Reducing Spreading: Removing the Impact of Irrelevant Dyes Improves Unmixed Flow Cytometry Data

Staining panels have become increasingly complex in recent years, with 40 to 50 dye panels regularly reported, particularly on “spectral” flow cytometers. It is universal practice to include all dyes in the mixing matrix when unmixing the data, even though it is well‐known that individual events within the sample only stain with a subset of dyes. Adding dyes to the mixing matrix increases the variance of the unmixed abundance distributions, even if those dyes are not present on particular events. This manuscript introduces a novel unmixing method called TRU‐OLS. TRU‐OLS utilizes a priori biological knowledge (i.e., unstained controls) to unmix each event with only the dyes present on that event. We show that TRU‐OLS decreases the variance of the unmixed abundances both statistically and visually in simple (4–6 color) and complex (40 color) staining panels.