Introduction
- Bilirubin is a yellow pigment formed during breakdown of hemoglobin.
- It is produced mainly in the reticuloendothelial system after destruction of old red blood cells.
- Hemoglobin released from RBCs is converted into biliverdin and then bilirubin.
- Newly formed bilirubin is water insoluble and circulates bound to albumin.
- This form is called indirect bilirubin (unconjugated bilirubin).
- In the liver, bilirubin is conjugated with glucuronic acid.
- Conjugated bilirubin becomes water soluble and is called direct bilirubin.
- Direct bilirubin is excreted through bile into intestine.
- In intestine, bacterial action converts bilirubin into urobilinogen and stercobilinogen.
- Bilirubin estimation is an important liver function test.
- It helps in diagnosis of jaundice, liver disease, bile duct obstruction, and hemolytic disorders.
Types of Bilirubin
- Total bilirubin = Direct bilirubin + Indirect bilirubin
- Direct bilirubin = Conjugated bilirubin
- Indirect bilirubin = Unconjugated bilirubin
Principle
- Bilirubin estimation is based on Modified Pearlman and Lee Diazo Method.
- Sulphodiazonium salt reacts with bilirubin to form colored azobilirubin.
- The color intensity produced is directly proportional to bilirubin concentration.
Direct Bilirubin Principle
- Direct bilirubin reacts directly with diazo reagent.
- Azobilirubin is formed immediately.
Total Bilirubin Principle
- Indirect bilirubin requires surfactant for solubilization.
- Cetrimonium bromide helps release indirect bilirubin from albumin.
- Both direct and indirect bilirubin then react to form azobilirubin.
Measurement
- Absorbance is measured at 546 nm
- Secondary wavelength: 630 nm or 670 nm
Specimen
Sample Type
- Serum is preferred specimen
- Plasma may also be used
- Heparinized plasma acceptable
- EDTA plasma acceptable
Precautions
- Use non-hemolyzed sample
- Protect sample from light
- Analyze quickly because bilirubin is light sensitive
Stability
Total Bilirubin
- 1 day at 15–25°C
- 7 days at 2–8°C
- 3 months at −20°C
Direct Bilirubin
- 2 days at 15–25°C
- 7 days at 2–8°C
- 3 months at −20°C
Reagents
Reagent 1
- Sulphanilic acid
- HCl
- Cetrimonium bromide
Reagent 2
- Sulphanilic acid
- HCl
Reagent 3
- Sodium nitrite
Role of Reagents
- Sulphanilic Acid – Forms diazo reagent
- Sodium Nitrite – Produces diazonium salt
- Cetrimonium Bromide – Solubilizes indirect bilirubin
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Colorimeter / semi-auto analyzer
- Cuvette
- Timer
- Distilled water
- Bilirubin reagent kit
Procedure
| Components | Blank | Standard | Test |
|---|---|---|---|
| Working reagent | 500 µL | 500 µL | 500 µL |
| Distilled water | 25 µL | — | — |
| Standard | — | 25 µL | — |
| Sample | — | — | 25 µL |
Incubation
- Mix properly
- Incubate at 37°C for 5 minutes
Reading
- Measure absorbance of standard and test against blank
- Read at 546 nm / 630 nm
Calculation
Formula Using Standard
Bilirubin (mg/dL) = Absorbance of Test / Absorbance of Standard × Standard concentration
Formula Using Factor
- Total Bilirubin = ΔA × 23
- Direct Bilirubin = ΔA × 17
Normal Reference Values
Total Bilirubin
| Group | Value |
|---|---|
| Adults | 0 – 2.0 mg/dL |
Direct Bilirubin
| Group | Value |
|---|---|
| Adults and infants | 0 – 0.2 mg/dL |
Newborn Values
Premature Newborn
- 0–1 day: 1.0–8.0 mg/dL
- 1–2 days: 6.0–12.0 mg/dL
- 3–5 days: 10.0–14.0 mg/dL
Full Term Newborn
- 0–1 day: 2.0–6.0 mg/dL
- 1–2 days: 6.0–10.0 mg/dL
- 3–5 days: 4.0–8.0 mg/dL
Clinical Significance
- Bilirubin estimation is one of the most important biochemical tests for evaluation of jaundice.
- It helps determine whether bilirubin elevation is due to excessive red blood cell destruction, liver cell injury, or bile duct obstruction.
- Bilirubin values must always be interpreted together with clinical findings and liver enzyme tests such as ALT, AST, and ALP.
- Both total bilirubin and its fractions (direct and indirect bilirubin) provide important diagnostic information.
Clinical Importance of Total Bilirubin
- Total bilirubin represents the sum of direct and indirect bilirubin.
- Increased total bilirubin indicates disturbance in bilirubin metabolism, transport, conjugation, or excretion.
- Elevated total bilirubin produces visible jaundice when serum concentration rises significantly.
- Clinical jaundice usually becomes visible when bilirubin exceeds 2 mg/dL.
Conditions Causing Increased Total Bilirubin
Hemolytic Disorders
- Excessive destruction of red blood cells produces large amounts of bilirubin.
- Liver cannot conjugate all bilirubin rapidly.
- This leads to rise in total bilirubin mainly due to indirect fraction.
Seen in:
- hemolytic anemia
- malaria
- transfusion reaction
- sickle cell disease
- thalassemia
Liver Cell Disease
- Damaged hepatocytes cannot conjugate or excrete bilirubin properly.
- Both direct and indirect bilirubin may increase.
Seen in:
- viral hepatitis
- toxic hepatitis
- cirrhosis
- drug-induced liver injury
- fatty liver disease
Obstructive Disorders
- Bile flow obstruction prevents bilirubin excretion.
- Conjugated bilirubin returns to blood.
Seen in:
- gall stones
- bile duct obstruction
- pancreatic tumor
- cholestasis
Clinical Importance of Direct Bilirubin
- Direct bilirubin is conjugated bilirubin formed in liver.
- It is water soluble.
- Increased direct bilirubin usually indicates post-hepatic or hepatocellular problem.
Increased Direct Bilirubin Seen In
Obstructive Jaundice
- Bile duct blockage prevents excretion of conjugated bilirubin.
- Direct bilirubin rises markedly.
Causes
- gall stone obstruction
- carcinoma head of pancreas
- biliary stricture
- cholangiocarcinoma
Hepatocellular Disease
- Liver cells conjugate bilirubin but cannot excrete properly.
Seen in
- hepatitis
- cirrhosis
- liver cell necrosis
Urine Finding
- Direct bilirubin appears in urine because it is water soluble.
- Dark urine is commonly seen.
Clinical Importance of Indirect Bilirubin
- Indirect bilirubin is unconjugated bilirubin.
- It is water insoluble and bound to albumin.
- It does not appear in urine.
Increased Indirect Bilirubin Seen In
Pre-hepatic Causes
- Excess RBC destruction produces excess bilirubin.
Seen in
- hemolytic anemia
- malaria
- incompatible blood transfusion
Defective Conjugation
- Liver cannot conjugate bilirubin normally.
Seen in
- Gilbert syndrome
- Crigler–Najjar syndrome
- neonatal jaundice
Bilirubin in Neonatal Jaundice
- Neonates commonly show increased indirect bilirubin because liver conjugation mechanism is immature.
- Physiological jaundice usually appears after birth.
- Bilirubin level rises during first few days.
Danger in Newborn
- High unconjugated bilirubin crosses blood-brain barrier.
- It deposits in brain tissue.
- Causes kernicterus.
Kernicterus May Cause
- brain damage
- convulsions
- deafness
- developmental delay
Clinical Importance
- Neonatal bilirubin monitoring is essential.
- Severe cases require phototherapy or exchange transfusion.
Bilirubin Pattern in Different Types of Jaundice
Hemolytic Jaundice
- Indirect bilirubin increased
- Direct bilirubin normal or slightly increased
- Urine bilirubin absent
Hepatocellular Jaundice
- Both direct and indirect bilirubin increased
- Urine bilirubin present
Obstructive Jaundice
- Direct bilirubin markedly increased
- Urine bilirubin strongly positive
- Stool becomes pale
Diagnostic Importance of Bilirubin Test
- Differentiates type of jaundice
- Assesses liver excretory function
- Detects bile duct obstruction
- Monitors hemolytic disease
- Evaluates neonatal jaundice
- Helps in prognosis of liver disease
