Blood Glucose Determination

Glucose levels are maintained in the body at 60-100 mg/dL and this is done through the body's homeostatic ability.

When the glucose levels increase, the primary hormone that decreases the level is insulin. On the other hand, the primary hormone that increases glucose levels when it is below normal, is glucagon.

Both of these hormones are produced by the pancreas in the beta and alpha cells of the Islets of Langerhans, respectively.

Since blood glucose levels are affected by diet through the food that we eat, fasting is required before a test is performed ( except in cases of RBS - Random Blood Sugar).

In the colorimetric method of glucose determination, The Orthotoluidine Method is the most common method utilized.

MANUAL PROCEDURE:

Precautions:

1. Patient should have fasted for 8-12 hours.
2. Unhemoylzed, non icteric and non turbid serum should be used to avoid interferences with the procedure.
3. The reagent contains sodium azide, which is corrosive, so PPE should be worn.
4. The solution should be brought to a boil to ensure proper reaction

MANUAL PROCEDURE:

Precautions:

1. Patient should have fasted for 8-12 hours.
2. Unhemoylzed, non icteric and non turbid serum should be used to avoid interferences
with the procedure.
3. The reagent contains sodium azide, which is corrosive, so PPE should be worn.
4. The solution should be brought to a boil to ensure proper reaction.

PROCEDURE PROPER:

1. Prepare Three tubes labeled, SA (Sample)/ C (Control), ST (Standard) and RB (Reagent Blank).

2. To the SA/C tube add 0.1 ml of the test specimen/ Control specimen.

3. To the ST tube add 0.1 ml of the standard stock solution.

4. To the RB tube add 0.1 ml of distilled water.

5. Add 4 ml of glucose reagent to all tubes. Cap and mix by gentle inversion.

6. Boil all tubes at 100 degrees centigrade for 8 minutes.

7. Cool tubes in running tap water for about 2 minutes.

8. Read absorbance of solutions in the spectrophotometer with appropriate cuvets at 636 nanometers against the RB (Reagent Blank).

9. Compute for the Cu (Concentration of the Unknown making use of the formula:

Cu= Absorbance of Unknown(Au) multiplied by the Concentration of the
Standard(Cs) divided by the Absorbance of the Standard (As)

Adapt the unit of the Cs

Normal Values = 60 - 100 mg/dL
to convert values to mmol/L multiply values in mg/dL with 0.0555

The clinical significance of the elevation of blood glucose denotes an abnormality in the body. One of the main causes of elevated blood levels is diabetes mellitus. It maybe Type 1, Type 2.

Further testing maybe done using OGTT (Oral Glucose Tolerance Test) or 2HPPT (2 Hour Post Prandial test). The 2HPPT is more sensitive as a test for diagnosis of DM (diabetes mellitus). There are two types of DM: Type 1 and Type 2.

ORTHOTOLUIDINE METHOD FOR BLOOD GLUCOSE DETER MINATION

MANUAL PROCEDURE:

Precautions:

1. Patient should have fasted for 8-12 hours.
2. Unhemoylzed, non icteric and non turbid serum should be used to avoid interferences
with the procedure.
3. The reagent contains sodium azide, which is corrosive, so PPE should be worn.
4. The solution should be brought to a boil to ensure proper reaction.

PROCEDURE PROPER:

1. Prepare Three tubes labeled, SA (Sample)/ C (Control), ST (Standard) and RB (Reagent Blank).

2. To the SA/C tube add 0.1 ml of the test specimen/ Control specimen.

3. To the ST tube add 0.1 ml of the standard stock solution.

4. To the RB tube add 0.1 ml of distilled water.

5. Add 4 ml of glucose reagent to all tubes. Cap and mix by gentle inversion.

6. Boil all tubes at 100 degrees centigrade for 8 minutes.

7. Cool tubes in running tap water for about 2 minutes.

8. Read absorbance of solutions in the spectrophotometer with appropriate cuvets at 636 nanometers against the RB (Reagent Blank).

9. Compute for the Cu (Concentration of the Unknown making use of the formula:

Cu= Absorbance of Unknown(Au) multiplied by the Concentration of the
Standard(Cs) divided by the Absorbance of the Standard (As)

Adapt the unit of the Cs

Normal Values = 60 - 100 mg/dL
to convert values to mmol/L multiply values in mg/dL with 0.0555

LABORATORY TESTS FOR GLUCOSE

Glucose is the most predominant carbohydrate and is the sugar tested in blood sugar
tests. There are certain preparations/precautions before an accurate laboratory test for glucose could be performed.

Whatever method is selected, the Medical Technologist must take the proper precautions in sample collection to prevent glucose utilization by leukocytes.

If the sample is left at room temperature, glucose in sample is reduced 10mg/dL/hour, therefore sample should be refrigerated within 30 minutes after extraction.

2 mg sodium fluoride per mL blood could be also added to prevent glycolysis for 24 hours.

Normal values:
Before meal: 90-130 mg/dL
After meal: < 180 mg/dL

There are Two General Methods for Glucose Detemination:

A. Chemical Methods - oxidation-reduction methods (ferric and cupric), condensation
methods
B. Enzymatic Methods - Hexokinase, Glucose Oxidase

Under the Chemical Methods: ( Alkalike copper reduction methods)

1. Folin-Wu - involves the conversion of phosphomolybdic acid to phosphomolybdenum blue

Reference value: 80-120 mg/dL

Precautions:
* Reagents should be freshly prepared
* Spectrophotometer: wavelength -520 nm
* Deprotienization should be done with the sample- removing of proteins from biologic fluids thru dilution

2 Nelson-Somogyi - involves the conversion of arsenomolybdic acid to arsenomolybdenum blue.
Spectrophotometer: 500-520 nm

3. Neocuproine - involves neocuproine reagent which produces a neocuproine complex.
• Spectrophotometer: 450 nm

4. Shaeffer-Hartmann-Somogyi - involves the use of iodine utilization as an indicator of the amount of glucose in the sample.
Spectrophotometer: 500-520 nm

5. Benedict’s

Reference value: 80-120 mg/dL
Precautions:
* Reagents should be freshly prepared

Chemical Methods (Alkaline Ferric Reduction Method)

1. Hagedorn-Jensen

Precautions:
* Traces of coagulum into the filtrate can cause increase in results.
* The stem of the funnel should have an ample amount of cotton.


Chemical Methods (Condensation Method)

1. Ortho-Toluidine
Gluconeer

Precautions:
* Non-icteric, non lipemic and unhemolyzed serum should be used.
* Patients should not be suffering from galactosemia
* Patients should not be under galactose load


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B. Enzymatic Methods
1. Glucose Oxidase Method
2. Hexokinase Method

Reference Values:
* Fasting Serum Glucose: 70-110 mg/dL

* CSF:
Infant: 60-80 mg/dL
Adult: 40-70 mg/dL

* Urine (24 hours): 1-15 mg/dL

Precautions:
* Do not ingest the reagent, toxicity has not been established
* Spectrophotometer: 340 nm
* Use fresh, unhemolyzed serum. Plasma from tubes containing heparin, citrate,
oxalate or EDTA.
* Separate RBC from serum or plasma ASAP
* Glucose in whole blood is decreased 7% per hour.
* Turbid or icteric sera require sample blank

3. Glycosylated Hemoglobin
– HbA1c
Reference value: 4.0-6.0%
Precautions:
* Ample amount of blood- 5ml
* Apply pressure to the puncture site

FBS (Fasting Blood Sugar),the patient must have fasted for 8- 14 hours. This is because a non fasting patient will have an inaccurately increased value because of the effect of the ingested food.

The best anticoagulant in the specimen collection is sodium fluoride. This is because aside from acting as an anticoagulant, it also prevents glycolysis which can decrease inaccurately, the values obtained.

There are other tests like OGTT, (Oral Glucose Tolerance Tests), IGTT (Intravenous Glucose Tolerance Tests), RBS (Random Blood Sugar), 2HPPT ( 2- Hour Post Prandial Test)