0003183742122c501V9.

AMYL2
α-Amylase EPS ver.2
Order information
Analyzer(s) on which cobas c pack(s) can be used
03183742 122 α‑Amylase EPS ver.2 (300 tests) System‑ID 07 6609 7 Roche/Hitachi cobas c 311, cobas c 501/502
10759350 190 Calibrator f.a.s. (12 x 3 mL) Code 401
10759350 360 Calibrator f.a.s. (12 x 3 mL, for USA) Code 401
12149435 122 Precinorm U plus (10 x 3 mL) Code 300
12149435 160 Precinorm U plus (10 x 3 mL, for USA) Code 300
12149443 122 Precipath U plus (10 x 3 mL) Code 301
12149443 160 Precipath U plus (10 x 3 mL, for USA) Code 301
10171743 122 Precinorm U (20 x 5 mL) Code 300
10171735 122 Precinorm U (4 × 5 mL) Code 300
10171778 122 Precipath U (20 x 5 mL) Code 301
10171760 122 Precipath U (4 × 5 mL) Code 301
05117003 190 PreciControl ClinChem Multi 1 (20 x 5 mL) Code 391
05947626 190 PreciControl ClinChem Multi 1 (4 x 5 mL) Code 391
05947626 160 PreciControl ClinChem Multi 1 (4 x 5 mL, for USA) Code 391
05117216 190 PreciControl ClinChem Multi 2 (20 x 5 mL) Code 392
05947774 190 PreciControl ClinChem Multi 2 (4 x 5 mL) Code 392
05947774 160 PreciControl ClinChem Multi 2 (4 x 5 mL, for USA) Code 392
04489357 190 Diluent NaCl 9 % (50 mL) System‑ID 07 6869 3

English Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all

the degradation products to p‑nitrophenol with the aid of α‑glucosidase
System information (100 % chromophore liberation). The results of this method correlate with
For cobas c 311/501 analyzers: those obtained by HPLC. This assay follows the recommendation of the
AMYL2: ACN 570 IFCC, but was optimized for performance and stability.
SAMY2: ACN 566 (STAT, reaction time: 7) Test principle10,11
For cobas c 502 analyzer: Enzymatic colorimetric assay acc. to IFCC.
AMYL2: ACN 8570 Defined oligosaccharides such as 4,6‑ethylidene‑(G7)
p‑nitrophenyl‑(G1)‑α‑D‑maltoheptaoside (ethylidene‑G7PNP) are cleaved
SAMY2: ACN 8566 (STAT, reaction time: 7) under the catalytic action of α‑amylases. The G2PNP, G3PNP and G4PNP
Intended use fragments so formed are completely hydrolyzed to p‑nitrophenol and
In vitro test for the quantitative determination of α‑amylase in human serum, glucose by α‑glucosidase.
plasma and urine on Roche/Hitachi cobas c systems. Simplified reaction scheme:
Summary1,2,3,4,5,6,7,8,9
α‑amylase
The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the
hydrolytic degradation of polymeric carbohydrates such as amylose, 5 ethylidene‑G7PNPa) + 5 H2O
amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In 2 ethylidene‑G5 + 2 G2PNP + 2 ethylidene‑G4 + 2 G3PNP +
polysaccharides and oligosaccharides, several glycosidic bonds are
hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted ethylidene‑G3 + G4PNP
into maltose and glucose, albeit very slowly. Two types of α‑amylases can α‑glucosidase
be distinguished, the pancreatic type (P‑type) and the salivary type
(S‑type). Whereas the P‑type can be attributed almost exclusively to the 2 G2PNP + 2 G3PNP +   5 PNP + 14 Gb)
pancreas and is therefore organ‑specific, the S‑type can originate from a G4PNP + 14 H2O 
number of sites. As well as appearing in the salivary glands it can also be
a) PNP p‑nitrophenol
found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the
epithelium of the fallopian tube. b) G Glucose

Because of the sparsity of specific clinical symptoms of pancreatic The color intensity of the p‑nitrophenol formed is directly proportional to the
diseases, α‑amylase determinations are of considerable importance in α‑amylase activity. It is determined by measuring the increase in
pancreatic diagnostics. They are mainly used in the diagnosis and absorbance.
monitoring of acute pancreatitis. Hyperamylasemia does not, however, only Reagents - working solutions
occur with acute pancreatitis or in the inflammatory phase of chronic
pancreatitis, but also in renal failure (reduced glomerular filtration), tumors R1 HEPES: 52.4 mmol/L; sodium chloride: 87 mmol/L; calcium
of the lungs or ovaries, pulmonary inflammation, diseases of the salivary chloride: 0.08 mmol/L; magnesium chloride: 12.6 mmol/L;
gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in α‑glucosidase (microbial): ≥ 66.8 µkat/L; pH 7.0 (37 °C);
the case of macroamylasemia. To confirm pancreatic specificity, it is preservatives; stabilizers
recommended that an additional pancreas‑specific enzyme ‑ lipase or
pancreatic‑α‑amylase ‑ also be determined. R2 HEPES: 52.4 mmol/L; ethylidene‑G7‑PNP: 22 mmol/L; pH 7.0
Numerous methods have been described for the determination of (37 °C); preservatives; stabilizers
α‑amylase. These either determine the decrease in the amount of substrate R1 is in position B and R2 is in position C.
viscometrically, turbidimetrically, nephelometrically and amyloclastically or
measure the formation of degradation products saccharogenically or Precautions and warnings
kinetically with the aid of enzyme‑catalyzed subsequent reactions. The For in vitro diagnostic use.
kinetic method described here is based on the well‑proven cleavage of Exercise the normal precautions required for handling all laboratory
4,6‑ethylidene‑(G7)‑1,4‑nitrophenyl‑(G1)‑α,D‑maltoheptaoside (Ethylidene
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AMYL2
α-Amylase EPS ver.2

reagents. Reagent pipetting Diluent (H2O)

Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request. R1 100 µL –

Reagent handling R2 20 µL –

Ready for use Sample volumes Sample Sample dilution
Storage and stability Sample Diluent (NaCl)
AMYL2 Normal 4 µL – –
Shelf life at 2‑8 °C: See expiration date Decreased 8 µL 15 µL 135 µL
on cobas c pack Increased 4 µL – –
label.
cobas c 501 test definition
On‑board in use and refrigerated on the analyzer: 12 weeks
Assay type Rate A
Diluent NaCl 9 %
Reaction time / 10 / 30‑47
Shelf life at 2‑8 °C: See expiration date
Assay points (STAT 7 / 30‑47)
on cobas c pack
label. Wavelength (sub/main) 700/415 nm
On‑board in use and refrigerated on the analyzer: 12 weeks Reaction direction Increase
Specimen collection and preparation9,12 Unit U/L (µkat/L)
For specimen collection and preparation only use suitable tubes or Reagent pipetting Diluent (H2O)
collection containers.
R1 100 µL –
Only the specimens listed below were tested and found acceptable.
Serum R2 20 µL –
Plasma: Li‑heparin plasma. Sample volumes Sample Sample dilution
The sample types listed were tested with a selection of sample collection
tubes that were commercially available at the time of testing, i.e. not all Sample Diluent (NaCl)
available tubes of all manufacturers were tested. Sample collection systems Normal 4 µL – –
from various manufacturers may contain differing materials which could
affect the test results in some cases. When processing samples in primary Decreased 8 µL 15 µL 135 µL
tubes (sample collection systems), follow the instructions of the tube Increased 4 µL – –
manufacturer.
Centrifuge samples containing precipitates before performing the assay. cobas c 502 test definition
Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay type Rate A
Assay promptly or adjust pH to alkaline range (just above pH 7) before
storage.13 Reaction time / 10 / 30‑47
Assay points (STAT 7 / 30‑47)
Stability in serum or plasma:13 7 days at 15‑25 °C
Wavelength (sub/main) 700/415 nm
1 month at 2‑8 °C
Reaction direction Increase
Unit U/L (µkat/L)
Stability in urine:14 2 days at 15‑25 °C
Reagent pipetting Diluent (H2O)
10 days at 2‑8 °C
R1 100 µL –
Materials provided R2 20 µL –
See “Reagents – working solutions” section for reagents.
Sample volumes Sample Sample dilution
Materials required (but not provided)
Sample Diluent (NaCl)
▪ See “Order information” section
Normal 4 µL – –
▪ General laboratory equipment
Assay Decreased 8 µL 15 µL 135 µL
For optimum performance of the assay follow the directions given in this Increased 8 µL – –
document for the analyzer concerned. Refer to the appropriate operator’s
manual for analyzer‑specific assay instructions. Calibration
The performance of applications not validated by Roche is not warranted Calibrators S1: H2O
and must be defined by the user.
S2: C.f.a.s.
Application for serum, plasma and urine
Calibration mode Linear
cobas c 311 test definition
Calibration frequency 2‑point calibration
Assay type Rate A • after reagent lot change
Reaction time / 10 / 22‑32 • as required following quality control
Assay points (STAT 7/ 22‑32) procedures
Wavelength (sub/main) 700/415 nm Traceability: This method has been standardized against Roche system
reagent using calibrated pipettes together with a manual photometer
Reaction direction Increase providing absolute values and substrate-specific absorptivity, ε.
Unit U/L (µkat/L)

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α-Amylase EPS ver.2

Quality control Limits and ranges

For quality control, use control materials as listed in the "Order information" Measuring range
section. Serum/plasma/urine
In addition, other suitable control material can be used. 3‑1500 U/L (0.05‑25.0 µkat/L)
The control intervals and limits should be adapted to each laboratory’s Determine samples having higher activities via the rerun function. Dilution
individual requirements. Values obtained should fall within the defined of samples via the rerun function is a 1:5 dilution. Results from samples
limits. Each laboratory should establish corrective measures to be taken if diluted using the rerun function are automatically multiplied by a factor of 5.
values fall outside the defined limits.
Lower limits of measurement
Follow the applicable government regulations and local guidelines for
quality control. Lower detection limit of the test
3 U/L (0.05 µkat/L)
Calculation
Roche/Hitachi cobas c systems automatically calculate the analyte activity The lower detection limit represents the lowest measurable analyte level
of each sample. that can be distinguished from zero. It is calculated as the value lying three
standard deviations above that of the lowest standard (standard 1 + 3 SD,
Conversion factor: U/L x 0.0167 = µkat/L repeatability, n = 21).
Limitations - interference Expected values9
A slight change in the yellow coloration of solution 2 does not interfere with
the performance of the test. Serum/plasma Men/Women 0.47‑1.67 µkat/L 28‑100 U/L
Do not pipette by mouth, and ensure that the reagent does not come into Spontaneously voided Men 0.27‑8.20 µkat/L 16‑491 U/L
contact with the skin. Saliva and sweat contain α‑amylase! urine Women 0.35‑7.46 µkat/L 21‑447 U/L
Criterion: Recovery within ± 10 % of initial value at an amylase activity of
100 U/L (1.67 µkat/L).
α‑amylase/ Men 0.97‑4.73 µkat/g 58‑283 U/g
Serum/plasma
creatinine quotient Women 1.25‑6.51 µkat/g 75‑390 U/g
Icterus:15 No significant interference up to an I index of 60 for conjugated
and unconjugated bilirubin (approximate conjugated and unconjugated
bilirubin concentration: 1026 µmol/L or 60 mg/dL). α‑Amylase/creatinine quotient
Hemolysis:15 No significant interference up to an H index of 500 To allow for fluctuations in the α‑amylase activity in urine, it is advisable to
(approximate hemoglobin concentration: 310 µmol/L or 500 mg/dL). determine the α‑amylase/creatinine quotient. To do this, determine the
Lipemia (Intralipid):15 No significant interference up to an L index of 1500. α‑amylase activity and creatinine concentration in spontaneously voided
There is poor correlation between the L index (corresponds to turbidity) and urine.
triglycerides concentration.
In rare cases, samples with a combination of elevated turbidity (L‑index) Quotient [U/g or µkat/mmol] = α‑amylase [U/L or µkat/L]
and high Amylase activity may cause a >React or >Abs flag. creatinine [g/L or mmol/L]
Highly turbid and grossly lipemic samples may cause Abs. flags.
Anticoagulants: Interference was found with citrate, fluoride, and EDTA.12 Amylase/Creatinine Clearance Ratio (ACCR)13
Glucose: No interference from glucose up to 111 mmol/L (2000 mg/dL). The ACCR is calculated from amylase activity and creatinine concentration.
Approximately 10 % higher recovery was found at glucose concentrations Both the serum and urine samples should be collected at the same time.
of 250 mmol/L (4500 mg/dL). ACCR [%] = urine amylase [U/L] × serum creatinine [mg/L] × 100
Ascorbic acid: No interference from ascorbic acid up to 5.68 mmol/L
(100 mg/dL). serum amylase [U/L] × urine creatinine [mg/L]
Drugs: No interference was found at therapeutic concentrations using The ACCR is approximately equal to 2‑5 %.
common drug panels.16,17 Each laboratory should investigate the transferability of the expected values
Exception: Icodextrin‑based drugs may lead to decreased amylase results. to its own patient population and if necessary determine its own reference
18 ranges.
In very rare cases, gammopathy, in particular type IgM (Waldenström’s Specific performance data
macroglobulinemia), may cause unreliable results.19 Representative performance data on the analyzers are given below.
Urine Results obtained in individual laboratories may differ.
Ascorbic acid: No interference from ascorbic acid up to 2.27 mmol/L Precision
(40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid Precision was determined using human samples and controls in an internal
concentrations of 22.7 mmol/L (400 mg/dL). protocol with repeatability (n = 21) and intermediate precision (3 aliquots
Drugs: No interference was found at therapeutic concentrations using per run, 1 run per day, 21 days). The following results were obtained:
common drug panels.17 Serum/plasma
For diagnostic purposes, the results should always be assessed in
conjunction with the patient’s medical history, clinical examination and other Repeatability Mean SD CV
findings.
U/L (µkat/L) U/L (µkat/L) %
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory Precinorm U 83.2 (1.39) 0.8 (0.01) 0.9
when certain test combinations are run together on Roche/Hitachi Precipath U 182 (3.09) 1 (0.02) 0.6
cobas c systems. The latest version of the carry‑over evasion list can be
found with the NaOHD/SMS/Multiclean/SCCS or the Human serum 1 34.5 (0.576) 0.4 (0.007) 1.2
NaOHD/SMS/SmpCln1+2/SCCS Method Sheets. For further instructions
refer to the operator’s manual. cobas c 502 analyzer: All special wash Human serum 2 97.9 (1.63) 0.7 (0.01) 0.7
programming necessary for avoiding carry‑over is available via the
cobas link, manual input is not required. Intermediate precision Mean SD CV
Where required, special wash/carry‑over evasion programming must U/L (µkat/L) U/L (µkat/L) %
be implemented prior to reporting results with this test.
Precinorm U 84.0 (1.40) 1.1 (0.02) 1.3

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AMYL2
α-Amylase EPS ver.2

Precipath U 184 (3.08) 3 (0.05) 1.5 8 Kruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al.
Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-
Human serum 3 35.1 (0.586) 0.9 (0.015) 2.4 (G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin
Human serum 4 98.9 (1.65) 1.6 (0.03) 1.6 Chem Clin Biochem 1989;27:103-113.
Urine 9 Junge W, Wortmann W, Wilke B, et al. Development and evaluation of
assays for the determination of total and pancreatic amylase at 37°C
Repeatability Mean SD CV according to the principle recommended by the IFCC. Clin Biochem
2001;34:607-615. Erratum Clin Biochem 2003;36:161.
U/L (µkat/L) U/L (µkat/L) %
10 Lorentz K. Approved recommendation on IFCC methods for the
Control level 1 50.6 (0.845) 0.5 (0.008) 0.9 measurement of catalytic concentration of enzymes. Part 9. IFCC
Control level 2 164 (2.74) 1 (0.02) 0.6 Method for α-Amylase. (1,4-α-D-Glucan 4-Glucanohydrolase, EC
3.2.1.1). Clin Chem Lab Med 1998;36(3):185-203.
Urine 1 21.4 (0.357) 0.2 (0.003) 1.1 11 Kurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the
Urine 2 68.5 (1.14) 0.7 (0.01) 0.9 determination of total and pancreatic α-amylase based on 100 %
cleavage of the protected substrate ethylidene-4-nitrophenyl-
Intermediate precision Mean SD CV maltoheptaoside. Clin Chem 1996;42(S6):98.
U/L (µkat/L) U/L (µkat/L) % 12 Young DS. Effects of Preclinical Variables on Clinical Laboratory Tests.
AACC Press 1997, 2nd edition 1997.
Control level 1 51.8 (0.865) 0.9 (0.015) 1.7 13 Tietz NW, ed. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia
Control level 2 168 (2.81) 2 (0.03) 1.1 PA: WB Saunders Company 1995;46-51.
Urine 3 24.5 (0.409) 0.5 (0.008) 1.9 14 Hohenwallner W, Hägele EO, Scholer A, et al. Bestimmung von alpha-
Amylase mit p-Nitrophenylmaltoheptaosid als Substrat. Ber Öster Ges
Urine 4 67.0 (1.12) 2.8 (0.05) 4.2 Klin Chem 1983;6:101-112.
Method comparison 15 Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of
Amylase values for human serum, plasma and urine samples obtained on a Interferences in Clinical Chemistry Instrumentation. Clin Chem
Roche/Hitachi cobas c 501 analyzer (y) were compared with those 1986;32:470-475.
determined using the same reagent on a Roche/Hitachi 917 analyzer (x). 16 Breuer J. Report on the Symposium “Drug effects in Clinical Chemistry
Serum/plasma Methods”. Eur J Clin Chem Clin Biochem 1996;34:385-386.
Sample size (n) = 79 17 Sonntag O, Scholer A. Drug interference in clinical chemistry:
recommendation of drugs and their concentrations to be used in drug
Passing/Bablok20 Linear regression interference studies. Ann Clin Biochem 2001;38:376-385.
y = 0.999x + 2.83 U/L y = 0.998x + 4.75 U/L 18 Gokal R, Moberly J, Lindholm B, et al. Metabolic and laboratory effects
of icodextrin. Kidney Int 2002;62(81):62-71.
τ = 0.969 r = 0.998
19 Bakker AJ, Mücke M. Gammopathy interference in clinical chemistry
The sample activities were between 51.7 and 1409 U/L (0.863 and assays: mechanisms, detection and prevention.
23.5 µkat/L). Clin Chem Lab Med 2007;45(9):1240-1243.
Urine 20 Bablok W, Passing H, Bender R, et al. A general regression procedure
Sample size (n) = 88 for method transformation. Application of linear regression procedures
for method comparison studies in clinical chemistry, Part III. J Clin
Passing/Bablok20 Linear regression Chem Clin Biochem 1988 Nov;26(11):783-790.
y = 0.986x + 0.423 U/L y = 0.982x + 2.03 U/L A point (period/stop) is always used in this Method Sheet as the decimal
separator to mark the border between the integral and the fractional parts of
τ = 0.987 r = 1.000 a decimal numeral. Separators for thousands are not used.
The sample activities were between 33.6 and 1248 U/L (0.561 and Symbols
20.8 µkat/L). Roche Diagnostics uses the following symbols and signs in addition to
those listed in the ISO 15223‑1 standard.
References
1 Greiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Contents of kit
Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.
Volume after reconstitution or mixing
2 Keller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed.
Stuttgart/New York: Georg Thieme Verlag 1991:354-361. GTIN Global Trade Item Number
3 Salt WB II, Schenker S. Amylase - its clinical significance: a review of
the literature [Review]. Medicine 1976;55:269-281. FOR US CUSTOMERS ONLY: LIMITED WARRANTY
Roche Diagnostics warrants that this product will meet the specifications
4 Steinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in
acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580. stated in the labeling when used in accordance with such labeling and will
be free from defects in material and workmanship until the expiration date
5 Tietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the
differential diagnosis of hyperamylasemia. Clin Chem printed on the label. THIS LIMITED WARRANTY IS IN LIEU OF ANY
1986;32(2):301-307. OTHER WARRANTY, EXPRESS OR IMPLIED, INCLUDING ANY IMPLIED
6 Junge W, Troge B, Klein G, et al. Evaluation of a New Assay for WARRANTY OF MERCHANTABILITY OR FITNESS FOR PARTICULAR
Pancreatic Amylase: Performance Characteristics and Estimation of PURPOSE. IN NO EVENT SHALL ROCHE DIAGNOSTICS BE LIABLE
Reference Intervals. Clin Biochem 1989;22:109-114. FOR INCIDENTAL, INDIRECT, SPECIAL OR CONSEQUENTIAL
7 Rauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected DAMAGES.
substrate for the assay of human α-amylase. Fresenius Z Anal Chem COBAS, COBAS C, PRECINORM, PRECIPATH and PRECICONTROL are trademarks of Roche.
1986;324:304-305. All other product names and trademarks are the property of their respective owners.
Significant additions or changes are indicated by a change bar in the margin.
© 2014, Roche Diagnostics

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AMYL2
α-Amylase EPS ver.2

Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim

www.roche.com
Distribution in USA by:
Roche Diagnostics, Indianapolis, IN
US Customer Technical Support 1-800-428-2336

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