General Introduction and Objectives 15 1 operate at a relatively high-throughput rate of thousands to millions of EVs per minute, 4) be used without the need for prior EV isolation (thus omitting sample selection biases whilst simultaneously reducing sample handling time), and 5) discriminate the identified EVs from other contaminating components in the biofluid of interest. Objectives of this thesis Sensitive and standardized methods for single EV analysis are needed if EVs are to be translated into clinical practice. In recent years, imaging flow cytometry (IFCM) has emerged as a technique that enables the discrimination and analysis of single EVs with increased sensitivity compared to conventional FC. The ability of IFCM to detect submicron particles has been demonstrated using fluorescent polystyrene beads 55-58 or the use of cell supernatant-derived EVs 50. At the moment of writing, several studies have reported the detection of EVs - obtained after performing isolation procedures - from plasma using IFCM 55, 56, 58, 59. However, the used isolation procedures may have changed some EV properties: ultrafiltration might disintegrate larger EVs (thus generating smaller particles which, in turn, skew EV quantification upward) 60 whereas ultracentrifugation might cause aggregation and encapsulation of EVs (skewing EV quantification downward) 61. Thus, it is unlikely that these results represent all EVs in plasma 62. The main objective of this thesis is to explore whether IFCM is a suitable platform for the direct detection of single EVs in molecular complex samples such as plasma without the need to perform EV isolation techniques. This thesis is composed of two parts: ⦁ Part I presents the development of a standardized IFCM-based methodology which allows for the direct detection, characterization and quantification of single EVs in molecular complex samples such as perfusate, plasma or urine. ⦁ Part II aims to validate the standardized methodology to detect EV subsets in the context of kidney transplantation. In chapter 2, we aim to validate the proof-of-concept that kidneys release nanoparticles (such as protein aggregates and EVs) ex-vivo. In this chapter, we examine the release of nanoparticles into the perfusion fluid by expanded-criteria donor (ECD) kidneys during normothermic machine perfusion (NMP). To this end,
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