Anemia, defined by WHO criteria as Hb < 13 g/dL in males, < 12 g/ dL in females, and < 11 g/dL in pregnant women, is absent in isolated iron deficiency; when present it may be mild, moderate, or severe. The absolute reticulocyte count is low, consistent with decreased erythrocyte production.

Circulating ferritin is an indirect estimate of body iron stores; it is an important tool to evaluate iron disorders in general, and especially iron deficiency. In the absence of inflammation serum ferritin <30  μg/L appears to be the most reliable and cost-effective test for the diagnosis of iron deficiency. The cut-off of 30 μg/L has 92% sensitivity and 83% specificity, as established comparing ferritin levels with bone marrow Perls’ staining (Fig. 1), the recognized gold standard for iron deficiency, although these results were obtained in a small study. Isolated iron deficiency without anemia is defined by serum ferritin <30 μg/L and normal Hb levels. Serum iron is reduced, while total iron binding capacity (TIBC), which measures the functional capacity of transferrin as iron carrier, or transferrin levels are increased, resulting in decreased transferrin saturation (the ratio of iron on TIBC). However, as discussed below transferrin saturation may be low also in anemia of inflammation. Thus for a precise evaluation of an individual case and for the differential diagnosis with other conditions characterized by altered iron parameters it is important to assess both serum ferritin and transferrin saturation.

Fig1. ASSESSMENT OF IRON STORES ON A BONE MARROW ASPIRATE. Iron stores are better assessed on the aspirate as opposed to the biopsy because the decalcification procedure required for processing the biopsy leaches out the iron and can lead to a false conclusion of absent stores. On the aspirate, a Prussian blue stain evaluates iron. This can demonstrate iron stores (blue reaction product), particularly in the cytoplasm of macrophages and histiocytes (A, B). Iron can also be seen in the cytoplasm of some erythroblasts (tiny blue cytoplasmic specks), which would allow these cells to be designated sideroblasts (C). These are in contrast to red blood cell precursors with abnormal mitochondria iron accumulation around the nucleus, or “ring sideroblasts” (C, inset).

When anemia develops serum iron and transferrin saturation are even lower than in isolated iron deficiency. Usually transferrin saturation is <16 % and serum ferritin <12 μg/L. However, the use of ferritin <30 μg/L as the lower limit of normal captures most cases with the exception of some situations in the elderly. Thresholds of serum ferritin to diagnose iron deficiency in the elderly should be higher, considering concomitant morbidities and inflammation.

Erythrocyte morphology on peripheral blood smear inspection is abnormal, with small, hypochromic erythrocytes showing the pale central area larger than usual (see Fig. 2). Automated counters better assess microcytosis and hypochromia by providing mean MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), and MCHC (mean corpuscular hemoglobin concentration) values. Erythrocyte indexes are not altered in all cases. Reduction of MCV (<80 fl), MCH (<27 pg), MCHC (<32 g/L) and increased hypo chromic red cells (HYPO, with MCH < 28 pg) are relatively late changes compared to iron parameters, due to the long erythrocyte lifespan. The same applies to the increased red cell distribution width (RDW), an index of anisocytosis, indicating the presence of erythrocytes of different size (Table 1). Iron deficiency anemia of recent onset is usually normocytic and normochromic and only Hb content of reticulocytes (CHr) may be reduced (<27 pg). This early indicator of iron deficiency may signal the need of iron supplementation after treatment with ESA, while an early increase in CHr (day 4) indicates a positive response to iron treatment. The percentage of circulating hypochromic red cells (>6%) is a sensitive marker of iron deficiency in patients with chronic kidney disease; experience in other conditions is limited. Erythrocyte indexes can be deceitfully normal in case of combined iron and vitamin B12 or folic acid deficiency.

Fig2. IRON DEFICIENCY ANEMIA. Peripheral blood smear (A, B), bone marrow (BM) aspirate (C), and Prussian blue stain of BM aspirate (D) from a 16-year-old girl with hemoglobin 6.7 g/dL, hematocrit 22.6%, and mean corpuscular volume 59.2 fL. Peripheral smear shows hypochromic microcytic red blood cells (A), with widening of the central pallor and “pencil” cells (B). Polychromatophilic erythroid precursors in the aspirated specimen have scanty cytoplasm that is irregular and vacuolated (C). The Prussian blue-stained aspirate shows no iron stores in multiple spicules (D).

Table1. Laboratory Tests for Differential Diagnosis of Iron Deficiency Anemia

In all cases of iron deficiency, zinc is preferentially chelated into protoporphyrin-IX (PPF). Elevated ZnPPF levels (>80 μg/dL) persist throughout the lifespan of erythrocytes, providing a screening test for iron deficiency. However, ZnPPF levels may be elevated in other conditions of low iron utilization, such as anemia of inflammation, hemolytic and sideroblastic anemia, and in chronic lead and other heavy metal poisoning.

Iron deficiency increases transferrin receptor (TFR) synthesis in all cells. However, when TFR is not stabilized by its ligand because diferric transferrin is low/absent, its extracellular domain is shed from the membrane and released into the circulation as a soluble component (sTFR). Erythroid precursors that express the largest number of receptors are the major contributors to sTFR concentration. Although its function remains unknown, serum sTFR levels measured by ELISA provide a sensitive and quantitative measure of iron deficiency (see Table 1) in the absence of erythroid hyperplasia. Unfortunately the use of this test is limited by the scarce availability and the absence of standardization and harmonization in different laboratories.

The diagnosis of iron deficiency in the setting of chronic inflammatory disorders is challenging since ferritin may increase as an acute phase protein. No specific test assesses tissue iron deficiency when ferritin levels are unreliable. It is generally agreed that iron deficiency can be ruled out only when ferritin levels are >100 μg/L; a higher cut-off to diagnose iron deficiency ( <300 μg/L) is accepted in special conditions, such as chronic heart failure, provided that transferrin saturation is low (<20 %). Inflammation is characterized by hypoferremia, hyperferritinemia, and normal/decreased levels of sTFR (see Table 1). The shedding of the receptor persists at an elevated level when inflammation is associated with iron deficiency. The discrepancy between levels of sTFR (high in iron deficiency and normal/ low in inflammation) and serum ferritin (low in iron deficiency and normal/high in inflammation) can be utilized to calculate the ratio between sTFR and log-transformed serum ferritin. This ratio is 1 when iron is normal, <1 in anemia of inflammation, and >2 in iron deficiency and in anemia of mixed type due to both iron deficiency and inflammation (see Table 1), representing a valuable diagnostic tool. Unfortunately, the reported limitations with regard to standardization present an obstacle to routine use.

Prussian blue (Perls’) staining directly assesses iron stores revealing the presence/absence of iron in bone marrow macrophages (see Fig. 1) and offering a semi-quantitative grading of iron. Absence of bone marrow iron at Perls’ staining is still considered the reference test for iron deficiency. However, the test is rarely used nowadays, unless other disorders that require bone marrow aspiration are suspected. Hepcidin levels are low/undetectable in absolute iron deficiency, except in patients with genetic IRIDA. The latter have low transferrin saturation, strikingly decreased MCV and MCH, and normal/ high hepcidin and ferritin levels. Serum hepcidin levels are high in inflammation as are serum ferritin and C-reactive protein (CRP). Whether the hepcidin concentration may help in assessing mixed anemia (due to both iron deficiency and inflammation) is still under investigation. Table 1 reports the results of tests currently used to differentiate iron deficiency from other conditions characterized by altered iron parameters. In individual patients it is important to evaluate the global clinical and laboratory picture rather than relying on single tests. The efficiency of iron absorption might be tested by an oral iron challenge. Finally the suspected diagnosis of iron deficiency may be retrospectively confirmed by the positive outcome of a therapeutic trial of iron supplementation.

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