Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 159 dd-EVs are detected in KTRs with stable allograft function Next, we examined whether dd-EVs could be directly detected in PPP of KTRs after transplantation. As stated previously, samples not passing the threshold of ≥95% reduction after detergent treatment were excluded from analysis; excluded samples were not associated with any specific time-point. Sample exclusion on the basis of detergent treatment resulted in the exclusion of 10 KTR sample series, leaving 26 KTR sample series in the analysis. Table 1 shows the patient characteristics corresponding to the samples included in our analysis, stratified into the four groups as described in the methods section. First, we compared the total concentrations of double-positive events measured in PPP samples taken before (n = 13) and 2-4 days (n = 17) after transplantation (Figure 4A). We observed a statistically significant difference between these time points which confirms the release of dd-EVs into KTR’s circulation. We then performed a longitudinal analysis of dd-EV concentrations comparing KTRs in the control group with KTRs who underwent a ‘for-cause’ kidney biopsy (‘Biopted’ group) (Figure 4B). In the control group, we observed significant increases in dd-EV concentrations at 2-4 days, 6-8 days and 6 months after transplantation compared to the concentrations detected before transplantation. These dd-EV concentrations were observed to be stable throughout follow-up, suggesting that stable allograft function (without a biopsy) leads to a detectable dd-EV signal in KTR plasma. This notion was further strengthened by the observation that dd-EV concentrations did not increase (compared to levels detected before transplantation) in individuals who did experience allograft complications (‘Biopted’ group); this effect was observed up to at least 6 months after transplantation (Figure 4C). To examine a potential diagnostic value of dd-EVs, we next compared the concentrations of CD9+HLA-A3+ EVs measured in ‘for-cause’ biopsy samples taken at 6 days after transplantation (median, range: 2 – 60) with the concentrations as detected in samples taken 6 – 8 days after transplantation from patients in the ‘control’ group. The concentration of dd-EVs was higher in time-matched KTR samples without a biopsy compared to concentrations detected at the moment of a for-cause biopsy (irrespective of their pathological classification, 5.36E6 ± 2.05E6 objects/mL vs 3.24E6 ± 8.55E5 objects/mL, respectively, p = 0.05). However, no statistical differences were
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