 After opening: 60 days when properly capped 7.

Calculate the mean of the results to obtain the

immediately after each use and stored at 2 – 8 °C. average change in absorbance per minute
 Store the vials tightly closed, protected from light (ΔA/min).
α-Amylase and prevented contaminations during the use. Avoid
Colorimetric enzymatic method contamination and recap the vials immediately after CALCULATIONS
Kinetic use. Serum, plasma
For In-Vitro and professional use only Discard If signs of deterioration appear: U/L = ΔA/min x 3178
Store at 2-8C  Presence of particles and turbidity.
 Blank absorbance (A) at 405 nm > 0.5 in 1 cm If results are to be expressed as SI units apply:
PRINCIPLE cuvette. U/L x 0.01667 = μkat/L.
In this direct method α-amylase catalyzes the
hydrolysis of 2- chloro-p-nitrophenyl-α-D- REAGENT PREPARATION REFERENCE VALUES
maltotrioside (CNP-G3) substrate at pH 6.0 forming The Monoreagent is ready-to-use. Serum, plasma
2-chloro-p-nitrophenol (CNP) and free glycosides. 23-88 U/L
The reaction is monitored kinetically at 405 nm SAMPLES
by the rate of formation of the colored CNP  Serum or plasma. It is recommended that each laboratory establishes
produced, proportional to the activity of the α-  Serum and plasma α-amylase is stable for 30 days at its own reference range.
amylase in the sample. 2-8ºC.
QUALITY CONTROL
α-AMYLASE INTERFERENCES To ensure adequate quality control (QC), each run
10 CNP-G3 9 CNP + 1CNP-G2 + G3 + G no interference up to: should include a set of controls (normal and
pH 6.0 ascorbic acid 50 mg/dL
abnormal) with assayed values handled as unknowns.
bilirubin 50 mg/dL
Elevated level of α-amylase . Assayed.
REAGENT COMPOSITION hemoglobin 500 mg/dL If the values are found outside of the defined
R1 Monoreagent: triglycerides 3000 mg/dL
range, check the instrument, reagents and procedure.
MES buffer 100 mmol/L (pH 6.0) Each laboratory should establish its own Quality
Calcium acetate 6 mmol/L PROCEDURE Control scheme and corrective actions if controls do
1. Preincubate working reagent, samples and
Sodium chloride 350 mmol/L not meet the acceptable tolerances.
controls to reaction temperature.
Potassium thiocyanate 900 mmol/L
2. Set the photometer to 0 absorbance with distilled
CNP-G3 2.27 mmol/L. CLINICAL SIGNIFICANCE
water.
Stabilizers and detergents <0.1% Amylase activity tests in serum and urine are
3. Pipette into a cuvette: mainly used in the diagnosis of diseases of the
37ºC pancreas and in the investigation of pancreatic
MATERIALS REQUIRED BUT NOT PROVIDED Reaction temperature
 Photometer or spectrophotometer with a Blank Sample function.
thermostatted cell compartment set at 37ºC, R1.Monoreagent 1.0 mL 1.0 mL Amylase is found chiefly in the saliva and in
capable of reading at 405 nm. Dist. Water or saline 25 μl - pancreatic tissue.
 Stopwatch, strip-chart recorder or printer. Normally, small amounts of amylase are present in
Serum/plasma - 25 μl the blood, but with various forms of pancreatic
 Cuvettes with 1-cm path length.
 Pipettes to measure reagent and samples. 4. Mix gently by inversion. Insert cuvette into the disturbance large amounts of amylase are secreted
cell holder and start stopwatch. into the blood by the pancreas.
STORAGE AND STABILITY Incubate at 37ºC for 1 minute and record initial The activity of the amylase in serum may fluctuate
 Store at 2-8ºC. absorbance reading. rapidly rising acutely during an attack and
 All the kit compounds are stable until the expiry 5. Read the absorbance (at 405nm) exactly after subsiding to normal levels shortly afterward.
date stated on the label. Do not use reagents over 1, 2 and 3 minutes. Increased levels are found associated with acute
the expiration date. 6. Calculate the difference between absorbances. pancreatitis, pancreatic duct obstrution, intra-
 abdominal diseases, mumps and bacterial parotitis. S2: Keep out of the reach of children. ATLAS Medical
A significant amount of the serum amylase is S13: Keep away from food, drink and animal Unit 4, William James House
excreted in the urine, and as a result elevation of feedingstuffs. Cowley Rd, Cambridge, CB4 0WX
serum activity is reflected in the rise of urinary S36/37: Wear suibable protective clothing and Tel: ++44 (0) 1223 858 910
amylase activity. Urine amylase appears to be more gloves. Fax: ++44 (0) 1223 858 524
frequently elevated, reaches higher levels, and S46: If swallowed, seek medical advice immediately PPI428A01
persists for longer periods. and show this container or label. Rev D (22.12.2014)
 Caution: Contains Potassium Thiocyanate.
ANALYTICAL PERFORMANCE Potassium thiocyanate is not compatible with strong
- Linearity : Up to 2000 U/L acids.
If a sample exceeds 2000 U/L, it should be diluted  Caution: Contains sodium azide, which may react
1:1 with normal saline and re-assayed. Multiply the with lead or copper plumbing to form potentially
result by 2. explosive metal azide. On disposal, flush drain with a
large volume of water to prevent build up.
- Precision:
Intra-assay Mean SD CV REFERENCES
n=20 (U/L) (U/L) (%) 1. Ranson, JHC, Curr, Prob. Surg., 16:1 (1979).
Sample 1 61 0.82 1.34 2. Salt, WB II and Schenker, S., Medicine, 55:269 (1976).
Sample 2 272 1.66 0.61 3. Stefanini, P., Ermini, M., and Carboni, M., J.Am.Surg.,
Sample 3 902 4.60 0.51 119:866 (1965).
Sample 4 1509 9.36 0.62 4. Henry, RJ, and Chiamori, N., Clin Chem., 6:434 (1960).
5. Kaufman, RA and Tietz, NW, Clin Chem., 26:846
Inter-assay Mean SD CV (1980).
n=40 (U/L) (U/L) (%) 6. Blair, HE, U.S. Patent No. 4,649,108.
Sample 1 c 1.0 1.7 7. Chavez, RG, et al, U.S. Patent 4,963,479.
Sample 2 273 2.2 0.8 8. NCCLS document “Procedures for the Collection of
Sample 3 917 8.3 0.9 Diagnostic Blood Specimens by Skin Puncture”, 3rd
Ed., 11:11 (1991).
Sample 4 1507 9.0 0.6
9. Demetriou, J., et al, “Clinical Chemistry: Principles and
Techniques”, 2nd Ed., Harper & Row (1974).
- Correlation:
10. Suelter, CH, “A Practical Guide To Enzymology”,
A comparison between α-Amylase (y) and a
Wiley & Sons (1985).
commercial obtainable assay (x) using 50 samples
11. Rosenblum, JL, et al, Clin Chem., 38 :9 (1992).
(28 –304 U/L) gave following results:
12. Tietz, NW ; “Clinical Guide to Laboratory Tests”;
y = 0.90 x – 2.50 U/L; r = 0.999.
W.B. Saunders Co., 54 (1983)
13. NCCLS document “Evaluation of Precision
WARNINGS AND PRECAUTIONS
Performance of Clinical Chemistry Devices”, 2nd Ed.,
 For In Vitro Diagnostic Use.
12:4 (1992).
 Take the necessary precautions for the use of
14. 14 Young, DS, “Effects of Drugs on Clinical
laboratory reagents.
Laboratory Tests”, 4th Ed., AACC Press, Washington
 Avoid contamination of the reagent with salivary α-
DC; 3-43 to 3-47 (1995).
amylase. Do not pipette by mouth, and ensure that
15. IFCC document “The Theory of Reference Values”,
the reagent does not come into contact with the
Stage 2, draft 2 J.Clin. Biohcem., 21:749 (1983).
skin. (Saliva and sweat contain α-amylase)
 Xn: Harmful
R22: Harmful if swallowed