Annals of the Royal College of Surgeons of England (1978) vol 6o

New perspectives on biliary atresia

R E Jenner FRCSSenior Surgical Registrar, King's College Hospital, London

SummaryAn investigation into the aetiology, diagnosis,and treatment of biliary atresia was carriedout because the prognosis remains so poor.

In an electron microscopical study no viralparticles or viral inclusion bodies were seen,nor were any specific ultrastructural featuresobserved. An animal experiment suggested thatobstruction within the biliary tract of newbornrabbits could be produced by maternal intra-venous injection of the bile acid lithocholicacid.A simple and atraumatic method of diag-

nosis was developed using 99mTc-labelled com-pounds which are excreted into bile. Twocompounds, 99mTc-pyridoxylidene glutamate(99mTc-PG) and 99mTc-dihydrothioctic acid("9mTc-DHT) were first assessed in normalpiglets and piglets with complete biliary ob-struction. Intestinal imaging correlated withbiliary tract patency, and the same correlationwas found in jaundiced human adults, in whomthe 99mTc-PG scan correctly determined biliarypatency in 2I out of 24 cases. The 99mTc-PGscan compared well with liver biopsy and .1S-Rose Bengal in the diagnosis of I I infants withprolonged jaundice.A model of extrahepatic biliary atresia was

developed in the newborn piglet so that dif-ferent methods of bile drainage could be as-sessed. Priorities in biliary atresia lie in a betterunderstanding of the aetiology and early diag-nosis rather than in devising new bile drainageprocedures.IntroductionAlthough biliary atresia was recognised as acause of neonatal jaundice in i 8oo, the progno-sis still remains gloomy. There are three mainreasons why today's results are so poor: thecause(s) are unknown, diagnosis is difficult, andonly a minority of infants are cured by radicalsurgery. This paper describes a tripartite studyinto the aetiology, diagnosis, and treatment ofbiliary atresia.HlunteIian Lecture delivered on i6th June 1977

AetiologyAt first glance the term congenital biliary atr'e-sia might suggest that the disease results froma mishap occurring during the intrauterinedevelopment of the biliary system, an assump-tion which is unlikely for -the following rea-sons: (i) biliary atresia is usually an isolatedlesion without other foregut defects; (2) biliaryatresia has rarely been reported in a stillborninfant; (3) the colour of the first meconium isusually normal; (4) histologically there is vari-able replacement of the intra- and extrahepaticbile ducts by fibrous tissues; and (5) among thevisceral abnormalities associated with thalido-mide was duodenal atresia, but biliary atresiawas not mentioned'. The pathology thuspoints to an acquired inflammatory pro-cess rather than an error in develop-ment. In a closely reasoned article Land-ing2 concluded that transplacental passage ofserum hepatitis virus was the most likely causeof both neonatal hepatitis and biliary atresia.Another hypothesis that has been suggested isthat excess synthesis of a toxic bile acid couldproduce the unusual and varied pathology ofbiliary atresia and possibly also neonatal hepa-titis3, although recent epidemiological evidencesuggests that biliary atresia and neonatal he-

e4.atitis are different diseasesELECTRON MICROSCOPICAL STUDYLiver biopsy specimens and segments of gall-bladders and bile ducts from infants with bili-ary atresia were examined with an AEI Corinth275 electron microscope. All the specimenswere taken at laparotomy once the diagnos'isof biliary atresia was confirmed and were pro-cessed using a standard technique. For comp-arison a specimen of normal infant liver wasobtained during a staging laparotomy fortumnour. Liver biopsy specimens from pigletswith complete biliary obstruction, the biliarytracts of two 5-month aborted human fetuses,and the bile duct from a 14-year-old transplantdonor were also examined.

368 R E Jenner

FIG. I Biliary atresia, liver cell X 2850. Bile-pigment-like material (arrowed) is seen in thecytoplasm. Equidistant from the 3 liver cellnuclei (N) is an essentially normal bile cana-liculus. Below it lie parallel arrays of granularendoplasmic reticulum (ger).

Findings The majority of liver cells in biliaryatresia appeared to have a normal structure.Viral particles or inclusion bodies were notidentified in the nuclei or cytoplasm. The cyto-plasm generally did not show any strikingabnormality except at the excretory pole of theliver cell, where bile-pigment-like material wasseen in all specimens (Fig. I). The bile canal-iculi showed variable dilatation with loss ofmicrovilli (Fig. 2a). These alterations, though,are not specific because they were also seen inpiglets with obstruction of the bile ducts (Fig.2b) and the same bile canalicular changes havebeen reported in experimental drug-induced

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jauiidice'. In the portal tracts bile-pigment-likematerial was also seen in both the cytoplasmand the lumen of the bile ductules, and sur-rounding these proliferated bile ductules therewas an increased amount of collagen. Theanatomical route by which bile is 'regurgitated'into the blood in obstructive jaundice is notknown, but passage of irritant bile at this sitecould explain the periductular fibrosis.

Outside the liver the ultrastructure of thegallbladder and common bile duct showed amassive replacement of the walls by collagen.Generally there was ulceration of the mucosa,but two specimens of common bile duct show-ed foci of epithelium (Fig. 3b). The epithelialcells of the fetal gallbladder looked mature andresembled the cells lining the adult commonbile 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 intravenouslyto pregnant New Zealand White rabbits individed doses. Control animals received thebile acid solvent only. Injections were startedIo days after mating, by which time it wasestimated that the fetal bile ducts had formed.Findings Gallbladder obstruction was found in2 out of I6 rabbits born to mothers who hadreceived lithocholic acid, but no obstructionwithin the biliary tract was seen in any of the28 animals born to control rabbits or the 25rabbits born to mothers who had received 3,8-hydroxy-5-cholenoic acid.

- ~ p

Al ~ ~~ ~ ~ ~ ~ ~ ~ ~ 4

M.~~~~~~~~~~~~~~~..

FIG. 2(a) Biliary atresia, liver cell X 7250. This bile canaliculus (bc) is dilated with almost com-plete loss of microvilli (arrowed). The tight junctions (tj) appear intact. (b) Piglet liver 6 weeksafter bile duct obstructon X IO 200. Similar bile canalicular changes are seen. g = Golgi ap-paratus; m = mitochondrion.

_viL

FIG. 3(a) Common bile duct epithelium of a

young transplant donor X 48oo. The epith-elium is composed of 'light' and 'dark' cells.Electron-lucid membrane-bound secretorygranules (S) are seen in the supranuclearzone. (b) Biliary atresia, common bile duct epi-thelium X 4800. 'Light' and 'datk' cells are

also seen in which the microvilli are reduced insize, and the secretory granules are reduced innumber as well.

DISCUSSION

Whereas these studies may serve as pointersfor future investigation, some caution is neededin their interpretation. Inability to find virusesdoes not necessarily exclude a viral aetiology-firstly, virus particles may be missed with trans-mission electron microscopy and secondly,absence of virus particles at the time of lapa-rotomy does not rule out the possibility of vi-

Net perspectives on biliary atresia 369

ruses being present ab initio. The electronmicrostopical study does, however, provide fur.ther evidence for an acquired aetiology bydemonstrating bile duct epithelial cells in bili-ary atresia.The bile acid experiment suggests that litho-

cholic acid can cause obstruction within thebiliary tract of a newborn animal and that3,1-hydroxy-5-cholenoic acid is not toxic in thisrespect. Both these 'secondary' bile acids havebeen identified in the urine of infants withbiliary atresia', a surprising finding becausemonohydroxy bile acids are normally producedin the colon by bacterial dehydroxylation of'primary' bile acids. In biliary atresia, of course,'primary'. bile acids cannot be excreted into thegut. Possible sources for these 'aberrant' bileacids are either the liver, in which an altern-ative synthetic pathway is Iused, or themother. Analysis of maternal 1erum in casesof intrahepatic cholestasis of pregnancy sug-gests that the first possibility is more likely.Toxicity studies using lithocholic acid in a widevariety of animals consistently point to hepato-biliary damage7 and it is tempting to speculatethat its presence in cases of neonatal jaundicemay be a cause rather than an effect of liverinjury.DiagnosisThe differentiation of 'medical' from 'surgical'jaundice is more difficult in the neonate thanin the adult. In the latter the type of jaundicecan be diagnosed in approximately 8o% ofcases with standard clinical and laboratorymethods8. A special problem in biliary atresiais the lack of dilatation of the intrahepatic bileducts, which limits the value of ultrasoundscanning and percutaneous cholangiography inthese small and frail patients. Nevertheless,early diagnosis is most important9.

Currently, percutaneous liver biopsy and the"'I-Rose Bengal faecal excretion test are themost widely used investigations for discrimin-ating neonatal hepatitis from biliary atresia.Unfortunately neither of these tests is comp-letely reliable and both of them require timeand expertise. There is therefore a need for asimple and atraumatic method of diagnosis anda study was undertaken to assess the usefulnessof two new hepatobiliary imaging agents.Both dihydrothioctic acid (DHT) and pyrido-

370 R E Jenner

xylidene glutamate (PG) are excreted by livercells into bile and they can be labelled with99mTc, which is an almost ideal isotope for-imaging with a gamma camera.

ANIMAL STUDY

99mTc-DHT and `OmTc-PG were injected intra-venously into healthy piglets as well as pigletsthat had undergone excisional and recon-structive procedures on the bile ducts. Witha gamma camera I7 scans were performed onio normal piglets, 3 on piglets with bile ductexcision, and 3 on piglets whose obstructivejaundice had been relieved by hepaticojejun-ostomy.

Results Sequential images of the liver, gall-bladder, and intestine were seen in all healthyanimals. 9smTc-DHT produced clearer liverimages, but 99mTc-PG produced more rapidand intense gallbladder images. Images of theintestines were not seen in animals with bileduct excision, but scans performed after re-

construction by hepaticojejunostomy demon-strated intestinal transit of isotope. It wasconcluded that a 9"mTc-PG scan might beuseful in the diagnosis of obstructive lesions ofthe biliary tract'0.

CLINICAL STUDY

With ethical committee approval 37 adultsand i i infants were scanned. Thirteen adultswere volunteers with no clinical evidence ofbiliary disease and 24 adults were jaundiced(mean serum total bilirubin concentration 255,Imol/l (14.9 mg/ioo ml)). The average ageof the infants was io- weeks. The jaundicedpatients were scanned at intervals up to andincluding i8 h after injection of 99mTc-PG,and it should be noted that over half of thesepatients were referred from other hospitalsfor investigation.

TABLE I Visualisation of the intestine with99mTc-PG in jaundiced adults and infantsCause of jaundice No of Intestine

patients imagedAdults

Complete biliary obstruction Io OHepatocellular disease* IO 9Partial common bile

duct obstruction 2 2Sclerosing cholangitis ItHepatic metastases it

InfantsExtrahepatic biliary atresia 6 it

Neonatal hepatitis 3 2a1-Antitrypsin deficiency I OIntrahepatic biliary hypoplasia i I

*8 Hepatitis, I drug jaundice, i septicaemiatInconclusive result

FIG. 4 99mTc-PG scan 32 min after injection inan adult volunteer. The liver, gallbladder,extrahepatic bile duct, and duodenum (arrow-ed) are seen.

TABLE II Comparison of "9mTc-PG scan with liver biopsy and "'I-Rose Bengalfaecal excretion test in neonatal jaundice.Result Liver biopsy '31I-Rose Bengal 99mTc PG scanCorrect 8 6 8Incorrect I 2 2Equivocal 2 I I

Total II 9 II

New perspectives on biliary atresia 37I

hepatobiliary imaging agents in the normalvolunteers". 99mTc-PG was rapidly excreted

I ___ I ILby the liver and produced clear biliary images(Fig. 4).

In jaundiced adults occlusion or patency ofthe bile duct was correctly determined in 2 1cases1 (Figs 5 and 6) and these results support

, _ r / S_ _ S _ _ Z Z l_X~~~~~~~~~~~~~11 1the findings of others 13,14* The scan did notshow details of the site of the obstruction orthe pathology, but the technique was simpleand was safely performed on patients withserious illness-for example, renal failure,coagulation defects, and septicaemia.

In infants the results of TmTc-PG scanningcompared well with those of the "'I-RoseBengal faecal excretion test and with liverbiopsy interpretations. "mTc-PG scans wererepeated postoperatively on 4 infants and de-monstrated bile drainage into the gut.

FIG. 5 "9Tc-PG scan i8 h after. injection in aman who had a stone impacted in the lowerend of the common bile duct. No isotope wasseen in the gut, suggesting complete biliaryobstruction. The lower edge of the liver andurinary bladder (Bl) are the only abdominalorgans seen. U, R, and L are external cobaltmarkers placed over the umbilicus and rightand left anterior superior iliac spines respect-ively.The scan diagnoses were made on the pre-

sence or absence of isotope in the intestine,the latter being interpreted as complete biliaryobstruction. Scans were reported independ-ently either from Polaroid images containing4oo-K counts or from a colour printoutobtained from the colour television display ofEl Scint gamma camera. A right lateral viewwas required in some instances to distinguishthe right kidney from the gallbladder.Results Some patients were seriously ill, but no FIG. 6 99mTc-PG scan in a patient with acuteadverse reaction to the procedure was noted relapsing hepatitis. Biliary patency is demon-in any patient. Tables I and II record the strated by isotope- in the ascending and trans-scan findings, verse colon .17 h after injection. X and R show

Apart from one apparent false-negative gall- positions of external cobalt markers over thebladder image, successive images of the liver, xiphoid and lateral extremity of the right costalgallbladder, and intestines were seen- with both margin respectively.

372 R E Jenner

TreatmentAlthough biliary atresia is a rare disease withan incidence of approximately i in I5 ooo livebirths, it is the commonest cause of 'surgical'jaundice in the newborn infant and most clini-cians are agreed that corrective surgery per-formed before the age of i o weeks offers thebest hope for cure. LTnfortunately, about 8o%of infants will be found at laparotomy to havethe non-correctable variety of atresia-that is,they possess no suitable proximal bile duct towhich an enteric anastomosis may be made.The operation of hepatic portoenterostomy(Kasai procedure)'5 has given the best resultsin this difficult situation, but initial enthusiasmhas been tempered by the fact that this radicaloperation will cure only a minority of infants'6.-A favourable prognostic feature is the presence

of microscopic tubules in the seemingly atreticbile duct remnant in the porta hepatis. Re-cently Schweizer" reported dramatically im-proved results in newborn piglets by drainingbile via the hepatic lymphatics at the same

time as bile duct excision. This technique was

re-evaluated in the same animal but with estab-lished extrahepatic bile duct obstruction.DEVELOPMENT OF ANIMAL MODEL OF

EXTRAHEPATIC BILIARY ATRESIA

The newborn piglet was used because its sizeand physiology are comparable to those of thehuman neonate.

Liver function tests were performed and theliver histology examined at the time of ope-

ration and one month later. In addition to a

detailed study of the histological changespresent and comparison with the liver biopsyfindings in IO infants with biliary atresia, a

semiquantitative analysis of the extent of fib-rous tissue deposition was carried out. Thismorphometric study was based on a differentialpoint-counting procedure"8 using a 42-pointWeibel graticule (Wild Heerbrugg Ltd) fittedin one eyepiece.

Results and discussion Weaning and anaesthe-sia" in the newborn pig were not difficult, butproducing a reliable model of extrahepaticbiliary atresia was not straightforward. Sixgroups of animals were used, each being sub-jected to a different procedure. Fig 7 andTable III illustrate the development of themodel and the results.

GROUP ILaparotomy

GROUP IVCholocystectomy +

Lign. of CBD + IB2-C

GROUP 11Ligation of CBD

GROUP VExcision of biliary tract

GROUP III

Cholecystectomy + IB2-G

GROUP VIExcision of biliary tract

+1B2-C

FIG. 7 Operative groups in development ofanimal model for biliary atresia. CBD = com-mon bile duct; IB2- C=isobutyl 2-cyanoacry-late.Young piglets quickly formed adhesions after

laparotomy and deaths due to intestinalobstruction were seen in all operative groups.Recanalisation of the ligated bile duct in 2animals in Group II (see Fig 7) was un-expected, but sporadic reports of this pheno-menon have followed Sir Benjamin Brodie'sobservations in cats with ligated bile ducts20.A similar result occurred in Group III animalswhen intra- and extrahepatic biliary occlusionwas attempted by injection of the acrylic cem-ent isobutyl 2-cyanoacrylate (1B2-C). Onlytemporary obstruction occurred, after whichbile flow was re-established around the biliarycast.A combination of bile duct ligation and intra-

biliary injection of IB2-C (Group IV) waseffective in producing biliary cirrhosis, butmortality was high. Cholangitis and intestinal

TABLE in Animal model: results one monthpostoperativelyGroup* No of animals Satisfactory resultt

I IO 8II 3 0III 2 , 0IV II 3V 13 4VI 24 I5

*See Figure 7.tIn which clear biochemical and histological evi-dence of biliary obstruction was observed.

New perspectives on biliary atresia 373

obstruction occurred in half the animals of thisgroup and the IB2-C almost certainly causedthe cholangitis and the foreign body giant cellswhich were seen close to the particles of injectedcement.

Complete excision of the extrahepatic biliarytract (Group V) was the model advocated bySchweizer21, but this cannot be recommendedbecause of the high incidence of bile leakagefrom the ligated bile duct remnant (6 out ofI3 animals). Nevertheless, all 4 Group V sur-vivors showed severe biliary cirrhosis. By apply-ing the rapidly adhesive cement over the bileduct remnant (Group VI) the incidence of bileperitonitis was almost completely eliminated.Biliary cirrhosis was seen in I4 of the I 5 sur-viving Group VI animals and extensive fibrosiswas seen in the remaining animal. Morpho-metric analysis showed a highly significant in-crease in liver fibrous tissue in Group VI ani-mals compared with controls (P<o.ooi).Gastric ulceration in the squamous portion ofthe stomach was the major complication, andhaemorrhage from the ulcer caused half thedeaths in this group, which could not be pre-vented by vitamin A prophylaxis.

Although histological appearances identicalto those of biliary atresia were seen in the por-tal tracts, differences were found in the paren-chyma, principal among which were the atro-phic appearance of the liver cells in the pigletas opposed to the swollen, pigment-laden, andmultinucleate cells seen in biliary atresia. Thissuggests that liver injury in biliary atresia can-not be explained solely on the basis of biliaryobstruction.COMPARISON IN AN ANIMAL MODEL OF TWOBILE DRAINAGE TECHNIQUESOne month after biliary obstruction had beenproduced laparotomy was performed and thehepatic hilar lymphatics were identified byintraparenchymal injection of methylene blue.These lymphatics with their connective tissuewere dissected and placed over the anti-mesenteric border of a loop of jejunum, theserosa over the apex of this loop having alreadybeen excised. Seven animals underwent thisprocedure and 9 animals underwent hepatico-jejunostomy.Results None of the 6 surviving animals inthe lymphatic group demonstrated any im-

provement in biochemistry or liver histology.On the other hand 6 out of 7 animals survivinghepaticojejunostomy lost their jaundice andone month later their liver histology was indi-stinguishable from that of the controls.This study shows that the fibrosis and cirrhosis

seen in a newborn animal with biliary obstruc-tion can be reversed when effective bile drain-age is obtained. The speed and extent of thisreversal was striking. However, the failure toobtain effective bile drainage in a jaundicedanimal by way of the hepatic hilar lymphaticvessels adds another unsuccessful operative at-tempt in biliary atresia.

ConclusionA fresh look has been taken at three importantaspects of biliary atresia. The popular view thatbiliary atresia is the outcome of a viral processhas been examined and no direct evidence hasbeen found to support this contention. On theother hand obstruction within the biliary tractof newborn rabbits was produced by maternalinjection of a bile acid with cholestatic and in-flammatory properties. More precise inform-ation could be obtained by injection of litho-cholic acid directly into a primate fetus. Thescans obtained after injection of 99mTc-labelledhepatobiliary imaging agents in animals and a'difficult' group of adults and infants withobstructive jaundice suggests that the techniquemay be a useful screening test for identifyingpatients with complete biliary obstruction. Inbiliary atresia a 99mTc-PG scan may be helpfulboth in diagnosis and postoperative assessmentof bile drainage. An animal model of extra-hepatic biliary atresia was developed in whichrapid and severe liver changes were observed.Interpretation of these suggested that liver in-jury in biliary atresia is not simply the resultof biliary obstruction. An equally rapid andstriking return of liver histology to normal wasseen when effective bile drainage was obtainedin the animal model. The failure of the lympha-tic bile drainage operation emphasises the needfor further studies in aetiology and earlydiagnosis.

I thank the clinicians and technicians both at King'sCollege Hospital and the Royal College of SurgeonsResearch Establishment, Downe, who willingly gaveassistance. Professor J G Murray, Mr C T Howe, andProfessor A Bennett have given helpful criticism; and

3 R E Jenner

I am grateful to Dr J Barrett and Dr B Portmanfor their expertise in nuclear medicine and liverhistology. I am indebted most of all to Mr E RHoward, who introduced me to the mysteries ofbiliary atresia and who nurtured this project with akindly and critical interest. This work was supportedby the Medical Research Council.

I'ables I and II and Figures 5 and 6 are reproducedby kind permission of the Editors of the BritishJournal of Radiology.

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