Platelets from patients with MPN are characterized by the cell surface expression of mutant calreticulin. Platelets were isolated from the blood samples of patients with mutant CRT-expressing MPN and healthy controls. (A) Gating strategy used for the analysis of unfixed CD41⁺ and CD62p⁺ platelets by flow cytometry. (B-C) Bar graphs indicate flow cytometry-based detection of staining for CD41 (B) and CRT (detected using an N-domain–specific antibody, anti-CRT[N]) (C) in healthy donor platelets either without fixation (surface) or after fixation and permeabilization (total) (n = 7). Relative MFI values after surface and total staining are plotted, assuming MFI values for total CD41 (B) or total CRT (C) as 100%. Paired t tests were used for determining the statistical significance. (D-G) Flow cytometry–based detection of surface CRT on unfixed CD41⁺ (D-E) and unfixed CD62p⁺ (F-G) platelets. Representative histograms and bar graphs show the MFI of surface CRT detected on platelets using either mutant CRT-specific antibody (anti-CRT [C_mut]) or anti-CRT (N) antibody. Platelets from patients with MPN and healthy controls (HC) that were processed simultaneously were analyzed as pairs for these measurements. Data are separately analyzed for Del52 (n = 12 for CD41⁺ platelets and n = 9 for CD62p⁺ platelets) and Ins5 (n = 8 for CD41⁺ platelets and n = 8 for CD62p⁺ platelets) patient samples. Error bars show the standard error of the mean. Statistical significance indicated by P values was determined using GraphPad Prism and paired t test analyses. The heterogeneity of CD41 expression in the representative panel shown in A most likely relates to the limiting amount of antibody, as similar results were obtained based on CD41^hi-gating vs gating on all single cells. FSC-H, Forward Scatter-Height; SSC-A, Side Scatter-Area.