Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

Estimation of Bilirubin

ESTIMATION OF SERUM BILIRUBIN (Modified DMSO Method)

Principle

• Blirubin in the presence of diazotised sulfanilic acid (& DMSO) reacts with sodium nitrite to form azobilirubin. In the absence of dimethyl sulfoxide (DMSO), only the direct bilirubin reacts to give azobilirubin.

H⁺ H⁺

• Bilirubin + Diazotised Sulfanilic acid Azobilirubin Red-Purple Complex

Sample

• Serum / plasma

Reagents

• Total bilirubin reagent (sulfanilic acid, HCl, DMSO) 
• Direct bilirubin reagent (sulfanilic acid, HCl)
• Total bilirubin activator
• Direct bilirubin activator
• Bilirubin Artificial Standard (10 mg/dl for Total bilirubin & 7.7 mg/dl for Direct bilirubin)

Procedure

• Pipette into clean dry test tubes labeled as T₁, T₂ (for total bilirubin) and D₁, D₂ (for direct bilirubin) as follows:

S.No.	Reagents	Total bilirubin		Direct bilirubin
		Sample blank T1	Test T2	Sample blank D1	Test D2
1.	Total bilirubin reagent	1.0 ml	1.0 ml	-	-
2.	Direct bilirubin reagent	-	-	1.0 ml	1.0 ml
3.	Activator – Total	-	20 m l	-	-
4.	Activator – Direct	-	-	-	20 m l
5.	Serum	50 m l	50 m l	50 m l	50 m l

• Mix well and incubate exactly for 5 minutes. Measure the absorbance of the tests and respective sample blanks at 546 nm setting zero with D.W.

Calculation:

                                                         OD (T₂) – OD (T₁) OD (D₂) – OD (D₁)

A. Total Bilirubin (mg/dl): = ------------------------------------------- x 10

B. Direct Bilirubin (mg/dl): = --------------------- x 7.7

                                                                  OD (S) OD (S)

C. Indirect Bilirubin (mg/dl): = (A) – (B)

Result: In the given serum

• Total bilirubin is (Direct + Indirect) 
• Direct bilirubin is (Conjugated)
• Indirect bilirubin is (unconjugated)

Clinical Significance:

• Hyperbilirubinaemia is a characteristic of jaundice.
• In hemolytic and neonatal jaundice, unconjugated bilirubin level increases without corresponding increase in conjugated bilirubin.
• In viral hepatitis, toxic hepatitis and cirrhosis, there is overall damage to liver cells and hence there is increase in both conjugated and unconjugated bilirubin levels.
• In obstructive jaundice, there is an increase in conjugated bilirubin and a normal level of unconjugated bilirubin.