CHAPTER 13 Childhood Liver Disease and Metabolic Disorders
Introduction
Paediatric liver biopsies present a unique set of diagnostic problems for the pathologist, many of which become clinically apparent in the first few months of life as neonatal cholestasis.1 Among the important disorders one must consider in evaluating neonatal liver biopsies are extrahepatic biliary atresia, paucity of intrahepatic bile ducts (syndromatic and non-syndromatic types), metabolic diseases, viral hepatitis and the hepatic effects of parenteral nutrition (Table 13.1). Common to many of these conditions are the histological features of cholestasis and giant-cell hepatitis (formation of multinucleated hepatocytes). Because these features are not specifically diagnostic of any one neonatal liver disease, the pathologist must be acquainted with other biopsy changes by which to establish or suggest the diagnosis. In many instances, assays of metabolic enzymes and products in serum and liver tissue take diagnostic precedence over routine histopathological interpretation. Electron microscopy may be required to assess the structure of organelles or storage material in hepatocytes or Kupffer cells, particularly when lysosomal storage disorders are being considered. Consultation with investigators dealing with mitochondriopathies,2,3 mutations of bile-salt transport proteins4 and expression of proteins involved in blood vessel and bile-duct morphogenesis (e.g. Jagged proteins and Notch receptors) should be considered if special studies are needed to determine the cause of neonatal cholestasis. Childhood liver tumours are discussed in Chapter 11.
Table 13.1 Liver biopsy interpretation in neonatal cholestasis*
| Aetiology | Histological features |
|---|---|
| Extrahepatic biliary atresia | Ductular reaction; ductular cholestasis; portal and periportal fibrosis |
| Paucity of intrahepatic bile ducts | Loss of interlobular bile ducts (bile duct–hepatic artery ratio <1) |
| Neonatal hepatitis | Portal and lobular mononuclear cell inflammation; apoptotic bodies |
| Metabolic disorders | Steatosis; fibrosis or cirrhosis; storage product in liver cells or Kupffer cells (see specific disorder) |
| Parenteral nutrition | Ductular reaction; portal fibrosis or cirrhosis |
* Many of the conditions shown in Table 13.1 are associated with histological cholestasis and formation of giant multinucleated hepatocytes, in addition to the diagnostic features listed.
Diagnostic approach to neonatal liver biopsy
1. Is the acinar structure normal for age? As described in Chapter 3, the hepatic plates are two cells thick until 5 or 6 years of age and should not be misconstrued as a pathological change. As with adult biopsies, the presence of fibrosis, nodularity or cirrhosis should be noted early in the biopsy evaluation and correlated with other histological features which may define the aetiology.
2. Are cholestasis and giant cells present? As indicated above, neither of these is diagnostically specific. If present, the next interpretive steps should be examination of portal tracts for evidence of biliary tract obstruction (atresia, etc.) and of portal tracts and parenchyma for evidence of hepatitis.
3. Are histological changes of hepatitis present? Mononuclear cell infiltrates within acini and portal tracts associated with liver-cell degeneration should be sought when considering cytomegalovirus, Epstein–Barr virus, rubella or hepatitis virus infections.
4. Are the interlobular bile ducts normal? This question has three major ramifications. Abundance of bile ducts usually signifies some form of biliary obstruction, such as extrahepatic atresia or choledochal cyst. Paucity of bile ducts (ductopenia, vanishing bile-duct syndrome) may be due to developmental, metabolic, or infectious causes. Last, malformations of bile ducts comprise a spectrum of problems related to abnormal remodelling of the embryonic bile-duct plate (fibropolycystic diseases).
5. Does the biopsy specimen contain iron or copper? Although rare, neonatal haemochromatosis5 and Indian childhood cirrhosis (copper toxicosis in young children) are serious liver diseases with high mortality rates that must be excluded. In the older child and adolescent, Wilson’s disease (Ch. 14) must not be overlooked. It should be noted, however, that fetal and neonatal liver contains much higher copper levels than adults, with an irregular tissue distribution.6 Mild siderosis is also within the spectrum of normal findings in the fetal and neonatal liver.7
6. Has the biopsy specimen been studied by diastase–periodic acid–Schiff (PAS) or immunoperoxidase staining to exclude α1-antitrypsin deficiency? The expression of α1-antitrypsin deficiency is variable, and biopsies may not show diagnostic staining of retained enzyme within liver cells prior to 13–15 weeks of age. This condition should be histologically excluded whenever possible.
7. Are storage cells present? Abnormal storage products in liver cells or Kupffer cells may be seen in various metabolic diseases which cause hepatomegaly and failure to thrive. These should be sought on routine haematoxylin and eosin (H&E) as well as special stains.
Neonatal hepatitis
Inflammation and hepatocellular damage in the neonatal period may result from infections and from inborn errors of metabolism. The former include type B hepatitis, cytomegalovirus infection and rubella among others, the latter α1-antitrypsin deficiency, galactosaemia and bile acid synthetic defects.8 Disorders at the ultrastructural or molecular level may also need to be considered, such as neonatal hepatitis due to depletion of mitochondrial DNA.9 A diagnosis of neonatal hepatitis is therefore a signal for further investigation. The histological picture is broadly similar whatever the cause. There is a variable degree of hepatocellular swelling and multinucleation, cholestasis and portal inflammation. Lobular inflammation may be mild. Liver-cell necrosis and swelling result in collapse and distortion of the reticulin framework (Fig. 13.1). Fibrosis is sometimes already well developed, as in neonatal haemochromatosis (see Ch. 14) or the severe perinatal liver disease which may rarely be seen in Down’s syndrome.10 Giant multinucleated hepatocytes are commonly seen, whatever the cause of the hepatitis (Fig. 13.2). The outcome of neonatal giant-cell hepatitis is resolution, liver failure, cirrhosis or a chronic cholestatic course. The variety of different outcomes is well illustrated in α1-antitrypsin deficiency.11
Extrahepatic biliary atresia
Extracellular biliary atresia results from inflammation and destruction of all or part of the extrahepatic bile-duct system in utero or in the perinatal period.12 Pathological studies of atretic bile-duct segments13–15 show chronic inflammation and obliterative fibrosis, sometimes with a few remaining bile-duct cells16 seen on routine stains or cytokeratin immunostaining. Satisfactory bile drainage and an improved outcome after the Kasai portoenterostomy17 have sometimes been associated with identification of bile ducts with lumens of 150 µm or greater at the proximal resection margin.14 Optimal surgical results are obtained if the Kasai procedure (hepatic portoenterostomy) is performed within the first 8 weeks of life.18 Many patients, nevertheless, later require liver transplantation.19
The trigger for the destructive process in extrahepatic atresia is unknown, but considerations have included viral infections (reovirus type 3, rotavirus), exposure to toxins, abnormal remodelling of the embryonic bile-duct plate and disorders of Jagged protein/Notch receptor signalling.12 DNA microarray studies suggest a gene profile of abnormal cell signalling and transcription regulation.20 There may be associated congenital abnormalities, including polysplenia21 and intrahepatic biliary cysts,22 in approximately 20% of cases with more severe and earlier disease (the ‘embryonic’ form of biliary atresia). In contrast, the majority of patients have the ‘perinatal’ form without such anomalies. Expression of various regulatory genes appears to differentiate the two types.23 The process of biliary atresia is a dynamic one which may also involve the intrahepatic bile ducts,24,25 even after Kasai surgery.26
Liver biopsy shows cholestasis and portal tract changes resembling those of large bile-duct obstruction in the adult (see Ch. 5). Portal tracts are enlarged by oedema, a striking ductular reaction and infiltrating neutrophils with fewer chronic inflammatory cells (Fig. 13.3). Native interlobular bile ducts are usually intact and can be identified near hepatic arterioles. Bile-containing portal macrophages are often also present. Ductular structures may contain inspissated bile (’ductular cholestasis’) and occasionally resemble the embryonic bile-duct plate described by Jörgensen27 (Figs 13.4, 13.5). A prominent ductular reaction is the major histological point of distinction from neonatal hepatitis.28 There is panlobular cholestasis with accentuation in zone 3. Giant cells are common, but not as numerous or as striking as in neonatal hepatitis. The degree of portal fibrosis29 depends on the duration of the obstruction and the age at diagnosis. The overall lobular architecture remains intact except in patients diagnosed late in the disease who may then show secondary biliary cirrhosis (see Fig. 5.11). The differential diagnosis includes biliary obstruction by a choledochal cyst, chronic injury associated with parenteral nutrition and various inborn errors of metabolism.
Paucity of intrahepatic bile ducts in childhood
Two varieties of intrahepatic bile duct paucity (formerly called intrahepatic biliary atresia) are recognised in childhood: syndromatic and non-syndromatic.30 In syndromatic paucity31,32 (Alagille’s syndrome, arteriohepatic dysplasia), loss of small intrahepatic bile ducts is associated with abnormal facies, vertebral anomalies and various other malformations. The pathogenesis is linked to mutations in the Jagged 1 gene that produce a structurally abnormal ligand for binding to the Notch 1 receptor which is involved in cell–cell interactions in differentiation and the development of intrahepatic bile ducts.33 There is associated impairment of branching and elongation of hilar ducts distally into the liver periphery.34 Increased mortality in these patients is linked to the presence of intracardiac congenital heart disease.35 In non-syndromatic paucity, duct loss is not associated with facial or other anomalies. In some patients it may be related to a definable cause such as α1-antitrypsin deficiency or cytomegalovirus infection,36 while in others there is no detectable aetiological factor. The exact time of onset of bile-duct injury is difficult to establish accurately and probably varies from case to case. Some patients have active destruction of ducts in the first few weeks of life32 and later stabilise, potentially with few symptoms or only mild chronic cholestasis, into young adulthood. In others, cirrhosis and liver failure may develop within months or many years later.37 It has been speculated that there may be a small subgroup of patients with non-syndromatic paucity in which cholestatic disease first presents in adulthood37 (‘idiopathic adulthood ductopenia’).38
Histologically, in both forms of intrahepatic duct paucity there is canalicular cholestasis and chronic periportal cholestasis. Portal tracts show a variable degree of fibrosis and small bile ducts are scanty or absent39 (Fig. 13.6). Step sections and cytokeratin 7 or 19 immunostaining may be needed for thorough assessment of duct numbers which, as in primary biliary cirrhosis, should approximately correspond to the number of arteries of similar size. A ductular reaction is usually not a prominent feature, in contrast to extrahepatic biliary atresia40 (see Fig. 1.8). Inflammation is often slight or even absent, but lymphoid aggregates may be seen in the place of bile ducts (Fig. 13.7). Secondary biliary cirrhosis develops in some patients.37,41 α1-Antitrypsin deficiency should be looked for in all patients with paucity of ducts. Duct paucity has also been described in association with Langerhans’ cell histiocytosis.42,43 As primary sclerosing cholangitis can also present in childhood,44 it should be considered in the differential diagnosis.
Fibropolycystic diseases
The term fibropolycystic diseases covers a number of congenital abnormalities involving bile ducts, many of them related to an abnormal remodelling of the embryonic ‘bile-duct plate’.45–48 They include congenital hepatic fibrosis, Caroli’s disease (congenital dilatation of the intrahepatic bile ducts), microhamartoma (von Meyenburg complex), choledochal cyst, and both infantile and adult forms of polycystic disease. The first four of these carry an increased risk of carcinoma of the biliary tree.49–52 The bile-duct plate, first seen at approximately 8 weeks of gestation, is a layer of primitive small cells encircling the portal tract mesenchyme (see Figs 13.4, 13.5). Progressive involution of most of these cells, with acquisition of strong cytokeratin 7 and 19 positivity in those remaining, is the process by which mature interlobular bile ducts of the portal tracts are formed.46 Persistence of portions of the ductal plate and abnormal remodelling (the ‘ductal plate malformation’ described by Jörgensen27) lead to ectatic and irregularly shaped bile ducts set in dense fibrous stroma, the basic histopathological feature common to all fibropolycystic diseases.
Congenital hepatic fibrosis
Congenital hepatic fibrosis is a recessively inherited condition, which presents as hepatomegaly or the effects of portal hypertension, usually in childhood but occasionally in adults.53 Some cases have been associated with phosphomannose isomerase deficiency in which the resultant hypoglycosylation may affect remodelling of the bile-duct plate.54 The liver is enlarged and very hard. Islands of normal liver parenchyma with unaltered vascular relationships are separated by broad and narrow septa of dense, mature fibrous tissue containing elongated or cystic spaces lined by regular biliary epithelium (Fig. 13.8). These represent cross-sections of the hollow structures constituting the ductal plate malformation. Two separate sets of duct-like structures can often be identified, one lying centrally in the septa, the other near the parenchyma. The lumens may contain inspissated bile. Portal-vein branches are small and inconspicuous in some cases. There is usually no cholestasis, necrosis, inflammation or hepatocellular regeneration. In older patients with congenital hepatic fibrosis, the abnormal duct-like structures may be less apparent because of atrophy.
Congenital hepatic fibrosis must be differentiated from cirrhosis, in which there is nodular regeneration and often inflammation and necrosis, and in which the abnormal biliary channels are not seen. The shape of the parenchymal islands in congenital hepatic fibrosis is very similar to that seen in secondary biliary cirrhosis (see Fig. 5.11). In this condition the septa contain irregular, newly proliferated bile ducts rather than congenitally abnormal plates; the connective tissue of the septa is loose and inflamed and there may be cholestatic features. Histological cholangitis, other types of inflammation or cholestasis in a liver with the characteristic features of congenital hepatic fibrosis should raise the possibility of coexisting Caroli’s disease. The combination constitutes Caroli’s syndrome.
Caroli’s disease (congenital dilatation of the intrahepatic bile ducts)
This cystic malformation can affect different parts of the intrahepatic biliary tree and is seen alone or in combination with other congenital abnormalities, notably congenital hepatic fibrosis.55 Because the cysts communicate with the rest of the biliary tree, there is a risk of ascending bacterial infection. Liver biopsy then shows the changes of cholangitis, with or without associated congenital hepatic fibrosis. The lesion of Caroli’s disease must be distinguished from the acquired cholangiectases sometimes found in primary sclerosing cholangitis.56
Microhamartoma
Microhamartomas (von Meyenburg complexes, bile-duct malformations) are rounded nodules closely related to portal tracts, containing multiple biliary channels lined by regular epithelium and set in a stroma of dense fibrous tissue (Fig. 13.9). They may be grossly visible on the liver surface as white nodules 1–2 mm across. The lumens of the biliary structures sometimes contain inspissated bile. Serial sectioning shows that they are interconnected.57 Microhamartomas are usually found incidentally and do not normally give rise to symptoms or abnormalities of liver function. They are often multiple, in which case they may very occasionally be associated with portal hypertension; distinction from congenital hepatic fibrosis is then difficult. If a small nodule on the liver surface is seen during surgery, frozen section may occasionally be requested in order to exclude metastatic carcinoma. The irregularly dilated duct structures, inspissated bile, and circumscription seen in microhamartomas are helpful in making this distinction.
Polycystic disease
The infantile type of polycystic disease is regularly associated with renal involvement.46,58 Portal tracts contain multiple cystic channels set in a fibrous stroma. In the adult type the cysts are lined by epithelium of biliary type (Fig. 13.10) but are not connected with the rest of the biliary tree. Solitary congenital cysts are histologically similar. The cuboidal or flattened epithelial lining helps distinguish these cysts from ciliated hepatic foregut cysts which are lined by ciliated cells and mucin-secreting goblet cells.59 The presence of microhamartomas and features of Caroli’s disease in individuals with polycystic disease favours a continuum in the expression of fibropolycystic disease.60–62
Inherited metabolic disorders
Cystic fibrosis
Cystic fibrosis is an inherited disease in which abnormally viscous exocrine secretions are present in the pancreas, salivary glands, alimentary tract and lungs; the prevalence of liver disease is variable and in part age-dependent.63–65 Subclinical liver disease may be significant.64 Jaundice in the neonatal period has been attributed to bile-duct obstruction by abnormally viscous bile and to gastrointestinal obstruction by meconium. Intercurrent hepatitis may also be responsible. Steatosis is common, although not always related to malnutrition.66 Paucity of intrahepatic bile ducts in cystic fibrosis has also been reported.67 In a proportion of older children a characteristic lesion of intrahepatic bile ducts is found.68 Dense plugs of PAS-positive material are seen within dilated, proliferated ducts (Fig. 13.11). Bile-duct cells may undergo degeneration and necrosis.66 There is surrounding fibrosis69 and a variable degree of inflammatory infiltration which may be associated with abnormal intrahepatic ducts on cholangiography.64 Eventually the fibrous areas may join, separating parenchymal islands. The term focal biliary fibrosis expresses the uneven involvement of the intrahepatic bile ducts in this process, with parts of the liver remaining unaffected. In some patients the disease evolves to secondary biliary cirrhosis.68
Storage disorders: general remarks
Inherited metabolic defects leading to the abnormal accumulation of lipids, proteins and carbohydrates in the liver are many and varied; for a full description of the morphological changes, recent reviews should be consulted.70–72 Ishak70 helpfully discusses the differential diagnosis of individual histological features. Liver biopsy is sometimes useful in diagnosis, though by no means always decisive. The following points are offered as practical suggestions for occasions when biopsy is contemplated in children suspected of having storage disorders.
1. Storage disorders can involve hepatocytes (e.g. glycogenoses, α1-antitrypsin deficiency), macrophages (e.g. Gaucher’s disease), or both (e.g. Niemann–Pick disease, mucopolysaccharidoses,73 cholesterol ester storage disease). When Kupffer cells are involved, they may swell to the size of hepatocytes and their involvement may not at first be apparent; the use of stains other than haematoxylin and eosin (H&E), especially PAS and trichrome stains, then usually makes the Kupffer-cell involvement obvious.
2. Suspicion of a possible storage disorder is one of the few indications for electron microscopy of part of the biopsy specimen as a diagnostic procedure, because characteristic ultrastructural appearances sometimes enable a correct diagnosis to be established quickly.74 Even when the changes are not diagnostic they can direct attention to a particular group of diseases, and suggest the next line of investigation. Arrangements for electron microscopy should be made beforehand, so that part of the specimen can be put into the correct fixative without delay. In centres wtihout facilities for electron microscopy, part of the specimen should still be correctly fixed and/or embedded, and sent to a referral centre later if light microscopic findings warrant this.
3. Arrangements should also be made to freeze part of the specimen and to store it in liquid nitrogen for possible biochemical analysis and histochemical staining of frozen sections. Speed is essential to avoid loss of enzyme activity. Again, a specialist centre may need to be consulted, because few centres or pathologists have the necessary expertise to investigate the rarer metabolic diseases.
Many inherited metabolic diseases affect the liver and several may lead to cirrhosis70 (glycogenosis types III, IV and VI, galactosaemia, tyrosinaemia type I, α1-antitrypsin deficiency, Wilson’s disease, hereditary haemochromatosis). Liver transplantation may be indicated in some patients.19,75,76 The discussion in this chapter will be limited primarily to the disorders mentioned under point 1 above. Haemochromatosis and Wilson’s disease are described in Chapter 14.
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