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Diagnostic value of transferrin
Clinica Chimica Acta 413 (2012) 1184–1189
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
Diagnostic value of transferrin Dominika Szőke ⁎, Mauro Panteghini Cattedra di Biochimica Clinica e Biologia Molecolare Clinica, Dipartimento di Scienze Cliniche ‘Luigi Sacco’, Università degli Studi, Milano, Italy
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Article history: Received 20 March 2012 Received in revised form 16 April 2012 Accepted 17 April 2012 Available online 23 April 2012 Keywords: Transferrin Iron deficiency Iron overload Risk marker
a b s t r a c t Despite the growing interest in hepcidin and other relatively new biomarkers, guidelines and clinical pathways continue to recommend traditional markers, such as serum transferrin (Tf) and ferritin, as laboratory tests for the diagnostic evaluation of iron-related disorders. In this study, we aimed to critically evaluate the diagnostic role of Tf relying on the highest level of available evidence by a comprehensive literature search. The role of Tf in iron deficiency (ID) and iron overload (IO) syndrome as well as a risk marker was evaluated. The low accuracy of Tf and Tf saturation (TS) in the diagnosis and management of ID conditions does not permit definitively recommending their use, even if recently published guidelines still consider the TS investigation as a complementary test for ferritin. If a tissue IO is suspected, TS is often used, even if it may not be the best test for detecting this condition. Nevertheless, clinical guidelines strongly recommend the use of TS as a first-level test for performing genetic diagnosis of hereditary hemochromatosis. Recently reported data indicating elevated TS as a risk factor for diabetes mellitus, cancer, and total mortality, may provide useful additions to the debate over whether or not to screen for IO using TS. © 2012 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . Role of transferrin in physiological iron homeostasis Diagnostic role of transferrin determination . . . . 3.1. Background . . . . . . . . . . . . . . . . 3.2. Transferrin in iron deficiency . . . . . . . . 3.3. Transferrin in iron overload . . . . . . . . 4. Transferrin as a risk marker . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Abbreviations: AASLD, American Association for the Study of Liver Diseases; CHD, Coronary heart disease; CI, Confidence interval; CKD, Chronic kidney disease; DM, Diabetes mellitus; EASL, European Association for the Study of the Liver; ESA, Erythropoiesis-stimulating agents; FID, Functional iron deficiency; Hb, Hemoglobin; HH, Hereditary hemochromatosis; ID, Iron deficiency; IDA, Iron deficiency anemia; IO, Iron overload; KDOQI, Kidney Disease Outcomes Quality Initiative; NTBI, Non-transferrin-bound iron; OR, Odds ratio; ROS, Reactive oxygen species; Tf, Transferrin; TIBC, Total iron-binding capacity; TfR, Transferrin receptor; TS, Transferrin saturation; UIBC, Unsaturated iron binding capacity. ⁎ Corresponding author at: Laboratorio Analisi Chimico-Cliniche, Ospedale ‘Luigi Sacco’, Via GB Grassi 74, 20157 Milano, Italy. Tel.: + 39 02 3904 2290; fax: + 39 02 5031 9835. E-mail address: [email protected] (D. Szőke). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.04.021
Iron is an essential micronutrient required for various biological processes, including erythropoiesis, oxidative metabolism, and cellular immune response [1]. At the same time, excess iron causes organ dysfunction through the production of reactive oxygen species (ROS) [2]. As a consequence, a strict regulation of iron homeostasis is mandatory, its maintenance being achieved by a sophisticated balance among mechanisms including iron absorption, transfer, utilization, storage, and loss. During the past few years, the understanding of molecular mechanisms controlling body iron metabolism has dramatically changed by the recognition of hepcidin as the main regulatory hormone of iron metabolism [3]. The introduction of hepcidin determination into clinical practice requires, however, further investigation [4]. Although a few commercial assays currently exist, the appropriate specimen type remains an unresolved issue [5,6]. In addition, excessive hepcidin
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production may occur in a number of acquired disease states, including infections, malignancies, and inflammatory conditions that makes its clinical assessment rather complex [7]. Although we can assume that hepcidin will very likely soon obtain an important role in the diagnostic evaluation of iron-related disorders, current guidelines and clinical pathways continue to recommend the measurement of more traditional biomarkers, such as serum transferrin (Tf) and ferritin. However, it is often difficult to understand if the clinical application of these markers is always evidence-based. The aim of this study was to conduct a comprehensive literature search regarding the diagnostic usefulness of Tf in iron-related disorders to derive available scientific evidence concerning the clinical role of Tf determination in serum. Particularly, we focused on meta-analyses and systematic reviews. Clinical guidelines were, although with precaution, also considered. 2. Role of transferrin in physiological iron homeostasis The human body absorbs 1–3 mg/day of dietary iron balanced with losses via sloughed intestinal epithelial cells, menstruation, and other blood losses. These iron losing mechanisms do not provide an effective iron excreting system and thus the regulation of dietary iron absorption from the duodenum plays a critical role in iron homeostasis. Dietary iron is found in hem and non-hem forms and their absorption occurs in Fe 2+ form at the apical surface of duodenal enterocytes via different mechanisms [8]. Once inside the intestinal epithelial cell, iron may remain in the cell for use or storage, never being absorbed but just lost when senescent enterocytes slough into the gut lumen, or it may be exported across the basolateral membrane of epithelial cells into the circulation [1]. When there is a body demand, iron is exported in reduced form through the basolateral membrane by ferroportin, then it has to be oxidized to Fe 3+ by hephaestin before being bound by Tf. However, the Tf iron pool is replenished mostly by iron recycled from effete red blood cells. Besides dietary iron absorption, iron reutilization is indeed another elementary mechanism providing iron to the body. Senescent red blood cells are cleared by reticuloendothelial macrophages, which metabolize hemoglobin and hem, releasing iron into the bloodstream. By analogy to enterocytes, macrophages export Fe 2+ via ferroportin in a process coupled by re-oxidation of Fe 2+ to Fe 3+ by ceruloplasmin and followed by the loading of Fe 3+ to Tf [9]. Iron is transported in the circulation bound to Tf, which maintains iron in a redox-inert state and delivers it to tissues. Tf has multiple functions; first of all, it is the main plasma transport protein responsible for iron distribution and serves as a storage sink for sequestering iron extracellularly until iron is needed, then allowing it to reach target tissues [10]. On the other hand, Tf represents a protective mechanism against the presence of free-iron in the plasma, which could be extremely toxic to cells. This protective action is dependent upon two features of Tf: its high affinity (Kd ≈ 10 − 20 M) to Fe 3+ and the fact that every Tf molecule has two iron binding sites and under physiological conditions transferrin saturation (TS) is only up to 30%–40% [11]. Developing erythroid cells, as well as most other cell types, acquire iron from plasma. Tf-bound iron enters target cells through receptor mediated endocytosis. Iron-loaded Tf binds with high affinity to Tf receptor (TfR) 1 and the complex undergoes endocytosis via clathrin-coated pits. The acidic pH of endosomes triggers the release of Fe 3+ from Tf. Following the dissociation of iron, the affinity of Tf to TfR 1 dramatically drops resulting in the break-up of the complex and secretion of apo-Tf into the bloodstream to recapture Fe 3+ [9]. The amount of iron bound to Tf (~ 3 mg) corresponds to less than 0.1% of total body iron, but it is highly dynamic and undergoes more than ten times daily turnover to sustain erythropoiesis [9]. In case of iron deficiency (ID), the concentration of Tf in plasma increases, but it reflects the iron status properly only when iron stores are exhausted and when serum iron concentration is b40–60 μg/dL, so
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it does not diagnose ID prior to ineffective erythropoiesis [12]. In iron overload (IO) states, when the whole iron binding capacity of Tf is saturated, an additional iron compartment called non-Tf-bound iron (NTBI) appears in the circulation. Unlike Tf-bound iron, the cellular uptake of NTBI is not dependent on TfR and NTBI is biologically toxic through the production of ROS to every tissue, especially liver, heart, and some endocrine tissues [2]. The activation of NTBI can be predicted by TS values and its increased levels may indicate developing organ toxicity in iron-overloaded patients [13]. 3. Diagnostic role of transferrin determination 3.1. Background Theoretically, the diagnostic gold standard for iron-related disorders is the technique that is able to estimate body iron stores most accurately. In this way, the bone marrow examination, establishing the absence of stainable iron, remains the gold standard for ID diagnosis. Bone marrow examination is, however, invasive, expensive, and requires technical expertise, and thus cannot be performed routinely in clinical practice [14]. In IO conditions, liver iron levels are considered to accurately reflect total body iron stores because liver is the dominant iron storage organ. Liver iron levels have also been used to estimate risk and predict outcomes, such as liver failure, diabetes mellitus (DM), heart failure, and death [13]. As with bone marrow examination, liver iron level quantifying methods, including liver biopsy and imaging techniques, are invasive and/or highly expensive, limiting their use in the screening and diagnosis of IO status. The use of surrogate biomarkers for evaluation of iron status is, therefore, inevitable, even if their diagnostic value can often be limited. Several serum markers are currently used to estimate the individual's body iron stores, including serum ferritin, iron, Tf, total iron-binding capacity (TIBC), and TS. In clinical practice, both Tf and derived TS are widely used parameters in iron status evaluation. Screening for either ID or IO traditionally includes the determination of TS rather than a simple measurement of serum Tf concentrations [15]. The measurement of TS is widely available and cheap. Basically, there are two ways for TS determination, one based on TIBC estimate, the other employing the determination of Tf protein [16]. TIBC is the capacity of plasma proteins to bind iron. Although not the only iron-binding protein in the blood, Tf is the most important and TIBC can be utilized to indirectly determine serum Tf concentrations. In practice, TIBC is determined by the addition of sufficient iron to saturate iron-binding sites of serum proteins in the sample. After removing the iron excess, the assay for iron is performed. The ratio in percentage of serum iron and TIBC, in turn, indicates how much Tf is saturated by iron, so defining TS. As unsaturated iron binding capacity (UIBC) measurement is easily automatable, TIBC is often not directly measured as described above, but calculated by summing the results of serum iron and UIBC. UIBC assays add a fixed amount of iron to saturate ironbinding sites in the sample; the excess iron is not removed, but measured and UIBC is calculated as the difference of added and measured iron. Tf can also be directly determined as protein mass concentration using immunoassays and TS estimated on the results of serum iron and Tf measurements. In a head-to-head comparison by Hawkins [17], the diagnostic value of the immunologic measurement of Tf and of TIBC in the ID diagnosis was evaluated. By means of ROC curve analysis, no difference in the diagnostic performance was found between direct Tf measurement (immunoturbidimetry) and two commercial TIBC formulations. Analytic, biologic, and pathologic factors may influence TS and limit its clinical utility. The within-subject biological variation of iron in serum is high (26.5% in average — http://www.westgard. com/biodatabase1.htm); furthermore, Tf may react as negative acute phase protein in acute inflammation. Additionally, the lack of
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an established higher-order reference method and materials for iron measurement contributes to the variability between different commercial assays and platforms, further limiting the clinical utility of TS as a generic reference interval and/or common decision thresholds cannot be derived [18]. Notwithstanding, Tf and derived saturation index remain tests that are generally applied in clinical practice for the evaluation of both ID and IO. Optimal use of a laboratory test in clinical practice requires an accurate estimate of pre-test probability, i.e. disease prevalence, and knowledge of the test characteristics, i.e. sensitivity, specificity, predictive values, and likelihood ratios associated with various test results. Accordingly, hereafter we critically evaluate the diagnostic value of Tf and TS in iron-related disorders relying on the highest level of available evidence. 3.2. Transferrin in iron deficiency ID and iron deficiency anemia (IDA) may result from the interplay of three distinct factors: increased iron requirements, limited external supply, and increased loss. In absolute ID, body iron stores are depleted, while in functional ID (FID), although the stores are replete, iron cannot be mobilized to the bone marrow fast enough to meet erythropoiesis demands. One of the most important clinical settings for FID is in patients with chronic kidney disease (CKD), who require parenteral iron supplementation to respond to administered erythropoiesisstimulating agents (ESA) for correcting anemia, but it also occurs in many chronic inflammatory diseases [19]. ID can exist in the absence of anemia, if ID has not been present for sufficient time or if it has not been severe enough to cause hemoglobin (Hb) concentrations in blood to fall below the specific thresholds for gender, age, and racial groups [20]. Laboratory evaluation of ID involves assessment of the body's iron depletion and measurements reflecting iron deficient red cell production [16]. As stated above, Hb concentrations in the physiological range do not exclude ID, because an individual with unaltered body iron stores has to lose a large portion of his body iron before Hb concentrations fall below the cut-off defining anemia. Although the red cell distribution width may be the earliest indicator of ID and iron deficient erythropoiesis without anemia, the main laboratory finding indicating ID/IDA is a low concentration of serum ferritin [20,21]. Already in 1992, the landmark overview by Guyatt et al. [22] demonstrated the diagnostic effectiveness of serum ferritin for ID detection across an extensive population of anemic patients with and without inflammatory, liver or neoplastic diseases. Historically, TS has widely been used as screening test for suspected ID [15]. Not long ago, some authors still suggested “a normal or elevated TS as useful for excluding IDA as a low value is for identifying it” [20]. However, even 20 years ago, the study by Guyatt et al. [22], which comprehensively reviewed the available literature, did not recommend TS as an appropriate test for the laboratory diagnosis of IDA. The area under the ROC curve obtained by cumulatively analyzing data from the recruited studies (0.74; 95% confidence interval (CI): 0.70–0.78) indicated the altogether low accuracy of the test for diagnosing ID [23]. On the other hand, using a comprehensive literature search we were unable to find any recent meta-analysis or systematic reviews that could support the clinical utility of TS for the diagnosis of ID and IDA. The only retrieved systematic analysis aimed to identify the best biomarker to monitor population response in large-scale iron fortification programs: unfortunately, TS was measured in only two out of nine selected randomized iron intervention trials, making it difficult to draw any conclusions about this indicator [24]. In spite of the lack of high-grade scientific evidence, recently published clinical guidelines concerning ID/IDA still recommend the TS investigation as a test complementary to ferritin for ID detection [19,25]. Particularly, the guideline by the British Society of
Gastroenterology put TS in the group of further laboratory tests “occasionally necessary” to confirm ID [19]. Similarly, the British Columbia Medical Association's guideline includes TS in the group of additional tests to be considered when clinical features and hematology profile are suggestive of ID, but serum ferritin concentrations are >100 μg/L [25]. Altogether, these guidelines seem to assign to TS the role of confirmation test with higher accuracy for ID than ferritin, something that does not seem supported by the available evidence. Patients with CKD may suffer from various forms of ID, including absolute ID, FID, and an extreme case of FID known as reticuloendothelial blockage. The particular type of ID may affect the validity and reliability of laboratory test results for iron status and often result in a dilemma regarding treatment decisions [26]. Although a number of international guidelines have been published for the evaluation and management of ID/IDA in CKD patients, their recommendations on which combination of laboratory biomarkers are required and how frequently should patients be tested often differ [27]. In all available guidelines the use of TS measurement for ID evaluation is, however, mentioned [28–31]. In clinical practice guidelines of the Canadian Society of Nephrology [28,29], the minimum evaluation for anemia in CKD patients includes both serum ferritin and TS, in addition to a complete blood count. This is, however, associated with the lowest grade of evidence [28]. In addition, recommendations for different CKD patient populations stratified by dialysis execution and ESA therapy administration include target ferritin and TS values, used for the evaluation of iron administration when necessary [29]. In the opinion of the Kidney Disease Outcomes Quality Initiative (KDOQI), the initial laboratory assessment of anemia in CKD patients should include, in addition to a complete blood count, serum ferritin (to assess body iron stores) and TS (or, when available, the content of Hb in reticulocytes) to assess the adequacy of iron for erythropoiesis [30]. However, the same work group also highlights that the clinical utility of TS is markedly impaired by the absence of a decision threshold for ID diagnosis, preventing from the inclusion of the biomarker in a recommendation statement. The KDOQI also suggests TS > 20% as the target of iron therapy during ESA treatment, even if, once again, the supporting evidence is of very limited quality, i.e. a single small randomized controlled trial [32]. The most recent clinical guideline on anemia management in CKD patients similarly recommends a TS >20% as goal for ESA maintenance therapy and iron supplementation, but still with the lowest grade of scientific evidence [31]. The UK National Institute for Health and Clinical Excellence also recommends that for CKD patients with suspected FID (serum ferritin > 100 μg/L), this condition should be defined by a percentage of hypochromic red cells > 6% or, only when this test is not available, by a TS b20% [31]. This is a class B recommendation, indicating that evidence was obtained from well-conducted diagnostic studies. 3.3. Transferrin in iron overload IO syndromes can be classified as hereditary or secondary (Table 1). Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasians [33]. First described in 1996, the most frequent genetic defect is a G→A mutation of the HFE gene, leading to a substitution of tyrosine for cysteine (C282Y) in HFE protein [34]. The C282Y mutation leads to the disruption of an intramolecular disulfide bond of the protein necessary for the interaction with β2-microglobulin, by which the MHC class I-like HFE protein is physically associated. As a result, the C282Y mutant HFE protein is abnormally processed, leading to accelerated degradation and reduced cell surface expression [35]. Various explanations have been proposed about the role of HFE protein in the pathomechanism of HH, including the ‘crypt-programming model’ and the ‘hepcidin model’ [36]. The
D. Szőke, M. Panteghini / Clinica Chimica Acta 413 (2012) 1184–1189 Table 1 Classification of iron overload syndromes. Clinical condition Hereditary iron overload Hereditary hemochromatosis Type 1 (HFE) Type 2A (HJV) Type 2B (HAMP) Type 3 (TFR2) Types 4A and 4B (SLC40AI) Hereditary hyperferritinemia Atransferrinemia Others
Secondary iron overload Thalassemias, congenital sideroblastic anemia, congenital dyserythropoietic anemias, myelodysplastic syndromes, chronic hemolytic anemias Thalassemia major Intravenous, oral, or dietary Alcoholic, viral, non-alcoholic steatohepatitis Porphyria, hemodialysis
Etiopathogenesis
HFE gene mutations: C282Y, H63D, S65C, others Hemojuvelin gene mutations Hepcidin gene mutations Transferrin receptor 2 gene mutations Ferroportin gene mutations Iron-responsive element mutation Transferrin gene mutation DMT1 mutations, aceruloplasminemia
Ineffective erythropoiesis
Transfusion for long periods Iron administration for long periods Liver dysfunction Other
intestinal iron absorption hypothesis is now regarded as unlikely; on the other hand, the mechanisms by which HFE may influence the iron-dependent regulation of hepcidin are still not fully understood [33]. Iron status regulates hepatic hepcidin expression by liver iron stores and by circulating iron levels [37]. One proposed explanation suggests that the complex of TfR1 and HFE acts as an iron sensor at the hepatocyte cell membrane [38]. Because HFE and diferric Tf have overlapping binding sites on TfR1, the bioavailability of HFE might be influenced by concentrations of both TfR1 and diferric Tf [35]. As TS increases, diferric Tf displaces HFE from TfR1, thereby making HFE available to bind to TfR2. It is postulated that the complex of HFE and TfR2 then influences hepcidin expression [33]. Loss of HFE (or TfR2) attenuates gene-mediated signaling to hepcidin [37]. As physiologically hepcidin acts to decrease the absorption of dietary iron and the release of recycled iron from macrophage stores, in case of lacking or decreased hepcidin expression systemic IO can develop [7]. Other common HFE gene mutations include H63D and S65C, but these mutations are generally not associated with IO, unless seen with C282Y as a compound heterozygote [33]. Koziol et al. [39] examined 7250 wild-type, 81 C282Y homozygote, and 438 C282Y/H63D compound heterozygote individuals and found that C282Y homozygotes had abnormally high TS and serum ferritin values relative to the HFE wild types, while compound heterozygotes appeared to be a mixture of individuals with physiologic TS and ferritin concentrations and those with abnormally high values similar to the homozygotes, with equal proportions of each. With the availability of genetic testing, the HFErelated HH is now frequently identified in asymptomatic relatives of HH patients. Accordingly, a genetic diagnosis can be applied to individuals who have not yet developed any phenotypic expression of disease [33]. In 2000, the European Association for the Study of the Liver (EASL) proposed to classify HH individuals detected as C282Y homozygotes in four disease stages as follows: 1) genetic predisposition, but no other abnormality; 2) IO, but without symptoms; 3) IO with early symptoms; and 4) IO with organ damage, e.g. cirrhosis [40]. However, the subsequent observation that a remarkable proportion of individuals who have genetic susceptibility may never develop IO has changed the way of thinking [41].
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Although the majority of C282Y homozygotes may have raised TS levels, this cannot be relied upon as solid evidence of IO, as sensitivity and specificity of TS for identifying C282Y homozygotes are not well documented. On the other hand, the estimate of the prevalence of C282Y homozygosity in individuals with increased values of serum iron markers depends from test cut-offs used for screening people for genetic examination, which are markedly variable in different studies. Elevated TS was found in 1.2%–7.0% of screened individuals in unselected populations (TS cut-off ranging from 45% to 55%, with and without partitioning by gender), 4.3%–21.7% of those were then found to be C282Y homozygotes [42]. Despite these limitations, TS is often recommended as screening test when IO is suspected [43]. A recently performed study aimed to evaluate the diagnostic accuracy of TS as a screening test for finding C282Y homozygotes in the general population [44]. Assuming Hardy–Weinberg equilibrium, the unbiased frequency estimate for C282Y homozygote genotypes in the screened population of 4804 randomly selected persons was 0.75%, somewhat higher than previous estimates in other populations [33,45]. Using 55% (men) and 50% (women) as decision limits on two consecutive TS measurements, the sensitivity was 90.0% and 55.0%, the specificity was 99.6% and 99.4%, the negative predictive value was 99.9% and 99.7%, and the positive predictive value was 64.3% and 43.0% for men and women, respectively [44]. The authors concluded that two consecutive measurements of TS represent an accurate screening test for C282Y homozygosity in men, but not in women. Although differences in inclusion criteria and in biochemical and clinical definitions in various studies exist, an average disease penetrance of 13.5% for C282Y homozygosity was found by a metaanalysis of 19 studies [42]. According to this low disease penetrance, neither EASL nor the American Association for the Study of Liver Diseases (AASLD) in their practice guidelines for HH recommends genetic screening of the general population [42,46]. EASL suggests the following case definition for the diagnosis of HFE gene-related (or type 1) HH: “C282Y homozygosity and increased body iron stores with or without clinical symptoms” [42]. Quite analogously, AASLD establishes that the clinical diagnosis of hemochromatosis should be based on documentation of increased iron stores and that HH can be further defined genotypically by the familial occurrence of IO associated with C282Y homozygosity or C282Y/H63D compound heterozygosity [46]. According to EASL, the detection of increased iron stores is possible by surrogate serum biomarkers (i.e. ferritin) and determination of the amount of iron removed, or by the direct assessment of tissue IO with liver biopsy or imaging techniques [42]. In general, EASL suggests that serum iron concentrations and TS do not quantitatively reflect body iron stores and should, therefore, not be used as surrogate markers of tissue IO [42]. However, EASL strongly recommends the use of TS, even if without any specific cut-off definition, as a screening test for performing the genetic diagnosis of HFE-related HH in: − subjects with suspected IO, who should first receive measurement of fasting TS (and serum ferritin) and the HFE testing performed only in those with increased TS (high quality of evidence); − patients from liver clinics, who should be screened for fasting TS (and serum ferritin) and receive genetic HFE testing if TS is increased (moderate quality of evidence). On the other hand, for EASL HFE testing for the C282Y and H63D mutations should be carried out in all patients with otherwise unexplained increases in serum ferritin and TS [42]. Likewise, AASLD recommends that individuals with abnormal iron studies, including serum ferritin and TS, should be evaluated for hemochromatosis, even in the absence of symptoms [46]. In patients with suggestive symptoms, physical findings, or family history, a combination of TS and ferritin should be obtained rather than relying on a single test. If either test is abnormal (TS > 45% or ferritin above the upper
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reference limit), then HFE mutation analysis should be performed [46]. Finally, to detect early disease and prevent complications AASLD recommends iron studies and HFE mutation analysis in all first-degree relatives of patients with HFE-related HH. 4. Transferrin as a risk marker Elevated TS has been proposed as a marker of the risk for various conditions, such as coronary heart disease (CHD), DM, cancer, or total mortality [47–50]. However, the number of available meta-analyses and systematic reviews evaluating this issue is limited. A metaanalysis of 12 prospective studies involving 7800 CHD cases showed for TS, similarly to other iron status indicators, a non significant CHD risk ratio (0.9, 95% CI: 0.7–1.1) [47]. Ellervik et al. [48] demonstrated by a meta-analysis performed on two general population studies and on an age- and gender-matched population-based case– control study, including 8535 cases and 37,039 controls altogether, that TS levels ≥ 50% were associated with a two- to threefold increased risk of developing DM. In the combined studies, the odds ratio (OR) was 2.1 (95% CI: 1.3–3.4; P = 0.003) for any form of DM, 2.6 (95% CI: 1.2–5.6; P = 0.01) for type 1 DM, and 1.7 (95% CI: 1.4–2.1; P = 0.001) for type 2 DM, respectively, in those individuals with TS ≥ 50% vs. b50%. The same group of authors found that elevated TS levels were associated with increased risk of any cancer [49]. From the meta-analysis, which was carried out using data of three studies with a total of 57,841 patients, an association between elevated TS (≥60%) and increased risk of any cancer was found in the overall population, certainly in women and, to a lesser degree, in men. Corresponding OR for random effect models were 1.5 (95% CI: 1.2– 1.8, P b 0.001) in overall population, 2.2 (95% CI: 1.2–3.8, P b 0.001) in women, and 1.3 (95% CI: 1.0–1.8, P = 0.08) in men alone, respectively [49]. Finally, the same authors carried out a further metaanalysis, including three studies involving 55,873 patients, striving to examine whether increased TS is associated with increased mortality in the general population [50]. The calculated OR for total mortality for TS of ≥50% vs. b50% was 1.4 (95% CI: 1.1–1.9; P b 0.005) under the random effects model. The authors suppose that the biological mechanism behind the association between elevated TS and either DM, cancer, and total mortality risk may be the iron-induced oxidative stress, which may cause β-cell destruction, promote the development of cancer cells, or affect survival. 5. Conclusions From many years Tf and TS have been widely used surrogate markers for the detection of both body iron depletion and IO. However, a general disadvantage of traditional biomarkers in comparison with more recently proposed diagnostic tests is that their use is seldom evidence-based. Our aim was therefore to evaluate the scientific evidence, if any, supporting the clinical usefulness of Tf and derived TS. ID is the most common hematological disorder encountered in general practice and, at the same time, it is one of the most common diseases worldwide, so its correct diagnosis is of utmost importance. Although therapeutic trial of iron has been suggested as a method to diagnose ID in an anemic patient, most clinicians prefer to make a definitive laboratory diagnosis prior to treatment: the lack of high-level evidences limits, however, the role of Tf and TS in the diagnosis of ID and IDA. Clinical guidelines recommend the use of TS only in specific situations, first of all in the management of CKD patients, and not as the test of choice in the diagnosis and management of ID in general. Moreover, the quality of evidence and the strength of the guidelines' recommendations, where indicated, are generally low and weak, and are often based only on the experts' opinion [28–31]. The course and prognosis of HH and most of its complications, including liver cancer, depend on the amount and duration of iron
excess and on the time of therapeutic intervention. Early diagnosis and therapy can certainly prevent the adverse consequences of IO [51]. In 2005, the American College of Physicians published a clinical practice guideline about the screening of primary care patients for HH using TS and serum ferritin determinations, not recommending for or against the screening for HH in the general population because of insufficient evidence [52,53]. More recent HH guidelines do not yet recommend screening for HH in the general population either by genetic testing or by evaluation of increased iron stores [42,46]. Nonetheless, studies examining raised TS levels as a risk factor for CHD, DM, cancer, and total mortality also provide conflicting results about the utility of screening for increased TS [47–50]. If a causal association exists between IO and risk for DM, cancer, and total mortality, then individualized screening programs could be developed and therapy could easily lower high iron concentrations. Morever, a cost/benefit analysis of screening for elevated TS in the general or in selected populations would be required to determine the public health consequences and whether to advice for or against iron supplement tablets and large-scale iron fortification programs. In conclusion, from the currently available data it seems that the role of TS in the diagnosis and management of ID/IDA is limited. On the other hand, TS appears to be a valid test for screening selected patients with suspected IO and/or liver disease for performing genetic tests for HH. Finally, the possible use of TS as risk marker clearly requires further evaluation. References [1] Muñoz M, Villar I, García-Erce JA. An update on iron physiology. World J Gastroenterol 2009;15:4617–26. [2] Kohgo Y, Ikuta K, Ohtake T, Torimoto Y, Kato J. Body iron metabolism and pathophysiology of iron overload. Int J Hematol 2008;88:7–15. [3] Franchini M, Montagnana M, Lippi G. Hepcidin and iron metabolism: from laboratory to clinical implications. Clin Chim Acta 2010;411:1565–9. [4] Moyer TP, Highsmith WE, Smyrk TC, Gross Jr JB. Hereditary hemochromatosis: laboratory evaluation. Clin Chim Acta 2011;412:1485–92. [5] Kemna EH, Tjalsma H, Willems HL, Swinkels DW. Hepcidin: from discovery to differential diagnosis. Haematologica 2008;93:90–7. [6] Singh B, Arora S, Agrawal P, Gupta SK. Hepcidin: a novel peptide hormone regulating iron metabolism. Clin Chim Acta 2011;412:823–30. [7] Finberg KE. Unraveling mechanisms regulating systemic iron homeostasis. Hematology Am Soc Hematol Educ Program 2011;2011:532–7. [8] Siah CW, Ombiga J, Adams LA, Trinder D, Olynyk JK. Normal iron metabolism and the pathophysiology of iron overload disorders. Clin Biochem Rev 2006;27:5–16. [9] Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J 2011;434:365–81. [10] MacKenzie EL, Iwasaki K, Tsuji Y. Intracellular iron transport and storage: from molecular mechanisms to health implications. Antioxid Redox Signal 2008;10: 997–1030. [11] Baker HM, Anderson BF, Baker EN. Dealing with iron: common structural principles in proteins that transport iron and heme. Proc Natl Acad Sci U S A 2003;100:3579–83. [12] World Health Organization. Centers for disease control and prevention. Assessing the iron status of populationsSecond edition. ; 2007. including Literature Reviews. [13] Wood JC. Diagnosis and management of transfusion iron overload: the role of imaging. Am J Hematol 2007;82:1132–5. [14] Zimmermann MB. Methods to assess iron and iodine status. Br J Nutr 2008;99(Suppl. 3):S2–9. [15] Beutler E, Hoffbrand AV, Cook JD. Iron deficiency and overload. Hematology Am Soc Hematol Educ Program 2003:40–61. [16] Higgins T, Eckfeldt JH, Barton JC, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 5th ed. St. Louis: Elsevier Saunders; 2012. p. 985–1030. [17] Hawkins RC. Total iron binding capacity or transferrin concentration alone outperforms iron and saturation indices in predicting iron deficiency. Clin Chim Acta 2007;380:203–7. [18] Miller WG, Myers GL, Ashwood ER, et al. State of the art in trueness and interlaboratory harmonization for 10 analytes in general clinical chemistry. Arch Pathol Lab Med 2008;132:838–46. [19] Goddard AF, James MW, McIntyre AS, Scott BB, on behalf of the British Society of Gastroenterology. Guidelines for the management of iron deficiency anaemia. Gut 2011;60:1309–16. [20] Cook JD. Diagnosis and management of iron-deficiency anaemia. Best Pract Res Clin Haematol 2005;18:319–32. [21] Muñoz M, García-Erce JA, Remacha ÁF. Disorders of iron metabolism. Part II: iron deficiency and iron overload. J Clin Pathol 2011;64:287–96. [22] Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory diagnosis of iron-deficiency anemia: an overview. J Gen Intern Med 1992;7:145–53.
D. Szőke, M. Panteghini / Clinica Chimica Acta 413 (2012) 1184–1189 [23] Swets JA. Measuring the accuracy of diagnostic systems. Science 1988;240: 1285–93. [24] Mei Z, Cogswell ME, Parvanta I, et al. Hemoglobin and ferritin are currently the most efficient indicators of population response to iron interventions: an analysis of nine randomized controlled trials. J Nutr 2005;135:1974–80. [25] British Columbia Medical Association Advisory Committee. Guidelines & protocols. Iron deficiency — investigation and management. Available at:http:// www.bcguidelines.ca/guideline_iron_deficiency.html Last access: 28.February.2012. [26] Wish JB. Assessing iron status: beyond serum ferritin and transferrin saturation. Clin J Am Soc Nephrol 2006(Suppl. 1):S4–8. [27] Agency for Healthcare Research, Quality. Evidence-based practice center systematic review protocol: laboratory biomarkers for iron deficiency anemia in late stage chronic kidney disease. Available athttp://www.effectivehealthcare.ahrq. gov/index.cfm/search-for-guides-reviews-and-reports/? productid=767&pageaction=displayproduct Last access: 28.February.2012. [28] White CT, Barrett BJ, Madore F, et al. Clinical practice guidelines for evaluation of anemia. Kidney Int 2008;74:S4–6. [29] Madore F, White CT, Foley RN, et al. Clinical practice guidelines for assessment and management of iron deficiency. Kidney Int 2008;74:S7–S11. [30] National Kidney Foundation. KDOQI clinical practice guidelines and clinical practice recommendations for anemia in chronic kidney disease. Am J Kidney Dis 2006;47(Suppl. 3):S1–S145. [31] NCGC National Clinical Guideline Centre. Anaemia management in chronic kidney disease. Rapid update 2011. London, UK: Royal College of Physicians; 2011. [32] Besarab A, Amin N, Ahsan M, et al. Optimization of epoetin therapy with intravenous iron therapy in hemodialysis patients. J Am Soc Nephrol 2000;11:530–8. [33] Adams PC, Reboussin DM, Barton JC, et al. Hemochromatosis and Iron Overload Screening (HEIRS) Study Research Investigators. Hemochromatosis and ironoverload screening in a racially diverse population. N Engl J Med 2005;352: 1769–78. [34] Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399–408. [35] Fleming RE, Britton RS, Waheed A, Sly WS, Bacon BR. Pathophysiology of hereditary hemochromatosis. Semin Liver Dis 2005;25:411–9. [36] Pietrangelo A. Hereditary hemochromatosis — a new look at an old disease. N Engl J Med 2004;350:2383–97. [37] Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med 2012;366: 348–59. [38] Goswami T, Andrews NC. Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing. J Biol Chem 2006;281:28494–8.
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[39] Koziol JA, Ho NJ, Felitti VJ, Beutler E. Reference centiles for serum ferritin and percentage of transferrin saturation, with application to mutations of the HFE gene. Clin Chem 2001;47:1804–10. [40] Adams P, Brissot P, Powell LW. EASL international consensus conference on haemochromatosis. J Hepatol 2000;33:485–504. [41] Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance of 845G→A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet 2002;359:211–8. [42] European Association for the Study of the Liver. EASL clinical practice guidelines for HFE hemochromatosis. J Hepatol 2010;53:3–22. [43] Swinkels DW, Jorna AT, Raymakers RA. Synopsis of the Dutch multidisciplinary guideline for the diagnosis and treatment of hereditary haemochromatosis. Neth J Med 2007;65:452–5. [44] Thorstensen K, Kvitland MA, Irgens WO, Hveem K, Asberg A. Screening for C282Y homozygosity in a Norwegian population (HUNT2): the sensitivity and specificity of transferrin saturation. Scand J Clin Lab Invest 2010;70:92–7. [45] Jackson HA, Carter K, Darke C, et al. HFE mutations, iron deficiency and overload in 10,500 blood donors. Br J Haematol 2001;114:474–84. [46] Bacon BR, Adams PC, Kowdley KV, Powell LW, Tavill AS. American association for the study of liver diseases. Diagnosis and management of hemochromatosis: 2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology 2011;54:328–43. [47] Danesh J, Appleby P. Coronary heart disease and iron status: meta-analyses of prospective studies. Circulation 1999;99:852–4. [48] Ellervik C, Mandrup-Poulsen T, Andersen HU, et al. Elevated transferrin saturation and risk of diabetes: three population-based studies. Diabetes Care 2011;34: 2256–8. [49] Ellervik C, Tybjaerg-Hansen A, Nordestgaard BG. Risk of cancer by transferrin saturation levels and haemochromatosis genotype: population-based study and meta-analysis. J Intern Med 2012;271:51–63. [50] Ellervik C, Tybjærg-Hansen A, Nordestgaard BG. Total mortality by transferrin saturation levels: two general population studies and a metaanalysis. Clin Chem 2011;57:459–66. [51] Niederau C, Fischer R, Pürschel A, Stremmel W, Häussinger D, Strohmeyer G. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110:1107–19. [52] Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med 2005;143:517–21. [53] Schmitt B, Bolub M, Green R. Screening primary care patients for hereditary hemochromatosis with transferrin saturation and serum ferritin level: systematic review for the American College of Physicians. Ann Intern Med 2005;143:522–36.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
Diagnostic value of transferrin Dominika Szőke ⁎, Mauro Panteghini Cattedra di Biochimica Clinica e Biologia Molecolare Clinica, Dipartimento di Scienze Cliniche ‘Luigi Sacco’, Università degli Studi, Milano, Italy
a r t i c l e
i n f o
Article history: Received 20 March 2012 Received in revised form 16 April 2012 Accepted 17 April 2012 Available online 23 April 2012 Keywords: Transferrin Iron deficiency Iron overload Risk marker
a b s t r a c t Despite the growing interest in hepcidin and other relatively new biomarkers, guidelines and clinical pathways continue to recommend traditional markers, such as serum transferrin (Tf) and ferritin, as laboratory tests for the diagnostic evaluation of iron-related disorders. In this study, we aimed to critically evaluate the diagnostic role of Tf relying on the highest level of available evidence by a comprehensive literature search. The role of Tf in iron deficiency (ID) and iron overload (IO) syndrome as well as a risk marker was evaluated. The low accuracy of Tf and Tf saturation (TS) in the diagnosis and management of ID conditions does not permit definitively recommending their use, even if recently published guidelines still consider the TS investigation as a complementary test for ferritin. If a tissue IO is suspected, TS is often used, even if it may not be the best test for detecting this condition. Nevertheless, clinical guidelines strongly recommend the use of TS as a first-level test for performing genetic diagnosis of hereditary hemochromatosis. Recently reported data indicating elevated TS as a risk factor for diabetes mellitus, cancer, and total mortality, may provide useful additions to the debate over whether or not to screen for IO using TS. © 2012 Elsevier B.V. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . Role of transferrin in physiological iron homeostasis Diagnostic role of transferrin determination . . . . 3.1. Background . . . . . . . . . . . . . . . . 3.2. Transferrin in iron deficiency . . . . . . . . 3.3. Transferrin in iron overload . . . . . . . . 4. Transferrin as a risk marker . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Abbreviations: AASLD, American Association for the Study of Liver Diseases; CHD, Coronary heart disease; CI, Confidence interval; CKD, Chronic kidney disease; DM, Diabetes mellitus; EASL, European Association for the Study of the Liver; ESA, Erythropoiesis-stimulating agents; FID, Functional iron deficiency; Hb, Hemoglobin; HH, Hereditary hemochromatosis; ID, Iron deficiency; IDA, Iron deficiency anemia; IO, Iron overload; KDOQI, Kidney Disease Outcomes Quality Initiative; NTBI, Non-transferrin-bound iron; OR, Odds ratio; ROS, Reactive oxygen species; Tf, Transferrin; TIBC, Total iron-binding capacity; TfR, Transferrin receptor; TS, Transferrin saturation; UIBC, Unsaturated iron binding capacity. ⁎ Corresponding author at: Laboratorio Analisi Chimico-Cliniche, Ospedale ‘Luigi Sacco’, Via GB Grassi 74, 20157 Milano, Italy. Tel.: + 39 02 3904 2290; fax: + 39 02 5031 9835. E-mail address: [email protected] (D. Szőke). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.04.021
Iron is an essential micronutrient required for various biological processes, including erythropoiesis, oxidative metabolism, and cellular immune response [1]. At the same time, excess iron causes organ dysfunction through the production of reactive oxygen species (ROS) [2]. As a consequence, a strict regulation of iron homeostasis is mandatory, its maintenance being achieved by a sophisticated balance among mechanisms including iron absorption, transfer, utilization, storage, and loss. During the past few years, the understanding of molecular mechanisms controlling body iron metabolism has dramatically changed by the recognition of hepcidin as the main regulatory hormone of iron metabolism [3]. The introduction of hepcidin determination into clinical practice requires, however, further investigation [4]. Although a few commercial assays currently exist, the appropriate specimen type remains an unresolved issue [5,6]. In addition, excessive hepcidin
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production may occur in a number of acquired disease states, including infections, malignancies, and inflammatory conditions that makes its clinical assessment rather complex [7]. Although we can assume that hepcidin will very likely soon obtain an important role in the diagnostic evaluation of iron-related disorders, current guidelines and clinical pathways continue to recommend the measurement of more traditional biomarkers, such as serum transferrin (Tf) and ferritin. However, it is often difficult to understand if the clinical application of these markers is always evidence-based. The aim of this study was to conduct a comprehensive literature search regarding the diagnostic usefulness of Tf in iron-related disorders to derive available scientific evidence concerning the clinical role of Tf determination in serum. Particularly, we focused on meta-analyses and systematic reviews. Clinical guidelines were, although with precaution, also considered. 2. Role of transferrin in physiological iron homeostasis The human body absorbs 1–3 mg/day of dietary iron balanced with losses via sloughed intestinal epithelial cells, menstruation, and other blood losses. These iron losing mechanisms do not provide an effective iron excreting system and thus the regulation of dietary iron absorption from the duodenum plays a critical role in iron homeostasis. Dietary iron is found in hem and non-hem forms and their absorption occurs in Fe 2+ form at the apical surface of duodenal enterocytes via different mechanisms [8]. Once inside the intestinal epithelial cell, iron may remain in the cell for use or storage, never being absorbed but just lost when senescent enterocytes slough into the gut lumen, or it may be exported across the basolateral membrane of epithelial cells into the circulation [1]. When there is a body demand, iron is exported in reduced form through the basolateral membrane by ferroportin, then it has to be oxidized to Fe 3+ by hephaestin before being bound by Tf. However, the Tf iron pool is replenished mostly by iron recycled from effete red blood cells. Besides dietary iron absorption, iron reutilization is indeed another elementary mechanism providing iron to the body. Senescent red blood cells are cleared by reticuloendothelial macrophages, which metabolize hemoglobin and hem, releasing iron into the bloodstream. By analogy to enterocytes, macrophages export Fe 2+ via ferroportin in a process coupled by re-oxidation of Fe 2+ to Fe 3+ by ceruloplasmin and followed by the loading of Fe 3+ to Tf [9]. Iron is transported in the circulation bound to Tf, which maintains iron in a redox-inert state and delivers it to tissues. Tf has multiple functions; first of all, it is the main plasma transport protein responsible for iron distribution and serves as a storage sink for sequestering iron extracellularly until iron is needed, then allowing it to reach target tissues [10]. On the other hand, Tf represents a protective mechanism against the presence of free-iron in the plasma, which could be extremely toxic to cells. This protective action is dependent upon two features of Tf: its high affinity (Kd ≈ 10 − 20 M) to Fe 3+ and the fact that every Tf molecule has two iron binding sites and under physiological conditions transferrin saturation (TS) is only up to 30%–40% [11]. Developing erythroid cells, as well as most other cell types, acquire iron from plasma. Tf-bound iron enters target cells through receptor mediated endocytosis. Iron-loaded Tf binds with high affinity to Tf receptor (TfR) 1 and the complex undergoes endocytosis via clathrin-coated pits. The acidic pH of endosomes triggers the release of Fe 3+ from Tf. Following the dissociation of iron, the affinity of Tf to TfR 1 dramatically drops resulting in the break-up of the complex and secretion of apo-Tf into the bloodstream to recapture Fe 3+ [9]. The amount of iron bound to Tf (~ 3 mg) corresponds to less than 0.1% of total body iron, but it is highly dynamic and undergoes more than ten times daily turnover to sustain erythropoiesis [9]. In case of iron deficiency (ID), the concentration of Tf in plasma increases, but it reflects the iron status properly only when iron stores are exhausted and when serum iron concentration is b40–60 μg/dL, so
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it does not diagnose ID prior to ineffective erythropoiesis [12]. In iron overload (IO) states, when the whole iron binding capacity of Tf is saturated, an additional iron compartment called non-Tf-bound iron (NTBI) appears in the circulation. Unlike Tf-bound iron, the cellular uptake of NTBI is not dependent on TfR and NTBI is biologically toxic through the production of ROS to every tissue, especially liver, heart, and some endocrine tissues [2]. The activation of NTBI can be predicted by TS values and its increased levels may indicate developing organ toxicity in iron-overloaded patients [13]. 3. Diagnostic role of transferrin determination 3.1. Background Theoretically, the diagnostic gold standard for iron-related disorders is the technique that is able to estimate body iron stores most accurately. In this way, the bone marrow examination, establishing the absence of stainable iron, remains the gold standard for ID diagnosis. Bone marrow examination is, however, invasive, expensive, and requires technical expertise, and thus cannot be performed routinely in clinical practice [14]. In IO conditions, liver iron levels are considered to accurately reflect total body iron stores because liver is the dominant iron storage organ. Liver iron levels have also been used to estimate risk and predict outcomes, such as liver failure, diabetes mellitus (DM), heart failure, and death [13]. As with bone marrow examination, liver iron level quantifying methods, including liver biopsy and imaging techniques, are invasive and/or highly expensive, limiting their use in the screening and diagnosis of IO status. The use of surrogate biomarkers for evaluation of iron status is, therefore, inevitable, even if their diagnostic value can often be limited. Several serum markers are currently used to estimate the individual's body iron stores, including serum ferritin, iron, Tf, total iron-binding capacity (TIBC), and TS. In clinical practice, both Tf and derived TS are widely used parameters in iron status evaluation. Screening for either ID or IO traditionally includes the determination of TS rather than a simple measurement of serum Tf concentrations [15]. The measurement of TS is widely available and cheap. Basically, there are two ways for TS determination, one based on TIBC estimate, the other employing the determination of Tf protein [16]. TIBC is the capacity of plasma proteins to bind iron. Although not the only iron-binding protein in the blood, Tf is the most important and TIBC can be utilized to indirectly determine serum Tf concentrations. In practice, TIBC is determined by the addition of sufficient iron to saturate iron-binding sites of serum proteins in the sample. After removing the iron excess, the assay for iron is performed. The ratio in percentage of serum iron and TIBC, in turn, indicates how much Tf is saturated by iron, so defining TS. As unsaturated iron binding capacity (UIBC) measurement is easily automatable, TIBC is often not directly measured as described above, but calculated by summing the results of serum iron and UIBC. UIBC assays add a fixed amount of iron to saturate ironbinding sites in the sample; the excess iron is not removed, but measured and UIBC is calculated as the difference of added and measured iron. Tf can also be directly determined as protein mass concentration using immunoassays and TS estimated on the results of serum iron and Tf measurements. In a head-to-head comparison by Hawkins [17], the diagnostic value of the immunologic measurement of Tf and of TIBC in the ID diagnosis was evaluated. By means of ROC curve analysis, no difference in the diagnostic performance was found between direct Tf measurement (immunoturbidimetry) and two commercial TIBC formulations. Analytic, biologic, and pathologic factors may influence TS and limit its clinical utility. The within-subject biological variation of iron in serum is high (26.5% in average — http://www.westgard. com/biodatabase1.htm); furthermore, Tf may react as negative acute phase protein in acute inflammation. Additionally, the lack of
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an established higher-order reference method and materials for iron measurement contributes to the variability between different commercial assays and platforms, further limiting the clinical utility of TS as a generic reference interval and/or common decision thresholds cannot be derived [18]. Notwithstanding, Tf and derived saturation index remain tests that are generally applied in clinical practice for the evaluation of both ID and IO. Optimal use of a laboratory test in clinical practice requires an accurate estimate of pre-test probability, i.e. disease prevalence, and knowledge of the test characteristics, i.e. sensitivity, specificity, predictive values, and likelihood ratios associated with various test results. Accordingly, hereafter we critically evaluate the diagnostic value of Tf and TS in iron-related disorders relying on the highest level of available evidence. 3.2. Transferrin in iron deficiency ID and iron deficiency anemia (IDA) may result from the interplay of three distinct factors: increased iron requirements, limited external supply, and increased loss. In absolute ID, body iron stores are depleted, while in functional ID (FID), although the stores are replete, iron cannot be mobilized to the bone marrow fast enough to meet erythropoiesis demands. One of the most important clinical settings for FID is in patients with chronic kidney disease (CKD), who require parenteral iron supplementation to respond to administered erythropoiesisstimulating agents (ESA) for correcting anemia, but it also occurs in many chronic inflammatory diseases [19]. ID can exist in the absence of anemia, if ID has not been present for sufficient time or if it has not been severe enough to cause hemoglobin (Hb) concentrations in blood to fall below the specific thresholds for gender, age, and racial groups [20]. Laboratory evaluation of ID involves assessment of the body's iron depletion and measurements reflecting iron deficient red cell production [16]. As stated above, Hb concentrations in the physiological range do not exclude ID, because an individual with unaltered body iron stores has to lose a large portion of his body iron before Hb concentrations fall below the cut-off defining anemia. Although the red cell distribution width may be the earliest indicator of ID and iron deficient erythropoiesis without anemia, the main laboratory finding indicating ID/IDA is a low concentration of serum ferritin [20,21]. Already in 1992, the landmark overview by Guyatt et al. [22] demonstrated the diagnostic effectiveness of serum ferritin for ID detection across an extensive population of anemic patients with and without inflammatory, liver or neoplastic diseases. Historically, TS has widely been used as screening test for suspected ID [15]. Not long ago, some authors still suggested “a normal or elevated TS as useful for excluding IDA as a low value is for identifying it” [20]. However, even 20 years ago, the study by Guyatt et al. [22], which comprehensively reviewed the available literature, did not recommend TS as an appropriate test for the laboratory diagnosis of IDA. The area under the ROC curve obtained by cumulatively analyzing data from the recruited studies (0.74; 95% confidence interval (CI): 0.70–0.78) indicated the altogether low accuracy of the test for diagnosing ID [23]. On the other hand, using a comprehensive literature search we were unable to find any recent meta-analysis or systematic reviews that could support the clinical utility of TS for the diagnosis of ID and IDA. The only retrieved systematic analysis aimed to identify the best biomarker to monitor population response in large-scale iron fortification programs: unfortunately, TS was measured in only two out of nine selected randomized iron intervention trials, making it difficult to draw any conclusions about this indicator [24]. In spite of the lack of high-grade scientific evidence, recently published clinical guidelines concerning ID/IDA still recommend the TS investigation as a test complementary to ferritin for ID detection [19,25]. Particularly, the guideline by the British Society of
Gastroenterology put TS in the group of further laboratory tests “occasionally necessary” to confirm ID [19]. Similarly, the British Columbia Medical Association's guideline includes TS in the group of additional tests to be considered when clinical features and hematology profile are suggestive of ID, but serum ferritin concentrations are >100 μg/L [25]. Altogether, these guidelines seem to assign to TS the role of confirmation test with higher accuracy for ID than ferritin, something that does not seem supported by the available evidence. Patients with CKD may suffer from various forms of ID, including absolute ID, FID, and an extreme case of FID known as reticuloendothelial blockage. The particular type of ID may affect the validity and reliability of laboratory test results for iron status and often result in a dilemma regarding treatment decisions [26]. Although a number of international guidelines have been published for the evaluation and management of ID/IDA in CKD patients, their recommendations on which combination of laboratory biomarkers are required and how frequently should patients be tested often differ [27]. In all available guidelines the use of TS measurement for ID evaluation is, however, mentioned [28–31]. In clinical practice guidelines of the Canadian Society of Nephrology [28,29], the minimum evaluation for anemia in CKD patients includes both serum ferritin and TS, in addition to a complete blood count. This is, however, associated with the lowest grade of evidence [28]. In addition, recommendations for different CKD patient populations stratified by dialysis execution and ESA therapy administration include target ferritin and TS values, used for the evaluation of iron administration when necessary [29]. In the opinion of the Kidney Disease Outcomes Quality Initiative (KDOQI), the initial laboratory assessment of anemia in CKD patients should include, in addition to a complete blood count, serum ferritin (to assess body iron stores) and TS (or, when available, the content of Hb in reticulocytes) to assess the adequacy of iron for erythropoiesis [30]. However, the same work group also highlights that the clinical utility of TS is markedly impaired by the absence of a decision threshold for ID diagnosis, preventing from the inclusion of the biomarker in a recommendation statement. The KDOQI also suggests TS > 20% as the target of iron therapy during ESA treatment, even if, once again, the supporting evidence is of very limited quality, i.e. a single small randomized controlled trial [32]. The most recent clinical guideline on anemia management in CKD patients similarly recommends a TS >20% as goal for ESA maintenance therapy and iron supplementation, but still with the lowest grade of scientific evidence [31]. The UK National Institute for Health and Clinical Excellence also recommends that for CKD patients with suspected FID (serum ferritin > 100 μg/L), this condition should be defined by a percentage of hypochromic red cells > 6% or, only when this test is not available, by a TS b20% [31]. This is a class B recommendation, indicating that evidence was obtained from well-conducted diagnostic studies. 3.3. Transferrin in iron overload IO syndromes can be classified as hereditary or secondary (Table 1). Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasians [33]. First described in 1996, the most frequent genetic defect is a G→A mutation of the HFE gene, leading to a substitution of tyrosine for cysteine (C282Y) in HFE protein [34]. The C282Y mutation leads to the disruption of an intramolecular disulfide bond of the protein necessary for the interaction with β2-microglobulin, by which the MHC class I-like HFE protein is physically associated. As a result, the C282Y mutant HFE protein is abnormally processed, leading to accelerated degradation and reduced cell surface expression [35]. Various explanations have been proposed about the role of HFE protein in the pathomechanism of HH, including the ‘crypt-programming model’ and the ‘hepcidin model’ [36]. The
D. Szőke, M. Panteghini / Clinica Chimica Acta 413 (2012) 1184–1189 Table 1 Classification of iron overload syndromes. Clinical condition Hereditary iron overload Hereditary hemochromatosis Type 1 (HFE) Type 2A (HJV) Type 2B (HAMP) Type 3 (TFR2) Types 4A and 4B (SLC40AI) Hereditary hyperferritinemia Atransferrinemia Others
Secondary iron overload Thalassemias, congenital sideroblastic anemia, congenital dyserythropoietic anemias, myelodysplastic syndromes, chronic hemolytic anemias Thalassemia major Intravenous, oral, or dietary Alcoholic, viral, non-alcoholic steatohepatitis Porphyria, hemodialysis
Etiopathogenesis
HFE gene mutations: C282Y, H63D, S65C, others Hemojuvelin gene mutations Hepcidin gene mutations Transferrin receptor 2 gene mutations Ferroportin gene mutations Iron-responsive element mutation Transferrin gene mutation DMT1 mutations, aceruloplasminemia
Ineffective erythropoiesis
Transfusion for long periods Iron administration for long periods Liver dysfunction Other
intestinal iron absorption hypothesis is now regarded as unlikely; on the other hand, the mechanisms by which HFE may influence the iron-dependent regulation of hepcidin are still not fully understood [33]. Iron status regulates hepatic hepcidin expression by liver iron stores and by circulating iron levels [37]. One proposed explanation suggests that the complex of TfR1 and HFE acts as an iron sensor at the hepatocyte cell membrane [38]. Because HFE and diferric Tf have overlapping binding sites on TfR1, the bioavailability of HFE might be influenced by concentrations of both TfR1 and diferric Tf [35]. As TS increases, diferric Tf displaces HFE from TfR1, thereby making HFE available to bind to TfR2. It is postulated that the complex of HFE and TfR2 then influences hepcidin expression [33]. Loss of HFE (or TfR2) attenuates gene-mediated signaling to hepcidin [37]. As physiologically hepcidin acts to decrease the absorption of dietary iron and the release of recycled iron from macrophage stores, in case of lacking or decreased hepcidin expression systemic IO can develop [7]. Other common HFE gene mutations include H63D and S65C, but these mutations are generally not associated with IO, unless seen with C282Y as a compound heterozygote [33]. Koziol et al. [39] examined 7250 wild-type, 81 C282Y homozygote, and 438 C282Y/H63D compound heterozygote individuals and found that C282Y homozygotes had abnormally high TS and serum ferritin values relative to the HFE wild types, while compound heterozygotes appeared to be a mixture of individuals with physiologic TS and ferritin concentrations and those with abnormally high values similar to the homozygotes, with equal proportions of each. With the availability of genetic testing, the HFErelated HH is now frequently identified in asymptomatic relatives of HH patients. Accordingly, a genetic diagnosis can be applied to individuals who have not yet developed any phenotypic expression of disease [33]. In 2000, the European Association for the Study of the Liver (EASL) proposed to classify HH individuals detected as C282Y homozygotes in four disease stages as follows: 1) genetic predisposition, but no other abnormality; 2) IO, but without symptoms; 3) IO with early symptoms; and 4) IO with organ damage, e.g. cirrhosis [40]. However, the subsequent observation that a remarkable proportion of individuals who have genetic susceptibility may never develop IO has changed the way of thinking [41].
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Although the majority of C282Y homozygotes may have raised TS levels, this cannot be relied upon as solid evidence of IO, as sensitivity and specificity of TS for identifying C282Y homozygotes are not well documented. On the other hand, the estimate of the prevalence of C282Y homozygosity in individuals with increased values of serum iron markers depends from test cut-offs used for screening people for genetic examination, which are markedly variable in different studies. Elevated TS was found in 1.2%–7.0% of screened individuals in unselected populations (TS cut-off ranging from 45% to 55%, with and without partitioning by gender), 4.3%–21.7% of those were then found to be C282Y homozygotes [42]. Despite these limitations, TS is often recommended as screening test when IO is suspected [43]. A recently performed study aimed to evaluate the diagnostic accuracy of TS as a screening test for finding C282Y homozygotes in the general population [44]. Assuming Hardy–Weinberg equilibrium, the unbiased frequency estimate for C282Y homozygote genotypes in the screened population of 4804 randomly selected persons was 0.75%, somewhat higher than previous estimates in other populations [33,45]. Using 55% (men) and 50% (women) as decision limits on two consecutive TS measurements, the sensitivity was 90.0% and 55.0%, the specificity was 99.6% and 99.4%, the negative predictive value was 99.9% and 99.7%, and the positive predictive value was 64.3% and 43.0% for men and women, respectively [44]. The authors concluded that two consecutive measurements of TS represent an accurate screening test for C282Y homozygosity in men, but not in women. Although differences in inclusion criteria and in biochemical and clinical definitions in various studies exist, an average disease penetrance of 13.5% for C282Y homozygosity was found by a metaanalysis of 19 studies [42]. According to this low disease penetrance, neither EASL nor the American Association for the Study of Liver Diseases (AASLD) in their practice guidelines for HH recommends genetic screening of the general population [42,46]. EASL suggests the following case definition for the diagnosis of HFE gene-related (or type 1) HH: “C282Y homozygosity and increased body iron stores with or without clinical symptoms” [42]. Quite analogously, AASLD establishes that the clinical diagnosis of hemochromatosis should be based on documentation of increased iron stores and that HH can be further defined genotypically by the familial occurrence of IO associated with C282Y homozygosity or C282Y/H63D compound heterozygosity [46]. According to EASL, the detection of increased iron stores is possible by surrogate serum biomarkers (i.e. ferritin) and determination of the amount of iron removed, or by the direct assessment of tissue IO with liver biopsy or imaging techniques [42]. In general, EASL suggests that serum iron concentrations and TS do not quantitatively reflect body iron stores and should, therefore, not be used as surrogate markers of tissue IO [42]. However, EASL strongly recommends the use of TS, even if without any specific cut-off definition, as a screening test for performing the genetic diagnosis of HFE-related HH in: − subjects with suspected IO, who should first receive measurement of fasting TS (and serum ferritin) and the HFE testing performed only in those with increased TS (high quality of evidence); − patients from liver clinics, who should be screened for fasting TS (and serum ferritin) and receive genetic HFE testing if TS is increased (moderate quality of evidence). On the other hand, for EASL HFE testing for the C282Y and H63D mutations should be carried out in all patients with otherwise unexplained increases in serum ferritin and TS [42]. Likewise, AASLD recommends that individuals with abnormal iron studies, including serum ferritin and TS, should be evaluated for hemochromatosis, even in the absence of symptoms [46]. In patients with suggestive symptoms, physical findings, or family history, a combination of TS and ferritin should be obtained rather than relying on a single test. If either test is abnormal (TS > 45% or ferritin above the upper
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reference limit), then HFE mutation analysis should be performed [46]. Finally, to detect early disease and prevent complications AASLD recommends iron studies and HFE mutation analysis in all first-degree relatives of patients with HFE-related HH. 4. Transferrin as a risk marker Elevated TS has been proposed as a marker of the risk for various conditions, such as coronary heart disease (CHD), DM, cancer, or total mortality [47–50]. However, the number of available meta-analyses and systematic reviews evaluating this issue is limited. A metaanalysis of 12 prospective studies involving 7800 CHD cases showed for TS, similarly to other iron status indicators, a non significant CHD risk ratio (0.9, 95% CI: 0.7–1.1) [47]. Ellervik et al. [48] demonstrated by a meta-analysis performed on two general population studies and on an age- and gender-matched population-based case– control study, including 8535 cases and 37,039 controls altogether, that TS levels ≥ 50% were associated with a two- to threefold increased risk of developing DM. In the combined studies, the odds ratio (OR) was 2.1 (95% CI: 1.3–3.4; P = 0.003) for any form of DM, 2.6 (95% CI: 1.2–5.6; P = 0.01) for type 1 DM, and 1.7 (95% CI: 1.4–2.1; P = 0.001) for type 2 DM, respectively, in those individuals with TS ≥ 50% vs. b50%. The same group of authors found that elevated TS levels were associated with increased risk of any cancer [49]. From the meta-analysis, which was carried out using data of three studies with a total of 57,841 patients, an association between elevated TS (≥60%) and increased risk of any cancer was found in the overall population, certainly in women and, to a lesser degree, in men. Corresponding OR for random effect models were 1.5 (95% CI: 1.2– 1.8, P b 0.001) in overall population, 2.2 (95% CI: 1.2–3.8, P b 0.001) in women, and 1.3 (95% CI: 1.0–1.8, P = 0.08) in men alone, respectively [49]. Finally, the same authors carried out a further metaanalysis, including three studies involving 55,873 patients, striving to examine whether increased TS is associated with increased mortality in the general population [50]. The calculated OR for total mortality for TS of ≥50% vs. b50% was 1.4 (95% CI: 1.1–1.9; P b 0.005) under the random effects model. The authors suppose that the biological mechanism behind the association between elevated TS and either DM, cancer, and total mortality risk may be the iron-induced oxidative stress, which may cause β-cell destruction, promote the development of cancer cells, or affect survival. 5. Conclusions From many years Tf and TS have been widely used surrogate markers for the detection of both body iron depletion and IO. However, a general disadvantage of traditional biomarkers in comparison with more recently proposed diagnostic tests is that their use is seldom evidence-based. Our aim was therefore to evaluate the scientific evidence, if any, supporting the clinical usefulness of Tf and derived TS. ID is the most common hematological disorder encountered in general practice and, at the same time, it is one of the most common diseases worldwide, so its correct diagnosis is of utmost importance. Although therapeutic trial of iron has been suggested as a method to diagnose ID in an anemic patient, most clinicians prefer to make a definitive laboratory diagnosis prior to treatment: the lack of high-level evidences limits, however, the role of Tf and TS in the diagnosis of ID and IDA. Clinical guidelines recommend the use of TS only in specific situations, first of all in the management of CKD patients, and not as the test of choice in the diagnosis and management of ID in general. Moreover, the quality of evidence and the strength of the guidelines' recommendations, where indicated, are generally low and weak, and are often based only on the experts' opinion [28–31]. The course and prognosis of HH and most of its complications, including liver cancer, depend on the amount and duration of iron
excess and on the time of therapeutic intervention. Early diagnosis and therapy can certainly prevent the adverse consequences of IO [51]. In 2005, the American College of Physicians published a clinical practice guideline about the screening of primary care patients for HH using TS and serum ferritin determinations, not recommending for or against the screening for HH in the general population because of insufficient evidence [52,53]. More recent HH guidelines do not yet recommend screening for HH in the general population either by genetic testing or by evaluation of increased iron stores [42,46]. Nonetheless, studies examining raised TS levels as a risk factor for CHD, DM, cancer, and total mortality also provide conflicting results about the utility of screening for increased TS [47–50]. If a causal association exists between IO and risk for DM, cancer, and total mortality, then individualized screening programs could be developed and therapy could easily lower high iron concentrations. Morever, a cost/benefit analysis of screening for elevated TS in the general or in selected populations would be required to determine the public health consequences and whether to advice for or against iron supplement tablets and large-scale iron fortification programs. In conclusion, from the currently available data it seems that the role of TS in the diagnosis and management of ID/IDA is limited. On the other hand, TS appears to be a valid test for screening selected patients with suspected IO and/or liver disease for performing genetic tests for HH. Finally, the possible use of TS as risk marker clearly requires further evaluation. References [1] Muñoz M, Villar I, García-Erce JA. An update on iron physiology. World J Gastroenterol 2009;15:4617–26. [2] Kohgo Y, Ikuta K, Ohtake T, Torimoto Y, Kato J. Body iron metabolism and pathophysiology of iron overload. Int J Hematol 2008;88:7–15. [3] Franchini M, Montagnana M, Lippi G. Hepcidin and iron metabolism: from laboratory to clinical implications. Clin Chim Acta 2010;411:1565–9. [4] Moyer TP, Highsmith WE, Smyrk TC, Gross Jr JB. Hereditary hemochromatosis: laboratory evaluation. Clin Chim Acta 2011;412:1485–92. [5] Kemna EH, Tjalsma H, Willems HL, Swinkels DW. Hepcidin: from discovery to differential diagnosis. Haematologica 2008;93:90–7. [6] Singh B, Arora S, Agrawal P, Gupta SK. Hepcidin: a novel peptide hormone regulating iron metabolism. Clin Chim Acta 2011;412:823–30. [7] Finberg KE. Unraveling mechanisms regulating systemic iron homeostasis. Hematology Am Soc Hematol Educ Program 2011;2011:532–7. [8] Siah CW, Ombiga J, Adams LA, Trinder D, Olynyk JK. Normal iron metabolism and the pathophysiology of iron overload disorders. Clin Biochem Rev 2006;27:5–16. [9] Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J 2011;434:365–81. [10] MacKenzie EL, Iwasaki K, Tsuji Y. Intracellular iron transport and storage: from molecular mechanisms to health implications. Antioxid Redox Signal 2008;10: 997–1030. [11] Baker HM, Anderson BF, Baker EN. Dealing with iron: common structural principles in proteins that transport iron and heme. Proc Natl Acad Sci U S A 2003;100:3579–83. [12] World Health Organization. Centers for disease control and prevention. Assessing the iron status of populationsSecond edition. ; 2007. including Literature Reviews. [13] Wood JC. Diagnosis and management of transfusion iron overload: the role of imaging. Am J Hematol 2007;82:1132–5. [14] Zimmermann MB. Methods to assess iron and iodine status. Br J Nutr 2008;99(Suppl. 3):S2–9. [15] Beutler E, Hoffbrand AV, Cook JD. Iron deficiency and overload. Hematology Am Soc Hematol Educ Program 2003:40–61. [16] Higgins T, Eckfeldt JH, Barton JC, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 5th ed. St. Louis: Elsevier Saunders; 2012. p. 985–1030. [17] Hawkins RC. Total iron binding capacity or transferrin concentration alone outperforms iron and saturation indices in predicting iron deficiency. Clin Chim Acta 2007;380:203–7. [18] Miller WG, Myers GL, Ashwood ER, et al. State of the art in trueness and interlaboratory harmonization for 10 analytes in general clinical chemistry. Arch Pathol Lab Med 2008;132:838–46. [19] Goddard AF, James MW, McIntyre AS, Scott BB, on behalf of the British Society of Gastroenterology. Guidelines for the management of iron deficiency anaemia. Gut 2011;60:1309–16. [20] Cook JD. Diagnosis and management of iron-deficiency anaemia. Best Pract Res Clin Haematol 2005;18:319–32. [21] Muñoz M, García-Erce JA, Remacha ÁF. Disorders of iron metabolism. Part II: iron deficiency and iron overload. J Clin Pathol 2011;64:287–96. [22] Guyatt GH, Oxman AD, Ali M, Willan A, McIlroy W, Patterson C. Laboratory diagnosis of iron-deficiency anemia: an overview. J Gen Intern Med 1992;7:145–53.
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