Methods of RBC Alloimmunization to ABO and Non-ABO Antigens, and Test Methodologies

CHAPTER 2

Methods of RBC Alloimmunization to ABO and Non-ABO Antigens, and Test Methodologies KRISTIN STENDAHL, MD • CHRISTOPHER A. TORMEY, MD • IAN L. BAINE, MD, PHD

INTRODUCTION Red blood cell (RBC) transfusions are a vital part of clinical patient care and can be a critical, life-saving procedure. According to Center for Disease Control and Prevention 2015 National Collection and Utilization Survey (NBCUS), 12,591,000 whole-blood derived and apheresis RBC units were collected and approximately 11,349,000 whole-blood derived and apheresis RBC units were transfused at acute care hospitals in the United States.1 Blood transfusions only became routine procedures less than 100 years ago. The first successful transfusion of human blood to a patient occurred in 1818 by a British obstetrician, James Blundell, for treatment of postpartum hemorrhage.2,3 The goal of this chapter is to provide readers with a basic foundation of blood group antigens, antibodies, and blood group systems. With this foundation, the process of RBC alloimmunization will be reviewed including various factors that impact the rate of alloimmunization as well as special considerations for populations at risk for high rates of alloimmunization. Lastly, a brief discussion of the most common testing methodologies as well as emerging molecular techniques will be reviewed.

BLOOD GROUP ANTIGENS Blood group antigens are clinically important due to their ability to react with antibodies that are formed in antigen-negative individuals after exposure to antigen-positive RBCs either through transfusion or pregnancy with the potential to cause hemolytic transfusion reactions or hemolytic disease of the fetus and newborn (HDFN). The RBC membrane has 23 major antigens present on the extracellular surface that comprised both proteins and carbohydrates. The

specificity of blood group antigen is determined either by oligosaccharide epitopes such as ABO antigens or by the amino acid sequence such as the Rh and Kell antigens. The variable RBC antigens arise due to polymorphisms in the genes encoding these proteins, such as single nucleotide polymorphisms, insertions/deletions, or gene rearrangements.4,5 RBC antigens are involved in several functions including RBC membrane structural integrity, transport proteins, cell adhesion, ligand and/ or microbe receptors, complement regulatory proteins, and numerous other functions.6 Many of the antigens are integrated into the membrane of the red cell; however, others are soluble antigens that are adsorbed onto the surface of the red cell. The portions of the antigens that are recognized by the immune system are termed immunodominant epitopes. The immunodominant epitopes consist of linear stretches of sugars or amino acids, or of amino acids that are distant from each other in primary sequence, but assume a threedimensional protein structure that brings the amino acid sequences near each other to form a “conformational” antigen (see Fig. 2.1). For carbohydrate antigens, this usually is up to seven residues long, and most often involves the terminal nonreducing sugar. One important example of such an epitope is the A/B/H glycoprotein/glycolipid antigens. For protein antigens, there are two major groups on the RBC membrane, termed Type 1 and Type 2 proteins. Type 1 proteins pass through the cell membrane once (they resemble a “stick floating upright in water”), and therefore have a single extracellular domain, a single transmembrane domain, and a single intracellular domain. By contrast, Type 2 transmembrane proteins make multiple passes through the cell membrane (resembling the “Loch Ness Monster in a lake”). As such, they have multiple extracellular, intracellular,

Immunologic Concepts in Transfusion Medicine. https://doi.org/10.1016/B978-0-323-67509-3.00002-0 Copyright © 2020 Elsevier Inc. All rights reserved.

15

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Immunologic Concepts in Transfusion Medicine

Antibody

Antibody

Linear Epitope (e.g. Kell)

Conformational Epitope (e.g. RhD)

Extracellular Space Red Blood Cell FIG. 2.1 Immunodominant Epitopes. (Adapted from Maitta RW. Clinical Principles of Transfusion Medicine. Elsevier; 2018.)

and transmembrane domains. Therefore, antibodies formed against a given protein may be directed against multiple extracellular loops of the same polypeptide chain, as it traverses in and out of the RBC membrane.

BLOOD GROUP ANTIBODIES AND THEIR RESPONSE TO RED BLOOD CELL ANTIGENS RBC antibodies are classified as either alloantibodies, those produced by individuals against antigens that are not present on their RBC surface, or autoantibodies, those produced by individuals against antigens present on their own RBC surface. In addition, antibodies can be divided into two major categories, naturally occurring antibodies, those formed after exposure to environmental antigens that are similar to RBC antigens, and induced antibodies, those formed after an immune response to red cells through transfusion or pregnancy. Naturally occurring antibodies, which are generally present at the time of birth or very early in life, arise either from exposure to environmental antigens that have similar structure to red cell antigens or spontaneously. Animal studies in the 1940s and 1950s by Landsteiner and Weiner showed that exposure to pathogens such as certain strains of Escherichia coli, which contain antigens similar to the group B RBC antigens resulted

in anti-B alloimmunization in mice who were never exposed to the B antigen directly.8 This is likely due to cross-reactivity of anti-Escherichia coli antibodies with the B antigen, which has further been supported by findings in humans as well.9e12 Examples of antibodies that are thought to arise spontaneously include those directed against the Rh-E antigen, as well as Lua, Dia, and others.13 In general, naturally occurring antibodies, whether due to exposure to an antigenic mimic or spontaneously, are formed against carbohydrate antigens (with the exception of anti-E). In addition, naturally occurring antibodies are predominantly IgM, but can contain at least a partial IgG component, with pure IgG antibodies being rare.14 Induced antibodies, such as those against the Rh-D, K, Jk, and Fy antigens are only produced after exposure to antigen-positive RBCs via transfusion or pregnancy. The alloimmune responses consist of a gradually forming, weak primary immune response followed by a rapid, strong secondary immune response, with the secondary response being responsible for the clinical manifestations of immunologic transfusion reactions. Studies using Rh-D alloimmunization as the model have demonstrated that alloantibodies are detected by standard serologic methods beginning at approximately 4 weeks after exposure, and reach maximum

CHAPTER 2 concentration between 6 and 10 weeks.15 Studies using other antigens have also demonstrated that the kinetics of primary and secondary responses vary by antigen, but follow the same paradigm as that seen with RhD.16 Consistent with our understanding of immunogenicity of antigens, there appears to be a minimum threshold model of exposure for RBC antigens, above which is sufficient to induce an alloantibody response, and below which does not.17,18 Whether a recipient will respond or not and the degree to which the recipient will respond to allogeneic RBC exposure will vary based on numerous factors intrinsic to the RBC as well as the patient, which will be discussed later in this chapter.

BLOOD GROUP SYSTEMS A blood group system consists of inherited RBC antigens defined by specific antibodies. There are over 30 blood group systems currently recognized, each encoded by individual genes.19 The following discussion is limited to the most clinically relevant blood group systems, including ABO, Rh, Kell, Kidd, Duffy, and MNS blood group systems because of their association with clinically significant antibodies.

ABO Blood Group System The ABO and H blood groups are the oldest and most important blood group system in transfusion medicine due to their high immunogenicity. The ABO system was originally discovered in 1900 by Austrian scientist, Karl Landsteiner, for which he later received the Nobel prize in 1930. Landsteiner used agglutination tests on glass slides to demonstrate the presence of groups A, B, and O (originally termed C) and later AB in 1902.3 The ABO antigens are carbohydrate antigens that are present not only on the surface of RBCs and other hematopoietic cells, but also on organ tissues throughout the body including the kidney, heart, and lung, thus are considered histocompatibility antigens for the purposes of organ transplantation. According to the central dogma of molecular biology, DNA is transcribed into RNA, which is subsequently translated to proteins. However, the ABO and H antigens are carbohydrates antigens attached to the terminal amino acid residues of multiple surface glycoproteins on tissues, including RBCs. The structural base on which both the A and B antigens are built is the H substance, also termed the O antigen. This represents an a1-2 linkage between the terminal galactose and an L-fucose sugar, catalyzed by the enzyme a-2-L-fucosyltransferase (H transferase), coded for by the H gene, an enzyme present in more than 99.99% of the

Methods of RBC Alloimmunization

17

general population. Once the H substance is formed, it can be further modified by the action of two other enzymes to produce either the A or B antigens. Rather than coding for the sugars themselves, the ABO genetic locus at 9q34 has three alternative major alleles, A, B and O, which codes for alternative forms of an enzyme that functions as glycosyltransferases. The alleles coding for A and B differ in just three critical amino acid residues G235S, L266M, and G268A.20 These mutations determine the substrate specificity of the enzyme, and therefore determine the type of sugar that the enzyme will add to the H substance and the resulting antigen formed. The enzyme a-3-N-acetylgalactosaminyltransferase (A transferase), coded by the A allele, can transfer a single N-acetylgalactosamine (GlcNAc) in an a1-3 linkage to the terminal galactose to make the A antigen. Alternatively, the enzyme a1,3-galactosaminyl transferase (B transferase), coded by the B allele, transfers a galactose in a a1-3 linkage to the terminal galactose to make the B antigen. If both isoforms of the A and B genes code for nonfunctional forms of the aforementioned two enzymes, then neither modification is made to the terminal galactose, and the H substance remains untouched; it is then termed the O antigen (Fig 2.2). Conversely, if both of the genes are expressed, both the A and B chains will be produced, and the patient will have the phenotype of Group AB. If the genotype is AA or AO, the patient will express the A antigen only. Similarly, the patient will express the B antigen only with a BB or BO genotype. The genes coding for these varying glycosyltransferases, and thus, for the different A/B/O phenotypes, is present at varying frequencies in the U.S. population depending on ethnicity, as outlined in Table 2.1. In addition to allelic variants that simply code for the A, B, or O forms of the glycosyltransferases, there are additional genetic mutations that can alter the level of catalytic activity of the enzyme giving rise to what is termed ABO “subgroups.” Subgroups are distinguished by decreased amounts of A, B, or O (H) antigens on the RBC surface. This bears the most relevance for how it affects the enzyme responsible for the formation of the A antigen, as A-antigen subgroups are the most common. Review of the A-antigen subgroup will be the primary focus here, as other reference texts cover this topic more comprehensively.21,22 In approximately 80% of individuals who express the A antigen, the a-3-N-acetylgalactosaminyltransferase has a relatively high level of enzymatic activity, resulting in the production of approximately 1
106 A antigens on the surface of a single RBC. This isoform of the enzyme, and its resulting high activity level, is termed A1. By contrast, the

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Immunologic Concepts in Transfusion Medicine

TABLE 2.1

Diversity of ABO Phenotypes in the United States ABO Type

Caucasian (%)

Black (%)

Asian (%)

Hispanic (%)

O

45

50

40

56

A

40

28

28

31

B

11

20

25

10

AB

4

4

7

3

remaining 20% of individuals express a relatively inefficient form of the a-3-N-acetylgalactosaminyltransferase enzyme, termed A2., resulting in approximately 2
105 A antigens per RBC.23 The difference between A1 and A2 is important, as individuals who express the A2 phenotype or A2B phenotype are capable of making an anti-A1 antibody. These individuals may demonstrate an anti-A on serological typing, potentially confounding results. Furthermore, this anti-A1 can sometimes be reactive at 37 C, making it clinically significant. Therefore, A2 patients should only receive transfusions from O or A2 donors, and A2B patients should only be transfused with O, A2, A2B, or B donors. There are also numerous other isoforms of the enzyme that catalyzes A antigen

formation, including A3, Ax, Am, and Ael, the details of which are beyond the scope of this text . The ABO antigens are present on RBCs as early as 5e 6 weeks of gestation, and reach adult levels of expression between 2 and 4 years old due to the increasing levels of branching with age in the precursor chains on which the ABO and H antigens are built. Critically important is the fact that antibodies to the A and/or B antigens form in children by 1 year old, even in the absence of deliberate exposure to these antigens; therefore, ABO antibodies are considered naturally occurring antibodies. It is thought that these antibodies develop spontaneously due to exposure to environmental bacteria with antigens similar to the A and B antigens. Thus,

GTA Enzymatic Activity

A Antigen

O Antigen (aka H antigen)

H Enzyme Activity

GTB Enzymatic Activity

B Antigen

FIG. 2.2 Structure of A/B/O antigens.

CHAPTER 2 natural anti-A and anti-B are likely antibodies to other antigens that cross-react with RBC ABO antigens. The ABO blood group system is defined by the presence of A and B antigens on the RBC surface and anti-A and anti-B antibodies in the serum. Therefore, patients expressing A antigen will make anti-B antibodies, patients expressing B antigen will make anti-A antibodies, patients expressing O will express both anti-A and antiB antibodies, as well as an additional third antibody that can react with either A or B, termed anti-A,B antibody. Patients expressing the AB phenotype will not make any anti-ABO antibodies, which is why their plasma is potentially valuable as universal donor units. Anti-A and anti-B antibodies are IgM class antibodies with a broad thermal amplitude, agglutinating cells most efficiently at around 25 C, but can also fix complement and lyse RBCs at 37 C. They cause rapidonset acute intravascular hemolytic reactions, with associated complications that arise from such a reaction including thrombosis from free hemoglobin exposure and acute renal failure. The anti-A,B antibody formed by Type O patients is an IgG class antibody. Therefore, while anti-A and anti-B cannot cross the placenta in ABO-mismatched maternalefetal scenarios, the IgGclass anti-A,B can, and in fact it is the most common cause of HDFN. Importantly though, it does not manifest as severely as other antigen mismatches such as anti-D or anti-K, and fetal anemia is often less severe or absent. Anti-A, anti-B, and anti-A,B antibodies are present at varying titers throughout life, but are always detectable, and do not demonstrate senescence.

Rh(D) Blood Group System After the ABO blood group, the Rh blood group system is the second most antigenic and clinically important of all RBC antigens, with the D antigen being the most clinically important within this system. Alloantibodies to Rh antigens represent some of the most common alloantibodies identified, and are generally considered clinically significant. There is up to an 80% risk of alloantibody formation when Rh(D)-negative patients are exposed to D antigen-positive blood, which is responsible for the most severe forms of HDFN.24,25 More than 50 independent Rh antigens have been described with the most important clinically including D, C/c, and E/e. These five major Rh antigens were identified in the 1940s, including Rh-D, C/c, and E/e, with C/c and E/e being alternative alleles. By contrast, there is no alternative allele to the D antigen; it is either expressed or not expressed on the RBC surface. Originally, it was uncertain as to whether these antigens were coded for by a single gene (RhDCE) or by three

Methods of RBC Alloimmunization

19

separate genes (RhD, RhC, and RhE), however, it was later discovered by Tippett in 1980s that all Rh antigens are coded for by two genes, RhD and RhCE, both located on chromosome 1.26e28 RhD and RhCE are highly homogenous genes, each comprising 10 exons and separated from each other by approximately 30 kb.29,30 The RhD gene encodes the D antigen, and the RhCE gene encodes the RhCE protein to produce the Cc and Ee antigens. The RhD and RhCE genes code for transmembrane proteins, each 417 amino acid residues in length, that are nearly identical in structure. Each protein makes 12 passes through the RBC membrane, resulting in six extracellular domains. Two point mutations in different extracellular loops of the RhCE protein, which result in two amino acid substitutions, S103P and P226A, differ between the C/c and E/e alleles, respectively.13 The phenotypic frequencies of Rh antigen expression vary by ethnic background, and are listed in Table 2.2. A third gene, RhAG, located on chromosome 6, encodes for an Rh-associated glycoprotein (RhAG), which is needed for expression of Rh antigens. As mentioned earlier, only the D allele (denoted by a capital D) can be expressed; a “d” allele does not exist; it is either deleted or mutated resulting in the silencing of its expression, which is denoted as a lowercase d when considering the genotype. Therefore, a patient’s genotype can have one expressed copy of RhD (D/d), two expressed copies of RhD (D/D), or no expressed copies (d/d). Phenotypically, patients with either the D/D or D/d genotype will express the D antigen, and are said to be RhDþ (“Rhþ”). In contrast, patient’s with the d/d genotype will not express any D antigen on the RBC surface, and will be RhD (“Rh”). In contrast to the D allele, the C and E alleles are inherited like normal allelic variants as CE, cE, Ce, or ce, with both alleles of the RhCE gene being expressed. It should also be noted that advances in molecular sequencing of the Rh genes have identified at least 58 different Rh phenotypic variants, some of which may be clinically significant in certain scenarios, and that genetic recombination events can create novel hybrid genes between RhD and RhCE; discussion of both of these concepts is beyond the scope here.21,31 Using the foundation built in earlier chapters, we can expect that an RhD patient who is transfused with RhDþ RBCs will form alloantibodies to the RhD antigen. Alloimmunization to the D antigen can occur to any of the extracellular loops or combination of loops that may function as linear or conformational epitopes. Numerous antigenic variants with at least 30 epitopes have been defined to date.32,33 The antigenicity of the D antigen depends on its density on the transfused RBC. Patients may express the D antigen at a

20

Immunologic Concepts in Transfusion Medicine

TABLE 2.2

Rh Antigen Frequency Based on Ethnicity. Rh Antigen

Caucasian (%)

Black (%)

Asian (%)

Hispanic (%)

D

85

92

99

93

C

68

27

93

71

E

29

22

39

41

c

80

96

47

64

e

98

98

96

81

much lower level; this is termed the “weak D” antigen.34 Although the extracellular portions of the weak D antigen are the same as those in normal RhDþ patients, the weak D antigen results from numerous amino acid changes that result in impaired efficiency of protein insertion in the cell membrane and decreased stability of the RhD protein in the RBC membrane, resulting in reduced expression of D protein epitopes at the surface. Weak D patients may test as RhD-negative by serologic methods; however, if weak D RBCs are transfused into a truly RhD patient, the patient may alloimmunize or experience a hemolytic transfusion reaction. Conversely, weak D patients who are pregnant with an RhD fetus will not need Rh immune globulin (RhIg) prophylaxis, despite serologic negative testing. The most common weak D phenotype, RhD weak type 1, is the result of the nucleotide substitution 89T > G.35 Alternatively, another mechanism by which patients may express lower levels of D antigen a phenomenon known as C-in trans/Cipellini effect, in which patients with the C allele present in trans to the D gene (i.e., on the other copy of chromosome 1), express lower levels of D protein on the RBC surface.21 These patients may also test serologically as RhD; however, alloimmunization or hemolytic reactions can occur if their RBCs are transfused into true RhD patients. Other forms of weak D expression exist such as Del; however, discussion is beyond the scope of this text. Most important clinically is that blood donors who express even small amount of D antigen must be labeled Rh(D)þ to prevent alloimmunization to RhD patients. As such, AABB Standards require that D-negative blood donors are tested for weak D.36 In addition, women of childbearing age with RhD phenotyping suggestive of a possible weak D should undergo RhD genotyping, as weak D types 1, 2, or 3 do not need prophylactic RhIg during pregnancy and can receive RhDþ transfusions.

In addition to the weak D antigen, there is another variant known as the “partial D” antigen. Partial D antigen is formed by a hybrid gene rearrangement between the RhD and RhCE, resulting in a novel RhDlike protein that is missing certain residues in the extracellular portions of the protein, present in the normal RhD protein. As a result, the host is able to form antibodies against these missing residues, and can develop an anti-RhD alloantibody if transfused with RhDþ cells. Partial D antigen can be identified via extended anti-D serologic testing with a panel of anti-D reagents, or by molecular sequencing. Clinically, patients with partial D should be considered D-negative and blood donors with partial D should be considered D-positive. The anti-C/c and anti-E/e antibodies are produced in response to different epitopes on the RhCE protein. Similar to RhD, the RhCE protein makes 12 passes through the RBC membrane, resulting in six extracellular domains. The C and c antigens differ by a single amino acid substitution, S103P, and the E and e antigens differ by P226A. As mentioned earlier, there are two alleles of RhCE, both of which are expressed. Hence, C/c/E/e antigen expression follows typical Mendelian rules of inheritance, but are codominantly expressed. Similar to RhD, there are several variants of RhCE, with a variant E antigen being the most common, however, still quite rare; these are discussed more fully in other resources.22,37,38 Antibodies to the Rh system can be directed against the D, C/c, and E/e antigens, or any variants thereof. For the purposes of simplicity, we will only focus on anti-RhD (anti-D), anti-RhC/c (anti-C or c), and anti-Rh-E/e (anti-E or e). The D antigen is the most immunogenic of the Rh system, with experimental studies showing up to an 85% alloimmunization rate.25 Transfusion of even a portion of 1 unit of Rh-incompatible cells is adequate to elicit an antibody response. Interestingly, the

CHAPTER 2 remaining 15% of patients that do not form detectable anti-D antibodies upon first exposure are termed “nonresponders”; however, half of this group will eventually form an anti-D antibody upon subsequent exposure to Rh-incompatible cells. In addition, retrospective studies of D-negative patients who received numerous intraoperative Dþ transfusions showed an alloimmunization rate of 95% when detected with enzyme-treated cells. Studies of patients who received fewer or a single unit show lower rates of alloimmunization, but still at significant rates, especially when “boosted” with a second antigen exposure 6 months later.15,39,40 Interestingly, immunosuppressed patients make anti-D at relatively low rates, underscoring the role of the recipient’s immune status at the time of antigen exposure. Anti-D antibodies can wane over a period of many years, but have been detected up to 38 years following exposure.41 Nonetheless, even if the antibody is not detected by serologic methods, the patient will remain immunized to the D antigen for the remainder of their life. Anti-D is an IgG class antibody, it can cross the placenta and cause a severe form of HDFN. Maternal formation of anti-D antibody can be seen as early as 2 months and as late 5 months, and does not form more rapidly if exposed multiple times. Although anti-D IgG1 and IgG3 subclasses have been demonstrated, anti-D generally does not fix complement efficiently, and more often results in a serologic transfusion reaction, with extravascular clearance of antigen-positive RBCs. Due to its IgG nature, anti-D is primarily detected via the indirect antiglobulin test (IAT), though some antisera can agglutinate RBCs directly. Formation of anti-D antibodies in patients has become increasingly rare due to the introduction of RhIg prophylaxis in the late 1960s combined with the standardized use of RhD typing to detect patients with weak D and partial D variants. RhIg acts by binding the RhDþ RBCs and shields them from immune recognition, thereby preventing the formation of antiD. There are a few scenarios, however, that still pose a risk of alloimmunization. Pregnant women who receive inadequate prenatal care and fail to receive RhIg prophylaxis are at increased risk of forming anti-D antibodies. In addition, release of emergent Rh-incompatible units to trauma patient’s presenting with massive bleeding can result in the formation of anti-D antibodies, although the rates of alloimmunization in this population are decreased as compared to healthy recipients.42 Another more commonly encountered scenario is a Rh-D negative patient receiving platelet transfusions, which contain a small amount of contaminating RBCs. One study of

Methods of RBC Alloimmunization

21

immunosuppressed patients receiving “Rhincompatible” platelet units showed that up to 8% of patients develop detectable anti-D, although other studies have demonstrated lower rates.43e45 The formation of anti-D following platelet transfusion can be mitigated with the coadministration of RhIg as a prophylactic measure. In addition to blood transfusions, anti-D can form following solid organ transplantations with one study showing 5% incidence of anti-D formation after a renal transplant.46 Clinically significant IgG class antibodies directed against the C, c, E, and e antigens have all been documented. Of these, the anti-E antibody is the most common with an incidence of 19.4%,47 however, virtually all cases are naturally occurring anti-E, making the E antigen a very weak antigen from the perspective of alloimmunization. Anti-C is the next most common antibody, with an incidence of less than 5% in a large study of over 18,700 transfused U.S. military veterans.47 This study population was largely male, and therefore the incidence in the general population is likely slightly higher due to exposure through pregnancy. Anti-c, while rare, is notable because it is solely formed because of antigenic exposure; no natural forms of this antibody exists. Anti-e has been reported, although studies show the e antigen to be weakly immunogenic. Anti-e antibody, however, is clinically significant once formed. Although most anti-Rh antibodies are alloreactive antibodies, resulting from antigen exposure via transfusion or pregnancy, well-documented cases of Rh-specific autoantibodies exist as well. By far, the most commonly encountered specificity is the autoanti-E, which is characterized as a natural antibody, appearing in patients without transfusion, and in both pregnant and nulliparous women. Generally speaking, the auto-anti-E is clinically insignificant, as these antibodies are only detected via the IAT, and do not cause destruction of E-antigen positive RBCs at 37 C. Coldreactive auto-anti-D IgG antibodies can be detected as well in up to 3% of patients in some studies; however, similar to the auto-anti-E, these antibodies are generally clinically insignificant, and only rare case reports exist of auto-anti-D antibodies reactive at 37 C. Auto-anti-C antibodies have also been characterized and are cold-reactive antibodies typically of the IgG class, and are not considered clinically significant.

Kell Blood Group System The Kell blood group system, which consists of at least 34 antigens,13 is the third most immunogenic, following the ABO and Rh systems. Compared to the RhD antigen, the K antigen is approximately 8e10

22

Immunologic Concepts in Transfusion Medicine

times less immunogenic. The Kell antigens are encoded by the KEL gene located on chromosome 7q33 and carried by the Kell glycoprotein, a 93-kd single-pass membrane protein. The following discussion will focus on the most frequently clinically encountered antigens including K, k, Kpa, Kpb, Jsa, and Jsb. A comprehensive review of all antigens can be found in numerous texts.21,22,48,49 Within the Kell system, the K antigen, and its alternate allele k, is encountered the most frequently clinically. The K and k antigens are encoded by a single protein that differs only by a single amino acid substitution, T193M, which disrupts the motif for N-glycosylation and may explain for the high immunogenicity of the K antigen.50 The K antigen is expressed in approximately 9% of whites and 1.5% of blacks, either in homozygous or heterozygous (Kk) forms. In contrast, the k antigen (also called as “Cellano”) is present in nearly 100% of patients. Additional K alleles, also on 7q33, code for the closely related antigens Kpa, Kpb, Jsa, and Jsb. The Kpa and Kpb antigens differ by single amino acid substitution, W281 and R281, respectively. Similarly, Jsa and Jsb antigens differ by single amino acid substitution, P597 and L597, respectively.13 After ABO and Rh antibodies, anti-K is the next most common alloantibody with an incidence ranging between 14% and 28%.51,52 Studies have estimated the risk of forming an anti-K at 10% if an individual is transfused with at least 1 unit of K-antigen positive blood.53,54 Similar to formation of anti-C antibodies, recipients who form anti-D antibodies are more likely to form anti-K antibodies as well, likely reflective of the general “responder” capability of the individual patient. Although anti-K antibodies are able to fix complement, they do so incompletely, and do not cause formation of the membrane attack complex. Nonetheless, anti-K is predominantly an IgG1 class antibody, therefore, capable of causing severe hemolytic transfusion reactions. In addition, severe HDFN can occur in K-antigen positive fetuses due to the ability of the antibody to cross the placenta and clear fetal red cells from circulation as well as bind and destroy fetal erythrocytic precursors, which express the K antigen early in lineage development.55e57 As a consequence, severe fetal anemia results due to suppressed fetal hematopoiesis. The frequency of anti-K in pregnant women is approximately 1/1000,58,59 and the incidence of HDFN due to anti-K is estimated at 1/20,000. Different from other cases of maternalefetal alloimmunization where the antibody titer correlates with the severity of fetal anemia, anti-K titers do not correlate with the degree of anemia. In the presence of maternal anti-K antibodies,

TABLE 2.3

Diversity of Kell Phenotypes by Ethnicity. Kell Phenotype

Caucasian (%)

Black (%)

Kkþ

91

98

K þ kþ

9

2

K þ k

0.2

<1

KpaKpbþ

98

100

Kpa þ Kpbþ

2

<1

JsaJsbþ

<1

0

Jsa þ Jsbþ

100

80

Js þ Js 

<1

19

a

b

the rate of hydrops or fetal death ranges from 15% to 38%.60e64 Cell-free fetal DNA testing assays now exist to test fetal K genotype in pregnant mothers with a known anti-K. Although anti-K is by far the most prevalent and severe cause of HDNF, Jsa and Jsb and Kpa antibodies have also been associated with HDFN. In contrast to anti-K, anti-k antibody is rare due to its status as a high-incidence antigen with expression in approximately 98% of patients. Conversely, the incidence of anti-k ranges from 0.005% of blacks, to up to 2% of whites in the United States.22 Anti-Kpa and Kpb are also rare, as are anti-Jsa and Jsb, about 0.5% and 0.4%, respectively.47 Similar to anti-K, they too are capable of mediating hemolytic transfusion reactions. The antigenic frequencies of the various Kell group antigens are outlined in Table 2.3.

Kidd Blood Group System The Kidd blood group system consists of three antigens, Jka, Jkb, and Jk3, encoded for by the Slc14a1 gene located on chromosome 18q12. The Kidd protein is a 10-pass transmembrane protein, with five extracellular loops.22 It is known to function as a urea transporter in the RBC membrane, transporting urea out of red cells as they pass through regions of high urea concentration thus preventing dehydration.65 The antigenic frequencies of the various Kidd group antigens are outlined in Table 2.4. Jka and Jkb antigens are codominantly expressed and differ by the p.D280N polymorphism.13 The Jk3 phenotype, which is present at an incidence of up to 1.4% in some Pacific Islander populations,66 represents a null phenotype, where the individual has an unexpressed allele at the Slc14a1 locus. This is most commonly due to a splice site mutation, or a p.S291P point mutation.67

CHAPTER 2

Methods of RBC Alloimmunization

23

TABLE 2.4

TABLE 2.5

Diversity of Kidd Phenotypes by Ethnicity.

Diversity of Duffy Phenotypes by Ethnicity.

Kidd Phenotype

Caucasian (%)

Black (%)

Asian (%)

Duffy Phenotype

Caucasian (%)

Black (%)

Asian (%)

Jka þ Jkb

26

52

23

Fya þ Fyb 

20

10

81

Jk þ Jk þ

50

40

50

Fy þ Fy þ

48

3

15

JkaJkb

24

8

27

FyaFybþ

32

20

4

FyaFyb

0

67

0

a

b

Most anti-Jka and Jkb antibodies are IgG1 and IgG3, but IgG2/4 and IgM components also exist. A large study of transfused U.S. veterans showed a frequency of 21% for anti-Jka antibody and approximately 1% for antiJkb.47 Two important features of anti-Jka and Jkb antibodies are their ability to fully activate the complement cascade, resulting in delayed acute and serologic hemolytic transfusion reactions. The latter feature is due to their ability to fall to undetectable levels by conventional serologic methods just several months following alloimmunization, a phenomenon known as antibody evanescence. In addition, Jka and Jkb antigens have a role as minor histocompatibility antigens in renal allografts due to the high concentration of the Jk antigens on renal medullary tissue. Studies have shown that mismatched renal allografts show a higher degree of cellular infiltration in early rejection.68

Duffy Blood Group System There are five antigens in the Duffy system including Fya, Fyb, Fy3, Fy5, and Fy6.13 Antigens within the Duffy blood group system are coded by the DARC gene, which codes for the (CD234) receptor protein that binds inflammatory cytokines interleukin-8, monocyte chemotactic peptide (MCP-1), and melanoma growth stimulatory activity (MGSA).69 The most commonly encountered antigens within the Duffy system are Fya and Fyb, which are expressed codominantly and differ by the G42D polymorphism. Approximately two thirds of blacks in the United States express neither antigen, and some populations worldwide show 100% of people with a Fya-/Fyb-phenotype. This is due to a mutation in the promoter of the DARC gene at the binding site of GATA-1, a transcription factor that drives Fy antigen transcription. The Fy antigen is still expressed on other tissue,70 preventing alloantibody formation in these patients. Cases also exist where Fya and Fyb antigens are missing due to deletions or other inactivating mutations; in these cases, Fy antigen expression is completely lost in all tissues, and these patients can form the anti-

a

b

Fy3 or anti-Fy5 antibodies, which react with Fya/b antigen positive RBCs. The antigenic frequencies of the various Duffy blood group antigens are outlined in Table 2.5. Anti-Fya is present in about 30% of patients,47 with anti-Fyb being much less common. There appears to be a strong association between anti-Fya formation and the human leukocyte antigen (HLA) DRB1*04 allele, indicating that presentation of the Fya antigen may be dependent on a very specific antigen presentation configuration.71 Anti-Fya and anti-Fyb are predominantly IgG1 class antibodies and are known to cause both acute and delayed hemolytic transfusion reactions, as well as HDFN.72 An association between Plasmodium vivax and Plasmodium knowlesi and patients with Fya/b null phenotype is well known.73,74 The Duffy glycoprotein is the receptor for the malarial parasite. It was discovered that the Fy antigens serve as a binding site and point of entry for these parasites into RBCs and patients with Fya/b null phenotype were resistant to RBC infection. This may explain selective evolutionary pressure toward the Fya/b null phenotype in malaria-endemic regions. Duffy antigens are destroyed by proteolytic enzymes, which is helpful in serologic methods to determine antibody specificity when multiple antibodies are under investigation.21,75

MNS Blood Group System The MNSs system is notable for marked antigenic diversity; however, most of the variants are not clinically significant. There are over 46 different alleles currently identified within the blood group system. The antigens are encoded by GYPA and GYPB homologous genes located on chromosome 4. GYPA contains seven exons and encodes the glycophorin A (GPA) molecule; GYPB contains five exons and encodes the glycophorin B (GPB) molecule.13,76 GPA and GPB are single-pass proteins that contain distinct intracellular, transmembrane, and extracellular domains, and serve as entry receptors for Plasmodium falciparum.77,78 Glycophorin B also

24

Immunologic Concepts in Transfusion Medicine

serves as a binding site for the C3b component of the complement cascade. The MNS system is divided into the MN and Ss loci, the two major allelic loci of most frequent clinical significance. Both of these alleles follow Mendelian inheritance, and can be expressed codominantly making all possible permutations of M/N/S/s expression possible. The M and N antigens are expressed on GPA, while the S and s (and U) antigens are expressed on GPB, with the differences between M and N, and S and s, consisting of alterations in the terminal amino acid sequence of their respective proteins. The S-s- phenotype is rare, and found in approximately 1.5%; these individuals also lack the high incidence U antigen (100% whites, 99% blacks), and are capable of developing an alloantibody to the U antigen,79 making it exceedingly difficult to obtain compatible units. Table 2.6 shows the frequency of MNS phenotypes by ethnicity. Anti-M and anti-N alloantibodies are common, typically naturally occurring, cold-reactive antibodies, all of which are at least partly IgM class; auto-anti-M may be IgG alone. Up to 4% of infants and adults are found to have anti-M that is reactive at 37 C; therefore, anti-M is a clinically significant antibody.47,80 Anti-M alloantibody has been demonstrated to form up to 12% of the time in the context of alloimmunization to other major alloantigens, such as RhD, indicating that the M antigen itself is not highly immunogenic. Although anti-M and anti-N do not bind complement or mediate intravascular hemolysis, there are case reports of delayed hemolytic reactions due to anti-M and anti-N, and therefore these antigens are still honored when providing compatible red cell units to patients. In addition, they are not implicated in HDFN as they are IgM class antibodies.

Anti-S and anti-s are typically IgG class antibodies that are reactive at 37 C, and can mediate both hemolytic transfusion reactions and HDFN. The highincidence U antigen is a shared epitope of the S and s antigens; patients that are S-s- are therefore capable of alloimmunizing to U as well and forming a clinically significant anti-U, which can cause hemolytic reactions and HDFN.

ALLOIMMUNIZATION TO BLOOD GROUP ANTIGENS Introduction Although potentially life-saving, RBC transfusions are not without risks. One of the most clinically significant risks of transfusions is alloimmunization and its consequences. In 2015, hemolytic transfusion reactions due to non-ABO incompatibilities (14% of fatalities) and ABO incompatibilities (7.5% of fatalities) accounted for the third leading cause of transfusion-associated deaths in the United States, following transfusionrelated acute lung injury and transfusion associated circulator overload.81 Therefore, an understanding of factors that impact alloimmunization rates, patient populations at greatest risk, and mitigation strategies to prevent and minimize alloimmunization is critical for transfusion medicine physicians as well as clinicians. RBC alloimmunization is the process of forming antibodies against nonself RBC antigens that occurs when an individual that lacks a particular antigen is exposed to the antigen through transfusion or pregnancy. Antigen exposure is necessary but not sufficient to trigger alloimmunization. There are numerous factors that are believed to impact whether an individual will alloimmunize. The main mechanism believed to underlie alloimmunization is the presentation of donor antigen peptides by APC to TCR on recipient CD4 T cells.

Factors that Impact Alloimmunization TABLE 2.6

Diversity of MNS Phenotypes by Ethnicity. MNS Phenotype

Caucasian (%)

Black (%)

M þ N

30

25

M þ Nþ

49

49

NeNþ

21

26

S þ s

10

6

S þ sþ

42

24

Ssþ

48

68

Ss

0

2

Some patients alloimmunize rapidly to an initial exposure to antigen, while others respond poorly. Thus, patients can be classified as “responders” and “nonresponders,” with those who do alloimmunize further classified as either a “strong” or “weak” responder to allogeneic RBCs. There are several factors that impact whether or not a patient will become alloimmunized, divided broadly into those related to the donor and recipient, those intrinsic to the RBC antigen, and those related to the transfused RBC unit.82 Donor and recipient factors include underlying disease state, degree of inflammation, number of previous transfusions, patient intrinsic “immune

CHAPTER 2 responder” status, ethnicity, prior pregnancies, HLA haplotype, and others. Patient inflammatory status at the time of transfusion is believed to have a large impact on the rate of alloimmunization. For example, in patients with sickle cell disease (SCD), alloimmunization rates have been shown to be higher when patients receive a transfusion during an acute illness.83 Other conditions with increased inflammation have also shown increased rates of alloimmunization such as inflammatory bowel disease and other chronic autoimmune diseases.84 Patients HLA haplotype has also been correlated with alloimmunization. A study looking at the impact of HLA haplotype suggested that the HLA-DRB1*15 phenotype may predispose transfused recipients to the formation of multiple different RBC alloantibodies. Specifically, the study found that HLA-DRB*15 was present in approximately 40% of individuals possessing multiple antibodies versus in approximately 25% of individuals with a single antibody or control population.85 Factors intrinsic to the antigen include the immunogenicity and copy number of the antigen. The immunogenicity of an antigen is estimated based on the number of individuals that produce an alloimmune response to that particular antigen combined with the probability that they were exposed to that antigen, which is based on the frequency of the antigen in the donor population. The density or copy number of antigens on the surface of the RBC also varies between the different antigens and is thought to contribute to immunogenicity. Animal studies investigating the role of copy number in immune responses to transfused transgenic murine RBCs expressing human KEL glycoprotein showed that recipients transfused with RBCs expressing KEL antigen at a greater copy number where more likely to generate anti-KEL antibodies after a single transfusion compared to recipients transfused RBCs with a lower copy number.86 Factors related to the RBC unit include the age of the unit and method of unit preparation such as washing, volume reduction, leukoreduction, irradiation, among others. Studies looking at the effect of the age of the RBC unit on the potential for alloimmunization in general show no effect of aged RBC units on RhD alloimmunization.87e89 One retrospective study evaluating the association between RBC unit age and alloimmunization rates in patients with SCD, however, found an association between age of the unit and risk of RBC antibody formation with an HR of 3.5 with units 7 days old and an HR of 9.8 with units 35 days old. Given that SCD patients are known to have a higher baseline risk for alloantibody formation, additional studies are

Methods of RBC Alloimmunization

25

needed to clarify whether this holds up for other patient populations, ideally factoring in patient disease and immune status.90 Other components of the overall blood transfusion process can contribute to potential alloimmunization including process errors that can occur during the manufacturing of the blood component, in which there is a breakdown in the process by which the correct compatible blood unit is matched to the correct patient. There are many steps in this, and can briefly be summarized as follows: collection of blood unit from correctly identified donor/testing of collected unit for ABO and Rh(D) antigens/correct labeling of the unit/duration and conditions of storage and transport of the unit/correct drawing and labeling of type and screen sample from the recipient/assuming unit is compatible by type and screen/crossmatch and antibody panel if needed, correct labeling of the unit with the intended transfusion recipient/transfusion of the unit within the allotted 4 hours upon exit from the blood bank. A breakdown in this process most commonly occurs in the clerical steps during which there is correct identification and labeling of patients, blood units, and type/ screen samples, both at the collection center and at the transfusion center. Lookback studies determined that 0.25% of blood was administered to the wrong patient91 and a large study of nearly 700,000 collected blood samples showed mislabeling in 1 out of every 165 samples. Newer additions to the process of collection/labeling, such as barcode scanner identification of both the patient and sample at the patient’s bedside, will help to mitigate this source of error.

Alloimmunization in Select Populations General hospital-based patients Approximately 1%e3% of the general hospital-based patient population will form an alloantibody;92e94 however, this data is based on retrospective analysis of patients at a single institution and thus alloimmunization rates are likely higher than these reports. Up to 7% of healthy adults in the general transfused population will form an alloantibody,95 reflecting the overall relatively low frequency of transfusions. In contrast, patient populations who chronically receive numerous transfusions, including patients with hemoglobinopathies as well as patients with hematologic malignancies, are more likely to form alloantibodies.

Patients with hemoglobinopathies Patients with SCD receive frequent transfusions, and alloimmunization in this population has critical clinical consequences including acute and delayed hemolytic

26

Immunologic Concepts in Transfusion Medicine

transfusion reactions, bystander hemolysis, delay in providing compatible blood, and HDFN. In addition, a recent study correlated RBC alloimmunization with overall worse survival in patients with SCD, with patients without alloimmunization showing life expectancies approximately a decade longer than did alloimmunized patients.96 Studies have estimated the alloimmunization rate in children and adults with SCD to be up to 29% and 47%, respectively, when partial or extended Rh antigen matching is not performed.97,98 The antibodies most prevalent in patients with SCD include those against Rh, K, and Jkb antigens.99 The high alloimmunization rates in SCD are thought to be due not only to the frequency of transfusions, but also to population-level discrepancies in the frequency of the Rh-system and K antigens. Therefore, many institutions will procure RhD/C/E and Kantigen matched units when transfusing SCD patients. Another related factor to the high alloimmunization rate in this population is the finding that patients with SCD have been shown to express unique variants of Rh-system antigens at a higher frequency than the general population, further complicating the identification of truly matched units.100 This may account for more recent studies showing persistent alloimmunization in SCD patients even when receiving phenotypically D/C/E/K-matched units.100 Future molecular RBC phenotyping and matching methods may address this important issue. In the case of SCD specifically, alloimmunization rates in children are lower than in adults, and studies have shown that alloimmunization rates are lower with RBC exposure via erythrocytapheresis as compared to simple transfusion.101,102 Interestingly, a subset of patients with SCD never become alloimmunized despite chronic transfusions. The degree of chronic inflammation at the time of transfusion is postulated to play a role, in addition to differences in immunologic or genetic signatures in “responder” and “nonresponders” patients. Studies have shown that patients with SCD presenting with acute chest syndrome or vaso-occlusive crises were more likely to alloimmunize than nonacute SCD patients.83,96 b-Thalassemia is another condition in which chronic transfusions are necessary for disease management, typically beginning early in life. Several studies have investigated the rate of alloimmunization in patients with thalassemia showing a range from 5% to 48%,103 with most antibodies directed against Rh or K antigens, although the overall rates vary depending on the region studied and the likely degree of donor/ recipient phenotype mismatch. One study showed

approximately 21% of patients alloimmunizing after 6 years of transfusion (average 18 units/year), with drastically lower rates present in the group receiving D/C/E/K matched units (3.7%) as compared to RhDonly matched units (15.7%).104e106

Patients with hematologic malignancies Patients with hematological disorders frequently require multiple transfusions placing this population at increased risk for alloimmunization. Several studies have investigated the rates alloimmunization in patients with various hematologic disorders. Studies investigating alloimmunization in patients with myelodysplastic syndrome (MDS) or MDS-related disorders and acute myeloid leukemia have shown rates ranging from 15% to 59%107,108 and 3% to 16%,108,109 respectively. Conversely, studies investigating the rate of alloimmunization in patients with lymphoid neoplasms have shown very lower rates of alloimmunization thought to be due to either underlying impaired immune function or the chemotherapy regimen. Studies have shown a 0% rate of alloimmunization in patients with acute lymphocytic leukemia and Hodgkin lymphoma.108,110 Rates ranging from 2% to 3% have been demonstrated in patients with non-Hodgkin lymphomas and plasma cell neoplasms.108

Preventing and Managing Red Blood Cell Alloimmunization Because the primary goal of the transfusion medicine service is to provide safe and effective blood components, the prevention of alloimmunization is essentially the chief safety role of the transfusion physician on a day-to-day basis. In addition to the short-term consequences of potential immunemediated transfusion reactions and their sequelae, the long-term consequences of developing novel alloantibodies are significant as well. Depending on the nature of the alloantibody, it can moderately or severely restrict the available pool of compatible RBC units going forward for the patient, potentially prolonging the process of finding safe blood for transfusion in time-sensitive clinical situations such as acute chest syndrome in patients with SCD. These considerations are of particular importance for populations who require chronic lifelong transfusions such as patients with SCD or b-thalassemia major. Thus, preventing alloantibodies from forming in the first place or mitigating the dangers of existing alloantibodies would lead to decreased transfusion-associated morbidity and mortality.

CHAPTER 2

Patient transfusion history Because many alloantibodies evanesce over time, obtaining a complete, accurate transfusion history for a patient is of critical, as it may reveal an undetectable clinically significant alloantibody. It is common for patients to receive transfusions at more than one institution, and to date there is no national centralized transfusion database or alloantibody identification records, leaving only the patient’s recollection as our best option for determining undetectable alloantibodies. One study of 100 patients undergoing hospital transfusion revealed a 64% prevalence of discrepancy in alloantibody records when comparing detection at the authors’ institution and records obtained from other local and distant hospitals.111 These data highlight the need for a centralized shared antibody database to ensure safe blood administration to patients.

RBC transfusion threshold One method to prevent the formation or sequela of alloantibodies is the adoption of a restrictive transfusion strategy, thereby preventing patients from exposure to RBC antigens. Currently, there still remains variation in clinical practice on thresholds used to transfuse patients. Based on review of several randomized control studies, the AABB has issued clinical practice guidelines recommending a restrictive transfusion approach with a hemoglobin threshold of 7 g/dL for hospitalized adult patients who are hemodynamically stable and a value of 8 g/dL for patients undergoing cardiac or orthopedic surgery and patients with preexisting cardiovascular disease. In patients with acute coronary syndrome, severe thrombocytopenia, and chronic transfusion-dependent anemia a threshold of 7 g/dL is likely comparable to 8 g/dL; however, no randomized control data is available for this population.112 According to the 2015 NBCUS, there has been a 13.9% decline in RBC transfusions in the United States compared to 2013, likely reflective in part due to this evidence supporting judicious use of blood components as well as increased awareness of appropriate transfusion thresholds.1

Antigen-matched red blood cells An additional strategy to mitigate alloimmunization is to provide partially antigen-matched RBC units between the donor and recipient. Current guidelines113 recommended that patients with SCD receive prophylactically matched RBCs for C/c, E/e, and K/k for reasons outlined previously. Currently, many institutions provide C/c, E/e, and K-antigen serologically matched units when transfusing patients with SCD. Currently, there

Methods of RBC Alloimmunization

27

are no guidelines regarding providing phenotypically matched units beyond Rh(D) for patients with b-thalassemia. Studies, however, have shown lower rates of alloimmunization in patients with thalassemia receiving RBCs matched at ABO, Rh, and K (3.7%) compared with patients receiving RBCs matched at ABO and Rh(D) (22.6%).114

Leukoreduction Leukoreduction is another method with potential to minimize RBC alloimmunization rates. Leukoreduction is the process of removing donor leukocytes from the RBC unit through a filter by action of size exclusion, with most of the RBCs and platelets passing through the filter and leukocytes remaining behind. In addition, adhesion between the filter and leukocytes further prevents leukocytes from passing through the filter. The process of leukoreduction can occur either at the time of blood component manufacturing, termed prestorage leukoreduction, or immediately before transfusion of the RBC unit, termed poststorage leukoreduction. There are approximately 5
108 leukocytes in one unit of RBCs before leukoreduction. After leukoreduction, this is decreased to at least 5
106 leukocytes or less per RBC unit.115 In addition to removing leukocytes, leukoreduction also has been shown to remove proinflammatory mediators such as IL-1 and IL-8, reduce HLA antibody production, and prevent transmission of certain infectious agents. Studies investigating the role of leukoreduction for prevention of alloimmunization have shown mixed results with some studies showing clinical benefit and reduced rates of alloimmunization,116 while others showing no benefit. Additional well-controlled studies are needed to clarify this further. In addition, it is thought that the timing of the leukoreduction processing may impact the number of leukocytes, cytokines, and other particles117 and therefore the rate of alloimmunization.

TESTING METHODOLOGIES The blood bank service performs various tests to ensure the safe administration of blood components to patients. This section will cover the most common immunohematologic tests performed by the transfusion medicine service for transfusion compatibility and antibody identification, including antibody screening, direct and indirect antiglobulin tests, red blood cell eluates, and minor antigen phenotyping. In addition, emerging molecular methods for genotyping will be briefly discussed.

28

Immunologic Concepts in Transfusion Medicine

Serological Typing Methods In 1907, Ludvig Hektoen realized that the safety of transfusions might be improved by cross-matching blood between donors and patients. Reuben Ottenberg, subsequently performed the first blood transfusion using blood typing and cross-matching. Today, the transfusion service performs various tests including ABO and Rh typing, direct antiglobulin testing (DAT), and antibody detection and identification procedures in addition to others to provide the most effective, safe blood components to patients. In addition to pretransfusion testing, red blood cell phenotyping is used to screen blood donors and donor RBC units for select antigens and in prenatal testing to type fathers of mothers with alloantibodies that can cause fetal hemolysis. ABO and Rh(D) antigens are the most important antigens for safe transfusion to avoid severe hemolytic complications; therefore, typing for these antigens is routinely performed for both transfusion recipients and blood donors. In 1947, it became standard practice to perform ABO blood-typing on each RBC unit. Typing for common minor blood group antigens is not typically performed; however, an antibody screen against selected antigens in the Rh, Kell, Kidd, Duffy, and MNS system is performed. Serologic typing is the most commonly used method of testing, which uses the principle of hemagglutination as the reaction end point. Hemagglutination is the recognition of red cell clumping because of antibody binding to antigen on the RBC surface when a patients’ specimen, either plasma or RBCs, is mixed with a reagent. A scale of 0e4 þ is used to grade the presence of hemagglutination, with 0 indicating no agglutination and 4 þ indicating a strong agglutination reaction. Hemagglutination reactions can be read and interpreted either manually or by automated methods. Serologic typing methods offer the benefit of being inexpensive, rapid, and accurate for most blood donors and recipients. Various serologic typing methods to detect hemagglutination have been developed for compatibility testing including tube typing, gel typing, solid phase adherence, among others. Tube typing involves visualizing a red cell serum mixture macroscopically and looking for the presence of hemagglutination. Grading of the strength of hemagglutination reaction using this method is manually performed by technologist and therefore is somewhat subjective and requires extra time by technologist to interpret. Gel typing, in contrast, has the potential for automation in addition to increased sensitivity compared to tube typing. The gel test was first developed in 1985 by Dr. Yves Lapierre.118

In this method, RBCs interact with antibodies at the top of a column. RBCs are forced into dextran-acrylamide gel matrix by centrifugation and based on the principle of size exclusion, agglutinated RBCs are too large to go through the matrix, therefore remain at the top of the gel column while nonagglutinated RBCs move to the bottom of the gel column. The gel typing method requires less technologist time as the gel cards can be read either using automated equipment or manually. Moreover, the gel typing provides a more stable reaction endpoint that can be reviewed up to 3 days and more reproducible results compared to the tube typing method. The most recently developed method, solid phase typing, is a method in which antihuman globulin is immobilized onto a solid medium and the patient’s serum is added, resulting in the binding to the Fc region of the patient’s serum antibodies. Then, reagent red cells are added and incubated, and the reaction is centrifuged. A positive reaction constitutes the spreading of the RBCs in a “carpet” pattern across the well indicating binding of reagent to RBCs, whereas a negative reaction constitutes an RBC “button” at the bottom of the well after centrifugation. Solid phase tests have shown to be more sensitive than conventional tube methods119 and can also be automated. Automation decreases the opportunity for human errors and allows personnel to perform other tasks. Modifications to these various standard serologic methods described earlier may be necessary in certain situations such as obtaining phenotypes in patients recently transfused; however, discussion of this topic is outside the scope of this text (Fig. 2.3). When the blood bank service receives a request for a blood component transfusion, a type and screen is performed. The type refers to determining the patients ABO and Rh(D) status, and the screen refers to detecting and identifying RBC autoantibodies or alloantibodies in the patient’s plasma or serum. A patient’s ABO status is determined by performing a forward grouping (also called as front type) for the presence or absence of A or B antigens and a reverse grouping (also called as back type) for the presence of anti-A or anti-B antibodies in plasma. For most patients, the Rh(D) status is determined by testing for the presence or absence of Rh(D), with phenotyping for other non-Rh(D) antigens reserved for select populations at increased risk for alloimmunization. Various tests are used to identify autoantibodies and alloantibodies for the purpose of identifying clinically significant antibodies associated with hemolytic transfusion reactions and HDFN. These include the IAT, DAT, RBC panel tests, and RBC elution, which will be discussed later (Table 2.7).

CHAPTER 2

Methods of RBC Alloimmunization

29

FIG. 2.3 Diagram of Tube (left), Gel (middle), and Solid Phase (right testing methodologies).

The purpose of the IAT is to identify possible antibodies by mixing patient plasma with various reagent screening cells expressing clinically significant nonABO antigens. Antihuman globulin (AHG) is added to detect the presence of agglutination. A positive IAT will be followed up with an RBC antibody panel to determine the specific antibody or antibodies causing the positive IAT reaction. RBC panel assays are performed by mixing patients’ plasma with several reagent screening cell lines. The DAT (also known as the Direct Coombs test) is a two-step process used to determine whether antibody and/or complement bound is bound to the surface of RBCs. In the first step, a polyspecific reagent to detect

IgG or C3 complement on the surface of RBCs is added to patient RBCs, either resulting in no agglutination (negative DAT) or agglutination (positive DAT). In the case of a positive DAT, AHG or anti-C3 is then added to determine if IgG (positive with addition of AHG) and/or C3 (positive with addition of anti-C3) is coating the surface of the RBC. Generally, a positive IgG DAT indicates a warm-reactive antibody and extravascular hemolysis, while a positive C3 DAT indicate a coldreactive antibody and intravascular hemolysis. There are various reasons for a positive DAT result, however, the most clinically relevant is hemolysis. Examples of causes of false positive DATs include previous transfusion, RBC rouleaux, and certain drugs.

TABLE 2.7

ABO Typing. REACTION WITH CELLS

REACTION WITH SERUM

INTERPRETATION

Anti-A

Anti-B

A1 cells

B cells

O cells

ABO group

0

0

þ

þ

0

O

þ

0

0

þ

0

A

0

þ

þ

0

0

B

þ

þ

0

0

0

AB

30

Immunologic Concepts in Transfusion Medicine

RBC elution studies are used to evaluate positive DATs with IgG reactivity to determine the specificity of antibody bound to the RBC surface. An acidic elution buffer is added to patient’s antibody-coated RBCs, which causes the antibodies to dissociate from the surface of the RBCs. The resultant concentrated antibody eluate is mixed with reagent panel cells to determine the antibody specificity. Note that C3 positive DATs are typically associated with IgM antibodies, which are poorly eluted from the RBC surface.

Molecular Typing Methods Although RBC phenotyping to evaluate antigen expression on the surface of RBCs has historically been performed using serologic methods, molecular typing has emerged in recent years as a potential alternative or supplemental method. Although serological typing involves directly determining a patient’s phenotype, molecular typing involves indirectly predicting a patient’s phenotype by genotype test. As discussed in the beginning of this chapter, most blood group antigens are inherited from single nucleotide gene polymorphisms via Mendelian inheritance. Molecular typing uses DNA methods to determine these polymorphisms and mutations controlling antigen expression to predict the antigen phenotype. Molecular typing is generally performed using genomic DNA extracted from whole blood or alternatively cellular samples. A polymerase chain reaction is used to amplify the region of interest with subsequent detection of sequence variations unique for the antigens. Although not currently widely adapted, molecular typing is now offered at several blood centers and reference testing laboratories. In addition, select hospital blood banks are starting to offer limited molecular testing. There are particular situations in which molecular typing can be especially useful such as fetal genotyping for HDFN, molecular testing of fetal and paternal samples in pregnant women with clinically significant antibodies, follow-up for patients with positive DAT, recently transfused patients, identifying variant antigens such as weak and partial D, resolving serologic typing discrepancies, and identification and matching for Rh variants more commonly seen in SCD populations. Molecular typing, however, is not without limitations and therefore for the time being is typically used in conjunction with serologic typing. Currently, molecular typing requires specialized equipment, trained personnel, and laboratory informatics platforms for processing results. In addition, because molecular testing only offers a prediction of the patient’s

likely phenotype based on polymorphisms, the patients’ true phenotype may be different if various changes that impact the protein are not taken into consideration. Therefore, molecular typing results should be interpreted with caution and in conjunction with serologic typing results.

SUMMARY In this chapter, we reviewed the most clinically significant blood group systems, as well as the various factors impacting alloimmunization. We hope that this chapter provided a basic understanding of the mechanism of RBC alloimmunization and the resulting clinical significance. Although many advances have been made in understanding factors that impact alloimmunization rates, much more work remains to understand the causes of differing alloimmunization rates observed between individuals. Lastly, we briefly covered the most common testing methodologies performed by the blood bank service including emerging molecular genotyping methods.

DISCLOSURE STATEMENT The authors (KS, CAT, ILB) have no direct financial interests in subject matter or materials discussed in this article/chapter.

REFERENCES 1. Ellingson KD, et al. Continued decline in blood collection and transfusion in the United States-2015. Transfusion. 2017;57:1588e1598. 2. Greenwalt TJ. A short history of transfusion medicine. Transfusion. 1997;37(5):550e563. 3. Giangrande PL. The history of blood transfusion. Br J Haematol. 2000;110(4):758e767. 4. Storry JR, Olsson MR. Genetic basis of blood group diversity. Br J Haematol. 2004;126:759e771. 5. Daniels G. The molecular genetics of blood group polymorphism. Hum Genet. 2009;126:729e742. 6. Reid ME, Mohandas N. Red blood cell blood group antigens: structure and function. Semin Hematol. 2004;41(2): 93e117. 7. Maitta RW. Clinical Principles of Transfusion Medicine. Elsevier; 2018. 8. Wiener AS. Origin of naturally occurring hemagglutinins and hemolysins; a review. J Immunol. 1951;66(2): 287e295. 9. Drach GW, Reed WP, Williams Jr RC. Antigens common to human and bacterial cells: urinary tract pathogens. J Lab Clin Med. 1971;78(5):725e735. 10. Drach GW, Reed WP, Williams Jr RC. Antigens common to human and bacterial cells. II. E. coli 014, the common

CHAPTER 2

11.

12.

13. 14.

15.

16.

17.

18.

19. 20.

21.

22.

23. 24.

25.

26. 27.

28. 29. 30. 31.

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