Estimation Of Bilirubin

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-

• Infants-

Total: 0.3-1.3 mg/dl. Direct: Adults 0.1-0.4 mg/dl. Indirect: 0.2 – 0.9mg/dl. 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)