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Beyond soluble transferrin receptor: Old challenges and new horizons
Best Practice & Research Clinical Endocrinology & Metabolism 29 (2015) 799e810
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Beyond soluble transferrin receptor: Old challenges and new horizons Kristian Harms, MD, Laboratory Medicine Resident Physician *, Thorsten Kaiser, MD, Laboratory Medicine Senior Physician Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany
a r t i c l e i n f o Article history: Available online 30 September 2015 Keywords: transferrin receptor iron metabolism disorders anaemia inflammation erythropoiesis hepcidin
Disturbances of iron metabolism are a frequent challenge in outpatient and inpatient care. Although several established biomarkers are commonly used by clinicians for differential diagnosis, the discrimination between latent or classic iron deficiency, anaemia of chronic disease or a combination of functional iron deficiency (iron-restricted erythropoiesis) with anaemia of chronic disease in patients affected by inflammatory disease can be demanding. Soluble transferrin receptor (sTfR) is a cleaved monomer of transferrin receptor 1 and correlates positively with tissue iron deficiency as well as with stimulated erythropoiesis. The ratio between sTfR and ferritin in combination with reticulocyte haemoglobin content further helps to identify different states of iron deficiency. In this review, we will focus on biological aspects of iron metabolism and sTfR, established clinical applications and limitations of sTfR and derived indices, and prospects of future research and applications. © 2015 Elsevier Ltd. All rights reserved.
Introduction The presence of iron is essential for several physiological and metabolic pathways. As surplus iron can result in cellular damage, iron homeostasis is subject to complex and tightly controlled regulation [1]. Furthermore, iron deficiency is known as the main cause of chronic anaemia worldwide, with an
* Corresponding author. Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Paul-List-Straße 13-15, 04103 Leipzig, Germany. Tel.: þ49 341 97 22458; Fax: þ49 341 97 22209. E-mail address: [email protected] (K. Harms). http://dx.doi.org/10.1016/j.beem.2015.09.003 1521-690X/© 2015 Elsevier Ltd. All rights reserved.
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estimated frequency of up to 15% in industrialized countries and 2 billion people worldwide [2]. Control of iron metabolism depends on several key elements related to acquisition, transport, utilization and storage of iron [3]. In plasma, iron is bound by transferrin and delivered into cells after interaction of the diferric ironetransferrin complex with the cell surface transferrin receptor 1 (TfR1) and subsequent internalization of ironetransferrineTfR1 via endocytosis. After release of iron in the acidic milieu (acidosome), iron is used or bound by ferritin (cellular iron storage), whereas the transferrineTfR1 complex is externalized and truncated, resulting in the appearance of a cleaved monomer of TfR1 (soluble transferrin receptor [sTfR]) [4]. Increased serum concentrations of sTfR are indicative of tissue iron deficiency and stimulated erythropoiesis [5]. Stimulated erythropoiesis is associated with haemolysis, ineffective erythropoiesis, or may be caused by therapeutic application of erythropoietin. In many uncomplicated cases, the determination of a low ferritin level is sufficient for a diagnosis of iron deficiency. Ferritin levels are increased in the presence of inflammation. Additional parameters independent of acute-phase response are, therefore, necessary. Under this condition, sTfR is considered a valuable, but not unrestricted, parameter for the differentiation of iron-deficient anaemia (IDA) and anaemia of chronic disease (ACD). New parameters and sTfR-derived indices have therefore been developed to help physicians to diagnose iron deficiency in complex clinical settings, with the peptide hormone hepcidin currently emerging in addition. Establishing a diagnosis of IDA with or without concurrent ACD is crucial, as iron deficiency may be a symptom of a potentially serious disease. Menstrual blood loss, gastrointestinal bleeding caused by malignancy or ulceration and iron malabsorption are typical differential diagnoses of iron deficiency [5,6]. Iron pools, TfR1 and the ironeTfeTfR1 cycle More than two-thirds (>2 g) of total body iron (3e5 g) is allocated to the haemoglobin of erythroid precursor cells and mature red blood cells. Even though only a small fraction (<0.1%, approximately 3 mg) of body iron is bound by plasma transferrin, this pool is considered the most active. To fulfil a daily requirement of at least 20e30 mg of iron for erythropoiesis, a plasma transferrin turnover occurs about 10 times a day. Transferrin has a high affinity for iron (Kd ¼ 1023 M), and, under physiological conditions, a transferrin saturation of about 30% of available iron binding sites is obtained. Major factors influencing transferrin saturation are dietary iron absorption in the duodenum and the upper part of the jejunum (physiologically only about 1e2 mg/day), recycled iron released by reticuloendothelial macrophages after phagocytosis of senescent erythrocytes (major source of plasma iron), and the amount of iron used (especially by bone marrow erythroblasts, >80% of transferrin-bound iron). Transferrin is mainly expressed in the foetal and adult liver, but small amounts arise also in the testes, brain, spleen and kidneys [3,7e9]. TfR1 (CD71) is the primary target of transferrin in the iron transport system, and is expressed as a type II transmembrane glycoprotein composed of a disulfide-bonded homodimer on the surface of nearly every cell type [10]. The receptor subunits are joined by two disulfide bonds at cysteines 89 and 98 [11] (Fig. 1). In humans, the TfR1 gene is located on human chromosome 3q29 [12,13]. High levels of TfR1 expression can be found in cells with increased proliferation rate, such as erythroid progenitor cells, placental syncytiotrophoblasts, and neoplastic cells [3,8]. Disruption of the TfR1 gene in mice leads to a severely impaired phenotype, affecting both erythropoiesis and neurologic development [14]. Each polypeptide subunit of the 90-kDa TfR1 protein containing 760 amino acids includes an Nterminal cytoplasmic domain, a single-pass transmembrane domain, and a large C-terminal extracellular domain, which includes a short glycosylated stalk region and three subdomains: protease-like, apical, and helical [10,15,16]. TfR1 belongs to the transferrin receptor/glutamate carboxypeptidase II family which, in addition to TfR1, includes transferrin receptor 2 (TfR2), folate hydrolase (prostatespecific membrane antigen) 1, N-acetylated alpha-linked acidic dipeptidase-like 1, N-acetylated alpha-linked acidic dipeptidase 2, and folate hydrolase 1B [10,15]. In its extracellular domain with TfR1, TfR2 shares a 45% identity and 66% similarity [17]. Compared with TfR1, the role of TfR2 in the uptake of transferring-bound iron into the cells is considered less significant, as TfR2 has a 25-fold lower affinity for iron-loaded (holo)transferrin than its homologue TfR1. In addition, the TfR2 mRNA expression pattern differs between both receptors. In contrast to TfR1, TfR2 shows much higher expression levels
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Fig. 1. Transferrin receptor 1 (transmembrane and soluble form).
in liver compared with other tissues. Mutations in the TfR2 gene mapping to 7q22 causes hereditary haemochromatosis type 3, a disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, especially the liver [18e22]. Two molecules of holo-transferrin are bound by the TfR1 dimer, and the resulting complex is internalized through the process of endocytosis via clathrin-coated pits [3,8,23] (Fig. 2). The release of iron from transferrin requires acidification (around pH 5.5) of the endosome [3,8,24]. Transferrin affinity to TfR1 depends on the iron status of transferrin; the highest affinity can be attributed to diferric transferrin, which is characterized by a 30-fold higher affinity to TfR1 compared with monoferric transferrin and a 500-fold higher affinity compared with non-iron-loaded (apo)transferrin [3,8,25]. After reduction of liberated insoluble ferric (Fe3þ) to soluble ferrous (Fe2þ) iron, iron is transported through the acidosomal membrane to the cytosol via DMT1 (divalent metal transporter 1 or, natural
Fig. 2. Iron metabolism with an emphasis on transferrin receptor 1 and soluble transferrin receptor.
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resistance-associated macrophage protein 2), where it can be used for metabolic functioning or can be stored in ferritin. Finally, the transferrin-TfR1 cycle is completed through return of the apo-transferrineTfR1 complex to the cell membrane where apo-transferrin is released from the cells [3,8,26,27] (Step 5a of Fig. 2). On a post-transcriptional level, cellular iron homeostasis is regulated by an interplay of ironresponsive elements (IRE) with iron regulatory proteins (IRP1 and IRP2). Iron-responsive elements constitute phylogenetically conserved hairpin structures within the untranslated regions (UTRs) of mRNAs encoding for key regulatory proteins of iron metabolism. The consequence of iron deficiency is the binding of IRPs to a single IRE in the 50 UTR of an mRNA, which leads to the repression of the translation and thus the reduction of the amount of the corresponding proteins in the cell. Examples of regulated proteins in this way, among others, are the iron storage protein ferritin, 50 -aminolevulinate synthase 2 (heme biosynthesis), ferroportin (mediates iron cell release to the plasma) and hypoxia inducible factor 2a. The binding of IRPs to multiple IREs in the 30 UTR of an mRNA leads to their stabilization. Therefore, the translation of mRNA lasts longer and the cell concentrations of the corresponding proteins increase. Examples of proteins regulated in this manner are TfR1 and DMT1. Taken together, as a consequence of iron deficiency, the regulation by the IREeIRP system leads to a decreased iron storage via ferritin and efflux via ferroportin as well as impaired heme biosynthesis, whereas cellular iron uptake and transport via TfR1 and DMT1 are supported. Iron excess leads to a reciprocal regulation of this system [3,28]. In contrast to this, the TfR2 transcript does not contain 30 UTR-IREs; therefore, TfR2 mRNA and protein levels vary little with changes in iron status [21,22]. IRPs are necessary for regulation of mammalian iron homeostasis. A complete loss of both proteins (IRP1 and IRP2) leads to embryonic lethality in mice [29]. Soluble transferrin receptor: biology and clinical applications In 1983, Pan and Johnstone [30,31] demonstrated the release of the transferrin receptor in vesicular form into culture medium during maturation of sheep reticulocytes. In addition, a polyclonal antibody against the transferrin receptor slowed down the release of the vesicles bearing the receptor, whereas transferrin accelerated vesicle release [32]. Electron microscopic analysis revealed large multivesicular bodies (MVBs, 1e1.5 mm) fusing with the plasma membrane and thus releasing their contents (50-nm bodies, exosomes) via shedding into the medium [33,34] (Step 5b of Fig. 2). In 1986, Kohgo et al. [4] described a truncated form of human TfR1 in human serum of healthy people and people with haematological malignancies (including acute leukaemia, multiple myeloma and malignant lymphoma). It could be shown by amino-terminal amino acid sequence analysis that residues 1e19 of serum receptor were identical to residues 101e119 of intact receptor. On the basis of this finding, the serum receptor was identified as a truncated monomer lacking the cytoplasmic and transmembrane domains (residues 1e100) of the intact receptor (Fig. 1). This cleaved fragment, which circulates in the form of a complex of transferrin and its receptor, has a molecular weight of 85 kD [35]. Production of sTfR is mediated by a membrane-associated serine protease [36], which has recently been identified as proprotein convertase subtilisin/kexin type 7 [37]. Proteolysis of TfR predominantly occurs from exosomes rather than the cell surface itself [38]. Furthermore, using surface 125I-labelled sheep reticulocytes as the experimental model, it was shown that, during invitro maturation of these cells, 125I-TfR of native size appears in exosomes (around 75%) before the soluble, truncated, exofacial domain of the receptor can be detected in the medium (around 25%, not exosome-associated). It was concluded that exosome formation is the major, if not the sole, route for shedding TfR, and that sTfR originates from exosomes followed by cleavage from the surface of exosomes [39]. In normal human sera, less than 1% of transferrin receptor is intact receptor. Consequently, almost all circulating transferrin receptor is the truncated monomeric form [40]. Recently, iron-dependent cleavage of cell surface TfR2 and release of a soluble form of this receptor (sTfR2) could also be described [41]. Tests used in clinical laboratories for measurement of sTfR are mostly latex-enhanced immunonephelometric/turbidimetric assays [5]. Challenges are the lack of international standards with different units and reference ranges, differing age and gender dependencies, and poor agreement among different kits [5,42e45]. The prospect of a recombinant sTfR World Health Organization
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reagent should help to resolve these issues [5,46]. One study showed age-related reference ranges among children (6 months to 8 years) with a slight decrease of upper and lower reference limits with increasing age [42]. Although its biological function remains unexplained, sTfR has been proposed as a marker of iron deficiency, total erythropoietic activity, or both [41]. Bone marrow erythropoiesis can cause variations up to eight times below and up to 20 times above average reference values. For this reason, it is considered the most important determinant of sTfR levels [47]. Patients with iron deficiency and autoimmune haemolytic anaemia present higher sTfR levels, whereas lower levels are detected in patients with aplastic anaemia [48]. In support of this finding, an inverse relationship between iron status and sTfR was also found in rat plasma, whereas a direct relationship existed between erythropoiesis and sTfR. It was assumed that the number of tissue receptors, mostly on erythroid cells, determines the number of sTfR [49]. In human plasma and by additional in-vivo experiments in rats, these observations of serum sTfR as a quantitative marker of the body mass of tissue TfR and regulation of TfR numbers particularly by iron deficiency and erythropoietin stimulation could be confirmed [50e52]. Even though the erythroblast compartment, not reticulocytes, constitutes the main source of sTfR, circulating reticulocytes can contribute significantly to serum sTfR levels, as shown by hypertransfusion with normal, reticulocyte-poor and reticulocyte-rich blood [52]. In summary, clinical applications of sTfR measurements in patients are comprehensive: soluble TfR levels are decreased in conditions characterized by erythroid hypoplasia, such as hypertransfusion, chronic renal failure, aplastic anaemia, or after intensive chemotherapy. Vice versa, sTfR levels are increased when erythropoiesis is stimulated, as seen in congenital dyserythropoietic anaemia, (autoimmune) haemolytic anaemia, hereditary spherocytosis, sickle cell anaemia, thalassaemia major or intermedia, megaloblastic or iron deficiency anaemia, and secondary polycythaemia. In patients with malignancies, with the possible exception of non-Hodgkin's lymphoma, chronic lymphocytic leukaemia and hepatocellular carcinoma, sTfR levels are not increased [47]. Furthermore, soluble TfR is suitable for monitoring erythropoietic response to various forms of treatment, such as the correction of megaloblastic anaemia with cobalamin, of iron deficiency anaemia with iron, or the recovery of erythropoiesis after renal transplantation [47,53e55]. The application of recombinant human erythropoietin is characterized by an extensive range of indications, and sTfR measurements can provide valuable information regarding response to recombinant human erythropoietin treatment [47]. For instance, sTfR levels are useful in assessing recombinant human erythropoietin response during treatment of chronic renal failure [56,57], aplastic anaemia [58], cancer [59], myelodysplastic syndrome [60], and in intensive care [61]. Diagnostical use of sTfR in a multiparameter approach Early recognition of latent subclinical iron deficiency is important to prevent systemic complications [62]. For instance, the most important systemic abnormality produced by iron deficiency in infancy is the alteration in cognitive performance, which might not be fully reversible [63]. Therefore, efforts have been made to further improve the diagnostic value of sTfR by developing multi-parameter interpretation aids. Skikne et al. [64] used a serial phlebotomy procedure in six men and eight women. They underlined the significance of sTfR measurements for the analysis of iron metabolism, especially combined with ferritin and haemoglobin measurements. Therefore, it is assumed that the iron status of patients without systemic inflammation can be fully assessed by using serum ferritin as a measure of iron stores, serum receptor as a measure of mild (functional) tissue iron deficiency, and haemoglobin concentration as a measure of advanced iron deficiency (iron deficiency anaemia). As a result of the reciprocal relationship between serum receptor and ferritin measurements, the logarithmic ratio of sTfR/ferritin portrays body iron in a linear fashion, showing an increased ratio from less than 100 in those with abundant iron stores to over 2000 in those with significant functional iron deficiency. A rise above 500 occurred when stores were fully depleted [64]. In contrast, on the basis of a study with a patient population consisting of 129 consecutive anaemic patients, Punnonen et al. [65] showed that sole calculation of the sTfR/ferritin ratio does not considerably improve diagnostic efficiency compared with sTfR alone. Logarithmic transformation of the
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ferritin values and calculation of the sTfR/log ferritin ratio (sTfR-F index, ferritin index), however, provided an outstanding indicator of iron depletion, as shown by analysis of receiver operating characteristic (ROC) curves (area under curveROC about 1.00). Therefore, by applying the ferritin index, the combination of sTfR and ferritin measurements provided the highest sensitivity and specificity compared with isolated measurements of sTfR and ferritin [65]. In another study, an attempt was made to further characterize the different stages of iron deficiency by means of serum ferritin, sTfR and ferritin index. A population sample of 65 apparently healthy nonanaemic adults (22 men and 43 women) was given oral iron supplementation for 3 months to produce an iron-replete population. A relatively large number of women of childbearing age were intentionally enrolled to permit inclusion of a larger proportion of potential participants with subclinical iron deficiency. A supplementation-induced change of sTfR, ferritin, and ferritin index did not occur in men, but, in a significant proportion of women, 25 were retrospectively judged to have had storage iron depletion (stage I) or depletion of the functional compartment (stage II). These changes were made most apparent by using the ferritin index, indicating that the non-anaemic stages of iron deficiency are readily detectable using this index. Especially in borderline cases, when the results of sTfR and ferritin assays are ambiguous, it is suggested that the ferritin index is more sensitive in the detection of irondeficient states [66]. Soluble transferrin receptor and inflammation Historically, bone marrow examination, along with Perls' Prussian blue stain, has been considered as the golden standard for deciding whether iron deficiency plays a role in the origin of anaemia, especially in cases of infection, chronic disease, malignancy or liver disease [67,68]. Hence, clinical demand for enabling a non-invasive distinction between IDA, which is due to a treatable cause of anaemia (e.g. bleeding, iron malabsorption or dietary lack of iron) and other entities in the differential diagnosis of anaemia is high. For the latter, this accounts especially for anaemia that accompanies infection, inflammation and cancer (ACD) or a combination of functional iron deficiency (iron-restricted erythropoiesis) with ACD [69]. Furthermore, it is also crucial to avoid overtreatment of patients with sufficient iron homeostasis, as iron excess poses a threat to cells and tissues owing to its ability to catalyse the generation of radicals, which attack and damage cellular macromolecules and promote cell death and tissue injury. In addition, most bacteria and virtually all pathogens have a significant iron requirement, which is essential for their growth [1,70]. Standard biochemical markers of iron metabolism show restricted validity under the influence of an acutephase response, with ferritin being an acute-phase reactant and transferrin a negative one [69]. Determination of sTfR has been proposed to identify IDA in patients with complicated anaemia affected by concurrent inflammatory disease that may lead to false-positive ferritin concentration [47,71]. Accordingly, levels of sTfR are not increased in patients with acute or chronic disorders (e.g. malaria, inflammatory disorders, acute hepatitis or chronic liver disease) [72,73]. Otherwise, elevated sTfR levels in patients with anaemia of chronic disease or acute inflammation can indicate true iron deficiency, which decrease after adequate iron supplementation, indicating functional iron deficiency [47,74]. Conflicting reports have been published on the effect of sTfR on differential diagnosis of complicated anaemia, which might be caused by heterogeneous study populations with different underlying mechanisms for iron-deficient erythropoiesis [5,47,71]. In general terms, the interpretation of individual sTfR values may be complicated in a patient in whom both changes in erythropoietic activity and iron status, both known to influence sTfR levels, occur simultaneously [47]. Accordingly, in other studies related to rheumatoid arthritis [75,76] or other inflammatory disorders [67], the contribution of sTfR measurements to determine presence or absence of coexistent functional iron deficiency was limited. Compared with patients with IDA, lower sTfR in patients with ACD (e.g. rheumatoid arthritis patients) may indicate a reduced erythropoietic activity [75]. Surprisingly, even cases of sTfR elevation in patients with normal or increased bone marrow iron have been described, lacking an alternative explanation for an increase (e.g. erythroid hyperplasia) [67,77]. These diagnostic difficulties have led to efforts to develop clinical laboratory tests that are capable of measuring functional iron availability at the site of haemoglobin synthesis in the erythron. Accordingly, the measurement of the proportion of
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hypochromic red cells has been shown to be of particular interest. Because of erythrocytes have a lifespan of about 120 days, hypochromic red cells constitute a late indicator of iron-restricted erythropoiesis, providing information over a several-month period. Furthermore, reticulocyte haemoglobin content has emerged as an early marker of functional iron deficiency, as reticulocytes exist in the circulation for only 1e2 days [69,78,79]. Thomas et al. [69,80] have shown that the relationship between functional iron deficiency (reticulocyte haemoglobin content) and iron supply to erythropoiesis (ferritin index, see above) can be represented in a diagnostic plot (ferritin index plot, Thomas plot) that corresponds to four different states of iron metabolism: ACD, latent iron deficiency, classic IDA, and ACD with concomitant functional iron deficiency/iron-restricted erythropoiesis (Table 1). As a consequence, the diagnostic plot can be used to derive therapeutic implications and to monitor response to erythropoietin therapy [69,80]. Soluble transferrin receptor and hepcidin: quo vadis? In recent years, more insight into the pathophysiology of ACD has been gained. In general, ACD results from activation of the immune and inflammatory systems, and is characterized by inadequate production of erythropoietin, inhibition of the proliferation of erythroid progenitor cells in the bone marrow, and disturbances in iron distribution [81]. In IDA, the iron supply depends on the amount of the iron stores, whereas in ACD, the supply depends on its rate of mobilization, with functional ID potentially occurring even in the presence of large iron stores when iron release is impaired [69]. Therefore, the relationship between iron status and sTfR levels in patients with inflammation depends on the severity of inflammation, the degree of anaemia, the adequacy of erythropoietin production, and the effect of cytokines on marrow activity [47]. Much effort has been made to further decipher the molecular mechanisms controlling iron homeostasis, and it has become evident that the peptide hormone hepcidin has emerged as the main regulator of iron recycling and iron balance, leading to speculation that excess of hepcidin may be the key pathogenic feature of ACD. By binding to the cellular iron exporter ferroportin and inducing its internalization and degradation, iron is trapped in enterocytes, macrophages, and hepatocytes, resulting in a reduced absorption of dietary iron, sequestration of iron in macrophages, and sequestration of iron in hepatic stores [82,83]. The bioactive form of hepcidin (peptide of 25 amino acids, hepcidin-25) is primarily produced in the liver, where its synthesis is stimulated by iron excess and inflammation and inhibited by anaemia and hypoxia [82,84]. Given that hepcidin-25 constitutes a pivotal role for systemic iron homeostasis, the prospect of further improving the classification of different iron states using this potential biomarker was recently addressed. In a single-centre study with 155 anaemic patients, patients with IDA could be differentiated from those with ACD and ACD/IRE, but not ACD from ACD/IRE based on hepcidin-25 alone. As hepcidin-25 showed a significant correlation with ferritin and the ferritin index, with most of the patients having normal to elevated ferritin concentrations but nevertheless suffering from iron deficiency or having iron-restricted erythropoiesis, it was concluded that hepcidin-25 is not a marker of iron-restricted erythropoiesis, but rather of iron supply and retention. The combination of hepcidin-25 with reticulocyte haemoglobin content CHr (hepcidin-25 plot) was useful for the differentiation of the iron states mentioned above. In addition, as hepcidin-25 is a real-time marker for the iron supply of erythropoiesis (responds within hours to changes in haematologic status) but the ferritin index a timedelayed marker (responds within days), this peptide might have the potential to eventually replace the sTfR-related ferritin index in the Thomas plot. This is further supported by the limitations of the ferritin index in some diseases (CLL) and the dual influence of erythropoietic activity and inflammation on sTfR and ferritin measurements [85e87]. Recent data confirm the limitation of sole measurement of hepcidin-25 to predict functional iron deficiency, with the Thomas plot as well as sTfR being better predictors of functional iron deficiency in patients with and without acute-phase response compared with hepcidin-25 and ferritin [88]. To further investigate the validity of hepcidin-25 for differential diagnosis of iron metabolism, important issues need to be addressed. At first, the standardization of hepcidin assays with appropriate reference material and suitable procedures is essential for valid interpretation of the results. Finally,
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State
1
3
<3.2 (2.0)
sTfR/log ferritin
>3.2 (2.0)
Iron supply [
Interpretation
No iron deficiency (iron repletion) Normal erythropoiesis Normal haemoglobinization About 80% of patients with ACD Patients with cancer-related anaemia, acute and chronic inflammation, end-stage renal failure
4 <28 pg (~1.74 fmol)
Iron demand by erythropoiesis Y (no functional iron deficiency)
Interpretation
State and patient characteristics
2 >28 pg (~1.74 fmol)
Reticulocyte haemoglobin content
Iron supply Y
Latent iron deficiency Reduced iron supply Erythropoiesis still normal Normal haemoglobinization Anaemic or non-anaemic patients with latent iron deficiency Patients with iron deficiency shortly after oral iron therapy Hyperproliferative erythropoiesis owing to acute haemorrhage, haemolysis, and in the third trimester of pregnancy
Iron demand by erythropoiesis [ (functional iron deficiency) >3.2 (2.0)
<3.2 (2.0)
Iron supply Y
Iron supply [
Classic iron deficiency Reduced iron supply to erythropoiesis Depletion of storage and functional iron Haemoglobinization Y No ACD
Functional iron deficiency Iron repletion Haemoglobinization Y About 20% of ACD patients Patients with iron deficiency who have anaemia accompanying infection, chronic inflammation, and cancer
Differential diagnosis of four different states of iron metabolism. The ferritin indices (sTfR/log ferritin) in parentheses indicate threshold values for C-reactive protein values above 5 mg/l, and all indices are assay-dependent [69,80,85]. ACD, anaemia of chronic disease; sTfR, soluble transferrin receptor. Adapted from Refs. [69,80,85].
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Table 1 Use of soluble transferrin receptor values within the ferritin index plot for the diagnosis of iron deficiency.
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realization of additional multicentre trials is necessary to define improved clinical applications [85,86,89e91].
Practice points Established biomarkers of iron metabolism show limited validity in cases of complicated anaemia (e.g. in cases of inflammation). Differential diagnosis of complicated anaemia or borderline cases should include the application of sTfR-derived indices (ferritin index; ferritin index plot) rather than measurement of sTfR alone. The ferritin index plot (Thomas plot) supports differential diagnosis of four different states of iron metabolism (ACD, latent ID, classic IDA, ACD with concomitant functional iron deficiency and iron-restricted erythropoiesis). Introduction of a recombinant sTfR World Health Organization reference reagent for standardization should improve the diagnostic value of sTfR assays.
Research agenda Physiological function and pathophysiological role of sTfR2 needs to be determined. As a possible alternative or addition for differential diagnosis of iron metabolism, standardization of hepcidin assays is necessary and should be brought into focus. The effect of iron regulatory peptide hepcidin on differential diagnosis of complicated anaemia, especially in combination with additional parameters of iron metabolism, needs further investigation.
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A WHO reference reagent for the Serum Transferrin Receptor (sTfR): international collaborative study to evaluate a recombinant soluble transferrin receptor preparation. Clin Chem Lab Med 2010;48: 815e20. [47] Beguin Y. Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clin Chim Acta 2003;329: 9e22. [48] *Kohgo Y, Niitsu Y, Kondo H, et al. Serum transferrin receptor as a new index of erythropoiesis. Blood 1987;70:1955e8. [49] Beguin Y, Huebers HA, Josephson B, et al. Transferrin receptors in rat plasma. Proc Natl Acad Sci U S A 1988;85:637e40. [50] Huebers HA, Beguin Y, Pootrakul P, et al. Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood 1990;75:102e7.
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Treatment of anemia in myelodysplastic syndromes with granulocyte [60] Hellstro colony-stimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients. Blood 1998;92:68e75. [61] van Iperen CE, Gaillard CA, Kraaijenhagen RJ, et al. Response of erythropoiesis and iron metabolism to recombinant human erythropoietin in intensive care unit patients. Crit Care Med 2000;28:2773e8. [62] Brugnara C, Zurakowski D, DiCanzio J, et al. Reticulocyte hemoglobin content to diagnose iron deficiency in children. J Am Med Assoc 1999;281:2225e30. [63] *Oski FA. Iron deficiency in infancy and childhood. N. Engl J Med 1993;329:190e3. [64] Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor: a quantitative measure of tissue iron deficiency. Blood 1990; 75:1870e6. €ki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron [65] Punnonen K, Irjala K, Rajama deficiency. Blood 1997;89:1052e7. 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Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia. J Lab Clin Med 1992;119:385e90. [73] Asobayire FS, Adou P, Davidsson L, et al. Prevalence of iron deficiency with and without concurrent anemia in population ^te d'Ivoire. Am J Clin Nutr 2001;74:776e82. groups with high prevalences of malaria and other infections: a study in Co € tto €nen T, Rajama €ki A, et al. Single values of serum transferrin receptor and transferrin receptor ferritin [74] Suominen P, Mo index can be used to detect true and functional iron deficiency in rheumatoid arthritis patients with anemia. Arthritis Rheum 2000;43:1016e20. [75] Zoli A, Altomonte L, Mirone L, et al. Serum transferrin receptors in rheumatoid arthritis. Ann Rheum Dis 1994;53: 699e701. [76] Baumann Kurer S, Seifert B, Michel B, et al. Prediction of iron deficiency in chronic inflammatory rheumatic disease anaemia. Br J Haematol 1995;91:820e6. [77] North M, Dallalio G, Donath AS, et al. Serum transferrin receptor levels in patients undergoing evaluation of iron stores: correlation with other parameters and observed versus predicted results. Clin Lab Haematol 1997;19:93e7. [78] Macdougall IC, Cavill I, Hulme B, et al. Detection of functional iron deficiency during erythropoietin treatment: a new approach. Br Med J 1992;304:225e6. [79] Brugnara C. Reticulocyte cellular indices: a new approach in the diagnosis of anemias and monitoring of erythropoietic function. Crit Rev Clin Lab Sci 2000;37:93e130. [80] *Thomas C, Kirschbaum A, Boehm D, et al. The diagnostic plot: a concept for identifying different states of iron deficiency and monitoring the response to epoetin therapy. Med Oncol 2006;23:23e36. [81] *Weiss G, Goodnough LT. Anemia of chronic disease. N. Engl J Med 2005;352:1011e23. [82] Franchini M. Hepcidin: the main regulator of iron homeostasis. Clin Chem Lab Med 2011;49:165e6. [83] *DeLoughery TG. Microcytic anemia. N. Engl J Med 2014;371:1324e31. 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Beyond soluble transferrin receptor: Old challenges and new horizons Kristian Harms, MD, Laboratory Medicine Resident Physician *, Thorsten Kaiser, MD, Laboratory Medicine Senior Physician Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany
a r t i c l e i n f o Article history: Available online 30 September 2015 Keywords: transferrin receptor iron metabolism disorders anaemia inflammation erythropoiesis hepcidin
Disturbances of iron metabolism are a frequent challenge in outpatient and inpatient care. Although several established biomarkers are commonly used by clinicians for differential diagnosis, the discrimination between latent or classic iron deficiency, anaemia of chronic disease or a combination of functional iron deficiency (iron-restricted erythropoiesis) with anaemia of chronic disease in patients affected by inflammatory disease can be demanding. Soluble transferrin receptor (sTfR) is a cleaved monomer of transferrin receptor 1 and correlates positively with tissue iron deficiency as well as with stimulated erythropoiesis. The ratio between sTfR and ferritin in combination with reticulocyte haemoglobin content further helps to identify different states of iron deficiency. In this review, we will focus on biological aspects of iron metabolism and sTfR, established clinical applications and limitations of sTfR and derived indices, and prospects of future research and applications. © 2015 Elsevier Ltd. All rights reserved.
Introduction The presence of iron is essential for several physiological and metabolic pathways. As surplus iron can result in cellular damage, iron homeostasis is subject to complex and tightly controlled regulation [1]. Furthermore, iron deficiency is known as the main cause of chronic anaemia worldwide, with an
* Corresponding author. Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Paul-List-Straße 13-15, 04103 Leipzig, Germany. Tel.: þ49 341 97 22458; Fax: þ49 341 97 22209. E-mail address: [email protected] (K. Harms). http://dx.doi.org/10.1016/j.beem.2015.09.003 1521-690X/© 2015 Elsevier Ltd. All rights reserved.
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estimated frequency of up to 15% in industrialized countries and 2 billion people worldwide [2]. Control of iron metabolism depends on several key elements related to acquisition, transport, utilization and storage of iron [3]. In plasma, iron is bound by transferrin and delivered into cells after interaction of the diferric ironetransferrin complex with the cell surface transferrin receptor 1 (TfR1) and subsequent internalization of ironetransferrineTfR1 via endocytosis. After release of iron in the acidic milieu (acidosome), iron is used or bound by ferritin (cellular iron storage), whereas the transferrineTfR1 complex is externalized and truncated, resulting in the appearance of a cleaved monomer of TfR1 (soluble transferrin receptor [sTfR]) [4]. Increased serum concentrations of sTfR are indicative of tissue iron deficiency and stimulated erythropoiesis [5]. Stimulated erythropoiesis is associated with haemolysis, ineffective erythropoiesis, or may be caused by therapeutic application of erythropoietin. In many uncomplicated cases, the determination of a low ferritin level is sufficient for a diagnosis of iron deficiency. Ferritin levels are increased in the presence of inflammation. Additional parameters independent of acute-phase response are, therefore, necessary. Under this condition, sTfR is considered a valuable, but not unrestricted, parameter for the differentiation of iron-deficient anaemia (IDA) and anaemia of chronic disease (ACD). New parameters and sTfR-derived indices have therefore been developed to help physicians to diagnose iron deficiency in complex clinical settings, with the peptide hormone hepcidin currently emerging in addition. Establishing a diagnosis of IDA with or without concurrent ACD is crucial, as iron deficiency may be a symptom of a potentially serious disease. Menstrual blood loss, gastrointestinal bleeding caused by malignancy or ulceration and iron malabsorption are typical differential diagnoses of iron deficiency [5,6]. Iron pools, TfR1 and the ironeTfeTfR1 cycle More than two-thirds (>2 g) of total body iron (3e5 g) is allocated to the haemoglobin of erythroid precursor cells and mature red blood cells. Even though only a small fraction (<0.1%, approximately 3 mg) of body iron is bound by plasma transferrin, this pool is considered the most active. To fulfil a daily requirement of at least 20e30 mg of iron for erythropoiesis, a plasma transferrin turnover occurs about 10 times a day. Transferrin has a high affinity for iron (Kd ¼ 1023 M), and, under physiological conditions, a transferrin saturation of about 30% of available iron binding sites is obtained. Major factors influencing transferrin saturation are dietary iron absorption in the duodenum and the upper part of the jejunum (physiologically only about 1e2 mg/day), recycled iron released by reticuloendothelial macrophages after phagocytosis of senescent erythrocytes (major source of plasma iron), and the amount of iron used (especially by bone marrow erythroblasts, >80% of transferrin-bound iron). Transferrin is mainly expressed in the foetal and adult liver, but small amounts arise also in the testes, brain, spleen and kidneys [3,7e9]. TfR1 (CD71) is the primary target of transferrin in the iron transport system, and is expressed as a type II transmembrane glycoprotein composed of a disulfide-bonded homodimer on the surface of nearly every cell type [10]. The receptor subunits are joined by two disulfide bonds at cysteines 89 and 98 [11] (Fig. 1). In humans, the TfR1 gene is located on human chromosome 3q29 [12,13]. High levels of TfR1 expression can be found in cells with increased proliferation rate, such as erythroid progenitor cells, placental syncytiotrophoblasts, and neoplastic cells [3,8]. Disruption of the TfR1 gene in mice leads to a severely impaired phenotype, affecting both erythropoiesis and neurologic development [14]. Each polypeptide subunit of the 90-kDa TfR1 protein containing 760 amino acids includes an Nterminal cytoplasmic domain, a single-pass transmembrane domain, and a large C-terminal extracellular domain, which includes a short glycosylated stalk region and three subdomains: protease-like, apical, and helical [10,15,16]. TfR1 belongs to the transferrin receptor/glutamate carboxypeptidase II family which, in addition to TfR1, includes transferrin receptor 2 (TfR2), folate hydrolase (prostatespecific membrane antigen) 1, N-acetylated alpha-linked acidic dipeptidase-like 1, N-acetylated alpha-linked acidic dipeptidase 2, and folate hydrolase 1B [10,15]. In its extracellular domain with TfR1, TfR2 shares a 45% identity and 66% similarity [17]. Compared with TfR1, the role of TfR2 in the uptake of transferring-bound iron into the cells is considered less significant, as TfR2 has a 25-fold lower affinity for iron-loaded (holo)transferrin than its homologue TfR1. In addition, the TfR2 mRNA expression pattern differs between both receptors. In contrast to TfR1, TfR2 shows much higher expression levels
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Fig. 1. Transferrin receptor 1 (transmembrane and soluble form).
in liver compared with other tissues. Mutations in the TfR2 gene mapping to 7q22 causes hereditary haemochromatosis type 3, a disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, especially the liver [18e22]. Two molecules of holo-transferrin are bound by the TfR1 dimer, and the resulting complex is internalized through the process of endocytosis via clathrin-coated pits [3,8,23] (Fig. 2). The release of iron from transferrin requires acidification (around pH 5.5) of the endosome [3,8,24]. Transferrin affinity to TfR1 depends on the iron status of transferrin; the highest affinity can be attributed to diferric transferrin, which is characterized by a 30-fold higher affinity to TfR1 compared with monoferric transferrin and a 500-fold higher affinity compared with non-iron-loaded (apo)transferrin [3,8,25]. After reduction of liberated insoluble ferric (Fe3þ) to soluble ferrous (Fe2þ) iron, iron is transported through the acidosomal membrane to the cytosol via DMT1 (divalent metal transporter 1 or, natural
Fig. 2. Iron metabolism with an emphasis on transferrin receptor 1 and soluble transferrin receptor.
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resistance-associated macrophage protein 2), where it can be used for metabolic functioning or can be stored in ferritin. Finally, the transferrin-TfR1 cycle is completed through return of the apo-transferrineTfR1 complex to the cell membrane where apo-transferrin is released from the cells [3,8,26,27] (Step 5a of Fig. 2). On a post-transcriptional level, cellular iron homeostasis is regulated by an interplay of ironresponsive elements (IRE) with iron regulatory proteins (IRP1 and IRP2). Iron-responsive elements constitute phylogenetically conserved hairpin structures within the untranslated regions (UTRs) of mRNAs encoding for key regulatory proteins of iron metabolism. The consequence of iron deficiency is the binding of IRPs to a single IRE in the 50 UTR of an mRNA, which leads to the repression of the translation and thus the reduction of the amount of the corresponding proteins in the cell. Examples of regulated proteins in this way, among others, are the iron storage protein ferritin, 50 -aminolevulinate synthase 2 (heme biosynthesis), ferroportin (mediates iron cell release to the plasma) and hypoxia inducible factor 2a. The binding of IRPs to multiple IREs in the 30 UTR of an mRNA leads to their stabilization. Therefore, the translation of mRNA lasts longer and the cell concentrations of the corresponding proteins increase. Examples of proteins regulated in this manner are TfR1 and DMT1. Taken together, as a consequence of iron deficiency, the regulation by the IREeIRP system leads to a decreased iron storage via ferritin and efflux via ferroportin as well as impaired heme biosynthesis, whereas cellular iron uptake and transport via TfR1 and DMT1 are supported. Iron excess leads to a reciprocal regulation of this system [3,28]. In contrast to this, the TfR2 transcript does not contain 30 UTR-IREs; therefore, TfR2 mRNA and protein levels vary little with changes in iron status [21,22]. IRPs are necessary for regulation of mammalian iron homeostasis. A complete loss of both proteins (IRP1 and IRP2) leads to embryonic lethality in mice [29]. Soluble transferrin receptor: biology and clinical applications In 1983, Pan and Johnstone [30,31] demonstrated the release of the transferrin receptor in vesicular form into culture medium during maturation of sheep reticulocytes. In addition, a polyclonal antibody against the transferrin receptor slowed down the release of the vesicles bearing the receptor, whereas transferrin accelerated vesicle release [32]. Electron microscopic analysis revealed large multivesicular bodies (MVBs, 1e1.5 mm) fusing with the plasma membrane and thus releasing their contents (50-nm bodies, exosomes) via shedding into the medium [33,34] (Step 5b of Fig. 2). In 1986, Kohgo et al. [4] described a truncated form of human TfR1 in human serum of healthy people and people with haematological malignancies (including acute leukaemia, multiple myeloma and malignant lymphoma). It could be shown by amino-terminal amino acid sequence analysis that residues 1e19 of serum receptor were identical to residues 101e119 of intact receptor. On the basis of this finding, the serum receptor was identified as a truncated monomer lacking the cytoplasmic and transmembrane domains (residues 1e100) of the intact receptor (Fig. 1). This cleaved fragment, which circulates in the form of a complex of transferrin and its receptor, has a molecular weight of 85 kD [35]. Production of sTfR is mediated by a membrane-associated serine protease [36], which has recently been identified as proprotein convertase subtilisin/kexin type 7 [37]. Proteolysis of TfR predominantly occurs from exosomes rather than the cell surface itself [38]. Furthermore, using surface 125I-labelled sheep reticulocytes as the experimental model, it was shown that, during invitro maturation of these cells, 125I-TfR of native size appears in exosomes (around 75%) before the soluble, truncated, exofacial domain of the receptor can be detected in the medium (around 25%, not exosome-associated). It was concluded that exosome formation is the major, if not the sole, route for shedding TfR, and that sTfR originates from exosomes followed by cleavage from the surface of exosomes [39]. In normal human sera, less than 1% of transferrin receptor is intact receptor. Consequently, almost all circulating transferrin receptor is the truncated monomeric form [40]. Recently, iron-dependent cleavage of cell surface TfR2 and release of a soluble form of this receptor (sTfR2) could also be described [41]. Tests used in clinical laboratories for measurement of sTfR are mostly latex-enhanced immunonephelometric/turbidimetric assays [5]. Challenges are the lack of international standards with different units and reference ranges, differing age and gender dependencies, and poor agreement among different kits [5,42e45]. The prospect of a recombinant sTfR World Health Organization
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reagent should help to resolve these issues [5,46]. One study showed age-related reference ranges among children (6 months to 8 years) with a slight decrease of upper and lower reference limits with increasing age [42]. Although its biological function remains unexplained, sTfR has been proposed as a marker of iron deficiency, total erythropoietic activity, or both [41]. Bone marrow erythropoiesis can cause variations up to eight times below and up to 20 times above average reference values. For this reason, it is considered the most important determinant of sTfR levels [47]. Patients with iron deficiency and autoimmune haemolytic anaemia present higher sTfR levels, whereas lower levels are detected in patients with aplastic anaemia [48]. In support of this finding, an inverse relationship between iron status and sTfR was also found in rat plasma, whereas a direct relationship existed between erythropoiesis and sTfR. It was assumed that the number of tissue receptors, mostly on erythroid cells, determines the number of sTfR [49]. In human plasma and by additional in-vivo experiments in rats, these observations of serum sTfR as a quantitative marker of the body mass of tissue TfR and regulation of TfR numbers particularly by iron deficiency and erythropoietin stimulation could be confirmed [50e52]. Even though the erythroblast compartment, not reticulocytes, constitutes the main source of sTfR, circulating reticulocytes can contribute significantly to serum sTfR levels, as shown by hypertransfusion with normal, reticulocyte-poor and reticulocyte-rich blood [52]. In summary, clinical applications of sTfR measurements in patients are comprehensive: soluble TfR levels are decreased in conditions characterized by erythroid hypoplasia, such as hypertransfusion, chronic renal failure, aplastic anaemia, or after intensive chemotherapy. Vice versa, sTfR levels are increased when erythropoiesis is stimulated, as seen in congenital dyserythropoietic anaemia, (autoimmune) haemolytic anaemia, hereditary spherocytosis, sickle cell anaemia, thalassaemia major or intermedia, megaloblastic or iron deficiency anaemia, and secondary polycythaemia. In patients with malignancies, with the possible exception of non-Hodgkin's lymphoma, chronic lymphocytic leukaemia and hepatocellular carcinoma, sTfR levels are not increased [47]. Furthermore, soluble TfR is suitable for monitoring erythropoietic response to various forms of treatment, such as the correction of megaloblastic anaemia with cobalamin, of iron deficiency anaemia with iron, or the recovery of erythropoiesis after renal transplantation [47,53e55]. The application of recombinant human erythropoietin is characterized by an extensive range of indications, and sTfR measurements can provide valuable information regarding response to recombinant human erythropoietin treatment [47]. For instance, sTfR levels are useful in assessing recombinant human erythropoietin response during treatment of chronic renal failure [56,57], aplastic anaemia [58], cancer [59], myelodysplastic syndrome [60], and in intensive care [61]. Diagnostical use of sTfR in a multiparameter approach Early recognition of latent subclinical iron deficiency is important to prevent systemic complications [62]. For instance, the most important systemic abnormality produced by iron deficiency in infancy is the alteration in cognitive performance, which might not be fully reversible [63]. Therefore, efforts have been made to further improve the diagnostic value of sTfR by developing multi-parameter interpretation aids. Skikne et al. [64] used a serial phlebotomy procedure in six men and eight women. They underlined the significance of sTfR measurements for the analysis of iron metabolism, especially combined with ferritin and haemoglobin measurements. Therefore, it is assumed that the iron status of patients without systemic inflammation can be fully assessed by using serum ferritin as a measure of iron stores, serum receptor as a measure of mild (functional) tissue iron deficiency, and haemoglobin concentration as a measure of advanced iron deficiency (iron deficiency anaemia). As a result of the reciprocal relationship between serum receptor and ferritin measurements, the logarithmic ratio of sTfR/ferritin portrays body iron in a linear fashion, showing an increased ratio from less than 100 in those with abundant iron stores to over 2000 in those with significant functional iron deficiency. A rise above 500 occurred when stores were fully depleted [64]. In contrast, on the basis of a study with a patient population consisting of 129 consecutive anaemic patients, Punnonen et al. [65] showed that sole calculation of the sTfR/ferritin ratio does not considerably improve diagnostic efficiency compared with sTfR alone. Logarithmic transformation of the
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ferritin values and calculation of the sTfR/log ferritin ratio (sTfR-F index, ferritin index), however, provided an outstanding indicator of iron depletion, as shown by analysis of receiver operating characteristic (ROC) curves (area under curveROC about 1.00). Therefore, by applying the ferritin index, the combination of sTfR and ferritin measurements provided the highest sensitivity and specificity compared with isolated measurements of sTfR and ferritin [65]. In another study, an attempt was made to further characterize the different stages of iron deficiency by means of serum ferritin, sTfR and ferritin index. A population sample of 65 apparently healthy nonanaemic adults (22 men and 43 women) was given oral iron supplementation for 3 months to produce an iron-replete population. A relatively large number of women of childbearing age were intentionally enrolled to permit inclusion of a larger proportion of potential participants with subclinical iron deficiency. A supplementation-induced change of sTfR, ferritin, and ferritin index did not occur in men, but, in a significant proportion of women, 25 were retrospectively judged to have had storage iron depletion (stage I) or depletion of the functional compartment (stage II). These changes were made most apparent by using the ferritin index, indicating that the non-anaemic stages of iron deficiency are readily detectable using this index. Especially in borderline cases, when the results of sTfR and ferritin assays are ambiguous, it is suggested that the ferritin index is more sensitive in the detection of irondeficient states [66]. Soluble transferrin receptor and inflammation Historically, bone marrow examination, along with Perls' Prussian blue stain, has been considered as the golden standard for deciding whether iron deficiency plays a role in the origin of anaemia, especially in cases of infection, chronic disease, malignancy or liver disease [67,68]. Hence, clinical demand for enabling a non-invasive distinction between IDA, which is due to a treatable cause of anaemia (e.g. bleeding, iron malabsorption or dietary lack of iron) and other entities in the differential diagnosis of anaemia is high. For the latter, this accounts especially for anaemia that accompanies infection, inflammation and cancer (ACD) or a combination of functional iron deficiency (iron-restricted erythropoiesis) with ACD [69]. Furthermore, it is also crucial to avoid overtreatment of patients with sufficient iron homeostasis, as iron excess poses a threat to cells and tissues owing to its ability to catalyse the generation of radicals, which attack and damage cellular macromolecules and promote cell death and tissue injury. In addition, most bacteria and virtually all pathogens have a significant iron requirement, which is essential for their growth [1,70]. Standard biochemical markers of iron metabolism show restricted validity under the influence of an acutephase response, with ferritin being an acute-phase reactant and transferrin a negative one [69]. Determination of sTfR has been proposed to identify IDA in patients with complicated anaemia affected by concurrent inflammatory disease that may lead to false-positive ferritin concentration [47,71]. Accordingly, levels of sTfR are not increased in patients with acute or chronic disorders (e.g. malaria, inflammatory disorders, acute hepatitis or chronic liver disease) [72,73]. Otherwise, elevated sTfR levels in patients with anaemia of chronic disease or acute inflammation can indicate true iron deficiency, which decrease after adequate iron supplementation, indicating functional iron deficiency [47,74]. Conflicting reports have been published on the effect of sTfR on differential diagnosis of complicated anaemia, which might be caused by heterogeneous study populations with different underlying mechanisms for iron-deficient erythropoiesis [5,47,71]. In general terms, the interpretation of individual sTfR values may be complicated in a patient in whom both changes in erythropoietic activity and iron status, both known to influence sTfR levels, occur simultaneously [47]. Accordingly, in other studies related to rheumatoid arthritis [75,76] or other inflammatory disorders [67], the contribution of sTfR measurements to determine presence or absence of coexistent functional iron deficiency was limited. Compared with patients with IDA, lower sTfR in patients with ACD (e.g. rheumatoid arthritis patients) may indicate a reduced erythropoietic activity [75]. Surprisingly, even cases of sTfR elevation in patients with normal or increased bone marrow iron have been described, lacking an alternative explanation for an increase (e.g. erythroid hyperplasia) [67,77]. These diagnostic difficulties have led to efforts to develop clinical laboratory tests that are capable of measuring functional iron availability at the site of haemoglobin synthesis in the erythron. Accordingly, the measurement of the proportion of
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hypochromic red cells has been shown to be of particular interest. Because of erythrocytes have a lifespan of about 120 days, hypochromic red cells constitute a late indicator of iron-restricted erythropoiesis, providing information over a several-month period. Furthermore, reticulocyte haemoglobin content has emerged as an early marker of functional iron deficiency, as reticulocytes exist in the circulation for only 1e2 days [69,78,79]. Thomas et al. [69,80] have shown that the relationship between functional iron deficiency (reticulocyte haemoglobin content) and iron supply to erythropoiesis (ferritin index, see above) can be represented in a diagnostic plot (ferritin index plot, Thomas plot) that corresponds to four different states of iron metabolism: ACD, latent iron deficiency, classic IDA, and ACD with concomitant functional iron deficiency/iron-restricted erythropoiesis (Table 1). As a consequence, the diagnostic plot can be used to derive therapeutic implications and to monitor response to erythropoietin therapy [69,80]. Soluble transferrin receptor and hepcidin: quo vadis? In recent years, more insight into the pathophysiology of ACD has been gained. In general, ACD results from activation of the immune and inflammatory systems, and is characterized by inadequate production of erythropoietin, inhibition of the proliferation of erythroid progenitor cells in the bone marrow, and disturbances in iron distribution [81]. In IDA, the iron supply depends on the amount of the iron stores, whereas in ACD, the supply depends on its rate of mobilization, with functional ID potentially occurring even in the presence of large iron stores when iron release is impaired [69]. Therefore, the relationship between iron status and sTfR levels in patients with inflammation depends on the severity of inflammation, the degree of anaemia, the adequacy of erythropoietin production, and the effect of cytokines on marrow activity [47]. Much effort has been made to further decipher the molecular mechanisms controlling iron homeostasis, and it has become evident that the peptide hormone hepcidin has emerged as the main regulator of iron recycling and iron balance, leading to speculation that excess of hepcidin may be the key pathogenic feature of ACD. By binding to the cellular iron exporter ferroportin and inducing its internalization and degradation, iron is trapped in enterocytes, macrophages, and hepatocytes, resulting in a reduced absorption of dietary iron, sequestration of iron in macrophages, and sequestration of iron in hepatic stores [82,83]. The bioactive form of hepcidin (peptide of 25 amino acids, hepcidin-25) is primarily produced in the liver, where its synthesis is stimulated by iron excess and inflammation and inhibited by anaemia and hypoxia [82,84]. Given that hepcidin-25 constitutes a pivotal role for systemic iron homeostasis, the prospect of further improving the classification of different iron states using this potential biomarker was recently addressed. In a single-centre study with 155 anaemic patients, patients with IDA could be differentiated from those with ACD and ACD/IRE, but not ACD from ACD/IRE based on hepcidin-25 alone. As hepcidin-25 showed a significant correlation with ferritin and the ferritin index, with most of the patients having normal to elevated ferritin concentrations but nevertheless suffering from iron deficiency or having iron-restricted erythropoiesis, it was concluded that hepcidin-25 is not a marker of iron-restricted erythropoiesis, but rather of iron supply and retention. The combination of hepcidin-25 with reticulocyte haemoglobin content CHr (hepcidin-25 plot) was useful for the differentiation of the iron states mentioned above. In addition, as hepcidin-25 is a real-time marker for the iron supply of erythropoiesis (responds within hours to changes in haematologic status) but the ferritin index a timedelayed marker (responds within days), this peptide might have the potential to eventually replace the sTfR-related ferritin index in the Thomas plot. This is further supported by the limitations of the ferritin index in some diseases (CLL) and the dual influence of erythropoietic activity and inflammation on sTfR and ferritin measurements [85e87]. Recent data confirm the limitation of sole measurement of hepcidin-25 to predict functional iron deficiency, with the Thomas plot as well as sTfR being better predictors of functional iron deficiency in patients with and without acute-phase response compared with hepcidin-25 and ferritin [88]. To further investigate the validity of hepcidin-25 for differential diagnosis of iron metabolism, important issues need to be addressed. At first, the standardization of hepcidin assays with appropriate reference material and suitable procedures is essential for valid interpretation of the results. Finally,
806
State
1
3
<3.2 (2.0)
sTfR/log ferritin
>3.2 (2.0)
Iron supply [
Interpretation
No iron deficiency (iron repletion) Normal erythropoiesis Normal haemoglobinization About 80% of patients with ACD Patients with cancer-related anaemia, acute and chronic inflammation, end-stage renal failure
4 <28 pg (~1.74 fmol)
Iron demand by erythropoiesis Y (no functional iron deficiency)
Interpretation
State and patient characteristics
2 >28 pg (~1.74 fmol)
Reticulocyte haemoglobin content
Iron supply Y
Latent iron deficiency Reduced iron supply Erythropoiesis still normal Normal haemoglobinization Anaemic or non-anaemic patients with latent iron deficiency Patients with iron deficiency shortly after oral iron therapy Hyperproliferative erythropoiesis owing to acute haemorrhage, haemolysis, and in the third trimester of pregnancy
Iron demand by erythropoiesis [ (functional iron deficiency) >3.2 (2.0)
<3.2 (2.0)
Iron supply Y
Iron supply [
Classic iron deficiency Reduced iron supply to erythropoiesis Depletion of storage and functional iron Haemoglobinization Y No ACD
Functional iron deficiency Iron repletion Haemoglobinization Y About 20% of ACD patients Patients with iron deficiency who have anaemia accompanying infection, chronic inflammation, and cancer
Differential diagnosis of four different states of iron metabolism. The ferritin indices (sTfR/log ferritin) in parentheses indicate threshold values for C-reactive protein values above 5 mg/l, and all indices are assay-dependent [69,80,85]. ACD, anaemia of chronic disease; sTfR, soluble transferrin receptor. Adapted from Refs. [69,80,85].
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Table 1 Use of soluble transferrin receptor values within the ferritin index plot for the diagnosis of iron deficiency.
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realization of additional multicentre trials is necessary to define improved clinical applications [85,86,89e91].
Practice points Established biomarkers of iron metabolism show limited validity in cases of complicated anaemia (e.g. in cases of inflammation). Differential diagnosis of complicated anaemia or borderline cases should include the application of sTfR-derived indices (ferritin index; ferritin index plot) rather than measurement of sTfR alone. The ferritin index plot (Thomas plot) supports differential diagnosis of four different states of iron metabolism (ACD, latent ID, classic IDA, ACD with concomitant functional iron deficiency and iron-restricted erythropoiesis). Introduction of a recombinant sTfR World Health Organization reference reagent for standardization should improve the diagnostic value of sTfR assays.
Research agenda Physiological function and pathophysiological role of sTfR2 needs to be determined. As a possible alternative or addition for differential diagnosis of iron metabolism, standardization of hepcidin assays is necessary and should be brought into focus. The effect of iron regulatory peptide hepcidin on differential diagnosis of complicated anaemia, especially in combination with additional parameters of iron metabolism, needs further investigation.
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