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 

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