In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on Roche/Hitachi cobas c systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene‑(G₇)‑ p‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (ethylidene‑G₇PNP)^a) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum/plasma/urine

3‑1500 U/L (0.05‑25.0 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

Lower detection limit:

3 U/L (0.05 µkat/L)

The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying 3 standard deviations above that of the lowest standard (standard 1 + 3 SD, repeatability, n = 21).

Values below the lower detection limit (< 3 U/L) will not be flagged by the instrument.

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

α‑Amylase/creatinine quotient

To allow for fluctuations in the α‑amylase activity in urine, it is advisable to determine the α‑amylase/creatinine quotient. To do this, determine the α‑amylase activity and creatinine concentration in spontaneously voided urine.

Amylase/Creatinine Clearance Ratio (ACCR)

The ACCR is calculated from amylase activity and creatinine concentration. Both the serum and urine samples should be collected at the same time.

The ACCR is approximately equal to 2‑5 %.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L (1.67 µkat/L).

Serum/plasma

Icterus:

Hemolysis:

Lipemia (Intralipid):

In rare cases, samples with a combination of elevated turbidity (L‑index) and high Amylase activity may cause a >React or >Abs flag.

Highly turbid and grossly lipemic samples may cause Abs. flags.

Anticoagulants: Interference was found with citrate, fluoride, and EDTA.

Glucose: No significant interference from glucose up to a concentration of 111 mmol/L (2000 mg/dL). Approximately 10 % higher recovery was found at glucose concentrations of 250 mmol/L (4500 mg/dL).

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 5.68 mmol/L (100 mg/dL).

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Exception:

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 2.27 mmol/L (40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid concentrations of 22.7 mmol/L (400 mg/dL).

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L (7.68 µkat/L).

Hemolysis: No significant interference up to a hemoglobin concentration of 311 µmol/L or 500 mg/dL.

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on Roche/Hitachi cobas c systems. All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases. The latest version of the carry‑over evasion list can be found with the NaOHD/SMS/SmpCln1+2/SCCS Method Sheet and for further instructions refer to the operator’s manual.

Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.

Materials required (but not provided):

AMYL2: ACN 8570

SAMY2: ACN 8566 (STAT, reaction time: 7)

Ready for use

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.

Precision was determined using human samples and controls in an internal protocol with repeatability (n = 21) and intermediate precision (3 aliquots per run, 1 run per day, 21 days). The following results were obtained:

Serum/plasma

Urine

Results for intermediate precision were obtained on the master system cobas c 501 analyzer.

Amylase values for human serum, plasma and urine samples obtained on a Roche/Hitachi cobas c 701 analyzer (y) were compared with those determined using the corresponding reagent on a Roche/Hitachi cobas c 501 analyzer (x).

Serum/plasma

Sample size (n) = 76

Urine

Sample size (n) = 82

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32:301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO et al.Fresenius Z Anal Chem 1986;324:304–5.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ-specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas-specific enzyme - lipase or pancreatic-α‑amylase - also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme-catalyzed subsequent reactions. The kinetic method described here is based on the well-proven cleavage of 4,6‑ethylidene‑(G₇)‑1,4‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p‑nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC. This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

R1 is in position B and R3 (STAT R2) is in position C.

For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.

The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

^,

^LREFYoung DS. Effects of Preclinical Variables on Clinical Laboratory Tests. AACC Press, 1997, 2nd edition.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.

Serum

Plasma: Li‑heparin plasma.

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay.

See the limitations and interferences section for details about possible sample interferences.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on the cobas c 111 system.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC

Defined oligosaccharides such as 4,6‑ethylidene-(G₇)p‑nitrophenyl-(G₁)-α,D‑maltoheptaoside (ethylidene-G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

The color intensity of the p-nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum/plasma
3‑1500 U/L (0.05‑25.1 µkat/L)

Urine
3‑2000 U/L (0.05‑33.4 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

Lower detection limit of the test:
3 U/L (0.05 µkat/L)

The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying 3 standard deviations above that of the lowest standard (standard 1 + 3 SD, repeatability, n = 21).

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

α-Amylase/creatinine quotient

To allow for fluctuations in the α‑amylase activity in urine, it is advisable to determine the α‑amylase/creatinine quotient. To do this, determine the α‑amylase activity and creatinine concentration in spontaneously voided urine.

Amylase/Creatinine Clearance Ratio (ACCR)

^LREFTietz NW, ed. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia PA: WB Saunders Company 1995;46-51.

The ACCR is calculated from amylase activity and creatinine concentration. Both the serum and urine samples should be collected at the same time.

ACCR is approximately equal to 2‑5 %

Limitations - interference

^LREFYoung DS. Effects of Drugs on Clinical Laboratory Tests. 2nd edition, AACC Press 1997.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Serum/plasma

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L (1.67 µkat/L).

Icterus:

Hemolysis:

Lipemia (Intralipid):

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Anticoagulants: Interference was found with citrate and fluoride.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L (7.68 µkat/L).

Hemolysis: No significant interference up to a hemoglobin concentration of 311 µmol/L (500 mg/dL).

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

Materials required (but not provided):

AMYL2: ACN 570

AMYU2: ACN 301

Ready for use

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the cobas c 111 analyzer are given below. Results obtained in individual laboratories may differ.

Precision was determined using human samples and controls in an internal protocol with repeatability (n = 21) and intermediate precision (3 aliquots per run, 1 run per day, 10 days). The following results were obtained:

Amylase values for human samples obtained on the cobas c 111 analyzer (y) were compared with those determined using the same reagent on a COBAS INTEGRA 400 analyzer (x).

The sample activities were between 12.9 and 1126 U/L (0.22 and 18.8 µkat/L).

The sample activities were between 4.75 and 1966 U/L (0.08 and 32.8 µkat/L) on the reference system (x).

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32(2):301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected substrate for the assay of human α-amylase. Fresenius Z Anal Chem 1986;324:304-305.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α-D-glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4-α-glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ-specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas-specific enzyme - lipase or pancreatic-α-amylase - also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme-catalyzed subsequent reactions. The kinetic method described here is based on the well-proven cleavage of 4,6-ethylidene-(G₇)-1,4-nitrophenyl-(G₁)-α,D-maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p-nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC.

This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

Serum/plasma

For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.

Urine
Quantitative urine controls are recommended for routine quality control.

The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC Clin Biochem/Erratum. Clin Biochem 2003;36:161.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Heparin (Li-, Na-, NH₄⁺-) or EDTA (K₂-, K₃-) plasma.

EDTA plasma values are approximately 5‑10 % lower than serum values.

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Urine
Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

See the limitations and interferences section for details about possible sample interferences.

Centrifuge samples containing precipitates before performing the assay.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on Roche/Hitachi cobas c systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene‑(G₇) p‑nitrophenyl‑(G₁)‑α‑D‑maltoheptaoside (ethylidene‑G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

Simplified reaction scheme:

5 ethylidene‑G7PNP FREFPNP p‑nitrophenol  + 5 H2O	α-amylase
2 ethylidene‑G5 + 2 G2PNP + 2 ethylidene‑G4 + 2 G3PNP  + ethylidene‑G3 + G4PNP

2 G2PNP + 2 G3PNP + G4PNP + 14 H2O

α‑glucosidase

5 PNP + 14 G FREFG Glucose

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum, plasma and urine

3‑1500 U/L (0.05‑25.0 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.

The Limit of Blank is the 95^th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the activity below which analyte‑free samples are found with a probability of 95 %.

The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low activity samples.

The Limit of Detection corresponds to the lowest analyte activity which can be detected (value above the Limit of Blank with a probability of 95 %).

The Limit of Quantitation is the lowest analyte activity that can be reproducibly measured with a total error of 20 %. It has been determined using low activity α‑amylase samples.

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Serum/plasma

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L.

Icterus:

Hemolysis:

Lipemia (Intralipid):

In rare cases, samples with a combination of elevated turbidity (L‑index) and high Amylase activity may cause a >React or >Abs flag.

Highly turbid and grossly lipemic samples may cause Abs. flags.

Anticoagulants: Interference was found with citrate, fluoride, and EDTA.

Glucose: No significant interference from glucose up to a concentration of 111 mmol/L (2000 mg/dL). Approximately 10 % higher recovery was found at glucose concentrations of 250 mmol/L (4500 mg/dL).

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 5.68 mmol/L (100 mg/dL).

Drugs: No interference was found at therapeutic concentrations using common drug panels.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L.

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 2.27 mmol/L (40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid concentrations of 22.7 mmol/L (400 mg/dL).

Hemolysis: No significant interference up to an H index of 500 (approximate hemoglobin concentration: 311 µmol/L or 500 mg/dL).

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas c systems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry-over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet. For further instructions refer to the operator’s manual.

AMYL2: ACN 20170 (Serum/plasma)

AMYL2U: ACN 20171 (Urine)

Ready for use

For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.

Application for urine (ACN 20171)
Transfer of calibration from serum/plasma application (ACN 20170)

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.

Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.

Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). Results for repeatability and intermediate precision were obtained on the cobas c 503 analyzer.

Amylase values for human serum, plasma and urine samples obtained on acobas c 503 analyzer (y) were compared to those determined using the corresponding reagent on a cobas c 501 analyzer (x).

The sample activities were between 10.3 and 1439 U/L.

The sample activities were between 6.90 and 1467 U/L.

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32(2):301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected substrate for the assay of human α-amylase. Fresenius Z Anal Chem 1986;324:304-305.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ‑specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas‑specific enzyme ‑ lipase or pancreatic‑α‑amylase ‑ also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme‑catalyzed subsequent reactions. The kinetic method described here is based on the well‑proven cleavage of 4,6‑ethylidene‑(G₇)‑1,4‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p‑nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC. This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

R1 is in position B and R3 is in position C.

For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines.
Safety data sheet available for professional user on request.

For USA: Caution: Federal law restricts this device to sale by or on the order of a physician.

This kit contains components classified as follows in accordance with the Regulation (EC) No. 1272/2008:

Warning

H317	May cause an allergic skin reaction.

Prevention:

P261	Avoid breathing dust/fume/gas/mist/vapours/spray.

P272	Contaminated work clothing should not be allowed out of the workplace.

P280	Wear protective gloves.

Response:

P333 + P313

If skin irritation or rash occurs: Get medical advice/attention.

P362 + P364

Take off contaminated clothing and wash it before reuse.

Disposal:

P501	Dispose of contents/container to an approved waste disposal plant.

Product safety labeling follows EU GHS guidance.

Contact phone: 1-800-428-2336

For quality control, use control materials as listed in the \"Order information\" section.

In addition, other suitable control material can be used.

The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 26 weeks. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

^,

^LREFYoung DS. Effects of Preclinical Variables on Clinical Laboratory Tests. AACC Press 1997, 2nd edition 1997.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li‑heparin plasma

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay.

Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

See the limitations and interferences section for details about possible sample interferences.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on Roche/Hitachi cobas c systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene‑(G₇) p‑nitrophenyl‑(G₁)‑α‑D‑maltoheptaoside (ethylidene‑G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

Simplified reaction scheme:

5 ethylidene‑G7PNP FREFPNP p‑nitrophenol  + 5 H2O	α-amylase
2 ethylidene‑G5 + 2 G2PNP + 2 ethylidene‑G4 + 2 G3PNP  + ethylidene‑G3 + G4PNP

2 G2PNP + 2 G3PNP + G4PNP + 14 H2O

α‑glucosidase

5 PNP + 14 G FREFG Glucose

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum, plasma and urine

3‑1500 U/L (0.05‑25.0 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.

The Limit of Blank is the 95^th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the activity below which analyte‑free samples are found with a probability of 95 %.

The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low activity samples.

The Limit of Detection corresponds to the lowest analyte activity which can be detected (value above the Limit of Blank with a probability of 95 %).

The Limit of Quantitation is the lowest analyte activity that can be reproducibly measured with a total error of 20 %. It has been determined using low activity α‑amylase samples.

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L.

Serum/plasma

Icterus:

Hemolysis:

Lipemia (Intralipid):

In rare cases, samples with a combination of elevated turbidity (L‑index) and high Amylase activity may cause a >React or >Abs flag.

Highly turbid and grossly lipemic samples may cause Abs. flags.

Anticoagulants: Interference was found with citrate, fluoride, and EDTA.

Glucose: No significant interference from glucose up to a concentration of 111 mmol/L (2000 mg/dL). Approximately 10 % higher recovery was found at glucose concentrations of 250 mmol/L (4500 mg/dL).

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 5.68 mmol/L (100 mg/dL).

Drugs: No interference was found at therapeutic concentrations using common drug panels.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 2.27 mmol/L (40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid concentrations of 22.7 mmol/L (400 mg/dL).

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L.

Hemolysis: No significant interference up to an H index of 500 (approximate hemoglobin concentration: 311 µmol/L or 500 mg/dL).

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on Roche/Hitachi cobas c systems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry-over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet for information. For further instructions refer to the operator’s manual.

Materials required (but not provided):

AMYL2: ACN 20170 (Serum/plasma)

AMYL2U: ACN 20171 (Urine)

Ready for use

For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.

Application for urine (ACN 20171)
Transfer of calibration from serum/plasma application (ACN 20170)

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.

Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.

Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). The following results were obtained:

Amylase values for human serum, plasma and urine samples obtained on a Roche/Hitachi cobas c 503 analyzer (y) were compared to those determined using the corresponding reagent on a Roche/Hitachi cobas c 501 analyzer (x).

The sample activities were between 10.3 and 1439 U/L.

The sample activities were between 6.90 and 1467 U/L.

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32(2):301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected substrate for the assay of human α-amylase. Fresenius Z Anal Chem 1986;324:304-305.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ‑specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas‑specific enzyme ‑ lipase or pancreatic‑α‑amylase ‑ also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme‑catalyzed subsequent reactions. The kinetic method described here is based on the well‑proven cleavage of 4,6‑ethylidene‑(G₇)‑1,4‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p‑nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC. This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

R1 is in position B and R3 is in position C.

For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.

The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 26 weeks. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

^,

^LREFYoung DS. Effects of Preclinical Variables on Clinical Laboratory Tests. AACC Press 1997, 2nd edition 1997.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li‑heparin plasma

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay.

Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

See the limitations and interferences section for details about possible sample interferences.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on Roche/Hitachi cobas c systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene‑(G₇) p‑nitrophenyl‑(G₁)‑α‑D‑maltoheptaoside (ethylidene‑G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

Simplified reaction scheme:

5 ethylidene‑G7PNP FREFPNP p‑nitrophenol  + 5 H2O	α-amylase
2 ethylidene‑G5 + 2 G2PNP + 2 ethylidene‑G4 + 2 G3PNP  + ethylidene‑G3 + G4PNP

2 G2PNP + 2 G3PNP + G4PNP + 14 H2O

α‑glucosidase

5 PNP + 14 G FREFG Glucose

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum, plasma and urine

3‑1500 U/L (0.05‑25.0 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

The Limit of Blank, Limit of Detection and Limit of Quantitation were determined in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP17‑A2 requirements.

The Limit of Blank is the 95^th percentile value from n ≥ 60 measurements of analyte‑free samples over several independent series. The Limit of Blank corresponds to the activity below which analyte‑free samples are found with a probability of 95 %.

The Limit of Detection is determined based on the Limit of Blank and the standard deviation of low activity samples.

The Limit of Detection corresponds to the lowest analyte activity which can be detected (value above the Limit of Blank with a probability of 95 %).

The Limit of Quantitation is the lowest analyte activity that can be reproducibly measured with a total error of 20 %. It has been determined using low activity α‑amylase samples.

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L.

Serum/plasma

Icterus:

Hemolysis:

Lipemia (Intralipid):

In rare cases, samples with a combination of elevated turbidity (L‑index) and high Amylase activity may cause a >React or >Abs flag.

Highly turbid and grossly lipemic samples may cause Abs. flags.

Anticoagulants: Interference was found with citrate, fluoride, and EDTA.

Glucose: No significant interference from glucose up to a concentration of 111 mmol/L (2000 mg/dL). Approximately 10 % higher recovery was found at glucose concentrations of 250 mmol/L (4500 mg/dL).

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 5.68 mmol/L (100 mg/dL).

Drugs: No interference was found at therapeutic concentrations using common drug panels.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 2.27 mmol/L (40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid concentrations of 22.7 mmol/L (400 mg/dL).

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L.

Hemolysis: No significant interference up to an H index of 500 (approximate hemoglobin concentration: 311 µmol/L or 500 mg/dL).

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on cobas csystems. All special wash programming necessary for avoiding carry-over is available via the cobas link. The latest version of the carry-over evasion list can be found with the NaOHD/SMS/SCCS Method Sheet for information. For further instructions refer to the operator’s manual.

Materials required (but not provided):

AMYL2: ACN 20170 (Serum/plasma)

AMYL2U: ACN 20171 (Urine)

Ready for use

For further information about the assay test definitions refer to the application parameters setting screen of the corresponding analyzer and assay.

Application for urine (ACN 20171)
Transfer of calibration from serum/plasma application (ACN 20170)

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the analyzers are given below. These data represent the performance of the analytical procedure itself.

Results obtained in individual laboratories may differ due to heterogenous sample materials, aging of analyzer components and mixture of reagents running on the analyzer.

Precision was determined using human samples and controls in accordance with the CLSI (Clinical and Laboratory Standards Institute) EP05‑A3 requirements with repeatability (n = 84) and intermediate precision (2 aliquots per run, 2 runs per day, 21 days). Results for repeatability and intermediate precision were obtained on the cobas c 503 analyzer.

The data obtained on cobas c 503 analyzer(s) are representative for cobas c 303 analyzer(s).

Amylase values for human serum, plasma and urine samples obtained on a cobas c 503 analyzer (y) were compared to those determined using the corresponding reagent on a cobas c 501 analyzer (x).

The sample activities were between 10.3 and 1439 U/L.

The sample activities were between 6.90 and 1467 U/L.

Amylase values for human serum, plasma and urine samples obtained on a cobas c 303 analyzer (y) were compared to those determined using the corresponding reagent on a cobas c 501 analyzer (x).

The sample activities were between 9.10 and 1460 U/L.

The sample activities were between 4.80 and 1444 U/L.

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32(2):301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected substrate for the assay of human α-amylase. Fresenius Z Anal Chem 1986;324:304-305.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ‑specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas‑specific enzyme ‑ lipase or pancreatic‑α‑amylase ‑ also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme‑catalyzed subsequent reactions. The kinetic method described here is based on the well‑proven cleavage of 4,6‑ethylidene‑(G₇)‑1,4‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p‑nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC. This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

R1 is in position B and R3 is in position C.

For quality control, use control materials as listed in the “Order information” section. In addition, other suitable control material can be used.

The control intervals and limits should be adapted to each laboratory’s individual requirements. It is recommended to perform quality control always after lot calibration and subsequently at least every 26 weeks. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

^,

^LREFYoung DS. Effects of Preclinical Variables on Clinical Laboratory Tests. AACC Press 1997, 2nd edition 1997.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li‑heparin plasma

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay.

Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

See the limitations and interferences section for details about possible sample interferences.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

In vitro test for the quantitative determination of α‑amylase in human serum, plasma and urine on Roche/Hitachi cobas c systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weitenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene‑(G₇) p‑nitrophenyl‑(G₁)‑α‑D‑maltoheptaoside (ethylidene‑G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

Simplified reaction scheme:

	α‑amylase
5 ethylidene‑G7PNP FREFPNP p‑nitrophenol  + 5 H2O
2 ethylidene‑G5 + 2 G2PNP + 2 ethylidene‑G4 + 2 G3PNP + ethylidene‑G3 + G4PNP
	α‑glucosidase
2 G2PNP + 2 G3PNP + G4PNP + 14 H2O		5 PNP + 14 G FREF G Glucose

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance.

Measuring range

Serum/plasma/urine

3‑1500 U/L (0.05‑25.0 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

Lower detection limit of the test

3 U/L (0.05 µkat/L)

The lower detection limit represents the lowest measurable analyte level 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, repeatability, n = 21).

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

α‑Amylase/creatinine quotient

To allow for fluctuations in the α‑amylase activity in urine, it is advisable to determine the α‑amylase/creatinine quotient. To do this, determine the α‑amylase activity and creatinine concentration in spontaneously voided urine.

Amylase/Creatinine Clearance Ratio (ACCR)

The ACCR is calculated from amylase activity and creatinine concentration. Both the serum and urine samples should be collected at the same time.

The ACCR is approximately equal to 2‑5 %.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

A slight change in the yellow coloration of solution 2 does not interfere with the performance of the test.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. Saliva and sweat contain α‑amylase!

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 100 U/L (1.67 µkat/L).

Serum/plasma

Icterus:

Hemolysis:

Lipemia (Intralipid):

In rare cases, samples with a combination of elevated turbidity (L‑index) and high Amylase activity may cause a >React or >Abs flag.

Highly turbid and grossly lipemic samples may cause Abs. flags.

Anticoagulants: Interference was found with citrate, fluoride, and EDTA.

Glucose: No significant interference from glucose up to a concentration of 111 mmol/L (2000 mg/dL). Approximately 10 % higher recovery was found at glucose concentrations of 250 mmol/L (4500 mg/dL).

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 5.68 mmol/L (100 mg/dL).

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Exception: Icodextrin‑based drugs may lead to decreased amylase results.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Ascorbic acid: No significant interference from ascorbic acid up to a concentration of 2.27 mmol/L (40 mg/dL). Approximately 15 % lower recovery was found at ascorbic acid concentrations of 22.7 mmol/L (400 mg/dL).

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L (7.68 µkat/L).

Hemolysis:

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on Roche/Hitachi cobas c systems. The latest version of the carry‑over evasion list can be found with the NaOHD-SMS-SmpCln1+2-SCCS Method Sheets. For further instructions refer to the operator’s manual. cobas c 502 analyzer: All special wash programming necessary for avoiding carry‑over is available via the cobas link, manual input is required in certain cases.

Where required, special wash/carry‑over evasion programming must be implemented prior to reporting results with this test.

Materials required (but not provided):

For cobas c 311/501 analyzers:

AMYL2: ACN 570

SAMY2: ACN 566 (STAT, reaction time: 7)

For cobas c 502 analyzer:

AMYL2: ACN 8570

SAMY2: ACN 8566 (STAT, reaction time: 7)

Ready for use

Calibration interval may be extended based on acceptable verification of calibration by the laboratory.

Traceability: This method has been standardized against Roche system reagent using calibrated pipettes together with a manual photometer providing absolute values and substrate-specific absorptivity, ε.

Representative performance data on the analyzers are given below. Results obtained in individual laboratories may differ.

Precision was determined using human samples and controls in an internal protocol with repeatability (n = 21) and intermediate precision (3 aliquots per run, 1 run per day, 21 days). The following results were obtained:

Serum/plasma

Urine

The data obtained on cobas c 501 analyzer(s) are representative for cobas c 311 analyzer(s).

Amylase values for human serum, plasma and urine samples obtained on a Roche/Hitachi cobas c 501 analyzer (y) were compared with those determined using the corresponding reagent on a Roche/Hitachi 917 analyzer (x).

Serum/plasma

Sample size (n) = 79

Urine

Sample size (n) = 88

The data obtained on cobas c 501 analyzer(s) are representative for cobas c 311 analyzer(s).

Summary

^LREFGreiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed. Stuttgart/New York: Schattauer Verlag 1995.

^,

^LREFKeller H, ed. Klinisch-chemische Labordiagnostik für die Praxis, 2nd ed. Stuttgart/New York: Georg Thieme Verlag 1991:354-361.

^,

^LREFSalt WB II, Schenker S. Amylase - its clinical significance: a review of the literature [Review]. Medicine 1976;55:269-281.

^,

^LREFSteinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985;102:576-580.

^,

^LREFTietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986;32(2):301-307.

^,

^LREFJunge W, Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals. Clin Biochem 1989;22:109-114.

^,

^LREFRauscher E, von Bülow S, Hägele EO, et al. Ethylidene protected substrate for the assay of human α-amylase. Fresenius Z Anal Chem 1986;324:304-305.

^,

^LREFKruse-Jarres JD, Hafkenscheid JCM, Hohenwallner W, et al. Evaluation of a New α-Amylase Assay Using 4,6-Ethylidene-(G7)-1-4-nitrophenyl-(G1)-α-D-maltoheptaoside as Substrate. J Clin Chem Clin Biochem 1989;27:103-113.

^,

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

The α‑amylases (1,4‑α‑D‑glucanohydrolases, EC 3.2.1.1) catalyze the hydrolytic degradation of polymeric carbohydrates such as amylose, amylopectin and glycogen by cleaving 1,4‑α‑glucosidic bonds. In polysaccharides and oligosaccharides, several glycosidic bonds are hydrolyzed simultaneously. Maltotriose, the smallest such unit, is converted into maltose and glucose, albeit very slowly. Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ‑specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube.

Because of the sparsity of specific clinical symptoms of pancreatic diseases, α‑amylase determinations are of considerable importance in pancreatic diagnostics. They are mainly used in the diagnosis and monitoring of acute pancreatitis. Hyperamylasemia does not, however, only occur with acute pancreatitis or in the inflammatory phase of chronic pancreatitis, but also in renal failure (reduced glomerular filtration), tumors of the lungs or ovaries, pulmonary inflammation, diseases of the salivary gland, diabetic ketoacidosis, cerebral trauma, surgical interventions or in the case of macroamylasemia. To confirm pancreatic specificity, it is recommended that an additional pancreas‑specific enzyme ‑ lipase or pancreatic‑α‑amylase ‑ also be determined.

Numerous methods have been described for the determination of α‑amylase. These either determine the decrease in the amount of substrate viscometrically, turbidimetrically, nephelometrically and amyloclastically or measure the formation of degradation products saccharogenically or kinetically with the aid of enzyme‑catalyzed subsequent reactions. The kinetic method described here is based on the well‑proven cleavage of 4,6‑ethylidene‑(G₇)‑1,4‑nitrophenyl‑(G₁)‑α,D‑maltoheptaoside (Ethylidene Protected Substrate = EPS) by α‑amylase and subsequent hydrolysis of all the degradation products to p‑nitrophenol with the aid of α‑glucosidase (100 % chromophore liberation). The results of this method correlate with those obtained by HPLC. This assay follows the recommendation of the IFCC, but was optimized for performance and stability.

R1 is in position B and R2 is in position C.

For quality control, use control materials as listed in the \"Order information\" section.

In addition, other suitable control material can be used.

The control intervals and limits should be adapted to each laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory should establish corrective measures to be taken if values fall outside the defined limits.

Follow the applicable government regulations and local guidelines for quality control.

Specimen collection and preparation

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

^,

^LREFYoung DS. Effects of Preclinical Variables on Clinical Laboratory Tests. AACC Press 1997, 2nd edition 1997.

For specimen collection and preparation only use suitable tubes or collection containers.

Only the specimens listed below were tested and found acceptable.
Serum
Plasma: Li‑heparin plasma.

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 available tubes of all manufacturers were tested. Sample collection systems from various manufacturers may contain differing materials which could affect the test results in some cases. When processing samples in primary tubes (sample collection systems), follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay.

See the limitations and interferences section for details about possible sample interferences.

Sample stability claims were established by experimental data by the manufacturer or based on reference literature and only for the temperatures/time frames as stated in the method sheet. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

Urine: Collect urine without additives. α‑Amylase is unstable in acid urine. Assay promptly or adjust pH to alkaline range (just above pH 7) before storage.

In vitro test for the quantitative determination of the catalytic activity of α‑amylase (1,4‑α‑D‑glucan: glucanohydrolase; EC 3.2.1.1) in human serum, plasma, and urine on COBAS INTEGRA systems.

Test principle

^LREFLorentz K. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC 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.

^,

^LREFKurrle-Weittenhiller A, Hölzel W, Engel D, et al. Method for the determination of total and pancreatic α-amylase based on 100 % cleavage of the protected substrate ethylidiene-4-nitrophenyl-maltoheptaoside. Clin Chem 1996;42(S6):S98.

Enzymatic colorimetric assay acc. to IFCC.

Defined oligosaccharides such as 4,6‑ethylidene-(G₇)-p‑nitrophenyl-(G₁)-α,D‑maltoheptaoside (ethylidene‑G₇PNP) are cleaved under the catalytic action of α‑amylases. The G₂PNP, G₃PNP and G₄PNP fragments so formed are completely hydrolyzed to p‑nitrophenol and glucose by α‑glucosidase.

The color intensity of the p‑nitrophenol formed is directly proportional to the α‑amylase activity. It is determined by measuring the increase in absorbance at 409 nm.

Measuring range

Serum/plasma/urine
3‑2000 U/L (0.05‑33 µkat/L)

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 5.

Lower limits of measurement

Lower detection limit of the test:
3 U/L (0.05 µkat/L)

The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying 3 standard deviations above that of a zero sample (zero sample + 3 SD, repeatability, n = 21).

Expected values

^LREFJunge W, Wortmann W, Wilke B, et al. Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC. Clin Biochem 2001;34:607-615. Erratum Clin Biochem 2003;36:161.

α‑Amylase/creatinine quotient

To allow for fluctuations in the α‑amylase activity in urine, it is advisable to determine the α‑amylase/creatinine quotient. To do this, determine the α‑amylase activity and creatinine concentration in spontaneously voided urine.

Amylase/Creatinine Clearance Ratio (ACCR)

^LREFTietz NW, ed. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia PA: WB Saunders Company 1995;46-51.

The ACCR is calculated from amylase activity and creatinine concentration. Both the serum and urine samples should be collected at the same time.

ACCR is approximately equal to 2‑5 %.

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.

Do not pipette by mouth, and ensure that the reagent does not come into contact with the skin. (Saliva and sweat contain α‑amylase!)

Criterion: Recovery within ± 10 % of initial value.

Serum/plasma

Icterus:

Hemolysis:

Lipemia (Intralipid):

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Anticoagulants: Interference was found with citrate and fluoride.

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

Urine

Drugs: No interference was found at therapeutic concentrations using common drug panels.

Criterion: Recovery within ± 10 % of initial value at an amylase activity of 460 U/L (7.68 µkat/L).

Hemolysis: No significant interference up to a hemoglobin concentration of of 311 µmol/L (500 mg/dL).

Phosphate: No significant interference from phosphate up to a concentration of 70 mmol/L (217 mg/dL).

Urea: No significant interference from urea up to a concentration of 1500 mmol/L (9009 mg/dL).

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are run together on COBAS INTEGRA analyzers. Refer to the CLEAN Method Sheet for further instructions and for the latest version of the Extra wash cycle list.
Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.

Materials required (but not provided):

Test AMYL2, test ID 0‑609 (serum, plasma)

Test AMYU2, test ID 0‑509 (urine)

Ready for use

Representative performance data on the COBAS INTEGRA analyzers are given below. Results obtained in individual laboratories may differ.

Precision was determined using human samples and controls in an internal protocol with repeatability (n = 21) and intermediate precision (1 aliquot per run, 1 run per day, 21 days). The following results were obtained:

α‑Amylase values for human serum, plasma and urine samples obtained on a COBAS INTEGRA 700 analyzer using the COBAS INTEGRA α‑Amylase EPS ver.2 (AMYL2) reagent (y) were compared with those determined using the corresponding reagent on a Roche/Hitachi 917 analyzer (x) and to the previous reagent (AMYLL) on a COBAS INTEGRA 700 analyzer (x).