SOP Number: CH007
Author: Sue Galindo
Version: 1
Effective Date: August 25, 2009

This SOP applies to all staff who perform clinical chemistry testing
The accurate measurement of glucose is important in the diagnosis and management of hyperglycemia and hypoglycemia. The hexokinase/glucose-6-phosphate dehydrogenase method developed by the American Association of Clinical Chemistry has been accepted as the reference method for glucose determination. It consists of a coupled chemical reaction. In the first reaction, hexokinase catalyzes the phosphorylation of glucose by ATP producing ADP and glucose-6-phosphate. In the second reaction, glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase to 6-phosphogluconate with the reduction of NAD+ to NADH as shown below:

Glucose + ATP glucose 6-phosphate + ADP

Glucose 6-phosphate + NAD+ 6-phosphogluconate + NADH + H+

The increase in NADH concentration is directly proportional to the glucose concentration and can be measured spectrophotometrically at 340 nm.
  1. Use Standard Precautions when handling body fluids
  2. Refer to the Chemical Hygiene Plan for the proper storage and use of chemicals
Serum, heparin plasma, or fluoride plasma may be used. Plasma or serum samples without preservatives should be separated from the cells or clot within a half hour of being drawn. Glucose in separated, unhemolyzed serum is stable up to four hours at 25°C and up to 24 hours at 4°C.
  1. Spectrophotometer
  2. Spectrophotometer cuvettes
  3. Distilled deionized water
  4. Physiological (0.9% NaCl) saline
  5. Pipettes
  6. Glucose hexokinase reagent obtained from Pointe Scientific, Inc. Reconstitute reagent with 15 mL distilled water. Swirl gently to dissolve. When reconstituted as described, the reagent contains Hexokinase 1,000 IU/L, G6PDH 1,000 IU/L, ATP 1.0 mM, NAD 1.0 mM, buffer pH 7.5. The un reconstituted reagent is stored at 2-8°C. Once reconstituted, the reagent is stable for 48 hours at 25°C and for 30 days at 2-8°C.
  7. Glucose standards 20 mg/dL, 100 mg/dL, 200 mg/dL, 400 mg/dL
  8. Paraffin squares
  9. Heating block or water bath 37°C
  10. Timer
  11. Graph paper (Click to Print)
Level one and level two serum controls are tested with each patient run. The level one control range is 70-85 mg/dL and the level two range is 271-306 mg/dL.
  1. Turn on the spectrophotometer and let warm up for at least 15 minutes.
  2. Set the wavelength to 340 nm.
  3. Label cuvettes 1 through 10.
  4. Add 3.0 mL of distilled deionized water to cuvette 1.
  5. Add 3.0 mL of Glucose hexokinase reagent to cuvette 2 through 10.
  6. Add 20 uL of 20 mg/dL glucose standard to cuvette 3.
  7. Add 20 uL of 100 mg/dL glucose standard to cuvette 4
  8. Add 20 uL of 200 mg/dL glucose standard to cuvette 5.
  9. Add 20 uL of 400 mg/dL glucose standard to cuvette 6.
  10. Add 20 uL of control Level One to cuvette 7.
  11. Add 20 uL of control Level Two to cuvette 8.
  12. Add 20 uL of the patient serums to be tested in the remaining cuvettes.
  13. Mix by inversion using the paraffin squares to prevent spillage.
  14. Incubate all cuvettes at 37°C for 5 minutes
  15. Place the cuvette 1 in the spectrophotometer and set the Absorbance to read 0.000.
  16. Read and record the Absorbance for cuvettes 2-10. Subtract the absorbance of cuvette 2 from each of the sample absorbances before plotting results.

The reference range for glucose is as follows:

Cord 45-96 mg/dL
20-60 mg/dL
40-60 mg/dL
1 wk
50-80 mg/dL
60-100 mg/dL
74-100 mg/dL
>60 yr
82-115 mg/dL
>90 yr
75-121 mg/dL

  1. Using graph paper, plot the Absorbance on the vertical (y axis) against the concentration on the horizontal (x axis) for each of the glucose standards.
  2. Draw a "best fit line" and use this standard curve to determine the glucose concentration for the controls and patient specimens.
  3. Verify that the control results are acceptable before reporting patient results.
  1. Examine the reconstituted glucose reagent for signs of deterioration. Do not use if the reagent develops turbidity or if it has an absorbance greater than 0.20 when measured against water at 340 nm.
  2. Extremely lipemic or icteric samples may give falsely high glucose results. In those cases, prepare a Sample Blank by adding 5ul to 1.0 physiological saline. Zero the spectrophotometer with the saline and read the absorbance of the Sample Blank. Subtract this absorbance reading from the assay reading and use this corrected absorbance when determining concentration.
  3. Once a standard curve has been constructed, it may be used for subsequent patient runs as long as the same batch of working reagent is used. If a new batch of reagent is used or the lamp on the spectrophotometer is changed, a new standard curve must be constructed.
  4. If the patient results exceed the linearity of the assay, dilute the patient serum 1:2 and repeat the test. Multiply the result by 2 before reporting results.
  5. Some glucose hexokinase methods use an enzyme preparation derived from yeast. In those methods, NADPH is formed as is directly proportional to the amount of glucose in the sample.
  6. Wavelength accuracy can be checked with a commercial filter, such as a didymium filter (IR and visible) or holmium oxide filter (UV and visible). Wavelength accuracy can also be checked with a prepared solution such as colbalt chloride, potassium dichromate, or nickel sulfate. Ultraviolet spectrometers are checked with a quartz mercury arc lamp or transmission standards.  To correct for poor results, realign exciter lamp with wavelength selector.
  7. Photometric linearity can be checked by running different concentrations of the same solution. Varying dilutions of a solution known to follow Beer's law are prepared and analyzed. Spectrophotometers that exhibit linearity inaccuracy should be tested for excess stray light (filter slit is too wide) or a failing photocell.
  8. Photometric accuracy can be checked with nickel sulfate solutions or with special filters. Corrections for problems with photometric accuracy include realigning the excitor lamp or cleaning a dirty excitor lamp or photocell window. Corrections may be needed to the filter slit width or a damaged diffraction grating may need to be replaced.
  9. Stray light can be detected in a spectrophotometer by utilizing a sharp cutoff filter.
  1. Serum and plasma must be separated from the red blood cells promptly to prevent glycolysis. Glucose will decrease approximately 7% per hour when left in contact with red cells. 
  2. Whole blood glucose is 12-15% less than serum glucose.
  3. Venous blood glucose is approximately 5 mg/dL less than arterial or capillary blood glucose.
  1. Burtis, Carl A et al. Tietz Fundamentals of Clinical Chemistry, 6th ed. Saunders: St Louis, Missouri, 2008.
  2. Naser, Najih. Clinical Chemistry Laboratory Manual. Mosby: St Louis, Missouri, 1998.
  3. Pointe Scientific, Inc package insert. February 2009.
Dr. QC
Susan Galindo