Abstract
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 adenocar-cinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM)] or platelet EVs from human plasma [integrin 3 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 b lactadherin The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 680-689 |
| Number of pages | 10 |
| Journal | Clinical chemistry |
| Volume | 64 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 2018 |
| Externally published | Yes |
Bibliographical note
Funding Information:Employment or Leadership: Z. Varga, Centre for Natural Sciences, HAS and Semmelweis University. Consultant or Advisory Role: None declared. Stock Ownership: E. van der Pol, Exometry B.V. Honoraria: None declared. Research Funding: L. de Rond, Perspectief CANCER-ID funded by Netherlands Organisation for Scientific Research - Domain Applied and Engineering Sciences (NWO-TTW), Perspectief CANCER-ID 14195; E. van der Pol, Perspectief CANCER-ID funded by Netherlands Organisation for Scientific Research - Domain Applied and Engineering Sciences (NWO-TTW); F.A.W. Coumans, Research program VENI 13681. Expert Testimony: None declared. Patents: E. van der Pol, PCT application 15192403.2; T.G van Leeu-wen, PCT/EP2016/076238.
Funding Information:
The authors thank Linda Rikkert from the Department of Medical Cell BioPhysics, University of Twente, Enschede, the Netherlands, for making the transmission electron microscopy images in this study.
Publisher Copyright:
© 2018 American Association for Clinical Chemistry.