Quantitative Fluorescence Measurements with Multicolor Flow Cytometry

  • Lili Wang
  • Adolfas K. Gaigalas
  • Ming Yan
Part of the Methods in Molecular Biology book series (MIMB, volume 699)


Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, ­monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure includes the following critical steps (a) quality control (QC) of the multicolor flow cytometer, (b) fluorescence calibration using hard dyed microspheres assigned with fluorescence intensity values in equivalent number of reference fluorophores (ERF), (c) compensation for correction of fluorescence spillover, and (d) application of a biological reference standard for translating the ERF scale to the ABC scale. The chapter also points out current efforts for implementing quantification of biomarkers in a manner which is independent of instrument platforms and reagent differences.

Key words

Multicolor flow cytometry Fluorescence calibration Equivalent number of reference fluorophores CD4+ lymphocytes Antibody bound per cell CS&T microspheres Instrument quality control Compensation 



We are indebted to Dr. Gerald Marti and Fatima Abbasi at Center for Biologics Evaluation and Research, FDA and Robert Hoffman at BD biosciences for their collaborative effort on quantitative multicolor flow cytometer measurements over the years.


Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Lili Wang
    • 1
  • Adolfas K. Gaigalas
    • 1
  • Ming Yan
    • 2
  1. 1.National Institute of Standards and TechnologyGaithersburgUSA
  2. 2.BD BiosciencesSan JoseUSA

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