A compendium of single extracellular vesicle flow cytometry

Joshua A. Welsh, Ger J. A. Arkesteijn, Michel Bremer, Michael Cimorelli, Françoise Dignat-George, Bernd Giebel, André Görgens, An Hendrix, Martine Kuiper, Romaric Lacroix, Joanne Lannigan, Ton G. van Leeuwen, Estefanía Lozano-Andrés, Shoaib Rao, Stéphane Robert, Leonie de Rond, Vera A. Tang, Tobias Tertel, Xiaomei Yan, Marca H. M. WaubenJohn P. Nolan, Jennifer C. Jones, Rienk Nieuwland, Edwin van der Pol

Research output: Contribution to journalReview articleAcademicpeer-review

37 Citations (Scopus)


Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.
Original languageEnglish
Article numbere12299
Pages (from-to)e12299
JournalJournal of extracellular vesicles
Issue number2
Publication statusPublished - 1 Feb 2023


  • MIFlowCyt-EV
  • calibration
  • extracellular vesicles
  • flow cytometry
  • microparticles
  • nanoparticles
  • standardization

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