Calibration of iron concentration in the presence of normal and pathologic sera.

A series of concentrations of iron, ranging from 0 to 50 μmol/L, was prepared as described in “Materials and methods.” A 20-μL sample from each iron solution was mixed with 20 μL of either 10 mg/mL BSA in HBS buffer (BSA; empty triangles) or sera from a healthy individual (Normal, empty squares), a patient with end-stage renal disease (ESRD, filled squares), a thalassemic patient (Thalassemic; filled triangles), and a hemochromatosis patient (HC; filled circles), and incubated for 20 minutes. To each mixture was then added 210 μL oxalate-Mn-Fe reagent (200 mmol/L Na-oxalate, 20 mmol/L MnCl₂, 1 μmol/L FeCl₃) to give a final volume of 250 μL; aliquots of 100 μL were transferred to DFO-coated plates and processed as before. The fluorescence was read after 2 hours of incubation with CA-Fe and plotted semilogarithmically against the concentration of iron in the original 20-μL input sample.