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AMYLASE (KINETIC) PROCEDURE
Intended for the Quantitative Determination of Amylase in Human Serum and Plasma
SUMMARY AND EXPLANATION
For many years, the levels of serum and urinary amylase in patients have provided needed evidence for the diagnosis of acute pancreatitis (1). The early assays referred to as amyloclastic were based on the absorption of a complex between starch and iodine as the amylase degraded the starch. These methods proved to be very reliable and popular. Other assays using a dye-labeled amylase substrate in a buffer appeared later. These chromogenic methods were based on the color developed when the added serum sample attacks the bound complex releasing the dye which could be measured on a spectrophotometer. These methods were hard to standardize and results varied by method (1). The use of better defined substrates and indicator enzymes in the assay has led to a more consistent reagent and better correlation between methods. These type methods are based on the production of p-nitrophenol from defined oligosaccharide substrates with blocking groups attached on the terminal sugar. The action of the amylase on the oligosaccharide yields a variety of chain lengths after hydrolysis. These methods then use a variety of coupling enzymes to hydrolyze the resulting short chain oligosaccharide to produce p-nitrophenol.
The EAGLE AMYLASE (KINETIC) PROCEDURE involves the use of a chromogenic
substrate, 2-chloro-4-nitrophenol linked with maltotriose (2). The direct reaction of amylase
with the substrate results in the formation of 2-chloro-4-nitrophenol, which is monitored
spectrophotometrically at 405 nm. The reaction proceeds rapidly and can easily be automated.
No coupling enzymes are required and the reaction is not readily inhibited by endogenous
factors. The use of this substrate allows for the direct colorimetric assay of amylase using a
stable liquid reagent.
PRINCIPLE
CNP-G3 CNP + CNP-G2 + MALTORIOSE + GLUCOSE
Amylase hydrolyzes the 2-chloro-4-nitrophenyl-a-D-maltotrioside (CNPG3) to release 2-chloro-4-nitrophenol and form 2-chloro-4-nitrophenyl-a-D-maltoside (CNPG2), maltotriose and
glucose. The rate of formation of the 2-chloro-4-nitrophenol can be measured at 405 nm to give
a direct measurement ofamylase in the sample.
REAGENTS: FOR IN-VITRO DIAGNOSTIC USE
Reagent Set Cat. No. 2310 provides:
AMYLASE (KINETIC) REAGENT - Cat. No. 2311
REACTIVE INGREDIENTS:
2-chloro-4-nitrophenyl-a-D-maltotrioside, 2.25 mM; Calcium Acetate, 6 mM; Potassium
Thiocyanate, 900 mM; Stabilizers, and activator in a buffered solution, pH = 6.0
REAGENT PREPARATION:
This reagent is provided as a liquid, ready to use. No further preparation is required.
PRECAUTIONS:
1. Do not pipette by mouth to avoid contamination by salivary amylase.
2. The reagent causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amount of water.
3. Contains Sodium Azide as a preservative, when disposing of reagent, flush with a large
amount of water to prevent azide build-up in copper plumbing causing a potentially explosive
metal azide.
DETERIORATION:
The reagent should be a slightly opaque, pale yellow solution. Presence of extreme turbidity or
signs of mold growth would indicate deterioration and the reagent should not be used. In
addition, if the reagent has an absorbance of greater than 0.50 when read against DH20 at 405
nm, it should not be used.
INSTRUMENTS
Use a spectrophotometer or colorimeter calibrated at 405 nm.
SPECIMEN COLLECTION
PRECAUTIONS:
1. Serum free from hemolysis is preferred.
2. Plasma containing heparin may be used. Anticoagulants such as EDTA or citrate should not be used.
3. Separate serum from clot as soon as possible after collection.
SAMPLE STORAGE:
Serum amylase appears stable for one week at room temperature or for several months when
stored refrigerated (3).
ADDITIVES:
No special additives or preservatives are needed.
INTERFERING SUBSTANCES:
Some drugs produce increased serum amylase levels. Young, et al (4) have reviewed drug effects
on amylase levels. Contamination of the specimen or reaction mixture will cause interference in
the assay.
PROCEDURE
MATERIALS PROVIDED:
AMYLASE (KINETIC) REAGENT (Cat. No. 2311) in a stable, liquid form.
MATERIALS REQUIRED BUT NOT PROVIDED:
1. 0.025 mL micropipettor
2. 1.0 pipettor
3. Incubator capable of maintaining 37ºC
4. Timer or stopwatch
5. Instrument calibrated at 405 nm and is capable of maintaining 37ºC in cuvette assay well
6. Test tubes and rack
7. Normal saline
REACTION CONDITIONS:
Wavelength 405 nm
Reaction Type Kinetic
Prewarm Substrate 5 minutes
Reaction Temperature 37º C
Preincubation Time 1 minute
Reaction Time 1 minute
Sample Volume 0.025 mL
Reagent Volume 1.00 mL
Total Volume 1.025 mL
Low Normal 25 U/L
High Normal 125 U/L
Calibration Factor 3178
AUTOMATED PROCEDURE:
Refer to specific instrument application for instructions.
MANUAL PROCEDURE:
PROCEDURAL NOTE: For instruments requiring >>1.0 mL total volume to read,
increase reagent volume to 2.0 mL and sample volume to 0.05 mL and proceed as directed.
1. Dispense 1.0 mL AMYLASE (KINETIC) REAGENT into tubes labeled Reagent Blank, Control, Sample 1, etc.
2. Prewarm the tubes at 37º C for 5 minutes.
3. Place 0.025 mL Specimen into appropriately labeled tube and mix well. Use Saline for Specimen in Reagent Blank.
4. Incubate at 37º C for 1 minute (60 seconds).
5. After the 1 minute preincubation, record the absorbance (A1 reading). Return the tube to the 37º C incubator.
6. After exactly 1 minute (60 seconds), read and record the absorbance (A2 reading). It is recommended that a second one minute reading be taken for better accuracy. Average the two readings to obtain change per minute.
7. Repeat procedure for each sample to be assayed.
NOTE: If the instrument used is equipped with a temperature controlled cuvette, the reaction
mixture may be left in the cuvette well while the readings are taken.
PROCEDURAL NOTE:
Linearity extends to 2000 U/L. Specimens exceeding the value should be diluted with 0.9%
sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain
the final answer.
STABILITY OF FINAL REACTION:
The reaction in ongoing. The timing of the readings must be accurate.
CALIBRATION:
The Amylase activity is calculated based on the millimolar absorptivity of 2-chloro-p-nitrophenol, which varies with pH, temperature and wavelength. The millimolar absorptivity
when measured at 405 nm, pH 6.0 and 37ºC is 12.9.
CALCULATION OF RESULTS
Calculate the amylase activity of each sample using the following formula:
amylase (U/L) =
(As / min - Ab / min) x T.V. x 1000
-------------------------------------------------------
E x S.V. d
Where:
As / min = Change in absorbance per minute of the Sample or Control.
Ab / min = Change in absorbance per minute of the Reagent Blank
T.V. = Total Volume of assay (1.025 mL)
1000 = Conversion factor (U/mL to U/L)
E = 12.9 (millimolar extinction coefficient of 2-chloro-4-nitrophenol at 405 nm)
S.V. = Sample Volume (0.025 mL)
d = Light path (1 cm)
or amylase (U/L) = (As / min - Ab / min) x 317
EXAMPLE:
Reagent Blank Ab / min = 0.006
Sample As / min = 0.026
U/L aAmylase - (0.026 - 0.006) x 3178 = 63.56 U/L
QUALITY CONTROL:
The reliability of test results should be monitored routinely using suitable quality control
materials (normal and abnormal) analyzed in the same manner as the Unknowns. EAGLE
DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL
ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve values of freshly prepared
control sera should be thoroughly investigated before patient values are reported.
LIMITATION
1. Amylase enzyme activity is temperature dependent. Assays must be performed at 37º C + 0.5.
2. Macroamylase will show increased activity when using an oligosaccharide substrate (5).
EXPECTED VALUES (3)
25 - 125 U/L
This range represents the 95% confidence interval from a clinically normal population. Each laboratory should establish its own range of expected values.
PERFORMANCE CHARACTERISTICS
LINEARITY:
This method is linear to 2000 U/L.
PRECISION:
Normal and abnormal control sera were assayed 20 times each for within run precision and for
10 working days to establish run to run precision.
WITHIN RUN
MEAN / ST. DEV. / %CV
Normal 54.5 / 0.75 / 4.6
Abnormal 1184 / 8.95 / 4.4
RUN TO RUN
Normal 52.7 / 0.95 / 5.1
Abnormal 1125 / 8.84 / 4.2
SPECIFICITY:
A comparison of the AMYLASE (KINETIC) PROCEDURE with another commercially
available method showed a correlation of 99.8%. The regression equation was y = 1.12x - 1.1.
SENSITIVITY:
This AMYLASE (KINETIC) PROCEDURE has a sensitivity of 3 U/L per 0.001 absorbance unit.
REFERENCES
1. Tietz, N.W., Textbook of Clinical Chemistry, 2nd ed., W.B. Saunders Co.,Philadelphia, 1994, p. 854.
2. Chavez, R.G., et al, U.S. Patent No. 4,963,479.
3. Tietz, N.W., Clinical Guide to Laboratory Tests, 2nd ed., W.B.Saunders Co., Philadelphia, 1990, p.50.
4. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin Chem Vol. 21,
5. Rosenblum, J.L., et al, Clin Chem Vol. 38, 1992, p. 9.
