Abstract
In the current dogma, immune hemolysis is considered an exclusively humorally-mediated state. However, in some cases, serological investigations are negative and without alternative explanations. In such a subset of unexplained seronegative hemolysis (SNH), we investigated for potentially attributable cellular mechanisms. Release of chromium-51(Cr51) was selected as the most sensitive method. Effector cells of the immune system (NK cells, neutrophils, monocytes) were purified by negative selection and evaluated as mediators of RBC lysis. Anti-D-opsonized R2R2 RBCs were selected as positive control RBCs for standardization of cytotoxicity assays and evaluation of the killing capacity of different immune cells. We observed that PBMCs were the least sensitive at lysing control RBC while NK cells, used at an effector/target ratio of 10:1, were the most efficient in killing control RBC, followed by monocytes and neutrophils. Neutrophils, used at an effector/target ratio of 25:1, are also capable of lysing control RBCs but required priming with GM-CSF. By informed consent, ten patients with unexplained SNH were tested for cellular-mediated hemolysis using autologous, purified immune cells (NK or neutrophils), against autologous RBCs with or without opsonization with autologous serum. Evidence of cell-mediated RBC lysis occurred in 60% (6/10) of the patients analysed (PBMCs, 1; NK, 4; neutrophils, 1). We also observed that RBC from patients with SNH display a significant reduction in the expression of CD47 when compared to healthy individuals. With CD47 being a "do not eat me" signal, its reduction is plausibly contributing to enhanced RBC clearance. These initial results suggest that cellular mechanisms beside extravascular antibody-mediated phagocytosis may play a role in seronegative hemolytic anemias.
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