Written by Tim Bushnell, PhD
To reproduce reliably in flow cytometry, one must control the gate.
The identification of the target cells of an experiment is the critical first step to performing the secondary analysis that will be used to judge the biological hypothesis and is done by peeling away the layers of cells that do not meet the criteria.
This involves the data reduction method of ‘gating’ with the researcher as gatekeeper, controlling what may pass and what shall not pass, based on the controls designed for the specific experiment.
It is disappointing to realize that in the paper, Maecker et al., the authors evaluated different models for conducting clinical trials and found that individual labs experienced a ~20% CV in the data analysis whereas a central lab showed only a ~4% variance in data analysis.
One of the best ways to improve gating is to ensure the most appropriate controls are identified and collected in the experiment.
How these controls are used to identify the population of interest is also critical to improving this process. There are 4 common gating controls that can be used for improving gating consistency and reproducibility:
1. Fluorescence Minus One (FMO control).
The term Fluorescence Minus One (FMO) was first introduced in this Cytometry paper in 2001. The FMO control is designed to identify the effects of spectral overlap of fluorochromes into the channel of interest.
This overlap can reduce the sensitivity of measurement in the channel of interest and make identifying the true positive population difficult. The FMO control is performed by staining the cells of interest with all fluorochromes except one. When the data is displayed, the spread of the data in the channel of interest becomes apparent, as shown in the figure below.
Here, human PBMCs were stained with FITC, PE, CY5.5 PE and APC. The left panel shows the unstained sample and the right panel, the fully stained sample. The middle panel shows the PE FMO control.
If the unstained control was used to set positivity, as shown by the red line, it would appear all the cells would be PE positive. However, when the same cells are viewed in the context of the FMO control, it becomes clear that there is spread of the signal, and based on the blue FMO bound line, it is clear these cells are not PE positive.
The FMO control is a valuable control and should be run with all combinations during panel development. Through this development cycle, the researcher will be able to identify the critical FMO controls that are necessary for proper gate placement.
The FMO control is especially essential when attempting to measure rare events, identify emergent markers, or where there is a continuum of expression.
2. Internal Negative Controls (INCs).
Internal Negative Controls (INCs) are those cells in the staining sample that do not express the marker of interest. Unlike the FMO control, where one reagent is left out, the INC is exposed to all the markers, but biologically does not express the marker of interest.
In this case, the INC can help identify and address proper gating when there is non-specific binding of the antibody. This control takes advantage of the fact that we know a bit of the biology of the system and do not expect that the INC cells will bind with the target marker. This, of course, needs to be confirmed in the literature and through experimentation, but leads to a powerful control for proper gate placement.
In this figure, the data on the left comes from the identified INC cells. They are plotted against CD4+, which is our population of interest.
To help set the gate, a quadrant marker can be used to help track the boundary of the INC. As can be seen, the target cells are clearly positive for the marker of interest, and the INC helps ensure we have identified the correct gate.
3. Unstimulated control.
A third control, useful for stimulation experiments, is the unstimulated control that Maecker and Trotter discuss in their paper from 2006.
The unstimulated control again relies on the biology of the system to assist in setting the proper gate. The unstimulated control also takes into account the background binding of the target antibody, since the unstimulated cells should not be expressing the target.
As shown in this figure, there is some background binding of the Activation Maker target on the un-stimulated cells. The FMO (left panel) is used to correct for issues of spectral spreading into the Activation Maker channel, but alone does not allow the proper gate placement. It is only when the FMO is combined with the unstimulated control that the best gate placement identified.
4. Isotype control.
The final control to consider is the isotype control. The concept is that one stains cells with an irrelevant antibody that has the same isotype as the target antibody and labeled with the same fluorochrome. This is supposed to allow for identification of the background binding caused by the specific antibody isotype.
The use of this control remains controversial.
Several papers, such as this one from Keeney et al., call into question the use of isotype controls for setting gates. Maecker and Trotter caution on reliance of the isotype control, and show an excellent figure (Figure 2) where PE-labeled isotype controls show wide variability of staining on small lymphocytes.
When using an isotype control, one makes several assumptions:
- That the affinity of the variable region on the isotype has similar characteristics for secondary targets as the target antibody.
- There are no primary targets for the isotype Ab to bind to (and do you know what the primary target is for the isotype?).
- The fluorochrome to protein (F/P) ratio is the same (and how do you titrate an isotype control?)
We cannot easily know the answer to #1 or #2 and must trust the vendor that the Ab target will not bind to the cells of interest.
Other than with large fluorochromes (PE, APC, etc.), where the F/P is usually 1:1 (due to the size of these fluorochromes), antibodies can have very dramatic optimal F/P ratios for FITC and the Alexa dyes (for example), that have to be optimized out during labelling.
This information therefore has to be collected by the vendor during QC and provided to the customer, something not always readily available on websites.
The isotype control becomes another variable to be tested, validated, and optimized for marginal gain as a gating control. As Maecker and Trotter state,
“…It is thus a hit-or-miss prospect to find an isotype control that truly matches the background staining of a particular test antibody. And, remembering that we are using the isotype control to help us define the true level of background staining, this becomes a circular proposition…”
Where isotype controls can assist researchers is in assessing the success of the blocking of the cells. In this case, if the cells are poorly blocked, the isotype control can reveal that, but should not be used to set gates.
In the continuing efforts to ensure consistent and reproducible data, the proper use of controls to establish the boundaries of gates is critical. With the exception of the isotype control, each of the controls discussed above serve a specific role in that process, and should be part of every experiment. This will help reduce the variability in the data in a given experiment, and when the use is communicated (or demonstrated) in publications, it will assist researchers seeking to reproduce the data in achieving similar results, while helping to reduce data analysis variability between institutions.
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My other passions include grilling, wine tasting, and real food. To be honest, my biggest passion is flow cytometry, which is something that Carol and I share. My personal mission is to make flow cytometry education accessible, relevant, and fun. I’ve had a long history in the field starting all the way back in graduate school.
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