Performance comparison of the Spectral Signature, Spillover Spreading Matrix, and Data Unmixing Quality between the spectral Cytek Aurora and the spectral BD FACS Symphony A5 SE by a 35-Color Human Broad Immunophenotyping Panel

Abstract

Spectral flow cytometry allows the collection of the entire spectral signature across all lasers. This aids in the overall workflow including improvements in data collection, finetuned visualization of autofluorescence and panel design. Moreover, by the process of spectral unmixing we can take the sum of the fluorescence and mathematically separate out each individual color, allowing us to further discriminate between the variations within the spectral signature of a molecule and use novel fluorochrome combinations that were historically not used in the same panel. In recent years, many cytometry manufacturers have developed their own instrumentation to perform such multicolor spectral cytometry experiments, most notably Cytek with their Aurora system as well as more recently by BD Biosciences with their FACS Symphony A5 SE (Spectrally Enabled). In this study, we independently compared the performance between the Cytek Aurora and the FACS Symphony A5 SE. First, we assessed human peripheral blood mononuclear cells (PBMCs) individually stained with anti-human CD4 antibodies (35 different colors, clone SK3) to compare the stain index for each fluorescent channel as well as the overall spillover spreading matrix, which visualizes the interactions between pairs of fluorochromes and estimates the spread. Secondly, based on instrument optical configurations, stain index, and spillover spread matrix, we designed a 35-color broad human immunophenotyping panel to stain lysed whole blood from 3 donors and compared the data quality and resolution of previously-described and anticipated immune cell populations between the instruments and have determined an accurate comparative analysis of both instruments.