Infinity™

Amylase Liquid Stable Reagent

PRODUCT SUMMARY Symbols in Product Labelling
Stability : Until Expiry at 2-8°C Authorized Representative Temperature Limitation
Linear Range : 10 - 2000 U/L (0.167 - 33.4 µkat/L)
For in vitro diagnostic use Use by/Expiration Date
Specimen Type : Serum or Urine
Method : Kinetic Batch code/Lot number CAUTION. Consult instructions
Reagent Preparation : Supplied ready to use. for use.
Catalogue number
Consult instructions for use Manufactured by

Prescription Use Only

INTENDED USE CAUTION: This product contains animal source material. Handle and dispose of this product
This reagent is intended for the in vitro diagnostic use only, for the quantitative as if it were potentially infectious.
determination of α‑Amylase (1,4-α-D-glucan glucanohydrolase EC3.2.1.1) in human
serum or urine on both manual and automated systems. REAGENT PREPARATION
Reagent is supplied ready to use.
CLINICAL SIGNIFICANCE 1, 2, 3

α-Amylase is derived mainly from the salivary glands and the exocrine pancreas. STABILITY AND STORAGE
α-Amylase catalyses the hydrolysis of α-1 → 4 glucosidic linkages of starch and Prior to Use:
other related polysaccharides to produce maltose and other oligosaccharides. The When stored at 2-8°C reagent is stable until the expiration date stated on the bottle and
enzyme is a relatively small molecule which is rapidly cleared by the kidneys and kit box label.
excreted in the urine. Once the Reagent is Opened:
α-Amylase is most frequently measured in the diagnosis of acute pancreatitis when When stored capped at 2-8°C, the reagent is stable until expiry.
serum levels may be grossly elevated. In acute pancreatitis α-amylase starts to Indications of Reagent Deterioration:
rise approximately 4 hours after the onset of pain, reaches a peak at 24 hours and • Turbidity;
remains elevated from 3‑7 days. • Reagent Absorbance >1.0 AU at 405nm (1cm); and/or
Hyperamylasaemia is also associated with other acute abdominal disorders, biliary • Failure to recover control values within the assigned range.
tract disease, diabetic ketoacidosis, severe glomerular dysfunction, salivary gland
disorders, ruptured ectopic pregnancy and macroamylasaemia. SPECIMEN COLLECTION AND HANDLING
Serum: Use non-haemolysed serum.4
METHODOLOGY Urine: Random or timed collections are valid specimens.4
Several procedures are available for the assay of serum α-amylase activity. Storage: α-Amylase is exceptionally stable and serum samples may be stored for at least
Amyloclastic methods measure the disappearance of substrate and include the 7 days at room temperature and for at least 1 month at 4°C or -20°C.2 Urine samples are
iodine-starch method. Saccharogenic methods measure the production of sugars stable for 7 days when stored at 4°C. If it is expected that there will be a delay in transporting
such as maltose and glucose. Both these methods lack linearity, sensitivity and the urine sample to the laboratory, the use of a chemical preservative such as Merthiolate
precision when compared to chromogenic methods which yield a colored product (0.24 mmol/L) is recommended.5
that can be measured spectrophotometrically.2
The Infinity Amylase Liquid Stable Reagent utilises Ethylidene-pNP-G7 (E-pNP-G7) ADDITIONAL EQUIPMENT REQUIRED but not provided
as the substrate. The use of ethylidene prevents exo-enzymes from breaking down • A clinical chemistry analyser capable of maintaining constant temperature (37°C) and
the substrate, so in the absence of α-amylase, no color change is observed. The measuring absorbance at 405nm (405 - 420nm).
substrate is also commonly referred to as EPS. Once the substrate has been cleaved • Analyser specific consumables, eg: sample cups.
by a-amylase, the smaller fragments produced can be acted upon by α‑glucosidase, • Normal and Abnormal assayed control material.
which causes the ultimate release of the chromophore.
assay procedure
The series of reactions involved in the assay system is as follows: The following system parameters are recommended. Individual instrument applications are
available upon request from the Technical Support Group.
1. 5 E-pNP-G7 + 5H2O
SYSTEM PARAMETERS
α amylase Temperature 37°C
Primary Wavelength 405 nm (405-420nm)
E-G3 + pNP-G4 + 2E-G4 + 2pNP-G3 + 2pNP-G2 Secondary Wavelength 480-600nm
Assay Type Rate/Kinetic
2. pNP-G4 + 2pNP-G3 + 2pNP-G2 + 14H2O Direction Increase
Sample : Reagent Ratio 1:40
α glucosidase eg : Sample Vol 7 µL
Reagent Vol 280 µL
5 pNP + 14G Delay/Lag Time 60 seconds
Read Time 2 minutes
Where: G = Glucose Reagent Blank Limits Low 0.0 AU
pNP = p-nitrophenol (405nm, 1cm lightpath) High 1.0 AU
Linearity 10 - 2000 U/L
The rate of formation of pNP is proportional to the α-amylase activity present in (refer to linearity section) (0.167 - 33.4 µkat/L)
the sample and is measured by the rate of increase in absorbance at 405 nm Analytical Sensitivity 0.195 ∆mA/min per U/L
(405-420nm). (405nm, 1cm lightpath) (11.7 ∆mA/min per µkat/L)

REAGENT COMPOSITION CALCULATIONS

Active Ingredients Concentration Results are calculated, usually automatically by the instrument, as follows:
E-pNP-G7 1.1 mmol/L
α-Glucosidase (microbial) >3500 U/L Activity in U/L = DAbs/min x Factor
NaCl 51 mmol/L TV x 1000 x 1.27
Buffer 50 mmol/L Factor = —————————
SV x E x P
pH 7.0 ± 0.1 at 20°C.
where:
WARNING: Do not ingest. Avoid contact with skin and eyes. If spilt, thoroughly TV = Total reaction volume in mL
wash affected areas with water. Reagent contains sodium azide which may react 1.27 = Method Conversion Factor (See note 2).
with copper or lead plumbing. Flush with plenty of water when disposing. For SV = Sample Volume in mL
further information consult the Infinity Amylase Liquid Stable Reagent Material E = millimolar extinction coefficient of pNP at 405nm
Data Safety Sheet. = 10.13 (See note 1).
P = Cuvette pathlength.
 Imprecision was evaluated over a period of 20 days using two levels of commercial control
Example: ∆Abs/min = 0.02 and following the NCCLS EP5-T procedure.9
Factor = 5140 LEVEL I LEVEL II
Amylase = 0.02 x 5140 = 103 U/L Number of data points 80 80
Mean (U/L / µkat/L) 42 / 0.701 341 / 5.69
NOTES Within Run: SD (U/L / µkat/L) 2.2 / 0.037 3.2 / 0.053
1. The millimolar extinction coefficient for pNP in this reagent system at 410nm CV (%) 5.3 0.9
= 9.80, 415nm = 9.17 and at 420nm = 8.30. Total: SD (U/L / µkat/L) 3.4 / 0.057 8.9 / 0.149
2. With this factor the values obtained with the Infinity Amylase Liquid Stable CV (%) 8.1 2.6
Reagent at 405nm/37°C are comparable to those obtained with the previously
available blocked PNPG7 method. METHOD COMPARISON:
3. The reagent and sample volumes may be altered proportionally to Comparison studies were carried out using another similar commercially available α-amylase
accommodate different spectrophotometer requirements. reagent. Serum and urine samples were assayed in parallel and the results compared by
4. If the change in absorbance is greater than 0.39/min repeat the assay with least squares regression. The following statistics were obtained.
less sample or dilute with saline. Remember to adjust the factor for the smaller Serum:
sample volume or multiply the final result by the dilution factor. Number of sample pairs 60
5. Valid results depend on accurately calibrated instrument, timing and Range of sample results 24 - 465 U/L (0.401 - 7.77 µkat/L)
temperature control. Mean of reference method results 67 U/L (1.12 µkat/L)
6. Unit conversion: U/L x 16.67 x 10-3 = µkat/L. Mean of Amylase Infinity results 69 U/L (1.15 µkat/L)
Slope 0.9425
CALIBRATION Intercept 1.8 U/L (0.030 µkat/L)
Calibration is not required. The rate of reaction is converted to U/L of activity by a Correlation coefficient 0.9970
calculation factor. Refer to the calculation section of this package insert. Urine:
Number of sample pairs 41
QUALITY CONTROL Range of sample results 5 - 595 U/L (0.084 - 9.94 µkat/L)
To ensure adequate quality control, normal and abnormal control with assayed Slope 1.187
values should be run as unknown samples:- Intercept -7.6 U/L (0.127 µkat/L)
• At least once per day or as established by the laboratory. Correlation coefficient 0.9953
• When a new bottle of reagent is used.
• After preventative maintenance is performed or a critical component is replaced. LINEARITY:
Control results falling outside the upper or lower limits of the established ranges When run as recommended, the assay is linear between 10- 2000 U/L (0.167 - 33.4 µkat/L).
indicate the assay may be out of control. The following corrective actions are Linearity on automated instruments will be dependent upon the ratio of sample volume to
recommended in such situations:- reagent volume used and the timing of measurements. The specific instrument application
• Repeat the same controls. should be consulted.
• If repeated control results are outside the limits, prepare fresh control serum
and repeat the test. ANALYTICAL SENSITIVITY:
• If results on fresh control material still remain outside the limits, then repeat When run as recommended the sensitivity of this assay is 0.195 ∆mA/min per U/L (11.7
the test with fresh reagent. ∆mA/min per µkat/L).
• If results are still out of control, contact Technical Services or your local
distributor. REFERENCES
1. JF Zilva and PR Pannall. “Plasma Enzymes in Diagnosis” in Clinical Chemistry in
LIMITATIONS Diagnosis and Treatment. Lloyd-Luke London 1979: Chapter XV: 341-2
1. Studies to determine the level of interference from bilirubin (free & conjugated), 2. Foo YA and Brosalki SB. Ann Clin. Biochem 1986; 23:624-37
haemoglobin and lipaemia were carried out using commercially available 3. Bais R. Am. Jnl of Clin. Path 1982; 78: 184-8
interference check products. The following results were obtained: 4. Clinical Chemistry Infobase: A Scientific & Management Cyclopedia. Pesce-Kaplan
Haemoglobin: No interference from haemoglobin up to a level of 522 mg/dL. Publishers 1996; 2619-2620.
Free bilirubin: No interference from free bilirubin up to a level of 265µmol/L 5. Shepherd MDS and Mazzachi RD. The Clin. Biochem 1983; 4: 61-7
(15.5mg/dL). 6. Young DS. Effects of Drugs on Clinical Laboratory Tests Third Edition 1990; 3: 34-6.
Conjugated bilirubin: No interference from conjugated bilirubin up to a level 7. Tietz Textbook of Clinical Chemistry. Burtis CA and Ashwood ER (Eds). Second Edition,
of 286µmol/L (16.7mg/dL). WB Saunders Company, 1994;2178.
Lipaemia: No interference from lipaemia, measured as an absorbance at 8. Wachtel M et al, Creation and Verification of Reference Intervals. Laboratory Medicine
630nm, up to 1.045AU. 1995; 26:593-7.
2. Young DS6 has published a comprehensive list of drugs and substances which 9. National Committee for Clinical Laboratory Standards. User evaluation of Precision
may interfere with this assay. Performance of Clinical Chemistry Devices. NCCLS, 1984, NCCLS Publication
3. To avoid the possibility of contamination with α-amylase, please ensure that EP5-T.
the reagent does not come in to contact with saliva and skin.

EXPECTED VALUES
Serum:* At 37°C 35 - 140 U/L (0.585 - 2.34 µkat/L)
At 30°C 27 - 108 U/L (0.451 - 1.80 µkat/L)

Urine: 1 - 17 U/hour (0.017 - 0.284 µkat/hour)7

*The quoted values were derived from a study of 59 normal samples and should
serve as a guide only. The 30°C values were calculated as a guide only, using a
temperature conversion factor of 0.77. We do not however recommend the routine
use of temperature conversion factors. It is recommended that each laboratory
verify this range or derives a reference interval for the population that it serves.8

PERFORMANCE DATA
The following data was obtained using the Infinity Amylase Liquid Stable Reagent
on a well maintained automated clinical chemistry analyser.

IMPRECISION:

Fisher Diagnostics
a division of Fisher Scientific Company, LLC © 2012 Thermo Fisher Scientific Inc. All rights reserved. Hitachi is a registered trademark of
Roche Diagnostics, Indianapolis, IN 46250. ILab 600 is a registered trademark of Instrumentation
a part of Thermo Fisher Scientific Inc. Laboratory Company, Lexington, MA 02421. All other trademarks are the property of Thermo
Middletown, VA 22645-1905 USA Fisher Scientific Inc. and its subsidiaries.
Phone: 800-528-0494
540-869-3200
Fax: 540-869-8132 Reorder Information
Catalogue No. Configuration
WMDE TR25421 2 x 125 mL
Bergerweg 18
6085 AT Horn
The Netherlands

JL840761-en (R1)