Clinical Pathology & Cytology

What parameters may be abnormal in liver disease, relating to substances produced in the liver?

• Cholesterol

• Urea

• Glucose

• Albumin

• Coagulation factors

• Some globulins

What parameters may be abnormal in liver disease, relating to substances excreted by the liver?

Bile acids

Bilirubin

What parameters are considered to be markers of hepatic function?

• Bilirubin

• Albumin

• Bile acids

• Urea

Describe the haematological changes that can be seen in liver disease?

Anaemia that is usually microcytic. This is usually due to iron deficiency (as a result of chronic external blood loss) but can also be caused by defective delivery of iron to the bone marrow if hepsidin is blocking this which can occur commonly in liver disease and portosystemic shunts.

Following acute liver disease, the magnitude of the rise in liver enzymes is roughly proportional to the number of hepatocytes affected and the severity of cholestasis. What does it NOT give you any information on?

Prognosis

Reversibility

Nature of the disease

What time of hepatic enzymes are ALT and AST?

They are hepatocellular or leakage enzymes

Where is ALT released from?

The cytosol of the hepatocytes

What causes ALT release from the cytosol?

Acute hepatic necrosis or changes in membrane permeability or metabolism.

ALT is considered reasonably liver specific, though a small amount is found where?

In RBC, heart and skeletal muscles

Which cause of ALT release will result in higher levels, hepatic necrosis or leakage from membranes?

Hepatic necrosis

The halflife of ALT is a few hours, but why does ALT fall slowly following hepatocellular injury?

Due to increased production by regenerating hepatocytes.

A reduction in ALT of 50% every 3-4d following a return to normal by 2-3 weeks can be expected. If the ALT falls very quickly following an acute episode, this is a negative prognostic indicator - why?

As it is suggestive of reduced hepatocyte regeneration

Why are small increases in ALT more significant in the cat than in the dog?

Because the half life is shorter

Give causes of ALT elevation

• Primary liver disease - infection, inflammation, neoplasia, toxic damage, ischaemia and trauma

• Drugs

• Secondary liver disease/reactive hepatopathy > hyperthyroidism, cardiac disease, gut disease, pancreatitis

• Bile stasis (usually mild with more marked increase in ALP and GGT) due to toxic effects of bile acids damaging hepatocyte membranes

• Severe muscle damage

What typical changes are seen in liver enzymes when there is bile stasis?

• Elevation in ALP and GGT then a mild-moderate elevation in ALP

Where is AST located in the hepatocyte?

Mainly in the mitochondria of the cell, some in the cytosol

Increased ALT reflects cell membrane damage and leakage, what does increased AST tend to reflect?

More serious hepatic damage

In general, increases of AST parallel those of ALT. What is this influenced by?

The location of liver disease

• ALT more concentrated in hepatocytes near the portal triad

• AST is located around the central vein.

AST is more markedly elevated than ALT in acute hepatic necrosis, when is ALT more markedly elevated than AST?

In diseases arising in periportal area eg. cholangiohepatitis

Glucocorticoids and what other drug may produce mild increases in AST?

Anti-convulsants

AST is not very liver specific. Where can it also be found in high concentrations?

Muscle

Red cells

Skeletal muscle inflammation or damage will lead to a large increase in AST as well as what other enzyme?

Creatinine kinase (with an absent or very mild increases in ALT)

Haemolysis can also cause an increase in what enzymes?

AST

CK

What type of enzymes are ALP and GGT?

They are cholestatic enzymes

Where can cholestatic enzymes be found?

On the bile canalicular membrane, they are secreted in small amounts into bile.

Increased amounts of ALP and GGT are synthesised on what membrane as a result of impaired bile flow?

Sinusoidal

Cholestatic enzymes are induced by cholestasis and then what happens?

Then they leak out through the sinusoidal membrane rather than go into the bile canaliculus.

What are the causes of a high ALP?

• Hepatic causes

  • Intrahepatic cholestasis eg cholangitis, cholangiohepatitis

  • Hepatitis due to hepatocyte swelling, occluding the biliary canaliculi eg with hepatic lipidosis

  • Neoplasia

  • Vascular hepatopathy

  • Hepatic nodular hyperplasia

• Non-hepatic causes

  • Extrahepatic bile duct obstruction eg due to pancreatitis or pancreatic neoplasia

  • Release from bone

    • Young animals have approx 2-5x the normal range due to increased osteoblastic activity in growing bones. Usually within adult ranges by 15mo

    • Fractures, osteomyelitis, secondary renal hyperparathyroidism

    • Hyperthyroid cats > increased bone turnover

    • Drug induction - steroids, anti-convulsants

• Secondary reactive hepatopthies > eg hyperthyoidism that leads to increase in ALP of up to 4x URL

• High levels of ALP in dogs with mammary tumours have been associated with myoepithelial expression on ALP

Why do steroid induced increases in ALP occur?

Due to synthesis of an isoenzymes called c’steroid induced ALP. Due to exogenous steroids, endogenous steroids (HAC) or in response to endogenous steroids produced in severe stress

How long can ALP remain elevated after cessation of steroid treatment?

Weeks to months

What other changes can be seen in steroid induced elevations of ALP?

Mild increase in ALT, AST, GGT

After prolonged high doses of steroids or severe, untreated HAC you can sometimes see moderate to marked increases in ALT, AST and GGT with ALP elevations being most marked. Why?

As the hepatocytes become distended with glycogen (“steroid hepatopathy”)

C-ALP may be distinguished from normal ALP using electrophoretic sepataion. Why is this NOT a useful means of distinguishing cholestasis from steroid induction?

As C-ALP is also increased in some hepatobiliary diseases and DM, hypoT, acute pancreatitis and after anticonvulsant therapy.

Describe the typical liver changes associated with phenobarbitone therapy

• Significant elevation in ALP, usually not <500iu/l. Increases about ref range by 5 weeks and can be up to 10x baseline

• GGT increases over baseline and exceeds the RR in 50% of dogs

• ALT increases from baseline but doesn’t exceed the RR

• AST, bile acids and bilirubin do not usually change

• Albumin may fall transiently from baseline but not usually out of the RR

What is nodular hyperplasia, and when can it develop from?

Multifocal nodular lesions containing vacuolated hepatocytes, sometimes surrounded by mild inflammation but no fibrosis. It can develop in dogs from 6-8 years and is seen in almost all dogs >14 y/o

What changes to liver parameters are usually seen with nodular hyperplasia?

ALP markedly elevated

Other enzymes mildly elevated

What is vacuolar hepatopathy?

This is a reversible parenchymal change that is characterised by swollen hepatocytes with clear cytoplasm due to glycogen accumulation in >25% of hepatocytes.

What is the most common cause of vacuolar hepatopathy?

Cushing’s disease

Aside from Cushing’s disease, what are the other causes of vacuolar hepatopathy?

• Cardiac

• Hepatic

• GI disease

• Neoplasia

• Stress induced hypercortisolaemia associated with acute or chronic illness.

What dogs are most affected by idiopathic vacuolar hepatopathy?

Middle-older aged, Scotties are predisposed

What clinical signs may be associated with idiopathic vacuolar hepatopathy?

PUPD (rarely)

What are the typical liver enzyme changes in a dog with Idiopathic Vacuolar Hepatopathy?

Elevated ALP 5-10x normal

Other bile acids and liver enzymes are normal or only mildly elevated

What changes are seen on histopathology in a dog with idiopathic vacuolar hepatopathy?

Excessive hepatic glycogen accumulation in hepatocytes.

In idiopathic vacuolar hepatopathy, there is no evidence of a stress induced illness, Cushings or a hepatic disease. It may be due to what?

17-hydroxyprogesterone, progesterone, estradiol, testosterone and androstenedione causing “atypical Cushings”

What ultrasound changes are associated with vacuolar hepatopathy?

Uniformly hyperechoic liver with rounded borders.

How can you make a diagnosis of idiopathic vacuolar hepatopathy?

• FNA > vacuolar change

• Exclusion of Cushing’s, nodular hyperplasia, hepatic neoplasia or cholestatic liver disease

Dogs with IVH are also thought to have an increased risk of what?

Biliary mucoceles

Why is it prudent to perform ultrasounds to monitor Scotties with IVH?

Because they are also at risk of developing nodular hyperplasia, hepatocellular carcinoma or adenoma.

Where is GGT located?

On the cell membranes of biliary epithelial cells and to a lesser extend on the hepatocyte membranes

What is GGT elevated in response to?

• Cholestasis due to choolangiohepatitis, cirrhosis and post hepatic obstructions

• It is less affected by cholestasis due to hepatocyte swelling

In dogs, ALP is a much more sensitive but much less specific marker of cholestasis than GGT. What is the situation in cats?

GGT is much more snsitive than ALP, except in hepatic lipidosis in which ALP increases more markedly than GGT, and GGT may be normal. Likely because hepatic lipidosis is a form of hepatocellular cholestasis and ALP is primarily associated with hepatocyte membranes, whilst GGT with biliary epithelial membranes.

GGT may be mildly increased after steroids in dogs and sometimes after phenobarbitone. What are the changes in cholestasis?

Increases in parallel with ALP

Why is GGT less affected by primary hepatocellular damage?

Because it is located further down the biliary tree.

GGT is also found on what other membrane?

Renal tubular cell membranes. Increased GGT is released into urine after tubular damage (but not released into blood). The GGT;creatinine ratio may be useful for monitoring early signs of tubular injury.

What takes longer to fall after an insult, ALT or ALP?

ALP

Cholestasis leads to a much more marked increase in ALP in the dog than the cat. In dogs ALP is a sensitive marker of cholestasis and increases before bilirubin. What is the situation in cats?

In cats, ALP is much less sensitive and can be normal in cats with biliary obstruction or jaundice. This means that small increased in ALP are therefore always significant.

How is bilirubin formed?

By the breakdown of haemoglobin from senescent red cells, degraded by macrophages in the liver, spleen and marrow.

What happens after haemoglobin is converted to unconjugated bilirubin?

It is released from macrophages and transported to the liver where it is taken up by hepatocytes where it conjugated and then released into the biliary system and then into the intestine. Intestinal bacteria convert it into a number of faecal pigments including urobilinogen which are excreted in faeces.

Can urobilinogen be found in the urine of normal animals? Why or why not?

Yes, of absorbed urobilinogen, most undergoes enterohepatic circulation (hepatic portal vein —> hepatocytes —> bile ducts —> intestine) and some enters the general circulation and passes into urine.

What are the causes of hyperbiilirubinaemia?

• Prehepatic

• Hepatic

• Post hepatic

• (septic)

What is pre-hepatic jaundice?

Increased cell breakdown due to haemolysis leads to increased bilirubin formation, exceeding the liver’s capacity to excrete it.

What changes can be seen to liver enzymes in pre-hepatic jaundice?

Normal, or mildly increased if there is hypoxic damage

Normal bile acids

Why does intracavitatory bleeding not usually cause jaundice? What kind of bleeding can lead to jaundice?

Because many rbc are reabsorbed into the circulation. Large haematomas or bleeding into tissues can lead to increased bilirubin.

What is hepatic jaundice?

This is when hepatic disease leads to abnormal uptake, conjugation and release of bilirubin

What liver enzyme changes are typically seen with hepatic jaundice?

Liver enzymes and bile acids are increased (may not be marked if end stage)

Cholesterol can be increased (but not cholesterol)

Why can a functional cholestasis occur in dogs and cats with sepsis or very severe tissue infection?

Due to decreased excretion of conjugated bilirubin from hepatocytes due to the effect of tumour necrosis factor leading to decreased transport proteins for bile acids and bilirubin on canalicular membranes

What enzyme changes are typically seen in functional cholestasis?

Mildly increased bilirubin (50umol/l) as well as mild to moderate increases in bile acids. Resolves when infection is treated.

Why can mild increases in bilirubin be seen in anorexic cats?

Due to mobilisation of fatty acids which compete for binding sites for unconjugated bilirubin on hepatocyte membranes.

What is post hepatic jaundice?

Where there is obstruction of the intra or post hepatic tree.

What enzyme changes are usually seen with post hepatic jaundice?

Marked increases in ALP, GGT, milder increases in ALT and AST. Increased bile acids (can be marked)

During recovery of post hepatic jaundice, what enzymes return to normal before ALP and GGT?

ALT, AST, bile acids and bilirubin

Why does only conjugated bilirubin usually pass through the glomerulus?

Because unconjugated bilirubin, which is protein bound, is too large to pass through.

In animals with what renal disease can result in unconjugated bilirubin also being present in the urine?

Proteinuria

Why can you see bilirubin in a healthy dog but no other animal?

Because canine renal tubular cells can conjugate and excrete bilirubin and it has a lower renal threshold.

What causes a protein rich transudate (in abdomen/ascites)?

Increased hydraulic pressure on veins and lymphatics in the abdomen

What are causes of a protein rich transudate in the abdomen?

• Liver disease with portal hypertension

  • NOT Prehepatic (eg due to a portal thrombus or periportal fibrosis) as this leads to a LOW protein fluid

  • Hepatic or post hepatic (eg due to ventral vein fibrosis or hepatic vein obstruction) leads to a protein rich effusion because the sinusoids and lymphatics are more permeable to albumin

• Cardiac disease

• Neoplasia

What are the primary bile acids?

Cholic acid and chenodeoxycholic acid

Where are cholic acid and chendeoxycholic produced?

In hepatocytes from cholesterol

What are cholic acid and chenodeoxycolic acid conjugated to?

Taurine or glycine

What do bile acids do?

They enter the intestine where they contribute to fat absorption.

On reaching what section of the intestine do primary bile acids metabolise into secondary bile acids?

In the ileum

What are the secondary bile acids?

Deoxycholic and lithocholic acid

Over what percentage of secondary bile acids are reabsorbed?

90%

Via what route are secondary bile acids reabsorbed?

They enter the hepatic portal vein and are returned to the liver recirculated

Are bile acids specific for primary liver disease?

No, secondary or reactive hepatopathies can also cause a mild to moderate increase in BAs too

List causes of increased bile acid concentrations

• Disruption of the enterohepatic circulation

• Cholestasis/bile duct obstruction

• PSS

• Reduced uptake or excretion of bile acids by the hepatocytes

• Reduced hepatocellular mass eg cirrhosis

• Impaired hepatocyte function due to cell damage eg hepatitis/lipidosis

Why will a jaundiced animal (due to hepatic or post hepatic issues) have abnormal bile acids?

Because bilirubin and bile acids have similar excretory pathways. If an animal is jaundiced due to hepatic or post-hepatic causes, they will have abnormal bile acids due to obstruction of bile flow.

How does a bile acid stimulation test increase sensitivity/why is it more sensitive than a fasting bile acid test?

Because eating stimulates the release of bile by gallbladder contraction

Post prandial bile acids of >80mmol/l indicates what?

Hepatic insufficiency. If neurological signs are present, its consistent with hepatic encephalopathy.

What changes in bile acids usually result from secondary reactive hepatopathies?

Normal, or mild elevations in pre and post BA (eg ip to 40mmol/l)

BAs values of up to what can be seen in Cushing’s disease?

80mmol/l

What BA changes are typically seen in PSS?

A marked increase post prandially >90mmol/l

What bile acid changes are typical of cholesasis?

High fasting bile acids with a small postprandial rise

Pre and post prandial bile acids may sometimes be transiently elevated (up to 40-50mmol/l) within 24h following what event, and return to normal after a few days?

seizures

Explain why a fasting bile acid value can be greater than the post value?

Unclear, but may be due to incomplete gallbladder contraction and the fact that not all newly produced bile is stored in the GP. A high pre-level may be due to a spontaneous gallbladder contraction prior to the first sample.

Why should a feeding challenge for a BAST consist of a relatively small amount of food?

To reduce the potential for marked lipaemia, as this can lead to falsely high readings. If lipoclear is used to clear the lipaemia, this may also clear BAs bound to lipoproteins and give a false low result

How is ammonia produced?

It is produced in the gut following bacterial breakdown of dietary protein and sloughed intestinal cells

How is ammonia converted into urea?

It is absorbed into the portal circulation and transported to the liver where it is converted into urea

What extra-hepatic disorders can cause an increase in liver enzymes?

• Dental disease

• Hypoxia - cardiac disease, anaemia

• GI disease

• Pancreatic disease

• Endocrine disease

• Extra-hepatic bacterial infection

• Septicaemia and sock

Why is the liver susceptible to hypoxia due to anaemia or cardiac disease?

Because only 20% of the blood flowing to the liver is oxygenated blood (from the hepatic artery) and the remainder is deoxygenated blood from the portal vein. The liver is very dependent on adequate oxygenation