Chapter 5 124 variety of protein markers depending on the cell source and the confinement of some markers on the luminal side of the vesicles 19, 20. In the absence of a specific marker, EVs are identified by their expression of common markers such as CD9, CD63 and CD81. These proteins have a broad tissue distribution, belong to the tetraspanin superfamily, and are enriched on EVs 21. Previously, our group was able to quantify the release of nanoparticles (such as protein aggregates and EVs) by ECD kidneys during NMP 22. Here we apply our recently developed Imaging Flow Cytometry (IFCM)-based methodology 23 to identify, phenotype and determine the concentration EVs ≤ 400 nm in diameter released by discarded human kidney grafts during NMP. We show the identification of distinct EV subsets based on their tetraspanin profile in combination with the detection of esterase activity, platelet endothelial cell adhesion molecule (CD31) or the pan-leukocyte protein (CD45). Additionally, in the absence of posttransplantation kidney function, we perform correlation analysis of the identified EV subsets with crude donor and NMP viability characteristics to explore the potential clinical implications of the identified EVs. RESULTS Kidneys release nanoparticles during NMP To study whether discarded kidneys release nanoparticles during NMP, perfusate samples drawn at 0 / 60 / 180 / 360 minutes were measured with NTA to determine the particle concentration and size distribution (Figure 1). We observed a baseline concentration of 2.05E9 ± 2.13E8 particles/mL (mean ± standard deviation, area under the curve) within the perfusate prior to contact with the kidney (T0, baseline perfusate). Total particle concentrations were observed to increase over time during NMP: 1.96E10 ± 7.21E8 / 2.54E10 ± 7.85E8 / 3.06E10 ± 6.27E8 objects/mL at 60, 180 and 360 minutes respectively. Average particle size was established to be < 400 nm irrespective of the time of sampling.
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