In vitro test for the quantitative determination of alanine aminotransferase (ALT), with or without pyridoxal phosphate activation, in human serum and plasma on the cobas c 111 system.
This assay follows the recommendations of the IFCC, but was optimized for performance and stability.
ALT catalyzes the reaction between L-alanine and 2-oxoglutarate. The pyruvate formed is reduced by NADH in a reaction catalyzed by lactate dehydrogenase (LDH) to form L-lactate and NAD+.
Pyridoxal phosphate serves as a coenzyme in the amino transfer reaction. It ensures full enzyme activation.
ALT | ||
L-alanine + 2-oxoglutarate | pyruvate + L-glutamate | |
LDH | ||
Pyruvate + NADH + H+ | L-lactate + NAD+ |
The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by measuring the decrease in absorbance.
2‑700 U/L (0.03‑11.7 µkat/L)
Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:10 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 10.
Lower detection limit of the test
2 U/L (0.03 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).
• Without PYP activation
Acc. to the optimized standard method (comparable to the IFCC method without pyridoxal phosphate activation
Males | up to 41 U/L | up to 0.685 µkat/L |
Females | up to 33 U/L | up to 0.551 µkat/L |
Calculated values: a factor of 1.85 is used for the conversion from 25 °C to 37 °C.
• With PYP activation
acc. to IFCC/Standard Method 94 with pyridoxal phosphate activation:
Measured at 37 °C: | ||
Males | 10-50 U/L | (0.167-0.835 µkat/L) |
Females | 10-35 U/L | (0.167-0.585 µkat/L) |
Consensus values with pyridoxal phosphate activation:
Males | up to 50 U/L | (up to 0.835 µkat/L) |
Females | up to 35 U/L | (up to 0.585 µkat/L) |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
• Without PYP activation
Criterion: Recovery within ± 10 % of initial value at an ALT activity of 30 U/L (0.501 µkat/L)
• With PYP activation
Criterion: Recovery within ± 10 % of initial value at an ALT activity of 35 U/L (0.585 µkat/L)
Icterus:
Hemolysis:
Lipemia (Intralipid):
Anticoagulants: Citrate and fluoride inhibit the enzyme activity.
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.
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 the cobas c 111 analyzer. For information about test combinations requiring special wash steps, please refer to the latest version of the carry over evasion list found with the CLEAN Method Sheet and the operator’s manual for further instructions.
Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which kit(s) can be used | |
04718569190 | Alanine aminotransferase acc. IFCC (4 × 100 tests) | cobas c 111 |
Materials required (but not provided):
04774221190 | Pyridoxal Phosphate (4 × 200 tests) | Code 953 | |
10759350190 | Calibrator f.a.s. (12 × 3 mL) | Code 401 | |
12149435122 | Precinorm U plus (10 × 3 mL) | Code 300 | |
12149443122 | Precipath U plus (10 x 3 mL) | ||
05117003190 | PreciControl ClinChem Multi 1 (20 × 5 mL) | Code 391 | |
05947626190 | PreciControl ClinChem Multi 1 (4 × 5 mL) | Code 391 | |
05117216190 | PreciControl ClinChem Multi 2 (20 × 5 mL) | Code 392 | |
05947774190 | PreciControl ClinChem Multi 2 (4 × 5 mL) |
ALTL: ACN 685
ALTPL: ACN 684
Ready for use
cobas c 111 test definition | |
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction direction | Decrease |
Wavelength A/B | 340/378 nm |
Calc. first/last | 20/35 |
Unit | U/L |
Reaction mode | R1-S-SR |
Pipetting parameters | ||
Diluent (H2O) | ||
R1 | 59 µL | 10 µL |
Sample | 11 µL | 26 µL |
SR | 17 µL | 9 µL |
Total volume | 132 µL |
cobas c 111 test definition | |
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction direction | Decrease |
Wavelength A/B | 340/378 nm |
Calc. first/last | 20/35 |
Unit | U/L |
Reaction mode | R1-DL-S-SR |
Pipetting parameters | ||
Diluent (H2O) | ||
R1 | 59 µL | 10 µL |
Sample | 11 µL | 8 µL |
Diluent (DL) | 18 µL | |
SR | 17 µL | 9 µL |
Total volume | 132 µL |
See expiration date on reagent | |
On-board in use and refrigerated on the analyzer: | 4 |
Calibrator | Calibrator f.a.s. |
Calibration mode | Linear regression |
Calibration interval | Each lot and as required following quality control procedures |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against the original IFCC formulation using calibrated pipettes together with a manual photometer providing absolute values and the 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:
Repeatability | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 39.6 (0.661) | 0.5 (0.008) | 1.1 |
Precipath U | 127 (2.12) | 1 (0.02) | 0.5 |
Human serum 1 | 27.9 (0.466) | 0.6 (0.010) | 2.0 |
Human serum 2 | 99.4 (1.66) | 0.5 (0.01) | 0.5 |
Intermediate precision | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 40.2 (0.671) | 0.7 (0.012) | 1.8 |
Precipath U | 124 (2.07) | 1 (0.02) | 0.9 |
Human serum 3 | 22.0 (0.367) | 0.6 (0.010) | 2.9 |
Human serum 4 | 272 (4.54) | 8 (0.14) | 3.0 |
Repeatability | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 41.5 (0.693) | 0.6 (0.010) | 1.5 |
Precipath U | 132 (2.20) | 1 (0.02) | 0.4 |
Human serum 1 | 32.1 (0.536) | 0.7 (0.012) | 2.1 |
Human serum 2 | 337 (5.63) | 2 (0.03) | 0.5 |
Intermediate precision | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 42.0 (0.701) | 0.7 (0.012) | 1.6 |
Precipath U | 130 (2.17) | 1 (0.02) | 0.7 |
Human serum 3 | 32.7 (0.546) | 0.9 (0.015) | 2.7 |
Human serum 4 | 331 (5.53) | 9 (0.15) | 2.6 |
ALT values for human serum and plasma 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).
Sample size (n) = 77 | |
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.000x + 0.753 U/L τ = 0.983 | y = 0.999x + 0.924 U/L r = 1.00 |
The sample activities were between 2.20 and 405 U/L (0.037 and 6.76 µkat/L). |
Sample size(n) = 74 | |
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.005x + 0.793 U/L τ = 0.987 | y = 1.011x + 0.672 U/L r = 1.00 |
The sample activities were between 3.04 and 399 U/L (0.051 and 6.66 µkat/L). |
The enzyme alanine aminotransferase (ALT) is present in highest concentrations in the liver, in the cytosol of the hepatocytes, although it is also found in the kidney, and, in much smaller quantities, in heart and skeletal muscle cells.
Although both serum aspartate aminotransferase (AST) and ALT become elevated whenever disease processes affect liver cell integrity, evidence suggests that ALT is a more specific marker of hepatic injury than AST. Moreover, elevations of ALT activity persist longer than elevations of AST activity.
In patients with vitamin B6 deficiency (insufficient endogenous pyridoxal phosphate), serum aminotransferase activity may be decreased. The addition of pyridoxal phosphate to this assay causes an increase in aminotransferase activity (activation higher for AST than for ALT). Pyridoxal phosphate activation prevents falsely low aminotransferase activity in patient samples with insufficient endogenous pyridoxal phosphate (vitamin B6 deficiency).
R1 | TRIS buffer: 224 mmol/L, pH 7.3 (37 °C); L-alanine: 1120 mmol/L; albumin (bovine): 0.25 %; LDH (microorganisms): ≥ 45 µkat/L; stabilizers; preservative |
PYP | Pyridoxal phosphate (DL): 730 µmol/L; preservative |
SR | NADH (yeast): ≥ 1.7 mmol/L; 2-oxoglutarate: 94 mmol/L; preservative; additives |
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
Safety data sheet available for professional user on request.
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.
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, K3-EDTA
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.
Separate the serum or plasma from the clot or cells promptly.
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
Stability: LREFWHO Publication: Use of anticoagulants in diagnostic laboratory investigations, WHO/DIL/LAB/99.1 Rev.2:Jan 2002. | 3 days at 20‑25 °C 7 days at 4‑8 °C 7 days at -20 |
Freeze only once.
In vitro test for the quantitative determination of alanine aminotransferase (ALT), with or without pyridoxal phosphate activation, in human serum and plasma on the cobas c 111 system.
This assay follows the recommendations of the IFCC, but was optimized for performance and stability.
ALT catalyzes the reaction between L-alanine and 2-oxoglutarate. The pyruvate formed is reduced by NADH in a reaction catalyzed by lactate dehydrogenase (LDH) to form L-lactate and NAD+.
Pyridoxal phosphate serves as a coenzyme in the amino transfer reaction. It ensures full enzyme activation.
ALT | ||
L-alanine + 2-oxoglutarate |
| pyruvate + L-glutamate |
LDH | ||
Pyruvate + NADH + H+ |
| L-lactate + NAD+ |
The rate of the NADH oxidation is directly proportional to the catalytic ALT activity. It is determined by measuring the decrease in absorbance.
2‑700 U/L (0.03‑11.7 µkat/L)
Determine samples having higher activities via the rerun function. Dilution of samples via the rerun function is a 1:10 dilution. Results from samples diluted using the rerun function are automatically multiplied by a factor of 10.
Lower detection limit of the test
2 U/L (0.03 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).
• Without PYP activation
Acc. to the optimized standard method (comparable to the IFCC method without pyridoxal phosphate activation
Males | up to 41 U/L | up to 0.685 µkat/L |
Females | up to 33 U/L | up to 0.551 µkat/L |
Calculated values: a factor of 1.85 is used for the conversion from 25 °C to 37 °C.
• With PYP activation
acc. to IFCC/Standard Method 94 with pyridoxal phosphate activation:
Measured at 37 °C: | ||
Males | 10-50 U/L | (0.167-0.835 µkat/L) |
Females | 10-35 U/L | (0.167-0.585 µkat/L) |
Consensus values with pyridoxal phosphate activation:
Males | up to 50 U/L | (up to 0.835 µkat/L) |
Females | up to 35 U/L | (up to 0.585 µkat/L) |
Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges.
• Without PYP activation
Criterion: Recovery within ± 10 % of initial value at an ALT activity of 30 U/L (0.501 µkat/L)
• With PYP activation
Criterion: Recovery within ± 10 % of initial value at an ALT activity of 35 U/L (0.585 µkat/L)
Icterus:
Hemolysis:
Lipemia (Intralipid):
Anticoagulants: Citrate and fluoride inhibit the enzyme activity.
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.
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 the cobas c 111 analyzer. For information about test combinations requiring special wash steps, please refer to the latest version of the carry-over evasion list found with the CLEAN Method Sheet and the operator’s manual for further instructions.
Where required, special wash/carry-over evasion programming must be implemented prior to reporting results with this test.
|
| Analyzer(s) on which kit(s) can be used | |
04718569190 | Alanine aminotransferase acc. IFCC (4 × 100 tests) | cobas c 111 |
Materials required (but not provided):
04774221190 | Pyridoxal Phosphate (4 × 200 tests) | Code 953 | |
Calibrator f.a.s. (12 × 3 mL) | Code 401 | ||
12149435160 | Precinorm U plus (10 × 3 mL) | Code 300 | |
12149443160 | Precipath U plus (10 x 3 mL) | Code 301 | |
05947626160 | PreciControl ClinChem Multi 1 (4 × 5 mL) | Code 391 | |
05947774160 | PreciControl ClinChem Multi 2 (4 × 5 mL) | Code 392 |
ALTL: ACN 685
ALTPL: ACN 684
Ready for use
cobas c 111 test definition | |
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction direction | Decrease |
Wavelength A/B | 340/378 nm |
Calc. first/last | 20/35 |
Unit | U/L |
Reaction mode | R1-S-SR |
Pipetting parameters | ||
Diluent (H2O) | ||
R1 | 59 µL | 10 µL |
Sample | 11 µL | 26 µL |
SR | 17 µL | 9 µL |
Total volume | 132 µL |
cobas c 111 test definition | |
Measuring mode | Absorbance |
Abs. calculation mode | Kinetic |
Reaction direction | Decrease |
Wavelength A/B | 340/378 nm |
Calc. first/last | 20/35 |
Unit | U/L |
Reaction mode | R1-DL-S-SR |
Pipetting parameters | ||
Diluent (H2O) | ||
R1 | 59 µL | 10 µL |
Sample | 11 µL | 8 µL |
Diluent (DL) | 18 µL | |
SR | 17 µL | 9 µL |
Total volume | 132 µL |
Shelf life at 2-8 °C: | See expiration date on reagent |
On-board in use and refrigerated on the analyzer: | 4 weeks |
Shelf life at 2-8 °C: | See expiration date on reagent |
On-board in use and refrigerated on the analyzer: | 4 weeks |
Calibrator | Calibrator f.a.s. |
Calibration mode | Linear regression |
Calibration interval | Each lot and as required following quality control procedures |
Calibration interval may be extended based on acceptable verification of calibration by the laboratory.
Traceability: This method has been standardized against the original IFCC formulation using calibrated pipettes together with a manual photometer providing absolute values and the 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:
Repeatability | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 39.6 (0.661) | 0.5 (0.008) | 1.1 |
Precipath U | 127 (2.12) | 1 (0.02) | 0.5 |
Human serum 1 | 27.9 (0.466) | 0.6 (0.010) | 2.0 |
Human serum 2 | 99.4 (1.66) | 0.5 (0.01) | 0.5 |
Intermediate precision | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 40.2 (0.671) | 0.7 (0.012) | 1.8 |
Precipath U | 124 (2.07) | 1 (0.02) | 0.9 |
Human serum 3 | 22.0 (0.367) | 0.6 (0.010) | 2.9 |
Human serum 4 | 272 (4.54) | 8 (0.14) | 3.0 |
Repeatability | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 41.5 (0.693) | 0.6 (0.010) | 1.5 |
Precipath U | 132 (2.20) | 1 (0.02) | 0.4 |
Human serum 1 | 32.1 (0.536) | 0.7 (0.012) | 2.1 |
Human serum 2 | 337 (5.63) | 2 (0.03) | 0.5 |
Intermediate precision | Mean U/L (µkat/L) | SD U/L (µkat/L) | CV % |
|---|---|---|---|
Precinorm U | 42.0 (0.701) | 0.7 (0.012) | 1.6 |
Precipath U | 130 (2.17) | 1 (0.02) | 0.7 |
Human serum 3 | 32.7 (0.546) | 0.9 (0.015) | 2.7 |
Human serum 4 | 331 (5.53) | 9 (0.15) | 2.6 |
ALT values for human serum and plasma 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).
Sample size (n) = 77 | |
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.000x + 0.753 U/L τ = 0.983 | y = 0.999x + 0.924 U/L r = 1.00 |
The sample activities were between 2.20 and 405 U/L (0.037 and 6.76 µkat/L). |
Sample size(n) = 74 | |
Passing/Bablok LREFBablok W, Passing H, Bender R, et al. A general regression procedure for method transformation. Application of linear regression procedures for method comparison studies in clinical chemistry, Part III. J Clin Chem Clin Biochem 1988 Nov;26(11):783-790. | Linear regression |
y = 1.005x + 0.793 U/L τ = 0.987 | y = 1.011x + 0.672 U/L r = 1.00 |
The sample activities were between 3.04 and 399 U/L (0.051 and 6.66 µkat/L). |
The enzyme alanine aminotransferase (ALT) has been widely reported as present in a variety of tissues. The major source of ALT is the liver, which has led to the measurement of ALT activity for the diagnosis of hepatic diseases. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction.
Although both serum aspartate aminotransferase (AST) and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver-specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity.
In patients with vitamin B6 deficiency, serum aminotransferase activity may be decreased. The apparent reduction in aminotransferase activity may be related to decreased pyridoxal phosphate, the prosthetic group for aminotransferases, resulting in an increase in the ratio of apoenzyme to holoenzyme.
The addition of pyridoxal phosphate to the assay causes an increase in aminotransferase activity. The activation is higher for AST than for ALT. Pyridoxal phosphate activation prevents falsely low aminotranferase activity in patient samples with insufficient endogenous pyridoxal phosphate (vitamin B6 deficiency).
R1 | TRIS buffer: 224 mmol/L, pH 7.3 (37 °C); L-alanine: 1120 mmol/L; albumin (bovine): 0.25 %; LDH (microorganisms): ≥ 45 µkat/L; stabilizers; preservative |
PYP | Pyridoxal phosphate (DL): 730 µmol/L; preservative |
SR | NADH (yeast): ≥ 1.7 mmol/L; 2-oxoglutarate: 94 mmol/L; preservative; additives |
For in vitro diagnostic use for health care professionals. Exercise the normal precautions required for handling all laboratory reagents.
Infectious or microbial waste:
Warning: handle waste as potentially biohazardous material. Dispose of waste according to accepted laboratory instructions and procedures.
Environmental hazards:
Apply all relevant local disposal regulations to determine the safe disposal.
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.
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.
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, K3-EDTA
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.
Separate the serum or plasma from the clot or cells promptly.
Centrifuge samples containing precipitates before performing the assay.
See the limitations and interferences section for details about possible sample interferences.
Stability: LREFWHO Publication: Use of anticoagulants in diagnostic laboratory investigations, WHO/DIL/LAB/99.1 Rev.2:Jan 2002. | 3 days at 20‑25 °C 7 days at 4‑8 °C 7 days at −20 °C |
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.
Alanine aminotransferase acc. IFCC with or without pyridoxal phosphate activation
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