Repeated MK proliferation, differentiation, and proplatelet-formation studies for a R1213* variant case. (A) Total amount of CFU-MK colonies derived from a total of 5000 peripheral blood CD34⁺ mononuclear cells per plate from a control and from case 21 (21) at day 12 of culture. This experiment (Exp) was repeated at 2 independent occasions (Exp 1 and Exp 2). (B) Representative images of cultured CFU-MK colonies from a control and from case 21 at day 12 of culture visualized by light microscopy after staining with May-Grünwald-Giemsa stain (Exp 2). Bars represent 50 μm. (C) MKs in triplicated liquid suspension cultures performed at 2 independent occasions were classified as proplatelet forming (PPF) MKs when proplatelet extensions were visible by light microscopy. The proportion of PPF MKs was lower in the cultures from case 21 compared with controls (1-way analysis of variance, ***P > .001). (D) Representative light microscopy images of cultured MKs showing formation of proplatelet extensions for the control. PPF MKs are almost absent for case 21, although they typically present in MK clusters that contain large and small cells. Bars represent 20 μm. (E) Immunofluorescence confocal microcopy images of differentiated fibrinogen-adhered MKs at day 12 of culture visualized by anti-integrin β3 (green; CD61) and phalloidin (red; F-actin) staining, showing colocalization in MKs from control, but not from case 21. Bars represent 20 μm. Numerous PPF MKs are present in the control (representative image), whereas MKs for case 21 form clusters. C, control.