Population analysis of platelet surface markers on the size-separated platelet fractions. Isolated, size-separated platelets were stained for CD36, CD42b, CD61, GPVI, and HLA for 15 minutes before fixation, and 10 000 events were recorded per sample. Clustering tool (FlowSOM) and dimensionality reduction algorithm were used to analyze and visualize these multicolor flow cytometry data. (A) Representative dimensionality reduction algorithm plots with a FlowSOM populations filter for the different platelet fractions. (B) Characterization of the FlowSOM clusters (3 populations; I-III). (C) Distribution profile of platelet populations resulting from FlowSOM analysis for the different platelet size fractions. (D) The proportion of RNA-positive platelets was determined by T-oligonucleotides in separated large and small platelets. (E) Correlation of RNA-content and HLA-I expression was determined by costaining using T-oligonucleotides and HLA-A,B,C-antibody in separated large and small platelets. (F) Relation between RNA content and GPVI expression. Mean ± standard error of mean; n ≥ 4. *P < .05, **P < .01. MFI, mean fluorescence intensity.