Comparison of traditional methods and mitogen-stimulated direct antiglobulin test for detection of anti-red blood cell autoimmunity

Abstract

The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed using various methods with different sensitivities. Recently, mitogen-stimulated (MS)-DAT was suggested to be able to identify latent anti-erythrocyte autoimmunity. Traditional methods (tube, microcolumn, and solid phase) and MS-DAT were compared in 54 consecutive cases of suspected AIHA, 28 idiopathic AIHA in clinical remission, and 12 difficult-to-diagnose cases of DAT-negative AIHA, and the results (all cases) were correlated with hematologic and hemolytic parameters. DAT tube was confirmed as the gold standard to diagnose AIHA since almost all positive cases showed hemolytic anemia and positive eluates; 10 out of 26 tube-negative cases were positive on microcolumn and solid phase antiglobulin tests, and 22 out of 26 using MS-DAT, although only half of them showed clear signs of hemolysis. Mitogen stimulation increased the amount of IgG bound to red blood cells in all groups; moreover, MS-DAT was the only positive test in 10 cases of AIHA, and mitogen stimulation facilitated the identification of autoantibody specificity in culture supernatants. We conclude that a battery of tests rather than a single test is useful for the diagnosis of AIHA, including MS-DAT as an additional test for selected cases, although the results have to be cautiously interpreted based on the overall clinical context.

Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients

“…1,2 We describe about 5% of DAT-negative cases, a percentage rather lower than previously reported (;10% to 20% 1,2 ), possibly due to the use of more sensitive tests, such as MS-DAT, which allowed the diagnosis in some DATnegative cases. 3,4 These cases showed a more severe pattern and may represent a diagnostic puzzle, causing troublesome delays in the start of therapy. This notion is clinically important, because an earlier onset of therapy has been related with lower probability of relapse.…”

“…However, diagnostic pitfalls are recognized with increasing frequency, due to IgA autoantibodies (not detectable by most polyspecific antiglobulin reagents), warm IgM, low-affinity IgG, or IgG below the threshold of sensitivity. In fact, despite the availability of various DAT techniques (microcolumn, solid phase, enzyme linked, flow cytometry, dual DAT, and mitogen-stimulated [MS] DAT [3][4][5] ), about 10% of AIHA remains DAT negative. In these atypical cases, the diagnosis, made after extensive laboratory workup and exclusion of other causes of hemolysis, is frequently deferred, with consequent delay in therapy.…”

Detection of red blood cell antibodies in mitogen‐stimulated cultures from patients with hereditary spherocytosis

“…We found that 61% of HS cases showed RBC antibody-bound IgG values above the mean plus 3 SD of the controls (150 ng/mL), and one-third of positive cases values above the threshold of AIHA (250 ng/mL). [25][26][27] The presence of RBC-bound IgG in HS patients was confirmed by flow cytometry. Moreover, Western blot experiments showed binding of purified IgG to a-and b-spectrin, Band 3, and dematin.…”

“…The quantification of RBC antibodies in mitogenstimulated cultures was assessed by MS-DAT, as previously described. [25][26][27] Heparinized blood samples were diluted 1:6 with RPMI 1640 (Gibco-Life Technologies, Foster City, CA) and either unstimulated or stimulated with 2 mg/mL phytohemagglutinin (Sigma, St Louis, MO), 20 ng/mL phorbol-12-myristate-13-acetate (Sigma), and 1% pokeweed (Sigma) for 48 hours in 24-well plates. A 96-well polyvinyl chloride assay plates (Costar, Cambridge, MA) was coated with 50 mL of human IgG (Sclavo, Siena, Italy) overnight at 148C, washed 33, and blocked with 200 mL of 2% fetal calf serum-phosphate-buffered saline (PBS).…”

Detection of erythroblast antibodies in mitogen‐stimulated bone marrow cultures from patients with myelodysplastic syndromes

“…Other more sophisticated techniques such as the enzyme-linked and radiolabeled tests, and the flow cytometry displayed the highest sensitivity, being able to detect up to 30 to 40 molecules of RBC autoantibodies. 3,4 Moreover, mitogen stimulation in culture, which probably amplifies the production of autoantibodies, was the most sensitive (and least specific) and has proven useful in the diagnosis of DAT-negative AIHA. 5 In addition, it was found positive in a fraction of patients with B-chronic lymphocytic leukemia or myelofibrosis without an overt diagnosis of AIHA and in more than a half of patients with hereditary spherocytosis.…”