TITRATION OF A CD45-FITC CONJUGATE TO DETERMINE THE LINEARITY AND DYNAMIC-RANGE OF FLUORESCENCE INTENSITY MEASUREMENTS ON LYMPHOCYTES

To produce biologic calibrators for relative fluorescence intensity (R FI) measurements, we stained leukocytes with serial dilutions of CD45- FITC conjugate and processed them using our regular whole blood lysis procedure. Cells were stained with conjugate concentrations ranging fr om twice recommended to a million-fold lower At the highest concentrat ions of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentra tion decreased, the RFI declined in a highly linear relationship betwe en the dilution factor and the histogram channel number. For channel n umbers corresponding to the lowest percentiles of the RFI distribution , Linearity persisted down to the first half decade. The slope of this relationship revealed a true dynamic range of 4.5 decades, which was comparable to the value obtained with microbead standards calibrated i n molecules of equivalent soluble fluorochrome (MESF). Our results sug gest that the lower Limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 ME SF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provi des an adroit way of characterizing the Linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly th e same matrix, stains, and preparation methods as those used for cellu lar analytes, (C) 1998 Wiley-Liss, Inc.(dagger).