Reducing Bias in Mass Cytometry Data through Corrections for Spillover and Nonspecific Binding

Area of Science:

• Biotechnology
• Immunology
• Cell Biology

Background:

• Mass cytometry enables high-dimensional analysis of over 40 protein markers in single cells.
• Isotopic spillover from metal-tagged antibodies complicates mass cytometry data interpretation.
• Accurate data analysis is crucial for understanding complex biological systems and rare cell populations.

Purpose of the Study:

• To introduce ccSpill, an instrument-specific matrix for correcting isotopic spillover in mass cytometry.
• To evaluate the combined efficacy of ccSpill and nonspecific binding (NSB) corrections.
• To demonstrate the enhanced identification of cell types and artifacts using these combined corrections.

Main Methods:

• Development of ccSpill using calibration curves of metal-labeled antibodies and pure metals.
• Application of ccSpill to single-cell mass cytometry data.
• Integration of ccSpill with isotype-based nonspecific binding (NSB) corrections.
• Analysis of phenotypic markers to assess cell type identification enhancement.

Main Results:

• ccSpill effectively removes isotopic spillover from mass cytometry data.
• Combined ccSpill and NSB corrections optimize sample preparation and identify cell artifacts.
• Low metal ion/antibody ratios were found to be detrimental to capturing cell distribution edges without correction.
• Combined corrections significantly enhanced the identification of cell types, including rare and subtypes.

Conclusions:

• Combined ccSpill and NSB corrections are essential for accurate mass cytometry analysis.
• These corrections facilitate the unequivocal characterization of rare cells, subtypes, and treatment effects.
• This approach is vital for advancing clinical and biomedical research using mass cytometry.