1.0 INTENDED USE
This reagent is intended for the quantitative
determination of aspartate aminotransferase (AST) in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Karmen (10.1) introduced a method for the determination of aspartate aminotransferase, formerly called glutamate oxalacetic transaminase (GOT). Henry et. al.(10.2) optimized the method. The procedure used in this kit is a modification of the method of Henry. The NADH concentration is increased to extend the linearity and oxamic acid is added to remove interference by serum pyruvate (10.3,10.4,10.5).
2.2 TEST PRINCIPLE
The AST catalyzes the reaction of 2‑oxoglutarate
and L-aspartate to L‑glutamate and oxalacetate. Then malate dehydrogenase (MDH) catalyzes the
oxidation of NADH to NAD.
AST
2‑oxoglutarate
+ L‑aspartate ----------> L‑glutamate + Oxalacetate
MDH
Oxalacetate
+ NADH + H+ -----------> Malate + NAD+
The rate of decrease in absorbance of the reaction
mixture at 340 nm, due to the oxidation of NADH is directly proportional to the
AST activity.
2.3 CLINICAL SIGNIFICANCE (10.8)
The principle causes of elevated aspartate
aminotransferase activity in serum are damage to or disease of heart and
liver. Decreased levels of AST activity
in serum may occur as a result of pyridoxal phosphate (vitamin B6)
deficiency. Low levels of this vitamin
can occur in patients undergoing dialysis.
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh,
clear, unhemolyzed serum is the preferred specimen.
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 5-200 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 should be kept refrigerated (2° to 8°C) and
analyzed within 24 hours. If this is not possible, serum samples may be stored
refrigerated (2° to 8°C) or frozen (-20° to 0° C) and are stable for up to 7
days. Frozen samples should be thawed at
room temperature and mixed completely before analysis. Thawed samples should not be refrozen.
4.0 MATERIALS (10 X 10 ml)
(6 X 50 ml)
Reagents
necessary for the determination of AST are included in the kit.
4.1 REAGENT
AST
REAGENT contains, after reconstitution with deionized water:
NADH 0.21
mM
malate dehydrogenase
(pig heart) ³ 600 U/L
2‑oxoglutarate 12
mM
L‑aspartic
acid 200
mM
tris buffer 100
mM (pH = 7.8)
preservative
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic Use. Not for Internal use in Humans or
Animals. In Vitro Diagnostics reagents
may be hazardous. Avoid ingestion and
skin or eye contact.
4.3 REAGENT PREPARATION
4.3.1
Add 10 ml of the deionized water to each of the required number of vials of AST reagent. 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.
4.3.2
Add 50 ml of the deionized water to each of the required
number of vials of AST reagent. 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.
4.4 REAGENT STORAGE AND STABILITY
Unopened reagents are stable at 2-8° C (refrigerated)
until the expiration date stated on the labels.
The reconstituted reagent is stable at 2-8° C (refrigerated) for 14 days
or at 18‑26° C (room temperature) for 8 hours.
The reconstituted reagent solution should be clear. Cloudiness indicates contamination and the
reagent should be discarded. The initial
absorbance of the reagent read against distilled water at 340 nm should be
1.100 or greater.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
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 4 ml.
4.5.4 Pipettes
capable of delivering 3.0 ml and 200 µl.
4.5.5 Timer with
one minute increments.
4.5.6 Constant
temperature source which can be adjusted to 30° C or 37° C.
4.5.7 Normal and
abnormal control 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 30°
C or 37° C
Pathlength 1.0
cm
Mode kinetic
Reaction time 2
‑ 4 min
Sample volume 200
µl
Reagent volume 3.0
ml
Total volume 3.2
ml
Sample to reagent
ratio 1/15
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
No reagent calibration is necessary as this procedure is
standardized based on the millimolar absorptivity of NADH which is taken as
6.22 at 340 nm under the test conditions described.
5.4 PROCEDURE
5.4.1 Prepare the
required volume of AST working reagent.
(See 4.3 Reagent Preparation section.)
5.4.2 Into separate
test tubes pipette 200 µl of serum to be assayed.
5.4.3 Add 3.0 ml of
reagent, mix, and incubate for one to three minutes at 30° C or 37° C. The lag
time will be decreased if the reagent is prewarmed to the incubation
temperature.
5.4.4 Record the
decrease in absorbance at 340 nm at one minute intervals until the absorbance
change is constant.
5.5 CALCULATION AND RESULTS
AST (U/L)
=
DA/min X assay volume (ml) X 1000
-------------------------------------------------------
= DA/min
X 2572
6.22
X light path (cm) X sample volume (ml)
DA/min = change in absorbance per minute
Assay
volume = total reaction volume expressed in ml
1000 =
converts U/ml to U/L
6.22 =
absorbance coefficient of NADH at 340 nm
Light path
= length of the light path expressed in cm (usually 1)
Sample
volume = sample volume expressed in ml
2572 =
factor derived from constants in the equation
Example: AST (U/L) =
.015 X 3.2
X 1000
--------------------------- = .015 X 2572 = 39 U/L
6.22 X 1 X 0.2
0.015 =
change in absorbance per minute
3.2 =
assay volume in ml
1 = light
path in cm
0.2 = sample volume in ml
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.7)
The range
of expected values is:
8 ‑ 20 U/L (30° C)
12 ‑ 31 U/L (37° C)
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 (10.9)
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
This procedure measures total AST. Red blood cells contain high concentrations
of AST, therefore hemolysis can elevate results. A summary of the influence of drugs on
clinical laboratory tests may be found by consulting Young, D.S., et. al,
(10.6).
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
No reagent calibration is necessary as this procedure is
standardized based on the millimolar absorptivity of NADH which is taken as
6.22 at 340 nm under the test conditions described.
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, 30 replicates of 2 control sera were run.
Mean
(U/L) SD (U/L) CV (%)
33.3 ± 1.1 3.3
96.0 ± 3.3 3.4
Between-Run
In this
study, 10 runs were made on 2 control sera.
Mean
(U/L) SD (U/L) CV
(%)
25.4 ± 2.4 9.3
73.5 ± 4.0 5.4
9.2 CORRELATION
A correlation study was done comparing this method and a
similar AST method. The samples range
between 7 and 103 U/L.
Number Regression Equation Correlation
of
Samples y=Biotron, x=Comparative Coefficient
42 y = .999 x - 1.314 .984
9.3 LINEARITY
This procedure is linear to 350 U/L. Procedures on automated instruments which use
greater than a one to fifteen dilution factor will have an extended linearity.
A sample with AST activity exceeding the linearity limit
should be diluted with 0.9% saline and reassayed incorporating the dilution
factor in the calculation of the result.
9.4 SENSITIVITY
An absorbance change of 0.001 DA/min corresponds to approximately 2.6 U/L AST activity.
10.0 REFERENCES
10.1 Karmen, A.,
Wroblewski, F., Ladue J., J. Clin.
Invest. 34, 126 (1955)
10.2 Henry, R.J.,
Chiamori, N., Gobub, O.J. and Berkman, S., Am. J. Clin. Pathology, 34,381(1960)
10.3 Novoa, W.B.,
Winter, A.D., Glaid, A.J., Schwert, G.W., J. Biol. Chem. 234,1143(1959)
10.4 Travedi,
R.C., Strong, L.J., Dickstein R., Desai, K., Clin. Chem. 23,1171(1977)
10.5 Lustig, V.,
Redman, L.W., Clin. Biochem. 12,254(1979)
10.6 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC
Press (1990).
10.7 Tietz, N.W.
(editor) Clinical Guide to Laboratory Tests (1983), W.B. Saunders Company,
Toronto. p. 74
10.8 Henry, R.J.,
Cannon, D.C. and Winkleman, J.W. (Editors), Clinical Chemistry Principles and
Technics, 2nd ed., Harper and Row.
10.9 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
10/99