Can I use the FlowAI script in FCS Express?

Yes, with FCS Express 7, you can run FlowAI directly within FCS Express using Pipelines. FlowAI is used to check the quality of the data based on the flow rate, signal acquisition, and dynamic range.

In FCS Express 6 you can run the FlowAI script via R Integration.  Due to the script’s reliance upon $TIMESTEP and $PnR keywords, it will need to be modified as necessary for each file to processed.

What is Flow AI?

FlowAI is an algorithm available to be used for quality control of flow cytometry data written in FCS3.0 file format or later.  Developed by Gianni Monaco et al, FlowAI can be used to detect and remove spurious events or regions automatically by evaluating flow rate, signal acquisition, and dynamic range. FlowAI checks data for:

  • Flow rate steadiness using the $TIMESTEP keyword
  • Signal acquisition stability by plotting each channel's median vs. time using equal-sized bins (typically displayed as Levy-Jennings-type plot)
  • Dynamic range - identification and removal of outliers from lower limit of $PnR and margin events from upper limit of $PnR

Executing FlowAI in FCS Express

With the January 2021 release of FCS Express 7, FlowAI has been added as a standalone Pipeline step. FlowAI can now be run directly within FCS Express as a pre-defined algorithm without any external software requirements (i.e. R Integration is no longer required).

FlowAI evaluates data using specific keywords and requires non-compensated parameters. Because of these requirements, modifications to the FCS Express Layout Options are needed to run FlowAI with Pipelines.

  • When running FlowAI with Pipelines, the uncompensated data, or raw fluorescence data, must be processed. To enable the use of uncompensated parameters on your layout:
    • Navigate to the FCS Express Layout Options.
    • In the Compensation Options, set the "New Compensated Parameters Should:" to "Append to data after uncompensated parameters".
    • Uncompensated parameters will now be accessible on the layout. Uncompensated parameters will be indicated by the absence of the suffix "- Comp" in the parameter list.
    • For further details on working with uncompensated data, see the Compensation Options section of our manual.

If you plan to use FlowAI with R Integration, modifications will need to be made to the FCS Express User Options and R script for each data file to be processed.

  • When running FlowAI with R Integration, the uncompensated data, or raw flourescence data, are processed.  Since plots are displaying compensated data by default in FCS Express, the compensated data are processed when executing FlowAI via the R Application Bridge in FCS Express.
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