Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Glucose – Glucose Oxidase Method

     Introduction

Glucose is a reducing monosaccharide that serves as the principal fuel of all the tissues. It enters the cell through the influence of insulin and undergoes a series of chemical reactions to produce energy.

Lack of insulin or resistance to its action at the cellular level causes diabetes. Therefore, in diabetes mellitus the blood glucose levels are very high. Some patients with very high blood glucose levels may develop metabolic acidosis and ketosis caused by the increased fat metabolism, the alternate source for energy. Hyperglycaemia is also noted in gestational diabetes of pregnancy and may be found in pancreatic disease, pituitary and adrenal disorders.

A decreased level of blood glucose, hypoglycaemia is often associated with starvation, hyper insulinaemia and in those who are taking high insulin dose for therapy.

     Principle of the method

Glucose present in the plasma is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid with the liberation of hydrogen peroxide, which is converted to water and oxygen by the enzyme peroxidase (POD).

4 aminophenazone, an oxygen acceptor, takes up the oxygen and together with phenol forms a pink coloured chromogen which can be measured at 515mm.

     Specimen type, collection and storage

Plasma is the specimen of choice for glucose estimation. Plasma glucose levels have been checked to be quite stable for 6 hours at room temperature (25 -35⁰C) in the author’s laboratory. It is important that plasma should be separated from the cells soon after collection, preferably within 1 hour.

About 2 ml of the patient’s blood should be collected by venipuncture into a tube containing a mixture of potassium ethylene diaminetetraacetate (EDTA) sodium fluoride at a ratio 1:2 (W/W). Five mg of the mixture is adequate for 2 ml of blood. The tube should be gently but thoroughly shaken for complete mixing.

Preparation of the anitcoagulant mixture: 100 g of potassium EDTA and 200 g of sodium fluoride should be mixed and ground into a fine powder using a blender. This should preferably be done in a fume cupboard. The mixture should be stored in a clean container.

A thin, long spatula that can scoop 5 mg when levelled, can be used for delivering the mixture into the tube.

     Reagents

All chemicals must be Analar grade

     Phosphate Buffer : 100 mmol/L. pH 7.0

To 800 ml of distilled water add the following in the order:

Disodium hydrogen phosphate dihydrate [Na2HPO4 2H2O] 12.95 g
Anhydrous potassium dihydrogen phosphate [KH2PO4] 4.95 g
Sodium azide [NaN3] 0.5 g

Add one by one, dissolve and finally make up to 1 litre with distilled water. Stable for 3-4 months, at 2-8⁰C. Check that the final pH is 7.0 + 0.05 with a pH meter.

     Colour Reagent

To 100ml of the above phosphate buffer add the following in the order and then mix to dissolve:

4 amino phenazone 16 mg
GOD [Sigma G 7016] 1800 units
POD [Sigma P 8250 ] 100 units
Phenol 105 mg
Tween 20 [Sigma P 1359] 50m l

Reconstitute the purchased GOD & POD powder with phosphate buffer. Dispense separately into vials so that each vial represents the requisite number of units. Store the vials frozen. Stable for 2 weeks at 2-8⁰C. Store in a brown bottle.

     Benzoic acid 1g/l.

Dissolve 1.0g of benzoic acid in water and make up to 1 litre with water. This solution is stable indefinitely at room temperature.

     Stock glucose solution, 1 g/l.

Before weighing, dry the glucose at 60-80⁰C for 4 hours. Allow to cool in a dessicator. Dissolve 1g of glucose in benzoic acid solution and make up to 100 ml in a volumetric flask. Stable for six months at room temperature (25-35⁰C).
DO NOT FREEZE THE STANDARD

     Working glucose standard 100 mg/dl.

Dilute 10 ml of stock glucose (use either a volumetric pipette or a burette) to 100 ml with benzoic acid in a 100 ml volumetric flask. Mix well. Stable for 6 months at room temperature (25-35⁰C).

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP

     Procedure

The protocol of the procedure is described below.

     Dilution of standards (S1-S5), Test & QC

Pipette the following into appropriately labelled 13 x 100 mm tubes

     Colour development

Pipette the following into another set of appropriately labelled tubes.

Mix all tubes well. Incubate at 37⁰C in a waterbath for 15 minutes.

Remove from waterbath and cool to room temperature. Set the spectrophotometer/ filter photometer to zero using blank at 510 nm/ green filter and measure the absorbance of Standards, Test and QC.

This protocol is designed for spectrophotometers / filter photometer that require a minimum volume of reaction mixture in the cuvette of 1 ml or less. Economical use of reagents is possible with this protocol, thus the cost per test can be kept to the minimum. However, if a laboratory employs a photometer requiring a large volume of the reaction mixture for measurement, viz. 5 ml, it is advisable to increase the volume of all reagents mentioned under Tabulation "(b) Colour development" proportionately.

     Calculation and calibration graph

Since the protocol for standard tube S1 and test is identical, the standard S1 will represent a concentration of 100 mg/dl. The glucose concentrations represented by other standard tubes are S2 =200; S3 = 300; S4 =400 & S5 = 500 (mg/dl).

Plot the absorbance values of standards against their respective concentrations. The measurable range with this graph is from 10 to 500 mg/dl.

Plot absorbance values of Test/QC on the calibration graph and read off the concentrations.

Once linearity is proved, it is not necessary to prepare the standard graph every time that patients’ samples are analysed. It will be adequate if standard S2 is taken every time and patients’ results are calculated using the formula :

Test absorbance
---------------------- x 200 mg/dl
Standard absorbance

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients’ results).

Since glucose is the most common analyte measured in a laboratory, it is advisable to include an internal QC (normal QC pool) with every batch of samples analysed in the day, irrespective of the number of samples in a batch. Further, even when a single sample is analysed as an "emergency" sample at any time of the day or night, it is essential to include an internal QC. From the QC results obtained for the day, mean, standard deviation and %CV can be calculated to ensure that within-day precision is well within the acceptable limit, i.e, 5%.

The mean value of internal QC for the day can be pooled with the preceding 10 or 20 mean values obtained in the previous days, and between–day precision can be calculated and expressed as % CV. Ensure that this is well within the acceptable limit, i.e, 8%.

At least once a day analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials.

      Hazardous materials

This procedure uses sodium azide and phenol, which are poisonous and caustic. Do not swallow, and avoid contact with skin and mucous membranes

      Reference range and clinical interpretation

Plasma glucose: Fasting: 70 –110 mg/dl
Post-prandial: 80 – 140 mg/dl
Random: 60 – 140 mg/dl

Elevated plasma glucose levels are expected in a variety of clinical conditions, especially diabetes mellitus, Cushing’s syndrome and hyperadrenalism. Decreased plasma glucose levels are observed in hyper-insulinism, anti-diabetic treatment and hypoadrenalism.

     Limitations

Any sample that gives aglucose value > 500 mg/dl should be diluted 1:2 with 0.9g% sodium chloride solution and the correct value obtained by multiplying the result by 3.

At high plasma levels, uric acid, glutathione and bilirubin may interfere with the assay by causing a decrease in glucose values. Ascorbic acid will decrease glucose values by retarding colour development. Do not report results from specimens with suspected interference. Inform the requesting physician of the problem.

     References

1.      Trinder, P. (1969). Annals of Clin. Biochem. 6: 24 – 27.

2.      Barham D and Trinder P. (1972). Analyst 97: 142 – 145