Figure 1.

Analysis of maturation and of proplatelet formation in suspension liquid cultures of megakaryocytes of the investigated patient. Megakaryocytes (Mk) were differentiated from peripheral blood progenitors through 14-day culture. Samples of the patient (PT) were processed in parallel with those of three healthy controls (HC). (A-C) Analysis of Mk maturation. (A) At the end of the culture, the proportion of mature Mk was measured by flow cytometry, as the percentage of CD41-positive cells co-expressing the CD42b antigen. 9 (B,C) Mk were also cytospun onto slides and stained with an anti-β1-tubulin antibody (red). Hoechst (blue) was used for counterstaining nuclei (C). Mk were then classified into maturation stages I to IV by morphological analysis according to standard criteria16 (B). Overall, the maturation profile of the patient’s Mk was not different from that of healthy controls. Scale bars: 10 mm. (D-E) Analysis of proplatelet formation in suspension liquid cultures, which measures the intrinsic ability of Mk to form proplatelets i.e., free from the engagement with proteins of the extracellular matrix. (D) Representative examples of proplatelet formation of Mk of the patient and controls (phase-contrast microscopy). Scale bars: 10 μm. (E) Proplatelet formation was quantified as the percentage of cells displaying at least one proplatelet with respect to the total number of cells. Overall, the rate of proplatelet formation and the morphology of proplatelets in suspension were similar in patient and controls. The data are expressed as means ± standard deviation.