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www.biomaghreb.com
IVD
TOTAL BILIRUBIN
Reagent for the quantitative determination of
total bilirubin (accelerator: DMSO) in human plasma
FT En 19 Date of updating : 10/ 2020 Version B
IN VITRO DIAGNOSTIC USE
CLINICAL SIGNIFICANCE
Bilirubin is a product of the degradation of hemoglobin. It is delivered in the blood in its «free» form,
then transformed by the liver into «conjugated bilirubin» to be filtered by the kidney. The increase in
bilirubin levels (bilirubinemia) causes jaundice, commonly called «jaundice», which can be caused
by either unconjugated (free) bilirubin, conjugated bilirubin, or both fractions. Unconjugated bilirubin
icters are primarily associated with increased red blood cell destruction (hemolysis) or decreased bile
conjugation in the liver. Cholestasis, the reduction or cessation of bile secretion, is the most common
cause of conjugated bilirubin icters.
PRINCIPLE
Sulfanilic acid reacts with sodium nitrite to give diazotized sulfanilic acid. In the presence of dimethyl
sulfoxide total bilirubin couples with diazotized sulfanilic acid to give azobilirubin.
REAGENT COMPOSITION
Reagent 1
Sulfanilic Acid
Hydrochloric Acid
Dimethylsulfoxyd
30 mmol/l
150 mmol/l
7 mmol/l
Reagent 3 Sodium Nitrite 20 mmol/l
Reagent 4
Standard
See Operating Procedure
(preparation of standard R4)
SAFETY CAUTIONS
Biomaghreb reagents are intended for use by qualified personnel for in vitro use (do not pipette by
mouth).
- Refer to the current SDS available on request or at www.biomaghreb.com;
- Verify the integrity of the reagents before use; and
- Disposal of waste: comply with the legislation in force.
For safety reasons, treat any specimen or reagent of biological origin as potentially infectious. Res-
pect the legislation in force.
SAMPLE PREPARATION
Serum or plasma collected on EDTA heparin, citrate or fluoride and stored in a dark place. Embarras-
sing hemolysis for Bilirubin.
ADDITIONAL EQUIPMENT
• Basic equipment of the medical analysis laboratory ;
• Spectrophotometer or Clinical Biochemistry Analyzer.
QUALITY CONTROL
External quality control program.
It is recommended to check in the following cases:
• At least one serial check;
• Changing the reagent bottle;
• After maintenance operations on the analyzer.
When a control value is outside the confidence limits, repeat the operation using the same control.
Use normal and pathological control sera.
LINEARITY
The method is linear up to 10 mg/dl-100 mg/l (170 µmol/l).
PROCEDURE
• Preparation of standard (R4)
Reconstitute the lyophilisate R4 with exactly 3 ml of distilled water. Wait 15 minutes. Complete the
dissolution of the lyophilisate by successively turning the vial over. The exact concentrations are
indicated on each vial.
Stability in the dark after reconstitution is:
2 days at 20° - 25°C ;
4 days at 2 - 8°C ;
6 weeks at minus 20°C.
It is essential to establish a calibration factor under laboratory conditions as soon as the R4 is
reconstituted.
F =
(Conc.Total Bilirubin ) standard
Abs (Standard) - Abs (Standard BIank )
• Calibration
- Wavelength: 555 nm (546 Hg) ;
- Temperature: 37°C ;
- Tank : 1 cm thick ;
- Analyzer zero: Standard Blank or sample Blank.
• Working Solution (T.B)
Mix 20 volumes R1 with 1 volume R3.
• Stability in the dark
6 Hours at 20 -25°C ;
2 days at 2-8°C.
Standard Sample
Blank Dosage Blank Dosage
Standard R4
50 μl 50 μl - - - -
Sample
- - - - 50 μl 50 μl
Reagent R1
1 ml - - 1 ml - -
Working Solution (T.B)
- - 1 ml - - 1 ml
Mix and incubate exactly 5 minutes at 37°C.
Read the absorbance (A) of the standard and the samples against their blanks.
N.B : Dilute newborn or very icteric samples 1/5 in a 9g/l NaCl solution.
CALCULATION
[Total.Bil ] =
Abs (A) Sample
Abs (A) Standard
x [Conc. standard] [Total.Bil] = Abs (A) sample x F
• NOTE :
0.585
µmol/I mg/l
1.710
REFERENCE VALUES
Total Bilirubin
0.2 - 1.0 mg/dl
2 - 10 mg/l
3.4 - 17 µmol/l
REFERENCES
Hijmans Van den Bergh A.A., Muller P., Biochem, 77, 90 (1916) ;
Walters M.l., Gerarde R.W., Microchem 15, 231 (1970).
Manufacturer Use by In Vitro Diagnostic Temperature
Limitation
Catalogue number See insert Store away from light Sufcient
for < n > essais
Batch number
