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Original articles

Assessment of liver biopsies in obese patients

a histological and immunohistochemical study

Mansy, Aisha E.a; Faruk, Eman M.a; Abd El Hamid, Mohamedb

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The Egyptian Journal of Histology: June 2013 - Volume 36 - Issue 2 - p 285-291
doi: 10.1097/01.EHX.0000429197.96746.ad
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Abstract

Introduction

Obesity has reached epidemic proportions with a prevalence that continues to increase. Obesity is the result of combined effects of multiple gene, lifestyle, and environmental factors. Paralleling the increase in the incidence of obesity and diabetes, nonalcoholic fatty liver disease (NAFLD) is emerging as one of the most common causes of chronic liver disease 1–3. NAFLD occurs in the setting of metabolic syndrome, in which insulin resistance plays a key role. Metabolic syndrome is a metabolic disorder related to obesity 4. In obesity, increased delivery of free fatty acids to the liver is observed along with an impairment of fatty acid metabolism, with accumulation of triglycerides in the liver of obese individuals, suggesting a defect in the mitochondrial lipid oxidative enzyme. Accumulation of tissue triglycerides and acyl COA enzyme derivatives may activate the serine kinase pathway that induces insulin resistance by blocking the insulin receptors, leading to type 2 diabetes mellitus 4,5.

Histological assessment will continue to play an important role in the diagnosis and management of people with NAFLD 6–8. Liver biopsy is an appropriate method to determine the severity of liver damage and may reveal an additional cause of liver damage 3,9,10. As the histological course of NAFLD remains undescribed, the aim of the present study was to determine the different changes in liver histology.

Aim of the work

The aim of this study was to determine the histological characteristics of NAFLD in obese patients.

Materials and methods

Patients

Twenty obese patients (10 male and 10 female) (BMI of ≥40 or ≥35) were enrolled prospectively and consecutively in the present study. Patients being treated with hepatotoxic or antineoplastic drugs and those with primary liver disorders other than fatty liver that could account for hepatocellular steatosis were excluded from the study. All patients underwent bariatric surgery at Benha University Hospital. The ethics committee of the hospital approved the study, and all patients provided written informed consent.

Histological study

Liver biopsies were obtained from obese patients at the time of bariatric surgery using an 18-G Tru-Cut needle, India. Only those samples longer than 14 mm on histological examination were included in the study 11,12. The biopsy specimens were immediately fixed in buffered formalin and processed for paraffin sectioning. The sections were then stained with H&E and Masson’s trichrome stain.

Immunohistochemical and quantitative study

To identify ‘surgical hepatitis’ and thus to obtain correct scores for lobular inflammation, immunoperoxidase techniques were used by means of an automated system (Lab Vision Autostainer 720, India) that utilizes primary antibodies (Ab) against myeloperoxidase (MPO) (polyclonal Ab, prediluted; Master Diagnostica, MD Spain) and CD68 (monoclonal Ab 512H12, prediluted, MD). Anti-p62 Ab (Monoclonal Ab 3P62LCK, 1/500 dilution, BD, Biosciences, USA, San Francisco, Transduction) was also applied to locate MDB.

Statistical analysis

The fields were estimated using the Olympus BX40, DOT meds Shipping Quote Service, UPS, (USA), image analyzer computer system at the Histology Department, Faculty of Medicine, Cairo University. Measurements were taken from 10 nonoverlapping fields at ×400 magnification.

All quantitative data were presented as mean (X) ±SD and were compared using the unpaired Student test. A P value less than 0.05 was considered statistically significant.

Results

Morphometric results

The area % of positive anti-CD68 immunostaining, anti-p62Ab immunostaining, and anti-MPO Ab immunostaining cells in the liver was significantly increased in the obese group when compared with the control group (Tables 1–3 and Histograms 1–3).

T1-2
Table 1:
Mean area % of anti-CD68 immunostaining and SD and P values in normal liver cells vs. fatty liver cells
T2-2
Table 2:
Mean area % of anti-p62Ab immunostaining and SD and P values in normal liver cells vs. fatty liver cells
T3-2
Table 3:
Mean area % of anti-MPO Ab immunostaining and SD and P values in normal liver cells vs. fatty liver cells
F1-2
Histogram 1. Mean area % of anti-CD68 immunostaining and SD and P values in normal liver cells vs. fatty liver cells.
F2-2
Histogram 2. Mean area % of anti-p62Ab immunostaining and SD and P values in normal liver cells vs. fatty liver cells.
F3-2
Histogram 3. Mean area % of anti-MPO Ab immunostaining and SD and P values in normal liver cells vs. fatty liver cells.

Histological results

On histological examination, the liver biopsy specimens of the control group showed hepatic lobules. At the center of the lobule was the central vein (Fig. 1). A higher magnification of the specimen revealed the portal tract. The hepatocytes were polygonal and had vesicular nuclei and granular acidophilic cytoplasm (Fig. 2). The hepatic cords were separated by sinusoids and showed minimal amount of collagen fibers around the central vein (Fig. 3).

F4-2
Figure 1:
A photomicrograph of a liver biopsy specimen from a control individual showing the central vein (C), radiating cords of liver cells separated by sinusoids (S), and the portal area (p). H&E, ×200.
F5-2
Figure 2:
A photomicrograph of a liver biopsy specimen from a control individual showing the portal vein (V), the bile duct (B), and hepatocytes with vesicular nuclei and granular acidophilic cytoplasm (higher magnification). H&E, ×400.
F6-2
Figure 3:
A photomicrograph of a liver biopsy specimen from a control individual showing minimal amount of collagenous fibers around the central vein (C). Note the thin collagen fibers delineating the wall of the sinusoids (↑↑). Masson’s trichrome, ×400.

Examination of the liver specimens from obese individuals revealed a liver loaded with a single large fat droplet (Fig. 4). A higher magnification of the specimens revealed a single fat droplet pushing the nucleus to the periphery of the cytoplasm (Fig. 5). Some hepatocytes showed glycogenated nuclei (Fig. 6). The examination also revealed a fatty liver with cellular infiltration (Fig. 7). Analysis of Masson’s trichrome-stained specimens revealed pericellular and perivascular fibrosis (Fig. 8), hepatic cell necrosis, cellular infiltration, and fibrosis (Fig. 9), and periportal fibrosis with an apparent reduction in the fatty changes in the liver (Fig. 10).

F7-2
Figure 4:
A photomicrograph of a liver biopsy specimen from an obese individual showing liver cells loaded with a single large fat droplet (arrow heads). Some hepatocytes contain multiple fat droplets. H&E, ×200.
F8-2
Figure 5:
A photomicrograph of a liver biopsy specimen from an obese individual showing some hepatocytes with multiple fat droplets (arrows) and others with a single fat droplet pushing the nucleus to the peripheral part of the cytoplasm (arrow head) (higher magnification). H&E, ×400.
F9-2
Figure 6:
A photomicrograph of a liver biopsy specimen from an obese individual showing some hepatocytes with glycogenated (very pale) nuclei (arrow). H&E, ×400.
F10-2
Figure 7:
A photomicrograph of a liver biopsy specimen from an obese individual showing a fatty liver with cellular infiltration (arrows). H&E, ×400.
F11-2
Figure 8:
A photomicrograph of a liver biopsy specimen from an obese individual showing a fatty liver with pericellular and perivascular fibrosis (arrow). Masson’s trichrome, ×400.
F12-2
Figure 9:
A photomicrograph of a liver biopsy specimen from an obese individual showing hepatic cell necrosis (G) (arrow), cellular infiltration, and fibrosis. Masson’s trichrome, ×400.
F13-2
Figure 10:
A photomicrograph of a liver biopsy specimen from an obese individual showing periportal fibrosis (p) and (arrow). Note the decreased severity of fatty changes (arrow). Masson’s trichrome, ×400.

Specimens from the control group showed few Kupffer cells in relation to blood sinusoids (Fig. 11), whereas those from obese individuals showed macrophage aggregation in necrotic areas of the liver (Fig. 12).

F14-2
Figure 11:
A photomicrograph of a liver biopsy specimen from a control individual showing branched Kupffer cells lining the sinusoids (↑). Anti-CD68 Ab immunostaining, ×400.
F15-2
Figure 12:
A photomicrograph of a liver biopsy specimen from an obese individual showing macrophage aggregation in the necrotic areas. Anti-CD68 Ab immunostaining, ×400.

The degenerated (ballooned) hepatocytes showed positive immunoreactive Mallory bodies around the nuclei (Fig. 13) as well as aggregations of polymorphonuclear leukocytes (Fig. 14).

F16-2
Figure 13:
A photomicrograph of a liver biopsy specimen from an obese individual showing Mallory–Denk bodies around the nuclei of ballooned degenerated hepatocytes (arrows). Anti-p62Ab immunostaining, ×400.
F17-2
Figure 14:
A photomicrograph of a liver biopsy specimen from an obese individual showing an aggregation of polymorphonuclear leukocytes around dying hepatocytes. Anti-MPO Ab immunostaining, ×400.

Discussion

Fatty liver is a metabolic syndrome related to an increase in the delivery of free fatty acids to the liver in obese individuals 5,6. The present study showed that the cytoplasm of a large number of hepatocytes was distended with a single large fat droplet that displaced the nucleus to the periphery (macrovesicular steatosis). Some hepatocytes showed microvesicular steatosis in the form of small multiple vesicles. These results were in accordance with those of other studies 10,13. In addition, hepatocellular injury in the form of ballooning and necrosis was also observed in the present study. Some authors report that when the capacity of liver cells to store fat decreases, the fatty acids become toxic to the cells, leading to cell death 14,15. In addition, lipid mediators secreted by adipocytes such as tumor necrosis factors and other metabolites could also lead to liver cell death 10.

Fatty liver disease is associated with an increased expression of membrane receptors, rendering the cells susceptible to apoptosis 16–18. In the present study, Mallory bodies were detected in the cytoplasm of some hepatocytes. Mallory bodies are irregular acidophilic structures composed of intermediate filament aggregates. These bodies are associated with steatohepatitis. Inflammatory cells such as neutrophils and macrophages were also detected in the liver parenchyma. In addition, mitochondrial dysfunction associated with NAFLD might lead to the release of reactive oxygen species.This further leads to the release of proinflammatory cytokines 19. Moreover, inflammatory mediators are released from adipose tissues, leading to inflammatory changes 19–21.

In the present study, hepatocytes with vacuolated (glycogenated) nuclei were observed. Some scientists report that the presence of these nuclei denotes glycogen accumulation occurring in the setting of insulin resistance 4.

Pericellular and periportal fibrosis were detected in the present study. Some investigators report that fibrosis is due to mitochondrial injury or due to mobilization of profibrogenic cytokines in response to hyperglycemia and inflammatory necrotic changes 10,16,17. According to some studies, the progression of liver fibrosis is dependent on the presence of diabetes 4,11,12. In addition, some studies have reported that fibrosis is an indicator of liver damage and is the best prognostic marker for morbidity and mortality 22,23. Moreover, in the present study, an aggregation of Kupffer cells secreting interleukin I and tumor necrosis factor-α was observed around necrotic areas of the liver in specimens from obese individuals.

The present study showed that periportal fibrosis was associated with an apparent reduction in the severity of steatosis and steatohepatitis.

Recommendation

Some scientists report that modest, sustained weight reduction in association with exercise decreases the severity of fatty liver, fatty inflammation, and fibrosis as well as reverses insulin resistance such that no medical treatment is required.

Conclusion

Liver biopsies are helpful in the diagnosis of NAFLD.

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Table:
No title available.

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

immunohistochemistry; NAFLD; obesity; steatohepatitis

© 2013 The Egyptian Journal of Histology