title: Model system evaluating fluorescein-labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers.
abstract: Fluorescence intensity calibration was evaluated in a model system for flow cytometers using commercially available fluorescein-labeled microbeads as internal standards and stabilized fluoresceinated thymus cell nuclei (Fluorotrol) as surrogates for stained mononuclear cells. Spectrophotometrically determined calibration values for the microbeads were used to generate a standard curve that converted green fluorescence histogram channels into molecular equivalents of soluble fluorescein (MESF). In 19 analyses repeated during a single run, the coefficients of variation (CVs) for the derived MESF values on both dimly and brightly stained Fluorotrol populations were less than 2%. In 26 separate determinations over 14 weeks, the CVs of the derived MESF values were less than 3%. The MESF values of the dim and bright Fluorotrol populations derived from the microbead standard curves were both about 50% lower than those determined by direct spectrophotometric analysis of Fluorotrol. The analytical imprecision of fluorescence intensity measurements in this idealized model system has a CV less than 3%, and the analytical inaccuracy shows that calibration in MESF units remains uncertain over about a two-fold range.
authors.affiliation: Division of Environmental Health Laboratory Sciences Centers for Disease Control, Atlanta, Georgia 30333.
authors.forename: R F
authors.initials: RF
authors.lastname: Vogt
authors.forename: G D
authors.initials: GD
authors.lastname: Cross
authors.forename: L O
authors.initials: LO
authors.lastname: Henderson
authors.forename: D L
authors.initials: DL
authors.lastname: Phillips
mesh_terms: D000818:Animals; D002138:Calibration; D002417:Cattle; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008863:Microspheres; D013950:Thymus Gland / Q000166:cytology*; D014894:Weights and Measures
chemical_list: D005456:Fluorescent Dyes