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 colorimeter measuring at 546 nm.
- 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 months at –20ºC.
REFERENCE VALUES
Direct bilirubin 0 – 0,2 mg/dL (0 – 3,42 mol/L)
These values are for orientation purpose; each laboratory should establish
its own reference range.
QUALITY CONTROL
Control sera are recommended to monitor the performance of assay
procedures: SPINTROL H Normal and Pathologic (Ref. 1002120 and
1002210). If control values are found outside the defined range, check the
instrument, reagents and calibrator for problems.
Each laboratory should establish its own Quality Control scheme and
corrective actions if controls do not meet the acceptable tolerances.
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.
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.
