Quantitative flow cytometry enables end-to-end optimization of cross-platform extracellular vesicle studies

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Flow cytometry (FCM) is widely used for characterizing extracellular particles (EPs) like viruses and extracellular vesicles (EVs).
• Existing reporting guidelines (e.g., MIFlowCyt-EV) address metadata and data reporting but lack tools for systematic FCM optimization for EPs.
• A need exists for quantitative methods to standardize FCM analysis of small particles across different platforms.

Purpose of the Study:

• To develop and present a suite of methods and software tools for optimizing FCM settings for EP analysis.
• To enable systematic and quantitative optimization of FCM for small particle characterization.
• To facilitate standardized cross-platform comparisons of EP data.

Main Methods:

• Introduction of an automated small-particle optimization (SPOT) pipeline to optimize fluorescence and light scatter detector settings for FCM.
• Application of quantitative FCM (qFCM) for optimizing fluorophore panels, laser power, pulse statistics, and window extensions.
• Validation through a cross-platform standardization study using a commercial EV reference material.

Main Results:

• The SPOT pipeline and qFCM provide systematic and quantitative optimization of FCM for EP analysis.
• Demonstration of optimized FCM settings and fluorophore panels for enhanced EP detection and characterization.
• Successful standardization of cross-platform EP comparisons, reducing variability due to instrument differences.

Conclusions:

• The developed methods and software tools significantly improve the optimization and standardization of FCM for EP analysis.
• qFCM is a valuable tool for enabling reliable cross-platform comparisons of extracellular particle data.
• This work addresses a critical gap in EP analysis, paving the way for more robust and reproducible research.