FlowClean makes use of additional R packages that must be installed via R prior to using in FCS Express.

• To install the FlowClean package, please run the following lines in your R console:

source("https://bioconductor.org/biocLite.R")
biocLite("flowClean")

• To confirm the installation was performed properly, please load the FlowClean package in your R console by running the following line:

library("flowClean")

• The following lines should be returned, which verifies the package was installed properly:

> library("flowClean")
Loading required package: flowCore
>

If any errors or problems are encountered, please contact us at support@denovosoftware.com.

FlowClean makes use of additional R packages that must be installed via R prior to using in FCS Express.

• To install the FlowClean package, please run the following lines in your R console:

if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")

BiocManager::install("flowClean")

• To confirm the installation was performed properly, please load the FlowClean package in your R console by running the following line:

library("flowClean")

• The following lines should be returned, which verifies the package was installed properly:

> library("flowClean")
Loading required package: flowCore
>

If any errors or problems are encountered, please contact us at support@denovosoftware.com.

FlowClean can be run in FCS Express via the steps below. Please ensure the R Application Bridge is set to be open in your FCS Express Options.

1. Open FCS Express.
2. Load a data file into the layout and create a plot.
3. 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).
4. Choose the FlowClean_FCSE.R script as the R Script File in the Transformations dialog.
5. 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.
6. Select Events as row in the Events order section of the Transformations dialog.
7. Drag the new Transformation from step 3 from the Transformation dialog 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 from the downstream analysis) as depicted below:

However, there are exceptions to this behavior outlined in the following section.

1. When no time parameter is available, the GoodVsBad parameter will contain only 0 (zero).
2. When the number of events is lower than 30000, the GoodVsBad parameter will contain only 0 (zero).
3. When the script encounters a fault or error due to the FlowClean algorithm failing, no parameter will be created and a warning message will be reported in FCS Express.  See example below: