Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry

The advent of mass cytometry increased the number of parameters measured at the single-cell level while decreasing the extent of crosstalk between channels relative to dye-based flow cytometry. Although reduced, spillover still exists in mass cytometry data, and minimizing its effect requires considerable expert knowledge and substantial experimental effort. Here, we describe a novel bead-based compensation workflow and R-based software that estimates and corrects for interference between channels. We performed an in-depth characterization of the spillover properties in mass cytometry, including limitations defined by the linear range of the mass cytometer and the reproducibility of the spillover over time and across machines. We demonstrated the utility of our method in suspension and imaging mass cytometry. To conclude, our approach greatly simplifies the development of new antibody panels, increases flexibility for antibody-metal pairing, opens the way to using less pure isotopes, and improves overall data quality, thereby reducing the risk of reporting cell phenotype artifacts. [Display omitted] •Signal spillover can impact quality of mass cytometry data•Spillover can be corrected by compensation throughout the linear range of the CyTOF•Compensation enables signal correction and data structure preservation•CATALYST is a new R package and a web tool to estimate and correct for spillover Signal spillover exists in mass cytometry and complicates the development of antibody panels and the interpretation of the data. Here, we characterize this spillover and its effects on the data. Further, we present an approach to estimate and correct for it, which was implemented in a newly developed R package called CATALYST. We show how this method can be used to correct for channel crosstalk in suspension and imaging mass cytometry data throughout the linear range of the instrument.