Rewind

• Bilirubin is formed from breakdown of _______.

• Bilirubin is formed in _______.
• Sources of heme :
• 1.________
• 2.________
• 3.________
• 4.________
• 5.________
• Heme : ________ + ________
Heme
Answers
• Bilirubin is formed from breakdown of heme (porphyrin ring).
• Bilirubin is formed in reticuloendothelial cells.
• Sources of heme :
• 1. hemoglobin
• 2. myoglobin
• 3. cytochrome
• 4. peroxidases
• 5. catalases
• Heme : iron + porphyrin
• Heme is also called : ferro-protoporphyrin
 Bilirubin formation
-in reticuloendothelial cell
 Biliverdin – linear tetrapyrrole
Porphyrin – tetrapyrrole ring
Biliverdin
Estimation of serum
bilirubin
Estimation of serum bilirubin

Total bilirubin

Direct/conjugated Indirect/unconjugated
bilirubin bilirubin

Soluble Insoluble
 Principle
Vandenbergh test: Diazo method
Total bilirubin estimation
• conjugated and unconjugated bil.
Bilirubin + sulphalinic acid + sod. Nitrite + DMSO→ azobilirubin
• { DMSO- dimethyl sulfoxide : polar organic compound- makes indirect
bilirubin soluble}

Direct bilirubin estimation

• Bilirubin + sulphalinic acid + sod. Nitrite → azobilirubin
 Total bilirubin estimation
• Using semi auto analyser
Reagents in kit:
• Reagent T1: Sulphalinic acid, HCL, DMSO
• Reagent T2: Sodium nitrite
Procedure :
Reagent Serum blank Test
REAGENT T1 500µL 500µL
REAGENT T2 - 10µL
SERUM 25µL 25µL
 Direct bilirubin estimation
Reagents in kit:
• Reagent D1: Sulphalinic acid, HCL
• Reagent D2: Sodium nitrite
Procedure : Reagent Serum blank Test
REAGENT D1 500µL 500µL
REAGENT D2 - 10µL
SERUM 25µL 25µL

Incubate on room temp (25-30°C): 5 min

Take reading at 546nm
• Normal levels

• Total bilirubin: 0.2- 0.8 mg/dl

• Direct bilirubin: 0.2 – 0.6 mg/dl
• Indirect bilirubin: 0 – 0.2 mg/dl
hyperbilirubinemia
• Total bilirubin > 1mg/dl – latent jaundice
• Total bilirubin > 2mg/dl – jaundice {clinically evident}

jaundice

Hemolytic / Obstructive/
Hepatocellular/
prehepatic Post hepatic
hepatic jaundice
jaundice jaundice
• Hemolytic jaundice
Unconjugated bil. rises

• Hepatocellular jaundice
Both unconjugated and conjugated bil.
rises

• Obstructive jaundice
Conjugated bilirubin rises
 jaundice

Hemolytic Hepatocellular jaundice Obstructive jaundice

Erythroblastosis Gall bladder

Viral hepatitis
fetalis stones

Congenital CA head of
Cirrhosis
spherocytosis pancreas

G6pd Hepatic Biliary

deficiency tumors cirrhosis

Autoimmune
Drugs
hemolytic anemia

toxins
Thank you