|SOP Number: CH007
Author: Sue Galindo
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.
CONSIDERATIONS/PERSONAL PROTECTIVE EQUIPMENT
- Use Standard Precautions when handling body fluids
- 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.
- Spectrophotometer cuvettes
- Distilled deionized water
- Physiological (0.9% NaCl) saline
- 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.
- Glucose standards 20 mg/dL, 100 mg/dL, 200 mg/dL,
- Paraffin squares
- Heating block or water bath 37°C
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
- Turn on the spectrophotometer and let warm up for at
least 15 minutes.
- Set the wavelength to 340 nm.
- Label cuvettes 1 through 10.
- Add 3.0 mL of distilled deionized water to cuvette
- Add 3.0 mL of Glucose hexokinase reagent to cuvette
2 through 10.
- Add 20 uL of 20 mg/dL glucose standard to cuvette 3.
- Add 20 uL of 100 mg/dL glucose standard to cuvette 4
- Add 20 uL of 200 mg/dL glucose standard to cuvette
- Add 20 uL of 400 mg/dL glucose standard to cuvette
- Add 20 uL of control Level One to cuvette 7.
- Add 20 uL of control Level Two to cuvette 8.
- Add 20 uL of the patient serums to be tested in the
- Mix by inversion using the paraffin squares to
- Incubate all cuvettes at 37°C for 5 minutes
- Place the cuvette 1 in the spectrophotometer and set
the Absorbance to read 0.000.
- 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:
- 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.
- Draw a "best fit line" and use this standard curve
to determine the glucose concentration for the
controls and patient specimens.
- Verify that the control results are acceptable
before reporting patient results.
- 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.
- 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.
- 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.
- 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
- 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.
- 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
correct for poor results, realign exciter lamp with
- 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
- 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.
- Stray light can be detected in a spectrophotometer
by utilizing a sharp cutoff filter.
- 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.
- Whole blood glucose is 12-15% less than serum
- Venous blood glucose is approximately 5 mg/dL less
than arterial or capillary blood glucose.
- Burtis, Carl A et al. Tietz Fundamentals of Clinical
Chemistry, 6th ed. Saunders: St Louis, Missouri, 2008.
- Naser, Najih. Clinical Chemistry Laboratory Manual.
Mosby: St Louis, Missouri, 1998.
- Pointe Scientific, Inc package insert. February