TY - JOUR
T1 - Platelet removal by single-step centrifugation
AU - Rikkert, Linda G.
AU - Coumans, Frank A. W.
AU - Hau, Chi M.
AU - Terstappen, Leon W. M. M.
AU - Nieuwland, Rienk
N1 - Funding Information: This work was supported by the Netherlands Organisation for Scientific Research?Domain Applied and Engineering Sciences (NWO-TTW), research programs VENI 13681 (FC) and Perspectief CANCER-ID 14198 (LR). Publisher Copyright: © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.
PY - 2021
Y1 - 2021
N2 - The study of extracellular vesicles (EVs) in plasma requires removal of cells including platelets. At present, a two-step centrifugation protocol is recommended and commonly used. A simpler protocol that is less operator dependent is likely to improve the quality of plasma samples collected for EV research. The objective of this study is to develop an easy, fast and clinically applicable centrifugation protocol to produce essentially platelet-free plasma with a high yield for EV research. We compared the two-step centrifugation protocol to a single-step protocol at 5,000 g for 20 minutes. The removal of platelets was computationally predicted and experimentally validated. Flow cytometry was used to detect residual platelets and platelet-derived (CD61+) EVs. The single-step protocol at 5,000 g (i) is less laborious and approximately ten minutes faster, (ii) removes platelets as effective as the two-step centrifugation protocol, and (iii) has a ~ 10% higher plasma yield, whereas (iv) the recovery of platelet-derived EVs is comparable. For future research on plasma EVs we recommend the newly developed, easy and fast single-step protocol for preparation of platelet-free plasma for research on plasma biomarkers including EVs.
AB - The study of extracellular vesicles (EVs) in plasma requires removal of cells including platelets. At present, a two-step centrifugation protocol is recommended and commonly used. A simpler protocol that is less operator dependent is likely to improve the quality of plasma samples collected for EV research. The objective of this study is to develop an easy, fast and clinically applicable centrifugation protocol to produce essentially platelet-free plasma with a high yield for EV research. We compared the two-step centrifugation protocol to a single-step protocol at 5,000 g for 20 minutes. The removal of platelets was computationally predicted and experimentally validated. Flow cytometry was used to detect residual platelets and platelet-derived (CD61+) EVs. The single-step protocol at 5,000 g (i) is less laborious and approximately ten minutes faster, (ii) removes platelets as effective as the two-step centrifugation protocol, and (iii) has a ~ 10% higher plasma yield, whereas (iv) the recovery of platelet-derived EVs is comparable. For future research on plasma EVs we recommend the newly developed, easy and fast single-step protocol for preparation of platelet-free plasma for research on plasma biomarkers including EVs.
KW - Blood
KW - blood platelets
KW - centrifugation
KW - extracellular vesicles
KW - flow cytometry
UR - http://www.scopus.com/inward/record.url?scp=85087066393&partnerID=8YFLogxK
U2 - https://doi.org/10.1080/09537104.2020.1779924
DO - https://doi.org/10.1080/09537104.2020.1779924
M3 - Article
C2 - 32552252
SN - 0953-7104
VL - 32
SP - 440
EP - 443
JO - Platelets
JF - Platelets
IS - 4
ER -