General Discussion and Future Perspectives 8 193 kidneys to examine potential differences. Alternatively, perfusion fluids obtained after hypothermic (4 degrees Celsius) machine perfusion of kidneys may be analyzed and compared with perfusion fluids obtained after NMP to determine if cellular activation has an influence on EV release. Ideally, future research should aim to correlate EV parameters to post-transplantation graft status such as delayed graft function and creatinine levels. The clinical kidney transplantation samples demonstrated that circulating donorderived EVs are present at higher concentrations in recipients with stable allograft function than in recipients with allograft dysfunction. These findings are in line with animal models 38, 39, 43, which demonstrated reduced concentrations of ddEVs before histological proof of allograft rejection. Mechanistically, it is currently unknown whether the lower concentrations of dd-EVs during allograft dysfunction is a consequence of decreased production of EVs by the allograft, increased consumption of dd-EVs by recipient immune cells, or a combination of both 43. These intriguing questions may be answered by future researchers ideally using a secondary patient cohort using both the same donor-recipient HLA mismatch (HLA-A3) to validate our findings (serving as a technical replicate), as well as using a different donor-recipient HLA mismatch (to validate the biological significance). In summary, although EV FC requires a higher level of expertise compared to conventional FC, its potential as a tool for single EV characterization in both health and disease is promising. It must be noted that EV analysis with IFCM has been performed in fields other than transplantation e.g., oncology (to characterize glioblastomaderived EVs 26, 44 or leukemia-derived EVs 45), diabetes (to examine the protective effects of EVs released by adipose-derived stem cells on obesity 46), or immunology (to study the interface between pathogen-derived EVs and host recipient cells 47). However only a few studies have performed calibration of their fluorescent signals, and none - other than those presented in this thesis - have performed calibration of light scattering intensities when using IFCM. As multicenter studies are needed to validate the clinical relevance of EVs during health and disease, e.g. with different instruments measuring the same concentration of cell-type specific EVs in a given sample 14, it is imperative that EV detection assays produce results in a standardized manner. The calibration of arbitrary signals generated by IFCM into standard units as presented in this thesis represents an important step towards increasing the reliability of EV measurements between instruments and laboratories, which are pre-requisites for understanding the biological and diagnostic significance of EVs.
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