An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, researchers at the University Medical Center Rotterdam have developed a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) the researchers found that >90% of double-positive fluorescent events represented single EVs. Through this work, they provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Schematic overview of the sample processing and staining protocol

EDTA whole blood (I) is centrifuged for 10 min at 1910 × g, after which the plasma top layer (II) is harvested and subjected to centrifugation for 10 minutes at 16,000 × g. The resulting platelet-poor plasma (PPP) is collected and stored as aliquots in cryovials containing an anti-protease inhibitor stock solution (4% v/v) at −80 °C (III). Prior to use, monoclonal antibodies and CFDA-SE stock solutions are centrifuged for 10 min at 16,000 × g to reduce the number of potential clumps of fluorescent particles (IV & V). mAbs are added to 30 µL of PPP, and samples are brought to a total volume of 130 µL using filtered PBS (VI) and incubated overnight (O/N) at 4 °C in the dark. CFDA-SE is added on the day of acquisition and incubated for 30 minutes at room temperature in the dark. Samples are brought to a total volume of 380 µL using filtered PBS and are assessed using a Luminex ISx MKII imaging flow cytometer (VII). After initial acquisition, detergent treatment is performed for each sample by adding 20 µL 10% (v/v) TritonX-100 solution followed by 30 minutes incubation at room temperature. Permission to use the image of the Imagestream (ISx) Imaging Flow cytometer (VII) was granted by Luminex.