CHEMISTRY REAGENT KITS
                                              ------->BILIRUBIN T & D
                                                                            (DMSO)End-Point
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CLINICAL SIGNIFICANCE
Increased levels are associated with various liver diseases, as well as haemolytic disorders in adults and new borns.

PRINCIPLE
The azobilirubin produced by the reaction between bilirubin and the diazonium salt of sulphanilic acid shows a maximum absorbance at 555 nM in an acid medium. The intensity of the colour produced is proportional to the quantity of bilirubin which has reacted. In the absence of an accelerator, only conjugated bilirubin reacts. In the presence of an accelerator, dimethylsulphoxide (DMSO) non-conjugated bilirubin also participates in the reaction.

Composition
1: BILIRUBIN DIRECT 100 mL
    Sulphanilic acid 29mMol/L
    Hydrochloric acid 145 mMol/L
2: BILIRUBIN TOTAL DMSO 100 mL
    Sulphanilic acid 29mMol/L
    Hydrochloric acid 145 mMol/L
Dimethylsulphoxide (DMSO) 7 Mol/L
3: SODIUM NITRITE 15 mL
Sodium nitrite 0.07 Mol/L
4: STANDARD (Artificial 0.4 mGs/dL) 50 mL
Concentration in assay  
Condition (1:22 dilution) 9.2 mGs/dL

"REAGENTS SUPPLIED   Cat. No. LS 315N

1.Bilirubin Direct 100 mL
2.Bilirubin Total 100 mL
3.Sodium Nitrite 15 mL
4.Standard (Artificial 0.4 mGs/dL)  50 mL
Concentration in assay
Condition (1:22 dilution) 9.2 mGs/dLy

STABILITY
All reagents are stable at 2-8°C until expiration date marked on the labels. The reactive mixtures are stable at 2-8°C for 3 days. Discard the reagent that > 0.1 A at 555. PROTECT ALL REAGENTS AND REACTIVE MIXTURES FROM LIGHT.

SAMPLE
Serum or plasma which has no sign of haemolysis. Specimen should be stored in dark till the test is being performed. No prior patient preparation is needed. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials).

 

1)
    Pipette into 4 Test Tubes Blank
(ml)
Standard
(ml)
Test
(ml)
 
    Direct Reagent No.1 1.0 1.0 - -
    Total Reagent No.2 - - 1.0 1.0
    Distilled Wate 0.1 - 0.1 -
    Sodium Nitrite Reagent No.3 - 0,1 - 0.1
    Serum or Plasma 0.05 0.05 0.05 0.05

Mix carefully
'Pipette serum only after pipetting all reagents

2. READING :
Direct Bilirubin: Read immediately, No delay, after adding serum. Total Bilirubin : Read after 10 minutes at RT or 5 minutes at 37C incubation.

Read the OD against blank at 555 nM (530-580 nM) or GREEN filter


NOTE: Programme the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request.

SYSTEM PARAMETERS
Reaction =  End-point
Temperature =  25-30°C (RT)
Wavelength =  555 nM [530-580 nM)
Standard =  9.2 mGs/dL
Absorbance Range =  0-1 ° A1
Cuvette Path Length =  1 cM
Reagent Volume (Working Reagent) =  1.0 mL
Sample Volume

=  0.05 mL

Reaction Time: Total Bilirubin 10 minutes
  Direct. Bilirubin      Immediate
Linearity =  20 mGs/dL
Max. limit of Blank Reagent. =  0.1000
Final Colour Stability =  30 Mins.

EXPECTED VALUES
Adults & Children
Total bilirubin: upto 1.0 mGs/dL (17.1 mmol/L)
Conjugated bilirubin            upto 0.4 mG/dL
(6.84 nmol/L).


As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations.

LIMITATIONS
This assay is linear upto 20 mGs/dL of TOTAL BILIRUBIN. Avoid pipetting of reagent by mouth or contact with broken skin. No reaction will be seen if the sample is added prior to Reagent No. 3 in the test. Haemolysis inhibits diazo reaction. Exposer to light
decreases bilirubin in the sample.

QUALITY CONTROL
To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both instrument and reagent function together. Factors which might affect the performance of this test include proper instrument function, temperature control, cleanliness of glasswares and accuracy of pippetting.
It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly, laboratory findings should be established by clinical
manifestations.

WARNING
This reagent system is for in vitro Diagnostic use only. This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such
incidents wash off with plenty of water, or consult a physician.

BIBLIOGRAPHY

WINSTENS,CEHELYK  B.  -CLIN.CHIM. Acte 169, 25, 441-446.
WALTERS M.,GERARDEH.-mICROCHEM,J.,1970,15,
231-243.