Red cell immunology and compatibility testing

CHAPTER 16 Red cell immunology and compatibility testing Connie M. Westhoff Laboratory of Immunohematology and Genomics, New York Blood Center, New York, NY, USA Introduction Immunohematology is the study of the immune response to blood cells. This chapter reviews the basic concepts of immunity as they apply to production of antibodies to red blood cells (RBCs), also called atypical alloantibodies. Antibodies to platelets and neutrophils, as well as autoantibodies and drug-induced antibodies, are discussed elsewhere. The relevance of immunohematology to transfusion medicine is addressed through discussion of the principles of serologic and DNA-based testing and the application to blood transfusion and donor recipient compatibility. Identification of the blood group specificity of alloantibodies (antibody identification) is performed by testing plasma or serum against red cells of known phenotypes. Importantly, modern immunohematology uses knowledge of the blood group antigen profile of the patient to aid and inform the process of antibody identification and selection of units for transfusion. This is feasible with the use of DNA-based testing, which provides an extended RBC antigen profile performed as a single assay. The chapter also includes a discussion of the steps and requirements for pretransfusion compatibility testing and release of blood components for transfusion, as well as efforts to track adverse transfusion events through biovigilance programs and to reduce alloimmunization by extended antigen matching. Red cell immunology The immune response Immune responses can be divided into two categories: humoral immunity and cell-mediated events. Humoral immunity is B-cell mediated and results in the production of antibody. Cell-mediated immunity involves the activation of T cells, which regulate the immune response through the production of cytokines and direct interactions with other elements and cells of the immune system. 1 Beginning during fetal development and continuing after birth, B and T cells produce receptors that enable lymphocytes to recognize foreign antigens from self. Antigen receptors on T cells and B cells The antigen receptors of both T and B cells consist of two polypeptide chains synthesized under the direction of two different chromosomal loci. In the early stages of lymphocyte development, the genetic material at each of these loci undergoes a process of rearrangement, known as somatic recombination, which is unique to each cell. 1 The loci consist of gene segments, grouped into families on the basis of sequence similarity, and the gene rearrangement process entails selection and joining of the segments for each portion of the immunoglobulin molecule (Figure 16.1) and elimination of unused genetic material. Each B-cell and T-cell receptor chain consists of a constant region, and a variable region associated with antigen binding and peptide MHC (major histocompatibility complex) recognition. For B cell receptors, which are immunoglobulin molecules, the amino acid sequences of the constant regions determine the isotype, and amino acid sequences of the variable region determine the idiotype and antigenic specificity. For both receptors, one chain contains three gene segments (V, D, and J), whereas the other chain contains rearrangements of V and J segments. The human immunoglobulin loci are polymorphic between individuals as far as the number of genomic V, D, and J segments, but the heavy chain locus has at least 51 V gene segments, 6 J region gene segments, and 27 D region gene segments, and the number of possible combinations for different antibody binding regions is over 8000. At the light chain loci, there are at least 71 V gene segments and 9 J gene segments. Further amino acid sequence variation is introduced by imprecise splicing of the gene segments and addition of extra nucleotides (called N nucleotides), which are not encoded in the genome, to V D and D J joints in the heavy chain variable region. Given all these mechanisms and assuming the combination of gene segments is random, the number of different antibody variable regions is virtually unlimited. 1 Exposure to foreign protein antigens If foreign protein antigens enter the host, antigen-presenting cells (APCs) process the antigen and display antigen-derived peptides in association with host Class II MHC molecules on the cell surfaces. This peptide MHC complex is recognized as foreign by the T-cell antigen receptor on T lymphocytes. In contrast, B-cell receptor antibody molecules on B lymphocytes recognize foreign epitopes on the protein. Cytokines produced by the activated T cells cause antigen-specific B cells to develop into immunoglobulin-secreting plasma cells. Plasma cells have a short but active life of antibody production. Some antigen-specific B cells develop into memory cells that persist in the circulation long after their initial activation. 1 Rossi s Principles of Transfusion Medicine, Fifth Edition. Edited by Toby L. Simon, Jeffrey McCullough, Edward L. Snyder, Bjarte G. Solheim, and Ronald G. Strauss. 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. 193

194 Section II: Part I: Red blood cells Unrearranged heavy chain gene VH1 VH2 VH3 VHn D1 D2 D3 Dn J1 J2 J3 J4 J5 J6 Cμ Cδ Cγ3 Cγ1 Cχ1 Cγ2 Cγ4 Cε Cγ4 D/J Joining VH1 VH2 VH3 VHn V/D/J Joining V/D/J Transcription Alternative RNA splicing V DJ Cμ Cμ Cδ V DJCδ Cμ Cδ Rearranged heavy chain gene Primary transcript μ/δ mrna Figure 16.1 Diagram of gene rearrangement generating antibody diversity at the immunoglobulin heavy chain locus on chromosome 14. The un-rearranged locus (top) shows the V, D, and J gene segments on the left and the constant region segments to the right. Gene rearrangement occurs by selection and joining of one D and one J segment (exon) to form a D/J unit. This is then joined by one V exon. The V/D/J unit, via alternative splicing, is joined to Cμ or Cδ heavy chain gene segments. In later generations, clonal progeny can undergo isotype switching in which the same variable region V/D/J is joined to other sequences from the constant region. Primary response The first immunoglobulin class to appear in the bloodstream is immunoglobulin M (IgM); the lag phase (time between antigen exposure and appearance of antibody) can vary from days to weeks. Shortly after the appearance of IgM antibody, IgG antibody of the same specificity usually becomes detectable. The IgM component disappears over time, whereas the IgG component persists indefinitely but can drop in titer to undetectable levels. Cytokines from activated T cells are essential for this immunoglobulin class switching and for generation of memory B cells. Secondary (anamnestic) response On re-exposure to a foreign antigen, a memory response is activated within a few hours or days, resulting in a sharp rise in the level of IgG antibody, higher than was produced in the primary response. T-cell independent response Antibodies produced in response to foreign carbohydrate antigens are primarily IgM. This is because B-cell antigen receptors can be activated by direct binding to repeating polysaccharide epitopes, such as the blood group A or B antigens. Structures that have multiple identical repeat carbohydrate chains can initiate B-cell proliferation and antibody secretion without T-cell help. In the absence of T-cell help, isotype switching from IgM to other isotypes does not occur. 1,2 Affinity maturation The affinity of IgG antibody for specific antigen increases progressively during the immune response, particularly after stimulation with low doses of antigen. Affinity maturation is most pronounced after secondary challenge with antigen and involves somatic mutation and clonal selection of B cells with higher affinity antibody receptors for the antigen. These two interrelated processes occur in the germinal centers of the secondary lymphoid organs: 1 Somatic hypermutation: Mutations in the variable, antigen-binding coding sequences (known as complementarity-determining regions [CDRs]) of the immunoglobulin genes. The mutation rate is up to 1,000,000 times higher than normal. Although the exact mechanism is not known, a major role for the activation-induced (cytidine) deaminase has been discussed. The increased mutation rate results in 1 2 mutations per CDR per cell generation, and these mutations alter the binding specificities and affinities of the resultant antibodies. 2 2 Clonal selection: B cells that have undergone somatic hypermutation must compete for antigen binding to survive. The follicular dendritic cells (FDCs) in the germinal centers present antigen to the B cells, and only those with the highest affinities for antigen will survive. B-cell progeny that have undergone somatic hypermutation but bind antigen with lower affinity or not at all will be deleted by programmed cell death or apoptosis. Over several rounds of selection, the resultant secreted antibodies produced will have effectively increased affinities for antigen. 2 Clonality Only antigen-activated B cells proliferate and mature into antibodysecreting plasma cells. The number of progenitor B cells that are initially activated will determine the heterogeneity of the immune response. A polyclonal response occurs when many different B cells are activated simultaneously, best exemplified by anti-d made in D-negative individuals who lack the RhD protein, which carries many foreign epitopes. A more restricted oligoclonal (pauciclonal) response occurs when the antibody-producing cells originate from a few B cells, exemplified by anti-fy a produced by a Fy(b+) individual, which represents a single amino acid change. Monoclonal antibodies, which are commonly used as reagents in the laboratory, are specific for a single epitope. Antigen-specific monoclonal antibody responses are rarely found in vivo and are usually associated with some leukemias and multiple myeloma. Rather, monoclonal antibodies are isolated in vitro from polyclonal responses using hybridoma technology. Immunoglobulin molecules Figure 16.2 portrays the basic structure of an antibody molecule, which consists of four polypeptide chains: two identical light chains of 211 217 amino acids, and two identical heavy chains of 440 or more amino acids. Antigenic specificity is conferred by the first half of the N-terminal amino acids of the light chains and the first quarter of the N-terminal amino acids of the heavy chains; these are described as variable regions. The remaining portions of both chains are referred to as constant regions. 1

Chapter 16: Red cell immunology and compatibility testing 195 V L Disulphide bond Antigen binding sites Variable Constant C L Table 16.1 Characteristics of blood group antibodies Immunoglobulin Characteristic IgM IgG IgA V H C H 1 Hinge region S S S S S S S S Light chain C H 2 C H 3 Carbohydrate Heavy chain H-chain isotype μ γ Α Subclasses 2 4 1 L-chain types κλ κλ Κλ Sedimentation constant 19 S 7 S 11 S Molecular weight 900 1000 kd 150 kd 180 500 kd Electrophoretic mobility Between β and γ γ Γ Serum concentration 85 205 1000 1500 200 350 (mg/dl) Antigen binding sites 10 (5) 2 4 Fixes complement Often Some No Placental transfer No Yes No Direct agglutinin Yes Usually not Usually not Hemolytic in vitro Often Usually not No Example Anti-A, anti-b Anti-D Anti-Lu a Figure 16.2 Basic structure of an immunoglobulin molecule. Light chains The constant region of the light chains can have one of two different amino acid sequences, designated kappa (κ) and lambda (λ). A single B cell will synthesize an immunoglobulin molecule containing either κ or λ chains, but never both. Heavy chains The constant region of heavy chains, designated by the Greek letters α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), gives rise to five types of immunoglobulin classes, or isotypes, termed IgA, IgD, IgE, IgG, and IgM, respectively. A single B cell will synthesize an immunoglobulin molecule containing only one type of heavy chain. Blood group antibodies Blood group antibodies are immunoglobulins that bind to antigens on the surface of red cells, platelets, and neutrophils. The focus of this chapter is primarily on antigens expressed on the RBC. The antibodies can be acquired either naturally, meaning from exposure to common environmental stimuli, or through exposure to foreign red cells via pregnancy or transfusion. Most blood group antibodies are either IgG or IgM; only occasionally are they IgA. 3 The IgD and IgE immunoglobulins have not been implicated as blood group antibodies. Physical properties The physical properties and serologic characteristics of the three immunoglobulin antibody classes with blood group specificity are summarized in Table 16.1. IgM antibodies are pentameric and have 10 antigen binding sites, are direct agglutinating, and often fix complement to the red cells. Antibodies to A and B antigens are predominantly IgM; they are found in persons whose red cells lack the corresponding antigen. They are stimulated by antigens present in the environment, including plants and bacteria that carry blood group A- and B-like polysaccharides. In adults with a normal immune system, these antibodies are almost always present when the corresponding antigens are absent on the red cells. In contrast to anti-a and anti-b, most other blood group antibodies are immune in origin and do not appear in plasma/ serum unless the host is exposed to foreign red cell antigens through blood transfusion or pregnancy. The majority are IgG, which have two sites for antigen binding (Figure 16.2). Most IgG antibodies do not activate complement, but this depends on both the subclass of IgG and the number and nature of the antigen on the cells. Antibodies to red cell antigens, other than naturally occurring anti-a and anti-b, are considered unexpected alloantibodies, and are directed to antigens absent on the RBCs of the individual. Antibodies in the recipient can cause accelerated destruction of antigen-positive transfused red cells, and some potent antibodies in donor plasma may destroy recipient RBCs. In pregnant women, IgG antibodies may cross the placenta and cause hemolytic disease of the fetus and newborn. 3 There are more than 300 different blood group antigens described. 4 The immunoglobulin class and clinical relevance of some of the antibodies encountered are shown in Table 16.2. Red cell antigen antibody interactions Red cells normally repel one another. The force of repulsion (zeta potential) at the surface depends not only on the electronegative surface charge, but also on the ionic cloud that surrounds it. The electronegative charge is imparted primarily by the carboxyl (C00 ) group of N-acetyl-neuraminic acid, which is a constituent of alkali-labile tetrasaccharides that are attached to MN and Ss sialoglycoproteins (glycophorins A and B, respectively). The magnitude of this charge is modified by the formation of an ionic cloud of positively charged (+) sodium ions and negatively charged ( ) chloride ions at the red cell membrane surface (Figure 16.3). The net effect of this ionic cloud is to decrease the electrostatic repulsion between cells. 5 For in vivo enhancement of the interaction of red cell antigens with blood group antibodies, three variables are considered: the surface charge of the red cells, the dielectric constant of the medium, and the mutual attraction between antigen and antibody. The latter involves electrostatic (Coulombic) and hydrogen bonds, and van der Waals interactions, and there must be structural complementarity between the antigen and its binding site on antibody molecules. There are two phases of red cell antigen antibody agglutination interactions; these often occur simultaneously. The first phase is one of association, involving binding of antibody to antigens on the red cell membrane. The second phase involves the formation of an agglutination lattice of antibody-coated cells. For the latter to occur,

196 Section II: Part I: Red blood cells Table 16.2 Blood group antibody, class, clinical significance, and % antigen-negative (compatible) donors 7 Antibody Ig Class y C3-Binding Effect of Ficin HDFN z HTR %Compatible Whites Blacks Comments A M, G Yes Moderate Yes 57 73 No dosage A 1 M rare No Rare 66 81 Present in 1% A 2, 25% A 2 B At a G No No Yes Rare Many 1 case mild HDFN B M, G Yes Moderate Yes 91 80 No dosage Bg G No No No 99 99 To HLA C G, M No Mild Yes 32 73 c G, M No Yes Yes 20 4 Often with anti-e Ch G No No No # 4 4 antibody to C4d # C w G, M No Yes Yes 98 99 Co a G No Mild Mild Rare Rare Co b G Rare Mild Yes 90 90 Cr a G No No Mild None Rare Cs a G No No No 2 4 D G, M No Yes Yes 15 8 Di a G No Yes Yes >99 >99 64% American Indians Di b G No Mild Yes Rare Rare 4% American Indians Do a G Yes No Yes 33 45 Do b G Yes No Yes 18 11 E G, M No Mild Yes 71 78 Often with anti-c e G, M No Rare Yes 2 2 f (ce) G, M No Mild Mild 35 8 Compatible with c Fy a G Rare Yes Yes 34 90 Fy b G Rare Mild Yes 17 77 Ge G, M Yes Some No Yes Rare Rare H M, G Yes No Yes Rare Rare In O h blood Hy G No No Mild Rare Rare In Blacks I M, G Yes No Yes Rare Rare In i adults Jk a G Yes Yes Yes 23 8 Jk b G Yes Mild Yes 26 51 JMH G No No No Rare Rare HTLA Js a G No Yes Yes >99 80 Js b G No Yes Yes Rare 1 K G, M Rare Yes Yes 91 98 k G No Yes Yes Rare Rare Kn a G No No No 2 1 Kp a G No Yes Mild 98 >99 Kp b G No Mild Mild Rare Rare Lan G Some Mild Yes Rare Rare Le a M Yes No Rare 78 77 Le b M Yes No No 28 45 Lu a G, A Rare Rare No 92 95 Lu b G, A Rare Mild Mild Rare Rare M M, G Rare Rare Rare 22 26 McC a G No No No 2 6 HTLA N M, G No Rare Rare 28 25 Potent in N U P 1 M Some No Rare 21 6 P+P 1 +P k M, G Yes Yes Yes Rare Rare Rg G No No # Rare 2 2 Antibody to C4d # S G Some Yes Yes 45 69 s G Rare Yes Yes 11 7 Sc1 G No No No Rare Rare Sc2 G No Yes No 99 99 Sl a G No No No 2 55 U G No Yes Yes Rare 1 V G No No Yes >99 70 Vel M, G Yes Yes Yes Rare Rare Wr a M, G No Yes Yes >99 >99 Xg a G No No No 23 23 Yk a G No No No 8 2 HTLA Yt a G No Some No Some Rare Rare y Predominant immunoglobulin class shown first. z HDFN = reported to cause hemolytic disease of the fetus and newborn. HLA = antibody to antigen present on white blood cells that is variably expressed on red cells. HTLA = antibody, usually of high titer, to antigen of low site density on RBC. Incorrectly called high-titer, low-avidity antibody. # C4d = antibody to epitopes found on the fourth component of human complement (C4). Hives and anaphylaxis reported with plasma containing products. Hemolytic Transfusion Reaction.

Chapter 16: Red cell immunology and compatibility testing 197 (A) (C) antibody molecules must be able to span the distance between adjacent red cells. Interaction between red cells and blood group antibodies can be observed either directly by examination of red cell and antibody mixtures for agglutination (clumping) and/or hemolysis, or indirectly by use of an antihuman globulin reagent in the antiglobulin test. These two serologic techniques are summarized in Table 16.3. Table 16.3 Types of red cell serologic tests Direct Tests Mix serum and red cells. y Centrifuge. Examine for agglutination and hemolysis. Indirect Tests Mix serum and red cells. Incubate (37 C). Centrifuge. + + - + - - - + + - + - - + - + (B) + + - + - - - + + - + - - + - + Figure 16.3 (A) Red cells carry a strong negative electric charge, imparted primarily by the carboxyl (C00 ) group of N-acetylneuraminic acid. (B) When suspended in saline, red cells are kept apart by virtue of their negative surface charge. The magnitude of this charge is modified by the formation of an ionic cloud of positively charged (+) sodium ions and negatively charged ( ) chloride ions at the red cell membrane surface. The force of repulsion that exists between red cells in suspension is referred to as the zeta potential. (C) In order for antibody molecules to cause agglutination of adjacent red cells, they must be able to span the intercellular distance. Pentameric IgM antibody molecules can readily bridge the distance between adjacent red cells causing agglutination. (D) IgG molecules (black) cannot readily cause direct agglutination. The bound IgG antibody can be detected by antihuman IgG antibody (e.g., rabbit antihuman IgG), depicted here in gray, causing agglutination. Examine for agglutination and hemolysis. y Wash, to remove unbound globulins. Add antihuman globulin reagent. Centrifuge. Examine for agglutination. z y Can incubate at room temperature. An enhancement reagent, to promote antibody uptake, may be incorporated here. z Confirm negative test results with IgG-coated red cells. (D) Agglutination reactions IgM antibody molecules can cause direct agglutination of RBCs carrying the corresponding antigen. Examination for agglutination may be performed either macroscopically or microscopically, but the latter may detect nonspecific reactivity and is not routinely performed. The strength of the observed reactions are graded, and numerical values are assigned based on a scoring system. 6 Hemolysis IgM antibodies, in particular anti-a and anti-b, can also cause direct lysis of antigen-positive red cells in the presence of complement. Hemolysis results from the action of complement, a series of α and β globulins that act in sequence as enzymes (e.g., esterases) to form a complex (membrane attack complex) that causes rupture of the red cell membrane through which intracellular hemoglobin canescape(seefigure16.4).for initiation of the complement cascade, the Fc portions of two immunoglobulin heavy chains must be in close proximity on the red cell surface. Initiation of the complement cascade readily occurs when a single pentameric IgM antibody molecule is bound; for it to occur with IgG antibodies, there must be closely adjacent heavy chain regions of two IgG molecules at the cell surface. Furthermore, not all IgM antibodies bind complement to red cells, and complement binding does not always proceed to complete red cell lysis (intravascular hemolysis). Rather, activated C3b may be cleaved to C3d, which remains bound to red cells and may be detected by the antiglobulin test (see the Antiglobulin Test section). 6 Red cells coated with C3b are also removed by cells of the reticuloendothelial system (extravascular hemolysis). Antiglobulin test IgG antibodies do not readily cause direct agglutination of red cells, although some IgG antibodies will cause direct agglutination if the red cell zeta potential is reduced, or if there are a large number of antigens on the RBC, for example the M antigen carried on glycophorin A. Zeta potential can be decreased by raising the dielectric constant of the red cell suspending medium by the addition of colloids such as bovine serum albumin, or by treating the cells with proteolytic enzymes such as papain or ficin, which cleave the proteins that carry the negatively charged neuraminic acid residues. IgG antibodies, as well as complement components bound to red cells by either IgM or IgG antibodies, are detected by the antiglobulin test. This test, once called the Coombs test, entails the use of antibodies raised in animals (usually rabbit), or prepared by hybridoma technology, to detect human IgG and complement bound to red cells. Antihuman globulin (AHG) will react with human globulins, either bound to red cells or free in plasma or serum. Red cells must be washed free of unbound globulins before testing with AHG to avoid false-negative tests caused by neutralization of AHG by unbound globulins. Direct antiglobuin test The direct antiglobulin test (DAT) is used to detect antibodies bound to red cells in vivo; such antibodies may be seen in patients with autoimmune hemolytic anemia, infants with hemolytic disease of the fetus and newborn, and patients manifesting an alloimmune response to a recent transfusion. 6 Indirect antiglobulin test An indirect antiglobulin test (IAT) is used to detect and identify unexpected antibodies in the serum or plasma. These antibodies

198 Section II: Part I: Red blood cells Antigen + Antibody Activated C1qr 2 s 2 Ca ++ C1qr 2 s 2 C2 C2a C3 Convertase C5 Convertase C4a C4 C4b C4b2a C4b2a3b C3a C3 C3b C5 C5a C3d C3bINA C3c Membrane attack complex C5b6789 C9 C8 C7 C5b C6 Figure 16.4 Classical pathway for complement activation. may be seen in blood donors, transfusion recipients, and prenatal patients who have been previously transfused or pregnant. 6 Table 16.4 Elements of pretransfusion testing 10 Element Requirement Compatibility testing Compatibility testing comprises pretransfusion procedures and laboratory tests with the goal to provide blood for transfusion that will have the optimal clinical effect. The elements of pretransfusion testing can be placed into one of three categories: those related to donor unit testing and processing, those related to patient sample collection and testing, and those that serve as a final check between the donor unit and intended recipient (Table 16.4). Proper performance of each element will enable the right unit of blood to be transfused to the right patient. The most important part of the process is proper identification of the patient, both at sample collection and at administration of the blood product. The most common cause of morbidity and mortality following blood transfusion is ABO incompatibility due to mislabeling of the sample, wrong blood in tube (WBIT), or misidentification and transfusion to the wrong patient. 8,9 Donor testing Collection facility ABO, Rh, and antibody detection tests on donor blood, tests for infectious diseases, and labeling of donor units are functions often carried out by a regional donor center; however, some hospitalbased transfusion services continue to procure a portion of their blood needs. Donations come from the population at large (allogeneic donors), but may also be collected from patients for their own use in elective surgical procedures (autologous donors); or, rarely, some patients request that they receive blood from relatives or friends (directed donations), but this practice is discouraged with the implementation of extensive tests for infectious disease and the safety of the blood supply (Chapter 62). The volume of tests performed at donor centers necessitates use of automated equipment. ABO grouping entails testing both donor red cells and serum/plasma, and the results must be concordant for the unit to be labeled. Red cells are tested with anti-a and anti-b; and the serum/plasma is tested against A 1 and B red cells. Anti-A,B Patient Consent Patient Sample Collection Identification Label Timing Donor Unit Confirmatory ABO and Rh Patient Sample Testing ABO and Rh Unexpected antibodies Donor/Patient Testing Crossmatch Electronic Blood bank or transfusion service medical director to participate in development of policies and processes Positive identification of intended recipient and blood sample at time of collection Mechanism to identify phlebotomist Two independent identifiers (e.g., first and last names, patient unique number) Date of collection Affixed to sample before leaving side of intended recipient Within 3 days of red cell transfusion if patient was transfused or pregnant within previous 3 months, or if patient history is uncertain or unavailable Testing from an attached segment, after the label has been affixed Anti-A and anti-b on RBCs of units labeled group A or group B Anti-A,B on units labeled group O Direct tests with anti-d for units labeled as Rh-negative Anti-A and anti-b on RBCs; serum or plasma with A 1 and B red cells; concordance between red cell and serum results Test with anti-d; test for weak expression optional To reduce risk of misidentification of the sample, perform ABO/Rh on second sample collected at a separate phlebotomy, or compare with previous records, or electronic identification verification Method that will demonstrate clinically significant antibodies using reagent red cells that are not pooled Comparison of current ABO, Rh, and antibody screen with historical records Antiglobulin test if clinically significant antibodies detected, currently or in the past Tests for ABO incompatibility only if no clinically significant antibodies detected, currently or in the past Computer system validated to ensure only ABOcompatible blood is selected may be used to detect ABO incompatibility

Chapter 16: Red cell immunology and compatibility testing 199 and/or A 2 red cells are often also used to detect subgroups of A that may be nonreactive in direct tests with anti-a alone. Rh typing for the D antigen on blood donor samples must be performed by a method that will detect weak expression of the D antigen as D is highly immunogenic 8 and weak expression of the antigen can evoke an immune response. Donor red cells that initially type as Rh-negative in direct agglutination tests can be tested further for weak D by an IAT, or more commonly at donor centers enzyme treatment of the RBCs is a used in automated tests to enhance reactivity of weak D antigen. All D-positive and weak D- positive blood is considered Rh-positive. 6,10 Some RBCs that express very weak D antigens, including those only detected by adsorption/elution of anti-d (termed Del), are not detected as Rhpositive. 6 This is a shortcoming of serologic methods as the units are labeled as Rh-negative. Hence, production of anti-d in a patient receiving RBCs labeled as Rh-negative should be reported to the donor center for follow-up testing of the blood donor. Transfusing facility The ABO group of units of whole blood or RBCs, and the Rh type of those labeled Rh-negative, must be confirmed by the transfusion facility on a segment from the unit before transfusion. 10(p35) This retyping for confirmation of ABO only requires testing of the RBCs with anti-a and anti-b, and units labeled group O can be tested with anti-a,b alone. 6 For confirmation of the D type of units labeled Rh-negative, only direct testing with anti-d is necessary; testing for weak expression of D is not required, nor is repeat testing for unexpected antibodies or for markers of infectious diseases. 6 For transfusion facilities that also collect blood, confirmatory testing for first-time donors must be performed after the original ABO and Rh label has been affixed using a sample from a segment attached to the donor unit. Patient testing Sample collection The major cause of fatal, hemolytic transfusion reactions is ABOincompatible transfusion resulting from patient and/or sample misidentification somewhere in the process from specimen collection to transfusion. Facilities must have validated processes and procedures that define each step in the process. Some considerations include: 1 Requisition. Forms requesting blood and blood components must contain two independent patient identifiers, usually the first and last names and a unique numerical identifier such as a hospital registration number. 10(p34) The name of the requesting physician, gender and date of birth of the patient, clinical diagnosis, and previous transfusion or pregnancy history are additional useful pieces of information. 2 Patient identity. The collection of a properly labeled blood sample for pretransfusion testing from the correct patient is critical to safe blood transfusion. The person collecting the sample must positively identify the patient from the wristband containing two unique patient identifiers (generally patient s full name and unique hospital registration number) that remains attached to the patient throughout the hospitalization and blood transfusion. The information on the requisition form must be compared with that on the wristband; blood samples should never be collected if there is a discrepancy. 10(p35) In the absence of a wristband, the nursing staff should follow hospital procedure for identifying and attaching a wristband to the patient before any samples are drawn. In an emergency, a temporary identification number should be used, and crossreferenced with the patient s name and hospital identification number once they are known. 3 Labeling. Blood samples must be clearly labeled at the bedside with the patient s unique identifiers. There must be a mechanism to identify the date of sample collection and the individual who collected the sample. Conventionally, the phlebotomist signs or initials the tube and signs or initials the requisition form so that there is a permanent record of the phlebotomist. 10(p35) 4 Confirmation of sample identity. Prior to testing, the information on the blood sample label must be compared with that on the requisition. A new sample must be obtained whenever there are discrepancies or if there is any doubt about the identity of the sample. It is unacceptable to correct a mislabeled sample. 10(p35) 5 Type of sample. Either serum or plasma may be used for pretransfusion testing. Anticoagulated (plasma) samples are more commonly used and are required if testing is automated. Some technologists prefer to use serum rather than plasma for compatibility testing to facilitate detection of antibodies that primarily coat red cells with the C3d component of complement. Bound C3d will not cause lysis but can be detected with AHG reagents containing anti-c3d. EDTA, citrate, and other commonly used anticoagulants chelate calcium ions that are essential for complement activation. 6 Age of specimen. To ensure that the specimen used for compatibility testing is representative of a patient s current immune status, serologic studies must be performed using blood collected no more than three days in advance of the transfusion when the patient has been transfused or pregnant within the preceding three months, or when such information is uncertain or unavailable. 10(p36) From a practical standpoint, it is often simpler to stipulate that all pretransfusion samples must be collected within three days before red cell transfusions, rather than ascertain whether or not each patient has been recently transfused or pregnant. 7 Storage. Blood samples used for compatibility testing, including donor red cells, must be stored at refrigerated temperatures for at least seven days after transfusion. 10(p35) This ensures that appropriate samples are available for investigational purposes should adverse responses occur. ABO typing Reagent antisera and red cells for ABO typing are available commercially. Anti-A and anti-b are monoclonal antibodies prepared by hybridoma technology. Reagent red cells are usually suspended in a preservative medium containing EDTA, to prevent lysis of the red cells by complement-binding anti-a and anti-b. ABO grouping is performed using a direct agglutination technique(table16.3).redcellsaretestedwithanti-aandanti-b, and the serum or plasma with A1 and B red cells. Use of anti-a,b and A2 red cells is optional, but it is generally considered unnecessary when routinely ABO typing potential transfusion recipients. 6 The expected findings for each of the four ABO phenotypes are shown in Table 16.5. When interpreting the results of ABO grouping tests, the reciprocal relationship that exists between the absence of A and/or B antigens on red cells and the presence of anti-a and/or anti-b in the serum is considered. If there is conflict between cell and serum ABO tests, group O blood can be provided for transfusion until the discrepancy is resolved and reliable conclusions of the patient s ABOtypecanbemade.

200 Section II: Part I: Red blood cells Table 16.5 The Expected Reactions of the Four Common ABO Phenotypes: Results of Blood Typing Tests Blood Type Anti-A Anti-B A 1 Red Cells B Red Cells O 0 0 4+ 4+ A 4+ 0 0 4+ B 0 4+ 4+ 0 AB 4+ 4+ 0 0 O = no agglutination; 4+ = strong agglutination. Rh typing In the United States, anti-d for testing patient samples is a blend of monoclonal IgM and monoclonal/polyclonal human IgG. With blended anti-d reagents, the IgM component causes direct agglutination of D-positive red cells and the IgG component permits detection of the weak expression of D by application of the antiglobulin test. 14 Only direct tests with blended anti-d reagents are required on patient samples; the test for weak D is not required. To avoid incorrect designation of a D-negative recipient as D-positive because of autoantibodies or abnormal serum proteins, a control system appropriate to the anti-d reagent is required. 6 A negative test with anti-a and/or anti-b is considered an appropriate control system, but apparent Group AB, D-positive samples require an Rh control reagent. Tests for unexpected antibodies Table 16.2 lists the majority of blood group alloantibodies encountered and provides the approximate percentage of compatible units that are likely to be encountered in Caucasian and black donor populations. Methods for testing samples for unexpected antibodies in the serum or plasma of prospective transfusion recipients must be those that detect clinically significant antibodies. 10(p36) An IAT after 37 C incubation of patient serum or plasma with reagent red cells is required. A number of options exist regarding the selection of methods for pretransfusion antibody detection (Table 16.6). Decisions relative to these options are within the purview of the blood bank medical director. They should be made based on the type of patients served, the causes and frequency of previous significant antibody-mediated transfusion reactions, and the availability of resources, with the realization that no one method will detect all clinically significant antibodies. Tube test methods A variety of red cell suspending media or additives are used either to enhance antibody uptake or to potentiate the agglutination phase of antibody antigen interactions (Table 16.6). Low-ionicstrength saline (LISS) solution, normal saline, or red cell preservatives (modified Alsever s solution) are used as red cell suspending media. In contrast, bovine serum albumin (22% or 30% w/v), LISS additives, or polyethylene glycol (PEG) are commonly added directly to serum red cell mixtures. 11 Antibody uptake is accelerated when red cells are suspended in LISS or PEG. The magnitude of the ionic cloud (Figure 16.3) surrounding negatively charged red cells suspended in LISS (approximately 0.03 M) is lower than that surrounding red cells suspended in normal saline (0.15 M NaCl). The ionic cloud, imparted by negatively charged carboxyl groups, hinders association between antibody and antigen; thus, reducing the magnitude of the ionic cloud promotes antibody association and also causes an increase in attraction between the negatively charged red cell membrane and positive charges on the antigen-binding sites of antibody molecules. The net effect of additives is that more antibody can be bound in a shorter period of time using LISS or PEG as opposed to saline. Consequently, incubation times can be reduced to 10 15 minutes for LISS, or 15 30 minutes for PEG, compared to 30 60 minutes for saline. 11 In contrast, albumin promotes the second stage of antigen antibody interactions, namely, the agglutination of antibody-coated red cells. Albumin can promote the direct agglutination of IgGcoated red cells; however, albumin is not used in the United States in a manner that promotes direct agglutination of antibody-coated red cells. Rather, the enhancing effect of albumin on red cell antigen interactions can be attributed to its formulation as a lowionic solution. 11 It is common practice to examine saline, albumin, and LISS tests for direct agglutination before subjecting them to an IAT. These examinations can be made immediately after mixing cells and serum followed by centrifugation (immediate-spin tests) and again after incubation at 37 C. For antiglobulin testing, either anti-igg or polyspecific AHG (containing anti-igg and anti-c3) may be used. However, use of polyspecific AHG may lead to the detection of a number of unwanted positives. PEG tests should not be examined for direct agglutination, and antiglobulin reagent containing anti-c3 is not routinely used. 11 Gel column testing A gel test for detecting red cell antigen antibody interactions was first described in 1990 by Lapierre and colleagues. 12 Cards consisting of six microcolumns, each containing agarose gel suspended in anti-igg, are commercially available. Atop each card is an incubation chamber in which reagent red cells and test plasma are dispensed. The cards are incubated at 37 C and then Table 16.6 Methods for Pretransfusion Antibody Detection Serum Red Cells Incubation AHG Saline 2 3 drops 1 drop, 3 5% 30 60 min; 37 C IgG/PS Albumin 2 3 drops 1 drop,3 5% 15 30 min; 37 C IgG/PS Low-ionic-strength saline (solution) 2 drops 2 drops, 2% 10 15 min; 37 C IgG/PS Gel 25 μl 50 μl, 0.8% 15 min; 37 C IgG Polyethylene glycol (PEG) 2 drops y 1 drop 15 30 min; 37 C IgG Solid-phase adherence 1 drop z 10 15 min; 37 C IgG Drop volumes should be equal. y Plus 4 volumes of 20% polyethylene glycol. z Predetermined by reagent supplier. AHG, antihuman globulin; PS, polyspecific AHG; RT, room temperature.

Chapter 16: Red cell immunology and compatibility testing 201 50μL 0.8% RBCs in LISS + 25μL sample GEL Test Antigen-coat Solid Phase Assays + Antibody 15' at 37 C + AHG-coated RBCs 10' Spin 4+ 3+ 2+ 1+ 0 mf Graded reactions Anti-IgG + GEL Negative Figure 16.6 Solid-phase adherence assays. Positive Figure 16.5 The gel test for detecting unexpected antibodies. centrifuged. As the red cells pass through the gel, they are separated from the serum/plasma and come into contact with anti-igg. If the red cells become coated with antibody during incubation, they will be agglutinated by anti-igg. The agglutinated red cells become trapped in the gel; unagglutinated red cells pellet to the bottom of the microcolumn (Figure 16.5). Column technologies offer several advantages over test tube procedures. When compared to tube tests, the time savings are reflected in no centrifugation/reading for direct agglutination after incubation, no addition of antiglobulin reagent, and no need to validate negative tests using IgG-coated red cells. Reproducibility is increased through use of measured volumes of reactants, elimination of the washing process, and less subjective reading of tests. The stability of reactions facilitates validation and review of results by a second technologist. Testing can be automated, or semiautomated using liquid sample handling devices. Solid-phase adherence methods Two forms of solid-phase adherence assays are available for red cell serologic testing. For direct tests, antibody is fixed to wells of a microplate (e.g., anti-a and anti-b for donor/recipient ABO typing), and red cells are added. Following centrifugation, red cells expressing the corresponding antigen will efface across the well; red cells lacking the antigen will pellet to the bottom of the well. For indirect tests (e.g., for detecting unexpected antibodies), red cell membranes are affixed to microplate wells; test serum or plasma are added, and the plates washed to remove unbound globulins. Indicator red cells, which are coated with anti-igg, are then added, and the plates are centrifuged. The indicator red cells efface across the well in a positive test, and pellet to the center of the well in a negative test (Figure 16.6). Automated pretransfusion testing Automated systems for pretransfusion testing are particularly suited for large hospital-based transfusion services, and include: 1 Gel column technology; 2 Solid-phase adherence technologies; and 3 Liquid microplates or microplates containing dried antisera for ABO/Rh typing, and modified solid-phase methods for antibody detection. Automated systems perform sample and reagent pipetting and analysis of agglutination reactions. Standard features include positive sample identification, process control through automated documentation of reagent lot numbers and expiration dates, batch and random access operating modes, STAT sample interrupt, and image analysis. The test results can be transmitted via an interface into the laboratory computer system. The benefits of automation include technologist time savings, positive sample identification, standardized testing (compliance with current good manufacturing practice), and increased workload capacity. Reagent red cells for antibody screening The FDA mandates that sets of reagent red cell samples licensed for use in pretransfusion antibody detection tests carry the C, c, D, E, e, Fy a,fy b,jk a,jk b,k,k,le a,le b,p 1 M, N, S, and s antigens. Reagent red cells for antibody detection are available commercially as sets of either two or three samples. The Rh phenotypes present in twosample sets are R 1 R 1 (D+C+c E e+) and R 2 R 2 (D+C c+e+e ). In three-sample sets, an rr (D C c+e e+) sample is also provided. Use of three red cell samples facilitates the identification of anti-d and enables inclusion of red cells from individuals homozygous for particular blood group genes. Such red cells often have a stronger expression of an antigen when compared to red cells from individuals heterozygous for the same gene; this phenomenon is known as dosage. The principles of antibody identification When unexpected antibodies are present, as indicated by positive antibody screening tests, they must be identified. This involves testing the patient s serum or plasma against a panel of fully phenotyped reagent red cell samples and the patient s own cells. A typical panel is shown in Table 16.7, which illustrates the results of antibody identification tests with a sample containing both anti- M and anti-k. Tests performed in this example include a reading for direct agglutination after room temperature incubation and after incubation at 37 C. Results of testing with LISS additive and tests with ficin-treated red cells are displayed. A typical approach follows: 1 The reactions with the patient s own red cells (AC) are examined. If the AC is positive, autoantibodies may be present and the patient may have a positive DAT. Therapy with certain drugs can also cause the AC and DAT to be positive. 6,11 Alternatively, the AC may react because alloantibodies have formed to recently transfused red cells that are still circulating in the recipient. The AC is negative in the case shown, so autoantibodies likely are not present.

202 Section II: Part I: Red blood cells Table 16.7 Results of tests between a panel of reagent red cells and a serum containing anti-m and anti-k RH MNS P1 LE KEL JK FY XG LISS FICIN Panel D C c C w E e f M N S s P 1 Le a Le b K k Kp a Js a Jk a Jk b Fy a Fy b Xg a RT 37 IAT 37 IAT 1 r r 0 + + 0 0 + + + + + 0 0 0 + 0 + 0 0 + + 0 + + 1 3+ 1+ 0 0 0 2 R 1 R 1 + + 0 0 0 + 0 0 + + + + + 0 0 + 0 0 + + + + + 2 0 0 0 0 0 3 R 1 R 1 + + 0 + 0 + 0 + 0 0 + + 0 + + + 0 0 + + + 0 + 3 4+ 3+ 3+ 0 3+ 4 R 2 R 2 + 0 + 0 + 0 0 0 + 0 + + 0 + 0 + 0 0 0 + + + + 4 0 0 0 0 0 5 r r 0 0 + 0 + + + + 0 + 0 + 0 + 0 + 0 0 + + 0 + + 5 4+ 3+ 3+ 0 0 6 rrv 0 0 + 0 0 + + + + 0 + + 0 0 0 + 0 0 0 + 0 + + 6 3+ 1+ 0 0 0 7 rr 0 0 + 0 0 + + 0 + 0 + + 0 + + 0 0 0 0 + + 0 0 7 0 0 4+ 0 4+ 8 rr 0 0 + 0 0 + + + + + + + + 0 0 + 0 0 + 0 + + 0 8 3+ 1+ 0 0 0 9 rr 0 0 + 0 0 + + + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + 9 4+ 3+ 3+ 0 0 10 rr 0 0 + 0 0 + + + + + + 0 + 0 + + 0 0 + + + 0 + 10 3+ 1+ 3+ 0 3+ 11 Ror + 0 + 0 0 + + + + 0 0 + 0 + 0 + 0 + + + 0 0 0 11 3+ 1+ 0 0 0 PATIENT AC 0 0 0 2 The graded reaction strengths are examined. Variability in reaction strength may be an indication of dosage, that is, the antibody reacts stronger with red cells from homozygotes (double-dose) than with red cells from heterozygotes (single-dose). Given the varying degrees of reactivity of positive red cell samples in Table 16.7, more than one antibody appears to be present, and M+N red cells react significantly stronger than M+N+ red cells. 3 A process of ruling out is undertaken by evaluating the nonreactive cells and the antigens present on the nonreactive cells. Only reagent red cell samples 2 and 4 are nonreactive. The presence of antibodies to antigens present on these two cells (D, C, c, E, e, N, S, s, P 1,Le a,le b,jk a,jk b,fy a,fy b, and Xg a ) can be eliminated from initial consideration. This leaves the possibility of antibodies to M, K, C w,f,kp a, and Js a. However, C w,kp a, and Js a are low-prevalence antigens not present on the reagent red cells used for antibody detection, and f antigen will not be present on a two-sample screening set of R 1 R 1 and R 2 R 2 cells, so these specificities are not responsible for the positive antibody screen. Thus, the results suggest the presence of anti-m and anti-k. 4 The test phase at which reactivity is observed is evaluated. Antibodies that are often IgM (Table 16.2), such as anti-m, -N, -Le a, and -P 1, react as direct agglutinins at room temperature. Antibodies that are usually IgG (e.g., anti-rh, -K, -Fy, -Jk, and -S) react preferentially by an IAT, although during the early stages of the immune response, IgM antibodies of these specificities can be encountered. With the example here, the anti-m appears to react best at room temperature, and the anti-k reacts best by IAT. This is consistent with the anti-m being IgM, although many examples of anti-m do have an IgG component, 3,7 whereas the anti-k is most likely IgG (Table 16.2). Anti-f does not appear to be present because f-positive cell samples 1, 6, 8, and 11 are nonreactive by IAT. 5 The results of tests with enzyme-treated red cells, if performed, are evaluated. Knowledge of the anticipated behavior of certain antibodies with enzyme-treated red cells can be very helpful when identifying antibodies especially when dealing with sera containing mixtures of alloantibodies. Antigens of the MNS, FY, and XG systems are cleaved by treatment of red cells with proteolytic enzymes such as papain or ficin, and negative or weak reactions are observed when antibodies to these antigens are tested with ficin- or papain-treated red cells. In contrast, treatment of red cells with proteolytic enzymes enhances reactivity with Rh antibodies, as well as antibodies such as anti-le a,-p 1, and -Jk a. The double-dose M-positive red cell samples (3, 5, and 9) react in LISS tests at 37 C and by IAT. These cells are nonreactive when pretreated with ficin, as expected. In contrast, K antigen is not affected by ficin treatment, so there is no difference in the reactions of ficin and LISS tests with cell samples 3, 7, and 10. Anti-f can now be completely eliminated because, if present, it should have reacted with all the K-negative ficin-treated cells from donors with r (ce) haplotype. 6 There must be sufficient negative test results with red cells that lack the corresponding antigen and sufficient positive test results with red cells that carry that antigen. To obtain a confidence level of >95% (p = 0.05), there should be at least three nonreactive antigen-negative red cell samples and at least three reactive antigen-positive red cells. This requirement has been met for both the anti-m and the anti-k. 7 The red cells from the patient are tested with anti-m and anti-k, and should lack both M and K antigens. There are exceptions of alloantibody formation when the corresponding antigen appears to be present on the autologous red cells. Most notably, this is seen in the Rh system in individuals with partial antigens who make antibody to the portions of antigen that are absent from their red cells. The above illustration represents a basic approach to antibody identification. More complex cases involving autoantibodies, multiple alloantibodies, mixtures of both auto- and alloantibodies, and antibodies to high-prevalence antigens often will require the resources of an immunohematology reference laboratory. Blood group DNA-based typing in pretransfusion testing Blood group antigens other than ABO and RhD are often referred to as minor blood group antigens. Testing for antigens beyond the ABO and RhD groups is referred to as performing an extended phenotype. For pretransfusion testing, knowing which common clinically significant antigens are expressed on the patient s RBCs and which antigens the patient lacks, and hence may have the corresponding antibody in the serum or plasma, is very useful for antibody identification. In the past, this information has not been readily available because performing extended typing for multiple minor RBC antigens by manual serologic methods is time consuming and costly. In contrast, with DNA-based testing, an extensive antigen profile on the patient (and blood donor) can be obtained in a single assay. This enables antigen matching of patients with donors for