The Liver, Gallbladder, and Biliary Tract

The Liver, Gallbladder, and Biliary Tract

Sarangarajan Ranganathan, M.D.

M. John Hicks, M.D., D.D.S., M.S., Ph.D.


Hepatobiliary morphogenesis occurs during the first 10 weeks of gestation (1). The liver primordium appears in week 3 as a tubular evagination of the future duodenal segment of the foregut endoderm. The hepatic diverticulum differentiates cranially into the proliferating hepatic cords and caudally into the extrahepatic bile ducts and the gallbladder. The hepatic diverticulum branches dichotomously, and thick anastomosing sheets of epithelial cells grow into the mesenchyme of the septum transversum, and the mesenchymal cells form the connective tissue elements of the hepatic stroma and capsule. As the hepatic sheets extend outward in the septum transversum, they are penetrated by the capillary plexus derived from the vitelline veins, which arise from the primitive hepatic sinusoids.

In the 10-mm embryo, bile canaliculi appear as intercellular spaces between sheets of presumptive hepatocytes. The epithelial lining of the extrahepatic bile ducts is continuous with the primitive hepatic sheets that give rise to the epithelium of the intrahepatic bile ducts. The epithelium of the intrahepatic bile ducts is probably generated by interaction of the primitive hepatic epithelium and the mesenchyme surrounding the developing and branching portal vein. The epithelial layer, which is in direct contact with the mesenchyme around the portal vein, transforms into bile duct-like cells, after which a second layer transforms into bile duct epithelial cells. At around 8 weeks’ gestation, the ductal plate develops, appearing as a cleft in the shape of a cylinder around the mesenchyme of the progressively developing and branching portal vein. The ductal plate (Figure 15-1) undergoes gradual remodeling to form the interlobular bile ducts in the portal tract, undergoing a balanced process of cell proliferation and apoptosis. Intrahepatic bile ducts are recognized in the 20- to 30-mm embryo. The hepatocytes and bile duct epithelial cells are structurally and functionally distinct. The canals of Hering, which connect the canaliculi to the bile ducts, consist of both typical hepatocytes and bile duct epithelial cells (1,2).

The development of the liver is associated with changes in the primordial vitelline veins, which give rise to the portal, hepatic, and umbilical veins. The definitive pattern of veins within the liver is established in the 10-mm embryo. The proximal end of the right vitelline vein forms the terminal part of the inferior vena cava. The portal vein arises from persistence of segments of both right and left vitelline veins and three anastomotic channels between the two. The right umbilical vein disappears, and all blood from the placenta enters the liver from the left umbilical vein. The coalescence of some of the hepatic sinusoids produces an oblique channel, the ductus venosus, which connects the left umbilical vein to the right vitelline vein, diverting some of the oxygenated blood directly to the heart.

The right side of the liver receives blood predominantly from the portal vein, and the left lobe is supplied mainly by oxygenated blood from the left umbilical vein. This may account for the difference in the appearance of the two lobes. At birth, the left lobe is larger relative to its size in later life. Moreover, the right lobe shows more hematopoiesis, and the hepatocytes contain more glycogen, lipid, and iron pigment than those in the left lobe. Fetal blood flow through the hepatic artery is insignificant compared with that delivered by the umbilical and portal veins.

The caudal part of the hollow diverticulum elongates and presumably becomes the common bile duct, hepatic duct, cystic duct, and the gallbladder between weeks 5 and 7.5. The liver is the site of hematopoiesis between weeks 6 and 7, and erythropoiesis dominates from week 12 until the beginning of the third trimester. During the third trimester, the bone marrow is the dominant site of hematopoiesis, and hepatic erythropoiesis decreases, although it continues in the newborn period and may persist into the first few weeks of life.


The conventional histologic unit of the liver is the hepatic lobule, which consists of a central efferent vein with cords of hepatocytes radiating to several peripheral portal tracts. The portal tract contains the interlobular bile ducts, branches of
the portal vein, and hepatic artery and lymphatics. The functional unit of the liver is the hepatic acinus (Figure 15-2). The hepatic acinus is a three-dimensional structure with the portal tract as the central point (zone 1) where blood flows from terminal branches of the portal vein and hepatic arteries into the sinusoids and empties into the terminal hepatic venules at the periphery of the acinus (centrilobular/zone 3). Bile is secreted into the canaliculi and flows toward the portal areas into the interlobular bile ducts that are connected to the canaliculi by canals of Hering. The acinus thus includes parts of several adjacent lobules.

FIGURE 15-1 • Ductal plate in the fetal liver is formed by a collar of epithelial cells at the periphery of the portal tract and abuts against zone 1 hepatocytes. Note the presence of extramedullary hematopoiesis (H&E stain, 200×).

The hepatocytes in children older than 5 or 6 years of age are organized into single-cell plates. In younger children, the liver cells are arranged in two-cell-thick plates. In the preterm infant, the lobular structure of the liver is poorly defined and hepatic plates are more than one cell thick. Canaliculi lie between adjacent hepatocytes and, ultrastructurally, tight junctions are present between the hepatocytes surrounding the canaliculus. Microvilli from the hepatocytes project into the canalicular lumen. The hepatocytes in childhood often have nuclear glycogen, and lipofuscin in the cytoplasm is usually scanty. The hepatic sinusoidal lining cells include endothelial and Kupffer cells. The endothelial cells are supported by reticulin fibers, and between the endothelial cells and hepatocytes is the space of Disse. Perisinusoidal cells (cells of Ito) are interstitial fat-storing cells and appear to play a significant role in hepatic fibrogenesis.

FIGURE 15-2 • Schematic view of hepatic lobule or acinus. The conventional view of the liver consisted of a hepatic lobule with a central vein (CV) surrounded by hepatocyte cords radiating to peripheral portal areas. The functional unit of the liver, the acinus, however, consists of a three-dimensional structure with a central portal tract surrounded by concentric zones of hepatocytes (I, II, and III), with the most peripheral zone (III) lying near the central vein.


Agenesis of the liver is incompatible with life and is usually associated with other severe congenital anomalies in stillborn fetuses. Agenesis of one lobe of the liver, usually the right, is seen infrequently and is rarely associated with clinical symptoms (3,4). In situs inversus totalis, the liver, its peritoneal and vascular connections, and the gallbladder and extrahepatic ducts have a mirror-image configuration to normal situs. In the asplenia-polysplenia syndromes, the liver may be midline and bilaterally symmetrical.

The liver may herniate through defects in the diaphragm (Figure 15-3). Diaphragmatic defects are more common on the left side, and the liver often herniates into the left pleural cavity (4,5). The herniated portion of the liver may be dusky, and a groove often marks the site of compression by the margin of the diaphragm. The right lobe of the liver may bulge into the right pleural cavity in association with eventration of
the right hemidiaphragm. In cases of omphalocele, the liver is often herniated into the omphalocele sac. In large omphaloceles, there is often distortion of the liver and its vascular and biliary connections. The liver may have signs of marked congestion and even hemorrhagic necrosis. Intrapericardial herniation of the liver occurs rarely and may result in massive pericardial effusion in neonates. Nearly all cases of the thoracopagus type of conjoined twins show connections between the two livers, ranging from a bridge to a common liver between the two twins.

FIGURE 15-3 • Herniation of the liver through diaphragmatic defect. A large defect in the left leaflet of the diaphragm has led to the herniation of the left lobe of the liver and intestines into the left hemithorax, resulting in mediastinal shift to the right and severe pulmonary hypoplasia.

FIGURE 15-4A: Ectopic liver tissue within the diaphragm B: Lung C: Umbilical cord (H&E, 40×) D: Ectopic liver tissue in the umbilical cord with bile ducts (H&E stain, 200×).

Hepatic ectopia or heterotopia is extremely unusual, with only rare reports of distinct lobules of hepatic tissue within the gallbladder wall, the substance of the diaphragm, lung, and umbilical cord (6) (Figure 15-4). Often times, this liver tissue is seen in conjunction with congenital diaphragmatic hernias and congenital heart disease. Ectopic pancreatic tissue within the liver or in the porta hepatis may also be seen, occasionally obstructing the common hepatic duct. Adrenal heterotopias are usually the result of adrenal-hepatic adhesion or fusion depending on the presence (adhesion) or absence (fusion) of a capsule between the organs. Liver tissue at these variable sites is at the same risk for viral hepatitis and subsequent hepatocellular carcinoma (HCC) as an orthotopic liver tissue infected with hepatitis viruses.


The most important aspect of providing an accurate diagnosis is appropriate triaging of tissue to allow for optimal evaluation (Figure 15-5). It is imperative that adequate tissue is obtained to perform all necessary tests for an appropriate diagnosis to guide future therapy and to avoid repeat biopsy. Fresh tissue can be obtained for microbiologic and viral cultures, polymerase chain reaction (PCR) testing, and cytogenetic studies. Tissue should be obtained for routine histology (formalin fixation), histochemical stains (frozen in optimal cryomatrix material [OCT] at -20°C and alcohol fixation), electron microscopy (glutaraldehyde), and genetic/molecular evaluation (frozen at -70°C). It is especially important
with glycogen storage diseases (GSDs) to maintain optimal preservation of glycogen. With formalin fixation, up to 70% of glycogen is lost due to the soluble nature of the predominant form of glycogen in the cytoplasm. Glycogen can be preserved with freezing and/or alcohol fixation, allowing for quantitative evaluation by analytical techniques (frozen tissue) and qualitative assessment by histochemical staining (periodic acid-Schiff [PAS], PAS diastase). Quantitative analysis of enzyme(s) responsible for suspected metabolic and mitochondrial diseases must be done on frozen tissue. Assessment of metals such as copper and iron may require use of metal-free containers to freeze liver tissue for proper quantification. Assessment of gene mutation and sequencing of the gene responsible for the enzyme defect or mitochondrial disease also requires frozen tissue. Preservation of the enzyme, enzyme activity, DNA, and RNA requires cryopreservation at -70°C and maintaining this temperature until the tissue reaches the appropriate reference laboratory. Depending on the testing required for a definitive diagnosis, tissue requirements may dictate an open biopsy of the liver and/or skeletal muscle. Obtaining fibroblast cultures from a skin biopsy may also be necessary for genetic and enzyme studies. The current trend in surgical and interventional radiology practice has been toward needle core biopsies for diagnosis. The pathologist should be aware of necessary tissue requirements (tissue weights and preservation methods) for appropriate testing to be completed. A single tissue core of 20 mm in length from a 16-gauge needle with a 1.5 mm diameter yields about 15 mg of tissue. Several metabolic disease tests require a minimum of 20 mg of tissue. With GSDs, 100 mg or more of tissue will be needed. This may necessitate numerous tissue cores or an open biopsy to obtain adequate tissue for all tests. This emphasizes the importance of active communication between the health care team and the pathologist. Because tissue will be preserved in a steady state with cryopreservation (-70°C), comprehensive workup (histopathology, histochemistry, electron microscopy) by the pathologist to determine which additional testing is most appropriate can be completed prior to performing specialized testing on the frozen tissue.

FIGURE 15-5 • Liver biopsy triaging consists of tissue submitted for formalin fixation and paraffin embedding, freezing tissue at -70°C, viral or microbiologic culture submission, and glutaraldehyde fixation for electron microscopy.


Hyperbilirubinemia in the neonatal period is one of the earliest postnatal events that requires clinical assessment to determine its clinical significance (7). In the majority of cases, it is assessed to be physiologic jaundice with an elevated unconjugated bilirubin, which resolves within the first 2 weeks of life. However, in the presence of conjugated hyperbilirubinemia and other concurrent hepatic enzyme abnormalities, a clinically serious underlying disorder must be given consideration. With infants and older children, development of jaundice is a sign of hepatic or biliary tract disease of diverse etiologies; requires thorough clinical, imaging, and laboratory evaluation; and may need liver biopsy to determine the exact nature of the underlying disease.

Physiologic jaundice is characterized by an increase in serum unconjugated bilirubin of 5 to 6 mg/dL by 2 to 4 days of age (8). This is a result of increased bilirubin production following breakdown of fetal red blood cells, combined with transient limitation in the conjugation of bilirubin by the liver. Levels of up to 12 mg/dL may be seen in Chinese, Japanese, Korean, or Native American infants. Other risk factors include maternal diabetes, prematurity, altitude, polycythemia, male sex, trisomy 21, cutaneous bleeding, cephalohematoma, oxytocin induction, and vitamin K use. Additional causes of unconjugated hyperbilirubinemia are listed in Table 15-1. Cholestasis is rarely present in the liver in the absence of other diseases.


Crigler-Najjar syndrome (CNS), an autosomal recessive or dominant disorder, results from a mutation in one of the five exons of the UGT1A1 gene coding for the enzyme bilirubin-UDP-glucuronosyltransferase (8,9). UGTIA1 mutation leads to elevated unconjugated bilirubin levels. In type 1 CNS, enzymatic activity is completely absent and the neonate presents with jaundice and frequently kernicterus with death by 1 year of age. Liver transplantation has been successfully used in management. The liver may show prominent canalicular bile or may appear normal. With type 2 CNS, there is only a partial deficiency of glucuronyl transferase, and this has a milder clinical course with most affected individuals being asymptomatic. Gilbert syndrome is a benign condition with minimal clinical manifestations, owing to greater preservation of enzyme activity. Although the condition is occasionally seen in children, the diagnosis is usually made incidentally in young adults or in later life.

Dubin-Johnson syndrome, an autosomal recessive trait, may present in the neonatal period with conjugated and unconjugated hyperbilirubinemia and severe cholestasis
(9,10). This syndrome has a mutation in the ABCC2 gene that is responsible for synthesis of MRP2/cMOAT, an organic ion transporter. Zone 3 hepatocytes contain deposits of a granular golden-brown pigment, with staining characteristics of melanin. Loss of cMOAT staining is demonstrated by immunohistochemistry (IHC), a valuable tool in diagnosis of early cases when pigment deposition may not be evident. Ultrastructurally, however, these granules do not have the features of melanosomes, but are lysosomes with a distinctive appearance.


Physiologic Features

Associated Conditions

Overproduction of bilirubin


Rh/ABO incompatibility

Erythrocyte defects


Hematoma, birth trauma

Polycythemia, maternal fetal or fetal maternal transfusion


Impaired transport of bilirubin






Impaired hepatic uptake

Decreased sinusoidal perfusion

Gilbert syndrome

Impaired conjugation

Breast milk jaundice



High intestinal obstruction

Glucuronyl transferase deficiency, types I and II


Impaired enterohepatic circulation

Low intestinal obstruction

Meconium ileus

Rotor syndrome is characterized by persistent elevation of conjugated and unconjugated serum bilirubin and presents infrequently in children (8). It differs from Dubin-Johnson syndrome clinically and morphologically and can be distinguished from Dubin-Johnson syndrome by elevated urinary coproporphyrin levels (2.5 to 5 times normal). The liver is normal histologically, but ultrastructurally immature bile canaliculi and osmiophilic lysosomal granules have been described.


Idiopathic Neonatal Hepatitis

Idiopathic neonatal hepatitis (INH), also known as neonatal giant cell hepatitis (NGCH), is largely a diagnosis of exclusion, because there are many infectious, metabolic, toxic, and anatomic etiologies to explain neonatal cholestasis (11) (Table 15-2). Once other disorders have been excluded, INH accounts for approximately 25% to 40% of cases, with an incidence of 1 in 4500 to 9000 live births. Although two subsets are seen, sporadic INH (85% to 90%) and familial INH (10% to 15%), it is likely that INH will become a better defined diagnostic category with the elucidation of addition etiologies for cases considered to be INH. For example, alpha-1-antitrypsin deficiency was included in the idiopathic category prior to the discovery of the clinical and genetic features of this disease. It is now a well-known separate and distinct entity, accounting for 25% to 30% of neonatal hepatitis cases. Other causes of cholestasis have now been included under the familial cholestasis group. It should be noted that there are some familial INH cases without a defined genetic pattern. A subgroup of NGCH infants has recently been recognized in the clinical setting of hypopituitarism (12). Still another group in this category is alloimmune gestational giant cell hepatitis that is due to complement activation (13). It is evident that more extensive neonatal testing will result in more etiologies being identified for INH.

Grossly, the liver in neonatal hepatitis may be enlarged, is usually smooth, and has a deep green bilious appearance. Microscopically, cholestasis is usually seen in zone 3 hepatocytes and canaliculi and rarely in the interlobular bile ducts. Giant cell transformation is usually prominent, hence the name, but is a nonspecific finding, because it may be seen in many disorders involving the neonatal liver (Figure 15-6). Hepatocytes
may show ballooning, acidophilic necrosis including necrosis of giant cells, and pseudoglandular or acinar formation. Lobular or portal mononuclear cells are generally sparse, but a prominent inflammatory component and extramedullary erythropoiesis should suggest an infectious etiology. The portal areas in INH are usually not expanded, except for the sometimes prominent myelopoiesis, and the bile ducts are normal or may be inconspicuous. Rarely, there may be mild proliferation of the interlobular bile ducts, better demonstrated with a cytokeratin stain. Histologic features comparing INH with those of extrahepatic biliary atresia (EHBA) are listed in Table 15-3.


Extrahepatic obstruction

Biliary atresia

Bile duct stenosis

Sclerosing cholangitis



Mucus/bile plug

Intrahepatic disorders

Giant cell hepatitis

Paucity of intrahepatic bile ducts

Syndromic (Alagille)


Progressive familial cholestasis

Byler disease (PFIC 1) (ATP8B1 defect)

BSEP disease (PFIC2) (ABCB11 defect)

MDR3 defect (PFIC3) (ABCB4 defect)

Other defects (TJP2, claudin defects)

Cholestasis associated with microvillus inclusion disease (MYO5B, RAB11)

Defects in bile acid metabolism (trihydroxycoprostanic acidemia)

Congenital abnormalities

Congenital hepatic fibrosis

Caroli disease


Total parenteral nutrition




Down syndrome

Trisomy 17,18

Inborn errors of metabolism

Amino acid





Glycogen storage disease, type IV


Gaucher disease

Niemann-Pick disease

Wolman disease


A1AT deficiency



Neonatal iron storage

Copper overload-Indian childhood cirrhosis

Cerebrohepatorenal syndrome of Zellweger






Hepatitis B

Herpes simplex











Infiltrative disorders

Langerhans cell histiocytosis

Familial erythrophagocytic lymphohistiocytosis

Juvenile xanthogranuloma



Cardiac failure

The prognosis of sporadic cases of INH is generally favorable (74% complete recovery, 7% chronic liver disease, 19% death). Infants with the familial form (family history of neonatal cholestasis) have a considerably poorer prognosis (22% recovery, 16% chronic liver disease, and 63% death).

Extrahepatic Biliary Atresia

EHBA is a disorder of infants that occurs worldwide with an incidence of 1 in 8000 to 12,000 live births (14,15). EHBA presents in two forms: an embryonic or fetal type (10% to 35%) and a perinatal form (65% to 90%). The embryonic or fetal form is characterized by early onset of neonatal cholestasis without a jaundice-free period, unlike neonatal physiologic jaundice. This form has been recently classified into three groups (16); group 1 presents as the perinatal form of the disease with late-onset neonatal cholestasis and has no associated anomalies. Group 2 is associated mainly with significant cardiovascular and gastrointestinal anomalies along with genitourinary abnormality (cystic kidney
and hydronephrosis) and group 3 is associated with laterality defects with cardiovascular, gastrointestinal, and splenic anomalies. Similar to neonatal hepatitis, EHBA is also considered to be a condition with more than one etiology. In fact, INH and EHBA have been seen as sequential processes in the same infant over a period of several weeks to months (17). While viral theories have been proposed as etiologies for EHBA, recent documentation of a high incidence of autoimmune diseases in first-degree relatives of all BA groups raises the possibility of an autoimmune mechanism (16). Defects in cilia have also been implicated in the pathogenesis of EHBA (18).

FIGURE 15-6A-C: Idiopathic neonatal hepatitis. Giant cell transformation with expansion of the portal region by chronic inflammatory cells, prominent bile ducts, and readily identified cholestasis (A at 100×, B at 200×, C at 200×, H&E).



Biliary Atresia

Neonatal Hepatitis

Giant cell transformation

Usually focal

Diffuse; occasionally focal

Hepatocellular necrosis



Lobular disarray

Usually mild

May be marked


Hepatocytes, canaliculi, and ducts

Hepatocytes and canaliculi; ducts (rare)

Portal fibrosis

In all portal areas

Absent early in the course

Bile ducts

Proliferation typically seen in all portal areas

Normal; rarely focal proliferation

Cellular infiltrate

Variable; mononuclear

Variable; mononuclear

Extramedullary hematopoiesis

Usually present

Usually present


Typically absent

Typically absent

The liver biopsy remains an integral component in the diagnosis of a neonate or young infant with persistent conjugated hyperbilirubinemia and is a highly reliable means of establishing the diagnosis of EHBA in 85% to 97% of cases (19,20). Most difficulties are encountered in making a definitive diagnosis in the very young (first 4 weeks) or older patients (more than 3 months old). In many instances, the biopsies are open biopsies done at the time of surgical exploration, but needle biopsies if done not too early in the disease may be diagnostic. Ductular proliferation is the most common finding and is considered a diagnostic feature of EHBA, although modest bile duct proliferation may be seen in other causes of neonatal hepatitis (Figures 15-7, 15-8, 15-9). The interlobular bile ducts are tortuous and have distorted contours, readily demonstrated with pancytokeratin or cytokeratin AE1/AE3. Resemblance to ductal plate malformation may be noted both in the interlobular bile duct arrangement as well as within the ductular reaction (21). The lining epithelium shows degenerative changes, and periductal reactive fibrosis may occur with plump fibroblasts surrounded by a loose edematous stroma. Lymphocytes and even neutrophils are found within the portal areas, with occasional infiltration of the bile duct epithelium. Portal lymphocytes, which are usually few in number, should not be confused with extramedullary hematopoiesis in younger infants. As the disease progresses in the first few weeks of life, nearly all portal areas are expanded by fibrosis, with type IV collagen deposition. Bridging fibrosis occurs, and early nodular transformation is evident as a prelude to the development of secondary biliary cirrhosis. The progression to cirrhosis varies considerably from one case to another, but there is some direct relationship with age.

FIGURE 15-7A: EHBA liver biopsy. Histopathologic features include diffuse bile duct proliferation in expanded portal region with canalicular cholestasis. B: Hepatocytes organized into pseudoacinar pattern C: Giant cell transformation adjacent to fibrotic portal region D: Cirrhosis may occur rapidly without surgical intervention (H&E stains, A, B, C at 200×, D 40×).

Hepatocellular alterations include cholestasis (canalicular, hepatocellular, ductular), feathery (pseudoxanthomatous) degeneration, pseudoacinar transformation, and focal giant cell transformation. These features overlap with those of neonatal hepatitis. Cholestasis in EHBA is usually severe and is most prominent in zone 3, but is also present within the ductules and bile ducts at the zone 1 interface. Hepatocytes may form gland-like structures around bile plugs, imparting a “pseudoacinar” configuration, the so-called cholestatic
rosettes. Bile “lakes” consisting of amorphous collections of bile surrounded by inflammatory cells and connective tissue are seen rarely in liver biopsies, unlike in adults with obstruction of the biliary tract. Hepatocytes may display mild enlargement and rarefaction of the cytoplasm (feathery degeneration), but fatty change is rarely seen. Giant cell transformation, if present, is generally restricted to zone 1 at the interface with the expanded portal tracts (Table 15-3). Instances of hepatocyte and giant cell necrosis may be encountered.

FIGURE 15-8 • Resection of residual common bile duct during Kasai procedure. A: Common bile duct remnant with orientation by surgeon. CHD, hepatic duct, GB; gallbladder, CBD; common bile duct; plate, liver plate. B: Near-total obliteration of common bile duct lumen with no residual epithelial lining (H&E, 20×). C: Microscopic residual common bile duct lumen with epithelial lining (H&E, 20×). D: Nests of bile duct epithelium in common bile duct wall (H&E, 200×). The latter side chain structures should not be mistaken as evidence of a patent bile duct.

The most frequently observed changes within the liver in EHBA are prominent cholestasis, portal fibrosis, and ductal/ductular proliferation. Other causes of obstruction (bile duct stenosis, choledochal cyst, mucous or bile plug) produce similar changes, as will disorders such as alpha-1 antitrypsin deficiency (A1AT) and total parenteral nutrition (TPN)-associated cholestasis. It is important to realize that other disorders can simulate patterns of liver injury similar to those for EHBA.

The extrahepatic ducts may display a wide variety of histopathologic changes, ranging from a mild degree of inflammation to complete obliteration (22,23) (Figure 15-8). The epithelium of large, medium, and small ducts shows nuclear irregularity and pyknosis with cellular degeneration and necrosis. Cellular debris and bile-stained macrophages may be present in the lumen. The duct lining is often infiltrated by neutrophils and is ulcerated, with intraluminal and extraluminal fibrosis distorting the lumen. As the epithelial inflammation and degeneration progresses, fibrosis increases and eventually obliterates the duct. With active ductular
destruction, the stroma around and between ducts becomes infiltrated by neutrophils, lymphocytes, and macrophages, along with a prominent fibroblastic proliferation. As the ductular inflammation diminishes and the ducts are destroyed, the stromal activity is replaced by dense fibrosis, containing a few residual inflammatory cells and remnants of bile ducts. Rarely, islands of hyaline cartilage may be found in the porta hepatis, suggesting a congenital malformation as the cause of the atresia in these selected cases. The gallbladder may be diminutive and exhibit varying degrees of fibrosis, epithelial degeneration and destruction, and luminal compromise.

FIGURE 15-9 • Explanted liver with prior Kasai procedure. A: Explanted liver with micronodular cirrhosis. B: Patent small-bowel anastomosis site at liver hilum. C: Liver in cross-section with close apposition of small-bowel anastomosis to liver hilum and micronodular liver parenchyma with diffuse bile staining. D: Small-bowel anastomosis separated by muscular wall of small bowel and fibrous tissue from the underlying liver parenchyma (H&E, 10×).

Biliary remnants have been classified by Gautier and Eliot (22) into three types:

  • Absence of any lumen lined by biliary epithelium, with little or no inflammatory cells in the connective tissue (Figure 15-8).

    FIGURE 15-9 • (continued) E: Large bile-filled lakes within the liver parenchyma and micronodular cirrhosis with diffuse bile staining. F: Bile plugs distending portal region with adjacent micronodular liver parenchyma (H&E, 40×).

  • Presence of lumina lined by cuboidal epithelium. The remnants may be numerous, have lumens less than 50 µm, and are surrounded by a neutrophilic infiltrate. Cellular debris and bile may be present in the lumen, and epithelial necrosis may be seen in ducts with a diameter exceeding 300 µm.

  • The presence of a central altered bile duct incompletely lined by columnar epithelium, in addition to smaller epithelial structures resembling those in the second type.

The size of the ducts tends to be larger in infants younger than 12 weeks of age, and beyond this age, total obliteration of ducts is the common finding. It has been observed that few or absent ductal remnants at the porta hepatis and absence of portal inflammation were predictors of a poor prognosis (23). Age at Kasai procedure surgery (improved outcome at <60 days of age), the surgical team’s experience, and the degree of liver disease are factors associated with prognosis. Liver transplantation is the only option available for children with failed Kasai procedures.

Persistent Intrahepatic Cholestasis

Once the presence of a normal biliary tract has been established through a variety of studies and procedures, the differential diagnosis of persistent conjugated hyperbilirubinemia shifts in the direction of inherited and infectious etiologies. The inherited disorders include those conditions of a primary nature affecting the structure of intrahepatic bile ducts or bile secretion with secondary effects on the intrahepatic ducts. The first category is represented primarily by the Alagille and progressive familial intrahepatic cholestasis (PFIC) syndromes and the second by a diverse group of infectious, metabolic, and inherited disorders.

Alagille Syndrome (Syndromic Paucity of Interlobular Bile Ducts, Arteriohepatic Dysplasia)

Alagille syndrome is an autosomal dominant disorder associated with abnormalities of the liver, heart, eye, skeleton, and a characteristic facial appearance (24,25) (Table 15-4). The genetic defect for this syndrome is the JAG1 gene locus on chromosome 20p12. JAG1 encodes a ligand for the Notch signaling pathway that is important in early cellular development, particularly in the liver, kidney, and heart (26,27). Alagille syndrome is the most frequent condition associated with paucity of intrahepatic bile ducts and has been referred to as syndromic paucity of interlobular bile ducts. The onset of cholestasis occurs in the first 3 months of life with unconjugated hyperbilirubinemia and an obstructive pattern on laboratory evaluation and hepatobiliary scintigraphy. Cutaneous manifestations occur later in the course and include pruritus (hyperbilirubinemia) and xanthomas (hypercholesterolemia). The typical facies includes a prominent forehead, hypertelorism, flattened malar eminence, and a pointed chin, although the specificity of the abnormal facies has been questioned. Characteristic eye findings include a posterior embryotoxon. The cardiovascular anomaly most often reported is pulmonic stenosis with a heart murmur (95%). Vertebral abnormalities (butterfly vertebrae, 60% to 70%) and foreshortened fingers are skeletal anomalies associated with the syndrome. Renal abnormalities leading to renal failure include interstitial nephritis and membranoproliferative glomerulonephritis with mesangial lipid deposits. Unilateral renal cystic dysplasia, renal hypoplasia, ureteropelvic obstruction, and renal artery stenosis may also be seen. Other features include neurodevelopmental delay, stunted growth, cerebrovascular accidents (15%), pancreatic insufficiency, moyamoya, and middle aortic syndrome. Incomplete forms of the syndrome have been described in which only some of the major features are present. The mortality rate is 17% to 28%, which is largely determined by the presence of cardiovascular disease or progressive liver disease (28).

Liver disease is noted in almost 95% of cases within the first year of life, with progression to cirrhosis. HCC is an infrequent complication. Transplantation has been performed in
approximately 50% of patients in some series, with approximately a 75% survival rate (29).



Age at Onset

Associated Anomalies



Pathologic Features


<3 mo

Facies, heart, eye, bone, kidneys

Autosomal dominant

Mild disease, cirrhosis in 12%-14%

Paucity of ducts, cholestasis, giant cells; pigment in Golgi, ER, and lysosomes


3-12 mo

Diarrhea, malabsorption pancreatitis

Autosomal recessive


Progression to cirrhosis in 2-7 y

Cholestasis, small hepatocytes, fibrosis, Byler bile on EM

BSEP disease

Birth-6 mo

None, development of HCC

Autosomal recessive ABCB11

Progression to cirrhosis 6 mo-10y

Cholestasis, giant cell hepatitis, no coarse bile on EM, absent BSEP stain

MDR3 disease (PFIC3)

1 mo-20 y


ABCB4 gene

Progression to cirrhosis 5 mo-20 y

Variable cholestasis, ductular proliferation, mimic atresia


<3 mo


Autosomal recessive

Mild disease

Cholestasis, portal fibrosis

Benign, recurrent

1-15 y



No disease


North American Indian

<3 mo


Autosomal recessive

Fatal cirrhosis

Cholestasis, giant cells, actin filament hyperplasia

ER, endoplasmic reticulum; EM, electron microscopy.

The characteristic histopathologic feature of Alagille syndrome is absence or paucity of interlobular bile ducts (Figure 15-10). Because normal numbers of bile ducts may be present in early biopsies and even ductal proliferation, it is assumed that the syndrome is characterized by progressive damage and subsequent loss of intrahepatic ducts, as noted in liver biopsies from older children. Loss of ducts through atrophy secondary to decreased bile flow is an alternative explanation for the paucity of bile ducts. An optimal diagnostic liver biopsy should contain 20 portal areas, which may require a wedge biopsy, but a needle biopsy containing at least six portal areas may be adequate. Portal triads may be diminished in size and number and show no or mild fibrosis. Cholestasis is usually present in zone 3, but may be seen in zone 1. Hepatocellular ballooning, pseudoacinar transformation, focal giant cell formation, and lobular disarray are other nonspecific features. A quantitative increase in hepatic copper may occur and is demonstrable by rhodamine or other copper stains in zone 1 hepatocytes, a finding also common in other obstructive or cholestatic hepatopathies. Ultrastructural changes are distinctive with bile pigment retention in the cytoplasm, especially in lysosomes and in vesicles in the outer convex region of the Golgi apparatus. Rarely, bile pigment is present in the bile canaliculi or immediate pericanalicular region, suggesting a block in the bile secretory apparatus (30).

Progressive Familial Intrahepatic Cholestasis

PFIC is a group of severe genetic cholestatic hepatopathies of early life, including the archetypical PFIC1 (Byler disease) first described in Amish children. This autosomal recessive disorder is heralded by infantile cholestasis, which leads to hepatic fibrosis and death (31). Children who have a clinically similar disorder, but are not members of the Amish kindred in which Byler disease was described, are said to have Byler syndrome (now called PFIC2 or BSEP disease). The gene for Byler disease (FIC1 gene) is at 18q21 locus of the ATP8P1 gene, which synthesizes an aminophospholipid translocating ATPase on the bile duct epithelium. This same gene mutation is implicated in benign recurrent intrahepatic cholestasis 1 (BRIC1), which is associated with recurrent cholestasis with pruritus, but a mild course. A second form of BRIC is also seen with BSEP gene mutations (BRIC2). PFIC type 1 (ATP8B1 gene mutation at 18q21) and PFIC type 2 (ABCB11 gene mutation at 2q24) are characterized by cholestasis and low serum gamma-glutamyltransferase (GGT) activity. With PFIC type 3, serum GGT is elevated and is associated with mutation of the ABCB4 gene (7q21) (32). This gene encodes the canalicular protein MDR3 responsible for translocation of phospholipids from hepatocytes to canalicular lumens. Intrahepatic cholestasis of pregnancy occurs in heterozygotes with an ABCB4 gene mutation and is associated with elevated aminotransferases, cholestasis with pruritus, and recurrent fetal losses. More recent studies have determined varying mutations within the respective genes causing this
familial cholestasis, which may explain the variable presentations and manifestations of the disease (33). There is still a group of familial cholestasis in which the exact genetic defect is still not known, and recent attempts have identified TJP2 and claudin genes as possible candidates (34). PFIC1 disease is associated with extrahepatic disease manifested by diarrhea, pancreatitis, and hearing loss, while BSEP disease (PFIC2) causes progressive liver failure and increased risk of malignancy with no extrahepatic disease (35,36,37). Gallstones may be seen in some of the BRIC2 patients (36).

FIGURE 15-10A-D: Alagille syndrome (paucity of interlobular bile ducts). Absence of bile ducts within the portal tracts and presence of proliferating cholangioles at the periphery of the liver lobules as identified with cytokeratin 7 immunostaining (H&E staining, A, C: 20×; cytokeratin 7 immunostaining, B, D: 20×). E: Micronodular cirrhosis with bile plugs and diffuse bile staining.

Histologically, PFIC1 defect (Byler disease) exhibits small uniform appearing hepatocytes with canalicular and hepatocellular cholestasis and progressive paucity rather than proliferation of bile ducts and no significant fibrosis in these patients (Figure 15-11). Giant cell transformation may be occasionally seen. Immunohistochemical analysis shows normal staining for BSEP and MRP2 with some variation in CD10 staining. The bile has a characteristic coarse granular appearance on electron microscopic examination (38). In contrast, non-Amish children have neonatal hepatitis, amorphous to finely filamentous bile, and a more benign course, but with recurrent cholestasis. PFIC type 2 is characterized by persistent neonatal cholestasis with features of NGCH, feathery degeneration of hepatocytes and progressive biliary cirrhosis that may manifest before 1 year of age. IHC is useful in the diagnosis due to the absence of staining for BSEP protein in the canaliculi in most cases. HCC and even cholangiocarcinoma have been reported incidentally in these livers at time of transplant (37,39). PFIC type 3 displays periportal inflammation, extensive bile duct proliferation, feathery hepatocyte degeneration, and fibrosis, which progresses to biliary cirrhosis. IHC may be used to facilitate diagnosis and shows alterations or absence of MRP2 protein staining in canaliculi with preservation of BSEP staining. Partial external biliary diversion and transplantation have been helpful in 80% of patients (40). Instances of recurrence of low GGT cholestatic disease in the liver graft posttransplant for BSEP disease have been documented and are thought to be due to de novo bile salt exporter protein antibodies (41). Liver biopsies in Amish and Mennonite
children with familial hypercholesterolemia have bland intracanalicular cholestasis and low GGT and improve with ursodeoxycholic acid treatment. The genetic defects in these children are associated with aberrant tight junction proteins (claudin, TJP2 gene) and a defective bile acid conjugation enzyme (gene BAAT) (42). More recent molecular methods have helped elucidate genetic defects in TJP2 gene in a larger subset of infants with progressive cholestatic liver disease who have low GGT and neither the ATP8B1 nor ABCB11 mutations (34).

FIGURE 15-11A, B: Progressive familial intrahepatic cholestasis (PFIC). Hepatic lobular disarray with giant cell transformation and focal canalicular cholestasis (H&E, A: 100×, B: 400×). C: Diffuse cytoplasmic cholestasis of hepatocytes with granular bile (H&E, C 400×). D: Central lobular fibrosis with fine feathery extension into the peripheral zone toward the portal region (Trichrome, D: 100×).

FIGURE 15-11 • (continued) E: Micronodular cirrhosis with portal to portal bridging fibrosis and loss of central veins (H&E, E, 100×). F: Electron microscopic appearance of coarse granular bile markedly distending a canalicular space between hepatocytes (electron microscopy, F: 20,000×). G: Gross appearance.

Other conditions may also present initially with cholestasis and end in cirrhosis. A disease that presents with neonatal cholestasis and may mimic EHBA is North American Indian childhood cirrhosis (43). This disease has progressive fibrosis and usually culminates in cirrhosis early in life. The genetic defect has been localized to a mitochondrial protein CIRHIN (CIRH1A, 16q22). A syndrome that is comprised of arthrogryposis, renal tubular dysfunction, and cholestasis (ARC) may present initially as cholestasis with a low GGT and is typically fatal in the first few years of life (44). Another form of progressive cholestatic disease has been associated with microvillus inclusion disease (MVID) mutations in MYO5B/RAB11A with low expression of BSEP in these individual by IHC (45). An element of paucity is also reported in this setting of MVID.

Nonsyndromic Paucity of Intrahepatic Ducts

Paucity of intrahepatic bile ducts has been reported in several sporadic cases of neonatal cholestasis with progressive liver disease, but rarely does the condition evolve into cirrhosis (46). A1AT has been associated with paucity of intrahepatic bile ducts in a subgroup of patients. Other conditions include congenital syphilis, Turner syndrome, Down syndrome, cytomegaloviral infection, hepatitis B antigenemia, hypopituitarism, medications, infections, toxins, immunemediated injury, and graft-versus-host disease (Table 15-5).
Ultrastructural evidence of bile duct destruction in nonsyndromic paucity of bile ducts has been regarded as representing a primary ductal injury.





Syndromic paucity—Alagille

Primary sclerosing cholangitis

Langerhans cell histiocytosis

Extrahepatic biliary atresia

Primary biliary cirrhosis

Hodgkin disease

Alpha-1 antitrypsin deficiency

Acute vanishing bile duct syndrome—posttransplant


Peroxisomal disorders

Chronic rejection

Hepatitis C and CMV

Familial cholestasis: MDR3 and Byler

Graft versus host disease

Idiopathic adult ductopenia

CHF and Caroli disease

EBV infection

Cystic fibrosis


Hereditary Cholestasis with Lymphedema (Aagenaes Syndrome)

Hereditary intrahepatic cholestasis with lymphedema (Aagenaes syndrome) is an autosomal recessive, inherited syndrome with more than 75% of the cases occurring in Norwegians and is associated with a genetic defect on chromosome 15q (47). Cholestasis with high serum GGT is present before or shortly after birth. With modern treatment, the cholestasis usually improves considerably during the first 2 years of life, but periods of recurrent cholestasis occur later. In some cases, lymphedema is present at birth, but this usually comes to light during childhood. The prognosis for the liver disease is good, but cirrhosis develops in about 15% of Norwegian cases.

Anatomic Anomalies and Disorders of Biliary and Hepatic Ducts

Agenesis of the Common Bile or Hepatic Duct

Agenesis of the common bile duct or hepatic duct is extremely rare. With common duct atresia, the hepatic duct enters directly into the gallbladder, and a long cystic duct drains into the duodenum.

Congenital Bronchobiliary Fistula

Congenital bronchobiliary fistula (CBBF) is a rare anomaly with varied presentations, including aspiration pneumonia and atelectasis, and may be associated with common bile duct abnormalities, including biliary atresia and diaphragmatic hernia (48). CBBF usually arises from the proximal part of the right main bronchus, a short distance below the carina, and joins the biliary system at the level of the left hepatic duct. The intrahepatic portion is usually lined by squamous or columnar epithelium, whereas the proximal section resembles a bronchus with respiratory epithelial lining and cartilage plates in the wall (see Chapter 12).

Ciliated Foregut Cyst

Ciliated hepatic foregut cyst is usually seen in adults, but may rarely present in childhood with abdominal pain or portal hypertension (49). The cyst is thought to arise from the migration of a bronchiolar bud of the foregut through the pleuroperitoneal canal. The cyst is subcapsular, measuring from 1 to 4 cm in diameter, and is composed of a lining of ciliated pseudostratified columnar epithelium overlying the connective tissue, a layer of smooth muscle bundles, and a fibrous capsule.

Congenital Dilatation of the Bile Ducts

Choledochal cyst is a presumed congenital anomaly of the intrahepatic and extrahepatic ducts characterized by segmental ductal dilatation, bile stasis, and hyperbilirubinemia (50,51,52). An association with malunion of the pancreatic and distal common bile ducts is a common finding. The prevalence of choledochal cysts is 1:15,000 live births and is higher in Asian populations. There is a female predilection. Secondary causes of bile duct dilatation include cholangitis, biliary perforation, biliary tract carcinoma, acute pancreatitis, and biliary cirrhosis. The cysts are classified (Figure 15-12) as follows:

Type Ia—large cystic or saccular dilatation of the choledochus

Type Ib—segmental dilatation with no pancreaticobiliary malunion

Type Ic—diffuse cylindrical or fusiform dilatation

Type II—diverticulum of the common bile duct or gallbladder

Type III—choledochocele of the distal common bile duct that usually extends into the wall of the duodenum

Type IVA—multiple choledochal cysts with intrahepatic and extrahepatic involvement (Caroli disease)

Type IVB—multiple extrahepatic cysts

Type V—single or multiple intrahepatic dilatations (may belong to Caroli disease—see later)

Choledochal cysts present most often with nonspecific symptoms. In 40% of patients, most of whom are children, a classic clinical triad of pain, jaundice, and right upper quadrant mass is seen. Irritability, nausea, vomiting, and a palpable abdominal mass may also be present. Affected infants often have large choledochal cysts, presenting as abdominal masses. Associated atresia or stenosis of the biliary tree is often present and has a greater risk for cirrhosis in infants. Diagnostic imaging studies, including isotope scan, ultrasonography, CT scan, and endoscopic or percutaneous cholangiography, are useful in establishing a preoperative diagnosis. With some, prenatal ultrasound examination may
identify dilatation of the bile ducts, suggesting choledochal cyst or extrahepatic biliary obstruction.

FIGURE 15-12 • Classification of congenital bile duct cysts.

Intrahepatic histopathology is similar to that seen with EHBA. There is bile ductular proliferation and periportal fibrosis, which may progress to biliary cirrhosis. Regression of biliary cirrhosis has been documented after drainage of the choledochal cyst. Total excision of the cyst is recommended to avoid ascending cholangitis, choledocholithiasis, chronic liver disease, pancreatitis, and carcinoma of the bile ducts, liver, or pancreatic ducts that may be associated with internal drainage alone. The excised cyst wall is usually 1 to 2 mm thick and bile stained (Figure 15-13). It consists of dense fibrous tissue containing a few to no inflammatory cells. Only a few smooth muscle fibers may be identifiable within the wall. An epithelium is generally lacking, but occasional foci of residual columnar epithelium may be identified and some cysts may even have a complex epithelial pattern.

Congenital polycystic dilatation of the larger intrahepatic bile ducts is known as Caroli disease and has a marked predisposition to cholangitis, liver abscess, and portal hypertension (53). Caroli disease occurs most frequently in adults, but may be seen in children and infants if there are severe clinical symptoms. The diagnosis is based on cholangiographic findings of polycystic segmental dilatation of the intrahepatic biliary tree, including multiple small saccular dilatations of the peripheral segments of the intrahepatic biliary ductal system. Histopathologically, a pattern of dysplastic portal ducts and fibrosis resemble congenital hepatic fibrosis (CHF) in 90% of cases, even though only 10% have clinical evidence of portal hypertension. The combination of intrahepatic bile duct cystic changes and CHF has been termed Caroli syndrome (54). Medullary sponge kidney or renal tubular ectasia may be present in approximately 50% of patients with Caroli disease. Isolated hepatic polycystic liver disease may also occur that histopathologically appears similar to autosomal dominant polycystic kidney disease (ADPKD). The genetic defect lies at the 19p13.2 locus and is associated with mutation in the PRKCSH gene (52). This gene is responsible for synthesis of hepatocystin, which modulates fibroblast growth factor receptor functions. Predominantly, young adult females are affected.

FIGURE 15-13 • Choledochal cyst. A: Choledochal cyst with portion of common bile duct. B: Choledochal cyst with smooth glistening lining of cystic cavity.

Congenital Hepatic Fibrosis

CHF presents in a seemingly healthy child or young adult with hematemesis from esophageal varices secondary to portal hypertension. Cholangitis may also be seen on occasion. CHF has been noted in association with a variety of
renal lesions (Figure 15-14; Table 15-6) (55,56) including ARPKD (mutation of PKHD1—fibrocystin at 6p21 locus), ADPKD (mutation of PKD1—polycystin-1 or mutated PKD2—polycystin-2), and rarely in Meckel-Gruber syndrome (mutation of MKS1 at 17q, MKS2 at 1q, or MKS3 at 8q) and Jeune syndrome. The causative gene for ARPKD is PKDH1, which encodes for fibrocystin/polyductin that is located on the primary cilium together with other proteins that are defective in other renal cystic disease syndromes. Together, these constitute the group of diseases known as “ciliopathies.” Because Caroli disease and choledochal cyst are associated with CHF in a small proportion of cases, a common pathogenesis is worth consideration. CHF has also been seen in association with a variety of other syndromes including Joubert syndrome (agenesis or hypoplasia of the cerebellar vermis, retinal dystrophy, chorioretinal colobomata, oculomotor abnormalities, episodic hyperpnea, ataxia, neurodevelopmental delay), Ivemark syndrome (renal, pancreatic, hepatic dysplasia), Down syndrome, Laurence-Moon-Biedl syndrome (mental retardation, retinitis pigmentosa, obesity), and COACH syndrome (hypoplasia of the cerebellar vermis, oligophrenia, congenital ataxia, coloboma, hepatic fibrosis, (MKS3, CC2D2A, and RPGRIP1L genes) (55,57) (see Chapter 12).

FIGURE 15-13 • (continued) C, D: Lumen of choledochal cyst lacking an epithelial lining with the wall formed by dense connective tissue with scattered chronic inflammatory cells and no residual smooth muscle (H&E, C: 20×, D: 40×).

Desmet (56) suggested that CHF is caused by faulty development of the interlobular bile ducts with a disturbance in epithelial-mesenchymal inductive interactions. As a result, the ducts are subject to progressive destructive cholangiopathy of variable progression and duration that leads to biliary fibrosis. In addition, HCC has been reported to arise in a case of CHF.

There is nearly a 1:1 correlation between the frequency of liver and kidney disease in ARPKD, although the degree of kidney involvement may vary considerably. The majority of ARPKD patients present in utero or shortly after birth with abdominal masses, anuria, and oligohydramnios and frequently die within days. With the milder (juvenile) ARPKD form in older children, the clinical picture may be dominated by cholestasis in the newborn period or symptoms related to CHF (portal hypertension, bleeding esophageal varices). A number of diagnostic imaging studies are available for the diagnosis of CHF (see Chapter 17).

The liver in ARPKD displays a gross pattern of interweaving white “streaks” beneath the capsule (58). The cut surface may also show small cysts of a few millimeters in diameter. Microscopically, the portal areas contain increased numbers of bile duct structures usually arranged in concentric rings around the portal area (Figure 15-14). The anastomosing and branching ducts are associated with an increase in connective tissue, which is minimal at first but expands to form broad fibrous bands over time. Unlike cirrhosis, the fibrosis does not have a bridging appearance, and there are no regenerative nodules. However, there is the potential for the misinterpretation of CHF for cirrhosis. The portal bile ducts in infants are lined by cuboidal to columnar epithelium, which may form small polypoid projections. Pink or orange secretions are often present in bile duct lumina.

Unlike in ARPKD, CHF is rare in ADPKD (55,59). Hepatic involvement varies widely from one kindred to another, with CHF reportedly causing death shortly after birth in one ADPKD family. Other ADPKD families have shown little
tendency for progression of the hepatic manifestations over long periods of clinical follow-up.

FIGURE 15-14 • CHF associated with autosomal recessive polycystic kidney disease. A, B: Ductal plate abnormality with dilated and concentric arrangement of bile ducts in expanded and fibrotic portal regions (H&E, A: 20×, B: 40×). C: Autosomal recessive polycystic kidney disease with markedly enlarged kidney due to numerous thin cysts extending from the cortical to medullary regions.

Several syndromes of inherited renal dysplasia are characteristically associated with hepatic changes that are identical to CHF and carry the designation of biliary dysgenesis (59,60,61). These include Meckel-Gruber syndrome, chondrodysplasia (short rib-polydactyly), Jeune asphyxiating thoracic dysplasia, trisomy 21, Bardet-Biedl syndrome, Ivemark syndrome (renal-hepatic-pancreatic dysplasia), Zellweger cerebrohepatorenal syndrome, and type II glutaric aciduria. Central nervous system, ocular, and pancreatic abnormalities are additional components of these syndromes. Compared with CHF, the differences in the hepatic lesions in these syndromes are a matter of degree rather than type, with less severe fibrosis and bile duct abnormalities being a general observation. The essential saclike structure of the biliary passages is similar, and ductal dilatation resembling Caroli disease has been seen. Large intrahepatic cysts may be present. A similar hepatic lesion has been described in some cases of vaginal atresia syndrome and tuberous sclerosis (58). Another condition that is associated with CHF is nephronophthisis (mutation in NPHP1 [nephrocystin], NPHP2 [inversin], NPHP3, or NPHP4). Ductal plate abnormalities in the liver and marked tubulointerstitial kidney
disease are the features of this familial condition. Progressive renal failure occurs during the first two decades of life (62).


Infantile polycystic disease

Hereditary renal dysplasia

Meckel syndrome and congeners

Chondrodysplasia syndromes

Majewski and Saldino-Noonan short rib-polydactyly syndromes

Jeune asphyxiating thoracic dystrophy syndrome

Ellis-van Creveld chondroectodermal dysplasia

Elejalde acrocephalopolydactylous dysplasia

Trisomy 9 syndrome

Trisomy 13 syndrome

Zellweger cerebrohepatorenal syndrome

Ivemark renal-hepatic-pancreatic dysplasia

Glutaric aciduria, type II

Hereditary tubulointerstitial nephritis

Juvenile nephronophthisis

Bardet-Biedl syndrome

Adult polycystic disease

Isolated nonsyndromic disease



Histologic Features


Moderate-to-severe fatty change, fibrosis, no other distinctive features


Moderate-to-severe fatty change, fibrosis, EM shows characteristic “fructose holes”


Moderate-to-severe fatty change and fibrosis; regenerative nodules and hepatocellular dysplasia are suggestive.

A1AT deficiency

Stored A1AT in hepatocytes PAS-positive diastase-resistant globules; IHC and EM are diagnostic aids, especially in the neonate.

Glycogenosis (IV)

Early cirrhosis; stored structurally abnormal glycogen (diastase-resistant PAS-positive material with characteristic fibrillar nonmembrane-bound dense material by EM) in hepatocytes

CF (mucoviscidosis)

“Focal biliary cirrhosis” manifests as proliferated bile ducts with intraluminal inspissated material in expanded and fibrotic portal areas; rarely seen in the newborn

Niemann-Pick disease

Stored sphingolipids in Kupffer cells demonstrable histochemically; EM shows characteristic pleomorphic lamellar inclusions in the lysosomes.

Idiopathic iron storage

Hepatocellular necrosis with collapse; fibrosis; massive amounts of iron in hepatocytes and duct epithelial cells with negligible amounts in Kupffer cells

EM, electron microscopy.


A wide variety of metabolic disorders involve the liver, in addition to other organs, and a number of these disorders may present in the neonatal period as cholestasis and with neonatal hepatitis-like changes (Table 15-7). The overall incidence of metabolic disease is approximately 4 per 10,000 live births (63). The following disorders are considered in some detail because of their association with significant liver disease (see Chapter 5).

Carbohydrate Metabolism Disorders


Hereditary galactosemia, an autosomal recessive disorder, is most commonly due to deficiency of galactose-1-phosphate uridyl transferase, an enzyme encoded on the GALT gene on chromosome 9q13 (64,65). Genetic defects in the enzymes galactokinase and uridyl diphosphate galactose-4-epimerase are less common causes of galactosemia. These three enzymatic defects impair conversion of galactose to glucose. The incidence varies from approximately 1.5 to 4 per 100,000 Whites to 1 per 400,000 in Chinese (63). The disorder exhibits considerable allelic heterogeneity, and more than 150 different mutations have been identified in 24 different populations and ethnic groups in 15 countries. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent (65). The clinical features in neonates include hepatomegaly, jaundice, hypoglycemia, generalized aminoaciduria, presence of reducing substances in the urine, diarrhea, and vomiting. The differential diagnosis includes inborn errors of metabolism that manifest as neonatal hepatitis (Table 15-7). Histopathologically, canalicular and intracellular cholestasis, pseudoacinar transformation, bile ductular proliferation, focal giant cell transformation of hepatocytes, and presence of lipid are the notable features (Figure 15-15). These features in the neonatal period are similar to those seen in tyrosinemia and fructosemia. Fibrosis occurs early and progresses to cirrhosis if left untreated within the first 3 to 6 months of life (66). The ultrastructural features are diagnostic, although the individual features are not specific. The diagnosis is made by demonstration of enzyme deficiency in erythrocytes. Neonatal screening tests are available. Liver effects may be reversible with dietary galactose restriction.


Hereditary fructose intolerance is caused by catalytic deficiency of aldolase B (fructose-1,6-bisphosphate aldolase) in the liver, intestines, and kidneys and is a recessively inherited condition. Aldolase B deficiency inhibits gluconeogenesis and glycogenolysis. Two mutations on chromosome 9q (A149P and A174D) account for more than 70% of cases (67). The disease becomes manifest when fructose is introduced into the diet and presents with vomiting, diarrhea, failure to thrive, jaundice, renal failure, and hepatomegaly. Liver failure may be severe, with hepatic necrosis during the acute phase (66). More commonly, there is variable fibrosis involving the portal and lobular regions as well as microsteatosis and macrosteatosis. Chronic disease may be associated with severe fibrosis, potentially leading to cirrhosis. A neonatal hepatitis-like pattern may be seen with diffuse hepatocellular steatosis, a frequent feature. Electron microscopy of hepatocytes shows endoplasmic reticulum degranulation with membrane profiles. The response to dietary exclusion of fructose is rapid, and when so treated, the disease is compatible with a normal life span (68).

Glycogen Storage Diseases

The GSDs are a group of metabolic disorders with specific enzyme defects resulting in accumulation of abnormal amounts of structurally normal or abnormal glycogen in the
liver and other tissues (69,70). The GSDs most frequently associated with hepatic manifestations include types I, II, III, IV, VI, and IX. The mode of inheritance is autosomal recessive in all the types described, with the exception of type IXb, which is inherited as a sex-linked recessive trait. Some forms of the disease present in infancy and others in early childhood, with failure to thrive, developmental delays, acidosis, hypoglycemia, and hepatomegaly. The morphologic changes have been reviewed by McAdams et al. (69), and the ultrastructural features have been illustrated by Phillips et al. (71). Features of those glycogenoses that significantly involve the liver are summarized in Table 15-8.

FIGURE 15-15 • Galactosemia. A, B: Hepatocytes display medium to large droplet fatty metamorphosis, along with pseudoglandular transformation (H&E, A: 100×, B: 200×).

Type I Glycogenosis (von Gierke Disease)

GSD type I is the most common form of glycogenosis and potentially the most severe (70,72). There is no gender predilection. Children with this disease present in infancy with hypoglycemia and hepatomegaly. There is lactic acidosis, seizures, and failure to thrive. The subsequent course is characterized by the development of hyperlipidemia, xanthomata, hyperuricemia, cyclic neutropenia with recurrent infections, nephropathy, and chronic bowel inflammation with type 1b GSD. Type 1a GSD is due to a deficiency in the enzyme glucose-6-phosphatase (17q21). Type 1b GSD has a deficiency in a transmembrane protein required for glucose-6-phosphate transport (11q23) into microsomes. Type 1c has a deficiency in a phosphatase transporter (11q23-24.2). These deficiencies result in the accumulation of large amounts of normal glycogen in the liver, kidney, and intestine.



Light Microscopic Findings

Electron Microscopic Findings

Type I von Gierke disease

Excess glycogen in enlarged hepatocytes and in nuclei; uniform mosaic pattern; lipid droplets

Accumulation of cytoplasmic glycogen and lipid; nuclear glycogen

Type II Pompe disease

Nonuniform, mild distension, and vacuolation of hepatocytes

Lysosomal monoparticulate glycogen

Type III Cori glycogenosis

Uniform mosaic pattern, resembling type I; portal fibrosis

Similar to type I; lipid and nuclear glycogen less pronounced

Type IV Andersen disease

Inclusions in periportal hepatocytes; cirrhosis

Nonmembrane-bound inclusions of fibrillary material, glycogen, and tubules

Type VI Her disease

Nonuniform enlargement of hepatocytes; periportal fibrosis

Glycogen and finely granular material in cytoplasm


Glycogen deposition


Type IX

Glycogen deposition

Glycogen deposition with “starry sky” pattern

Type X



Histopathologically, the liver shows marked distension of the hepatocytes with glycogen, resulting in a diffuse mosaic pattern with compression of the sinusoids (Figure 15-16). Intranuclear glycogen is a common feature (70). The glycogen is best demonstrated by PAS stains of unfixed
frozen sections or alcohol-fixed tissue. Lipid is also present, while fibrosis is typically absent. Rarely, Mallory bodies may be seen. Ultrastructurally, there are pools of monoparticulate glycogen in the cytoplasm and nuclei of hepatocytes. Lipid vacuoles are found in the cytoplasm. The organelles are displaced, and the size of the mitochondria may be increased. Hepatic adenomas have been reported with some frequency, and cases of HCC and hepatoblastoma have been described in type 1 glycogenosis (73,74). Hepatic transplantation has been used in the treatment of type I, III, and IV GSDs (75).

FIGURE 15-16 • Glycogen storage disease, type I. A: Hepatocytes distended by glycogen with obliteration of the sinusoids, forming a mosaic pattern, and glycogenated nuclei. (H&E, 400×.) B: Hepatocyte with glycogenated nuclei, cytoplasmic monoparticulate glycogen, large lipid droplet, and abnormally shaped mitochondria (electron microscopy, 7500×).

Type II Glycogenosis (Pompe Disease, Generalized Glycogenosis, or Acid Maltase Deficiency)

GSD type II is classified as a lysosomal storage disorder in contrast to the cytoplasmic storage disorder that occurs in the other GSDs. It is the result of a deficiency in acid maltase caused by mutations in the alpha-1,4-glucosidase (GAA, 17q25.2-25.4) gene (76,77). The major manifestations are muscular and cardiac, and the liver shows changes as a component of the generalized involvement. Three clinical types have been described. Infantile or classic Pompe disease manifests in infancy with hypotonia and cardiomyopathy, leading to death in infancy. The second type presents in childhood with predominant involvement of the skeletal musculature, and a third type is described with an onset in the second to fourth decades. Cardiac involvement in these latter variants is minimal. Affected hepatocytes are mildly enlarged and have finely vacuolated cytoplasm (70) (Figure 15-17). Ultrastructural features are characterized by the presence of monoparticulate glycogen within membrane-bound lysosomal vacuoles. Acid phosphatase activity is associated with the lysosomal vacuoles. Currently, enzyme replacement therapy has achieved great success in treatment of these children (76).

Type III Glycogenosis (Cori Disease, Forbes Disease, Limit Dextrinosis, Debranching Enzyme Disease)

GSD type III is the result of a deficiency in the amylo-1, 6-glucosidase (debrancher enzyme, 1p21) activity (70,72). This deficiency leads to abnormal glycogen formation with increased branching points that accumulate in the liver and muscle. Hypoglycemia develops during stress or fasting due to lack of conversion of the abnormal glycogen to glucose. Hepatic morphologic features are very similar to those seen in type I GSD with panlobular cytoplasmic distension by glycogen and a uniform mosaic pattern. Accumulated glycogen is demonstrable by the presence of diastase digestible PAS-positive material in the cytoplasm. Nuclear glycogen is not as prominent as in type I GSD, but is a distinguishing feature from other types of GSD, especially types VI and IX. Hepatomegaly with hepatic fibrosis may be prominent and may progress to cirrhosis by the third or fourth decade of life.

Type IV Glycogenosis (Andersen Disease, Amylopectinosis; Glycogen Branching Enzyme Disease)

GSD type IV manifests at birth or in early infancy with failure to thrive and hepatosplenomegaly (70,72,78). In the absence of transplantation, there is progression to cirrhosis and death in early childhood (79). The brancher enzyme 1,4-1,6-glucon:1-4-glucan, 6-glycosyl transferase (3p12, GBE1 gene) is absent, resulting in abnormal glycogen with decreased branch points that resembles amylopectin or starch. Deposits of the abnormal glycogen are generalized with significant involvement of the liver, skeletal and cardiac muscles, and intestine. Microscopically, the hepatocytes in the periportal region (zone 1) contain pale eosinophilic hyaline inclusions surrounded by a clear halo, resembling Lafora bodies. These inclusions resist diastase digestion,
but are digestible with pectinase treatment (Figure 15-18). Colloidal iron staining is also seen in the cytoplasmic inclusions. Ultrastructurally, the inclusions consist of central fibrillar glycogen surrounded by polyparticulate glycogen rosettes (69). Rare instances of diffuse reticuloendothelial involvement have also been reported (80). Prenatal diagnosis is possible.

FIGURE 15-17 • Glycogen storage disease, type II. A: Hepatocytes demonstrating mosaic pattern with obliteration of sinusoids (H&E, 40×). B: Hepatocytes in close proximity to one another, with thickened cell membranes, fine cytoplasmic vacuolization, and indistinct sinusoids (H&E, 400×). C: Monoparticulate glycogen within membrane-bound lysosomes (electron microscopy, 20,000×).

Type VI Glycogenosis (Hers Disease)

GSD type VI, a deficiency in hepatic phosphorylase E activity (14q21-22), presents with hepatomegaly in the absence of the serious complications seen in other glycogenoses (70,72). Histopathologically, there is a mosaic pattern of hepatocellular distension with glycogen in zone 1 hepatocytes. Mild portal fibrosis may be seen. Ultrastructurally, pools of monoparticulate glycogen with interspersed glycogen rosettes displace the cytoplasmic organelles. A finely granular material of low electron density that is devoid of organelles may be scattered in the glycogen aggregates, imparting a starry-sky appearance (70).

Type VIII Glycogenosis

GSD type VIII, a deficiency in phosphorylase kinase (16q12-13), is accompanied by progressive neurologic deterioration leading to death in early childhood secondary to glycogen accumulation within the central nervous system (72,81). Hepatic changes are those of glycogen accumulation with nonspecific features, although a rare case of cirrhosis and adenomatous hyperplasia has been reported.

Type IX, X, and XI Glycogenoses

Other glycogenoses with hepatic manifestations are GSD types IX (phosphorylase b kinase deficiency, Xp22.2-22.1) and X (cyclic 3,5 AMP-dependent kinase deficiency,
17q23-24) (72,81). Hepatic glycogenosis with stunted growth (type XI, Fanconi-Bickel syndrome) is associated with renal glycogen deposition. Type XI glycogenosis is caused by mutations in the glucose transporter 2 (GLUT2, 3q26.1-26.3) gene (82). Generalized glycogen deposition is accompanied by cirrhosis, but with normal glycogen metabolism.

FIGURE 15-18A: Glycogen storage disease, type IV. Hepatocytes with pale hyaline inclusions surrounded by indistinct halos, resembling Lafora bodies (H&E, 200×). B, C: The inclusions stain intensely with PAS (400×) and colloidal iron (400×). D:Hepatocyte with hyaline inclusion comprised of fibrillary glycogen (electron microscopy, 6000×).

Other Glycogenoses

Hepatic involvement is not a feature of GSD types V (McArdle disease, muscle glycogen phosphorylase [myophosphorylase], 11q13) and VII (Tarui disease, phosphofructokinase enzyme deficiency, 12q13), in which skeletal muscle is primarily affected (72,81). GSD type 0 (aglycogenosis) is an autosomal recessive disease with a deficiency in glycogen synthase (chromosome 12p12.2) (72,81). Deficiency in glycogen synthase leads to a marked reduction in liver glycogen stores. The symptoms of GSD type 0 are those associated with hypoglycemia and include lethargy, pallor, nausea, vomiting, and, rarely, seizures in the early morning before breakfast. Liver biopsy will demonstrate moderate steatosis and small amounts of glycogen (0.5% versus 1.6% for normal wet liver weight) on quantitative analysis. There have also been reports of liver fibrosis in some GSD type 0 cases.

Amino Acid Metabolism Disorders


Tyrosinemia results from a deficiency of fumarylacetoacetate hydrolase (FAH, 15q23-25) and presents as acute fulminant disease in infancy or as a chronic liver disease later in childhood (83,84). Diagnosis is based upon serologic or urinary determination of succinylacetone level and FAH assays. Liver biopsy in the acute form reveals cholestasis, pseudoacinar transformation of hepatocytes, fatty change, marked intralobular fibrosis, and variable giant cell transformation (Figure 15-19) (66). These features are indicative of a metabolic hepatopathy, but are not specific for tyrosinemia.
Regenerative nodules may already be present in early liver biopsies. The chronic form is characterized by cirrhosis with variable-sized nodules separated by thick bands of fibrous connective tissue with little inflammation or bile duct proliferation. Hepatocytes may demonstrate nuclear hyperchromasia, dysplasia, or adenomatous hyperplasia. The incidence of HCC is quite high with tyrosinemia. Liver transplantation is advisable soon after diagnosis and before 2 years of age, because of the high risk of HCC (85,86). Treatment with NTBC [2(-nitro-4-trifluoromethylbenzoyl)-1-3-cyclohexanedione] prevents the accumulation of the toxic metabolites of FAH and the subsequent liver and neurologic effects, but does not entirely eliminate the risk of HCC (87).

FIGURE 15-19 • Tyrosinemia. A, B: Hepatocytes with macrovesicular steatosis and indistinct sinusoid spaces (H&E, A: 200×, B: 400×). C: Micronodular cirrhosis in chronic form of tyrosinemia (H&E, 40×).

Lysosomal Storage Diseases


Wolman Disease

Wolman disease and cholesterol ester storage disease (CESD) are rare autosomal recessive lipoprotein-processing disorders caused by mutations in the gene encoding human lysosomal acid lipase (10q24-25; Table 15-9) (88). Wolman disease is fatal in early life, presents with failure to thrive and diarrhea, and is characterized by generalized accumulation of foam cells and adrenal calcifications. Because there is partial enzyme activity, CESD is a milder clinical form of the disorder, generally limited to the gastrointestinal tract and the liver. Liver pathology includes steatosis and numerous foamy macrophages that contain cholesterol and lipid (Figure 15-20) and are similar in both diseases, although cirrhosis may occur in CESD. Cholesterol accumulation is demonstrated with frozen sections using polarized light microscopy. Ultrastructurally, hepatocytes, Kupffer cells, and portal macrophages are engorged with membrane-bound lipid vacuoles with dense membranes. Cholesterol clefts are seen in the cytoplasm (89).


The mucolipidoses are a group of disorders caused by defects of various lysosomal hydrolases and include sialidosis (ML I, neuraminidase gene at 6p21.3), I-cell disease (ML II, GNPTAB gene at 12q23.3), pseudo-Hurler disease (ML III, GNPTAB gene at 12q23.3), and sialolipidosis (ML IV, mucolipin-1 gene
at 19p13.3) (90,91). Many of the clinical stigmata of mucopolysaccharidoses may be seen, but mucopolysaccharides are not excreted in the urine. I-cell disease and pseudo-Hurler polydystrophy are autosomal recessive disorders. Coarse facies, skeletal changes, hepatosplenomegaly, and delayed growth and development are some of the clinical features. The primary histopathologic and ultrastructural changes are cytoplasmic vacuolization of hepatocytes, Kupffer cells, and, less frequently, biliary epithelial cells. Inclusions within clear vacuoles can be demonstrated within fibroblasts and peripheral nerves in skin and conjunctival biopsies (71). Glomeruli and renal tubular epithelium contain similar inclusions, and the inclusions are also present in the urine.



Enzyme Deficiency

Light Microscopic Findings

Electron Microscopic Findings



Gaucher cells with striated cytoplasm; fibrosis

Membrane-bound inclusions with twisted tubules in Kupffer cells



Finely vacuolated cytoplasm of Kupffer cells

Myelin figures in Kupffer cells and hepatocytes

Wolman and cholesterol ester storage

Acid lipase

Steatosis of hepatocytes and Kupffer cells; cholesterol clefts, fibrosis

Lipid droplets and lipolysosomes and cholesterol clefts in hepatocytes and histiocytes

Mucopolysaccharidoses, Hurler, Hunter, Scheie

Iduronidases sulfatases

Swollen clear cytoplasm of hepatocytes and Kupffer cells; fibrosis; cirrhosis

Membrane-bound, sharply delimited, electron-lucent inclusions with some granular material

Mucolipidoses, I-cell disease

Acid hydrolases

Vacuolated hepatocytes, Kupffer cells

Membrane-bound vacuoles with flocculent material


Sialidase, mannosidase, fucosidase

Vacuolated hepatocytes and Kupffer cells

Membrane-bound vacuoles with finely granular material

Metachromatic leukodystrophy

Aryl sulfatase A

Metachromatic granules in portal macrophages

Lamellar prismatic inclusions within macrophages, hepatocytes, and Kupffer cells


Acid ceramidase

Lipogranulomatous infiltrates

Curvilinear lysosomal material

Gangliosidosis GM1


Vacuolated hepatocytes and Kupffer cells

Membrane-bound vacuoles with granular material

FIGURE 15-20A, B: Cholesterol ester storage disease. Hepatocytes with diffuse microsteatosis (H&E, A 200×, B 400×).

Oligosaccharidoses (Glycoproteinoses)

Disorders of glycoprotein degradation resulting from defects in specific lysosomal enzymes lead to the accumulation of oligosaccharides in tissues and urinary excretion of these substances. These are rare autosomal recessive conditions with a phenotypic similarity to the mucopolysaccharidoses (92). These disorders include sialidosis (neuraminidase gene at 6p21.3), mannosidosis (mannosidase 2B1 gene at
19cen-q12), fucosidosis (FUCA1 gene at 1p34), and aspartylglycosaminuria (aspartylglucosaminidase gene at 4q32-33) (93). The liver is involved in all forms and has enlarged vacuolated hepatocytes. Ultrastructurally, the foamy appearance is due to cytoplasmic membrane-bound clear vacuoles (71). The vacuoles are of variable sizes, may be molded by adjacent vacuoles, and fuse to form larger vacuoles. They are composed of finely granular to flocculent material intermingled with membrane material. Kupffer cells, biliary epithelial cells, and endothelial cells show similar vacuoles.

FIGURE 15-21 • Metachromatic leukodystrophy. A: Gallbladder with markedly thickened mucosa with fine cobblestone to papillary surface. B, C: Papillary fronds lined by columnar epithelial cells with amphophilic cytoplasm (H&E, B: 100×, C: 200×). D: Lysosomal inclusions with closely packed herringbone appearance (electron microscopy, 25,000×).

Metachromatic Leukodystrophy

Metachromatic leukodystrophy is an autosomal recessive condition caused by a deficiency in lysosomal aryl sulfatase activity (arylsulfatase A gene at 22q13.31-qter) (93,94). This results in accumulation of galactosyl sulfatide in the tissues and excessive urinary excretion of the metachromatic material. Demyelination occurs with excess storage of the substrate in the central and peripheral nervous system (93). The storage material is metachromatic and shows brown granules with a characteristic birefringence in cresyl violet-stained, unfixed frozen sections. By light microscopy, foam cells are seen in the nervous system, liver, kidneys, pancreas, adrenal cortex, and gallbladder. The gallbladder may show papillary fronds lined by epithelial cells and with foam cells in the subepithelial stroma (Figure 15-21). Ultrastructurally, the lysosomal inclusions consist of prismatic structures with closely packed periodic leaflets that display a herringbone pattern. In the liver, the inclusions are found in portal macrophages, fibroblasts, and Kupffer cells.

Farber Disease (Farber Lipogranulomatosis)

Farber disease is an autosomal recessive condition in which ceramide, a sphingolipid, accumulates in the tissues due to a deficiency of the lysosomal enzyme acid ceramidase
(N-acylsphingosine amidohydrolase gene at 8p22-21.3) (95). Disseminated lipogranulomata are the morphologic findings. The liver is mildly affected, with clear vacuoles in the hepatocytes similar to the membrane-bound vacuoles in mucopolysaccharidoses. The Kupffer cells and portal macrophages have lysosomal comma-shaped, banana-shaped, and curvilinear inclusions in common with other tissues. Death occurs in adolescence or early adulthood (96).

Fabry Disease

Fabry disease is an X-linked recessive disorder caused by mutations in the alpha-galactosidase A gene (GLA gene, Xp22) and results in globotriaosylceramide accumulation in the liver and other organs (97). Endothelial cells are the most commonly affected cell type. Ultrastructural findings are characterized by pleomorphic, membrane-bound, osmiophilic lamellar and concentric inclusions (Figure 15-22).


The gangliosidoses are a group of autosomal recessive disorders with impairment of ganglioside metabolism (93,98,99). GM1 and GM2 gangliosidoses have several clinical variants in each group. Five types of GM1 gangliosidosis have been described. The infantile type presents in infancy with coarse facies, skeletal abnormalities, retinal cherry-red spot, hepatosplenomegaly, and progressive deterioration (beta galactosidase-1 at 3p21.33). Lysosomal beta galactosidase is deficient, and the substrate accumulates in the brain and the viscera. Hepatocytes and Kupffer cells are foamy and vacuolated. Ultrastructurally, the cells are distended with large lysosomes that appear as electron lucent vacuoles filled with reticular granular (71). Lamellar, concentric, membrane-bound bodies may also be seen. GM2 gangliosidosis is a group of heterogeneous disorders that includes Tay-Sachs disease (hexosaminidase A gene at 15q23-24) with a hexosaminidase A deficiency and Sandhoff disease with hexosaminidase A and B deficiencies (beta subunit hexosaminidase at 5q13). In Tay-Sachs disease, the central nervous system is primarily affected. The liver appears normal by light microscopy, but concentric membrane-bound inclusions (“zebra bodies”) may be seen on electron microscopic examination (Figure 15-23).

FIGURE 15-22 • Fabry disease. Membrane-bound lysosomal inclusions with lamellar and concentric pattern (electron microscopy, 12,000×).


The mucopolysaccharidoses are a group of distinct genetic disorders with accumulation of acid mucopolysaccharides (glycosaminoglycans), dermatan sulfate, heparan sulfate, chondroitin sulfate, and keratin sulfate in the tissues with excretion of these substances in the urine (93,100). Multiple clinical types have been described, each associated with a specific enzyme defect. With the exception of Hunter disease (type II), which is an X-linked recessive condition (Xq28), mucopolysaccharidoses are inherited in an autosomal recessive pattern. The major clinical manifestations are caused by involvement of the brain, skeletal system, liver, cornea, and other organ systems. Because the histopathologic and ultrastructural features are identical, the various syndromes cannot be differentiated on morphologic grounds alone.

The liver is involved in all types with marked cytoplasmic vacuolization of the hepatocytes, Kupffer cells, and Ito cells. Stored acid mucopolysaccharide can be demonstrated with colloidal iron staining, but requires frozen sections or nonaqueous fixatives. Numerous electron lucent membrane-bound vacuoles are seen with electron microscopic examination, corresponding to acid mucopolysaccharides that are extracted with routine tissue processing. Finely granular to flocculent material may be seen in some of the vacuoles arranged in concentric whorls. Hepatic fibrosis may occur.


Niemann-Pick Disease

Niemann-Pick disease is an autosomal recessive lysosomal disorder associated with a deficiency of sphingomyelinase (type IA and 1B [type A and B], sphingomyelin phosphodiesterase-1 gene at 11p15.4-15.1) or a defect in cholesterol
esterification (type II or type C, NPC gene at 18q11-12) (70,93). This disease is characterized by sphingomyelin storage in various organs. Sphingomyelin accumulation varies in extent, but it is most pronounced in type A (type 1A), the acute neuropathic form, and in type B (type 1B), the chronic nonneuropathic form. Sea blue histiocytes are seen in the bone marrow. The liver is enlarged and pale on gross examination. The lobular structure of the liver is not disorganized, and fibrosis is generally not a feature. However, cirrhosis may rarely occur.

FIGURE 15-23 • Tay-Sachs disease. “Zebra bodies” comprised of concentric membrane-bound lysosomal inclusions (electron microscopy, 20,000×).

FIGURE 15-24 • Niemann-Pick disease, type C. A: Hepatocytes and Kupffer cells with swollen, granular to foamy vacuolated cytoplasm (H&E, 400×). B: Large pleomorphic membrane-bound lysosomal inclusions with concentric to parallel lamellae (electron microscopy, 15,000×).

Type C (type II) disease usually presents with neurologic symptoms between 2 and 4 years of age (70,93,101). However, it may present in the neonatal period with jaundice, hepatosplenomegaly, and failure to thrive and progress to death in months. Foamy macrophages and Kupffer cells may be infrequent initially, but there is progression to the more classic swollen, foamy vacuolated appearance of the cytoplasm (Figure 15-24). Hepatocytes show similar alterations. Ceroid pigment, cholesterol, and phospholipids accumulate in the cells. The stored material is best demonstrated by the Baker hematin reaction for phospholipids. Histochemical staining for acid phosphatase activity reveals a reticular pattern. Ultrastructurally, the appearance is distinctive (71). Large, pleomorphic, membrane-bound inclusions composed of concentric or parallel osmiophilic lamellae are seen in the Kupffer cells and to a lesser extent in the hepatocytes. Bone marrow transplantation has been reported to reverse the amount of storage material in the liver, spleen, lung, and bone marrow, but it does not prevent progression of the neurologic changes.

Gaucher Disease

Gaucher disease is caused by glucocerebrosidase deficiency (acid beta glucosidase gene at 1q21) and leads to glucosylceramide accumulation in various organs (102,103). The disorder is inherited in an autosomal recessive manner, and three clinical types have been described. Type I, the most common, is the adult or chronic nonneuropathic form; type II is the acute neuropathic or infantile form; and type III is the juvenile or subacute neuropathic form. The liver has a similar appearance in all three clinical types. There is massive hepatosplenomegaly with portal hypertension. Gaucher cells are the hallmark. These cells are distended and have a characteristic striated, “wrinkled tissue paper” appearance of the cytoplasm (Figure 15-25). The striations are accentuated with the PAS stain, and acid phosphatase activity can be demonstrated histochemically. Hemosiderin and lipofuscin are frequently present. These macrophages are also seen within the spleen and bone marrow. Clusters of Gaucher cells in the lobule and in portal areas may be associated with fibrosis and cirrhosis in some cases. The ultrastructural features are distinctive with closely apposed, irregular lysosomal inclusions, which correspond to the wrinkled tissue paper light microscopic appearance of Gaucher cells. The inclusions are composed of innumerable tubules with circular profiles on cross-section. “Pseudo-Gaucher” cells have been described in association with benign and malignant hematologic diseases and HIV and mycobacterial infections.

Bile Acid Metabolism Disorders

Bile acid synthesis defects are inherited in an autosomal recessive manner, have low to normal GGT, present in infancy, and are progressive (104). These diseases typically present with neonatal hepatitis and mimic other etiologies for this nonspecific disease process. In older children, there is a more chronic hepatitis-like picture. Clinical signs and symptoms include pruritus with hyperbilirubinemia, difficulty with lipid absorption, and failure to thrive. With some conditions, bile acid substitution will reverse the clinical and histopathologic effects of the bile synthesis deficiencies. The three characteristic clinical findings that distinguish bile acid defects from other causes of neonatal cholestasis are a normal or low serum bile acid levels (as opposed to elevated
levels in all neonatal cholestasis), normal or only minimally elevated GGT levels, and absence of pruritus. Diagnosis is made by measurement of serum and urinary bile acid levels and detection of specific enzyme defects and/or genetic defects.

FIGURE 15-25 • Gaucher disease. A, B: Markedly enlarged Kupffer cells with cytoplasm with a striated, wrinkled tissue paper appearance (H&E, A: 100×, B: 800×). C, D: Kupffer cells with cytoplasmic tubular inclusions with a circular profile on cross-section (electron microscopy, C: 6000×, D: 24,000×).

Zellweger Syndrome (Cerebrohepatorenal Syndrome)

Zellweger syndrome (cerebrohepatorenal syndrome) is an autosomal recessive disorder characterized clinically by multiple congenital abnormalities, including craniofacial abnormalities, hypotonia, and psychomotor retardation. Renal cortical cysts, cerebral dysgenesis, and hepatic abnormalities are present (105). Several different genes involved in peroxisome biogenesis occur in different forms of Zellweger syndrome, including peroxin-1 (PEX1 at 7q21-q22), peroxin-2 (PEX2, 8q21.1), peroxin-3 (PEX3 6q23-q24), peroxin-5 (PEX5 12p13.3), peroxin-6 (PEX6 6p21.1), peroxin-12 (PEX12 on chromosome 17), peroxin-14 (PEX14 1p36.2), and peroxin-26 (PEX26 22q11.21). Absence of peroxisomes in hepatocytes and renal tubular cells is a distinctive feature (70,104). Death occurs in early infancy. The liver shows hepatocellular disarray, biliary dysgenesis, portal inflammation, and striking iron deposition in Kupffer cells and hepatocytes. Giant cell transformation, steatosis, and hepatic fibrosis or cirrhosis may be seen.

Other Bile Acid Synthesis Defects

Many genetic defects of bile acid synthesis are known (104,106,107). The most common defect among these is 3-beta-hydroxy dehydrogenase deficiency caused by a mutation in the gene encoding 3-beta-hydroxy-delta-5-C27-steroid oxidoreductase (HSD3B7 gene at 16p12-p11.2). This entity is referred to as congenital bile acid synthesis defect type 1. This leads to neonatal hepatitis and will progress to chronic liver disease without appropriate diagnosis and bile acid substitution. Another form of a congenital defect in bile acid synthesis is due to delta(4)-3-oxosteroid 5-beta-reductase
deficiency (congenital bile acid synthesis defect type 2). This is caused by mutation in the AKR1D1 gene (7q32-33). Congenital bile acid synthesis defect type 4 is caused by mutation in the alpha-methylacyl-CoA racemase (AMACR) gene located at 5p13.2-q11.1. Neonatal hepatitis with bile duct proliferation is associated with a bile synthesis defect in oxysterol 7-alpha-hydroxylase (CYP7B gene at 6p21.1-p11.2). Cholesterol is converted into one of several oxysterols prior to being 7-alpha-hydroxylated by an oxysterol 7-alpha-hydroxylase. Lack of this enzyme leads to neonatal hepatitis, with the potential for progressive liver disease, that can lead to infantile death. A deficiency in 25-hyroxylase (gene at 10q23) results in a bile acid synthesis defect. This is due to the role of this enzyme in expression of genes involved in cholesterol and lipid metabolism. Liver fibrosis is somewhat variable, with a more prolonged course of fibrosis in affected neonates and children. Early detection of bile acid synthetic defects can prevent progressive liver disease due to the good response to oral bile acid replacement therapy in most defects.

Bile acid conjugation defects such as familial hypercholanemia is characterized by elevated serum bile acid concentrations, itching, and fat malabsorption (BAAT gene at 9q22.3) (42). The defect in this condition is associated with the enzyme bile acid CoA amino acid N-acyltransferase (BAAT). This enzyme produces N-acyl conjugates of cholanoates (C24 bile acids) with glycine or taurine. The resulting bile acid-amino acid conjugates serve as detergents in the gastrointestinal tract. Those affected with bile acid conjugation defects may present as neonatal hepatitis with fibrosis or as mild chronic liver disease. There are several other conditions that may also have bile acid synthesis defects, such as peroxisome diseases (Zellweger syndrome, Refsum disease, hyperpipecolic anemia, adrenolipodystrophies) and cerebrotendinous xanthomatosis (CYP27A1 gene encoding sterol 27-hydroxylase at 2q33-qter).

Alpha-1 Antitrypsin Deficiency

Liver disease associated with A1AT was initially described by Sharp et al. and has been extensively reviewed (81,108,109). This is an autosomal recessive disease caused by mutations in the protease inhibitor gene (Pi) on chromosome 14. Both liver and lung diseases (emphysema) occur due to lack of neutralization of neutrophil elastase secondary to absent or decreased protease inhibitor activity. Liver disease without pulmonary emphysema occurs when a mutant but functional form of protease inhibitor is present that inhibits neutrophil elastase activity. This mutant form of AIAT has a defect that does not allow for proper folding, resulting in failure of the material to be translocated from the endoplasmic reticulum to the Golgi for further processing before release from the hepatocyte. The AIAT continues to accumulate in the rough endoplasmic reticulum, leading to hepatocyte injury and liver disease. Clinical presentations vary from neonatal hepatitis with cholestatic jaundice, to young adults with recurrent hepatitis that may lead to chronic hepatitis and cirrhosis, and to older adults with a silent clinical course and cirrhosis development.

A close association of A1AT deficiency has been noted with neonatal cholestasis, accounting for over 10% of cases of neonatal cholestasis, making it the most common genetic cause of neonatal liver disease (70). Bleeding diathesis, including intracranial hemorrhage, may be the presenting manifestation in the newborn, probably related to an associated vitamin K deficiency.

A1AT is a glycoprotein that is synthesized in the liver and secreted into the serum. Its biosynthesis is controlled by a pair of genes at the protease inhibitor (Pi) locus (81,110). More than 25 alleles have been described and are responsible for A1AT variant molecules. The normal genotype is PiMM. PiZZ is the most clinically significant genotype with respect to liver disease and is due to a point mutation with substitution of Lys for Glu. PiMZ genotype patients have 50% normal A1AT and 50% mutant A1AT. Other mutant gene alleles include PiS with reduced A1AT level and no clinical disease and PiNull with no detectable A1AT. With electrophoresis, PiZ is the slowest of the A1AT variants. In the homozygous (PiZZ) state, there is a marked reduction in the serum A1AT levels. Homozygous PiZZ A1AT has an incidence of 1 in 1600 to 2000 live births, making it nearly as frequent as cystic fibrosis (CF). A few cases of liver disease have been reported in association with PiSZ. Neonatal liver injury has occurred with the PiZ null phenotype. The risk of HCC is increased, especially in homozygous patients, with most cases being reported in adults (111).

Liver morphology varies in the early phase of the disease. Hepatocellular injury is manifested principally as cholestasis, pseudoacinar transformation, and giant cell transformation, similar to other metabolic hepatopathies (Figure 15-26). Extramedullary hematopoiesis is usually seen. Cholestasis is hepatocellular and present in the form of plugs within the canaliculi. Three morphologic patterns with prognostic significance have been described for the early cholestatic phase. In group 1, portal areas show mild portal fibrosis and no bile duct proliferation, which has a neonatal hepatitis-like appearance (Figure 15-26). In group 2, the portal triads are fibrotic and expanded and contain proliferating bile ducts in which bile may be present. This pattern may be mistaken for the obstructive changes seen in EHBA and is associated with persistent hepatic disease leading to cirrhosis with a higher frequency. With group 3, there is a paucity of intrahepatic ducts. The prognosis of this group is uncertain. Extensive hepatocellular necrosis may also occur and lead to fulminant hepatic failure.

The morphologic hallmark of the disease is the presence of A1AT in the hepatocytes, predominantly in zone 1 and occasionally in bile duct epithelium. The stored material appears in the form of eosinophilic hyaline globules that are PAS positive and resist diastase digestion. The globules progressively increase in number and may not be visible by hematoxylin-eosin sections in biopsy specimens obtained in the first

3 months of life (112). The stored material may be demonstrable by IHC, even in the absence of appreciable globules. Ultrastructurally, the stored material appears as flocculent, moderately electron-dense material within dilated cisternae of rough endoplasmic reticulum.

FIGURE 15-26A: Alpha-1 antitrypsin deficiency. Zone 1 hepatocytes with reactive changes and portal areas with chronic inflammation and mild bile duct proliferation (A: H&E, 200×). B: Cirrhosis in late stage of disease detection (B: H&E, 40×). C, D: Zone 1 hepatocytes with PAS-positive (C: 400×) cytoplasmic globules that are diastase resistant (D: 400×). E: Immunostaining for alpha-1 antitrypsin reacts with the cytoplasmic globules (E: 400×). F: Granular, flocculent material distends cisternae of the rough endoplasmic reticulum (F: electron microscopy, 10,000×).

The frequency of progression to cirrhosis after neonatal presentation with prolonged cholestasis is variable. Only about 15% of the PiZZ population develops liver disease in the first 20 years of life. If A1AT deficiency is manifested in the neonatal period, as many as 50% of cases progress to cirrhosis, typically micronodular type (66,70) (Figure 15-26). The presence of PAS-positive diastase-resistant globules is the pathologic hallmark, differentiating micronodular cirrhosis associated with A1AT deficiency from micronodular cirrhosis associated with other disorders. The extrahepatic bile ducts are usually normal. A few cases of hypoplasia of the extrahepatic bile ducts with A1AT deficiency have been described, and this hypoplasia has been ascribed to a low-flow state (20).

Cystic Fibrosis

CF is caused by mutations in the CFTR gene (CF transmembrane conductance regulator, 7q31.2) that regulates a cyclic AMP-dependent chloride channel (113). CFTR gene mutation results in decreased sodium and water content of bile with an increase in bile viscosity and reduction in bile low, leading to intrahepatic bile duct obstruction and injury. The incidence of hepatic involvement in CF has increased over the past several decades with increased life expectancy of CF patients. Although pulmonary complications are the predominant clinical manifestations, up to 5% of CF patients may have substantial hepatic dysfunction and an even larger proportion have the typical histologic lesions of CF in the liver without abnormal liver function tests (113,114). The liver may have multiple capsular depressed scars, with a resemblance to hepar lobatum. Histopathologically, there are focal irregular areas of fibrosis with bile duct proliferation and intraluminal inspissated eosinophilic or pale orange secretions (Figure 15-27). This pathognomonic hepatic lesion is the so-called focal biliary cirrhosis. Mononuclear cell infiltration may be seen. Steatosis is confined to zone 3 or shows a panacinar distribution, especially in infants with newly diagnosed CF whose pancreatic enzyme replacement has not yet been initiated.

FIGURE 15-27 • Cystic fibrosis. A: Appendix with dilated lumen and dense eosinophilic mucin in the lumen (H&E, 20×). B: Appendiceal glands with inspissated densely eosinophilic mucin (H&E, 200×). C: Hepatocytes with diffuse microsteatosis and focal macrosteatosis (H&E, 200×).

The disease may present in the neonate with cholestatic changes with giant cell transformation and steatosis as the feature of a metabolic hepatopathy. A liver biopsy in an infant with CF may not show the distinctive bile duct lesion (focal
biliary cirrhosis). The progression from neonatal cholestasis to focal biliary cirrhosis is not clear.

FIGURE 15-27 • (continued) D: Bile ducts in fibrotic portal region with luminal inspissated densely eosinophilic secretions (H&E, 400×). E-G: Explanted liver with macronodular and micronodular cirrhosis (H&E, G 40×).

A coarsely nodular cirrhosis is present in 4% to 10% of CF cases, with the prevalence increasing through childhood (113). Interestingly, there is a diminished prevalence of cirrhosis in those surviving to young adulthood, suggesting that liver disease may influence premature respiratory death in teenagers. At the cirrhotic stage, the liver shows multiple, large nodules, with areas between the nodules appearing depressed and presenting a finely nodular appearance. Portal hypertension and its complications may occur, and, rarely, death may ensue acute bleeding from esophageal varices. Combined liver and lung transplantation are necessary in a minority of cases.

The gallbladder is frequently abnormal. The prevalence of gallbladder abnormalities increases with age (115). The gallbladder may be small, with the epithelium frequently having mucinous metaplasia. Diagnostic imaging may show a diminutive or nonfunctioning gallbladder. Cholesterol gallstones are seen in 6% to 12% of patients over 12 years of age, with the risk of developing calculi increasing with age.

Iron Storage Disease

Primary and secondary disorders of iron metabolism are characterized by excessive iron accumulation in the liver (Figure 15-28) as a component of increased total body iron stores (116,117,118). Secondary iron overload may be the result of multiple transfusions for hemoglobin disorders such as thalassemia and sickle cell disease or may be due to excessive iron intake. Inherited iron storage disease or hemochromatosis is most often an autosomal recessive condition characterized by a defect in the regulation of iron absorption in the intestine and may present in childhood. There are several inherited forms of hemochromatosis caused by different gene mutations. The clinical features of hemochromatosis include cirrhosis of the liver, diabetes, hypermelanotic pigmentation of the skin, and heart failure. Pancreatic deposition of iron leads to diabetes, and congestive cardiomyopathy is the result of iron deposition in the myocardium. Primary HCC, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. Because hemochromatosis is a relatively easily treated disorder if diagnosed
early, this is a form of preventable cancer. At least five iron overload disorders labeled hemochromatosis have been identified on the basis of clinical, biochemical, and genetic characteristics (6,119). Classic hemochromatosis (HFE), an autosomal recessive disorder, is most often caused by a mutation in a gene designated HFE on chromosome 6p21.3. It has also been found to be caused by a mutation in the gene encoding hemojuvelin (HJV), which maps to 1q21. Juvenile hemochromatosis or hemochromatosis type 2 (HFE2) is also autosomal recessive. One form, designated HFE2A, is caused by a mutation in the HJV gene (1q21). A second form, designated HFE2B, is caused by a mutation in the gene encoding hepcidin antimicrobial peptide, which maps to 19q13. Hemochromatosis type 3, also an autosomal recessive disorder, is caused by mutation in the gene encoding transferrin receptor 2 (TFR2), which maps to 7q22. Hemochromatosis type 4, an autosomal dominant disorder, is caused by a mutation in the SLC40A1 gene, which encodes ferroprotein and maps to 2q32. Most affected children and adolescents are asymptomatic with periportal iron accumulations extending toward the central lobule during adolescents. With juvenile hemochromatosis, organ failure with severe iron overload presents before age 30. Both the inherited (hemochromatosis) and secondary (transfusion) forms of iron storage differ from hemosiderosis in that the iron, in addition to being deposited in mononuclear phagocytic cells, is also deposited in the parenchymal cells. Iron deposition in biliary epithelial cells is seen more often in inherited iron storage disease.

FIGURE 15-28 • Secondary hemosiderosis due to chronic transfusion therapy. A: Occasional Kupffer cells with iron pigment in their cytoplasm (H&E, 400×). B: Abundant iron storage in Kupffer cells revealed with Prussian blue histochemical stain for iron (400×).

Alloimmune Gestational Hepatitis (Neonatal Iron Storage Disease)

Alloimmune gestational hepatitis (AGH), previously known as neonatal iron storage disease (NISD), is a fatal neonatal disorder, characterized by massive iron overload (13,118). The liver is the predominant organ affected, but iron is also deposited in the pancreas, thyroid, adrenals, pituitary, heart, intestinal mucosa, salivary glands, and sweat glands. The condition should be differentiated from other disorders such as tyrosinemia, galactosemia, and Zellweger syndrome, in which excess iron is usually present and is not related to neonatal hemochromatosis. In the last few years, the etiopathogenesis of AGH has been better understood. It is now considered a gestational disease in which maternal IgG antibodies cross the placenta and induce fetal liver injury leading to severe liver disease with a neonatal hepatitis pattern in the neonate. AGH recurrence rate in siblings after the index case is 60% to 80%, implicating maternal alloimmune damage to fetal liver. Pregnant mice injected with human IgG from women with AGH offspring had pups with extensive hepatic injury and liver necrosis. It is now believed that this maternal antibody induces activation of the complement cascade in the neonate with the resultant C5b-9 complex (membrane attack complex, MAC) causing the hepatocyte necrosis and loss (118). Clinical investigations have evaluated treatment of pregnant women with a prior AGH neonate with intravenous immunoglobulin (IV Ig) (120). In these clinical studies, prior gestational histories indicated a high risk for AGH occurrence, with 92% of at-risk pregnancies resulting in intrauterine fetal demise, neonatal death, or liver failure necessitating transplant (121). With IV Ig gestational therapy during pregnancy, there were only three failures, while 52 infants did not experience AGH (118).

In AGH, the hepatic architecture is markedly disorganized with lobular collapse and early fibrosis (Figure 15-29). Scattered nests of hepatocytes with heavy iron deposits, pseudoacinar profiles, and multinucleated hepatocytes are other microscopic features. The diagnosis has now been supported by the diffuse staining of the cytoplasm of hepatocytes with C5b-9 demonstrated by a MAC immunohistochemical stain. This deposition is noted to be prominent in most cases of NGCH and can result in death in the neonatal period. Similar deposition of MAC has not been found in any other causes of neonatal cholestasis or normal livers. Increased iron deposition is noted within the hepatocytes and Kupffer cells as well
as bile duct epithelial cells. With other organ systems, the iron deposits tend to be within the reticuloendothelial system, with sparing of the parenchymal cells. Minor salivary glands in the oral mucosa show iron deposition in AGH and may be biopsied for diagnosis in suspected cases while awaiting genetic testing results for hemochromatosis.

FIGURE 15-29 • Alloimmune gestational hepatitis. A, B: Hepatocytes and bile duct epithelium with readily identified iron pigment accumulation in cytoplasm, note giant cell hepatocytes (H&E, 400×). C: Explanted liver with micronodular cirrhosis and green and brown pigmentation from bile and iron accumulation, respectively.

Wilson Disease

Wilson disease is an inborn error of copper metabolism with an autosomal recessive pattern of inheritance. A genetic defect in ATP7B on chromosome 13q14-21 has been described. This gene encodes a transmembrane copper-transporting adenosine triphosphatase (ATPase) that is located on the canalicular membrane of hepatocytes and is also homologous with Menkes disease gene (122,123). The genetic defect results in reduced copper excretion in the bile and decreased copper incorporation into ceruloplasmin. There are many different mutations in ATP7B, which account for the variable clinical phenotypes.

In normal metabolism, copper is taken up by the stomach and duodenum, weakly bound to albumin, and transferred to hepatocytes (122). Within the hepatocytes, copper is incorporated into the alpha-2-globulin of ceruloplasmin and released into the bloodstream. Senescent ceruloplasmin is reabsorbed by the hepatocytes and undergoes lysosomal degradation and excreted into the bile. In Wilson disease, copper accumulation occurs in the liver, brain, eyes, and other organs. Elevated levels of serum and hepatic copper, increased urinary copper excretion, and diminished levels of serum ceruloplasmin are the common laboratory abnormalities. In some cases, serum ceruloplasmin values may be within normal limits. A normal serum level of copper excludes Wilson disease, but an elevated level is not always diagnostic, because elevations in copper may be seen in other forms of liver disease, especially of cholestatic type, and in chronic hepatitis. Genetic analysis is available for the diagnosis of Wilson disease in patients and their families (123). The mitochondria have been shown to be the main storage house for copper within hepatocytes, and progressive accumulation leads to shutdown of the respiratory cycle and release of reactive oxygen species resulting in cell death and in severe cases acute liver failure (124).

The clinical presentation varies according to the age of the patient and the stage of the disease. The most frequent symptoms are related to hepatic involvement. Liver disease may be chronic, and cirrhosis or chronic hepatitis may be evident at clinical presentation. Acute hepatitis and fulminant hepatic failure may be the presenting features in a minority of cases, especially in the first two decades of life. Hemolytic anemia is frequent. Central nervous system signs, neuropsychiatric symptoms associated with basal ganglia involvement, and Kayser-Fleischer rings develop in the course of the disease. The latter are characterized by green-brown deposits of copper in Descemet membranes of the corneal limbus.

Penicillamine therapy has been reported to alter the natural course of the disease and, when instituted early, can prevent progression of liver disease (122,125). Controversy, however, exists as to the timing of the use of penicillamine in treatment. Treatment using zinc and trientine has also been studied. Transplantation including human hepatocyte transplantation may be necessary in some cases (126,127).

Histopathologic features in the liver vary from mild-to-moderate fatty changes, focal cytoplasmic swelling, glycogenated nuclei, and occasional acidophilic bodies in the early stages (128) (Figure 15-30). Generally, portal tract inflammation, lobular chronic inflammation, and fibrosis are not seen at this stage. Copper is diffusely dispersed in the cytoplasm and usually cannot be demonstrated histochemically. In the symptomatic stage, the liver may have features of chronic hepatitis (interface hepatitis, portal inflammation with plasma cells, fibrosis, and spotty acidophilic necrosis of hepatocytes). Mallory bodies may be seen, especially in the zone 1 hepatocytes. Glycogenated nuclei are a frequent, but nonspecific, feature. A mixed micronodular-macronodular cirrhosis is the consequence of the chronic hepatitis. Rarely, massive liver necrosis is seen.

FIGURE 15-30 • Wilson disease. A: Hepatocytes with variable cytoplasmic swelling and decreased cytoplasmic eosinophilia (H&E, 200×). B: Infrequent hepatocytes with glycogenated nuclei, apoptotic (acidophil) bodies, and fine granular cytoplasm with a certain degree of cytoplasmic swelling (H&E, 400×).

Copper can be demonstrated histochemically and is most pronounced in the periportal hepatocytes. The rhodamine stain gives a brick red reaction product, while rubeanic acid stains the copper gray-black. The Shikata orcein stain demonstrates associated copper-binding protein. Copper may be irregularly distributed in the hepatocyte nodules and may be absent in the regenerative nodules by histochemical methods. Biochemical quantitation of hepatic copper typically demonstrates marked elevations (>250 µg/g dry weight). This can be performed on fresh tissue collected in metal-free containers or a paraffin block. Ultrastructurally, the mitochondria show characteristic alterations appearing enlarged and pleomorphic. Separation of the inner membranes, widening of the intracristal space with microcystic formations at the tips of the cristae, crystalloid inclusions, disoriented cristae, and increased granules in the matrix of the mitochondria are regarded as diagnostic of the disorder (71,124). Copper deposits are seen in the lysosomes of zone 1 hepatocytes and appear extremely electron dense. Peroxisomal deposition of copper has also been described.

Other copper overload diseases in children include the Indian childhood cirrhosis (ICC), endemic Tyrolean infantile cirrhosis (ETIC), and idiopathic copper toxicosis (ICT) (129). All of these are characterized by elevated liver copper levels, but have normal or elevated ceruloplasmin levels. They are characterized by progressive fibrosis leading to micronodular cirrhosis in the first 2 years of life, especially in ICT. ICC is characterized by variable mixed inflammation, large bands of fibrosis, and hepatocellular damage but not steatosis as in Wilson disease. Mallory hyaline may also be present. Cholestasis may be prominent. A link to dietary copper ingestion has been implicated in these copper-related diseases as opposed to Wilson disease, which usually affects older children.

FIGURE 15-30 • (continued) C: Cytoplasmic copper detection in periportal hepatocytes (Rhodamine stain, 400×). D: Wilson disease with cirrhosis of the liver (H&E, 100×). E: Variably sized and relatively pleomorphic mitochondria and dense lysosomal deposits in Wilson disease (electron microscopy, 3000×).


Porphyrias are a group of disorders of porphyrin and heme biosynthesis (93,117). Porphyria may be inherited or acquired and is characterized by increased excretion of porphyrins and storage of abnormal types of porphyrin pigments within tissues. Hepatic abnormalities may be seen in acute intermittent porphyria (hydroxymethylbilane synthase 11q23.3), porphyria cutanea tarda (hemochromatosis gene at 6p21.3, uroporphyrinogen decarboxylase gene at 1p34), and congenital erythropoietic protoporphyria (uroporphyrinogen III synthase gene at 10q25.2-q26.3). The changes in acute intermittent porphyria and porphyria cutanea tarda are similar, although the severity of hepatic injury is greater in porphyria cutanea tarda. Fatty changes and iron overload are evident. Cirrhosis and hepatic failure may occur in porphyria patients, and HCC has been described as a complication in later life. The uroporphyrin crystals are water soluble and needle shaped and have a red fluorescence on examination under ultraviolet light. The needle-shaped inclusions are seen in the hepatic cells by electron microscopy. Additional ultrastructural features include abnormal mitochondria, autophagic vacuoles, and myelin figures. In congenital erythropoietic protoporphyria, the hepatic findings consist of focal accumulation of dark brown pigment in the canaliculi, bile duct epithelium, Kupffer cells, and connective tissue. The pigment is birefringent with bright granules and central Maltese crosses. An intense red fluorescence is seen in frozen sections examined under ultraviolet light. Ultrastructurally, the crystals are electron dense, straight or curved, and arranged singly or in a radiating starburst pattern.

Urea Cycle Disorders

Hyperammonemia is characteristic of this group of disorders and should be differentiated from other conditions with elevated ammonia levels (93,130). In the newborn, hyperammonemia may be found in premature infants or infants with birth asphyxia. In utero hepatic necrosis of undetermined cause has been found to be associated with hyperammonemia. Defects of the urea cycle include deficiency of ornithine transcarbamylase (Xp21.1), deficiency of carbamoyl synthetase (2q35), citrullinemia associated with deficiency of
argininosuccinic acid synthetase (9q34.1), argininosuccinic aciduria due to deficiency of argininosuccinase lyase (7cenq11.2), argininemia associated with arginase deficiency (6q23), and deficiency of N-acetyl-glutamate synthetase (17q21.31) (130,131). With the exception of ornithine transcarbamylase deficiency, which is inherited as an X-linked dominant condition, the other conditions have an autosomal recessive pattern of inheritance. Prenatal diagnosis is possible. Liver biopsy in urea cycle defects may be normal or may have only mild nonspecific changes including steatosis, cholestasis, individual cell necrosis, and early fibrosis (132) (Figure 15-31). Liver transplantation may be necessary depending upon the specific urea cycle defect disorder (133). The liver being normal during transplant, these explanted livers can be used as donor organs for other patients with liver disease (domino transplant).

FIGURE 15-31 • Urea cycle disorder—ornithine transcarbamylase deficiency. A, B: Explanted liver with no gross abnormalities in ornithine transcarbamylase deficiency. C: Portal triad and zone 1 and 2 hepatocytes with no histopathologic abnormalities (H&E, 200×).

Hepatic Steatosis and Steatohepatitis

Fatty change of the liver is a frequent, nonspecific finding associated with a variety of metabolic and nutritional disorders (134). Diagnosis of the specific metabolic disorder associated with this change requires the demonstration of pathognomonic biochemical and morphologic features of that disease. Disorders of lipid and lipoprotein metabolism include abetalipoproteinemia, hypercholesterolemia, congenital lipodystrophy, and fatty acid oxidation defects. The ultrastructural features of these diseases have been described elsewhere (71). Various chemical agents, drugs (valproate, asparaginase, steroids, amiodarone), and toxins (alcohol) are known to induce hepatosteatosis. Other causes of hepatosteatosis in childhood include protein malnutrition, kwashiorkor, obesity, chronic illnesses, type I diabetes, hepatitis C, TPN, mitochondrial disease, inborn errors of metabolism, and severe infection. In the case of obesity, fatty change may be accompanied by inflammation in the lobules and portal tracts as features of steatohepatitis (135).

This is a reversible form of cellular injury. The lipid may accumulate in the form of small droplets of microvesicular fat or in the form of large (macrovesicular) droplets that occupy most of the cytoplasm and displace the nucleus to the periphery (Figure 15-32). Microvesicular fat leads to a foamy or clear appearance of the hepatocyte cytoplasm, without displacement of the nucleus, and may not be obvious as fat in the usual preparation. Fat stains on frozen sections and electron microscopy conclusively demonstrate the fat.

FIGURE 15-32 • Nonalcoholic fatty liver disease. A: Hepatocytes with macrovesicular and microvesicular steatosis in an azonal pattern (H&E, 100×). B: Macrosteatosis and occasional hepatocytes with glycogenated nuclei (H&E, 400×). C: Portal expansion by chronic inflammatory cells with extension into zone 1 (H&E, 200×).

The marked increase in childhood obesity and type II diabetes has significantly increased the prevalence of nonalcoholic fatty liver disease (NAFLD) in the pediatric population (136). In fact, NAFLD has emerged as the leading cause of chronic liver disease in children and adolescents in the United States. Elevated insulin, ALT levels, and hyperlipidemia (increased cholesterol and triglyceride) are commonly present in these children and constitute a metabolic syndrome. Further, cardiovascular risk and morbidity in children and adolescents are associated with fatty liver. Studies have shown that age, gender, and ethnicity are important factors for NAFLD and NASH in children (134,137). There is a higher incidence in male children and also a higher incidence in Asian and Hispanic children than White children and least in African American children.

The characteristic histologic features of NAFLD range from steatosis alone to steatohepatitis (NASH) with or without fibrosis to cirrhosis (Figure 15-32). Liver biopsy remains the gold standard for the diagnosis of NASH. NAFLD grading systems are based upon the proportion of hepatocytes demonstrating macrovesicular steatosis, hepatocyte injury (ballooning degeneration), lobular inflammation, and stage of fibrosis (138). In adults, the histologic features of NAFLD have been well described and include macrovesicular steatosis, perisinusoidal or pericellular fibrosis, foci of lobular inflammation, lipid granulomas, Mallory hyaline, and megamitochondria (138). The combination of macrovesicular steatosis with ballooning change of hepatocytes and/or perisinusoidal fibrosis constitutes a pattern of histology considered diagnostic of NASH in an appropriate clinical context. However, pediatric fatty liver disease often displays a histologic pattern distinct from that found in adults (134,139,140). In a large study of 100 children with biopsy-proven NAFLD, Schwimmer et al. demonstrated two different forms of steatohepatitis. While both types showed steatosis, “type 1” was characterized by ballooning degeneration and perisinusoidal fibrosis (as in adults) affecting 17% of subjects, while “type 2” was more common (affecting 51% of subjects) and was characterized by portal inflammation and portal fibrosis. Boys were significantly more likely to have type 2 NASH than girls. Further, type 1 NASH was more common in White children, whereas type 2 NASH was more common in children of Asian, Native American, and Hispanic ethnicity. In cases of advanced fibrosis, the pattern was generally that of type 2 NASH (139). Recently, attempts have been made to understand the molecular basis of pediatric NAFLD (141). The hedgehog (Hh) pathway has been implicated especially in the prepubertal/adolescent age group and may play an
important role in the progression of liver disease with a portal-based pattern leading to liver fibrosis, especially in males. This may explain the prevalence of this disease in the pediatric age group and hence the need to identify and reverse childhood obesity to prevent progressive liver disease.

Reye Syndrome

Reye syndrome (acute encephalopathy with hepatic fatty degeneration) is an acute disease of childhood that presents as an encephalopathy, which may progress rapidly to irreversible coma and death (142). The disease has decreased dramatically since its association with salicylate use was described and warnings issued about the use of salicylates in febrile children. The disease has a biphasic clinical course with an initial febrile illness, usually associated with an upper respiratory viral infection, followed by apparent recovery and the abrupt onset of protracted vomiting, delirium, and stupor. The basic damage appears to be a widespread mitochondrial injury, especially in the liver, brain, and muscle, leading to abnormal metabolism of lipids. Children with symptoms mimicking Reye syndrome may have metabolic disorders, such as organic acid and beta-oxidation defects, and urea cycle disorders. This emphasizes the need to evaluate these children thoroughly, setting aside tissue appropriate for metabolic disorder investigations and molecular genetic studies.

Liver dysfunction is manifested by elevations in transaminases, hypoprothrombinemia, and hyperammonemia (142,143). Hypoglycemia may be present. Serum amino acid and free fatty acid levels may be elevated. Grossly, the liver is enlarged and is yellow to pale due to increased parenchymal lipid. Microscopically, the hepatocytes either appear normal or contain finely vacuolated microvesicular steatosis, which does not displace the nucleus. Oil red O stains on frozen sections reveal the panlobular distribution of lipid, and virtually all hepatocytes contain small droplets of lipid. Characteristically, there is no hepatocellular necrosis or inflammation. Severe decrease or absence of succinate dehydrogenase enzyme activity is demonstrable histochemically.

FIGURE 15-33 • Fatty acid oxidation defect—carnitine deficiency. A-C: Variable lipid deposition from fine cytoplasmic vacuolization (A) to microsteatosis (B) to macrosteatosis (C) within hepatocytes (H&E, A: 400×, B: 200×, C: 400×). D: Nonmembrane-bound lipid droplets within the hepatocyte cytoplasm (electron microscopy, 4000×).

The ultrastructural features of microvesicular lipid droplets and typical mitochondrial abnormalities are considered virtually diagnostic of the syndrome (71). The changes are reversible, and in children who recover, the liver may show normal morphology, except for the presence of lipid in some hepatocytes and Kupffer cells, and occasional large mitochondria.

Lipid accumulation is also seen in other organs, notably renal tubular epithelium, myocardial and skeletal muscles, lungs, and pancreatic islets. The brain is edematous, and mitochondrial changes similar to those in the liver have been described.

Defects in Fatty Acid Oxidation

Defects in fatty acid oxidation, such as carnitine deficiency (SLC22A5 gene at 5q31.1) and acyl-CoA-dehydrogenase deficiency (gene locus at 12q22-qter), may be associated with clinical features resembling Reye syndrome. Episodes of a recurrent Reye-like illness or siblings similarly affected should raise the distinct possibility of fatty acid oxidative disorder (see Chapter 5).

Carnitine has a role in the beta-oxidation of fatty acids by aiding in their transport across the inner mitochondrial membranes (144,145). Three clinical types of carnitine deficiency (SLC22A5 gene at 5q31.1) have been described: myopathic, systemic, and mixed. In the systemic form, carnitine levels are reduced in the serum, liver, and muscle. During the acute episode, often initiated by a relatively minor clinical event such as gastroenteritis, the liver shows microvesicular steatosis with panacinar distribution (Figure 15-33) (93). Ultrastructurally, there is nonmembrane-bound lipid and proliferation of smooth endoplasmic reticulum, increased numbers of lysosomes, and accumulation of lipofuscin. Mitochondria may be abnormal in a nonspecific manner.
Between clinical episodes, the liver may appear normal. It is important to keep this group of metabolic disorders in mind when a child dies rather abruptly during a seemingly innocuous febrile illness. Tissue and fluids should be obtained at the time of autopsy and be appropriately preserved for possible biochemical and genetic analysis.

FIGURE 15-33 • (continued)

Glutaric aciduria type II (type IIA, ETFA gene at 15q23-25; type II B, ETFB gene at 4q32-qter; type IIC, ETFDH gene at 19q13.3) is associated with deficiency of several mitochondrial acyl-CoA-dehydrogenases and is characterized by acidosis, nonketotic hypoglycemia, organic aciduria, hyperammonemia, and accumulation of lipid in the liver, myocardium, and renal tubular epithelium (93,146). One of the unique aspects of this inherited metabolic disorder is the presence of several congenital malformations, including renal cortical and medullary cysts, cerebral pachygyria, pulmonary hypoplasia, and facial dysmorphism. A familial syndrome of hepatosteatosis, jaundice, and kernicterus has been described. Death occurs in the first 3 months of life. Histologically, the liver shows panlobular steatosis with variable cholestasis and portal fibrosis. Lipid is also demonstrable in the renal tubular epithelium and myocardium. The basic mechanism of this disease has not been defined, and there is a possibility that the disease may not be a distinct entity.

Mitochondrial DNA Depletion syndromes

Liver involvement can be the predominant presentation in some of these mitochondrial DNA depletion syndromes (147,148). These are a group of genetic diseases caused by defects in the polymerase genes (POLG, POLG2, and PEO1) or mtDNA maintenance genes such as TP1, TK, DGUOK, and MPV17. This group of disease has been known under the rubric of Alpers syndrome, but is now recognized to represent different entities in this group identified by their genetic defect, all causing defects in electron transport chain function. They are frequently associated with cerebral symptoms especially epilepsy. Of these, liver involvement has been most commonly reported with POLG mutations though some of the others can also cause liver involvement. A characteristic feature is the precipitation of liver failure due to valproic acid used for the treatment of epilepsy. Liver pathology in Alpers disease (POLG mutation) is characterized by at least three of the following eight features: microvesicular steatosis, bile ductular proliferation, hepatocyte necrosis or dropout, liver plate collapse, parenchymal disarray, bridging fibrosis or cirrhosis, regenerative changes, and oncocytic change of hepatocytes. Fulminant liver failure may be seen in some cases (149,150).

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Sep 23, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on The Liver, Gallbladder, and Biliary Tract

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