bilirubin practicals for first year mbbs

bilirubin practicals for first year mbbs

1. Dr. Dinesh kumar Ravi M.B.B.S., MD., Fellowship in Diab., PGDEM CONSULTANT EMERGENCY PHYSICIAN DIABETOLOGIST TUTOR, BIOCHEMISTRY Exp No: Date: ESTIMATION OF BILIRUBIN   Competency BI 11.12 Demonstrate the estimation of serum bilirubin

2. AIM & METHOD AIM: To determine the amount of total, direct and indirect bilirubin present in the given serum/ plasma sample. METHOD: Modified DMSO/Diazo method

3. principle Sulfanilic acid reacts with sodium nitrite to form diazotized sulfanilic acid. Bilirubin reacts with diazotized sulfanilic acid in the presence of DMSO to form reddish purple Azobilirubin (Van Den Berg Reaction). Water soluble, Conjugated bilirubin react very fast with Diazo reagent. Whereas unconjugated bilirubin present in serum is complexed with albumin and reacts very slowly with Diazo reagent and it requires an activator, methanol (alcohol).

4. Total bilirubin: Activator - Methanol Serum + diazotized sulfanilic acid Azobilirubin

5. Direct bilirubin: Serum + diazotized sulfanilic acid Azobilirubin

6. PROCEDURE

7. Total Bilirubin For Total Bilirubin: Take 2 test tubes and label them as Test (T1) and Blank (T2), add the following reagents. S No REAGENT Total Bilirubin     T1 (Test) T2 (blank) 1 Diazo reagent ( Sulfalinilic acid + HCl+ Na 2 S) 0.5 ml --- 2 Diazo blank (Conc. HCl) --- 0.5 ml 3 Methanol ( Activator) 2.5ml 2.5ml 4 Distilled water 1.8ml 1.8ml 5 Serum 0.2ml 0.2 ml   Mix well and incubate for exactly 5 minutes. Measure the absorbance of test against respective sample blank at 532 nm .

8. Direct Bilirubin For Direct Bilirubin: Take 2 test tubes and label them as Test (D1) and Blank (D2), add the following reagents. S No Reagent Direct Bilirubin     D1 (Test) D2 (blank) 1 Diazo reagent ( Sulfanilic acid + HCl+ Na 2 S) 0.5 ml --- 2 Diazo blank (Conc. HCl) --- 0.5 ml 3 Methanol ( Activator) --- - 4 Distilled water 4.3ml 4.3ml 5 Serum 0.2 ml 0.2 ml   Mix well and incubate for exactly 5 minutes. Measure the absorbance of test against respective sample blank at 532 nm .

9. CALCULATIONS: Total Bilirubin = OD of test - OD of sample blank X Factor Direct Bilirubin = OD of test - OD of sample blank X Factor ( Note: factor will be provided by the kit manufacturer) Indirect bilirubin = Total bilirubin - Direct bilirubin

10. RESULT: The amount of total bilirubin in the given serum sample is __________mg/dl The amount of direct bilirubin is _________mg/dl The amount of indirect bilirubin is __________mg/dl.

11. NORMAL VALUES: Total bilirubin: Adults : up to 1.2 mg/dl Infants : 0.2- 8 mg/dl Direct bilirubin : 0 to 0.2 mg/dl. Indirect bilirubin : 0.2 to 0.8 mg/dl

12. CLINICAL SIGNIFICANCE: Causes of Unconjugated Hyperbilirubinemia: Increased production of unconjugated bilirubin from heme Hemolysis Ineffective erythropoiesis Rapid turnover of increased RBC mass 2. Decreased delivery of unconjugated bilirubin to hepatocyte Right sided congenital heart failure Post caval shunt

13. Causes of Unconjugated Hyperbilirubinemia: 3. Reduced uptake of conjugated bilirubin across hepatocyte membrane Gilbert syndrome Sepsis 4 . Reduced storage in cytosol 5. Decreased biotransformation Neonatal jaundice Crigler Najjar syndrome Hepato cellular dysfunction

14. Causes of Conjugated Hyperbilirubinemia: Decreased secretion of conjugated bilirubin in to canalicular tract Hepatocellular jaundice Dubin Johnson and Rotor syndrome Drug induced reaction 2 . Decreased drainage Extrahepatic obstructive stones, carcinoma, stenosis Sclerosis cholangitis Intrahepatic obstruction – granuloma, drugs, primary biliary cirrhosis, tumor.

15. Thank you