Chapter 8 186 detergent treatment. However, it should be noted that detergent treatment is nondiscriminatory between EVs or other lipid-composed particles (such as lipoproteins) – which stipulates the importance of the assay controls described above. Standardized reporting Like conventional flow cytometry (FCM), the signals generated by IFCM are expressed in arbitrary units, which hinders data interpretation and comparison of measurement results between instruments and laboratories. As multicenter studies are needed to validate the clinical relevance of EVs during health and disease, e.g. with different instruments measuring the same concentration of cell-type specific EVs in a given sample 14, it is imperative that EV detection assays produce results in a standardized manner. One way to achieve standardization is through calibration, which is a conversion of arbitrary units into standard units 15. While the calibration of fluorescence signals can be readily achieved by measuring beads with known fluorescent intensities (e.g., expressed Equivalent number of Reference Fluorophores (ERF – chapter 3) or in units of Molecules of Equivalent Soluble Fluorochrome (MESF – chapter 4) 16, the calibration of light scattering signals into particle size presents an ongoing challenge for IFCM. In this thesis, we demonstrated – for the first time – the calibration of light scatter signals into particle size for IFCM using Mie theory. The high degree of correlation between predicted and measured side scatter (SSC) intensities for polystyrene beads beads (R2 = 0.91) underlines the utility of the SSC channel to relate scatter signals to standard units. This development enhances reproducibility between different IFCMs, and even across FC platforms with different optical configurations and settings. However, it should be noted that heterogeneity of the membrane and cargo composition of EVs affects their refractive index (and thus their level of scattering) and may potentially introduce errors in the estimated EV sizes. In summary, following the application of strict assay controls, and calibration of both fluorescence and SSC signals for IFCM, we developed a standardized methodology which allows analysis of single EVs in complex biofluids such as platelet-poor plasma, urine, and perfusion fluids. Our methodology does not require prior isolation of EVs (thus limiting EV modulation), is able to simultaneously analyze EV concentration, phenotype, and size, and reports these parameters in standardized units.
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