Bilirubin estimation .ppt

Bilirubin estimation
Different methods used to estimate bilirubin

1. Clinical chemistery2
nd
class
Practical NO (2)
Estimation of total and direct
bilirubin-1

2. Serum Bilirubin
•A break down product of porphyrinring 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 bilirubinis formed per day

3. •Plasma bilirubinexceeds 1mg/dl –
hyperbilirubinemia
•B/w 1-2 mg/dl –latent jaundice
•>2 mg/dl –yellowish discolourationof sclera,
conjunctiva, skin , mucous memresulting in
jaundice

4. Aim :
Determination of serum bilirubin.
Method :
Malloy and evelyn:
Principle: the method is based on van den bergh
reaction . When bilirubinreacts with diazo
reagent purple coluredazobilirubinis formed .
Methanolis 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
bilirubinand direct bilirubingives mesureof
indirect bilirubin.

5. Reagent component :
1.diazoA:(sulphanalicacid /hydro chloricacid )
2.diazoB: sodimenitrite
3.Diazoblank reagent: 1.5% hydro chloricacid
4.methanol

6. Jendrassicand groft:
Aim : detrminationof serum bilirubinon auto
analysers.
PRINSIPLE : in determination of total bilirubinin
presence of caffiene, bilirubinis coupled with
diazotisedsulphanalicacid form an azodye .
NO caffieneis added when direct bilirubinis
determined .

7. •Reagents:
•Suphanalicacid / hydrochloric acid
•Sodium nitrite
•Caffeine reagent / sodium benzoat
•Tartaratereagent (sodium-potassium tartarate
and sodium hydroxide )

8. DMSO
Dimethylsulfoxide:
•Principle:
the bilirubinpresent in sample react with
diazotized sulphanilicacid to form azobilirubin
which has pink to purple color measured by
colorimeter at 540nm.

9. •The conjugated bilirubinpresent in sample
react directly with diazotized sulphanilicacid
to form azobilirubin, while unconjugated
bilirubinrequires solubilizationwith
dimethylsulfoxide(DMSO) to react with
diazotized sulphanilicacid to form azobilirubin

10. •Reagent:
1.R1: Sulfanilicacid /Hydrochloric acid (HCl)
2.R2: Sulfanilicacid / 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
bilirubinconcmg/dl
•For D.bilirubin:
Sample –(A) sample blank X factor(14) = direct
bilirubinconcmg/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
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