Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

Area of Science:

• Biotechnology
• Cell Biology
• Immunology

Background:

• Extracellular vesicles (EVs) are crucial biomarkers for various diseases.
• Flow cytometry is a common tool in clinical labs for analyzing cell surface markers.
• Accurate characterization of EVs is essential for diagnostic and therapeutic applications.

Purpose of the Study:

• To evaluate the efficacy of conventional flow cytometry for analyzing extracellular vesicles (EVs) of different sizes.
• To identify limitations in detecting and quantifying small and medium-sized EVs using standard flow cytometers.
• To assess the impact of technical challenges on the reliability of EV immunophenotyping.

Main Methods:

• Purification of EVs from the HT29 human colorectal adenocarcinoma cell line.
• Analysis of small (~70 nm) and medium (~250 nm) EVs using a conventional flow cytometer with a 405 nm laser.
• Utilizing general EV labels (Calcein-green, Calcein-violet) and surface marker staining.
• Investigating issues such as swarm detection, high abort rates, and coincident events.

Main Results:

• Immunophenotyping of small EVs failed due to insufficient antibody binding sites.
• Medium EVs exhibited swarm detection and high abort rates, affecting concentration measurements.
• Swarm detection generated false positive events, reduced but not eliminated by sample dilution.
• Analysis of diluted samples required extended cytometer run times.

Conclusions:

• Conventional flow cytometry presents significant challenges for the reliable analysis of small and medium extracellular vesicles.
• Technical limitations like swarm detection and high abort rates hinder accurate EV quantification and characterization.
• The routine applicability of conventional flow cytometers for comprehensive EV analysis is questionable.