Summaries 7 177 unsuitable to further characterize the released nanoparticles, and thus unsuitable to confirm that the released nanoparticles represent EVs. In chapter 3 we present, following the criteria established in chapter 1, an IFCM method for the detection of single EVs in plasma samples which omits the use of EV isolation techniques. Therefore, our method is able to directly show the status of an individual (as reflected by EV concentrations and phenotypes), which is greatly beneficial in the monitoring of EVs in health and disease. As plasma is considered to be the most complex bio fluid for single EV detection (due to the interference of protein aggregates, cell debris and lipoproteins), this methodological development has the potential to push the EV-field further. Additionally, this chapter demonstrates that light scattering signals generated by IFCM can be correlated to particle size through Mie theory. This latter achievement represents a much needed step towards the calibration of IFCM light scattering signals and this standardization improves the reproducibility of EV measurements. Ultimately, the methodology as described in this chapter allows the analysis of single EVs ≤ 400 nm in diameter. The direct detection of single EVs in urinary samples required an adaptation of the IFCM methodology as developed for the direct detection of single EVs in plasma, as ‘Auto-Fluorescent’ particles in urine interfere with true EV signals (chapter 4). These ‘Auto-Fluorescent’ particles are not representative of uEVs as they are not lysed by detergent treatment, and consequently should be removed from analysis. We designed a gating strategy which removes such particles from analysis, whilst maintaining events representative for uEVs. Chapter 5 returns to the clinical setting of organ preservation with NMP (chapter 2). In a new ECD kidney cohort, we validate that ECD kidneys release nanoparticles (<400 nm in diameter) into the perfusion fluids. Using our developed IFCM methodology, we 1) are able to show that these nanoparticles are indeed representative of EVs, 2) identify different EV subsets based on the detection of different antigens on the EV surface, and 3) show that some of the identified EV subsets correlate with well-established indicators of transplant outcome. This suggests that EVs might represent new potential candidates for the assessment of kidney graft quality prior to transplantation. As a first step towards clinical applicability, chapter 6 sets out to determine whether the developed IFCM methodology is able to detect and follow-up single,
RkJQdWJsaXNoZXIy MTk4NDMw