Bilirubin Total Biochemistry Reagent

PRINCIPLE

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 to calculate 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

RT: Sulfanilic acid 29 mmol/L, hydrochloric acid 0.24 mol/L, Duposol® 3% (w/v). Xi R:36/37/38 S:26.

 RN: Sodium nitrite 11.6 mmol/L.

Bilirubin Total Biochemistry Reagent- Labkit Calibrator.
Bilirubin freeze-dried into a protein matrix traceable to the
SRM 916a. The concentration of total T and direct D
Bilirubin is stated on the label is lot-specific. The target
values are derived using LINEAR reagents on the Cobas Mira

STORAGE AND STABILITY: Store at 2-8ºC.
The reagents are stable until the expiry date stated on the label.
Discard reagent RN if it develops a yellow coloration

REAGENT PREPARATION: Working reagents. Mix 1 mL RN + 4 mL RT (Total)  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, EDTA or heparinized plasma. Store in the dark until use.

Samples can be frozen at –15ºC or below in which case bilirubin is stable for 2 months.

INTERFERENCES:

Lipemic samples interfere with the assay. The interference can be corrected by preparing a sample blank before applying the general formula of

The effect of hemoglobin may be negligible up to a level of 1g/dL.

A number of drugs and substances are known to affect bilirubin ³

MATERIALS REQUIRED:

1.Photometer or colorimeter capable of measuring absorbance at 540 ± 20 nm.

2.Constant temperature incubator set at 37ºC. Use the same temperature for assay of calibrator, controls and

3.Pipettes to measure reagent and samples

PROCEDURE:

Bilirubin Total Biochemistry Reagent- Labkit:

1.pipette into labelled tubes:

1. Mix thoroughly and let the tubes stand for 2 minutes at room
2. Read the absorbance (A) of the sample blanks at 540 nm against distilled
3. Read the absorbance (A) of the samples at 540 nm against the reagent

The color is stable for at least 60 minutes at room temperature.

CALCULATIONS:

Samples with concentrations higher than 20 mg/dL should be diluted 1:2 with saline and assayed again. Multiply results by 2. If results are to be expressed as SI units apply: mg/dL x 17.1 = µmol/L

REFERENCE VALUES⁴

 ADULTS:

NEWBORNS (TOTAL BILIRUBIN):

QUALITY CONTROL:

 The use of a standard to calculate results allows to obtain an accuracy independent of the system or instrument used.

To ensure adequate quality control (QC) each run should include a set of controls (normal and abnormal) with assayed values handled as unknowns.

CLINICAL SIGNIFICANCE:

 Hyperbilirubinemia (an abnormal elevation of bilirubin, whether conjugated or unconjugated) in plasma is an indication of a disturbance in bilirubin metabolism. This condition is caused either by an overproduction of bilirubin or by an impairment in the metabolic pathway. The increase in bilirubin production is usually caused by a rapid destruction of erythrocytes, resulting from blood diseases such as haemolilytic anemia. In newborns the increase in bilirubin may be caused by Rh, ABO, or other blood group incompatibility, by sepsis, hepatic immaturity, or by a variety of hereditary defects in bilirubin conjugation.

An impairment in the bilirubin metabolism is caused either by an enzyme deficiency or by a physical obstruction in bilirubin flow such as biliary (bile duct) obstruction. The hyperbilirubinemia leads to kernicterus (deposition of unconjugated bilirubin in brain and nerve cells) or jaundice (discoloration of mucus membranes, sclera and skin caused by the deposition of bilirubin pigment).

NOTES:

For bilirubin determination in newborns pipette 50 mL of sample or standard, and follow the exposed

These reagents may be used in several automatic

Instructions for many of them are available on request

ANALYTICAL PERFORMANCE:

Linearity: Up to 20 mg/dl

Precision:

Replicates: 10 for each level.

Instrument: CECIL CE 2021

Replicates: 6 for each level for 8 days.

• Sensitivity. Using a 1:10 sample/reagent at 540 nm, 1 mg o bilirubin will produce a net absorbance of approximately 081.
• Correlation. This assay (y) was compared with a similar commercial method (x). The results were:

N = 40       r = 0.998      y = 0.993 + 0.004

REFERENCES:

1. Walters, I. and Gerarde, H.W. Microchemical Journal. 15, 231 (1970).
2. Pearlman, C. and Lee, R.T.Y. Clin. Chem. 20/4, 447 (1974).
3. Young S. Effects of drugs on clinical laboratory tests, 3rd ed. AACC Press (1997).
4. Tietz, W., Fundamentals of Clinical Chemistry,  p.940.

W.B. Saunders Co., Philadelphia , 1987.