Chapter 6 152 transplant recipients (KTRs) on the basis of donor-recipient Human Leukocyte Antigen (HLA) mismatching by analyzing samples obtained before (control, no donor EVs present) and after (signal validation, detection of dd-EVs originating from the allograft) transplantation. Dd-EVs were measured against a background of recipient EVs in longitudinally collected KTR samples, and the dynamics of ddEVs was monitored over time, as well as at time of ‘for-cause’ biopsy samples. RESULTS Direct detection of single EVs ≤ 400 nm in donor and KTR plasma samples Compared to donor samples, the plasma composition of KTRs contains multiple elements (e.g., elevated creatinine and urea, (medicinal) waste products) which may interfere with single EV detection. Therefore, we first tested the ability of our protocol to directly detect single EVs ≤ 400 nm in diameter in plasma samples obtained from KTRs before transplantation. To this end, PPP samples from living donors (used as healthy controls) and KTRs were diluted in fPBS, and stained with either anti-CD9 (as common plasma EV marker) or isotype control (to infer labelling specificity) before analysis with IFCM. After initial acquisition, detergent treatment was applied on each anti-CD9 labeled sample to discriminate between vesicular and non-vesicular events. Total concentrations of CD9+ objects/mL (Figure 1A) as measured in donor and KTR plasma samples were compared (Figure 1B). In the donor group, we detected 1.26E8 ± 6E7 objects/mL before, and 2.74E6 ± 6.7E6 objects/mL after detergent treatment. This represented an approximate 98% reduction – which implies that the majority of CD9+ events analyzed represent EVs. In the plasma samples obtained from KTRs, we detected 1.07E8 ± 5.3E7 objects/mL before, and 1.6E6 ± 5.1E6 objects/mL after detergent treatment (~97% reduction). No significant differences in total CD9+ EV concentrations were observed between both groups. Additionally, isotype staining resulted in 4.16E5 ± 3.85E5 objects/mL and 5.27E5 ± 1.22E5 objects/mL for donor and KTR samples, respectively, illustrating the high specificity of our labelling strategy (~300-fold difference with mAb labelling). Buffer only controls (measured each day of sample acquisition) showed no detectable events – indicating that no signals derived from antibody aggregates were detected.
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