Hemochromatosis is the most common type of primary iron overload disease, but it remains under-diagnosed because of the lack of awareness of it, its long latency period, and its nonspecific symptoms [2, 3]. Recently, increased emphasis has been placed on early detection, shifting the case definition and diagnosis to earlier stages of the disease. This has led to various views on the best diagnostic methods and the essential components of the diagnostic evaluation. In the next few years, these issues should become clearer as we gain insight into the natural history and expression of hemochromatosis. In this article, we update the description of hemochromatosis and the tests used to diagnose it.

Background

Hemochromatosis was first recognized more than a century ago as a condition with a triad of symptoms-diabetes, skin bronzing, and cirrhosis-associated with hepatic iron overload [1]. The condition was first called hemochromatosis in 1889 [4], and it was first proposed as an inherited disorder in 1935 [5]. Its inheritability remained controversial for four decades [6], until Simon and colleagues [7] demonstrated the close association between HLA-linked hemochromatosis and HLA-A3 and established that the responsible gene was tightly linked to the HLA-A locus on the short arm of chromosome 6. In recent years, a candidate gene for HLA-linked hemochromatosis, HFE, has been cloned, and a single G-to-A mutation resulting in a cysteine-to-tyrosine substitution (C282Y) has been identified in 60% to 100% of study patients with hereditary hemochromatosis [8-11]. A second mutation, H63D, was linked to an additional 1% to 10% of cases in one series [11], but no large population-based studies have been done to definitively establish the prevalence of this mutation in the general population. Cases of and families with hemochromatosis not associated with either the C282Y or the H63D mutation (non-HFE-associated hemochromatosis) have been reported from studies of European populations, and the genetic basis for these cases is being studied [10, 11].

Historically, hemochromatosis was a clinical and pathologic diagnosis. Diagnosis relied on the classic features of cirrhosis: pigmentation, diabetes, and arthralgia. As a result, hemochromatosis was described as rare, with an estimated frequency of 1 case in 20 000 hospital admissions in the United States [12]. However, autopsy studies [13, 14] found a much higher frequency: 1 to 2 cases per 1000 persons. More recently, population-based screening studies in several western countries [15-18] have established the prevalence of hemochromatosis as approximately 1 case per 300 persons. With the advent of genetic testing, earlier diagnosis is possible. In addition, some long-standing cases of hemochromatosis have been reviewed and found to be due to the heterozygous form of the C282Y mutation [11, 19]. To date, it seems that in case series of patients with hemochromatosis, 0.5% to 14% of patients have actually been heterozygous [11].

Expression and Natural History

The natural history of hemochromatosis begins with a genetic potential (Table 2). This condition expresses itself as a tendency to overabsorb iron from the gastrointestinal tract. At least 50% of male and 25% of female persons homozygous for hemochromatosis are likely to develop potentially life-threatening complications of the disease [1, 18, 19], especially in countries with high dietary intake of iron [19-24].

The first phenotypic expression of disease is an elevation in serum transferrin saturation, which represents the transport of excess iron from the intestine and occurs before significant iron loading (Table 2). As iron accumulates in tissue, the serum ferritin concentration increases in direct linear relation to total-body iron stores [1, 24]. Patients usually begin to have symptoms between age 30 and 50 years. This natural history varies; symptoms occur much earlier in some patients. Early symptoms and signs of hemochromatosis include severe fatigue, impotence, arthralgia, arthritis, and an elevated concentration of liver enzymes [1]. Later, patients may experience skin bronzing; arthropathy; cardiomyopathy; and endocrine disorders, including diabetes and hypogonadism [1, 21-26]. Once the hepatic iron concentration reaches 400 µmol per g dry weight, cirrhosis is common and the risk for hepatocellular carcinoma and death are markedly increased [26]. However, this threshold may be lower if cofactors, such as ethanol intake and chronic hepatitis, are present [1].

Although persons who are heterozygous for hemochromatosis sometimes have phenotypic expression, they do not generally develop overt clinical disease [1]. Of persons detected through family screening who are established as heterozygous for hemochromatosis (for example, by HLA typing), approximately 25% have mild biochemical abnormalities and increased body iron stores (as assessed by liver biopsy or quantitative phlebotomy) but do not develop clinical disease from progressive iron loading or the consequent organ damage [1, 18, 27, 28]. If a patient is heterozygous for the C282Y mutation and has a coexisting condition (such as hepatitis, alcoholism, or porphyria cutanea tarda) that increases hepatic iron stores, however, symptoms of organ damage may appear [19, 25]. Thus, consideration of coexisting conditions is important for heterozygous as well as homozygous patients.

The expression of hemochromatosis is affected by environmental factors. The use of supplementary iron and vitamin C (which increases iron absorption) may lead to earlier phenotypic expression. On the other hand, blood donation, physiologic blood loss (through menstruation and pregnancy), and pathologic blood loss (for example, through peptic ulceration or inflammatory bowel disease) may delay phenotypic expression and decrease the amount of iron stored in the liver. The belief that premenopausal women cannot develop symptomatic or even life-threatening hemochromatosis is a misconception [29-31].

Diagnosis

The basis for the early diagnosis of hemochromatosis has shifted from clinical symptoms to biochemical tests. This shift has spared patients the sequelae of protracted iron overload and chronic disease, although it has also spawned differences of opinion about the role and necessity of certain diagnostic tests, particularly liver biopsy. As more information about the disease is gathered, the case definition for hemochromatosis is likely to continue to evolve in this rapidly changing field.

Clinical Features

Hemochromatosis has many clinical presentations, and heightened awareness on the part of the physician is required for early diagnosis [1]. Fatigue and arthralgia are the most common symptoms prompting a visit to a physician. Patients may also present with hepatomegaly, diabetes mellitus, arthritis, heart failure, increased skin pigmentation, or abdominal pain, any of which might lead to referral to a specialist. The prevalence of hemochromatosis in patients attending diabetes and rheumatology clinics is greater than that expected in the general population [25, 32, 33]. Another mode of presentation may be cardiomyopathy, particularly in younger patients [1, 2]. Patients may present with congestive heart failure or arrhythmia. Occasionally, no clinical symptoms are seen even when hemochromatosis is advanced and cirrhosis is present [24].

Biochemical Tests Indicating Phenotypic Expression

Biochemical measures of iron status are used to screen for hemochromatosis (Table 2); tests for transferrin saturation (serum iron concentration divided by total iron-binding capacity, multiplied by 100) and serum ferritin level are recommended. A persistently elevated transferrin saturation in the absence of other causes of iron overload strongly suggests hemochromatosis. A fasting transferrin saturation of 45% or more is typically used as the screening threshold because it identifies 98% of affected persons while producing relatively few false-positive results [34]. An alternative screening strategy may use the test for unsaturated iron-binding capacity, which is inexpensive and may be best used in population screening. However, this test has yet to be thoroughly evaluated. The follow-up evaluation also includes physical examination, estimation of the serum ferritin level, complete blood count, and liver function tests.

A high transferrin saturation is the earliest phenotypic evidence of hemochromatosis. If a patient has a transferrin saturation of more than 45% but less than 55% on a repeated test and the elevation has no other evident cause, such as inflammatory liver disease, hemochromatosis may be present. If the serum ferritin level is normal, the patient should have repeated tests after 2 years to identify any change. The patient may be either homozygous or heterozygous for hemochromatosis. We do not have enough information to know the usual course of disease detected at this stage.

If the transferrin saturation is 55% or more on a repeated test, the first step is to check for the presence of increased body iron stores. Patients who have an elevated transferrin saturation on repeated tests but have a normal serum ferritin level may be classified as having nonexpressed hemochromatosis. These patients warrant annual or biennial assessment to watch for increases in iron stores [24, 35]. The serum ferritin level defines the point at which hemochromatosis is expressing iron overload and treatment should be initiated. When transferrin saturation on a repeated test is 55% or more, an elevated serum ferritin level ( 200 µg/L in premenopausal women and>300 µg/L in men or postmenopausal women) or evidence of liver disease (elevated liver enzyme concentrations or hepatomegaly) indicates primary iron overload due to hemochromatosis. What remains unclear is the serum ferritin level at which organ damage, particularly cirrhosis of the liver, occurs. In patients in whom hemochromatosis is indicated by an elevated transferrin saturation or genetic testing, organ damage is unlikely if the serum ferritin level and liver function are normal. Phlebotomy therapy is initiated if serum ferritin levels increase beyond normal limits, and patients whose serum ferritin levels are known to have recently gone from the normal to the abnormal range can be assured that they have a very low likelihood of cirrhosis and other irreversible conditions.

Serum ferritin is an acute-phase reactant. The C-reactive protein level or erythrocyte sedimentation rate can be obtained to establish whether elevated serum ferritin levels are due to inflammation, infection, or cancer rather than elevated iron stores. However, mild iron overload has been described in patients with normal transferrin saturation and elevated serum ferritin levels alone [36].

Another biochemical test sometimes used in screening for hemochromatosis is a test for serum iron concentration, but the result of this test alone is an unreliable marker for hemochromatosis. The test is often available on large screening panels, but the levels are labile. Marked diurnal variation is normal; concentrations are highest in the morning and are elevated after meals [1].

Serum liver biochemical test results for alanine aminotransferase and aspartate aminotransferase are often, but not always, abnormal in patients with overt hemochromatosis [26]. However, abnormal results suggest the presence of fibrosis, cirrhosis, or other complicating factors in combination with hemochromatosis [26, 32]. Conversely, another cause of hepatic inflammation, particularly alcoholic liver disease or nonalcoholic steatohepatitis, should be considered if liver biochemical test results are abnormal [1].

Liver Biopsy

Liver biopsy with histologic evaluation and biochemical quantification of hepatic iron concentration has long been the gold standard for the diagnosis of hemochromatosis [1, 37-39]. Recent emphasis on the early detection of presymptomatic cases has called into question the need for liver biopsy in evaluating the early stages of iron overload due to hemochromatosis. Nonetheless, most experts believe that liver biopsy is still central to the evaluation of most patients with hemochromatosis [1, 26, 39]. It is used to identify not only the degree of iron overload but also the presence of cirrhosis, which is an important determinant of prognosis and management. When cirrhosis is documented, the patient should be monitored for the development of primary hepatic carcinoma, which can be treated effectively if detected in its earliest stages. However, liver biopsy may not be necessary for all patients, particularly younger patients who may not have accumulated hepatic iron stores large enough to cause liver damage.

It is generally agreed that liver biopsy is essential for patients who present with the physical signs of cirrhosis or with a history of excessive alcohol intake. Liver diseases are common in the general population and are at least as common in persons with hemochromatosis. For example, hepatitis C exacerbates liver damage from hemochromatosis; conversely, excessive iron in the liver resulting from hemochromatosis may decrease the effectiveness of interferon therapy for hepatitis [40].

The liver biopsy specimen is evaluated for histochemical characteristics and hepatic iron concentration to determine whether iron overload due to hemochromatosis is present (Table 3). In patients with hemochromatosis, Perls stain typically reveals grade 3 to 4 iron stores in hepatocytes; the greatest concentration is in periportal hepatocytes [1]. In contrast, in patients with alcoholic liver disease, liver biopsy specimens show fatty infiltration, alcoholic hyaline, and Kupffer-cell iron deposition. Dividing the hepatic iron concentration in µmol per g dry weight by the patient's age in years gives a hepatic iron index score. Hepatic iron concentration can be measured from fresh tissue obtained at the time of biopsy or, retrospectively, from formalin-fixed paraffin-embedded tissue. A hepatic iron index score of 1.9 or more has been shown to reliably differentiate patients with hemochromatosis who are presumed to be homozygous from those who are heterozygous for hemochromatosis and those who have alcoholic or other chronic liver diseases [37-41]. In some homozygous patients with hemochromatosis, however, the hepatic iron index may be less than 1.9, particularly if physiologic or occult pathologic blood loss has occurred [37].

Liver biopsy may be contraindicated because of concomitant medical conditions or may be refused by the patient for religious or other reasons. In such cases, careful follow-up with clinical assessment and laboratory testing should be used to look for symptoms or signs of cirrhosis. If cirrhosis is suspected, screening with testing for serial -fetoprotein levels and ultrasonography may be appropriate.

Quantitative Phlebotomy

The amount of mobilizable iron removed from the body by weekly or biweekly phlebotomy is an accepted criterion for measuring the degree of iron overload and confirming the diagnosis of hemochromatosis. In patients without hemolytic diseases or other causes of secondary iron overload, removal of 4 g or more of mobilizable iron stores (16 phlebotomies, each removing 500 mL of blood [250 mg of iron per 500 mL]) before the development of iron-limited erythropoiesis confirms the presence of primary iron overload due to hemochromatosis.

Maintaining complete phlebotomy records is necessary to establish the presence of iron overload by quantitative phlebotomy. Assessment of iron stores may also be confounded by sporadic blood donation or other blood loss.

Tissue Typing

In siblings of a proband who has been proven to have hemochromatosis by other means (such as liver biopsy or quantitative phlebotomy), HLA typing has been used to detect homozygous hemochromatosis. In this setting, a sibling who is HLA-A-identical and HLA-B-identical to the proband is considered homozygous; a sibling who shares only one haplotype with the proband is considered heterozygous [1, 23]. However, direct genetic testing for the C282Y mutation will probably ultimately replace HLA typing as a means of genetic testing in families with HFE-associated hereditary hemochromatosis.

Genetic Testing and DNA-Based Diagnostic Tests

The identification of the C282Y mutation in the HFE gene responsible for most cases of hereditary hemochromatosis has led to a DNA-based test for at least one form of hemochromatosis, seen particularly in European populations; a second mutation, H63D, may account for a smaller proportion of cases. Genetic testing for HFE mutations may play an important role in confirming the diagnosis of hemochromatosis, particularly in young patients detected through screening programs. In such patients, cirrhosis is unlikely to be present and liver biopsy to detect organ damage may be unnecessary. HFE genotyping may be adequate as a confirmatory test but cannot provide any information about the degree of increased body iron stores or organ damage. In addition, DNA-based testing cannot replace liver biopsy in confirming the presence of end-stage organ damage in patients whose clinical picture includes other forms of liver disease or patients with an elevated serum ferritin level.

Although the diagnostic algorithm for hemochromatosis presented here (Figure 1) does not consider genetic testing to be a central factor in decision making, genetic testing may be helpful for a patient with an elevated transferrin saturation, a normal serum ferritin level, normal liver enzyme concentrations, and no hepatomegaly (Table 2). If a patient is found to be homozygous for the C282Y mutation, he or she can be tested annually until the serum ferritin level increases; this increase should prompt initiation of phlebotomy therapy. Depending on age, serum ferritin level, presence of hepatomegaly or liver enzyme abnormalities, and other clinical features, a patient who is homozygous for C282Y or H63D or is compound heterozygous can be further assessed by quantitative phlebotomy or liver biopsy. Approximately 20% to 25% of persons heterozygous for C282Y have an elevated transferrin saturation; fewer have iron stores in the range expected for homozygotes. In these cases, liver biopsy is helpful in differentiating heterozygous patients with normal iron stores from persons with increased iron stores who may be compound heterozygous. In addition, heterozygotes should be evaluated for hepatitis C, porphyria cutanea tarda, and nonalcoholic steatohepatitis.

View larger version (12K): [in this window] [in a new window]   Figure 1. Algorithm for screening for and diagnosing hemochromatosis. *At the time of the second transferrin saturation test, tests for serum ferritin level and liver function, physical examination, and complete blood count should also be done. A genetic test to show whether a patient is a homozygote or a mixed heterozygote for HFE gene mutations may be useful for risk assessment, but the positive and negative predictive values of this test have not been established. If a patient is heterozygous, the physician may want to evaluate for such conditions as hepatitis C virus infection, nonalcoholic steatohepatitis, and porphyria cutanea tarda. Homozygotes with normal iron measures might have follow-up with annual serum ferritin tests. HC = hemochromatosis; HIC = hepatic iron concentration; HII = hepatic iron index; LFT = liver function test; SF = serum ferritin; TS = transferrin saturation.

The use of DNA-based tests alone may fail to identify 20% to 40% of white patients and most African-American patients with clinical evidence of hemochromatosis who lack the C282Y mutation. Genetic testing cannot yet play a role in family screening when the proband lacks an HFE mutation. Presumably, other (as yet unidentified) genetic mutations are responsible for at least some cases of non-HFE-associated hemochromatosis. For example, as noted above, at the time of discovery of the C282Y mutation of the HFE gene for hemochromatosis, a second mutation, H63D, was found in approximately 4% of patients with hemochromatosis [8]. Most laboratories that perform genetic testing for hemochromatosis provide information about the presence of both mutations, but the clinical significance of H63D is still unclear. There is uncertainty about the penetrance of this mutation, both when it is present alone or in association with heterozygosity for C282Y and when it is combined with other pathologic conditions, such as porphyria cutanea tarda or hepatitis. Therefore, genetic testing for and clinical decision making based on H63D status cannot be recommended as routine clinical practice at this time.

Finally, it should be emphasized that some persons with clinical hemochromatosis lack the C282Y mutation and some are heterozygous for it. The place of DNA testing in hemochromatosis awaits widespread test availability and further information on the test's operating characteristics. Population screening for the C282Y mutation is not currently recommended [11].

Discussion

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The diagnosis of hemochromatosis requires a high index of suspicion on the part of the physician. In most patients, elevation of the serum ferritin level in the absence of inflammatory causes is considered to be consistent with clinical expression of hemochromatosis. The serum ferritin level reflects the degree of iron overload, but the critical threshold at which the serum ferritin level is associated with cirrhosis is currently unknown. Thus, liver biopsy is the only method with which to establish or exclude the presence of hepatic cirrhosis. The role of DNA testing for the recently described C282Y and H63D mutations is currently under investigation, and population screening is currently considered premature.

Early diagnosis and treatment of hemochromatosis can lead to normal life expectancy. It is therefore appropriate that medical subspecialty organizations and patients are calling for the routine evaluation of iron status as part of adult health care.

Conclusions

The earliest phenotypic expression of hemochromatosis is an elevated transferrin saturation; this is seen before significant iron loading occurs. The test for transferrin saturation is recommended for population screening. After this test is done, further evaluation is instituted to confirm the presence of hemochromatosis or to rule out the presence of other diseases that may elevate the transferrin saturation, such as primary liver diseases or secondary iron overload. The increasing interest in and availability of a genetic test for hemochromatosis promises to simplify the diagnosis of this disease in relatives of affected persons and may be helpful as a confirmatory test in patients with very early disease. The role of DNA-based testing in confirming or diagnosing hemochromatosis is still being reviewed. The use of DNA testing for population screening cannot be advocated until appropriate studies have been done.