Direct EV Measurement in Complex Biofluids Using Single Vesicle Flow Cytometry (vFC) and the CytoFLEX

Welcome, I'm John Nolan representing Cellarcus Biosciences, and we’re pleased to be partnering with Beckman Coulter to tell you about single vesicle analysis using vesicle flow cytometry. In the next few minutes, I’m going to tell you about the needs and challenges for single vesicle analysis and show you how vesicle flow cytometry enabled sensitive and specific analysis of EV number, size and molecular cargo, including measurement of EVs directly in plasma. First, let's consider this diagram, which depicts a cell releasing several different types of EVs into the extracellular space that can deliver cargo to other cells and affect their function. The EVs that can be collected from the extracellular fluid are diverse, but potentially carry informative biomarkers or therapeutic cargo. One goal of EV researchers is to determine exactly which EVs carry what cargo and understanding the implications of this information for biomarker and therapeutic development. If we look at a simple biofluid, like cell culture media, we find that in addition to the various types of vesicles, cells also release a myriad of different biological nanoparticles. If we look in a complex biofluid such as plasma, we find abundant lipoproteins and other similarly sized nanoparticles that might be more abundant than EVs in the biofluid. This complexity poses a challenge to EV analysis. The conventional approach to EV analysis typically involves biofluid fractionation, often by centrifugation, followed by analysis using Western blot, mass spectrometry, PCR, or other bulk biochemical analysis methods. This approach reports the total amount of cargo associated with an EV-containing fraction, but does not provide information about the distribution of that cargo amongst individual EVs. It is increasingly appreciated that in order to understand EVs and potential biomarkers or therapeutics, you need to analyze EVs as individuals, counting them, estimating their size and measuring their cargo on a vesicle-by-vesicle basis, to allow us to define and enumerate EV subpopulations within a sample. NTA and RPS are two popular single particle analysis methods that have their uses in EV research, but they both suffer from a lack of specificity in terms of discriminating EVs from other nanoparticles and the inability to measure molecular cargo. Flow cytometry is widely used to measure individual cells, but is challenged to measure EVs due to their small size and dim signals. A typical EV is a hundred times smaller than a cell, which means it scatters much less light and has ten thousand times smaller surface area, which means that instead of carrying a few tens of thousands of molecules of a surface antigen, it might only carry a few. ENA/RNA might be present in only a single copy per vesicle. Conventional flow cytometers and conventional assays designed to measure cells, like the sensitivity and specificity to measure single vesicles. Moreover, calibration and reporting guidelines designed to enable reproducibility are often not followed, resulting in artifacts and poor performance overall. However, these lessons have taught us that it is possible to sensitively and quantitatively measure EVs using suitably sensitive instruments and well-designed assays that incorporate the best practices in instrument and assay calibration, validation and reporting. The key issues in EV flow cytometry have recently been described and discussed in this recent paper in the journal of extracellular vesicles, which sets out a series of guidelines that cover not only how to determine and report features of instrument operation, but also the other important aspects of the assay, including sample preparation and staining, and data analysis and reporting that are critical for reproducibility. Now I’m going to describe the practical implementation of those guidelines in an assay that involves the Beckman Coulter CytoFLEX, a very sensitive instrument, combined with the Cellarcus vFC assay, which provides the reagent standards and protocols to use that instrument to measure EVs. vFC measures EVs directly and dilute the biofluids by using a fluorogenic membrane dye, vFRed, which stains membrane particles in proportion to their surface area, along with VTag antibodies that are validated for use in vFC. After staining, sample is diluted and introduced into the flow cytometer, where the red membrane florescence triggers detection of vesicles and measurement of immunofluorescence. After acquisition, the data are calibrated to report VFRed fluorescence in terms of vesicle size, and immunofluorescence in terms of antibody molecules per vesicle. In practice, vFC starts with qualification and calibration of the instrument using nano rainbow beads. These nano rainbow beads allow us to measure the sample flow rate, assess the laser alignment, and evaluate the fluorescence resolution of the instrument to ensure performance is up to spec. These nano rainbow beads can also serve as a calibrator, allowing the fluorescence intensities to be expressed in either units of mean equivalent soluble fluors, antibodies per vessel, or equivalent surface area. Once the instrument has been qualified and calibrated, EV analysis begins with a dilution series, which is performed for all new sample types. This lets us determine the EV concentration, the dynamic range of the assay, and the sample dilution, which will put us in the middle of that dynamic range. It also will allow us to demonstrate the absence of coincidence, which is responsible for the dreaded swarm artifact. The vFC size estimate is derived from the fluorescence of a synthetic lipid vesicle with a well-defined size distribution that allows us to determine the fluorescence response of the instrument in terms of equivalent surface area. EV light scatter depends not only on particle size, but on refractive index, which can reveal features of the internal structure and cargo of the EVs. And it's interesting to note that EVs from different sources can scatter different amounts of light. Once the optimal dilution of an EV sample is determined, immunofluorescence can be used to detect specific surface cargo using a well-standardized protocol consisting of the necessary controls and calibrators to demonstrate and quantify specific binding in terms of molecules per EV. EV cargo measurements by vFC are facilitated by EV standards or reference materials which serve as positive controls for immunostaining, as demonstrated here in the measurement of cell-specific markers on red cell and platelet-derived EVs. The optimization and validation of multicolor vFC assays uses EV reference materials, as well as calibrated antibody capture beads, which can serve as spectral reference samples for compensation or spectral unmixing, as well as immunofluorescence calibration particles, which allow the brightness of different immunofluorescent labels to be understood and accounted for. This collection of validated reagent standards and protocols will then let us measure EVs specifically, even in complex fluids like plasma, by employing cytoplasmic or cell surface markers to identify EVs even against the background of lipoproteins and other particles. And this approach can be extended to the identification of cell-specific markers on EVs in plasma, as we recently demonstrated in this paper in the Journal of Extracellular Vesicles. In summary, while EVs are small, dim and difficult to measure, a sensitive instrument like the CytoFLEX combined with a specific assay, like the vFC assay, can enable EVs to be measured quantitatively and reproducibly, including directly in complex biofluids, such as diluted plasma. Thank you for your time.