Titration of a CD45-FITC conjugate to determine the linearity and dynamic range of fluorescence intensity...

To produce biologic calibrators for relative fluorescence intensity (RFI) 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 from twice recommended to a million-fold lower. At the highest concentrations of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentration decreased, the RFI declined in a highly linear relationship between the dilution factor and the histogram channel number. For channel numbers 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 in molecules of equivalent soluble fluorochrome (MESF). Our results suggest that the lower limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provides an adroit way of characterizing the linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular analytes.