Estimation of Serum Bilirubin

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Introduction

  • Bilirubin is a yellow pigment formed during the breakdown of hemoglobin from senescent red blood cells.
  • It is produced mainly in the reticuloendothelial system, especially spleen, liver, and bone marrow.
  • Hemoglobin breakdown produces heme, which is converted into biliverdin and then bilirubin.
  • Bilirubin circulates in blood in two major forms:
  • unconjugated bilirubin (indirect bilirubin)
  • conjugated bilirubin (direct bilirubin)
  • Unconjugated bilirubin is transported to the liver bound to albumin.
  • In the liver, bilirubin is conjugated with glucuronic acid and excreted into bile.
  • Serum bilirubin estimation is one of the most important liver function tests.
  • It is essential for diagnosis of jaundice, hemolytic disorders, and hepatobiliary disease.

Principle

  • Serum bilirubin estimation is based on Diazo method (Jendrassik–Grof method).
  • Bilirubin reacts with diazotized sulfanilic acid to form azobilirubin, a colored compound.

Reaction

Bilirubin + Diazotized Sulfanilic Acid → Azobilirubin

  • Direct bilirubin reacts directly in aqueous medium.
  • Total bilirubin requires accelerator such as caffeine-benzoate for reaction.
  • The intensity of color formed is directly proportional to bilirubin concentration.
  • Absorbance is measured at 546 nm.

Reagents

  1. Diazo reagent A: Dissolve 1 g sulphanilic acid in 15 ml conc. HCl; and make volume 1L with distilled water.
  2. Diazo reagent B: 0.5% sodium nitrite.
  3. Diazo color reagent: Mix 5 ml diazo A in 0.15 ml diazo B (prepare fresh).
  4. 0.15 N HCl, (e) Methanol, (f) Standard bilirubin in chloroform (1 ml ≡ 0.1 mg).

Sample

Sample Type

  • Serum is preferred specimen

Precautions

  • Use non-hemolyzed sample
  • Protect sample from light
  • Fresh sample preferred

Important Note

  • Bilirubin is light sensitive and decreases on exposure to light

Materials Required

  • Test tubes
  • Micropipette
  • Pipette tips
  • Colorimeter / spectrophotometer
  • Cuvette
  • Timer
  • Bilirubin reagent kit

Procedure

Label six test tubes as TT (total test), TC (total control), DT (direct test), DC (direct control), S (standard) and B (blank).

Components Blank Standard Test
Working reagent 1000 µL 1000 µL 1000 µL
Distilled water 100 µL — —
Standard — 100 µL —
Sample — — 100 µL

Incubation

  • Mix properly
  • Incubate for 5 minutes at room temperature

Reading

  • Measure absorbance against blank
  • Read at 546 nm

Calculation

  1. Direct bilirubin (mg/ dl) = OD of D test – OD of D blank / OD of std. × 10
  2. Total bilirubin (mg/dl) = OD of total test – OD of total blank / OD of std. × 10

Precautions

  1. A hemolyzed sample should not be used because hemoglobin interferes with the diazo reaction and absorbs at 540 nm.
  2. Serum for bilirubin estimation must be kept away from bright light since bilirubin is destroyed by ultraviolet light. Therefore, all tubes should be kept in the dark.

Reference range

Normal total bilirubin: 0.2 – 1.2 mg/dl

Indirect bilirubin: 0.2 – 0.7 mg/dl

Direct bilirubin: 0.1 – 0.4 mg/dl.

Hyperbilirubinemia of more than 3 mg/dl results in clinical jaundice.

Clinical Interpretation

  1. Assessing Liver Function
    • Elevated bilirubin levels can indicate liver diseases such as:
      • Hepatitis (inflammation of the liver)
      • Cirrhosis (scarring of the liver)
      • Liver failure
      • Liver tumors
  1. Identifying Jaundice
    • High bilirubin levels result in jaundice (yellowing of the skin and eyes).
    • Common causes:
      • Neonatal jaundice in newborns (often due to immature liver function)
      • Hemolytic jaundice from excessive breakdown of red blood cells
      • Obstructive jaundice from bile duct blockage (e.g., gallstones, tumors)
  1. Diagnosing Hemolytic Disorders
    • Elevated bilirubin can result from conditions that cause rapid red blood cell destruction, such as:
      • Hemolytic anemia
      • Sickle cell disease
      • G6PD deficiency
  1. Evaluating Biliary System Disorders
    • High bilirubin may indicate problems in bile flow, including:
      • Gallstones
      • Cholestasis (reduced bile flow)
      • Biliary atresia in infants
  1. Monitoring the Effectiveness of Treatments
    • Bilirubin tests are used to monitor patients with known liver diseases or conditions like hepatitis or post-surgery bile duct obstruction.

Types of Bilirubin Measured

  1. Total Bilirubin: Includes both direct and indirect bilirubin.
  2. Direct (Conjugated) Bilirubin: Processed in the liver and water-soluble.
    • Elevated levels suggest liver or bile duct issues.
  3. Indirect (Unconjugated) Bilirubin: Not yet processed by the liver.
    • Elevated levels suggest hemolysis or liver immaturity.

Common Conditions and Bilirubin Results

Condition Bilirubin Type Elevated Cause
Liver disease (e.g., hepatitis) Direct & indirect Impaired liver processing
Hemolytic anemia Indirect Excessive RBC breakdown
Gallstones or bile duct block Direct Obstruction of bile excretion
Neonatal jaundice Indirect Immature liver in newborns

Significance in Neonates

  • Neonatal jaundice is common and usually benign but requires monitoring to prevent complications like kernicterus (bilirubin toxicity in the brain).

MCQs

1. Bilirubin is formed from breakdown of:

A. Albumin
B. Hemoglobin
C. Cholesterol
D. Glucose
Answer: B. Hemoglobin

2. Bilirubin is mainly produced in:

A. Kidney
B. Spleen
C. Lung
D. Brain
Answer: B. Spleen

3. Bilirubin is a:

A. Protein
B. Yellow pigment
C. Enzyme
D. Hormone
Answer: B. Yellow pigment

4. Serum bilirubin estimation is mainly part of:

A. Lipid profile
B. Liver function test
C. Renal function test
D. Thyroid profile
Answer: B. Liver function test

5. Principle commonly used for bilirubin estimation:

A. GOD-POD method
B. Diazo method
C. Jaffe method
D. Biuret method
Answer: B. Diazo method

6. Another name of bilirubin method:

A. IFCC method
B. Jendrassik–Grof method
C. Kinetic method
D. Endpoint glucose method
Answer: B. Jendrassik–Grof method

7. Bilirubin reacts with:

A. Picric acid
B. Diazotized sulfanilic acid
C. Arsenazo III
D. Urease
Answer: B. Diazotized sulfanilic acid

8. Colored compound formed:

A. Azobilirubin
B. Quinoneimine
C. Picrate complex
D. Indigo dye
Answer: A. Azobilirubin

9. Wavelength used:

A. 340 nm
B. 405 nm
C. 546 nm
D. 650 nm
Answer: C. 546 nm

10. Direct bilirubin means:

A. Unconjugated bilirubin
B. Conjugated bilirubin
C. Free bilirubin
D. Protein bilirubin
Answer: B. Conjugated bilirubin

11. Indirect bilirubin means:

A. Conjugated bilirubin
B. Unconjugated bilirubin
C. Direct bilirubin
D. Free bile salt
Answer: B. Unconjugated bilirubin

12. Unconjugated bilirubin is transported bound to:

A. Globulin
B. Albumin
C. Hemoglobin
D. Fibrinogen
Answer: B. Albumin

13. Bilirubin conjugation occurs in:

A. Kidney
B. Liver
C. Spleen
D. Intestine
Answer: B. Liver

14. Conjugating substance:

A. Glucose
B. Glucuronic acid
C. Urea
D. Cholesterol
Answer: B. Glucuronic acid

15. Preferred sample:

A. Serum
B. Urine
C. Whole blood
D. Saliva
Answer: A. Serum

16. Sample should be protected from:

A. Heat
B. Light
C. Cold
D. Air
Answer: B. Light

17. Hemolyzed sample:

A. Preferred
B. Avoided
C. Frozen
D. Heated
Answer: B. Avoided

18. Accelerator used for total bilirubin:

A. Urease
B. Caffeine-benzoate
C. Peroxidase
D. Picrate
Answer: B. Caffeine-benzoate

19. Sulfanilic acid belongs to:

A. Reagent 1
B. Reagent 2
C. Standard
D. Sample
Answer: A. Reagent 1

20. Sodium nitrite belongs to:

A. Accelerator
B. Diazo reagent
C. Standard
D. Buffer
Answer: B. Diazo reagent

21. Total bilirubin normal value:

A. 0.2–1.2 mg/dL
B. 2–5 mg/dL
C. 5–10 mg/dL
D. 10–15 mg/dL
Answer: A. 0.2–1.2 mg/dL

22. Direct bilirubin normal value:

A. 0–0.3 mg/dL
B. 1–2 mg/dL
C. 3–5 mg/dL
D. 5–7 mg/dL
Answer: A. 0–0.3 mg/dL

23. Indirect bilirubin normal value:

A. 0.2–0.9 mg/dL
B. 2–4 mg/dL
C. 5–7 mg/dL
D. 10 mg/dL
Answer: A. 0.2–0.9 mg/dL

24. Increased unconjugated bilirubin seen in:

A. Hemolytic jaundice
B. Obstructive jaundice
C. Cholestasis
D. Hepatitis only
Answer: A. Hemolytic jaundice

25. Increased direct bilirubin seen in:

A. Hemolysis
B. Obstructive jaundice
C. Anemia
D. Malnutrition
Answer: B. Obstructive jaundice

26. Both direct and indirect bilirubin increase in:

A. Hemolysis
B. Hepatocellular jaundice
C. Renal disease
D. Diabetes
Answer: B. Hepatocellular jaundice

27. Bilirubin rises in newborn causing:

A. Hyperglycemia
B. Neonatal jaundice
C. Acidosis
D. Polyuria
Answer: B. Neonatal jaundice

28. Bilirubin is excreted through:

A. Urine only
B. Bile
C. Sweat
D. Saliva
Answer: B. Bile

29. Serum bilirubin is useful in:

A. Jaundice diagnosis
B. Liver disease
C. Hemolysis
D. All of the above
Answer: D. All of the above

30. Bilirubin color is:

A. Green
B. Yellow
C. Blue
D. Red
Answer: B. Yellow

Short MCQs (31–50)

  1. Bilirubin precursor = Heme
  2. Biliverdin converts to = Bilirubin
  3. Method = Diazo method
  4. Colored product = Azobilirubin
  5. Main wavelength = 546 nm
  6. Sample type = Serum
  7. Light sensitive = Yes
  8. Direct bilirubin soluble in water = Yes
  9. Indirect bilirubin soluble in water = No
  10. Bilirubin test part of LFT = Yes
  11. Obstructive jaundice direct bilirubin ↑ = Yes
  12. Hemolytic jaundice indirect bilirubin ↑ = Yes
  13. Liver disease raises both bilirubin types = Yes
  14. Accelerator used = Caffeine-benzoate
  15. Diazo reagent contains nitrite = Yes
  16. Conjugation organ = Liver
  17. Transport protein = Albumin
  18. Bilirubin in urine indicates conjugated bilirubin = Yes
  19. Neonatal jaundice bilirubin increases = Yes
  20. Bilirubin routine biochemical test = Yes
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