BILIRUBIN: QUANTITAIVE COLORIMETRIC REAGENT STORAGE AND STABILITY:

DETERMINATIONNOF TOTAL AND DIRECT BILIRUBIN IN

- store at 15-30 C
SERUM OR PLASMA
- Direct bilirubin reagent (15-30 C)
SUMMARY AND PRINCIPLE: - Oxidant discard if a dark yellow
color develops
 Most routine clinical procedures for the
determination of serum bilirubin are based on SPECIMEN COLLECTION AND PREPARATION:
the classical Diazo reaction of Ehrlich
- Serum or plasma collected in the
- was first applied to the estimation of bile
fasting state
pigment in serum in 1913 by Van den Berg
- sample should be protected from
and Snapper
sunlight and white artificial light
 Winkelman and Co-workers – has an excellent
(50% of bilirubin can be lost to
review of many bilirubin methodologies
sunlight for 1 hour)
- avoid anticoagulants containing
1. Nitrous acid: formed when a solution of
ammonium or fluoride salts
sulfanilic acid in dilute hydrochloric acid is
combined with sodium nitrite SAMPLE STABILITY
- this unstable acid reacts to form diazotized
- bilirubin is stable in serum or
sulfanilic acid
plasma 4-7 days (2-8 C), 3 months (-
2. Diazotized sulfanilic acid ( p-benzenediazonium
20 C)
sulfonate) couples with bilirubin to produce:
azobilirubin INTERFERING SUBSTANCES: Gross hemolysis ( can
- color intensity is equal to bilirubin cause falsely low bilirubin values due to inhibition of
concentration diazo reaction by oxyhemoglobin) Turbid or lipemic
3. Calibrator – a aqueous solution of N-1- serum (may cause falsely elevated bilirubin values)
naphthylethylenediamine dihydrochloride
(Bilissis and Speer)
- this coupled with diazotized sulfanilic acid
to give an azo dye
- color equivalent at 540 nm

REAGENTS:

Total Bilirubin reagent

 Sulfanilic acid, 32 mmol/L in dilute hydrochloric

acid, Accelerator and stabilizer added

Direct Bilirubin Reagent

 Sulfanilic acid, 32 mmol/L in dilute hydrochloric

acid and stabilizer added

Bilirubin Oxidant

 Sodium nitrite, 20mmol/L, aqueous, stabilizer

added

Bilirubin Calibrator (10mmol/L)

 N-naphthylethylenediamine dihydrochloride,
0.346 mmol/L, stabilizer added. Equivalent to
10mg/dL Bilirubin in method described