1.0 INTENDED USE
This reagent is intended for the quantitative
determination of a‑Amylase activity in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
The classic method of determining a‑Amylase
activity is enzymatic hydrolysis of a starch substrate followed by product
analysis. The saccharogenic analysis
method (10.2,10.3) measures the quantity of reducing sugars formed. There are difficulties associated with this
method such as inconsistent results due to variations in substrate preparation
and treatment.
Other methods of analysis include the dye‑starch
substrate methods(10.4) and the amyloclastics methods (10.5). Each has drawbacks (10.6,10.7). Modified saccharogenic methods have been
introduced recently which utilize a defined oligosaccharide substrate. These substrates produce colorimetric
products when coupled with p‑nitrophenyl.
Wallenfels et al(10.11) introduced p-nitrophenylglycosides as defined
substrates for a-amylase determination in a procedure that eliminates
interference from endogenous glucose and pyruvate.
2.2 TEST PRINCIPLE
The present method is based on the use of a chromogenic
substrate, 2-chloro-p-nitrophenol linked with maltotriose. The reaction of amylase with this substrate
results in the formation of 2-chloro-p-nitrophenol, which can be measured
spectrophotometrically at 405nm. This
reaction proceeds very rapidly, no coupling enzymes are required, and the
reaction is not readily inhibited by endogenous factors.
a-amylase
10 CNPG3
----‑‑--------‑----->
9CNP + CNPG2 + 9G3 + G
a-Amylase hydrolyzes the
2-chloro-p-nitrophenyl-a-D-malthoheptaoside (CNPG3) to release
2-chloro-nitrophenol and form 2-chloro-p-nitrophenyl-a-D-maltoside (CNPG2),
maltotriose (G3) and glucose (G). The
rate of increase in absorbance is measured at 405nm and is proportional to the
a-amylase activity in the sample.
2.3 CLINICAL SIGNIFICANCE
Assay for the a‑Amylase activity are of interest
for the evaluation of pancreatic function of the diagnosis of pancreatic
disease. The greatest elevation in serum
a‑Amylase activity is seen in acute pancreatitis and obstruction of
pancreatic duct (10.1).
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
3.2.1 Unhemolyzed
serum is the specimen of choice.
Specimens should be colleted as per NCCLS document H4-A3(10.12).
3.2.2 Anticoagulants,
such as Citrate and EDTA, bind calcium which is needed for amylase
activity. Therefore, plasma with these
anticoagulants should not be used.
3.3 SPECIMEN STORAGE
Amylase in serum is reported stable for one week at room
temperature (18-26°C) and for two months when stored refrigerated at
2-8°C.(10.1)
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 (1 X 60 ml)
(2 X 125 ml)
Reagents necessary for the determination of a‑Amylase
are included in the kit.
4.1
REAGENT
a‑Amylase reagent contains:
MES Buffer ph
6.0 ± 0.1
2-Chloro-p‑nitrophenyl‑a‑D‑maltotrioside 1.8 mM
sodium chloride 350
mM
calcium acetatee 6
mM
Potassium
thiocyanate 900
mM
sodium azide as
preservative 0.01%
4.2 WARNINGS AND PRECAUTIONS
4.2.1 This reagent
kit is intended for in vitro diagnostic use only.
4.2.2 This reagent
contains potassium thiocyanate.
POISON. Do not ingest.
4.2.3 This reagent
contains sodium azide (0.01%) as a preservative. Do not ingest. May react with lead and copper plumbing to
form highly explosive metal azides. Upon
disposal, flush with a large volume of water to prevent azide build up.
4.2.4 All specimens
and controls should be handled as potentially infectious. using safe laboratory
procedures. (NCCLS M29-T2)(10.13).
4.3 REAGENT PREPARATION
The reagent is provided as a ready-to-use liquid. No preparation is required.
4.4 REAGENT STORAGE AND STABILITY
4.4.1 Store reagent
at 2°-8°C.
4.4.2 The reagent
is stable until the expiration date if stored as directed. When stored at 2°-8°C unopened reagents are
stable until the expiration date printed on the label.
4.4.3 Do not use if
the absorbance of the reagent is greater than 0.600 when measured at 405nm
against water in a cuvette with a 1cm path length or if the reagent fails to
meet stated parameters of performance.
4.4.4 Do not use if
the reagent is turbid or displays other evidence of bacterial contamination.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 A
spectrophotometer or colorimeter capable of reading absorbance accurately at
405 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 405 nm.
4.5.3 Test tubes
capable of holding 2 ml.
4.5.4 Pipettes
capable of delivering 1 ml and 25 µl.
4.5.5 Timer with 30
second increments.
4.5.6 Constant
temperature heat source which can be adjusted to 37° C.
4.5.7 King 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 405
nm
Temperature 37°
C
Pathlength 1
cm
Mode Kinetic
Reaction Time 2
min.
Sample Volume 25
µl
Reagent volume 1.0
ml
Total Volume 1.025
ml
Sample to reagent
ratio 1/40
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 405 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 method is
standardized by means of the molar absorptivity of 2-chloro-p‑nitrophenyl
taken as 12.9 at 405nm under the test conditions described.
5.4 PROCEDURE
5.4.1 Pipette 1.0ml
of reagent into tubes labelled "control", "patient",
etc. DO NOT PIPETTE BY MOUTH.
5.4.2 Pre-incubate
all tubes at 37°C for at least five minutes.
5.4.3 Zero
spectrophotometer with water at 405nm.
5.4.4 Add 0.025ml
(25µl) of sample and read after 30 seconds.
5.4.5 Record the
absorbance at 30 second intervals for 2 minutes.
5.5 CALCULATION AND RESULTS
Amylase
U/L =
DA/min
X assay volume (ml) X 1000
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑-------------
= DA/min X 3178
12.9 X light path (cm) X sample volume
(ml)
DA/min = change in absorbance per minute
assay
volume = 1.25 (ml)
1000 =
converts U/ml to U/L
12.9 =
absorbance coefficient of 2-chloro-p‑nitrophenyl at 405 nm
lightpath
= 1 (cm)
sample
volume = 0.025 (ml)
3178 =
factor derived from constants in the equation
Example:
0.019
X 1.02 X 1000
Amylase
U/L = ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑-------
= 0.019 X 3178 = 60 U/L
12.9
X 1 X 0.02
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.9)
The range
of expected values is:
25
‑ 125 U/L (37 degrees 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 (10.10)
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
Young (10.8) gives a list of drugs and other substances
that interfere with the determination of amylase activity.
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.
Biotron 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 routine reagent calibration is necessary as this
method is standardized by means of the molar absorptivity of 2-chloro-p‑nitrophenyl
taken as 12.9 at 405nm under the test conditions described.
The results obtained when measuring the activity of a
kinetic reaction are based on the change in absorbance per minute. In order to accurately monitor and report
this reaction rate, the operating parameters of the spectrophotometer
(wavelength, temperature of the reaction and timing of the test) must be known
and controlled.
9.0 PERFORMANCE CHARACTERISTICS
9.1 PRECISION
The estimates of precision shown below were performed
following the guidelines contained in NCCLS document EP5-T2(10.14).
Within-Run
Mean
(U/L) SD
(U/L) CV (%)
14 ±
0.5 3.6
106 ±
0.7 0.7
418 ±
1.1 0.3
1392 ± 7.8 0.6
Between-Run
Mean
(U/L) SD
(U/L) CV (%)
15 ±
0.7 4.7
109 ±
2.3 2.1
421 ±
5.6 1.3
1413 ± 16.6 1.2
9.2 CORRELATION
A correlation study was done comparing this method (y)
and a similar comparative method (x).
The samples (n=125) ranged from 32 to 2112 U/L. The study yielded a regression curve of x =
0.98x + 5.4 with a correlation of 0.999.
Note: CNPG3 values greater than 2000 U/L were run after
dilution with an equial volume of saline.
9.3 LINEARITY
Linearity: This
procedure is linear through 2000 U/L beyond which the specimen should be
diluted with an equal volume of saline.
Reassay the specimen and multiply the results by 2.
9.4 SENSITIVITY
A change in absorbance of 0.003 DA/min at 405nm at 37° C corresponds to 9.53 U/L.
10.0 REFERENCES
10.1 Tietz, N.W.,
(Editor) Fundamentals of Clinical Chemistry, W.B. Saunders Company,
Philadelphia (1982).
10.2 Somogyi, M.,
J. Biol. Chem., 125:399 (1938).
10.3 Henry, R.J.,
Chiamori, N., Clin, Chem., 6:434 (1960).
10.4 Klein, B.,
Foreman, J., Searry, R., Clin. Chem., 16:32 (1970).
10.5 Somogyl, M.,
Clin. Chem., 6:23 (1960).
10.6 Kaufman,
R.A., Tietz, N.W., Clin. Chem., 26:846 (1980).
10.7 Rauscher, E.,
Neumann, U., Schaich, E., von Bulow, S., Wahlefeld, A.W., Clin. Chem., 31:14
(1985).
10.8 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC
Press (1990).
10.9 Tietz, N.E.,
(Editor) Clinical Guide to Laboratory Tests, W.B. Saunders Company, Philadelphia
(1983) p. 54.
10.10 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
10.11 Wallenfels,
K., et al, Carbohydrate Research 61:359 (1978).
10.12 NCCLS
documents "Procedures for the Collection of Diagnostic Blood Specimens by
Abin Puncture" 3rd Ed (1991).
10.13 NCCLS
documents "Protection fo Laboratory Workers from Infectious Disease
Transmitted by Blood, Body Fluids and Tissues" 2nd Ed (1991).
10.14 NCCLS document
"Evaluation of Precision Performance of Clinical Chemistry Devices"
2nd Ed (1992).