I am trying to optimize my flow experiments by titrating my antibodies down. I found the formula to calculate the Separation Index (SI) in this article (http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.20790/abstract) but I don't understand how to use it. The formula to calculate SI is:
(median positive signal - median background signal) / ((84th percentile median background signal - median background signal) / 0:995)
Wherein signal is the Median Fluorescent Intensity (MFI).
The problem I am having is that I dont know how to know what my 84th percentile median background signal is. I am using Venturi software to analyse my data, but Venturi only provides the mean, median, Var, SD and %CV.
Does somebody has experience with the SI, and knows how to calculate the 84th percentile? I would really appreciate some help :) Thanks, Leroy
Hi Leroy,
The MFI (median fluorescence intensity) is the median value of fluorescence for a given fluorescence parameter. For example, if I have cells stained with a FITC conjugated antibody, my FITC+ cells may have an MFI of 10^4, whereas my negative might have an MFI of 10^2. All of the values for this (or other calculations below) are values that relate to the fluorescence measurements (median fluorescence, SD of fluorescence, etc).
In most of the metrics that are designed to show the "separation" of a stained population, the difference between the median positive and negative signal is divided by a value that represent the spread of the negative population. The "separation index" uses the "((84th percentile median background signal - median background signal) / 0:995)" component to calculate rSD (robust standard deviation) as the denominator (the value under the division line). Other methods such as the "staining index" simply use 2xSD as the denominator.
There are two options here:
a) I'm not familiar with using Venturi, but if it can't give you percentile values then you can use another software package that does. Alternatively, you could even export your data from Venturi (your gated positive and negative populations) and look at in excel or a statistical program like 'R'.
b) use the "staining index" approach instead, as the values needed for this calculation are evidently available in your Venturi software. I have detailed the equation for "staining index" below, and a link to a brief animated page on how it is calculated.
Staining index:
MFI(positive population) - MFI(negative population) / 2xSD(negative population)
MFI = median fluorescence intensity
SD = standard deviation
This is the MFI and SD of the relevant fluorescence parameter you are investigating. If you are titrating a FITC conjugated antibody, then you are looking at the MFI of FITC signal on the positive and negative population, and the SD of FITC on the negative population.
http://www.biolegend.com/newsdetail/1245/
Hope this helps.
Thanks for your in dept answer Thomas!
While I was waiting to figure out how to do calculate the 84th percentile I worked on calculating the stain index for my antibodies just like you said.
I will contact Venturi in the near future and let you know if there is an option do 84th percentile calculations in their software. This might be interesting for other people to know as well.
Thomas Myles Ashhurst, If I want to stain IgM cells, in this case, gating should be only on a lymphocyte population or the whole PI negative population?
Thomas Myles Ashhurst Could you explain me the difference between separation index to staining index?
Thank you.
Amanda Rocha Staining Index in flow cytometry generally refers to a metric that evaluates the separation between positive and negative populations based on fluorescence intensity.
Separation Index, when used in the context of flow cytometry, may also refer to a parameter that assesses the distinction between positive and negative populations. It typically involves comparing the mean fluorescence intensity of positive and negative populations, considering the spread or standard deviation of the negative population.
In summary, while the Staining Index focuses more on the intensity of staining through different fluorescents, the Separation Index considers both intensity and the ability to differentiate between stained and unstained components in a sample e.g. in the context of Ab titration. Both indices contribute to the overall assessment of the fluorescence staining quality in biological samples.
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