Name, signalment:

Freya, 4 year old female spayed Labrador Retriever

Clinical history:

Freya had an abnormal looking liver observed during laparoscopic spay. Serum biochemistry revealed decreased urea. Multiple biopsies were obtained from the liver and submitted for histology.

Histology:

Depending on the biopsy evaluated, there was significant distortion of the lobular architecture of the liver. Large regions of hepatocytes were lost (parenchymal collapse) and replaced by portal to portal, bridging fibrosis and biliary hyperplasia (ductular reaction). This created a macroscopically irregular contour, as observed during laparoscopy. Significant lymphoplasmacytic, histiocytic, and neutrophilic inflammation was present within the portal tracts and extended into the parenchyma, frequently associated with hepatocyte apoptosis and necrosis (portal to interface hepatitis). Hepatocytes frequently golden to dark brown, finely granular pigment. Randomly within the biopsies, there were small nodular foci of necrotic hepatocytes, macrophages, neutrophils, and pigmented material (pigment granulomas). Bile plugs were present in canaliculi.

Figure 1: Widespread replacement of hepatocytes by bridging fibrosis (outlined in black arrow heads), ductular reaction (green arrow heads), and mixed inflammation (blue circles).

Figure 2: Pigment granulomas (black circles), bile plugs in canaliculi (blue arrow), and random hepatocyte necrosis (green box)

Interpretation:

Chronic hepatitis with intrahepatocellular and intrahistiocytic pigment, parenchymal collapse, bridging fibrosis, and ductular reaction

What is chronic hepatitis?

Chronic hepatitis is defined as an inflammatory condition of the liver which is associated with hepatocyte necrosis (“piecemeal necrosis” or “interface hepatitis”) and varying degrees of fibrosis. The type of inflammation varies, but typically includes a combination of lymphocytic, plasmacytic, histiocytic (macrophagic), and neutrophilic infiltrates. Chronic hepatitis may progress into cirrhosis, which is defined as severe loss of the lobular architecture with bridging fibrosis and nodular regeneration (micronodular or macronodular). This is an end-stage phase of chronic hepatitis and is irreversible. It is very important to note that the degree of fibrosis, nodular regeneration, and inflammation can vary greatly throughout the liver lobes. The periphery of the lobes tend to be the worst affected. Determination of cirrhosis thus requires clinical input and sampling from multiple different lobes. Complications of hepatitis include coagulopathies, portal hypertension, ascites, hepatic encephalopathy, gastroduodenal ulceration, and coagulation.

The underlying cause of chronic hepatitis is often unidentified (idiopathic). Breed predispositions, toxicities (particularly copper accumulation), immune-mediated inflammation, and some infectious organisms (such as atypical Leptospirosis) are observed. Breeds with a suspected hereditary predisposition to chronic hepatitis include English Springer Spaniels, Cocker Spaniels (English and American), West Highland White Terriers, Labrador retrievers, and Dobermans. Female dogs are often overrepresented in these breeds, but not always (Cocker Spaniels).

When chronic hepatitis is identified histologically, further testing for copper should be performed because copper accumulation is one of the few known causes of chronic hepatitis and can potentially be treated. Pathologists typically recommend a combination of special stains for copper (Rubeanic acid or Rhodanine stain) and copper quantification. Assessment of the degree of fibrosis is also crucial and can be achieved by performing stain(s) such as Sirius Red, Trichrome, and/or Reticulin.

If the inflammation is pyogranulomatous, the pathologist will often perform special stains to investigate for microorganisms, such as mycobacterium (Ziehl-Neelsen), fungi (PAS, GMS), and other bacteria (Gram stain). There is an atypical variant of Leptospirosis which is associated with a pyogranulomatous hepatitis. The pathologist may suggest performing FISH (Fluorescence In Situ Hybridization), PCR, +/- Leptospirosis serology to investigate this differential.

What next for Freya?

Copper staining (Rubeanic acid) was recommended and a Sirius Red stain was also performed. Rubeanic acid staining revealed significant copper accumulation within hepatocytes. Additionally, it was frequently observed in the pigment granulomas and in Kupffer cells. Within the Sirius Red, there were large regions of parenchymal collapse and fibrosis.

Figure 3: Rubeanic acid. The dark brown to black colour is copper, which can be seen throughout this entire biopsy. This section was severely affected by parenchymal collapse and fibrosis (see figure 5).

Figure 4: Rubeanic acid. Higher magnification. The central vein is in the upper left corner and the portal tract is in the lower right corner. Centrilobular to periportal hepatocytes contain variable amounts of dark brown to black, finely stippled to coarsely granular copper.

Figure 5: Sirius Red. Same section as the sample in Figure 3. In regions of parenchymal collapse, there is severe replacement of hepatocytes (which are yellow) by fibrillar collagen (bright red).

Toxicology:

Because of the severity of the copper accumulation revealed with the copper staining, copper quantification was recommended. Copper quantification can be performed on liver samples that are stored fresh, frozen, or formalin fixed. It can also be performed after processing for histology.

The amount of copper in Freya’s liver was 2250ppm (dry weight).

The normal amount of copper in the canine liver (dry weight) is 120-400ppm. Copper amounts of greater than 1500ppm are considered toxic.

Final diagnosis:

Chronic hepatitis with toxic copper accumulation and likely cirrhosis

A bit about copper hepatitis:

The causes of pathologic copper accumulation are multifactorial. Heritable defects in copper metabolism are identified (Bedlington Terriers) or suspected (Labrador retrievers, Dalmatian, Doberman Pinscher, and West Highland White Terrier) in many breeds. Excessive dietary copper is also being investigated, as the average dog food contains 2-4 (or more!) times the recommended amount of copper by national nutrition boards. Cholestasis may play a role in copper accumulation, although this is more commonly seen in cats than dogs.

Copper staining allows the pathologist to evaluate the distribution of copper accumulation within the hepatic lobule. Centrilobular accumulation of copper is typically indicative of a pathologic accumulation. Periportal accumulation may indicate a secondary or reactive copper accumulation in dogs with cholestasis. In severe cases, copper may be observed throughout the lobule (centrilobular, midzonal, and periportal). The pathologist will give a semi-quantitative analysis of the severity of copper accumulation through a copper score, from 0-5. Zero indicates no copper accumulation and five indicates copper is present in every hepatocyte at moderate to severe levels.

Copper toxicology provides a numerical amount of copper present in a given liver sample, as parts per million (ppm) per dry weight (dw). Liver tissue may be fresh, frozen, fixed, or processed for histology and embedded in a paraffin block. A minimum of 10mg dry weight (50mg wet tissue) is required as small sample sizes may affect the test accuracy. This corresponds to a pea-sized wedge of liver or 3 x 1cm long 14g needle biopsies.

How should I approach sampling in patients with suspected chronic hepatitis?

A recent consensus statement (Webster et al. ACVIM consensus statement on the diagnosis and treatment of chronic hepatitis in dogs. 2019;33:1173-1200) provides an excellent review of the guidelines for liver sampling in patients with suspected chronic hepatitis.

A minimum of 12-15 portal tracts is required for assessment of chronic hepatitis. This is best achieved with guillotine wedge or laparoscopic liver biopsies. However, these sampling methods are more invasive than Tru-cut (needle) biopsies. Determination of the biopsy type (Tru-cut, laparoscopic, guillotine wedge) requires assessment of the patient’s coagulation parameters, index of clinical suspicion for chronic hepatitis, distribution of the liver lesions on ultrasound, and the veterinarian’s tools. For Tru-cut needle biopsies, diagnostic accuracy is increased with a larger gauge needle (14 or 16) and by obtaining biopsies from multiple sites.

At least 2 different lobes should be sampled, with a minimum of 5 laparoscopic, surgical, or Tru-cut biopsies. At least 3 samples should be placed in formalin for histology, 1 sample in transport media for culture (aerobic and anaerobic), and 1 in an empty glass tube for copper quantification. Note that samples placed in formalin cannot be cultured.

At VPG Histology, we will routinely perform haematoxylin and eosin (H&E) staining on all liver biopsies. Collagen staining is performed at the pathologist’s discretion. Copper staining is performed at an additional charge, usually following the recommendation of the pathologist. We also offer a specific liver evaluation test (HISL), which includes the copper and collagen staining. Because pathologic copper accumulation is a common and potentially treatable cause of chronic hepatitis, if chronic hepatitis is suspected, copper analysis should be performed.