Hematological and biochemical evaluation
All participants underwent complete blood count evaluation using automated cell count (UniCel®DxH 800, Beckman Coulter, Brea, USA), hemoglobin typing by automated capillary electrophoresis analyzer (MINICAP, Sebia, Lisses, France), iron parameters including serum iron (SI), total iron-binding capacity (TIBC) using a fully automated quantitative assay, and serum ferritin by electrochemiluminescence immunoassay (ECLIA or Elecsys® technology, Roche Diagnostics, Penzberg, Germany). The assays of SI, TIBC, and serum ferritin were performed using ROCHE COBAS BIO centrifugal analyzer according to the manufacturer’s instruction.27 Serum hepcidin was determined by a competitive inhibition enzyme-linked immunosorbent assay (cELISA)28,29, with detection ranges of 2.47-200 ng/mL, according to the manufacturer’s instructions (Catalog No. CEb979Hu, Cloud-Clone Corp., Uscn Life Science Inc., Wuhan, China), using afternoon blood sampling to prevent diurnal variation.28,30 In the assay, a monoclonal antibody specific to hepcidin was pre-coated onto a microplate. A competitive inhibition reaction was launched between biotin-labeled hepcidin and unlabeled hepcidin (standards or samples) with the pre-coated antibody specific to hepcidin. After incubation, the unbound conjugate was washed off. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well and incubated. The amount of bound HRP conjugate was reversely proportional to the concentration of hepcidin in the sample. After the addition of the substrate solution, the intensity of color developed was reversely proportional to the concentration of hepcidin in the sample.