Although the hepatotoxicity of chronic iron overload is indisputable, the specific mechanisms by which the cellular injury and fibrosis develop remain a mystery. Several possibilities have been advanced with recent attention centering on peroxidation of intracellular membrane phospholipids by iron-induced free radicals.^10,92 This peroxidation, in turn, can cause loss of membrane fluidity, changes in membrane potential, and leakage of ions and other molecules, producing damage to organelles and ultimately resulting in cell injury or death. Iron-loaded lysosomes, in particular, become unusually fragile and may rupture and release their destructive hydrolytic enzymes into the cytoplasm. An additional proposition suggests that iron, as a required cofactor in collagen synthesis, may directly stimulate fibrogenesis, independently of hepatocyte injury.⁹⁶ Hereditary Hemochromatosis

This preeminent and prototypic cause of iron overload is an autosomal recessive disorder characterized by the continued absorption of excessive iron with progressive accumulation in the liver and other organs. Also referred to as primary or idiopathic hemochromatosis, the disorder was long suspected on the basis of clinical observation to have a genetic basis, but its inheritance pattern remained unclear until the late 1970's when an association was discovered with human leukocyte antigen (HLA) class I haplotypes, particularly HLA-A3, and, to a lesser extent, HLA-B7 and B14. This association definitively established the autosomal recessive transmission, with clinical disease occurring only in homozygous individuals.^38,87

The susceptibility gene for hemochromatosis has thus been localized to the short arm of chromosome 6, in the close vicinity of the HLA-A locus, although its function and product are yet to be defined. Prevalence studies have demonstrated that hereditary hemochromatosis is one of the most common genetic disorders: In populations of predominantly Northern European descent, the gene frequency is estimated at about 5%, indicating that approximately 10% of individuals are heterozygous and that about 1 in every 250 to 400 is homozygous for hemochromatosis.^37,87 The prevalence in nonwhite populations may be less, but has not been rigorously studied.

In homozygous individuals, increased iron absorption occurs throughout life and results in the gradual accumulation of excessive, and eventually toxic, iron stores. Initially the accumulation is silent, but when sufficient levels are reached, tissue damage develops and clinical manifestations become evident. The sites involved include the liver, heart, pancreas, heart, joints, and pituitary gland. Since only minor quantities of surplus iron, in range of 1 or 2 mg, are absorbed daily, many years are required for the 15 gm to 40 gm typically present in symptomatic patients to accrue. Moreover, the expression of the disease is further modified by such factors as alcohol usage, coexisting blood loss, dietary iron content, and genetic heterogeneity.^4,35,77 Heterozygous subjects do not develop consequential iron overload, but about a third of them exhibit a minor increase in iron stores with abnormal serum iron studies and minor siderosis on liver biopsy.^23,77,90

The pathogenesis of hereditary hemochromatosis is not well understood, and the relationship to alterations in the structure or function of the hemochromatosis gene is obscure. The primary defect is believed to reside at the level of the duodenal mucosa and to involve disrupted regulation of intestinal iron absorption: Even in the presence of gross iron overload, the small bowel of affected patients continues to actively absorb iron. Recent evidence suggests a failure of proper control over transferrin receptor expression with enhanced transport of iron from the absorptive cell to the plasma.^66,68 Another possibility is a generalized defect in macrophages that renders them incapable of iron retention and thus promotes parenchymal deposition.⁶⁷ The liver does not appear play a direct role and instead represents a passive victim of the process.⁵

Clinical Features. ^36,43,70 Despite their gradually expanding iron stores, most patients with hereditary hemochromatosis are asymptomatic for many years, and clinical manifestations usually do not appear until the fifth or sixth decade. Increasingly, however, affected individuals are being identified early in their course, having been detected largely because of biochemical screening of the general population or relatives of recognized patients.^7,37 The clinical disease tends to occur more commonly and more prominently in men than women, who are presumably protected by iron loss during menstruation and pregnancy and a lower dietary intake of iron. Sporadic cases have also been described in young adults and even children; although such instances have usually been considered to be early examples of hereditary hemochromatosis, they may also represent a distinct inherited disorder.^39,47

The clinical manifestations are many and varied, reflecting both the direct effects and secondary complications of iron overload as well as nonspecific constitutional complaints.^7,27,35,71 Common features include weakness and fatigue, weight loss, abdominal pain, hepatomegaly, arthropathy, skin pigmentation, and glucose intolerance. Advanced hepatic involvement may be evident because of portal hypertension or hepatic failure, and, in some cases, cardiac dysfunction with heart failure or arrhythmias or hypogonadism evinced by impotence, decreased libido, or premature menopause, may dominant the clinical picture. Particularly among young patients, the presentation may comprise a syndrome of accelerated cardiomyopathy and endocrine failure.⁴⁷

Laboratory abnormalities include mild increases in serum aminotransferase levels, which may serve as the first clue to the diagnosis, but other routine liver tests are frequently normal, even in the presence of cirrhosis.^53,94 More helpful are the serum iron tests, indirect indices of iron overload that include the serum iron concentration, the transferrin saturation (which is calculated from the serum iron and the total iron-binding capacity), and the serum ferritin concentration. The transferrin saturation is a sensitive marker of parenchymal iron accumulation, although false negative and false positive tests occur; similarly, the serum ferritin concentration provides a measure of the total body iron stores, but, as an acute phase reactant, it can be elevated in various inflammatory conditions.^6,15,28,42 Despite their lack of complete sensitivity and specificity, the combination of a transferrin saturation greater than 50% with an abnormal serum ferritin level strongly suggests excess iron accumulation, and conversely, when both tests are normal, significant iron overload is unlikely.^15,42

The standard treatment for hereditary hemochromatosis is the removal of excess iron by therapeutic venesection. Since each 500 ml unit of blood contains about 250 mg of iron, an average of about 70 units must usually be removed to restore normal iron stores, after which lifelong maintenance phlebotomy is necessary.⁶ This therapy alters the natural history of the disease, prolongs survival, and may improve some clinical manifestations, although cirrhosis, arthropathy, and hypogonadism are not generally affected. This underscores the need for early diagnosis and treatment before such complications develop.

The overall ten-year survival of treated patients approximates 80%, and in the absence of cirrhosis or diabetes, patients have a normal life expectancy.^8,71 Most deaths result from hepatic or cardiac failure and their complications, but an ominous outcome is the development of hepatocellular carcinoma, which occurs with an 200-fold increased risk.^27,30,71 The carcinomas generally develop in cirrhotic livers, in some cases following depletion of iron stores, although rare examples have also been described in patients without cirrhosis.^18,40 An increased risk of extrahepatic malignancies has also been described, but this has been disputed by other studies.^71,89,93

Pathologic Features. The early stages of hereditary hemochromatosis are characterized by gradually increasing hepatocellular siderosis. The extent of accumulation progresses from minor and nondiagnostic levels, in the grade 1+ or 2+ range, to more substantial grade 3+ or 4+ deposition. The periportal hepatocytes are predominantly affected, although as the iron burden grows, the midzonal and centrilobular regions also become involved. Even when the accumulation is heavy, however, a portal-central distribution gradient can frequently be identified (Figure 9-3).^31,84 At this stage, the liver is otherwise largely unremarkable, and iron is absent or scanty in Kupffer cells and portal tracts.

Further progression of the disease is accompanied by periportal and bridging fibrosis. The portal tracts become enlarged with small fibrous spurs that extend into the adjacent parenchyma and confer a spiked or serrated contour (Figure 9-4). This eventually results in the formation of irregular fibrous septa that join portal tracts and begin to envelop the lobules. The resulting pattern of portal-based bridging fibrosis with small islands of relatively unaltered parenchyma resembles that seen with chronic biliary obstruction (Figure 9-5). Bile ductular proliferation may be prominent and mild fatty change, glycogenated nuclei, or scattered foci of hepatocyte necrosis noted, but lobular and portal inflammation, notable hepatocyte necrosis, and cholestasis are generally absent. The siderosis continues to be predominantly hepatocellular in distribution, although the biliary epithelium, portal macrophages, and Kupffer cells also gain deposits, and these become more noticeable as the disease progresses.

The parenchyma eventually undergoes nodular hyperplasia, initially in an uneven fashion with small irregular nodules of hepatocytes variably combined with partially preserved lobules but ultimately the classic cirrhosis of hemochromatosis ("pigment cirrhosis") becomes apparent.⁷⁶ Heavy iron deposition is noted in the hepatocytes, with appreciable accumulation in biliary epithelium within the fibrous septa, although some hyperplastic parenchymal foci are striking by their lack of iron deposition. Kupffer cell iron is present in varying degrees and, reflecting sites of hepatocyte necrosis, is irregularly distributed.³¹ Over time, remodeling of the cirrhotic liver is accompanied by an increase in the size of the nodules and the appearance of thin, compressed fibrous septa.⁷⁶

Therapeutic phlebotomy leads to a gradual loss of siderosis, leaving behind conspicuous lipofuscin pigment within remnants of iron-loaded lysosomes; iron in biliary epithelium or collagen fibers is often the last to disappear. Preexisting fibrosis generally persists, although its progression may be slowed. Regression of severe fibrosis or cirrhosis has been described in occasional reports, but these are difficult to evaluate because the evolution of cirrhosis may be misinterpreted on needle biopsy specimens as histologic improvement.^76,93

In heterozygous individuals, mild hepatocellular siderosis in the grade 1+ to 2+ range may be noted, but this increases only minimally with time and does not lead to substantial iron deposition or progressive fibrosis.^16,23,33,90

Differential Diagnosis. Hereditary hemochromatosis should be suspected whenever severe (grade 3+ or 4+) hepatocellular siderosis is encountered, and the suspicion should be especially strong if the characteristic periportal pattern of fibrosis is also present. Although the various causes of secondary iron overload enter into the differential diagnosis, they can generally be distinguished on the basis of clinical history and the distribution of the siderosis. In their advanced stages, however, many iron overload conditions have a similar appearance and therefore may not be histologically distinctive. The most troublesome discrimination involves alcoholic cirrhosis with secondary siderosis. In this setting, as discussed below, the degree of iron accumulation does not generally reach the histologic or biochemical levels found in hereditary hemochromatosis, and, in fact, the presence of severe iron deposition suggests that the patient suffers from both hereditary hemochromatosis and alcoholic liver disease.^63,82 Specific features of alcoholic hepatitis such as pericellular fibrosis, Mallory bodies, and neutrophil infiltration may also be present to aid in the distinction.

Hepatic siderosis may also complicate an assortment of disorders of varying etiology. The pathogenesis of this iron accumulation is frequently multifactorial; HLA typing and family studies have indicated that heterozygosity for the hemochromatosis gene may contribute in some instances, although contrary results have also been reported.^13,24,86 As a general rule, secondary iron overload is characterized by lesser degrees of siderosis and more conspicuous Kupffer cell iron than is hereditary hemochromatosis.⁵⁷ However, there are numerous exceptions and, in certain situations, the histologic alterations and the clinicopathologic consequences are similar to those of the more common primary form of the disease.

Chronic anemias of various types may be accompanied by iron overload. Perhaps the most common cause is thalassemia, but other anemias characterized by active erythropoiesis may also be responsible, including sideroblastic anemias, congenital dyserythropoietic anemias, and an assortment of anemias associated with defective heme synthesis. Hemolytic anemias are less common causes, but cases have been noted with sickle cell disease, hereditary spherocytosis, and pyruvate kinase deficiency, and rare instances are associated with aplastic anemias or myelofibrosis.^64,83,92

These conditions can be separated into two general groups depending on the degree of erythropoietic activity. In the first group, active -- but often ineffective -- erythropoiesis is present and provides a stimulus for increased intestinal absorption of iron.^73,74 The presence of a single hemochromatosis allele may, in some cases, play an additional role.^13,24 The histologic consequence is hepatocellular siderosis, which may eventually be deposited in a pattern and extent similar to that seen in hereditary hemochromatosis. Furthermore, these patients may require repeated blood transfusions, adding to their iron overload and leading to Kupffer cell siderosis. When the combined accumulation is severe, as seen in thalassemia major, for example, early hepatic fibrosis and cirrhosis may develop, and the presence of clusters of iron-laden Kupffer cells and macrophages assists in the distinction from hereditary hemochromatosis (Figure 9-6).⁸⁰ An additional complicating factor in the hepatic injury is transfusion-related chronic viral hepatitis.⁸⁶

The second group of conditions encompass the various aplastic anemias and is characterized by hypoplastic erythropoiesis. Intestinal iron absorption is normal in this situation, and the excess iron derives solely from the transfusion of blood products. The primary histologic finding therefore is Kupffer cell siderosis; however, since iron can be redistributed to hepatocytes after as few as 10 to 15 units of blood, a combination of hepatocellular and Kupffer cell iron may also be present. Portal fibrosis may appear, but the development of cirrhosis is an uncommon event.^64,83

Alcoholic liver disease is commonly accompanied by hepatocellular siderosis: Up to 66% of liver biopsy specimens from alcoholic patients in various series demonstrate iron accumulation.⁵⁵ Generally, the degree of siderosis is only slight to moderate (grade 1+ to 2+), and the hepatic iron concentration is normal or mildly increased. (In this setting, the histochemical assessment is apt to overstate the amount of iron accumulation actually present). The distinction from early hereditary hemochromatosis may sometimes be a problem, but, in borderline cases, quantitative determinations, especially when adjusted for age, are usually discriminatory.^16,82 The cause of this common but mild alcoholic siderosis is unclear, but suggested possibilities have implicated the high iron content of certain alcoholic beverages, increased intestinal absorption by alcohol ingestion, or impaired iron utilization resulting from associated folate deficiency; heterozygosity for hemochromatosis does not appear to contribute.⁵⁴

On the other hand, when substantial (grade 3+ or 4+) siderosis and high hepatic iron concentrations (> 10,000 mg/g dry weight) are encountered, the likelihood is that both alcoholic liver disease and hereditary hemochromatosis are present. This conclusion has been borne out by several studies utilizing family evaluation and HLA typing.^25,63 Alcohol abuse by individuals homozygous for hemochromatosis appears to potentiate the iron accumulation, accelerate the hepatic injury, and hasten the onset of clinical disease, perhaps accounting for the high prevalence of alcoholism in series of patients with symptomatic hemochromatosis.⁵⁴ The pathogenesis thus reflects the contributions of both alcohol and iron, but the histologic appearances are determined largely by the extent and severity of the alcoholic liver disease. Although marked, the siderosis is often irregularly distributed and may be prominent in Kupffer cells and portal macrophages.⁷⁶

Dietary iron overload is best exemplified by the distinctive entity referred to as Bantu siderosis or African iron overload. Encountered in areas of sub-Saharan Africa, this disorder results from the ingestion of large quantities of iron by genetically susceptible individuals; the source of the iron is a traditional home-brewed beer with a high content of readily-absorbed ferrous iron, and the genetic predisposition is not well defined but appears to be unrelated to hereditary hemochromatosis.^44,45 The histologic features include a combination of hepatocellular and Kupffer cell siderosis, typically marked in degree, with prominent involvement of portal macrophages and the development of periportal fibrosis or cirrhosis. Unlike hereditary hemochromatosis, a portal-central gradient of hepatocellular siderosis is not apparent, and little iron collects in biliary epithelium.^21,45 In the advanced stages, parenchymal iron accumulates in other organs including the pancreas, thyroid, adrenal, and heart.

Iron overload resulting from immoderate dietary intake is otherwise a rare and controversial event: A few isolated cases associated with prolonged medicinal iron intake have been reported, but the possibility of concomitant hereditary hemochromatosis was not excluded.^46,51

Neonatal (or perinatal) hemochromatosis, also known as neonatal iron storage disease, is an unusual clinicopathologic syndrome characterized by severe hepatic injury and massive parenchymal siderosis. The basic defect is not known, but the iron accumulation probably represents a consequence, rather than a cause, of the liver damage, which develops during the prenatal period and may well have several different origins.^52,59,98 The clinical course is characterized by intrauterine growth retardation, prematurity, early failure to thrive, and acute hepatic failure, with progressive deterioration and death within the first few weeks of life. A familial predilection has been noted in some instances and an autosomal recessive transmission suggested, but many sporadic cases are also reported and a genetic basis is not established; moreover, there is no apparent relationship with hereditary hemochromatosis.^12,29,50

Histologically, the liver shows extensive confluent necrosis with varying degrees of canalicular cholestasis, reticulin collapse, and ductular proliferation (Figure 9-7).^17,60,69,85 Fibrosis and nodular hyperplasia may develop, and multinucleated giant hepatocytes and cholestatic rosettes can be noted. Iron is abundant in hepatocytes, with lesser quantities found in Kupffer cells, biliary epithelium, and portal macrophages. Excess iron is additionally found in other visceral sites including the pancreas, thyroid and other endocrine organs, kidney, and heart; the spleen and lymph nodes are commonly spared. Although hepatic siderosis is common in infants less than six months of age and may be evident, usually in mild degrees, in a variety of neonatal liver disorders, neonatal hemochromatosis is distinguished by the extrahepatic deposition.⁹⁸ Since minor salivary glands are also affected, oral mucosal biopsies have be suggested as means of diagnosis.⁶¹

A variety of other conditions may be associated with hepatic iron accumulation. Such metabolic disorders as porphyria cutanea tarda, tyrosinemia, and Zellweger's syndrome may be responsible for mild siderosis and are discussed elsewhere. Cirrhosis of almost any cause can, on occasion, exhibit iron deposition, but, unlike advanced hemochromatosis, the degree is usually mild and the iron is generally spares the fibrous septa, suggesting that the deposition followed the development of the cirrhosis. On occasion, portacaval shunt procedures may prompt an increase in iron accumulation and, in isolated cases, may lead to severe systemic iron overload.^26,65 Siderosis is also common in patients with chronic renal failure treated by long-term hemodialysis or renal transplantation; varying degrees and combinations of hepatocellular and Kupffer cell accumulations may be present.^9,48,62,78 Rare reports also describe siderosis in association with congenital atransferrinemia, an extremely rare autosomal-recessive disorder, and following long-term cimetidine therapy.^19,92

The liver is a central participant in normal copper homeostasis. After absorption from the bowel, the metal is transported to the liver, incorporated into various copper-bearing enzymes, bound to the carrier protein ceruloplasmin for export to the plasma, and stored for physiologic reserve. Within the hepatocyte, retained copper is joined to metallothionein (and other binding proteins) and eventually sequestered in lysosomes in the form of insoluble polymerized aggregates of metallothionein referred to as copper-associated protein. Excess absorbed copper is eliminated through the bile, and, although the exact mechanisms are not well understood, normal copper balance is regulated chiefly through this biliary excretion.¹⁴⁰

Copper accumulation can be detected by several histochemical procedures that demonstrate the presence of either ionic copper or copper-associated protein. Rhodanine or rubeanic acid stains directly react with cupric ions to produce a visible colored precipitate, red or black-green respectively. Although copper diffusely distributed within the cytoplasm may sometimes be identified, granular staining reflecting lysosomal deposition is more typically observed (Figure 9-8).¹¹² However, since copper ions may be leeched from the tissue during aqueous fixation, these methods may not always give predictable or reliable results; in addition, the staining is hampered by fixation with unbuffered formalin or Bouin's, Zenker's, or Lillie's B5 fixatives.^113,115,121 Alternatively, copper-associated protein can be demonstrated with orcein, aldehyde fuchsin, or Victoria blue stains. All of these give comparable results, and the staining has a granular lysosomal-based appearance. Because the protein is insoluble and resists fixation, these stains are often more dependable than the direct copper stains.^112,113,117 Metallothionein may itself be specifically identified through immunohistochemical procedures.^106,127

Unfortunately, histochemistry is not an entirely sensitive means of detecting copper overload, and, as a result, only positive staining is diagnostically meaningful. Absent staining does not exclude copper accumulation, and, in fact, stains may sometimes be negative in the face of greatly elevated hepatic copper concentrations; this is particularly true when the deposition is predominantly cytosolic rather than lysosomal in distribution.¹¹²

A variety of conditions may be responsible for copper deposition in the liver. The predominant cause is Wilson's disease, an inborn error in biliary copper excretion that leads to progressive hepatic injury. Other childhood syndromes of hepatic copper overload and cirrhosis are also described, but they are uncommon -- at least in the United States -- and their pathogenesis is incompletely understood. The other setting in which copper accumulation is frequently encountered is chronic cholestasis; a diagnostically useful secondary feature, copper deposition is noted in such disorders as primary biliary cirrhosis, primary sclerosing cholangitis, chronic biliary obstruction, and the various cholestatic conditions of children (Chapters 4, 5, and 6). In addition, mild focal copper accumulation can be noted in cirrhosis or extensive fibrosis of almost any cause as well as in a variety of hepatic neoplasms.^113,114,144 As a curiosity, the use of copper-containing fungicides by vineyard workers has been reported to produce hepatic copper deposition, Kupffer cell proliferation, and granuloma formation.¹³⁰

Although excess copper is clearly implicated in the hepatic injury found in certain of these conditions, the responsible cellular mechanisms are not well-understood. The general pathogenetic concept is that, when the accumulation of copper exceeds the hepatocyte's binding capacity, free copper ions are released and cause hepatocyte damage. Possible explanations for this copper-induced toxicity include the inhibition of critical intracellular enzymes or other proteins and the induction of free radicals with subsequent oxidant injury to cellular membranes or cytoskeleton.^101,140

Wilson's disease is an inherited disorder of copper metabolism characterized by the excessive accumulation of copper in the liver and other organs. Also known as hepatolenticular degeneration, it is an rare autosomal-recessive condition, occurring with a prevalence of only about 1 in 30,000 individuals, but assumes disproportionate importance because of the availability of effective therapy.

The gene for Wilson's disease is assigned to chromosome 13, specifically to the 13q14-q21 region, and is linked to the loci for erythrocyte esterase D and retinoblastoma. The responsible gene has not been isolated or characterized, and its normal product and function remain elusive. Nonetheless, molecular genetics techniques utilizing the recognized genetic linkages should permit noninvasive detection of both heterozygous carriers and affected individuals, perhaps even prenatally.^142,148

The basic pathogenetic abnormality in Wilson's disease involves the liver's inability to export copper effectively: Both biliary excretion and incorporation into ceruloplasmin are impaired. However, the specific defect responsible for the disrupted transport is not known.¹³⁵ A conspicuous feature of the disease is a low serum level of ceruloplasmin, and attention has thus been drawn to the possible role played by this protein, although clearly the disease does not result from mutation of the ceruloplasmin gene, which is carried on chromosome 3. However, abnormalities in the translation or post-translational modification of ceruloplasmin have been demonstrated and may frustrate delivery of the protein into blood or bile.^103,116 Other possibilities include the production of an abnormal copper-binding protein, an alteration in a hypothetical carrier protein involved in copper export processes, or the aberrant preservation of the fetal mode of copper metabolism.^107,135,142

Whatever the exact mechanisms involved, the consequence of the genetic abnormality is the accumulation of hepatic copper. Beginning in infancy and gradually progressing over time, this deposition eventually leads to hepatocyte damage of some degree, and fibrosis and cirrhosis may consequently develop. The liver is the main target organ and is universally affected, although its involvement may remain subclinical. Once the liver's binding capacity is exceeded, copper is released into the circulation and accumulates in extrahepatic sites including the brain, cornea (yielding Kayser-Fleischer rings), kidneys, striated muscle, bones, and joints. The excess copper deposited in these organs then presumably leads to tissue injury and functional derangement. This general sequence accounts for the diverse clinicopathologic expressions of Wilson's disease, and the evolution likely depends upon the distribution, extent, and tempo of the copper accumulation and subsequent tissue damage.^104,105,134 In addition, the progression of the disease may be influenced by such factors as allelic heterogeneity or variations in dietary copper intake.

Clinical Features. ^101,135 The clinical manifestations of Wilson's disease vary greatly in their nature, severity, and time of onset. The diagnosis is generally one of older childhood to early adulthood, with most patients presenting between the ages of 5 and 30 years, although occasional individuals do not present until the fifth or sixth decade.^134,142 The clinical features encompass a broad spectrum of hepatic, neurologic, psychiatric, and hematologic abnormalities, often with an insidious onset, but most patients present in either a hepatic or neuropsychiatric fashion.

Hepatic presentations are particularly common among younger patients from 8 to 15 years of age, and a variety of clinical pictures may be seen.¹⁴⁷ Most often, patients present with evidence of chronic liver disease, including chronic active hepatitis and cirrhosis, and may thus manifest variously with fatigue, jaundice, splenomegaly, variceal hemorrhage, progressive hepatic insufficiency, or ascites. In this setting, the serum aminotransferase levels may be misleadingly modest in the face of severe hepatic damage.^136,137 In other cases, an acute hepatitis-like episode of jaundice, abdominal pain, and malaise may occur and may be accompanied by hemolytic anemia and hypouricemia. This may clinically appear as a self-limited process and may be mistaken for viral hepatitis or infectious mononucleosis, although the accompanying biochemical abnormalities persist. An dramatic but unusual presentation is one of fulminant hepatic failure with encephalopathy, marked jaundice, ascites, and edema. Biochemically, this may be distinguished by high serum bilirubin and copper levels with surprisingly lesser elevations of alkaline phosphatase and aminotransferases.^99,123 In other instances, asymptomatic hepatomegaly or biochemical abnormalities are the only evidence of disease.

The neuropsychiatric manifestations, which typically begin during the second or third decades, entail a wide range of findings. Movement disorder predominate and may include tremor, incoordination, dystonia, dysarthria, ataxia, or spasticity; the psychiatric disturbances can range from subtle behavioral or personality alterations to schizophreniform psychosis. Other presentations are unusual, but occasional cases are associated with hemolytic anemia, renal tubular defects, osteoporosis, cardiac findings, or unexplained endocrine abnormalities.¹²²

When untreated, Wilson's disease typically leads to death within a few years. This outcome was greatly altered, however, by the introduction of penicillamine therapy in the 1950's. This chelating agent acts to reduce and detoxify the copper burden and is effective in suppressing disease activity, relieving clinical symptoms, and, in asymptomatic patients, preventing manifestations.^136,142,146 Alternative drugs such as trientine and zinc acetate have also been used with success, and the comparative therapeutic roles of the various agents are debated.^101,142 The prognosis depends largely on the extent of organ damage before treatment is started: In the absence of cirrhosis or severe neurologic disease, most treated patients will have a normal life span; early recognition and prompt treatment are therefore imperative. The outcome is less favorable for patients suffering from fulminant hepatic failure or end-stage liver disease; liver transplantation remains a treatment option in these cases.^128,132,133

Hepatocellular carcinoma is described in sporadic cases of Wilson's disease, but this appears to be an uncommon complication.^102,131

Pathologic Features. ^135,143 The early stages of Wilson's disease are characterized by a variety of minor lobular alterations, including fatty change, which may be of either macrovesicular or microvesicular types and glycogenated nuclei together with reactive changes such as scattered acidophilic bodies and focal hepatocyte necroses (Figure 9-9). The liver cells and their nuclei may vary in size and appearance, and lipofuscin pigment may be conspicuous, sometimes appearing as large irregular granules in periportal hepatocytes. Kupffer cells are often enlarged and may contain hemosiderin. Portal inflammation is not usually present, but, on occasion, thin slips of fibrous tissue may extend from the portal tracts.

The hepatic copper concentration is generally elevated at this stage, but stainable copper is not usually detectable because much of the accumulated metal is dispersed within the cytoplasm.^112,127 On occasion, however, faint cytoplasmic staining may be noted, and focal positivity involving the abnormal lipofuscin granules can be seen. Ultrastructural examination demonstrates certain distinctive changes: The mitochondria are heterogeneous in appearance and display increased matrix density, separation of the inner and outer membranes, enlarged intracristal spaces, and crystalline and vacuolated inclusions.² These alterations are characteristic and are considered virtually diagnostic, although similar features have been described in other settings.¹¹¹

An early acute hepatitis pattern is also described but is rarely biopsied. It is characterized by hepatocyte swelling with acidophilic degeneration and necrosis, mononuclear inflammatory infiltration, and cholestasis.¹³⁵

As the disease progresses, a pattern of chronic active hepatitis develops. The portal tracts acquire an inflammatory infiltrate composed primarily of lymphocytes with sparse neutrophils, plasma cells, or eosinophils. This infiltrate spreads into the periportal regions, where it is accompanied by piecemeal necrosis with varying degrees of destruction of the adjacent parenchyma (Figure 9-10). The associated fibrosis ranges in extent from mild periportal involvement to the formation of bridging fibrous septa.

Mallory bodies are a distinctive embellishment of the injured liver cells, being particularly abundant in cases with more active hepatocyte damage (Figure 9-11). Often accompanied by a neutrophil infiltrate, they are found primarily in the periportal (or periseptal) regions. In addition, any of the minor lobular alterations noted above may persist in the chronic hepatitis stage. Copper accumulation can commonly be demonstrated at this stage, appearing as granular deposits in periportal hepatocytes (Figure 9-12).^112,143

The eventual consequence of advanced disease is the development of cirrhosis. This may adopt several histologic appearances: Although often predominantly macronodular in type, parenchymal nodules of varying sizes, including micronodules, are frequently seen (Figure 9-13). The encompassing septa may be slender and delicate or, alternatively, may comprise broad, thick zones with proliferated bile ductules and scattered mononuclear inflammation. Any of the inflammatory and reactive changes noted in the precirrhotic phases may also be present, including fatty change, glycogenated nuclei, focal hepatocyte necrosis, and Mallory bodies. In some instances, occlusive venular fibrosis and endophlebitis have also been identified. Stainable copper is generally present, but its distribution varies considerably among nodules; some nodules demonstrate heavy deposition, diffusely or focally arrayed, while other nodules may be completely negative.¹⁴³

Confluent hepatocyte necrosis and parenchymal collapse may complicate either the chronic active hepatitis or cirrhosis of Wilson's disease (Figure 9-14). This histologic picture corresponds to the clinical presentation of fulminant hepatic failure. Varying degrees of cholestasis and fatty change are noted, Mallory bodies may be identified, and copper accumulation involving hepatocytes, Kupffer cells, and portal macrophages is a distinguishing feature.^103a Mild, subclinical episodes of confluent necrosis and subsequent fibrosis have been suggested as the histogenetic pathway to cirrhosis, although others propose that the fibrosis may arise in the absence of hepatocyte injury.^138,141

Differential Diagnosis. None of the histologic patterns seen in Wilson's disease is completely specific, and the final diagnosis is therefore usually based upon the clinical findings and laboratory data. The morphologic changes may, however, suggest or confirm the diagnosis and are useful in gauging the extent and degree of hepatic damage. As a general rule, Wilson's disease must be considered in any case of unexplained liver disease -- especially in patients under age 40 years -- and a high index of suspicion is requisite.

The various nonspecific alterations seen in early Wilson's disease may be seen in several settings, including normal livers, but a combination of such changes in a young patient should raise the diagnostic possibility. In the later stages, the other causes of chronic active hepatitis lead the list of differential considerations: Although Wilson's disease may not be distinguishable on histologic grounds alone, Mallory bodies and copper accumulation lend support to that diagnosis. Steatohepatitis may be suggested by the fatty change, Mallory bodies, and fibrosis, but the presence of copper accumulation and lack of pericellular or perivenular patterns of fibrosis favor Wilson's disease.

The massive hepatic necrosis seen in Wilson's disease differs from that of other etiologies by the presence of heavy copper deposition and a cirrhotic or fibrotic background. Copper accumulation and Mallory bodies may also be seen in chronic cholestatic conditions, including primary biliary cirrhosis and primary sclerosing cholangitis, as well as in other syndromes of childhood copper overload; these can be distinguished on the basis of clinical history and the presence of more distinctive histologic findings such as bile duct injury and loss.

Several reports describe childhood syndromes, distinct from Wilson's disease, that are characterized by severe hepatic copper overload and cirrhosis. The best known of these syndromes is Indian childhood cirrhosis, which represents one of the major causes of pediatric chronic liver disease throughout the Indian subcontinent.¹¹⁸ Similar cases have also been noted, however, in Europe and North America.^{119,120,125,126} Whether these cases all represent the same entity is unclear, but they have similar clinicopathologic features and are therefore discussed together. The pathogenesis is obscure, but excessive copper deposition appears to be a prime factor: Some cases demonstrate a familial predisposition, and an inherited abnormality in copper metabolism has been proposed; in other cases, the combination of excessive dietary intake, perhaps from household utensils, and an immature biliary excretory mechanism has been implicated.^109,129

Affected patients are generally between four months and five years of age, with a peak prevalence of about 2 years. Symptoms including jaundice, hepatomegaly, and fever develop insidiously and rapidly progress to hepatic failure with ascites, splenomegaly, and encephalopathy. Unlike Wilson's disease, Kayser-Fleischer rings are absent and serum ceruloplasmin levels are normal or elevated. The mortality rate is high with death usually following within six months of onset; treatment with penicillamine has been successful, however, in reversing this dire outcome.^100,118

This histologic pattern is characterized by the presence of intracytoplasmic bodies of varying size, shape, and appearance. These bodies occur in a wide range of conditions and commonly represent the abnormal accumulation of proteins or other materials within the cell. They range from dense, homogeneous eosinophilic bodies to pale, finely granular masses, typically described as ground-glass in nature, and may occupy all or part of the cytoplasm and be enclosed by a clear artifactual halo.

In some cases the appearance alone is sufficient distinctive to establish the nature of the inclusion, but in most instances, special stains and immunohistochemical procedures are necessary. The differential considerations are summarized in Table 9-4.

Alpha-1-antitrypsin, the predominant component of the alpha-1-globulins, is the major circulating inhibitor of the serine proteases. Despite its name, its primary target is the potent elastase found in neutrophils, and it serves physiologically to protect tissues against injury during acute inflammatory reactions. Alpha-1-antitrypsin deficiency, an autosomal hereditary condition in which serum levels are of less than 35% of normal, is an important and wide-ranging disease entity characterized by the development of emphysema and liver disease.^152,163

Alpha-1-antitrypsin is a glycoprotein of 52,000 daltons and synthesized in the liver under the control of codominant alleles at a locus on the long arm of chromosome 14. These genes are highly polymorphic, with more than 75 known alleles, and, consequently, numerous biochemical variants of the alpha-1-antitrypsin molecule can be recognized. These variants comprise the Pi (protease inhibitor) system and can be distinguished by various electrophoretic procedures and, more recently, by monoclonal antibodies and molecular biology techniques. Many of the Pi variants are associated with normal serum concentrations and are of little clinical consequence, but a few result in low circulating levels of alpha-1-antitrypsin and thereby gain pathologic significance.^152,169

The most common variant is PiM, which is found in over 90% of most populations; the usual homozygous PiMM phenotype is associated with normal serum alpha-1-antitrypsin levels in the range of 1.5 to 3.5 g/l by standard determinations. In contrast, PiZ is the classic deficient variant, and, in the homozygous state, yields a markedly decreased serum concentration of some 15% of normal. Accounting for most instances of severe alpha-1-antitrypsin deficiency, the PiZZ phenotype occurs in less than 0.2% of the population, is largely restricted to whites, and tends to be more frequent in individuals of Northern European extraction.^163,169 The PiZ protein differs from PiM by an single amino acid substitution -- lysine for glutamine -- at position 342, an alteration that results in defective secretion of the molecule.¹⁵² Other rare deficient variants include M_malton, M_durate, and the curious null alleles (PiQ0) distinguished by the complete absence of circulating alpha-1-antitrypsin.^152,195

The leading complication of alpha-1-antitrypsin deficiency is the development of pulmonary emphysema. This may occur in early adult life, particularly in patients who smoke, and is characteristically panacinar and basilar in distribution.^163,179 Liver disease in children and adults is a less frequent complication and is associated almost exclusively with the homozygous PiZZ phenotype. The exact frequency of hepatic injury is difficult to determine, but only a minority of such patients develops clinically evident disease: In prospective studies, up to 70% of PiZZ infants are found to have biochemical evidence of hepatic dysfunction, which usually resolves in most cases, but overt liver disease occurs in only about 10% to 20%.^151,208 In addition, adults with the PiZZ phenotype are at increased risk of cirrhosis; in various series, up to 10% of such patients are affected, giving rise to an eight-fold excess risk.^164,168,182

In isolated cases, liver disease has also been associated with the rare Pi alleles M_malton and M_durate, but has not been described with the null (PiQ0) alleles.^165,201 The relationship between liver disease and the heterozygous alpha-1-antitrypsin phenotypes such as PiMZ and PiSZ remains controversial: Chronic liver disease is overrepresented among these patients and the increased risk estimated at approximately 1.8, but it is uncertain whether this reflects a causal association or an coincidental finding, as these phenotypes are found in up to 4% of the population.^{150,160,176,196}

The pathogenesis of the hepatic injury seen with alpha-1-antitrypsin deficiency is a subject of active but unresolved investigation.^158,195,207 The leading proposal, the engorgement theory, focuses on the aberrant accumulation of mutant alpha-1-antitrypsin molecules in the hepatocyte: Because of its amino acid substitution, the PiZ protein cannot be secreted effectively and becomes trapped in the endoplasmic reticulum; through unknown mechanisms possibly involving heat shock proteins, this accumulation is thought to trigger hepatocyte injury.^158,195 This suggestion explains the absence of liver disease noted with the null alleles, in which no alpha-1-antitrypsin is produced, and is supported by studies with transgenic mouse models, but it does not indicate why most patients with alpha-1-antitrypsin deficiency fail to develop hepatic disease.^170,195 Possibly the engorged hepatocytes are disposed to injury, but an additional hepatic insult, such as chronic hepatitis virus infection or alcohol abuse, is required.^180,197

Clinical Features. The hepatic manifestations of alpha-1-antitrypsin deficiency encompass an broad spectrum, involving both children and adults and exhibiting wide variation in clinical presentation and course.

The most common picture is one of neonatal cholestasis, characterized by jaundice, hepatomegaly, and elevated serum transaminase levels developing within a few months after birth. Splenomegaly, acholic stools, low birth weight, or failure to thrive may also be noted. Overall, alpha-1-antitrypsin deficiency accounts for some 5% to 10% of cholestatic disease occurring in infants and may be confused with other forms of this syndrome including biliary atresia and neonatal hepatitis.^178,183,191 In other children, cholestasis may be absent with hepatomegaly or abnormal liver function tests as the sole manifestation.

In most cases, the disease spontaneously regresses by six months of age, and the affected children appear clinically well. However, in other instances, hepatomegaly and biochemical abnormalities persist, and, although the long-term outcome of these cases is not well delineated, they may form the basis for liver disease evolving in later adult life.^149,179,208

Less commonly, the disease does not resolve, but progresses to more severe involvement with cirrhosis and liver failure. This may occur early, within the first six months of life, or may evolve more slowly and not become evident until childhood or adolescence.^{149,172,186,187,198} The frequency of progression varies in differing reports, in part related to biologic differences in the severity of disease, but also reflecting referral bias and variation in case ascertainment among published series. Overall, cirrhosis develops in approximately 20% to 30% of the infants presenting with cholestasis, thus representing about 3% of all alpha-1-antitrypsin deficiency cases.^151,208

Adults with hepatic involvement associated with alpha-1-antitrypsin deficiency manifest primarily with chronic, advanced liver disease. Most patients are men over 50 years of age and have no history of neonatal cholestasis or prior hepatic disease. Clinically, the disease can be asymptomatic or well-compensated, but patients frequently present with complications of portal hypertension or hepatic failure including encephalopathy or esophageal varices. The clinical course is often one of rapid progression with death often following within two years of diagnosis.^{164,168,180,200,210}

Some patients with alpha-1-antitrypsin deficiency develop hepatocellular carcinoma, although the overall risk appears to be small. In most cases, this complication is seen in elderly males and occurs in a background of established cirrhosis.^{160,168,182,201,206}

Given its protean manifestations, alpha-1-antitrypsin deficiency must be considered in all cases of jaundice in newborns or unexplained chronic liver disease in patients of any age. Although the disorder may be suspected by the diminished alpha-1-globulin peak on routine serum electrophoresis, a more specific determination of the serum concentration by immunologic assay is usually necessary. The diagnosis is confirmed by Pi phenotyping, which is usually performed by polyacrylamide gel isoelectric focusing.¹⁶³

As there is, as yet, no specific therapy for the liver disease associated with alpha-1-antitrypsin deficiency, treatment consists largely of managing the complications of cirrhosis and chronic cholestasis. Liver transplantation has been successfully employed for advanced liver disease, particularly in pediatric patients; this results in a conversion to a normal alpha-1-antitrypsin phenotype and an elevation of the serum levels.¹⁷⁷ Other experimental approaches include pharmacologic induction of alpha-1-antitrypsin secretion or the use of gene transfer technology to introduce the PiM gene into affected hepatocytes; although of apparent benefit in treating the pulmonary disease, replacement therapy with purified alpha-1-antitrypsin is not useful in hepatic disease.^151,163

These globules represent accumulations of abnormal alpha-1-antitrypsin, and, by ultrastructural examination, represent dilated sacs of endoplasmic reticulum filled with electron-dense proteinaceous material. Immunohistochemical techniques can therefore be used to establish the presence and facilitate the detection of these globules, which may appropriately be referred to as alpha-1-antitrypsin globules, as well as to help distinguish them from other cytoplasmic inclusions (Figure 9-17).^156,193,199

The globules basically serve as a marker of the Z allele: They are seen in PiMZ and PiSZ heterozygotes as well as homozygous PiZZ individuals, and their presence is independent of any underlying hepatic disease. The same globules may also be noted in the rare deficiency alleles associated with defective alpha-1-antitrypsin synthesis, PiM_malton and Pi_duarte, and this may account for some reports globules in patients with PiM phenotypes.^201,204

The underlying histologic features vary considerably depending upon the natural history and course of the disease. At one extreme are those patients who do not develop hepatic disease; alpha-1-antitrypsin deficiency globules may then represent the sole morphologic abnormality seen on liver biopsy specimens.¹⁷³

In affected infants, the appearances are most often those of neonatal hepatitis: Varying degrees of hepatocyte injury manifested by cytoplasmic swelling, focal necroses, and giant cell transformation are present and are accompanied by canalicular cholestasis, sometimes with the formation of cholestatic rosettes (Figure 9-18). The portal tracts are typically expanded by an mononuclear inflammatory infiltrate, but portal fibrosis is generally minor and the lobular architecture preserved. Extramedullary hematopoiesis may also be seen. Diastase-PAS-positive globules may be small and difficult to detect in this age group, and immunohistochemistry and electron microscopy may help in confirming their presence.^{166,174,186,209}

On occasion, bile ductular proliferation can be a prominent feature, and portal and periportal fibrosis may develop. By contrast, in other cases, the number of interlobular bile ducts is markedly diminished, and thus alpha-1-antitrypsin deficiency becomes one of the causes of nonsyndromic paucity of intrahepatic ducts (Chapter 6). These latter two histologic patterns have been correlated with a greater risk of prolonged disease and cirrhosis in some, but not all, studies.^{149,172,174,186}

In affected adults, the histologic pattern may be one of chronic active hepatitis, characterized by portal and periportal inflammation, piecemeal necrosis, and fibrosis of varying severity, but cirrhosis is frequently established at the time of diagnosis (Figure 9-19). The cirrhosis in either children or adults is typified by a variable and often nondescript appearance with a range of nodular sizes.¹⁶⁷ Alpha-1-antitrypsin deficiency globules are found in hepatocytes at the periphery of the nodules and are helping in suggesting the diagnosis. Mild canalicular cholestasis or fatty change is occasionally present, and the degree of inflammatory activity varies greatly.

Differential Diagnosis. Because of the broad range of possible histologic patterns seen, alpha-1-antitrypsin deficiency enters into the differential diagnosis of many conditions including neonatal hepatitis, biliary atresia, paucity of intrahepatic bile ducts, chronic active hepatitis, and cirrhosis in both children and adults. The only histologic clue to the diagnosis is the presence of alpha-1-antitrypsin deficiency globules, and these should be searched for in such settings. Determination of the serum alpha-1-antitrypsin concentration and the Pi phenotype are the confirmatory tests.

Additional cytoplasmic inclusions derive from the retention of hepatic proteins other than alpha-1-antitrypsin within the endoplasmic reticulum of the affected hepatocytes. Although few such cases have been reported, the accumulation presumably occurs because of defects in either the protein or its secretory mechanisms, and these disorders, together with alpha-1-antitrypsin deficiency, have been termed endoplasmic reticulum storage diseases.¹⁵⁹ Fibrinogen inclusions have been described in cases of familial hypofibrinogenemia and are recognized as pale, weakly eosinophilic ground-glass bodies that are PAS-negative but strongly positive for fibrinogen by immunohistochemical methods.^155,188 Accumulation of complement components has also been described, and, in one case of cirrhosis associated with partial alpha-1-antichymotrypsin deficiency, small granules in periportal hepatocytes were immunostained for alpha-1-antichymotrypsin.^184,188

Lafora's disease is an autosomal recessive disorder characterized by seizures, myoclonus, progressive dementia, and intracytoplasmic inclusions in the brain, liver, and other organs. The responsible defect is not yet defined, but the stored material is a polyglycosan and an abnormality of polysaccharide metabolism appears likely. The inclusions are pale, well-defined masses, round or kidney-shaped, that displace the nuclei and reside primarily in periportal hepatocytes. They are weakly positive with diastase-PAS, stain with methenamine silver and colloidal iron stains, and, by electron microscopy, are composed of a nonmembrane-bound combination of interwoven fibrils (6 to 10 nm diameter), aggregates of smooth endoplasmic reticulum, and granular material with glycogen rosettes.^2,190

Drug-related inclusions indistinguishable from those of Lafora's disease have been described with cyanamide and disulfiram therapy.^154,212 In addition, a ground-glass cytoplasmic appearance can results from the proliferation of smooth endoplasmic reticulum induced by therapeutic agents such as phenobarbital or phenytoin, although discrete inclusions are not typically seen.

Cytoplasmic inclusions discussed elsewhere include the ground-glass inclusions of chronic hepatitis B infection and IV glycogenosis and two distinctive eosinophilic masses, Mallory bodies and megamitochondria. Sporadic cases with Lafora-like inclusions of unknown significance have also been described.¹⁸⁹

In certain conditions, the abnormally accumulated substances are either removed during tissue processing or are not well-visualized in histologic sections. As a result, the affected cells appear enlarged and with pale-staining cytoplasm, often faintly vacuolated. The nature of the stored material can sometimes be inferred from the histologic changes, but, in other cases, the pathologist is only able to raise the possibility of a storage disorder, and the diagnosis depends upon appropriate biochemical assays.

Fatty change represents an additional example of cytoplasmic rarefaction, but it carries different clinical connotations and is discussed in Chapter 7. Usually fatty change can be distinguished from other forms of cytoplasmic rarefaction by the sharply defined outlines of its lipid vacuoles, although the distinction may be difficult in some instances of microvesicular fatty change and supplementary information may be required. Furthermore, minor degrees of cytoplasmic rarefaction may be difficult to recognize, and the liver may mistakenly be considered normal.

In this group of disorders, specific enzymatic defects in glycogen metabolism result in the accumulation of excess or structurally abnormal glycogen in the liver and other organs. Seven major disease entities are distinguished, encompassing at least ten different enzyme deficiencies. These are uncommon conditions with an overall frequency of approximately one in 50,000, and, with the exception of an X-linked variant of type VI, are inherited as autosomal recessive traits.^228,230

The liver is the primary site of involvement in types I, III, and VI and is also affected as part of a generalized process in types II and IV. (Types V and VII are primarily disorders of the skeletal muscle and spare the liver). The clinical manifestations vary greatly, with some patients presenting in infancy or early childhood and others during adult life. Hepatomegaly is the chief, and often the exclusive, feature of hepatic involvement, and only type IV glycogenosis is regularly associated with the development of progressive liver disease.

Types I, II, III, and VI share the same basic histologic pattern, although there are minor differences in certain features, and, except for type II, demonstrate similar electron microscopic findings. Type IV glycogenosis, on the other hand, exhibits more distinctive histologic alterations and has a characteristic ultrastructural appearance, thereby justifying a separate discussion. In all cases, the diagnosis is confirmed by demonstrating the responsible enzyme deficiency.

Histologically, these four types are distinguished by varying degrees of hepatocyte enlargement with empty, pale-staining cytoplasm resulting from the accumulated glycogen (Figure 9-21).²³⁵ The changes are especially prominent in types I and III, where the liver cells become markedly and uniformly distended and compress the sinusoids to form a mosaic pattern. In addition, glycogenated nuclei of extreme degree can be seen, and variable fatty change also noted, particularly in type I glycogenosis.²¹⁹ In types II and VI, the hepatocyte enlargement tends to be less prominent and less regular, and a delicate cytoplasmic vacuolation may be discerned. The stored glycogen can be demonstrated with PAS stains, but since reliable quantitative determinations cannot be made with routine sections, this is of little practical use.²³³

The hepatocellular changes may be complicated by portal and periportal fibrosis, especially in types III and VI, and progression to cirrhosis has been recorded on rare occasion.²²⁸ Centrilobular fibrosis and Mallory bodies have also been described in type Ia glycogenosis.²³¹

A further complication of type I and, to a lesser degree, type III is the development of hepatocellular adenomas. Typically these arise as multiple lesions in patients in their second or third decades; some have regressed with appropriate dietary management, while other have progressed to hepatocellular carcinoma. Examples of hepatocellular carcinoma arising without an associated adenoma are also described.^{221,228,242,244}

The rare type IV glycogenosis is caused by a deficiency of the branching enzyme and characterized by the generalized accumulation of an abnormally organized and poorly-soluble glycogen. Affected patients present in infancy with hepatosplenomegaly, failure to thrive, and nonspecific gastrointestinal symptoms, and progress to cirrhosis and its complications, with death usually occurring before the third year of life.²³⁰ Liver transplantation has been successful in some cases.²⁴⁶

The histologic changes are distinctive: The hepatocytes, particularly in the periportal region, contain rounded cytoplasmic inclusions that stain amphophilic or faintly eosinophilic and are often enclosed by an artifactual halo (Figure 9-22).^216,235 These inclusions, which consist of the abnormal amylopectin-like glycogen, are PAS-positive and partially resistant to diastase digestion; they are completely digested, however, by pectinase pretreatment and can be nonspecifically stained with Lugol's iodine or colloidal iron stains. Ultrastructurally, the inclusions consist of non-membrane-bound aggregates of delicate, haphazardly-arrayed fibrils of 5 nm diameter together with glycogen particles and finely granular material.² The background histology includes periportal fibrosis that becomes progressively more extensive with a micronodular cirrhosis as the final outcome.

Lysosomes are the organelles responsible for degrading phagocytosed material, and the acid hydrolases contained within are the active agents, mediating the cleavage of sugar, sulfate, or fatty acid moieties from complex macromolecules. When one of these enzymes is deficient or defective, the appropriate catabolic mechanisms are interrupted, and excessive quantities of the corresponding substrate collects within the lysosomes of the affected cells. Numerous such inherited deficiencies have been recognized and are distinguished by the type of accumulated substance.²²⁷

The liver is involved in many lysosomal storage diseases, but typically this manifests solely as hepatomegaly and signficant hepatic dysfunction develops in only a few . In most instances, both hepatocytes and Kupffer cells are affected and exhibit cellular enlargement and rarefaction, often with a poorly-defined vacuolated appearance (Figure 9-23). Although affected Kupffer cells may, on first examination, be mistaken for engorged hepatocytes, the dual population can usually be identified on PAS or trichrome stains. Overall, the changes are often sufficiently distinctive to suggest a storage disorder of some type, but the individual disorders cannot generally be distinguished on a histologic basis. The specific diagnosis is established by identifying the accumulated metabolite or the responsible enzyme deficiency in fibroblasts or blood cells.

Sphingolipidoses. These conditions are characterized by defects in the degradation of sphingomyelin, ceramide, and other sphingolipids. The best recognized and most common variety is Gaucher's disease, in which a deficiency of glucocerebrosidase leads to the accumulation of glucosylceramide in the macrophages of various organs.^218,227 This disease most often presents as a relatively benign process manifest by hepatosplenomegaly, thrombocytopenia, and pathologic bone fractures, but in infants and children, central nervous system involvement may result in severe and progressive neurologic dysfunction and early death.²¹⁷

Histologically, the Kupffer cells and portal macrophages are markedly distended and have the distinctive striated or wrinkled cytoplasm of the classic Gaucher's cell (Figure 9-24). These striations may be faint on routine sections, but are more obvious with diastase-PAS or trichrome stains, and intense acid phosphatase activity can be demonstrated by enzyme histochemistry. Erythrophagocytosis or mild siderosis may also be seen. Ultrastructurally, these cells are distinguished by crowded and irregular lysosomal inclusions that correspond to the histologic striations and comprise disordered, twisted bundles of elongated tubules measuring 10 nm to 30 nm in diameter.²

The Gaucher's cells may be dispersed singly within the sinusoids, aggregated into clusters that favor the centrilobular regions, or distributed diffusely across the lobule, and the number of cells roughly correlates with the clinical severity of the disease.²³² The hepatocytes do not accumulate the glucosylceramide and therefore remain largely uninvolved, in contrast to other sphinogolipidoses. However, with encroachment by the Gaucher's cells, liver cells may become degenerated or atrophic. Varying degrees of fibrosis may develop, usually in association with aggregates of Gaucher's cells, and portal hypertension or a progression to cirrhosis have been described in some cases.^232,248 Hepatic calcification is also reported.²⁵⁰

Other sphingolipidosis are characterized by foamy cytoplasmic vacuolation involving both hepatocytes and Kupffer cells. At times, the degree of metabolite accumulation is so pronounced that the two cell types cannot be clearly differentiated on routine stains. The most important examples are Niemann-Pick disease and G_M1 gangliosidosis; liver involvement is a minor feature of Fabry's disease, metachromatic leukodystrophy, Farber's disease, and G_M2 gangliosidosis. Although these several disorders are not histologically distinguishable, electron microscopy may provide some diagnostic clues (Table 9-5).

Niemann-Pick disease comprises a wide spectrum of clinical expression: Most patients have hepatomegaly and varying degrees of neurologic impairment, but the scope of the disease ranges from infants with rapidly progressive deterioration to adults with more indolent disease and a protracted clinical course.²⁴⁹ The foamy hepatocytes and Kupffer cells may be sparse in young infants, but they become more prevalent with advancing age. These cells often develop a prominent mulberry-like vacuolation, and they may accumulate PAS-positive ceroid pigment and appear as so-called sea-blue histiocytes; in frozen sections, stains for phospholipid and cholesterol are positive. Minor degrees of periportal and intralobular fibrosis may be noted, but cirrhosis is an unusual development.²⁴⁸ In infants with the so-called type C variant, the histologic picture includes giant multinucleated hepatocytes, canalicular cholestasis, and cholestatic rosettes with sparse foamy cells, and the appearances mimic those of any type of neonatal hepatitis.²⁴⁷

The infantile form of generalized G_M1 gangliosidosis is characterized by coarse facies, hepatosplenomegaly, cherry-red macular spots, retarded motor development, and progressive deterioration and death by two years of age. Histologically, finely vacuolated hepatocytes and Kupffer cells are noted and, by electron microscopy, large lysosomes with lamellar concentric membranes or nonspecific granular or fibrillar material are identified.^2,243 In the G_M2 gangliosidoses, which includes Tay-Sachs disease, the liver is histologically normal, but ultrastructurally, lamellar concentric membranes known as "zebra bodies" may be seen.

Fabry's disease is an X-linked disorder that results in the accumulation of globotriasylceramide in the endothelial, perithelial, and smooth muscle cells of blood vessels. Among the predominantly involved sites are the skin, peripheral nerves, heart, and kidneys, and the corresponding clinical manifestations include cutaneous and mucosal angiokeratomas, paresthesias and pain in the extremities, and corneal opacities with the later development of renal, cardiac, or cerebrovascular complications. The abnormal metabolite also accumulates within Kupffer cells, portal macrophages, and the endothelial and perithelial cells of hepatic blood vessels; these cells consequently become enlarged, tan-colored, and diastase-PAS positive and, by electron microscopy, contain concentrically laminated inclusions and amorphous material.^218,236

Metachromatic leukodystrophy is an inherited disorder in myelin metabolism and its manifestations are chiefly neurologic in character: ataxia, mental deterioration, blindness, and peripheral neuropathy. The liver may also be affected, and light brown granules of sulfatide are noted within swollen Kupffer cells, portal macrophages, and biliary epithelial cells; these granules are PAS-positive and metachromatic with cresyl violet or toluidine blue stains. More dramatic involvement is seen in the gallbladder, however, with the formation of mucosal polyps packed with subepithelial foam cells.^2,220

In Farber's disease, a degradation product of sphingolipids, ceramide, is deposited in tissues in the form of granuloma-like collections of lipid-laden macrophages. The joints, subcutaneous tissues, and larynx are among the favored sites of involvement, and the liver may be incidentally affected. Foamy Kupffer cells and histiocytes may gather into loose aggregates or massively permeate the sinusoids, producing a picture that simulates malignant histiocytosis.^214,218

Mucopolysaccharidoses. This family of disorders is caused by deficiencies in the lysosomal enzymes needed to degrade the glycosaminoglycans, namely dermatan sulfate, heparan sulfate, and keratan sulfate. As a consequence, partially degraded molecules accumulate within lysosomes of various tissues and are excreted into the urine. Six clinical syndromes with their subtypes have been described, corresponding to ten individual enzyme deficiencies, but there is a wide variation in the clinical features and severity of each type. In general, all the mucopolysaccharidoses share similar clinical features, including abnormal facies, organomegaly, skeletal abnormalities referred to as dysostosis multiplex, and a chronic, progressive course; mental retardation, corneal clouding, joint stiffness, and aortic valve disease may also be present.^227,239

The liver is affected in several of the mucopolysacchardioses, all with similar histologic features. The hepatocytes and Kupffer cells demonstrate varying, and sometimes subtle, degrees of cytoplasmic rarefaction and vacuolation (Figure 9-25). The accumulated mucopolysaccharide can be demonstrated by stains for mucosubstances such as colloidal iron or Alcian blue stains, but, since these substances may be largely extracted from tissues during processing, special fixatives give more reliable results. Fibrosis may extend from the portal tracts and along the sinusoids and outline the lobular periphery; cirrhosis may develop in children or adults .²⁴¹ Ultrastructural examination demonstrates membrane-bound lysosomal vacuoles containing abundant electron-dense material with various fibrillar, granular, and irregular structures.²

Mucolipidoses. These rare conditions, which include I-cell disease and pseudo-Hurler polydystrophy, are now known to be caused by derangements in the intracellular transport of lysosomal enzymes with a resulting generalized defect in macromolecular degradation.²⁴⁰ The clinical picture resembles that of the mucopolysaccharidoses with coarse facies, psychomotor retardation, skeletal abnormalities, and hepatosplenomegaly as prominent features. Histologically there is variable enlargement and vacuolation of the hepatocytes, Kupffer cells, and portal tract fibroblasts, sometimes with mild involvement of biliary epithelium.^2,245 By electron microscopy, the membrane-bound vacuoles contain nonspecific flocculent or fibrillogranular material likely representing various oligosaccharides, mucopolysaccharides, and lipids.

Oligosaccharidoses. These disorders are characterized by defective lysosomal degradation of glycoproteins and, depending upon the particular enzyme affected, include mannosidosis, fucosidosis, sialidosis, and aspartylglycosaminuria.²²⁷ Inherited as autosomal recessive traits, these conditions are phenotypically similar to the mucopolysaccharidoses. The liver is regularly affected and exhibits enlarged, vacuolated hepatocytes and Kupffer cells, occasionally with vacuoles in endothelial cells, Ito cells, or biliary epithelial cells.^1,215 On ultrastructural examination, this appearance derives from a large population of electron-lucent vacuoles with a heterogenous content of granular or flocculent material and membrane remnants.²

Acid Lipase Deficiency. The deficiency of lysosomal acid lipase is associated with two conditions that are phenotypic variants, Wolman's disease and cholesteryl ester storage disease. Both conditions are characterized by the extreme accumulation of cholesteryl esters and triglycerides in lysosomes. Wolman's disease represents the severe form; affected individuals present with hepatomegaly, steatorrhea, failure to thrive, and adrenal calcifications, with deterioration and death during infancy. In cholesteryl ester storage disease, the clinical course is more moderate, and hepatomegaly and premature atherosclerosis are common manifestations.²²⁷

The hepatic changes are similar in both variants. On gross examination, the liver is enlarged and strikingly yellow to orange in color. Histologically, both the hepatocytes and Kupffer cells are swollen, pale-staining, and vacuolated (Figure 9-26). In the hepatocytes, numerous small, well-defined vacuoles may be discerned, and the appearance recognized as microvesicular fatty change; on occasion, ordinary large lipid droplets are also seen. The foamy Kupffer cells may additionally acquire PAS-positive ceroid pigment, which imparts a tan to brown tint on routine stains, and scattered portal macrophages with a similar foamy appearance are also often present. Proliferated bile ductules or mononuclear inflammatory infiltration of the portal tracts may be evident, and varying degrees of periportal or pericellular fibrosis can develop, in some cases progressing to cirrhosis.^{222,223,225,237}

When frozen sections are available, the lipid nature of the accumulation can be demonstrated by appropriate stains such as oil red or Sudan black, and the presence of cholesterol confirmed by finding needle-shaped birefringent crystals with polarized light.²³³ Ultrastructurally, the cells contain numerous lipid droplets of varying size together with elongated cholesterol clefts lying free within the cytoplasm; unexpectedly, these changes are noted in biliary epithelial cells, pericytes, and endothelial cells as well as in hepatocytes, Kupffer cells, and portal macrophages.^2,223

Several therapeutic drugs may cause cytoplasmic rarefaction unassociated with inflammatory reactions and outside the setting of fatty change. For example, the antiarrhythmic agent amiodarone consistently causes phospholipid accumulation in the liver: vacuolated hepatocytes and Kupffer cells, resembling those of Niemann-Pick disease, are seen histologically and whorled lamellar lysosomal inclusions are noted by electron microscopy.^229,234 In some patients, this change is complicated by the development of steatohepatitis. Other drugs implicated in hepatic phospholipidosis include perhexilene maleate, trimethoprin-sulfamethoxazole, and 4,4'-diethylaminoethoxyhexestrol.²³⁸ The plasma extender hydroxyethyl starch has also caused hepatocyte cytoplasmic rarefaction.²²⁴

The combination of marked fatty change and canalicular cholestasis typifies three metabolic disease of childhood, namely galactosemia, tyrosinemia, and hereditary fructose intolerance (Figure 9-27). This histologic pattern, although not discrete or specific, is distinctive and may be sufficient to suggest the diagnosis in the proper clinical setting.²⁵⁸

Galactosemia actually represents three separate autosomal recessive disorders of galactose metabolism, each involving the deficiency of one of several enzymes that act to convert galactose to glucose. The classic and most severe form results from inadequate galactose-1-phosphate uridyl transferase encoded on chromosome 9. Because of the disruption in the process, the metabolic products of galactose, some of which are toxic, accumulate in the liver, kidneys, lungs, and other organs.

The clinical presentation includes hepatomegaly, jaundice, vomiting, and diarrhea beginning with the onset of lactose feeding, and lenticular cataracts and mental retardation may follow. The correct diagnosis is usually suspected by the finding of a non-glucose reducing substance in the urine, and confirmation depends on demonstrating deficiency of transferase activity in red blood cells.^259,260,270 The histologic alterations comprise marked fatty change, canalicular cholestasis, and bile ductular proliferation, sometimes with ductular cholestasis. Cholestatic liver cell rosettes may be a prominent feature, and multinucleated giant hepatocytes are occasionally seen. Additional findings include extramedullary hematopoiesis and mild siderosis. Periportal fibrosis develops early and progresses through the formation of portal-portal fibrous septa to cirrhosis, which is usually present by six months of age.^258,264,271 However, even extensive fibrosis may regress with institution of a galactose-free diet.²⁵¹ Exceptional cases of hepatocellular adenoma or carcinoma have been reported.²⁶⁷

Several inborn abnormalities of tyrosine metabolism are described, but the only one associated with liver disease is hereditary tyrosinemia (tyrosinemia type I). This is an autosomal recessive disorder that probably results from a deficiency of fumaryl acetoacetase. Toxicity is presumably due to some injurious metabolite produced because of the deficiency, but the exact species involved and its mechanism of action are unknown.^257,261

Clinically, tyrosinemia presents in either an acute or a chronic form, likely reflecting differences in the responsible molecular defects. The acute form manifests as rapidly progressive hepatic failure in infancy. Vomiting, diarrhea, failure to thrive, abdominal pain, and jaundice are the corresponding symptoms, and death usually occurs by eight months. The slowly evolving chronic form presents with renal tubular defects, growth retardation, rickets, and evidence of chronic liver disease developing during childhood. The diagnosis in either case is established by identifying succinylacetone, a metabolic product of the substrates of fumaryl acetoacetase, in the urine or blood. The enzyme deficiency can also be directly assayed.^257,261 The primary treatment is dietary restriction, but liver transplantation is being increasingly employed to definitively correct the metabolic abnormality.^265,268

The histologic changes in the acute form include canalicular cholestasis with cholestatic rosettes, severe fatty change, and occasional giant multinucleated hepatocytes with variable zones of parenchymal collapse. Fibrosis with a periportal or pericellular distribution is commonly present, and nodular hyperplasia may be noted.^253,269,273 The chronic form is characterized by well-established cirrhosis of varying pattern and differing degrees of fatty change and canalicular cholestasis. The hepatocytes have a variegated appearance and may display clear glycogen-rich cytoplasm, cytoplasmic globules, cholestatic rosettes, or variably-sized nuclei. Pericellular fibrosis additionally dissects the parenchymal nodules, and proliferated bile ductules and scattered mononuclear inflammatory cells populate the fibrous septa. Additional findings include mild siderosis, extramedullary hematopoiesis, and, in some cases, liver cell dysplasia.^{255,258,263,269}

An ominous complication is the development of hepatocellular carcinoma. The risk increases with advancing age, occurring in up to 37% of children who survive beyond two years, and provides an additional justification for liver transplantation.^255,274

This autosomal recessive disorder results from deficiency of fructose-1-phosphate aldolase B, one of the enzymes catalyzing the conversion of fructose into intermediates of the glycolytic pathway. The result is the buildup of fructose-1-phosphate, a potentially toxic substance that can inhibit glycogenolysis and gluconeogenesis and deplete high energy phosphates such the adenine nucleotides.^254,256

Affected patients remain well and symptom-free as long as they do not ingest fructose, but once the sugar is introduced, vomiting, failure to thrive, jaundice, and hypoglycemia develop. In severe cases, acute hepatic failure and lactic acidosis occur and may lead to death. In other instances, patients learn to avoid fructose ingestion, and the diagnosis may be delayed until childhood or adulthood, when hepatomegaly, abdominal pain, and growth retardation are the main manifestations.^252,256

The histologic features vary considerably but frequently include fatty change and canalicular cholestasis. This picture may be embellished by cholestatic rosettes or multinucleated giant hepatocytes, and confluent hepatocyte necrosis may be evident in extreme examples. The portal tracts are enlarged, variably fibrotic, and contain proliferated bile ductules. In advanced cases, pericellular fibrosis may be seen, and, in some instances, a progression to cirrhosis is described.^258,262,266 When fructose is removed from the diet, these changes largely disappear, although minor fibrosis can persist.²⁶⁶ Ultrastructural abnormalities, which may arise within hours of a single dose of fructose in susceptible individuals, comprise concentric layers of endoplasmic reticulum known colorfully as fructose holes.²

The porphyrias are a group of disorders associated with inherited or acquired disturbances in heme biosynthesis. Seven different such disorders are recognized, each representing the deficiency of a specific enzyme, but significant liver involvement occurs in only three.

In porphyria cutanea tarda, distinctive needle-shaped cytoplasmic crystals of uroporphyrin accumulate in the hepatocytes (Figure 9-28).^284,317 These crystals are yellow-brown, birefringent, and display red autofluorescence, but because of their solubility in water, they may be sparse or absent from routine sections. They are best visualized in unstained sections examined under polarized light and are demonstrated by the ferric ferricyanide reduction test.^282,284,287

The background histologic changes are variable and nonspecific: fatty change, hepatocellular siderosis of mild to moderate grade, focal hepatocellular necrosis, and mild portal infiltration by mononuclear cells. Various forms of fibrosis may be present, and cirrhosis has been noted in up to a third of reported cases.^284,300 These alterations may, in part, reflect the contributions of alcohol abuse or iron overload, two common pathogenetic cofactors in porphyria cutanea tarda.^276,299 Hepatocellular carcinoma has occasionally been recognized as a complication, usually in cases with underlying cirrhosis.

Erythropoietic porphyria is characterized by deposits of dense, dark brown protoporphyrin pigment within bile canaliculi, hepatocytes, Kupffer cells, portal macrophages, and, in some instances, interlobular bile ducts (Figure 9-29). The pigment is strikingly birefringent and, in particular, the larger deposits appear bright red with a central Maltese-cross configuration.^285,298 In addition, the deposits display red autofluorescence when examined by ultraviolet microscopy. Portal and pericellular fibrosis can develop, and, in a small percentage of patients, progression to extensive fibrosis or cirrhosis may occur and result in liver failure and death.^278,280 In some advanced cases, liver transplantation has been successfully employed.²⁷⁹

In acute intermittent porphyria, fatty change and mild hepatocyte siderosis may be identified.²⁷⁷

Amyloid is recognized by its homogenous eosinophilic appearance and its classic apple-green birefringence after positive Congo red staining. Although it may be confused with pericellular fibrosis, its amorphous character and staining qualities are generally discriminatory. Primary myeloma-associated (AL) amyloidosis cannot be distinguished from the secondary (AA) form by the distribution pattern of the amyloid, but various histologic procedures may aid the distinction: AL amyloid remains Congo red-positive after potassium permanganate pretreatment and is positive by immunoglobulin light chains immunohistochemistry.^281,302

Hepatic complications of cystic fibrosis have become increasingly prominent as the life expectancy of the patients has improved. Although the exact frequency of involvement is difficult to evaluate, up to 40% of affected adolescents have clinical or pathologic evidence of liver disease.^307,309

Several histologic features may be noted. The most common is fatty change, which is seen in about one-third of patients, but the most distinctive finding is so-called focal biliary fibrosis.^295,306 This lesion is characterized by proliferated and dilated bile ductules containing inspissated eosinophilic concretions and embedded in an expanded, fibrotic portal tract with a variable inflammatory component (Figure 9-31). These ductular concretions represent the abnormal secretion typical of cystic fibrosis and are PAS-positive but negative with Alcian blue or mucicarmine stains. Focal biliary fibrosis varies greatly in its extent and distribution, but with progression, the lesions multiply and coalesce, eventually forming a scarred, irregular liver with nodular zones of preserved parenchyma. This appearance is referred to as multilobular biliary cirrhosis, and, although all cases do not qualify by strict criteria as being cirrhotic, hepatic failure and portal hypertension may nonetheless follow.^306,309

Cystic fibrosis is also associated with bile duct strictures involving either the extrahepatic and large intrahepatic bile ducts. This complication, noted with a frequency ranging from 13% to 96% in differing series, can yield a cholangiographic appearance suggesting sclerosing cholangitis, and the liver may accordingly demonstrate the histologic changes of biliary obstruction.^290,304,313 In addition, cystic fibrosis may present during infancy and exhibit the histologic picture of neonatal hepatitis with canalicular cholestasis, hepatocyte injury, and giant multinucleated hepatocytes; the presence of fatty change or inspissated material in bile ducts may suggest the correct diagnosis.^307,308

The peroxisomal disorders are a recently recognized group characterized by defects in the biogenesis or function of peroxisomes and including Zellweger's syndrome (also called cerebrohepatorenal syndrome), infantile Refsum's disease, and neonatal adrenoleukodystrophy. The basis of the defects is unknown, but chromosomal microdeletions have been identified. The clinical picture is diverse and encompasses a variety of craniofacial, neurologic, renal, and hepatic manifestations with developmental retardation and early death in most instances.^{305,310,311,318}

The liver may show any of a number of nondiagnostic histologic changes including cholestasis, fatty change, prominent Kupffer cell siderosis, focal hepatocyte necrosis, portal fibrosis, and cirrhosis.^286,296,310 Paucity of interlobular ducts has also been described in rare instances. None of these changes is distinctive, and the characteristic features are instead noted at ultrastructural examination: Peroxisomes are typically absent, but they may also be decreased in number or size or appear abortive and malformed.^{2,292,303,316}

In many other metabolic diseases, the histologic findings are completely nonspecific and do not provide much diagnostic leverage. Fatty change is the most common of these inconclusive features and represents the major finding in a wide range of conditions including the organic acidemias, the aminoacidopathies other than hereditary tyrosinemia, the urea cycle disorders, and various disturbances in lipid metabolism such as abetalipoproteinemia, Tangier's disease, and primary carnitine deficiency.^{1,291,293,315}

Another type of storage disorder is represented by the accumulation of exogenous materials in Kupffer cells and portal macrophages. Among the identified foreign substances are talc, silica, Thorotrast, anthracotic pigment, silicone, barium sulfate, and polyvinyl pyrrolidone.^275,300,312 In addition, these cells may collect endogenous material, including as lipofuscin, ceroid, bile, or iron, but this is usually as part of another recognizable process. One exception, however, is cystinosis, in which the centrilobular Kupffer cells acquire colorless birefringent cystine crystals unassociated with hepatic injury.¹