Reliable measurements of extracellular vesicles by clinical flow cytometry

Abstract

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.

Enrichment and characterization of extracellular vesicles from ex vivo one‐sided human placenta perfusion

“…Particle count depends on the instrument sensitivity, configuration, and EV labeling, causing a wide variation among experiments. In contrast, protein quantification seems to be subject to less variation 48 …”

Extracellular Vesicles in Allergic Rhinitis and Asthma and Laboratory Possibilities for Their Assessment

“…Here, we focus on a sensitive and reproducible method for EVs flow cytometry detection that allows characterization of small amounts of microparticles in a routine laboratory. Flow cytometry enables the detection of EVs in the tested sample, as well as to determine the size and phenotype of particles in a short time [45,46].…”

“…The difficulties in the detecting of EVs are their small size-most often below 200 nm, and the presence of very different composition together with concomitant particles of similar size [45].…”

“…So far, the best feature for this purpose has been demonstrated by HOBs [50]. Only these beads are considered to enable calibration and evaluation of the EVs size [45].…”

“…In 2020, MIFlowCyt-EVs, a framework for standardized reporting of EVs flow cytometry experiments [53,54], was released. Further educational articles and seminars for researchers are being prepared (EV Flow Series) [45]. Calibration and standardization attempts based on the appropriate setting of cytometer parameters (fluorescence-triggered) and appropriate analysis are also undertaken by individual research centers [55,56].…”