Bilirubin

Clinical Significance :-

                        Bilirubin is mainly formed from the heme portion of aged or damaged RBC's. It then combines with albumin to from a complex which is not water soluble . This is referred to as indirect or unconjugated Bilirubin . In the liver this Bilirubin complex is combined with glucuronic acid into a water soluble conjugate. This is referred to as conjugated or direct Bilirubin. Elevated levels of bilirubin are found in liver disease ( Hepatitis, cirrhosis) excessive hemolysis / destruction of RBC (hemolytic ( ) obstruction of biliary tract and in drug induced reactions. The differentiation between the direct and indirect bilirubin is important in diagnosis the cause of hyperbilirubinemia. 

Principle :-

            Bilirubin react with diazotised sulphanilic acid to form a coloured azobilirubin compound. The unconjugated bilirubin couples with sulphanilic acid in the presence of a caffein- benzoate accelerator . The intensity of the colour formed is directly proportional to the amount of bilirubin present in the sample.

 Material required :-

• Clean and dry glassware.                                     
• Laboratory glass Pipettes or  micro Pipettes and tips. 
• Colorimeter.

 Normal Value :- 

                             0.3  - 1.2 mg/dl   

Sample :-

           Serum

Procedure :-

Direct Bilirubin 

Pipette into clean dry test tubes labelled as Blank(B),and Test(T):

Addition Sequence	B	T
Direct  Bilirubin  Reagent	1.0 ml	1.0 ml
Direct  Nitrite Reagent	-	0.05 ml
Sample	0.1 ml	0.1 ml

 Mix well and incubate at R.T. for exactly 5 min. Measure the absorbance of the Test Sample (Abs.T)immediately against their respectively Blanks. 

Total  Bilirubin 

Pipette into clean dry test tubes labelled as Blank(B),and Test(T):

Addition Sequence	B	T
Direct  Bilirubin  Reagent	1.0 ml	1.0 ml
Direct  Nitrite Reagent	-	0.05 ml
Sample	0.1 ml	0.1 ml

 

Mix well and incubate at R.T. for exactly 10 min. Measure the absorbance of the Test Sample (Abs.T)immediately against their respectively Blanks. 

  Calculation  :-

                                              

                Total or Direct Bilirubin  ( mg/dl ) = Abs.T * 13

                                                

Preparation and reagent and stability :-

                                        All Reagent are ready to use and are stable till the expiry date mentioned on the label , when stored at 2' to 8' C.

Quality Control :-

                  For accuracy it is necessary to run known controls with every assay.

Limitation & Precautions :-

 

1. Storage conditions as mentioned on the kit to be adhered.
2. Do not freeze or expose the reagent to high temperature and protect from direct as it may affect the performance of the kit.
3. Use clean glassware free from dust .
4. Before the assay bring all reagents to room temperature.

 General System Parameters:-

          Reaction type           : End point 

          Wave length             : 546 nm 

          Temperature             : Room Temperature

          Incubation              : 05/10 minutes 

          Reagent Volume          : 1.0 ml

          Sample Volume           : 20 ul 

          Standard concentration  :  -

          Zero setting            : Reagent Blank

          Light Path              : 1.0 cm 

          Factor                  : 13