As a consequence, large, highly complex datasets (from as many as eight plates, comprising 768 samples and over 200 million events) can be acquired in a single experimental day. Similarly, an increasing number of parameters can be acquired for each sample, with the availability of 20-parameter flow cytometry technology 1. High-throughput acquisition platforms, which allow the collection of at least 96 samples automatically, are common. In recent years, the volume of data from a flow cytometry experiment has increased dramatically. flowClean is available as an R package on Bioconductor, as a module on the free-to-use GenePattern web server, and as a plugin for FlowJo X. The algorithm flags events that are suspicious by visual inspection, as well as those showing more subtle effects that might not be consistently flagged by investigators reviewing the data manually, and out-performs the current state-of-the-art. We apply this method to proof-of-concept datasets and also to a subset of data from a recent vaccine trial. Aberrant time periods are reported as a new parameter and added to a revised data file, allowing users to easily review and exclude those events from further analysis. Here, we present flowClean, an algorithm to track subset frequency changes within a sample during acquisition, and flag time periods with fluorescence perturbations leading to the emergence of false populations. Therefore, we hypothesized that tracking cell populations (which represent a summary of all parameters) in centered log ratio (CLR) space would provide a sensitive and consistent method of quality control. In particular, fluorescence measurements for a sample over the collection time may not remain stable due to fluctuations in fluid dynamics the impact of instabilities may differ between samples and among parameters. Our examination of 29.228 publicly available FCS files from laboratories worldwide indicates 13.7% have a fluorescence anomaly. Quality control of the data remains challenging, however, and is further complicated when a large number of parameters is measured in an experiment. These systems allow hundreds of samples to be analyzed in a single day. Modern flow cytometry systems can be coupled to plate readers for high-throughput acquisition.
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