Biochemistry 2 : practical

Biochemistry test :bilirubin total

Biochemistry method test : colorimetric test

Principle
Bilirubin in the presence of sulphanilic acid diazonium salt forms a red coloured
azobilirubin in alkaline solutions.Of the two fractions present in serum, bilirubin-
glucuromide and free bilirubin loosely bound to albumin, only the former reacts
directly in aqueous solution (bilirubin direct), while free bilirubin requires
solubilization with dimethylsulphoxide (DMSO) to react (bilirubin indirect)The
intensity of the colour formed is proportional to the bilirubin concentration in the
sample.

Reagent composition

Reagent T Sulphanilic acid......................30 mmol/l

Reagent N Hydrochloric acid (HCl)...........50 mmol/l
Dimethyl sulfoxide (DMSO)........7 mol/l
Sodium Nitrite..........................29 mmol/l

Storage and stability

All the components of the kit are stable at 2-8°C up to the date of expiration as
specified, when stored tightly closed, protected from light and contaminations
prevented during their use. The reagent should be a clear solution. . If turbidity or
precipitation has occurred or if colour development has occurred in Reagent N, the
reagent should be discarded.

Materials
Cyanstart analyser, vortex, test tube, mircopipette,beaker,distilled water,water bath,
liquid reagent.

Reagent and preparation

Cod.HB0030.The reagent is ready for use.
All the components of the kit are stable at 2-8℃ up to the date of expiration at
specified, when stored tightly closed,protected fromlight and contaminations
prevented during their use.the reagent should be discarded.if turbidity or precipitation
has occured or if the blank absorbances is out of range.handle standard very carefully
to prevent contamination .

Calibration and quality control

Calibration by means of factor,only when quality controls are within the defined
ranges. Otherwise,use the human calibrator(HBC03)for calibration.use the value
indicated on the with sample blank. Control sera are recommened to monitor the
performances of essay procedures. Use normaland pathological human (HBC01),
(HBC02) sera. Prepare and measure these controls the same as samples. If the control
values are found outside the defined range ,mentioned on the insert of the control.
Check the instrument reagents and calibration for problems.

Sample and preparation

Make sure the reagents and samples are the room temperature ,then pipette into the
test tube.

Blank sample blank sample calibrator blank calibrator

Calibrator 100ul 100ul
Sample 100ul 100ul
Reaagent N 25ul 25ul
Reagent T 750ul 750ul 750ul 750ul 750ul

Conclusion

As this test is normal and reagent can be used.actually Bilirubin is a metabolite of the
heme portion of heme proteins, mainly haemoglobin. Normally it is excreted into the
intestine and bile from the liver.Elevation of total serum bilirubin may occur due to
excessive haemolysis or destruction of the red blood cells e.g. haemolytic disease of
the newborn, liver diseases e.g., hepatitis and cirrhosis obstruction of the biliary tract
e.g., gallstones.

References
1. cyanstart analyser insert or manual
2. Cyanpress reagent insect