Comparison of standard curves performed in serum-free buffer or in 1:20 dilution of sera from 6 iron-deficient patients. On 3 different days (red, blue, and black lines), standard curves were performed in buffer (solid line) and in sera from 2 of the 6 patients (dotted and dashed lines). The OD values for all plots were normalized by dividing with the “no hepcidin” buffer OD for that day. The differences between serum and paired buffer OD ranged from −1% plus or minus 4% to −13% plus or minus 8%. The points were fitted with a sigmoidal logistic 4-parameter curve optimized through 200 iterations (Sigmaplot; Systat Software, San Jose, CA).