Normal Liver

Nalini Bansal Gupta; Apurba  Rajbongshi

SECTION 1

1Liver Biopsy

Normal Liver

Nalini Bansal Gupta, Apurba Rajbongshi
Important Aspects of Liver Biopsy

Nalini Bansal Gupta
Simple Approach to Liver Biopsy Interpretation

Nalini Bansal Gupta, Chhagan Bihari
Special Stains and Immunohistochemistry

Kaushik Majumdar, Puja Sakhuja
Acute or Chronic Liver Failure and Clinical Scores

Nalini Bansal Gupta

Normal LiverChapter 1

Nalini Bansal Gupta,

Apurba Rajbongshi

GROSS ANATOMY

Liver has grossly four lobes (Figs 1A and B):

Right lobe—largest
Left lobe
Caudate lobe—drains directly into inferior vena cava
Quadrate lobe.

Figs 1A and B: Liver anatomy, (A) Anterior view; (B) Undersurface view

FUNCTIONAL SEGMENTS

For surgical resections liver is divided into eight segments (Fig. 2) by Couinaud system with each lobe having independent vascular pedicle (portal venous, arterial and lymphatic branches) and biliary drainage facilitating easy surgical segmental resection.^1,2

MICROSCOPY

Liver on histology has hexagonal lobules described by Kiernan in 1833³ comprising of portal tracts constituting the periphery of the hexagonal lobule, occupying three of the six apices of the hexagon (Fig. 3). The effluent hepatic vein is at the center of the lobule, hence its name central vein.⁴

Normal liver biopsy shows:

Hepatocytes arranged in plates (liver cell plates)
Central vein
Portal tract showing triad of hepatic artery, bile duct, portal vein.

Fig. 2: Segmental anatomy of liver

Fig. 3: Hexagonal lobules with portal tracts at periphery

Hepatocytes

Constitute 80% of all cells in normal liver
Hepatocyte—polygonal cell with a small round nucleus and abundant pink finely granular cytoplasm due to abundant glycogen contents and mitochondria. Binucleate forms can be seen
Hepatocytes are aligned in uniform single cell plate arrangement from central vein to portal tract (Fig. 4)
In children up to 5–6 years of age, hepatocyte plate can be two cells thick
They directly abutt on central vein with no significant connective tissue between them.

Hepatocytes contain following organelles:

Mitochondria
Peroxisomes
Lysosomes
Golgi complexes
Rough/smooth endoplasmic reticulum.

Hepatocyte Domains/Surfaces^5,6

Hepatocytes are polygonal cells and have three domains/surfaces.

Basolateral Domain/Vascular Pole/Sinusoidal
- 35% of hepatocyte surface faces sinusoids and perisinusoidal space of Disse (SOD)
- It has microvilli that protrude into sinusoids and maintain integrity of SOD
- It has N-K ATPase pump, NTCP (Sodium taurocholate cotransporting peptide) and OATP (Organic anion transporting polypeptide C). NTCP involved in sodium dependent uptake of bile acid from sinusoids.
  
  Fig. 4: Normal hepatocyte cords (H & E ×20) & (MT ×20)
  6
Apical/Canalicular Domain

It comprises 15% of hepatocyte surfaces and faces bile canaliculi. It has several ATP dependent export pumps belonging to ATP binding casette (ABC). Their main function is bile secretion in canaliculi.

ABC include following transporters:
- Bile salt export pump (BSEP) transport bile salt into bile canaliculi
- Multidrug resistant protein 3 (MDR3)—is a phospholipid transporter, translocates phosphatidylcholine from inner to outer membrane of hepatocyte and release it into bile canaliculi which is then taken up by bile salts
- Multidrug resistance-associated protein 2 (MRP2)—transport glutathione into bile.
Lateral Domain

50% of total hepatocyte surface faces adjacent hepatocytes.

Immunohistochemistry

Hepatocyte Paraffin 1 (Hep Par 1): It is antibody against urea cycle enzyme (carbamoyl phosphate synthetase 1) present in mitochondria and take fine granular cytoplasmic stain. It is useful for identifying hepatocyte differentiation.

HEPATOCYTE COUPLET (FIG. 5)

It is a model for studying bile physiology
It comprises of two adjacent hepatocyte with a bile canaliculus in between.

ZONATION (FIGS 6A AND B)

For better understanding of liver histological and metabolic perspective, liver lobule is divided into three zones.

Zone 1: Periportal
- Nearest to the incoming vascular supply and receives the most oxygenated blood
- Least sensitive to ischemic injury while making it very susceptible to chronic viral hepatitis, toxins and heavy metals injury⁷
- Specialized for oxidative liver functions, such as gluconeogenesis, β-oxidation of fatty acids, urea cycle functions and cholesterol synthesis.
  
  Fig. 5: Hepatocyte couplet
  7
  Figs 6A and B: Zones of liver (MTX10)
Zone 2: Midzonal
Zone 3: Perivenular
- Is least oxygenated
- Most commonly affected during ischemic injury
- Zone III cells are more important for glycolysis, lipogenesis and cytochrome P-450-based drug detoxification and rich for glutamine synthetase.⁸

Zonal Heterogeneity in the Liver⁹

As different zones of liver perform different function there is zonal heterogeneity in the liver (Fig. 7).

Two type of heterogeneity are seen:

Gradient Zonation: Present in all zones but expression differs—In the gradient type, all hepatocytes are able to express a particular gene, but the level of expression depends on the position of the hepatocyte along the portocentral radius like enzymes of carbohydrate metabolism, cytosolic phosphoenol-pyruvate carboxykinase I (PCK) and glucokinase8

Fig. 7: Billiary drainage system
Compartment Zonation: Present only in specific zones: In the compartment type of zonation, the expression of genes has been thought to be restricted to either the periportal or the pericentral compartment, e.g. key enzymes of ammonia metabolism, carbamoyl phosphate synthetase I (CPS) and glutamine synthetase (GS), which in normal liver exhibit strict localization to hepatocytes rimming the terminal hepatic vein.

SPACE OF DISSE

Very important space as it contains vital cells of liver pathology
The space of Disse (SOD) was named after German anatomist Joseph Disse¹⁰
It is fluid space between basolateral domain of hepatocyte and sinusoidal endothelial cells (Fig. 8)
Also known as perisinusoidal extravascular space.

Components of SOD

It has four different cell types and extracellular matrix (ECM). Four cell types in SOD are:

Hepatic stellate cells (HSC)
Sinosoidal endothelial cells
Kupffer cells
Liver-associated lymphocytes/pit cells (NKT cells).

Extracellular matrix of SOD differ in various zones.^11,12

Zone 1/periportal—ECM comprises of laminin, type IV collagen and heparin sulphate9

Fig. 8: Space of Disse
Zone 3/perivenular—ECM comprises of fibronectin, type III collagen and dermatan sulphate.

Hepatic Stellate Cells (HSC)

First described by von Kupffer as ‘Sternzellen’ or star cells¹³
Toshito Ito named them as fat storing cells/Ito cells¹⁴
Named as perisinusoidal cells containing vitamin A by Kenjiro Wake¹⁵
Store 80% of retinoids in the whole body as retinyl palmitate in lipid droplets in the cytoplasm
Play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP) on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis
Regulate sinusoidal blood flow
Immunohistochemistry—positive for glial fibrillary acidic protein (GFAP) and calretinin in quiescent stage and when activated they show positivity for Desmin, and Alpha smooth muscle actin^16–18
They transform into myofibroblast during liver injury and initiate fibrosis^19–22
Important regulators for regeneration and controlling the process of regeneration
It helps in neoangiogenesis and myofibroblast migration
They are also involved in epithelial mesenchymal transition (EMT) during liver injury.

Sinusoidal Endothelial Cells (SEC)

Discontinuous fenestrated endothelial cells
It known as sinusoidal as they have no basement membrane and form a fenestrated lining
Diameter of fenestrae is 150–175 nm
Fenestrate are more in zone 3/perivenular region than in zone 1 as zone 3 receives relatively less oxygenated blood²³
Immunohistochemistry—Do not express usual vascular markers like CD34, CD31, VEGF and do not bind UEL; however they are positive for CD16, CD14, CD11 B, CD 32 and ICAM^24,2510
They are different from endothelial cells lining glomerular capillaries in kidney by absence of basement membrane.²⁶

Kupffer Cells

There are two subgroup of liver macrophages—resident macrophages also known as Kupffer cells and infiltrating macrophages.
Liver macrophages derived from monocytes—mononuclear group of macrophages²⁷
Located within the lumen of sinusoids
Most numerous in the portal regions²⁸
When activated → produce large amounts of chemokines/cytokines
Acute liver inflammation begins with activation of Kupffer cells.

Liver-associated Lymphocytes

65% lymphocytes in liver are NK cells expressing TCR and remaining are T and B cells (minority)
NKT cells also known as Pit cells are large granular lymphocytes in subendothelial layers in space of Disse
Liver comprises of largest number of gamma+delta+T cells in body and hence it is the site for delta, gamma T cell lymphoma.^29,30

Portal Tract (Fig. 9)

Comprises of hepatic artery, bile duct and portal vein
Hepatic artery and bile duct are of same caliber
Portal vein has the largest caliber
Portal mesenchyme
Normal ratio of bile duct to portal tract 0.9–1.8
Limiting plate—discontinuous lining of hepatocytes around the portal tract
Periportal hepatocytes—Hepatocytes adjacent to limiting plate are called periportal hepatocytes.

Fig. 9: Normal portal tract with hepatic artery, bile duct and portal vein

Space of Mall

Fluid space between hepatocytes along the limiting plate in the periportal area, and portal tract fibrous tissue; it is in continuity with the perisinusoidal space of Disse.

Sinusoids

Are present between the hepatocyte plates. Through the sinusoids blood comes from portal tract through hepatic artery and portal vein and drains into central vein. Periportal region thus receives more oxygenated blood as is near to portal tract.

Central Vein

Terminal hepatic venule (THV), also known as central vein, is the smallest tributary of hepatic vein. It collects venous blood draining through sinusoids of a hepatic lobule (Fig. 10).
It joins with similar veins of other lobules and forms the main hepatic vein.

BILIARY SYSTEM

Billiary system comprises of bile canaliculi which collect bile from hepatocytes and near portal interface → Canaliculi drain into canals of Hering (lined partly by hepatocytes and partly by cholangiocytes) → Canal of Hering drains into bile ductules (or Cholangioles) which begin at the limiting plate (interface bertween parenchyma and portal tract) → Medium-sized interlobular ducts → Septal ducts → Area ducts → Segmental ducts → Left and right hepatic ducts. Intrahepatic biliary duct system (IHBD) comprises of large duct including area, segmental and right and left hepatic duct which also form main hepatic ducts intrahepatic biliary ductal system (Table 1).³¹

Fig. 10: Central vein with hepatocyte cords (H & E ×20)

Table 1 Intrahepatic biliary ductal system

Branching	Diameter (micron)	Terminology
Large bile duct
First generation	>800	Right and left hepatic duct
Second generation	400–800	Segmental duct
Third generation	300–400	Area ducts
Small intrahepatic bile duct
	>100	Septal duct
	15–100	Interlobular duct
12th generation	<15	Ductules (cholangiole) Canal of Hering

Larger ducts are lined by peribiliary mucous glands which are of two types:

Intramural—Directly draining in duct lumen
Extramural—Drain through secretory duct.

Cholangiocyte

Cholangiocytes are flattened or cuboidal in small branches of biliary tree
Columnar in large branches
Contribute to bile secretion via net release of bicarbonate and water^32–34
Mounts IgA and IgM response (not IgG)
Capable of proliferating after liver injury.

Canalicular Network

Canalicular network can be highlighted with immunostains for PCEA, CD 10, MDR3 and MUC1

Fig. 11: IHC showing canalicular positivity of pCEA
13
Polyclonal carcino embryonic antigen (pCEA)—Antibody react and stains a CEA like cross reactive substance called biliary glycoprotein present in bile canaliculi and ductal epithelium but not hepatocytes (Fig. 11).

Where can we get normal Biopsy

Received mainly from perspective liver donors for assessment of steatosis, inflammation or significant fibrosis.

LIVER BIOPSY SIGN OUT FOR NORMAL BIOPSY

No significant inflammation/fibrosis/steatosis seen.

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Chapter Notes

Normal LiverChapter 1