Diagnostic Laboratory Section

Method of estimation

VAN DEN BERGH METHOD

Jaundice is a condition in which the mucous membrane becomes yellowish in colour. It is classified into three types.

• Haemolytic / prehepatic jaundice – increase in indirect type
• Hepatic / toxic / intrahepatic jaundice – increase in both direct / indirect
• Post hepatic / obstructive jaundice – increase in direct type Based on this test, bilirubin is classified into two types
• Direct bilirubin – Conjugated bilirubin
• Indirect bilirubin – Unconjugated bilirubin

Conjugated bilirubin in the serum reacts with diazo reagent and gives immediately purple color. Therefore, it is called as direct bilirubin or immediate type. While unconjugated bilirubin in the serum does not reacts with diazo reagent and will not give purple color. It requires addition of alcohol. Then only it gives the purple color. So it is called as indirect bilirubin or delayed type.

Specimen: Serum/Plasma

Reagents

• Sulfanilic acid (Solution A)
• Sodium nitrite -5%
• Sodium nitrite - 0.5%  (Solution B)
• Diazo reagent - Mix 0.3 ml 0f solution B with 10 ml of solution A. Make a fresh just prior to use.
• Dilute HCl (1.5 ml HCl by adding distilled water to a volume of 100 ml).
• Methanol
• Artificial standard (=10mg% Bilirubin)
• Control solution
• Diazo blank (60 ml of conc  HCl to a volume of 1000 ml of water)

Procedure

• Total bilirubin
  • Label two test tubes, one blank and one sample for each unknown, standard and control
  • Add 5 ml methyl alcohol and 1 ml diazo blank solution to blank tube.
  • Add 5 ml methyl alcohol and 1 ml diazo blank solution to sample tube.
  •  Dilute 1 ml of serum or plasma by addding 9 ml distilled water.
  • Add 4 ml diluted serum or plasma to each  tube.
  • Let stand for exatly 30 minutes
  • Read the OD of all the tubes at 540 nm against the blank
• Direct bilirubin
  • Label two test tubes, one blank and one sample with standard and control, one blank and one sample
  • Add 5 ml distilled water and 1 ml diazo blank solution to the test tube.
  • Add 5 ml distilled water and 1 ml diazo reagent to sample tube.
  • Add 4 ml diluted serum or plasma to each  tube.
  • Shake content well and let stand for exatly 5 minutes
  • Read the OD of all the tubes at 540 nm against the blank

Calculation