Estimation of Bilirubin

Structure of Bilirubin
 Types of Bilirubin

• Conjugated
• Un-conjugated
• Total
Bilirubin Metabolism
Clinical Significance
Jaundice
Causes for Jaundice
 Estimation of Bilirubin

• By general colourimetric analysis

• By photometric analyzer
 Estimation of Bilirubin: History

• Biliverdin measurement
• Icteric index
• Van den Bergh and Muller method
• Jendrassik and Grof
• Diazyme assay
By Photometric Analyzer
Absorption of Bilirubin
Total and Direct Bilirubin Estimation
by
Quantitative, Colourimetric Method
 Principle
 Direct bilirubin (conjugated)
+
diazotized sulfanilic acid ---- > azobilirubin (blue at
alkaline pH)

 Total bilirubin (conjugated +unconjugated0

+
caffeine-benzoate-acetate mixture + diazotized
sulfanilic acid ------→ azobilirubin (blue at alkaline
pH)
 Requirements

• Apparatus
• Reagents
• Serum samples
Apparatus
Reagent: Bilirubin Estimation Kit
 Reagent Kit: Components

Caffeine Reagent
Alkaline Tartrate
HCl
Diazo Reagent
Cysteine Reagent
Standard Bilirubin
 Preparation of Kit Reagents

• Diazo reagent--13 mg. of sulfanilate and

0.457 mg. of sodium nitrite in 6ml HCl.
• Caffeine reagent--25 g./liter of caffeine and
38 g./liter of sodium benzoate in sodium
acetate solution.
• Cysteine reagent--Prepared by adding 10.5 ml
water
• Sodium potassium tartrate (350 g./liter) in
2.5 N sodium hydroxide
• HCl--200 ml. of 0.05 N
Preparation of Standard Bilirubin
Serum
 Method

• Preparation of reaction assay systems

• Incubation
• Measurement
• Preparation of calibration curve
Working Table
 Parameters

Wave length : 540-600nm

Optical pathway : 1 cm
Temperature : 37°C
Measurement : Against reagent blank
Method : End point analysis
 Result

• Prepared calibration curve is used to

determine the concentration of unknown
samples.
• Total and direct bilirubin levels are
determined from the curve.
• The indirect bilirubin is the difference
between the total and the direct.
• All results are tabulated in worksheet.
Calculation by Standard Formula
 Example

• Absorbance of Unknown = 0.052

• Absorbance of Calibrator = 0.180
• Calibrator Value = 5.2 mg/dL
0.052
• Total Bilirubin = ———— x 5.2 = 1.5 mg/dL
0.180
Calculation—Calibration Curve
 Analytical Reliabilities

• Precession
• Accuracy
 Precaution

• Blood should be drawn with aseptic precaution.

Pouring of blood from syringe to cuvette, niddle must
be avoided.
• Hazardous materials: Alkaline tartrate is strong base
so caution should be taken (for eyes and clothes),
This method uses sulphanilic acid and sodium
hydroxide. Avoid contact with eyes, skin and mucous
membranes.
• During incubation appropriate temperature and
time should be maintain.
• Appropriate filter should be used.
 Advantages
• Specific
• Sensitive
 Limitation
• Dilution
• Interference
Specific Performance Characteristics

• Reproducibility studies
• Recovery studies
• Numerous variations
 Standard Reference Range
Clinical Interpretation

• Adult- Total: 0.3-1.3 mg/dl.

Direct: Adults 0.1-0.4 mg/dl.
Indirect: 0.2 – 0.9mg/dl.
• Infants- Total: Infants 1.0-12.0 mg/dl.
 Clinical Symptoms

•Plasma bilirubin exceeds 1mg/dl – hyperbilirubinemia

•B/w 1-2 mg/dl – latent jaundice
•>2 mg/dl – yellowish discolouration of sclera, conjunctiva, skin , mucous
membrane resulting in jaundice.
Diagnosis and Therapy (Infants)
Diagnosis and Therapy (Adults)