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The Most Common Mistake Researchers Make When Designing Flow Cytometry Antibody Panels

Written by Tim Bushnell, PhD

Pairing highly expressed antigens (like CD3) with dimmer fluorochromes, and the antigens of interest with the brightest fluorochromes, is a key part of panel design with few tools to help.

With early generation instruments, this was relatively easy to determine, since fluorochrome choice was limited. With the advent of instruments capable of measuring more than 4 fluorochromes, there is a need to characterize the relative brightness of different fluorochromes under actual experimental conditions, rather than as free fluors.

Bigos et al (2004) first reported this in an abstract and it was later simplified in Maecker et al (2004). This equation (Figure 1) compares the  differences between the means of the positive and negative and corrects this value by dividing by two times the spread (as measured by the standard deviation) of the negative population.

This measure — the Staining Index (or SI) allows for the comparison of the relative brightness of fluorochromes.  As shown in Figure 2 below, if the different fluorochromes are attached to the same antigen, cells can be stained and the SI compared. Researchers who fail to consider SI values are making a significant mistake that can negatively impact their flow cytometry experiments. 

The above calculations reveals that PE > APC > FITC > PerCP   Thus, providing the researcher with critical information necessary to design the polychromatic panel.

There were some limitations to this Staining Index. Most notably was the fact that the data was corrected by the standard deviation of the noise. With many fluorochromes on digital instruments, at least part of the negative signal is background noise the can be attributed in part to the electronic noise in the flow cytometer. A modification of the SI was pulsed by Telford et al. (2009).  The equation for this correction is shown in Figure 3.

In this equation the difference between the medians of the positive and negative populations and divides that by the right hand of the negative distribution, as reflected by the 84% of the negative minus the median of the negative. This Separation Index (or SI) provides a similar metric to the Bigos/Maecker Staining Index as shown in Figure 4.

Be it Staining or Separation, the SI is a critical parameter and the experiment is straightforward, and can be performed using any highly-expressed antigen that is available in many fluorochromes.

Tim Bushnell, PhD

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