Chapter 8 188 category. At the moment of writing, IFCM as a platform has been commercially available for over a decade, yet its ability to detect EVs has only relatively recently been discovered 23-26. Key features or advantages that contribute to IFCM being a more powerful platform for EV analysis compared to conventional FC include the slower flow rate, CCD-camera based detection (enabling higher quantum efficiency compared to conventional photon multiplier tubes), and integration of detected signals over time using Time Delayed Integration 27. Additionally, IFCM does not require an experienced operator with technical-expertise, allows standardized reporting following calibration (this thesis), and can be used for the analysis of both cells and/or EVs (even simultaneously) 27. As promising as this platform seems, there are some inherent limitations. First, due to the recording of images associated with detected events, the size of individual data files are large compared to conventional FC data files. As clinical trials often involve the measurement of large sample cohorts, the accumulation of many large data files will lead to an increased need for computational storage space, as well as analysis power. Second, the low flow rate (approximately 0.3 µL/minute as used in our setup) hinders the rate of sample throughput. This becomes especially apparent when increasing sample acquisition times to identify rare EV subpopulations. However, this can be resolved by automating the process of sample acquisition with a 96-well plate reader and performing overnight measurements. PART 2 – SINGLE EV-DETECTION: VALIDATION The Promise of Perfusion At the moment of writing, the field of organ transplantation is suffering from a shortage of donor organs. To increase the donor-pool, increasing numbers of expanded-criteria donor (ECD) kidneys as well as kidneys procured from donation after circulatory death (DCD) 28 are used in kidney transplantation (chapter 1). ECD kidneys are, however, associated with poorer transplant outcomes when compared to kidneys from standard criteria donors 29, 30, and as such the field is exploring options to infer kidney quality prior to transplantation. Normothermic machine perfusion (NMP) is extensively being researched as a platform which may allow assessment, preservation, and even treatment of marginal quality donor kidneys 29, 31, 32. Because metabolism is activated at 37 oC, NMP offers the possibility to assess kidney status prior to transplantation through monitoring of
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