Test ID: FET
Iron, Liver Tissue

Diagnosis of hemochromatosis

See Hereditary Hemochromatosis Algorithm in Special Instructions.

• Hereditary Hemochromatosis Algorithm

Dynamic Reaction Cell-Inductively Coupled Plasma-Mass Spectrometry (DRC-ICP-MS)

Container/Tube:

Preferred: Mayo metal-free specimen vial (blue label) (Supply T173)

Acceptable: Paraffin block, if not more than 1 or 2 cuts have been made to it for slides.

Specimen Volume: 2 mg

Collection Instructions:

1. Two mg of liver tissue is required. This is typically a piece of tissue from a 22-gauge needle biopsy at least 2 cm long. If an 18-gauge needle is used, the tissue must be at least 1 cm in length.

2. Any specimen vial other than a Mayo metal-free vial used should be plastic, leached with 10% nitric acid for 2 days, rinsed with redistilled water, and dried in clean air.

Additional Information:

1. Patient's date of birth is required to calculate iron index.

2. If tissue is other than liver tissue, see MSCM/20522 Miscellaneous Metals Testing.

3. Paraffin blocks will be returned 3 days after analysis.

Forms: If not ordering electronically, submit a General Request Form (Supply T239) with the specimen.

Hemosiderosis is the condition of excessive iron accumulation in tissues. Liver is the first organ affected in iron-overload diseases. Transient increases in iron first appear in Kupffer cells. This finding is commonly related to sideroblastic anemia, excessive iron consumption, or chronic alcohol ingestion. Persistent hemosiderosis, as seen in hemochromatosis, causes iron accumulation in hepatocytes, and is usually concentrated in biliary cells.

Hereditary hemochromatosis is an autosomal recessive disease with estimated prevalence in the population of 2 in 1,000 in Caucasians, with lower incidence in other races. The gene responsible for hereditary hemochromatosis (HFE) is located on chromosome 6; the majority of hereditary hemochromatosis patients have mutations in this HFE gene. Hereditary hemochromatosis is characterized by an accelerated rate of intestinal iron absorption and progressive iron deposition in various tissues that typically begins to be expressed in the third to fifth decades of life, but may occur in children. The most common presentation is hepatic cirrhosis in combination with hypopituitarism, cardiomyopathy, diabetes, arthritis, or hyperpigmentation. Because of the severe sequelae of this disease if left untreated and recognizing that treatment is relatively simple, early diagnosis before symptoms or signs appear is important.

Screening for hemochromatosis is best done by measuring serum iron and transferrin saturation (FEC/34624 Iron and Total Iron-Binding Capacity, Serum). If the serum iron concentration is >175 mcg/dL and the transferrin saturation is >55%, analysis of serum ferritin concentration (FERR/8689 Ferritin, Serum) is indicated. A ferritin concentration >400 ng/mL is suggestive of hemochromatosis, but also can indicate other forms of hepatocyte injury such as alcoholic or viral hepatitis, or other inflammatory disorders involving the liver. HFE analysis (HHEMO/81508 Hemochromatosis HFE Gene Analysis, Blood) may be used to confirm the clinical diagnosis of hemochromatosis, to diagnose hemochromatosis in asymptomatic individuals with blood tests showing increased iron stores, or for predictive testing of individuals who have a family history of hemochromatosis. The alleles evaluated by HFE gene analysis are evident in approximately 80% of patients with hemochromatosis; a negative report for HFE gene does not rule-out hemochromatosis. In a patient with negative HFE gene testing, elevated iron status for no other obvious reason, and family history of liver disease, additional evaluation of liver iron concentration is indicated.

Diagnosis of hemochromatosis may also be based on biochemical analysis and histologic examination of a liver biopsy. In this assay, FET/8350 Iron Liver Tissue, results are reported as the hepatic iron index (HII) and dry weight of iron. The HII is considered the "gold standard" for diagnosis of hemochromatosis. This test is appropriate when:

-Serum iron is >160 mcg/dL 

-Transferrin saturation is >55% 

-Ferritin is >400 ng/mL (males) or >200 ng/mL (females)

-HFE gene test is negative for HFE variants

See Hereditary Hemochromatosis Algorithm in Special Instructions.

IRON

Males: 200-2,400 mcg/g dry weight

Females: 400-1,600 mcg/g dry weight

IRON INDEX

Reference values have not been established for patients that are <13 years of age.

<1.0 mcmol/g/year (> or =13 years)

A hepatic iron concentration >10,000 mcg/g dry weight is diagnostic for hemochromatosis.

Hepatic iron concentrations >3,000 mcg/g are seen when there is iron overload without cellular injury and cirrhosis. Hepatic iron concentrations greater than the reference range are associated with hemosiderosis, thalassemia, and sideroblastic anemia. Some patients with hepatitis or cirrhosis without significant fibrosis will have hepatic iron concentrations at the top end of normal or just slightly above the normal range.

Iron accumulates in the liver normally with aging. The hepatic iron index (HII) normalizes hepatic iron concentration for age. The HII is calculated from the hepatic iron concentration by converting the concentration from mcg/g to mcmol/g dry weight and dividing by years of age. The normal range for HII is <1.0. Patients with homozygous hemochromatosis have HII >1.9. Patients with heterozygous hemochromatosis often have HII ranging from 1.0 to 1.9. Patients with hepatitis and alcoholic cirrhosis usually have HII <1.0, although a small percentage of patients with alcoholic cirrhosis have HII in the range of 1.0 to 1.9. Patients with hemochromatosis who have been successfully treated with phlebotomy will have HII <1.0.

Liver specimens collected from patients with cirrhosis containing a high degree of fibrosis have results near the low end of the reference range, even though they will show significant iron staining in hepatocytes. While it is true that iron accumulates in hepatocytes in advanced alcoholic cirrhosis with fibrosis, there are relatively few hepatocytes compared to other inert (fibrotic) tissue, so the quantitative iron determination, which is expressed as mcg of iron per gram of dry weight tissues, yields a low result. Histologic examination of all tissue specimens should be performed to facilitate correct interpretation. When structural heterogeneity is apparent histologically, variation in measured iron should be anticipated. We have observed, in approximately 2% of cases, a high degree of hepatic heterogeneity that makes quantitation highly variable.

Formalin-fixed, paraffin-embedded tissue can be used when histologic examination (including iron stains) is requested, in addition to the hepatic iron concentration and calculated hepatic iron index. However, fresh or frozen tissue is preferred.

Paraffin blocks from which many slides have been previously cut often appear to contain sufficient quantity of tissue, however, the specimen is often very thin and <2 mg in weight.

1. Brandhagen DJ, Fairbanks VF, Baldus W: Recognition and management of hereditary hemochromatosis. Am Fam Physician 2002;65:853-860, 865-866

2. Summers KM, Halliday JW, Powell LW: Identification of homozygous hemochromatosis subjects by measurement of hepatic iron index. Hepatology 1990;12:20-25

3. Ludwig J, Batts KP, Moyer TP, et al: Liver biopsy diagnosis of homozygous hemochromatosis: a diagnostic algorithm. Mayo Clin Proc 1993;68:263-267

4. Pietrangelo A: Hemochromatosis: an endocrine liver disease. Hepatology 2007;46:1291-1301

5. Ashley EA, Butte AJ, Wheeler MT, et al: Clinical assessment incorporating a personal genome. Lancet 2010;375:1525-1535

6. McLaren CE, Barton JC, Eckfeldt JH, et al: Heritability of serum iron measures in the hemochromatosis and iron overload screening (HEIRS) family study. Am J Hematol 2010;85:101-105

After digestion of the liver tissue with nitric acid and hydrogen peroxide, the digest is diluted and tissue copper concentration is determined using an inductively coupled plasma-mass spectrometer in dynamic reaction cell mode. Calibrating standards and blanks are diluted with an aqueous acidic diluent containing internal standard(s). Quality control specimens and patient samples are diluted in an identical manner. In turn, all diluted blanks, calibrating standards, quality control specimens, and patient specimens are aspirated into a pneumatic nebulizer and the resulting aerosol directed to the hot plasma discharge by a flow of argon. In the annular plasma the aerosol is vaporized, atomized, then ionized. The ionized gases plus neutral species formed in the annular plasma space are aspirated from the plasma through an orifice into a quadrupole mass spectrometer. The mass range from 1 to 263 amu is rapidly scanned multiple times and ion counts tabulated for each mass of interest. Instrument response is defined by the linear relationship of analyte concentration vs. ion count ratio (analyte ion count/internal standard ion count). Analyte concentrations are derived by reading the ion count ratio for each mass of interest and determining the concentration from the response line. Results are reported in mcg copper/g dry weight of tissue.(Bush VJ, Moyer TP, Batts KP, Parisi JE: Essential and toxic element concentrations in fresh and formalin-fixed human autopsy tissues. Clin Chem 1995:41[2];284-294; Hanley MM: Copper and Iron Liver Tissue Analysis: A Comprehensive Platform Comparison [Abstract 1600-7P]. PittCon 2008, New Orleans, LA, March 2-7, 2008)

Monday through Friday; 11 a.m.

83540