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

Mass cytometry can facilitate the study of over 40 protein markers in single cells, making it an ideal technique to investigate complex biological systems. The use of antibodies tagged with metal isotopes as probes makes mass cytometry measurements susceptible to isotopic spillover. In this report, we introduce ccSpill, an instrument-specific matrix built from calibration curves of metal-labeled antibodies and solutions of pure metals that can be applied to single-cell data to remove spillover. ccSpill is unique to each lab, and each set of reporter antibodies and is compatible with isotype-based nonspecific binding (NSB) corrections. We demonstrate that combining ccSpill with NSB corrections is integral to the optimization of sample preparation procedures that involve challenging antibodies and that such a correction identifies and excludes cell artifacts in single-cell analyses. Furthermore, the combined correction demonstrated that low metal ion/antibody ratios are detrimental to capturing features at the edges of single-cell distributions, which are misassigned without a correction. Lastly, by applying the combined correction to phenotypic markers, the identification of cell types was highly enhanced. Combined corrections of spillover and NSB provide a gateway to use mass cytometry unequivocally to characterize rare cells (e.g., senescent), subtypes (e.g., endothelial), or treatment (e.g., rapamycin), which is highly needed in clinical and biomedical research.