Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry

Peer-reviewed article
L. de Rond, E. van der Pol, C.M. Hau, Z. Varga, A. Sturk, T.G. van Leeuwen, R. Nieuwland, and F.A.W. Coumans
Accepted January 12th, 2018
Clinical Chemistry
de Rond 2018 Clin.Chem. Fluorescent EV markers.pdf (411 kB)
de Rond 2018 Clin.Chem. Fluorescent EV markers suppl.pdf (1,313 kB)
de Rond 2018 Clin.Chem. Fluorescent EV markers MIFlowCyt.pdf (100 kB)


Background:Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry.

Methods: Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM+)] or platelet EVs from human plasma [integrin β3 positive (CD61+)]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated.

Results: Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM+ MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61+ EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection.

Conclusions: None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.