Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 157 IFCM discriminates single CD9+HLA-A3+ EVs down to 1% above recipient-specific background To determine the discriminative power of our IFCM protocol to detect CD9+HLA-A3+ EVs above (recipient-self) background signals in KTR samples, we serially diluted (2-fold dilutions) HLA-A3+ PPP into HLA-A3- PPP (5 matched donor-recipient couples), labelled these samples with anti-CD9 and anti-HLA-A3, and analyzed these with IFCM. For each of the 5 donor-recipient couples, we quantified the concentrations of CD9+HLA-A3+ EVs as detected for each dilution step (Figure 3A). Similar to the previous observation, we observed that each of the 5 HLA-A3- PPP recipient samples (without any spiked-in HLA-A3+ PPP) yielded varying background concentrations. These were interpreted to represent the recipient-self samplespecific background (indicated with dashed green lines, Figure 3A). We were able to detect CD9+HLA-A3+ EVs above their respective sample-specific background concentrations down to ~100-fold dilution, thus with CD9+HLA-A3+ EVs being detectable ~1% above the recipient ‘self’ signal. Next, we examined the accuracy of our IFCM protocol to detect single CD9+HLA-A3+ EVs in mixed plasma samples – as opposed to the coincidence detection of multiple particles recorded as a single event. For each individual (donor-in-recipient) dilution experiment, the concentrations of CD9+HLA-A3+ EVs as detected for each dilution step (range: 100% to 1.56% HLA-A3+ PPP) were analyzed using a linear regression model (Figure 3B). We observed that the concentrations of CD9+HLA-A3+ EVs were linearly proportional to the dilution factor, as indicated by the R2 values. Additionally, analysis of the (standardized) fluorescent intensities as detected in both detection channels revealed that the fluorescent intensities were unaffected by serial dilution; mean: 1315 (range 1085 – 1604) ERF for BV421 (HLA-A3), and 29.5 (range 25.7 – 35.8) ERF for APC (CD9) (Figure 3C). Taken together, these data indicate that IFCM is able to detect single dd-EVs directly in recipients’ circulation if their fraction exceeds ~1% above their recipient specific background.
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