Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

CSF Glucose – Glucose Oxidase Method

     Principle of the method

Glucose present in the CSF is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid with the liberation of hydrogen peroxide, which is converted into 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 515nm.

GOD

                                Glucose 4 Gluconic acid + H₂O_{2    -------------------> 4} Gluconic acid _{+ H2O2}                         

POD
      H₂0₂--------------> 4 H₂0 + [0]

                  [0] + 4 aminophenazone + phenol ------>   chromogen (pink)

     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 one litre with distilled water. Stable for 3-4 months, at 2-8⁰C. Check final pH 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 GOD and POD powder with phosphate buffer. Dispense separately into vials so that each vials 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 one 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

Mix well

     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 photometers that require a minimum volume of reaction mixture in the cuvette of one ml. or less. Since economical use of reagents is possible with this protocol, 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 advised that the volume of all reagents mentioned under Tabulation "(b) Colour development", be increased 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 and 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 CSF analysis is carried out infrequently in intermediate laboratories, one QC for glucose should be included as and when CSF glucose is analysed. Hence it will not be possible to analyse several QC samples and calculate within-day precision. However, even if only a single QC sample is analysed in a day, this value can be pooled with the preceding 10 or 20 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

Concentrations of analytes in the CSF should always be compared with those in plasma. Normal CSF glucose is about 60% of the plasma value.

Normal range for CSF glucose 50-80 mg/dl

Decreased CSF glucose levels are observed in tuberculosis, benign lymphocytic chronic meningitis and in hypoglycemia. Increased levels are observed in encephalitis, poliomyelitis and in cerebral abscess.

     Limitations

Grossly bloody CSF may give spuriously elevated values for glucose. Undue delay in analysis may give low values. The report to the requesting physician should include the appearance of the CSF before and after centrifugation.

     References

1.      Trinder, P. (I 969). Annals of Clin. Biochem. 6: 24 - 27.

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