Rate zonal centrifugation can partially separate platelets from platelet-derived vesicles

Abstract

Background: Centrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co-isolation with extracellular vesicles (EVs). Objective: To assess the ability of rate zonal centrifugation (RZC) to separate platelets from co-isolated EVs. Methods: Using a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet-derived EVs (n = 3). After RZC, all fractions were collected and stained with anti-CD61-Alexa 488 to measure the concentrations of platelets and platelet-derived EVs by flow cytometry. Results: We confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8-19, whereas the majority of platelet-derived EVs are detectable in fractions 1-7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. Conclusion: RZC can partially separate platelets from EVs.