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An Educational Supplement prepared by ALQEP October 2003 The antiglobulin test was first described by Coombs, Mourant and Race in 1945 as the indirect antiglobulin test, a test to detect non-agglutinating antibodies in serum.1,2 A year later, they used the test to detect Rh antibodies on the red blood cells (rbcs) of babies suffering from hemolytic disease of the newborn.3 The same year, Boorman, Dodd and Loutit demonstrated the presence of autoantibodies on the rbcs of patients with acquired hemolytic anemia and the absence of rbc-bound antibodies in patients with congenital hemolytic anemia.4 This test to detect antibodies that had sensitized rbcs in vivo became known as the direct antiglobulin test (DAT). A positive DAT is generally caused by the attachment of immunoglobulin (IgG, IgM, IgA) and / or components of complement (C3d, C3, C4 etc.) to the red cell surface. The DAT has been applied in many ways. Although never a required element of pretransfusion testing, it became common practice to include a DAT or an autocontrol as part of screening tests for unexpected antibodies. The test was performed as a mechanism to detect the early manifestations of an immune response to a previous recent transfusion, as well as screening for clinically unsuspected cases of autoimmune or drug-induced hemolysis.5 The Test By tube: Traditionally, the DAT has been performed by tube agglutination, initially with a polyspecific antihuman globulin reagent capable of detecting cell-bound IgG and C3d. If positive, tests with specific anti-IgG and anti-complement (C3d) reagents are employed. The test is rapid, easy to perform and the agglutination is easy to observe and interpret. The test has been shown to detect 100-500 molecules of IgG / red cell and 400 1100 molecules of C3d / red cell.6 By gel technique: This method is based on column technology, in which red cells are filtered through a column containing a gel medium, which is contained in a card or strip of microtubes. The gel medium contains anti-IgG, anti-C3 or polyspecific antiglobulin sera which binds to red cells sensitized with immunoglobulin, consequently trapping them in the gel column. Unsensitized cells pellet at the bottom of the column. Gel media containing anti-IgM or -IgA may also be produced.6 Some studies have shown that DATs by gel technique may be more sensitive than tube technique DATs for the detection of cell-bound IgG.7,8 However, other similar studies have concluded that the gel DAT shows lower sensitivity, due mainly to a failure to detect C3d-coated red cells.9 By flow cytometry: Immunofluorescence techniques have been utilized in flow cytometry to detect and measure low levels of cell-bound IgG. While studies have shown that DATs by flow cytometric assays are more precise and reproducible than those by tube agglutination tests, a threshold of clinical significance has not been detemined.10
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DAT Results on Blood Donors The number of positive DATs reported in healthy blood donors ranges from 1 in 1000 (USA) to 1 in 14,000 (UK).31 This result usually indicates the presence of warm-reactive autoantibodies without evidence of immune hemolysis, although in some individuals, it may foreshadow AIHA or other autoimmune phenomena.5 George Garratty reports that very few donors would benefit from being informed of the positive DAT and having a subsequent medical / hematological evaluation. He does, however, argue that perhaps it would be of benefit to recommend evaluation of the rare donor with a strongly (3+ or 4+) positive DAT.31 Most hospitals do not issue DAT-positive donor rbcs for transfusion; however, as many hospitals no longer perform autocontrols or antiglobulin crossmatches and blood centres do not perform DATs routinely on blood donors, it is very possible that a hospital might fail to identify a DAT-positive donor. It has been reported that 72% of DAT-positive units have shortened rbc survival49 and the conclusion would be that it is not appropriate to transfuse any DAT-positive rbcs. However, no other study has confirmed these results and it has been reported that a positive DAT on donor rbcs is often a false-positive result.50
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Investigation of a Positive DAT The investigation of a positive DAT should be based on clinical considerations. The results of the serologic tests must be assessed in conjunction with clinical information and other laboratory tests such as hematocrit, bilirubin, haptoglobin and reticulocyte count. The AABB Technical Manual6 recommends the following evaluation:
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1. Is there any evidence of in-vivo red cell destruction? Testing to evaluate a possible immune etiology is appropriate if laboratory tests on an anemic patient with a positive DAT show evidence of increased red cell destruction. However, if there is no evidence of red cell destruction, no further studies are necessary unless the patient needs transfusion and the serum contains incompletely identified antibodies to red cell antigens. 2. Has the patient been recently transfused? A positive DAT may be indicative of a delayed hemolytic transfusion reaction and therefore, should be investigated if a patient has been recently transfused. Also, a post-transfusion positive DAT may the first indication of a developing immune response. Elution performed to evaluate the positive DAT often concentrates antibody activity and may facilitate identification of weakly reactive serum antibodies. 3. Is the patient receiving any drugs, such as procainamide, -methyldopa, cephalosporins or intravenous penicillin? If a positive DAT is found in a patient receiving such drugs, the attending physician should be alerted so that appropriate surveillance for red cell destruction can be maintained. If red cell survival is not shortened, no further studies are necessary. Judd questions the need to investigate even those cases where hemolysis is indicated. As these investigations can be extremely time-consuming, difficult to perform and hard to control, he recommends switching the patient to a pharmacologically unrelated drug.5 4. Has the patient received a marrow or an organ transplant? Passenger lymphocytes of donor origin produce antibodies directed against ABO or other antigens on the recipients cells, causing a positive DAT. 5. Is the patient receiving IVIG or IV RhIG? IVIG may contain ABO, Rh or other antibodies. IV RhIG causes development of a positive DAT in Rh-positive patients. The list of techniques available to investigate a positive DAT is extensive. Excellent references exist for the techniques. These include: 1. 2. 3. 4. 5. 6. Elution Techniques6,23,24,57 Auto- and alloadsorptions6,23,24,58 Drug-induced hemolytic anemia investigations6,23,24 Testing rbcs for the presence of low affinity antibodies58 Testing rbcs for the presence of low levels of IgG58 Subclassing of rbc-bound IgG.58
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