Calibration of Fe concentration versus fluorescence.

A series of concentrations of Fe, ranging from 0 to 200 μM (described in “Patients, materials, and methods”), was prepared by serial dilution of Fe:NTA in HBS buffer (input sample). From each dilution 20 μL was transferred to 96-well plates followed by 100 μL 2.5 μM Fl-DFO in HBS. Replicate wells were treated with the same reagents containing in addition 100 μM DFO, to give the “maximal” fluorescence for each sample. The fluorescence ratio (expressed as “% of maximal value” obtained in the presence of excess DFO) was calculated for each sample (see “Patients, materials, and methods”) and plotted semilogarithmically against the concentration of Fe in the original 20-μL input sample (A). The linear region of the calibration curve (0-6.25 μM Fe) is shown in panel B. The values are averages ± SD of 6 separate calibration curves. The boxed area labeled “48 control sera” represents the range of values obtained for serum samples of 48 individuals without Fe overload.