Chapter 6 150 ABSTRACT Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recentlydeveloped calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma (PPP) was generated from 36 HLA-A3 mismatched donor (HLA-A3+) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before ‘for-cause’ kidney transplant biopsies were stained with anti-CD9 (plasma EVmarker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9+ EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9+HLA-A3+. Serial dilution experiments of HLA-A3+ in HLA-A3- PPP showed that single CD9+HLA-A3+ EVs were detectable down to ~1% above the recipient ‘self-signal’. After transplantation, CD9+HLA-A3+ EVs were detected above pretransplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.
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