You are on page 1of 9

BILIRUBIN

CEFFEINE METHOD
Clinical Significance:
Determination for differential diagnostic of
Jaundice;
3 types of Jaundice according to Bilirubin
concentration:
1. Hemolytic unconjugated Bilirubin,
2. Obstructive conjugated Bilirubin,
3. Hepatic combine of Hemolytic & Obstructive;
Other test done along with Bilirubin are: SGPT,
ALP etc.
Principle:
Bilirubin reacts with di-azotized sulphanilic
acid forms a colored azo compound,
measured at 546 nm (530 560 nm) after
5 minutes reaction & is directly
proportional to the concentration of
Bilirubin.
For total Bilirubin the reaction is
accelerated by Caffeine reagent.
Reagent presentation
Preparation for Working Solution:
WS should be prepared fresh as per need.
i. WS 1 Equal volume of Reagent 1&2 i.e
(T1:T2/D1:D2),
ii. WS 2 - 1 volume of saline with 2 volume of
Reagent 2 i.e 1ml saline + 2 ml T2/D2
Reagent 1 : Total (T1) Reagent 1 : Direct (D1)
Reagent 2 : Total (T2) Reagent 2 : Direct (D2)
Reagent 3 : Total (T3)
Procedure 2 methods
Monochromatic & Bichromatic
Monochromatic Parameters:
Reaction type End Point
Reaction slope Increasing
Wavelength 546 nm
Flow cell temperature 30 C
Incubation 5 min at Room Temp.
Sample Volume 50 ml
Reagent Volume 1.1 ml
Factor 26.312 (for both Total &
Direct)
Zero setting Distilled Water
Sample Blank - Yes
Bichromatic Parameter:
Reaction type End Point
Reaction slope Increasing
Wavelength 546 nm & 630 nm
Flow cell temperature 30 C
Incubation 5 min at Room Temp.
Sample Volume 50 ml
Reagent Volume 1.1 ml
Factor For Total - 23 & for
Direct - 17
Zero setting Distilled Water
Sample Blank - No

Procedure for Monochromatic method
Incubation 5 min at room temperature.
Mix & read after 5 min.
Calculation:
Total Bilirubin in mg% = ( Test Blank )*26.312
Direct Bilirubin in mg% = ( Test Blank )*26.312
For Total & Direct Bilirubin
Reagents Test Blank
WS 1 Total/Direct 100 ml -
WS 2 Total/Direct - 100 ml
Sample 50 ml 50 ml
R3 -T3 for Total/Saline for Direct 1 ml 1 ml
Procedure for Bichromatic method
Incubation 5 min at room temperature.
Mix & read after 5 min.
Calculation:
Total Bilirubin in mg% = ( Test Blank )*23
Direct Bilirubin in mg% = ( Test Blank )*17
Expected values :
Total & Direct Bilirubin Upto 1.0 & 0.3 mg% respectively
For Total & Direct Bilirubin
Reagents Test
WS 1 Total/Direct 100 ml
Sample 50 ml
R3 -T3 for Total/Saline for Direct 1 ml
The molar absorptivity of unconjugated bilirubin in
caffeine reagent is independent of the protein matrix.
Together with the simplicity of a dilution step for direct
spectrophotometry, will improve the calibration methods
of bilirubin and make more nearly accurate.
Encouraging for the development of a new method for
bilirubin determination in neonates;
The caffeine reagent has a clearing influence on the
turbidity of human sera.
These findings should also be important for
standardization,
Therefore, the introduction of one reliable, inexpensive,
"universal" standard of bilirubin in bovine serum albumin
will be of importance for both methods.
Comparison chart
Company
name
Method Reagent Parameter Pack Size
Accurex

Caffeine T1,T2,T3;
D1,D2
End Point; Increasing;
Temp 30 C; Incubation
5 Min
T & D
2*60ml
Bayer J & G
method
R1,R2,R3 End Point; Increasing;
Temp 30 C; Incubation
5 Min
T & D
2*30ml
6*30ml
Merck J & G
method
R1,R2,R3 End Point; Increasing;
Temp 30 C; Incubation
5 Min
T & D
2*125ml
Transasia

Diazo
Pearlma
n &Lee
R1,R2,R3

End Point; Increasing;
Temp 30 C; Incubation
5 Min
T & D
2*50m
Crest

J & G
method

L1,L2 End Point; Increasing;
Temp 30 C; Incubation
5 Min
T & D
L1 75/150ml
L2 4 ml