Flow Cytometry Protocols pp 53-65
Quantitative Fluorescence Measurements with Multicolor Flow Cytometry
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 wordsMulticolor flow cytometry Fluorescence calibration Equivalent number of reference fluorophores CD4+ lymphocytes Antibody bound per cell CS&T microspheres Instrument quality control Compensation
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