Light-scattering gating and characterization of plasma microparticles

Area of Science:

• Biophysics
• Analytical Chemistry
• Cell Biology

Background:

• Flow cytometry method (FCM) is a key technique for analyzing cell-derived microparticles (MPs).
• Standardization of FCM measurements across different instruments remains a significant challenge.
• Accurate characterization of MPs is vital for understanding various biological processes.

Purpose of the Study:

• To push the limits of FCM characterization for microparticles.
• To develop an instrument-independent method for MP analysis using Mie theory.
• To establish standardized gating strategies for MPs based on absolute size and refractive index.

Main Methods:

• Utilized a scanning flow cytometer (SFC) to measure forward- and side-scatter (FSC/SSC) signals and light-scattering profiles (LSPs).
• Applied Mie theory to evaluate instrument detection limits and construct absolute FSC/SSC gates.
• Determined size and refractive index (RI) of spherical MPs and differentiated particle shapes using LSPs.

Main Results:

• Developed an absolute FSC/SSC gating method that is naturally standardized for any FCM instrument.
• Successfully determined the size and RI of individual spherical MPs.
• Demonstrated the utility of LSPs for advanced characterization, including differentiating spherical from nonspherical particles.

Conclusions:

• The proposed absolute FSC/SSC gating method enables instrument-independent analysis of MPs.
• Mie theory provides a robust framework for accurate MP characterization, though limitations exist for small or low RI particles.
• Incorporating LSPs allows for more comprehensive MP analysis, relaxing the assumption of particle sphericity.