Plasma extracellular vesicle test sample to standardize flow cytometry measurements

Area of Science:

• Biotechnology
• Biomarker Discovery
• Analytical Chemistry

Background:

• Extracellular vesicles (EVs) in body fluids are promising disease biomarkers.
• Current flow cytometry (FCM) measurements of EV concentrations lack comparability.
• Standardized methods are needed for reliable EV analysis.

Purpose of the Study:

• To develop a human plasma EV test sample (PEVTES) for validating reference materials and procedures.
• To create a ready-to-use, stable, and FCM-compatible EV sample.
• To improve the comparability of EV concentration measurements.

Main Methods:

• Prepared PEVTES from human plasma, staining EVs with specific antibodies.
• Isolated stained EVs using size-exclusion chromatography and filtered to remove platelets.
• Stored PEVTES in cryopreservation agents at -80°C for 12 months and measured concentrations via FCM.

Main Results:

• Developed PEVTES resembles plasma subcellular particles when analyzed by FCM.
• PEVTES demonstrated stable concentrations of platelet-derived, erythrocyte-derived, and lactadherin+ EVs after 12 months of storage in trehalose.
• The sample is compatible with flow cytometry analysis.

Conclusions:

• The developed PEVTES is ready-to-use, stable, and flow cytometry-compatible.
• PEVTES accurately reflects plasma subcellular particles.
• This test sample is ideal for validating new reference materials and procedures in EV research.