1.Tests based on abnormalities of pigment metabolism
a. Determination of serum Bilirubin
b. Van Den Bergh’s reaction
c. Detection of bile pigments in urine
d. Determination of fecal and urinary urobilinogen

2. Tests based on abnormalities of protein metabolism
a. Determination of total serum proteins
b. Estimation of serum albumin
c. Determination of albumin / globulin ratio
d. Detection of amino acids in urine
e. Determination of prothrombin time
f. Determination of α feto protein

3. Tests based on serum enzyme activities
a. Serum alkaline phosphatase
b. Serum amino tranferases
c. Serum 5 – nucleotidase
e. γ – glutamyl transferase
f. LDH

4. Tests based on abnormalities of lipid metabolism
a. Serum cholesterol
b. Ester / free cholesterol ratio

5. Tests based on abnormalities of carbohydrate metabolism
a. GTT
1.Tests based on abnormalities of pigment metabolism
a. Determination of serum Bilirubin
Estimation of serum bilirubin gives a measure of intensity of jaundice
Normal range : Total 0.2 – 1.2 mg/dl
Direct 0 – 0.4 mg/dl
Higher values are found in obstructive jaundice than in haemolytic jaundice

b. Van Den Bergh’s reaction
Bilirubin forms a reddish compound with diazotized sulfanilic acid and can be estimated colorimetrically.Three different responses may be observed
1. Direct reaction : Given by conjugated bilirubin. Immediate  development of color proceeding rapidly to a maximum (with
in a minute)
2. Indirect reaction : Unconjugated bilirubin being water  insoluble , color develops only when alcohol is added to the solution
3. Delayed direct reaction : Colour begins to appear only after  5 – 30 minutes and develops slowly to a maximum.

Haemolytic jaundice : Due to excessive destruction of RBC , the  main pigment in circulation is unconjugated bilirubin – gives indirect reaction
Obstructive jaundice : Due to regurgitation of conjugated bile into  the hepatic veins and lymphatics , immediate reaction is obtained
Hepatocellular jaundice : Mixture of conjugated and unconjugated pigment in circulation – gives biphasic reaction

c. Detection of bile pigments in urine
Haemolytic jaundice : unconjugated bilirubibn is carried in plasma attached to albumin , hence it cannot pass through   glomerular filter.So urine is -ve for bilepigments and salts
Obstructive jaundice: conjugated bilirubin can pass through glomerular  filter. So urine is +ve for bile pigments and salts
Hepatocellular jaundice: urine is +ve for bile pigments and salts d.Determination of fecal and urinary urobilinogen,urobilinogen is formed in the intestine by the reduction of bilirubin The  amount of fecal urobilinogen depends primarily on the amount of bilirubin entering the intestine.
Haemolytic jaundice : Faecal urobilinogen is increased in haemolytic  jaundice .Hence faeces dark in colour
Obstructive jaundice: Fecal urobilinogen is decreased or absent if there is  obstruction to flow of bile . So clay coloured faeces is passed . Complete obstruction is found in tumors , where as obstruction due to gall stone is  intermittent . A Complete absent of fecal urobilinogen is strongly suggestive of malignant obstruction.

Hepatocellular jaundice: decrease may occur in extreme cases affecting hepatic parenchyma.

Urinary urobilinogen
Normally only traces of urobilinogen is seen in urine.
Haemolytic jaundice: increased production of bilirubin leads to  increased production of urobilinogen which appears in urine in large amounts .
Obstructive jaundice: In cases of complete obstruction no urobilinogen is seen in urine
Hepatocellular jaundice: Increased urinary urobilinogen may be seen .

2. Tests based on abnormalities of protein metabolism
a.Determination of total serum proteins
Normal range : Total protein 5 – 8 gm /dl
b. Estimation of serum albumin
Normal range : serum albumin 3 – 5.5 gm /dl
Decrease in total serum proteins and serum albumin may occur in extreme  cases affecting hepatic parenchyma due to decreased synthesis.
c .Determination of albumin / globulin ratio
Normal ratio 2:1
Decrease in serum albumin and a rise in globulins occur in liver disease This results in reversal of albumin / globulin ratio and in extreme cases it becomes 1:2
Obstructive jaundice: normal values are the rule as long as obstructive jaundice is not associated with accompanying liver damage.
Acute heapatitis: Quantitative estimation of albumin and globulin may give normal values in the early stages. But qualitatative changes may be present .In the early stages β - globulins and in later stages γ – globulins may show rise

d .Detection of amino acids in urine
In severe liver disease such as advanced cirrhosis , crystals of certain amino  acids may be found in urine sediments on microscopic examination.
Tyrosine crystal: tufts of fine needle
Leucine crystal:spherical crystal

e .Determination of prothrombin time
Prothrombin is a protein synthesized by liver from inactive pre prothrombin in presence of vitamin K prothrombin time is the time required for clotting to take place in citrated plasma to which calcium and thromboplastin are added Normal prothrombin time is 10 – 16 seconds prothrombin time is increased both in obstructive jaundice and hepatocellular jaundice

In parenchymatouus liver disease , depending on the degree of liver cell  damage , prothrombin time may be increased from 22 to as much as 150 seconds

In obstructive jaundice , due to deficiency or absence of bile salts , there  may be defective absorption of of vitamin K leading to prolongation of prothrombin time

However , when prothrombin time is increased as a result of nutritional  deficiency or intestinal malabsorption of vitamin K , parenteral administration of vitamin K should correct it with in 12 hours .
Unresponsiveness suggest hepatic disease.

f. Determination of α feto protein
α feto protein is an α globulin present in high concentration in fetal blood  during mid pregnancy.
Normal range in adult blood is less than 1μg/dl
It is a tumour marker useful in detecting presence of hepatocellular carcinoma and teratoblastomas

3. Tests based on serum enzyme activities
a. serum alkaline phosphatase
Alkaline phosphatase is most plentiful in bone and liver
Normal range : 3 – 13 KA units/100 ml
The level of alkaline phosphatase is increased in both parenchymatouus liver disease and in obstructive jaundice .But the rise is more in case of obstructive jaundice . A value higher than 35 KA units / 100 ml is is strongly suggestive of obstructive jaundice . Higher values are occasionally found in space occupying lesions of liver.
Increase in alkaline phosphatase in liver disease is not due to hepatic cell  destruction or due to failure of clearance , but it is due to increased synthesis  of hepatic alkaline phosphatase.The stimulus for this increased synthesis is the bile duct obstruction .
The activity of alkaline phosphatase varies considerably with age especially the isoenzyme that originate in bone. An isolated increase in alkaline phosphatase is always benign , if other LFT results are normal.

b. Serum amino tranferases
Normal range : AST 7 – 35 units/ml
ALT 6 – 32 units/ml
The determination of serum amino tranferases is of extreme use in assessing  the severity and prognosis of parenchymatous liver disease such as acute hepatitis , toxic injury , following blunt trauma of abdomen etc where values
in thousands of units are seen .In acute hepatitis , rise in ALT may be greater where as in alcohol induced liver disease , in fulminant echovirus infection and in various metabolic disease , predominant rise in AST have been reported In chronic liver disease rise in serum amino tranferases is less marked .
In obstructive jaundice rise in serum amino tranferases occur but usually do not exceed 200 – 300 IU/L

c. Serum 5 – nucleotidase
Normal range : 2 – 17 IU/L
Level of serum 5 – nucleotidase is raised along with that of alkaline  phosphatase in liver disease . This enzyme has a biliary origin and hence levels are not elevated in bone disease

c. γ – glutamyl transferase
The activity of this enzyme is influenced by drugs , alcohol retc . It is useful in screening alcohol abuse.

d. LDH
Normal range : 70 – 240 IU/L
LDH enzyme is widely distributed Increased levels are seen in acute hepatitis , leukemia , renal disease etc . It is less specific.

4. Tests based on abnormalities of lipid metabolism
a. Serum cholesterol
Liver is concerned with the synthesis , esterification , oxidation and excretion of cholesterol .
Normal range : 150 – 250 mg/100ml

b. Ester / free cholesterol ratio is 2:1
In obstructive jaundice there is gross increase in cholesterol content with  out any alteration in ester/ free cholesterol ratio as the ester fraction is also increased.
In parenchymatous liver disease , there is no rise in cholesterol and the ester fraction is always reduced

5. Tests based on abnormalities of carbohydrate metabolism
GTT
In hepatic disease, due to poor glycogen storage there is fasting  hypoglycemia . Following glucose ingestion , there is a steep rise in blood glucose to hyperglycemic level due to decreased uptake as liver glycogen .
But muscle take up glucose as usual and brings down glucose in a short time.

Liver biopsy
Liver biopsy when combined with a clinical data can indicate an etiological diagnosis in most cases. Usually done in long standing cases when other non invasive measures are . The usual percutaneous approach is transcostal or transdiaphragmatic
Contraindication include prolonged prothrombin time , thrombocytopenia suspicion of vascular , cystic or infectious lesions in the path of the needle and severe ascites.

Hepatic imaging procedures
It help in defining the size , shape and architecture of the liver including intrahepatic and extrahepatic biliary tree.

Plain X ray study: may suggest hapatomegaly. The liver may appear less dense than normal with fatty infiltration and more dense with deposition of heavy metal such as iron. Calcification may be evident in liver in cases of parasitic or neoplastic disease , gall stones etc

USG: Helps in assessing gall bladder size , dilatation of biliary tract , in detecting gall stones , ascitic fluid etc

CT Scan: is slightly more accurate for detection of focal lesions such as tumors , cysts , abcess etc. When hepatic tumor is suspected CT Scanis the best method to define the anatomic extend , solid or cystic nature and vascularity.

Hepatic scintiscan: It relies on selective uptake of radio pharmaceutical agents .Anatomical resolution is generally less than that obtained with CT scan or USG. Commonly used agents include

1. Technitium 99 – m labeled sulphur colloid- which undergoes phagocytoses by Kupffer cells of liver . Is used to detect focal lesions more than 2-3 cm in diameter.

2. Technitium 99 – m iminodiacetic acid agents – which are taken up by hepatocytes and excreted in to bile which is normally detected with in 1-2 hours after i.v injection of the isotope . In presence of cholestasis , excretion of the isotope is delayed.

3. Gallium 67 which is concentrated in inflammatory cells and neoplastic cells

Cholangiography
It evaluates the cause , location or extend of biliary obstruction . It includes PTC and ERCP
Selective angiography: of celiac , superior mesenteric and hepatic artery may be employed to visualize portal circulation. It is mainly used in patients with portal hypertension and to define the blood supply to tumors prior to surgery.