Chapter 6 154 Identification and discrimination of single dd-EVs based on HLA phenotype In order to identify dd-EVs in the circulation of KTRs after kidney transplantation, we assessed whether IFCM is capable of discriminating EVs based on HLA phenotype. To this end, we labelled the same PPP samples (donors – HLA-A3+, and recipients (before KTx) – HLA-A3-) with anti-CD9 and anti-HLA-A3, and performed the same control experiments as described above. Figure 2A shows representative scatter plots for both a donor and recipient PPP sample analyzed with IFCM, before and after detergent treatment. Visual examination of events representative for each fluorescent population (before detergent treatment) demonstrated the selection and analysis of particles showing single spot fluorescence (no coincidence events), indicating the selection and analysis of single EVs 23 (Figure 2B). For HLA-A3+ single-positive events, we observed only marginal reduction by detergent treatment, showing that these events are not representative of biological particles. Moreover, buffer only controls showed a high degree of fluorescent events, indicating that these signals are most likely representative of antibody aggregates (Figure 2C). Therefore, HLA-A3 single-positive fluorescent events were interpreted to not be representative of EVs, and were excluded from further analysis. Analysis of CD9+HLA-A3+ double-positive fluorescent event concentrations in the KTR group showed that our assay produced background signals that varied among the different recipients (recipient-specific background) despite recipients all being HLA-A3 negative. Luminex single antigen assay for the anti-HLA-A3 antibody showed no cross reactivity of the antibody with other HLA epitopes (Supplementary Data S1 online), thus validating the specificity of the antibody. Comparison of CD9+HLA-A3+ concentrations between the donor and recipient groups (1.60E7 ± 1.03E7 objects/mL vs 4.29E6 ± 4.48E6 objects/mL, respectively) showed that these double-positive fluorescent events can be used to discriminate samples on the basis of HLA phenotype. Concentrations of CD9+HLA-A3+ double-positive events as measured in donor PPP samples showed an approximately 96% reduction after detergent treatment, with concentrations after detergent treatment residing in the range of the isotype and buffer controls (Figure 2D). Consequently, CD9+HLA-A3+ double-positive fluorescent events were identified to represent EVs.
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