Abstract
Introduction:
Extrahepatic biliary atresia (EHBA) is a fibroinflammatory disorder characterised by the fibrosed development of extrahepatic biliary ducts, resulting in cholestatic jaundice. Inflammation and inflammatory cytokines have been implicated as causative factors in the development of fibrosis. The disease, in essence, is a rare entity with an incidence varying from 1 in 5000 to 1 in 18,000 live births in India. The role of inflammatory cells, such as mast cells, in causing fibrosis in liver biopsies is being researched in recent times to determine their impact on patient prognosis.
Aims and Objectives:
The aim of the study was to assess the number of mast cells in cases diagnosed as EHBA and correlate that with the degree of fibrosis and other histopathological findings in order to indicate the role of mast cells as fibrogenic inflammatory mediator.
Material and Methods:
The department of pathology carried out a retrospective observational study on 7 cases of suspected EHBA, in samples received from cases with ages from 8 weeks to 2 years, which were sent to the lab from the department of pediatric surgery. Extrahepatic biliary atresia was confirmed by histopathological examination of the liver biopsy specimen and intraoperative cholangiography taken during Kasai’s portoenterostomy. Multiple stains were done on sections, and the common scoring systems mentioned by Lee.WS and Russo P in their respective papers, were used in reporting the cases for EHBA. Fibrosis was scored as per the modified Scheuer’s system.
Results:
All the cases showed similar findings with a fair number of mast cells, variable degree of fibrosis accompanying inflammation, biliary bile plugs and degeneration in the hepatocytes. A positive trend was noted between an increase in mast cells and the degree of fibrosis in the biopsies, which were reported out as EHBA. Eosinophils were present in all the cases analyzed.
Conclusion:
Presence of Mast cells in all cases of EHBA with an increasing number of mast cells with a higher degree of fibrosis can be a potential or probable indicator of prognosis and scoring in EHBAs, however in view of the study being a pilot study with a small sample size, a larger cohort with control arm needs to be studied to confirm the findings of our study.
Keywords: Cell, extrahepatic biliary atresia, fibrosis, mast, new, count, scoring
Résumé
Introduction:
L’atrésie biliaire extrahépatique (AEBH) est une maladie fibro-inflammatoire caractérisée par le développement fibrosé des voies biliaires extrahépatiques, entraînant un ictère cholestatique. L’inflammation et les cytokines inflammatoires sont impliquées dans le développement de la fibrose. Cette maladie est par essence rare, son incidence variant de 1 sur 5 000 à 1 sur 18 000 naissances vivantes en Inde. Le rôle des cellules inflammatoires, telles que les mastocytes, dans la fibrose observée lors des biopsies hépatiques fait l’objet de recherches récentes afin de déterminer leur impact sur le pronostic des patients.
Objectifs:
L’objectif de l’étude était d’évaluer le nombre de mastocytes chez les patients diagnostiqués comme AEBH et de le corréler avec le degré de fibrose et d’autres observations histopathologiques afin d’indiquer le rôle des mastocytes comme médiateurs inflammatoires fibrogènes. Matériel et méthodes : Le service d’anatomopathologie a mené une étude observationnelle rétrospective sur sept cas suspects d’AEBH, à partir d’échantillons provenant de patients âgés de 8 semaines à 2 ans, envoyés au laboratoire par le service de chirurgie pédiatrique. L’atrésie biliaire extrahépatique a été confirmée par l’examen histopathologique de la biopsie hépatique et par une cholangiographie peropératoire réalisée lors de la porto-entérostomie de Kasai. De multiples colorations ont été réalisées sur les coupes, et les systèmes de score courants mentionnés par Lee, WS et Russo P dans leurs articles respectifs ont été utilisés pour décrire les cas d’AEBH. La fibrose a été évaluée selon le système de Scheuer modifié.
Résultats:
Tous les cas ont présenté des résultats similaires : un nombre important de mastocytes, un degré variable de fibrose accompagnant l’inflammation, des bouchons biliaires et une dégénérescence des hépatocytes. Une tendance positive a été observée entre l’augmentation du nombre de mastocytes et le degré de fibrose dans les biopsies, lesquelles ont été rapportées comme EHBA. Des éosinophiles étaient présents dans tous les cas analysés.
Conclusion:
La présence de mastocytes dans tous les cas d’EHBA, avec un nombre croissant de mastocytes présentant un degré de fibrose plus élevé, peut être un indicateur potentiel ou probable du pronostic et du score des EHBA. Cependant, compte tenu du caractère pilote de cette étude avec un échantillon réduit, une cohorte plus large avec un groupe témoin doit être étudiée pour confirmer les résultats de notre étude.
Mots-clés: Cellule, atrésie des voies biliaires extrahépatiques, fibrose, mastocytome, nouveau
INTRODUCTION
Extrahepatic biliary atresia (EHBA) is a fibroinflammatory disorder where extrahepatic biliary ducts become fibrosed, leading to cholestatic jaundice. Inflammation and inflammatory cytokines have been implicated as causative agents for fibrosis to occur.[,2,3] The disease, in essence, is a rare entity with an prevalence varying from 1 in 5000 to 1 in 18,000 live births in India.[1] EHBA is a common cause of neonatal cholestasis, which is surgically reversible. The importance of studying this disorder and its pathogenesis lies in the fact that this disease is purely surgical, and once a successful surgery is performed, neonatal cholestasis receives complete correction. Neonatal cholestasis is a commonly encountered problem in the surgical OPD. There are 2 forms of neonatal jaundice, the perinatal form and the fetal/embryonic form. Perinatal form includes causes such as genetic, toxic, vascular, and infectious and is the most common.[1] The embryonic or fetal form accounts for 10%–20% of the cases and is associated with congenital abnormalities like ductal plate malformation (DPM). This group has severe disease and poor outcomes even after surgical intervention is done.[2] 25%–30% cases of this cholestasis are attributable to EHBA.[1]
The diagnostic radiological workup done in such cases includes cholecintigraphy with Hepatobiliary Imidoacetic Acid (HIDA) scan that shows a lack of bile excretion. Kasai’s portoenterostomy is the primary treatment of choice for EHBA, and a 10-year survival rate postsurgery is around 30%.[3,4] Histopathological examination of liver biopsy is the gold standard in diagnosing EHBA, showing evidence of extrahepatic obstructive liver disease with extensive bile duct proliferation and biliary fibrosis, which is the classical presentation.[5] In 67% of the postsurgical cases, chronic liver disease will set in as the patient reached adulthood, and treatment of choice will be liver transplantation.[6]
The pathogenesis of EHBA has generated a lot of curiosity among researchers due to its early age of onset and poor outcome in pediatric population. The important aspect of research in the field of EHBA is to study the correlation of severity and outcome with fibrosis. There are some inflammatory markers and cells being attributed to the pathogenesis of EHBA, like eosinophils, mast cells, and cytokines. There is increasing evidence to show that mast cells play a key role in disease progression in cases of EHBA.
There are two types of mast cells at the molecular level or biologically based on their origin and function: connective tissue type and mucosal type. Mast cells degranulate and release many mediators, e.g., transforming growth factor-β1, tumor necrosis factor-α–α, histamine, etc., all chemicals mediating the progression of liver disease. Changes in histopathology akin to microvesicular steatosis, ductal reaction, biliary senescence, inflammation, angiogenesis, and liver fibrosis, have been reported due to mast cell degranulation.[7] Hence, the study was aimed at correlating the presence of a degree of inflammation (specifically, mast cells) with the degree of fibrosis in cases of EHBA.
MATERIALS AND METHODS
The department of pathology conducted a retrospective observational pilot study involving 7 cases of suspected EHBA, with ages ranging from 8 weeks to 2 years, that were received from the department of pediatric surgery.
The diagnosis of EHBA was confirmed by histopathological examination of the liver biopsy specimen and intraoperative cholangiography taken during Kasai’s portoenterostomy.
The samples collected in formalin were processed routinely in the histopathology lab, and H and E, Reticulin, Masson trichome, and Toluidine blue staining were performed on the biopsy to visualize various histopathological points that are to be reported in cases of EHBA. Common scoring systems mentioned by Lee. WS and Russo P, in their respective papers, were used in reporting the cases for EHBA.[4,6] The scoring system by Lee and Ruso contains multiple points, like the presence of inflammatory cells, neutrophils, and lymphocytes, the degree of fibrosis, bile ductular reaction, and each feature is given a specific score. As per te scoring system used on histopathological scoring of EHBA there are multiple parts to the scoring system. Once all parts are scored for a single case a total is done and a score>8 favours EHBA over other causes of pediatirc cholestasis(eg:neonatal cholestasis). Grading of fibrosis was done using Modified Scheuer’s grading system.[4]
Mast cell count has not yet been standardized in any scoring system; hence, the authors divided one low-power field area (0.0314 mm2) into 4 quadrants and calculated the number of mast cells in each quadrant and then averaged out the value for 1 field. Similarly, 10 fields were calculated, and a mean value of mast cell count was obtained. This was done for all cases by a single pathologist on the same microscope (Nikon E–100). The mast cells were seen scattered throughout the field, with some lying in small clusters of 2–3 mast cells. These areas were identified and counted by the pathologist [Tables 1 and 2].
Table 1.
On tabulating the data for histopathological features, the following findings were observed
| Case number | Mast cells/10 hpf | Degree of fibrosis | Other inflammatory cells | Other changes in the tissue |
|---|---|---|---|---|
| Case 1 | 10 | Grade 3 | Lymphocytes predominant with occasional eosinophil | Mild feathery degeneration in hepatocytes |
| Intracanalicular biliary plugs | ||||
| Bile ductular proliferation | ||||
| Case 2 | 7 | Grade 2 | Mild mixed inflammation comprising of lymphocytes and neutrophils, few eosinophil | Hepatic ballooning |
| Intracanalicular biliary plugs | ||||
| Mild ductular reaction | ||||
| Case 3 | 12 | Grade 2 | Lymphocytic predominant inflammation, occasional eosinophil | Intracanalicular biliary plugs |
| Mild ductular reaction | ||||
| Case 4 | 5 | Grade 1 | Mild lymphocytic inflammation, occasional eosinophil | Mild periportal inflammation |
| Case 5 | 15 | Grade 2 | Mild lymphocytic infiltration with occasional eosinophil | Marked ductular proliferation |
| Periportal swelling of hepatocytes | ||||
| Bile plugs in the portal tracts | ||||
| Case 6 | 30 | Grade 3 | Moderate lymphocytic inflammation with few neutrophils and eosinophils | Hepatocellular swelling |
| Bile ductular reaction mild | ||||
| Bile plugs present | ||||
| Case 7 | 20 | Grade 3 | Mild mixed inflammation (lymphocyte and neutrophils, few eosinophils) | Mild ballooning degeneration |
| Few multinucleated hepatocytes |
Table 2.
The association between the number of mast cells and the degree of fibrosis
| Mast cell/10 hpf | Cases | Degree of fibrosis |
|---|---|---|
| 5–10 | 2 | Fibrosis Grade 1–2 |
| 11–19 | 3 | Fibrosis Grade 2–3 |
| >20 | 2 | Fibrosis Grade 3 |
The sample size, when calculated using the formula (z2 × p (1–p)/q2 = n) with the prevalence of EHBA taken as 1 in 5000 cases of pediatric cholestasis in India, comes to be 30 cases.
All the findings were tabulated for analysis.
RESULTS
All the cases showed similar findings with a fair number of mast cells, a variable degree of fibrosis accompanying inflammation, biliary bile plugs, and degeneration in the hepatocytes [Figures 1-4]. Occasional mast cells showing degranulation (empty vacuole-like space in cytoplasm) were noted; however, no definite importance of the finding was noted to the histopathological features.
Figure 1.
H and E staining of liver biopsy at 100X shows periportal fibrosis
Figure 4.
Hand E staining at 100X shows biliary cholestasis with periportal inflammation (lymphocytes)
Figure 2.
H and E staining of liver biopsy at 400X shows bile ductular proliferation in the case of EHBA
Figure 3.
H and E staining at 400X shows intrabiliary cholestasis
A positive trend can be seen between the increasing number of mast cells [Figures 5 and 6] and the degree of fibrosis in the biopsies, which were reported out as EHBA. One of the cases with Grade 3 fibrosis showed degranulated mast cells. Eosinophils were present in all the cases analyzed. Supporting changes of inflammation and fibrosis, like feathery or ballooning degeneration of hepatocytes, ductular reaction, and portal bile plugs [Figures 1-4] were also noted in all cases; however, the degree of change varied as per degree of disease severity (fibrosis) [Figures 7 and 8].
Figure 5.
Touledene blue stain at 400X showing mast cells in histopathology section
Figure 6.
Toulidene blue stain showing cluter of mast cell at 400X
Figure 7.
Reticulin stain at 200X showing pericellular fibrosis
Figure 8.
Masson trichome stain at 100X showing areas of fibrosis
DISCUSSION
A common cause of obstructive cholangiopathy in neonates is EHBA, which accounts for 25%–30% of the neonatal cholestasis cases.[5] Various clinical features and laboratory tests like liver function tests, ultrasonography of the abdomen, HIDA scan, and histopathological examination of the liver can help us arrive at the correct diagnosis. Combined histopathological, clinical, and radiological findings lead to a comprehensive management of the patient.[6,7]
Variability in the overall accuracy of reporting histopathological findings in liver biopsy is 60%–95%. The variability occurs due to interobserver variation in reporting EHBA as well as non availability of standard scoring system in diagnosing a case of EHBA. The common histopathological findings in a biopsy of EHBA are lobular cholestasis, variable degrees of bile duct proliferation, and portal edema. Other features, like focal hepatocytic multinucleated giant cells, may also be seen. The most consistent features of biliary atresia are the bile duct proliferation and portal fibrosis, as said in various studies. A similar study done on 43 cases of EHBA collected over 4 years highlighted similar histopathological findings in their cases; however, to the best of the authors’ knowledge, no study has attempted a study associating the number of mast cells with the degree of fibrosis in these cases.[8]
In EHBA, fibrosis is considered a progressive and irreversible process, and it impairs the flow of bile, leading to jaundice and a worse outcome even after surgery.[6] The role of mast cell degranulation has been studied by multiple Western authors in inducing inflammation-mediated changes, and as already discussed, mast cells favor the development of fibrosis in the tissue. It has been reported that the onset of liver fibrosis can lead to liver cirrhosis, leading to poor patient prognosis. High-grade fibrosis has been reported to lead to poor patient outcomes even after a liver transplant.[8]
In our study, 3 out of 7 cases showed mild portal inflammation (lymphocytes). Inflammatory cells were admixed with mast cells and a few eosinophils in all our cases. As per a study, the absence of portal inflammation was associated with poor prognosis.[2] However, due to the smaller sample size of our study, a definite role of other inflammatory cells in prognosis could not be studied. Hepatocyte giant cell transformation is seen in 20%–50% of biliary atresia cases but is not as prominent as seen in neonatal hepatitis. In a study, it was present in 13.95% of cases, while in this study, 1 out of 7 cases (14.28%) showed the presence of a few giant hepatocytes. The presence of mast cells and degranulating mast cells highlights their role in causing fibrosis and leading to worsening of disease prognosis. In this study, the authors noted a worsening degree of fibrosis with an increase in the number of mast cells.[9]
In a study done by Lewandowska et al. in 2020, the authors noted that mast cells accumulated near the periportal area and the septa, and they hypothesized their role in liver fibrosis. They found a strong positive correlation between the number of mast cells, especially those located in the portal vein and fibrous septum, and the degree of liver fibrosis (r = 0.736, P < 0.0001).[10]
In our study, we found scattered mast cells [Figures 7 and 8] near septa and periportal areas as well, and such distribution was supported by the presence of fibrosis in the specimen.
In a study done by Hargrove et al. in 2017 on knockout mice, they concluded that mast cells were profibrogenic, promoted ductular reaction, inflammation, and secreted vascular endothelial growth factor, causing vascular activation.[11] González et al., 2022, in their study, established that mast cells secrete pro-angiogenic factors and release histamine after activation, causing fibrosis and ductular reaction. These findings support our findings of mast cells increasing in number with the grade of fibrosis.[12] However, since the sample size is limited for our pilot study; hence, we need more extensive research in establishing the probable role of mast cells in causing fibrosis, as well as incorporating it in a robust scoring system.
DPM and Bile ductular reaction are important features in cases of EHBA. These findings are attributable to the degree of fibrosis, leading to the proliferation of bile ductules. Intrahepatic bile ducts develop from fetal duct plate by the process of duct plate remodeling. Multiple studies report association of EHBA with DPM, with one study showing 51.29% of their cases showing DPM. This might be because of a lack of strict diagnosing criteria for differentiating DPM from biliary proliferation. In another study the cases with DPM had high grade fibrosis with nodular effacement of liver architecture with biliary proliferation. DPM in such cases has a poor prognosis.[13]
In almost all our cases, slides with Grade 3 fibrosis had a very prominent ductular reaction. However, due to the small sample size, the authors cannot comment upon the significance of this finding with patient prognosis. However, many studies have analyzed importance of bile duct proliferation in EHBA, and while most studies do not give a conclusive opinion on the same, some studies state that severe bile duct proliferation is associated with worst prognosis.[7] In the study on 43 cases of EHBA, the authors noted that a lesser degree of bile duct proliferation led to better patient prognosis.
Intrahepatic cholestasis was seen in all our cases; however, there was an increase in the number of portal ductules showing bile plugs in cases with a higher degree of fibrosis. Studies claim that intrahepatic bile pigments (cholestasis) are a persistent feature of biliary atresia. One study reported a better prognosis in cases with lesser cholestasis.[6,9]
Similar to an Indian study on EHBA showing predominance in the male population, this study also showed 5 males and 2 females, i.e., 71.4% of the cases under study were males. This finding was in contrast to other studies that stated EHBA occurs more commonly in females when compared to male patients.[8,9]
The disease occurs in neonates, and correlating age demographics with the course of disease, studies have seen that early lesions present with a lesser degree of severity as compared to lesions biopsied at a later stage. In our study, the case operated at 8 weeks of age had a milder degree of fibrosis with no porto-portal bridging, while the case operated at 2 years showed a higher grade of fibrosis. Multiple studies have shown that the higher the age of surgical intervention poorer is the patient outcome.[9,10] In an Indian study done on 43 cases of EHBA, the authors noted that age at surgery did not show any correlation with prognosis, as cases operated even at <60 days had poor survival.[14]
The limitation of the study is the small number of cases and lack of control group (non-EHBA liver biopsies); however, the role of mast cells in causing fibrosis and worsening patient outcome is being studied extensively in western literature and hence this study paves a way for similar research in a larger sample size to establish mast cell as an important criterion in histopathological scoring of EHBA to determine disease prognosis. The above study is a pilot study, and a larger study in collaboration with other hospitals receiving pediatric liver biopsies is being planned to address our limitations of sample size and absence of control groups in the present study.
CONCLUSION
The authors emphasize the probable role of mast cells in causing liver fibrosis, a concept warranting further research on a larger sample size and also emphasizing the development of more robust and standardized scoring systems for histopathological grading of EHBA, as well as highlighting the importance of the presence of inflammatory cells like mast cells in such cases, affecting patient prognosis.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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