Protocol for the Isolation and Analysis of Extracellular Vesicles From Peripheral Blood: Red Cell, Endothelial, and Platelet-Derived Extracellular Vesicles.

This protocol describes the isolation and flow cytometric analysis of extracellular vesicles (EVs) derived from red blood cells, endothelial cells, and platelets in human peripheral blood. The protocol includes steps for preparing platelet-free plasma, fluorescent antibody staining, gating strategies, and technical controls. This protocol was developed within a study on EV release in snakebite-associated thrombotic microangiopathy; the protocol addresses challenges such as variable autofluorescence and heterogeneity in EV origin. It is flexible and can be adapted for alternative antibody panels targeting different cell populations or EV subtypes, including leukocyte-derived EVs. Key features • Bead-free, two-step plasma preparation enhances extracellular vesicle yield, reduces platelet contamination, and improves purity compared with conventional isolation methods for small-volume clinical samples. • Reduced autofluorescence by compensation strategy using flow cytometry. • Gating strategies to detect distinct EV populations derived from red cells, endothelial cells, and platelets. • Validated in healthy donors and patients, enabling reproducible detection of EVs with broad downstream compatibility for flow cytometric applications.