Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 155 It should be noted that some samples showed low/minimal reduction in concentrations of fluorescent events detected after detergent treatment (as illustrated by 1 recipient sample in Figure 2D). These events were interpreted to not represent membrane-delimited vesicular events, but rather be indicative for protein complexes (which are unaffected by detergent treatment). To identify and exclude such samples from future analysis, we established a threshold of ≥95% reduction (in concentration) after detergent treatment for samples to be included in future analysis. This cut-off value was established to ensure that only samples are included that are representative (≥95%) of membrane-delimited particles (as opposed to protein complexes). These observations show that our IFCM protocol is able to identify and discriminate single EVs in diluted PPP samples on the basis of their HLA phenotype by analyzing events that are both CD9+ (plasma EV marker) and HLA-A3+ (to discriminate between HLA-A3+ and HLA-A3- individuals).
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