What is “Stain Index” and how do I calculate it with FCS Express?

What is the Stain Index?

The Stain Index is a method to help determine the relative intensity for a fluorochrome on a given instrument and is calculated for a fluorescent parameter by subtracting the Median of the negative population from the Median of the positive population, then dividing that quantity by the variance of the negative population, multiplied by 2.

Stain Index = (Median of Positive - Median of Negative) / (SD of Negative * 2)

By determining the relative brightness of a series of fluorochromes, flow cytometry users can simplify the process by which they design an experiment and use the Stain Index metric to match fluorochromes with a higher Stain Index to antigens that are of a lower density or a lower affinity, thus maximizing resolution sensitivity. 

Calculating the Stain Index in FCS Express

With FCS Express, you can easily create your own Stain Index to help optimize your experiment for the best possible results. View our example Stain Index data in FCS Express using the free reader, a demonstration download, or your current FCS Express license. You can also view the layout as a PDF.

Resolution sensitivity (the ability to resolve a dim positive signal from background) depends upon the difference between positive and background peak means and the spread of the background peak. The stain index is a metric that captures both of these factors.

Stain index is easily calculated in FCS Express by using integrated spreadsheets. Simply drag and drop statistics from your plots or tables to enter the positive and negative median values in spreadsheet cells a as well as the negative population standard deviation. Once complete, your vales for Stain Index will update in real time as gates or data change in your analysis. View an example PDF of stain index analysis.

The short webinar below describes the theory behind the Stain Index metric and how to use FCS Express to quickly calculate and report Stain Index.

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