Chapter 6 162 DISCUSSION EVs have great potential value as (minimally invasive) biomarkers 26, but sensitive and reproducible methods for single EV analysis are essential to understand the role of EVs in human health and disease 22. In the current work, we assessed the applicability of our recently developed IFCM-based methodology 23 to directly measure EV subsets in human patient plasma samples – as exemplified by the detection of single dd-EVs in KTR plasma samples after kidney transplantation. In addition to their physical properties and current technological challenges, the detection of EV subsets during health and disease may – depending on the disease – be hampered by the presence of ‘contaminating’ agents in the sample matrix. In the current study, components such as urea and creatinine are elevated as a consequence of kidney failure and may interfere with single EV detection. We show that our IFCM protocol is able to detect single CD9+ EVs in plasma samples obtained from both donors (healthy controls) and KTRs before transplantation, thereby suggesting that single EV detection by our IFCM protocol is uninfluenced by such contaminating components. The concentration of EVs in urine is related to nephron mass, explaining a lower concentration of total CD9+ EVs in KTRs compared to donors 27. Here, we did not observe a difference in total CD9+ EVs between both groups in blood plasma, which is most likely due to the contribution of multiple organs to the total plasma CD9+ EV pool. The concept of plasma circulating dd-EV detection and characterization with IFCM has been presented previously 4. Mastoridis et al. showed that by analyzing CFSE+ events in combination with an exosome-specific marker (CD63) and an origin-specific marker (donor-HLA), circulating dd-EVs could be detected in the circulation of a liver transplantation recipient 4. However, our protocol provides several improvements over the approach presented in previous work: 1) our IFCM platform is both SSC (size) and fluorescence calibrated – which enhances reproducibility, 2) no EV isolation was performed – thus the ‘full spectrum’ of detectable EVs was analyzed, and 3) by analyzing CD9+ EVs (shown to be highly prevalent in plasma samples) 4, 23 in combination with donor-HLA, we were able to analyze a broad spectrum of circulating dd-EVs – as opposed to the analysis of donor-derived CFSE+CD63+ exosomes (a donor EV subset). Additionally, we prove the detection of single dd-EVs by our IFCM protocol and determined its sensitivity through serial dilution of HLA-A3+ plasma into HLA-A3- plasma demonstrating a linear correlation with the dilution factor and stable ERF 22.
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