TY - JOUR
T1 - Rate zonal centrifugation can partially separate platelets from platelet-derived vesicles
AU - Rikkert, Linda G.
AU - Engelaer, Mendel
AU - Hau, Chi M.
AU - Terstappen, Leon W. M. M.
AU - Nieuwland, Rienk
AU - Coumans, Frank A. W.
N1 - © 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.
PY - 2020/8/1
Y1 - 2020/8/1
N2 - 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.
AB - 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.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85103803564&origin=inward
UR - https://www.ncbi.nlm.nih.gov/pubmed/32864556
U2 - https://doi.org/10.1002/rth2.12366
DO - https://doi.org/10.1002/rth2.12366
M3 - Article
C2 - 32864556
SN - 2475-0379
VL - 4
SP - 1053
EP - 1059
JO - Research and practice in thrombosis and haemostasis
JF - Research and practice in thrombosis and haemostasis
IS - 6
ER -