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.