Splenectomy in Liver Cirrhosis with Splenomegaly and Hypersplenism

*Adianto Nugroho*

#### **Abstract**

Spleen is a "mysterious" organ since with unique functions, and might be related to other pathology in the human body. Splenomegaly and hypersplenism can manifest following the development of portal hypertension in liver cirrhosis through fibrogenesis, immune and microenvironment dysregulation. Cirrhotic patients are generally considered as immunocompromised and prone to infections. Splenectomy in cirrhotic patients has produced concern over decrease immunity and elevated risk of infection, namely overwhelming post splenectomy pneumococcal sepsis. This review discus the splenectomy effect to the liver and how it can play a role in cirrhotic patients with portal hypertension without readily available access to liver transplantation.

**Keywords:** splenectomy, liver cirrhosis, hypersplenism, splenomegaly, liver transplantation

#### **1. Introduction**

The spleen is a unique organ with many functions, including its crosstalk with the liver in cirrhotic patients. This review aims to answer a clinical question "Should splenectomy be done in liver cirrhosis with hypersplenism and splenomegaly?".

#### **2. The spleen**

The spleen is an organ full of mystery, as stated by Galen. From the ancient times until the Renaissance, descriptions of the gross anatomy of the spleen were relatively accurate, yet the physiology of this organ remains incomplete and inaccurate. Even until today, much of spleen's function are still yet to be discovered [1].

Spleen comprised of two distinct compartments, both functional and morphological, namely red pulp and white pulp. The red pulp filters blood to remove foreign material and damaged erythrocytes. It also serves as iron, erythrocytes and platelets storages. With one fourth of body's lymphocytes stores in the spleen, it is the largest secondary organ which initiate immune response to blood-borne antigens [2]. It exerts important effects on local and systemic immune responses, which have the potential to affect different tissues and organs [3]. The white pulp, composed by periarteriolar lymphoid sheath (PALS), the follicles and the marginal zones, are the one responsible for this so called immune functions [2].

In addition, the spleen also produces opsonins, a substances that bind to the foreign antigen, which in turn enhance their uptake and phagocytosis by macrophages. Furthermore, the B-lymphocytes within the germinal centers of the spleen are also sites for the production of antibody activated by foreign antigen. The realization of this important immunological function has promoted the desire for splenic preservation [4].

#### **3. Liver cirrhosis and the spleen**

The association between the liver and spleen are shown in three different categories. Both organ, anatomically important in the portal circulation. Histologically, they share similar possession of reticuloendothelial structures, participating in substance exchange and cellular migration. And immunologically, both organs plays essential roles in immune homeostasis and pathogen clearance [2].

The first recorded encounter between spleen and cirrhosis could be trace back to Carl Freiderich Quittenbaum (1793–1852) of Rostock, Germany, who removed the spleen of a woman with cirrhosis and ascites "more from the patient's urgent entreaty rather than the surgeon's judgment." Unfortunately the woman lived only 6 h after the surgery [5].

The palpable spleen has long been considered as an obvious signs of liver cirrhosis, frequently occurs in parallel with hypersplenism, to be the major cause of cytopenia and thrombocytopenia. This condition are relatively sub-fatal, even in the absence of a bleeding varices. During the progression of liver cirrhosis, the spleen-derived immune cells and cytokines may travel into the injured liver via portal blood flow. Together with the portal hypertension and congestion, this will result in splenomegaly and hypersplenism. Furthermore, the chemokines, DAMPs like HMGB1, or exosomes, are also release into the circulation, which will trigger the activation and/or migration of splenocytes. This mechanism are known as the liver and spleen crosstalk pathways during liver cirrhosis [2].

Spleen size in patients with cirrhosis varies by the etiology of the disease. While in healthy adults, the size of the spleen in usually less than 12 cm, in cirrhotic patients it is relatively larger, as shown in the study by Kashani et al. This study revealed that the mean spleen size in the alcohol group (13.1 ± 2.5 cm) was significantly smaller than in the hepatitis C (15.0 ± 3.4 cm) and nonalcoholic steatohepatitis (15.2 ± 3.0 cm) groups (95% confidence intervals of the mean difference, 0.6 to 3.3 and 0.8 to 3.4 cm, respectively), sonographically [6].

#### **4. Splenectomy effects to the liver**

Cirrhotic patients are generally considered as immunocompromised, mainly due to the development of bacterial infection and community-acquired infections. Since the spleen is the largest lymphoid organs with large amount of T and B cells, macrophages, and dendritic cells, splenectomy in cirrhotic patients has produced concern over decrease immunity and elevated risk of infection, namely overwhelming post splenectomy pneumococcal sepsis.

However, a study by Hirakawa et al., showed the possibility of reducing suppressive cell fractions and enhancement of the effector cell population and functions by means of splenectomy, thus ameliorate the impaired immune status of cirrhotic patients [7].

Yamada et al. demonstrated that splenectomy improved hepatic functional reserves and nutritional metabolism, together with improvement in thrombocytopenia and

**227**

*Splenectomy in Liver Cirrhosis with Splenomegaly and Hypersplenism*

to metabolize bilirubin, are also reduced after splenectomy [8].

this inhibition and enhanced the regeneration of hepatocytes [9].

treatment of liver cirrhosis can be achieved through [2]:

interferons and other pharmaceuticals.

with a relative contraindications that preclude surgery.

gastric varices

of HCC

**cirrhotic liver**

cardiomyopathy [14].

and accelerate liver regeneration as well as reduce the risk of HCC [10].

leukopenia in cirrhotic patients. Splenectomy is thought to induce a decrease in platelet pooling or breakdown in the spleen of thrombocytopenic patients, and as a result, increase blood platelet counts. Bilirubinemia secondary to hypersplenism, which is caused by an increase in bilirubin production, that overloads the capacity of the liver

In a study by Ueda et al. of rats undergoing major liver resection with or without splenectomy, early stage splenic red pulp TGF-β1 production and secretion into the portal blood exert an inhibitory effect on liver regeneration. Splenectomy reversed

Study by Huang et al., unveiled serum cytokine profiles in HBV-related cirrhosis patients with PH and hypersplenism, indicating a potential role of the hypertensive spleen in the progression of liver disease. Furthermore, the changes in cytokine levels following splenectomy maybe potential advantageous to reduce liver fibrosis

Splenectomy also enhanced the repopulation of adoptively transferred bone marrow cell in cirrhotic liver and decreased collagen deposition through the upregulation of MM9 expression in transferred bone marrow cells, as suggested by Iwamoto et al. [11], and improved the efficiency of adipose tissue-derived mesenchymal cell transplant into the liver by enhancing liver SCF-1 and HGV expressions [12]. Considering all of the above mention mechanism, targeting spleen for the

• amelioration of cirrhosis' fatal complications such as bleeding esophageal or

• efficiently improving liver function and the prognosis of esophageal varices

• increasing the efficacy of liver transplantation and improving the prognosis

Surgery in a patient with liver disease carries specific and higher risks, compare to those with normal populations. Perioperative care including assessment and optimalization is the key to a safe surgery. Many cirrhosis patients present themselves

The predictors for complications including Child-Pugh class B or C, ascites, etiology of cirrhosis other than PBC, elevated creatinine, preoperative infection, COPD, preoperative upper GI bleeding, invasiveness of surgical procedure, intraoperative hypotension, and ASA status 4–5. While the predictors of mortality including male gender, Child-Pugh class B or C, ascites, etiology of cirrhosis other than PBC, preoperative infection, ASA status 4–5 and respiratory surgery. The presence of 1 risk factors carries a 9.3% risk of complications, and this increase with the more numbers of risk factors. A total of 7–8 risk factors carries a 100% risk of complications [13]. Friedman proposed the following list of contraindication to elective surgery in patients with liver disease, including acute viral hepatitis, alcoholic hepatitis, acute liver failure, acute renal failure, severe coagulopathy, hypoxemia and

• supplementary treatment for anti-HCV therapy in combination with

**5. Technical and perioperative consideration for splenectomy in** 

*DOI: http://dx.doi.org/10.5772/intechopen.94337*

#### *Splenectomy in Liver Cirrhosis with Splenomegaly and Hypersplenism DOI: http://dx.doi.org/10.5772/intechopen.94337*

*Liver Pathology*

for splenic preservation [4].

6 h after the surgery [5].

**3. Liver cirrhosis and the spleen**

In addition, the spleen also produces opsonins, a substances that bind to the foreign antigen, which in turn enhance their uptake and phagocytosis by macrophages. Furthermore, the B-lymphocytes within the germinal centers of the spleen are also sites for the production of antibody activated by foreign antigen. The realization of this important immunological function has promoted the desire

The association between the liver and spleen are shown in three different categories. Both organ, anatomically important in the portal circulation. Histologically, they share similar possession of reticuloendothelial structures, participating in substance exchange and cellular migration. And immunologically, both organs plays

The first recorded encounter between spleen and cirrhosis could be trace back to Carl Freiderich Quittenbaum (1793–1852) of Rostock, Germany, who removed the spleen of a woman with cirrhosis and ascites "more from the patient's urgent entreaty rather than the surgeon's judgment." Unfortunately the woman lived only

The palpable spleen has long been considered as an obvious signs of liver cirrhosis, frequently occurs in parallel with hypersplenism, to be the major cause of cytopenia and thrombocytopenia. This condition are relatively sub-fatal, even in the absence of a bleeding varices. During the progression of liver cirrhosis, the spleen-derived immune cells and cytokines may travel into the injured liver via portal blood flow. Together with the portal hypertension and congestion, this will result in splenomegaly and hypersplenism. Furthermore, the chemokines, DAMPs like HMGB1, or exosomes, are also release into the circulation, which will trigger the activation and/or migration of splenocytes. This mechanism are known as the

Spleen size in patients with cirrhosis varies by the etiology of the disease. While

Cirrhotic patients are generally considered as immunocompromised, mainly due to the development of bacterial infection and community-acquired infections. Since the spleen is the largest lymphoid organs with large amount of T and B cells, macrophages, and dendritic cells, splenectomy in cirrhotic patients has produced concern over decrease immunity and elevated risk of infection, namely overwhelm-

However, a study by Hirakawa et al., showed the possibility of reducing suppressive cell fractions and enhancement of the effector cell population and functions by means of splenectomy, thus ameliorate the impaired immune status of cirrhotic

Yamada et al. demonstrated that splenectomy improved hepatic functional reserves and nutritional metabolism, together with improvement in thrombocytopenia and

in healthy adults, the size of the spleen in usually less than 12 cm, in cirrhotic patients it is relatively larger, as shown in the study by Kashani et al. This study revealed that the mean spleen size in the alcohol group (13.1 ± 2.5 cm) was significantly smaller than in the hepatitis C (15.0 ± 3.4 cm) and nonalcoholic steatohepatitis (15.2 ± 3.0 cm) groups (95% confidence intervals of the mean difference, 0.6 to

essential roles in immune homeostasis and pathogen clearance [2].

liver and spleen crosstalk pathways during liver cirrhosis [2].

3.3 and 0.8 to 3.4 cm, respectively), sonographically [6].

**4. Splenectomy effects to the liver**

ing post splenectomy pneumococcal sepsis.

**226**

patients [7].

leukopenia in cirrhotic patients. Splenectomy is thought to induce a decrease in platelet pooling or breakdown in the spleen of thrombocytopenic patients, and as a result, increase blood platelet counts. Bilirubinemia secondary to hypersplenism, which is caused by an increase in bilirubin production, that overloads the capacity of the liver to metabolize bilirubin, are also reduced after splenectomy [8].

In a study by Ueda et al. of rats undergoing major liver resection with or without splenectomy, early stage splenic red pulp TGF-β1 production and secretion into the portal blood exert an inhibitory effect on liver regeneration. Splenectomy reversed this inhibition and enhanced the regeneration of hepatocytes [9].

Study by Huang et al., unveiled serum cytokine profiles in HBV-related cirrhosis patients with PH and hypersplenism, indicating a potential role of the hypertensive spleen in the progression of liver disease. Furthermore, the changes in cytokine levels following splenectomy maybe potential advantageous to reduce liver fibrosis and accelerate liver regeneration as well as reduce the risk of HCC [10].

Splenectomy also enhanced the repopulation of adoptively transferred bone marrow cell in cirrhotic liver and decreased collagen deposition through the upregulation of MM9 expression in transferred bone marrow cells, as suggested by Iwamoto et al. [11], and improved the efficiency of adipose tissue-derived mesenchymal cell transplant into the liver by enhancing liver SCF-1 and HGV expressions [12].

Considering all of the above mention mechanism, targeting spleen for the treatment of liver cirrhosis can be achieved through [2]:


#### **5. Technical and perioperative consideration for splenectomy in cirrhotic liver**

Surgery in a patient with liver disease carries specific and higher risks, compare to those with normal populations. Perioperative care including assessment and optimalization is the key to a safe surgery. Many cirrhosis patients present themselves with a relative contraindications that preclude surgery.

The predictors for complications including Child-Pugh class B or C, ascites, etiology of cirrhosis other than PBC, elevated creatinine, preoperative infection, COPD, preoperative upper GI bleeding, invasiveness of surgical procedure, intraoperative hypotension, and ASA status 4–5. While the predictors of mortality including male gender, Child-Pugh class B or C, ascites, etiology of cirrhosis other than PBC, preoperative infection, ASA status 4–5 and respiratory surgery. The presence of 1 risk factors carries a 9.3% risk of complications, and this increase with the more numbers of risk factors. A total of 7–8 risk factors carries a 100% risk of complications [13].

Friedman proposed the following list of contraindication to elective surgery in patients with liver disease, including acute viral hepatitis, alcoholic hepatitis, acute liver failure, acute renal failure, severe coagulopathy, hypoxemia and cardiomyopathy [14].

Regarding the preferred method for splenectomy, recently laparoscopic has become technically feasible, safe and effective procedure for hypersplenism secondary to cirrhosis, and contributes to less blood loss, shorter length of stay and less impairment of liver function. However, this methods are generally more costly and might not readily available in every hospital. Thus the choice of splenectomy method must be personally selected for each patient, surgeon and hospital [15].

#### **6. Splenectomy as a bridge to liver transplant**

It is already a general consensus that liver transplantation is the preferred treatment options for patient with end stage liver disease. However, the waiting time for liver transplantation is also long due to the shortage of donor organs, even in living donor liver transplantation setting. Moreover, in some countries, liver transplantation still not a feasible option for all patients.

One among many alternatives is by doing a splenectomy prior to liver transplantation in patient with liver cirrhosis and subsequent splenomegaly-hypersplenism. A study by Kong et al., studied 833 patient patients underwent liver transplantation, of which 88 patients had splenectomy before liver transplantation. They found that postoperative infection and 90-days mortality in the splenectomy and non-splenectomy group were not statistically difference. Furthermore, the posttransplant thrombocytopenia and early allograft dysfunctions is significantly lower in splenectomy group compare to non-splenectomy group. They suggested that pretransplantation splenectomy is recommended in cases with risky patients without appropriate source of liver for LT. Taking into consideration the possibility of more difficult operation due to adhesion when transplantation is being done. One thing to note is that as a "re-operation" the splenectomy is often as- sociated with more difficult dissection due to adhesions [16].

#### **7. Summary**

Splenectomy is beneficial in reversal of the pathologic process through live regeneration and pre-transplant splenectomy could be an alternative in patients without appropriate source of liver for liver transplantation. However, perioperative considerations should be thoroughly assessed to allow a safe surgery.

#### **Conflict of interest**

"The authors declare no conflict of interest."

#### **Notes/thanks/other declarations**

Part of this article was presented in APASL Congress, Bali, March 2020.

**229**

**Author details**

Adianto Nugroho

General Hospital, Jakarta, Indonesia

provided the original work is properly cited.

\*Address all correspondence to: adiyusuf97@gmail.com

Department of Surgery, HPB Section, Digestive Division, Fatmawati Central

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Splenectomy in Liver Cirrhosis with Splenomegaly and Hypersplenism*

*DOI: http://dx.doi.org/10.5772/intechopen.94337*

*Splenectomy in Liver Cirrhosis with Splenomegaly and Hypersplenism DOI: http://dx.doi.org/10.5772/intechopen.94337*

*Liver Pathology*

Regarding the preferred method for splenectomy, recently laparoscopic has become technically feasible, safe and effective procedure for hypersplenism secondary to cirrhosis, and contributes to less blood loss, shorter length of stay and less impairment of liver function. However, this methods are generally more costly and might not readily available in every hospital. Thus the choice of splenectomy method must be personally selected for each patient, surgeon and hospital [15].

It is already a general consensus that liver transplantation is the preferred treatment options for patient with end stage liver disease. However, the waiting time for liver transplantation is also long due to the shortage of donor organs, even in living donor liver transplantation setting. Moreover, in some countries, liver transplanta-

One among many alternatives is by doing a splenectomy prior to liver transplantation in patient with liver cirrhosis and subsequent splenomegaly-hypersplenism. A study by Kong et al., studied 833 patient patients underwent liver transplantation, of which 88 patients had splenectomy before liver transplantation. They found that postoperative infection and 90-days mortality in the splenectomy and non-splenectomy group were not statistically difference. Furthermore, the posttransplant thrombocytopenia and early allograft dysfunctions is significantly lower in splenectomy group compare to non-splenectomy group. They suggested that pretransplantation splenectomy is recommended in cases with risky patients without appropriate source of liver for LT. Taking into consideration the possibility of more difficult operation due to adhesion when transplantation is being done. One thing to note is that as a "re-operation" the splenectomy is often as- sociated with more

Splenectomy is beneficial in reversal of the pathologic process through live regeneration and pre-transplant splenectomy could be an alternative in patients without appropriate source of liver for liver transplantation. However, perioperative

Part of this article was presented in APASL Congress, Bali, March 2020.

considerations should be thoroughly assessed to allow a safe surgery.

"The authors declare no conflict of interest."

**Notes/thanks/other declarations**

**6. Splenectomy as a bridge to liver transplant**

tion still not a feasible option for all patients.

difficult dissection due to adhesions [16].

**7. Summary**

**Conflict of interest**

**228**

## **Author details**

Adianto Nugroho Department of Surgery, HPB Section, Digestive Division, Fatmawati Central General Hospital, Jakarta, Indonesia

\*Address all correspondence to: adiyusuf97@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Paraskevas, G.K., Koutsouflianiotis, K.N., Nitsa, Z. *et al.* Knowledge of the anatomy and physiology of the spleen throughout Antiquity and the Early Middle Ages. *Anat Sci Int* 2016; **91,** 43-55

[2] Cesta MF. Normal structure, function, and histology of the spleen. *Toxicol Pathol*. 2006;34(5):455-465.

[3] Li, L, Duan, M, Chen, W *et al.* The spleen in liver cirrhosis: revisiting an old enemy with novel targets. *J Transl Med* 2017;**15,** 111

[4] Stricland A, Lloyd D. The spleen and indications for splenectomy. Surgery 2007;25(2): 98-101

[5] Morgenstern L. A history of splenectomy. In: Hiat JR, Phillips EH, Morgenstern L, ed. Surgical disease of the spleen. Springer, 1997.

[6] Kashani A, Salehi B, Anghesom D, Kawayeh AM, Rouse GA, Runyon BA. Spleen size in cirrhosis of different etiologies. *J Ultrasound Med*. 2015; 34(2):233-238.

[7] Hirakawa Y, Ogata T, Sasada T, Yamashita T, Itoh K, Tanaka H and Okuda K: Immunological consequences following splenectomy in patients with liver cirrhosis. Exp Ther Med 18: 848-856, 2019

[8] Yamada S, Morine Y, Imura S, et al. Liver regeneration after splenectomy in patients with liver cirrhosis. *Hepatol Res*. 2016;46(5):443-449.

[9] Ueda S, Yamanoi A, Hishikawa Y, et al. Transforming growth factor-b1 released from the spleen exerts a growth inhibitory effect on liver regen- eration in rats. Lab Invest. 2003;83(11):1595-603.

[10] Huang N, Ji F, Zhang S, et al. Effect of Splenectomy on Serum Cytokine Profiles in Hepatitis B Virus-Related Cirrhosis Patients with Portal Hypertension. *Viral Immunol*. 2018;31(5):371-378.

[11] Iwamoto T, Terai S, Mizunaga Y, et al. Splenectomy enhances the antifibrotic effect of bone marrow cell infusion and improves liver function in cirrhotic mice and patients. *J Gastroenterol*. 2012;47(3):300-312.

[12] Tang WP, Akahoshi T, Piao JS, et al. Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats. *Liver Int*. 2016;36(8):1151-1159

[13] Rai R, Nagral S, Nagral A. Surgery in a patient with liver disease. *J Clin Exp Hepatol*. 2012;2(3):238-246

[14] Friedman L.S. Surgery in the patient with liver disease. Trans Am Clin Climatol Assoc. 2010;121:192-205

[15] Zhan XL, Ji Y, Wang YD. Laparoscopic splenectomy for hypersplenism secondary to liver cirrhosis and portal hypertension. *World J Gastroenterol* 2014; 20(19): 5794-5800

[16] Kong, L., Li, M., Li, L. *et al.* Splenectomy before adult liver transplantation: a retrospective study. *BMC Surg 2017;* **17,** 44

**231**

**Chapter 12**

**Abstract**

*Nese Karadag Soylu*

psychiatric symptoms.

**1. Introduction**

Histopathology of Wilson Disease

Wilson Disease (WD) is a genetic metabolic disease of copper metabolism. The implicated gene is ATP7B, encodes a P-type ATPase which transports copper. The resultant defective metabolism of copper results in copper accumulation in multiple tissues especially liver, eye and central nervous system. WD occurs worldwide, usually between 5 and 35 years; a wider age range is also reported. Clinical presentations are diverse and include combinations of hepatic, neurological, ophthalmic and psychiatric manifestations. Other organs or tissues may also be affected. Biochemical abnormalities such as serum ceruloplasmin and 24-h urinary copper excretion are important for the diagnosis but are not always abnormal in WD. The liver histopathology has several different patterns from mild nonspecific changes to acute fulminant hepatitis and cirrhosis. Copper histochemistry is helpful in diagnosis. Genetic testing is another diagnostic tool. It is important to diagnose WD because it is fatal when overlooked, curable when diagnosed. The diagnosis should be keep in mind at all ages in patients with hepatic disease, neurological disease, or

**Keywords:** Wilson disease, copper, liver, histopathology, histochemistry

satisfying genetic diseases to be diagnosed and treated.

Wilson Disease (WD) is an autosomal recessive genetic metabolic disease of copper metabolism. Its incidence is vary in different geographic areas with an average incidence of 1 in 30,000 individuals worldwide. Recent studies suggest a considerably higher prevalence of 1:1500–1:3000 for WD. It is caused by mutations in the ATP7B gene encoding a copper transporting P-type ATPase required for copper excretion into the bile [1, 2]. WD is first described by the American neurologist Samuel Alexander Kinnier Wilson in 1912. There are earlier case reports mostly by neurologist in mid 1800s [3]. Kayser and Fleischer mentioned the pigmented corneal rings, in 1902 and 1903 respectively In 1911, Wilson presented his monograph describing the "progressive lenticular degeneration". Bramwell, in 1916, was the first to realize the importance of liver pathology in WD. In 1948, Cumings described the copper abnormalities in WD and in 1952, Scheinberg and Gitlin showed that the ceruloplasmin levels were low in most of WD patients. In 1956, Walshe introduced the penicillamine as a chelating agent, the first effective treatment for the condition [3, 4]. This discovery of successful chelation therapy makes WD one of the most

Originally WD was described as a neurodegenerative disease associated with cirrhosis of the liver. Later, WD was observed in children and adolescents with acute or chronic liver disease without any neurologic symptoms [5]. Now, WD is considered a multi-systemic disorder, in which hepatic, neurological and

## **Chapter 12** Histopathology of Wilson Disease

*Nese Karadag Soylu*

## **Abstract**

Wilson Disease (WD) is a genetic metabolic disease of copper metabolism. The implicated gene is ATP7B, encodes a P-type ATPase which transports copper. The resultant defective metabolism of copper results in copper accumulation in multiple tissues especially liver, eye and central nervous system. WD occurs worldwide, usually between 5 and 35 years; a wider age range is also reported. Clinical presentations are diverse and include combinations of hepatic, neurological, ophthalmic and psychiatric manifestations. Other organs or tissues may also be affected. Biochemical abnormalities such as serum ceruloplasmin and 24-h urinary copper excretion are important for the diagnosis but are not always abnormal in WD. The liver histopathology has several different patterns from mild nonspecific changes to acute fulminant hepatitis and cirrhosis. Copper histochemistry is helpful in diagnosis. Genetic testing is another diagnostic tool. It is important to diagnose WD because it is fatal when overlooked, curable when diagnosed. The diagnosis should be keep in mind at all ages in patients with hepatic disease, neurological disease, or psychiatric symptoms.

**Keywords:** Wilson disease, copper, liver, histopathology, histochemistry

#### **1. Introduction**

Wilson Disease (WD) is an autosomal recessive genetic metabolic disease of copper metabolism. Its incidence is vary in different geographic areas with an average incidence of 1 in 30,000 individuals worldwide. Recent studies suggest a considerably higher prevalence of 1:1500–1:3000 for WD. It is caused by mutations in the ATP7B gene encoding a copper transporting P-type ATPase required for copper excretion into the bile [1, 2]. WD is first described by the American neurologist Samuel Alexander Kinnier Wilson in 1912. There are earlier case reports mostly by neurologist in mid 1800s [3]. Kayser and Fleischer mentioned the pigmented corneal rings, in 1902 and 1903 respectively In 1911, Wilson presented his monograph describing the "progressive lenticular degeneration". Bramwell, in 1916, was the first to realize the importance of liver pathology in WD. In 1948, Cumings described the copper abnormalities in WD and in 1952, Scheinberg and Gitlin showed that the ceruloplasmin levels were low in most of WD patients. In 1956, Walshe introduced the penicillamine as a chelating agent, the first effective treatment for the condition [3, 4]. This discovery of successful chelation therapy makes WD one of the most satisfying genetic diseases to be diagnosed and treated.

Originally WD was described as a neurodegenerative disease associated with cirrhosis of the liver. Later, WD was observed in children and adolescents with acute or chronic liver disease without any neurologic symptoms [5]. Now, WD is considered a multi-systemic disorder, in which hepatic, neurological and

**230**

*Liver Pathology*

**References**

43-55

2017;**15,** 111

2007;25(2): 98-101

34(2):233-238.

848-856, 2019

2016;46(5):443-449.

2003;83(11):1595-603.

[1] Paraskevas, G.K., Koutsouflianiotis, K.N., Nitsa, Z. *et al.* Knowledge of the anatomy and physiology of the spleen throughout Antiquity and the Early Middle Ages. *Anat Sci Int* 2016; **91,**

[10] Huang N, Ji F, Zhang S, et al. Effect of Splenectomy on Serum Cytokine Profiles in Hepatitis B Virus-Related Cirrhosis Patients with Portal Hypertension. *Viral Immunol*.

[11] Iwamoto T, Terai S, Mizunaga Y, et al. Splenectomy enhances the antifibrotic effect of bone marrow cell infusion and improves liver function in cirrhotic mice and patients. *J Gastroenterol*. 2012;47(3):300-312.

Piao JS, et al. Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats. *Liver Int*.

[13] Rai R, Nagral S, Nagral A. Surgery in a patient with liver disease. *J Clin Exp* 

[14] Friedman L.S. Surgery in the patient with liver disease. Trans Am Clin Climatol Assoc. 2010;121:192-205

Laparoscopic splenectomy for hypersplenism secondary to liver cirrhosis and portal hypertension. *World J Gastroenterol* 2014; 20(19): 5794-5800

2018;31(5):371-378.

[12] Tang WP, Akahoshi T,

2016;36(8):1151-1159

*Hepatol*. 2012;2(3):238-246

[15] Zhan XL, Ji Y, Wang YD.

[16] Kong, L., Li, M., Li, L. *et al.* Splenectomy before adult liver transplantation: a retrospective study.

*BMC Surg 2017;* **17,** 44

[2] Cesta MF. Normal structure, function, and histology of the spleen. *Toxicol Pathol*. 2006;34(5):455-465.

[3] Li, L, Duan, M, Chen, W *et al.* The spleen in liver cirrhosis: revisiting an old enemy with novel targets. *J Transl Med*

[4] Stricland A, Lloyd D. The spleen and indications for splenectomy. Surgery

[6] Kashani A, Salehi B, Anghesom D, Kawayeh AM, Rouse GA, Runyon BA. Spleen size in cirrhosis of different etiologies. *J Ultrasound Med*. 2015;

[7] Hirakawa Y, Ogata T, Sasada T, Yamashita T, Itoh K, Tanaka H and Okuda K: Immunological consequences following splenectomy in patients with liver cirrhosis. Exp Ther Med 18:

[8] Yamada S, Morine Y, Imura S, et al. Liver regeneration after splenectomy in patients with liver cirrhosis. *Hepatol Res*.

[9] Ueda S, Yamanoi A, Hishikawa Y, et al. Transforming growth factor-b1 released from the spleen exerts a growth inhibitory effect on liver regen- eration in rats. Lab Invest.

[5] Morgenstern L. A history of splenectomy. In: Hiat JR, Phillips EH, Morgenstern L, ed. Surgical disease of

the spleen. Springer, 1997.

psychiatric symptoms are often associated with renal, endocrine, osteoarticular, corneal and myocardial disturbances, all related to abnormal copper metabolism ending with systemic accumulation of the copper [6, 7].

Ultrastructural findings of WD have also been studied. The mitochondrial changes are the most distinctive and pathogenetically significant and include heterogeneity of size and shape, increased matrix density, separation of inner from outer membranes, enlarged intercristal spaces and various types of inclusions. Importantly, ultrastructural mitochondrial changes in WD cannot be considered pathognomonic; although exceedingly rare with cholestatic liver disease, such changes are found with mtDNA depletion disorders [4, 8].

WD has considerable variation in clinical presentations, the most common ones being liver disease and neuropsychiatric disturbances [9]. There is considerable phenotypic variation in WD: Some patients present with hepatic disease during the first decade of life, some with neurological degeneration in adolescence or adult life, with or without overt liver disease. In a study by Ferenci et al., the severity of liver disease did not show correlation with the mutation status. Rather, they reported that the prevalence of cirrhosis increased with age in pediatric patients. They found that hepatic disease was more common among females, whereas neurological presentation occurred more frequently in males [10]. The wide range of disease patterns cannot be explained just by different mutations. Environmental, epigenetic, and other genetic factors are also contribute pathogenesis of WD [6, 10].

Classically low serum copper and low ceruloplasmin levels with high urinary copper content make a triad which is usually associated with WD diagnosis. But this triad may be absent or incomplete in 3% of genetically confirmed WD cases [7].

Early diagnosis of WD is important. But it is also important to make the diagnosis of WD prior to transplantation. Because organ transplant networks make special provision for acute liver failure (ALF) due to WD when considering the urgency of transplantation and the terminology relating to acute presentations of liver disease become relevant when listing a case of WD [11].

A large variability in the age of onset and in the clinical presentation of WD exists. Hepatic manifestations of WD at presentation can be extremely variable, and range from asymptomatic hepatomegaly, isolated splenomegaly, persistent or intermittent elevation of serum aminotransferases, jaundice, fatty liver or pseudoautoimmune hepatitis, acute hepatitis, compensated or decompensated cirrhosis to acute liver failure (ALF). The varied clinical manifestations of WD due to pathological copper accumulation in different organs, even in the early course of the disease, often pose a diagnostic challenge [7].

The main therapeutic strategy is using chelating agents, particularly D-penicillamine. Liver transplantation (LT) is reserved for patients unresponsive to medical therapy or with fulminant hepatic failure. LT for neurological complications is highly controversial and generally cannot be recommended [8].

#### **2. Pathogenesis and clinical manifestations**

Copper is an essential element for cellular function. Dietary copper is absorbed in the stomach and duodenum and reaches the liver by the portal vein [1]. Intestinal uptake is regulated by the Menkes ATPase (ATP7A). The ATP7A gene is expressed in most tissues except the liver. Menkes disease, an X-linked copper deficiency disorder, results from mutations in this gene. The abnormal gene in Wilson disease is ATP7B (the Wilson ATPase) which shows 56% homology to ATP7A [8]. It is expressed mainly in the liver but its expression is not restricted to liver cells. This data suggests that ATP7B dysfunction might be responsible for the systemic

**233**

investigation [1].

*Histopathology of Wilson Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

disturbances of copper trafficking in the whole human body [1, 6]. The hepatic protein ATP7B encodes a copper-transporting P-type ATPase, transporting copper into the secretory pathway for incorporation into apoceruloplasmin, forming ceruloplasmin. ATP7B moves copper into the trans-Golgi network, where ceruloplasmin peptide acquires its complement of copper, assumes its folded state and is then released into the circulation [12]. Excess is excreted eventually into the bile. Without the normal complement of copper, the peptide folds differently and has a decreased circulating half-life, leading to a low level of serum ceruloplasmin. Biliary excretion of copper is necessary for its homeostasis. When ATPB7 is defective, excess copper accumulates in the hepatocytes. Eventually the excess copper exceeds the storage capacity causing hepatocellular injury and release of copper into the circulation. Most WD patients have a low level of circulating ceruloplasmin which is a direct result of defective copper handling in hepatocytes as a result of mutation of the ATP7B gene. Free copper is extremely toxic and can produce irreversible cellular damage. The functional consequences of pathogenic ATP7B mutation are increased intracellular copper levels. This produces oxidative stress and free radical formation as well as mitochondrial dysfunction, which results in cell death in the liver, brain, kidneys, heart, eyes, and joints. As this disease damages multiple systems at one time, it poses a diagnostic challenge [2]. Over 600 gene alteration in ATP7B were recognized [6, 12]. The most common ones are single-nucleotide missense and nonsense mutations, chased by insertions/deletions, and, rarely, splice site mutations. H1069Q is the most common mutation around the world, seen in most of the WD carriers in Europe and USA, with some absence for this mutation in some countries [6]. Correlation of phenotype with specific mutations (genotype) is difficult in Wilson disease because the vast majority of affected individuals are compound heterozygotes, possessing one copy each of two different mutations. Differences in clinical features of various mutations between siblings and even identical twins suggests that other genes or environmental factors are important [6, 8]. In a study by Ferenci et al., it was suggested that the HSD17B13:TA allele may modulate the phenotype and outcome of WD by reducing the transition from copper induced hemolysis to fulminant WD. Furthermore, it is associated with milder histological changes [10]. When testing for mutations of the WD gene ATP7B becomes inexpensive and rapid, genetic testing may become the starting point for diagnostic

WD has a myriad of clinical presentations, hepatic, neurological, ophthalmic and psychiatric, that mimic other conditions. WD may present at any age. Although most patients present between ages 5 and 35, the age range is much wider. There are cases reported as early as 9 months and as late as the eighth decade [1, 2, 13]. So far,

Approximately one half of the patients with WD present with liver disease. In the majority of cases, WD manifests its presence during childhood or teenage years in the form of liver symptoms [7]. Hepatic symptoms and presentations of WD are very variable from asymptomatic cases to cases with overt cirrhosis or with ALF. Liver disease may mimic all forms of common liver conditions. All children with an apparent diagnosis of autoimmune hepatitis should also be investigated for WD, and adults with a presumptive diagnosis of autoimmune hepatitis failing to respond rapidly and appropriately to corticosteroid therapy must be carefully evaluated for WD. In terms of the rate of progression of the disease, cirrhosis is usually diagnosed in the second decade of life, although some individuals do not develop cirrhosis, even after the fourth decade of life [15]. Hepatic manifestations usually present earlier than neurological symptoms by 5 years. The most common hepatic signs and symptoms are jaundice, hepatomegaly and abdominal pain [1]. In a subset of patients focal liver lesions may show up, showing with a wide run of imaging

the oldest patient in English literature is a 77- year-old Turkish woman [14].

#### *Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

*Liver Pathology*

psychiatric symptoms are often associated with renal, endocrine, osteoarticular, corneal and myocardial disturbances, all related to abnormal copper metabolism

Ultrastructural findings of WD have also been studied. The mitochondrial changes are the most distinctive and pathogenetically significant and include heterogeneity of size and shape, increased matrix density, separation of inner from outer membranes, enlarged intercristal spaces and various types of inclusions. Importantly, ultrastructural mitochondrial changes in WD cannot be considered pathognomonic; although exceedingly rare with cholestatic liver disease, such

WD has considerable variation in clinical presentations, the most common ones being liver disease and neuropsychiatric disturbances [9]. There is considerable phenotypic variation in WD: Some patients present with hepatic disease during the first decade of life, some with neurological degeneration in adolescence or adult life, with or without overt liver disease. In a study by Ferenci et al., the severity of liver disease did not show correlation with the mutation status. Rather, they reported that the prevalence of cirrhosis increased with age in pediatric patients. They found that hepatic disease was more common among females, whereas neurological presentation occurred more frequently in males [10]. The wide range of disease patterns cannot be explained just by different mutations. Environmental, epigenetic,

and other genetic factors are also contribute pathogenesis of WD [6, 10].

Classically low serum copper and low ceruloplasmin levels with high urinary copper content make a triad which is usually associated with WD diagnosis. But this triad may be absent or incomplete in 3% of genetically confirmed WD cases [7]. Early diagnosis of WD is important. But it is also important to make the diagnosis of WD prior to transplantation. Because organ transplant networks make special provision for acute liver failure (ALF) due to WD when considering the urgency of transplantation and the terminology relating to acute presentations of liver disease

A large variability in the age of onset and in the clinical presentation of WD exists. Hepatic manifestations of WD at presentation can be extremely variable, and range from asymptomatic hepatomegaly, isolated splenomegaly, persistent or intermittent elevation of serum aminotransferases, jaundice, fatty liver or pseudoautoimmune hepatitis, acute hepatitis, compensated or decompensated cirrhosis to acute liver failure (ALF). The varied clinical manifestations of WD due to pathological copper accumulation in different organs, even in the early course of the disease,

The main therapeutic strategy is using chelating agents, particularly D-penicillamine. Liver transplantation (LT) is reserved for patients unresponsive to medical therapy or with fulminant hepatic failure. LT for neurological complica-

Copper is an essential element for cellular function. Dietary copper is absorbed in the stomach and duodenum and reaches the liver by the portal vein [1]. Intestinal uptake is regulated by the Menkes ATPase (ATP7A). The ATP7A gene is expressed in most tissues except the liver. Menkes disease, an X-linked copper deficiency disorder, results from mutations in this gene. The abnormal gene in Wilson disease is ATP7B (the Wilson ATPase) which shows 56% homology to ATP7A [8]. It is expressed mainly in the liver but its expression is not restricted to liver cells. This data suggests that ATP7B dysfunction might be responsible for the systemic

tions is highly controversial and generally cannot be recommended [8].

ending with systemic accumulation of the copper [6, 7].

changes are found with mtDNA depletion disorders [4, 8].

become relevant when listing a case of WD [11].

**2. Pathogenesis and clinical manifestations**

often pose a diagnostic challenge [7].

**232**

disturbances of copper trafficking in the whole human body [1, 6]. The hepatic protein ATP7B encodes a copper-transporting P-type ATPase, transporting copper into the secretory pathway for incorporation into apoceruloplasmin, forming ceruloplasmin. ATP7B moves copper into the trans-Golgi network, where ceruloplasmin peptide acquires its complement of copper, assumes its folded state and is then released into the circulation [12]. Excess is excreted eventually into the bile. Without the normal complement of copper, the peptide folds differently and has a decreased circulating half-life, leading to a low level of serum ceruloplasmin. Biliary excretion of copper is necessary for its homeostasis. When ATPB7 is defective, excess copper accumulates in the hepatocytes. Eventually the excess copper exceeds the storage capacity causing hepatocellular injury and release of copper into the circulation. Most WD patients have a low level of circulating ceruloplasmin which is a direct result of defective copper handling in hepatocytes as a result of mutation of the ATP7B gene. Free copper is extremely toxic and can produce irreversible cellular damage. The functional consequences of pathogenic ATP7B mutation are increased intracellular copper levels. This produces oxidative stress and free radical formation as well as mitochondrial dysfunction, which results in cell death in the liver, brain, kidneys, heart, eyes, and joints. As this disease damages multiple systems at one time, it poses a diagnostic challenge [2]. Over 600 gene alteration in ATP7B were recognized [6, 12]. The most common ones are single-nucleotide missense and nonsense mutations, chased by insertions/deletions, and, rarely, splice site mutations. H1069Q is the most common mutation around the world, seen in most of the WD carriers in Europe and USA, with some absence for this mutation in some countries [6]. Correlation of phenotype with specific mutations (genotype) is difficult in Wilson disease because the vast majority of affected individuals are compound heterozygotes, possessing one copy each of two different mutations. Differences in clinical features of various mutations between siblings and even identical twins suggests that other genes or environmental factors are important [6, 8]. In a study by Ferenci et al., it was suggested that the HSD17B13:TA allele may modulate the phenotype and outcome of WD by reducing the transition from copper induced hemolysis to fulminant WD. Furthermore, it is associated with milder histological changes [10]. When testing for mutations of the WD gene ATP7B becomes inexpensive and rapid, genetic testing may become the starting point for diagnostic investigation [1].

WD has a myriad of clinical presentations, hepatic, neurological, ophthalmic and psychiatric, that mimic other conditions. WD may present at any age. Although most patients present between ages 5 and 35, the age range is much wider. There are cases reported as early as 9 months and as late as the eighth decade [1, 2, 13]. So far, the oldest patient in English literature is a 77- year-old Turkish woman [14].

Approximately one half of the patients with WD present with liver disease. In the majority of cases, WD manifests its presence during childhood or teenage years in the form of liver symptoms [7]. Hepatic symptoms and presentations of WD are very variable from asymptomatic cases to cases with overt cirrhosis or with ALF. Liver disease may mimic all forms of common liver conditions. All children with an apparent diagnosis of autoimmune hepatitis should also be investigated for WD, and adults with a presumptive diagnosis of autoimmune hepatitis failing to respond rapidly and appropriately to corticosteroid therapy must be carefully evaluated for WD. In terms of the rate of progression of the disease, cirrhosis is usually diagnosed in the second decade of life, although some individuals do not develop cirrhosis, even after the fourth decade of life [15]. Hepatic manifestations usually present earlier than neurological symptoms by 5 years. The most common hepatic signs and symptoms are jaundice, hepatomegaly and abdominal pain [1]. In a subset of patients focal liver lesions may show up, showing with a wide run of imaging

highlights. The lion's share of focal liver lesions in patients with WD are benign nodules, but there are reports that have depicted malignant liver tumors or dysplastic nodules in these patients. Although rare in WD compared to other liver diseases, hepatocellular carcinoma occurs in patients of all ages. Cholangiocarcinoma may also occur in WD [8].

Neurologic manifestations include tremor, gait disturbances, choreiform movements, Parkinsonism or akinetic rigid syndrome i.e., partial parkinsonism, dysarthria, pseudobulbar palsy, rigid dystonia, seizures, migraine headaches, and insomnia. In WD cohorts, neurological presentation is associated with a significantly longer time from onset of symptoms to diagnosis than hepatic presentation, ranging from 2.5 to 6 years. In large case series, mean age at onset of neurologic problems extends from 15 to 21 a long time of age, a decade after onset of liver disease, but a number of patients have been analyzed with a starting neurologic onset earlier than age 10 [7]. Psychiatric manifestations encompass depression, neuroses, personality changes, psychosis and poor performance at school. It was reported that 30—40% of patients have psychiatric symptoms at diagnosis and 20% had seen a psychiatrist prior to their WD diagnosis [12]. WD should be ruled out in any teenager with unexplained cognitive, psychiatric, or movement disorder [13]. Neuropsychiatric signs are the predominant presentation in adults but also may be present in up to 50% of teenagers. WD should also be included in the differential diagnosis work-up of unclear neuropsychiatric syndromes in patients after age 60 years [9].

Ocular findings include the Kayser–Fleischer (KF) ring, due to copper accumulation in Desçemet's membrane, and sunflower cataracts, due to copper accumulation in the lens. They are diagnosed by slit lamp examination. In known cases of hepatic WD, the rings are present in just over half of patients. KF rings are usually absent in children with liver disease. KF rings are rarely observed in other conditions such as in patients with chronic cholestatic diseases, monoclonal gammopathies, multiple myeloma, arci senilis, and pulmonary carcinoma and are thus not specific for WD [1, 7]. Of note, KF rings are not so easy to diagnose without experience, some authors suggest that anterior segment Scheimpflug imaging (Pentacam, Oculus) could be more helpful to diagnose or confirm KF rings by ophthalmologists with little experience in patients with WD. In general it is said that when neurological symptoms are present, KF rings is present in almost all WD patients at disease diagnosis [7]. But there are reports of cases with neurological involvement without KF rings [8].

Other presentations and clinical findings are intermittent bouts of jaundice caused by haemolysis, gynaecomastia, amenorrhoea, repeated spontaneous abortion, cardiac complications including ECG abnormalities, ventricular fibrillation, cardiomyopathy, orthostatic hypotension, urolithiasis, renal tubular disease, hypoparathyroidism, pancreatitis and rhabdomyolysis [8].

#### **3. Laboratory findings**

Elucidation of some straightforward biochemical tests have been appeared to be both touchy and decently particular for WD. Two such records incorporate a proportion of alanine aminotransferase (ALT) by aspartate aminotransferase (AST), and a proportion of alkaline phosphatase (ALP) by total bilirubin (TB). An ALT/AST proportion of more than 2.2 contains a sensitivity of 94% and a specificity of 86%; the ALP/TB proportion of less than 4 encompasses a sensitivity of 94% and a specificity of 96% [4].

**235**

*Histopathology of Wilson Disease*

thought [8].

levels [1, 7, 13].

in children with liver disease [13].

borderline levels [7].

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

In Wilson disease, the 24-hour urine copper excretion is usually >100 μg (1.6 μmol) and almost always exceeds 40 μg (0.6 μmol). When penicillamine 500 mg is administered by mouth at the beginning and 12 h later during a 24-hour urine collection, copper excretion >25 μmoles (1587 μg) per 24 h is taken as diagnostic. This test has been validated only in children, and its sensitivity is not as great as originally

Ceruloplasmin is the major carrier for copper in the blood. Testing for serum ceruloplasmin is often done when searching for the cause of unexplained liver disease. There are physiologic variations in the serum level of ceruloplasmin. It is very low in early infancy to the age of 6 months, peak at higher than adult levels in early childhood, and then decrease to the normal adult range [1]. A serum ceruloplasmin level < 200 mg/L (<20 mg/dL) has been considered consistent with WD, and diagnostic if associated with KF rings. Except WD, conditions such as marked renal or enteric protein loss, severe end stage liver disease of any etiology, neurologic diseases copper deficiency, and Menkes disease can show low ceruloplasmin

Total serum copper (which incorporates non–ceruloplasmin bound copper or "free copper" and copper joined in ceruloplasmin) is ordinarily diminished in extent to the diminished serum ceruloplasmin. However, in patients with WD with extreme liver damage, serum copper may be inside the ordinary extend or uniquely hoisted within the setting of ALF due to the discharge of copper from liver tissue stores and the increase in free copper in the blood [13]. A novel approach is the direct specification of labile copper (non-Cp-bound copper), called interchangeable copper (CuEXC). It permits to calculate the "relative replaceable copper" (REC) which alludes to the proportion of CuEXC to total copper. REC was assessed as a convenient diagnostic appliance for WD with a high sensitivity and specifity allows the calculation of relative interchangeable copper (REC) that compares to the proportion between CuEXC and total serum copper. It is represented that REC is a great diagnostic biomarker with a specifity and specificity near to 100% for the determination of WD when its value is >18.5%. It allows a separation of Wilsonian liver disease from other types of liver disorders such as autoimmune, infectious. Moreover, REC can make a great aid to family screening, because it is possible to make a distinction between WD patients and heterozygous carriers or healthy subjects. The CuEXC value at diagnosis indicates of extrahepatic involvement and its seriousness [7]. But further studies are needed to evaluate its diagnostic accuracy

The urine copper shows to the sum of non-ceruloplasmin bound copper within the circulation. Urinary copper concentration is measured per 24 h since there's noteworthy changeability within the copper substance of spot urine collections for them to be utilized. The customary level taken as demonstrative of WD is >100 μg/24 h (>1.6 μmol/24 h) in symptomatic patients [1]. In asymptomatic children or children with mild liver disease, urinary copper values are often normal [13]. However, high urinary copper values may be seen in other sorts of liver disorders (e.g., autoimmune hepatitis, unremitting active liver disease, or cholestasis and in specific during acute liver failure of any etiology). Heterozygotes may too have

The diagnosis is not fundamentally straightforward indeed even when the disease is effectively being considered. In a patient within the age-range 5–50 years who has liver disease or characteristic neurological symptoms, finding serum caeruloplasmin underneath 5 mg/dL is profoundly compatible with WD; association too a Kayser–Fleischer (KF) ring affirms the diagnosis. In nearly one-third of patients, serum caeruloplasmin can be within normal limits. As a sole, serum caeruloplasmin

#### *Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

*Liver Pathology*

60 years [9].

KF rings [8].

**3. Laboratory findings**

a specificity of 96% [4].

also occur in WD [8].

highlights. The lion's share of focal liver lesions in patients with WD are benign nodules, but there are reports that have depicted malignant liver tumors or dysplastic nodules in these patients. Although rare in WD compared to other liver diseases, hepatocellular carcinoma occurs in patients of all ages. Cholangiocarcinoma may

Neurologic manifestations include tremor, gait disturbances, choreiform movements, Parkinsonism or akinetic rigid syndrome i.e., partial parkinsonism, dysarthria, pseudobulbar palsy, rigid dystonia, seizures, migraine headaches, and insomnia. In WD cohorts, neurological presentation is associated with a significantly longer time from onset of symptoms to diagnosis than hepatic presentation, ranging from 2.5 to 6 years. In large case series, mean age at onset of neurologic problems extends from 15 to 21 a long time of age, a decade after onset of liver disease, but a number of patients have been analyzed with a starting neurologic onset earlier than age 10 [7]. Psychiatric manifestations encompass depression, neuroses, personality changes, psychosis and poor performance at school. It was reported that 30—40% of patients have psychiatric symptoms at diagnosis and 20% had seen a psychiatrist prior to their WD diagnosis [12]. WD should be ruled out in any teenager with unexplained cognitive, psychiatric, or movement disorder [13]. Neuropsychiatric signs are the predominant presentation in adults but also may be present in up to 50% of teenagers. WD should also be included in the differential diagnosis work-up of unclear neuropsychiatric syndromes in patients after age

Ocular findings include the Kayser–Fleischer (KF) ring, due to copper accumulation in Desçemet's membrane, and sunflower cataracts, due to copper accumulation in the lens. They are diagnosed by slit lamp examination. In known cases of hepatic WD, the rings are present in just over half of patients. KF rings are usually absent in children with liver disease. KF rings are rarely observed in other conditions such as in patients with chronic cholestatic diseases, monoclonal gammopathies, multiple myeloma, arci senilis, and pulmonary carcinoma and are thus not specific for WD [1, 7]. Of note, KF rings are not so easy to diagnose without experience, some authors suggest that anterior segment Scheimpflug imaging (Pentacam, Oculus) could be more helpful to diagnose or confirm KF rings by ophthalmologists with little experience in patients with WD. In general it is said that when neurological symptoms are present, KF rings is present in almost all WD patients at disease diagnosis [7]. But there are reports of cases with neurological involvement without

Other presentations and clinical findings are intermittent bouts of jaundice caused by haemolysis, gynaecomastia, amenorrhoea, repeated spontaneous abortion, cardiac complications including ECG abnormalities, ventricular fibrillation, cardiomyopathy, orthostatic hypotension, urolithiasis, renal tubular disease,

Elucidation of some straightforward biochemical tests have been appeared to be both touchy and decently particular for WD. Two such records incorporate a proportion of alanine aminotransferase (ALT) by aspartate aminotransferase (AST), and a proportion of alkaline phosphatase (ALP) by total bilirubin (TB). An ALT/AST proportion of more than 2.2 contains a sensitivity of 94% and a specificity of 86%; the ALP/TB proportion of less than 4 encompasses a sensitivity of 94% and

hypoparathyroidism, pancreatitis and rhabdomyolysis [8].

**234**

In Wilson disease, the 24-hour urine copper excretion is usually >100 μg (1.6 μmol) and almost always exceeds 40 μg (0.6 μmol). When penicillamine 500 mg is administered by mouth at the beginning and 12 h later during a 24-hour urine collection, copper excretion >25 μmoles (1587 μg) per 24 h is taken as diagnostic. This test has been validated only in children, and its sensitivity is not as great as originally thought [8].

Ceruloplasmin is the major carrier for copper in the blood. Testing for serum ceruloplasmin is often done when searching for the cause of unexplained liver disease. There are physiologic variations in the serum level of ceruloplasmin. It is very low in early infancy to the age of 6 months, peak at higher than adult levels in early childhood, and then decrease to the normal adult range [1]. A serum ceruloplasmin level < 200 mg/L (<20 mg/dL) has been considered consistent with WD, and diagnostic if associated with KF rings. Except WD, conditions such as marked renal or enteric protein loss, severe end stage liver disease of any etiology, neurologic diseases copper deficiency, and Menkes disease can show low ceruloplasmin levels [1, 7, 13].

Total serum copper (which incorporates non–ceruloplasmin bound copper or "free copper" and copper joined in ceruloplasmin) is ordinarily diminished in extent to the diminished serum ceruloplasmin. However, in patients with WD with extreme liver damage, serum copper may be inside the ordinary extend or uniquely hoisted within the setting of ALF due to the discharge of copper from liver tissue stores and the increase in free copper in the blood [13]. A novel approach is the direct specification of labile copper (non-Cp-bound copper), called interchangeable copper (CuEXC). It permits to calculate the "relative replaceable copper" (REC) which alludes to the proportion of CuEXC to total copper. REC was assessed as a convenient diagnostic appliance for WD with a high sensitivity and specifity allows the calculation of relative interchangeable copper (REC) that compares to the proportion between CuEXC and total serum copper. It is represented that REC is a great diagnostic biomarker with a specifity and specificity near to 100% for the determination of WD when its value is >18.5%. It allows a separation of Wilsonian liver disease from other types of liver disorders such as autoimmune, infectious. Moreover, REC can make a great aid to family screening, because it is possible to make a distinction between WD patients and heterozygous carriers or healthy subjects. The CuEXC value at diagnosis indicates of extrahepatic involvement and its seriousness [7]. But further studies are needed to evaluate its diagnostic accuracy in children with liver disease [13].

The urine copper shows to the sum of non-ceruloplasmin bound copper within the circulation. Urinary copper concentration is measured per 24 h since there's noteworthy changeability within the copper substance of spot urine collections for them to be utilized. The customary level taken as demonstrative of WD is >100 μg/24 h (>1.6 μmol/24 h) in symptomatic patients [1]. In asymptomatic children or children with mild liver disease, urinary copper values are often normal [13]. However, high urinary copper values may be seen in other sorts of liver disorders (e.g., autoimmune hepatitis, unremitting active liver disease, or cholestasis and in specific during acute liver failure of any etiology). Heterozygotes may too have borderline levels [7].

The diagnosis is not fundamentally straightforward indeed even when the disease is effectively being considered. In a patient within the age-range 5–50 years who has liver disease or characteristic neurological symptoms, finding serum caeruloplasmin underneath 5 mg/dL is profoundly compatible with WD; association too a Kayser–Fleischer (KF) ring affirms the diagnosis. In nearly one-third of patients, serum caeruloplasmin can be within normal limits. As a sole, serum caeruloplasmin is not an adequate diagnostic test for WD. KF rings are diagnostic, but they can also be seen in patients who have persistent cholestasis of other etiology. Lack of KF rings happens in around 50% of adult patients with liver disease and hence does not run the show out WD. KF rings may not be determined even when there's neurological involvement.

### **4. Histopathology and histochemistry**

Liver biopsy is typically performed when clinical and laboratory findings are not diagnostic or for evaluation of unexplained liver disease or abnormal liver tests. Another aim is to determine the degree of hepatic inflammation and for hepatic copper quantitation [1]. The spectrum of hepatic pathological changes occurring in WD is very broad, ranging from elementary changes typical of a toxic pathology, to inflammatory changes typical of viral or autoimmune etiology [6]. The main features are microvesicular and macrovesicular steatosis, glycogenated hepatocyte nuclei, inflammation, and variable hepatocellular anisonucleosis [16, 17].

The manifestations of liver involvement have a varied spectrum depending on the stage of the disease. In the earlier steps, hepatocyte injury may at first manifest as simple steatosis (**Figure 1**) with frequent association of glycogenated nuclei. Steatosis, Mallory-Denk bodies (MBDs), lipogranulomas and glycogenated nuclei have been represented as characteristic morphologic findings in liver biopsies with WD. This picture frequently imitates alcoholic and non-alcoholic fatty liver disease [6]. The distinction from nonalcoholic steatohepatitis (NASH) depends upon the demonstration of accumulated copper in the hepatocytes by histochemical stains. Lipofuscin accumulates in periportal areas, and some of the granules are large, irregular in shape and vacuolated. The intermediate stage of the disease shows

**237**

**Figure 2.**

*Mallory-Denk bodies in a hepatectomy specimen (H&E).*

*Histopathology of Wilson Disease*

phages in the cirrhotic stage [8].

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

histological features similar to those of chronic hepatitis of any etiology including viral or autoimmune hepatitis, with the arrival of the portal and periportal inflammation composed of lymphocytes and plasma cells, which results in the destruction of the limiting plate, and parenchymal necrosis followed by bridging fibrosis [4]. Because of low-titer autoantibodies (mainly antinuclear antibodies) are commonly found in patients with WD, differential diagnosis with autoimmune hepatitis (AIH) can be more complicated. Also, cases of WD and concomitant AIH have been reported [13]. More than 50% of cases may show the presence of intra-cytoplasmic eosinophilic MBDs (**Figure 2**). The literature suggests that steatosis, glycogenated nuclei and MBDs in periportal hepatocytes are features that may be used to distinguish the chronic hepatitis of WD from other more common etiologies [1]. In the cirrhotic stage which is usually macronodular but can be mixed or even micronodular, the histologic features are non-specific, and usually little or no inflammation is present. Some cases may show mild steatosis or features of steatohepatitis. Clusters of large hepatocytes with a granular eosinophilic cytoplasm (oncocytic or oxyphil cells), resulting from an increased number of mitochondria, are often seen but this is not specific for WD [8]. The distribution of copper is quite variable, with some of the cirrhotic nodules containing a lot and others containing little or none. Defining widespread copper deposits by histochemistry can help for the diagnosis. It should be noted that the distribution of copper is variable: some nodules with prominent staining, others with minimal or none (**Figure 3**). This could generate false negative impression in biopsy specimens, and it has been suggested that two liver cores may be needed for copper detection and diagnosis. Cases which present with ALF or fulminant hepatitis, the histology includes portal and parenchymal inflammatory infiltrate, associated with hepatocyte injury, swelling and necrosis. There may be massive or submassive necrosis. Copper can be demonstrated in hepatocytes and when there has been significant necrosis, in Kupffer cells and portal macrophages [1, 8]. In contrast, copper is rarely demonstrable in Kupffer cells or portal macro-

Excess copper storage in the hepatocytes is a relevant sign of WD, and determination of hepatic copper content in the liver biopsy, is important in the diagnosis of WD. This may be accomplished by utilizing special histochemical stains for copper

**Figure 1.** *Steatosis and anisonucleosis in a hepatectomy specimen (H&E).*

#### *Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

*Liver Pathology*

logical involvement.

anisonucleosis [16, 17].

**4. Histopathology and histochemistry**

is not an adequate diagnostic test for WD. KF rings are diagnostic, but they can also be seen in patients who have persistent cholestasis of other etiology. Lack of KF rings happens in around 50% of adult patients with liver disease and hence does not run the show out WD. KF rings may not be determined even when there's neuro-

Liver biopsy is typically performed when clinical and laboratory findings are not diagnostic or for evaluation of unexplained liver disease or abnormal liver tests. Another aim is to determine the degree of hepatic inflammation and for hepatic copper quantitation [1]. The spectrum of hepatic pathological changes occurring in WD is very broad, ranging from elementary changes typical of a toxic pathology, to inflammatory changes typical of viral or autoimmune etiology [6]. The main features are microvesicular and macrovesicular steatosis, glycogenated hepatocyte nuclei, inflammation, and variable hepatocellular

The manifestations of liver involvement have a varied spectrum depending on the stage of the disease. In the earlier steps, hepatocyte injury may at first manifest as simple steatosis (**Figure 1**) with frequent association of glycogenated nuclei. Steatosis, Mallory-Denk bodies (MBDs), lipogranulomas and glycogenated nuclei have been represented as characteristic morphologic findings in liver biopsies with WD. This picture frequently imitates alcoholic and non-alcoholic fatty liver disease [6]. The distinction from nonalcoholic steatohepatitis (NASH) depends upon the demonstration of accumulated copper in the hepatocytes by histochemical stains. Lipofuscin accumulates in periportal areas, and some of the granules are large, irregular in shape and vacuolated. The intermediate stage of the disease shows

**236**

**Figure 1.**

*Steatosis and anisonucleosis in a hepatectomy specimen (H&E).*

histological features similar to those of chronic hepatitis of any etiology including viral or autoimmune hepatitis, with the arrival of the portal and periportal inflammation composed of lymphocytes and plasma cells, which results in the destruction of the limiting plate, and parenchymal necrosis followed by bridging fibrosis [4]. Because of low-titer autoantibodies (mainly antinuclear antibodies) are commonly found in patients with WD, differential diagnosis with autoimmune hepatitis (AIH) can be more complicated. Also, cases of WD and concomitant AIH have been reported [13]. More than 50% of cases may show the presence of intra-cytoplasmic eosinophilic MBDs (**Figure 2**). The literature suggests that steatosis, glycogenated nuclei and MBDs in periportal hepatocytes are features that may be used to distinguish the chronic hepatitis of WD from other more common etiologies [1]. In the cirrhotic stage which is usually macronodular but can be mixed or even micronodular, the histologic features are non-specific, and usually little or no inflammation is present. Some cases may show mild steatosis or features of steatohepatitis. Clusters of large hepatocytes with a granular eosinophilic cytoplasm (oncocytic or oxyphil cells), resulting from an increased number of mitochondria, are often seen but this is not specific for WD [8]. The distribution of copper is quite variable, with some of the cirrhotic nodules containing a lot and others containing little or none. Defining widespread copper deposits by histochemistry can help for the diagnosis. It should be noted that the distribution of copper is variable: some nodules with prominent staining, others with minimal or none (**Figure 3**). This could generate false negative impression in biopsy specimens, and it has been suggested that two liver cores may be needed for copper detection and diagnosis. Cases which present with ALF or fulminant hepatitis, the histology includes portal and parenchymal inflammatory infiltrate, associated with hepatocyte injury, swelling and necrosis. There may be massive or submassive necrosis. Copper can be demonstrated in hepatocytes and when there has been significant necrosis, in Kupffer cells and portal macrophages [1, 8]. In contrast, copper is rarely demonstrable in Kupffer cells or portal macrophages in the cirrhotic stage [8].

Excess copper storage in the hepatocytes is a relevant sign of WD, and determination of hepatic copper content in the liver biopsy, is important in the diagnosis of WD. This may be accomplished by utilizing special histochemical stains for copper

**Figure 2.** *Mallory-Denk bodies in a hepatectomy specimen (H&E).*

#### **Figure 3.**

*Heterogenous copper accumulation in a hepatectomy specimen (Rhodanine).*

#### **Figure 4.** *Diffuse cytoplasmic staining pattern (Timm).*

which are rhodanine, rubeanic acid and Timm's silver stains, and for copper related protein of which are orcein, aldehyde fuchsin and Victoria blue. None of these stains is fully sensitive nor specific. Orcein reveals the accumulation of metallothioneins, the proteins involved in excess copper sequestration. Positive staining appears as large irregular granules dark-brown in color. In the Timm's stained slides, if there is mild accumulation copper shows up small black or greenish-black granules in the intracytoplasmic perinuclear area or canalicular side of hepatocytes, and when there is heavy accumulation, the whole cytoplasm of the hepatocyte stuffed with coarse granules. With rhodanine stain copper accumulation appears as

**239**

WD [18].

**Figure 5.**

small red granules [1, 6, 8]. Out of granular staining, diffuse cytoplasmic staining pattern (**Figure 4**) can be seen with copper stains, which is frequently reported in

*Different sensitivities of copper stains in the same case (A. Timm, B. Rhodanine, C. Orcein).*

*Histopathology of Wilson Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

*Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

*Liver Pathology*

**238**

**Figure 4.**

**Figure 3.**

*Diffuse cytoplasmic staining pattern (Timm).*

*Heterogenous copper accumulation in a hepatectomy specimen (Rhodanine).*

which are rhodanine, rubeanic acid and Timm's silver stains, and for copper related protein of which are orcein, aldehyde fuchsin and Victoria blue. None of these stains is fully sensitive nor specific. Orcein reveals the accumulation of metallothioneins, the proteins involved in excess copper sequestration. Positive staining appears as large irregular granules dark-brown in color. In the Timm's stained slides, if there is mild accumulation copper shows up small black or greenish-black granules in the intracytoplasmic perinuclear area or canalicular side of hepatocytes, and when there is heavy accumulation, the whole cytoplasm of the hepatocyte stuffed with coarse granules. With rhodanine stain copper accumulation appears as

small red granules [1, 6, 8]. Out of granular staining, diffuse cytoplasmic staining pattern (**Figure 4**) can be seen with copper stains, which is frequently reported in WD [18].

The most effective method is vary in different reports. In our study with transplant hepatectomies, we found that positivity rates of Timm, rhodanine and orcein are 85%, 82%, and 48% respectively (**Figure 5A-C**). We thought that pannodular (prominent diffuse staining of nodule), staining is a powerfull indicator of WD. In this context, we suggested that pannodular staining is a more convincing staining pattern for the histopathologic diagnosis of WD and against other diseases with copper accumulation [16]. In our routine practice we do Timm's stain for every liver biopsy and hepatectomy. Next to evaluating copper accumulation for diagnosis WD disease, it can help to define late stage fibrosis [18]. It should be keep in mind, copper accumulation can be seen in other diseases such as cholestatic liver diseases, alcoholic liver disease and idiopathic copper toxicosis [6]. In chronic cholestasis and non WD cirrhosis, copper staining is usually limited to periseptal areas with a patchy/focal distribution (**Figure 6**). It is suggested that that in the absence of advanced fibrosis (or WD), a positive rhodanine stain for copper argues strongly in favor of chronic biliary diseases and against other liver diseases [19]. Of note, marked hepatic copper overload mimicking WD has been described in children with MDR3 deficiency [8]. It is important to remember that negative staining for both copper and copper-associated protein does not exclude the diagnosis of WD.

In equivocal cases, measurement of liver copper content is recommended as the next step for diagnosis of WD. A 5-fold increase of hepatic copper concentration is considered as diagnostic for diagnosis of hepatic WD [5]. In a more strict definition, a copper content >250 μg/g dry weight (normal value <50 mg/g dry weight) in adult patients without cholestasis is accepted as diagnostic for WD. Probably depending on sampling error due to nonhomogeneous copper distribution in the liver, lower values are reported in up to 20% of patients with WD. The exactness of liver copper estimation is moved forward with an optimal measured biopsy sample (ideally >1 cm long, min. 0.5 cm) that ought to be put on a little piece of paper for drying,

**241**

**Author details**

**Thanks**

tests [5].

**5. Conclusion**

Nese Karadag Soylu

cryptogenic adult cases.

Department of Pathology, Medical School, Inonu University, Malatya, Turkey

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The author would like to thank to Dr. Emine Samdanci as a team member of hepatopathology and to Dr. Seyma Eren and Dr. Meryem Uludag for photography.

\*Address all correspondence to: nesekaradag@yahoo.com

provided the original work is properly cited.

*Histopathology of Wilson Disease*

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

and in a dry plastic copper-free holder for atomic absorption analysis on fresh tissue [13]. Hepatic copper levels in advanced stage chronic biliary diseases in adults and children often exceed 250 mg/100 g of dried liver, sometimes reaching levels higher than those observed in WD [19]. In spite of the fact that utilize of dried tissue has been proposed for tissue copper quantitation, the utilize of formalin-fixed, paraffin-embedded (FFPE) tissue is said fair as valuable. Utilizing FFPE tissue specimens evacuates the specialized troubles related to dried unfixed tissue, as well as gives the same tissue for histopathological and quantitative assessments. Since copper accumulation may well be non homogenious indeed even in most progressed cases of WD, the availability of light microscopy on the same tissue being evaluated for copper may well be a really valuable tool in mostly tending to this potential examining inclination tissue quantitation of copper is subject to [17]. Although liver copper content is a useful parameter, but a value below 250 μg/g does not exclude WD. Diagnosis requires the combination of a variety of clinical and biochemical

WD is a curable disease, but early diagnosis is essential to stop the progression to cirrhosis or worsening of the neurological and psychiatric conditions. As a treatable disease, WD should be detected by any health professionals at any care level. If WD is not recognized and adequately treated, the progression of liver disease to cirrhosis and liver failure can be rapid or irreversible brain damage can occur. Unfortunately, even though of all advances, the diagnosis of WD shows up frequently compelling, due to the variability of its clinical manifestation and to the complexity of the microscopic findings within the liver biopsy. Liver histopathology, in reality, does not show a unique morphology, but it may appear in different patterns. From a pathologist's perspective, when evaluating the liver biopsies, WD should be included in the differential diagnosis especially in pediatric age and also

**Figure 6.** *Periseptal copper accumulation in a non WD cirrhosis (Timm).*

#### *Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

and in a dry plastic copper-free holder for atomic absorption analysis on fresh tissue [13]. Hepatic copper levels in advanced stage chronic biliary diseases in adults and children often exceed 250 mg/100 g of dried liver, sometimes reaching levels higher than those observed in WD [19]. In spite of the fact that utilize of dried tissue has been proposed for tissue copper quantitation, the utilize of formalin-fixed, paraffin-embedded (FFPE) tissue is said fair as valuable. Utilizing FFPE tissue specimens evacuates the specialized troubles related to dried unfixed tissue, as well as gives the same tissue for histopathological and quantitative assessments. Since copper accumulation may well be non homogenious indeed even in most progressed cases of WD, the availability of light microscopy on the same tissue being evaluated for copper may well be a really valuable tool in mostly tending to this potential examining inclination tissue quantitation of copper is subject to [17]. Although liver copper content is a useful parameter, but a value below 250 μg/g does not exclude WD. Diagnosis requires the combination of a variety of clinical and biochemical tests [5].

#### **5. Conclusion**

*Liver Pathology*

the diagnosis of WD.

The most effective method is vary in different reports. In our study with transplant hepatectomies, we found that positivity rates of Timm, rhodanine and orcein are 85%, 82%, and 48% respectively (**Figure 5A-C**). We thought that pannodular (prominent diffuse staining of nodule), staining is a powerfull indicator of WD. In this context, we suggested that pannodular staining is a more convincing staining pattern for the histopathologic diagnosis of WD and against other diseases with copper accumulation [16]. In our routine practice we do Timm's stain for every liver biopsy and hepatectomy. Next to evaluating copper accumulation for diagnosis WD disease, it can help to define late stage fibrosis [18]. It should be keep in mind, copper accumulation can be seen in other diseases such as cholestatic liver diseases, alcoholic liver disease and idiopathic copper toxicosis [6]. In chronic cholestasis and non WD cirrhosis, copper staining is usually limited to periseptal areas with a patchy/focal distribution (**Figure 6**). It is suggested that that in the absence of advanced fibrosis (or WD), a positive rhodanine stain for copper argues strongly in favor of chronic biliary diseases and against other liver diseases [19]. Of note, marked hepatic copper overload mimicking WD has been described in children with MDR3 deficiency [8]. It is important to remember that negative staining for both copper and copper-associated protein does not exclude

In equivocal cases, measurement of liver copper content is recommended as the next step for diagnosis of WD. A 5-fold increase of hepatic copper concentration is considered as diagnostic for diagnosis of hepatic WD [5]. In a more strict definition, a copper content >250 μg/g dry weight (normal value <50 mg/g dry weight) in adult patients without cholestasis is accepted as diagnostic for WD. Probably depending on sampling error due to nonhomogeneous copper distribution in the liver, lower values are reported in up to 20% of patients with WD. The exactness of liver copper estimation is moved forward with an optimal measured biopsy sample (ideally >1 cm long, min. 0.5 cm) that ought to be put on a little piece of paper for drying,

**240**

**Figure 6.**

*Periseptal copper accumulation in a non WD cirrhosis (Timm).*

WD is a curable disease, but early diagnosis is essential to stop the progression to cirrhosis or worsening of the neurological and psychiatric conditions. As a treatable disease, WD should be detected by any health professionals at any care level. If WD is not recognized and adequately treated, the progression of liver disease to cirrhosis and liver failure can be rapid or irreversible brain damage can occur. Unfortunately, even though of all advances, the diagnosis of WD shows up frequently compelling, due to the variability of its clinical manifestation and to the complexity of the microscopic findings within the liver biopsy. Liver histopathology, in reality, does not show a unique morphology, but it may appear in different patterns. From a pathologist's perspective, when evaluating the liver biopsies, WD should be included in the differential diagnosis especially in pediatric age and also cryptogenic adult cases.

#### **Thanks**

The author would like to thank to Dr. Emine Samdanci as a team member of hepatopathology and to Dr. Seyma Eren and Dr. Meryem Uludag for photography.

#### **Author details**

Nese Karadag Soylu Department of Pathology, Medical School, Inonu University, Malatya, Turkey

\*Address all correspondence to: nesekaradag@yahoo.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Guindi M. Wilson disease. Seminars in Diagnostic Pathology. 2019;36: 415-422. DOI: 10.1053/j.semdp.2019. 07.008

[2] Chaudhury S, Patkar P. Wilson's Disease: An Update. Med J DY Patil Vidyapeeth. 2018;11:92-3.11 (2018): 92-3. DOI: 10.4103/MJDRDYPU. MJDRDYPU\_139\_17

[3] Barbosa ER, Machado AAC, Cançado ELR, Deguti MM, Scaff M. Wilson's disease: a case report and a historical review. Arq Neuropsiquiatr. 2009;67(2b): 539-43. DOI: 10.1590/ S0004-282X2009000300036

[4] Madakshira MG, Das A, Umair M, Dutta U. Liver histology and histochemistry in Wilson disease. Autops Case Report [Internet]. 2018;8(3): e2018026. DOI: 10.4322/ acr.2018.026

[5] Ferenci P, Steindl-Munda P, Vogel W, et al. Diagnostic Value of Quantitative Hepatic Copper Determination in Patients With Wilson's Disease. Clin Gastroenterol Hepatol. 2005; 3: 811-8. DOI: 10.1016/s1542-3565(05)00181-3

[6] Gerosa C, Fanni D, Congiu T, Piras M, Cau F, Moi M, Faa G. Liver pathology in Wilson's disease: From copper overload to cirrhosis. Journal of Inorganic Biochemistry. 2019;193: 106-111. DOI: 10.1016/j. jinorgbio.2019.01.008

[7] Poujois A, Woimant F. Challenges in the diagnosis of Wilson disease. Ann Transl Med. 2019;7(Suppl 2):: S56. DOI: 10.21037/atm.2019.02.10

[8] Quaglia A, Roberts EA, Torbenson M. Disorders of copper metabolism. In: Burt AD, Ferrell LD, Hübscher SG, editors. MacSween's Pathology of the Liver. 7th ed. Elsevier; 2018. p. 200-206. ch3

[9] Žigrai M, Vyskoči M, Tóthová A, Vereš P, Bluska P, Valkovič P. Late-Onset Wilson's Disease. Front Med (Lausanne). 2020;(7): 26. DOI: 10.3389/ fmed.2020.00026

[10] Ferenci P, Stremmel W, Czlonkowska A, et al. Age and sex but not ATP7B genotype effectively influence the clinical phenotype of Wilson. Hepatology. 2019;69: 1464-76. DOI: 10.1002/hep.30280

[11] Shribman S, Webb G, Taylor R, Warner TT, Duckworth A, Gimson A, et al. Liver transplantation for late-onset presentations of acute liver failure in Wilson's disease: the UK experience over 2 decades. JHEP Rep. 2020;2(3): 100096. DOI: 10.1016/j.jhepr.2020.100096

[12] Poujois A, Woimant F. Wilson's disease: A 2017 update. Clinics and Research in Hepatology and Gastroenterology. 2018;42: 512—520. DOI: 10.1016/j.clinre.2018.03.007

[13] Socha P, Janczyk W, Dhawan A, et al. Wilson's Disease Wilson's disease in children: a Position paper by the Hepatology Committee of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr. 2018;66:334-344. DOI: 10.1097/MPG.0000000000001787

[14] Sohtaoglu M, Ergin H, Özekmekçi S, et al. Patient with lateonset Wilson disease: deterioration with penicillamine. Mov Disord. 2007;22:290-1. DOI: 10.1002/mds.21201

[15] Sini M, Sorbello O, Sanna F, et al. Histologic evolution and long-term outcome of Wilson's disease: results of a single-center experience. Eur J Gastroenterol Hepatol. 2013;25(1): 111- 7. DOI: 10.1097/MEG.0b013e328358f7

[16] Karadag N, Tolan K, Samdanci E, et al. Effect of Copper Staining in Wilson

**243**

*Histopathology of Wilson Disease*

10.6002/ect.2015.0319

*DOI: http://dx.doi.org/10.5772/intechopen.95105*

Disease: A Liver Explant Study. Exp Clin Transplant. 2017;15(5): 542-546. DOI:

[17] Hafezi-Bakhtiari S, Adeyi OA. Metabolic Disorders of the Liver. Diagnostic Histopathol. 2014;20: 125- 133. DOI: 10.1016/j.mpdhp.2014.01.012

[18] Miyamura H, Nakanuma Y, Kono N. Survey of copper granules in liver biopsy specimens from various liver abnormalities other than Wilson's disease and biliary diseases. Gastroenterol Jpn. 1988;23: 633-638.

DOI: 10.1007/BF02782948

[19] Mounajjed T, Oxentenko AS, Qureshi H, Smyrk TC. Revisiting the topic of histochemically detectable copper in various liver diseases with special focus on venous outflow impairment. Am. J. Clin. Pathol. 2013;139: 79-86. DOI: 10.1309/ AJCPDZR4OHDQNG3L

*Histopathology of Wilson Disease DOI: http://dx.doi.org/10.5772/intechopen.95105*

Disease: A Liver Explant Study. Exp Clin Transplant. 2017;15(5): 542-546. DOI: 10.6002/ect.2015.0319

[17] Hafezi-Bakhtiari S, Adeyi OA. Metabolic Disorders of the Liver. Diagnostic Histopathol. 2014;20: 125- 133. DOI: 10.1016/j.mpdhp.2014.01.012

[18] Miyamura H, Nakanuma Y, Kono N. Survey of copper granules in liver biopsy specimens from various liver abnormalities other than Wilson's disease and biliary diseases. Gastroenterol Jpn. 1988;23: 633-638. DOI: 10.1007/BF02782948

[19] Mounajjed T, Oxentenko AS, Qureshi H, Smyrk TC. Revisiting the topic of histochemically detectable copper in various liver diseases with special focus on venous outflow impairment. Am. J. Clin. Pathol. 2013;139: 79-86. DOI: 10.1309/ AJCPDZR4OHDQNG3L

**242**

*Liver Pathology*

**References**

07.008

[1] Guindi M. Wilson disease. Seminars in Diagnostic Pathology. 2019;36: 415-422. DOI: 10.1053/j.semdp.2019.

[9] Žigrai M, Vyskoči M, Tóthová A, Vereš P, Bluska P, Valkovič P. Late-Onset Wilson's Disease. Front Med (Lausanne). 2020;(7): 26. DOI: 10.3389/

fmed.2020.00026

[10] Ferenci P, Stremmel W, Czlonkowska A, et al. Age and sex but not ATP7B genotype effectively influence the clinical phenotype of Wilson. Hepatology. 2019;69: 1464-76.

DOI: 10.1002/hep.30280

[11] Shribman S, Webb G, Taylor R, Warner TT, Duckworth A, Gimson A, et al. Liver transplantation for late-onset presentations of acute liver failure in Wilson's disease: the UK experience over 2 decades. JHEP Rep. 2020;2(3): 100096.

DOI: 10.1016/j.jhepr.2020.100096

[12] Poujois A, Woimant F. Wilson's disease: A 2017 update. Clinics and Research in Hepatology and Gastroenterology. 2018;42: 512—520. DOI: 10.1016/j.clinre.2018.03.007

[13] Socha P, Janczyk W, Dhawan A, et al. Wilson's Disease Wilson's disease in children: a Position paper by the Hepatology Committee of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr. 2018;66:334-344. DOI: 10.1097/MPG.0000000000001787

[14] Sohtaoglu M, Ergin H,

Özekmekçi S, et al. Patient with lateonset Wilson disease: deterioration with penicillamine. Mov Disord. 2007;22:290-1. DOI: 10.1002/mds.21201

[15] Sini M, Sorbello O, Sanna F, et al. Histologic evolution and long-term outcome of Wilson's disease: results of a single-center experience. Eur J Gastroenterol Hepatol. 2013;25(1): 111- 7. DOI: 10.1097/MEG.0b013e328358f7

[16] Karadag N, Tolan K, Samdanci E, et al. Effect of Copper Staining in Wilson

[2] Chaudhury S, Patkar P. Wilson's Disease: An Update. Med J DY Patil Vidyapeeth. 2018;11:92-3.11 (2018): 92-3. DOI: 10.4103/MJDRDYPU.

[3] Barbosa ER, Machado AAC, Cançado ELR, Deguti MM, Scaff M. Wilson's disease: a case report and a historical review. Arq Neuropsiquiatr. 2009;67(2b): 539-43. DOI: 10.1590/ S0004-282X2009000300036

[4] Madakshira MG, Das A, Umair M, Dutta U. Liver histology and histochemistry in Wilson disease. Autops Case Report [Internet]. 2018;8(3): e2018026. DOI: 10.4322/

[5] Ferenci P, Steindl-Munda P, Vogel W, et al. Diagnostic Value of Quantitative Hepatic Copper Determination in Patients With Wilson's Disease. Clin Gastroenterol Hepatol. 2005; 3: 811-8. DOI: 10.1016/s1542-3565(05)00181-3

[6] Gerosa C, Fanni D, Congiu T, Piras M, Cau F, Moi M, Faa G. Liver pathology in Wilson's disease: From copper overload to cirrhosis. Journal of Inorganic Biochemistry. 2019;193: 106-111. DOI: 10.1016/j.

[7] Poujois A, Woimant F. Challenges in the diagnosis of Wilson disease. Ann Transl Med. 2019;7(Suppl 2):: S56. DOI:

jinorgbio.2019.01.008

10.21037/atm.2019.02.10

2018. p. 200-206. ch3

[8] Quaglia A, Roberts EA,

Torbenson M. Disorders of copper metabolism. In: Burt AD, Ferrell LD, Hübscher SG, editors. MacSween's Pathology of the Liver. 7th ed. Elsevier;

acr.2018.026

MJDRDYPU\_139\_17

**245**

**Chapter 13**

**Abstract**

**1. Introduction**

Towards the Study of Liver Failure:

Studies have shown that extended hepatectomy mimics post-hepatectomy liver failure (PHLF) and could also be used to study other small-for-flow syndromes. Extended hepatectomy can be defined as the removal of more than 70% of liver volume. At the molecular level, there seems to be a delayed entrance to the cell cycle, and thus liver dysfunction ensues. Therefore, there is an imperious need to study the mechanisms of such delay to understand how it can be regulated. While the classical 70% hepatectomy model to study liver regeneration has been previously described thoroughly, there are no protocols describing the surgical procedure for a 90% extended hepatectomy (90% EHx). Therefore, we here describe a detailed and reproducible protocol for such model, defining specific aspects that must be considered as well as the most common complications and troubleshooting strategies.

Protocol for a 90% Extended

*Maria J. Lizardo Thiebaud, Eduardo Cervantes-Alvarez* 

**Keywords:** liver regeneration, 90% extended hepatectomy, liver failure

in the portal blood that promoted liver regeneration began [4].

study of liver regeneration and liver failure [8–10].

Liver regeneration is the process by which lost tissue is replaced through compensatory hyperplasia of the remaining healthy tissue [1–3]. The regenerative capacity of the liver has been studied since the early nineteenth century [4], when scientists observed changes in liver tissue after surgical procedures. By using portosystemic shunts, they first speculated that overall flow was important for liver regeneration, and not specifically portal blood flow. Later on, a combined model including lobectomies and shunts was used as the main model for liver regeneration [4]. Finally, the acknowledgment that portal blood flow was crucial for liver homeostasis gave rise to the "humoral theory," and with this, the race to find factors

Most of what we currently know about liver regeneration is thanks to the results obtained with surgical models. These models are the most precise, since timing and volume removal can be controlled. In fact, the surgical technique for a 2/3 hepatectomy in rats as a model for liver regeneration has been described and perfected since first published by Higgins and Anderson in 1931 [5–7]. With advances in anesthesia and analgesia, the extension of the 2/3 hepatectomy provides a useful model for the

Hepatectomy in Mice

*and Nalu Navarro-Alvarez*

#### **Chapter 13**
