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bioRxiv · 10.1101/2025.05.27.656370

cytoFlagR: A comprehensive framework to objectively assess high-parameter cytometry data for batch effects

Eswar, S.·Koenig, Z. T.·Tursi, A. R.·Cobena-Reyes, J.·Tilburgs, T.·Andorf, S.
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Abstract

MotivationHigh-parameter cytometry is widely used in longitudinal studies, but technical variation across batches can confound biological signals. However, tools that objectively identify problematic batches and markers are limited. ResultsWe introduce cytoFlagR, a comprehensive tool to flag batch-related problems at the marker and cell cluster level based on robust statistical evaluations. Batch and marker variations are assessed based on median signal intensities of negative and positive cell populations and positive cell frequencies, along with Earth Movers Distance (EMD) of signal intensity distributions. Additionally, cytoFlagR identifies cell type specific batch problems via unsupervised clustering and is suitable for mass and spectral cytometry datasets where it objectively detects distinct types of batch issues. We demonstrated cytoFlagRs utility for assessing datasets that include or lack reference controls. Thus, cytoFlagR improves quality control by objective identification of technical variations that may impact downstream analysis. Availability and ImplementationcytoFlagR is freely available as R scripts with documentation and an example at https://github.com/AndorfLab/cytoFlagR. Contactandorfsa@ucmail.uc.edu

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Eswar, S., Koenig, Z. T., Tursi, A. R., Cobena-Reyes, J., Tilburgs, T., Andorf, S.. 2025-05-31. cytoFlagR: A comprehensive framework to objectively assess high-parameter cytometry data for batch effects. https://doi.org/10.1101/2025.05.27.656370

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