8) Soluble Transferrin Receptor (sTfR) : It is a normal proteolytic cleavage product of transferrin receptor derived from erythroid precursor cells. An increase in serum transferrin receptors is a sensitive response during the early development of iron deficiency. Serum transferrin receptor levels increase progressively as the supply of iron to the tissues become deficient. The advantages are that it does not get affected by infection or inflammatory process; does not vary with age, sex or pregnancy. However, serum transferrin receptor level may be elevated in ineffective erythropoiesis (thalassemia syndrome) (27).

9) TfR-index : Ratio of sTfR to the log of serum ferritin; values > 1.5 suggest iron deficiency alone or in combination with inflammatory conditions. Value < 1.5 is characteristic of anemia of chronic diseases. TfR index is also sensitive enough to detect iron deficiency, before iron deficient erythropoiesis is clinically apparent. Both sTfR and TfR index are decreased in iron overload (28, 29).

10) Erythrocyte protoporphyrin (EPP) : (14) In deficiency EPP increases moderately as there is less iron available to bind to protoporphyrin for conversion to heme. However does not differentiate from chronic inflammatory anemia. EPP shows marked increase in lead poisoning. Lead directly inhibits ferrochelatase the last enzyme in heme biosynthesis. In contrast, in thalassemia less globin chain synthesis, leads to commensurate decrease in protoporphrin synthesis (EPP). Studies in African children showed significant ethnic differences in sTfR and zn-PP(zinc protoporphyrin) levels suggesting separate cut off (30).

11) Iron stores : a) serum ferritin concentration gets altered independently of change in body iron burden. It predicts hepatic iron content to 95%. Unlike serum iron levels, serum ferritin value is not affected by recent iron therapy. However, it is also an acute phase reactant protein that is elevated in response to infection. Therefore, only an unequivocal low ferritin level in CRP negative (< 5 mg/L) patient can be used to make the diagnosis of iron deficiency. In India prevalent infections and protein energy malnutrition limit its use. Conditions like ascorbic acid deficiency, fever, infection/inflammation (chronic inflammation like rheumatoid arthritis), acute or chronic hepatic damage (release of intracellular ferritin), hemolysis and ineffective erythropoiesis (thalassemia major) increase the ferritin levels. The sensitivity of serum ferritin in diagnosis of iron deficiency was 80%, compared to 57% for HyPom and CHr, respectively (69% for both the RBC indices) and 26% to hemoglobin; taking TfR-index as the gold standard. Serum ferritin in relation to iron stores is depicted in Table 1.

Table 1: Serum ferritin in relation to iron stores

	Serum Ferritin in ug/L in children < 5 years	Serum Ferritin in ug/L in children > 5 years
Depleted iron stores	< 12	< 15
Depleted iron stores in presence of infection	< 30	< 30
Severe risk of iron overload	Adult male >200 and female >150

High values of serum ferritin at birth indicate abundant iron stores. Infant, young children and pregnant women (diminishes in late pregnancy, even when bone marrow iron is present) usually have serum ferritin values near or in the range reflective of depletion; however a low level does not imply functional iron deficiency. Further it is of limited value in diagnosis of iron overload and reliance on this modality alone may be faulty.

12) Bone marrow iron : (14) remains the gold standard for diagnosis of iron status. In iron deficiency, marrow hemosiderin is absent; in anemia of chronic disorders iron is always present. In early childhood it may not be reliable as bone marrow stainable iron generally not seen due to rapid growth. Iron stores are greatly increased in thalassemia major and sideroblastic anemia.

13) Hepatic iron : (32) is of value but single needle biopsy may not be of help. This parameter is used in animal experiments to produce iron depletion (33). It is useful in diagnosis of iron overload.