Chapter 8 190 A last observation was the correlation of the identified EV-subsets with crude donor kidney and NMP viability characteristics cold ischemia times (CIT) and renal blood flow. Both CIT and renal blood flow are parameters in a recently developed scoring system to aid clinicians in determining kidney quality during NMP 37. We therefore postulate that EV release during NMP is not random, but rather indicative of the biological status of the (ECD) kidneys. The needle in the haystack Compared to perfusion fluids (which contain multiple components released by a single organ), the biological complexity of platelet-poor plasma (containing multiple components from multiple organs) makes single-EV detection much more challenging. To examine the applicability of the protocol (as developed in part I of this thesis) in the detection of low abundant EVs, we utilized the unique setting of clinical organ transplantation. In chapter 6 we aimed to directly analyze donor tissue-derived EVs (dd-EVs) in plasma samples of kidney transplant recipients (KTRs). To detect dd-EVs, we took advantage of two concepts: 1) plasma EVs express surface MHC antigens, and 2) donor-recipient MHC mismatch enables identification of transplant organderived EVs from recipient bodyfluids 38, 39. These concepts enabled the detection of a relatively low abundant population of dd-EVs with the recipient serving as its own control (pre-transplantation) to rule out non-specific antibody targeting, and confirm the detection of dd-EVs post transplantation (signal validation). Although the detection of human dd-EVs through IFCM has been demonstrated by other research groups 25, 40, our protocol provides several improvements over the approach presented in previous work. First, the full calibration (both size and fluorescence) of our IFCM platform enhances the reproducibility of our findings. Second, as no EV isolation (and thus modulation) was performed, our protocol allows the analysis of the ‘full spectrum’ of detectable EVs representative of their natural biological state. Third, we omitted the use of CFDA-SE as ‘pan-EV’ marker as to date no marker capabale of identifying all EV has been reported. Fourth, we demonstrated the analysis of single dd-EVs by our IFCM protocol and determined its sensitivity through serial dilution of donor plasma into KTR plasma.
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