BILIRUBIN (TOTAL AND DIRECT)

BILIRUBIN (TOTAL AND DIRECT)

PRINCIPLE

TOTAL and DIRECT
Colorimetric method
END POINT

Bilirubin is converted to coloured azobilirubin by diazotized
sulfanilic acid and is measured photometrically. Of the two bilirubin
fractions in serum –bilirubin-glucuronide and free bilirubin which is
bound to albumin– only the former reacts directly, while free
albumin reacts after being displaced from protein by an accelerator.
The difference of two measurements total bilirubin (with
accelerator) and direct bilirubin (without accelerator) enables the
calculation of indirect bilirubin. The terms «direct» and «indirect»
bilirubin refers exclusively to the reaction characteristics in the
presence or absence of an accelerator or solubilizer and are only
approximate equivalents of the two bilirubin fractions.

REAGENT COMPOSITION

2 x 100 ml

RT Sulfanilic acid 29 mmol/L, hydrochloric acid 0.24 mol/L,
Duposol® 3% (w/v). 2 x 80 ml.
RD Sulfanilic acid 29 mmol/L, hydrochloric acid 0.24 mol/L.
RN Sodium nitrite 11.6 mmol/L. 1 x 20 ml.
Ancillary kit:
CAL Bilirubin Calibrator. REF. 1912005
Bilirubin freeze-dried into a protein matrix. Concentration value
is traceable to Standard Reference Material 916a. The
concentration of total T and direct D Bilirubin is stated on the
label and is lot-specific. The target values are derived using
LINEAR reagents on the Cobas Mira.

STORAGE AND STABILITY
Store at 2-30ºC.

REAGENT PREPARATION
Working reagents. Mix 1 mL RN + 4 mL RT (Total) or 1 mL RN
+ 4 mL RD (Direct). Stable for 8 days at 2-8ºC.
Calibrator. Reconstitute the vial by adding exactly 1.0 mL of
distilled water. Mix carefully and let stand for 5-10 minutes before
use. The stability of bilirubin in the dark is: 8 hours 16-25ºC, 2 days
2-8ºC and 28 days –20ºC frozen once.
SAMPLES
Fresh hemolysis-free serum. Store in the dark until use.
Samples can be frozen at –15ºC or below in which case bilirubin is
stable for 2 months.

MATERIALS REQUIRED

• Photometer or colorimeter capable of measuring absorbance
  at 540 ± 20 nm.
• Constant temperature incubator set at 37ºC. Use the same
  temperature for assay of calibrator, controls and samples.
• Pipettes to measure reagent and samples.

BILIRUBIN (TOTAL AND DIRECT) Reagents per box