An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles in Unprocessed Human Plasma

Abstract

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, we present 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) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we 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 new corners of EV as cellular messengers in healthy and pathological conditions.

Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics

“…Perfusates were stained with monoclonal antibodies (mAbs) and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as extensively described in our previous work 23 and detailed in SDC , http://links.lww.com/TP/C460 , Materials and Methods; Sample Labelling.…”

“…All samples were acquired on an ImageStreamX MkII instrument (IS x ; Luminex). Settings as extensively described in our previous work 23 and detailed in SDC , http://links.lww.com/TP/C460 , Materials and Methods; Acquisition were used.…”

“…To ensure the analysis of EVs we (1) selected all particles with side scatter intensities ≤900 a.u., and (2) identified and excluded coincidence detection by counting the number of fluorescent spots within the pixel grid for each event acquired; events showing multiple spots were excluded from the analysis. 23 This gating strategy ensures the selection and analysis of single spot fluorescent particles ≤400 nm. Gating areas and cutoffs were established through the identification of (fluorescent) populations in unstained and single stained samples, and arbitrary fluorescent intensities were converted into equivalent molecules of fluorescence (ERF) values based on previously published calibration data.…”

“…Gating areas and cutoffs were established through the identification of (fluorescent) populations in unstained and single stained samples, and arbitrary fluorescent intensities were converted into equivalent molecules of fluorescence (ERF) values based on previously published calibration data. 23 Lower and upper gating area cutoffs were defined as 677–112,201 ERF for BV421; 35.40–3776 ERF for carboxyfluorescein succiminidyl ester (CFSE); 206–14,770 ERF for phycoerythrin; and 6.40–123 ERF for allophycocyanin.…”

“…21 Previously, our group was able to quantify the release of nanoparticles (such as protein aggregates and EVs) by ECD kidneys during NMP. 22 Here we apply our recently developed imaging flow cytometry (IFCM)-based methodology 23 to identify, phenotype and determine the concentration of EVs ≤400 nm in diameter released by discarded human kidney grafts during NMP. We show the identification of distinct EV subsets based on their tetraspanin profile in combination with the detection of esterase activity, platelet endothelial cell adhesion molecule (CD31), or the pan-leukocyte protein (CD45).…”