Chapter 1 16 perfusate samples taken before, during, and after the NMP procedure are analyzed with nanoparticle tracking analysis (NTA) to quantitate and determine the size distribution of nanoparticles released during NMP. Though NTA currently is a gold-standard technique for EV-quantitation and size analysis, it is unsuitable for complex samples such as plasma or urine (due to its limited phenotyping capabilities). In chapter 3, we aim to provide a standardized (size- and fluorescence calibrated) IFCM-based methodology which is able to discriminate, phenotype, and determine the concentration of individual human plasma-derived EVs ≤400 nm in diameter – without prior isolation of EVs. This methodology aims to discriminate EVs from contaminating agents such as lipoproteins and protein aggregates in molecular complex samples such as plasma, and forms the backbone of this thesis. In chapter 4, we present an adaptation of this methodology aiming to detect single EVs in urine – another complex bio fluid with its own set of challenges in the context of EV detection. In chapter 5, we characterize the nanoparticles released by ECD kidneys during NMP, and confirm that these are representative of EVs. Following the identification of EVs in the perfusion fluids, we aim to identify distinct EV subsets and examine whether these are potentially correlated with donor and NMP viability characteristics. As a first step towards clinical applicability, we next set out to determine whether the developed methodology is able to detect and follow-up single, (donor) tissuederived EVs in plasma samples of kidney transplant recipients (chapter 6). Chapter 7 provides a summary of the results described in this thesis. Chapter 8 discusses these results and provides a perspective on future implications of our findings.
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