LIQUIZYME

BILIRUBIN (TOTAL AND DIRECT)

SUMMARY
Bilirubin is mainly fomed from the heme portion of aged or
Total Blliubin
Addition sequence (B) (T)
damaged RBC'S. It then combines with albumin to form a
complex, which is not water-soluble. This is referred to as indirect Total Bilirubin Reagent 1.0 ml 1.0 ml
or unconjugated Bilirubin. In the liver this Bilirubin complex is
combined with glucuronic acid into a water-soluble conjugate. Total Bilirubin Activator 20 I
This is refered to as conjugated or direct Bilirubin. Elevated 50 50
Serum/ Plasma
levels of bilirubin ane found in liver diseases (Hepatitis, cirthosis)
excessive haemolysis / destruction of RBC (hemolytic jaundice)
obstruction of the biliary tract (obstuctive jaundice) and in drug Direct Blirubin
induced reactions. The differentiation between the direct bilirubin (B) (T)
hyperbilirubinemia. |Addition sequence
Direct Bilinubin Reagent 1.0 ml 1.0 ml
PRINCIPLE:
Bilirubin reacts with diazotized sulphanilic acid to produce Direct Bilirubin Activator 20
azobilirubin (violet colour). DMSO catalyzes the formation of Serum/ Plasma 50 50
azobilirubin from free bilinubin. The violet color is proportional to
blirubin concentration measured at 546 nm (530-550nm).
Mix well and incubate at 37°C for exactly 5 minutes.
Measure the absorbance of the Test Samples
REACTION:
Total Bilirubin
(Abs. T) immediately against their respective Blanks.
DMSO
CALCULATION
Bilirubin+Sulphanilic acid+Sodium Nitrite Azobilirubin Bilirubin mg/d! (Total or Direct)=Abs. of Test-Abs. Blank x Factor
Direct Bilirubin Factor = 20
LINEARITY:
Bilinubin Suiphanilic acid+ Sodium Nitrite obilirubin
OOD This procedure is linear upto 20 mg/dl. If the values exceed this
limit, dilute the sample with normal saline (NaCI 0.9%]and
CONTENTS: repeat the assay.Multiply result by dilution factor.
Reagent 1 Total Bilinubin Reagent
Reagent 2 : Direct Bilinubin Reagent
Reagent 3: Total Bilirubin Activator NORMAL VALUE
Serum
Reagent 4:Direct Bilirubin Activator
Reagent 5: Blirubin Arificial Standard (10.0 mg/dl) Total Bilirubin: upto 1.0 mg/dl
Direct Bilirubin: upto 0.3 mg/dl
Each Laboratory should establish it's own nomal range
MATERIALSs REQUIRED BUT NOT PROVIDED:
representing its patient populatlon.
Clean & Dry Glassware.
Laboratory Glass Pipettes or Micropipettes& Tips.
Bio-Chemistry Analyzer. QUALITY CONTROL
For accuracy it is necessary to run known controls with every
SAMPLES: assay.
Serum. Bilirubin is reported to be stable in the sample for 4 days
at2-8°C protected from light, as it is photosensitive. LIMITATION & PRECAUTIONS
1. It is important to ensure through mixing of Bilinubin reagent
and nitrite reagent before assay.
PREPARATiON OF REAGENT&
STABILITY:
All reagents are stable at room temperature tll expiry date
2. Blirubin lovols may bo roduced if tho sample ard exposed to

llght.
mentioned on the label.
BIBLIOGRAPHY:
1. H. varley, A. H. Gowen lock, Maurice Bell et. al. Practical
GENERAL SYSTEM PARAMETERS: clinical Biochemistry. 5th adi. P 1022 to 1024
Reacton type End polnt 2. Jendrasalk L. & Grof P. Blochem 2.297, 81 (1938)
Wave length 546nm(530 550nm) GODE NO. PACK aZE Neagent1 Reagent 2 Roagent 3 Reagent4 Reagent 5
Temperature 37°C
2 100 mt 100 m s h0 m 100 mi 150m 1x5.0 m
Incubation :6 minutes
Reagent volume :1.02ml
Sample volume 50
Standard 10.0 mg/dl
: Deionised water
IVD O 9O01 2016J RSCIG
Zero setting
Light path : 1.0 cm.