PRINCIPLE OF THE METHOD
Direct bilirubin (conjugated) couples with the diazo reagent in the presence
of sulfamic acid to form azobilirubin. The intensity of color formed is
proportional to the bilirubin concentration in the sample tested. The
increase of absorbance at 546 nm is directly proportional to the direct
bilirubin concentration.
CLINICAL SIGNIFICANCE
Bilirubin is caused by the degradation of hemoglobin and exists in two
forms. Unconjugated bilirubin is transported to the liver bound by albumin
where it becomes conjugated (direct) with glucuronic acid and excreted.
Hyperbilirubinemia is the result of an increase of bilirubin in plasma.
Possible causes:
Total bilirubin: Increase hemolysis, genetic, neonatal jaundice, ineffective
erythropoiesis and presence of drugs.
Direct bilirubin: Hepatic cholestasis, genetic, hepatocellular damage.
Clinical diagnosis should not be made based on a single test result; it
should integrate clinical and other laboratory data.
STORAGE AND STABILITY
The reagents are stable until the expiry date stated on the label when
stored at
2-8ºC, protected from light and contaminations are prevented during their
use. Do not use reagents over the expiration date.
Signs of reagent deterioration:
- Presence of particles and turbidity.
ADDITIONAL EQUIPMENT
- Spectrophotometer or analyzer capable of measuring absorbance at 546nm.
- Cuvettes with 1,0 cm light path.
- General laboratory equipment.
SAMPLES
Serum or plasma, free of hemolysis. Protect samples from light.
Stability of the sample: 4 days at 2-8ºC or 2 month at –20ºC.
INTERFERENCES
Interferences from hemolysis, lipemia and ascorbic acid were evaluated for this
direct bilirubin method on a Spintech 240 analyzer. Two concentrations of direct
bilirubin were evaluated. No interferences were observed for lipemia (Intralipid)
up to 350 mg/dL and ascorbic acid up to 40 mg/L. Hemolysis causes decreased
direct bilirubin values, therefore hemolytic samples should be discarded.
A list of drugs and other interfering substances with bilirubin has been reported
by Young et. al 4,5.
NOTES
1. SPINREACT has instruction sheets for several automatic analyzers.
Instructions for many of them are available on request.
