• • • In flow cytometry (FCM) experiments, investigators usually rely on instrument manufacturers and “black box” commercial software to transform cellular marker expressions into cell populations on 2D dot plots. Techniques behind these systems and their limitations have not been sufficiently addressed or disclosed.

Currently, a Flow Cytometry Standard (FCS) file can be FCS2.0 or FCS3.0 (N.B. Newer standards like FCS3.1 and ACS1. Huawei B593 Qos. 0 have been proposed and might be generated by some manufacturers in the future.).

According to the Becton Dickinson (BD, ) acquisition software manual [], by default FCS2.0 fluorescence data are log-transformed, whereas FCS3.0 files keep the raw outputs from the instrument in a linear mode. Therefore, FCS3.0 format provides more control to bioinformaticians and FCM software developers in data processing (e.g., changing data compensation after acquisition). However, it remains unclear how the linear-mode fluorescence data in FCS3.0 files should be transformed before events can be plotted for population identification. Figure A–C shows a comparison on FCS3.0 data conversion and transformation we conducted between FlowJo (Tree Star, ), flowTrans [] of Bioconductor, and FCS2CSV [] on three FCS3.0 files collected from different labs (details of FCS files and software systems can be found in Supporting Information, File 1; headers of the three BD FCS file can be found in Supporting Information, File 2).

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It seems that under the default setting FCS2CSV and flowTrans seemed to generate insufficient data modality for population segregation in these FCS files. The conversion techniques (and transformation parameters) used in the three systems seem to be inconsistent, reflected by the output data range and the data distribution characteristics. These issues have brought in uncertainty not only in FCM data analysis but also in data sharing and interpretation and, therefore, made us believe it was necessary to develop a single open source software system that would transform different types of FCS files appropriately, robustly, and consistently.

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