Brazilian Journal of Medical and Biological Research (1998) 31: 911-919 Histological diagnosis of neonatal cholestasis 911 ISSN 0100-879X

Histopathological diagnosis of intra- and extrahepatic neonatal cholestasis

J.L. Santos¹, Departamentos de 1Pediatria, 2Cirurgia and 3Patologia, H. Almeida², Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil C.T.S. Cerski3 and T.R. Silveira¹

Abstract

Correspondence The histopathology of the is fundamental for the differential Key words J.L. Santos diagnosis between intra- and extrahepatic causes of neonatal cholestasis. • Liver biopsy Rua Fernandes Vieira, 501, apto. 02 However, histopathological findings may overlap and there is dis- • Neonatal cholestasis 90035-091 Porto Alegre, RS agreement among authors concerning those which could discriminate • Brasil between intra- and extrahepatic cholestasis. Forty-six liver biopsies Fax: 55 (051) 312-2090 E-mail: [email protected]. (35 wedge biopsies and 11 percutaneous biopsies) and one specimen from a postmortem examination, all from patients hospitalized for Part of a Master’s thesis neonatal cholestasis in the Service of Hospital de Clínicas presented by J.L. Santos to the de Porto Alegre, were prospectively studied using a specially designed Departamento de Gastroenterologia, histopathological protocol. At least 4 of 5 different stains were used, UFRGS, Porto Alegre, RS. and 46 hepatic histopathological variables related to the differential diagnosis of neonatal cholestasis were studied. The findings were scored for severity on a scale from 0 to 4. Sections which showed less Received June 4, 1997 than 3 portal spaces were excluded from the study. Sections were Accepted March 26, 1998 examined by a pathologist who was unaware of the final diagnosis of each case. Bile tract permeability was defined by scintigraphy of the bile ducts and operative cholangiography. The F test and discriminant analysis were used as statistical methods for the study of the hepatic histopathological variables. The chi-square method with Yates correc- tion was used to relate the age of the patients on the date of the histopathological study to the discriminatory variables between intra- and extrahepatic cholestasis selected by the discriminant function test. The most valuable hepatic histopathological variables for the dis- crimination between intra- and extrahepatic cholestasis, in decreasing order of importance, were periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, foci of myeloid metaplasia, and portal-portal bridges. The only variable which pointed to the diagnosis of intrahepatic cholestasis was myeloid metaplasia. Due to the small number of patients who were younger than 60 days on the date of the histopathological study (N = 6), no variable discriminated between intra- and extrahepatic cholestasis before the age of 2 months and all of them, except for the portal expansion, were discriminatory after this age. In with cholestasis, foci of myeloid metaplasia, whenever present in the liver biopsy, suggested intrahepatic cholestasis. Periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis and portal-portal bridges suggested extrahepatic obstructive cholestasis.

Braz J Med Biol Res 31(7) 1998 912 J.L. Santos et al.

Introduction according to Balistreri (8), one-third of neo- natal cholestasis cases are due to BA. After Neonatal cholestatic is defined 1968, with the spread of the Kasai technique by the persistence of jaundice of variable (portoenterostomy) in the Western world, intensity, choluria and hypocholic or acholic many cases which were classified as “non- stools for more than 10 days, occurring dur- correctable BA” became surgically correct- ing the first months of life (1). Its reported able with good results (9-13). Other authors frequency is variable. In Australia, the preva- agree that it is of fundamental importance to lence was of 1:5000 liveborn infants (2) and perform the before eight weeks of in Norway, Henriksen et al. (3) reported 1 life in order for it to be effective and that the case of neonatal cholestasis per 9000 live- results of performed after this pe- born infants. In the Pediatric Gastroenterol- riod are poor. Thus, the differential diagno- ogy sector of Hospital de Clínicas de Porto sis between obstructive extrahepatic causes, Alegre, about 25% of the outpatient visits particularly BA, and intrahepatic causes, es- are related to neonatal cholestasis (4). Neo- pecially INH, is of great importance. Among natal cholestasis is caused by two large groups the tests used for this purpose, the liver of diseases, i.e., extrahepatic (EHC) and in- biopsy is of great importance. trahepatic (IHC) conditions. This division The differential histopathological study based on the presence or absence of extrahe- of neonatal cholestasis is performed by de- patic obstruction to bile flow is useful with termining the presence or absence of vari- respect to the intervention strategy to be ables which characterize either group of dis- used, since in terms of management, the eases - IHC or EHC. The histopathological patient belonging to the first group needs to characterization of INH by Craig and Land- undergo surgery, whereas those belonging to ing (6) introduced the perspective of mor- the second group are subjected to nonsurgi- phological differentiation between INH and cal treatment. Although there is a large num- BA. At large centers, the histopathological ber of possible causes, the relative frequency study is the method of choice, among others of idiopathic neonatal (INH) and of such as scintigraphy of the bile ducts, duode- biliary atresia (BA) far exceeds that of any nal intubation, ultrasonic echography and other etiology. In the United States, these cholangiography. Correction rates of about two disorders account for 70 to 80% of all 90% after this differential diagnosis through cases of neonatal cholestasis (5). In Brazil, a liver biopsy have been reported in the Silveira (4), in a study of 287 neonatal literature (14-17). Previous experience of cholestasis cases, found a higher frequency the pathologist with this group of diseases is of intrahepatic than extrahepatic causes of essential, since there are more similarities neonatal cholestasis. Taken together, INH than differences between IHC and EHC from and BA accounted for about 54% of all an anatomopathological point of view. The cases. INH is an inflammation of the neona- difficulties for a correct differential diagno- tal liver of unknown etiology which was sis, according to Dahms (18), include: a) a histopathologically defined by Craig and specimen with few portal triads, b) the over- Landing in 1952 (6). Balistreri (7) estimated estimation of the mild bile duct hyperplasia its frequency at 1.25 cases per 10,000 in INH, c) the nonrecognition that infants livebirths. BA is a disease occurring only in younger than 4 weeks with BA may not yet childhood whose cause is still unknown and show portal duct proliferation, and d) the which consists of total obliteration of part or nonrecognition that this proliferation is not all of the extrahepatic bile ducts. Its fre- exclusive of BA. quency is 1:8,000 to 1:15,000 livebirths and, In 1985, Zerbini (19) assessed 49 hepatic

Braz J Med Biol Res 31(7) 1998 Histological diagnosis of neonatal cholestasis 913 histopathological variables in 100 biopsies moderate-severe; 4 - severe). The stains used from 78 infants with neonatal cholestasis in were hematoxylin-eosin, trichromic green, terms of their discriminant value in the dif- picrosirius, PAS and reticulin. Sections of ferential diagnosis between IHC and EHC. percutaneous biopsies which had less than 3 She divided the variables into two classes, portal spaces were excluded from the study. “indicating variables” and “guiding vari- Slides were scored by a pathologist who was ables” of the diagnosis of EHC. The former, unaware of the final diagnosis of each case. with a higher discrimination power The cases were divided into 2 groups ac- (P≤0.0001), includes portal ductal prolifera- cording to the diagnostic histopathologic tion, distribution of the portal proliferation impression: extrahepatic biliary pattern in the portal spaces, overall degree of (EHBP) or parenchymatous pattern (PP). cholestasis, cholestasis in the portal Biliary permeability was defined in each neoductules, cholestasis in the bile capillar- case by scintigraphy of the bile ducts and, in ies, and portal fibrosis. The second class, the absence of excretion of the radiotracer to with lower discriminatory power (P between the duodenum by this method, by operative 0.0001 and 0.05), includes myeloid metapla- cholangiography. sia, portal-portal bridges, periductal fibrosis, neutrophils in the general inflammatory in- Data analysis filtrate, swelling of hepatocytes and giant cells. Discriminant analysis was used as a sta- The aim of the present study was to de- tistical method for the study of the histopath- fine the discriminant value of the hepatic ological variables in order to determine pos- histopathological variables in the differen- sible significant differences between the EHC tial diagnosis of neonatal cholestasis in pa- and IHC groups based on information of tients with this disorder seen at the sector of independent variables, and which independ- Pediatric of the Pediatrics ent variables would contribute to the differ- Service of Hospital de Clínicas de Porto entiation between the 2 groups. In order to Alegre, and to identify the hepatic histopath- use this technique, the variables were origi- ological variables which discriminated be- nally treated as being continuous. Due to the tween IHC and EHC before and after the age high number of variables being studied, a of two months. previous selection of these variables by means of the F test was carried out, and only those Material and Methods which were found to be significant by this test were subjected to the discriminant anal- The slides of 46 biopsies (35 wedge biop- ysis, with the level of significance set at 5%. sies and 11 percutaneous biopsies) and one After selection by the F test, 22 variables of from a postmortem examination, all from 47 patients were assessed by the discrimi- patients hospitalized for neonatal cholestasis nant analysis test. In this test the total sample at the Pediatrics Service of Hospital de (N = 47) was divided into two groups: the Clínicas de Porto Alegre, were prospectively first group (N = 30) was used to obtain the studied from December 1987 to January discriminant function, and the second (N = 1991. At least 4 of 5 different stains were 17) for the discriminant function test (accu- used, and 46 histopathological variables from racy). A “stepwise” computational model the protocol introduced by Zerbini (19) were was used to assess the independent variables assessed, and the findings were scored on a 0 in each step, including or excluding them to 4 scale according to the degree of severity from the model according to their discrimi- (0 - absent; 1 - mild; 2 - moderate-mild; 3 - natory power. Thus, it was possible to estab-

Braz J Med Biol Res 31(7) 1998 914 J.L. Santos et al.

lish which variables were most significant discriminant hepatic histopathological vari- for the distinction between the EHC and IHC ables between IHC and EHC selected by the groups. discriminant function test. The chi-square method with Yates cor- The significance level adopted for the rection was used to relate the age of the statistical tests was 0.05 (two tailed). patients at the time of liver biopsy to the Results

Table 1 - The most significant hepatic histopatho- logical variables for the distinction between IHC The following variables were considered and EHC according to the discriminant function to be the most significant according to the test in this study. discriminant analysis test in the discrimina- Histopathological Canonical discriminant tion between IHC and EHC, in decreasing variable function coefficient order of the coefficient value of the canoni- cal discriminant function: periportal ductal 1. Periportal ductal proliferation -0.44342 2. Portal ductal proliferation -0.32899 proliferation, portal ductal proliferation, por- 3. Portal expansion -0.30354 tal expansion, cholestasis in neoductules, 4. Cholestasis in neoductules -0.26593 portal cholestasis, foci of myeloid metapla- 5. Portal cholestasis -0.25241 6. Foci of myeloid metaplasia +0.24917 sia, portal-portal bridges, focal necrosis, 7. Portal-portal bridges -0.22414 cholestasis in canaliculi, periductal fibrosis 8. Focal necrosis -0.19082 and portal-central bridges (Table 1). Figure 9. Cholestasis in canaliculi -0.17636 10. Periductal fibrosis -0.14359 1a represents the variables which indicate 11. Central portal bridges -0.10213 intrahepatic cholestasis. Since coefficients around 0.3000 (20) are considered signifi- cant in the discriminant analysis test, the

Table 2 - Discriminant hepatic histopathological variables from 8 to 11 in Table 1 contribute variables between IHC and EHC in relation to pa- little to the histopathological differential di- tient age. agnosis of neonatal cholestasis. The canoni-

No variable discriminated between IHC and EHC cal correlation of the test was 0.9237 and the in patients younger than 60 days. Chi-square with test significance was 88.24%. We can see Yates correction. that the sixth variable, foci of myeloid meta- plasia, shows a canonical discriminant func- Variable Patients older than 60 days (N = 41) tion coefficient with a plus, thus being the only variable related to the diagnosis of a χ2 Significance nonobstructive intrahepatic cause; the re- level maining variables, with a minus, point to an Periportal ductal 11.99648 0.00053 EHC diagnosis. Figure 1b represents the proliferation myeloid metaplasia variable. Portal ductal 16.08518 0.00006 Table 2 presents the age of the patients at proliferation the time of the liver biopsy related to the Myeloid 8.82869 0.00297 significance of the variables selected in the metaplasia foci discriminant analysis test for the patients Portal 13.96010 0.00019 studied. Only six patients were younger than cholestasis 60 days on the date the histopathological Cholestasis in 17.89688 0.00002 material was collected. No hepatic histo- neoductules pathological variables discriminated between Portal-portal 11.30285 0.00077 IHC and EHC before 2 months of age. After bridges 60 days of life, all variables selected by the

Braz J Med Biol Res 31(7) 1998 Histological diagnosis of neonatal cholestasis 915 discriminant function test, except for portal (19), a severe degree of myeloid metaplasia expansion, were important for the differen- was only found in the extrahepatic cholestasis tial diagnosis between intra- and extrahe- group between 2 and 4 months of age. Be- patic cholestasis. The variables with the high- fore this time, myeloid metaplasia was milder est significance level were portal ductal pro- and occurred in both intra- and extrahepatic liferation and cholestasis in neoductules. cholestasis. The author found no histologi- cal parameter for the diagnosis of intrahe- Discussion patic cholestasis. Our finding is consistent with the observations by Allagille (1), which Most authors find more support for a include extramedullary hematopoiesis among differential diagnosis among variables which those variables which characterize IHC. Re- point to EHC (Table 3). Very few histopath- cently, Romero (23), analyzing the histo- ological variables indicate IHC and, accord- pathological variables which are useful in ing to some (14,19,21), no variable suggests the discrimination among different causes of this group of diseases. However, in our study neonatal cholestasis, included foci of ex- there was a relationship between the pres- tramedullary hematopoiesis among those ence of myeloid metaplasia and the exist- which characterize idiopathic neonatal hepa- ence of IHC. According to Desmet (22), titis. In our study and in Zerbini’s study (19), myeloid metaplasia is a nonspecific finding. giant cell transformation was a nonspecific However, this author found that in cases of finding. Giant cell transformation originates early severe BA, i.e., 1/4 of the cases of BA, from a syncytial fusion of several hepato- myeloid metaplasia or gigantocyte prolifera- cytes (22) and its biological meaning is un- tion is not found. In the study by Zerbini known. In contrast to data reported by Craig

1a 1b exps exps exps ps

1 3 cv

cv

ps ps 5 exps 2 4 exps

6 1 gig dis

Figure 1 - Hepatic histopathological variables which indicate the intra- and extrahepatic forms of cholestasis in this study. 1a, Extrahepatic cholestasis: 1 - periportal ductal proliferation; 2 - portal ductal proliferation; 3 - portal expansion; 4 - cholestasis in neoductules; 5 - portal cholestasis; 6 - portal-portal bridges. 1b, Intrahepatic cholestasis: 1 - myeloid metaplasia. dis, Lobular disarray; ps, portal space; exps, expanded portal space; gig, gigantocytes; cv, central vein.

Braz J Med Biol Res 31(7) 1998 916 J.L. Santos et al.

Table 3 - Hepatic histopathological variables which are discriminatory between IHC and EHC.

References Data indicating EHC Data indicating IHC

Kasai et al. (25) Portal fibrosis, portal ductal proliferation, More marked hepatocytic degenerative neoductule cholestasis, Kupffer cell alterations and giant cell transformation cholestasis, severe portal expansion, marked lobular fibrosis with disarray of the lobular architecture even before the age of 6 months

Bennett (26) Portal ductal proliferation (particularly Intralobular fibrosis, early lobular disarray before the first 3 or 4 weeks of life), portal fibrosis. “Biliary infarcts” (pathognomonic of extra-hepatic biliary atresia)

Brough and Diffuse portal ductal proliferation - - Bernstein (14) the most important criterion. Others: proliferation of ducts on the periphery of the portal spaces

Zerbini (19) Portal ductal proliferation, cholestasis - in neoductules, marked canalicular cholestasis, marked portal fibrosis

Shiraki et al. (21) Cholestasis in neoductules - (the most specific variable)

Alagille (1) Neoductule proliferation, portal and Lobular disarray, giant cell proliferation, perilobular fibrosis, portal “biliary hepatocellular necrosis, minimal fibrosis, plugs”, normal lobular architecture, rare formation of neo-ductules, steatosis, little inflammatory response extra-medullary hematopoiesis

Present study Periportal ductal proliferation, Foci of myeloid metaplasia portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis, portal-portal bridges

and Landing (6), it is a histopathological and ductal portal proliferation (18,19). variable which is more specific for age than Chandra (24) found portal expansion in all for causal disease, thus not serving as an IHC cases of BA, with distribution in all portal marker. triads and with a varying extent of fibrosis. According to several authors, the changes Kasai et al. (25) presented it as a feature of which indicate EHC more often are portal BA. Bennett (26) differentiates the fibrosis ductal proliferation, cholestasis in neoduc- pattern of both groups of disease as being tules and portal fibrosis (Table 3). In our predominantly portal in EHC and lobular in study, we found that portal ductal prolifera- IHC. However, in the present study the most tion and cholestasis in neoductules were dis- important indicator of EHC was periportal criminatory variables between IHC and EHC. ductal proliferation, whose importance had Concerning portal fibrosis, we should clarify already been demonstrated by Brough and that it is one of the elements which charac- Bernstein in 1974 (14). Portal ductal prolif- terize portal expansion, in addition to edema eration originates from the mitotic prolifera-

Braz J Med Biol Res 31(7) 1998 Histological diagnosis of neonatal cholestasis 917 tion of the preexisting ducts (particularly in stasis in neoductules in the group of the complete mechanical biliary obstruction) and indicating variables. Portal fibrosis, assigned from ductular metaplasia of hepatocytes of by Zerbini to this group, was included in the zone 1 of the Rappaport acinus (mainly in portal expansion variable found by us. A- cases of incomplete obstruction of the bil- mong the guiding variables, our findings iary flow) (22). The study by Cocjin et al. agree in terms of portal-portal bridges and (27) seems to confirm the idea that in BA myeloid metaplasia. In the present study, the there is a coexistence of an important ductular two latter variables had the lowest canonical metaplasia with mitotic proliferation of the discriminant function coefficients among the biliary ductules on a smaller scale. Accord- variables selected (Table 1), which seems to ing to Tan et al. (28), the portal ductal prolif- agree with Zerbini’s study, in which they eration, at least on the hilar hepatic area, were placed in the group with less discrimi- would be a reactive phenomenon to the pro- natory capacity between IHC and EHC. gressive obstruction of the biliary flow due Romero (23) includes among the histo- to fibrosis. pathological variables which characterize BA Cholestasis in neoductules is considered the proliferation of bile ducts, portal expan- by Brough and Bernstein (14) to be a non- sion, fibrosis in portal tracts, the presence of specific finding, and by Shiraki et al. (21) as biliary cylinders, the disarray of hepatic pa- the most specific discriminatory element renchyma and the absence of a marked pro- between EHC and IHC. Desmet (22) calls liferation of giant cells. Among the charac- this variable “ductular bilirubinostasis”, plac- teristics of INH, the author points to the loss ing it among those findings characteristic of of parenchyma organization, the existence BA in the histopathological test. The fifth of important giant cell proliferation, the variable selected in the present study, portal bilirubinic pigmentation of the hepatocytes, cholestasis, is directly correlated to chole- the presence of biliary cylinders to a lesser stasis in neoductules. extent and foci of myeloid metaplasia. As The seventh selected variable, portal-por- previously discussed, this theoretical descrip- tal bridges, originates from what Desmet tion does not reflect the complexity of the (22) calls “fibrosing piecemeal necrosis”, differential diagnosis of neonatal cholestasis i.e., the periportal fibrosis process which by histopathology, since there is a broad leads to the formation of fibrous septa amid overlap of the findings for the intra- and portal spaces, which are still potentially re- extrahepatic cholestasis groups. Elements versible lesions, since the intrahepatic vas- such as disarray of the hepatic parenchyma, cular relationships are preserved. In our study, proliferation of giant cells and the presence as in Zerbini’s (19), this finding favored the of biliary cylinders have been found to be EHC diagnosis. In Zerbini’s work there was nonspecific. no IHC case in patients younger than 2 A criterion the pathologist has to take months. According to Cocjin’s study (27), into account in this diagnostic differentia- the fibrogenesis process seems to be due to tion is the age of onset of these histopatho- the proliferation of Ito cells for the formation logical changes in both IHC and EHC. The of the periductular stroma surrounding the proliferation of ducts in portal spaces is a neoductules at the periphery of the portal later finding in INH than in BA (19). How- spaces. ever, even in children with BA portal ductal By comparing the present findings with proliferation may be absent in patients Zerbini’s indicating and guiding variables younger than 4 weeks (18). The histopatho- (19), we can see an agreement relative to the logical variables which indicate EHC, but portal ductal proliferation and portal chole- which may be present in cases of IHC, usu-

Braz J Med Biol Res 31(7) 1998 918 J.L. Santos et al.

ally appear later and in a more focal way in dynamic process, with different characteris- this second group of diseases. In our study, tics in distinct age groups, a fact which should only 6 patients underwent biopsy before the be taken into account by the pathologist who age of two months, a fact that did not permit is investigating it. the use of this criterion as an adjuvant in the To conclude, in this study seven histo- differential diagnosis between IHC and EHC. pathological variables indicated EHC, These data stress the need for early investi- namely, periportal ductal proliferation, por- gation of patients with neonatal cholestasis tal ductal proliferation, portal expansion, through a liver biopsy to provide a more cholestasis in neoductules, portal cholestasis adequate diagnosis (29). The histopatholog- and portal-portal bridges. Only myeloid meta- ical changes in neonatal cholestasis reflect a plasia pointed to a diagnosis of IHC.

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