Absolute sizing and label-free identification of extracellular vesicles by flow cytometry.

Blood contains extracellular vesicles (EVs), which are biological nanoparticles with clinical applications. In blood plasma, EVs are outnumbered by similar-sized lipoprotein particles (LPs), leading to controversial data such as non-specific binding of antibodies to LPs. Flow cytometry is a clinically applicable technique to characterize single EVs in body fluids. However, flow cytometry data have arbitrary units, impeding standardization, data comparison, and data interpretation, such as differentiation between EVs and LPs. Here we present a new method, named flow cytometry scatter ratio (Flow-SR), to relate the ambiguous light scattering signals of flow cytometry to the diameter and refractive index (RI) of single nanoparticles between 200-500 nm in diameter. Flow-SR enables label-free differentiation between EVs and LPs and improves data interpretation and comparison. Because Flow-SR is easy to implement, widely applicable, and more accurate and faster than existing techniques to size nanoparticles in suspension, Flow-SR has numerous applications in nanomedicine.

Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles.

BACKGROUND: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface. METHODS: We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM). RESULTS: For cells, the SPRi and FCM signals for antigen exposure correlated (RHS578T cells2 = 0.66, RMCF7 cells2 = 0.78, RSKBR3 cells2 = 0.60). With regard to EVs, SPRi detected 31 out of 33 tested antibody-EV pairs, whereas our flow cytometer detected 5 antibody-EV pairs because of high blank and isotype control signals. For HS578T-derived EVs, the SPRi and FCM signals correlated (R2HS578T EVs = 0.98). However, on MCF7- and SKBR3-derived EVs, insufficient antigens were detected by our flow cytometer. To confirm that the SPRi responses correlated with mean antigen density on EVs, the SPRi responses of EVs were correlated with antigen density on parental cells as measured by FCM (RHS578T2 = 0.77, RMCF72 = 0.49, RSKBR32 = 0.52). CONCLUSIONS: SPRi responses correlate with mean antigen density. Moreover, SPRi detects lower antigen-exposure levels than FCM because SPRi measures an ensemble of EVs binding to the sensor surface, whereas FCM detects antigens of single EV.

Bulk immunoassays for analysis of extracellular vesicles.

There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.