Th34 - 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 Text: 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, fine-tuned 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 compare 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
staining index for each fluorescent channel as well as the spillover
spreading matrix, which visualize the interactions between pairs of
fluorochromes and estimate the spread. Secondly, we designed a 35-color
broad human immunophenotyping panel to stain PBMCs from 3 donors
and compared the data quality and resolution of canonical immune cell
populations between the instruments. The outcome of this study will help
inform and elucidate the selection of appropriate platforms to support
immunological research.