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.

Extracellular vesicles (EVs) are cell‐derived particles with a phospholipid membrane present in all body fluids. Because EV properties change in health and disease, EVs have excellent potential to become biomarkers for diagnosis, prognosis, or monitoring of disease. The only technique capable of detecting, sizing, and phenotyping a million of EVs within minutes is (clinical) flow cytometry. A flow cytometer measures light scattering and fluorescence signals of single EVs. Although these signals contain valuable information about the presence and composition of EVs, the signals are expressed in arbitrary units, which make the comparison of measurement results impossible between instruments and laboratories. Additionally, unintended and undocumented variations in the source, preparation, and analysis of the sample lead to orders of magnitude variations in the measured EV concentrations. Here, we will explain the basics, challenges, and common misconceptions of EV flow cytometry. In addition, we provide an overview of recent standardization initiatives, which are a prerequisite for comparison of clinical data and thus for clinical biomarker exploration of EVs.