Introduction: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies promote the destruction and underproduction of platelets. Recent evidence suggests that immune-mediated destruction of bone marrow megakaryocytes is associated with the pathogenesis of this disease. In addition, the attenuation of megakaryocyte apoptosis can lead to platelet underproduction since this process appears to be involved in thrombopoiesis. In the current study, we investigated megakaryocyte apoptosis as a possible mechanism in the pathogenesis of ITP.
Patients/Methods: Bone marrow biopsy sections from ITP patients and controls were stained with anti-human CD61 to enumerate megakaryocytes. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining was performed as a measure of megakaryocyte apoptosis. The samples were coded and reviewed by an experienced hematopathologist who was blinded to the diagnosis. Adult primary ITP patients had isolated thrombocytopenia with no underlying cause and a platelet count less than 100 x109/L at the time of bone marrow biopsy procurement. Patients with secondary ITP (in the context of medications, lymphoproliferative disease, HIV, hepatitis B or C infection) were excluded, as were patients who received prior treatment with thrombopoietin receptor agonists or other hematopoietic growth factors. Thrombocytopenic controls were patients with myelodysplastic syndrome (MDS) who had platelet counts below 100 x109/L. Control patients with normal platelet counts had bone marrow biopsies performed as part of investigations for lymphoma or plasma cell dyscrasia with negative test results.
Results: The average platelet count for ITP patients, MDS patients, and controls were 18 x109/L (range 2-76 x109/L), 29 x109/L (range 9-60 x109/L), and 281 x109/L (range 171-400 x109/L), respectively. Elevated megakaryocyte counts were observed in the bone marrow sections of 5/14 (36%) ITP patients, 0/8 (0%) MDS patients, and 2/11 (18%) controls. Megakaryocyte apoptosis was comparable between ITP patients and MDS patients [2/14 (14%) vs 1/8 (13%) (p=1.00)], while fewer ITP patients exhibited megakaryocyte apoptosis compared to controls with normal platelet counts [2/14 (14%) vs 7/11 (64%) (p=0.02)]. In the entire study cohort, the average platelet counts of patients with negative and positive TUNEL staining were 66 x109/L (range 2-378 x109/L) and 206 x109/L (range 20-400 x109/L), respectively (p=0.01). In addition, the normalized megakaryocyte counts (per high powered field) were 8.2 ± 5.5 and 5.4 ± 2.5 in patients with negative and positive TUNEL staining, respectively (p=0.05).
Conclusion: Megakaryocyte apoptosis was reduced in ITP bone marrow samples compared to controls with normal platelet counts, but was also low in thrombocytopenic MDS patients. Reduced megakaryocyte apoptosis was found to be associated with a low platelet count, and may be related to thrombocytopenia regardless of etiology. Our study is consistent with the hypothesis that attenuated megakaryocyte apoptosis is relevant in the context of platelet underproduction in ITP.
Arnold: Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; UCB: Consultancy; Amgen: Consultancy, Research Funding.