COD 11582 COD 11534 α-AMYLASE-EPS

1 x 25 mL 1 x 40 mL
STORE AT 2-8ºC
Reagents for measurement of α-amylase concentration α-AMYLASE-EPS
Only for in vitro use in the clinical laboratory IFCC

PRINCIPLE OF THE METHOD These ranges are given for orientation only; each laboratory should establish its own reference
ranges.
Amylase catalyzes the hydrolysis of 4-nitrophenyl-maltoheptaoside-ethylidene to smaller
oligosacharides which are hydrolyzed by α-glucosidase liberating 4-nitrophenol. The catalytic QUALITY CONTROL
concentration is determined from the rate of 4-nitrophenol formation, measured at 405 nm1,2 .
It is recommended to use the Biochemistry Control Serum level I (cod. 18005, 18009 and
CONTENTS 18042) and II (cod. 18007, 18010 and 18043) to verify the performance of the measurement
COD 11582 COD 11534 procedure.

A. Reagent 1 x 20 mL 1 x 32 mL
Each laboratory should establish its own internal Quality Control scheme and procedures for
B. Reagent 1 x 5 mL 1 x 8 mL corrective action if controls do not recover within the acceptable tolerances.

METROLOGICAL CHARACTERISTICS
COMPOSITION
− Detection limit: 3.0 U/L = 0.05 µkat/L.
A. Reagent. HEPES 50 mmol/L, calcium chloride 0.075 mmol/L, sodium chloride 90 mmol/L,
magnesium chloride 13 mmol/L, α-glucosidase > 4 U/mL, pH 7.1. − Linearity limit: 1300 U/L = 21.6 µkat/L (serum and plasma) and 2600 U/L = 43.2 µkat/L
B. Reagent. HEPES 50 mmol/L, 4-Nitrophenyl-maltoheptaoside-ethylidene 18 mmol/L, pH 7.1. (urine). For higher values dilute sample 1/5 with distilled water and repeat measurement.
− Repeatibility (within run):
STORAGE
Serum. Mean Concentration CV n
Store at 2-8ºC.
70 U/L 1.3% 20
Reagents are stable until the expiry date shown on the label when stored tightly closed and if
666 U/L 0.6% 20
contaminations are prevented during their use.
Indications of deterioration: Urine. Mean Concentration CV n
− Reagents: Presence of particulate material, turbidity, absorbance of the blank over 0.300 at 460 U/L 0.7% 20
405 nm (1 cm cuvette). 950 U/L 0.6% 20

REAGENT PREPARATION − Reproducibility (run to run):

Working Reagent: Pour the contents of the Reagent B into the Reagent A bottle. Mix gently.
Other volumes can be prepared in the proportion: 4 mL Reagent A + 1 mL Reagent B. Stable for Serum. Mean Concentration CV n
20 days at 2-8ºC. 70 U/L 1.9% 25
666 U/L 1.7% 25
ADDITIONAL EQUIPMENT
− Analyzer, spectrophotometer or photometer with cell holder thermostatable at 30 or 37ºC and Urine. Mean Concentration CV n
able to read at 405 nm. 460 U/L 0.8% 25
− Cuvettes with 1 cm light path. 950 U/L 1.2% 25

SAMPLES − Sensitivity: 0.309 ∆mA⋅L/U⋅min = 18.6 ∆mA ⋅L/µkat⋅min.

Serum, plasma or urine collected by standard procedures. − Trueness: Results obtained with this reagent did not show systematic differences when
α-Amylase in serum or plasma is stable for 1 month at 2-8ºC. Use heparin or EDTA as compared with reference reagents. Details of the comparison experiments are available on
anticoagulant. request.
α-Amylase in urine is stable for 1 month at 2-8ºC if pH is adjusted to approximately 7 before − Interferences: Lipemia (triglycerides 10 g/L) and bilirubin (20 mg/dL) do not interfere.
storage. Hemoglobin (10 g/L) interfere. Other drugs and substances may interfere4.

PROCEDURE These metrological characteristics have been obtained using an analyzer. Results may vary if a
different instrument or a manual procedure are used.
1. Bring the Reagent and the instrument to reaction temperature.
2. Pipette into a cuvette: (Notes 1,2) DIAGNOSTIC CHARACTERISTICS
Serum or plasma Urine α-Amylase catalyzes the hydrolysis of α-1,4-linkages of carbohydrates constituted of α-D-
37ºC 30ºC 37ºC 30ºC glucose units. The result is the formation of dextrins, maltose and some glucose molecules. α-
Working Reagent 1.0 mL 1.0 mL 1.0 mL 1.0 mL Amylase is produced mainly by the exocrine pancreas (P-type) and the salivary glands (S-type)
Sample 30 µL 60 µL 15 µL 30 µL but it is also found in other tissues.
Assays of amylase activity in serum and urine are largely of use in the diagnosis of pancreatic
3. Mix and insert the cuvette into the photometer. Start the stopwatch. diseases such as acute or chronic pancreatitis. Hyperamylasemia can also be due to renal
4. Record initial absorbance and at 1 minute intervals thereafter for 3 minutes. insufficiency, acute pain of the abdomen, tumors of the lungs and the ovaries, salivary glands
5. Calculate the difference between consecutive absorbances, and the average absorbance lesions, macroamylasemia, diabetic ketoacidosis, biliary tract disease, cerebral trauma, chronic
difference per minute (∆A/min). alcoholism and drugs (opiates)5,6.

CALCULATIONS Clinical diagnosis should not be made on the findings of a single test result, but should integrate
both clinical and laboratory data.
The α-Amylase concentration in the sample is calculated using the following general formula:
Vt x 106 NOTES
∆A/min x = U/L 1. Saliva and skin do contain α-amylase, therefore never pipette by mouth and avoid skin
ε x I x Vs contact with the reagents.
The molar absorbance (ε) of 4-nitrophenol at 405 nm is 10600 and the lightpath (l) is 1 cm. For 2. This reagent may be used in several automatic analysers. Instructions for many of them
serum and plasma samples, the total reaction volume (Vt) is 1.030 at 37ºC and 1.060 at 30ºC are available on request.
and the sample volume (Vs) is 0.030 at 37ºC and 0.060 at 30ºC. For urine samples, the total
reaction volume (Vt) is 1.015 at 37ºC and 1.030 at 30ºC and the sample volume (Vs) is 0.015 at BIBLIOGRAPHY
37ºC and 0.030 at 30ºC. 1 U/L are 0.0166 µkat/L. The following formulas are deduced for the 1. Lorentz K. Approved recommendation on IFCC methods for the measurement of calytic
calculation of the catalytic concentration: concentration of enzymes part 9. IFCC method for α-amylase (1,4-α-D-glucan 4-
37ºC 30ºC glucanohydrolase, EC 3.2.1.1). Clin Chem Lab Med 1998;36:185-203.
x 3239 = U/L x 1667 = U/L 2. Lorentz K. Routine α-amylase assay using protected 4-nitrophenyl-1,4-α-D-maltoheptaoside
Serum, plasma
x 53.8 = µkat/L x 27.7 = µkat/L and a novel α-glucosidase. Clin Chem 2000;46:644-649.
∆A/min
x 6384 = U/L x 3239 = U/L 3. Junge W, Werner W, Wilke B et al. Development and evaluation of assays for the
Urine
x 105.9 = µkat/L x 53.8 = µkat/L determination of total and pancreatic amylase at 37 ºC according to the principle
recommended by the IFCC. Clin Biochem 2001;34:607-615.
REFERENCE VALUES 4. Young DS. Effects of drugs on clinical laboratory tests, 4th ed. AACC Press, 1995.
Reaction Serum, plasma Urine 5. Tietz Textbook of Clinical Chemistry, 2nd edition. Burtis CA, Ashwood ER. WB Saunders
temperature U/L µkat/L U/L µkat/L Co., 1994.
30ºC, up to1 25-65 0.41-1.08 - - 6. Friedman and Young. Effects of disease on clinical laboratory tests, 3th ed. AACC Press,
37ºC, up to3 28-100 0.47-1.67 16-491 0.26-8.15 1997.

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