CHAPTER 14 Disturbances of Copper and Iron Metabolism

Wilson’s disease (hepatolenticular degeneration)

Wilson’s disease is an autosomal recessive disorder due to mutations in the gene ATP7B for copper-transporting ATPase located in the trans-Golgi network of the liver.¹ It is uncommon but important and treatable. Normal hepatic copper transport² is disrupted owing to various ATP7B mutations,³ leading to the accumulation of copper in hepatocytes and liver disease. The large number and diverse mutations identified currently precludes simple genetic testing,⁴ in contrast to hereditary haemochromatosis (discussed later). Liver biopsy is important for histological diagnosis and monitoring.^4a

Chemical quantitation of copper concentration in the biopsy sample helps to establish the diagnosis and is sometimes used for determination of the genetic status of a patient’s siblings.⁵^,⁶ Copper determination can be made from specimens obtained by routine liver biopsy or retrieved from paraffin blocks, without special copper-free solutions or instruments.⁷ Homozygous individuals have increased liver copper levels from an early age but do not develop symptoms of liver disease in the first few years of life. Increased liver copper levels precede the development of histological abnormalities. Hepatic copper levels are typically greater than 4 µmol/g dry weight (>250 µg/g dry weight).⁷

Histological lesions develop before the disease is clinically apparent. In the early, pre-cirrhotic phase there is fatty change,⁶ sometimes with the formation of fat granulomas.⁵ Slender fibrous septa extend from portal tracts (Fig. 14.1). There may be unusually abundant lipofuscin pigment in hepatocytes and glycogen vacuolation of hepatocyte nuclei, but neither feature is easy to evaluate; both are found in normal individuals, and nuclear vacuolation is particularly common in the young. Lipofuscin granules may be larger and less regular in outline than normal.⁸ Inflammation is absent or mild in the early stages. Kupffer cells are sometimes enlarged and may stain for iron as a result of haemolysis. Electron microscopy helps in the diagnosis of both early and late disease because of characteristic changes in mitochondria and lysosomes (see Ch. 17).

Figure 14.1 Wilson’s disease.

At this early stage slender septa extend from portal tracts (P) but acinar architecture is intact. There is steatosis, just visible in this reticulin preparation. (Wedge biopsy, reticulin.)

In some patients a phase of chronic hepatitis develops next that is difficult to distinguish histologically from chronic viral hepatitis. Stains for copper and copper-associated protein may be helpful, as discussed below. Cirrhosis develops in untreated patients, with or without a recognisable preceding phase of chronic hepatitis. A common though not invariable pattern is of active cirrhosis with fatty change, ballooned hepatocytes, focally dense eosinophilic cytoplasm and glycogen vacuolation of nuclei (Fig. 14.2). Cholestasis may be present. Hepatocytes often contain Mallory bodies and these are sometimes very abundant. They are associated with an infiltrate rich in neutrophils, as in steatohepatitis (Fig. 14.3). Partial fibrous occlusion of efferent veins has been reported.⁸ Hepatocellular carcinoma is a rare sequel of cirrhosis in Wilson’s disease.⁹^,¹⁰

Figure 14.2 Wilson’s disease.

Active cirrhosis with liver-cell swelling, steatosis (arrowheads) and nuclear vacuolation (arrow). (Wedge biopsy, H&E.)

Figure 14.3 Wilson’s disease.

Numerous Mallory bodies (arrowheads) are seen within hepatocytes. (Post-mortem liver, H&E.)

Fulminant hepatic failure may be the first manifestation of Wilson’s disease and is a major indication for liver transplantation.¹¹ The presence of haemolysis in a young individual with acute liver failure should therefore prompt consideration of Wilson’s disease.¹² Cirrhosis is usually already present in such cases¹³^,¹⁴ in contradistinction to acute liver failure, owing to viral or drug hepatitis where recent massive necrosis is evident. The cirrhotic nodules are frequently small and separated by septa containing abundant ductular structures and variable chronic inflammatory cells (Fig. 14.4). The death of hepatocytes in the fulminant disease occurs by both apoptosis and necrosis,¹⁵ resulting in new zones of confluent necrosis superimposed on the underlying cirrhotic architecture. Cholestasis is often striking and hepatocytes may contain large- or small-droplet fat. The presence of much stainable copper and/or copper-associated protein in hepatocytes and Kupffer cells distinguishes Wilson’s disease from other causes of fulminant hepatic failure.

Figure 14.4 Fulminant liver failure in Wilson’s disease.

Fulminant hepatitis in Wilson’s disease usually develops on a background of already developed cirrhosis, as seen in this case. A: Cirrhotic nodules (N) are surrounded by inflamed fibrous septa with numerous bile ductular structures. The acute illness is related to progressive hepatocyte necrosis, inflammation and ductular reaction at the septal–parenchymal interface as seen in the upper right field. B: Severe bile canalicular and hepatocellular cholestasis with both small- and large-droplet steatosis are present. (Explant liver, H&E.) C: Copper-binding protein is present in both periportal hepatocytes and sinusoidal Kupffer cells. (Explant liver, Victoria blue.)

Staining for copper and copper-associated protein plays a part in the diagnosis of Wilson’s disease, though staining results (as well as the copper concentration) can vary considerably throughout the liver.¹⁶ Failure to stain in either case is common at some stages of the disease and does not therefore exclude the diagnosis. Conversely, both copper and copper-associated protein are found in other liver diseases, usually as a result of failure to secrete copper into the bile. Thus, in a child with liver disease strong staining for copper might reflect loss of bile ducts rather than Wilson’s disease. Other copper storage disorders have been described, including Indian childhood cirrhosis (see Ch. 13, Fig. 13.22), which is also occasionally seen elsewhere in the world.¹⁷^–¹⁹ Furthermore, neonatal liver is normally rich in copper.²⁰

In the early phases of Wilson’s disease, liver copper levels are high, but the copper is difficult to demonstrate histochemically. This is because it is diffusely distributed in hepatocytes and not concentrated in lysosomes. Sensitive histochemical methods (e.g. Timm’s silver method or rhodanine) may show faint cytoplasmic staining. Later in the course of the disease copper begins to accumulate in liver-cell lysosomes and is then more easily stained. Once cirrhosis has developed, the distribution of copper is typically uneven, some nodules staining strongly while others are negative (Fig. 14.5). Staining for copper and copper–protein may be dissociated, although in most cases both are positive.²¹^,²² Timm’s silver stain appears to be the most sensitive staining method for demonstrating copper in this disease.²³

Figure 14.5 Wilson’s disease.

The upper nodule is strongly positive for copper, stained orange-red. The lower nodule is completely negative. (Wedge biopsy, rhodanine.)

Because of the great variety of histological lesions in the liver, Wilson’s disease can easily be mistaken for other liver disorders. Clinicians and pathologists should consider Wilson’s disease in the differential diagnosis of hepatocellular disease especially in the young, but also at all ages, including (uncommonly) older-aged individuals.²⁴ The disease can be arrested by treatment and its development prevented in siblings. The penalties for missing the diagnosis are therefore very great.

Iron overload

Siderosis

Siderosis (or haemosiderosis) means the presence of demonstrable iron in tissues, irrespective of cause. The main forms of iron in hepatocytes are ferritin, haemosiderin and haem.²⁵ Stainable iron is mainly haemosiderin which is principally located in lysosomes and is seen as granules concentrated towards the biliary poles of the cells. Ferritin gives rise to more diffuse staining, imparting a bluish hue to the liver-cell cytoplasm on iron staining. Hepatocellular siderosis almost always shows a diminishing gradient of intensity from the periphery of lobules towards the central (efferent) veins. It is most severe in periportal regions (acinar zones 1) near small portal tracts, and least severe in centrilobular regions (acinar zones 3). The normal adult liver is usually negative on iron staining or at best shows minimal siderosis.²⁶ This is also true of the neonatal liver, although some cases may show mild periportal liver-cell siderosis (residual iron storage from the active period of hepatic haemopoiesis of the third trimester).²⁷

Since iron stains of liver tissue are expected to be negative in most instances, a positive stain requires explanation. In this regard, two major categories of hepatic iron storage disease need to be considered, designated as primary and secondary iron overload disorders²⁸ (Table 14.1). The primary disorders are predominantly forms of hereditary haemochromatosis in which genetic mutations alter iron homeostasis in the gastrointestinal tract and liver. The secondary disorders are acquired conditions in which increased iron in the liver is due to exogenous sources of iron, abnormal erythrocyte destruction or changes in iron absorption and distribution related to underlying liver disease. The pathologist may be able to suggest the reason for the siderosis, based on the distribution of the stainable iron. For example, in most of the primary iron overload disorders, such as classic HFE-related haemochromatosis, the excess iron is mainly hepatocellular. In thalassaemia both hepatocytes and macrophages are positive, while exogenous iron overload leads to Kupffer-cell storage in the first instance. Various types of underlying liver disease are also associated with siderosis. Cirrhotic livers of varied aetiology may contain much iron,²⁹^–³¹ even within macroregenerative nodules.³² In viral hepatitis and alcoholic liver disease small amounts of stainable iron are often found. Siderosis in the setting of non-alcoholic fatty liver disease (dysmetabolic hepatic iron overload) is increasingly recognised.³³ Dense, iron-positive granules are common in endothelial cells in a variety of conditions including acute hepatitis,³⁴ chronic hepatitis B and C³⁵ and alcoholic liver disease, but their significance is not known.

Table 14.1 Primary and secondary iron overload disorders

^* Types 1–4 are classified as forms of hereditary haemochromatosis in the OMIM (Online Mendelian Inheritance in Man) database.⁴⁴

The siderotic liver should be evaluated for the distribution of stainable iron among the various cell types, the grade of siderosis, the presence of any related tissue damage (fibrosis, cirrhosis, necrosis or even hepatocellular carcinoma) and coexisting liver disease of other aetiology. Various numerical methods of assessing the degree of siderosis (discussed below) are also helpful in evaluating causation and the effectiveness of therapeutic iron removal.

Numerical assessment of tissue iron

Many different systems have been devised for the quantification of iron in tissue sections.³⁶ Histological grading of hepatocellular iron can be simply scored on a scale from 1 to 4, with grade 1 representing minimal deposition (recognisable only with a high-power objective), grade 4 massive deposits with obliteration of the usual lobular gradient, and grades 2 and 3 intermediate amounts. Examples are shown in various illustrations to this chapter. The alternative comprehensive grading system of Deugnier and colleagues³⁷ measures iron not only in hepatocytes, but also in mesenchymal cells, bile-duct epithelium, blood vessels and connective tissue, generating a score between 0 and 60. This has proved helpful in the assessment of patients with hereditary haemochromatosis.

Tissue for measuring the iron concentration can be taken separately at the time a specimen is obtained for histology or by fine-needle aspiration biopsy,³⁸ or the actual paraffin block can be analysed³⁹ after histological examination is complete. This has the advantage that the nature of the sample is known.⁴⁰ Once the hepatic iron concentration is determined, the hepatic iron index (HII) described by Bassett and colleagues⁴¹ can be calculated: HII = tissue iron in µmol/g dry weight, divided by the age of the patient in years (or tissue iron in µg/g dry weight divided by [55.8 × age in years]).⁴⁰^,⁴¹ Because of the progressive accumulation of iron in classic HFE-related hereditary haemochromatosis, this enables patients with the disease (who have an iron index of 1.9 or more) to be distinguished from heterozygous patients and those with siderosis from other causes. However, in some patients with cirrhosis due to alcohol or other aetiologies unrelated to hereditary haemochromatosis, secondary accumulations of large amounts of iron in the liver may result in a calculated HII of 1.9 or greater, thereby mimicking the hereditary disease.²⁹^,³⁰ Conversely, a small percentage of individuals with hereditary haemochromatosis may have an iron index of less than 1.9.⁴² In recent years genetic analysis has diminished the importance of these calculations.

The HII has been found to correlate well with a similar index derived from histological assessment and age, using the grading system of Deugnier and colleagues described above.⁴³ Use of this histological iron index avoids destruction of the tissue block and can be performed when the hepatic iron concentration is not available. Computerised image analysis is another approach to measuring iron deposition which correlates well with biochemical assay.³⁶

Primary iron overload disorders

Molecular genetic studies have now defined a variety of heritable disorders affecting iron handling by the gastrointestinal tract and liver.⁴⁴ Several of these are listed in Table 14.1 and the reader is encouraged to consult the section on General reading at the end of this chapter for further details. The best understood of the primary iron overload disorders was first described in 1889 by von Recklinghausen⁴⁵ and is the disease referred to as ‘hereditary haemochromatosis’. The majority of these cases are examples of what is currently known to be classic HFE-related hereditary haemochromatosis, which is discussed below. However, the identical picture of predominantly periportal hepatocellular iron overload can be found in patients with various combinations of the gene defects listed in Table 14.1. There is thus a pathological pattern of classic haemochromatosis with more than one possible cause.⁴⁴

Classic HFE-related hereditary haemochromatosis

This autosomal recessive disorder is associated with progressive accumulation of iron in the liver, heart, pancreas and other organs. The frequency of homozygous disease is approximately 1 person in 300,⁴⁶ while heterozygotes are found in about 1 person in 8–10.⁴⁷ Overt disease may be found in as few as 1 in 5000,²⁵ and even within families homozygous persons may show different rates of iron accumulation.⁴⁸ The HFE gene, the gene for this type of haemochromatosis, is located on the short arm of chromosome 6 at some distance from the HLA-A locus.^47,⁴⁹^–⁵² A mis-sense mutation in HFE known as Cys282Tyr (C282Y) has been identified which results in tyrosine substitution for cysteine at position 282 of the gene protein product.⁵² The majority (80–100%) of individuals with the typical phenotype of hereditary haemochromatosis are homozygous for this mutation (designated C282Y/C282Y).⁴⁶^,⁵² Genetic tests for C282Y can be performed on peripheral blood or on paraffin-embedded tissue.⁵³ Expression of the mutated HFE protein on duodenal crypt epithelium is one of several factors that have been considered important in the pathogenesis of iron overload in haemochromatosis.⁵⁴ A second mutation, His63Asp (H63D), has been identified in fewer patients with haemochromatosis, either in homozygous form or as compound heterozygotes in conjunction with C282Y or the wild-type (normal) protein.⁵² Other HFE mutations such as S65C (serine to cysteine) are also reported.⁵⁵ Non-HFE hereditary haemochromatosis²⁸ and other genetic disorders associated with iron overload are discussed later.

Until recently, a comprehensive panel of diagnostic tests combined with liver biopsy findings could be expected to provide a firm diagnosis of hereditary haemochromatosis (Table 14.2). However, the availability of genetic testing for HFE-related and other forms of haemochromatosis now sometimes obviates the need for liver biopsy, particularly if certain criteria indicate that the likelihood of hepatic fibrosis is low⁵⁶ (i.e. the patient is less than 40 years old, ferritin is less than 1000 ng/ml, serum liver tests are normal and hepatomegaly is absent). However, when there are coexisting liver diseases such as chronic hepatitis C or alcoholism that may accelerate hepatic fibrosis in the presence of a genetic iron overload disorder⁵⁷ or there are other reasons for direct morphological assessment of liver tissue, liver biopsy continues to offer considerable information. Moreover, understanding the pathological progression of classic HFE-related hereditary haemochromatosis (discussed below) provides a useful comparative model of iron-related liver damage.

Table 14.2 Characteristic diagnostic profile in HFE homozygous hereditary haemochromatosis

Diagnostic modality	Typical result(s)
Serum transferrin saturation	>62% (screening threshold is >45%⁴⁶)
Serum ferritin	≥300 µg/l (men); ≥200 µg/l (women)
Hepatic iron concentration	>2200 µg/g dry weight (men);
Hepatic iron concentration	>1600 µg/g dry weight (women)
Hepatic iron index	≥1.9
Genetic testing	C282Y/C282Y
Liver biopsy	Hepatocellular iron ≥grade 2
Liver biopsy	Minimal or no Kupffer-cell iron

The first histological abnormality in homozygous HFE-related haemochromatosis is the appearance of stainable iron in periportal hepatocytes. This may be found incidentally in the course of investigation for other diseases. The unexplained presence of more than very small amounts of iron in hepatocytes should always raise the possibility of early hereditary haemochromatosis. The diagnosis can then be confirmed or refuted by means of genetic testing and/or calculating the hepatic iron index, as discussed previously. Early diagnosis is most important, because cirrhosis can be prevented by appropriate treatment both in patients and in their homozygous relatives, and life expectancy returned to normal.⁵⁸ In heterozygotes, stainable liver iron is either absent or very scanty.⁴³

As iron stores increase, fibrosis begins to expand the portal tracts and slender septa extend from these to give a pattern of fibrosis resembling holly leaves (Fig. 14.6). The enlarged tracts contain iron-rich macrophages and a ductular reaction, but usually show little or no inflammatory infiltration. Iron may be seen in the ductular structures and in the epithelium of interlobular ducts in small amounts; larger quantities are not found until a later stage, when parenchymal siderosis is severe. It is a challenging paradox that in early haemochromatosis most of the iron is in hepatocytes but there is little or no evidence of liver-cell damage, liver-cell function remains virtually unimpaired, and the progressive lesion is portal in location. However, with increasing iron overload foci of sideronecrosis³⁷ are found, comprising eosinophilic or lytic necrosis of iron-laden hepatocytes, often in close association with clusters of macrophages. The ratio of non-hepatocytic to hepatocytic iron, as assessed histologically, rises progressively. The ultrastructural progression of iron overload has also been examined.⁵⁹