The ideal screening test would be capable of identifying ID in the absence of anemia. This would help in the treatment of ID in the pre-anemic stage, preventing IDA and it's associated mental, motor and behavior effects. Such test is not widely used at this time .
In our study, urinary hepcidin levels were significantly lower in all stages of ID than in control group, more significant reduction in its level was observed with the progress in severity of ID. That coincided with Cherian et al et al  who found that urinary hepcidin levels were significantly lower in ID and IDA. Hepcidin is homeostatically regulated by iron and erythropoietic activity. Hepcidin is suppressed in ID, allowing increased absorption of dietary iron and replenishment of iron stores . The feedback loop between iron and hepcidin ensures stability of plasma iron concentrations . Hepcidin production is also regulated by the process which consumes most iron, erythropoiesis . Increased erythropoietic activity suppresses hepcidin production which allows the release of stored iron from macrophages and hepatocytes, and increased iron absorption, all resulting in greater supply of iron for hemoglobin synthesis .
In our study, we determined three cutoff points for urinary hepcidin level to differentiate ID in its different stages (stage-1, stage-2 and stage-3) from healthy children. These three cutoff points had strong confidence intervals and valuable predictive potentials.
Guyatt et al  calculated the predictive value and area under the receiver operating characteristic (ROC) curve for serum ferritin in detection of IDA. Area under the receiver operating characteristic (ROC) was 0.95 (p < 0.001), compared to 0.77 for MCV, 0.74 for transferrin saturation, and 0.62 for absolute red cell distribution wideness (RDW).
In our study, urinary hepcidin levels at cutoff point ≤0.08 nmol/mmol Cr could predict ID stage-3 with Sensitivity 96% and specificity 100%. Furthers, urinary hepcidin levels at cutoff point ≤0.42 nmol/mmol Cr could predict ID stage-2 with sensitivity 96% and specificity 92%.
There is a shortage of iron available to the erythroid precursors in the bone marrow for hemoglobin synthesis in the second stage of ID . The second stage may be characterized by abnormalities in particular iron parameters, including low Tsat and elevation in ZnPP level. Hemoglobin levels may be reduced but the resulting mild anemia may not be detectable using normal cutoff values for hemoglobin. Iron deficient erythropoiesis may be undetectable by using traditional laboratory parameters. In iron deficient erythropoiesis (second stage ID), storage iron may be normal or even increased due to impaired release of iron into the circulation .
In our study, urinary hepcidin levels at cutoff point ≤0.94 nmol/mmol Cr could predict ID stage-1 with sensitivity 88% and specificity 88%.
Beutler et al  stated that there is no overt effect on erythropoiesis in the first stage of ID; blood hemoglobin levels are usually normal, and ID generally can escape detection by hemoglobin or hematocrit screening. We obtained these relatively low values due to presence of three false positive cases and three false negative cases at the cutoff level ≤0.94 nmol/mmol Cr. We could not exactly explain if this result was due to fallacies in ferritin assay, which might be associated with missed cases (false negative) in control group and over estimation (false positive) in ID stage-1 group, or it might be related to limitation in hepcidin at this relatively high (≤0.94 nmol/mmol Cr) cutoff level. An explanation for our findings may stem from the elucidation of Kis et al  in a retrospective study of 101 patients, who had undergone bone marrow aspiration, as they found that a ferritin of ≤100 μg/l had 64.9% sensitivity and 96.1% specificity for IDA. It is noteworthy that ferritin level increases with age, and is an acute-phase reactant that may be falsely elevated in the setting of chronic inflammation, infection, malignancy and chronic renal failure [23, 31]. In this situation, performing bone marrow aspiration may provide more explanation about this finding through estimation of stainable tissue iron.
In our study, urinary levels of hepcidin showed significant positive correlation with Hb, MCV, MCHC, hematocrit value, serum iron level, ferritin level and Tsat (P < 0.01). On the other hand urinary levels of hepcidin showed significant negative correlation with serum transferrin and TIBC (P < 0.01).
That agreed with the study which carried by Cherian et al , they demonstrated that hepcidin was positively associated with hemoglobin, MCV, iron, ferritin and Tsat levels. In contrary, hepcidin was negatively associated with transferrin.
One of our limitations in this study was the small number of cases as we tried to select demographic matched groups. To the best of our knowledge, this was the first trial to determine cutoff level for hepcidin in diagnosis of iron deficiency.