Bilirubin estimation .ppt
- 1. Clinical chemistery 2nd class Practical NO (2) Estimation of total and direct bilirubin -1
- 2. Serum Bilirubin • A break down product of porphyrin ring of heme – containing proteins , found in blood in 2 fractions – conj/unconj • Conjugated : water soluble , so excreted by kidneys • Unconjugated : insoluble in water , bound to albumin in blood • About 300 mg of bilirubin is formed per day
- 3. • Plasma bilirubin exceeds 1mg/dl – hyperbilirubinemia • B/w 1-2 mg/dl – latent jaundice • >2 mg/dl – yellowish discolouration of sclera, conjunctiva, skin , mucous mem resulting in jaundice
- 4. Aim : Determination of serum bilirubin . Method : Malloy and evelyn : Principle : the method is based on van den bergh reaction . When bilirubin reacts with diazo reagent purple colured azobilirubin is formed . Methanol is used as reaction accelerator .since total bilirubin ( indirect and direct type )is soluble in it . By using only distilled water , direct bilirubin is determined . The difference between total bilirubin and direct bilirubin gives mesure of indirect bilirubin .
- 5. Reagent component : 1. diazoA :(sulphanalic acid /hydro chloric acid ) 2. diazoB : sodime nitrite 3. Diazo blank reagent: 1.5% hydro chloric acid 4. methanol
- 6. Jendrassic and groft : Aim : detrmination of serum bilirubin on auto analysers . PRINSIPLE : in determination of total bilirubin in presence of caffiene , bilirubin is coupled with diazotised sulphanalic acid form an azo dye . NO caffiene is added when direct bilirubin is determined .
- 7. • Reagents: • Suphanalic acid / hydrochloric acid • Sodium nitrite • Caffeine reagent / sodium benzoat • Tartarate reagent (sodium-potassium tartarate and sodium hydroxide )
- 8. DMSO Dimethylsulfoxide : • Principle: the bilirubin present in sample react with diazotized sulphanilic acid to form azobilirubin which has pink to purple color measured by colorimeter at 540nm.
- 9. • The conjugated bilirubin present in sample react directly with diazotized sulphanilic acid to form azobilirubin, while unconjugated bilirubin requires solubilization with dimethylsulfoxide(DMSO) to react with diazotized sulphanilic acid to form azobilirubin
- 10. • Reagent: 1. R1: Sulfanilic acid /Hydrochloric acid (HCl) 2. R2: Sulfanilic acid / Hydrochloric acid (HCl) /Dimethylsulphoxide (DMSO) 3. R 3: Sodium nitrite
- 11. • Sample: Serum or plasma, free of hemolysis ,Protect samples from direct light.
- 12. • Procedure: • Mix and incubate at R.T 5min,read the absorbance (A) at 540nm. Blank Total.B Blank Direct.B R1(D) ----- ----- 1.5ml 1.5ml R2(T) 1.5ml 1.5ml ----- ------ R3 ----- 50μl ----- 50μl Sample 100μl 100μl 100μl 100μl
- 13. • Calculation: • For total bilirubin: Sample – (A) sample blank X factor(19.1) = total bilirubin conc mg/dl • For D.bilirubin: Sample – (A) sample blank X factor(14) = direct bilirubin concmg/dl • Indirect bilirubin = Total bilirubin – direct bilirubin
- 14. • Units: Old unit: mg/dl SIU: μmol/L Reference value: Total bilirubin: Up to 1mg/dl Direct bilirubin: up to 0.25mg/dl Indirect bilirubin: Up to 0.75 mg/dl Comments :