Background: Direct antiglobulin test (DAT)-negative warm autoimmune hemolytic anemia (AIHA) is mainly caused by three mechanisms: red blood cell (RBC)-bound immunoglobulin (Ig)G below the detection limit of routine DAT; RBC-bound IgA or IgM; or low-affinity autoantibodies. Although most cases of DAT-negative AIHA are thought to be caused by RBC-bound IgG, and combinatory serological analyses are recommended, the relative ratios of each mechanism have not been clarified. Methods: Two groups of patients with undiagnosed hemolytic anemia and negative conventional tube method-DAT (TM-DAT) were investigated using anti-IgA and anti-IgM sera, or column agglutination method-DAT (CM-DAT), respectively, in addition to radioimmunological quantitation of RBC-bound IgG. Results: Three of 73 patients with DAT-negative AIHA showed positive RBC-bound IgA and normal amounts of RBC-bound IgG. Another group of 3 patients were RBC-bound IgM-positive, but only one of these showed normal amounts of RBC-bound IgG. In another group of patients with DAT-negative AIHA, 4 of the 20 showed positive CM-DAT and negative CM-DAT after washing RBCs. Three of these patients had normal amounts of RBC-bound IgG. Five patients with positive CM-DAT both before and after washing RBCs had high amounts of RBC-bound IgG. Conclusion: Relative ratios of patients with DAT-negative AIHA resulting from RBC-bound IgG, RBC-bound IgA, RBC-bound IgM, and low-affinity IgG were estimated as 80, 4, 1 and 15%, respectively. A new classification and diagnostic algorithm for DAT-negative AIHA were proposed.

Petz LD, Garratty G: Immune Hemolytic Anemias (ed 2). Philadelphia, Churchill Livingstone, 2004.
Engelfriet CP, Overbeeke MA, von dem Borne AE: Autoimmune hemolytic anemia. Semin Hematol 1992; 29: 3–12.
Evans RS, Weiser RS: The serology of autoimmune hemolytic disease; observations on forty-one patients. AMA Arch Intern Med 1957; 100: 371–399.
Worlledge SM, Blajchman MA: The autoimmune haemolytic anaemias. Br J Haematol 1972; 23: 61–69.
Chaplin H Jr: Clinical usefulness of specific antiglobulin reagents in autoimmune hemolytic anemias. Prog Hematol 1973; 8: 25–49.
Issitt PD, Gutgseu NS: Clinically significant antibodies not detected by routine methods; in Nance S (ed): Immune Destruction of Red Blood Cells. Arlington, American Association of Blood Banks 1989, pp 93–99.
Kamesaki T, Oyamada T, Omine M, Ozawa K, Kajii E: Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia. Am J Hematol 2009; 84: 98–101.
Garratty G: Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005; 42: 156–164.
Jeje MO, Blajchman MA, Steeves K, Horsewood P, Kelton JG: Quantitation of red cell-associated IgG using an immunoradiometric assay. Transfusion 1984; 24: 473–476.
Omine M, Kajii E, Kamesaki T, Karasawa M: A reference guide for diagnosis and treatment of autoimmunne hemolytic anemia. Jpn J Clin Hematol 2006; 47: 117–136.
Sugano N, Obata T, Onozaki F, et al: Hemagglutination reaction by column agglutination: comparison of the conventional tube, bead and gel techniques. Jpn J Med Technol 2000; 49: 951–955.
Kamesaki T, Toyotsuji T, Kajii E: Characterization of direct antiglobulin test-negative autoimmune hemolytic anemia: a study of 154 cases. Am J Hematol 2013; 88: 93–96.
Segel GB, Lichtman MA: Direct antiglobulin (“Coombs”) test-negative autoimmune hemolytic anemia: a review. Blood Cells Mol Dis 2014; 52: 152–160.
Leger RM, Co A, Hunt P, Garrity G: Attempts to support an immune etiology in 800 patients with direct antiglobulin test-negative hemolytic anemia. Immunohematology 2010; 26: 156–160.
Lai M, Rumi C, D’Onofrio G, Voso MT, -Leone G: Clinically significant autoimmune hemolytic anemia with a negative -direct antiglobulin test by routine tube test and positive by column agglutination method. Immunohematology 2002; 18: 109–113.
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