Incidence

Biliary atresia occurs worldwide, affecting an estimated one in 8,000–15,000 live births. There is a slight female predominance in most published series. One population-based birth defects surveillance system for infants with biliary atresia in metropolitan Atlanta calculated an incidence rate of 0.73 per 10,000 live births [10]. In one report there was significant seasonal clustering of the disease, with rates three times higher in infants born between December and March; reported rates were significantly higher among non-white infants. The demonstration of significant seasonal clustering in this and other studies supports the theory that biliary atresia may be caused by environmental exposure (consistent with a viral cause) during the perinatal period [6].

Clinical Forms

At least two different forms of biliary atresia are recognized, with disparate pathogenesis: a syndromic or embryonic form and a non-syndromic or perinatal form. In patients with the less common syndromic form (10–20% of all patients), cholestasis is present from birth, with no jaundice-free interval. Bile duct remnants may not be detectable in the hepatic hilum and there is a high frequency of associated malformations such as polysplenia, which may be associated with cardiovascular defects, asplenia, situs inversus, intestinal malrotation, and positional anomalies of the portal vein and hepatic artery. Extrahepatic anomalies reported in patients with biliary atresia are outlined in Table 11.1.

In patients with the syndromic form of biliary atresia genetic factors likely predominantly contribute to the pathogenesis, in the form of defective embryogenesis. These two forms have not been distinguished on the basis of histology of porta hepatis specimens; both forms may have inflamed and obliterated bile duct segments in this resected tissue mass.

In the series of Davenport et al. [11] of 308 patients with biliary atresia, 23 (7.5%) had polysplenia, two had double spleens, and two had asplenia. All 27 splenic anomalies were grouped into the term “biliary atresia splenic malformation syndrome (BASM).” Infants with this syndrome had a lower birth weight and a higher incidence of maternal diabetes compared with non-syndromic cases. The extrahepatic anatomy of the biliary tract also reportedly was different, including instances of what they termed biliary agenesis. These findings suggest that either the timing or the nature of the biliary lesion of this subgroup may differ from the more common non-syndromic form, in which a virus- or toxin-induced injury to cholangiocytes causes a progressive obliteration of extrahepatic bile ducts, with some degree of intrahepatic bile duct injury [6, 12]. In both clinical forms, the injury appears to involve an ongoing inflammatory process that produces complete sclerosis of extrahepatic bile ducts and progression to cirrhosis. Whether the lesion noted in the intrahepatic bile ducts results from an extension of the extrahepatic lesion or is a consequence of cholestasis is not defined. It is believed that ductal plate malformation and segmental agenesis of bile ducts in porta hepatis specimens are identified more commonly in the syndromic form, but an analysis of portal tracts from eight infants with the non-syndromic form of biliary atresia and six infants with biliary atresia splenic malformation syndrome identified no association between ductal plate malformation and either clinical form of disease [13].

A third form or clinical variant is defined by the presence of cystic dilatation of extrahepatic bile ducts, in addition to the fibrosing obstruction of duct segments. Some of the biliary cysts are detected prenatally during routine ultrasound examination of the fetus; jaundice and acholic stools may present soon after birth or after a variable period of time. In a review of a large cohort with biliary atresia, biliary cysts occurred in approximately 8% of patients [14]. Infants with this “cystic variant” were younger at presentation, but a delay in performing a portoenterostomy beyond 70 days of age was associated with poor long-term survival with the native liver. The anatomical details of this variant, the earlier age at presentation, and the differences in outcome raise the possibility that the pathogenic mechanism of disease is unique.

Clinical Features

Despite the presumed multifactorial pathogenesis, there is consistency in the clinical features of biliary atresia: affected infants present with jaundice (conjugated hyperbilirubinemia) and acholic stools. The presence of hepatomegaly, failure to thrive, pruritus, and coagulopathy depends on the level of progression of disease. Affected infants usually are born at term, are of normal birth weight, and weight gain is appropriate early in the course. Patients with biliary atresia occasionally present with bleeding as a result of vitamin K deficiency. Examination may reveal hepatomegaly and splenomegaly. Ascites and wasting may be seen as late manifestations if biliary cirrhosis has supervened. Increased awareness to ensure early diagnosis and development of methods to prevent progressive hepatic fibrosis are essential. Early recognition of babies who have biliary atresia is particularly critical for optimal intervention; ideally, biliary atresia should be identified by the time of the two-week well-baby visit. The importance of a prompt and precise diagnosis must be stressed to all pediatric healthcare providers. In the UK, an educational effort (the Yellow Alert campaign) was established to indicate the significance of jaundice persisting after 14 days of age. Population screening also has been considered, including the use of stool color cards to identify at-risk infants. In Taiwan, population screening with these cards resulted in an increase in the national rate of portoenterostomy before 60 days of age from 60% to 74% [15].

Cause and Pathogenesis

The ultimate goals are to define the pathogenesis of biliary atresia and establish preventive strategies. Although our understanding of the cause of biliary atresia has remained unchanged for several decades, there is now greater knowledge regarding the pathogenesis of the disease from patient-based studies and the use of experimental models of bile duct injury [6, 7]. Theoretic considerations of the cause of biliary atresia have been based on epidemiologic and clinical features. Two critical clinical features offer potential clues to the chief biologic processes. The first is that the onset of disease is restricted to the perinatal or immediate postnatal period (<4 months). The second is the presence of inflammation and fibrosis of the extrahepatic bile ducts. In the “typical” patient, the structural changes present in the hepatobiliary tract suggest a progression of the lesion from acute cholangitis to fibrotic obliteration of the ducts (Figure 11.1). The dynamic nature of the obliterative process is illustrated by the fact that atresia has been found at autopsy or re-exploration in infants previously shown to have patent extrahepatic ducts or “neonatal hepatitis.”

Figure 11.1 Stages of biliary atresia. (a) Patent bile duct in a specimen from the porta hepatis exhibits periductal inflammation and epithelial erosion; elsewhere in this patient, the duct was obliterated by reactive tissue (magnification 37×). (b) Detail of eroded bile duct shows regressive epithelial change, periductal edema, and mild inflammation (magnification 250×). (c) At the autopsy of a patient with biliary atresia, a minute remnant of common bile duct has intact epithelium (magnification 100×). (d) Minute fibrous cord represents the final stage of biliary atresia (magnification 100×). In many patients, the atretic duct is not visible to the naked eye. (Hematoxylin and eosin staining.)

Multiple studies have focused on normal and altered bile duct morphogenesis and the role of various factors (infectious or toxic agents and metabolic insults) in isolation or in combination with a genetic or immunologic susceptibility to biliary atresia [4, 6]. The absence of documented recurrence in siblings of infants with biliary atresia and reports of dizygotic and monozygotic twins discordant for biliary atresia appears to exclude simple Mendelian inheritance in the vast majority of patients [4, 6]. The concept that an acquired obliterative process underlies biliary atresia is attractive and suggests that a virus- or toxin-related inflammation may initiate the sequence that leads to fibrosis and luminal obstruction. In support of this concept, giant multinucleate hepatocytes have been noted in up to 40% of liver biopsy samples obtained early in life from patients with biliary atresia.

Viral Infection

A favored theory implicates subclinical viral infection as the inciting mechanism. The demonstration of significant seasonal clustering provides support for the theory that biliary atresia may be caused by environmental exposure (consistent with a viral cause) during the perinatal period. Multiple potential etiopathogenic viruses have been ruled out as “suspects.” Hepatitis A, B, and C virus infections are not related to biliary atresia [6], and there was no apparent increase in the incidence of biliary atresia during rubella epidemics. Cytomegalovirus (CMV), which characteristically infects the biliary epithelium, has been suggested as a cause of biliary atresia. For example, a Swedish study showed a higher prevalence of CMV antibodies in mothers of infants with biliary atresia, and CMV DNA was present in livers from nine of 18 infants with biliary atresia [16]. A role for CMV in the pathogenesis of biliary atresia, however, was not supported by studies examining porta hepatis specimens by in situ hybridization and polymerase chain reaction using CMV DNA probes [17]. Yet, exposure to CMV remains a potential causative factor because of the viral tropism to the bile duct epithelium, but future studies will need to employ new approaches that detect virus-specific epitopes in memory lymphocytes. A high prevalence of human papillomavirus has been detected in liver tissue and in cervical swabs from mothers of patients with biliary atresia. There is no animal model, however, demonstrating the consequences of human papillomavirus infection in an immature liver, nor have these findings been confirmed. Mason et al. [18] detected retroviral antibody reactivity in patients with biliary atresia attributable either to an autoimmune response to antigenically related cellular proteins or to an immune response to uncharacterized viral proteins that share antigenic determinants with these retroviruses. Further studies are needed.

The viral agents most frequently implicated in the pathogenesis of biliary atresia include reovirus and rotavirus. Serologic reactivity to reovirus type 3 and reovirus particles in the porta hepatis has been found in children with biliary atresia [6]. It had been known for some time that this virus can cause an obliterative cholangiopathy in weanling mice; a similarity exists between the hepatitis with biliary tract inflammation induced by reovirus type 3 infection in the weanling mouse model and the progressive postnatal fibrotic obliteration of the extrahepatic bile ducts and liver cell injury noted in biliary atresia [6]. Reovirus type 3 infection, therefore, has been implicated as the initial insult in the sequence of events resulting in the observed lesions. Murine reovirus infection may lead to necrosis of the bile duct epithelium and hepatocytes, inflammatory infiltration, and possibly identifiable viral inclusions in bile duct epithelial cells. Pathologic changes in reovirus-infected mice, including distal stenosis of the common bile duct and dilation of the proximal bile duct, remain after infectious virus or viral antigens can no longer be detected [19]. Infection of newborn mice with reovirus in the first days of life, however, did not produce obstruction of extrahepatic bile ducts [20]. Previous attempts to show an association between reoviruses and human hepatobiliary disease have yielded conflicting results.

Reoviruses have not been isolated from human hepatobiliary tissue, but reovirus antigen was detected in the bile duct remnant resected from an infant with biliary atresia, and reovirus-like virion particles were seen in this tissue by electron microscopy [19]. Using reverse transcriptase polymerase chain reaction, reovirus was found in hepatobiliary samples of 55% of patients with biliary atresia and 78% with choledochal cyst, whereas the virus was present in tissues of only 8–21% of appropriately matched controls [21]. More work is needed to establish or exclude a causative relationship between reovirus and biliary atresia.

Riepenhoff-Talty et al. [22] reported the development of extrahepatic biliary obstruction in newborn mice orally inoculated with group A rotavirus. These investigators also presented evidence for polymerase chain reaction amplification of group C rotavirus sequences from livers of patients with biliary atresia, for immunoreactivity to group C rotavirus in serum of patients with biliary atresia, and for group C rotavirus particles in the stool of patients with biliary atresia [23]. Additional studies in infected newborn mice have clearly shown the induced injury to reside in the biliary epithelium. This model of rotavirus-induced biliary injury has proved valuable in studying the mechanisms of biliary atresia because it recapitulates two consistent clinical features of the disease in humans: the onset of disease in the immediate neonatal period and the progressive cholangiopathy [6].

Additional studies are needed to further investigate a relationship between reovirus, rotavirus, or any other virus in the pathogenesis of biliary atresia. Investigation into the contribution of virus-initiated immune or autoimmune mechanisms of hepatobiliary injury may yield information essential for development of treatment or prevention strategies. One example is the observation that the incidence of biliary atresia appeared to decrease in Taiwan after the introduction of rotavirus vaccine [24]. It is unlikely, however, that antiviral therapy alone would alter the natural history of biliary atresia if the pathologic process is an immunologic reaction to a preceding viral injury, without ongoing viral replication.

Defect in Morphogenesis

The hypothesis that a defect in morphogenesis of the biliary tract underlies the pathogenesis of biliary atresia is appealing, particularly considering the co-existence of other anomalies, such as anomalies of visceral organ symmetry (Table 11.1) that occur in 10–20% of infants with biliary atresia. Tan et al. [25] compared the developing biliary system of normal human embryos and fetuses with the resected extrahepatic biliary remnants from 205 patients with biliary atresia. At the porta hepatis level, the primary biliary ductal plate underwent a specific sequence of remodeling between 11 and 13 weeks after fertilization, resulting in the formation of large tubular bile ducts surrounded by thick mesenchyme. Luminal continuity with the extrahepatic biliary tree was maintained throughout gestation. Contrary to previous speculation, no “solid phase” was documented during the development of the extrahepatic bile duct. Examination of the biliary remnants from patients with biliary atresia showed that the porta hepatis was encased in fibrous tissue, with a variable pattern of obliteration of the common hepatic and common bile ducts. There were similarities on anti-cytokeratin immunostaining between the abnormal ductules within the porta hepatis in biliary atresia and the normal developing bile ducts during the first trimester. The investigators proposed that biliary atresia may be caused by failure of the remodeling process at the hepatic hilum, with persistence of fetal bile ducts poorly supported by mesenchyme. They further postulated that, as bile flow increases perinatally, bile leakage from these abnormal ducts may trigger an intense inflammatory reaction, with subsequent obliteration of the biliary tree. It remains to be demonstrated whether these processes are causative or whether the histological features result from the activation of cellular circuits in response to poorly defined insults. Among these are infectious or immune insults that can interfere with the normal remodeling process at the hepatic hilum and with ductal plates within the liver.

Genetic Factors

Several genes have been implicated in the abnormal development of the biliary system and potentially in the pathogenesis of biliary atresia [6, 7, 26]. Anomalies of visceral organ symmetry, including complete abdominal situs inversus, severe jaundice, and death within the first week of life, have been reported in transgenic mice that have a recessive insertional mutation in the proximal region of mouse chromosome 4 and deletion of the inversin (inv) gene, but the role in pathogenesis of biliary atresia remains undefined in view of the inability to detect abnormalities of INV in children with laterality defects and biliary atresia [27].

Gene sequencing studies have reported variants associated with biliary atresia; FOXA2 variants were found in members of a single family and ADD3 and GPC1 variants in surveys of cohorts of children with biliary atresia [4]. Other variants reported in larger cohorts include PKD1L1 (encoding a ciliary protein) and EFEMP1 (encoding the elastic fiber protein fibulin-3) [4].

A series of observations clarified the morphogenesis and differentiation of the intrahepatic bile ducts [2, 28]. In a study of human liver samples from different stages of fetal development and immunostaining with anti-cytokeratin antibodies specific for bile duct epithelial cells, investigators showed that bile ducts arise within the mesenchyme surrounding portal vein radicals. Presumed primitive hepatic precursor cells differentiate into a single layer of cytokeratin-staining cells and then form a double layer. At focal points, these cells then scatter and remodel as a single layer around a lumen. In livers from some infants with biliary atresia, there was evidence for an arrest in remodeling such that lumens are not formed (ductal plate malformation) [2, 28]. Studies in mice showed that extrahepatic bile ducts arise from a PDX1+/SOX17+ domain and then segregate into SOX17+ bile ducts and PDX1+ ventral pancreas; the requirement of Sox17 for normal development was supported by the report of abnormal bile ducts in Sox17 heterozygous mice [4]. Histological and functional abnormalities in the biliary tract have been reported in mice with genetic mutations in Jagged, Notch, Hes1, Hnf6, Hnf1b, Foxm1b, Foxf1, Foxa1/Foxa2, Sox17, and Lgr4, which raises questions about the potential role of these genes as susceptibility factors or modifiers of disease in humans [6, 7, 26].

Pro-Inflammatory Mechanisms of Disease

Several lines of evidence point to a pro-inflammatory response that targets the bile ducts in patients with biliary atresia. One theory holds that a viral or toxic insult to the biliary epithelium leads to newly expressed antigens on the surface of bile duct epithelia, which in the proper genetically determined immunologic milieu (e.g., the presence of major histocompatibility molecules) are recognized by T-lymphocytes that elicit a cellular immune injury. In support of this notion, Silveira et al. [29] reported an association of the human leukocyte antigen (HLA)-B12 allele and haplotypes HLA-A9B5 and HLA-A28B35 with biliary atresia. Other haplotypes of potential involvement include HLA-Cw4/7, HLA-A33, HLA-B44, and HLA-DR6 and a higher prevalence of specific polymorphisms in selected genes, such as SNP rs17095355 on 10q24 of ADD3 gene [30]. This path of inquiry will benefit from future validation studies in a large patient population.

Histological and immunostaining analyses of the liver and extrahepatic remnants suggest that lymphocytes, dendritic cells, and Kupffer cells play key roles in the regulation of inflammation and destruction of bile ducts in infants with biliary atresia. Both CD4 T-cells and natural killer cells are increased in livers at diagnosis and are associated with epithelial cell pyknoses within intrahepatic portal tracts, porta hepatis, and common bile duct remnants [7, 31]. The liver expresses high levels of cytokines and receptors, such as tumor necrosis factor-α, interleukin-2 and its receptor, the transferring receptor CD71, and interferon-γ. More direct evidence for an effector role of T-lymphocytes emerged from a report that liver and bile duct remnants of patients with biliary atresia have oligoclonal expansion of CD4 and CD8 T-cells [32]. These technically challenging experiments add functional relevance to this group of antigen-specific T-cells, and set the stage for future studies investigating their relationship to molecular epitopes in cholangiocytes. One example is the detection of elevated levels of anti-enolase antibodies in ~35% of infants with biliary atresia [33].

The prevailing hypothesis that pro-inflammatory cytokines are important to the pathogenesis of biliary atresia has been tested in the rotavirus-mouse model. In this model, blocking of signals regulated by interferon-γ, α₂-integrin and interleukin- 15 prevented bile duct obstruction and the phenotype of biliary atresia [7, 34]. Searching for the cellular basis of cytokine production and biliary obstruction, individual mononuclear cells were depleted in newborn mice to examine their contribution to the atresia phenotype. While the loss of CD4 cells had no obvious influence on the biliary injury, individual depletion of CD8 lymphocytes, NK lymphocytes, dendritic cells, or macrophages decreased the epithelial injury and/or prevented the obstruction of extrahepatic bile ducts, with improved growth and long-term survival in experimental mice [3, 7, 35]. The similarities between the phenotypes produced by the loss of these cell types and cytokines suggest that they work in concert to promote duct obstruction, and may constitute therapeutic targets to block progression of liver disease.

One feature that deserves special note is the restriction of onset of disease to the first few months of life in infants and the first few days of life in mice, suggesting that the mechanisms of disease are substantially influenced by developmental factors. This feature may be explained by the influence of genes regulating embryogenesis, such as PKD1L1, FOXA2, EFEMP1, and others as discussed above [4, 29]. However, the lack of congenital malformations in the majority of patients opens the possibility of a greater influence of other biological processes. Among them is an inflammatory response triggered by the presence of rare maternal cells in the liver of affected infants (maternal chimerism). Evidence for this process is largely circumstantial. Another relates to the paucity of regulatory T-lymphocytes in the liver and other peripheral tissues in the first three days of life in mice. These regulatory cells have an important immunomodulatory function; their absence leads to an array of autoimmune phenotypes. Regulatory T-cells were reported to be nearly absent from mouse livers following rotavirus challenge in the first three days of life [36]. In contrast, when rotavirus was injected at seven days of age, a time when the liver was populated by regulatory T-cells, mice were resistant to the biliary atresia phenotype. How these results apply to susceptibility to biliary injury in humans, however, is unknown.

The combined genetic and cell depletion studies in mice uncover a continuum of biological events that produce obstruction of extrahepatic bile ducts in a fashion that recapitulates some features of the disease in humans. The events begin with a viral infection (e.g., rotavirus) that targets the bile duct epithelium and primes macrophages and dendritic cells (“initiating” phase). This is followed by activation of NK cells that injure cholangiocytes and disrupt mucosal continuity (phase of epithelial injury). An amplification of the adaptive immune response by CD4 and CD8 T-cells and by the release of pro-inflammatory cytokines form a cellular plug at the site of epithelial injury (phase of obstruction), followed by the evolution to collagen deposition to produce the atresia phenotype (Figure 11.2).

Figure 11.2 Proposed model of the pathogenesis of biliary atresia identifies a continuum of disease, in which an initial insult targets the bile duct epithelium and activates an immune response that obstructs the duct lumen (inflammatory plug) and rapidly progresses to fibrosis and atresia. At the stage of epithelial injury, macrophages (M), neutrophils (N), dendritic cells (DC) and natural killer (NK) cells work collaboratively to injure cholangiocytes via several effector cytokines. Regulatory T (Treg) lymphocytes are proposed to suppress the response of dendritic cells and lymphocytes. At the stage of inflammatory plug, the adaptive immune system makes use of DC, NK, and CD8 T-cells and cytokines to amplify the inflammatory response. In the later stages of atresia, alternatively activated macrophages and fibrosis-related cytokines promote tissue fibrosis. IFNγ: interferon-γ; IL: interleukin; TNFα: tumor necrosis factor-α; NKG2D: activating receptor on NK cells; RAE-1: mRNA export factor; MIP-2: macrophage inflammatory protein 2; TGFβ: transforming growth.

Environmental Toxic Exposure

To date, the only supportive patient-based evidence for a toxic insult as a causative factor of biliary atresia is the time–space clustering of cases. In animals, unusual outbreaks of hepatobiliary injury in lambs and calves in New South Wales, Australia, occurred in 1964 and 1988, with pathologic specimens displaying features akin to the pathology seen in humans with biliary atresia [37]. Investigators have been able to isolate and synthesize a toxin (named “biliatresone”) from plants eaten by pregnant rams and showed that it causes selective extrahepatic biliary damage in larval zebrafish and in mice. There is no evidence that patients with biliary atresia have been exposed to biliatresone, but the identification of key structural motifs may allow detection of toxins with human relevance.

Vascular Abnormalities

Developmental abnormalities in the position of the portal vein and in hepatic artery anatomy at the porta hepatis are common in patients with biliary atresia. There are no consistent experimental data, however, to confirm the hypothesis that there is a vascular basis, such as ischemia, as a cause of the progressive duct injury seen in biliary atresia [38]. In utero devascularization or ligation of the extrahepatic bile duct has been attempted in some animal models, and lesions similar to the less common “correctable” variants of biliary atresia have been produced; however, other studies have been inconclusive.

Evaluation

Our approach to the work-up of an infant with cholestasis is shown in Box 11.1. We recommend the following sequential approach:

1. 
   

   1. Prompt recognition of cholestasis is essential. Jaundice in an infant must not be attributed erroneously to physiologic hyperbilirubinemia or to breast-feeding. Fractionation of the serum bilirubin usually separates out these later conditions, which cause a predominant elevation (>80%) of unconjugated bilirubin levels.

   
   
2. 
   

   2. The evaluation should be expeditiously performed to rule out potentially devastating illnesses such as sepsis, endocrine disorders, and nutritional hepatotoxicity attributable to metabolic disease (e.g., galactosemia). Definitive detection is usually straightforward, and institution of appropriate treatment for these conditions may prevent further liver injury. Early recognition of specific, treatable primary causes of neonatal cholestasis also allows particular clinical issues to be addressed. For example, hypoprothrombinemia may be present regardless of the cause of cholestasis; administration of vitamin K may prevent spontaneous, life-threatening bleeding, such as intracranial hemorrhage.

   
   
3. 
   

   3. “Idiopathic” neonatal intrahepatic cholestasis must be differentiated from biliary atresia because the prognosis and management differ significantly. In infants with biliary atresia, progressive fibrosis rapidly occurs; therefore, a significant delay in diagnosis or treatment must be avoided.

No single test is entirely satisfactory in discriminating intrahepatic cholestasis from biliary atresia; however, historical and clinical features may aid in the differential diagnosis. Neonatal hepatitis is reported to have a familial incidence of 15–20%; in contrast, the intrafamilial recurrence risk is negligible for biliary atresia. Infants with biliary atresia may look well, become clinically jaundiced at three to six weeks of age, and have slowly progressive elevation of serum bilirubin levels but seldom have pruritus or skin xanthoma as seen in forms of intrahepatic cholestasis). The liver is enlarged and firm; splenomegaly occurs as cirrhosis develops. Stools of patients with biliary atresia are acholic at presentation, but early in the course, during the evolving process of bile duct obliteration, they may contain some bile pigment. Acholic stools are either intermittent or delayed in onset in a quarter of patients with biliary atresia and are present in some patients with neonatal hepatitis. The consistent presence of pigmented stools excludes biliary atresia.

Hepatobiliary scintigraphy using iminodiacetic acid analogues has been used to provide discriminatory data. In biliary atresia, hepatocyte function is intact early in the disease; therefore, uptake of the imaging agent is unimpaired but excretion into the intestine is absent. Conversely, in intrahepatic cholestasis, tracer uptake is sluggish or impaired but excretion into the bile and intestine eventually occurs. Oral administration of phenobarbital, 5 mg/kg daily for five days before the study, is required to enhance biliary excretion of the isotope and, therefore, the sensitivity of the procedure. However, in our experience, this may delay the evaluation process and is rarely definitive, therefore we do not recommend the use of scintigraphy. Techniques that are used extensively in evaluation of adults with cholestatic disease, such as percutaneous transhepatic and endoscopic retrograde cholangiography, are not of proven value in children. Ultrasonography may detect dilation of the biliary tract, the presence of a choledochal cyst, or, in patients with biliary atresia, absence of the gallbladder. Ultrasound can also identify a sonographic finding known as a triangular cord, a fibrous cone of tissue at the bifurcation of the portal vein that has been associated with biliary atresia [39].

Numerous diagnostic algorithms incorporating these features have been proposed in an attempt to select those infants who are surgical candidates and to avoid unnecessary surgery. Discriminatory analysis of clinical, biochemical, and histological data obtained from 288 infants younger than three months presenting with neonatal cholestasis allowed for an accurate differentiation in 85% of the patients [40]. The following features occurred significantly more frequently in infants with intrahepatic disease, than in infants with biliary atresia: male gender, low birth weight, later onset of jaundice (mean of 23 vs. 11 days of age), later onset of acholic stools (mean of 30 vs. 16 days), and pigmented stools within ten days after admission (79% vs. 26%). Patients with biliary atresia more frequently had hepatomegaly, and the liver usually had a firm or hard consistency. Despite the use of scoring systems such as this, about 10% of infants with intrahepatic cholestasis cannot be distinguished from those with biliary atresia. Unnecessary surgical explorations are to be avoided; however, delay in establishing a diagnosis also is unwarranted because the data suggest that the success rate for surgical management of patients with biliary atresia rapidly declines with age.

Role of Liver Biopsy

In our experience, clinical examination, careful and repeated examination of the stool, and needle biopsy of the liver correctly identify the majority of patients with biliary atresia. In most patients, biopsy can be performed safely via percutaneous route with or without ultrasound guidance with sedation and local anesthesia or general anesthesia. An accurate, biopsy-based diagnosis is possible in up to 95% of patients and avoids unnecessary surgery in patients with intrahepatic disease. Early in the progression of biliary atresia, the liver shows preservation of the basic hepatic architecture, with bile ductular proliferation, canalicular and cellular bile stasis, and portal or perilobular edema and fibrosis (Figure 11.3). Bile plugs in the portal ducts are relatively specific but are found in only 40% of biopsy specimens. Portal fibrosis with wide swaths of connective tissue extending into the liver substance develops in older infants but may be already present at diagnosis. Approximately 25–40% of infants have portal inflammatory infiltration and hepatocyte giant cell transformation indistinguishable from neonatal hepatitis. These portal tract findings contrast with those of neonatal hepatitis, in which variable, often severe, intralobular cholestasis may be accompanied by focal hepatocellular necrosis. Bile ducts show little or no alteration in idiopathic intrahepatic cholestasis. Portal inflammatory infiltrates are present in both conditions and tend to be more prominent in idiopathic intrahepatic cholestasis. Portal area stroma is more likely to show edema in patients with biliary atresia. Giant cell transformation and extramedullary hematopoiesis, particularly sinusoidal erythropoiesis, are found in a large percentage of infants with either condition and have no diagnostic specificity. In very young infants, the initial biopsy may be inconclusive; re-biopsy after 7–14 days may be more definitive.

Figure 11.3 Histological changes in patients with biliary atresia. (a) Portal tract expansion with increased bile duct profiles, edema, and a bile plug (arrow) (magnification 200×). (b) Biopsy with a predominant inflammatory infiltrate (magnification 200×). (c) Biopsy with porto-portal fibrosis (with minimal inflammatory cells, magnification 100×). All biopsy slides were stained with hematoxylin and eosin.

The scoring systems discussed above have been evaluated in the differential between obstructive and non-obstructive forms of neonatal cholestasis [41, 42]. The accuracy, sensitivity, and specificity rates reported by Zerbini et al. were all 94% [42]. The model then was applied to a new sample of 74 needle liver biopsy specimens. The accuracy, sensitivity, and specificity rates were 91%, 100%, and 76%, respectively. In a multicenter study, analysis of 97 liver biopsy samples was carried out by a group of pediatric pathologists; the histological features that best predicted biliary atresia on the basis of logistic regression were bile duct proliferation, portal fibrosis, and absence of sinusoidal fibrosis, with a positive predictive value of 91% [41]. This suggests that if extrahepatic obstruction cannot be ruled out, limited exploration with cholangiography and repeat needle or wedge biopsy of the liver should be performed; if atresia is apparent, the biliary tract can be explored further.