Annals of the Royal College of Surgeons of England (1978) vol 6o
New perspectives on biliary atresia R E Jenner FRCS Senior Surgical Registrar, King's College Hospital, London
Summary An investigation into the aetiology, diagnosis, and treatment of biliary atresia was carried out because the prognosis remains so poor. In an electron microscopical study no viral particles or viral inclusion bodies were seen, nor were any specific ultrastructural features observed. An animal experiment suggested that obstruction within the biliary tract of newborn rabbits could be produced by maternal intravenous injection of the bile acid lithocholic acid. A simple and atraumatic method of diagnosis was developed using 99mTc-labelled compounds which are excreted into bile. Two compounds, 99mTc-pyridoxylidene glutamate (99mTc-PG) and 99mTc-dihydrothioctic acid ("9mTc-DHT) were first assessed in normal piglets and piglets with complete biliary obstruction. Intestinal imaging correlated with biliary tract patency, and the same correlation was found in jaundiced human adults, in whom the 99mTc-PG scan correctly determined biliary patency in 2I out of 24 cases. The 99mTc-PG scan compared well with liver biopsy and .1SRose Bengal in the diagnosis of I I infants with prolonged jaundice. A model of extrahepatic biliary atresia was developed in the newborn piglet so that different methods of bile drainage could be assessed. Priorities in biliary atresia lie in a better understanding of the aetiology and early diagnosis rather than in devising new bile drainage procedures. Introduction Although biliary atresia was recognised as a cause of neonatal jaundice in i 8oo, the prognosis still remains gloomy. There are three main reasons why today's results are so poor: the cause(s) are unknown, diagnosis is difficult, and only a minority of infants are cured by radical surgery. This paper describes a tripartite study into the aetiology, diagnosis, and treatment of biliary atresia. HlunteIian Lecture delivered on i6th June 1977
Aetiology At first glance the term congenital biliary atr'esia might suggest that the disease results from a mishap occurring during the intrauterine development of the biliary system, an assumption which is unlikely for -the following reasons: (i) biliary atresia is usually an isolated lesion without other foregut defects; (2) biliary atresia has rarely been reported in a stillborn infant; (3) the colour of the first meconium is usually normal; (4) histologically there is variable replacement of the intra- and extrahepatic bile ducts by fibrous tissues; and (5) among the visceral abnormalities associated with thalidomide was duodenal atresia, but biliary atresia was not mentioned'. The pathology thus points to an acquired inflammatory process rather than an error in development. In a closely reasoned article Landing2 concluded that transplacental passage of serum hepatitis virus was the most likely cause of both neonatal hepatitis and biliary atresia. Another hypothesis that has been suggested is that excess synthesis of a toxic bile acid could produce the unusual and varied pathology of biliary atresia and possibly also neonatal hepatitis3, although recent epidemiological evidence suggests that biliary atresia and neonatal heatitis are different diseasese4. ELECTRON MICROSCOPICAL STUDY
Liver biopsy specimens and segments of gallbladders and bile ducts from infants with biliary atresia were examined with an AEI Corinth 275 electron microscope. All the specimens were taken at laparotomy once the diagnos'is of biliary atresia was confirmed and were processed using a standard technique. For comparison a specimen of normal infant liver was obtained during a staging laparotomy for tumnour. Liver biopsy specimens from piglets with complete biliary obstruction, the biliary tracts of two 5-month aborted human fetuses, and the bile duct from a 14-year-old transplant donor were also examined.
368
R E Jenner
FIG. I Biliary atresia, liver cell X 2850. Bilepigment-like material (arrowed) is seen in the cytoplasm. Equidistant from the 3 liver cell nuclei (N) is an essentially normal bile canaliculus. Below it lie parallel arrays of granular endoplasmic reticulum (ger).
Findings The majority of liver cells in biliary atresia appeared to have a normal structure. Viral particles or inclusion bodies were not identified in the nuclei or cytoplasm. The cytoplasm generally did not show any striking abnormality except at the excretory pole of the liver cell, where bile-pigment-like material was seen in all specimens (Fig. I). The bile canaliculi showed variable dilatation with loss of microvilli (Fig. 2a). These alterations, though, are not specific because they were also seen in piglets with obstruction of the bile ducts (Fig. 2b) and the same bile canalicular changes have been reported in experimental drug-induced W
4A
jauiidice'. In the portal tracts bile-pigment-like material was also seen in both the cytoplasm and the lumen of the bile ductules, and surrounding these proliferated bile ductules there was an increased amount of collagen. The anatomical route by which bile is 'regurgitated' into the blood in obstructive jaundice is not known, but passage of irritant bile at this site could explain the periductular fibrosis. Outside the liver the ultrastructure of the gallbladder and common bile duct showed a massive replacement of the walls by collagen. Generally there was ulceration of the mucosa, but two specimens of common bile duct showed foci of epithelium (Fig. 3b). The epithelial cells of the fetal gallbladder looked mature and resembled the cells lining the adult common bile duct (Fig. 3a). BILE ACID EXPERIMENT
Two monohydroxy bile acids, lithocholic acid (40-I00 mg) and 3,8-hydroxy-5-cholenoic acid (15-60 mg) were administered intravenously to pregnant New Zealand White rabbits in divided doses. Control animals received the bile acid solvent only. Injections were started I o days after mating, by which time it was estimated that the fetal bile ducts had formed. Findings Gallbladder obstruction was found in 2 out of I6 rabbits born to mothers who had received lithocholic acid, but no obstruction within the biliary tract was seen in any of the 28 animals born to control rabbits or the 25 rabbits born to mothers who had received 3,8-
hydroxy-5-cholenoic
2...
-
Al
~ ~~
~
~
acid.
p
~
~
~
~
~
~
4
M.~~~~~~~~~~~~~~~..
FIG. 2(a) Biliary atresia, liver cell X 7250. This bile canaliculus (bc) is dilated with almost complete loss of microvilli (arrowed). The tight junctions (tj) appear intact. (b) Piglet liver 6 weeks after bile duct obstructon X IO 200. Similar bile canalicular changes are seen. g = Golgi apparatus; m = mitochondrion.
Net perspectives on biliary atresia
369
ruses being present ab initio. The electron microstopical study does, however, provide fur. ther evidence for an acquired aetiology by demonstrating bile duct epithelial cells in biliary atresia. The bile acid experiment suggests that lithocholic acid can cause obstruction within the biliary tract of a newborn animal and that 3,1-hydroxy-5-cholenoic acid is not toxic in this respect. Both these 'secondary' bile acids have been identified in the urine of infants with biliary atresia', a surprising finding because monohydroxy bile acids are normally produced in the colon by bacterial dehydroxylation of 'primary' bile acids. In biliary atresia, of course, 'primary'. bile acids cannot be excreted into the gut. Possible sources for these 'aberrant' bile acids are either the liver, in which an alternative synthetic pathway is Iused, or the mother. Analysis of maternal 1erum in cases of intrahepatic cholestasis of pregnancy suggests that the first possibility is more likely. Toxicity studies using lithocholic acid in a wide variety of animals consistently point to hepatobiliary damage7 and it is tempting to speculate that its presence in cases of neonatal jaundice may be a cause rather than an effect of liver
injury. _viL
FIG. 3(a) Common bile young transplant donor
duct epithelium of a X 48oo. The epithelium is composed of 'light' and 'dark' cells. Electron-lucid membrane-bound secretory granules (S) are seen in the supranuclear zone. (b) Biliary atresia, common bile duct epithelium X 4800. 'Light' and 'datk' cells are also seen in which the microvilli are reduced in size, and the secretory granules are reduced in number as well. DISCUSSION
Whereas these studies may serve as pointers for future investigation, some caution is needed in their interpretation. Inability to find viruses does not necessarily exclude a viral aetiologyfirstly, virus particles may be missed with transmission electron microscopy and secondly, absence of virus particles at the time of laparotomy does not rule out the possibility of vi-
Diagnosis The differentiation of 'medical' from 'surgical' jaundice is more difficult in the neonate than in the adult. In the latter the type of jaundice can be diagnosed in approximately 8o% of cases with standard clinical and laboratory methods8. A special problem in biliary atresia is the lack of dilatation of the intrahepatic bile ducts, which limits the value of ultrasound scanning and percutaneous cholangiography in these small and frail patients. Nevertheless, early diagnosis is most important9. Currently, percutaneous liver biopsy and the "'I-Rose Bengal faecal excretion test are the most widely used investigations for discriminating neonatal hepatitis from biliary atresia. Unfortunately neither of these tests is completely reliable and both of them require time and expertise. There is therefore a need for a simple and atraumatic method of diagnosis and a study was undertaken to assess the usefulness of two new hepatobiliary imaging agents. Both dihydrothioctic acid (DHT) and pyrido-
R E
370
Jenner
xylidene glutamate (PG) are excreted by liver TABLE I Visualisation of the intestine with cells into bile and they can be labelled with 99mTc-PG in jaundiced adults and infants 99mTc, which is an almost ideal isotope for Cause of jaundice No of Intestine -imaging with a gamma camera. patients imaged Adults Complete biliary obstruction Io O 99mTc-DHT and `OmTc-PG were injected intraHepatocellular disease* IO 9 venously into healthy piglets as well as piglets Partial common bile duct obstruction 2 2 that had undergone excisional and reconSclerosing cholangitis It structive procedures on the bile ducts. With Hepatic metastases it a gamma camera I7 scans were performed on Infants Extrahepatic biliary atresia 6 normal piglets, 3 on piglets with bile duct it Neonatal hepatitis 3 2 excision, and 3 on piglets whose obstructive I a1-Antitrypsin deficiency O jaundice had been relieved by hepaticojejunI i Intrahepatic biliary hypoplasia ostomy. *8 Hepatitis, I drug jaundice, i septicaemia tInconclusive result
ANIMAL STUDY
io
Results Sequential images of the liver, gallbladder, and intestine were seen in all healthy animals. 9smTc-DHT produced clearer liver images, but 99mTc-PG produced more rapid and intense gallbladder images. Images of the intestines were not seen in animals with bile duct excision, but scans performed after reconstruction by hepaticojejunostomy demonstrated intestinal transit of isotope. It was concluded that a 9"mTc-PG scan might be useful in the diagnosis of obstructive lesions of the biliary tract'0. CLINICAL STUDY
With ethical committee approval 37 adults and infants were scanned. Thirteen adults were volunteers with no clinical evidence of biliary disease and 24 adults were jaundiced (mean serum total bilirubin concentration 255 ,Imol/l (14.9 mg/ioo ml)). The average age of the infants was io- weeks. The jaundiced patients were scanned at intervals up to and including i8 h after injection of 99mTc-PG, FIG. 4 99mTc-PG scan 32 min after injection in and it should be noted that over half of these an adult volunteer. The liver, gallbladder, patients were referred from other hospitals extrahepatic bile duct, and duodenum (arrowfor investigation. ed) are seen. i i
TABLE II Comparison of "9mTc-PG scan with liver biopsy and "'I-Rose Bengal faecal excretion test in neonatal jaundice. Result Liver biopsy 99mTc PG scan '31I-Rose Bengal Correct Incorrect Equivocal Total
8 I 2 II
6 2 I 9
8 2 I II
New perspectives on biliary atresia
I
37I
hepatobiliary imaging agents in the normal volunteers". 99mTc-PG was rapidly excreted ___theI liver by IL and produced clear biliary images
(Fig. 4). In jaundiced adults occlusion or patency of the bile duct was correctly determined in 2 1 cases1 (Figs 5 and 6) and these results support , _ r / S_ _ S _ _ Z Z l_X~~~~~~~~~~~~~11 1 the findings of others 13,14* The scan did not show details of the site of the obstruction or the pathology, but the technique was simple and was safely performed on patients with serious illness-for example, renal failure, coagulation defects, and septicaemia. In infants the results of TmTc-PG scanning compared well with those of the "'I-Rose Bengal faecal excretion test and with liver biopsy interpretations. "mTc-PG scans were repeated postoperatively on 4 infants and demonstrated bile drainage into the gut.
FIG. 5 "9Tc-PG
scan i8 h after. injection in a who had a stone impacted in the lower end of the common bile duct. No isotope was seen in the gut, suggesting complete biliary obstruction. The lower edge of the liver and urinary bladder (Bl) are the only abdominal organs seen. U, R, and L are external cobalt markers placed over the umbilicus and right and left anterior superior iliac spines respectman
ively.
The scan diagnoses were made on the presence or absence of isotope in the intestine, the latter being interpreted as complete biliary obstruction. Scans were reported independ-
ently either from Polaroid images containing 4oo-K counts or from a colour printout obtained from the colour television display of El Scint gamma camera. A right lateral view was required in some instances to distinguish the right kidney from the gallbladder. Results Some patients were seriously ill, but no FIG. 6 99mTc-PG scan in a patient with acute adverse reaction to the procedure was noted relapsing hepatitis. Biliary patency is demonin any patient. Tables I and II record the strated by isotope- in the ascending and transverse colon .17 h after injection. X and R show scan findings, Apart from one apparent false-negative gall- positions of external cobalt markers over the bladder image, successive images of the liver, xiphoid and lateral extremity of the right costal gallbladder, and intestines were seen- with both margin respectively.
372
R E Jenner
Treatment Although biliary atresia is a rare disease with in I5 ooo live an incidence of approximately births, it is the commonest cause of 'surgical' jaundice in the newborn infant and most clinicians are agreed that corrective surgery performed before the age of weeks offers the best hope for cure. LTnfortunately, about 8o% of infants will be found at laparotomy to have the non-correctable variety of atresia-that is, they possess no suitable proximal bile duct to which an enteric anastomosis may be made. The operation of hepatic portoenterostomy (Kasai procedure)'5 has given the best results in this difficult situation, but initial enthusiasm has been tempered by the fact that this radical operation will cure only a minority of infants'6. -A favourable prognostic feature is the presence of microscopic tubules in the seemingly atretic bile duct remnant in the porta hepatis. Recently Schweizer" reported dramatically improved results in newborn piglets by draining bile via the hepatic lymphatics at the same time as bile duct excision. This technique was re-evaluated in the same animal but with established extrahepatic bile duct obstruction.
GROUP I Laparotomy
GROUP 11 Ligation of CBD
GROUP
GROUP IV Cholocystectomy + Lign. of CBD + IB2-C
GROUP V Excision of biliary tract
GROUP VI Excision of biliary tract +1B2-C
III
Cholecystectomy + IB2-G
i
i o
DEVELOPMENT OF ANIMAL MODEL OF EXTRAHEPATIC BILIARY ATRESIA The newborn piglet was used because its and physiology are comparable to those of
size the
FIG. 7 Operative groups in development of animal model for biliary atresia. CBD = common bile duct; IB2- C=isobutyl 2-cyanoacrylate. Young piglets quickly formed adhesions after laparotomy and deaths due to intestinal obstruction were seen in all operative groups. Recanalisation of the ligated bile duct in 2 animals in Group II (see Fig 7) was unexpected, but sporadic reports of this phenomenon have followed Sir Benjamin Brodie's observations in cats with ligated bile ducts20. A similar result occurred in Group III animals when intra- and extrahepatic biliary occlusion was attempted by injection of the acrylic cement isobutyl 2-cyanoacrylate (1B2-C). Only temporary obstruction occurred, after which bile flow was re-established around the biliary cast. A combination of bile duct ligation and intrabiliary injection of IB2-C (Group IV) was effective in producing biliary cirrhosis, but mortality was high. Cholangitis and intestinal
human neonate. Liver function tests were performed and the liver histology examined at the time of operation and one month later. In addition to a detailed study of the histological changes present and comparison with the liver biopsy findings in IO infants with biliary atresia, a semiquantitative analysis of the extent of fibrous tissue deposition was carried out. This morphometric study was based on a differential TABLE in Animal model: results one month point-counting procedure"8 using a 42-point Weibel graticule (Wild Heerbrugg Ltd) fitted postoperatively No of animals Group* Satisfactory resultt in one eyepiece. Results and discussion Weaning and anaesthe8 I IO 0 II 3 sia" in the newborn pig were not difficult, but 0 2 III , producing a reliable model of extrahepatic II IV 3 biliary atresia was not straightforward. Six V 4 13 groups of animals were used, each being subVI 24 I5 jected to a different procedure. Fig 7 and *See Figure 7. Table III illustrate the development of the tIn which clear biochemical and histological evimodel and the results. dence of biliary obstruction was observed.
New perspectives on biliary atresia
obstruction occurred in half the animals of this group and the IB2-C almost certainly caused the cholangitis and the foreign body giant cells which were seen close to the particles of injected cement. Complete excision of the extrahepatic biliary tract (Group V) was the model advocated by Schweizer21, but this cannot be recommended because of the high incidence of bile leakage from the ligated bile duct remnant (6 out of I3 animals). Nevertheless, all 4 Group V survivors showed severe biliary cirrhosis. By applying the rapidly adhesive cement over the bile duct remnant (Group VI) the incidence of bile peritonitis was almost completely eliminated. Biliary cirrhosis was seen in I4 of the I 5 surviving Group VI animals and extensive fibrosis was seen in the remaining animal. Morphometric analysis showed a highly significant increase in liver fibrous tissue in Group VI animals compared with controls (P
New perspectives on biliary atresia R E Jenner FRCS Senior Surgical Registrar, King's College Hospital, London
Summary An investigation into the aetiology, diagnosis, and treatment of biliary atresia was carried out because the prognosis remains so poor. In an electron microscopical study no viral particles or viral inclusion bodies were seen, nor were any specific ultrastructural features observed. An animal experiment suggested that obstruction within the biliary tract of newborn rabbits could be produced by maternal intravenous injection of the bile acid lithocholic acid. A simple and atraumatic method of diagnosis was developed using 99mTc-labelled compounds which are excreted into bile. Two compounds, 99mTc-pyridoxylidene glutamate (99mTc-PG) and 99mTc-dihydrothioctic acid ("9mTc-DHT) were first assessed in normal piglets and piglets with complete biliary obstruction. Intestinal imaging correlated with biliary tract patency, and the same correlation was found in jaundiced human adults, in whom the 99mTc-PG scan correctly determined biliary patency in 2I out of 24 cases. The 99mTc-PG scan compared well with liver biopsy and .1SRose Bengal in the diagnosis of I I infants with prolonged jaundice. A model of extrahepatic biliary atresia was developed in the newborn piglet so that different methods of bile drainage could be assessed. Priorities in biliary atresia lie in a better understanding of the aetiology and early diagnosis rather than in devising new bile drainage procedures. Introduction Although biliary atresia was recognised as a cause of neonatal jaundice in i 8oo, the prognosis still remains gloomy. There are three main reasons why today's results are so poor: the cause(s) are unknown, diagnosis is difficult, and only a minority of infants are cured by radical surgery. This paper describes a tripartite study into the aetiology, diagnosis, and treatment of biliary atresia. HlunteIian Lecture delivered on i6th June 1977
Aetiology At first glance the term congenital biliary atr'esia might suggest that the disease results from a mishap occurring during the intrauterine development of the biliary system, an assumption which is unlikely for -the following reasons: (i) biliary atresia is usually an isolated lesion without other foregut defects; (2) biliary atresia has rarely been reported in a stillborn infant; (3) the colour of the first meconium is usually normal; (4) histologically there is variable replacement of the intra- and extrahepatic bile ducts by fibrous tissues; and (5) among the visceral abnormalities associated with thalidomide was duodenal atresia, but biliary atresia was not mentioned'. The pathology thus points to an acquired inflammatory process rather than an error in development. In a closely reasoned article Landing2 concluded that transplacental passage of serum hepatitis virus was the most likely cause of both neonatal hepatitis and biliary atresia. Another hypothesis that has been suggested is that excess synthesis of a toxic bile acid could produce the unusual and varied pathology of biliary atresia and possibly also neonatal hepatitis3, although recent epidemiological evidence suggests that biliary atresia and neonatal heatitis are different diseasese4. ELECTRON MICROSCOPICAL STUDY
Liver biopsy specimens and segments of gallbladders and bile ducts from infants with biliary atresia were examined with an AEI Corinth 275 electron microscope. All the specimens were taken at laparotomy once the diagnos'is of biliary atresia was confirmed and were processed using a standard technique. For comparison a specimen of normal infant liver was obtained during a staging laparotomy for tumnour. Liver biopsy specimens from piglets with complete biliary obstruction, the biliary tracts of two 5-month aborted human fetuses, and the bile duct from a 14-year-old transplant donor were also examined.
368
R E Jenner
FIG. I Biliary atresia, liver cell X 2850. Bilepigment-like material (arrowed) is seen in the cytoplasm. Equidistant from the 3 liver cell nuclei (N) is an essentially normal bile canaliculus. Below it lie parallel arrays of granular endoplasmic reticulum (ger).
Findings The majority of liver cells in biliary atresia appeared to have a normal structure. Viral particles or inclusion bodies were not identified in the nuclei or cytoplasm. The cytoplasm generally did not show any striking abnormality except at the excretory pole of the liver cell, where bile-pigment-like material was seen in all specimens (Fig. I). The bile canaliculi showed variable dilatation with loss of microvilli (Fig. 2a). These alterations, though, are not specific because they were also seen in piglets with obstruction of the bile ducts (Fig. 2b) and the same bile canalicular changes have been reported in experimental drug-induced W
4A
jauiidice'. In the portal tracts bile-pigment-like material was also seen in both the cytoplasm and the lumen of the bile ductules, and surrounding these proliferated bile ductules there was an increased amount of collagen. The anatomical route by which bile is 'regurgitated' into the blood in obstructive jaundice is not known, but passage of irritant bile at this site could explain the periductular fibrosis. Outside the liver the ultrastructure of the gallbladder and common bile duct showed a massive replacement of the walls by collagen. Generally there was ulceration of the mucosa, but two specimens of common bile duct showed foci of epithelium (Fig. 3b). The epithelial cells of the fetal gallbladder looked mature and resembled the cells lining the adult common bile duct (Fig. 3a). BILE ACID EXPERIMENT
Two monohydroxy bile acids, lithocholic acid (40-I00 mg) and 3,8-hydroxy-5-cholenoic acid (15-60 mg) were administered intravenously to pregnant New Zealand White rabbits in divided doses. Control animals received the bile acid solvent only. Injections were started I o days after mating, by which time it was estimated that the fetal bile ducts had formed. Findings Gallbladder obstruction was found in 2 out of I6 rabbits born to mothers who had received lithocholic acid, but no obstruction within the biliary tract was seen in any of the 28 animals born to control rabbits or the 25 rabbits born to mothers who had received 3,8-
hydroxy-5-cholenoic
2...
-
Al
~ ~~
~
~
acid.
p
~
~
~
~
~
~
4
M.~~~~~~~~~~~~~~~..
FIG. 2(a) Biliary atresia, liver cell X 7250. This bile canaliculus (bc) is dilated with almost complete loss of microvilli (arrowed). The tight junctions (tj) appear intact. (b) Piglet liver 6 weeks after bile duct obstructon X IO 200. Similar bile canalicular changes are seen. g = Golgi apparatus; m = mitochondrion.
Net perspectives on biliary atresia
369
ruses being present ab initio. The electron microstopical study does, however, provide fur. ther evidence for an acquired aetiology by demonstrating bile duct epithelial cells in biliary atresia. The bile acid experiment suggests that lithocholic acid can cause obstruction within the biliary tract of a newborn animal and that 3,1-hydroxy-5-cholenoic acid is not toxic in this respect. Both these 'secondary' bile acids have been identified in the urine of infants with biliary atresia', a surprising finding because monohydroxy bile acids are normally produced in the colon by bacterial dehydroxylation of 'primary' bile acids. In biliary atresia, of course, 'primary'. bile acids cannot be excreted into the gut. Possible sources for these 'aberrant' bile acids are either the liver, in which an alternative synthetic pathway is Iused, or the mother. Analysis of maternal 1erum in cases of intrahepatic cholestasis of pregnancy suggests that the first possibility is more likely. Toxicity studies using lithocholic acid in a wide variety of animals consistently point to hepatobiliary damage7 and it is tempting to speculate that its presence in cases of neonatal jaundice may be a cause rather than an effect of liver
injury. _viL
FIG. 3(a) Common bile young transplant donor
duct epithelium of a X 48oo. The epithelium is composed of 'light' and 'dark' cells. Electron-lucid membrane-bound secretory granules (S) are seen in the supranuclear zone. (b) Biliary atresia, common bile duct epithelium X 4800. 'Light' and 'datk' cells are also seen in which the microvilli are reduced in size, and the secretory granules are reduced in number as well. DISCUSSION
Whereas these studies may serve as pointers for future investigation, some caution is needed in their interpretation. Inability to find viruses does not necessarily exclude a viral aetiologyfirstly, virus particles may be missed with transmission electron microscopy and secondly, absence of virus particles at the time of laparotomy does not rule out the possibility of vi-
Diagnosis The differentiation of 'medical' from 'surgical' jaundice is more difficult in the neonate than in the adult. In the latter the type of jaundice can be diagnosed in approximately 8o% of cases with standard clinical and laboratory methods8. A special problem in biliary atresia is the lack of dilatation of the intrahepatic bile ducts, which limits the value of ultrasound scanning and percutaneous cholangiography in these small and frail patients. Nevertheless, early diagnosis is most important9. Currently, percutaneous liver biopsy and the "'I-Rose Bengal faecal excretion test are the most widely used investigations for discriminating neonatal hepatitis from biliary atresia. Unfortunately neither of these tests is completely reliable and both of them require time and expertise. There is therefore a need for a simple and atraumatic method of diagnosis and a study was undertaken to assess the usefulness of two new hepatobiliary imaging agents. Both dihydrothioctic acid (DHT) and pyrido-
R E
370
Jenner
xylidene glutamate (PG) are excreted by liver TABLE I Visualisation of the intestine with cells into bile and they can be labelled with 99mTc-PG in jaundiced adults and infants 99mTc, which is an almost ideal isotope for Cause of jaundice No of Intestine -imaging with a gamma camera. patients imaged Adults Complete biliary obstruction Io O 99mTc-DHT and `OmTc-PG were injected intraHepatocellular disease* IO 9 venously into healthy piglets as well as piglets Partial common bile duct obstruction 2 2 that had undergone excisional and reconSclerosing cholangitis It structive procedures on the bile ducts. With Hepatic metastases it a gamma camera I7 scans were performed on Infants Extrahepatic biliary atresia 6 normal piglets, 3 on piglets with bile duct it Neonatal hepatitis 3 2 excision, and 3 on piglets whose obstructive I a1-Antitrypsin deficiency O jaundice had been relieved by hepaticojejunI i Intrahepatic biliary hypoplasia ostomy. *8 Hepatitis, I drug jaundice, i septicaemia tInconclusive result
ANIMAL STUDY
io
Results Sequential images of the liver, gallbladder, and intestine were seen in all healthy animals. 9smTc-DHT produced clearer liver images, but 99mTc-PG produced more rapid and intense gallbladder images. Images of the intestines were not seen in animals with bile duct excision, but scans performed after reconstruction by hepaticojejunostomy demonstrated intestinal transit of isotope. It was concluded that a 9"mTc-PG scan might be useful in the diagnosis of obstructive lesions of the biliary tract'0. CLINICAL STUDY
With ethical committee approval 37 adults and infants were scanned. Thirteen adults were volunteers with no clinical evidence of biliary disease and 24 adults were jaundiced (mean serum total bilirubin concentration 255 ,Imol/l (14.9 mg/ioo ml)). The average age of the infants was io- weeks. The jaundiced patients were scanned at intervals up to and including i8 h after injection of 99mTc-PG, FIG. 4 99mTc-PG scan 32 min after injection in and it should be noted that over half of these an adult volunteer. The liver, gallbladder, patients were referred from other hospitals extrahepatic bile duct, and duodenum (arrowfor investigation. ed) are seen. i i
TABLE II Comparison of "9mTc-PG scan with liver biopsy and "'I-Rose Bengal faecal excretion test in neonatal jaundice. Result Liver biopsy 99mTc PG scan '31I-Rose Bengal Correct Incorrect Equivocal Total
8 I 2 II
6 2 I 9
8 2 I II
New perspectives on biliary atresia
I
37I
hepatobiliary imaging agents in the normal volunteers". 99mTc-PG was rapidly excreted ___theI liver by IL and produced clear biliary images
(Fig. 4). In jaundiced adults occlusion or patency of the bile duct was correctly determined in 2 1 cases1 (Figs 5 and 6) and these results support , _ r / S_ _ S _ _ Z Z l_X~~~~~~~~~~~~~11 1 the findings of others 13,14* The scan did not show details of the site of the obstruction or the pathology, but the technique was simple and was safely performed on patients with serious illness-for example, renal failure, coagulation defects, and septicaemia. In infants the results of TmTc-PG scanning compared well with those of the "'I-Rose Bengal faecal excretion test and with liver biopsy interpretations. "mTc-PG scans were repeated postoperatively on 4 infants and demonstrated bile drainage into the gut.
FIG. 5 "9Tc-PG
scan i8 h after. injection in a who had a stone impacted in the lower end of the common bile duct. No isotope was seen in the gut, suggesting complete biliary obstruction. The lower edge of the liver and urinary bladder (Bl) are the only abdominal organs seen. U, R, and L are external cobalt markers placed over the umbilicus and right and left anterior superior iliac spines respectman
ively.
The scan diagnoses were made on the presence or absence of isotope in the intestine, the latter being interpreted as complete biliary obstruction. Scans were reported independ-
ently either from Polaroid images containing 4oo-K counts or from a colour printout obtained from the colour television display of El Scint gamma camera. A right lateral view was required in some instances to distinguish the right kidney from the gallbladder. Results Some patients were seriously ill, but no FIG. 6 99mTc-PG scan in a patient with acute adverse reaction to the procedure was noted relapsing hepatitis. Biliary patency is demonin any patient. Tables I and II record the strated by isotope- in the ascending and transverse colon .17 h after injection. X and R show scan findings, Apart from one apparent false-negative gall- positions of external cobalt markers over the bladder image, successive images of the liver, xiphoid and lateral extremity of the right costal gallbladder, and intestines were seen- with both margin respectively.
372
R E Jenner
Treatment Although biliary atresia is a rare disease with in I5 ooo live an incidence of approximately births, it is the commonest cause of 'surgical' jaundice in the newborn infant and most clinicians are agreed that corrective surgery performed before the age of weeks offers the best hope for cure. LTnfortunately, about 8o% of infants will be found at laparotomy to have the non-correctable variety of atresia-that is, they possess no suitable proximal bile duct to which an enteric anastomosis may be made. The operation of hepatic portoenterostomy (Kasai procedure)'5 has given the best results in this difficult situation, but initial enthusiasm has been tempered by the fact that this radical operation will cure only a minority of infants'6. -A favourable prognostic feature is the presence of microscopic tubules in the seemingly atretic bile duct remnant in the porta hepatis. Recently Schweizer" reported dramatically improved results in newborn piglets by draining bile via the hepatic lymphatics at the same time as bile duct excision. This technique was re-evaluated in the same animal but with established extrahepatic bile duct obstruction.
GROUP I Laparotomy
GROUP 11 Ligation of CBD
GROUP
GROUP IV Cholocystectomy + Lign. of CBD + IB2-C
GROUP V Excision of biliary tract
GROUP VI Excision of biliary tract +1B2-C
III
Cholecystectomy + IB2-G
i
i o
DEVELOPMENT OF ANIMAL MODEL OF EXTRAHEPATIC BILIARY ATRESIA The newborn piglet was used because its and physiology are comparable to those of
size the
FIG. 7 Operative groups in development of animal model for biliary atresia. CBD = common bile duct; IB2- C=isobutyl 2-cyanoacrylate. Young piglets quickly formed adhesions after laparotomy and deaths due to intestinal obstruction were seen in all operative groups. Recanalisation of the ligated bile duct in 2 animals in Group II (see Fig 7) was unexpected, but sporadic reports of this phenomenon have followed Sir Benjamin Brodie's observations in cats with ligated bile ducts20. A similar result occurred in Group III animals when intra- and extrahepatic biliary occlusion was attempted by injection of the acrylic cement isobutyl 2-cyanoacrylate (1B2-C). Only temporary obstruction occurred, after which bile flow was re-established around the biliary cast. A combination of bile duct ligation and intrabiliary injection of IB2-C (Group IV) was effective in producing biliary cirrhosis, but mortality was high. Cholangitis and intestinal
human neonate. Liver function tests were performed and the liver histology examined at the time of operation and one month later. In addition to a detailed study of the histological changes present and comparison with the liver biopsy findings in IO infants with biliary atresia, a semiquantitative analysis of the extent of fibrous tissue deposition was carried out. This morphometric study was based on a differential TABLE in Animal model: results one month point-counting procedure"8 using a 42-point Weibel graticule (Wild Heerbrugg Ltd) fitted postoperatively No of animals Group* Satisfactory resultt in one eyepiece. Results and discussion Weaning and anaesthe8 I IO 0 II 3 sia" in the newborn pig were not difficult, but 0 2 III , producing a reliable model of extrahepatic II IV 3 biliary atresia was not straightforward. Six V 4 13 groups of animals were used, each being subVI 24 I5 jected to a different procedure. Fig 7 and *See Figure 7. Table III illustrate the development of the tIn which clear biochemical and histological evimodel and the results. dence of biliary obstruction was observed.
New perspectives on biliary atresia
obstruction occurred in half the animals of this group and the IB2-C almost certainly caused the cholangitis and the foreign body giant cells which were seen close to the particles of injected cement. Complete excision of the extrahepatic biliary tract (Group V) was the model advocated by Schweizer21, but this cannot be recommended because of the high incidence of bile leakage from the ligated bile duct remnant (6 out of I3 animals). Nevertheless, all 4 Group V survivors showed severe biliary cirrhosis. By applying the rapidly adhesive cement over the bile duct remnant (Group VI) the incidence of bile peritonitis was almost completely eliminated. Biliary cirrhosis was seen in I4 of the I 5 surviving Group VI animals and extensive fibrosis was seen in the remaining animal. Morphometric analysis showed a highly significant increase in liver fibrous tissue in Group VI animals compared with controls (P
