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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 572-580
Ultra-structural and histopathological features of liver biopsy taken during laparotomy to confirm the diagnosis of biliary atresia


1 Department of Pathology, Malatya Education and Research Hospital, Malatya, Turkey
2 Department of Histology and Embryology, Faculty of Medicine, İnönü University, Malatya, Turkey
3 Department of Biostatistics, Faculty of Medicine, Erciyes University, Kayseri, Turkey
4 Department of Pediatric Surgery, Faculty of Medicine, İnönü University, Malatya, Turkey
5 Pediatric Gastroenterology Hepatology and Nutrition, Faculty of Medicine, İnönü University, Malatya, Turkey

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Date of Submission28-Oct-2021
Date of Decision21-Feb-2022
Date of Acceptance23-Feb-2022
Date of Web Publication08-Jun-2022
 

   Abstract 


Background: Neonatal cholestasis is caused by a group of diseases that cause jaundice, which can be encountered in the neonatal period. Biliary atresia (BA) and idiopathic neonatal hepatitis (INH) are among neonatal cholestasis diseases. Aims: The aim of this study was to perform histopathological and ultra-structural examinations of liver biopsy tissue samples from BA and INH patients with liver biopsies taken during laparotomy to confirm the diagnosis of biliary atresia. Settings and Design: A total of patients undergoing Kasai surgery before the age of 60 days were included in an “early” group (n = 7), whereas patients undergoing surgery after the age of 60 days were included in a “late” group (n = 11). The control group (n = 11) included INH patients. Materials and Methods: For histopathological examinations, liver tissue samples obtained intra-operatively were subjected to routine histopathological procedures after being stained with caspase-3 and cytokeratin-7 antibodies. Ultra-structural evaluations were also performed. Statistical analysis used: For comparisons between the groups, a one-way analysis of variance (ANOVA) test and the Mann–Whitney U test were used for continuous variables. Results: Histopathological findings reflected the specific liver pathologic findings seen in biliary atresia. Although there was no significant difference between the BA groups, these parameters were not detected in the control group. The histopathological evaluations revealed no significant differences in the findings of liver parenchyma damage between the early, late, and control groups. Electron microscopic examinations showed that the patients in the late group had more severe signs of intra-cellular damage to the liver. Conclusions: Although the histopathological examination revealed no significant differences in liver damage between the three groups, in ultra-structural evaluation, intra-cellular damage was found to be less in groups with better prognosis. Electron microscopy evaluations of intra-cellular damage may be more useful in this respect.

Keywords: Biliary atresia, comparative histology, electron microscopy, liver, pathology

How to cite this article:
Gürünlüoğlu S, Gül M, Zararsız G, Akpınar N, Varol FI, Demircan M, Gürünlüoğlu K. Ultra-structural and histopathological features of liver biopsy taken during laparotomy to confirm the diagnosis of biliary atresia. Indian J Pathol Microbiol 2022;65:572-80

How to cite this URL:
Gürünlüoğlu S, Gül M, Zararsız G, Akpınar N, Varol FI, Demircan M, Gürünlüoğlu K. Ultra-structural and histopathological features of liver biopsy taken during laparotomy to confirm the diagnosis of biliary atresia. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:572-80. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/572/351590





   Introduction Top


Biliary atresia (BA) is a disease that causes cholestasis in infants and is characterized by idiopathic fibro-inflammatory obstruction in the extra-hepatic bile ducts.[1] According to the Japanese Association of Pediatric Surgeons, BA is classified into three main types: type I, atresia at the site of the common bile duct (a relative frequency of 11.9%); type II, atresia at the site of the hepatic duct (2.5%); and type III, atresia at the porta hepatis (84.1%).[1] Intra-operative cholangiography is the gold standard in the diagnosis of BA, which is fatal if untreated.[1],[2] Hepatic portoenterostomy, also known as the Kasai procedure, remains the most effective standard treatment for BA.[1] The age at the time of surgery is one of the crucial factors determining the prognosis of BA patients. The 10-year survival rate is significantly higher in BA patients undergoing Kasai surgery before rather than after the age of 60 days.[1] It has also been reported that the characteristics of liver histology at the time of Kasai surgery are key prognostic factors.[1],[2],[3],[4]

Although there is no identifiable cause in clinical, laboratory, and genetic studies, liver disease in infants with cholestasis findings is called idiopathic neonatal hepatitis (INH).[5] Although there are signs of parenchyma damage in the liver histology of INH patients, the prognosis is expected to be excellent.[6]

Few studies have reported electron microscopy findings obtained from liver biopsy samples collected during Kasai surgery or from liver biopsy samples of INH patients. In this study, we performed histopathological and electron microscopic ultra-structural examinations of liver biopsy tissue samples collected intra-operatively from both BA patients undergoing Kasai surgery before and after the age of 60 days and INH patients to investigate the degree of intra-cellular damage and assess the prognostic value of the findings.


   Materials and Methods Top


Patients

This was a single-center retrospective study of patients treated at Turgut Özal Medical Center Education and Research Hospital Malatya, Turkey between January 2015 and February 2020. Ethics approval was obtained from İnönü University's Scientific Research and Publication Ethics Committee (No. 2020/1279). The study included i) patients undergoing Kasai portoenterostomy, ii) patients with type 3 BA confirmed through intra-operative cholangiography (atresia at the porta hepatis), iii) patients aged 0–12 months at the time of surgery, iv) patients from whom liver biopsy samples were collected intra-operatively for histopathological and ultra-structural examinations, and v) patients who were followed up regularly. Patients with polysplenia or asplenia, situs inversus, intestinal malrotation, intra-abdominal vascular anomalies, or congenital heart disease and patients with post-operative complications were excluded.

In the study, INH patients were used as a control group because cholestasis was present without extra-hepatic biliary obstruction and with an excellent prognosis. For the diagnosis of all cases with cholestasis symptoms, a biopsy was taken from the liver and intra-operative cholangiography was performed to display the bile ducts. Cases which can be defined as extra-hepatic and intra-hepatic bile ducts but are not a cause of laboratory and clinical and genetically identifiable cholestasis according to the findings, giant cell transformation, lobular cholestasis, and extra-medial hematopoesis in liver histology were considered INH. A total of 18 patients undergoing standard Kasai portoenterostomy were included in the study as BA patients. Patients undergoing surgery before the age of 60 days were included in an “early” surgery group (n = 7), whereas patients undergoing surgery after the age of 60 days were included in a “late” surgery group (n = 11).

As a control group, 11 non-BA INH patients were included in the study (n = 11).

All patients underwent pre-operative clinical, radiological, and laboratory examinations as recommended by Altman et al.[1] Moreover, all BA and INH patients underwent standard intra-operative cholangiography.[1] Adequate wedge biopsy specimens were obtained from the livers of all patients. Liver biopsies were taken from all groups from the right lobe anterior superior region about 1 x 2 cm diameters and 1.5 cm deep. The Glisson capsule was excluded. The 0.5 cm surface area under the capsule was not included in fibrosis assessments. Thus, the bias that may arise was tried to be blocked. All biopsy samples contained a minimum of 10 portal areas that could be evaluated.

Histopathological evaluations

Liver wedge biopsy specimens were fixed in 10% formaldehyde for 24 hours and embedded in paraffin blocks. Sections 5 μm thick were cut and subjected to routine hematoxylin and eosin and Masson's trichrome staining. Sections placed separately on polylysine glasses were stained with caspase-3 and cytokeratin-7 (CK-7) antibodies.

The all-stained liver sections were examined using an Eclipse Ni-U light microscope (Nikon Corp., Tokyo, Japan). Each liver section was measured using the image analysis functions of NIS-Elements Documentation 5.02 (Nikon Corp.). Sharma et al.'s[7] grading system was used with modifications. Hepatocellular damage was graded as 1, ballooning; 2, feathery degeneration [Figure 1]a; and 3, hepatocellular necrosis. Cholestasis was graded as 1, canalicular; 2, hepatocellular; and 3, centrilobular. Bile duct proliferation was graded as 1, 4–6 ducts per portal area; 2, 7–10 ducts per portal area; and 3, more than 10 ducts per portal area. Bile duct inflammation was graded as 1, few inflammatory cells; 2, more inflammatory cells with no lymphoid aggregates; and 3, more inflammatory cells with lymphoid aggregates. Portal edema was graded as 1, small amount of edema fluid; 2, moderate amount of edema fluid; and 3, sufficient fluid to separate collagen [Figure 1]b and [Figure 1]c. Portal inflammation was graded as 1, few inflammatory cells in the portal area; 2, moderate inflammation with no lymphoid aggregates; and 3, severe inflammation with lymphoid aggregates.[7] Portal fibrosis was graded according to the Ishak (from 0 to 6) and Metavir (from 0 to 4) scoring systems [Figure 1]d and [Figure 1]e.[8]
Figure 1: (a) Hepatocellular necrosis (black arrows), feathery degeneration (dashed arrows), and hepatocellular cholestasis (arrowheads) (hematoxylin and eosin; 200x). (b and c) Portal edema. Mild amount of the edema fluid within the connective tissue of a portal area (b). Severe amount of the edema fluid separating the collagen fibers within a portal tract (c) (hematoxylin and eosin; 200x). (d and e) Portal fibrosis. Mild degree of fibrosis causing fibrous expansion of the portal area (d) and severe degree of fibrosis resulting in portal bridges (e) (Masson's trichrome; 200x). (f) Bile duct caliber. The longest diameter of the largest bile duct within a portal area (f) (black line segment) (hematoxylin and eosin; 400x). (g) DPM (black arrows) ((hematoxylin and eosin; 200x). (h) Ductular bile plugs (black arrows) (hematoxylin and eosin; 200x)

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During histopathological examination of the groups in the study, bile duct loss was evaluated. The total number of bile ductuli in every portal tract was counted and divided by the total number of portal tracts; this ratio was used as an indicator of bile duct loss. The ratio above 0.5 was considered none.[9]

For the calculation of the mean bile duct caliber of each case, in 10 randomly chosen portal tracts, the longest external diameter of the widest bile ducts within each portal tract was measured, starting from the sub-epithelial basal membrane on one side and ending on the other side, and an average of these measurements was calculated for each case [Figure 1]f.[7],[9] We have calibrated the software scales to 40× objective, and pictures were taken from each chosen portal tract. The diameters were measured from the JPEG format of each picture by using the image analysis functions of NIS-Elements Documentation 5.02 (Nikon Corp).

During histopathological examination of the groups in the study, the presence of ductal plate malformation (DPM) was evaluated as exist/none [Figure 1]g.[10]

Ductular bile plugs were evaluated as absent, mild, moderate, and prominent (0–+3); the cases with prominent (+3) bile plugs were determined in each group, and a comparison was made between the groups in terms of the ratio of these cases (1) [Figure 1]h.[10]

Immunohistochemical methods

The tissue sections were de-paraffinized and placed on adhesive slides. Citrate buffer (pH 7.6) was applied, and antigen retrieval was then performed in a pressure cooker at 121°C for 15 min. After the sections were cooled and the citrate buffer was removed, phosphate-buffered saline (PBS) was added. The sections were washed with PBS at every stage except for the primary antibody application and protein blockade stages. Then, a hydrogen peroxidase kit (Thermo Fisher Scientific, Kalamazoo, MI, USA) was used for immunohistochemical staining at room temperature. Subsequently, 3% hydrogen peroxide was dropped and left for 10 min. Then, the caspase-3 primary antibody [anti-caspase-3 monoclonal antibody (ABM1C12) ab208161; Abcam, Cambridge, UK; dilution ratio 1:300] and CK-7 [cytokeratin-7 mouse monoclonal antibody (OV-TL 12/30); Cell Marque, Hague, Netherlands; dilution ratio 1:300] were incubated separately according to the manufacturers' instructions, and a protein-V blocking solution was applied for 5 min. The glass slides were then incubated in the biotinylated goat anti-polyvalent secondary antibody for 10 min, followed by streptavidin peroxidase for 10 min and aminoethyl carbazole chromogen for 15 min. Finally, the slides were washed with distilled water.

Bile duct proliferation was evaluated in CK-7-stained sections.[11] CK-7 expression was evaluated in randomly selected portal areas. Photographs of ten randomly selected high-magnification (20×) fields were taken, keeping the halogen lamp voltage constant, and were saved in the Tag Image File Format. The photographs were quantitatively evaluated for CK-7 staining using Adobe Photoshop CS6 Extended 10.0 (Adobe Systems Inc., San Jose, CA, USA). In each image, the total number of pixels in each measured area was kept constant, and the CK-7-positive structures were measured in pixels using the “Magic Wand” tool. To obtain a ratio for each area, the number of pixels in each CK-7-positive area was divided by the total number of pixels in that area. Then, an overall ratio was obtained by calculating the average of ten fields for each patient. The overall ratios were statistically compared between the two groups.[11] For caspase-3 staining evaluations, hepatocytes stained positive and all the hepatocytes in ten high-power fields (400×) were counted. A score was then obtained for each patient by dividing the number of caspase-3-positive hepatocytes by the number of total hepatocytes.[12] The scores were statistically compared between the two groups.[12],[13]

Transmission electron microscopic examinations

Liver tissue samples were fixed in 2.5% glutaraldehyde buffered with 0.2 M NaH2PO4 + NaHPO4 (pH 7.2) and then post-fixed in 0.1 M buffered 1% OsO4. Subsequently, they were dehydrated in acetone and embedded in epoxy resin (Araldite CY212, Hatfield, CA, USA). Thin (70 nm) sections were cut from the araldite blocks with an ultra-microtome and contrasted with uranyl acetate and lead citrate. The prepared samples were examined under a Zeiss Libra 120 (Carl Zeiss NTS, Oberkochen, Germany) transmission electron microscope.

Statistical analysis

Histograms, q–q plots, and the Shapiro–Wilk test were used to assess data normality. Levene's test was used to assess homogeneity of variance. For comparisons between the groups, one-way ANOVA and the Kruskal–Wallis H test were used for continuous variables, and the Fisher–Freeman–Halton exact test was used for categorical variables. Bonferroni-adjusted Dunn's and z-tests were applied for multiple comparisons. The Wilcoxon t-test was used for intra-group comparisons. The ratio of the number of patients who survived during the study period to the total number of patients in each group was calculated. Kaplan–Meier curves were generated to estimate the survival probability of each group, and the log-rank test was used for survival probability comparisons between the two groups. All analyses were performed using the TURCOSA statistical software (Turcosa Analytics Ltd. Co., Kayseri, Turkey). Values of P <.05 were considered statistically significant.


   Results Top


The surgical age during biopsy, gender, and follow-up times and the life status of patients in all groups are shown in [Table 1]. The patient characteristics are shown in [Table 2]. There were no significant differences between the three groups in terms of gender and the need for liver transplantation. The survival rate was significantly higher in the early group and control group than in the late group.
Table 1: Detailed characteristics and the follow-up status of patients

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Table 2: Patient characteristics and comparison of laboratory parameters among control and biliary atresia patient groups

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Laboratory and histopathological parameter results

The analysis results of the pre- and post-operative laboratory parameters of the two groups are shown in [Table 2]. There were no significant differences between the three groups in terms of pre-operative total bilirubin (TB) or serum glutamic oxaloacetic acid transaminase (SGOT) values. The seventh-day post-operative TB value, TB7–TB0 difference value, and TB7/TB0 ratio were significantly lower in the early group and control group than in the late group. The seventh-day post-operative and pre-operative serum glutamic pyruvic transaminase (SGPT) levels did not differ significantly between the three groups. Conversely, the SGPT7–SGPT0 difference was significantly greater in the early group. In this group, the seventh-day post-operative TB, SGOT, and SGPT levels were significantly lower than the pre-operative levels. Conversely, in the late group, the pre-operative and seventh-day postoperative laboratory marker levels did not differ significantly. In the control group, the seventh-day post-operative TB levels were significantly lower than the pre-operative levels.

The statistical analysis results of the histopathological parameters of the two groups are shown in [Table 3]. Portal edema was significantly higher in the late group [Table 3] [Figure 2]a and [Figure 2]b. There were no statistically significant differences in the other histopathological parameters, caspase-3 activity, or CK-7 expression between the early and late groups [Table 3] [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h, [Figure 2]i, [Figure 2]j, [Figure 2]k, [Figure 2]l, [Figure 2]m, [Figure 2]n, [Figure 2]o, [Figure 2]p, [Figure 2]q, [Figure 2]r. Among patients in both the early group and the late group, bile plugs were detected. Prominent (+3) ductular bile plugs were present in four patients from the early group and six patients from the late group. There were no prominent ductular bile plugs in the control group. Portal fibrosis and bile duct fibrosis, bile duct proliferation, and CK-7 expression were significantly less in the control group than in the other groups. [Table 3].
Table 3: Comparison of histopathological variables among control and biliary atresia patient groups

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Figure 2: (a-d) Portal edema (black arrows) in (a) a case belonging to the early surgery group and (b) a case belonging to the late surgery group (hematoxylin and eosin; 200×). Hepatocellular necrosis (black arrows), feathery degeneration (dashed arrows), hepatocellular cholestasis (arrowheads), and canalicular cholestasis (short arrows) in (c) a case belonging to the early surgery group and (d) a case belonging to the late surgery group (hematoxylin and eosin; 200×) (e-h) Portal inflammation (black arrows) in (e) a case belonging to the early surgery group and (f) a case belonging to the late surgery group (hematoxylin and eosin; 200×). Portal fibrosis (white arrows) in (g) a case belonging to the early surgery group and (h) a case belonging to the late surgery group (Masson's trichrome; 200×). (i and j) Bile duct inflammation (black arrows) in (i) a case belonging to the early surgery group and (j) a case belonging to the late surgery group (hematoxylin and eosin; 200×). (k and l) Bile duct proliferation (black arrows) in (k) a case belonging to the early surgery group and (l) a case belonging to the late surgery group (hematoxylin and eosin; 200×). (m and n) Caspase-3-positive cells (black arrows) in (m) a case belonging to the early surgery group and (n) a case belonging to the late surgery group (caspase-3 staining; 400×). (o-r) Proliferating bile ducts (black arrows) and hepatocytes (black arrowheads) reacting with cytokeratin-7 (CK7) antibodies in (o) a case belonging to the early surgery group and (p) a case belonging to the late surgery group (CK7 staining; 200×). Bile ducts (black arrows) and hepatocytes (black arrowheads) reacting with cytokeratin-7 (CK7) antibodies in (r) a case belonging to the control group (CK7 staining; 200x)

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DPM was evaluated in all groups. DPM was detected in one and two patients, respectively, from the early and late groups; there was no statistically significant difference between two groups. However, there was no DPM in the control group [Table 3].

When evaluated between groups, the ductular caliber did not show a statistically significant difference between the early and late groups, but the result was significantly higher in the control group [Table 3].

When liver tissue samples were examined histopathologically, common multi-nucleate giant cell formation was detected in all INH patients. In both early and late groups, multi-nucleate giant cell formation was detected in the focal area.

Malory denk bodies were identified in both patients in the early group and the patients in the late group in histopathological examination and showed a similar pattern of distribution. Malory–Denk bodies were not identified in the control group. There was no evidence of duct loss in any of the groups in the study.

When histopathological findings were examined reflecting the specific portal damage seen in biliary atresia, such as portal fibrosis, peri-ductal fibrosis, ductular bile plugs, the ductular caliber, and CK-7 expression values, there were signs of serious damage in the early and late groups. Although there was no significant difference in damage severity between the BA groups, these parameters were different and lower in the control group [Table 3].

There was no statistically significant difference between the groups in terms of hepatocellular damage and caspase-3 staining (i.e., parameters indicating parenchyma damage). There was no significant difference between the groups in terms of bile duct inflammation and portal inflammation parameters either.

Transmission electron microscopy findings

In the liver sections of the early, late, and control groups, the liver ultra-structure exhibited profound changes in hepatocytes. Many vacuoles of different diameters and circular membrane-bound hydropic vacuoles, intra-cytoplasmic edema, autophagosomes containing glycogen granules, membranous materials, electron-dense degenerate organelle residues, and myelinated structures were observed. Furthermore, loss of cristae and mitochondrial matrix condensation were observed in some mitochondria. Among the intra-cellular damage findings, hydropic vacuoles were more numerous and more severe in the late group than in both the early group and control group [Figure 3]a, [Figure 3]b, [Figure 3]c. Intra-cellular damage in both the control group and early groups was minimally detected [Figure 3]a and [Figure 3]c.
Figure 3: (a) Hepatocyte nuclei (n), autophagosomes containing glycogen granules (+) and membranous materials (*), loss of mitochondrial cristae (white outlined arrow), and myelinated structures (black outlined arrow) in hepatocytes of the early surgery group. (b) Hepatocyte nuclei (n), intra-cytoplasmic hydropic vacuole (v), intra-cytoplasmic edema (e), electron-dense degenerate organelle residues (♦), loss of mitochondrial cristae (black outlined arrow), and mitochondrial matrix condensation (white outlined arrow) in hepatocytes of the late surgery group. (c) Hepatocyte nuclei (n), mitochondria (black outlined arrow), lysosome (white outlined arrow), and the dilated endoplasmic reticulum (arrow head) in hepatocytes of the control group (uranyl acetate–lead citrate; transmission electron microscopy, scale bar: 5 μm, original magnification: 8000×)

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   Discussion Top


In determining the prognosis of BA, it is most essential to know when liver damage starts and when it reaches a critical threshold. Considerable research on this subject has been conducted. The age at the time of Kasai portoenterostomy is decisive. Undergoing surgery before the 60th day of life is of prognostically crucial importance.[1]

In this study, in terms of hepatocellular damage, the histopathological parameters did not differ significantly between the early, late, and control groups. It can be concluded that histopathological parameters indicating the severity of parenchyma damage are not important prognostic factors. However, the prognosis of the patients in the early and control groups was significantly better than that of the patients in the late group, most of whom died during the follow-up period, and some had liver transplantation. The electron microscopic examinations showed that the patients in the late group had more severe signs of intra-cellular damage to the liver. Although the histopathological examinations revealed no significant differences in liver damage between the three groups, we speculate that liver damage did not exceed the irreversible threshold in the early and control groups, in which the liver's regeneration capacity may have been maintained. This may be because the hepatocytes in that group exhibited less intra-cellular damage at the ultra-structural level.

Histopathological examinations of liver biopsy samples of neonatal cholestasis patients were obtained at the time of laparotomy surgery and are particularly helpful in determining the prognosis of the BA disease. However, studies on the role of these histopathological findings in determining the prognosis of patients in the follow-up period have reported conflicting results.[14] Czubkowski et al.[15] found that the severity of fibrosis in the liver was associated with neither the age at the time of Kasai surgery nor the pre-operative bilirubin levels and concluded that intra-operative liver histology had limited prognostic value. Other studies have found that ductular proliferation, DPM, bile stasis, and giant cell transformation are associated with the prognosis of BA.[11],[15],[16],[17],[18],[19],[20],[21] Similarly, Gupta et al.,[17] Low et al.,[18] and Shimadera et al.[19] reported that DPM with structures of fetal bile duct remnants was associated with a poor prognosis. In a valuable study, Safwan et al.[22] claimed that the presence of DPM in BA patients had an adverse outcome effect on prognosis, even if it had been operated on. Weerasooriya et al.[23] found no correlation between age at the time of Kasai surgery and the severity of liver fibrosis. Lampela et al.[8] found that more lymphocytes and fewer macrophages in the area of inflammation were associated with a higher risk of requiring liver transplantation within 2 years. In our study, we found that DPM was present in one patient in the early group of BA patients and in two patients in the late group. We did not detect the loss of ducts in any groups' patients. Ductular plugs were only available in some of the patients in both BA groups. These findings may be valuable parameters in the diagnosis of BA. However, in our study, we found that these parameters did not differ significantly between the early and late groups.

Although the histology of the liver taken from BA patients has been claimed to have limited value in determining the prognosis,[15] Ramachandran et al.[24] claimed in their study that α-smooth muscle actin expression in the liver has the potential to be a more successful marker in predicting post-operative prognosis. We did not evaluate the expression of that α-smooth muscle actin in the histopathological evaluation in our study.

Altman et al.[25] found that an age at surgery of more than 70 days posed a serious risk of failure after Kasai portoenterostomy. Moreover, they reported that bile ducts in liver specimens smaller than 150 μm in diameter were poor prognostic predictors. However, they concluded that hepatic histology was not an independent risk factor. Their study included 266 patients and analyzed 20 years of follow-up results. In our study, we found that although there was no significant difference between the early and late groups of bile duct diameters, they were larger in the control group. Among the patients of control groups in our study, the bile duct calibers were over 150 μm. The number of patients in our study was considerably smaller, and our longest follow-up period was 36 months. Because of these limitations, we did not perform a multi-variate risk factor analysis.

Nio et al.[26] also found that the age at surgery was decisive and that the prognosis of patients undergoing surgery before the age of 60 days was more favorable. Conversely, Sharma et al.[7] found no correlation between the age at surgery and total histopathological scores and 1 year survival. In our study, although there were no significant differences in liver parenchyma damage findings of histopathology between the early, late, and control groups, the prognosis of the patients in the early and control groups was better.

Santos et al.[11] investigated the relationship between portal ductus proliferation and its marker, CK-7 expression, and the prognosis of BA and determined a cutoff of 10.18% for biliary proliferation in the liver tissue. They found that patients with a high CK-7 positivity percentage either died or underwent liver transplantation. Conversely, in our study, we found no statistically significant difference between the late group, which had a poorer prognosis, and the early and control groups in terms of portal proliferation. In our study, CK-7 expression was lower in the control group. However, the prognoses of the control group and early groups were better. We speculate that CK-7 expression severity may not be of prognostic significance but a valuable marker for BA diagnosis.

Gunadi et al.[3] reported that post-Kasai survival was associated with the severity of bile duct proliferation, portal inflammation, and cholestasis. Their patients were between 75 and 142 days old, whereas no patients were younger than 60 days old. In our study, we found no relationship between the severity of these histopathological signs and survival. In our opinion, the main factor determining post-Kasai survival is the age at surgery, with the 60th day of life being a critical threshold, because the symptoms of intra-cellular damage in the control group, which had a perfect prognosis, were similar to the ultra-structural findings in the early group.

Cheng et al.[4] found that among eight histopathological parameters, only liver fibrosis and DPM were associated with the prognosis of BA. Similarly, Muthukanagarajan et al.[27] found that DPM, increased liver fibrosis, bile duct proliferation, and cholestasis were associated with a poor prognosis. In our study, we used the same parameters and found no related differences between the early and late groups, although the prognosis of the patients in the early group was better. We believe that even if differences in the liver's regeneration capacity are observed in intra-operative liver biopsy samples, histopathological examinations may detect damage of the same severity in patients with good and poor prognoses. We speculate that this can be seen either in electron microscopic examinations or in examinations of multiple post-operative biopsy samples. In our study, we did not take biopsies from the hilar region. We took liver biopsy samples from the right lobe. We could not find an important relationship between early and late groups' patients' prognoses and DPM. We also found no significant difference between all groups in terms of DPM presence.

Hukkinen et al.[28] investigated the relationship between histopathological changes in liver tissue samples and the prognosis of BA. They examined samples collected not only at the time of surgery but also at 1, 5, and 10 years post-operatively. In post-operative examinations, they found that the dissolution of cholestasis, elimination of portal inflammation, and recovery of fibrosis were important survival predictors. Similarly, Lampela et al.[8] reported that the evaluation of liver biopsy samples obtained during the follow-up period may be helpful for more reliable prognosis predictions. In our study, we found no significant differences in intra-operative liver tissue samples' parenchyma damage findings of histopathology between the early, late, and control groups, although the prognosis was better in the early and control groups. We speculate that differences in histopathological parameters between liver biopsy samples collected intra-operatively and post-operatively can be effective in determining the prognosis of BA. Although liver tissue samples exhibited severe damage in the early group and the control group, the liver's regeneration capacity in these groups may have been preserved. This hypothesis is supported by our electron microscopic examinations, which showed less intra-cellular damage in the early and control groups than in the late group. This may explain the better prognoses of the former and control groups. If they had been conducted, evaluations of post-operative liver biopsy samples may have confirmed this hypothesis.

Our study has certain limitations. First, the number of patients was small. Second, it was a retrospective study. Also, we evaluated only intra-operative liver biopsy samples.

Evaluations of changes in histopathological findings in the post-operative period may enable more reliable predictions. In our next study, we plan to investigate post-operative follow-up period liver biopsy samples.


   Conclusion Top


In this study, although the prognosis of patients in the early and control groups was better than that of patients in the late group, histopathological findings from intra-operative liver biopsy samples did not differ significantly between the three groups in terms of liver damage. However, electron microscopic examinations revealed more severe intra-cellular damage in the late group than in the early and control groups. Therefore, in this study, we believe that the histopathological parameters reflecting parenchyma damage do not differ significantly between all groups. It can be concluded that histopathological parameters indicating the severity of parenchyma damage are not important prognostic factors. An ultra-structural examination with the electron microscope found that intra-cellular damage was low in the early and control groups, whereas in the late group with a poor survival status, the damage findings were significantly higher. This may be because the hepatocytes in that group exhibited less intra-cellular damage at the ultra-structural level.

Ethical approval

The study protocol was approved by the İnonu University's Scientific Research and Publication Ethics Committee (No. 2020/1279).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Correspondence Address:
Kubilay Gürünlüoğlu
Department of Pediatric Surgery, Faculty of Medicine, İnönü University, Malatya - 44315
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1057_21

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