AutoSpill is a principled framework that simplifies the analysis of multichromatic flow cytometry data.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Immunology

Background:

• Flow cytometry data analysis faces challenges with fluorescence spillover, even in spectral cytometry.
• Current methods for calculating spillover coefficients are outdated and struggle with high-parameter flow cytometry.

Purpose of the Study:

• To introduce AutoSpill, a novel, automated method for calculating spillover coefficients in flow cytometry.
• To improve the accuracy and robustness of spillover compensation, particularly for high-parameter experiments.

Main Methods:

• AutoSpill combines automated cell gating with robust linear regression for initial spillover matrix calculation.
• Iterative refinement is employed to minimize errors in spillover coefficient calculation.
• Autofluorescence is compensated by treating it as an endogenous dye in unstained controls.

Main Results:

• AutoSpill provides an alternative to traditional spillover coefficient calculation methods.
• The method is compatible with standard flow cytometry software and uses single-color controls.
• AutoSpill simplifies workflows and significantly reduces compensation errors in high-parameter flow cytometry.

Conclusions:

• AutoSpill offers a more robust and accurate approach to spillover compensation in flow cytometry.
• The method enhances data analysis for high-parameter flow cytometry, enabling simpler workflows.
• This advancement addresses a critical limitation in fluorescence-based flow cytometry data interpretation.