Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 165 Sample collection and processing From both donors and KTRs, whole blood samples (EDTA) were collected before transplantation, and, for KTRs, 3 days, 7 days, and 6 months after transplantation. Additionally, blood samples were collected on the morning of (or the day preceding) a ‘for-cause’ kidney transplant biopsy. Blood was drawn from each individual into two BD Vacutainer K3-EDTA-coated collection tubes (BD Biosciences, San Jose, USA). Whole blood was centrifuged at 1910 x g for 10 minutes at room temperature and the plasma layer was then collected at 16,000 x g for 10 minutes at room temperature in 1 mL aliquots using Safe-Lock Eppendorf tubes (Eppendorf AG, Hamburg, Germany). The resulting platelet-poor plasma (PPP) was first pooled before being divided into 700-µL aliquots in cryovials containing 28 µL of a 25x concentrated protease inhibitor cocktail solution (4% v/v) (cOmplete Protease inhibitor cocktail tablets, Roche, Mannheim, Germany) according to the manufacturers’ instructions and stored at -80 °C. Stratification of KTRs based on biopsy scores Biopsies were scored by an experienced renal pathologist, and KTRs were divided into 4 groups based on 1) no ‘for-cause’ biopsy and no anti-rejection therapy (‘Control’ group), 2) acute tubular necrosis (ATN) as the main finding in the biopsy and no anti-rejection therapy (‘ATN’ group), 3) no histopathological signs of rejection but treated with anti-rejection therapy on the basis of clinical suspicion of rejection (‘Presumed Rejection’ group), and 4) biopsy-proven acute rejection (BPAR – aABMR or aTCMR2A) in combination with anti-rejection therapy (‘Rejection’ group). In case patients underwent multiple biopsies over the course of the study, only the first biopsy was used in the analysis. Sample labelling PPP samples were stained with monoclonal antibodies (mAbs) and isotype controls as extensively described in our previous work 23. In the absence of a specific marker, EVs are identified by their expression of common markers such as CD9, CD63 and CD81 41. In this study, we used CD9 (which has been shown to be highly prevalent on plasma-derived EVs) as common EV marker 23. Additionally, following the characterization of kidney-derived EVs released during normothermic machine perfusion, we determined that CD9 co-localizes to a higher degree with MHC
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