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Volume: 15 Issue: 5 October 2017

FULL TEXT

ARTICLE
Effect of Copper Staining in Wilson Disease: A Liver Explant Study

Objectives: Wilson disease is a rare genetic disease with clinical and histopathologic differential diagnostic challenges. In this study, we evaluated the histo­pathologic findings of explanted livers in Wilson disease, with special emphasis on copper histochemistry.

Materials and Methods: Our study group was recruited by reviewing archived histopathology reports and the liver transplant clinic patient records retrospectively for patients who had liver transplant for Wilson disease between January 2010 and June 2015, at Turgut Ozal Medical Center. Archival slides were reevaluated. When needed, relevant clinical and laboratory data were obtained from patient medical records.

Results: During the selected period, there were 33 patients fitting the study criteria (22 male, 11 female, mean age of 22 ± 11 y). All patients had mild to moderate septal inflammation. We found that 29 patients (88%) showed glycogenated hepatocyte nuclei and 27 patients (79%) showed nuclear pleomorphism. Other histopathologic findings were cholestasis (48%) and macrovesicular steatosis (39%). There was no special finding in hilar regions except for 2 patients who had recanalized portal vein thrombosis. In terms of copper histochemistry, 2 copper stains, Timm silver sulfide and rhodanine, were performed in all cases, with orcein staining only done for 25 of the cases. Positivity rates for these copper stains were 85%, 82%, and 36%. Periodic acid-Schiff-diastase- and periodic acid-Schiff-positive granules were detected in 7 of 33 patients (21%). Iron deposition was seen in 12 patients (focal and/or minimal in 11, more than focal in 1). There was no dysplasia or malignancy in any of the patients.

Conclusions: On routine hematoxylin and eosin-stained slides, detection of glycogenated hepatocyte nuclei and the finding of the nuclear pleomorphism should alert the pathologist for the possibility of Wilson disease, especially with cryptogenic liver disease. Timm stain is a more convenient histo­chemical stain in revealing copper deposition in liver.


Key words : Copper histochemistry, Transplant, Wilson disease

Introduction

Wilson disease is a rare inborn metabolic disease involving copper metabolism. The spectrum of clinical findings is variable. More often, the clinical picture is as liver disease shown at early ages (especially in children and young adults). Liver disease can present both as acute and chronic forms. Most of the symptoms are nonspecific, as with the liver histologic findings on routine hematoxylin and eosin-stained slides. Histologic abnormalities include steatosis, glycogenated hepatocyte nuclei, inflammation, and variable hepatocellular aniso­nucleosis.1,2 From a pathologist’s perspective, when evaluating the liver biopsies, especially in cryptogenic cases and with chronic hepatitis and cirrhosis, Wilson disease should be included in the differential diagnosis when these findings are observed.1

Evaluation of copper deposition by histo­chemistry is an important step in histologic examination. Unfortunately, because of uneven accumulation of copper in liver and due to technical variations,3 histochemistry results can be false negative. However, different stains are available with variable diagnostic accuracy to reveal copper (or copper-binding protein) in routine processed tissue. In this study, we reevaluated the histopathology of explanted livers in patients with Wilson disease, with a special emphasis on suggestive histopathologic features in Wilson disease and the copper histochemistry.

Materials and Methods

Patients who underwent liver transplant for Wilson disease between January 2010 and June 2015 at Turgut Ozal Medical Center were selected retro­spectively from the pathology department histo­pathology report archive and from transplant clinic patient medical records. Archival slides from patients were reevaluated. When needed, the relevant clinical and laboratory data were obtained from patient medical records. Cases with different histopathologic diagnosis were excluded. In patients who had no stainable copper or inconclusive copper accu­mulation on histochemistry; we checked for other supporting clinical and laboratory data (family history, Kayser-Fleischer rings, low serum cerulo­plasmin level, elevated 24-hour urine copper excretion), and patients without these supporting findings were also excluded from the study group.

Information regarding age and sex was extracted from histopathology reports. Archival slides were evaluated for inflammation, steatosis, glycogenated hepatocyte nuclei, cholestasis, nuclear pleomorphism, and evidence of dysplasia and/or malignancy. For the first 3 parameters, semi-quantitative scoring was performed. Inflammation was scored from 0 to 3, with 0 = absent, 1 = mild, 2 = moderate, and 3 = severe. Steatosis was scored from 0 to 2, with 0 = less than 5% 1 = 5% to 15% (mild), and 2 = more than 15% (moderate to severe). Presence of glycogenated hepatocyte nuclei was scored as 1 or 2, with 1 = rare and 2 = prominent (noticed at ×4 magnification). Cholestasis and nuclear pleomorphism were noted as absent or present. Positivity of copper stains was noted as negative, focal/periseptal positive, and nodular positive (panlobular/global or near global staining in a cirrhotic nodule). Iron deposition was evaluated on Prussian blue-stained slides, and results were recorded from 0 to 3, with 0 = absent, 1 = focal/minimal, 3 = widespread, and 2 = between 1 and 3. Periodic acid-Schiff-diastase- and periodic acid-Schiff-positive-stained slides were reviewed for any eye-catching staining. Hilar structures were evaluated in case of any thrombus.

Results

Explanted livers from a total of 33 patients (22 male, 11 female) comprised the study cases. Tables 1, 2, and 3 summarize the age, sex, and liver histology, including copper histochemistry results of the patients. Mean age was 22 ± 11 years (range, 7-57 y).Thirty-two of the 33 patients had end-stage liver fibrosis (31 cirrhosis [6/6 per Ishak scale] and 1 incomplete cirrhosis [5/6 per Ishak scale]). The noncirrhotic case was diagnosed as Wilson disease at another center (outside case) and showed minimal fibrosis (1/6 per Ishak scale). All patients had mild to moderate septal inflammation. There was no severe inflammation or striking lobular activity. Glycogenated hepatocyte nuclei were seen in 29 cases (88%, with 23/29 being scattered and 6/29 being widespread, noticed at ×4 magnification). Results from 27 patients (79%) showed nuclear pleomorphism (Figure 1). Cholestasis, mostly hepa­tocellular, was noticed in 17 patients. Steatosis, predominantly mild, was seen in 13 patients (39%). There was no dysplasia or malignancy in any patient. Hilar regions were normal, except for 2 cases who had recanalized portal vein thrombosis. In terms of copper histochemistry, Timm and rhodanine staining was performed in all patients; however, orcein-stained slides were available only in 25 patients. There was no detectable copper staining in 5 patients (cases 3, 4, 6, 8, and 15). Three of these 5 patients (cases 4, 8, and 15) had all 3 copper stains and 2 only had Timm- and rhodanine-stained slides. Of the 28 patients with positive copper histochemistry, all were positive with Timm and 27 were positive with rhodanine. Of 25 cases evaluated with orcein, 13 were negative and 12 were positive (Figure 2). The nodular positivity rate was 76% for Timm, 52% for rhodanine, and 27% for orcein (Figure 2). Iron deposition was seen in 12 patients (focal/minimal in 11, moderate in 1). We found periodic acid-Schiff-diastase- and periodic acid-Schiff-positive granules in 7 of 33 patients (21%).

Discussion

Wilson disease is a rare metabolic disease with a prevalence of approximately 30/1 000 000. It can present clinically as acute or chronic disease. Symptoms are frequently nonspecific.1,2 Most of the patients present with chronic liver disease and cirrhosis. Age at presentation is usually between 5 and 35 years. In our study group, mean age was 22.1 years with a range of 7 to 57 years. Diagnosis of Wilson disease can be problematic because of nonspecific clinical findings and probability of common test results, ie, elevated hepatic copper, with chronic cholestatic conditions. Differential diagnosis among these disorders depends on the combination of the clinical findings, histologic features, and laboratory results. Liver biopsy is one of the important diagnostic modalities. Histopathologic features of Wilson disease are variable between “almost normal” liver and cirrhosis. The biopsy findings include steatosis, inflammation, glycogen nuclei, hepatocellular pleomorphism anisonucleosis, and cholestasis.2,4 These findings are nonspecific. Although the results can be negative, demonstration of excess copper by histochemistry is important for histologic diagnosis.5 There are multiple histochemical stains for tissue copper evaluation, including rhodanine, Timm silver sulfide, orcein, and Victoria blue. The rhodanine and Timm stains detect copper; orcein and Victoria blue detect copper-binding protein. Rhodanine is the most commonly used copper stain,4 accepted as more reliable and simple; however, Timm is known to be more sensitive.3,5 For our patients, we observed positivity rates of Timm of 85%, rhodanine of 82%, and orcein of 48%. Because copper accumulation can also be seen with chronic cholestasis, the distribution and severity of staining have to be taken into account. In chronic cholestasis, copper staining is usually limited to periseptal areas with a patchy/focal distribution. In this context, we suggest that nodular staining is a more convincing staining pattern for the histopathologic diagnosis of Wilson disease. In this study, we observed nodular staining rates with Timm of 76%, rhodanine of 52%, and orcein of 36%.

In terms of hematoxylin and eosin-stained slides, the most prevalently observed features were glyco­genated hepatocyte nuclei and hepatocellular nuclear pleomorphism/anisonucleosis. Portal vein thrombosis is a frequent finding of liver cirrhosis, with a prevalence range of 0.6% to 15.8%.6 In the present study, we found 2 cases with portal vein thrombosis (6%). We detected periodic acid-Schiff-diastase- and periodic acid-Schiff-positive granules in 7 of 33 cases (21%). We assume that, without copper histochemistry, these granules may cause a challenge in the differential diagnosis. Hemosiderin can be detected in Wilson disease because of hemolysis. In our patients, iron deposition was seen in 12 of 33 patients (46%; focal/minimal in 11 and moderate in 1).

In conclusion, although hepatocellular nuclear pleomorphism/anisonucleosis is not a commonly defined feature of Wilson disease, we think it is an important suggestive finding for Wilson disease in liver biopsies having glycogenated hepatocyte nuclei. This finding should bring into mind the consideration of copper histochemistry to look for presence of copper. According to our findings, Timm should be the first choice of stain in order to reveal copper in the liver tissue.


References:

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  3. Nemolato S, Serra S, Saccani S, Faa G. Deparaffination time: a crucial point in histochemical detection of tissue copper. Eur J Histochem. 2008;52(3):175-178.
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Volume : 15
Issue : 5
Pages : 542 - 546
DOI : 10.6002/ect.2015.0319


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From the 1Department of Pathology, the 2Liver Transplantation Institute, Department of Surgery, and the 3Departments of Pediatric Gastroenterology, Hepatology, and Nutrition, Inonu University, Malatya, Turkey
Acknowledgements: The authors have no sources of funding and no conflicts of interest to declare.
Corresponding author: Nese Karadag, Department of Pathology, Inonu University, Malatya, Turkey
Phone: +90 422 341 0660
E-mail: nesekaradag@gmail.com