Mitogen-stimulated direct antiglobulin test in patients with hereditary sperocytosis

Background: Hereditary spherocytosis (HS) is an hemolytic anemia due to defects in the red cell membrane proteins (band 3, spectrin, ankyrin, or protein 4.2) that cause loss of membrane surface area, reduced deformability, trapping and destruction of RBCs in the spleen. Naturally occurring autoantibodies (NAbs) against band 3 are thought to be involved in the removal of senescent and damaged erythrocytes. Mitogen-stimulated direct antiglobulin test (MSDAT) is a functional and quantitative method for the detection of anti-RBC antibodies in mitogen-stimulated whole blood cultures, found positive in some DATnegative autoimmune Hemolytic anemia (AIHA), and in a fraction (about 40- 50%) of patients with chronic lymphocytic leukemia and myelofibrosis.Aims: To investigate the occurrence of anti-erythrocyte antibodies by MS-DAT in HS, and to relate their presence with the degree of hemolysis and with the type of membrane defect. Methods: Ninety-one consecutive HS patients (51 male and 40 female, median age 38 yrs, range 2-87 yrs) were investigated; 13 patients had been splenectomized at the moment of the study. Diagnosis was made on the basis of clinical history, physical examination and the results of laboratory tests: complete blood counts, blood smear examination, reticulocyte counts, hemolytic parameters, eosin-5′-maleimide (EMA)-binding test, and red blood cell osmotic fragility tests. All patients underwent SDS-PAGE analysis of the red cell membrane proteins. MS-DAT was performed, by competitive solid phase ELISA, stimulating whole blood with mitogens (PHA, PMA and PWM). Two-hundred and ten healthy blood donors were studied as control group and 43 patients with AIHA were included as positive controls for MS-DAT. Results: We found that 61% of HS cases were MS-DAT positive: 29 (53%) with band 3 deficiency, 17 (30%) spectrin deficiency, and 9 (17%) with no detectable defect (Figure 1, open symbols for splenectomized cases). The amount of RBCbound IgG was clearly greater in HS compared to controls (P<0.0001), albeit lower than that observed in AIHA patients (P<0.0001); there was no difference in IgG bound among various HS groups. Since almost all (42/43) AIHA patients showed RBC-bound IgG values higher than 250 ng/mL, 26 HS patients (13 band 3 and 8 spectrin deficiency, and 5 no detectable defect) with values above this threshold were considered separately in the analysis. These cases displayed a significantly higher number of spherocytes (11%) than patients with RBC bound-IgG 150-250 (6.5%, P=0.04) and MS-DAT negative cases (5%, P<0.01); they showed also a more evident hemolytic pattern and a significantly greater number of reticulocytes compared with MS-DAT negative cases (230x103/mmc vs 125x103/mmc, median value, P<0.01).Summary and Conclusions: These findings suggest that in 60% of patients with HS, mitogen-stimulation is able to induce the production of auto-antibodies which might be responsible for immune-mediated destruction of erythrocytes, independently of the membrane structural alteration. These auto-antibodies may be naturally occurring anti-band 3 antibodies, as suggested by their low concentration compared with that found in AIHA, and their pathogenic role in enhancing the hemolytic process in HS.