AUTOANTIBODIES AND AUTOREACTIVE B CELLS IN THE BONE MARROW AND THE PERIPHERAL BLOOD OF PATIENTS WITH IMMUNE THROMBOCYTOPENIA

Abstract

Introduction: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a platelet count less than 100 x 109/L. Platelet-bound autoantibodies are detected in the peripheral blood of only 40-50% of ITP patients. The subset of ITP patients who do not have detectable autoantibodies may truly be seropositive, but autoantibodies may not be detected due to limitations of the assays. Studies have also suggested that autoantibodies might be sequestered in the bone marrow where autoantibodies may impair platelet production. In addition, detecting autoreactive antibody-secreting B cells using the Enzyme-linked Immunospot (ELISPOT) assay was shown to be highly sensitive. In this study, the bone marrow and the peripheral blood compartments of ITP patients were tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies and autoreactive B cells. Methods: Bone marrow aspirate and peripheral blood samples were collected from ITP patients (n=12), non-immune thrombocytopenic patient controls (n=3), and healthy controls (n=5). Mononuclear cells were isolated and tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoreactive B cells before stimulation and after stimulation with R848 and IL-2 using the ELISPOT assay. Anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies were detected in the bone marrow and the peripheral blood using the direct antigen capture assay. Results: In our study, we detected autoantibodies and autoreactive B cells of known specificity in the bone marrow of a subset of ITP patients. Detecting anti-GPIIbIIIa and anti-GPIbIX autoantibodies in the bone marrow or the peripheral blood had a sensitivity of 42% and testing both compartments increased the sensitivity to 58%, while maintaining 100% specificity. Autoreactive B cells were detected at low frequencies with low specificity in the bone marrow and the peripheral blood of a subset of ITP patients. The majority of the ITP patients without detectable autoantibodies in the peripheral blood did not have autoantibodies in the bone marrow, and autoreactive B cells were not detected in either compartment. Conclusion: Examining both the bone marrow and the peripheral blood compartments for autoantibodies or autoreactive B cells increased the sensitivity. Furthermore, detecting autoantibodies using the antigen capture assay is more sensitive and specific than detecting autoreactive B cells using the ELISPOT assay.