Histopathology 1990, 17, 521-527
Abnormalities of intrahepatic bile ducts in extrahepatic biliary atresia E.A.RAWEILY, A.A.M.GIBSON* & A.D.BUKT University Department of Pathology, Western Infirmary and *Department of Pathology, Royal Hospital for Sick Children, Glasgow, Scotland, UK Date of submission 1 June 1990 Accepted for publication 1 1 July 1990
R A W E I L Y E . A . , GIBSON A . A . M . & B U R T A . D .
(1990) Histopathology 17, 521-527
Abnormalities of intrahepatic bile ducts in extrahepatic biliary atresia The infantile cholangiopathies are a group of conditions associated with neonatal jaundice, which include extrahepatic biliary atresia, paucity of intra-hepatic bile ducts and disorders associated with persistence of fetal biliary structures, the so-called ductal plate malformations. Although previously regarded as distinct entities, it has recently been suggested that they may represent parts of a disease spectrum in which the principal process is one of bile duct destruction, the morphological manifestations in individual cases being influenced by the stage of intra-uterine development at which such injury occurs and by the site within the biliary system at which
there is maximum damage. To further examine this concept, we have studied liver biopsy specimens from 3 7 neonates with extrahepatic biliary atresia, with particular reference to abnormalities of the intrahepatic bile ducts. Paucity of intrahepatic ducts, defined as a bile duct: portal tract ratio of less than 0.9, was identified in six cases (16.2%). In eight cases (21.60/,) we found concentric tubular ductal structures similar to those observed in ductal plate malformations. In one case, both abnormalities could be demonstrated. Our findings support the concept that there is overlap between the various types of infantile cholangiopathy.
Keywords: extraheptic biliary atresia, infantile cholangiopathy, paucity of intrahepatic bile ducts, ductal plate malformation
Introduction Neonatal cholestasis with conjugated hyperbilirubinaemia occurs in approximately 2.35 per lo4live births': over a third of these are associated with disorders of the bile ducts. These infantile cholangiopathies include conditions in which there is atresia of either the extrahepatic or intrahepatic bile ducts. Although true agenesis may occur, there is increasing acceptance of the concept that these disorders result from injury to, and destruction of. ducts that have already A further group of congenital bile duct disorders is characterized by the persistence of fetal biliary structures, an appearance which has been referred to as the ductal plate malformation5. This group includes infantile polycystic disease and congenital hepatic fibrosis. Address for correspondence: Dr A.Ll.Rurt. Division of Pathology. School of Pathological Sciences. University of Newcastle-upon-Tyne. Royal Victoria Infirmary. Newcastle-upon-Tyne N E 4 YHE. IJK.
Several investigators have indicated that there may be overlap between the extrahepatic and intrahepatic atresias, and between the atresias and the ductal plate malformations 3.h-8, although the frequency with which these conditions co-exist has not been documented. On the basis of these observations, D e ~ m e thas ~ . ~proposed a hypothesis which suggests that the infantile cholangiopathies represent a disease spectrum in which the principal pathological process is that of active destruction of bile duct radicles. The morphological appearances in individual cases would thus be dependent upon the stage of fetal development, and the site within the biliary system, at which such injury occurred. Those characterized by the ductal plate malformation may represent cases in which the injury had occurred early in fetal life, with destruction and ineffective regression of immature bile ducts, while those characterized by atresia represent examples of injury at a later stage, with destruction of fully formed extra- or intrahepatic ducts.
52 1
522
E.A. Raweily. A.A.M.Gibson and A.D.Rurt
In order to further examine this concept, we have investigated the intrahepatic morphological changes occurring in 37 neonates in which a diagnosis of extrahepatic biliary atresia was made on the basis of surgical or radiological findings.
Materials and methods liver biopsy specimens from 3 7 neonates seen over a 2 5year period were obtained from the files of the Department of Pathology, Koyal Hospital for Sick Children, Glasgow. In each case a diagnosis of extrahepatic biliary atresia was based on operative findings and/or cholangiography. None of the patients exhibited clinical features of Alagille’s syndrome. In some of the cases, serial biopsies were obtained following operative intervention (Kasai procedure). However, this study was limited to a n assessment of the histological changes in the initial wedge biopsies obtained at laparotomy. In all cases, the initial biopsy was taken before 32 weeks of age (mean = 1 1.05; range = 4-32 weeks). Sections, 5 pm thick, were stained with haematoxylin and eosin, Masson’s trichrome. periodic acid-Schiff (with and without diastase), Shikata’s orcein and Perk’ reagent. In some cases, in order to enhance visualization of biliary structures, sections were stained with antibodies which detect cytokeratin sub-types expressed by bile duct epithelium but not hepatocytes”’. For these immunohistochemical studies, sections were treated with 0.1% trypsin (type I1 porcine T8 128, Sigma, Poole, Dorset. UK) prior to the application of the primary antibodies. Two primary antibodies were used: (i) polyclonal anti-keratin (Dako, High Wycombe, UK)” and (ii) mouse monoclonal antibody AE1 (ICN, High Wycombe. UK)”. A peroxidase-anti-peroxidase method’ was used with the polyclonal antiserum and a n avidin-biotin methodI4 with the monoclonal antibody. In each biopsy the presence of the following features was ahsessed by two independent observers (A.D.B. & E.R.): bilirubinostasis: bile duct dilation: cholangitis; cholangiolitis: bile ductular proliferation: biliary piecemeal necrosis: giant cell transformation: Mallory bodies. Particular attention was paid to the presence of ductal structures with an abnormal architecture, such as those described for the ductal plate m a l f ~ r m a t i o n ~In, ~each . biopsy, all portal tracts were examined and the number of bile duct radicles counted: biopsies with a bile duct :portal tract ratio of less than 0.9l5were considered to show paucity of intrahepatic ducts.
Results The frequency of the various histological features in the initial biopsies from the 3 7 cases is outlined in Table 1.In
Table 1. Histological features in 37 cases ofextrahepatic biliary atresia No. of cases (o(,)
Bilirubinostasis Dilatation of bile ducts Cholangitis
Cholangiolitis Bile ductular proliferation Biliary piecemeal necrosis Giant cell transformation Paucity of intrahepatic ducts Abnormal ductal structures (concentric tubular, reduplicated)
36
(97.3)
1 1 (29.7) 12 ( 3 2 . 5 ) 3 5 (94.5) 37 ( 1 0 0 ) 32 (86.4) 19 ( 5 1 . 3 ) h ( 16.2) 8 (21.6)
all cases there was bile ductular proliferation at the limiting plate and in periportal zones. The degree to which this occurred varied between cases: in some this was a marked feature (Figure 1): in most cases it was associated with a cholangiolitis. Cholangitis with a periductal and intraluminal polymorph infiltration was seen in 12 cases: 1 1 of these showed foci of bile duct dilatation. Accumulation of copper-associated protein was frequently observed in periportal hepatocytes using Shikata’s orcein stain (Figure 2 ) but Mallory bodies were not identified in any case. Giant cell transformation was seen in 1 9 cases, but was generally mild, predominantly involving acinar zone 111. In six cases there was a bile duct: portal tract ratio of less than 0.9, indicating paucity of intrahepatic ducts (Figure 3). In three of these, necro-inflammatory lesions involving medium-sized bile ducts were observed (Figure 4); no fibro-obliterative lesions were seen. In eight cases, abnormal ductal structures were noted. These lesions were present in only some portal tracts, their prevalence varying between cases. In some they were seen to comprise a single tubular structure encompassing a central fibrovascular core (Figures 5 & 6): in others a more complex pattern was observed, with reduplication ofthe tubular structures (Figures 7 & 8).In one case in which such features were observed, there was also evidence of paucity of intrahepatic ducts.
Discussion The infantile cholangiopathies include conditions in which there is loss of intrahepatic or extrahepatic bile ducts and disorders characterized by persistence of fetal biliary structures4. The latter group are best understood by considering normal hepatic development in the fetus.
-
Figure 1. Ductular proliferation: this was a n almost invariable feature. The ductules are surrounded by a mixed inflammatory cell infiltrate (cholangiolitis). H & E. Figure 2. Retention of copper-associated protein in periportal hepatocytes. Shikata’s orcein.
Figure 3. Paucity of intrahepatic ducts; note the absence of bile duct radicle in this medium-sized portal tract. H & E. Figure 4. Necroinflammatory lesion: this was seen in three cases in which there was paucity of intrahepatic ducts. Note degenerative changes in the ductal epithelium. H & E.
ligure 6. Concentric tubular structures were readily identified in immunohistochemical preparations: here ductal epithelium is stained with monoclonal antibody AEI. ABC method.
Figure 7. Complex reduplicated ductal pattern. This resembles so-called ductal plate malformation. H & iti
.
Figure 8. Low-power micrograph illustrating complex reduplicated tubular structures reacted with monoclonal antibody AEI . ABC method.
fritraheputic bile ducts in extrahepatic biliarg atresia
The intrahepatic bile ducts are derived from primitive hepatocytes which abut onto the mesenchyme of developing portal tracts. From around 9 weeks of gestation these cells undergo a series of phenotypic changes; they become flattened and exhibit altered expression of cytokeratins and other cytoskeletal proteins'".'h. This cell layer, referred to as the ductal plate5, subsequently becomes reduplicated with the formation of a tubular structure which surrounds the portal tract. From around 12 weeks of gestation, there is remodelling of this structure with gradual incorporation into the connective tissue surrounding portal vein branches. Parts of the ductal plate are then resorbed, leading to the development of individual 'mature' bile ducts. This process of remodelling proceeds until term and, by 4 weeks of life, the appearances are similar to those of adult liver'". Several conditions, including congenital hepatic fibrosis and infantile polycystic disease, are characterized by persistence and even duplication of the tubular structures. It has been suggested that such 'ductal plate malformations' are the result of a n intra-uterine insult leading to arrested development of the bile ducts3,9. Traditionally, extrahepatic atresia, paucity of intrahepatic bile ducts and the ductal plate malformations have been regarded as distinct entities. However, Desmet 1.9 has suggested that these may form part of disease spectrum, sharing a common pathogenetic mechanism of intra-uterine bile duct destruction. If this is true it may be expected that paucity of intrahepatic ducts and ductal plate malformation should occur in some neonates with extrahepatic biliary atresia. The aim of the present study was to reassess the intrahepatic changes occurring in extrahepatic biliary atresia to further examine this concept. Previous studies delineating the morphological features in the livers of neonates with extrahepatic biliary atresia have concentrated on changes associated with extrahepatic biliary obstruction' '. In the present study we have confirmed that bile ductular proliferation, bilirubinostasis, cholangiolitis and biliary piecemeal necrosis are almost invariably present at the time of initial biopsy. These findings indicate that the neonatal liver responds in a similar manner to adult liver when there is cholestasis. It is of interest, however, that we did not observe Mallory bodies; it may be that the duration of cholestasis in our cases was insufficient to induce this change. Several investigators have drawn attention to changes occurring in the porta hepatis in extrahepatic biliary atresia. W i t ~ l e b r e n ' ~and , ' ~ KasaiI9 noted that there is frequently active bile duct epithelial degeneration and that this is often associated with a mononuclear inflammatory cell infiltrate. Such observations are in
52 5
keeping with the concept that the atresia occurs as a result of progressive destruction of previously formed hepatic and common bile ducts"'. The stimulus for these necro-inflammatory lesions is at present uncertain. Morecki et al." demonstrated reovirus type 3 in the porta hepatis of an infant with extrahepatic biliary atresia and subsequently established an experimental model of the condition using this virus". However, the role of reovirus has recently been challengedl Other postulated mechanisms include chemicals14 and vascular insufficiency with ischaemic injury to bile duct epitheIium15. In our series, we have demonstrated that there is evidence of some loss of intrahepatic ducts in 16.2% of patients with extrahepatic biliary atresia at the time of the initial biopsy; in some cases this is associated with the presence of necro-inflammatory lesions involving ~ degemedium-sized bile ducts. Ito et ~ 1 . ' demonstrated nerative features within intrahepatic bile duct epithelial cells in 1 5 of their 1 7 patients with extrahepatic biliary atresia at the time of presentation: their study was, however, confined to ultrastructural observations. Progressive loss of ducts has previously been reported by light microscopyx,2i,but was observed in patients who had already undergone a surgical porto-enterostomy (Kasai procedure), occurring after 8-10 months. It has been assumed that paucity in such cases represents a secondary cicatrizing phenomenon consequent on progressive fibrosis. This mechanism is unlikely to explain our findings; in cases where paucity of intrahepatic ducts was observed, fibrosis was absent or mild. Furthermore, the mean age at initial biopsy in our six cases with paucity did not significantly differ from that of the group without this change. We suggest that paucity may represent continuation of the primary bile duct injury, with involvement of intrahepatic as well as extrahepatic ducts. However, we cannot exclude the possibility that intrahepatic paucity results from toxic injury because of retained bile acids. The abnormal ductal structures observed in 2 1.6%of our cases conformed to the descriptions of the ductal plate malformation. Such lesions have previously been described in extrahepatic biliary atresiaj but, to our knowledge, their frequency has not been documented. Although such structures may occasionally be seen in the normal newborn liver, they are absent from 4 weeks of life'". As all of our cases had their initial biopsy at a later stage we believe that the presence of ductal plate structures is of pathological significance. We cannot entirely exclude the possibility that these structures may represent a form of ductular proliferation which is peculiar to the neonate. However, the appearances are quite distinct from those of marginal ductular prolifer-
'.
526
E.A.Kuweily, A.A.M.Gibson and A.D.Burt
Table 2. P a t t e r n s ofintrahepatic cholangiopathy in 37 cases of extrahepatic biliary atresia
'me A
u
c D
Description Obstructive features o n l y Obstructive features and paucity of intrahepatic d u c t s Obstructive features a n d ductal plate malformation Obstructive features, paucity, d u c t a l plate malformation
No. of cases
(x)
2 4 (65) 5 (1 3.5)
7 (19) l(2.5)
ation and we are not aware of having observed such lesions in other forms of neonatal cholestasis, such as that associated with total parenteral nutrition2'. Their similarity to the lesions observed in congenital hepatic fibrosis is striking and we believe that they represent evidence of persistence of fetal structures. On the basis of our findings we have identified four patterns of intrahepatic cholangiopathy in extrahepatic biliary atresia (Table 2).In 65%of cases the predominant features are those associated with biliary obstruction, including bile ductular proliferation and cholangiolitis. In a smaller number these changes are accompanied by evidence of paucity of intrahepatic ducts, while in another group they are associated with ductal plate malformations. In one of our patients paucity and ductal plate malformations co-existed with features of obstruction. Clinical follow-up on some of our earlier cases is incomplete. Furthermore, only 1 5 of our cases were referred for surgical porto-enterostomy. The prognostic importance of these patterns is therefore uncertain and merits further investigation in prospective studies. Nevertheless, the demonstration of paucity of intrahepatic ducts and ductal plate malformations in some cases of extrahepatic biliary atresia suggests that there may be overlap between the infantile cholangiopathies, and lends support to Desmet's hypothesis3.
Acknowledgements Dr Raweily gratefully acknowledges the support of the Jordan University of Science and Technology.
References I . Balisteri WF. Neonatal cholestasis: lessons from the issues for the future. Srrniir. Liver. Dis. 1987: 7; Foreword. 2. Baptista A. Bianchi 1,. de Groote j et al. Histopathology of the intrahepatic biliary tree. Liver 1983; 3; 161-175.
3. Desmet VJ. Cholangiopathies: past, present and future. Srmiri. Liver. Dis. 1987: 7; 67-76. 4. MacSween RNM, Burt AD. Pathology of the intrahepatic bile ducts. In: Anthony PP. MacSween RNM eds. Recent Advances in Histopathology Volume 1 4 . Edinburgh: Churchill Livingstone. 1989; 161-184. 5 . Jorgensen M].The ductal plate malformation. Artu Puthol. Mirrobid. Scund. (Suppl.) 1977; 257; 1-88. 6. Landing BH. Considerations of the pathogenesis of neonatal hepatitis. biliary atresia and cholendochal cyst: thc concept of infantile obstructive cholangiopathy. Pro$. Pnl. Sirrg. 1974: 6; 113-1 39. 7 Hass J. Bile duct and liver pathology in biliary atresia. World/. Surg. 1978: 2: 561-569. 8. Alagille D. Closing remarks. In: Kasai M ed. Biliary Atreisa arid its Related Disorders. Amsterdam: Excerpta Medica: 30 1-302. 9. Desmet VJ. Intrahepatic bileductsunderthe 1ens.j. Hrpatol. 1985: 1 ; 545-559. 1 0 . Van Eyken P. Schiot R. Callea F et al. The development of the intrahepatic bile ducts in man: A keratin imniunohistochemical study. HPpatologg 1988: 8 ; 1586-1 595. 1 1. Schlegel R, Banks-Schlegel S. Pinkus GS. Immunohisto-chemical localisation of keratin in normal human tissues. Lab. Iriwst. 1980; 42; 91-96. 12. Woodcock-Mitchell I. Eicher R. Nelson WC. Sun'lT. lmmunolocalisation of keratin polypeptides in human epidermis using monoclonal antibodies. ]. Cell Biol. 1982; 95; 580-5.88. 13. Sterberger LA, Cucullis IS. Methods of enzymatic identification of immunocytochemical reaction without use of labelled antibodies. ]. Histochem. Cytochem. 1969: 17; 190-193. 14. Hsu M, Raine L. Fanger M. Llse of avidin-tiiotin peroxidase complex (ABC) in immunoperoxidase techniques. /. Histurhm. Cgtochem. 1981: 29: 577-580. 1 5. Alagille D. Intrahepatic biliary atresia (hepatic: ductular hypoplasia). In: Berenberg SR ed. L i v u Disease in Itijatqj arid Chilrlhomf. Baltimore: Williams & Wilkins. 1976; 129-142. 16. Burt AD, Stewart JA. Aitchison M. MacSween RNM. Expression of tissue polypeptide antigen (TPA) in fetal and adult liver: changes in liver disease. 1. Clin. Puthol. 1987: 40; 719-7'24. 17. Witzlebren CL. Extrahepatic biliary atresia: concepts of cause, diagnosis and management. In: Rosenberg HS. Bolande RP eds. Perspectives in Pediatric Pathology. New York: klasson. 1979; 5; 41-62. 18. Witzlebren CL. The pathogenesis of biliary atresia. In: Javitt N H ed. Neonatal Hepatitis and Bi1iar.q Atresia. London: Castle House. 198 1 ; 3 39-3 50. 19. Kasai M. Intra- and extrahepatic bile ducts in biliary atresia!.In: lavitt NB ed. Neonatal Hepatitis and Biliary Atresiu. London: Castle House. 1981: 351-364. 20. Perez-Soler A. The inflammatory and atresia inducing disease of the liver and bile ducts. Monogr. Paediatr. 197b; 8; 1-245. 21. Morecki R. Glaser jH. Johnson AB. Kress Y. Detection of reovirus type 3 in the porta hepatis of an infant with extrahepatic biliary atresia: ultrastructural and immunohistocheniical study. Hepatology 1984; 4; 1137-1141. 22. Glaser JH. Morecki R. Reovirus type 3 and neonatal cholestasis. Semin. Liver Dis. 1987: 7; 100-1 07. 23. Brown WR, Sokol RJ, Levin MJ et al. Lack of correlation between infection with reovirus 3 and extra-hepatic biliary atresia or neonatal hepatitis. j . Paediatr. 1988; 1 1 3; 670-676. 24. Ogawa T. Suruga K, Kojima Y, Kitahar T Kuwabara N. Experimental study of the pathogenesis of infantile obstructive cholangiopathy and its clinical evaluation. ]. Pediatr. Surg. 198 3; 18;131-135.
lntrahepputic bile ducts in extrahepatic biliary atresia
2 5. Kippel C. A new theory of biliary atresia. /. Pediatr. Surg. 1972; 7; 651-654. 26. Ito T. Horisawa M. Ando H. Intrahepatic bile ducts in biliary atresia-a possible factor determining prognosis. /. Prdiatr. Surg. 1983: 18; 124-130.
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27. Ohi R. Kasai M. Takahashi T. Intrahepatic biliary obstruction in congintal bile duct atresia. Tohokir /. Exp. Med. 1969: 99; 129135. 28. Bower RH. Hepatic complications of p a r e n t e d nutrition. Srmin. Liver Dis. 1983; 3 ; 2 16-224.
Abnormalities of intrahepatic bile ducts in extrahepatic biliary atresia E.A.RAWEILY, A.A.M.GIBSON* & A.D.BUKT University Department of Pathology, Western Infirmary and *Department of Pathology, Royal Hospital for Sick Children, Glasgow, Scotland, UK Date of submission 1 June 1990 Accepted for publication 1 1 July 1990
R A W E I L Y E . A . , GIBSON A . A . M . & B U R T A . D .
(1990) Histopathology 17, 521-527
Abnormalities of intrahepatic bile ducts in extrahepatic biliary atresia The infantile cholangiopathies are a group of conditions associated with neonatal jaundice, which include extrahepatic biliary atresia, paucity of intra-hepatic bile ducts and disorders associated with persistence of fetal biliary structures, the so-called ductal plate malformations. Although previously regarded as distinct entities, it has recently been suggested that they may represent parts of a disease spectrum in which the principal process is one of bile duct destruction, the morphological manifestations in individual cases being influenced by the stage of intra-uterine development at which such injury occurs and by the site within the biliary system at which
there is maximum damage. To further examine this concept, we have studied liver biopsy specimens from 3 7 neonates with extrahepatic biliary atresia, with particular reference to abnormalities of the intrahepatic bile ducts. Paucity of intrahepatic ducts, defined as a bile duct: portal tract ratio of less than 0.9, was identified in six cases (16.2%). In eight cases (21.60/,) we found concentric tubular ductal structures similar to those observed in ductal plate malformations. In one case, both abnormalities could be demonstrated. Our findings support the concept that there is overlap between the various types of infantile cholangiopathy.
Keywords: extraheptic biliary atresia, infantile cholangiopathy, paucity of intrahepatic bile ducts, ductal plate malformation
Introduction Neonatal cholestasis with conjugated hyperbilirubinaemia occurs in approximately 2.35 per lo4live births': over a third of these are associated with disorders of the bile ducts. These infantile cholangiopathies include conditions in which there is atresia of either the extrahepatic or intrahepatic bile ducts. Although true agenesis may occur, there is increasing acceptance of the concept that these disorders result from injury to, and destruction of. ducts that have already A further group of congenital bile duct disorders is characterized by the persistence of fetal biliary structures, an appearance which has been referred to as the ductal plate malformation5. This group includes infantile polycystic disease and congenital hepatic fibrosis. Address for correspondence: Dr A.Ll.Rurt. Division of Pathology. School of Pathological Sciences. University of Newcastle-upon-Tyne. Royal Victoria Infirmary. Newcastle-upon-Tyne N E 4 YHE. IJK.
Several investigators have indicated that there may be overlap between the extrahepatic and intrahepatic atresias, and between the atresias and the ductal plate malformations 3.h-8, although the frequency with which these conditions co-exist has not been documented. On the basis of these observations, D e ~ m e thas ~ . ~proposed a hypothesis which suggests that the infantile cholangiopathies represent a disease spectrum in which the principal pathological process is that of active destruction of bile duct radicles. The morphological appearances in individual cases would thus be dependent upon the stage of fetal development, and the site within the biliary system, at which such injury occurred. Those characterized by the ductal plate malformation may represent cases in which the injury had occurred early in fetal life, with destruction and ineffective regression of immature bile ducts, while those characterized by atresia represent examples of injury at a later stage, with destruction of fully formed extra- or intrahepatic ducts.
52 1
522
E.A. Raweily. A.A.M.Gibson and A.D.Rurt
In order to further examine this concept, we have investigated the intrahepatic morphological changes occurring in 37 neonates in which a diagnosis of extrahepatic biliary atresia was made on the basis of surgical or radiological findings.
Materials and methods liver biopsy specimens from 3 7 neonates seen over a 2 5year period were obtained from the files of the Department of Pathology, Koyal Hospital for Sick Children, Glasgow. In each case a diagnosis of extrahepatic biliary atresia was based on operative findings and/or cholangiography. None of the patients exhibited clinical features of Alagille’s syndrome. In some of the cases, serial biopsies were obtained following operative intervention (Kasai procedure). However, this study was limited to a n assessment of the histological changes in the initial wedge biopsies obtained at laparotomy. In all cases, the initial biopsy was taken before 32 weeks of age (mean = 1 1.05; range = 4-32 weeks). Sections, 5 pm thick, were stained with haematoxylin and eosin, Masson’s trichrome. periodic acid-Schiff (with and without diastase), Shikata’s orcein and Perk’ reagent. In some cases, in order to enhance visualization of biliary structures, sections were stained with antibodies which detect cytokeratin sub-types expressed by bile duct epithelium but not hepatocytes”’. For these immunohistochemical studies, sections were treated with 0.1% trypsin (type I1 porcine T8 128, Sigma, Poole, Dorset. UK) prior to the application of the primary antibodies. Two primary antibodies were used: (i) polyclonal anti-keratin (Dako, High Wycombe, UK)” and (ii) mouse monoclonal antibody AE1 (ICN, High Wycombe. UK)”. A peroxidase-anti-peroxidase method’ was used with the polyclonal antiserum and a n avidin-biotin methodI4 with the monoclonal antibody. In each biopsy the presence of the following features was ahsessed by two independent observers (A.D.B. & E.R.): bilirubinostasis: bile duct dilation: cholangitis; cholangiolitis: bile ductular proliferation: biliary piecemeal necrosis: giant cell transformation: Mallory bodies. Particular attention was paid to the presence of ductal structures with an abnormal architecture, such as those described for the ductal plate m a l f ~ r m a t i o n ~In, ~each . biopsy, all portal tracts were examined and the number of bile duct radicles counted: biopsies with a bile duct :portal tract ratio of less than 0.9l5were considered to show paucity of intrahepatic ducts.
Results The frequency of the various histological features in the initial biopsies from the 3 7 cases is outlined in Table 1.In
Table 1. Histological features in 37 cases ofextrahepatic biliary atresia No. of cases (o(,)
Bilirubinostasis Dilatation of bile ducts Cholangitis
Cholangiolitis Bile ductular proliferation Biliary piecemeal necrosis Giant cell transformation Paucity of intrahepatic ducts Abnormal ductal structures (concentric tubular, reduplicated)
36
(97.3)
1 1 (29.7) 12 ( 3 2 . 5 ) 3 5 (94.5) 37 ( 1 0 0 ) 32 (86.4) 19 ( 5 1 . 3 ) h ( 16.2) 8 (21.6)
all cases there was bile ductular proliferation at the limiting plate and in periportal zones. The degree to which this occurred varied between cases: in some this was a marked feature (Figure 1): in most cases it was associated with a cholangiolitis. Cholangitis with a periductal and intraluminal polymorph infiltration was seen in 12 cases: 1 1 of these showed foci of bile duct dilatation. Accumulation of copper-associated protein was frequently observed in periportal hepatocytes using Shikata’s orcein stain (Figure 2 ) but Mallory bodies were not identified in any case. Giant cell transformation was seen in 1 9 cases, but was generally mild, predominantly involving acinar zone 111. In six cases there was a bile duct: portal tract ratio of less than 0.9, indicating paucity of intrahepatic ducts (Figure 3). In three of these, necro-inflammatory lesions involving medium-sized bile ducts were observed (Figure 4); no fibro-obliterative lesions were seen. In eight cases, abnormal ductal structures were noted. These lesions were present in only some portal tracts, their prevalence varying between cases. In some they were seen to comprise a single tubular structure encompassing a central fibrovascular core (Figures 5 & 6): in others a more complex pattern was observed, with reduplication ofthe tubular structures (Figures 7 & 8).In one case in which such features were observed, there was also evidence of paucity of intrahepatic ducts.
Discussion The infantile cholangiopathies include conditions in which there is loss of intrahepatic or extrahepatic bile ducts and disorders characterized by persistence of fetal biliary structures4. The latter group are best understood by considering normal hepatic development in the fetus.
-
Figure 1. Ductular proliferation: this was a n almost invariable feature. The ductules are surrounded by a mixed inflammatory cell infiltrate (cholangiolitis). H & E. Figure 2. Retention of copper-associated protein in periportal hepatocytes. Shikata’s orcein.
Figure 3. Paucity of intrahepatic ducts; note the absence of bile duct radicle in this medium-sized portal tract. H & E. Figure 4. Necroinflammatory lesion: this was seen in three cases in which there was paucity of intrahepatic ducts. Note degenerative changes in the ductal epithelium. H & E.
ligure 6. Concentric tubular structures were readily identified in immunohistochemical preparations: here ductal epithelium is stained with monoclonal antibody AEI. ABC method.
Figure 7. Complex reduplicated ductal pattern. This resembles so-called ductal plate malformation. H & iti
.
Figure 8. Low-power micrograph illustrating complex reduplicated tubular structures reacted with monoclonal antibody AEI . ABC method.
fritraheputic bile ducts in extrahepatic biliarg atresia
The intrahepatic bile ducts are derived from primitive hepatocytes which abut onto the mesenchyme of developing portal tracts. From around 9 weeks of gestation these cells undergo a series of phenotypic changes; they become flattened and exhibit altered expression of cytokeratins and other cytoskeletal proteins'".'h. This cell layer, referred to as the ductal plate5, subsequently becomes reduplicated with the formation of a tubular structure which surrounds the portal tract. From around 12 weeks of gestation, there is remodelling of this structure with gradual incorporation into the connective tissue surrounding portal vein branches. Parts of the ductal plate are then resorbed, leading to the development of individual 'mature' bile ducts. This process of remodelling proceeds until term and, by 4 weeks of life, the appearances are similar to those of adult liver'". Several conditions, including congenital hepatic fibrosis and infantile polycystic disease, are characterized by persistence and even duplication of the tubular structures. It has been suggested that such 'ductal plate malformations' are the result of a n intra-uterine insult leading to arrested development of the bile ducts3,9. Traditionally, extrahepatic atresia, paucity of intrahepatic bile ducts and the ductal plate malformations have been regarded as distinct entities. However, Desmet 1.9 has suggested that these may form part of disease spectrum, sharing a common pathogenetic mechanism of intra-uterine bile duct destruction. If this is true it may be expected that paucity of intrahepatic ducts and ductal plate malformation should occur in some neonates with extrahepatic biliary atresia. The aim of the present study was to reassess the intrahepatic changes occurring in extrahepatic biliary atresia to further examine this concept. Previous studies delineating the morphological features in the livers of neonates with extrahepatic biliary atresia have concentrated on changes associated with extrahepatic biliary obstruction' '. In the present study we have confirmed that bile ductular proliferation, bilirubinostasis, cholangiolitis and biliary piecemeal necrosis are almost invariably present at the time of initial biopsy. These findings indicate that the neonatal liver responds in a similar manner to adult liver when there is cholestasis. It is of interest, however, that we did not observe Mallory bodies; it may be that the duration of cholestasis in our cases was insufficient to induce this change. Several investigators have drawn attention to changes occurring in the porta hepatis in extrahepatic biliary atresia. W i t ~ l e b r e n ' ~and , ' ~ KasaiI9 noted that there is frequently active bile duct epithelial degeneration and that this is often associated with a mononuclear inflammatory cell infiltrate. Such observations are in
52 5
keeping with the concept that the atresia occurs as a result of progressive destruction of previously formed hepatic and common bile ducts"'. The stimulus for these necro-inflammatory lesions is at present uncertain. Morecki et al." demonstrated reovirus type 3 in the porta hepatis of an infant with extrahepatic biliary atresia and subsequently established an experimental model of the condition using this virus". However, the role of reovirus has recently been challengedl Other postulated mechanisms include chemicals14 and vascular insufficiency with ischaemic injury to bile duct epitheIium15. In our series, we have demonstrated that there is evidence of some loss of intrahepatic ducts in 16.2% of patients with extrahepatic biliary atresia at the time of the initial biopsy; in some cases this is associated with the presence of necro-inflammatory lesions involving ~ degemedium-sized bile ducts. Ito et ~ 1 . ' demonstrated nerative features within intrahepatic bile duct epithelial cells in 1 5 of their 1 7 patients with extrahepatic biliary atresia at the time of presentation: their study was, however, confined to ultrastructural observations. Progressive loss of ducts has previously been reported by light microscopyx,2i,but was observed in patients who had already undergone a surgical porto-enterostomy (Kasai procedure), occurring after 8-10 months. It has been assumed that paucity in such cases represents a secondary cicatrizing phenomenon consequent on progressive fibrosis. This mechanism is unlikely to explain our findings; in cases where paucity of intrahepatic ducts was observed, fibrosis was absent or mild. Furthermore, the mean age at initial biopsy in our six cases with paucity did not significantly differ from that of the group without this change. We suggest that paucity may represent continuation of the primary bile duct injury, with involvement of intrahepatic as well as extrahepatic ducts. However, we cannot exclude the possibility that intrahepatic paucity results from toxic injury because of retained bile acids. The abnormal ductal structures observed in 2 1.6%of our cases conformed to the descriptions of the ductal plate malformation. Such lesions have previously been described in extrahepatic biliary atresiaj but, to our knowledge, their frequency has not been documented. Although such structures may occasionally be seen in the normal newborn liver, they are absent from 4 weeks of life'". As all of our cases had their initial biopsy at a later stage we believe that the presence of ductal plate structures is of pathological significance. We cannot entirely exclude the possibility that these structures may represent a form of ductular proliferation which is peculiar to the neonate. However, the appearances are quite distinct from those of marginal ductular prolifer-
'.
526
E.A.Kuweily, A.A.M.Gibson and A.D.Burt
Table 2. P a t t e r n s ofintrahepatic cholangiopathy in 37 cases of extrahepatic biliary atresia
'me A
u
c D
Description Obstructive features o n l y Obstructive features and paucity of intrahepatic d u c t s Obstructive features a n d ductal plate malformation Obstructive features, paucity, d u c t a l plate malformation
No. of cases
(x)
2 4 (65) 5 (1 3.5)
7 (19) l(2.5)
ation and we are not aware of having observed such lesions in other forms of neonatal cholestasis, such as that associated with total parenteral nutrition2'. Their similarity to the lesions observed in congenital hepatic fibrosis is striking and we believe that they represent evidence of persistence of fetal structures. On the basis of our findings we have identified four patterns of intrahepatic cholangiopathy in extrahepatic biliary atresia (Table 2).In 65%of cases the predominant features are those associated with biliary obstruction, including bile ductular proliferation and cholangiolitis. In a smaller number these changes are accompanied by evidence of paucity of intrahepatic ducts, while in another group they are associated with ductal plate malformations. In one of our patients paucity and ductal plate malformations co-existed with features of obstruction. Clinical follow-up on some of our earlier cases is incomplete. Furthermore, only 1 5 of our cases were referred for surgical porto-enterostomy. The prognostic importance of these patterns is therefore uncertain and merits further investigation in prospective studies. Nevertheless, the demonstration of paucity of intrahepatic ducts and ductal plate malformations in some cases of extrahepatic biliary atresia suggests that there may be overlap between the infantile cholangiopathies, and lends support to Desmet's hypothesis3.
Acknowledgements Dr Raweily gratefully acknowledges the support of the Jordan University of Science and Technology.
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