Estimation of Bilirubin in serum – liver function tests

Estimation of Bilirubin in serum

Estimation of Bilirubin in serum
Introduction

  • Bilirubin is an orange – yellow pigment , a waste product primarily produced by the normal breakdown of heme
  • This form of Bilirubin is called unconjugated (indirect) bilirubin ,not soluble form ,to convert it to soluble form and eliminate it ,The unconjugated bilirubin is carried by Albumin to the liver , where it converted to conjugated (direct) form , soluble , through UDP – glucuronyl transferase with glucuronic acid
  • Because the bilirubin is chemically different after it goes through the conjugation in the liver , lab tests can be differentiate between the unconjugated(indirect) or conjugated (direct) bilirubin
  • Conjugate bilirubin is water soluble form and reacts directly when dyes are added to the blood sample so , it called also direct bilirubin
  • Unconjugated bilirubin is non-water soluble , free form ,doesn’t react to the reagents until alcohol  is added to the solution so , it called indirect bilirubin
  • Total bilirubin measures both direct and indirect bilirubin


Principle

  • Diazotization is a principle of bilirubin test
  • Total bilirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an azodye
  • Normal saline is used to determination of direct bilirubin instead of caffeine
  • Sulfanilic acid + HNO2 (NaNO2 + dil Hcl) → diazotized – sulfanilic acid
  • Bilirubin + diazo reagent + (caffeine + benzoate) → Acid – Azo bilirubin ( pink color)
  • Acid – azo bilirubin + Alkaline tartarate soln → Alkaline – Azo bilirubin (blue green color) , read at 578 nm

Normal range

  • Total Bilirubin : up to 1 mg % ( 0.3 – 1 ) mg %
  • Direct Bilirubin : 0.1 – 0.4 mg %
  • Indirect Bilirubin : 0.2 – 0.7 mg %

Reagents

  • Sulfanilic acid
  • Sodium nitrite (NaNO2)
  • Caffeine and sodium benzoate ( act as a catalyst)
  • Alkaline tartarate
  • Saline solution (used in determination of direct bilirubin instead of caffeine)


Procedure with kits in lab

  • Depend on colorimetric technique 
Reagents Total bilirubin Direct bilirubin Reagents
Test Blank test Test Blank test
R1 100µ 100µ 100µ 100µ R1
R2 25µ 25µ R2
R3 500µ 500µ 1000µ 1000µ Saline
Serum 100µ 100µ 100µ 100µ Serum
Mix , wait for 10 min at RT then add R4 Mix , wait for 5 min at RT , read at 545 nm
R4 500µ 500µ
Mix , wait for 5 min at RT , read at 545 nm
  • R1 : Sulfanilic acid 
  • R2 : NaNO2
  • R3 : Caffeine
  • R4 : Tartarate solution


Calculations

  • Total bilirubin (mg %) =Asample X 10.8
  • Direct bilirubin (mg %) =Asample X 14.4
  • Indirect bilirubin (mg %) = Total – Direct

Direct bilirubin level increased in:

  • Hepatocellular disease as cirrhosis and hepatitis
  • Blockage of bile ducts due to gall stones or tumers
  • Drug reaction

Indirect bilirubin level increased in:

  • Hemolytic anemia
  • Transfusion reaction
  • Cirrhosis
  • Gilbert syndrome (low levels of glucoronyl transferase)


Q: Why make sample blank in bilirubin test?

  • To avoid color interference where serum is yellow color and alkaline azo-bilirubin color from yellow to blue green so , make sample blank by adding all contents except NaNO2

Q:The media must converted from acidic to basic = Role of alkaline tartarate solution?

  • Pink color low in sensitivity and can’t measure low concentration
  • Maybe due to presence of high turbidity , but blue green color have high sensitivity and measure low concentrations
  • Pink color low in sensitivity and can’t measure low concentration
  • Maybe due to presence of high turbidity , but blue green color have high sensitivity and measure low concentrations

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