*Purpose: By characterizing and quantifying the dynamical release of kidney Extracellular Vesicles (EVs) during Normothermic Machine Perfusion (NMP), we aim to gain a better understanding of the value of EVs in 1) kidney quality pre-transplantation and 2) as potential biomarker in recipient’s circulation post transplantation.

*Methods: Eight discarded Extended Criteria Donor kidneys (6 DCD, 2 DBD, mean warm ischemia times of 12 ± 8 minutes followed by ~13 ± 5 hours of cold ischemia, age 68 ± 7, all male) were perfused in a closed system at 37 ⁰C for 6 hours. Perfusate samples were taken before and at 1/3/6 hours intervals, stained with a mix of common EV markers (tetraspanins CD9/CD63/CD81) or one of these tetraspanins in combination with either CFSE (enzymatic activity) or HLA-A2. Samples were measured using Imaging Flow Cytometry to identify, quantify and characterize single small EVs (< 300 nm, ssEVs).

*Results: CFSE and tetraspanin double-positive ssEVs were quantified at each of the collected time points. We observed a ~700 / 740 / 560 fold increase compared to ssEV levels before perfusion at 1/3/6 hours of NMP, respectively. ssEVs levels were found to be positively correlated with donor age and kidney weight, whilst negative correlations were found for ischemia times. Tetraspanin CD81 was found to represent the majority (~70%) of the excreted ssEVs (CD9: ~15% / CD63 <5%). Furthermore, using HLA-A2 mAbs we were able to discriminate and quantify ssEV populations between NMP samples on the basis of HLA phenotype. Again, the majority of ssEVs expressing HLA-A2 was found to be colocalized with CD81.

*Conclusions: During NMP small EVs are excreted and this excretion is highest during the first hour of perfusion. The characterization of excreted small EVs, especially in combination with an HLA phenotype marker, provides a starting point to identify allograft specific EVs and map their clinical implications.

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