ESTIMATION

OF SERUM
BILIRUBIN
LEVEL

PRACTICAL NO: 06
INTRODUCTION/CLINICAL SIGNIFICANCE
bilirubin, a brownish yellow pigment of bile, secreted by the liver in vertebrates, which gives to solid
waste products (feces) their characteristic colour. It is produced in bone marrow cells and in the liver as
the end product of red-blood-cell (hemoglobin) breakdown. The amount of bilirubin manufactured relates
directly to the quantity of blood cells destroyed. About 0.5 to 2 grams are produced daily. It has no known
function and can be toxic to the fetal brain. Bilirubin in the bloodstream is usually in a free, or
unconjugated, state; it is attached to albumin, a protein, as it is transported. Once in the liver it conjugates
with glucuronic acid made from the sugar glucose. It is then concentrated to about 1,000 times the
strength found in blood plasma. Much bilirubin leaves the liver and passes to the gallbladder, where it is
further concentrated and mixed with the other constituents of bile. Bile stones can originate from
bilirubin, and certain bacteria can infect the gallbladder and change the conjugated bilirubin back to free
bilirubin and acid. The calcium from the freed bilirubin can settle out as pigment stones, which may
eventually block the passageway (common bile duct) between the liver, gallbladder, and small intestine.
When blockage occurs, conjugated bilirubin is absorbed into the bloodstream, and the skin becomes
yellow in colour (see jaundice).
Normally, conjugated bilirubin passes from the gallbladder or liver into the intestine. There, it is reduced by
bacteria to mesobilirubinogen and urobilinogen. Some urobilinogen is reabsorbed back into the blood; the
rest goes back to the liver or is excreted from the body in urine and fecal matter. In humans, bilirubin is
believed to be unconjugated until it reaches the liver. In dogs, sheep, and rats, there is no bilirubin in the
blood, though it is present in the liver.
EQUIPMENT
 Required specimen: Serum
 Bireagent Method:
Reagent 1 (R1)
Reagent 2 (R2)
Distilled water

 Other equipments:
Centrifuge
Incubator
Micropipettes and micropipette tips
Test tubes
Cuvettes
UV Spectrophotometer
PRINCIPLE/METHOD

 Two types of bilirubin level tests can be done

 Total bilirubin
 Direct bilirubin

 In both tests, conjugated bilirubin in serum, is coupled/mixed with di-azotized “Sulphanilic acid”, to
form a red colored “Azobilirubin” compound.
Azo = Nitrogen (in French)

Sulphanilic Acid
The increase of absorbance in the bilirubin α Bilirubin concentration in the sample
sample, due to the formation of the azobilirubin
compound
The absorbance of the bilirubin sample can be measured, using the spectrophotometer, and multiplying the
absorbance by a constant called, “the conversion factor”, the bilirubin concentration can be taken.

Direct Bilirubin Test Total Bilirubin Test

R1 R2 R1 R2

Sulphanilic Acid NaNO2 / NaNO3 Sulphanilic Acid NaNO2 / NaNO3

HCl Citric Acid / HCl

Caffeine
 Bilirubin can react with diazotized sulphanilic acid, if the bilirubin is dissolved in water.
 Therefore, in direct bilirubin test, which measures only the conjugated bilirubin, the conjugated
bilirubin can directly react with the diazotized sulphanilic acid.
 But, in the total bilirubin test, which measures both the conjugated and unconjugated bilirubin,
the unconjugated bilirubin must be dissolved in water.

 In the total bilirubin test, caffeine (in R1 reagent), is added to separate the “Albumin protein complex”
(helps the unconjugated bilirubin to travel through blood, due to its water insolubility.) from the
unconjugated bilirubin, accelerating the reaction with diazotized sulphalinic acid.

 In order to create diazotized sulphanilic acid, the sulphanilic acid must react with HNO 2 or HNO3. But,
HNO2 or HNO3, are not available in nature, due to them being highly unstable.
 Therefore, HCl in R1 and NaNO2 / NaNO3 in R2, react with each other to form HNO2 or HNO3, on
the spot.
PROCEDURE
 The blood samples were collected into red-top/plain tubes in order to separate the serum.
 After allowing the blood to clot, the tube was centrifuged at 2500 rpm for 15 minutes.
 And then serum was collected into an Eppendorf tube using a pipette.
 Two test tubes were taken and labelled as sample(EC) and the blank(EBC).
 1200μl of R1 was added to both tubes and 100 μl of distilled water was added to the blank tube. Then
100 μl of serum was added to the sample tube using the micropipette.
 The solutions were mixed separately and incubated at 37 C for 5 minutes.
 After the incubation, R2 reagent was added to each tube and incubated again at 37 c for 5 minutes.
 Then the blank sample was added to the to the spectrophotometer and calibrated the
spectrophotometer.
 After that the samples were added to the spectrophotometer and recorded the absorbance of the
samples at 546nm wavelength.
 After the calculation of Total Bilirubin and Direct Bilirubin levels, they were compared with the
reference values
 100
100micr Distilled
ol water
serum

Sample Blank

Incubate at 37C for

Incubate at 37C for
5min
5min
Add 300microliter R2 Add 300microl
Reagent R2 Reagent
Incubate at 37C for Incubate at 37C for
5min 5min
Measure the Measure the
absorbance of absorbance of blank
sample at 546nm at 546nm
wavelength wavelength
OBSERVATION
CALCULATION

Total Bilirubin (mg/dL) = [Absorbance of the sample (EC)–

Absorbance of the Blank (EBC)] × Conversion Factor

 Absorbance of the blank is usually zero (0), due to ‘Blank’ being the calibrator.
 The conversion factor is provided on the assay kit, by the manufacturer.
 Reference values are also provided by the manufacturer.

 Conversion factor = 14.5

 Reference values
 Adults 0.3-1.2 mg/dL (5.1-17.1 µmol/L)
DISCUSSION
 Use clean glassware and pipette tips
 Do not freeze or expose the reagents to higher temperatures as it might affect their performance. Store
the reagents under conditions mentioned on the box.
 Do not keep the sample out in the light for too long as bilirubin is light sensitive and degrades when
exposed to the light.
 Sequence in which reagents are added should be maintained.
 The only acceptable anticoagulants are heparin and oxalate.
 Excessive hemolysis should be avoided because it may falsely lower the bilirubin value
 Lipemic specimens interfere with the test
 Total bilirubin and direct bilirubin reagents have similar procedures and only the reagents used in the two
tests are slightly different. To find the direct bilirubin, the direct bilirubin in the serum sample must react
with sulphanilic acid, indirect bilirubin is water insoluble and therefore cant react.
IMPORTANT POINTS
THANK YOU!