Accurate assessment of iron status is critical to prescribing treatments for anemia in chronic kidney disease (CKD) and assessing the effectiveness of those treatments. Iron status can be assessed by a wide range of tests, including invasive procedures or through biomarkers in the blood. But which of these tests are most useful? Both the presence of storage iron and the availability of iron to support ongoing erythropoiesis must be determined. Further, once the results of the test are received, what level constitutes iron deficiency in patients with end-stage renal disease (ESRD) and indicates a need for an adjustment in treatment?
We have relied on the use of serum ferritin and transferrin saturation (TSAT) to guide iron therapy but increasingly, we face the clinical scenario of high serum ferritin to imply iron overload coupled with low TSAT to imply iron deficiency. This paradox is challenging our management of anemia of chronic renal disease.
Trends in iron therapy: Emerging treatments for anemia in patients with CKD, ESRD
This article presents a brief description of the most well-known iron status testing methods and discusses factors to consider, such as accuracy, reliability, potential for confounding, and burden on the patient and health care provider. Next, the clinical and epidemiological evidence supporting the recommended levels of biomarkers to indicate iron sufficiency are reviewed.
Tests involving invasive procedures
The gold standard for iron assessment is iron staining of bone marrow obtained by aspiration. But this method is invasive, expensive, and rarely done. The absence of stainable iron in the bone marrow is defined as absolute iron deficiency and correlates with a percent TSAT of < 20%. A liver biopsy provides a snapshot into the amount of iron stored in the liver. When performed by a skilled health care provider, these tests are highly accurate and reliable but interobserver variability may exist. Given the high burden of the invasive procedures, both in terms of cost and health care resource utilization, it is not feasible to conduct these tests every 3 months as is normally required to assess the efficacy of prescribed treatments. Superconducting Quantum Interference Devices (SQUIDs) detect liver iron content in a non-invasive manner through magnetic resonance, but this is not widely available and therefore not presently clinically useful.
Comparatively, blood or serum samples are more easily procured and a wide range of tests for biomarkers are available to determine iron status. Serum iron, hemoglobin (Hb), ferritin, and TSAT are the most commonly utilized tests while soluble transferrin receptor, reticulocyte hemoglobin content (CHr), and hepcidin are less common and some are thought to be less reliable. Tests for serum iron determine the levels of iron in the blood. Levels of iron in the blood drop dramatically during inflammation when iron becomes trapped in the reticuloendothelial system in macrophages, preventing the iron from being available for Hb synthesis and making it essentially biologically unavailable.
Ferritin is a large intracellular protein that stores iron. Most of the iron stored in ferritin is accessible for metabolic needs but it also serves as the iron storage depot during inflammation-induced iron sequestration. Anemia in patients with CKD is thought to be a highly inflammatory condition and this inflammatory state may inhibit the mobilization of iron from the reticuloendothelial stores. Consequently, serum ferritin, which is an acute-phase reactant, may reach very high levels. Ferritin helps to regulate iron absorption. Mucosal ferritin receives iron from within the gastrointestinal tract, which is then released to serum transferrin via mucosal transferrin when iron is needed by the body. The iron in the mucosal cells is excreted when those cells are shed as they undergo turnover every 3 days. The iron holding capacity of these cells provides a buffer against short-term changes in iron need or supply.
Apoferritin is a protein in the intestinal wall that binds iron in a ferric hydroxide-phosphate compound to form ferritin. Ferritin is measured in the plasma to indicate iron stores (1 ng ferritin/mL ≈ 10 mg total iron stores), but may not be a reliable indicator of total body iron load.1 Ferritin is commonly tested to evaluate iron stores but a recent study found 15.1% intrapatient biological variability. Researchers estimated 3 sample days were necessary to determine ferritin levels within ± 20% of the true mean with 95% probability.2 Ferritin is critical to cellular defense against oxidative stress and inflammation in its role as an acute phase reactant; ferritin translation is induced by pro-inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α.3,4
Transferrin is the major transporter of iron trafficking through the plasma and the protein to which virtually all iron in the blood is bound. Transferrin binds to its receptor, a transmembrane protein present on most cells but dense on erythroid precursors and placental cells, and it is rapidly internalized into the cell. Circulating transferrin is normally about 1/3 saturated with iron. Transferrin saturation is calculated as plasma iron concentration divided by total iron binding capacity (TIBC) × 100, which corresponds to circulating iron. TSAT is a common test to evaluate iron stores available to support erythropoiesis but the percent saturation is reduced during inflammation when the plasma iron is sequestered in macrophages. In a study of 15 dialysis patients, TSAT was a poor indicator of iron body stores.1 TSAT also has been reported to have a high degree (38.2%) of biological variability and requires an estimated 15 sample days to achieve a level of closeness within ± 20% of the true mean with 95% probability. Gaweda et al. recently concluded that the maximum Hb response is at 34% TSAT with little incremental effect above that level. TSAT had modifying effect on Hb response but ferritin did not.5
Reticulocyte hemoglobin content (CHr) or reticulocyte hemoglobin equivalent (RetHe) is a measure of the amount of Hb in the reticulocytes, red blood cells that are just 1 or 2 days old. It is a relatively new measurement of iron status that provides an indirect measure of iron available for erythropoiesis. CHr/RetHe can determine the need for iron repletion in patients with functional iron deficiency because it provides an accurate measurement of iron available for erythropoiesis in the past 3-4 days. Preliminary studies show the test is reliable, reproducible, and accurate but it is affected by changes in cellular volume and requires special diagnostic equipment.6
Soluble transferrin receptors (sTfR) are circulating receptors from bone marrow erythroid precursors. The sTfR concentration in the serum is proportional to the erythropoietic rate and inversely proportional to tissue iron bioavailability. While there are mixed reviews on the reliability of sTfR in assessing iron deficiency because it may be elevated in patients with increased erythropoietic rate, a ratio of sTfR to the log of ferritin concentration may be useful in determining iron stores.7
The percentage of hypochromic erythrocytes (% HYPO) is a test of the concentration of Hb in red blood cells and red blood cell size, as opposed to the Hb content as in CHr. It is used as a measure of functional iron deficiency because accounts for both size of and absolute amount of Hb in a red blood cell. The test for % HYPO must be performed within hours of sample collection because the test is dependent on cell size and red blood cells expand after sample collection. The test is used widely in Europe where swelling is not an issue since samples are measured in local laboratories with a short storage time. Most dialysis labs in the United States are national, so there is considerable time delay (up to 18-24 hrs) and RBC swelling between specimen collection and testing. Erythrocyte zinc protoporphyrin (ZPP) measures iron incorporation into heme because zinc is used to replace iron when iron stores are low.
Hepcidin is a peptide that is produced in the liver for iron homeostasis. It is an acute phase reactant protein that is up-regulated in response to high iron levels, infection, and inflammation, and down-regulated by erythropoiesis.8.9 It is an important mediator for iron absorption and mobilization. Hepcidin is important in regulating iron status during inflammation but its level has not been shown to be clinically useful or superior to other methods in determining iron sufficiency. 10
Reticuloendothelial blockade is a state in which iron is sequestered in the macrophages during inflammation. Functional iron deficiency is defined as the inability to efficiently mobilize iron from the liver and other storage sites despite having sufficient iron stores according to tests. In both states, TSAT is ≤ 20% and ferritin is elevated, usually > 800 ng/ml. Patients with absolute iron deficiency have insufficient iron stores to meet the erythropoietic demand, commonly defined as serum ferritin < 100 ng/ml for non-dialysis CKD and peritoneal dialysis and < 200 ng/ml for hemodialysis (HD).
When assessing iron status, alternative reasons for anemia should be considered, such as acute inflammation via C-reactive protein (CRP), and vitamin B12 and folate levels. Additionally, patients on HD have increased blood loss from blood left in the dialyzer, frequent blood sampling, bleeding from access sites, or gastrointestinal bleeding.
TSAT and ferritin remain the most useful markers for assessment of iron status because of their widespread availability and support in the literature. The clinical paradox of high ferritin and low TSAT, however, has made us focus on identifying other markers that can be used to predict a clinical response to iron administration. CHr and percent of % HYPO appear to have better predictive ability (sensitivity and specificity) for response to iron supplementation than TSAT or ferritin, according to a comparative effectiveness review.11 Due to the typical time delay due to sample shipping, the use of % HYPO is limited in the U.S. By guiding iron management with CHr rather than TSAT and ferritin, the number of iron status tests might be reduced but given the small number of available studies. This conclusion, however, may be preliminary.12, 13 CHr may be an effective additional test result or “tie breaker” for assessing patients with high ferritin levels and low TSAT.
How often should we test?
2012 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines suggest evaluating iron status more frequently than 3 months for patients who are new to ESA therapy, with marginal iron status, or who require their response to IV iron evaluated. For patients with adequate iron stores on maintenance ESA and iron, every 3 months is sufficient. In HD patients treated with a loading dose of IV iron, TSAT stabilized in 7 days and ferritin stabilized in 14 days indicate iron indices stabilize quickly.14
What are ideal levels for ferritin and TSAT?
In combination, serum ferritin and TSAT should be the primary tools and are the most commonly used measurements to determine iron status in patients with CKD. But the specific values or ranges to define iron sufficiency and deficiency are controversial. The 2012 Clinical Practice Guidelines for Anemia in CKD from the KDIGO Work Group define anemia as Hb < 13 g/dL in males and < 12 g/dL in females and recommend a 1–3 month trial of IV iron supplementation in CKD patients (or oral iron supplementation in CKD ND patients) if Hb is deemed too low, TSAT is ≤ 30%, and ferritin is ≤ 500 ng/ml for patients not already on iron or ESA therapy.10 Once end-stage renal disease patients are on erthryopoiesis-stimulating agent (ESA) therapy, the same thresholds apply but they also suggest iron if a decrease in ESA dose is desired. Previously, the 2006 guidelines from National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI)15 suggested administering iron to maintain ferritin > 200 ng/mL and TSAT > 20%. At that time, they found insufficient evidence to recommend IV iron when ferritin > 500 ng/mL.
The DRIVE study found that providing IV iron with concomitant increase in ESA dose to patients with high ferritin (500-1,200 ng/ml) and low TSAT levels (≤ 25%) increased hemoglobin levels in the short term.16 In the study, TSAT, ferritin, sTfR, and CRP were not good predictors of response to anemia treatment.17 Importantly, in the CHOIR study the patients treated to a target Hb level of 13.5 g/dL had a higher risk of adverse events with no improvement in quality of life.18 A number of new studies have been published since the KDIGO guidelines were developed. A small retrospective analysis of HD patients found providing iron to patients to target TSAT > 30% unless ferritin levels were > 1,200 ng/ml decreased ESA requirements but did not significantly increase infectious complications.19
Iron overload may occur in patients who have a serum ferritin > 800 ng/ml; however, this is not a validated marker for iron overload in CKD patients and is highly variable. In the pre-ESA era, when patients received multiple transfusions, serum ferritin levels were found to be in the 1,000–2,000 ng/ml range. However, autopsy evidence revealed very little tissue iron deposition in spite of these very high values. In addition, patients with hemochromatosis with documented liver deposition in major organs have serum ferritin levels > 2,000 ng/ml, which is 2.5 times greater than the levels which we see in most of our dialysis patients.
Hemodialysis patients often receive high doses of intravenous iron. The continuous injection of intravenous iron further contributes to the prooxidant state of HD patients. In a recent analysis of patients on HD with serum ferritin > 1,000 ng/ml, 90% of patients had increased malonyldialdehdye, a byproduct of lipid peroxidation and indication of oxidative stress. Oxidative stress was detected in the spleen and liver. Mild to severe iron overload was present in 70% of dialysis patients even though most had serum ferritin levels < 500 ng/ml.20 Importantly, iron overload, referring to hemosiderosis or too much iron in storage organs, differs from parenchymal organ damage from iron. During inflammation, iron should be in the reticuloendothelial cells; however, due to constant intravenous iron injections, the binding capacity of transferrin protein is overwhelmed. This causes the non-transferrin bound iron to become catalytically active. This phenomenon is a vicious cycle that will continue to repeat until a therapeutic alternative to intravenous iron is provided to clinicians.
What level of ferritin is clinically relevant?
Table 2 shows our interpretation of serum ferritin levels in dialysis patients. The clinical significance of the serum ferritin measurement in dialysis patients is somewhat different than in the general population, and the current guidelines to use serum ferritin for iron overload screening might be flawed when applied to the dialysis population. Using such arbitrary upper limits as 800 ng/ml to recommend withholding oral or IV iron administration in CKD patients may be scientifically flawed, because moderately high levels of serum ferritin do not necessarily indicate iron overload but most likely inflammation, infection, malnutrition, liver disease and/or other non-iron related factors in dialysis patients. However, if serum ferritin rises in response to oral or IV iron administration, serum ferritin may also reflect iron store expansion. Hence, the following conceptual equation was developed by Kalantar-Zadeh et al. to reflect multi-faceted features of serum ferritin variation in dialysis patients:21
Ferritin = iron + inflammation + other factors
Given the undeniable scientific evidence pertaining to the significant contribution of non-iron related factors, such as inflammation and hyperferritinemia in CKD, the utility of measuring serum ferritin as a routine measure of iron stores in CKD patients is highly questionable. Moderately high levels of serum ferritin are not, per se, indicators of iron-overload and should not be regarded as means to restrict iron supplementation for optimal anemia management. A low iron status may be as harmful as, if not more harmful, than the hyperferritinemia. In the previously reported cases of iron overload in dialysis patients, the reported serum ferritin levels were well above the currently observed ranges in CKD patients, usually in the 2,000 to 10,000 ng/mL range.22 Instead of focusing on a specific number or level from a laboratory test, a holistic approach of caring for the entire patient, including comorbidities and quality of life, is recommended. -by Ellie Kelepouris, MD, FAHA; Kamyar Kalantar-Zadeh, MD, MPH, PhD
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