Do you analyze a lot of samples? If so, data quality control may be challenging, especially when a large number of parameters is measured. In particular, fluorescence measurements for a sample over the collection time may not remain stable due to fluctuations in fluid dynamics. As many as 13.7% of publicly available FCS files have been shown to have this problem. But don’t worry, we are here to help!
As you may know, our latest release, FlowJo v10.2, contains new and improved architecture for plugins. One of our featured plugins—FlowClean—has been designed to address exactly this issue. It automatically identifies and flags fluorescence anomalies in your FCS files by tracking cell populations in the centered log ratio space. This has been shown to provide a sensitive and consistent method of quality control. Do you want to give it a try?
How to get started? First, you want to make sure to have our latest FlowJo, v10.2. Second, because the plugin relies on functionality developed in R as part of the FlowClean BioConductor library, you need to install a recent version of R. R is freely available for Windows, Mac OS X, and Linux. Please review our last week’s article on Tips for Troubleshooting Plugins and R. You will need to install the flowCore and flowClean libraries within your R installation. Generally, this can be done by copying and pasting the following three lines to your R console:
source("https://bioconductor.org/biocLite.R")
biocLite("flowCore")
biocLite("flowClean")
Finally, you will need the FlowClean FlowJo plugin; this is a FlowClean.jar file that you can download from GitHub and save to the plugins directory of your FlowJo installation. Once you have done that, the plugin will be detected the next time you restart FlowJo.
With one or more samples selected, navigate to the FlowClean menu item located under Plugins on the Workspace ribbon, as shown in the figure below.
Figure 1. The FlowJo Plugins menu, showing FlowClean installed.
This will open a dialog with FlowClean input parameters where you should select FCS channels that you want FlowClean to inspect.
Figure 2. FlowClean parameters.
Selecting all fluorescence channels provides good results in most cases. If you see both, compensated and uncompensated versions of the fluorescence channels then we recommend selecting the uncompensated data. The other input parameters have reasonable default values, which you don’t need to alter unless you are not satisfied with FlowClean’s results on your sample. Details about how these values affect the results are included in the flowClean package manual with additional explanation in Fletez-Brant, et al., Cytometry A, 2016. Also, since FlowClean is based on reviewing fluorescence expression values over the sample collection time, the Time channel needs to be present in the input FCS file. This channel will be used automatically even if you do not select it.
Depending on the size of your FCS file, the plugin should take from a few seconds to a few minutes to complete the sample evaluation. Results will appear as one or two clearly distinct cell populations. Specifically, a single population will be returned if no problems were identified, and two populations will be returned if FlowClean identifies fluorescence anomalies in your data. The population with FlowClean results around 6,000 is the population of events with fluorescence anomalies that you want to gate out, the other population with FlowClean peak around 2,000 is the one that you want to keep for further analysis.
Figure 3. FlowClean results, showing the two populations.
In order to manually review the results of the plugin, we suggest plotting time vs. fluorescence as shown in the example below. Although FlowClean with the default parameter settings seem to have worked well for most researchers, you can use these types of plots to determine optimal settings for your specific dataset.
Figure 4. FlowClean results, showing the ungated population of all events on the left, and the automatically gated “clean” population on the right.
Normally, for each fluorescence channel, one would expect the fluorescence measurements for a sample to remain stable over the data collection time. However, fluctuations in fluid dynamics and other factors may lead to instabilities and to the emergence of false populations. When FlowClean identifies such anomalies, there should be one or more fluorescence channels showing a non-stable behavior when plotted against time. For demonstration purposes, we picked an example where this is clearly apparent. Please note fluctuations in your data may not be as clearly visible as in our example.
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