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

Adamova, P ORCID: 0000-0002-7213-0547, Sloan, L ORCID: 0009-0001-6537-4383, Powell, AK ORCID: 0000-0002-3680-9384 and Dykes, IM ORCID: 0000-0002-1030-3721 (2026) Flow Cytometric Detection of Biomarker Changes in CFDA‐SE‐Labelled Plasma Extracellular Vesicles Using a Rodent Pregnancy Model of Prenatal Diagnostics. Journal of Extracellular Biology, 5 (5). ISSN 2768-2811

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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).

Item Type:	Article
Subjects:	R Medicine > RM Therapeutics. Pharmacology R Medicine > RS Pharmacy and materia medica
Divisions:	Pharmacy and Biomolecular Sciences
Publisher:	Wiley
Date of acceptance:	14 April 2026
Date of first compliant Open Access:	6 May 2026
Date Deposited:	06 May 2026 11:13
Last Modified:	06 May 2026 11:13
DOI or ID number:	10.1002/jex2.70145
URI:	https://researchonline.ljmu.ac.uk/id/eprint/28524

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