Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay

Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold.

Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production.

Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34⁺ HSCs directly from patients with ITP and healthy controls. HSPC (CD45⁺ CD34⁺) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis.

Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte).

Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

No relevant conflicts of interest to declare.