TABLE 1.

Diagnostic markers' status as reviewed for clinical iron deficiency in anaemic patients ¹¹ , ⁴⁵ , ⁴⁶ , ⁴⁷ , ⁴⁸ , ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ , ⁵⁶ , ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² , ⁶³ , ⁶⁴ , ⁶⁵ , ⁶⁶

Diagnostic variables	Significance in body iron detection	Advantages	Limitations
Serum ferritin	Iron storage marker and an acute phase reactant. Measures stored iron in liver and spleen.	Inversely correlates with iron assimilation in erythrocytes, cut‐offs for which, vary considerably among experts. Limited evidence suggests that a serum ferritin > and < than 15 μg/L marks high and low iron stores, respectively in patients aged over 5 years.	Sensitive only until body iron stores are depleted and is significantly influenced by infection and inflammation. May be independent of changes in iron stores, thus challenging the interpretation of infection‐driven body iron index. False normal levels may appear in cancer and other chronic conditions.
Soluble transferrin receptor (sTfR)	Regulates Fe‐Tf uptake by erythrocytes. Measures biologically available iron.	Marks the magnitude of erythropoiesis and iron requirement in the body. sTfR levels increase with depletion of iron reserves in IDA, indicating severe iron deficit, therefore, applicable for FID estimation, post iron store depletion.	Remains a critical erythropoietic determinant. sTfR‐logarithmic ferritin ratio opted as a promising index to determine changes in both iron stores and functional iron. The sTfR‐ferritin determination, however, includes high cost and lack of normalisation.
Transferrin saturation (TSAT)	TSAT < and > 20% indicate iron deficiency and overload, respectively. Measures the number of iron‐binding sites on Tf‐Fe.	May be applied to uninflamed patients to identify a normal iron supply to tissues and bone marrow.	Remains an older diagnostic tool, due to its sensitivity to inflammation.
Hepcidin	Hepatic peptide which acts by binding to FPN. High plasma hepcidin indicates iron‐restricted anaemia, including anaemia associated with inflammation, CKD and metastasis in cancers. Deficient hepcidin leads to iron overload conditions in HH, and inefficient erythropoiesis. Systemic hepcidin production is transcriptionally coordinated by plasma iron content, leading to its overproduction in conditions of iron abundance.	Preferred in clinical applications for erythrocytic iron assimilation.	Greatly affected by a range of stimuli, such as hypoxia and/or inflammation, thus making it a dynamic component.
Total iron‐binding capacity (TIBC)	Measures serum Tf (apo, mono and diferric) responsible for iron transport to RBCs or body stores.	Tool in immunological assays used for iron status determination.	May appear as false deficient in conditions of coexisting acute and chronic infections. False normal levels may appear due to hypoproteinaemia.
Gamma‐glutamyl transpeptidase/ gamma‐glutamyltransferase (GGT)	A liver enzyme involved in glutathione metabolism and transport of amino acids and peptides. Correlates with iron toxicity, disease risk and mortality.	Early predictive marker for a multitude of chronic diseases.	Significantly altered in a wide range of diseases and dependent on several iron markers, hence cannot be used as a single‐point measurement.
Complete blood count (CBC)	Measures RBC, white blood cells (WBC), platelet count, Hb and haematocrit (Hct). Also includes mean corpuscular volume (MCV) as a characteristic to RBC size, mean corpuscular Hb concentration (MCHC), indicating Hb amount per RBC.	Evaluates overall health and underlying diseases. MCV determines the underlying aetiology of specific anaemia type.	Individually unable to assess acute changes in iron availability, secondary to ESA therapy. Iron replenishment from RBC pool gets significantly compromised causing suboptimal iron supply to erythroid marrow due to iron store depletion. Inefficient iron supply causes impairment of Hb production, thus leading to low MCHC detectable post weeks of Hb decline.
Serum erythropoietin concentration	Principal regulator of RBC production. Correlates with body iron, serum unsaturated iron‐ binding capacity and ferritin in haemodialysis patients	Identifies the underlying causes of blood conditions.	Iron or vitamin deficiencies in patients may interfere with results.
Percentage of hypochromic red cells (% HRC)	Provides an accurate determination of FID and latent iron deficiency (LID) through the Hb production by RBCs and reticulocytes.	A long‐life span of mature circulating RBCs allows iron status of RBCs. Iron‐restricted erythropoiesis can be determined by %HRC in ACD patients.	Analysis must be done in a fresh sample to avoid errors resulting from RBC swelling.
Reticulocyte haemoglobin content (CHr)	Predicts FID in patients undergoing ESA therapy, determining response to intravenous iron administration in CKD patients, independent of acute phase.	Cost‐effective diagnostic factor for early detection of iron availability for erythropoiesis alongside quality assessment of reticulocytes.	Lacks consistency in cut‐off values determining outcomes in patients, therefore requiring further investigation.
RBC protoporphyrin	Measures compromised iron supply in peripheral RBCs.	Reflects iron supply during RBC production, derived through its accumulation in RBC precursors as a response to low iron.	Accumulation of its levels are also recorded for lead poisoning, hence requires exclusion to lead exposure. Its levels are also raised only several weeks post low‐iron erythropoiesis.
Zinc protoporphyrin	Stable screening marker for iron deficiency measured through systemic iron supply to bone marrow erythrocytes.	Instant, sensitive and cost‐effective measurement and can be used in combination with haemoglobin concentrations for assessment of iron status in population‐based studies.	Its evaluation in iron‐deficiency associated with certain chronic diseases such as uraemia is mainly derived from cross‐sectional studies, and hence, may produce conflicting results, therefore failing to validate its significance as a determinant in such chronic conditions.
Bone marrow aspiration with iron staining	Evaluative measure for elimination of condition of IDA rather than a diagnostic factor.	Used as a confirmatory test in conditions where low TSAT and ferritin levels are at borderline levels associated with disorders other than IDA	Invasive, and decreased predictive accuracy from bone marrow samples with low or unavailable iron in bone marrow samples.

Abbreviations: ACD, Anaemia of chronic diseases; CKD, chronic kidney disease; CBC, complete blood count; ESA, erythropoiesis‐stimulating agent; FPN, ferroportin; FID, functional iron deficiency; GGT, gamma‐glutamyl transpeptidase/gamma‐glutamyltransferase; Ht, haematocrit; HH, hereditary hemochromatosis; IDA, iron deficiency anaemia; LID, latent iron deficiency; MCHC, mean corpuscular Hb concentration; MCV, mean corpuscular volume; % HRC, percentage of hypochromic red cells; RBCs, red blood cells; CHr, reticulocyte haemoglobin content; sTfR, soluble transferrin receptor; TIBC, total iron‐binding capacity; Tf, transferrin; TSAT, transferrin saturation; WBC, white blood cells.