HOW TO ESTIMATE LEVEL OF GLUCOSE IN BLOOD SERUM - Code

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Wednesday, March 28, 2012

HOW TO ESTIMATE LEVEL OF GLUCOSE IN BLOOD SERUM

































AIM:  To estimate level of glucose in Blood serum.

REAGENT USED: 
Sample:
Serum/Plasma (must be separated from the cells within 30 minutes. Excessive concentration of sodium
fluoride may affect result by inhibiting color development).
Reagents: 
Reagent 1: Glucose Reagent
Reagent 2: Glucose diluent.
Reagent 3: Glucose standard
Working reagent preparation: 
Add 50 ml of reagent 2 to reagent 1 vial and store at 2-8C in dark colour bottle because it is light sensitive. 

APPARATUS USED: 
Test tubes, Test tube stand, Micropipettes, Spectrophotometer

CLINICAL SIGNIFICANCE: 
Glucose estimation provides valuable information about the course, severity and therapeutic control of
Diabetes Mellitus. Fasting Glucose levels exceeding 120 mg/dL and 2 hours Post-Prandial Glucose levels
exceeding 140 mg/dL indicate a strong possibility of Diabetes Mellitus. If in an oral Glucose Tolerance
Test, the Plasma Glucose level of 2 hours Sample exceeds 200 mg/dL, the diagnosis of Diabetes Mellitus is
established. In impaired tolerance, the 2 hours Plasma Glucose lies between 140 mg/dL and 199 mg/dL. 
Increased concentration: Hyperglycemia may occur in Diabetes Mellitus, in patients receiving intravenous
fluids containing Glucose and during severe stress and Cerebrovascular accidents. 
Decreased concentration: Hypoglycemia may be the result of an Insulinoma, Insulin ad ministration,
inborn errors of Carbohydrate metabolism or fasting. 

ASSAY PRINCIPLE: 
Glucose Oxidase (GOD) oxidizes glucose to gluconic Acid and hydrogen peroxide. In presence of enzyme
Peroxidase, released hydrogen peroxide is coupled with phenol and 4.-Aminoantipyrine (4-AAP) to form
coloured Quinoneimine dye. Absorbance of coloured dye is measured at 505 nm and is directly proportional
to glucose concentration In the Sample. 

    Glucose Oxidase
Glucose + O2 + H2O                                          Gluconic acid + H2O 2


 Peroxidase
H2O 2 + Phenol + 4-AAP               Quinoneimine dye + H2O

PROCEDURE:
For automatic analyser:
Mark the tubes as blank, standard and test and add the content according to the table given below:
Pipettes into tube marked   Blank   Standard        Test 
Serum / Plasma     10 µL
Reagent 3                                          10 µL  
Working Glucose Reagent  1000 µL  1000 µL  1000 µL

Mix well incubates at 370 C for 10 minutes or at room temperature (15-300 C) fir 30 minutes. 
Programme the analyser as per assay parameters.
1.  Blank the analyser with Reagent blank.
2.  Measure absorbance of Standard followed by the test
3.  Calculate results as per given calculation formula

For spectrophotometers/Colorimeters:
Mark the tubes as blank, standard and test and add the content according to the table given below:
Pipettes into tube marked   Blank   Standard        Test 
Serum / Plasma     20 µL
Reagent 3                                          20 µL  
Working Glucose Reagent  1500 µL  1500 µL  1500 µL

 Mix well, incubates at 370 C for 10 minutes or at room temperature (15-300 C) for 30 minutes. Now add
1500 µL of distilled water in each tube and mix well. 
1.  Blank the spectrophotometers/Colorimeters with Reagent blank.
2.  Measure absorbance of Standard followed by the test at 505 nm (read absorbance at 490-550 nm).
3.  Calculate results as per given calculation formula.

CALCULATION:

Serum / Plasma glucose (mg/ dL) =        Absorbance of Test        X    100
          Absorbance of Standard

OBSERVATION TABLE:
S. No.  Reading for tubes  Optical density (OD) at 505 nm
1. Blank                                                
2. Standard                                                
3. Test                                                 

PRECAUTIONS:
1.  Use clean and dry glassware.
2.  For best results, it is recommended to bring required reagents to selected assay temperature before
use.
3.  To avoid reagent deterioration, take required amount of reagent for assay and immediately store the
reagent bottle tightly capped, in refrigerator.
4.  Over the time, reagent may develop light pink colour which does no affect the reagent performance
up to reagent blank absorbance of 0.20.A.U. 

1 comment:

  1. nice post and also it too usefull....
    thanks a lot more

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