ABSTRACT Circulating extracellular vesicles (EVs) offer a promising source of non‐invasive biomarkers for congenital disease diagnostics but robust assays for their detection are lacking. We used a rodent pregnancy model as a proxy for a prenatal diagnostics assay to test whether we could detect a difference in EV tetraspanin display between pregnant rats and non‐pregnant controls using standard flow cytometry. Carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE) was used as a positive EV marker. CFDA‐SE positive particle count was linear but both size and concentration were below those measured by nanoparticle tracking analysis. Plasma proteins activated CFDA‐SE non‐specifically. Fractional analysis of eluates revealed that plasma protein is efficiently separated from EVs by size‐exclusion chromatography (SEC). We identify albumin within antibody storage buffers as the main source of false positives. Attempts to reduce background by quenching (trypan blue) and clean‐up (SEC) methods were ineffective. We then probed CFDA‐SE‐labelled EVs with antibodies to assay surface biomarker display using a quadrant method to measure double‐positive EVs. We observed a reduction in CD63 display in the pregnant condition but no change in CD9 or CD81. Using CD63 display level as a diagnostic test allowed detection of the pregnant condition with a sensitivity of 0.83 at specificity of 1 (AUC = 0.99).
Adamova et al. (Fri,) studied this question.