Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 163 Currently, it is consensus that CD9+ EVs detected in PPP are most likely plateletderived. As platelets express HLA Class I antigens on their surface, one might expect a high degree of co-localization of HLA Class I antigens with CD9 on these EVs. However, we found that CD9+HLA-A3+ particles are only a fraction (~16%) of the total CD9+ particles in HLA-A3+ donors. This observation may be explained by 1) different modes of EV biogenesis influencing whether HLA Class I antigens are present on EVs and/or whether their epitope topology is directed to the vesicular surface (and thus detectable with our setup), and 2) CD9 (but not HLA Class I antigens) may also be found on lipoproteins 18. The identification and quantification of single dd-EVs with IFCM as presented here is also subject to limitations. First, IFCM needs a minimum of 3 pixels before an event is recorded 23. Consequently, EVs with a low HLA-A3 epitope-density might be missed by our assay. Second, although we calibrated the arbitrary fluorescent SSC intensities of our platform to reflect particle-size, we obtained a goodnessof-fit measure (R2) of 91%, which implies a 9% error when selecting particles with SSC intensities corresponding to EVs ≤400 nm 23. For reference, (conventional) clinical-grade Flow Cytometers typically result in R2 values >0.99 – although lacking the resolution to detect EVs <300 nm 18, 28, 29. Additionally, we excluded samples not passing the threshold of ≥95% reduction after detergent treatment from analysis, which imposes an analytical bias towards samples containing ‘high’ concentrations of fluorescent events. However, by applying this selection criteria on our samples we ensured the analysis of fluorescent events of biological origin well above the (fluorescent) background of our assay. An alternative to the detergent treatment threshold could be the selective analysis of samples with a minimum number of events in the gate of interest (exceeding the number of events acquired in negative control samples e.g., buffer + reagents, unstained plasma, plasma + isotype staining) after acquisition. In transplantation, the potential of EVs as biomarker for the detection of allograft rejection has been reported by a few groups30-36. Most notably, animal models of heterotopic heart transplantation (mouse into mouse)24, islet xenotransplantation (human into mouse)14, and a lung transplantation model between rats (Wistar into Lewis)25, have provided evidence that concentrations of allograft-derived EVs diminish during rejection well before alterations in classical biomarkers occur or histologic manifestations of injury were observed. In the current study, we observed a stable release of dd-EVs in KTRs who did not experience allograft dysfunction
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