PRINCIPLE OF THE METHOD
Bilirubin (both conjugated and unconjugated) couples with the diazo reagent
in the presence of a surfactant 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 total
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 alteration, neonatal anemia,
erythropoiesis alterations and presence of drugs.
Direct Bilirubin: cholestasis liver, liver abnormalities and genetic.
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
Total bilirubin 0,2-1,2 mg/dL (3,4 – 20,5 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 a. ascorbic were evaluated for
this total bilirubin method on a Spintech 240 analyzer. Two concentrations
of total bilirubin were evaluated. No interferences were observed for lipemia
(Intralipid) up to 1800 mg/dL, hemoglobin up to 2000 mg/dL and ascorbic
acid up to 40 mg/L.
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.
BIBLIOGRAPHY
1. David G Levitt and Michael D Levitt. Quantitative assessment of the
multiple processes responsible for bilirubin homeostasis in health and
disease. Clin Exp Gastroenterol. 2014; 7: 307–328.
2. Malloy H T. et al. The determination of bilirubin with the photoelectric
colorimeter. J. Biol Chem 1937; 112, 2; 481-491.
3. Martinek R. Improved micro-method for determination of serum bilirubin.
Clin Chim 1966: Acta 13: 61-170.
4. Young DS. Effects of drugs on Clinical Lab. Tests, 4th ed AACC Press,
1995.
5. Young DS. Effects of disease on Clinical Lab. Tests, 4th ed AACC 2001.
6. Burtis A et al. Tietz Textbook of Clinical Chemistry, 3rd ed AACC 1999.
7. Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed AACC 1995.
