PRINCIPLE OF THE METHOD
Bilirubin is converted to colored azobilirubin by diazotized sulfanilic acid
and measured photometrically. Of the two fractions presents in serum,
bilirubin-glucuromide and free bilirubin loosely bound to albumin, only the
former reacts directly in aqueous solution (bilirubin direct), while free
bilirubin requires solubilization with dimethylsulphoxide (DMSO) to react
(bilirubin indirect). In the determination of indirect bilirubin the direct is also
determined, the results correspond to total bilirubin.
The intensity of the color formed is proportional to the bilirrubin
concentration in the sample1,2,3.
CLINICAL SIGNIFICANCE
Bilirubin is a breakdown product of hemoglobin.
It is transported from the spleen to the liver and excreted into bile.
Hyperbilirubinemia results from the increase of bilirubin concentrations in
plasma.
Causes of hyperbilirubinemia:
Total bilirubin: Increase hemolysis, genetic errors, neonatal jaundice,
ineffective erythrpoiesis, and drugs.
Direct bilirubin: Hepatic cholestasis, genetic errors, hepatocellular
damage1,6,7.
Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.
STORAGE AND STABILITY
All the components of the kit are stable until the expiration date on the
label when stored tightly closed at 2-8ºC, protected from light and
contaminations prevented during their use. Do not use reagents over the
expiration date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
- Color development in R 2.
ADDITIONAL EQUIPMENT
- Spectrophotometer or colorimeter measuring at 555 nm.
- Matched cuvettes 1.0 cm light path.
- General laboratory equipment.
SAMPLES
Serum or plasma, free of hemolysis1
. Protect samples from direct light.
Stability: Bilirubin is stable at 2-8ºC for 4 days and 2 months at –20ºC.
