Mk. Powell et al., TITRATION OF A CD45-FITC CONJUGATE TO DETERMINE THE LINEARITY AND DYNAMIC-RANGE OF FLUORESCENCE INTENSITY MEASUREMENTS ON LYMPHOCYTES, Cytometry, 33(2), 1998, pp. 219-224
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).