Separation Index: An Easy‐to‐Use Metric for Evaluation of Different Configurations on the Same Flow Cytometer

Martin Bigos1

1 Gladstone Institute of Virology and Immunology, San Francisco, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.21
DOI:  10.1002/0471142956.cy0121s40
Online Posting Date:  April, 2007
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Abstract

A quick and understandable method, using material readily available in a biology laboratory, for evaluating the measurement sensitivity of flow cytometers is presented in detail. Two examples of its use, in determining an optimal emission filter and in evaluating compensation, are also presented.

Keywords: configuration; filters; sensitivity; evaluation

     
 
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Table of Contents

  • Figures
     
 
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Materials

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Figures

  •   FigureFigure 1.21.1 Synthetic Gaussian distributions are displayed as histograms on a logarithmic intensity scale typical of much cytometric data. The blue distribution models autofluorescent objects and the red distribution models the stained objects. A is the median of the autofluorescent, B is the 95th percentile of that distribution. D is the median of the stained distribution and C is the 5th percentile of that distribution.
  •   FigureFigure 1.21.2 Same synthetic data as in Figure , with different intensity positions of the stained distribution. The corresponding SI values are shown.
  •   FigureFigure 1.21.3 FITC emission spectrum (partial) with various filter options. The emission of FITC is shown between 480 nm and 560 nm. The 532‐nm laser line is shown in yellow. The 515/20 bandpass filter is shown in gray. The 532‐nm notch filter is shown in purple. The 525/50 bandpass filter is shown in pink.
  •   FigureFigure 1.21.4 Results of LSR SI measurements. The standard, proposed, and optimal configurations for detecting FITC in the presence of a 532‐mm laser are shown left to right.
  •   FigureFigure 1.21.5 Effects of compensation on SI. In both panels all three populations are BD capture beads. Population 1 has no staining, population 2 is stained with CD4 PE‐TR, and population 3 is stained with CD28 PE. Panel A has no compensation applied, panel B is compensated for the PE spillover into PE‐TR. The SI in panel A, using populations 1 and 2, is 5.9. The SI in panel B, using populations 3 and 2, is 4.3.

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Literature Cited

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