Blood group serology

Abstract

A blood type (also called a blood group) is a classification of blood based on the presence or absence of inherited antigenic substances on the surface of red blood cells (RBCs). In the early 20th century, Karl Landsteiner, an Austrian scientist, observed the surface of the RBCs and found two distinct chemical molecules. A series of tests reported by Karl Landsteiner in 1900 led to the discovery of the ABO blood groups and the development of routine blood grouping procedures [1]. ABO remains the most significant for transfusion practice. It is the only system in which the reciprocal (or antithetical) antibodies (see Table 1) are consistently and predictably present in the sera of normal people who have had no exposure to human RBCs. Because of these antibodies, transfusion of ABO-incompatible blood may cause severe intravascular haemolysis as well as the other manifestations of an acute haemolytic transfusion reaction. Red blood cell antigens may be proteins, carbohydrates, glycoproteins or glycolipids, depending on the blood group system; some of these antigens are also present on the surface of other types of cells of various tissues. Several of these RBC surface antigens that stem from one allele (or very closely linked genes), collectively form a blood group system [1]. Three common alleles (A, B and O) are located at the ABO locus on chromosome 9. The A and B genes encode glycosyltransferases that produce the A and B antigens, respectively. The O gene is considered to be non-functional, because its protein product determines no detectable blood group antigen. The RBCs of group O persons lack A and B, but carry an abundant amount of H antigen, the precursor material on which A and B antigens are built. Direct agglutination tests are used to detect A and B antigens on RBCs. Reagent antibodies frequently produce weaker reactions with RBCs from newborns than with RBCs from adults. Although they can be detected on the RBCs of 5–6-week-old embryos, A and B antigens are not fully developed at birth, presumably because the branching oligosaccharide structures develop gradually. By the time a person is 2–4 years old, A and B antigen expression is fully developed, and remains fairly constant throughout life. Anti-A and anti-B, the common immunoglobulin M (IgM) antibodies to the RBC surface antigens of the ABO blood group system, are sometimes described as being ‘naturally occurring’; however, this is a misnomer, because these antibodies are formed in infancy by sensitization in the same way as other antibodies. The theory that explains how these antibodies are developed states that antigens similar to the A and B antigens occur in nature, including in food, plants and bacteria. After birth an infant gut becomes colonized with normal flora that expresses these A-like and B-like antigens, causing the immune system to make antibodies to those antigens that the RBCs do not possess. Therefore, people who are blood type A will have anti-B, blood type B will have anti-A, blood type O will have both anti-A and anti-B, and blood type AB will have neither. Because of these so called ‘naturally occurring’ and expected antibodies, it is important to correctly determine a patient's blood type prior to transfusion of any blood component. These naturally occurring antibodies are of the IgM class, which have the capability of agglutinating (clumping) and damaging RBCs within the blood vessels, possibly leading to death. It is not necessary to determine any other blood groups, because almost all other RBC antibodies can only develop through active immunization, which can only occur through either previous blood transfusion or pregnancy. A test called the antibody screen is always performed on patients who may require RBC transfusion, and this test will detect most clinically significant RBC antibodies. Subgroups are ABO phenotypes that differ in the amount of antigen carried on RBCs and, in secretors, present in the saliva (see Table 2). Subgroups are more commonly encountered and more significant for A than for B. The two principal subgroups of A are A1 and A2. Approximately 80% of group A or group AB individuals have RBCs that are agglutinated by anti-A1 and thus are classified as A1 or A1B. The remaining 20%, whose RBCs are agglutinated by anti-A but not by anti-A1, are called A2 or A2B. Anti-A1 occurs as an alloantibody in the serum of 1–8% of A2 persons and 22–35% of A2B persons [2]. Anti-A1 can cause discrepancies in ABO testing and incompatibility in cross-matches with A1 or A1B RBCs. Subgroups of B are even less common than subgroups of A. Tests that use anti-A and anti-B to determine the presence or absence of the antigens are often described as direct or RBC tests. The use of reagent A1 and B RBCs to detect anti-A and anti-B in serum is called serum or the reverse testing. Routine tests on donors and patients must include both RBC and serum tests, each serving as a check on the other [3]. To confirm the ABO type of donor units that have already been labelled, or to test blood of infants less than 4 months of age, ABO testing on RBCs only is permissible. Procedures for ABO typing can be done by slide, tube, gel cards/cassettes (column agglutination technique, CAT) and microplate. Some ABO RBC typing reagents are prepared from pools of sera from individuals who have been stimulated with A or B blood group substances to produce antibodies of high titre. Other ABO grouping reagents are manufactured from monoclonal antibodies derived from cultured cell lines. Both types of reagents agglutinate most antigen-positive RBCs on direct contact, even without centrifugation. Slide testing cannot, however, be recommended because of the likelihood of spillage and contact with blood. Anti-A and anti-B in the serum of most patients and donors are usually too weak to agglutinate RBCs without centrifugation, so serum tests should be performed by tube, CAT or microplate techniques, not on slides. The donor's or patient's RBCs should be tested in a cell (forward) group using monoclonal anti-A and anti-B reagents. Anti-A,B or anti-A+B reagents can be used in conjunction with anti-A and anti-B, but are not essential. Reverse grouping should be performed using A1 and B reagent RBCs. The results and interpretations of routine RBC and serum tests for ABO are shown in Table 1. A discrepancy exists when the results of RBC tests do not complement that of serum tests, and the expected two positives and two negatives are not observed. When a discrepancy is encountered, the discrepant results should be recorded, but interpretation must be delayed until the discrepancy is resolved. If the specimen is from a donor unit, the unit may not be released for transfusion until the discrepancy is resolved. When the blood is from a potential recipient, it may be necessary to administer group O RBCs of the appropriate Rh type before the investigation is completed. It is important to obtain a sufficient amount of the patient's pre-transfusion blood to complete any additional studies that may be required. Red blood cell and serum test results may be discrepant because of intrinsic problems with RBCs or serum, because of test-related problems, or because of technical errors. Discrepancies may be signalled either because negative results are obtained when positive results are expected, or positive results are found when tests should have been negative. The first step in resolving an apparent problem should be to repeat the tests on the same sample. If initial tests were performed on RBCs suspended in serum or plasma, repeat testing should use a saline suspension of washed cells. If the cause of the discrepancy is a failure to obtain a reliable reverse group due to hypogammaglobulinaemia or insufficient sample, the cell group should be repeated. Any groups showing unexpected mixed-field reactions should be repeated and/or investigated prior to the authorization of the group. These reactions may represent an ABO/D incompatible transfusion (planned or unplanned), bone marrow/stem cell transplant, an A3 or B3 or a twin chimera (extremely rare). Variants of groups A and B may give much weaker than normal reactions with monoclonal anti-A and anti-B. For example, Ax or Bx gives very variable reaction strengths with different reagents, with some anti-A and anti-B reagents failing to react with Ax and Bx cells. The most likely finding is a negative reaction with anti-A or anti-B and a missing agglutinin on the reverse group (though anti-A1 is commonly detected in A variants). Absorption/elution studies with anti-A or anti-B may be beneficial in identifying the variant. Due to the difficulties in identifying weak A/B antigens, it is prudent to transfuse these patients with group O RBCs. Some anti-B reagents may react strongly with the acquired B antigen. This usually leads to a discrepancy between cell and reverse groups. Anti-B reagents found to react with acquired B cells should not be used in routine ABO grouping. For several months post delivery, neonates who have received intrauterine transfusions may appear to be the same ABO and D group as the transfused cells due to bone marrow suppression. An unexpected reaction with the reverse grouping cells may be observed, if these cells carry an antigen to a cold reactive alloantibody (other than anti-A and anti B) that is present in the patient's plasma. In these cases, the reverse group should either be repeated at 37°C with the same cells or tested with A1 and B cells that lack the implicated antigen. If a sample shows evidence of strong autoagglutination, washing the cells in pre-warmed saline may be of benefit. In severe cases pre-warming the patient cells, serum/plasma and grouping reagents prior to mixing and incubation of the tests at 37°C may be the only option. The Rh system is the second most significant blood group system in human blood transfusion. There are five major antigens within the Rh blood group system: D, C, E, c and e. The most significant Rh antigen is the RhD antigen, because it is the most immunogenic of the five main Rh antigens. It is common for RhD-negative individuals not to have any anti-RhD IgG or IgM antibodies, because anti-RhD antibodies are not usually produced by sensitization against environmental substances. However, RhD-negative individuals can produce IgG anti-RhD antibodies following a sensitizing event: possibly a fetomaternal transfusion of blood from a fetus in pregnancy or occasionally a blood transfusion with RhD-positive RBCs. The first human example of the antibody against the D antigen was reported in 1939 by Levine and Stetson [4], who found it in the serum of a woman whose fetus had haemolytic disease of the newborn and who experienced a haemolytic reaction after transfusion of her husband's blood. In 1940, Landsteiner and Wiener [5] described an antibody obtained by immunizing guinea pigs and rabbits with the RBCs of Rhesus monkeys; it agglutinated the RBCs of approximately 85% of humans tested, and they called the corresponding determinant the Rh factor. In the same year, Levine and Katzin [6] found similar antibodies in the serum of several recently delivered women, and at least one of these sera gave reactions that paralleled those of the animal anti-Rhesus sera. Also in 1940, Wiener and Peters observed antibodies of the same specificity in the serum of persons whose RBCs lacked the determinant, who had received ABO-compatible transfusions in the past. Later evidence established that the antigen detected by animal anti-Rhesus and human anti-D were not identical, but by that time the Rh blood group system had already received its name. It is important to note that monoclonal anti-D reagents vary widely in their ability to detect both partial D and weak D. When two different reagents are used, it is helpful to use those of a similar reactivity with partial D and weak D RBCs, in order to reduce the number of discrepancies. If a discrepancy occurs, the patient should be treated as D negative until the D status is resolved. Patients should not be classified as D positive on the basis of a weak reaction with a single anti-D reagent. If clear positive results are not obtained with two monoclonal anti-D reagents, it is safer to classify the patient as D negative. Patients of category DVI are the most likely to produce anti-D. Reagents used for D grouping patients should not detect category DVI. Patients with known partial D status should be regarded as D negative. Reagents used for D grouping donors should detect category DVI. Rh phenotyping is done by testing the unknown cells with anti-D, anti C, anti E, anti c and anti e reagents. It is important to phenotype the patients for Rh to insure that they are not transfused with Rh incompatible donor blood. The phenotyping results are shown in Table 3. The H system has two genes, H and h, and one antigen, H, which serves as the precursor molecule on which A and B antigens are built. On group O RBCs, there is no A or B, and the membrane expresses abundant H. Individuals of the rare Oh phenotype, whose RBCs lack H, have anti-A and anti-B, and a potent and clinically significant anti-H in their serum. The term ‘Bombay’ has been used for the phenotype in which RBCs lack H, A and B, because examples of such RBCs were first discovered in Bombay, India. The Oh phenotype becomes apparent when serum from the Oh individual is tested against group O RBCs, and strong, immediate reactions occur. The International Society of Blood Transfusion currently recognizes 30 blood group systems (including the ABO ISBT No: 001 and Rh systems ISBT No: 004) (see Table 4). Thus, in addition to the ABO antigens and Rh antigens, many other antigens are expressed on the RBC surface membrane. A complete blood type would describe a full set of 30 substances on the surface of RBCs, and an individual's blood type is one of the many possible combinations of blood group antigens. Across the 30 blood groups, over 600 different blood group antigens have been found, but many of these are very rare or are mainly found in certain ethnic groups. Almost always, an individual has the same blood group for life; but very rarely an individual's blood type changes through addition or suppression of an antigen in infection, malignancy or autoimmune disease. Antibody screening is performed to determine whether the serum of the patient or the donor contains irregular antibodies against RBC antigens that might be either due to previous exposure to that antigen through previous blood transfusion or pregnancy. Irregular antibodies are more likely to be seen in multi-transfused patients. The donor's or patient's serum or plasma should be tested by an appropriate technique against an identification panel of reagent RBCs. An indirect antiglobulin test should be used as the primary method for the screening of donor/patients’ plasma for the presence of clinically significant RBC antibodies. Test systems using tubes (liquid-phase), microplates (liquid or solid phase), or gel cards/cassettes (CAT) are suitable. If a tube method is used for antibody screening, then this should involve the suspension of RBCs in a low ionic strength solution. When an alloantibody is detected in the screening procedure, the specificity should be determined and its likely clinical significance should be assessed. It is essential to determine the specificity of all clinically significant antibodies present. It is important to employ a systematic approach to antibody exclusion in the process of antibody identification. Several reagent RBC panels may be needed to identify some combinations of antibodies [7]. The patient's serum or plasma should be tested by an appropriate technique against an identification panel of reagent RBCs. As a starting point, the technique by which the antibody was detected during screening should be used. Inclusion of the patient's own RBCs may be helpful, for example, in the recognition of an antibody directed against a high frequency antigen or the presence of an autoantibody. The specificity of the antibody should only be assigned when it is reactive with at least two examples of reagent RBCs carrying the antigen and non-reactive with at least two examples of reagent RBCs lacking the antigen. Note that, wherever possible, the presence of anti-Jka, -Jkb, -S, -s, -Fya and -Fyb should be excluded using RBCs having homozygous expressions of the relevant antigen. When one antibody specificity has been identified, it is essential that the presence of additional clinically significant antibodies has not been missed. Multiple antibodies can only be confirmed by choosing cells antigen negative for the recognized specificity, but positive for other antigens to which clinically significant antibodies may arise. Knowing the phenotypes of the patient can aid in cell selection for the identification and exclusion process. The requirements detailed above should be met for each antibody detected, but in some circumstances this may not be possible.