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Liver architecture

1.     Normal liver architecture

http://www.vivo.colostate.edu/hbooks/pathphys/digestion/liver/histo_lobule.html

http://en.wikipedia.org/wiki/Lobules_of_liver

  • The basic structural unit of the liver is the lobule hexagonal arrangement of plates of hepatocytes radiating outward from a central vein (CV) in the centre. Central vein are quite prominent and makes orientation in understanding liver histology easy for beginners. The term “hepatic lobule”, without qualification, typically refers to the classical lobule or anatomical lobule. It is hexagonal; divided into concentric centrilobular, midzonal, periportal parts
  • Normal hepatocytes are arranged in narrow plates, which are usually no more than 2 cells thick, with intervening sinusoids radiating out from central vein to periphery
  • At the vertices of the lobule are regularly distributed portal triads (also known as portal tracts) – containing an artery, vein and bile duct. Due to plane of section, one can often observe more than one of each of these structures in a given portal tract or absence of one or more structures.
  • On the other hand from a metabolic perspective, the functional unit is the hepatic acinus elliptical or diamond-shaped; divided into zone I (periportal), zone II (transition zone), and zone III (centrilobular). each of which is centred on the line connecting two portal triads and extends outwards to the two adjacent central veins. The periportal zone I is nearest to the entering vascular supply and receives the most oxygenated blood. Conversely, the centrilobular zone III has the poorest oxygenation and is relatively more sensitive to ischemic injury.
  • Delicate bands of collagen invest hepatocytes– reticulin stains highlight the 2 cell thick trabeculae.
  • Starting point is an adequate biopsy  characterised by 20-25mm in length, 1.4mm in diameter, and contains at least 11 Portal tracts
  • Attempt an  initial assessment ‘blind’ to the clinical information and then review in light of clinical information which should include the following depending on the circumstances- BMI, alcohol intake, hepatitis screen results, US findings and clinical presentation and any particular questions from the treating clinicians. Special informations like vascultitic screen (hepatic vasculitis), presence of lymph nodes  or organomegaly ( lymphoma or malignancies) are needed in special situations. Remember better the information , better the interpretation
  • Special stains:

1.     Reticulin – stains Type III collagen fibres- to assess architecture

2. Trichrome/ Haematoxylin Van Gieson (HVG)- stains Type I collagen fibres-to assess fibrosis

3.     Orcein – stains elastic fibres- to assess HBsAg, copper associated protein and elastic fibres- found in long standing fibrosis

4.     PAS p to identify glycogen (also useful to look for small foci of necrosis or granulomas)

5.     d-PAS (PAS diastase) to identify ceroid pigment within kupffer cells, mucin in bile ducts, some hepatic neoplasms and α1antitrypsin granules

6.     Perl’s stain to assess iron

7.     Immunohistochemical stains (where appropriate)

§  Viral antigen markers (HBsAg, HBcAg, HDV, CMV, EBV)

§  Hepatocyte inclusions (ubiquitin for MH, α1antitrypsin)

§  Biliary cks (CK7, 19, AE1/AE3) to assess bile duct loss and ductular reaction in and around pts

§  Liver tumour markers (PCEA, CD10, heppar-1, αfp etc) to distinguish between primary and metastatic neoplasms

§  Proliferation marker Ki67 and CD34 used to distinguish between regenerative, dysplastic and neoplastic nodules

  • The order of analysis of a liver biopsy:

1.     Liver Architecture: connective tissue stains are required for accurate assessment of liver architecture

  • Are the normal vascular relationships maintained?
  • Is there any evidence of fibrosis/cirrhosis?
  • Any subtle architectural abnormalities eg. Atrophy, NRH

2. Portal Tracts And Periportal Regions

  • Bile ducts – present in normal numbers, any bile duct lesions (eg. Granulomas in pbc, fibrosing cholangitis in psc)?
  • Hepatic arteries – look for inflammation (eg. Pan, drug reactions), amyloid
  • Portal veins – look for pv lesions (eg. Obliteration and/or dilatation in non-cirrhotic portal ht, hepatoportal sclerosis, inflammation in liver allograft rejection
  • Then assess for other abnormal features:

§  Inflammatory cells (type, density, distribution)

§  Interface hepatitis

§  Ductular reaction (common reaction to many forms of liver injury, especially those with cholestasis)

3. Liver Parenchyma

  • Hepatocytes – degenerative changes (eg. steatosis, ballooning, bilirubinostasis), liver cell death (apoptosis or necrosis), and severity thereof (spotty, confluent, bridging, panacinar, multiacinar), nuclear or cytoplasmic inclusions (Mallory hyaline, eosinophilic globules) and whether these are zonal (eg. zone 1 MH in cholate stasis, and zone 3 MH in ALD)
  • Sinusoidal cells – Kupffer cell inclusions may be seen in some metabolic storage diseases and parasitic infections, Kupffer cell enlargement with ceroid pigment accumulation is non-specific reaction to previous hepatocellular injury
  • Then look for other abnormal features:

§  Inflammatory cells (type, density and distribution)

§  Sinusoidal dilatation, sickle cells, intravascular malignancy

§  Deposits eg. Amyloid

§  Note the presence/absence of liver cell dysplasia or neoplasia

4. Hepatic Veins

  • Is there evidence of endothelial inflammation?
  • Are veno-occlusive lesions present?
  • Is there inflammation within the vein wall?
  • Are there any lesions in the tissue surrounding the hepatic veins (inflammation, necrosis, fibrosis)?
  • Try to  identify the main patterns of injury/damage present. eg. steatosis, steatohepatitis, acute hepatitis, chronic hepatitis, chronic biliary disease with bile duct loss
  • Beware of the possibility of sampling error due to patchy distribution of changes
  • In some cases , might need to use of semi-quantitative scoring systems eg Ishak’s score , Metavir score etc.
  • Electron microscopy might need to be considered  in some cases of metabolic disease (glycogen storage, Niemann-Pick), certain viral infection (paramyxovirus in giant cell hepatitis)
  • Non-histological tests eg. biochemical measurements of liver iron or copper, detection of low levels of viral RNA or DNA by PCR is also sometime needed

2.     Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC)

  • Classically middle-aged female , anti-mitochondrial Ab (AMA) in 95% and may be associated with may be associated with Sjögrens, progressive systemic sclerosis, coeliac disease etc
  • PBC is characterised histologically by progressive destruction of intrahepatic (interlobular) bile ducts by lympho-plasma cellular infiltrate and granulomatous inflammation (~30%).
  • Sequence of events are- portal inflammation (by T lymphocytes, plasma cells, histiocytes, neutrophils and eosinophils) leading to interface hepatitis. Bile stasis causes ductular reaction (cytoplasmic eosinophilia, cell loss, nuclear pseudo-stratification, lymphocyte infiltration) and bile duct damage causing ductopenia- ultimately leading to periportal fibrosis with P-P linkage with preservation of normal vascular relationships.
  • Epithelioid granulomas (usually without giant cells) centred on damaged bile ducts is nearly always due to primary biliary cirrhosis (Fig. 8a to 8e). Sarcoidosis granulomas for comparison are-better-formed, more numerous and randomly distributed .
  • Biliary pattern of cirrhosis results in a ‘jigsaw puzzle’ arrangement of nodules and peripheral pallor around nodules due to cholate stasis.
  • Remember there are other diseases of bile ducts associated with vanishing bile duct syndrome in developmental anomaly ( extrahepatic biliary atresia,α1antitrypsin deficiency), Immune mediated diseases (autoimmune sclerosing cholangitis, sarcoidosis, liver allograft rejection, GVHD) , Vascular causes (ischaemic cholangitis, portal vein thrombosis), Infective causes ( cryptosporidia, CMV, recurrent pyogenic cholangitis) , Neoplastic (Hodgkin’s lymphoma)
  • PSC differs from PBC in that it affects not just small interlobular bile ducts, but also medium (septal), large (hilar) and extrahepatic bile ducts (but this distinction is not usually made on liver biopsies)
  • The patients in PSC show a lymphocytic infiltrate associated with atrophy of bile duct epithelium, luminal obliteration and concentric ‘onion-skin’ fibrosis (Fig. 9a to 9e). Similar lesions can occur in 2° causes of sclerosing cholangitis
  • Cholestasis is marked in both PSC and PBC , reflected in the deposition of copper binding protein (Orcein positive) (Fig. 9g and 9h).

3.     Liver infiltrates and deposits

  • Steatosis refers to the accumulation of lipid within hepatocytes and is typically described as macrovesicular (single large cytoplasmic vacuole filling the cell, Fig. 6a) or microvesicular (multiple small vacuoles imparting a bubbly appearance to the cytoplasm of hepatocytes).
  • Iron

1.     Iron is  one of the more common deposits seen in the liver (after fat) and occurs in the setting of hereditary haemochromatosis and acquired forms of siderosis (eg. Transfusion-related).

2.     The iron is deposited as haemosiderin, which has a fine, golden-yellow, refractile appearance on H&E and is not to be confused with lipofuscin (which is deposited in ‘aging cells’) Fig. 5a, 5b and 5c.

3.     The amount of haemosiderin deposition can be graded (1-4), depending on how much of the lobule is involved – initially the iron is deposited within the central portions of the lobule (in haemochromatosis) and as this progresses it is deposited across the entire lobule (a Perls histochemical stain confirms the presence of iron which is stained blue) (grade 4, Fig. 5d).

  • Amyloid is an abnormal protein which may be deposited in the liver as part of systemic amyloidosis (Fig. 6b and 6c) and typically has an amorphous (‘structure-less’), eosinophilic appearance, confirmed by performing Congo red or Thioflavin-T histochemical stains (‘apple-green’ birefringence under polarised light noted with the former).
  • Α-1 antitrypsin deficiency results in the accumulation of AAT globules in hepatocytes, which have a pink colour with the diastase-Periodic acid Schiff stain and can be highlighted with the aid of a specific immunohistochemical marker (Fig. 10a to 10c).

4.     Alcoholic and non-alcoholic fatty liver disease

  • fatty liver disease (whether alcoholic or non-alcoholic) comprises a spectrum of changes ranging from simple steatosis (fatty change) to steatohepatitis and fibrosis, to cirrhosis.
  • mainly macrovesicular steatosis is seen (Fig. 6a), but minor microvesicular change may be seen (Fig. 11a).
  • the inflammatory infiltrate consists of neutrophil polymorphs and hepatocyte damage is manifested by ballooning of hepatocytes and deposition of Mallory’s hyaline (Fig. 11b).
  • with continued hepatocyte damage, increasing amounts of pericellular and portal fibrosis are seen (Fig. 11c and 11d) eventually culminating in micronodular cirrhosis.

NAFLD

•          Microvesicular steatosis

•          Macrovesicular steatosis

•          Mallory bodies- eosinophilic accumulations of intracellular material

•          Ballooning degeneration

•          Perisinusoidal zone 3 fibrosis

•          Scattered, predominantly lobular, neutrophilic or mixed inflammation

•          In children, portal inflammation may be more prominent than in adults

5.     Metastases to the liver

  • The liver is a common site of metastatic disease, the commonest of which is metastatic adenocarcinoma from a variety of sites, but in particular the gastrointestinal tract and pancreas.
  • Metastatic adenocarcinoma varies from well-differentiated forms demonstrating well-formed glandular structures, to poorly differentiated tumours showing little morphological evidence of their glandular origin (fig. 4a and 4b).
  • Metastatic small cell carcinoma has a distinctive appearance (but may be confused with lymphoma and other ‘small round blue cell tumours’) , fig. 4c and 4d.
  • Immunohistochemistry is usually employed to confirm the site of origin of metastatic tumour, but may not necessarily be diagnostic (final diagnosis requiring correlation with clinical and/or radiological findings).

6.     Cirrhosis

  • With early cirrhosis, increased amounts of collagen are noted within, and later, between portal tracts (Fig. 2a shows a vaguely nodular outline, which is better seen on the HVG and reticulin stains – the fibrous septa do not completely link the portal tracts, therefore established cirrhosis cannot be confirmed histologically).
  • Fig. 2f reveals linking fibrosis and a micronodular architecture, indicating established cirrhosis is present (these biopsies are often fragmented).
  • Focal nodular hyperplasia is not to be confused with cirrhosis (usually easily distinguished due to the focal nature of the architectural abnormality on imaging and the lack of fibrosis)

7.     FNH (Focal nodular hyperplasia)

  • Benign, non-neoplastic hepatocellular lesion usually seen in young subjects (30-50 yrs)
  • Usually presents with pain or as incidental finding, very low risk of haemorrhage
  • Solitary (multiple in 20-30%) in a background of normal liver
  • Related to ?change in blood flow around arterial malformation with resulting hyperplastic growth response
  • Macroscopically – nodular appearance, often near capsule ,unencapsulated but well-demarcated because of nodularity, small (usually <5cm), but varies and may involve entire lobe and normally there is a central fibrous scar
  • Microscopically -normal-appearing hepatocytes arranged in incomplete nodules partially separated by fibrous tissue, intact reticulin framework (similar to normal liver) .key features include  variable numbers of bile ductular structures within the fibrous stroma at the edge of the nodules and  presence of medium to large, thick-walled muscular vessels .normal pts are not found in the lesion

8.     NRH (Nodular regenerative hyperplasia)

  • Innumerable nodules throughout the liver (sometime focal) varying from 1mm to 1cm in size, hyperaemic rim around larger nodules, nodules of uniform, bland hepatocytes without fibrous septa (distinguishes from cirrhosis),
  • associated with PV obstruction, polycythaemia vera, rheumatoid arthritis, Budd-Chiari, lymphoma and myeloproliferative disorders etc
  • often mistaken macroscopically for cirrhosis or metastatic tumour

9.     HCC (Hepatocelluar carcinoma)

  • The deranged liver architecture of hepatocellular carcinoma (HCC) is evident in Fig. 3a – solid sheets of atypical hepatocytes with occasional acinar (ring-like) structures are seen (the presence of bile within these acinar structures is a helpful clue to the presence of HCC, Fig. 3b).
  • Immunohistochemical staining with monoclonal CEA is another helpful indicator of HCC, highlighting the canalicular architecture of these tumours.
  • The fibrolamellar variant of hepatocellular carcinoma is characterised by increased amounts of collagen which is arranged in parallel bands around and between malignant cells showing focal bile production and occasional eosinophilic cytoplasmic bodies (Fig. 3f and 3g).

10.  Acute hepatitis

  • Acute hepatitis = <6 months and chronic hepatitis= >6 months
  • distinguishing between the two may be difficult, but useful differentiating features include: Acute hepatitis – the pattern of inflammation is usually mixed portal and parenchymal whereas in chronic (Fig. 7a -7d) it is usually portal/periportal. Cholestatic features are common in acute and rare in chronic (except in endstage disease). Fibrosis is mild and reversible in acute and more prominent in chronic hepatitis.
  • liver biopsy is performed in setting of hepatitis when the clinical presentation is atypical or the cause of the acute hepatitis is uncertain from the clinical, biochemical or serological findings. The three questions facing the pathologists are – Is this acute or chronic damage? How severe is the damage? What is the aetiology?

MICROSCOPICALLY

è  Inflammatory infiltrates within the liver are described according to the location of the infiltrate (portal or lobular) and the nature of the inflammatory cells

è  Inflammatory infiltrate à mainly lymphocytes (t more than b), but plasma cells may be seen in aih, neutrophils in alcoholic hepatitis and eosinophils in drug reactions (in chronic hepatitis, the inflammation is mainly mononuclear and there may be lymphoid aggregates eg. HCV, AIH, PBC ± interface hepatitis)

è  Exception = acute hav infection with plasma cell-rich portal/periportal infiltrate mimicking aih

è  Hepatocellular damage often most marked in the perivenular regions -is indicated by ballooning, bile pigment accumulation (bilirubinostasis), lobular disarray and cell death (either necrosis or apoptosis)

è  Bilirubinostasis is rarely a feature of chronic hepatitis except in decompensated end stage disease

è  Different degrees of severity of cell death may be identified:

a.     Spotty necrosis à apoptosis of individual hepatocytes resulting in  formation of intensely eosinophilic ‘councilman’ bodies which represent necrotic hepatocytes (fig. 7d).

b.    Confluent necrosis (zone 3) à loss of groups of adjacent liver cells

c.     Bridging necrosis à confluent necrosis linking vascular structures (c-c or c-p bridging)

d.    Panacinar necrosis à loss of hepatocytes within an entire acinus

e.     Multiacinar necrosis à panacinar necrosis involving several adjacent acini

è  Ductular reaction is common (associated with severity of cholestasis) and neutrophils may be present (‘cholangiolitis’) à ductular reaction is less common in chronic hepatitis and is associated with the severity of the fibrosis

è  Fibrosis (if present) is mild and may be reversible

a) Clues to aetiology:

1.     Viral à A, B, C, D, E and other (CMV, EBV, HSV)

2.     Drugs

3.     Autoimmune hepatitis (AIH)

4.     Idiopathic (‘seronegative hepatitis)

a.     Viral

i.    HAV infection à perivenular cholestasis with little inflammation, plasma cell-rich portal/periportal inflammation

ii.    Certain viral infections are associated with characteristic (but non-specific) patterns of inflammation (eg. formation of lymphoid nodules with steatosis is suggestive of Hepatitis C virus infection, Fig.7a) and with certain cytopathic effects (eg. ground glass cytoplasmic inclusions (Orcein positive) in Hepatitis B virus infection, Fig. 7e and 7f).

iii.    HEV infection suggested by à prominent cholestasis, microvesicular steatosis and prominent cholangiolitis

iv.    EBV and CMV may be associated with erythrophagocytosis by sinusoidal KCs and portal histiocytes (mistaken occasionally for sinus histiocytosis with massive lymphadenopathy or for malignant histiocytic neoplasm), EBV also  associated with atypical lymphocytes in sinusoids/PTs and occasionally epithelioid granulomas, CMV, HSV, VZV and adenovirus infections in the immunocompromised host are associated with typical nuclear inclusions

b.    Drugs

Paracetamol toxicity à discrete, centrilobular coagulative necrosis with little inflammation

c.     Autoimmune hepatitis (AIH)

1.     Plasma cell-rich portal infiltrate (sometimes parenchymal) with prominent interface hepatitis (need to exclude HAV)

2.     Centrilobular ballooning and hepatocyte necrosis, often with bridging necrosis

3.     Rosetting, fibrous entrapment of periportal hepatocytes and giant cell transformation of hepatocytes is sometime seen

4.     Differential includes AMA negative PBC, but in AIH the inflammation is more uniform and intense, shows lobular activity and lacks the typical bile duct damage

11.  Chronic hepatitis:

1.     The common differential diagnoses of chronic hepatitis include  chronic viral hepatitis B, C or D, autoimmune hepatitis (AIH), steatohepatitis, PBC, PSC, α1antitrypsin deficiency and Wilson disease and drug reaction.

2.     The inflammatory infiltrate is mainly mononuclear (± lymphoid follicles) and is predominantly portal/periportal ± interface hepatitis, much less lobular inflammation is seen (Fig. 7a -7d).

3.     Ductular reactions are less common and fibrosis to some degree is usually seen (the most reliable criteria for diagnosing chronic hepatitis)

4.     Role of liver Bx in chronic hepatitis:

a)     Establish histological diagnosis

b)    Identify or confirm aetiology

c)     Assess necro-inflammatory activity (grade) and extent of fibrosis (stage)

d)    Identify additional lesions

a)     Hepatitis B (HBV) infection

  • HBV and HCV infections are diagnosed serologically, but the liver is Bx for prognostic and/or therapeutic reasons
  • A characteristic and diagnostic feature of chronic HBV infection is the expression of HBsAg or HBcAg in hepatocytes
  • Cytoplasmic HBsAg expression corresponds to ground-glass hepatocytes seen on H&E (Orcein+ inclusions) Fig. 7e and 7f
  • HBcAg expression may be cytoplasmic, membranous or nuclear and infers active replication of virus

b) Hepatitis C (HCV) infection

  • METAVIR and modified Ishak staging systems are the most widely used, but suffer problems with inter-observer variation and sampling variation. Agreement is generally good for fibrosis, but less so for inflammation. Histological concordance (ie. Sampling) is good for inflammation but less so for fibrosis (the opposite to above)

  • Metabolic and HCV-related (genotype 3) pathways lead to steatosis and the severity of the steatosis affects the response to antiviral Rx

  • Minor degrees of siderosis are common in HCV infection (hepatocytes and kcs) but more severe siderosis in an hepatocellular distribution raises the possibility of genetic haemochromatosis. Siderosis also has an adverse effect on antiviral Rx

  • Microscopically the biopsy typically shows

a)     Mononuclear portal inflammation

b)    Lymphoid aggregates are typically prominent, but may also be seen in AIH, PBC (Fig 7a)

c)     Interface hepatitis is usually mild

d)    Lobular inflammation is often mild and confluent necrosis is not seen with HCV alone, except in the setting of immunocompromised patients (eg. Post-Tx) or if coexistent AIH present

e)     Fatty change is another feature, but is usually mild and mainly macrovesicular

Metavir score:

The fibrosis is graded on a 5-point scale from 0 to 4.

Fibrosis score:
F0 = no fibrosis
F1 = portal fibrosis without septa
F2 = portal fibrosis with few septa
F3 = numerous septa without cirrhosis
F4 = cirrhosis

The activity, which is the amount of inflammation (specifically, the intensity of necro-inflammatory lesions), is graded on a 4-point scale from A0 to A3.

Activity score:
A0 = no activity
A1 = mild activity
A2 = moderate activity
A3 = severe activity

Knodell Scores

The Knodell scoring system, also called the Histologic Activity Index (HAI), classifies liver biopsy specimens according to scores into four categories of histologic features:

• Periportal and/or bridging necrosis (scores from 0 to 10)

• Intralobular degeneration and focal necrosis (scores from 0 to 4)

• Portal inflammation (scores from 0 to 4)

• Fibrosis (scores from 0 to 4)

The Knodell Necroinflammatory Score is the sum of scores from parts I-III, hence a range of 0 to 18, and measures the degree of acute necroinflammatory activity in the liver.

The Knodell Fibrosis Score (part IV, above) measures the degree of scarring in the liver. Scarring builds up over time due to chronic necroinflammatory activity, ultimately leading to cirrhosis.

Recently, the Ishak fibrosis score has become the preferred method for evaluating liver fibrosis because it rates fibrosis according to seven categories on a continuous integer scale, as opposed to the discontinuous 4-point Knodell fibrosis score

Ishak’s Fibrosis Score

A scoring system that measures the degree of fibrosis (scarring) of the liver, which is caused by chronic necroinflammation.

A score of 0 represents no fibrosis,

Score of 1 and 2 indicate degrees of portal fibrosis;

Score  3 and 4 indicate bridging fibrosis.

A score of 5 indicates nodular formation and incomplete cirrhosis.

Score of  6 is established cirrhosis.


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