BIOLABO

www.biolabo.fr AMYLASE E-PNPG7 Method

MANUFACTURER:
BIOLABO SAS, Reagent for quantitative determination of alpha-amylase
[EC 3.2.1.1] in human serum and plasma, or urines
Les Hautes Rives
02160, Maizy, France REF 80023 R1 20 x 3 mL
REF 80123 R1 8 x 20 mL
REF 80223 R1 10 x 100 mL

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TECHNICAL SUPPORT AND ORDERS
Tel: (33) 03 23 25 15 50
IN VITRO DIAGNOSTIC
Fax: (33) 03 23 256 256

CLINICAL SIGNIFICANCE (1) (2) SAFETY CAUTIONS

α-amylase is most frequently measured in the diagnostic of acute BIOLABO reagents are designated for professional, in vitro diagnostic
pancreatitis. In this case, a transient rise in serum amylase activity use.
occurs within 2 to 12 h of the onset and maximal levels are attained 12 • Verify the integrity of the contents before use.
to 72 h later. However, elevation of α-amylase activity in serum is also • Use adequate protections (overall, gloves, glasses). Do not pipette
associated with other disorders (abdominal disorders, biliary tract with mouth.
diseases, diabetic ketoacidosis, severe glomerular dysfunction, • Avoid contact with skin and eyes. If spilt, thoroughly wash affected
salivary glands disorders…). The organ source can sometimes be areas with plenty of water.
identified by determining whether the major isoenzyme present is type • Reagents contain sodium azide (concentration < 0.1%) which may
P (pancreatic) or S (salivary). Diagnostic specificity and sensitivity of react with copper and lead plumbing. Flush with plenty of water
elevation of alpha-amylase activity in urine remain disputed. Renal when disposing.
clearance of amylase, as related to the reasonably constant clearance • Material Safety Data Sheet is available upon request.
of creatinine, has been found useful as a diagnostic concept. • Waste disposal: Respect legislation in force in the country.
All specimens should be handled as potentially infectious, in
accordance with good laboratory practices using appropriate
PRINCIPLE (4)
precautions. Respect legislation in force in the country.
Several procedures are available to assay alpha-amylase activity
(Amyloclastic methods, saccharogenic methods). Both these methods
lake linearity, sensitivity and precision when compared to
REAGENT PREPARATION
E-PNPG7 method. Reaction scheme is as follows: REF 80023: Use a non-sharp instrument to remove aluminium cap.
Add promptly the amount of demineralised water stated on the label.
1) 5 E-PNPG7 + 5 H2O Avoid contamination with salivary amylase.
Mix gently and wait for complete dissolution before using reagents
α-amylase (approximately 2 min.).
E-G3 + 1 pNP-G4 + 2 E-G4 + 2 pNPG3 + 2 E-G5 + 2 pNPG2
STABILITY AND STORAGE
Store away from light, well cap in the original vial at 2-8°C.
2) 1 pNP-G4 + 2 pNPG3 + 2 pNPG2 + 14 H2O
• Unopened, reagents are stable until expiry date stated on the label.
α-glucosidase of the kit when stored and used as described in the insert.
• Once reconstituted, working reagent is stable for 3 months when free
5 pNP + 14 G from contamination.
• Discard working reagent if cloudy or if absorbance measured at
E = Ethyliden, PNP = Paranitrophenol, G = Glucose
405 nm is > 0.600.
The rate of formation of pNP (p-Nitrophenol), directly proportional to • Don’t use working reagent after expiry date stated on the label of the
the α-amylase activity in the specimen, is measured at 405 nm. Kit.

REAGENT COMPOSITION SPECIMEN COLLECTION AND HANDLING (1) (2)

Unhemolysed serum or heparinised plasma.
Vial R1 WORKING REAGENT α-amylase activity is stable in serum/plasma for:
E-PNPG7 1.1 mmol/L • at least 7 days at room temperature.
α-Glucosidase 1500 IU/L • 1 month at 2-8° C.
NaCl 51 mmol/L Urines: Adjust pH to alkalin range before storage.
Buffer pH 7.0 at 20° C α-amylase activity is stable in urines for 7 days at 2-8° C.
Preservative In case of delay in transporting urines to the laboratory, use a
Before reconstitution: T, R25-R32 R38: Toxic if swallowed – preservative as merthiolate (thimerosal) 0.24 mM or 0.1 g/L.
irritant. Contact with acid liberates toxic gas – Irritating to skin.
Once reconstituted: None INTERFERENCES (3)
S22/S38: Do not breathe dust. After contact with skin, rinse with
plenty of water Hemoglobin no interference up to 522 mg/dL
Free bilirubin no interference up to 15.5 mg/dL (265 µmol/L)
Conjugated bilirubin no interference up to 16.7 mg/dL (286 µmol/L)
Lipemia no interference from lipemia measured as an
absorbance at 630 nm up to 1.180 Abs.
For a more comprehensive review of factors affecting this assay refer
to the publication of Young D.S.

Made in France Latest revision: www.biolabo.fr Revision: 26/07/2011

MATERIAL REQUIRED BUT NOT PROVIDED
MANUAL PROCEDURE
1. Basic medical analysis laboratory equipment.
2. Normal and pathological control sera Let stand reagent and specimens at room temperature.
3. Demineralised water for reagent preparation
Pipette into 1 cm path length cuvette:

CALIBRATION Reagent 1 mL
Results will depend on the accuracy of the instrument calibration, the Bring to 37° C (30° C) then add:
time counting, the respect of reagent/specimen ratio and the
temperature control. Specimen 25 µL
• Use the theoretical calibration factor (§ CALCULATION)
• Or REF 95015 BIOLABO Multicalibrator (calibration value Mix. Start a timer. Record initial absorbance after 1 minute at 405 nm.
determined with validated statistical technics and metrologically Record the absorbance again every minute during 3 minutes.
controlled instrument) Calculate absorbance change per minute (∆Abs/min.).
• or a multiparametric calibrator traceable to a reference method or
material. Notes: Specific procedures are available upon request for
automated instruments. Please contact BIOLABO technical
QUALITY CONTROL support.

• BIOLABO EXATROL-N Level I REF 95010

• BIOLABO EXATROL-P Level II REF 95011
CALCULATION
• Other assayed control sera referring to the same method. Calculate the result as follows:
• External quality control program. With theoretical factor:
It is recommended to control in the following cases:
• At least once a run.
• At least once within 24 hours. IU/L = (∆Abs/min) x 5140
• When changing vial of reagent.
• After maintenance operations on the instrument.
If control is out of range, apply following actions: µkat/L = IU/L
1. Repeat the test with the same control. 60
2. If control is still out of range, prepare a fresh control serum and
repeat the test.
3. If control is still out of range, verify analysis parameters: With seric multicalibrator
wavelength, temperature, specimen/reagent ratio, time counting, (∆Abs/min) Assay
calibration factor. α-amylase (IU/L) = x Calibrator Concentration
(∆Abs/min) Calibrator
4. If control is still out of range, use a new vial of reagent and
reassay
5. If control is still out of range, please contact BIOLABO technical
support or your local Agent.
REFERENCES
EXPECTED VALUES (1) (1) TIETZ N.W. Text book of clinical chemistry, 3rd Ed. C.A. Curtis, E.R.
Ashwood, W.B. Saunders (1999) p. 689-698, 1284, 1286.
th
(2) Clinical Guide to Laboratory Test, 4 Ed., N.W. TIETZ (2006) p. 100-107
Serum (37° C) α-amylase (IU/L) α-amylase (µKat/L) (3) YOUNG D.S., Effect of Drugs on Clinical laboratory Tests, 4th Ed. (1995) p.
22-80 [0.38-1.36] 3-43 to 3-46
(4) FOO YA and Brosalki SB, Ann. Clin. Biochem.(1986), 23, 624-637
(5) National Committee for Clinical Laboratory Standards. User evaluation of
Urines (37° C) 24-408 IU/24 h [0.41-6.94]/24 h Precision Performance of Clinical Chemistry Devices. NCCLS, 1984,
NCCLS Publication EP5-T

Each laboratory should establish its own normal ranges for the
population that it serves.

PERFORMANCE CHARACTERISTICS (5)

Within-run Run to run

N = 40 Normal level High level N = 40 Normal level High level

Mean IU/L 80.3 185 Mean IU/L 87.5 462

S.D. IU/L 3.4 4.6 S.D.IU/L 3.5 16.8
C.V. % 4.3 2.5 C.V. % 3.95 3.6
Detection limit: approximately 10 IU/L (0.2 µKat /L)
Sensitivity for 1 IU/L: 0.195 mAbs/min.
Comparison studies with commercially available reagent:
y = 0.9987 x – 1.8 r = 0.9805

LINEARITY
The assay is linear up to 2000 IU/L (33 µKat/L).
If ∆Abs./min > 0.390, dilute specimen with saline solution and reassay
taking into account the dilution factor. Linearity depends on the
specimen/reagent ratio.

IVD REF LOT →

Manufacturer Use by In vitro diagnostic Temperature limitation Catalogue number See insert Batch number Store away from light sufficient for dilute with

Made in France Latest revision: www.biolabo.fr Revision: 26/07/2011