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Stain Index Calculations using Flow Cytometry Software

The Stain Index (SI)1 is a normalized signal to background metric useful in quantitating the brightness of immunofluorescent reagents on cells or capture beads, and uses percentiles, the 50th percentile being the median. The idea is to normalize the distance between the positives and negatives by the width of the negative distribution as an expression index. The Stain Index is basically D/W, as shown below, where D is the difference between positive and negative and W may be the width for unstained cells, negative stained cells, or a compensated population from another dye dimension.

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Calculating SI in FlowJo

The Stain Index can be calculated in FlowJo using the percentile function in FlowJo for the 5th, 50th, and 95th percentiles (percentile is "%ile" in the FlowJo dialog).

Selecting %iles in FlowJo


Calculating SI in FACSDiVa™

FACSDiVa™ does not currently have any variable percentiles function implemented to generate printed output for percentile metrics other than the median. As of version 6.0, however, some relevant robust (nonparametric) statistics will be available. The robust standard deviation (rSD) is calculated in a nonparametric manner, and may be used with the median to estimate a nonparametric equivalent to the percentile method for the Stain Index:

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Obtaining the median and rSD in FACSDiVa™6.0

After creating a statistics window for a particular set of data, by selecting the "Edit Statistics View…" item in the popup menu from a statistics window, one may select median and rSD to be included for output:


This provides an acceptable method to calculate the Stain Index using FACSDiVa™ 6.0 that makes no assumptions as to whether or not the populations are normally distributed.

1 Presented by Dave Parks of Stanford at ISAC conference in Montpellier, France, 2004.

This document was adapted from Joe Trotter's (April, 2007) and reviewed by Dave Parks.
-March, 2014