Bilirubin estimation Different methods used to estimate bilirubin

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
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