Chapter 3 52 A common artefact encountered when measuring sub-micron particles with conventional FC is swarm detection, which is defined as a special case of coincidence detection where instead of two or a few particles, multiple (tenths to hundreds) of particles at or below the detection limit are simultaneously and continuously present in the laser beam of the flow cytometer and measured as single counts. This may occur during detection of EV in highly concentrated samples, and can lead to erroneous data interpretation 34. While swarm detection can be prevented by dilution of highly concentrated samples, coincidence detection may still occur (albeit at lower frequencies). To identify coincidence detection, and exclude potential multiplets from our analysis, we designed a gating strategy that selects all events displaying 0 or 1 fluorescent spot on acquired images, thus ascertaining the analysis of events representative for single (and not multiple) particles. The identification of multiple, spatially separated fluorescent particles within acquired images provides insight into the degree of coincidence detection in a given sample - which is not possible with conventional FC. To demonstrate that our methodology correctly identifies and quantifies single EV, we performed coincidence testing through serial dilution 35. Analysis of the concentration of CFSE+/Tetraspanin+ EV upon serial dilution yielded a linear correlation with the dilution factor while ERF remained stable. In this work we examined two fluorescent labeling strategies to identify and discriminate EV: 1) application of CFDA-SE staining in conjunction with an antitetraspanin antibody mixture, and 2) staining with two mAbs targeting two different EV surface proteins. With both approaches, single EV were identified through the colocalization of two fluorescent markers, thus excluding the possibility of soluble protein detection. The combination of isotype and detergent treatment controls demonstrated the specificity of the mAbs for EV labelling (and not lipoproteins), and the dissociation of lipid structures, respectively. Therefore, both of these controls are highly recommended, if not mandatory, for the correct interpretation of acquired results. Single EV concentrations as reported in this work are in line with concentrations reported by other groups obtained after the purification/ isolation of PPP samples 29,36. This shows the advantage of our methodology over existing analytical techniques as no isolation, and therefore less manipulation, of EV are performed in our approach. Another FC-based method to directly measure EV in plasma, performed on a Beckman Coulter CytoFlex and using a strategy that encompasses the labelling of EV with a fluorescent lipid probe (vFRed) in combination with CFDA-SE or an anti-
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