“Very nice, and remarkably easy to implement flowClean! [in FCS Express]…the fact that you supplied the proper code ready-made doesn't hurt…..Who knew you already had it implemented so cleanly and with such well-written implementation instructions? ”
- Robert W. Baer, Ph.D.
Professor of Physiology
Kirksville College of Osteopathic Medicine
A T Still University of Health Sciences
FlowClean via R Integration in FCS Express
FlowClean is an algorithm and tool for quality control, designed to identify fluorescence abnormalities caused by fluidic issues at the time of acquisition. FlowClean tracks subset frequency changes within a sample during acquisition, and "flags" time periods with fluorescence perturbations that could possibly lead to the emergence of false populations. The result of the FlowClean R script, when used in FCS Express, is a new parameter, called FCSE_GoodVsBad. The end user can utilize the newly created parameter to easily exclude events that are within aberrant time periods.
The FlowClean algorithm can be run from within the R Integration tools of FCS Express 6 by using an adapted version of the original (flowClean_1.12.0) code. For the sake of simplicity, the original script has been adapted so that a matrix is used as input parameter instead of a standard FCS file while the remaining portions of the code and algorithm are unchanged.
Please see the comments within the script or contact us at firstname.lastname@example.org for further information.
Please Note: The FlowClean R implementation may fail on a wide variety of data files do to the algorithm itself failing. Please use these tools at your own risk.
Installation FlowClean for use with R Integration in FCS Express
Running FlowClean via R Integration in FCS Express
- Installing the required packages
FlowClean makes use of additional R packages that must be installed via R prior to using with FCS Express.
To install the FlowClean package please run the following lines in your R console:
- To check that the installation was performed properly please load the FlowClean package in your R console by running the following lines:
- The following lines should be returned confirming the package was installed properly
If any errors are problems are encountered please contact us at email@example.com.
Loading required package: flowCore
FlowClean can be run in FCS Express 6 via the steps below. Please ensure the R Application Bridge is set to be open in your FCS Express Options
- Open FCS Express.
- Load a data file into the layout and create a plot.
- Create a new Transformation for R Add Parameters (please refer to the FCS Express manual to learn how to create an R Add Parameter transformation).
- Choose the FlowClean_FCSE6.R script as the R Script File in the Transformations dialog.
- Check all of the Fluorescence parameters and the time parameter in the parameter list check box section. Note that Scatter parameters are generally not included.
- Select Events as row in the Events order section of the Transformation dialog.
- Drag the newTransformation from step 3 from the Transformation window onto the plot you inserted in step 2.
The result of the FlowClean algorithm will be now available as an additional parameter called FCSE_GoodVsBad that will allow the discrimination between “Good” and “Bad” events, where “Bad” means perturbations caused by the fluidics. The algorithm will assign a numeric value to each event. When default settings of the script are used, numbers from 1 to 100 are assigned to Good events while numbers from 10000 to 20000 are assigned to Bad events. Once the FCSE_GoodVsBad parameter is available, gates can be drawn around Good events (to include them in the downstream analysis) or around Bad events (to exclude them form the downstream analysis) as depicted below.
However, there are exceptions to this behavior outlined below
|Above is an example of the FCSE_GoodVsBad parameter when viewed in linear (left) and logarithmic (right) scaling. Gates around the "good" and "bad" events have been created.
The user should be aware of the following possible results and errors:
- When no time parameter is available the GoodVsBad parameter will contain only 0 (zero).
- When the number of events is lower than 30000 the GoodVsBad parameter will contain only 0 (zero).
- When the script encounters a fault or error when the FlowClean algorithm fails, no parameter will be created and a warning message will be reported in FCS Express log as seen below
The FCS Express error log will indicate if the algorithm has failed via warning message.