Coping with artifact in the analysis of flow cytometric data

Area of Science:

• Cellular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Flow cytometry is a powerful tool for cell analysis.
• Artifacts in flow cytometric data can compromise accurate results.
• Solid tissues and cell cultures present unique challenges due to debris and autofluorescence.

Purpose of the Study:

• To identify common sources of artifact in flow cytometry.
• To propose strategies for improving flow cytometric data analysis.
• To demonstrate artifact removal techniques for challenging samples.

Main Methods:

• Hierarchical gating strategy: artifact removal, population definition, outcome measurement.
• Utilizing time parameter for fluidic perturbations.
• Doublet discrimination for cell clusters.
• DNA content measurement for debris and apoptotic cells.
• Boolean gating for autofluorescence.
• Dump gates for interfering cell populations.

Main Results:

• Identified specific artifacts in bone marrow, breast cancer, lung cancer, and adipose tissue.
• Demonstrated effective removal of subcellular debris, doublets, and autofluorescent cells.
• Showcased the utility of hierarchical gating for complex samples.

Conclusions:

• Artifact removal is crucial for optimal flow cytometry.
• Hierarchical gating and specific tools enhance data quality.
• Improved methods expand flow cytometry applications for diverse tissues.