DIRECT ANTIGLOBULIN TEST

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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Test controls: The DAT is considered positive when agglutination is observed as an endpoint of the procedure and negative when no agglutination is observed. Appropriate controls must be included with the test to ensure valid results: 1. IgG coated red cells must be added to negative tests when using polyspecific antiglobulin sera (AGS) or anti-IgG to verify that the AGS was functional.6 2. A controversy surrounds the use of complement-coated red cells to validate the complement component of polyspecific AGS or anti-C3B, -C3d reagents. Although this requirement has recently been incorporated into the CAP Inspection Checklist11, neither AABB12 or CSTM Standards13 specifically address this item. The CSTM Guidelines for Serologic Control in the Transfusion Medicine Laboratory14 require validation of anti-C3 upon receipt in the laboratory but do not require validation of negative tests. All standards do, however, require that manufacturers instructions be followed, and therefore, laboratories should consult the reagent package insert to ensure that their procedure meets the requirements of the manufacturer.12,13 3. The AABB Technical Manual advises including a control (e.g. phosphate buffered saline) when performing a DAT. No interpretation of the test can be made if the results with all antisera used to perform the DAT and control are positive. This indicates spontaneous agglutination, which must be resolved before further testing is performed.6 Many facilities do not routinely include this control but do repeat reactive tests and include a parallel control. Variables in DAT test results: A false positive result may be due to: 1. colloidal silica or metallic ions leached from the storage container 2. dirty glassware 3. polyagglutinable cells 4. in vitro complement attachment 5. procedural errors improper centrifugation reagent problems15 A false negative DAT result may be due to: 1. procedural errors inadequate washing delay between washing and reading improper centrifugation improper cell suspension too vigorous shaking of test tube reagent problems sample problems 2. antibody of low avidity (elutes off during washing) 3. low levels of bound immunoglobulin 4. interference by cryoglobulins15

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DAT and the Autocontrol: Although a positive autocontrol is usually thought to equate to a positive DAT, this is not always the case. A positive autocontrol in the presence of a negative DAT may be due to: 1. Agglutination dependent on enhancement media, either non-specific attachment or weak specific attachment. 2. Improper technique. 3. Preservative-dependent reactivity.15 A negative autocontrol may occasionally be encountered in the presence of a positive DAT. The addition of enhancement media and the incubation at 37C may cause the elution of the rbc bound antibody in the presence of a low avidity autoantibody.15 Positive DAT and Red Blood Cell Typing: The presence of a positive DAT may cause problems when performing antigen typing of patient rbcs. Red cells strongly coated with globulins may undergo spontaneous agglutination with high-protein blood typing reagents, and occasionally even with low-protein reagents.6,15,16 As well, any tests which require the use of antiglobulin techniques will yield false positive results. A heavily coated red cell may result in a blocking phenomenon of the antigen sites on the cell, causing false negative typing results.17 Recognition of the potential typing problems caused by sensitized rbcs is vital in laboratory testing. As the DAT is often not routinely included in pretransfusion testing, appropriate controls must be performed with red cell typing. Manufacturers instructions for typing antisera must be followed. However, manufacturers of monoclonal typing reagents do not always require the routine use of an Rh control as immunoglobulin-coated rbcs rarely yield false positive results with low protein reagents. Because it has been proven that rbcs which are strongly sensitized may give unreliable results with monoclonal reagents, it is recommended that a concurrent control be performed on all patient cells that show agglutination in all tubes (i.e. give the reactions of AB, D-positive). 6,16 Several techniques are available to assist in cell typing in the presence of a positive DAT. The aim of these methods is to dissociate immunoglobulin from the rbc surface without interfering with the antigen site. These methods include: 1. Monoclonal reagents: The lower protein level of monoclonals make them the ideal testing reagent for red cells that have positive DATs.15 However, the restrictions previously mentioned must be considered. 2. Warm washing: Warm washing (37-42C saline) the red cells 3-6 times will often resolve the problems caused by a positive DAT.15 3. Chloroquine diphosphate: This reagent dissociates IgG from antibody-coated rbcs;18 however, it does not remove C3d. Therefore, anti-IgG must be used for typing the chloroquine diphosphate treated cells. 4. Dithiothreitol (DTT): DTT will remove both IgM and IgG from rbcs.19 However, some antigens are destroyed, such as those in the Kell blood group system,20 and DTT-treated cells can become sticky making it difficult to interpret typings with weaker antisera.21 5. EDTA glycine: EDTA glycine dissociates IgG from the rbc surface in part by destroying some of the rbc antigens including Kell antigens.22 6. Cell separation: If the antibody is bound to transfused cells, use of separation techniques including reticulocyte separation or hypotonic cell separations may be useful.6,23
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These methods or chemicals are described in reference books6,23,24 and / or are commercially available. Detection of a Positive DAT A positive DAT is usually detected when a laboratory is investigating one or more of the following: 1. Positive autocontrol on a panel or antibody screen 2. Test ordered by a clinician to investigate anemia and / or unexplained hemolysis in a patient 3. Positive Rh control on an Rh(D) typing 4. Transfusion reaction 5. Hemolytic Disease of the Newborn (HDN) 6. Unexplained incompatibility in an antiglobulin crossmatch (i.e., donor unit may have a positive DAT) 7. Typing results which do not make sense. As many laboratories do not routinely perform an Rh control, autocontrol, or antiglobulin crossmatch, many positive DATs may go undetected in both donors and patients. DAT Results on Hospital Patients Studies report approximately 1 to 3.5% of unselected hospital patients will have a positive DAT.25,26,27,28 These positive DATs may or may not be associated with evidence of hemolysis. Conditions associated with a positive DAT include: 1. Autoimmune Hemolytic Anemia (AIHA): The diagnosis of AIHA depends on the demonstration of autoantibodies directed against red cells. The DAT has been recognized as the hallmark test for this diagnosis as a positive DAT has a high predictive value (PV) for an immune etiology in a patient with hemolytic anemia (HA).29 Approximately 2 4% of patients with a clinical picture of warm AIHA will present with a negative DAT. This may be due to IgA inducing the immune hemolysis or the presence of low affinity autoantibodies which are capable of inducing significant hemolysis by are undetectable by laboratory methods.30 3. Hemolytic Disease of the Newborn: Garratty reports that in his experience almost all babies with HDN (i.e., evidence of a hemolytic anemia being present) have a positive DAT.31 The DAT is usually strongly positive in HDN due to anti-D or other alloantibodies. In ABO HDN, the DAT may be much weaker or in some cases, negative. However, the strength of the DAT does not always correlate with the severity of the disease, especially in ABO HDN.6,32 4. Drug-Induced Protein Adsorption: Approximately 10% of positive DATs are drug induced.30 A wide variety of drugs are known to cause positive DATs and/or immune hemolysis. Garratty reports that all patients proven by his group to have drug induced hemolytic anemia have had a positive DAT (sometimes very weakly positive).31 Second and third generation cephalosporins are increasingly associated with severe and sometimes fatal immune hemolytic anemia. It has been reported that both drug dependent and drug independent cephalosporin antibodies may exist in the same serum and a patient with a positive DAT and hemolysis may be diagnosed with warm AIHA due to detection of the drug independent antibodies. Therefore, treatment may not include discontinuation of the drug. The authors of the study suggest that because cephalosporins are so commonly used, it would be prudent to consider their role in every case of immune hemolytic anemia.30
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5. Hemolytic Transfusion Reaction (HTR): It has been reported that 89% of HTRs have a positive DAT;33 the negative DATs were all found in ABO HTRs associated with acute intravascular hemolysis with subsequent hemoglobinuria. Therefore, AABB12 and CSTM13 Standards mandate two laboratory tests, a DAT and inspection of the plasma for hemoglobin, following a suspected HTR. The most recent edition of the AABB Standards12 also requires confirmation of the patient ABO type on a post-transfusion sample. A recent study by Alvarez et al. concluded that flow cytometry and antibody detection in eluates are more sensitive techniques than the DAT for revealing minor populations of IgG-coated rbcs and could be included in the investigation of suspected cases of HTR.34 It is of interest to note that in some cases of HTR, the patients autologous red cells are also DAT positive, possibly due to complement fixation or the production of autoantibodies in conjunction with alloantibodies.35,36,37 High Gamma Globulin Levels: Most eluates from the DAT-positive rbcs of hospital patients are non-reactive. The rbc-bound IgG is not usually a blood group antibody but rather cytophilic IgG nonspecifically adsorbed to the rbcs from the plasma of patients with high gamma globulin levels.27,28 Sickle Cell Disease (SCD) and Thalassemia: Patients with SCD and thalassemia have increased amounts of rbcbound IgG. This may lead to a positive DAT.37 Immune System Disorders: An increased incidence of positive DATs occurs in autoimmune diseases, congenital and acquired immunodeficiency syndromes and malignancy.31 Transplantation: Transplantation of ABO incompatible bone marrow or solid organs may result in a positive DAT due to production of anti-A and / or B by transplanted lymphocytes.38,39 Technical factors: Variables in sample procurement,40 storage,41 and sensitivity of the test method used for the test42,43 have been shown to result in positive DAT results. Miscellaneous: Transfusions of non-ABO compatible plasma products44 IVIG may contain blood group alloantibodies44 Increased incidences of positive DATs have been reported in patients following cardiac surgery,45 multitransfused hemodialysis patients,46 and patients with malaria.47 A positive DAT was reported in a boy struck by lightening.48

6.

7. 8. 9. 10. 11.

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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Use of DAT in the Hospital Transfusion Service Pretransfusion testing: Judd et al. report two studies which demonstrate the non-specificity of the DAT when performed as part of routine pretransfusion testing.51,52 They found that the predictive value (PV) of a positive DAT, when screening tests for unexpected antibodies are negative, is 0.29%. This led them to abandon the pretransfusion DAT. They continued with a further study which compared the risk of eliminating the pretransfusion DAT with the risks associated with eliminating the antiglobulin phase of the crossmatch and the risk of omitting the 37C reading for direct agglutination from screening tests for unexpected antibodies. They concluded that the risk of transfusing incompatible blood by eliminating the DAT, IAT-XM and 37C reading is 1:13,000, 1:2000 and 1:2400 units transfused, respectively. The cumulative risk from eliminating all three tests is 1:1000 units. This study confirmed their conclusion as to the futility of performing a DAT as part of routine pretransfusion testing.53 Diagnostic testing: The most appropriate use of the DAT is in a patient with a hemolytic anemia of suspected immune etiology.31 The patient may have: 1. autoimmune hemolytic anemia (AIHA) 2. hemolytic disease of the newborn (HDN) 3. hemolytic transfusion reaction (HTR). As the DAT has a very low PV for diagnosing AIHA, HDN or HTR in unselected patients,31 the DAT should never be used as a screening test; it should be applied to answer a specific question (e.g. is this an immune hemolytic anemia?).29 A retrospective study by Cid et al on the use of DATs in their facility supported their conclusion that a complete laboratory evaluation should be performed on a patient with anemia to determine if the anemia appears to be hemolytic in nature, prior to performing a DAT. They then supported the use of a DAT to determine if the hemolytic anemia has an immune basis.54 John Judd recommends performing a DAT whenever hemolysis, especially immune-mediated hemolysis, is suspected as the DAT has a good PV in this setting. He also states that a history check and laboratory data must be collated and included in the diagnostic evaluation.5 Neonates: Current standard DAT testing appears to be a reliable screening test for HDN. However, studies show that the DAT fails to identify over half of the cases of significant newborn hemolysis and end-tidal carbon monoxide concentration measurement provides a more sensitive index for predicting significant jaundice in a newborn.55 Current guidelines do not recommend performing a DAT on all newborns. The test should be undertaken when the mother has potentially significant antibodies or when observing neonatal jaundice and anemia.5,56

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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Prepared by: Beverly Padget Transfusion Medicine Program Coordinator Laboratory Proficiency Testing Program College of Physicians and Surgeons of Alberta References: 1. Coombs RRA, Mourant AE, Race RR. Detection of weak and incomplete Rh agglutinins: A new test. Lancet 1945;ii:15-16. Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br J Exp Pathol 1945;26:255-66. Coombs RRA, Mourant AE, Race RR. In-vivo isosensitisation of red cells in babies with haemolytic disease. Lancet 1946;i:264-6. Boorman KE, Dodd BE, Loutit JF. Haemolytic icterus (acholuric jaundice). Congenital and acquired. Lancet 1946;i:812-4. Judd WJ. The clinical insignificance of a positive direct antiglobulin test. In: Direct antiglobulin testing in the new millennium program. Bethesda: American Association of Blood Banks; 1999. Brecher ME, ed. Technical Manual 14th edition. American Association of Blood Banks. Bethesda MD; 2002. Ditmar K, Procter JL, Cipolone K, et al. Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures. Transfusion 2001;41:1258-62. Nathalang O, Chauansumrit A, Prayoonwiwat W, et al. Comparison between the conventional tube technique and the gel technique in direct antiglobulin test. Vox Sang 1997;71:169-71. Tissot JD, Kiener C, Burnand D, et al. The direct antiglobulin test: still a place for the tube technique? Vox Sang 1999;77:223-6. Garratty G, Nance SJ. Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry. Transfusion 1990;30:617-21. College of American Pathologists, Transfusion medicine checklist. December 2002. American Association of Blood Banks. Standards for blood banks and transfusion services. 22nd edition. Bethesda, MD: AABB, 2003. Canadian Society for Transfusion Medicine. Standards for transfusion medicine. 5th edition. Saskatoon SK: CSTM, 1997.
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2.

3.

4.

5.

6. 7.

8.

9.

10.

11. 12.

13.

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14. Canadian Society for Transfusion Medicine. Guidelines for serologic quality control in the transfusion medicine laboratory 2nd edition. Saskatoon SK: CSTM, 1998. Harris, T. Positive direct antiglobulin tests: serological problem solving tips. In: Direct antiglobulin testing in the new millennium program. Bethesda: American Association of Blood Banks; 1999. Padget BJ, Hannon JL. Discrepancies in Rh(D) typing of sensitized red blood cells using monoclonal/polyclonal anti-D reagents: case report and review. Immunohematology 2001;17:10-13. Rodberg K, Tsuneta R, Garratty G. Discrepant phenotyping results when testing IgG-sensitized RBCs with monoclonal Rh reagents. Transfusion, 1995;35:10S,67S (Abstracts). Edwards JM, Moulds JJ, Judd WJ. Chloroquine diphosphate dissociation of antigen-antibody complexes: a new technique for phenotyping rbcs with a positive direct antiglobulin test. Transfusion, 1982;22:59-61. Reid ME. Autoagglutination dispersal utilizing sulphydryl compounds. Transfusion 1978;18:353-5. Branch DR et al. Disulfide bonds are a requirement for Kell and Cartwright (Yta) blood group antigen integrity. Br J Haematol, 1983;54:574-8. Johnson ST, Rudmann SV, Wilson (ed). Antibody identification: Harris T. Serological problem-solving strategies: a systematic approach, 1996:136-137. Kosanke J et al. Treatment of DAT positive red cells with EDTA-glycine acid for antigen typing. Transfusion 1989;29:57S (Abstract). Judd WJ. Methods in Immunohematology, 2nd edition. Montgomery Scientific, 1994. Mallory D et al. Immunohematology Methods and Procedures, 1993. Bohnen RF, Ultmann JE, Gorman JG et al. The direct Coombs test: Its clinical significance. Ann Intern Med 1968;68:19-32. Lau P, Haesler WE, Wurzel HA. Positive direct antiglobulin reaction in a patient population. Am J Clin Pathol 1975;65:368-75. Huh YO, Lichtiger B. Evaluation of a positive autologous control in pretransfusion testing. Am J Clin Pathol 1985;84:632-6. Toy PTCY, Chin CA, Reid ME et al. Factors associated with positive direct antiglobulin tests in pretransfusion testing: a case-control study. Vox Sang 1985;49:215-20. Kaplan HS, Garratty G. Predictive value of direct antiglobulin tests. Diag Med 1985;8:29-33.
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15.

16.

17.

18.

19. 20.

21.

22.

23. 24. 25.

26.

27.

28.

29.
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30. 31. Manny N, Zelig O. Laboratory diagnosis of autoimmune cytopenias. Current Opinion in Hematology 2000;7:414-19. Garratty G. The clinical significance of a positive direct antiglobulin test. In: Direct antiglobulin testing in the new millennium program. Bethesda: American Association of Blood Banks; 1999. Herschel M, Karrison T, Wen M et al. Isoimmunization is unlikely to be the cause of hemolysis in ABO incompatible but direct antiglobulin test-negative neonates. Pediatrics 2002;110:127-30. Pineda AA, Brzica SM JR, Taswell HF. Hemolytic transfusion reaction: recent experience in a large blood bank. Mayo Clin Proc 1978;53:378-90. Alvarez A, Rives S, Montoto S, et al. Relative sensitivity of direct antiglobulin test, antibodys elution and flow cytometry in the diagnosis of immune hemolytic transfusion reactions. Haematologica 2000;85:186-8. Salama A, Mueller-Eckhardt C. Delayed hemolytic transfusion reactions. Evidence for complement activation involving allogeneic and autologous red cells. Transfusion 1984;24:188-93. Ness PM, Shirley RS, Thoman SK, et al. The differentiation of delayed serologic and delayed hemolytic transfusion reactions: incidence, long-term serological findings and clinical significance. Transfusion 1990;30:688-93. Garratty G. The significance of IgG on the red cell surface. Trans Med Rev 1987;1:47-57. Mangal AK, Growe GH, Sinclair M et al. Acquired hemolytic anemia due to auto-anti-A or auto-anti-B induced by group O homograft in renal transplant recipients. Transfusion 1984;24:201-5. Salerno CT, Burdine J, Perry EH, et al. Donor-derived antibodies and hemolysis after ABO-compatible but nonidentical heart-lung and lung transplantation. Transplantation 1998;65:261-4. Grindon AJ, Wilson MJ. False-positive DAT caused by variables in sample procurement. Transfusion 1981;21:313-4. Reid ME, Ellisor SS, Avoy DR. Positive direct antiglobulin test on thawed deglycerolized units of erythrocytes: prediction and prevention. Amer J Hematol 1979;7:293-8. de Figueiredo M, Lima M, Morais S, et al. The gel test: some problems and solutions. Transfus Med 1992;2:115-8. Shanwell A, Sallander S, Bremmer K, et al. Clinical evaluation of a solid-phase test for red cell antibody screening of pregnant women. Transfusion 1999;39:26-31. Garratty G. Problems associated with passively transfused blood group alloantibodies. Am J Clin Pathol 1998;109:769-77.

32.

33.

34.

35.

36.

37. 38.

39.

40.

41.

42. 43.

44.

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45. 46. Polesky HF, Smith R, Weirich F. Positive antiglobulin tests in cardiac surgery patients. Transfusion 1969;9:43-6. Lasky LC, Rose RR, Polesky HF. Incidence of antibody formation and positive direct antiglobulin tests in a multitransfused hemodialysis population. Transfusion 1984;24:198-200. Abdalla S, Weatherall DJ. The direct antiglobulin test in P. falciparum malaria. Br J Haematol 1982;51:415-25. McCarthy LJ, Parker C. Positive antiglobulin tests in a boy struck by lighting. N Eng J Med 1981;305:283. Habibi B, Muller A, Lelong F et al. Auto-immunisation erythrocytaire dans la population normale. La Nouvelle Presse Medicale 1980;43:3253-7. Mougey R, Martin J. Positive direct antiglobulin tests in stored donor samples: an avoidable case of discarded units. Transfusion 1986;26:550 (abstract). Judd WJ, Butch SH, Oberman HA, et al. The evaluation of a positive direct AHG test in pretransfusion testing. Transfusion 1980;20:17-23. Judd WJ, Barnes BA, Steiner EA, et al. The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited. Transfusion 1986;26:220-4. Judd WJ, Fullen DR, Steiner EA, et al. Revisiting the issue: can the reading for serologic reactivity following 37C incubation be omitted? Transfusion 1999;39:295-9. Cid J, Ortin X, Beltran V, et al. The direct antiglobulin test in a hospital setting. Immunohematology 2003;19:16-8. Herschel M, Karrison T, Wen M, et al. Evaluation of the direct antiglobulin (Coombs) test for identifying newborns at risk for hemolysis as determined by end-tidal carbon monoxide concentration (ETCOc); and comparison of the Coombs test with ETCOc for detecting significant jaundice. Journal of Perinatology 2002;22:341-7. Judd WJ. Practice guidelines for prenatal and perinatal immunohematology revisited. Transfusion 2001;41:1445-52. Snyder EL, Falast GA. Significance of the direct antiglobulin test. Lab Med 1985;16:89-96. Leger RM, Garratty G. Evaluation of methods for detecting alloantibodies underlying warm autoantibodies. Transfusion 1999;39:11-16.

47. 48. 49.

50.

51.

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54. 55.

56. 57. 58.

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