Author, as appears in the article.: van der Pol E, Sturk A, van Leeuwen T, Nieuwland R, Coumans F, ISTH-SSC-VB Working group

Department: Medicina i Cirurgia

URV's Author/s: Porras Ledantes, Jose Antonio

Keywords: Thrombosis Standardization Sensitivity Resolution Nanoparticle tracking analysis Multicenter Microparticle enumeration Flow cytometry Extracellular vesicles Exosomes Collaborative workshop Cells Cell-derived microparticles Calibrated beads Blood platelets flow cytometry extracellular vesicles exosomes cell-derived microparticles blood platelets

Abstract: © 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis. Essentials Platelet extracellular vesicles (EVs) concentrations measured by flow cytometers are incomparable. A model is applied to convert ambiguous scatter units to EV diameter in nanometer. Most included flow cytometers lack the sensitivity to detect EVs of 600 nm and smaller. The model outperforms polystyrene beads for comparability of platelet EV concentrations. Summary: Background Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights into the diameter of detected EVs. Objectives To evaluate gates based on the estimated diameter of EVs instead of beads. Methods A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates in order to measure the concentration of CD61–phycoerythrin-positive platelet EVs. Results Of the 46 evaluated FCMs, 21 FCMs detected the 600–1200-nm EV diameter gate. The 1200–3000-nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL min −1 differed six-fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with previous approaches. Of the evaluated FCMs, 24% could not detect 400-nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity.

Thematic Areas: Saúde coletiva Peripheral vascular disease Medicine (miscellaneous) Medicina veterinaria Medicina iii Medicina ii Medicina i Hematology General medicine Farmacia Ciências biológicas iii Ciências biológicas ii Ciências biológicas i Biotecnología

licence for use: https://creativecommons.org/licenses/by/3.0/es/

ISSN: 15387933

Author's mail: joseantonio.porras@urv.cat

Author identifier: 0000-0001-6418-1822

Record's date: 2023-02-22

Papper version: info:eu-repo/semantics/publishedVersion

Link to the original source: https://onlinelibrary.wiley.com/doi/full/10.1111/jth.14009

Licence document URL: http://repositori.urv.cat/ca/proteccio-de-dades/

Papper original source: Journal Of Thrombosis And Haemostasis. 16 (6): 1236-1245

APA: van der Pol E, Sturk A, van Leeuwen T, Nieuwland R, Coumans F, ISTH-SSC-VB Working group (2018). Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation. Journal Of Thrombosis And Haemostasis, 16(6), 1236-1245. DOI: 10.1111/jth.14009

Article's DOI: 10.1111/jth.14009

Entity: Universitat Rovira i Virgili

Journal publication year: 2018

Publication Type: Journal Publications