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bilirubin practicals for first year mbbs

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dinesh kumar

bilirubin practicals for first year mbbs

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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
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
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).
Indirect bilirubin is calculated as follows Concentration of Indirect bilirubin = Total bilirubin - Direct bilirubin For the estimation of total bilirubin serum is treated with diazo reagent and methanol. For the estimation of direct bilirubin serum is treated with only reagent.
Total bilirubin: Activator - Methanol Serum + diazotized sulfanilic acid Azobilirubin
Direct bilirubin: Serum + diazotized sulfanilic acid Azobilirubin
PROCEDURE
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+ Na2S) 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 .
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+ Na2S) 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 .
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
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.
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
• CLINICAL SIGNIFICANCE: • Causes of Unconjugated Hyperbilirubinemia: 1. 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
Causes of Unconjugated Hyperbilirubinemia: 3. Reduced uptake of conjugated bilirubin across hepatocyte membrane ï‚· Gilbert syndrome ï‚· Sepsis 4. Reduced storage in cytosol 5. Decreased biotransformation ï‚· Neonatal jaundice ï‚· Crigler Najjar syndrome ï‚· Hepato cellular dysfunction
• Causes of Conjugated Hyperbilirubinemia: 1. 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.
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bilirubin practicals for first year mbbs

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bilirubin practicals for first year mbbs

  • 1. 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. Indirect bilirubin is calculated as follows Concentration of Indirect bilirubin = Total bilirubin - Direct bilirubin For the estimation of total bilirubin serum is treated with diazo reagent and methanol. For the estimation of direct bilirubin serum is treated with only reagent.
  • 5. Total bilirubin: Activator - Methanol Serum + diazotized sulfanilic acid Azobilirubin
  • 6. Direct bilirubin: Serum + diazotized sulfanilic acid Azobilirubin
  • 8. 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+ Na2S) 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 .
  • 9. 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+ Na2S) 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 .
  • 10. 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
  • 11. 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.
  • 12. 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
  • 13. • CLINICAL SIGNIFICANCE: • Causes of Unconjugated Hyperbilirubinemia: 1. Increased production of unconjugated bilirubin from heme ï‚· Hemolysis ï‚· Ineffective erythropoiesis ï‚· Rapid turnover of increased RBC mass 2. Decreased delivery of unconjugated bilirubin to hepatocyte ï‚· Right sided congenital heart failure ï‚· Post caval shunt
  • 14. Causes of Unconjugated Hyperbilirubinemia: 3. Reduced uptake of conjugated bilirubin across hepatocyte membrane ï‚· Gilbert syndrome ï‚· Sepsis 4. Reduced storage in cytosol 5. Decreased biotransformation ï‚· Neonatal jaundice ï‚· Crigler Najjar syndrome ï‚· Hepato cellular dysfunction
  • 15. • Causes of Conjugated Hyperbilirubinemia: 1. Decreased secretion of conjugated bilirubin in to canalicular tract ï‚· Hepatocellular jaundice ï‚· Dubin Johnson and Rotor syndrome ï‚· Drug induced reaction 2. Decreased drainage ï‚· Extrahepatic obstructive stones, carcinoma, stenosis ï‚· Sclerosis cholangitis ï‚· Intrahepatic obstruction – granuloma, drugs, primary biliary cirrhosis, tumor.