REPORT : ESTIMATION OF TOTAL BILIRUBIN AND DIRECT BILIRUBIN.

NAME : KATHERSAN A/L SUBRAMANIAM. ID NO: DML/0430/11. TUTOR : PN.MELOR BINTI AHMAD.

Title : Estimation of Total Bilirubin and Direct Bilirubin. Aim : To determine the concentration of Total Bilirubin, Direct Bilirubin and Indirect Bilirubin in sample X. Principle : Procedure (A) : Estimation of Total Bilirubin. Total bilirubin, both conjugated and free, are measured by using a stabilized diazonium salt of 3, 5-dichloroaniline which reacts with bilirubin to form azobilirubin with maximum absorbance at 540 nm. Procedure (B) : Estimation of Direct Bilirubin. This methods are based on the reaction between direct (conjugated) bilirubin and diazotized sulphanilic acid solutions. Direct Bilirubin reagent utilizes an acid diazo method. Conjugated bilirubin will reacts with diazotized sulphanilic acid to produce an acid azobilirubin, the absorbance of which is proportional to the concentration of direct bilirubin in the sample and can be measured at 550 nm.

Materials : 1. 2. 3. 4. 5. Total bilirubin reagent. Direct bilirubin reagent. Calibrator. Sample X. Appropriate number of tube(s) for: a. Blank b. Calibrator c. Sample X 6. Cuvettes. 7. Spectrophotometer.

Methods : Procedure (A) : Estimation of Total bilirubin. 1. All the tubes was labeled accordingly to blank, calibrator and sample X. 2. 1500 L of Total bilirubin was pipettes into each tubes. 3. All tubes was incubated at 37oC for 5 minutes.

4. 75 L Calibrator and Sample X was pipettes into appropriate labeled tubes. The blank tube was leaved without add any sample. 5. All tubes was incubated at 37oC for 5 minutes. 6. The reading of Absorbance of blank tube in a spectrophotometer at 540 nm wavelength was recorded. This was the Blank Absorbance. 0 ABS/100%T was pressed to set blank. 7. The reading of Absorbance for Calibrator and Sample X in spectrophotometer at 540 nm wavelength was pointed. These absorbance are the Reaction Absorbance for each tube.

Procedure (A) : Estimation of Direct bilirubin. 1. 2. 3. 4. All the tubes was labeled accordingly to blank, calibrator and sample X. 1500 L of Direct bilirubin was pipettes into each tubes. All tubes was incubated at 37oC for 5 minutes. 75 L Calibrator and Sample X that used in procedure (A), was pipettes into appropriate labeled tubes. The blank tube was leaved without add any sample. 5. All tubes was incubated at 37oC for 5 minutes. 6. The reading of Absorbance of blank tube in a spectrophotometer at 550 nm wavelength was recorded. This was the Blank Absorbance. 0 ABS/100%T was pressed to set blank. 7. The reading of Absorbance for Calibrator and Sample X in spectrophotometer at 550 nm wavelength was pointed. These absorbance are the Reaction Absorbance for each tube.

Data Reduction : (A) Absorbance for Total bilirubin. Tubes Absorbance at 540 nm Blank Sample X Calibrator 0.000 0.127 0.146

(B) Absorbance for Direct bilirubin. Tubes Absorbance at 550 nm Blank Sample X Calibrator 0.000 0.070 0.129

Result : (a) Calibration Graph

(b)

Conclusion i. From the graph, the concentration of Total Bilirubin is 3.74 mg/dL and Direct Bilirubin is 0.88 mg/dL. ii. Conventional unit 3.75 mg/dL 0.88 mg/dL Conversion Factor 17.1 0.0585 Calculation 3.75 mg/dL 17.1 = 64.13 0.88 mg/dL 0.0585 = 0.0515 SI unit 64.13 umol/L 0.0515 umol/L

Analyse Total Bilirubin Direct Bilirubin

iii. [Total Bilirubin] = [Direct Bilirubin] + [Indirect Bilirubin] [Indirect Bilirubin] = [Total Bilirubin] [Direct Bilirubin] = [64.14 umol/L] [0.0515 umol/L] = [64.08 umol/L]