Chapter 6 156 Figure 2 – Discrimination of EVs based on HLA phenotype. A) Representative scatter plots of an HLA-A3+ PPP sample (left) and an HLA-A3- PPP sample (right) stained with anti-HLA-A3 (X-axis) and anti-CD9-APC (Y-axis) before and after detergent treatment (top and bottom row, respectively). B) Visual examination of events representative for each fluorescent population demonstrated the selection and subsequent analysis of particles showing single spot fluorescence (no coincidence events), indicating the selection and analysis of single EVs. C-D) Concentrations of HLA-A3+ and CD9+HLA-A3+ fluorescent events in all pre-transplantation donor, recipient, and buffer only (control) samples before and after detergent treatment, and after isotype staining (grey, orange, and blue boxes, respectively). HLA-A3+ events were found to not be indicative of EV signals and were excluded from analysis. Double-positive events were found to be indicative of EV signals, as well as to be discriminative between HLA-A3+ and HLA-A3- PPP samples.
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