EASL Clinical Practice Guidelines

How should HFE-HC be diagnosed?

The EASL CPG panel agreed on the following case definition for diagnosis of HFE-HC:

C282Y homozygosity and increased body iron stores with or without clinical symptoms.

The following section will address the genetic tests and tools for assessing body iron stores.

Genetic testing – Methodology

C282Y homozygosity is required for the diagnosis of HFE-HC, when iron stores are increased (see diagnostic algorithms). Any other HFE genotype must be interpreted with caution. The available methods are reported in Table 7. The intronic variant c.892+48 G>A may complicate amplification refractory mutation system (ARMS) – PCR for genetic testing [183]. The common S65C polymorphism may complicate interpretation of real-time PCR and melting curve analysis tests [184]. Finally, in cis inheritance of rare genetic variants [185] must be considered when gene tests are interpreted.

Table 7
Methods for HFE genotyping.

Increased body iron stores

Serum ferritin

The most widely used biochemical surrogate for iron overload is serum ferritin. According to validation studies where body iron stores were assessed by phlebotomy, serum ferritin is a highly sensitive test for iron overload in hemochromatosis [21]. Thus, normal serum concentrations essentially rule out iron overload. However, ferritin suffers from low specificity as elevated values can be the result of a range of inflammatory, metabolic, and neoplastic conditions such as diabetes mellitus, alcohol consumption, and hepatocellular or other cell necrosis.

Serum iron concentration and transferrin saturation do not quantitatively reflect body iron stores and should therefore not be used as surrogate markers of tissue iron overload.

Therefore, in clinical practice, hyperferritinemia may be considered as indicative of iron overload in C282Y homozygotes in the absence of the confounding factors listed above.


Magnetic resonance imaging (MRI): The paramagnetic properties of iron have been exploited to detect and quantify iron by MRI. The 'gradient recalled echo techniques' are sensitive when using a well-calibrated 1.5 Tesla device. There is an excellent inverse correlation between MRI signal and biochemical hepatic iron concentration (HIC) (correlation coefficient: −0.74 to −0.98) allowing for the detection of hepatic iron excess within the range 50–350 μmol/g with a 84–91% sensitivity and a 80–100% specificity according to cut-off levels of HIC ranging from 37 to 60 μmol/g wt [[196], [197], [198]]. MRI may also help to (i) identify heterogeneous distribution of iron within the liver, (ii) differentiate parenchymal (normal splenic signal and low hepatic, pancreatic, and cardiac signals) from mesenchymal (decreased splenic signal) iron overload, and (iii) detect small iron-free neoplastic lesions. However, only a few patients with HFE-proven HC were studied [197].

Superconducting quantum interference device (SQUID) susceptometer: The SQUID susceptometer allows for in vivo measurement of the amount of magnetization due to hepatic iron. Results are quantitatively equivalent to biochemical determination on tissue obtained by biopsy. However, the device was not specifically validated in HFE-HC patients. In addition, it is not widely available, which restricts its use in clinical routine [[199], [200], [201]].

Liver biopsy

Liver biopsy used to be the gold standard for the diagnosis of HC before HFE genotyping became available. Now that this is readily available, homozygosity for C282Y in patients with increased body iron stores with or without clinical symptoms is sufficient to make a diagnosis of HFE-HC.

Where there is hyperferritinemia with confounding cofactors, liver biopsy may still be necessary to show whether iron stores are increased or not [98]. Liver biopsy still has a role in assessing liver fibrosis. The negative predictive value of serum ferritin <1000 μg/L and normal AST in absence of hepatomegaly for the presence of severe fibrosis or cirrhosis averaged 95% [[202], [203]].

Serum hyaluronic acid is reported to correlate with the degree of hepatic fibrosis in HC, and if validated may provide an alternative approach to liver biopsy for the diagnosis of advanced fibrosis [204]. Transient elastography can also be helpful for determination of advanced fibrosis and cirrhosis [205].

Amount of iron removed

The total number of phlebotomies required to achieve low concentrations of serum ferritin may be a useful retrospective surrogate marker for the excess body iron stores in HFE-HC. The assumption that 1 L of blood contains 0.5 g of iron allows for an estimate of the amount of iron removed by phlebotomies. This broadly correlates with pre-therapeutic hepatic iron concentration. Allowing for the amount of absorbed iron during therapy and taking into account the initial and post-therapeutic haemoglobin levels improves the reliability of the calculation, especially when the interval between phlebotomies exceeds one week [203].

Family screening

Siblings of patients with HFE-related HC must undergo screening, since they have a 25% chance of being susceptible. Serum ferritin, and transferrin saturation should be assessed. Ideally HFE mutation analysis should be encouraged after appropriate counseling with regard to the pros and cons of testing (mortgage, insurance issues).

Whether they are screened with the above procedure depends upon their age, health status, and the attitude of the family.

Individuals who are C282Y homozygotes, or have HFE-related HC, frequently ask for advice on the evaluation of the susceptibility of their children who are often younger than the age of consent. In this situation, HFE genotyping of the unaffected spouse is valuable [206], so that the likelihood of genetic susceptibility and thus the need for testing of children later in life can be established.

Method Simultaneous detection of multiple mutations bf Detection of novel/rare genetic variations Specialized equipment required Amenable for high throughput Ref.
RFLP PCR amplification followed by restriction fragment length polymorphism +/− [ [148], [149], [150], [151], [152], [153], [154], [155], [156], [157], [158], [159], [160], [161], [162], [163], [164], [165], [166], [167], [168], [169], [170], [171], [172], [173], [174], [175], [176], [177], [178], [179], [180], [181], [182], [183], [184], [185], [186], [187], [188], [189], [190], [191], [192], [193], [194], [195], [196], [197], [198], [199], [200]