Serum bilirubin

Introduction

• The Serum bilirubin test is a crucial diagnostic tool to evaluate the health of the liver, bile ducts, and red blood cells.
• Bilirubin is a yellow-orange pigment produced during the natural breakdown of red blood cells.
• It is processed by the liver and excreted through bile into the intestines.
• Abnormal bilirubin levels in the blood can indicate various medical conditions, including liver diseases, hemolytic disorders, or bile duct obstruction.
• This test is particularly significant in diagnosing jaundice, a condition characterized by the yellowing of the skin and eyes caused by elevated bilirubin levels.
• Healthcare providers can assess liver function, identify underlying conditions, and monitor treatment effectiveness by measuring bilirubin levels.

 

Method

Malloy and Evelyn

 

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Principle

Bilirubin reacts with diazotized sulphanilic acid to form purple-colored azobilirubin. The water-soluble bilirubin glucuronides (conjugated bilirubin) react fast (within one minute) with the diazo reagent (direct reaction). The free bilirubin (unconjugated bilirubin) in serum complexed with albumin reacts very slowly and requires an accelerator or solubilizer as methanol (indirect reaction).

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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).

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Sample

Serum (from blood without anticoagulants).

Procedure

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

	TT	TC	DT	DC	S	B
Distilled water	1.8 ml	1.8 ml	4.3 ml	4.3 ml	1.8 ml	2.0 ml
Serum	0.2 ml	0.2 ml	0.2 ml	0.2 ml	–	–
Diazo color reagent	0.5 ml	–	0.5 ml	–	0.5 ml	0.5 ml
0.15N HCL	–	0.5 ml	–	0.5 ml	–	–
Methanol	2.5 ml	2.5 ml	–		2.5 ml	2.5 ml
Bilirubin standard	–	–	–		0.2 ml	–

 

Mix, keep the tubes for 30 minutes in the dark, and read the OD using a colorimeter with the green filter of wavelength 540 nm.

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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

 

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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.

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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.

 

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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

2. 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)

3. 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

4. Evaluating Biliary System Disorders

• • High bilirubin may indicate problems in bile flow, including:
    • Gallstones
    • Cholestasis (reduced bile flow)
    • Biliary atresia in infants

5. 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).