Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry

Abstract

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 503 more Annexin-A5 binding EVs (Anx51 EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD411 and CD235a1 EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 403 for Anx51 EVs, 753 for CD411 EVs, and 153 for CD235a1 EVs. We found that about 30% of Anx51 EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx51 EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers. V C 2015 International Society for Advancement of Cytometry

Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles

“…Fluorescence triggering Recently, several groups have shown that the sensitivity of flow cytometers can be increased by triggering on fluorescence [2,3,23,41,42]. Clearly, fluorescence triggering is a valid approach, although it is hampered by two problems.…”

High sensitivity flow cytometry of membrane vesicles

“…Appropriate calibration using commercially available reagents and well documented protocols allows us to report surface marker intensities in absolute units of MESF, an important step in comparing data over time and between labs. Brisson and colleagues (40,42) have estimated that a 150 nm EV covered to saturation with annexin V, would bear only approximately 2,500 molecules per vesicle while a 100 nm EV might bear only approximately 1,000 molecules, which is near the resolution limit of a typical commercial flow cytometer. Our high sensitivity system has a resolution limit of approximately 240 MESF of fluorescein, which provides improved sensitivity, but many less abundant vesicle markers might be present in number below this limit, making it important to calibrate the fluorescence measurements and report particle intensities in absolute units.…”

Extracellular Vesicles in Renal Diseases

“…This approach has the potential to offer better separation from background noise and is less likely affected by aggregation of particles and might detect particles as low as 100-200 nm. 51, 52 The addition of imaging to FCM can also significantly increase the sensitivity of MP detection. 53,54 Imaging flow cytometry (IFCM) can also provide morphologic confirmation and the ability to distinguish true single events from aggregates and cell debris (Figure 2).…”