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
The glucose oxidase and
peroxidase method was first reported in 1956 by Keston (10.1) and Teller
(10.2). In 1958, Beach and Turner
reported on the specificity of the reaction for beta-d-glucose, and determined
its accuracy by comparative tests with other glucose determination
methods. The Biotron Diagnostics Glucose Test is a
modification of the Meites and Gochman (10.3, 10.4) glucose determination.
2.2 TEST PRINCIPLE
D-glucose in the sample is
oxidised bu the enzyme glucose oxidase to gluconic acid and hydrogen
peroxide. The hydrogen peroxide, in the
presence of peroxidase, reacts with hydroxybenzoate and 4-aminophenazone to
form a quinone complex. The intensity of
the color formed is proportional to the glucose concentration in the sample.
Glucose
oxidase
d-Glucose ---------------------> gluconic acid + H2O2
Peroxidase
H2O2 + 4-aminophenazone
------------------> quinone complex
+ hydroxybenzoate
2.3 CLINICAL SIGNIFICANCE
(10.8)
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. The
specimen should be collected following a 12 hour fast. Plasma prepared from blood collected with an
anticoagulant containing fluoride may be used.
Samples must be separated
within 30 minutes of collection as glycolysis occurs in whole blood at a rate
of 7% per hour.(10.6)
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
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. (10.6)
Fluoridated plasma for
glucose assay is stable at 2°-8° C (refrigerated) for 5 days (10.5.)
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 (5 X 50 ml)
Reagents necessary for the
determination of glucose are included in the kit.
4.1 GLUCOSE ENZYME REAGENT
Each vial contains, after reconstitution with deionized
water:
glucose oxidase ³ 20,000 U/L
peroxidase ³ 5000 U/L
4-aminophenazone ³ 0.2 mM
p-hydroxybenzoate ³ 10 mM
stabilizer, and a
preservative
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 working reagent is
prepared by adding 50 ml of deionized water to each glucose reagent vial. Replace the rubber stopper and allow 5
minutes for reconstitution. Swirl gently
until the contents of the vial are completely dissolved. Record the date and time of reconstitution.
and time of reconstitution.
4.4 STORAGE AND STABILITY
When stored at 2°-8°C
unopened reagents are stable until the expiration date printed on the
label. The working reagent is stable for
30 days if kept at 2-8° C (refrigerated) and protected from light.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 Spectrophotometer or colorimeter capable of reading absorbance at
500-540 nm.
4.5.2 1 cm cuvettes or a flow cell capable of transmitting light at
500-540 nm.
4.5.3 Test tubes capable of holding 3 ml.
4.5.4 Pipettes capable of delivering 2.5 ml and 20 µl.
4.5.5 Deionized or distilled water for preparing the reagent blank.
4.5.6 Timer for a 3 minute incubation.
4.5.7 Constant temperature source for 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 500-540
nm
Temperature 37°
C
Pathlength 1.0
cm
Mode endpoint
Reaction time 3
min at 37° C
Sample volume 20
µl
Reagent volume 2.5
ml
Total volume 2.52
ml
Sample to reagent ratio 1/125
5.2 INSTRUMENT
Any instrument capable of
reading absorbance accurately with a sensitivity of 0.001 absorbance at 500-540
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 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 working reagent to room
temperature. (See 4.3 Reagent
Preparation Section.)
5.4.2 Into separate test tubes pipette 20 µl of distilled water,
calibrator, or serum to be assayed.
5.4.3 Add 2.5 ml of working reagent to each test tube and mix.
5.4.4 Following incubation for 3 minutes at 37° C determine the
absorbance of the calibrator (As) and of each serum (A) at 500-540 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 (10.5)
The range of expected values is:
60 - 110 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
(10.8)
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.
6.3 LIMITATIONS OF PROCEDURE
6.3.1 A comprehensive list of drugs and other substances which can affect
the glucose concentration in serum is given by Young. (10.7)
6.3.2 Bilirubin has no effect if present in concentrations of less than 8
mg/dl.
6.3.3 Uric acid has no effect if present in concentrations of less than
12 mg/dl.
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 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, 20 replicates of 2 control sera were run.
Mean (mg/dl) SD
(mg/dl) CV (%)
89 ± 2.8 3.1
298 ±
8.5 2.8
Between-Run
In this study, 8 runs were made with a pooled serum sample.
Mean (mg/dl) SD
(mg/dl) CV (%)
97 ± 3.3 3.4
9.2 CORRELATION
A correlation study was done
on the SMA 12/60 (registered trademark of Technicon Corp.) comparing this
method and a similar glucose method.
Number Regression
Equation Correlation
of Samples y=Biotron,
x=Comparative Coefficient
20 y
= 1.014 x - 2.39 0.98
9.3 RECOVERY STUDY
In this study, known aqueous
glucose standards of varying concentration were added to a pooled serum sample.
With this method, recovery was in the range of 90-99%.
9.4 LINEARITY
This method is linear through
400 mg/dl, beyond which the specimen should be diluted 1 to 1 with deionized
water. Reassay the specimen and multiply the result by 2.
10.0 REFERENCES
10.1 Keston, A., Abstracts of
Paper Presented at the 129th Meeting of the Am. Chem. Soc., Section 69C, 1956.
10.2 Teller, J., Abstracts of Paper Presented at the 130th Meeting of
the Am. Chem. Soc., Section 69C, 1956.
10.3 Meites, S., and Saniel-Barrey, K.,
Clin. Chem. 19, 308(1973).
10.4 Gochman, N., and Schmitz, J.M., Clin. Chem. 18, 943(1972).
10.5 Henry, Cannon and Winkleman, Clinical Chemistry - Principles and
Techniques, 2nd. ed., Harper & Row, 1974, 1275-1291.
10.6 Tietz, N.W., (ed.), Fundamentals of Clinical Chemistry, 2nd ed.,
W.B. Saunders Co., Toronto, 1213 (1982).
10.7 Young, D.S., Effects of Drugs on Clinical Laboratory Tests, 3rd
ed., Washington DC, AACC Press (1990).
10.8 G.J. Kost, "Critical Limits for Urgent Clinician Notification
at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
Rev
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