Section 1 -

Neonatal Cholestasis: Diagnostic Issues for Biliary Atresia, Neonatal Hepatitis and Look-alikes Grace E. Kim Associate Professor of Pathology University of California San Francisco

Introduction Many normal term newborns have transient neonatal jaundice with elevated unconjugated bilirubin levels. The unconjugated hyperbilirubinemia in some neonates results from an immature hepatic enzyme glucuronosyl transferase activity or can be associated with breast-feeding. Other etiologies include hemolysis, sepsis, hypothyroidism or pyloric stenosis and, inherited disorders such as Crigler-Najjar and Gilbert's syndromes. In contrast, conjugated hyperbilirubinemia nearly always reflects hepatic dysfunction. The current practice is to investigate jaundice in any infant who is greater than 14 days old to determine whether unconjugated or conjugated hyperbilirubinemia is present, and if the latter, an evaluation for a cause. Unfortunately, jaundiced infants escape clinical attention until the first well-baby examination at 6 to 8 weeks of age. The clinical investigation to establish a specific diagnosis may include a percutaneous liver biopsy. While the differential diagnosis of neonatal cholestasis is lengthy, the most frequent disorders in infants with conjugated hyperbilirubinemia are biliary atresia and idiopathic neonatal hepatitis. These two diagnoses can have overlapping features, but the distinction is important since early surgical intervention is required in biliary atresia. Biliary atresia is an idiopathic, necroinflammatory process of the bile ducts that leads to ductal fibrosis and obliteration and secondary biliary cirrhosis. Numerous etiological mechanisms, including congenital, infectious, immunologic, vascular, and toxins have been proposed; however, the cause of biliary atresia still remains unknown. Since it is the intrahepatic biliary lesion that determines the overall prognosis and outcome, the term extrahepatic has been dropped from its name. Biliary atresia – Clinical Generally, two clinical forms are recognized, the fetal, embryonic, or congenital form and the postnatal, perinatal, or acquired form. Approximately 10-30% of patients with biliary atresia have other congenital anomalies, such as cardiovascular defects, polysplenia, and situs inversus. This embryonic form of biliary atresia has an early onset (first 3 weeks of life) of cholestasis and an absence of a jaundice-free period after physiological jaundice. The majority of biliary atresia cases (65-90%) are of the acquired form. Typically these patients present later (2nd to 4th week of life) with jaundice and normally pigmented stools which become progressively alcoholic. Biochemical evidence of cholestasis and hepatocellular damage is present with variable increase in aminotransferases, gamma-glutamyltranspeptidase, and alkaline phosphatase levels. The serum level of conjugated bilirubin is elevated. These markers are not specific. The following imaging modalities can be utilized: ultrasound to visualize the gallbladder, extrahepatic bilary tree and liver, hepatobiliary scintigraphy (hepatobiliary iminodiacetic acid scan) to determine the patency of the biliary system by tracing the production and flow of bile from the liver through the biliary system into the small intestine, and magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography to delineate the extrahepatic biliary anatomy. Each of these diagnostic studies has limitations. The most reliable information is obtained by review of the hepatic histopathology followed by direct visualization of the obliterated extrahepatic bile ducts by intraoperative cholangiography. Untreated biliary atresia leads to biliary cirrhosis, hepatic failure, and death by 3 years of age. The first line of surgical therapy is hepatoportoenterostomy (Kasai's procedure). When performed by 2-3 months of age, this procedure successfully restores bile flow with resolution of jaundice in 30-80% of patient. Up to 70% of biliary atresia patients who had portoenterostomy performed before 60-70 days of life achieve bile flow. Long-term survival is similarly associated with age at the time of portoenterostomy. The atretic extrahepatic bile duct and gallbladder is resected and replaced by an intestinal conduit by anastomosing the jejunum to the patent bile duct remnant at the porta of the liver. Previously, a bile duct diameter of more than 150 µm within the ductal remnant was critical in determining post-operative bile flow. Subsequent studies have refuted this criterion and hence, surgeons should not request frozen sections on bile duct remnant to determine the luminal diameter. Approximately one third of patients who have had hepatoportoenterostomy survive 10 years without a liver transplantation. One third of the patients drain bile but develop complications of cirrhosis and require transplantation before age ten. Of the remaining one third of patients, bile flow is inadequate following heptoportoenterostomy and the children develop progressive fibrosis. When cirrhosis develops, liver transplantation is curative and offers a 5 year survival rate over 85%. Biliary atresia represents the most common indication for pediatric liver transplantation and accounts for 40-50% of all liver transplants performed in children. Biliary atresia – Pathology The histology is similar in both clinical forms. The liver usually shows morphologic signs of biliary obstruction with cholestasis, ductular reaction, and portal edema or fibrosis. The reported accuracy of the liver biopsy to determine an obstructive form of neonatal cholestasis approaches 95% with the awareness of clinical factors and when 5 to 7 portal tracts are present. Biliary atresia is a dynamic process and the histology transforms with the time course of the disease. During the neonate's first 4 to 6 weeks of life, non-specific features of cholestasis are present that can be accompanied by ballooned hepatocytes. Giant cell transformation can be found in at least 25% of patients with biliary atresia. Ductular reaction, the most reliable criterion in diagnosing biliary obstruction, develops around 6 to 8 weeks of age. The presence of bile plugs within interlobular ducts is a helpful feature when present. During this time, the portal tracts may show a variable density of lymphocytes and neutrophils, damaged interlobular duct epithelium, similar to that observed in the extrahepatic ducts, and portal edema or early fibrosis. At 8 weeks of age, the periportal fibrosis begins to progress into portal-to-portal bridging fibrosis. The amount of portal inflammation may decrease and damaged interlobular ducts can have concentric fibrosis. Cholestasis persists in the parenchyma with pigment-laden Kupffer cells. Eventually, secondary biliary cirrhosis, the so-called "jigsaw" pattern of cirrhosis, forms at 1 to 6 months of age with loss of interlobular ducts. The suggested timeline is certainly not absolute and can be quite variable. Differential diagnosis The features demonstrated in the liver biopsy are not specific for biliary atresia, but rather show an obstructive picture. Therefore, entities with ductular reaction and possible portal fibrosis, including choledochal cyst, inspissated bile syndrome, total parental nutrition-associated liver disease, cholestasis associated sepsis, alpha-1-antitrypsin deficiency, paucity of intrahepatic bile ducts and cytomegaloviral infection may histologically mimic biliary atresia. Correlating the biopsy findings with the knowledge of clinical data is essential. When evaluating a neonatal liver biopsy and the clinical indication is to rule out biliary atresia, I typically always ask the clinician the following questions: 1) Was the neonate born premature? If so, at what gestational age? 2) Has this neonate been on total parental nutrition? If so, for how long? 3) Is this child septic? Differential diagnosis Important considerations Alpha-1-antitrypsin Fully developed globules may be difficult to detect on H&E if <12 weeks of age Paucity of intrahepatic bile ducts Minimum of 6 portal tracts is suggested to evaluate for duct loss Cytomegaloviral infection Review all levels and special stained slides as viral inclusions can be focal Neonatal hepatitis Idiopathic neonatal hepatitis is an intrahepatic cholestatic disease of unknown cause and specific etiologies, including metabolic, genetic, endocrine and infectious diseases and structural causes must be excluded. The terminology giant cell hepatitis has also been used, because of the characteristic giant cells found in the lobule. These "giant cells" typically have greater than four nuclei that are often centrally clustered. Giant cell transformation is a non-specific finding that can be seen in other neonatal liver disorders. In addition to the diffuse giant cell transformation, the liver biopsy is characterized by hepatocanalicular cholestasis, extramedullary hematopoiesis, sparse lobular and/or portal tract inflammation, and apoptotic hepatocytes. The table below displays useful features to aid in distinguishing idiopathic neonatal hepatitis from biliary atresia. The treatment of idiopathic neonatal hepatitis is largely supportive. Histological features Biliary atresia Idiopathic neonatal hepatitis Giant cell Transformation Absent/focal Diffuse Lobular inflammation Absent Present Necrotic hepatocytes Absent/few Present Extramedullary Hematopoiesis Absent/some Present/extensive Ductular reaction Present Absent/rare/minimal Portal tract edema Present Absent Portal tract fibrosis Present Absent References Introduction Roberts EA. The jaundiced baby. In: Kelly DA, Ed. Diseases of the liver and biliary system in children. Malden, MA: Blackwell Science, Inc., 1999:11. Suchy JF. Approach to the infant with cholestasis. In: Suchy JF, Ed. Liver disease in children. St. Louis, MO: Mosby-Year Book, Inc., 1994: 351. Mieli-Vergan G, Portman B, Howard ER, et al. Late referral for biliary atresia-missed opportunities for effective surgery. Lancet 1989;1:412-423. Balistreri WF. Neonatal cholestasis: lessons from the past, issues for the future. Semin Liver Dis 1987;7(2):61-6. 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