Flow Cytometric Detection of Biomarker Changes in CFDA‐SE‐Labelled Plasma Extracellular Vesicles Using a Rodent Pregnancy Model of Prenatal Diagnostics

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).