| General Overview of Testing Tests for evaluating iron metabolism are generally used as initial or screening tests for hereditary hemochromatosis (HH) as they will detect the phenotypic expression of HH. These tests include serum iron (SI), transferrin (Tf) or total iron binding capacity (TIBC), serum ferritin (SF), and unsaturated iron binding capacity (UIBC).The serum ferritin assay is also used to assess the effectiveness of HH treatment.Molecular (DNA) analyses for HFE mutations are considered to be confirmatory tests for HH which may be ordered reflexively in patients with elevated iron results. Laboratories should establish their own reference intervals for assays of iron metabolism. In general, reference intervals vary by sex and by method used for the assays discussed in the following section. Typical reference intervals are included in the following sections for instructive purposes only and should not be used for evaluating actual patient data.The results of laboratory tests assessing iron metabolism should be interpreted with caution because a number of pre-analytical and physiologic factors can affect the results. Repeating elevated test results on fasting specimens is often advisable. | View Page |
| Which laboratory assay is considered to be a confirmatory test for hereditary hemochromatosis (HH)? | View Page |
| References 1. Beutler E. Iron storage disease: Facts, fiction and progress. Blood Cells Mol Dis. 2007;39:140-7.2. Higgins T, Beutler E, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, editor. Teitz Fundamentals of Clinical Chemistry. 6th ed. Saunders Elsevier, 2008.3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation. Blood 2003;102(3):78-8.4. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiolo. 2007;69:69-85.5. Murtagh LJ, Whiley M, Wilson S, et al. Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis. Am J Gastroenterol. 2002;97(8):2093-9.6. Haddy TB, Castro OL, Rana SR. Hereditary hemochromatosis in children, adolescents, and young adults. Am J Pediatr Hematol Oncol 1988;10:23-4.7. Edwards CQ, Ajoika RS, Kushner JP. Hemochromatosis: A genetic definition. In Barton JC, Edwards CQ, eds. Hemochromatosis: Genetics, Pathophysiology, Diagnosis and Treatment. Cambridge, UK:Cambridge Univ Pr 2000:8-11.8. Whitlock EP, Garlitz BA, Harris EL , et al. Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force. Ann Intern Med. 2006; 145: 209-23.9. Wallace DF, Subramaniam VN. Non-HFE haemaochromatosis. World J Gastroenterol. 2007;13(35):4690-8.10. Tavill AS. Diagnosis and management of hemochromatosis. Hepatology. 2001;33:1321-811. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK, et al. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2005;143:517-21.12. Phatak PD, Bonkovsky HL, and Kowdley KV. Hereditary Hemochromatosis: time for targeted screening. Ann Intern Med. 2008; 149(4): 270 – 2.13. Brissot P, deBels F. Current approaches to the management of hemochromatosis. Hematology Am Soc Hematol Educ Program. 2006:36-41. 14. Guidance for industry: Variances for blood collection from individuals with hereditary hemochromatosis. http://www.fda.gov/cber/gdlns/hemchrom.htm Accessed 12/17/08. | View Page |
| What is a mobilizable, water-soluble form of storage iron that is bound to protein? | View Page |
| Regulation of Iron Equilibrium Regulation of iron equilibrium occurs mainly through the process of absorption. Iron is absorbed through the mucosal cells lining the duodenum. A variety of proteins are involved in this process. Hepcidin, an antimicrobial protein primarily produced in the liver, has been recently found to be a major (negative) regulator of dietary iron absorption by disrupting cellular iron transport in the intestine. Decreased levels of hepcidin are related to increased iron absorption into the bloodstream. Hepcidin is increased in response to iron overload and inflammation. (4)Additional proteins involved in iron metabolism include transferrin (Tf), transferrin receptor (TfR), ferroportin, HFE protein, hemojuvelin, and others. Their roles in iron absorption are complex and in some instances incompletely understood.Factors affecting iron absorption include: Tissue stores, e.g., decreased stored iron is associated with a decrease in hepcidin and increase in iron absorption. Rate of hematopoietic activity, e.g., an increased rate of erythropoiesis is associated with a decrease in hepcidin and an increase in iron absorption. Oxygen concentration in tissues, e.g., hypoxia decreases hepcidin and increases iron absorption, thereby promoting increased erythopoiesis. Dietary intake, including form of iron ingested, e.g., heme iron is more readily absorbed than non-heme forms of iron. Condition of GI tract mucosal cells Intraluminal factors, e.g. intestinal motility | View Page |
| Which of the following is NOT considered to be an important protein regulator of iron metabolism? | View Page |
| Iron Transport Once absorbed through the mucosal cells of the duodenum, iron is bound to a carrier plasma protein, transferrin (Tf), for movement to sites of utilization. Almost all iron in plasma is bound to Tf, and most Tf-bound iron is carried to the bone marrow to be incorporated into developing erythrocytes. Transferrin is normally about 20% to 40% saturated with iron. (5)Transferrin releases iron to specific transferrin receptors (TfRs) for movement into cells. Transferrin receptors are found on all cells, but are found in relatively high concentration in erythroid precursors, hepatocytes, and placental cells. When the capacity of plasma Tf to bind iron is exceeded, i.e., transferrin saturation (TS) is higher than normal, excess iron is taken up by hepatocytes and other cells. A brief summary of iron metabolism is illustrated. | View Page |
| What is the protein that carries iron in the blood plasma? | View Page |
| Altered Iron Absorption Hereditary hemochromatosis (HH) is a genetic disorder characterized by iron overload as a result of increased iron absorption. As iron absorption increases, the amount of iron bound to transferrin and transported in the plasma subsequently increases.With no available mechanism for excreting excess absorbed iron, normal iron storage sites become overloaded, resulting in ferritin levels that far exceed normal. As a result, iron is deposited in the parenchymal cells of the liver, pancreas, pituitary, heart, synovium, and other tissues with high concentrations of transferrin receptors. Iron in excess of normal cellular ferritin stores contributes to the generation of free radicals and reactive oxygen intermediates that cause cell damage to organs and tissues. This process results in the clinical condition known as iron overload, a hallmark feature of HH. | View Page |
| HFE and Other Genes A hemochromatosis gene, HFE, was identified in 1996. Mutations in the HFE gene are found in the majority of patients diagnosed with hereditary hemochromatosis (HH). The locus for the gene is on the long arm of chromosome 6 where it codes for a membrane protein, HFE. The exact mechanism of the role of HFE protein in iron metabolism is incompletely understood. It is thought that HFE, along with a second protein, beta-2 microglobin, interacts with transferrin receptors (TfR) on cell membranes. This interaction supresses the affinity of transferrin for TfR, thus lowering the uptake of transferrin--and its attached iron--into the cell. Transferrin receptors have been found on the surface of a variety of cells, with the greatest concentration on cell membranes of intestinal cells, hepatocytes, and RBC precursors. In addition to HFE, HH is also associated with mutations in other genes involved in iron homeostasis, including hemojuvelin (HJV), TfR, hepcidin, and ferroportin. Hepcidin production is reduced in HH due to all of these genetic causes, with a resulting increase in iron absorption. Mutations in HFE are the most common cause of HH. | View Page |
| Mutations in which gene account for the majority of cases of hereditary hemochromatosis (HH)? | View Page |
| Specific HFE Mutations Several mutations of the HFE gene have been described. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations. | View Page |
| How is the function of HFE protein altered in the C282Y mutation? | View Page |
| Incomplete Penetrance For reasons as yet unknown, not all individuals who are homozygous for the C282Y mutation display phenotypic features of HH, and persons with H63D polymorphisms rarely develop iron overload. The penetrance (percentage of individuals with a specific genotype who express the associated phenotype) of HFE mutations is generally considered to be low. Results of a recent meta analysis by the US Preventive Services Task Force conclude that 38% to 50% of all C282Y homozygotes develop some evidence of iron overload, but that only 10% to 33% develop clinical disease due to HH. (8) In other words, some individuals may have elevated iron test results such as transferrin saturation, but do not demonstrate significant organ damage. Estimates of penetrance in some studies have found it to be even lower. Penetrance of HFE mutations is currently a controversial subject among experts, and the significance of finding HFE mutations in a given individual is often unclear. The probability that a given individual with HFE mutations will develop clinical disease from iron overload cannot be determined at this time. | View Page |
| Non-HFE Mutations Genes in addition to HFE have been linked to hereditary hemochromatosis (HH). They include the hepcidin, hemojuvelin, transferrin receptor, and ferroportin genes. Mutations of some are linked with dominantly inherited HH, juvenile HH, and African iron overload. Unlike HH due to HFE mutations, these clinical disorders are rarely observed in the US population.(9) | View Page |
| Diagnosing HH The diagnosis of hereditary hemochromatosis (HH) is made through a combination of laboratory tests and medical evaluation of a patient's signs and symptoms. Iron overload is identified by tests that evaluate iron metabolism, while molecular assays are needed to document mutations in the HFE gene or others such as hepcidin, hemojuvelin, or transferrin receptor. Individuals with documented iron overload who exhibit signs and symptoms consistent with HH and who possess HFE or other mutations are considered to have HH. Other causes of secondary iron overload may need to be ruled out.An example of a testing algorithm is shown. | View Page |
| Serum Iron Serum iron (SI) is a measure of circulating iron bound to transferrin and is reflective of total body iron. SI is elevated in hereditary hemochromatosis (HH) and acute hepatitis. SI is decreased in iron deficiency anemia and chronic inflammation. SI concentrations exhibit diurnal variation, with the lowest values occurring around midnight. In addition, specimens collected from the same individual at the same time of the day may exhibit day to day variations as high as 40%. SI determinations are also affected by diet, menstrual cycle, pregnancy, ingestion of iron supplements, and oral contraceptive use. SI levels alone are considered insensitive indicators of HH. SI is typically measured on automated analyzers using spectrophotometric methods. Iron in the sample is released from transferrin with an acid reagent, reduced to the ferrous state, and reacted with a chromogen such as bathophenanthroline or ferrozine. The intensity of the color change is proportional to the iron concentration. Interference can arise from the use of a hemolyzed sample and contamination of reagents and water with iron. A typical reference interval for SI is 60 - 150 micrograms/dL. SI is usually ordered along with its companion test, the total iron binding capacity (TIBC), or with transferrin (Tf).(2) | View Page |
| What laboratory test reflects circulating iron that is bound to transferrin? | View Page |
| Transferrin and Total Iron Binding Capacity The test for transferrin (Tf) measures the concentration of the primary carrier protein for iron. Measuring total iron binding capacity (TIBC) is an indirect method of assessing transferrin and provides comparable information. The TIBC (or transferrin) are typically performed along with the SI. Taken together, these determinations are useful in the differential diagnosis of many disorders affecting iron metabolism, including hereditary hemochromatosis (HH) and iron deficiency anemia. Tf and TIBC are typically low-normal or decreased in HH and are increased in iron deficiency. Serum transferrin can be measured directly using immunochemical methods such as nephelometry and turbidimetry. TIBC is performed in a 2-step method by adding ferric iron to the specimen in sufficient quantity to completely fill all of the iron binding sites on transferrin. Excess, unbound iron is removed by adsorption with magnesium carbonate, alumina, or ion resin. The iron content of the saturated binding protein is then measured as described for SI. Serum is the specimen of choice for Tf and TIBC. TIBC is less subject than SI to day-to-day variation and other causes of variability.A typical reference interval for TIBC is 300 - 360 micrograms/dL.(2) | View Page |
| Transferrin Saturation Transferrin saturation (TS) is usually reported along with the SI and TIBC. TS indicates the percent of iron binding sites on transferrin that are carrying iron. TS is derived from a calculation using the formula:TS =(SI/TIBC) x 100TS results are reported as percentages. Typical reference intervals for TS are 20% to 55% for males and 15% to 50% for females. TS is generally considered to be the most sensitive laboratory test for detecting altered iron metabolism in hereditary hemochromatosis (HH). It may be elevated prior to significant deposition of tissue iron. TS levels increase as additional iron is accumulated.A drawback to using the TS is that it is dependent on performing both the SI and TIBC. The UIBC (see section below) may be a lower cost alternative.The optimal TS criterion for detecting HH is controversial. Using a TS of >60% for males and >50% for females has been found highly accurate in detecting abnormal iron metabolism in persons with HH. Others studies suggest using lower TS levels, e.g. 45%, as a criterion indicating further testing is warranted. Current guidelines from the American College of Physicians include a TS cutoff level of >55% for identifying iron overload. (11)Patients with initially increased TS should be followed by performing a second TS from a fasting morning specimen. The patient should also be advised not to take vitamins supplemented with iron or oral contraceptives for several days prior to the repeated test. TS levels may be affected by diurnal variation, dietary factors, and co-existing disease states such as inflammation and hepatitis. Patients with HH may have falsely normal TS if chronic blood loss or inflammatory disease is present. | View Page |
| What is the transferrin saturation (TS) for a person with a serum iron of 200 micrograms/dL and a TIBC of 250 micrograms/dL? | View Page |
| What is the American College of Physicians' recommended criterion level for transferrin saturation when testing for hereditary hemochromatosis (HH)? | View Page |
| UIBC Unsaturated iron binding capacity (UIBC) may also be used as a marker for altered iron metabolism. UIBC represents the portion of iron binding sites on transferrin that are not occupied by iron. Therefore, a low UIBC indicates that transferrin is highly saturated with iron, a finding consistent with hereditary hemochromatosis (HH). HH may be suspected when the UIBC is less than 143 micrograms/dL, a criterion suggested by the results of one study.(5)UIBC may be a lower cost alternative to the more complex transferrin saturation (TS). UIBC and SI are both fully automated procedures that are available on widely used laboratory instruments. The TIBC can be calculated by adding UIBC and SI, resulting in a value for TIBC that can be used for determining TS: TS = SI/(SI + UIBC) X 100 | View Page |