Biosystems Bilirubin Total And Direct
Biosystems Bilirubin Total And Direct , each run should include assayed normal and abnormal controls. If commercial controls are not available it is recommended that known value samples be aliquoted.
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Biosystems Bilirubin Total And Direct
INTRODUCTION
Bilirubin is mainly formed from the heme portion of aged or damaged RBC’S. It then combines with albumin to form 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 diseases
(Hepatitis, cirrhosis), excessive hemolysis / destruction of RBC (hemolytic jaundice) obstruction of the biliary tract (obstructive jaundice) and in drug induced reactions. The differentiation between the direct and indirect bilirubin is important in diagnosing the cause of
hyperbilirubinemia.
METHOD PRINCIPLE
Bilirubin reacts with diazotized sulphanilic acid to form a coloured azobilirubin compound. The unconjugated bilirubin couples with the sulphanilic acid in the presence of caffein – benzoate accelerator. The intensity of the colour formed is directly proportional to the amount of bilirubin present in the sample.
Biosystems Bilirubin Total And Direct
DMSO
Bilirubin + Diazotized Sulphanilic acid + Sodium Nitrite ………>
Azobilirubin Compound
DIRECT BILIRUBIN
Bilirubin + Diazotized Sulphanilic acid + Sodium Nitrite ………>
Azobilirubin Compound
REAGENTS
Biosystems Bilirubin Total And Direct
BILIRUBIN | BILIRUBIN | |
R1-Direct Reagent |
1 x 50 ml |
1 x 100 ml |
R2-Direct Nitrite |
1 vial |
1 vial |
R3-Total Reagent |
1 x 50 ml |
1 x 100 ml |
R4-Total Nitrite |
1 vial |
1 vial |
Working reagent preparation and stability
The reagents are stable at at R.T till expiry date printed on the package.
Concentrations in the test
TOTAL BILIRUBIN | DIRECT BILIRUBIN | |
Sulphanilic acid – 32 mmol/l |
Sulphanilic acid |
– 32 mmol/l |
Conc. HCL – 165 mmol/L |
Conc. HCL |
– 165 mmol/L |
Sodium Nitrite – 290 mmol/L |
Sodium Nitrite |
– 290 mmol/L |
DMSO – 7 mmol/L |
DMSO |
– NIL |
Warnings and notes
Product for in vitro diagnostic use only.
Reagents are ready to use. Do not pipette with mouth
ADDITIONAL EQUIPMENT
-
• automatic analyzer or photometer able to read at 546 nm
-
• thermostat at 25°C or 37°C;
-
• general laboratory equipment;
-
SPECIMEN
Serum. Bilirubin is reported to be stable in the sample for 4 days at 2-8o C. protected from light as it is photosensitive.
PROCEDURE
These reagents may be used both for manual assay and in several automatic analysers.
Applications for them are available on request.
Manual procedure – Direct Bilirubin
Biosystems Bilirubin Total And Direct
wavelength temperature cuvette Pipette into the cuvette: |
546 nm 25°C/37°C 1 cm |
||
Addition Sequence |
Blank (B) |
Test (T) |
|
R1 Direct Reagent |
1000 pl |
1000 pl |
|
R2 Direct Nitrite |
– |
20 pl |
|
Sample |
50 pl |
50 pl |
Mix well and incubate at R.T. for exactly 5 min. Measure absorbance of the Test Samples (Abs.T) immediately against their respective Blanks within 8 mins.
Manual procedure – Total Bilirubin
wavelength |
546 nm |
temperature |
25°C/37°C |
cuvette |
1 cm |
Pipette into the cuvette:
Addition Sequence |
Blank (B) |
Test (T) |
R3 Total Reagent |
1000 pl |
1000 pl |
R4 Total Nitrite |
– |
20 pl |
Sample |
50 pl |
50 pl |
Mix well and incubate at R.T. for exactly 5 min. Measure absorbance of the Test Samples (Abs.T) immediately against their respective Blanks within 8 mins.
Calculation
Total or Direct Bilirubin in mg/dl = (Abs.T – Abs.B) X 20.20 (Factor)
Serum |
value |
Direct Bilirubin |
upto 0.3 mg/dl |
Total Bilirubin |
upto 1.0 mg/dl |
It is recommended for each laboratory to establish its own reference ranges for local population.
Biosystems Bilirubin Total And Direct
QUALITY CONTROL
To ensure adequate quality control, each run should include assayed normal and abnormal controls. If commercial controls are not available it is recommended that known value samples be aliquoted, frozen and used as controls.
Bilirubin T&D (EP) page 1
PERFORMANCE CHARACTERISTICS
Linearity: The procedure is linear up to 20 mg/dl. If values exceed this limit, dilute the sample with distilled water and repeat the assay. Calculate the value using the proper dilution factor.
WASTE MANAGEMENT
Please refer to local legal requirements.
Biosystems Bilirubin Total And Direct
NOTE
1. In case of Cuvette Volume is more than 1.0 ml requisite volume of reagents and sample can be multiplied keeping reagent to sample ratio same.
2. Sequence of reagent addition should be followed strictly as per the procedure.
SYSTEM PARAMETERS
LITERATURE
1. Jendrassik, L., Grof, P., (1938) Biochem. 2,297:81
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