Abstract
The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Here, we examined the preanalytical variations of extracellular vesicles (EVs) and cell-free mRNA (cf-mRNA) profiles in human plasma and how post-freeze/thaw processing removes and retains EVs and cf-mRNA subtypes. Using multiparametric nanoscale flow cytometric analysis, which utilized forward light scatter, side scatter, and fluorescence intensity, we characterized the effect of specific preanalytical variables on vesicle subpopulations in human plasma. Cf-mRNA levels were measured by multiplexed RT-qPCR with a panel including housekeeping, platelet, and tissue-specific genes. We found that blood processing centrifugation and temperature strongly impacted the levels of residual platelets and platelet microvesicles. Intriguingly, exosome-sized events were more resistant to processing methods. We also examined the morphology of quiescent and activated platelets and platelet-derived EVs by electron microscopy. We demonstrated that platelet activation and fragmentation generated ex vivo vesicles similar to EVs in size, morphology, and canonical surface markers. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of new EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze-thaw. In summary, we identified distinct subpopulations of EVs and cf-mRNA in human plasma that are differentially influenced by platelet activation and fragmentation during sample collection, processing, and banking via post freeze/thaw processing.