General Introduction and Objectives 13 1 33. Consequently, an elevation of these markers is often followed by a kidney transplant biopsy, which, despite being the gold standard to diagnose rejection, is an invasive procedure with a risk of complications including bleeding and infection 34. Combined, these issues reveal the critical need for more accurate, early and minimally invasive biomarker platforms to diagnose kidney allograft rejection. The potential of EVs as biomarker for the detection of allograft rejection has been described – in animal models – by a few groups 35-40. These studies have shown that donor-derived EVs are released into the circulation post transplantation, and provide indications that concentrations of donor-derived EVs diminish during rejection well before alterations in classical biomarkers or histologic manifistation of injury can be observed. These findings suggest that detection and monitoring of donor-derived circulating EVs may herald rejection in a more time-sensitive manner compared to classical markers. Challenging to measure Despite the interest in and clinical relevance of EVs as biomarker, EV analysis is hampered by a variety of factors. First of all, their physical characteristics, such as their small size, low epitope copy number 41, the variety of protein markers depending on the cell source, the confinement of some markers to the luminal side of the EV, and the low abundance of pathological EVs 11, 42 all contribute to the complexity of EV analysis. Additionally, no unique antigens representative for specific EV classes and subpopulations have been reported to date. Instead, tetraspanins (CD9/CD63/CD81) are recognized as common EV antigens. These proteins are enriched on EVs and are involved in EV biogenesis, cargo selection, and cell targeting 43, 44. Second, the identification of EVs in blood plasma is further hindered by the molecular complexity of plasma, which contains multiple elements (e.g. protein aggregates, cell debris and the far more abundant lipoproteins) that interfere with EV analysis 11, 45. Lipoproteins are submicron structures of lipids and apolipoproteins that are excreted into the circulation by the liver and intestines. They are classified into several subgroups, and their biophysical properties in terms of size and density largely overlap with those of EVs. However, a distinguishing feature is the presence of an aqueous core in EVs, whereas the core of lipoproteins is comprised of lipids 11.
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