Small Particle Fluorescence and Light Scatter Calibration Using FCM_PASS Software

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Flow cytometry is increasingly used for small particle analysis, including extracellular vesicles (EVs).
• Lack of standardization in EV flow cytometry data reporting hinders interpretation, validation, and reproducibility.
• Established methods exist for fluorescence and light scatter standardization.

Purpose of the Study:

• To present FCM_PASS, a software package for standardizing small particle flow cytometry.
• To enable fluorescence and light scatter calibration for small particles.
• To generate reports conforming to the MIFlowCyt-EV standard.

Main Methods:

• The study details a workflow for implementing calibration using FCM_PASS.
• This includes acquiring and cataloging calibration materials, creating cytometer databases, and importing FCS files.
• The process involves inputting fluorescence and light scatter calibration parameters and applying them to FCS files.

Main Results:

• FCM_PASS provides a standardized approach to small particle flow cytometry calibration.
• The software facilitates the generation of reports aligned with the MIFlowCyt-EV standard.
• This promotes better data interpretation, validation, and reproducibility in EV research.

Conclusions:

• FCM_PASS addresses the critical need for standardization in small particle flow cytometry.
• Implementation of this software can significantly improve the quality and comparability of EV research data.
• Standardized calibration is essential for advancing the field of extracellular vesicle analysis.