1.0 INTENDED USE
The reagent is intended for
the quantitative determination of Glucose in serum or plasma.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Enzymatic methods for the determination
of glucose were described by Keilin and Hartree (10.1). Passey, et. al.
(10.3) have reviewed 10 glucose methods and have used
the hexokinase method as the reference procedure.
2.2 TEST PRINCIPLE
Glucose is converted to
glucose-6-phosphate (G-6-P) by hexokinase (HK) in the presence of adenosine
triphosphate (ATP). G-6-P is converted
to 6-phosphogluconate and NADH in the presence of glucose-6-phosphate
dehydrogenase (G-6-PDH) and nicotinamide adenine dinucleotide (NAD) and the
increase in optical density at 340 nm is measured.
HK
Glucose + ATP -----> G-6-P + ADP
G-6-PDH
G-6-P + NAD+ ---------------> 6-phosphogluconate + NADH
+ H+
NADH causes an increase in
absorbance at 340 nm which is directly proportional to the concentration of glucose
in the sample.
2.3 CLINICAL SIGNIFICANCE
(10.4)
Elevated levels of glucose
(hyperglycemia) are found in uncontrolled diabetes mellitus, hyperthyroidism,
hyperadrenalism, uremia and bacterial or viral infections.
Low levels of glucose
(hypoglycemia) are found in certain adrenal and pituitary disorders, and in
cases of high levels of insulin or other antidiabetic drugs.
3.0 SPECIMEN COLLECTION AND
HANDLING
3.1 PATIENT PREPARATION
The patient should be fasting for 12 hours prior to
specimen collection.
3.2 SPECIMEN COLLECTION.
Fresh, clear, unhemolyzed
serum is the preferred specimen. Plasma
may be used if citrate, oxalate, heparin or EDTA are
employed as the anticoagulant. Hemolyzed
samples must not be used since erythrocytes contain glucose-6-phosphate which
is an intermediate product in this reaction.
Use a standard venipuncture tube to draw patient sample.
The amount of sample required
will depend on the analyzer used. The
amount of serum required is in the range of 5-25 µl. Call Biotron's technical service department
at 1-800-595 8766 for the recommended sample volume for your analyzer.
Record the patient's name,
date and time of sample collection and preparation.
3.3 SPECIMEN STORAGE
Samples must be separated
within 30 minutes of collection as glycolysis occurs in whole blood at a rate
of 7% per hour.(10.4)
Serum samples may be stored
refrigerated (2°-8° C) for 1 week , or frozen (-20° C) for 1 month prior to analysis. Frozen samples should be thawed at room
temperature and mixed thoroughly before analysis. Thawed samples should not be refrozen. (10.4)
It is recommended that
testing be done as soon as possible after sample collection and
preparation. If testing cannot occur
immediately, store the sample properly using the guidelines above.
4.0 MATERIALS (2 X 125 ml)
Reagents necessary for the
determination of glucose are included in the kit.
4.1 GLUCOSE (HK) REAGENT
The glucose reagent contains:
phosphate buffer 100
mM
magnesium chloride 2mM
ATP 4mM
NAD 2mM
HK (L.mesenteroides) 2000
U/L
G-6-PDH (yeast) 4000
U/L
4.1.1 Standard/Control/Calibrator
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic
Use. Never pipette by mouth. Exercise the normal precautions required for
handling all laboratory reagents.
4.3 REAGENT PREPARATION
The reagent is ready to use as is.
4.4 STORAGE AND STABILITY
When stored at 2°-8°C
reagents are stable until the expiration date printed on the label.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 Spectrophotometer or colorimeter capable of reading absorbance at
340 nm.
4.5.2 1 cm cuvettes or a flow cell capable of transmitting light at 340
nm.
4.5.3 Test tubes capable of holding 3 ml.
4.5.4 Pipettes capable of delivering 2.5 ml and 25 µl.
4.5.5 Deionized or distilled water for preparing the reagent blank.
4.5.6 Timer for a 5 minute or 10 minute incubation.
4.5.7 Constant temperature source which can be adjusted to 37° C.
4.5.8 Calibrator .
4.5.9 Normal and abnormal controls for quality control.
5.0 TEST PROCEDURE
The following is a general procedure for use on a manual
instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 340
nm
Temperature 37°
C, or 18-26° C
Pathlength 1.0
cm
Mode endpoint
Reaction time 5
min at 37° C
10
min at 18-26° C
Sample volume 25
µl
Reagent volume 2.5
ml
Total volume 2.525
ml
Sample to reagent ratio 1/100
5.2 INSTRUMENT
Any instrument capable of
reading absorbance accurately with a sensitivity of 0.001 absorbance at 340 nm
may be used. The band width should be 10
nm or less, stray light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
The Glucose (HK) assay is calibrated by
referencing the absorbance of the unknown sample to the absorbance of the
calibrator.
5.4 PROCEDURE
5.4.1 Warm the required volume of reagent to room temperature.
5.4.2 Into separate test tubes pipette 25 µl of distilled water,
calibrator, or serum to be assayed.
5.4.3 Add 2.5 ml of reagent to each test tube and mix.
5.4.4 Following incubation for 5 minutes at 37° C in a heat block or 10
minutes at 18-26° C (room temperature), determine the absorbance of the
calibrator (As) and of each serum (A) at 340 nm using the distilled water
sample as the reagent blank.
5.5 CALCULATION AND RESULTS
A
Glucose (mg/dl) = ------ X concentration of calibrator
As
A = absorbance of sample, As = absorbance of calibrator
Example:
.295
Glucose (mg/dl) = --------- X 100 mg/dl = 83 mg/dl
.355
with A = .295 and As = .355, concentration of calibrator =
100 mg/dl
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.4)
The range of expected values
in adults for fasting glucose using the glucose (HK) method is: 70-105 mg/dl
These values are suggested
guidelines. It is recommended that each
laboratory establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES
Each laboratory should establish
low and high values beyond which the patient would require immediate attention
by a physician. If a "medical alert
value" is reached, always repeat the test to confirm the result and notify
a physician if the result is confirmed.
Reference 10.6 provides a discussion of some critical
values.
6.3 LIMITATIONS OF PROCEDURE
6.3.1 High levels of fructose in serum, as may occur in essential
fructosuria or after excessive ingestion of fruits, may produce falsely
elevated glucose levels, i.e., 200mg of fructose will react as 1mg of
glucose. Greater than 500mg of fructose
are inhibitory. (10.4)
6.3.2 Hemoglobin itself is not an interfering substance, however,
hemolyzed cells can release enzymes which catabolize glucose as well as
glucose-6-phosphate, the latter of which is a reaction intermediate. This effect is minimized when samples are
stored in a refrigerator at 2°-8°C especially in the presence of fluoride ion.
(10.4)
6.3.3 A comprehensive list of drugs and other substances which can affect
the glucose concentration in serum is given by Young. (10.5)
6.3.4 As with any chemical reaction, users should be alert to the
possible effect on results caused by unknown interferences from medications or
endogenous substances. All patient
results should be evaluated in light of the total clinical status of the
patient.
6.3.5 Use of this test has not been evaluated in neonatal
hypoglycemia. Thus this test is not
recommended for use with neonatal (birth to 4 weeks) specimens.
7.0 QUALITY CONTROL
Standard practice for quality
control should be applied to this system.
Commercially available lyophilized controls can be used to monitor the
daily acceptable variations. Normal and
abnormal controls should be assayed at the beginning of each run of patient
samples, whenever a new reagent or a different lot number is being used, and
following any system maintenance.
A satisfactory level of
performance is achieved when the analyte values obtained are within the
"acceptable range" established by the laboratory.
8.0 CALIBRATION PROCEDURES
The Glucose (HK) assay is
calibrated by referencing the absorbance of the unknown sample to the
absorbance of the calibrator. Refer to
your instrument manual for more details.
Calibration is required with
the use of a new lot of reagent, any system maintenance or whenever indicated
by quality control data.
9.0 PERFORMANCE
CHARACTERISTICS
9.1 PRECISION
The estimates of precision
shown below were obtained from assays of human control serum.
Within-Run
In this study, 15 replicates of 2 control sera were run.
Mean (mg/dl) SD
(mg/dl) CV (%)
79 ±
0.57 0.73
309 ±
4.08 1.33
Between-Run
In this study, 5 runs were
made, each run consisting of 5 replicates of 2 control sera.
Mean (mg/dl) SD
(mg/dl) CV (%)
96 ±
1.42 1.49
332 ±
4.73 1.43
9.2 CORRELATION
A correlation study was done
on the Technicon RA-500 system at 37° C comparing King Glucose (HK) method and
a similar glucose (HK) method. The
samples range between 35 mg/dl and 559 mg/dl.
Number Regression
Equation Correlation
of Samples y=Biotron,
x=Comparative Coefficient
44 y
= .987 x + .470 0.999
9.3 LINEARITY
This method is linear to 500
mg/dl. A sample with glucose beyond the
linearity limit should be diluted 1 to 1 with deionized water. Reassay the specimen and multiply the results
by 2.
10.0 REFERENCES
10.1 Keilin, D., Hartree, E.F., Biochem. J. 42,250 (1948).
10.2 United States Department of Health, Education and Welfare, Food
and Drug Administration. In vitro
Diagnostic Products for Human Use, Proposed Establishment of Product Class
Standard for Detection or Measurement of Glucose, Fed. Regist. 39, No. 126,
24136-24147 (1974).
10.3 Passey, R.B., Gillum, R.L., Fuller, J.B., Urry, F.M., Giles, M.L.,
Evaluation and Comparison of Ten Glucose Methods and the Reference Method
Recommended in the Proposed Product Class Standard (1974), Clin. Chem. 23,
131-139 (1977).
10.4 Tietz, N.W., (ed.), Fundamentals of Clinical Chemistry, 2nd ed.,
W.B. Saunders Co., Toronto, 1213 (1982).
10.5 Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 3rd
ed., Washington DC, AACC Press (1990).
10.6 G.J. Kost, "Critical Limits for Urgent Clinician Notification
at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704