Cholestasis is a common manifestation of hepatic disease in pediatric patients, and most children with liver disease will present in infancy with this manner. Although many of the responsible disorders are the same as those in adults, cholestasis in younger patients, particularly newborns, differs in both its etiologic and pathologic aspects and therefore merits a separate discussion. This chapter deals with the cholestatic disorders peculiar to this population.

Neonatal cholestasis is estimated to occur with a frequency of 2.35 cases per 10,000 liver births.³ The list of diagnostic possibilities is extensive, ranging alphabetically from Alagille's syndrome to Zellweger's syndrome; this poses a challenging diagnostic puzzle to both the pediatrician and the pathologist. Careful clinical, biochemical, and radiologic evaluations can help exclude the known causes, including various infections, certain metabolic and endocrine diseases, and anatomic anomalies, but these are uncommon.^1,2,7,8 The differential diagnosis therefore usually focuses on three disorders -- biliary atresia, neonatal hepatitis, and paucity of intrahepatic bile ducts -- which are of largely unknown etiology yet comprise over 80% of all cases of neonatal cholestasis. Liver biopsy interpretation often plays an important role in distinguishing and diagnosing these disorders.

These three disorders exemplify the general classification of cholestatic conditions based on anatomic level (Chapter 4). Biliary atresia constitutes the major large bile duct disorder, although there are also other, less common, causes of biliary obstruction in children. The hepatic histology in biliary atresia is characteristic, but sometimes not conclusive, just as with biliary obstruction in adults. The small bile duct category is primarily filled by paucity of intrahepatic bile ducts, and the hepatocellular conditions are represented by neonatal hepatitis. Neither of these diagnoses, however, designate specific entities, but instead they constitute broad, histologically-defined responses of the neonatal liver to a variety of insults. Most cases remain idiopathic, although, in a few instances, the morphologic features may suggest a specific cause.

These three disorders overlap in many clinical and histological respects, and distinguishing among them is often a diagnostic problem. This overlap has suggested to some investigators that these conditions are not distinct entities, but rather are different expressions of the same spectrum of hepatobiliary disease. In 1974, Landing proposed that neonatal hepatitis, biliary atresia, and choledochal cysts represent a single process referred to as infantile obstructive cholangiography.⁹ By this hypothesis, a common etiologic agent is capable of damaging both hepatocytes and biliary epithelium, and the evolution and outcome of the disease depends on the localization of the injury. If the bile duct is the predominant site, the result is biliary atresia, but, on the other hand, principal damage of liver cells is recognized as neonatal hepatitis. Desmet has expanded this concept by focusing on the embryogenesis of the biliary system.^4,5 He submits that biliary atresia, paucity of intrahepatic bile ducts, and the congenital fibropolycystic liver diseases (including congenital hepatic fibrosis and infantile polycystic disease) all arise from intra-uterine damage to bile ducts, and the exact outcome depends on where in the biliary tract and when in fetal development that the damages occurs.

These unifying theories serve to emphasize the dynamic, evolving nature of these conditions and help explain their overlapping features. Corroborating support is provided by the observations that certain injuries, such as cytomegalovirus or alpha-1-antitrypsin deficiency, may be associated with any of the conditions.^{38,61,125,149} In addition, transitions from one to another condition have been noted, although the nonspecificity of the diagnostic criteria make these cases difficult hard to evaluate.^6,134 The ultimate validity of the concepts, however, rests upon identifying the responsible etiologic agents; unfortunately, none is definitively recognized. Therefore, for diagnostic and mangagement purpose, these disorders are best considered as distinct entities, since the are treated differently and have disparate prognostic implications.

Biliary atresia is defined by the complete obliteration of all or a portion of the extrahepatic bile ducts. Although an infrequent condition with an incidence of one in 8,000 to one in 14,000 live births, it represents a major cause of neonatal cholestasis, accounting for 25% to 30% of all cases.³

Rather than representing a congenital failure of bile duct development, this condition is now generally considered to be acquired and the result of a progressive inflammatory sclerosis of the ducts. This notion is supported by several observations. Histologic examination of the remnants of the ducts reveals active inflammation and fibrosis throughout, involving the non-atretic, as well as the atretic, portions of the ducts.^76,77 In addition, biliary atresia is rare in stillborn infants, and the exceptional instances of true biliary agenesis lack any ductal remnants or inflammatory component.⁶⁸ Furthermore, in unusual cases, the progression from patent to obliterated ducts has been directly demonstrated.^54,59 The disease consistently and intially affects the large bile ducts, but the small intrahepatic ducts are not spared. As the disease evolves, they are affected by a similar inflammatory process and are also eventually destroyed.

The cause of biliary atresia is unknown. Perinatal viral infections have often been leading etiologic candidates, but most searches have been unsuccessful in identifying a culprit. Recently, reovirus type 3 has been implicated on the basis of serologic, immunohistochemical, and animal model evidence, but other studies have questioned its etiologic role.^18,33,58 A cytomegalovirus infection has been identified in some cases, but a causal relationship is not proved.³⁸ Additional proposed mechanisms include the secretion of abnormal bile acids, an immune-mediated biliary insult, ischemic damage resulting from vascular insufficiency, and chemical injury due to pancreatic enzymes refluxing from an anatomically deviant pancreatico-biliary junction.^37,57,76 In occasional cases, biliary atresia has been associated with abnormal cilia and polysplenia, suggesting a pathogenetic defect in embryogenesis.^32,71 Biliary atresia is possibly the end-result of different causes acting at various stages of fetal and perinatal life.

Clinical Features^12,42,59

Affected infants present with jaundice, usually beginning at birth or within the first few weeks of life, but they otherwise appear suprisingly well with normal postnatal development. Most are full-term babies with normal birth weights and gestational histories; the sex ratio is roughly equal with a modest female predominance in some series. The liver is characteristically large and firm on physical examination, and laboratory studies reveal a conjugated hyperbilirubinemia and variably elevated alkaline phosphatase and transaminase levels. Ongoing disease is manifest by worsening jaundice, pruritus, malnutrition, and severe failure to thrive; eventually biliary cirrhosis develops with subsequent hepatic failure and portal hypertension. If untreated, death usually follows after one or two years.^11,12

Most cases of biliary atresia are sporadic and lack any family history of infantile liver disease, although exceptions are reported.²² Some 20% of cases are distinguished by developmental anomalies, most commonly including polysplenia, intestinal malrotation, and abnormal abdominal vasculature.^10,19,32,52 The pathogenetic implications of these anomalies are uncertain, but these patients may constitute a distinct subgroup with bile duct obliteration originating during early fetal life.⁵

Prompt and accurate diagnosis of biliary atresia is essential because the success rate of surgical reconstruction drops dramatically after 90 days of age. Although the disorder is often strongly suspected on clinical grounds, other procedures are necessary to eliminate other causes of neonatal cholestasis and establish the diagnosis.^2,8,67 For example, a normal excretion pattern on radioisotope scintigraphy or the presence of bilirubin in aspirated duodenal fluid suggest that the biliary tract is patent, and atresia is accordingly excluded. Percutaneous or endoscopic cholangiography has also been employed to directly evaluate the biliary tract, but these are difficult techniques in the newborn and have thus seen limited use.⁷⁵ Liver biopsy may be one of the most useful diagnostic tools. The appropriate histologic features are highly suggestive of biliary atresia, and, in experienced hands, provide the correct diagnosis in 85% to 95% of cases.^17,27,53,60

None of these procedures is infallible, but a combination of abnormalities sugesting biliary atresia furnish sufficient grounds for surgical exploration, the definitive diagnostic procedure. At laparotomy, the diagnosis is often obvious from the gross appearances alone: the gallbladder is shrunken and fibrous and the liver coarse, nodular, and green. In questionable cases, an operative cholangiography can be performed. The surgeon may be misled by the small but patent bile ducts sometimes associated with severe intrahepatic cholestasis ("bile duct hypoplasia").^13,67

In 85% to 90% of patients, the proximal extrahepatic ducts are obliterated up to the capsule of the liver, and a traditional biliary-intestinal anastomosis cannot be performed. These cases were considered uncorrectable before the advent of hepatoportoenterostomy. This procedure, which was originally described by Kasai and now has numerous modifications, involves complete excision of the biliary tract and connection of the exposed hepatic hilum to the intestine through a Roux-en-Y anastamosis.^40,42,45 Bile can then drain through the tiny bile channels that persist at the porta hepatis. The other 10% to 15% of patients with "correctable" atresia have distal obliteration with patent hepatic ducts (or duct-like structures), and a conventional surgical anastomosis is feasible. The results in these cases have been disappointing, however, and portoenterostomy is often recommended for this group as well.⁴⁹

Portoenterostomy can reestablish bile flow in 45% to over 80% of patients. The rate of success depends largely on the patient's age at the time of operation, and the best results are found with infants less than 60 days old.^34,41,50,55 By restoring bile flow and relieving high-grade biliary obstruction, the procedure may slow hepatic deterioration and thereby improve longevity. Unfortunately, the intestinal anastamosis predisposes to bacterial cholangitis, and furthermore, the ongoing destruction of the intrahepatic bile ducts is not prevented; these problems lead to biliary cirrhosis and portal hypertension in many patients and ultimately compromise long-term survival. Overall, the five-year survival rate following portoenterostomy approximates 50%, and some 20% to 30% of patients attain normal, or near-normal, liver function.^{34,41,45,47,50}

Liver transplantation is increasingly employed in the treatment of biliary atresia, which now constitutes the most common indication for liver replacement in the pediatric population.^14,25 Generally transplantation has been regarded as secondary therapy, reserved for patients who have progressive end-stage disease following portoenterostomy. However, some have advocated its use as initial therapy in infants apt to suffer a poor outcome because of delayed diagnosis or advanced disease. The comparative role of portoenterostomy and transplantation is thus a controversial and much discussed issue.^69,78

Examples of hepatocellular carcinoma complicating biliary atresia have been noted, most in patients older than three years.⁷⁴

The pathologic foundation of biliary atresia is the progressive inflammatory destruction of bile ducts. The entire biliary system may be involved, but the tempo and course of destruction differ among patients. Nevertheless, the large bile ducts (especially the extrahepatic ducts) suffer the major initial injury and, by definition, are completely obliterated at some level along their course.

The extent and location of this luminal obliteration vary considerably.^40,45 Often the entire biliary tract is entirely obliterated, and the hepatic ducts at the porta hepatis either disappear within a fibrous mass or appear simply as thin fibrous cords. In less than 15% of cases (the so-called "correctable" type), the proximal ducts are patent and only the distal common or hepatic ducts are atretic. In most cases, the gallbladder is additionally affected and appears shrunken or absent. The major intrahepatic ducts remain patent for several weeks in most patients, but they also eventually undergo similar inflammatory alterations. As a result, these ducts become irregularly distorted, narrowed, and reduced to multiple small biliary radicles.^{20,23,35,39,43}

Ductal fibrosis and obliteration represent the culmination of the inflammatory process, and a range of lesser histologic alterations can be appreciated in the biliary specimens excised during hepatoportoenterostomy (Figure 6-1). The early changes include damage and necrosis of the ductal epithelium together with an inflammatory infiltrate of neutrophils, mononuclear cells, and occasional eosinophils. With continued epithelial destruction, progressive periductal fibrosis and chronic inflammation begin to dominate the scene; increased numbers of small ductular structures and biliary glands may form clusters of cuboidal-lined tubules within the fibrous tissue. The lumen is gradually narrowed, and eventually fibrous scarring totally effaces the duct, whereupon the inflammatory element then often fades. These various changes are not uniformly distributed along the length of the duct, but vary in severity and breadth; the most active disease often centers on the distal end of the common hepatic bile duct.^28,29,56,77

The hepatic histology of biliary atresia mirrors that of biliary obstruction of any cause, and the histologic progression is similar. Canalicular cholestasis is an early and prominent feature, often accompanied by hepatocyte injury, focal hepatocellular necrosis, and lobular inflammation of variable degree. Giant multinucleated hepatocytes are noted in 15% to 50% of cases; they are typically scattered and few in number, but may be confusingly prominent in some instances (Figure 6-2). Occasional foci of extramedullary hematopoiesis may also be present.

The most characteristic features are found in the portal tracts and become increasingly pronounced after about four weeks of age.^17,20,35,77 Portal fibrosis and bile ductular proliferation involving the portal tracts diffusely across the liver are the most conspicuous findings. These proliferated ductules are elongated, angulated tubules that display small well-defined lumens and sometimes contain intraluminal bile plugs; usually four or more profiles are present in any portal tract (Figure 6-3). Furthermore, portal tracts become enlarged and edematous with a mixed population of inflammatory cells. The interlobular bile ducts often exhibit epithelial degeneration and inflammation similar to that seen in the large bile ducts (Figure 6-4), exemplifying the interlobular component of the basic ductal inflammation of biliary atresia.^16,35,66 Acute cholangitis or ductal cholestasis may be seen. In occasional cases, abnormal ductal structures are found within the portal tracts (Figure 6-5). These appear as concentric tubular rings surrounding the portal vein and suggest the persistence and abnormal remodeling of the fetal ductal plate.^5,66

Progressive disease is characterized by increasing fibrosis and the development of chronic cholestasis. Fibrous wedges extend irregularly into periportal areas, eventually linking adjacent portal tracts (Figure 6-6). Chronic cholestasis is characterized by periportal hepatocytes swelling and copper accumulation, but the Mallory bodies seen in adults are scarce.⁶² Bile lakes and bile infarcts are occasionally noted, and portal veins become sclerotic and sometimes reduced in number, contributing to the development of portal hypertension.⁶⁴ Moreover, the bile ducts and ductules gradually begin to disappear. This may start as early as the fourth month, and, by morphometric studies, rapidly accelerates at about seven or eight months.^46,66 Biliary atresia is therefore one of the conditions associated with loss of intrahepatic bile ducts (Chapter 5).

Ultimately, the bridging fibrosis is joined by nodular hyperplasia of hepatocytes to yield biliary cirrhosis with few remaining bile ducts (Figure 6-7). This development may start as early as two months in some patients, but there is a wide range of progression, and generally cirrhosis does not appear until at least 12 weeks. Residual intrahepatic ducts that have escaped destruction often dilate, sometimes forming bile duct cysts that may range up to several centimeters in diameter.^26,30

Several studies have examined the histologic changes seen after hepatoportoenterostomy or similar drainage procedures. The successful restoration of bile flow is often accompanied by an initial improvement in the degree of cholestasis, hepatocyte damage, inflammatory infiltration, and fibrosis.²⁴ However, longer term follow-up biopsies generally show persistent and progressive fibrosis and chronic cholestasis. Often these changes are irregularly distributed in the liver, and sampling errors therefore become a problem with needle biopsy specimens. Loss of intrahepatic bile ducts continues, and portal vein branches shrink and show medial hypertrophy. Despite successful surgery, cirrhosis eventually develops in many patients.^12,31,36,47

Several histologic features have been examined as predictors of prognosis following portoenterostomy. The extent of fibrosis and degree of hepatocellular damage have correlated with outcome in some studies, but, in general, hepatic histology is not a strong prognostic indicator.^20,65,70,73 The size of ductal remnants at the transsected porta hepatis has also received considerable attention as a predictor of surgical success. Several investigations have shown that effective restoration of bile flow is most likely when these residual biliary structures have diameters of at least 150 µm to 200 µm, especially when only the remaining bile ducts -- rather than biliary glands and ductules -- are included in the assessment.^20,21,45,63 Nevertheless, the correlation is not exact, and other studies have reached contrary conclusions.^29,48,70 In fact, bile flow returns postoperatively in some patients despite the complete absence of ducts at the porta hepatis. The size of ductal remnants is therefore, at best, a incomplete guide to prognosis.

Although biliary atresia can often by suspected on clinical grounds, liver biopsy plays an important role in confirming the diagnosis. In general, the most helpful histologic features are prominent and diffuse bile ductular proliferation and portal fibrosis. These changes are not specific, but in the proper context, the correct diagnosis can be made in over 85% of cases, an accuracy sufficient to guide further management.^17,27,53,60 In an occasional case, ductular proliferation is not prominent, and the correct diagnosis may be missed; this is particularly a problem with infants less than four weeks of age who have yet to develop a vigorous portal reaction.

Many causes of neonatal cholestasis are potentially differential considerations, but most have been excluded by clinical evaluation before the biopsy is obtained. From the pathologist's perspective, therefore, the differential diagnosis therefore centers primarily on idiopathic neonatal hepatitis and paucity of intrahepatic bile ducts (Table 6-1). Neonatal hepatitis can typically be distinguished by its prominent parenchymal changes together with relatively minor ductular proliferation and portal tract alterations. Difficulties arise with the occasional example of neonatal hepatitis that shows more conspicuous ductular proliferation; often this involves only scattered portal tracts, rather than being uniformly distributed as in the usual biliary atresia, but exceptions occur.

Paucity of intrahepatic bile ducts is characterized by loss of bile ducts. In some early cases, however, ductular proliferation may be noted and mistaken for biliary atresia. The distinction can only be made by documenting obliteration of the extrahepatic bile ducts in biliary atresia, but this may be difficult in some instances.¹³⁶ Furthermore, loss of intrahepatic ducts is a feature held in common by both bile duct paucity and advanced biliary atresia, although the time course and progression of the disease differs.

In addition, several specific intrahepatic disorders may mimic biliary atresia. For example, some examples of alpha-1-antitrypsin deficiency exhibit conspicuous ductular proliferation and portal fibrosis.^17,174 The PAS-positive, diastase-resistant globules characteristic of this disorder are unfortunately not a consistent feature in newborns; measurement of serum levels and Pi phenotyping are necessary to establish the diagnosis. This issue is further confounded, however, by the rare cases in which both biliary atresia and alpha-1-antitrypsin deficiency are concurrently present.^61,72 Cytomegalovirus hepatitis and total parenteral nutrition may also present with a similar histologic picture.^15,51

Other than biliary atresia, few pediatric disorders affect the large bile ducts. Biliary obstruction may rarely be caused by gallstones, isolated strictures, developmental anomalies of the ducts, or heterotopic gastric tissue.^{1,79,81,82,88} Primary sclerosing cholangitis has also been described in children (Chapter 5). Virtually the only neoplasm causing obstruction in pediatric patients is embryonal rhabdomyosarcoma of the bile ducts, but intrahepatic masses may rarely be responsible.^85,87

Another unusual but distinctive disorder that occurs in early infancy is spontaneous perforation of bile ducts.^83,89 The cause is not known, but the perforation commonly arises at the junction of the cystic and common hepatic bile ducts, suggesting an intrinsic weakness of the wall at that point. Most affected infants develop jaundice, acholic stools, and abdominal distension, but in some cases the clinical picture is of an acute abdominal catastrophe with bile peritonitis and shock. Surgical therapy is generally required.

Choledochal cysts -- congenital cystic dilatations of the biliary tract -- may be discovered at any age, but most present during childhood and exceptional cases have even been identified in utero (Chapter 15).^84,86 The clinical presentation varies: classic features include jaundice, abdominal pain, an abdominal mass, and cholangitis, but the clinical picture in infants may be indistinguishable from biliary atresia. Liver biopsies show nonspecific changes of biliary obstruction, and the chief means of diagnosis is radiographic imaging, especially ultrasonography. Surgical resection is generally recommended whenever technically possible.^68,80,86

Neonatal hepatitis is a general term that designates hepatocellular cholestasis occuring in infancy. It thus represents a heterogenous group of nonobstructive disorders that share common clinical and histologic features. The label of hepatitis is somewhat misleading, since inflammation is typically a minor component, although hepatocellular injury is a dominant feature. Neonatal hepatitis accounts for more than 35% of all cases of neonatal cholestasis and has been estimated to occur with an incidence between one in 4,800 and one in 9,000 live births.^3,97,99 The importance of the diagnosis rests largely in its differentiation from biliary atresia.

The cause of most cases of neonatal hepatitis is not known, and its etiologic relationship with biliary atresia remains a controversial topic. However, specific etiologic entities have occasionally been associated with the picture of neonatal hepatitis, and additional investigation will presumably define other responsible conditions, further shrinking the idiopathic group. The known causes include a variety of infectious agents: cytomegalovirus, rubella, herpes simplex, toxoplasma, varicella, Coxsackie virus, echovirus, human immunodeficiency virus, and trepomena.^{38,91,92,102,111,115} Hepatitis viruses were originally considered to be likely candidates, but subsequent serologic testing shows that they play only a slight role with unusual examples of hepatitis B or non-A, non-B viruses (presumably hepatitis C virus) being recognized. Bacterial sepsis, particularly with Gram-negative organisms, may also yield this pattern.

In addition, several metabolic disorders may give rise to neonatal hepatitis. The best recognized is alpha-1-antitrypsin deficiency, but some instances of Niemann-Pick type C disease and neonatal hypopituitarism are also implicated.^100,101,109 In exceptional familial cases of neonatal hepatitis, inborn errors in bile acid synthesis have been identified.^93,110

The unadorned term neonatal hepatitis is ambiguous. Sometimes it is used to designate any nonobstructive neonatal cholestasis, even if one of the recognized causes is identified, whereas at other times, it is restricted to the idiopathic group alone. In a pathologic sense, neonatal hepatitis is best considered a characteristic but nonspecific reaction of the newborn liver to a variety of insults; usually an etiology is not defined, but occasionally an infectious or metabolic derangement may be identified clinically or histologically.

Clinical Features^27,60,90

Infants with neonatal hepatitis tend to be males who are born prematurely and have low birth weights. Most cases are sporadic, but about 14% of patients have affected siblings.⁹⁵ The typical presentation includes jaundice that begins within several weeks of birth, hepatomegaly, splenomegaly, and, in severe cases, acholic stools. A conjugated hyperbilirubinemia is found, and serum transaminase and alkaline phosphatase levels are commonly elevated. Tests for specific causes of neonatal hepatitis should be performed, including, for example, alpha-1-antitrypsin levels (and perhaps phenotyping) and TORCH (toxoplasmosis, rubella, cytomegalovirus, and herpes simplex virus) titers. Unfortunately the differential diagnosis is often reduced to idiopathic neonatal hepatitis and biliary atresia, and these clinical and laboratory features do not specifically distinguish the two; radionuclide scintigraphy and liver biopsy are often indicated.

As there is no specific therapy for neonatal hepatitis, supportive medical management and nutritional support are employed. Given the heterogeneity of the disorder, data about outcome are varied, and the overall prognosis is correspondingly difficult to assess. Recovery is the general rule in the sporadic cases: Jaundice slowly resolves over weeks to months in 65% to over 90% of infants, although biochemical abnormalities may persist longer.^92,96,99,103 However, patients with particularly severe disease, who constitute 5% to 25% of cases, may die from liver failure or sepsis during the first year; chronic liver disease including cirrhosis develops in the remaining 5% to 10%. Familial cases have a worse prognosis with liver failure and death within the first year in about 60% and recovery in approximately 25%; this may reflect an underlying inborn error in metabolism, as recently described in a few instances.

Neonatal hepatitis is histologically defined by diffuse hepatocellular injury and cholestasis that is distinguished by the presence of giant multinucleated hepatocytes.^94,107 The liver cell plates appear distorted and disorganized by the hepatocellular changes, but the overall lobular architecture remains intact (Figure 6-8). The hepatocytes become enlarged and swollen and characteristically undergo giant cell transformation (Figure 6-9); focal necroses and acidophilic bodies are scattered across the lobule. This hepatocyte injury can lead to irregular collapse of the parenchymal framework and give rise to focal intralobular fibrosis. (In rare instances, massive hepatic necrosis may result.) Canalicular cholestasis is generally prominent and may be associated with the formation of cholestatic liver cell rosettes. Lobular inflammation is typically minor in degree and usually mononuclear in nature with scattered hypertrophied Kupffer cells. Neutrophils may conspicuously accumulate around necrotic giant liver cells (Figure 6-10). Foci of extramedullary hematopoiesis are common, and iron deposition can often be appreciated with appropriate stains.

The portal tracts typically demonstrate a modest reaction: a lymphocytic inflammatory infiltrate accumulates, but is generally mild in degree. Ductular proliferation can be seen in some cases, although it is usually patchy and not as dominant a finding as in biliary atresia (Figure 6-11). Ductular cholestasis with bile plugs within ducts is sometimes noted.

The most notorious feature of neonatal hepatitis is the giant multinucleated hepatocyte. These massive cells of over 200 µm in diameter bear at least three or four nuclei, but may contain ten times that number (Figure 6-12). Their granular cytoplasm is frequently decorated with brown pigment that may represent bilirubin, hemosiderin, or lipofuscin. Giant hepatocytes are found throughout the lobule, but favor the centrilobular zone.

The pathogenesis of these cells is debated; they may derive either by fusion of hepatocytes in cholestatic rosettes or by amitotic division.^102,108 Whatever their origin, they represent a nonspecific reaction of the infantile hepatocyte and can be found in almost all neonatal liver diseases. Thus, by themselves, they have little diagnostic significance, although they are particularly prominent in neonatal hepatitis. Similar cells are occasionally noted in adult liver diseases such as various forms of acute and chronic hepatitis and rare drug reactions.^{104,106,112-114} Most recently, they have been described in cases of severe sporadic hepatitis attributed to paramyxovirus.¹⁰⁵

With recovery, the cholestasis resolves and the giant cells gradually vanish. In progressive cases, intralobular and portal fibrosis slowly increase, and changes of chronic cholestasis appear; cirrhosis subsequently develops.^97,103 In some instances, paucity of interlobular bile ducts may evolve.⁹⁸

Prognostic factors have been difficult to define in neonatal hepatitis, in part because of the heterogeneity of the syndrome. Clinical indicators suggesting a poorer outcome include a positive family history, marked or prolonged jaundice, and peak serum bilirubin levels over 15 mg/dl.^92,96,97,103 No histologic features are clearly established as prognostic markers, although patients with more severe portal and periportal fibrosis, conspicuous bile ductular proliferation, or diffuse giant cell transformation have been associated with a poor outcome in some studies.^92,103

The histologic pattern of neonatal hepatitis is nonspecific, and appropriate studies should be done to exclude known causes of the pattern. Generally the clinician's diagnostic workup will have already eliminated the major differential considerations, and, for the pathologist, the usual problem is distinguishing neonatal hepatitis from biliary atresia and paucity of intrahepatic bile ducts (Table 6-1). Biliary atresia generally exhibits less hepatocyte damage, giant cell transformation, and inflammation, but the most useful discriminating features are the prominent portal tract abnormalities -- uniform bile ductular proliferation and portal fibrosis -- found in biliary atresia, but typically missing from neonatal hepatitis. On the basis of these criteria, the two conditions can be distinguished with an accuracy of 85% to 95%.^17,27,53,60 However, there is some histologic overlap, and the histologic examination may not be definitive. For example, some instances of biliary atresia, often those biopsied early in the course, lack characteristic portal changes and cannot be distinguished from neonatal hepatitis.

Another, but less common, consideration is paucity of interlobular bile ducts. This disorder is characterized by a decrease in bile duct numbers, but that may be a difficult finding to establish, especially in a small specimen. In some cases of neonatal hepatitis, bile ducts are small and partially obscured by the portal inflammation. On the other hand, interlobular bile ducts may be present early in the course of bile duct paucity; the histologic features are then indistinguishable from neonatal hepatitis and the correct diagnosis becomes clear only with follow-up.^122,129

After a diagnosis of neonatal hepatitis is contemplated, the known causes should be considered. These are typically diagnosed by clinical evaluation, as the histologic features are generally not discriminatory. As a general rule, cases with a recognized cause tend to have less conspicuous giant cell transformation than does the idiopathic subgroup, but this is an not absolute finding. In addition, a few disorders exhibit distinctive histologic features.

Cytomegalovirus infection is manifest by characteristic nuclear and cytoplasmic viral inclusions, which are primarily found in biliary epithelial cells. In herpes simplex infections, irregular zones of coagulative-type necrosis are found, and distinctive viral inclusions are noted within the viable hepatocytes at the margins of the necrosis. Immunohistochemistry can help to specifically identify the viral antigens in these two infections. Congenital syphilis is characterized by widespread lobular fibrosis and prominent plasmacytic inflammation; numerous spirochetes can be recognized with special stains. Alpha-1-antitrypsin deficiency characteristically displays PAS-positive globules in periportal hepatocytes, as discussed below. Finally, several metabolic causes of a neonatal hepatitis pattern, including galactosemia and tyrosinemia, demonstrate additional histologic features such as fatty change and prominent cholestatic rosettes; these are detailed later.

This term gathers together a heterogenous collection of disorders characterized by a reduction in the number of small intrahepatic bile ducts. It is a an uncommon cause of neonatal or childhood cholestasis and represents less than 5% of all such cases.³ Several other names have been employed as synonyms, including intrahepatic biliary atresia and hepatic ductular hypoplasia, but the phrase "paucity of interlobular bile ducts", although cumbersome, has the virtue of being purely descriptive and pathogenetically neutral.

The most common and best defined condition within this group is usually known as Alagille's syndrome or arteriohepatic dysplasia. In addition to the hepatic change, particular cardiac, facial, ocular, and skeletal anomalies are also variably present, as detailed below.^118,148 This syndromic form of bile duct paucity occurs with an incidence estimated at one in 70,000 live births.⁹⁵ It is a familial disorder; pedigree studies suggest an autosomal dominant inheritance pattern with low penetrance and variable expressivity.^{135,137,138,145} A deletion in the short arm of chromosome 20 has been discovered in several patients, and the syndrome has been tentatively assigned to that site.^121,151 Often, however, there is no family history, possibly because of spontaneous mutations or because, given the clinical variability, minimally affected relatives are not recognized.

Paucity of intrahepatic ducts can also be occur without extrahepatic manifestations. This nonsyndrome group is a diverse assortment; many cases lack any associations, but some are affiliated with other conditions. These include alpha-1-antitrypsin deficiency, cytomegalovirus infection, congenital rubella, and several chromosomal alterations such as trisomy 17-18, Turner's syndrome, and Down's syndrome.^125,131,149 Rare familial cases have demonstrated an abnormality in bile acid synthesis with elevated levels of trihydroxycoprostantic acid; these cases may represent mild variants of Zellweger's syndrome, which shows similar metabolic alterations.^124,128,144

The pathogenesis of the decrease in bile ducts is unknown. Originally it was thought to result from a failure of duct development, but normal or damaged intrahepatic bile ducts are found to be present early in infancy, only to disappear in subsequent biopsies. The process therefore appears to be one of progressive duct destruction.^122,130,131 The cause of the destruction remains obscure. One possibility is raised by electron microscopic studies of Alagille's syndrome that suggest a primary defect in bile secretion by the hepatocyte; one consequence of this would be diminished bile flow, which presumably could produce disuse atrophy and loss of the intrahepatic ducts.¹⁴⁷ This proposal has been challenged, however, because of observations indicating that bile flow does occur, at least in some patients.¹⁵⁰

Clinical Features^{1,117,119,123,142}

The hepatic manifestations are similar in both syndromic and nonsyndromic types. Most patients develop clinical evidence of cholestasis during the first three months of life, but there is a wide spectrum of presentations and some patients do not manifest until months or years later. A few cases have even been recognized in adulthood, frequently following the diagnosis in an affected relative. Jaundice is the usual initial symptom and may be either intermittent or persistent. Pruritus and the manifestations of chronic cholestasis, including malabsorption and growth retardation, evolve later.

The extrahepatic features are often the clinical clues to the syndromic form of the condition.^137,142 Included are a broad range of congenital abnormalities of the heart, facies, eyes, and skeleton that occur with variable frequency even with a single family. Cardiovascular abnormalities may range from the common -- but inconsequential -- peripheral pulmonary artery stenosis to severe anomalies such as tetralogy of Fallot. Characteristic facies, described in over 70% of patients, comprise a prominent forehead, wide-spread eyes, flattened nose, and a small pointed chin. This appearance often become more obvious with advancing age, but it may simply reflect the presence of early persistent cholestasis.^143,146 Other major features include posterior embryotoxon (an abnormality of the formation of the anterior chamber of the eye) and vertebral anomalies prominently including nonfused anterior arches that yield a butterfly appearance on radiographs. Renal anomalies, neurologic abnormalities, and growth retardation are also seen.

Treatment of bile duct paucity is primarily symptomatic and directly towards the management of chronic cholestasis. Liver transplantation has also been employed in patients with progressive liver failure. The prognosis is extremely variable, as befits the heterogeneous nature of this group of disorders, although conclusion are limited by the lack of outcome data. Many patients with syndromic disease have persistent, but apparently non-progressive, hepatic disease. Their cholestasis tends to wane in severity, but they continue to suffer the consequences of chronic cholestasis.^{98,117,120,123,137} Approximately 25% of patient develop periportal fibrosis on follow-up liver biopsies, and cirrhosis is noted in an estimated 14%. Accordingly, some deaths are attributed to portal hypertension or hepatic failure.^140,145 However, much of the early mortality is caused by the concomitant cardiovascular abnormalities. The prognosis of the various nonsyndromic forms is less well defined and probably depends on the particular underlying association. Overall, cirrhosis develops in a reported 40% to 55% of patients, although many patients have a benign course with prolonged survival and even spontaneous resolution.^90,140

Hepatocellular carcinoma is a rare complication, noted in both children and adults. This has been associated with cirrhosis in some, but not all, cases.^116,133,141

Pathologic Features^{119,122,123,130,131,147,149}

The histologic hallmark of this condition, as the name indicates, is an absence or marked decrease in interlobular bile ducts (Figure 6-13). Bile ducts and hepatic arteries are normally paired within the portal tract; a reduction in bile duct numbers can be recognized by finding arteries that are unescorted by ducts of similar size. The portal tracts, sometimes small and relatively inapparent, show variable fibrosis, and inflammation is usually mild or minimal. Loose aggregates of lymphocytes may collect at the site vacated by the ducts.

Although bile duct paucity can usually be identified by careful, directed observation, its presence is decisively established by an actual count of bile duct numbers. The normal ratio of bile ducts to portal tracts is between 0.9 and 1.8; a ratio of less than 0.4 clearly confirms a reduction in bile ducts.^127,149 These figures only apply to infants of more than 38 weeks gestation, since premature infants may normally have lower ratios.¹³² Bile duct numbers can also be assessed by the percentage of portal tracts without a bile duct: normally this value is about 10% to 15%, and when 30% to 100% of tracts lack ducts, bile duct paucity is suggested.¹²⁹ Furthermore, morphometric and radiographic studies demonstrate that the portal tracts are reduced in number and size.^126,127,129

Associated lobular changes include canalicular cholestasis, hepatocyte swelling with occasional multinucleated giant hepatocytes, and focal deposits of extramedullary hematopoiesis (Figure 6-14). Also commonly noted is chronic cholestasis, which is characterized by swollen, rarefied periportal liver cells with copper accumulation and sparse bile pigment. Hepatocyte necrosis and lobular inflammation are variably present, but typically mild in degree, and intralobular fibrosis is sometimes seen.

Although a reduction in bile ducts is the earmark of this disorder, it represents the end-result of a progressive process of duct destruction.^122,123,130 In early biopsy specimens, therefore, the numbers of interlobular bile ducts may be normal, and a diagnosis of bile duct paucity accordingly impossible to establish. Bile ductular proliferation also develops in some instances, adding to the diagnostic difficulty (Figure 6-15).^6,123,139 Evidence of ductal damage is occasionally noted: The epithelium is irregular and degenerated with cytoplasmic vacuolization, reactive nuclear changes, and inflammatory infiltration. A concentric layer of plump mesenchymal cells may encircle injured ducts, which are eventually obliterated and replaced by loose connective tissue. The time course of this destructive process varies: in some patients, ducts have vanished by four or five weeks of age, whereas in others, duct paucity is found only after three months. Some studies suggest that an earlier development of paucity is more characteristic of nonsyndromic than syndromic disease.¹³¹

Late histologic changes include severe cholestasis and an obvious loss of bile ducts together with portal and periportal fibrosis of varying degree (Figure 6-16). This fibrosis is irregularly distributed but is often most marked near the hepatic hilum.^129,130 Fibrous septa may develop, sometimes with accompanying ductular proliferation, and cirrhosis evolves in occasional patients (Figure 6-17). No histologic indicators of progression are recognized.

Paucity of intrahepatic bile ducts is primarily a pathologic diagnosis, although the extrahepatic features of the syndromic form -- while not definitive -- can be suggestive.¹³⁴ Unfortunately, the histologic findings are not always conclusive, and rendering a correct diagnosis may pose considerable difficulty. Every cholestatic biopsy warrants careful scrutiny of the bile ducts. One obvious necessity is a liver biopsy with an adequate sampling of portal tracts; the recommended minimum number has ranged from six to 20 portal tracts.¹⁴⁹ A surgical wedge biopsy is therefore helpful, although needle biopsy specimens can suffice if the histology is distinctive and the clinical setting appropriate.

Neonatal hepatitis demonstrates the same lobular changes as bile duct paucity, although usually with greater intensity, but the distinction can be made by the normal number of interlobular ducts (Table 6-1). However, bile duct loss may not be evident in some cases of bile duct paucity, and the two conditions cannot always be distinguished. In addition, the interlobular ducts in neonatal hepatitis can be inconspicuous or obscured by inflammation and may mistakenly be considered absent. Fortunately, this distinction does not have any particular therapeutic implications.

Biliary atresia can present a differential diagnostic problem for several reasons. Histologically, it is distinguished by its bile ductular proliferation, although destruction and loss of interlobular ducts develops in the later stages (Table 6-1). The occasional case of bile duct paucity that demonstrates ductular proliferation is therefore apt to be misdiagnosed; typically, however, the degree of portal edema, inflammation, and fibrosis seen in biliary atresia is absent. An additional difficulty is that the extrahepatic bile ducts in bile duct paucity, although patent, are often narrowed and hypoplastic; biliary atresia may be erroneously suggested, even with operative cholangiography, and inappropriate surgery can result.¹³⁶

Alpha-1-antitrypsin deficiency is one of the few known causes of bile duct paucity with specific histologic features, namely PAS-positive intracytoplasmic globules in periportal hepatocytes, as discussed below. Other causes of bile duct disappearance in children are similar to those of adults and include primary sclerosing cholangitis, graft-versus-host disease, and hepatic allograft rejection (Chapter 5).

PROGRESSIVE FAMILIAL CHOLESTASIS OF CHILDHOOD^{142,154,157,160,162}

Several unusual cholestatic disorders have been reported that, despite minor clinical differences, share certain important attributes: a familial occurrence, an onset early in life, and a chronic, progressive course leading to fibrosis and cirrhosis. Although they are poorly understood and likely have disparate etiologies, these conditions can be considered together because of their broadly similar natural history. A better understanding of their cause and pathogenesis will permit a more rational classification.

Examples have been reported from throughout the world, but the early descriptions were from an Amish kindred descended from Jacob Byler, and the name Byler's syndrome is often applied to this group.^156,157 Since siblings are affected in many instances and a history of consanguinity is sometimes noted, an autosomal recessive mode of inheritance is likely. The implication is that certain enzyme mutations may be responsible, but none have been specifically identified. However, abnormalities in bile acid metabolism have been recognized in some cases, while dysfunction of actin microfilaments has been suggested in others.^{93,110,159,165}

Patients present with jaundice, pruritus, and pale stools within the first year of life, typically after the neonatal period. Hepatomegaly is commonly present, and pertinent laboratory findings include elevated bilirubin and alkaline phosphatase levels whereas the serum gamma-glutamyl transpeptidase is curiously normal.¹⁵⁵ The subsequent course is variable with recurrent or persistent cholestasis that results in malabsorption, vitamin deficiencies, failure to thrive, and growth retardation. Most patients have no extrahepatic manifestations other than those secondary to chronic cholestasis, but some cases have been associated with lymphedema of the lower extremities (so-called Norwegian cholestasis).^152,153 End-stage liver disease with ascites, encephalopathy, and hepatic failure eventually develops and leads to death, usually by 15 years of age. Treatment is limited to supportive care, although biliary diversion may provide symptomatic palliation, and liver transplantation has been employed for advanced cases.¹⁶⁶ Hepatocellular carcinoma has complicated some instances.^158,164

The histologic features are not specific. Early changes include canalicular cholestasis, sometimes with cholestatic rosettes, and multinucleated giant hepatocytes (Figure 6-18). The portal tracts are typically enlarged and inflamed; proliferated bile ducts are common, and, rarely, bile duct paucity is described. Periportal and intralobular fibrosis progressively develop in later stages, and chronic cholestasis with swollen periportal hepatocytes containing accumulated copper and Mallory bodies becomes prominent. Increasing fibrosis eventually results in cirrhosis, usually in childhood or early adolescence.^161,163

A few other causes of pediatric cholestasis have distinctive histologic findings.

Alpha-1-antitrypsin deficiency is associated with a broad range of liver disease, both in children and adults (Chapter 9). It is responsible for 7% to 10% of cases of neonatal cholestasis, and, conversely, between 10% and 20% of infants with a deficient (PiZZ) phenotype develop clinical evidence of liver disease, although many others have minimal disease only characterized by laboratory abnormalities.^178,181 The outcome is variable: some patients develop early cirrhosis and die of liver failure, a few may progress to chronic disease later in life, and many appear to recover, demonstrating only minimal evidence of hepatic disease.^167,173,176

The histologic hallmark of alpha-1-antitrypsin deficiency is the presence of eosinophilic globules in periportal hepatocytes (Figure 6-19). These PAS-positive and diastase-resistent globules represent dilated sacs of endoplasmic reticulum stuffed with alpha-1-antitrypsin. They may be small and difficult to see in neonatal livers, but are consistently present after about three months and have even been noted in a 20 week fetus.^{171,174,179,182} Alpha-1-antitrypsin globules can be confused with other PAS-positive material, but immunohistochemical techniques can be used to specifically confirm their presence.¹⁶⁹ Measuring serum levels and determining the phenotype can definitively establish the diagnosis.

Three other metabolic disorders -- galactosemia, hereditary tyrosinemia type I, and hereditary fructose intolerance -- are all characterized by a broadly similar histology (Chapter 9). Although not specific, these changes are suggestive and should prompt appropriate biochemical analysis. In addition to canalicular cholestasis, early changes include prominent fatty change and acinar transformation of liver-cell plates, often in the form of cholestatic rosettes (Figure 6-20). Giant multinucleated hepatocytes are variably present, and portal fibrosis and bile ductular proliferation also noted. With advancing disease, intralobular fibrosis and fibrous septa progressively develop and, without appropriate treatment, cirrhosis may result, particularly in tyrosinemia and galactosemia.^{168,175,177,180}

Liver dysfunction associated with total parenteral nutrition occurs most frequently in premature infants. The pathologic picture is variable and nonspecific, but may include canalicular cholestasis, fatty change, giant multinucleated hepatocytes, and portal inflammation and fibrosis, which may progress to cirrhosis.^15,170,172 The overlap with neonatal hepatitis and biliary atresia may lead to differential diagnostic problems. Benign recurrent intrahepatic cholestasis is characterized by nonprogressive, repetitive attacks of acute cholestasis and may have an onset in childhood (Chapter 4). The histologic pattern is that of pure cholestasis -- canalicular cholestasis unaccompanied by other morphologic changes.