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Cyto-Feature Engineering: A Pipeline for Flow Cytometry Analysis to Uncover Immune Populations and Associations with Disease


Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel fow cytometry analysis pipeline to identify a plethora of cell populations efciently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population diferences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a fltering step refnes the data from all identifed cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline’s modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confrmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing fow cytometry regardless of cytometer or panel design. The code is available at engineering.

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