The iron content of the body and its distribution among the various proteins is summarized in Table 9.1. Most of the iron is present in the oxygen-carrying protein of the red blood cell, haemoglobin, the synthesis and breakdown of which dominates iron turnover. Haem of haemoglobin is synthesized in nucleated red cells in the bone marrow by a pathway ending with the incorporation of iron into protoporphyrin IX by ferrochelatase. Haem breakdown from haemoglobin takes place mainly in phagocytic cells, largely those in the spleen, liver and bone marrow. Iron is released from haem by haem oxygenase and is largely reused for haem synthesis. Every day about 30 mg iron is used to make new haemoglobin and most of this is obtained from the breakdown of old red cells.

Table 9.1 Distribution of iron in the body in a 70 kg man ^a

Protein	Location	Iron content (mg)
Haemoglobin	Red blood cells	3000
Myoglobin	Muscle	400
Cytochromes and iron sulphur proteins	All tissues	50
Transferrin	Plasma and extravascular fluid	5
Ferritin and haemosiderin	Liver, spleen and bone marrow	100–1000

a The numbers are different for women.

Relatively little iron is lost from the body (about 1 mg/d in men) and these losses are not influenced by body iron content or the requirement of the body for iron. The body iron content is maintained by variation in the amount of iron absorbed. In women, menstruation and childbirth increase iron losses to about 1.5 mg/d. Iron absorption may not increase sufficiently to compensate for these iron losses and this may eventually lead to the development of iron deficiency anaemia. In most men and postmenopausal women there is some ‘storage’ iron. This is iron in ferritin or its insoluble derivative haemosiderin, which is available for haem synthesis if necessary. Many young women and children have little or no storage iron.

The iron-binding protein transferrin is responsible for extracellular transport. Each molecule of transferrin can bind up to two molecules of ferric iron. Most cells obtain iron from diferric transferrin which binds to transferrin receptors (TfR) on the cell surface. This is followed by internalization into vesicles, release of iron, transport of iron into the cytoplasm and recycling of the apotransferrin (the protein without iron) into the plasma (Fig. 9.1).

Figure 9.1 Iron exchange within the body. Numbers in boxes refer to amount of iron (mg) in the various compartments and the numbers alongside arrows indicate transfer in mg/d. The iron in parenchymal tissues is largely haem in muscle and ferritin/haemosiderin in hepatic parenchymal cells. The dotted line indicates the small daily loss of iron (0.5 mg) into the gut from red cells; the remaining iron loss is mostly in exfoliated gut cells and bile.

Dietary Iron Absorption

Iron absorption ¹ depends on the amount of iron in the diet, its bioavailability and the body’s need for iron. A normal Western diet provides approximately 15 mg iron daily. Of that iron, digestion within the gut lumen releases about half in a soluble form, from which only about 1 mg (5–10% of dietary iron) is transferred to the portal blood in a healthy adult male.

Dietary and Luminal Factors

Most of the dietary iron is non-haem iron derived from cereals (often fortified with additional iron), with a lesser, but well-absorbed, component of haem iron from meat and fish. With iron deficiency, the maximum iron absorption from a mixed Western diet is no more than 3–4 mg daily. This amount is much less in the more vegetarian, cereal-based diets of many populations.

Non-haem iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestine. It is maximally absorbed from the duodenum and less well from the jejunum, probably because the increasingly alkaline environment leads to the formation of insoluble ferric hydroxide complexes. Many luminal factors enhance (e.g. meat and vitamin C) or inhibit (e.g. phytates and tannins) non-haem iron absorption. Therapeutic ferrous iron salts are well absorbed (approximately 10–20%) on an empty stomach, but when taken with meals, absorption is reduced by the same dietary interactions that affect non-haem food iron.

Iron Absorption at the Molecular Level

Several membrane transport proteins, regulatory proteins and associated oxidoreductases involved in iron transport through the intestinal cell have been identified. Non-haem iron is released from food as Fe³⁺ (ferric iron) and reduced to Fe²⁺ (ferrous iron) by a membrane-bound, ferrireductase, duodenal cytochrome b (DCYTB). Iron is then transported across the brush-border membrane by the divalent metal transporter, DMT1. Some iron is incorporated into ferritin and lost when the cells are exfoliated. Iron destined for retention by the body is transported across the serosal membrane by ferroportin-1. Before uptake by transferrin, Fe²⁺ is oxidized to Fe³⁺ by hephaestin (a membrane protein homologous to the plasma copper-containing protein caeruloplasmin) or by plasma caeruloplasmin.

Haemoglobin and myoglobin are digested in the stomach and small intestine. Haem is initially bound by haem receptors at the brush border membrane and the iron is released intracellularly by haem oxygenase before entering the labile iron pool and following a common pathway with iron of non-haem origin.

Regulation of Iron Absorption

Iron absorption may be regulated both at the stage of mucosal uptake and at the stage of transfer to the blood. Recent studies show that in mice HIF-2α, a mediator of cellular adaptation to hypoxia, regulates DMT1 transcription and thus mucosal uptake of iron.² As the epithelial cells develop in the crypts of Lieberkuhn, their iron status reflects that of the plasma (transferrin saturation) and this programmes the cells to absorb iron appropriately as they differentiate along the villus. Transfer to the plasma depends on the requirements of the erythron for iron and the level of iron stores. This regulation is mediated directly by hepcidin, a peptide synthesized in the liver in response to iron and inflammation.³

Cellular Iron Uptake and Release

Diferric transferrin binds with high affinity to the transferrin receptor (TfR1) on the membrane of the cell to form a complex which initiates clathrin-dependent endocytosis of the local membrane. The resulting endosome contains the holotransferrin–transferrin receptor complex.⁴ The pH of the endosome is reduced by a proton pump and a conformational change in holotransferrin is induced causing iron release. Iron is then reduced by a membrane-bound ferrireductase (six transmembrane epithelial antigen of the prostate 3, STEAP3 in erythroid cells⁵) and transported into the cytoplasm by DMT1. This iron is then either stored as ferritin or used within the cell. Iron destined for haem and mitochondrial iron sulphur (Fe-S) clusters is transported into mitochondria by mitoferrin for incorporation into protoporphyrin by ferrochelatase⁶ and for Fe-S cluster assembly on the scaffold protein ISCU.⁷ Export by ABCB7 of an as-yet unknown component to the cytoplasm stimulates Fe-S cluster protein assembly there, essential for cellular iron homeostasis.⁷ Apotransferrin and the transferrin receptor of the endosome return rapidly to the cell surface where they dissociate at neutral pH so that the cycle can start again.^7a

The reticuloendothelial macrophages play a major role in recycling iron resulting from the degradation of haemoglobin from senescent erythrocytes. They engulf red blood cells and release the iron within using haem oxygenase. The iron is rapidly released to plasma transferrin or stored as ferritin. The protein transporting iron to plasma is ferroportin or SLC40A1. Ferroportin activity is regulated in a negative manner by the mainly liver-derived peptide, hepcidin that binds to ferroportin externally, inducing endocytosis and degradation.

Hepcidin synthesis in the hepatocyte is controlled at the transcriptional level through the alteration of levels within the nucleus of different transcription factors able to bind to the gene. Interaction of diferric transferrin, bone morphogenetic proteins (BMPs), interleukin (IL)-6 and other inflammatory cytokines with cell surface receptors TfR1, TfR2, hemojuvelin (HJV) and IL-6 receptor lead to upregulation of the hepcidin gene through different and sometimes interacting signalling pathways. TMPRSS6, a plasma membrane serine protease, inhibits the HJV-BMP-SMAD signaling pathway by cleaving HJV from the surface of the cell, thus preventing overproduction of hepcidin and maintaining iron homeostasis.^8,⁹ A soluble form of HJV (sHJV) produced within the cell by the enzyme furin and released into the plasma antagonizes and also inhibits this pathway.¹⁰

The mechanism of action of the HFE protein is less clear. HFE binds to both TfR1 and TfR2, decreasing the affinity of each for transferrin. Stabilization and endocytosis of TfR2 stimulates hepcidin production and there is evidence that diferric transferrin displaces the protein HFE from TfR1, leaving it free to interact with TfR2, thus stimulating hepcidin production in response to plasma iron levels.¹¹^–¹³

Increased erythropoiesis causes decreased hepcidin, increased iron absorption and increased iron availability for haemoglobin production. So far, two plasma antagonists of the BMP-HJV-SMAD pathway, GDF15 and TWSG1, produced by erythroid cells and associated with expanded ineffective erythropoiesis, have been found.^14,¹⁵ Alteration of the function of key regulators leads to body iron overload or iron deficiency (see later). The extent and the manner in which these regulatory pathways vary between different tissues await further investigation.

Iron Storage

All cells require iron for the synthesis of proteins but have the ability also to store excess iron. There are two forms of storage iron: a soluble form, known as ferritin and insoluble haemosiderin.¹⁶ Ferritin consists of a spherical protein (molecular mass 480 000) enclosing a core of ferric-hydroxy-phosphate, which may contain up to 4000 atoms of iron. Haemosiderin is a denatured form of ferritin in which the protein shells have partly degraded, allowing the iron cores to aggregate. Haemosiderin deposits are readily visualized with the aid of the light microscope as areas of Prussian-blue positivity after staining of tissue sections with potassium ferrocyanide in acid (see Chapter 4). Ferritin is found in all cells and in the highest concentration in liver, spleen and bone marrow.

Regulation of Iron Metabolism

The expression of a number of iron proteins involved in both transport and storage is largely controlled posttranscriptionally by iron regulatory proteins (IRP1 and 2).⁴ The conformation of IRP1 required for binding to mRNA iron-responsive elements (IREs) and the turnover of IRP2 are directly affected by the amount of iron within a cell. Depending on where the target IRE elements are found these proteins may inhibit or enhance translation of many proteins involved in iron metabolism.

IREs are regions of mRNA that form hairpin-like stem-loop structures. These consist of a base-paired stem of variable length interrupted by an asymmetrical region including a 5′ unpaired cytosine, followed by an upper stem of five paired bases and a six-membered loop. When the labile iron pool is deficient of iron, IRP1 has an available binding site for IRE. When the labile iron pool is saturated with iron, the iron binds to IRP1 to produce a 4Fe-4S cluster which blocks the IRE binding site and prevents IRP1 binding to the IRE. Fe-saturated IRP1 is then able to function as the enzyme aconitase. In the presence of iron, IRP2 (which is not an Fe-S protein) is degraded. The turnover of IRP1 is also affected by iron levels.

Different iron proteins are regulated by IRP in different ways, depending on where the IRE is located. If the IRE is at the 3′ UTR of the mRNA, IRP binding will stabilize translation by protecting the translation product from endonucleolytic cleavage (e.g. TfR1 and DMT1). If the IRE is at the 5′ UTR of the mRNA, IRP binding will inhibit the translation of mRNA. Both L and H ferritin subunits have 5′ UTR IRE. When iron is abundant, the IRP does not bind to the 5′ IRE so ferritin expression is not inhibited and excess iron can be stored adequately. When iron is scarce, the IRP binds to the IRE and inhibits ferritin synthesis.

Plasma Iron Transport

Almost all the iron in plasma is tightly bound to transferrin, which is usually less than 50% saturated with iron. Transferrin, when incompletely saturated with iron, exists in four forms: apo, monoferric (iron bound at the ‘A’ site), monoferric (B) and diferric. The distribution may be determined by urea-polyacrylamide electrophoresis.¹⁷ Delivery to cells requires specific binding to transferrin receptors. The plasma iron pool (transferrin-bound iron) is about 3 mg, although the daily turnover is more than 10 times his amount. In addition, smaller amounts of iron are carried in the plasma by other proteins.

Haptoglobin is a serum glycoprotein that avidly binds haemoglobin αβ dimers released into the bloodstream by haemolysis. The haemoglobin–haptoglobin complex is rapidly removed from the plasma by a specific receptor, CD163, highly expressed on tissue macrophages.¹⁸

Hemopexin¹⁹ is a plasma glycoprotein of molecular mass approximately 60 kDa that binds haem and transports the haem to cells by a process that involves receptor-mediated endocytosis and recycling of the intact protein.

Low concentrations of ferritin are found in the plasma and ferritin concentrations in healthy subjects reflect body iron stores. Much of this ferritin appears to be glycosylated and has a relatively low iron content.²⁰ Such ferritin has a half life (T_½) of approximately 30 h. Ferritin is also released into the circulation as a result of tissue damage (most strikingly after necrosis of the liver). Tissue ferritin is cleared rapidly from the circulation (T_½ in approximately 10 min) by the liver.

Non-transferrin-bound iron describes a form of iron that is not bound to transferrin, is of low molecular mass and can be bound by specific iron chelators.²¹ Several assays have been described that have demonstrated such a fraction in plasma from patients with iron overload. The chemical form of this iron is unknown, but it is probably rapidly removed from the circulation by the liver. Recent studies implicate the involvement of the zinc transporter ZIP14 in this process.²²

Iron status

Normal iron status implies a level of erythropoiesis that is not limited by the supply of iron and the presence of a small reserve of ‘storage iron’ to cope with normal physiological needs. The ability to survive the acute loss of blood (iron) that may result from injury is also an advantage. The limits of normality are difficult to define and some argue that physiological normality is the presence of only a minimal amount of storage iron ²³ but the extremes (i.e. iron deficiency anaemia and haemochromatosis) are well understood.^23a

Apart from too little or too much iron in the body, there is also the possibility of a maldistribution (Fig. 9.2). An example is anaemia associated with inflammation or infection where there is a partial failure of erythropoiesis and of iron release from the phagocytic cells in liver, spleen and bone marrow, which results in accumulation of iron as ferritin and haemosiderin in these cells. Determination of iron status requires an estimate of the amount of haemoglobin iron (usually by measuring the haemoglobin concentration (Hb) in the blood; see Chapter 3) and the level of storage iron (measuring serum ferritin concentration). Iron deficiency should be suspected in hypochromic, microcytic anaemia, but in the early stages of iron deficiency red cells may be normocytic and normochromic. Another feature of iron deficiency is an increased concentration of protoporphyrin in the red cells; normally, there is a small amount present, but defective haem synthesis caused by lack of iron results in the accumulation of zinc protoporphyrin and occasionally free protoporphyrin may be increased as well.²⁴

Figure 9.2 Body iron content and distribution in different conditions. Additional assays are sometimes required. In genetic haemochromatosis, early iron accumulation is indicated by increased transferrin saturation alone. The serum ferritin concentration increases only later as the level of stored iron increases. In the anaemia of chronic disease, patients often have normal serum ferritin concentrations even when storage iron in the bone marrow is absent (see p. 336). In this situation, the assay of serum transferrin receptor may help detect tissue iron deficiency.

Disorders of iron metabolism

Clinical aspects of iron metabolism have been reviewed ²⁵^–²⁷ and are summarized in Table 9.2. A guideline on the management of genetic haemochromatosis is available.²⁸

Table 9.2 Disorders of iron metabolism

Iron deficiency

Deficient iron intake

Diet of low bioavailability

Increased physiological requirements due to rapid growth in early childhood and in adolescence

Blood loss: physiological (e.g. menstruation) or pathological (e.g. gastrointestinal)

Malabsorption of iron intake

Reduced gastric acid secretion (e.g. after partial gastrectomy)

Reduced duodenal absorptive area (e.g. in coeliac disease)

Rare, inherited iron-refractory iron deficiency anaemia

TMPRSS6 deficiency

Redistribution of iron

Macrophage iron accumulation

Inflammatory, infectious or malignant diseases (‘anaemia of chronic disease’)

Iron overload

Due to increased iron absorption

e.g. Hereditary haemochromatosis – commonly homozygosity for HFE C282Y but sometimes involving other genes (HAMP,HJV,TFR2,SLC40A1)
Substantial ineffective erythropoiesis (e.g. β thalassaemia syndromes of intermediate and major severity, some types of sideroblastic anaemia, congenital dyserythropoietic anaemia)
Sub-Saharan iron overload (‘Bantu siderosis’) – only in combination with increased dietary iron
Other rare inherited disorders (e.g. congenital atransferrinaemia, DMT1 deficiency)
Inappropriate iron therapy (rare)

Due to multiple blood transfusions in refractory anaemias

e.g. Thalassaemia major

Aplastic anaemia

Myelodysplastic syndromes

Methods for assessing iron status

The methods used to assess iron status are summarized in Table 9.3. Some are not generally applicable but have value in the standardization of indirect methods. The determinations of Hb and red cell indices are described in Chapter 3.

Table 9.3 Assessment of body iron status and confounding factors

Serum Ferritin Assay

With the recognition that the small quantity of ferritin in human serum (15–300 μg/l in healthy men) reflects body iron stores, measurement of serum ferritin has been widely adopted as a test for iron deficiency and iron overload. The first reliable method to be introduced was an immunoradiometric assay in which excess radiolabelled antibody was reacted with ferritin and antibody not bound to ferritin was removed with an immunoadsorbent.³¹ This assay was supplanted by the two-site immunoradiometric assay, which is sensitive and convenient. Since then the principle of this assay has been extended to non-radioactive labelling, including enzymes (enzyme-linked immunosorbent assay or ELISA). Most current laboratory immunoassay systems for clinical laboratories include ferritin in the assay repertoire. Factors to be considered when selecting an immunoassay system are discussed later. The method described in the next section is an ELISA. The most sophisticated equipment required is a microtitre plate reader.

Immunoassay for Ferritin

Preparation and Storage of Ferritin

Ferritin may be prepared from iron-loaded human liver or spleen obtained at operation (spleen) or postmortem. The informed consent of the patient or the patient’s relatives should be obtained for use in preparation of ferritin. Tissue should be obtained as soon as possible after death and may be stored at −20°C for 1 year. The usual precautions must be taken to avoid the risk of infection when handling tissues and extracts. Ferritin is purified by methods that exploit its stability at 75°C. Further purification is obtained by precipitation from cadmium sulphate solution and gel-filtration chromatography.³² Purity should be assessed by polyacrylamide gel electrophoresis,³³ and the protein content determined by the method of Lowry and colleagues, as described by Worwood.³³ Buffered solutions of human ferritin may be stored at 4°C, at concentrations of 1–4 mg protein/ml, in the presence of sodium azide as a preservative, for up to 3 years. Such solutions should not be frozen. Ferritin, from human liver or spleen, may be obtained from several suppliers of laboratory reagents. This should be used as an assay standard only after calibration against the international standard (see p. 183).

Antibodies to Human Ferritin

High-affinity antibodies to human liver or spleen ferritin are suitable. Polyclonal antibodies may be raised in rabbits or sheep by conventional methods,³⁴ and the titre checked by precipitation with human ferritin.³³ An immunoglobulin G (IgG)-enriched fraction of antiserum is required for labelling with enzyme in the assay. The simplest method is to precipitate IgG with ammonium sulphate.³⁵ Monoclonal antibodies that are specific for ‘L’ subunit-rich ferritin (liver or spleen ferritin) are also suitable. A mouse monoclonal antibody and a horseradish peroxidase (HRP)-labelled antibody are available from NIBSC (www.nibsc.ac.uk; codes 87/654 and 87/662). Suitable antibodies are available from commercial suppliers.

3. Interference by non-ferritin proteins in serum. This may occur with any method but particularly with labelled antibody assays. Serum proteins may inhibit the binding of ferritin to the solid phase when compared with binding in buffer solution alone. Such an effect may be avoided by diluting the standards in a buffer containing a suitable serum or by diluting serum samples as much as possible. For example, in the assay described earlier, the sample is diluted 20 times with buffer. Another cause of error, difficult to detect, is interference by antibodies to animal immunoglobulins.³⁷ These antibodies bind to the animal immunoglobulins used to detect the antigen and form artefactual ‘sandwiches’, thereby falsely elevating the reading. Such antibodies are found in up to 10% of patients and normal subjects. Interference may be reduced by adding the appropriate species of animal immunoglobulins to block the cross-reaction, but this is not always successful.³⁸ One solution is to use antibodies from different species as solid-phase and labelled antibodies. Thus one may use a polyclonal, rabbit antiferritin to coat plates in the ELISA with a polyclonal sheep antiferritin labelled with horseradish peroxidase as the second antibody. Rabbit serum (0.5%) replaces BSA in Buffer B.

4. Reproducibility. Most assays are satisfactory. With microtitre plate assays there may be ‘edge’ effects (differences between readings for inner and outer wells).

5. Dilution of serum samples. It should be established that both standard and serum samples dilute in parallel over a 100-fold range.

6. Accuracy. The use of the WHO standard ferritin preparation is recommended (see earlier discussion).

Interpretation

The use of serum ferritin for the assessment of iron stores has become well-established.³¹ In most normal adults, serum ferritin concentrations lie within the range of 15–300 μg/l. During the first months of life, mean serum ferritin concentrations change considerably, reflecting changes in storage iron concentration. Concentrations are lower in children (<15 years) than in adults and from puberty to middle life are higher in men than in women. In adults, concentrations <15 μg/l indicate an absence of storage iron. Reference ranges quoted by kit manufacturers vary and this is partly a result of the selection of ‘normal’ subjects. Sometimes subjects with iron deficiency are included and sometimes they are excluded. The interpretation of serum concentration in many pathological conditions is less straightforward, but concentrations <15 μg/l indicate depletion of storage iron. In children, mean levels of storage iron are lower and a 12 μg/l threshold has been found to be appropriate for detecting iron deficiency.³⁹

Iron overload causes high concentrations of serum ferritin, but these may also be found in patients with liver disease, infection, inflammation or malignant disease.³¹ In addition some rare inherited causes of increased ferritin production unlinked to iron stores occur. Careful consideration of the clinical evidence is required before concluding that a high serum ferritin concentration is primarily the result of iron overload and not a result of tissue damage or enhanced synthesis of ferritin. A normal ferritin concentration provides good evidence against iron overload but does not exclude genetic haemochromatosis. This is because haemochromatosis is a late-onset condition and iron stores may remain within the normal range for many years.

Serum ferritin concentrations are high in patients with advanced haemochromatosis, but the serum ferritin estimation should not be used alone to screen the relatives of patients or to assess re-accumulation of storage iron after phlebotomy. The early stages of iron accumulation are detectable by an increased serum iron concentration, a decreased unsaturated iron-binding capacity and increased transferrin saturation; the serum ferritin concentration may be within the normal range. In this situation, the measurement of serum iron and total iron-binding capacity provides useful clinical information not given by the ferritin assay.

In patients with acute or chronic disease, interpretation of serum ferritin concentrations is less straightforward and patients may have serum ferritin concentrations up to 100 μg/l despite an absence of stainable iron in the bone marrow. Ferritin synthesis is enhanced by interleukin-1 – the primary mediator of the acute-phase response.⁴⁰ In patients with chronic disease, the following approach should be adopted: low serum ferritin concentrations indicate absent iron stores, values within the normal range indicate either low or normal levels and high values indicate either normal or high levels. In terms of adequacy of iron stores for replenishing haemoglobin in patients with anaemia, the degree of anaemia must also be considered. Thus a patient with Hb 10 g/dl may benefit from iron therapy if the serum ferritin concentration is <100 μg/l because below this level there is unlikely to be sufficient iron available for full regeneration. Here, measurement of serum transferrin receptor concentration may be of value (see p. 190).

Immunologically, plasma ferritin resembles the ‘L-rich’ ferritins of liver and spleen and only low concentrations are detected with antibodies to heart or HeLa cell ferritin, ferritins rich in ‘H’ subunits. The heterogeneity of serum ferritin on isoelectric focusing is largely the result of glycosylation and the presence of variable numbers of sialic acid residues and not variation in the ratio of H to L subunits.⁴¹ Attempts to assay for ‘acidic’ (or ‘H’-rich) isoferritins in serum as tumour markers have not been successful.⁴¹ The iron content of serum ferritin is low⁴¹ and measurement of this iron has no diagnostic use.⁴²

Estimation of serum iron concentration

Iron is carried in the plasma bound to the protein transferrin (molecular mass 78 000). This molecule binds two atoms of iron as Fe³⁺ and delivers iron to cells by interaction with membrane transferrin receptors. The following method is a modification of that recommended by the International Council for Standardization in Haematology (ICSH) and is based on the development of a coloured complex when ferrous iron released by serum protein denaturation in the presence of reducing agent is treated with a chromogen solution.⁴³

Reagents and Materials

Reagents must be at least of analytical grade with the lowest obtainable iron content. Before handling any of the reagents and materials described below a risk assessment should be carried out. If there is significant risk of harm, the word ‘caution’ has been added.

Preparation of Glassware

It is essential to avoid contamination by iron. If possible, use disposable plastic tubes and bottles. If glassware is to be used, wash in a detergent solution, soak in 2 mol/l HCl (caution) for 12 h and finally rinse in iron-free water.

Protein Precipitant

100 g/l trichloroacetic acid (0.61 M) and 22.7 mmol/l ascorbic acid in 0.9 mol/l HCl. The original reducing agent was thioglycolic acid.⁴³ Ascorbic acid is an alternative reducing agent, although there may be more interference from copper. However, any benefit from reduced copper interference is usually outweighed by the associated health and safety problems of working with thioglycolic acid. To 45 ml 1 mol/l HCl in a 50 ml screwcap polypropylene tube add 5 ml 6.1 mol/l trichloroacetic acid solution (Sigma 490–10; caution). Add 200 mg ascorbic acid and mix. Make a fresh solution when required and discard after 4 h.

Chromogen Solution (Ferrozine)

In 100 ml 1.5 mol/l sodium acetate dissolve 25 mg ferrozine [Sigma-Aldrich 82950, monosodium 3-(2-pyridyl)-5, 6-bis(4-phenylsulphonic acid)-1, 2, 4-triazine] (caution). Store in the dark for up to 4 weeks.

Iron-free Water

Use deionized water for the preparation of all solutions.

Iron Standard 80 μmol/l

Add 22.1 ml deionized water to a universal container (the easiest way is by weight). Add 200 μl 2 mol/l HCl and mix. Add 100 μl iron standard solution (1000 μg Fe/ml in 1% v/v HCl, Aldrich No. 30, 595–2) and mix. Store for up to 2 months at room temperature.

Method

Place 0.5 ml serum (free of haemolysis), 0.5 ml working iron standard and 0.5 ml iron-free water (as a blank), respectively, in each of three 1.5 ml polypropylene microcentrifuge tubes with lids. Add 0.5 ml protein precipitant to each and replace the lid. Mix the contents vigorously (e.g. with a vortex mixer) and allow to stand for 5 min. Centrifuge the tube containing the serum at 13 000 g for 4 min (in a microfuge) to obtain an optically clear supernatant. To 0.5 ml of this supernatant and to 0.5 ml of each of the other mixtures, add 0.5 ml chromogen solution with thorough mixing. After standing for 10 min, measure the absorbance in a spectrophotometer against water at 562 nm. If a micro-centrifuge is not available, use double the volume of serum and reagents in a 3 ml plastic tube with lid and centrifuge at 1500 g for 15 min in a bench centrifuge.

If EDTA-plasma is used, the colour develops more slowly and the preparation should be allowed to stand for at least 15 min before measuring the absorbance. The use of EDTA-plasma is not recommended. Iron chelators (e.g. deferoxamine) also delay colour development.⁴⁴

Calculation

Alternative procedure: serum iron without protein precipitation

This is a microtitre plate method developed from the assay of Persijn et al.⁴⁵

Reagents and Materials

Iron Standards 80 μmol/l and 40 μmol/l

Dilute with an equal volume of water to make the 40 μmol/l standard.

Phosphate–Ascorbate Buffer (Stock)

Add approximately 200 ml deionized water to an acid-washed plastic beaker. Add 17.5 g sodium dihydrogen orthophosphate (NaH₂PO₄2H₂O) to the water and dissolve fully by stirring (plastic stirrer). Adjust the pH to 4.9 using 2 M NaOH solution (2 g NaOH in 25 ml water; caution). Make the volume up to 250 ml and add 25 ml of the buffer to 10 universal containers. Store for up to 1 month at room temperature. Prior to use, add 50 mg of ascorbic acid to each universal container required and shake to dissolve. Discard after 4 h.

Chromogen Solution

Add 50 mg ferrozine (see p. 185) to 25 ml deionized water and shake to dissolve. Store for up to 1 month in the dark at room temperature.

Microtitre Trays

Microtitre trays should be optical grade, with flat-bottomed wells.

Control Serum

Suitable control sera are Lyphochek Assayed Chemistry Control (Bio-Rad).

Method

Add 80 μl deionized water (‘O’), standard solution (40, 80 μmol/l), controls (C1, C2) and samples (S1, S2, etc.) to the microtitre plate (see plate map, Table 9.4).

Table 9.4 Plate map for serum iron determination

Add 80 μl phosphate–ascorbate buffer to each well, using a multichannel pipette. Tap the tray to mix. Leave for 20 min at room temperature. During this time, take an initial absorbance reading of the tray at 560–570 nm on a microtitre plate reader. Add 40 μl of chromogen solution to each well, then tap the tray to mix. Cover with a film or lid. Incubate for 40 min at 37°C. Take a second absorbance reading. Calculate the net absorbance increment values.

Calculations

Calculate the difference in absorbance (δA) between the final and initial readings for the water blank (δA₀), each standard (δA₄₀, δA₈₀) and serum sample (δA_sample).

The approximate values are 0.015–0.03 for the water blank (zero standard) and 0.25–0.28 for the 40 μmol Fe/l standard. Subtract the mean net value of the zero standard (δA₀) from each standard or sample (δA_sample). The net value of the 80 μmol/l standard δA₈₀ should be 2× that of the 40 μmol/l standard δA₄₀.

Serum iron concentrations are:

The data may be downloaded from the plate reader and imported into a suitable spreadsheet or statistical program (e.g. Excel or Minitab) for these calculations.

Automated Methods for Serum Iron

Procedures for measuring serum iron are available for most clinical chemistry analysers. A non-precipitation method similar to that described above is available from Randox Ltd (www.randox.com). The performance of several methods was reviewed by Tietz et al.⁴⁶ who found differences between the various methods, particularly at low values of serum iron concentration. More recently, Blanck et al.⁴⁷ carried out an interlaboratory comparison and found no significant differences in results generated by methods currently in use. Variability across laboratories and across methods was low. Serum iron concentrations may be measured by atomic absorption spectroscopy, but this has the disadvantage of measuring any haem iron present as a result of haemolysis and is not used for diagnostic purposes.

Serum iron concentrations in health and disease

Jacobs et al.⁴⁸ measured serum iron concentrations in a random sample of 517 women and 499 men in the general population of Wales. Serum iron concentrations⁴⁴ approximated to a normal distribution. The mean (±SD) of 16.1 ± 7.4 μmol/l in women was slightly lower than for men (18.0 ± 6.3 μmol/l). These figures do not refer to ‘iron replete’ subjects because subjects with absent iron stores or with frank anaemia were included. Using the microtitre plate method described earlier, Jackson and co-workers⁴⁹ determined serum iron concentrations in 10 500 blood donors from South Wales. For 1502 ‘first time’ donors, mean age of 28 years, the mean (±SD) was 16.7 ± 6.0 μmol/l for men and 14.4 ± 6.7 μmol/l for women. Again subjects included potential donors who failed the screening test for anaemia and were not permitted to give blood on that occasion. In the first month of life mean concentration of serum iron is higher (22 μmol/l) than in adults, falls to about 12 μmol/l by the age of 1 year and remains at that level throughout childhood.^50,⁵¹

Measurement of the serum iron concentration alone provides little useful clinical information because, although methodological variation is low, there is considerable variation from hour to hour and day to day in normal individuals (see p. 193). Low concentrations are found in patients with iron deficiency anaemia; in chronic disease (including inflammation, infection and cancer); and during the acute-phase response, including after surgery. Therefore, low serum iron concentrations do not necessarily indicate an absence of storage iron. High concentrations are found in liver disease, hypoplastic anaemias, ineffective erythropoiesis and iron overload.

Iron-binding capacity, serum transferrin and transferrin saturation

Estimation of Total Iron-Binding Capacity

In the plasma, iron is bound to transferrin and the total iron-binding capacity (TIBC) is a measure of this protein. The additional iron-binding capacity of transferrin is known as the unsaturated iron-binding capacity (UIBC). The serum iron concentration plus the UIBC together give TIBC.

Iron-binding capacity is usually measured by adding an excess of iron and measuring the iron retained in solution after the addition of a suitable reagent such as ‘light’ magnesium carbonate or an ion-exchange resin that removes excess iron. All methods are empiric and none is completely satisfactory. The method described as follows was developed by the ICSH.⁵²

Principle

Excess iron as ferric chloride is added to serum. Any iron that does not bind to transferrin is removed with excess magnesium carbonate. The iron concentration of the iron-saturated serum is then measured.

Reagents

Basic magnesium carbonate, MgCO₃, ‘light grade’.

Saturating solution (100 μmol Fe/l).

Add 17.7 ml deionized water to a universal container (by weight is most convenient). Add 100 μl of 1 mol/l HCl. Add 100 μl commercial iron standard solution (see p. 185. Mix and store for up to 2 months at room temperature. The ‘saturating iron solution’ contains 5.6 μg Fe/ml (100 μmol Fe/l).

Method

Place 0.5 ml serum (EDTA-plasma should not be used) in a 1.5 ml polypropylene microcentrifuge tube and add 0.5 ml saturating iron solution. Mix carefully by hand and leave at room temperature for 15 min. Use a plastic scoop or tube to add 100 mg (± 15 mg) light magnesium carbonate and cap the tube. Shake vigorously and allow to stand for 30 min with occasional mixing. Centrifuge at 13 000 g for 4 min in a microcentrifuge. If the supernatant contains traces of magnesium carbonate, remove the supernatant and re-centrifuge. Carefully remove 0.5 ml supernatant and treat as serum for the iron estimation described earlier (p. 185). Multiply the final result by 2.

Determination of unsaturated iron-binding capacity

The UIBC may be determined by methods that detect iron remaining and able to bind to chromogen, after adding a standard and excess amount of iron to the serum.⁴⁵ The UIBC is the difference between the amount added and the amount binding to the chromogen.

Reagents and Materials

Saturating Solution

2000 μmol iron/l. Add 7.95 ml deionized water to a universal container (by weight is most convenient). Add 1.0 ml Commercial Iron Standard Solution (1000 μg Fe/ml; see p. 185). Mix. Store for up to 2 months at room temperature.

Tris Buffer (Stock)

0.22 M, pH 7.8. Add approximately 200 ml of deionized water to a weighed acid-washed plastic beaker. Add 6.8 g Tris to the water and fully dissolve by stirring with a magnetic stirrer. Adjust the pH to 7.8 using 2 M HCl. Adjust the volume to 250 ml with water (by weight), mix and dispense 24.5 ml (24.5 g) into universal containers. Store for up to 1 month at room temperature.

Tris–Ascorbate–Iron Buffer

Immediately prior to use, add 50 mg ascorbic acid to each universal container of Tris buffer required and dissolve by mixing. Add 0.5 ml saturating solution (2000 μmol Fe/l) and mix. Discard after 4 h.

Chromogen Solution

See p. 185.

Microtitre Trays

See p. 186.

Control Serum

See p. 186.

Method

Add 80 μl deionized water (‘O’), control (C1, C2) and sample (S1, S2, etc.) to the microtitre plate (Table 9.5). Add 160 μl Tris–ascorbate–iron buffer to each well, using a multichannel pipette. Tap the tray to mix. Leave the tray for 20 min. During this time, take an initial reading (A_initial) of the A_{560–570 nm}. Add 40 μl of chromogen solution to each sample and tap the tray to mix; cover with a film or lid. Incubate for 40 min at 37°C. Take a final absorbance reading (A_final).

Table 9.5 Plate map for UIBC determination

Calculations

The saturating solution added to each well (160 μl) contains 6.4 nmol Fe. Calculate the absorbance reading corresponding to 6.4 nmol Fe from the mean value of the readings in column 1 as A_final – A_initial (A_s). (Note: this absorbance reading should be within 5% of the 80 μmol/l value for the iron determination.)

Once A_s has been calculated, it is used in the following equation:

For controls 1 and 2 and samples:

Data may be imported into a spreadsheet for calculation. As with the serum iron determination, protocols for clinical chemistry analysers sometimes include a method for UIBC.

Determination of total iron-binding capacity from UIBC:

Fully Automated Methods

A number of methods to determine the TIBC using clinical chemistry analysers require a pretreatment step. Direct (fully automated procedures) have been developed.⁵³ A non-precipitation method (UIBC) similar to that described above is available from Randox Ltd (Cat. No. S1250; www.randox.com).

Serum transferrin

An alternative approach is to measure transferrin directly by an immunological assay. This avoids some of the spuriously high values of TIBC found when the transferrin is saturated and non-transferrin iron is measured.⁴³ Rate immunonephelometric methods are rapid and precise. There is generally a good correlation between the chemical and immunological TIBC,^54,⁵⁵ although when TIBC was calculated as the sFe + UIBC, values were lower than the direct TIBC.⁵³ Transferrin concentrations (g/l) may be converted to TIBC (μmol/l) by multiplying by 25.

Normal Ranges of Transferrin and Total Iron-Binding Capacity

In health, the serum transferrin is 2.0–3.0 g/l and 1 mg of transferrin binds 1.4 μg of iron. The normal serum TIBC (mean ± SD) was 68.0 ± 12.6 μmol/l in a random sample of 517 women and 63.2 ± 9.1 μmol/l for 499 men.⁴⁸ For 890 first-time, female blood donors of mean age 27 years the mean TIBC (determined by the UIBC method described earlier) was 56.7 ± 12.1 μmol/l. In 612 first-time, male blood donors of mean age 28 years the mean was 54.2 ± 10 μmol/l (mean ± SD).⁴⁹ In both surveys, the sample included some individuals with iron deficiency. Note the comment earlier about lower values given by the colorimetric UIBC method. The TIBC is increased in iron deficiency anaemia and in pregnancy; it is lower than normal in infections, malignant disease and renal disease. In pathological iron overload, the TIBC of the serum is reduced.

Diagnostically, although a raised TIBC is characteristic of iron deficiency anaemia, the TIBC is usually used to calculate the transferrin saturation. The UIBC has attracted little diagnostic use, but is being evaluated as a screening test for iron overload in genetic haemochromatosis. In genetic haemochromatosis (subjects homozygous for HFE C282Y), a UIBC (determined as described earlier) of <20 μmol Fe/l was found in most men and in about 50% of women.⁴⁹ UIBC and transferrin saturation were equally sensitive and specific. For UIBC methods, optimum thresholds for detecting subjects with genetic haemochromatosis have varied. Murtagh and co-workers⁵⁶ determined the optimum threshold to be 25.6 μmol/l (sensitivity 0.91 and specificity 0.95). In this case, fasting blood samples were taken. UIBC was as reliable as transferrin saturation in detecting HFE haemochromatosis in both reports.

Transferrin saturation

The transferrin saturation is the ratio of the serum iron concentration and the TIBC expressed as a percentage. If transferrin is measured immunologically, then the corresponding TIBC (μmol/l) may be calculated by multiplying the transferrin concentration (g/l) by 25. In a sample of the Welsh population, the mean transferrin saturation in 499 men was 29.1 ± 11.0%; in 517 women it was 24.6 ± 11.8%.⁴⁸ For first-time blood donors from South Wales the mean transferrin saturation was 31.1 ± 10.9% for 612 men and 25.5 ± 12.9% for 890 women.⁴⁹ A transferrin saturation of <16% is usually considered to indicate an inadequate iron supply for erythropoiesis.⁵⁷ The most valuable use of transferrin saturation is for the detection of genetic haemochromatosis. Even in the early stages of the development of iron overload,^49,⁵⁸ an elevated transferrin saturation is indicative of the disorder (suggested thresholds vary but >55% for men and >50% for women are an appropriate compromise),²⁸ although a more recent guideline suggests >50% for men and >45% for women.^23a

Transferrin Index

Beilby and co-workers ⁵⁹ have recommended that the transferrin saturation is replaced by the ‘transferrin index’. This is the serum iron concentration (μmol/l) divided by the transferrin concentration (determined immunologically and expressed as μmol/l). They claimed that the transferrin index had better precision that the transferrin saturation and showed greater specificity for detecting iron overload than the transferrin saturation. However, the transferrin index has attracted little use.

Serum transferrin receptor

Almost all cells in the body obtain iron from the plasma protein transferrin, but transferrin has a very high affinity for iron at neutral pH and iron release takes place through a specific membrane receptor. There are two types of transferrin receptors – TfR1 and TfR2 – encoded by different genes. TfR1 is essential for tissue iron delivery and is a homodimer consisting of two identical protein subunits of molecular mass 95 kDa. Transferrin binds to TfR1, the complex is internalized and iron is released when the pH of the internal vesicles is reduced to about 5.5. After iron release, apotransferrin remains bound to the receptor until exposed to an alkaline pH at the cell surface, returns to the circulation and can undertake further cycles of iron uptake and delivery.⁶⁰ The cells that require most iron are the nucleated red cells in the bone marrow which synthesize haemoglobin and have the greatest number of transferrin receptors. TfR1 synthesis is also controlled by iron supply. The mechanism involves five IREs (see p. 178) at the 3′ untranslated region of the receptor mRNA. In the absence of iron, an IRP binds to each RNA IRE, thus stabilizing it and permitting synthesis of the protein chain. In the presence of adequate iron concentrations, binding of iron by the IRP changes the conformation of the protein and prevents its binding to the mRNA. The mRNA is rapidly broken down and synthesis of transferrin receptors is reduced. The second receptor, TFR2, also binds transferrin, but it is not required for iron delivery to cells and its synthesis is not regulated by IRPs.⁶¹

In 1986, Kohgo and co-workers ⁶² reported that transferrin receptors were detectable in the plasma by immunoassay. Since then, there has been much investigation of the physiological and diagnostic significance of circulating transferrin receptors.⁶³ The protein is derived by proteolysis at the cell membrane and circulates bound to transferrin. Plasma concentrations reflect the number of cellular receptors and, in patients with adequate iron stores, the number of nucleated red cells in the bone marrow. Because the number of cellular transferrin receptors per cell increases in iron deficiency, concentrations also increase when erythropoiesis becomes iron limited. Table 9.6 summarizes the conditions associated with reduced or elevated levels of circulating transferrin receptor.

Table 9.6 Serum transferrin receptor (sTfR) concentrations in human disease

sTfR concentration	Condition
Increased	Increased erythroid proliferation:
		Autoimmune haemolytic anaemia
		Hereditary spherocytosis
		β Thalassaemia intermedia or major
		β Thalassaemia/HbE
		Haemoglobin H disease
		Sickle cell anaemia
		Polycythaemia vera
	Decreased tissue iron stores:
		Iron deficiency anaemia
Normal to increased	Idiopathic myelofibrosis
	Myelodysplastic syndrome
	Chronic lymphocytic leukaemia
Normal	Haemochromatosis (but see text)
	Acute and chronic myeloid leukaemia
	Most lymphoid malignancies
	Solid tumours
	Anaemia of chronic disease
Decreased	Chronic renal failure
	Aplastic anaemia
	After bone marrow transplantation

Modified from Feelders et al (1999).⁶⁰

Assays for the Serum Transferrin Receptor

There has been no agreement about the source of transferrin receptor as standard or as an antigen for the raising of antibodies. Transferrin receptors have been purified from placenta and from serum. The receptor may or may not be bound to transferrin as a standard or to raise antibodies. No ‘reference’ method is therefore described here. Three enzyme immunoassay kits (Orion; Ramco; R&D) for the determination of serum transferrin receptor concentrations have been evaluated for the Medical Devices Agency.⁶⁴ All have been approved for diagnostic purposes in the USA by the Food and Drug Administration. Assays for fully automated, diagnostic, immunoassay systems are now being introduced and offer improved sensitivity, reproducibility and speed.

The different reference ranges in the available commercial assays reflect the differences in preparations of transferrin receptor used to raise antibodies and as a standard in the various assays. For the Orion, Ramco and R&D kits Akesson et al.⁶⁵ and Worwood et al.⁶⁴ noted some assay drift but found acceptable intra-assay coefficient of variance (CV) values. The determined sensitivity was adequate for clinical purposes for the three assay systems. Four different units (nmol/l, μg/ml, mg/l and ku/l) and different normal ranges are in use.⁶⁶ At the present time, serum transferrin receptor (sTfR) is not included in the national external quality control schemes for the UK (NEQAS) and the Welsh External Quality Assessment Scheme (WEQAS). A WHO reference reagent for the serum transferrin receptor has now been established and an international collaborative study showed that using this reagent as a standard markedly improved agreement between methods. Currently available commercial assays are listed in this report.⁶⁷

Reference Ranges

sTfR concentrations are high in neonates and decline until adult concentrations are reached at 17 years. Concentrations are similar in normal men and non-pregnant women. During pregnancy sTfR levels increase, returning to non-pregnant values 12 weeks after delivery.⁶⁸ sTfR concentrations obtained with different assay systems cannot be directly compared because reference ranges differ.

Samples

The information provided by the manufacturers shows good recovery of standard and linearity but some problems with interference.⁶⁴ Although serum is the preferred matrix, the R&D and Ramco assays give the same results with EDTA, heparin and citrate plasma. Orion states that EDTA-plasma is not acceptable. It is recommended that sera are stored for no more than 2 days at room temperature, 7 days at 2–8°C, 6 months at –20°C and 1 year at –70°C. Repeated freezing and thawing are not advisable. Moderate haemolysis is not a problem.

Transferrin Receptor Concentrations in Diagnosis

Erythropoiesis

The function of the TfR1 in delivering iron to the immature red cell immediately suggested an application in the clinical laboratory for the assay of circulating TfR. The use of the assay to monitor changes in the rate of erythropoiesis has been explored by several authors.⁶⁹ When iron supply is not limiting, the assay can provide a replacement for ferrokinetic investigations that required the injection of radioactive iron.

Iron Deficiency

The major application of the serum transferrin receptor assay has been to detect patients with an absence of stored iron (ferritin and haemosiderin in cells). In infants (age 8–15 months) sTfR concentration increases with increased severity of iron deficiency.⁷⁰ When normal subjects undergo quantitative phlebotomy, serum ferritin concentrations decrease steadily as iron stores are depleted, but there is little change in sTfR concentration. As iron stores become exhausted (serum ferritin <15 μg/l), sTfR levels increase and continue increasing as haemoglobin concentrations decrease.⁷¹ In this study the increased rate of erythropoiesis during phlebotomy had little effect on sTfR levels as long as iron stores were adequate so that most of the increase in sTfR level was the result of iron deficiency rather than increased erythropoiesis. However, the rate of phlebotomy was only 250 ml per week (about 500 ml per week is usually removed during treatment of haemochromatosis) and higher rates might cause an immediate increase in sTfR levels during phlebotomy. The log[sTfR/serum ferritin] gives a linear relationship with storage iron that has considerable potential for assessing iron stores in epidemiological studies.⁷²

Circulating transferrin receptor levels increase, not only in patients with simple iron deficiency but also in patients with the anaemia of chronic disease who lack stainable iron in the bone marrow.⁷³ Identifying a lack of storage iron in patients with the anaemia of chronic disease is difficult without this measurement because serum iron concentrations are low regardless of iron stores and serum ferritin concentrations are higher than in patients not suffering from chronic disease who have similar levels of stainable iron in the bone marrow (see p. 336). Unfortunately, the sTFR has not proved to be superior to serum ferritin for detecting iron deficiency in all studies (Table 9.7).

Table 9.7 Overall variability (%) of assays for iron status (within subject, day-to-day coefficient of variation for healthy subjects)

In both iron deficiency anaemia and the anaemia of chronic disease, sTfR levels are also influenced by changes in the rate of erythropoiesis. Ineffective erythropoiesis – an increase in the proportion of immature red cells destroyed within the bone marrow – increases in iron deficiency anaemia.⁸⁶ In the anaemia of chronic disease, erythropoiesis is normal or depressed⁸⁷; nevertheless, iron deficiency increases the number of receptors.

Although it has been claimed that sTfR measurements provide a sensitive indicator of iron deficiency in pregnancy,⁸⁸ questions remain about the decreased erythropoiesis in early pregnancy because this may mask iron deficiency at this time⁸⁹ and increases in sTfR in later pregnancy appear to relate to increased erythropoiesis rather than iron depletion.⁶⁸ Measurement of sTfR did not enhance the sensitivity and specificity for the detection of iron deficiency anaemia in pregnant women from Malawi, where anaemia and chronic disease are both common.⁹⁰

Iron Overload

Normal concentrations of sTfR have been reported for patients with genetic haemochromatosis (although some had been venesected) and also for patients with African iron overload.⁹¹ In contrast, lower mean values of sTfR were found in subjects with a raised transferrin saturation.^92,⁹³ However, there is considerable overlap with the normal range of sTfR concentration and measurement of sTfR in iron overload is unlikely to be of diagnostic value.

Erythrocyte protoporphyrin

Protoporphyrin IX is the immediate precursor to haem. The enzyme ferrochelatase is able to insert ferrous iron to produce haem or zinc cation to form zinc protoporphyrin (ZPP). When iron supply to ferrochelatase is limiting, ZPP increases. When ferrochelatase is limiting, free protoporphyrin accumulates. Different assays differ in the extent to which they measure and/or discriminate between these two forms of erythrocyte protoporphyrin (EP). Assay of EP ⁹⁴ has been performed for many years as a screening test for lead poisoning. More recently, there has been considerable interest in its use in evaluating the iron supply to the bone marrow. The protoporphyrin concentration of red blood cells increases in iron deficiency. Usually, more than 95% is present as ZPP. The original method converts zinc to free protoporphyrin, requires a chemical extraction and use of a fluorescence spectrometer.⁹⁴ This has now been largely replaced for detection of iron deficiency by the direct measurement of the fluorescence of ZPP (μmol/mol haem) in an instrument called a haematofluorometer.²⁴

Analysers

Two dedicated analysers are available: the Proto Fluor Z from Helena Laboratories, Beaumont, Texas (www.helena.com) and the ZPP 206D from Aviv Biomedical, Inc (www.avivbiomedical.com). These should be operated exactly as described by the manufacturer. The small sample size (about 20 μl of venous or skin-puncture blood), simplicity, rapidity and reproducibility within a laboratory are advantages. Furthermore, the test has an interesting retrospective application. Because it takes weeks for a significant proportion of the circulating red blood cells to be replaced with new cells, it is possible to make a diagnosis of iron deficiency anaemia some time after iron therapy has commenced. Chronic diseases that reduce serum iron concentration, but do not reduce iron stores, also increase protoporphyrin levels.⁹⁵

Diagnostic Applications

The measurement of EP levels as an indicator of iron deficiency has particular advantages in paediatric haematology and in large-scale surveys in which the small sample size and simplicity of the test are important. The normal range in adults is less than 70 μmol/mol haem. Mean values in normal women are slightly higher than in men.⁹⁶ One potential confounder is the contribution of other fluorescent compounds (including drugs) in the plasma and concentrations are lower if washed red cells are assayed.⁸⁴ However, washing is a tedious process and is rarely undertaken. Paediatric reference ranges have been determined in 6478 subjects (ages 0–17 years).⁹⁷ Mean ZPP values were higher in females than males and declined slightly with age. A diurnal variation was noted, with ZPP concentrations being higher between 18.00 h and midnight. No explanation was offered.

The WHO ³⁹ has recommended levels for the detection of iron deficiency. For children younger than age 5 years, levels should be >61 μmol/mol haem; for all other subjects, levels should be >70 μmol/mol haem. These are higher than the 97.5 percentile established from surveys of healthy children⁹⁷ and are based on the sensitivity and specificity for detecting the absence of storage iron.

Units

To convert between the various units used to express protoporphyrin levels, the following calculations apply:

μg EP/dl red cell = (μg EP/dl whole blood)/haematocrit

From μg EP/dl red cell to μg EP/g Hb multiply by 0.037

From μg EP/dl red cell to μmol EP/mol haem multiply by 0.87.

These factors are based on an assumed normal mean cell haemoglobin concentration, although this may be measured in individual samples and an appropriate factor calculated.

Infection and inflammation, lead poisoning and haemolytic anaemia all cause significant elevation of ZPP. Measurement of ZPP is most useful when iron deficiency is common and the other conditions are rare. In the general clinical laboratory, therefore, ZPP provides less information about iron storage levels in patients with anaemia than does the serum ferritin assay.⁹⁸

Although blood samples may be taken at any time of day, fresh blood is required (samples must not be frozen) and the method has not been automated.

Hepcidin

Reliable assays for serum hepcidin have now been developed but their role in the differential diagnosis of both iron deficiency and iron overload is not yet clear although a number of useful diagnostic applications are being investigated.⁹⁹ An international survey of urinary and plasma hepcidin assays demonstrated that absolute concentrations vary widely. However, analytical variation was generally low and similar for the six methods included in the study.¹⁰⁰

Methodological and biological variability of assays

The blood assays vary greatly in both methodological and biological stability. Haemoglobin concentrations are stable and a simple and well-standardized method ensures relatively low day-to-day variation in individuals (Table 9.7). Automated cell counters analyse at least 10 000 cells and thus increase precision. ZPP values also appear to be relatively stable. The more complicated procedures involved in immunoassays result in higher methodological variation for serum ferritin assays (CV of about 5%) and this, coupled with some physiological variation, gives an overall CV for serum ferritin for an individual over a period of weeks of the order of 15%. There is, however, little evidence of any significant diurnal variation in serum ferritin concentration.⁷⁴ On the other hand, the serum iron determination has a reasonably low methodological variation coupled with extreme physiological variability, giving an overall ‘within subject’ CV of approximately 30% when venous samples are taken at the same time of day. A diurnal rhythm has been reported with higher values in the morning than in late afternoon, when the concentration may fall to 50% of the morning value.⁴⁴ However, variations are not consistently diurnal.¹⁰¹ The circadian fluctuation is largely the result of variation in the release of iron from the reticuloendothelial system to the plasma. It should be noted that for the studies summarized in Table 9.7 the type of blood sample, length of study period and statistical analysis vary. The somewhat higher variability for Hb and ferritin reported by Borel et al.⁸¹ may be a result of their use of capillary blood and plasma. Pootrakul et al.¹⁰² have demonstrated that mean plasma ferritin concentration is slightly higher in capillary specimens than venous specimens and that within- and between-sample variation was approximately three-fold greater. Variability was less for capillary serum than plasma but still greater than for venous serum. However, the increased variability of capillary samples may be related to sampling technique, because Cooper and Zlotkin⁸³ found little difference in variability between venous and capillary samples.

The effect of menstruation on iron-status indicators was examined in 1712 women aged 18–44 years after adjusting for potential confounders.¹⁰³ Adjusted mean values of Hb, transferrin saturation and serum ferritin were lowest for women whose blood was drawn during menses and highest for women examined in luteal or late luteal phase of the menstrual cycle (Hb, 13.0 versus 13.3 g/dl; transferrin saturation, 21.2% versus 24.8%, p<0.01 for both; serum ferritin, 17.2 versus 24.0 μg/l, p<0.05). The prevalence estimate of impaired iron status was significantly higher for women whose blood was drawn during the menstrual phase than for women whose blood was drawn during the luteal and late luteal phases. The authors concluded that cyclic variations in indicators of iron status are a potential source of error when iron status is assessed in large population surveys that include women of reproductive age.¹⁰³

Starvation, or even fasting for a short period, can cause elevation of the serum ferritin concentration,¹⁰⁴ and vitamin C deficiency can reduce ferritin concentration.¹⁰⁵ Moderate exercise has little effect on serum ferritin concentration,¹⁰⁶ although exhausting exercise leads to increases in serum ferritin concentration as a result of muscle damage and inflammatory reactions.^107,¹⁰⁸ Seasonal changes in red cell parameters have been reported¹⁰⁹ and Maes et al.⁸² found statistically significant seasonal patterns for serum iron, transferrin, serum ferritin and sTfR. The peak–trough difference in the yearly variation, expressed as a percentage of the mean, was greatest for serum ferritin (39%) and least for sTfR and transferrin (12%).

These results have clear implications for the use of these assays in population studies. For accurate diagnosis, either a multiparameter analysis is required or several samples should be assayed. For haemoglobin, one sample was required for 95% confidence and 20% accuracy;¹¹⁰ for sTfR, one sample was required,^83,¹¹¹ but for ferritin one to three samples were required.^83,¹¹⁰

Predictive value of blood tests for iron deficiency

The major diagnostic use of the various measures of iron status is in the differential diagnosis of hypochromic anaemia. The large amount of iron present as haemoglobin means that the degree of any anaemia must always be considered in assessing iron status. There is an overall reduction in body iron in iron deficiency anaemia. No single measurement of iron status is ideal for all clinical circumstances because all are affected by confounding factors (Table 9.3). The anaemia of chronic disorders is associated with normal or increased iron stores (normal or increased serum ferritin), accompanied by a reduced tissue iron supply (low serum iron and low to normal TIBC). Although the serum ferritin is an acute-phase reactant, values below 50 μg/l are usually associated with absent iron stores in rheumatoid arthritis, renal disease and inflammatory bowel disease. Serum transferrin receptor levels may also provide valuable diagnostic information on iron deficiency in chronic disease.

Despite years of investigations, there is little definitive evidence of how different measurements compare in their ability to diagnose iron deficiency. The main reason for this is the difficulty of distinguishing between the presence and absence of storage iron. Most investigators have used the grade of storage iron in the bone marrow as a ‘gold standard’. This is an invasive procedure and therefore limits drastically the number of patients investigated. It is often difficult to justify bone marrow aspiration to determine a patient’s iron status and even more difficult in the case of normal volunteers. Furthermore, bone marrow aspiration followed by staining for iron is not a reproducible procedure. Observer error,¹¹² inadequate specimens and lack of correlation with response to iron therapy¹¹³ have been described. Demonstrating a response in Hb to oral iron therapy has been the method of choice to diagnose iron deficiency retrospectively in paediatric practice.

Iron Deficiency Anaemia in Adults

Almost all measures show a high sensitivity and specificity for distinguishing between subjects with iron deficiency and those with iron stores and normal haemoglobin levels in the absence of any other disease process. Guyatt et al.¹¹⁴ conducted a systematic review of the diagnostic value of the various laboratory tests for iron deficiency. They concluded that serum ferritin was the most powerful test for simple iron deficiency and also for iron deficiency in hospital patients. However, this analysis did not include measurements of sTfR.

Detection of Iron Deficiency in Acute or Chronic Disease

Table 9.8 summarizes a number of studies in which bone marrow iron was assessed and the sensitivity and specificity of various assays was compared. Despite very different results between studies, some general points may be made.

Table 9.8 Sensitivity/specificity of methods for diagnosis of iron deficiency in the presence of chronic disease

Conventional red cell parameters, mean cell volume (MCV) and mean cell haemoglobin (MCH), do not distinguish between the presence or absence of bone marrow iron in patients with chronic disease. The serum iron concentration is almost invariably low in chronic disease and, although the TIBC (or transferrin concentration) is higher for patients with no storage iron, neither this measurement, nor the transferrin saturation derived from the serum iron and TIBC, provides useful discrimination.

In chronic disease serum ferritin concentrations reflect storage iron levels but are higher than in normal subjects with the same amount of storage iron. It is necessary to set a threshold of 30–50 μg/l to distinguish between the presence and absence of storage iron. Even with this limit sensitivity is low.

Combinations of serum ferritin, erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) either in a discriminant analysis ¹¹¹ or logistic regression¹²² provide only marginal improvement in the ability to detect a lack of storage iron.

The serum transferrin receptor level discriminates between the presence and absence of storage iron, although there is disagreement as to whether the assay is superior to serum ferritin. Several studies show that the sTfR/log₁₀ ferritin ratio provides superior discrimination to either test on its own. The use of log ferritin decreases the influence of serum ferritin (and thus the acute-phase response) on the overall ratio. Although the log[sTfR/ferritin] is an excellent measure of iron stores in healthy subjects,⁷² this transformation (i.e. after calculating the ratio of sTfR/ferritin) may not provide the best discrimination for clinical applications. When the assay of sTfR is readily available on high-throughput immunoanalysers the sTfR/log ferritin ratio may provide the best discriminator for identifying the coexistence of iron deficiency in chronic disease. However, this will also require standardization of units and ranges for the various sTfR assays if the use of the ratio is to gain wide acceptance.

Functional Iron Deficiency

Functional iron deficiency is the situation in which iron stores are apparently adequate but iron supply for erythropoiesis remains inadequate. This often occurs during the treatment of patients with anaemia and renal failure with erythropoietin. The diagnostic question is to identify those patients with a functional iron deficiency who will require parenteral iron therapy to respond to erythropoietin with an acceptable increase in Hb. The percentage hypochromic erythrocytes is a good predictor of response.¹²³ Fishbane and colleagues¹²⁴ concluded that reticulocyte haemoglobin content (CHr) was a markedly more stable analyte than serum ferritin or transferrin saturation and it predicted functional iron deficiency more efficiently. They did not include percentage hypochromic cells in their analysis. Fernandez-Rodriguez and colleagues¹²⁵ assessed the sensitivity and specificity of ferritin, TIBC, transferrin saturation index, red blood cell ferritin and sTfR in 63 patients with anaemia and chronic renal failure undergoing dialysis, who were not being treated with erythropoietin. Storage iron was assessed by bone marrow iron staining. For serum ferritin, a cut-off value of 121 μg/l gave a sensitivity and specificity of 75%. Efficiency was lower for sTfR and RBC ferritin. MCV, transferrin saturation index and TIBC showed the lowest values for sensitivity and specificity.

Iron Deficiency in Infancy and Childhood

In infants, thresholds for the diagnosis of iron deficiency and iron deficiency anaemia are not universally agreed. There are rapid changes in iron status in the first year of life as fetal haemoglobin is replaced by adult haemoglobin. The serum ferritin concentration is a less useful guide to iron deficiency than in adults partly because of the rapid decline in concentration in the first 6 months and the low concentrations generally found in children older than 6 months of age. Domellof et al.¹²⁶ have suggested revised cut-offs for iron deficiency, including serum ferritin and sTfR, for infants up to age 1 year.

In children, the reason for detecting iron deficiency is to identify those who will respond to iron therapy. Margolis et al.¹²⁷ found that the best predictor of response was the initial Hb, although sensitivity was only 66% and specificity was 60%. Serum ferritin, transferrin saturation and erythrocyte protoporphyrin (EP) had even lower efficiencies and combination of the various measures made little improvement. Hershko et al.¹²⁸ studied children in villages from the Golan Heights (Israel) and concluded that EP was a more reliable index of iron deficiency than serum ferritin. They suggested that a significant incidence of chronic disease affected both ferritin and iron values. ZPP provides a useful indicator of iron-deficient erythropoiesis, although high values may indicate lead poisoning rather than iron deficiency. The small sample volume for ZPP determination is also an advantage in paediatric practice.

A report published in 2003 confirms the effect of low-level infection on measures of iron status. Abraham et al.¹²⁹ studied 101 healthy, 11-month-old infants. On the morning of blood sampling, slight clinical signs of airway infection were observed for 42 infants. Extensive blood analyses were done, including a high sensitivity one for C-reactive protein (CRP). CRP measured by the routine methods gave values of <6 mg/l for all infants, but with the high sensitivity assay values were higher for many infants with symptoms of airway infection. Serum iron concentration was depressed in these children and correlated significantly with CRP level. When a further blood sample was taken, serum ferritin concentration was higher for the children with the higher CRP level, serum iron was reduced, but sTfR and transferrin levels were unaffected.