Blank spaces indicate a lack of sufficient data for generalization about antibody behavior.

4 280 AABB Technical Manual two glycophorin molecules, or hybrid molecules of the two proteins. However, the antibodies most commonly encountered are directed at the M, N, S, and s antigens. Anti-M and Anti-N The M (MNS1) and N (MNS2) antigens were discovered in 1927, when Landsteiner and Levine obtained the antibodies defining them by immunizing rabbits with human red cells (reviewed by Unger and Laird-Fryer 5 ). Anti-M Anti-M is detected frequently in human sera, usually as a saline agglutinin in tests performed at room temperature, and usually in persons who have had no exposure to human red cells. Although M antibodies are generally thought to be predominantly IgM, examples that are partly or wholly IgG are frequently found. Agglutination of M-positive red cells in a saline test does not necessarily indicate that a given example of anti-m is wholly IgM. As with the A and B antigens of the ABO system, the M antigen has sufficient density on red cells that saline-suspended cells may be agglutinated by an antibody that is wholly IgG. Some examples of anti-m cause stronger agglutination if the ph of the test system is reduced to 6.5. Anti-M is rarely clinically significant, although examples that react at 37 C or at the antiglobulin phase of testing should be considered potentially significant. Compatibility testing performed by a strictly prewarmed method (see Method 3.3) should eliminate reactivity of most examples of anti-m. In a few exceptional cases, anti-m detectable at the antiglobulin phase has caused hemolytic disease of the newborn (HDN) or hemolysis of transfused cells. Anti-N Anti-N, in contrast to anti-m, is comparatively rare. Examples are almost invariably IgM, and they typically behave like weakly reactive cold agglutinins. Some powerful and potentially significant IgG examples have been observed in a few persons of the rare phenotypes M+N S s U and M+N S s U+ w, because these individuals lack the glycophorin that expresses the N antigen. N-positive and N-negative red cells of normal SsU phenotypes bear the N antigen (to be discussed later in this chapter). A potent anti-n-like agglutinating antibody has been observed in some hemodialysis patients. These patients had used formaldehyde-sterilized dialyzer membranes, and the formaldehyde appears to have induced immunogenic alteration of the N and N antigens. 6 Antibodies Showing Dosage The M and N antigens behave as products of paired genes, in that they bear an obvious allelic relationship. With rare exceptions, red cells type as M+N, M N+, or as M+N+, representing, respectively, homozygosity for M, homozygosity for N, and heterozygosity for both alleles, which are expressed codominantly. Some examples of anti-m and anti-n give significantly greater reaction strengths and higher titration scores against red cells from homozygotes than against red cells from heterozygotes, a phenomenon described as showing dosage. Many examples of anti-m react only with red cells from homozygotes, in which case the specificity of the antibody may not be immediately apparent from the reaction patterns obtained with a panel of red cells. This occurs much less commonly with anti-n. Reagent anti-m and anti-n rarely demonstrate dosage, having been selected and standardized to give strong positive reactions with all

5 Chapter 14: Other Blood Groups 281 red cells possessing the relevant antigen. The lectin obtained from extracts of Vicia graminea seeds has anti-n-like reactivity and, at a suitable dilution, can be used as a reliable anti-n reagent. Antibodies to S, s, and U Unlike anti-m and anti-n, antibodies to S (MNS3), s (MNS4), and U (MNS5) usually occur following red cell stimulation. All are capable of causing hemolytic transfusion reactions (HTRs) and HDN. Although a few saline-reactive examples have been reported, antibodies to S, s, and U are usually detected in the antiglobulin phase of testing. Anti-S occurs about as infrequently as anti-n, and anti-s even less often. The s-negative phenotype is less frequent than S-negative, so antibody occurrence may reflect phenotypic susceptibility as much as or more than inherent immunogenicity. Anti-U is rare but should be considered when serum from a previously transfused or pregnant Black person contains antibody to a high-incidence antigen. It may not be possible to type the patient s red cells for U, but the probability that the red cells are U-negative can be established by showing they are S s. M, N, S, s, and U Antigens M, N, S, s, and U are the most important antigens of the MNS system with regard to transfusion medicine and have been important in our understanding of biochemistry and genetics. Although the M and N antigens are located on glycophorin A (GPA) and S, s, and U are located on glycophorin B (GPB), there is interaction at the gene level that is manifest in the phenotypes observed. Table 14-3 shows the frequencies of the different common phenotypes of the MNS system. There is considerable linkage disequilibrium between M, N and S, s, with the gene complex producing N with s being much more common than that producing N with S. Although the M and N antigens are encoded by GYPA and the S and s antigens are encoded by GYPB, the two genes are in very close proximity on chromosome 4. 7 Red cells that lack S and s are also negative for a high-incidence antigen called U; persons who lack U may make anti-u when exposed to U-positive red cells. Some S s red cells are U-positive, but the U antigen may be so weakly expressed that an adsorption/elution technique is needed to demonstrate its presence. Table Phenotypes and Frequencies in the MNS System Reactions with Anti- Phenotype Frequency % M N S s U Phenotype Whites Blacks + 0 M+N M+N M N S+s U S+s+U S s+u S s U 0 Less than (+) S s U+w 0 Rare

6 282 AABB Technical Manual Biochemistry of MNS Blood Group System Antigens of the MNS system are carried on GPA and GPB, which are single-pass transmembrane glycoproteins. The carboxyl (C) terminus of each glycophorin extends into the cytoplasm of the red cell, while a hydrophobic segment consisting of 23 amino acids is embedded within the lipid bilayer. An amino (N) terminal segment extends into the extracellular environment. The molecules are sensitive to cleavage at varying positions by certain proteases. (See Fig 14-1.) M and N antigens are found on GPA, of which approximately 500,000 copies are present on each red cell. GPA is a 31 kda molecule; it consists of 131 amino acids, with attached carbohydrates con- Figure Diagrammatic representation of MN and Ss sialoglycoproteins in the red cell membrane including antigen locations and enzyme cleavage sites.

7 Chapter 14: Other Blood Groups 283 tributing approximately 60% of its total mass. Blood group antigen activity resides on the extracellular segment, a sequence of 72 amino acids with carbohydrate side chains attached within the first 50 residues of the amino terminus. When GPA carries M antigen activity (GPA M ), the first amino acid residue is serine and the fifth is glycine. When it carries N antigen activity (GPA N ), leucine and glutamic acid replace serine and glycine at positions one and five, respectively. (See Table 14-4.) Red cells that lack most or all of GPA are described as En(a ). En(a ) individuals may produce antibodies (collectively called anti-en a ) that react with various portions of the extracellular part of the glycoprotein. Some En(a ) persons may produce an antibody, against an antigen called Wr b, which is thought to arise from a salt bridge between GPA and the anion exchange molecule, AE-1 (also known as band 3). In the absence of GPA, Wr b is not expressed and the antibody, anti-wr b, is virtually indistinguishable from anti-en a unless other Wr(b ) cells are tested. The gene that determines Wr b is independent of those of the MNS system. The polymorphism that gives rise to both the Wr a and Wr b antigens exists on AE-1. 8 GPB is a smaller protein than GPA, and there are fewer copies per red cell. Approximately 100,000 copies of this molecule are present, in contrast to 500,000 for GPA. GPB carries S, s, and probably U antigens and possesses, at the amino terminus, a segment consisting of 26 amino acids that duplicate the sequence of GPA N. This accounts for the presence of an N antigen (known as N ) on all red cells of normal MNS types. Red cells that lack GPB altogether lack not only S, s, and U activity, but also N. Immunized individuals of the M+N S s U phenotype can produce anti-n that will react with all red cells of normal MNS types whether N-positive or N-negative. GPB that expresses S activity has methionine at position 29; GPB with s activity has threonine at that position. The Effect of Proteolytic Enzymes on MNS Antigens Because proteolytic enzymes such as ficin or papain cleave red cell membrane sialoglycoproteins (SGPs) at well- Table Partial Structures of Sialoglycoproteins (SGPs) of Common and Variant MNS System Antigens Amino Acid Position Antigen M-active GPA SER SER * THR * THR * GLY R N-active GPA LEU SER * THR * THR * GLU R N -active GPB LEU SER * THR * THR * GLU R S-active GPB R THR * ASN GLY GLU MET GLY R s-active GPB R THR * ASN GLY GLU THR GLY R *=attachment site for alkali-labile oligosaccharide. R=organic radical; ASN=asparagine; GLU=glutamic acid; GLY=glycine; LEU=leucine; MET=methionine; SER=serine; THR=threonine.

8 284 AABB Technical Manual defined sites, reactivity with anti-m and anti-n is abolished by commonly used enzyme techniques. The effect of different enzymes on the expression of MNS system antigens reflects the point at which the particular enzyme cleaves the antigen-bearing SGP and the position of the antigen relative to the cleavage site. (See Fig 14-1 and Table 14-2.) Sensitivity of the antigens to proteases may help in the identification of antibodies to M and N antigens, but the effects of proteases on tests for the S and s antigens are less firmly established. Most investigators have found that papain or ficin destroy the reactivity of S-positive red cells with anti-s. Depending on the enzyme solution employed, the reactivity of anti-s with s-positive red cells may be little affected by these enzymes. Rare examples of anti-s agglutinate ficin- or papain-treated red cells that are U-positive, irrespective of their S antigen status. If enzyme-treated S U cells are tested, however, no agglutination occurs. As a consequence, these examples of anti-s could be mistaken for anti-u in enzyme testing. 9 Most examples of anti-u react equally with untreated and ficin- or papain-treated red cells, but there have been examples of anti-u that detect an enzyme-sensitive determinant. Genes Encoding Glycophorins The genes encoding the MNS system antigens are located on chromosome 4 at 4q28-q31. The gene that encodes GPA is called GYPA and the gene that encodes GPB is GYPB. The similarities in amino acid sequences of GPA and GPB suggest both genes derive from a common ancestral gene. GYPA and GYPB consist of seven and five exons, respectively. The genes share >95% identical sequences from the 5 flanking region to the region approximately 1 kilobase downstream from the exon encoding the transmembrane regions. While the genes are highly homologous, GYPB results in a shorter protein because a point mutation at the 5 splicing site of the third intron that prevents translation of exon 3. Following the homologous sequences, GYPA and GYPB differ significantly in the 3 end sequences. Low-Incidence Antigens of the MNS System The MNS system includes a number of low-incidence antigens. Recent biochemical data attribute the reactivity of various low-incidence determinants to one or more amino acid substitutions; to variation in the extent or type of glycosylation; or to the existence of a hybrid SGP. Unexpected phenotyping results may occur when variants of the MNS antigens are present. For example, the M g (MNS11) antigen, product of a rare allele at the MNS locus, reacts neither with anti-m nor with anti-n reagents. The red cells of a person with the genotype M g N will give the reactions M N+, leading to the false conclusion that the genotype is actually NN. Similarly, the red cells of a person of the genotype M g M will give the reactions M+N. Parentage can be falsely excluded if persons with M g M or M g N genotypes are interpreted as being MM or NN, respectively. Anti-M g occurs as a saline agglutinin in serum from a person who has had no known exposure to human red cells. The rarity of the M g antigen makes it unlikely that anti-m g will be detected if present. Hybrid Molecules Pronounced SGP modifications occur in hybrid molecules that may arise from unequal crossing over or gene conversion between GYPA and GYPB. Such hybrids have occasionally been noted to carry low-incidence antigens that are due to novel amino acid sequences (reviewed by Reid and Tippett 10 ).

9 Chapter 14: Other Blood Groups 285 Hybrid SGPs may carry the aminoterminal portion of GPA and the carboxy terminus portion of GPB, or vice versa. Other hybrids appear to be a GPB molecule with a GPA insert. The low-incidence antigens Hil (MNS20), St a (MNS15), Dantu (MNS25), and Mur (MNS10), among others, are associated with hybrid SGPs. Some variants are found in specific ethnic groups. For example, the Dantu antigen occurs predominantly in Blacks. Miltenberger Antigens Many of the MNS low-incidence antigens were categorized, based on reactivity with selected sera, into a subsystem, named the Miltenberger system. As more antigens have been identified and knowledge of the genetic events that give rise to these novel antigens has increased, it is clear that the definition of Miltenberger is outdated and these antigens should simply be considered to be glycophorin variants. 11 Many of the corresponding antibodies occur as non-red-cell-stimulated agglutinins, although occasional antiglobulin-reactive examples have been implicated in cases of HDN. Kell Blood Group System (ISBT 006) The K (K1) antigen was first identified in 1946, because of an antibody that caused HDN. The gene responsible (K) ispresent in 9% of Whites and approximately 2% of Blacks. The existence of the expected allele k (determining the antigen K2) was confirmed when an antithetical relationship was established between K and the antigen detected by anti-k, that reacted with the red cells of over 99% of the random population. Kell system antigens are expressed on the red cell membrane in low density (K = 3500 sites, k = sites). The Kell system has been reviewed in Laird-Fryer, Daniels, and Levitt. 12 Anti-K and Anti-k The K antigen is strongly immunogenic, and it is therefore not surprising that anti-k is frequently found in sera from transfused patients. Rare examples of anti-k have appeared as a saline agglutinin in serum from subjects never exposed to human red cells. Most examples are of immune origin and are reactive on antiglobulin testing; some bind complement. Some workers have observed that examples of anti-k react less well in tests that incorporate low ionic strength saline (LISS) solutions (notably the Polybrene test) than in saline tests or tests that include albumin. Others, testing many examples of anti-k in low ionic systems, have not shown differences in antibody reactivity. Anti-K has caused HTRs on numerous occasions, both immediate and delayed. Since over 90% of donors are K-negative, it is not difficult to find compatible blood for patients with anti-k. Anti-k has clinical and serologic characteristics similar to anti-k; it occurs much less frequently, since only about one person in 500 lacks the k antigen, and finding compatible blood is correspondingly more difficult. Other Kell Blood Group Antigens Other antithetical antigens of the Kell system include Kp a (K3), Kp b (K4), and Kp c (K21); Js a (K6) and Js b (K7); K11 and K17; and, possibly, K:14 and K:24. These antigens, along with K and k, are inherited as if produced by alleles at closely linked loci, somewhat analogous to the situation that exists between the major antigens of the Rh system. Not all the theoretically possible genotype combi-

10 286 AABB Technical Manual nations have been recognized in the Kell system. For example, Kp a and Js a have never been found together on the same chromosome. Kp a is predominantly a gene found in Whites, and Js a is found predominantly in Blacks. The haplotype containing K and Kp a has also not been found. Table 14-5 shows some phenotypes of the Kell system. The table also includes the K o phenotype, a null phenotype in which the red cells lack all of the antigens of the system. As in the Lutheran system, several high-incidence antigens have been assigned to the Kell system because the identifying antibodies were found to give negative reactions with the K o red cells, the null phenotype of the Kell system. For simplicity, various high-incidence and low-incidence Kell antigens are not included in the table. Other Antibodies Anti-Kp a, anti-kp b,anti-js a, and anti-js b are all much less common than anti-k but show similar serologic characteristics and are considered clinically significant. Any of them may occur following transfusion or fetomaternal immunization. Antibody frequency is influenced by the immunogenicity of the particular antigen and by the distribution of the relevant negative phenotypes among transfusion recipients and positive phenotypes among donors. In Black patients frequently transfused with blood from Black donors, anti-js a is relatively common in sera that contain multiple antibodies, since the incidence of Js a in a Black population may approach 20%. (See Table 14-5.) Ordinarily, however, these antibodies are rare. Assistance from a rare donor file is usually needed to find compatible blood for patients immunized to the high-incidence antigens k, Kp b,andjs b. Anti-Ku (anti-k5) is the antibody characteristically seen in immunized K o persons. It appears to be directed at a single determinant, since it has not been separable into other Kell specificities. However, antibodies to other Kell system antigens may be present in serum containing anti-ku. Phenotypes with Depressed Kell Antigens K mod is an umbrella term used to describe phenotypes characterized by weak Table Some Phenotypes and Frequencies in the Kell System Reactions with Anti- Frequency % K k Kp a Kp b Js a Js b Phenotype Whites Blacks + 0 K+k 0.2 Rare + + K+k K k Kp(a+b ) Rare Kp(a+b+) 2.3 Rare 0 + Kp(a b+) Js(a+b ) Js(a+b+) Rare Js(a b+) K 0 Exceedingly rare

11 Chapter 14: Other Blood Groups 287 expression of Kell system antigens. Adsorption/elution tests are often necessary for their detection. Some people of the K mod phenotype have made an anti- Ku-like antibody. The K mod phenotype is thought to arise through the inheritance of two recessive genes. Unlike McLeod red cells (see page 288), K mod red cells may exhibit Kx antigen activity elevated even beyond that on K o cells. Red cells of persons with some Gerbich-negative phenotypes also exhibit depressed Kell phenotypes (see Gerbich system, later in this chapter). Persons of the Ge: 2, 3 and Ge: 2, 3, 4 (Leach) phenotypes have depression of at least some Kell system antigens but to a lesser degree than occurs in the McLeod phenotype. The Kp a gene modifies the expression of other Kell genes when in cis position and acts as a down regulator. For example, the k antigen of affected red cells reacts more weakly than expected and, with weaker examples of anti-k, may be interpreted as absent. The gene interaction can be recognized only under certain conditions, ie, when K is present on the opposite chromosome (Kp a is in cis position with k) orwhenthereisak o gene in trans. If serologic tests are performed carefully, the cis modifying effect can also be seen when Kp a is present on both chromosomes. Biochemistry The Kell system antigens are carried on a 93-kDa single-pass red cell membrane protein. Kell system antigens are easily inactivated by treating red cells with sulfhydryl reagents such as 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or 2-aminoethylisothiouronium bromide (AET). Such treatment is useful in preparing red cells artificially lacking Kell system antigens, for identification of Kell-related antibodies. Treatment with sulfhydryl reagents may impair the reactivity of other antigens (LW a, Do a, Do b,yt a, and others), however, so identification of an antibody as being associated with the Kell system because of reduced reactivity with sulfhydryl-treated red cells should only be tentative. As expected, Kell system antigens are also destroyed by ZZAP, a mixture of DTT and cysteine-activated papain that is widely used in immunohematology. This susceptibility to sulfhydryl reagents suggests that disulfide bonds are essential to maintain activity of the Kell system antigens. This hypothesis has been supported by biochemical characterization of Kell proteins deduced from cloned DNA 13 ; these exhibit a number of cysteine residues in the extracellular region. Cysteine readily forms disulfide bonds which contribute to the folding of a protein. Antigens that reflect protein conformation will be susceptible to any agent that interferes with its tertiary structure. The 2.5-kb gene sequence that has been cloned predicts a 732 amino acid protein. The junction of the Kell protein is unknown but it has structural similarities to a family of zinc-binding neutral endopeptidases. It has most similarity with the common acute lymphoblastic leukemia antigen (CALLA or CD10), a neutral endopeptidase on leukocytes. The Kx Antigen (ISBT 019) Two proteins, encoded by different genes, have been associated with Kell system antigens. One gene is autosomal and has been assigned to chromosome This gene gives rise to the 93-kDa Kell protein. The other is X-linked, located on the short arm at Xp21. The normal X-linked allele, designated XK1, encodes a 37-kD protein that carries the Kx antigen. Only trace amounts of Kx are found on red cells of normal Kell phenotypes, but elevated levels of Kx are present on K o red cells. The seeming recip-

12 288 AABB Technical Manual rocal relationship between Kx and Kell antigen expression has suggested that Kx was a precursor of Kell system antigens but no biochemical evidence has emerged to support this theory. The McLeod Phenotype Red cells that lack Kx have, in addition to markedly depressed expression of Kell system antigens, shortened survival, decreased permeability to water, and acanthocytic morphology. This constellation of red cell abnormalities is called the McLeod phenotype, after the first person in whom these observations were made. The term McLeod phenotype may be rather misleading, because possession of different Kell genes causes different antigens to be depressed in different people. Persons with McLeod red cells also have a poorly defined abnormality of the neuromuscular system, characterized by persistently elevated serum levels of the enzyme creatine phosphokinase and, in older people, disordered muscular function. The McLeod phenotype may arise through deletion of the XK locus of chromosome X. The biochemical origins of the molecular lesion responsible for the abnormalities of McLeod red cells are still unclear. In a few instances, the McLeod phenotype has been found in patients with chronic granulomatous disease (CGD), in which granulocytes exhibit normal phagocytosis of microorganisms but inability to kill ingested pathogens. The McLeod phenotype associated with CGD appears to result from deletion of a part of the X chromosome that includes the XK locusaswellasx-cgd.patientswith the McLeod syndrome may be immunized by red cell transfusion and produce anti-kl. This antibody can be shown by adsorption/elution studies to contain anti-kx and anti-km. K o individuals, who lack Kell system antigens, are not suitable donors for McLeod individuals, because K o cells have high Kx expression. Duffy Blood Group System (ISBT 008) The antigens Fy a and Fy b are encoded by a pair of codominant alleles at the Duffy locus on chromosome 1. Anti-Fy a and anti-fy b define the four phenotypes observed in this blood group system, namely: Fy(a+b ), Fy(a+b+), Fy(a b+) and Fy(a b ) (see Table 14-6). In Whites, the first three phenotypes are common and Fy(a b ) individuals are extremely rare. However, the incidence of the Fy(a b ) phenotype among Blacks is 68% and approaches 100% in some areas of West Africa. 7 Homozygosity for a silentduffygene(fy) causes absence of Fy a and Fy b antigens in these individuals. The Duffy gene encodes a glycoprotein that is expressed in other tissues, including brain, kidney, spleen, heart, and lung. In Fy(a b ) individuals, the Duffy gene is selectively turned off in the bone marrow and all Duffy blood group antigens are absent from the red cells. However, the Fy b polymorphism of the protein is expressed in nonerythroid cells. 15 A rare inherited form of weak Fy b called Fy x has been described, although Table Phenotypes and Frequencies in the Duffy System Reactions with Anti- Adult Phenotype Frequency % Fy a Fy b Phenotype Whites Blacks + 0 Fy(a+b ) Fy(a+b+) Fy(a b+) Fy(a b ) V. rare 68

13 Chapter 14: Other Blood Groups 289 the genetic mechanism for the weakened expression is not understood. The Fy x antigen may go undetected unless potent anti-fy b is used in testing. Antibodies to Fy Antigens Anti-Fy a is quite common and may cause HDN and HTRs. Anti-Fy b is rare and is, in general, weakly reactive. Although no HDN has been attributed to anti-fy b,the antibody has been responsible for mild HTRs. Both antibodies are usually IgG and react best by antiglobulin testing. The glycoprotein that expresses the antigens is cleaved by most proteases used in serologic tests, so anti-fy a and anti- Fy b are usually nonreactive in enzyme test procedures. Weak examples of anti-fy a or anti-fy b may give convincing reactions only with red cells that have a double dose of the antigen. In Whites, red cells that express only one of the two antigens are assumed to come from persons homozygous for thegeneandtocarryadoubledoseofthe antigen. In Blacks, however, the Fy gene is so common that red cells having only one of the two antigens are usually from persons heterozygous for Fy. Such cells express the antigen only in single dose, and do not give the expected strong reaction with antibodies that show dosage. Rarely Encountered Antibodies Anti-Fy3 was first described in the serum of a White person of the Fy(a b ) phenotype. It is directed at the high-incidence antigen Fy3; the only cells with which it is nonreactive are Fy(a b ). Unlike Fy a and Fy b, the Fy3 antigen is unaffected by protease treatment and anti-fy3 reacts well with enzyme-treated cells positive for either Fy a or Fy b. Anti-Fy3 is rare, but is sometimes made by Black Fy(a b ) patients who have been immunized by multiple transfusions. Two other rare antibodies have been described, both reactive with papaintreated red cells. One example of anti- Fy4 has been reported. It reacted with red cells of the Fy(a b ) phenotype, and with some Fy(a+b ) and Fy(a b+) red cells from Blacks, but not with Fy(a+b+) red cells, suggesting reactivity with a putative product of the Fy gene. However, the serum gave equivocal results in different reference laboratories and evidence for the existence of the Fy4 antigen is weak. Anti-Fy5 is similar to anti-fy3, except that it fails to react with Rh null red cells that express Fy3, as well as being nonreactive with cells from Fy(a b ) Blacks. It did, however, react with the red cells of the White Fy(a b ) proposita, thus providing a hitherto unrecognized distinction between the Fy(a b ) phenotype so common in Blacks and the one that occurs, but very rarely, in Whites. It has been postulated that Fy5 is formed by the interaction of Rh and Duffy gene products. Anti-Fy6 is a murine monoclonal antibody that describes a high-incidence antigen in the same region as Fy a and Fy b. The antibody reacts with all Fy(a+) and/or Fy(b+) red cells and it is nonreactive with Fy(a b ) red cells. Biochemistry In red cells, the Duffy gene encodes a multipass membrane glycoprotein. The antigens Fy a,fy b, and Fy6 are located on the N-terminus of the Duffy glycoprotein and are sensitive to denaturation by proteases such as ficin, papain, and α- chymotrypsin. The location of Fy3 is not known but the antigen is unaffected by protease treatment (reviewed in Pierce and MacPherson 16 ). The glycoprotein is the receptor for the malarial parasite Plasmodium vivax, and persons whose red cells lack Fy a and Fy b are resistant to that form of the disease. In sub-saharan

14 290 AABB Technical Manual Africa, notably West Africa, the resistance to P. vivax malaria conferred by the Fy(a b ) phenotype has favored its natural selection and most individuals are Fy(a b ). The Fy locus is located on chromosome 1, near the centromere. It is syntenic with the Rh locus. The gene has been cloned 14 and the Duffy glycoprotein has been identified as an erythrocyte receptor for a number of chemokines, notably IL Because chemokines are very biologically active molecules, it has been postulated that Duffy acts as a sponge for excess chemokines, without ill effect on the red cells. Kidd Blood Group System (ISBT 009) Anti-Jk a and Anti-Jk b Anti-Jk a was first recognized in 1951 in the serum of a woman who had given birth to a child with HDN. Two years later, anti-jk b was found in the serum of a patient who had suffered a transfusion reaction. Both antibodies react best on antiglobulin testing, but saline reactivity is sometimes observed in freshly drawn specimens. These antibodies are often weak when first detected and, perhaps because they are detected indirectly through the complement that they bind to red cells, some may become undetectable on storage. Serum containing weak anti-jk a or anti-jk b, even when freshly drawn, may manifest a dosage effect, reacting only with red cells expressing a double dose of the antigen. Effects of Complement Some workers report no difficulties in detecting anti-jk a and anti-jk b antibodies in low ionic tests that incorporate anti-igg, but others find that an antiglobulin reagent containing an anticomplement component is important for the reliable detection of these inconsistently reactive antibodies. Many serologists believe that red cells carrying a double dose of Jk a or Jk b are needed for reliable detection in antibody screening tests. Anti-Jk a and anti-jk b sera that have lost their reactivity during storage can sometimes be revived by the addition of fresh human serum as a source of complement, or the use of enzyme-treated red cells in antiglobulin testing. Clinical Significance Kidd system antibodies occasionally cause HDN, but the HDN is usually mild. These antibodies are notorious, however, for involvement in severe HTRs, especially delayed hemolytic reactions (DHTRs). DHTRs occur when antibody develops so rapidly in an anamnestic response to antigens on transfused red cells that it destroys the still-circulating red cells. In many cases, retesting the patient s pretransfusion serum confirms that the antibody was, indeed, undetectable in the original tests. This highlights the importance of consulting previous records before selecting blood for transfusion. Patients whose antibody has previously been detected and identified can, by review of past records, be protected against repeated contact with the known immunizing stimulus. Genes and Phenotypes The four phenotypes defined by the reactions of anti-jk a and anti-jk b are shown in Table The Jk(a b ) phenotype is extremely rare, except in some populations of Pacific Island origin, but two different mechanisms have been shown to produce it. 18 One is, apparently, the homozygous presence of the silent Jk allele. The other is the action of a dominant inhibitor gene called In(Jk). The dominant suppression of Kidd antigens

15 Chapter 14: Other Blood Groups 291 Table Phenotypes and Frequencies in the Kidd System Reactions with Anti- Adult Phenotype Frequency % Jk a Jk b Phenotype Whites Blacks + 0 Jk(a+b ) Jk(a+b+) Jk(a b+) Jk(a b ) Exceedingly rare is similar to the In(Lu) suppression of the Lutheran system. (See next section.) Sera from some rare JkJk persons have been found to contain an antibody that reacts with all Jk(a+) and Jk(b+) red cells but not with Jk(a b ) red cells. Although a minor anti-jk a or anti-jk b component is sometimes separable, most of the reactivity has been directed at an antigen called Jk3, considered to be present on both Jk(a+) and Jk(b+) red cells in a relationship analogous to that of Fy3 on Fy(a+) and Fy(b+) red cells. Anti-Jk3 is usually red-cell-stimulated. The Jk a and Jk b antigens are located on the urea transporter and Jk(a b ) red cells are resistant to lysis by 2M urea. Red cells of normal Jk phenotype swell and lyse rapidly in a solution of 2M urea. In contrast, Jk(a b ) red cells are resistant to hemolysis for long periods of time. The Jk blood group locus has been assigned to chromosome 18. Other Blood Group Systems So far this chapter has been devoted to systems of red cell antigens of which the principal antibodies may be seen fairly frequently in the routine blood typing laboratory. The other blood group systems listed in Table 14-1 will be reviewed here only briefly; the interested reader should refer to other texts and reviews for greater detail. Antibodies directed at antigens of these blood groups occur rarely, largely because the phenotypes of the paired antigens reveal one antigen to have high incidence and the other a low incidence, which greatly restricts opportunities for immunization. The antigens and their distribution may be important in genetic investigations and population or family studies. Lutheran Blood Group System (ISBT 005) The first example of anti-lu a (-Lu1) was found in 1945 in a serum that contained several other antibodies. The phenotypes of the Lutheran system as defined by anti-lu a and anti-lu b (-Lu2) are shown in Table The Lu(a b ) phenotype is very rare and may arise from one of three distinct genetic circumstances (reviewed in Pierce and MacPherson 15 ). In the first, a presumably amorphic Lutheran gene (Lu) isinherited from both parents. In the second, the negative phenotype is inherited as a dominant trait attributed to the independently segregating inhibitor gene Table Phenotypes and Frequencies in the Lutheran System in Whites Reactions with Anti- Phenotype Lu a Lu b Phenotype Frequency % + 0 Lu(a+b ) Lu(a+b+) Lu(a b+) Lu(a b ) Very rare Insufficient data exist for the reliable calculation of frequencies in Blacks.

16 292 AABB Technical Manual In(Lu), which prevents the normal expression of Lutheran and certain other blood group antigens (notably P 1, I, AnWj, In a,andin b ). The third Lu(a b ) phenotype is due to an X-borne suppressor, recessive in its effect. Anti-Lu a and anti-lu b are not often encountered. They are produced in response to pregnancy or transfusion, but have occurred in the absence of obvious red cell stimulation. Lutheran antigens are poorly developed at birth, so it is not surprising that anti-lu a has not been reported as the cause of HDN; neither has it been associated with HTRs. Anti- Lu b has been reported to shorten survival of transfused red cells but causes no, or at most very mild, HDN. Most examples of anti-lu a and some anti-lu b will agglutinate saline-suspended red cells possessing the relevant antigen, characteristically producing a mixedfield appearance with small to moderately sized, loosely agglutinated clumps of red cells interspersed among many unagglutinated red cells. Other Lutheran Blood Group Antigens A series of high-incidence antigens (LU4, LU5, LU6, LU7, LU8, LU11, LU12, LU13, LU16, LU17, and LU20) have been assigned to the Lutheran system because the corresponding antibodies do not react with Lu(a b ) red cells that reflect any of the three genetic backgrounds. Two lowincidence antigens, LU9 and LU14, have gained admission to the Lutheran system because of their apparent antithetical relationship to the high-incidence antigens LU6 and LU8, respectively. Au a (LU18), an antigen of high incidence [80% of Whites are Au(a+)] and its antithetical partner, Au b (LU19), present in 50% of Whites, were once considered an independent polymorphism but have now been shown to belong to the Lutheran system. 19 Gene Linkage and Chromosome Assignment The Lu and Se (secretor) loci were shown to be linked in 1951, the first recorded example of autosomal linkage in humans. The two loci have been assigned to chromosome 19. The gene encoding the Lu glycoproteins has been cloned. 20 Biochemistry of Lutheran Lutheran antigens are carried on two glycoproteins carrying both N-linked and O-linked oligosaccharides. The antigens are destroyed by trypsin, α-chymotrypsin and sulfhydryl-reducing agents. 21 These results, along with results of immunoblotting experiments, suggest the existence of interchain or intrachain disulfide bonds. Tests performed with monoclonal anti-lu b suggest that the number of Lu b antigen sites per red cell is low, approximately per Lu(a+b+) red cell and per Lu(a b+) red cell. 22 Diego Blood Group System (ISBT 010) The Diego system consists of two independent pairs of antigens, called Di a /Di b and Wr a /Wr b. Each pair contains a lowincidence antigen and an antithetical high-incidence determinant. (See Table 14-9.) The antigens are located on the anion transporter (AE-1) which is encoded by a gene on chromosome 17. The Di a /Di b antigens are useful as anthropological markers since the Di a antigen is almost entirely confined to populations of Mongoloid origin, including Native Americans, where incidence of Di a can be as high as 36%. Anti-Di a may cause HDN or destruction of transfused Di(a+) red cells. Anti-Di b is rare, but clinically significant when encountered. The Wr a and Wr b antigens are located on AE-1 in close association with GPA; expression of Wr b is dependent upon the presence of GPA (see MNS Blood Group

17 Chapter 14: Other Blood Groups 293 Table Phenotypes and Frequencies in the Diego System in Whites Reactions with Anti- Di a Di b Phenotype Phenotype Frequency % + 0 Di(a+b ) 0 * + + Di(a+b+) very rare 0 + Di(a b+) 100 Wr a Wr b + 0 Wr(a+b ) Wr(a+b+) Wr(a b+) 99 *The Di a antigen has a much higher incidence in Orientals and Native Americans. System). Anti-Wr a occurs without red cell stimulation; and it may be fairly common but passes undetected unless the cells used for antibody screening are Wr(a+). It is a rare cause of HTR or HDN. Yt Blood Group System (ISBT 011) The Yt blood group system consists of two antigens, Yt a and Yt b, the occurrence of which parallels the incidence of K and k in a White population (see Table 14-10). The antigens are encoded by a gene on chromosome 7. Most examples of anti-yt a are benign, although in a few cases, accelerated destruction of transfused Yt(a+) red cells has been observed. Prediction of clinical outcome by the Table Phenotype Frequencies in Other Blood Group Systems with Codominant Antithetical Antigens Phenotype Frequency % in System Reactions with Anti- Phenotype Whites * Yt Yt a Yt b + 0 Yt(a+b ) Yt(a+b+) Yt(a b+) 0.2 Dombrock Do a Do b + 0 Do(a+b ) Do(a+b+) Do(a b+) 33.3 Colton Co a Co b + 0 Co(a+b ) Co(a+b+) Co(a b+) Co(a b ) very rare Scianna Sc1 Sc2 + 0 Sc:1, Sc:1, Sc: 1,2 very rare 0 0 Sc: 1, 2 very rare Indian In a In b + 0 In(a+b ) very rare + + In(a+b+) < In(a b+) >99 * There are insufficient data for reliable calculation of frequencies in Blacks.

18 294 AABB Technical Manual monocyte monolayer assay has proved successful. 23 Anti-Yt a is not known to cause HDN. Anti-Yt b is rare, and has not been implicated in HTR or HDN. The Yt antigens are located on red cell acetylcholinesterase (AChE), 24 an enzyme important in neural transmission but whose function on red cells is unknown. Table Frequencies of the Xg(a+) and Xg(a ) Phenotypes in White Males and Females Phenotype Frequency % Phenotype Males Females Xg(a+) Xg(a ) Frequencies are based on combined results of testing nearly 7000 random blood samples from populations on Northern European origin. There are insufficient data for reliable calculation of frequencies in Blacks. Xg Blood Group System (ISBT 012) In 1962, an antibody was discovered that identified an antigen more common among women than among men. This would be expected of an X-borne characteristic, since females inherit an X chromosome from each parent, whereas males inherit X only from their mother. The antigen was named Xg a in recognition of its X-borne manner of inheritance. Table gives the phenotype frequencies among White males and females. Anti-Xg a is an uncommon antibody that usually reacts only on antiglobulin testing, although at least three examples are known that agglutinated saline-suspended red cells. Enzymes, such as papain and ficin, denature the antigen, so negative reactions are to be expected in enzyme test systems. Anti-Xg a has not been implicated in HDN or HTRs. One example has been reported as an autoantibody. Anti- Xg a may be useful for tracing the transmission of genetic traits associated with the X chromosome, although linkage with the Xg locus has been demonstrated for few traits to date. The gene encoding Xg a has been cloned. 25,26 Scianna Blood Group System (ISBT 013) Three antigens, Sc1, Sc2, and Sc3, are recognized by the ISBT working party as belonging to the Scianna blood group system. Sc1 is a high-incidence antigen, whereas Sc2 occurs very infrequently. Sc1 and Sc2 behave as products of antithetical alleles. (See Table ) Sc3 is thought to be present on the red cells of any individual who inherits a functional Sc1 or Sc2 gene, analogous to Fy3. The antigens are resistant to enzymes routinely used in blood group serology. The gene encoding the Scianna antigens is located on chromosome 1. The antibodies are rare. Anti-Sc1 has not been reported to cause HTR or HDN. Anti-Sc2 has caused mild HDN. The Scianna blood group antigens have been assigned to a glycoprotein of kda, but as yet, the function of the protein is unknown. The low incidence antigen Rd (700015) is encoded by a gene on chromosome 1 at the same locus as SC and there is some biochemical evidence that the antigen is part of the Scianna blood group system. Dombrock Blood Group System (ISBT 014) Initially, this blood group system consisted of a pair of antithetical antigens, Do a and Do b, with phenotype frequency as shown in Table The discovery that red cells negative for the high-incidence antigen Gy a were Do(a b ) has led to the recent expansion of the Dombrock blood group system. 27 Three high-incidence antigens are now included: Gy a,

19 Chapter 14: Other Blood Groups 295 Hy, and Jo a. The interrelationship of the phenotypes is shown in Table Gy(a ) red cells represent the null phenotype. The Gy(a+ w ) Hy, and the Jo(a ) phenotypes have been found exclusively in Blacks. Reactivity may be enhanced by papain or ficin treatment of red cells; however, the antigens are weakened or destroyed by pronase, trypsin, α-chymotrypsin, and sulfhydryl reagents. Anti-Do a is an uncommon antibody, rarely found as a single specificity. It has caused mild HTR and HDN. Anti-Do b is also uncommon but may be found in sera containing multiple specificities, which can make its detection and identification difficult. Some workers have reported enhanced antibody detection by the use of ficin-treated red cells. 28 HDN due to anti-do b has not been reported, but examples have caused HTR. Antibodies to Gy a, Hy, and Jo a may cause shortened survival of transfused antigen-positive red cells or mild HDN. The Dombrock antigens are located on glycoprotein of kda, the function of which is unknown. 22 The location for the determining gene is unknown. Colton Blood Group System (ISBT 015) The Colton blood group system consists of: Co a, a high-incidence antigen; Co b,a low-incidence antigen; and Co3, an antigen (like Fy3) considered to be the product of either the Co a or Co b gene. The antigens are products of a gene on chromosome 7. The phenotype frequencies in Whites are shown in Table Enzyme treatment of red cells enhances reactions with the defining antibodies. Anti-Co a and anti-co3, though rare, have been implicated in HTR and HDN. 29 Anti-Co b has caused a HTR, 29 but there are no reports of HDN. The Colton antigens have been located on the membrane protein CHIP 28 which functions as the water transporter. 30 The LW Blood Group System (ISBT 016) In early investigation of the Rh system, evidence accumulated that the antigen identified in the animal studies of Landsteiner and Wiener was not identical to that defined by the human serum described by Levine and Stetson. Specificity appeared at first to be similar because D-positive red cells reacted significantly stronger with animal antisera than did D-negative red cells, which the original investigators accordingly interpreted as negative. It is now recognized that nearly all human red cells possess the antigen on Rhesus monkey red cells that stimulated the original animal antibodies. D-negative red cells from adults have comparatively weak expression but Table Relationship of the Dombrock Blood Group Antigens Phenotype Do a Do b Gy a Hy Jo a Normal Do(a+b ) Normal Do(a+b+) Normal Do(a b+) Gy(a ) Hy (+) (+) Jo(a ) (+) + (+) (+)=weak antigen expression