INSTRUCTIONS FOR USE ALT

VITROS Chemistry Products ALT Slides Alanine

Aminotransferase

153 7513
165 5281

Intended Use
For in vitro diagnostic use only.
VITROS ALT Slides quantitatively measure alanine aminotransferase (ALT) activity in serum and plasma.

Summary and Explanation of the Test

Alanine aminotransferase is present in high activity in liver, skeletal muscle, heart, and kidney. Serum ALT increases rapidly in
liver cell necrosis, hepatitis, hepatic cirrhosis, liver tumors, obstructive jaundice, Reye’s syndrome, extensive trauma to skeletal
muscle, myositis, myocarditis, and myocardial infarction. 1

Principles of the Procedure

The VITROS ALT Slide method is performed using the VITROS ALT Slides and the VITROS Chemistry Products Calibrator
Kit 3 on VITROS Chemistry Systems.
The VITROS ALT Slide is a multilayered, analytical element coated on a polyester support.
A drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. The
spreading layer contains the ALT substrates L-alanine and sodium α-ketoglutarate. Alanine aminotransferase catalyzes the
transfer of the amino group of L-alanine to α-ketoglutarate to produce pyruvate and glutamate. Lactate dehydrogenase (LDH)
then catalyzes the conversion of pyruvate and NADH to lactate and NAD+.
The rate of oxidation of NADH is monitored by reflectance spectrophotometry. The rate of change in reflection density is
proportional to enzyme activity.

Reaction Sequence
ALT
alanine + α-ketoglutarate pyruvate + glutamate
pyridoxal-5-phosphate

+ LDH +
pyruvate + NADH + H lactate + NAD

Test Type and Conditions

Test Type and Conditions for ALT
Approximate Sample Drop
Test Type VITROS System Incubation Time Temperature Wavelength Volume
Multiple-point rate 5,1 FS, 950, 750, 550, 250 5 minutes 37°C (98.6°F) 340 nm 11 µL

Warnings and Precautions

For in vitro diagnostic use only.
Take care when handling materials and samples of human origin. Since no test method can offer complete assurance that
infectious agents are absent, consider all clinical specimens, controls, and calibrators potentially infectious. Handle specimens,
solid and liquid waste, and test components in accordance with local regulations and NCCLS Guideline M29 2 or other
published biohazard safety guidelines.
For specific warnings and precautions for calibrators, quality control materials, and other components, refer to the Instructions
for Use for the appropriate VITROS product, or to other manufacturer’s product literature.

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 ALT INSTRUCTIONS FOR USE
Alanine Aminotransferase Reagents

Reagents
Slide Diagram
Slide Ingredients 1. Upper slide mount
2 2. Spreading layer (BaSO4)
Reactive ingredients per cm • sodium α–ketoglutarate
• L-alanine
Lactate dehydrogenase (porcine muscle, E.C.1.1.1.27) 0.12 U; 3. Reagent layer
L-alanine 0.86 mg; sodium α-ketoglutarate 54 µg; nicotinamide adenine • buffer, pH 8.0
dinucleotide, reduced 35 µg; and sodium pyridoxal-5-phosphate 11 µg. • lactate dehydrogenase
• NADH
Other ingredients • pyridoxal-5-phosphate
4. Support layer
Pigment, binders, buffer, surfactants, cross-linking agent and stabilizer. 5. Lower slide mount

Cartridge Handling
CAUTION: Do not use slide cartridges with damaged or incompletely sealed packaging.

• Inspect the packaging for signs of damage.

• Be careful when opening the outer packaging with a sharp instrument so as to avoid damage to the individual product
packaging.

Cartridge Preparation
IMPORTANT: The slide cartridge must reach room temperature, 18°–28°C (64°–82°F), before it is
unwrapped and loaded into the slide supply.

1. Remove the slide cartridges from storage.

2. Warm the wrapped cartridge at room temperature for 30 minutes when taken from the refrigerator or 60 minutes from the
freezer.
3. Unwrap and load the cartridge into the slide supply.
NOTE: Load the cartridges within 24 hours after they reach room temperature,
18°–28°C (64°–82°F).

Slide Storage and Stability

VITROS ALT Slides are stable until the expiration date on the carton when they are stored and handled as specified.

Slide Storage and Stability for ALT

Slide Cartridges Storage Condition Stability
Unopened Refrigerated 2°–8°C (36°–46°F) Until expiration date
Frozen ≤-18°C (≤0°F) Until expiration date
Opened On-analyzer System turned on ≤4 weeks
On-analyzer System turned off ≤2 hours

• Verify performance with quality control materials:

– If the system is turned off for more than 2 hours.
– After reloading cartridges that have been removed from the slide supply and stored for later use.

Specimen Requirements
WARNING: Handle specimens as biohazardous material.

Specimens Recommended
• Serum
• Plasma: EDTA
Heparin

IMPORTANT: Certain collection devices have been reported to affect other analytes and tests. 3
Confirm that your collection devices are compatible with this test.

Specimens Not Recommended

• Do not use hemolyzed specimens. 4

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 INSTRUCTIONS FOR USE ALT
Testing Procedure Alanine Aminotransferase

Serum and Plasma

Specimen Collection and Preparation
Collect specimens using standard laboratory procedures. 5, 6
NOTE: For details on minimum fill volume requirements, refer to the operating instructions for
your VITROS Chemistry System.

Patient Preparation
• No special patient preparation is necessary.
Special Precautions
• Centrifuge specimens and remove the serum or plasma from the cellular material within 3 days of collection. 7
Specimen Handling and Storage
WARNING: Handle specimens as biohazardous material.

• Handle and store specimens in stoppered containers to avoid contamination and evaporation.
• Mix samples by gentle inversion and bring to room temperature, 18°–28°C (64°–82°F), prior to analysis.
IMPORTANT: Do not freeze the specimen.

Specimen Storage and Stability for ALT: Serum and Plasma 7

Storage Temperature Stability
Room temperature 18°–28°C (64°–82°F) ≤3 days
Refrigerated 2°–8°C (36°–46°F) ≤1 week
Frozen ≤-18°C (≤0°F) Not recommended

Testing Procedure
Materials Provided
• VITROS Chemistry Products ALT Slides

Materials Required But Not Provided

• VITROS Chemistry Products Calibrator Kit 3
• Quality control materials, such as VITROS Chemistry Products Performance Verifier I and II
• VITROS Chemistry Products 7% BSA
• VITROS Chemistry Products FS Diluent Pack 2 (BSA/Saline) (for on-analyzer dilution)

Operating Instructions
• Check reagent inventories at least daily to ensure that quantities are sufficient for the planned workload.
• For additional information, refer to the operating instructions for your VITROS Chemistry System.
IMPORTANT: Bring all fluids and samples to room temperature, 18°–28°C (64°–82°F), prior to
analysis.

Sample Dilution
Serum and Plasma
If alanine aminotransferase activities exceed the system’s reportable (dynamic) range:
Manual Sample Dilution
1. Dilute the sample with VITROS 7% BSA.
2. Reanalyze.
3. Multiply the results by the dilution factor to obtain an estimate of the original sample’s alanine aminotransferase activity.
On-Analyzer Sample Dilution (VITROS 5,1 FS and VITROS 250 only)
Refer to the VITROS Chemistry System operating instructions for more information on the On-Analyzer Dilution Procedure. For
VITROS 5,1 FS, use VITROS Chemistry Products FS Diluent Pack 2 for the dilution.

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 ALT INSTRUCTIONS FOR USE
Alanine Aminotransferase Calibration

Calibration
Required Calibrators
VITROS Chemistry Products Calibrator Kit 3

Calibrator Preparation, Handling, and Storage

Refer to the Instructions for Use for VITROS Calibrator Kit 3.

Calibration Procedure
Refer to the operating instructions for your VITROS Chemistry System.

When to Calibrate
Calibrate:
• When the slide lot number changes.
• When critical system parts are replaced due to service or maintenance.
• When government regulations require.
– For example, in the USA, CLIA regulations require calibration or calibration verification at least once every six months.
The VITROS ALT test may also need to be calibrated:
• If quality control results are consistently outside acceptable range.
• After certain service procedures have been performed.
For additional information, refer to the operating instructions for your VITROS Chemistry System.

Calculations
Based on sequential readings of the slide’s reflectance at 340 nm over the defined incubation period, a rate of change in
reflectance is determined. This rate is used in the software-resident multi-point rate calibration model to compute enzyme
activity. Once a calibration has been performed for each slide lot, alanine aminotransferase activity in unknown samples can be
determined from the rate of change in reflectance measured for each unknown test slide.

Validity of a Calibration
Calibration parameters are automatically assessed by the VITROS Chemistry System against a set of quality parameters
detailed in the Coefficients and Limits screen (for VITROS 5,1 FS, see the Review Assay Data screen). Failure to meet any of
the pre-defined quality parameters results in a failed calibration. The calibration report should be used in conjunction with
quality control results to determine the validity of a calibration.

Reportable (Dynamic) Range

Reportable (Dynamic) Range for ALT
Conventional and SI Units Alternate Units
(U/L) (µkat/L)
3–1000 0.05–16.70

For out-of-range samples, refer to “Sample Dilution.”

Traceability of the Calibration

Values assigned to the VITROS Chemistry Products Calibrator Kit 3 for alanine aminotransferase are traceable to the alanine
aminotransferase method recommended by the International Federation of Clinical Chemistry (IFCC), 8 adapted to a centrifugal
analyzer at 37°C.

Quality Control
Procedure Recommendations
WARNING: Handle quality control materials as biohazardous material.

• Choose control levels that check the clinically relevant range.

• Analyze quality control materials in the same manner as patient samples, before or during patient sample processing.
• To verify system performance, analyze control materials:
– After calibration.
– According to local regulations or at least once each day that the test is being performed.
– After specified service procedures are performed. Refer to the operating instructions for your VITROS Chemistry
System.
• If control results fall outside your acceptable range, investigate the cause before deciding whether to report patient results.

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 INSTRUCTIONS FOR USE ALT
Expected Values and Reporting Units Alanine Aminotransferase

• For general quality control recommendations, refer to Statistical Quality Control for Quantitative Measurements: Principles
and Definitions; Approved Guideline-Second Edition 9 or other published guidelines.
• For additional information, refer to the operating instructions for your VITROS Chemistry System.

Quality Control Material Selection

IMPORTANT: VITROS Performance Verifiers are recommended for use with the VITROS Chemistry
System. Evaluate the performance of other commercial control fluids for compatibility
with this test before using for quality control.

• Control materials other than VITROS Performance Verifiers may show a difference when compared with other alanine
aminotransferase methods if they:
– Depart from a true human matrix.
– Contain high concentrations of preservatives, stabilizers, or other nonphysiological additives.
• Enzyme activity might also vary with enzyme source, diluent temperature, and activation time during reconstitution.
• Do not use control materials stabilized with ethylene glycol.

Quality Control Material Preparation and Storage

Refer to the Instructions for Use for VITROS Chemistry Products Performance Verifier I and II or to other manufacturer's
product literature.

Expected Values and Reporting Units

Reference Interval
These reference intervals are the central 95% of results from an internal study of 2444 apparently healthy adults (547 females
and 1897 males).

Reference Interval for ALT

Conventional and SI Units Alternate Units
(U/L) (µkat/L)
Adult 13–69 0.2–1.2
Females 9–52 0.2–0.9
Males 21–72 0.4–1.2

Each laboratory should confirm the validity of these intervals for the population it serves.

Reporting Units and Unit Conversion

The VITROS Chemistry System may be programmed to report ALT results in conventional, SI, and alternate units.

Reporting Units and Unit Conversion for ALT

Conventional/SI Units Alt Units
U/L µkat/L (U/L x 0.0167)

Limitations of the Procedure

Known Interferences
None identified.

Other Limitations
Certain drugs and clinical conditions are known to alter alanine aminotransferase activity in vivo. For additional information,
refer to one of the published summaries. 10, 11

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 ALT INSTRUCTIONS FOR USE
Alanine Aminotransferase Performance Characteristics

Performance Characteristics
Method Comparison
The plot and table show the results of a comparison of samples analyzed on the VITROS 750 System with those analyzed
using the IFCC comparative method, 8 adapted to a centrifugal analyzer at 37°C. Testing followed NCCLS Protocol EP9. 12
The table also shows the results of comparisons of the VITROS 250 and 950 Systems with the VITROS 750 System, and
comparisons of the 5,1 FS System with the 950 System.

Method Comparison for ALT: Serum

Conventional and SI Units Alternate Units

VITROS 750 System (µkat/L)

VITROS 750 System (U/L)

Comparative Method: Modified IFCC at 37°C Comparative Method: Modified IFCC at 37°C
(U/L) (µkat/L)

Method Comparison for ALT: Serum

Conventional and SI Units (U/L) Alternate Units (µkat/L)
Correlation Range of Range of
n Slope Coefficient Sample Activity Intercept Sy.x Sample Activity Intercept Sy.x
750 System vs.
195 1.02 0.997 4–911 +9.2 20.3 0.1–15.2 +0.15 0.34
comparative method
250 System vs.
60 1.01 0.999 14–373 -0.6 2.6 0.2–6.2 -0.01 0.04
750 System
950 System vs.
122 0.99 0.999 9–988 -0.9 2.4 0.2–16.5 -0.02 0.04
750 System
5,1 FS System vs.
134 1.00 1.000 11–886 +1.7 2.3 0.2–14.8 +0.03 0.04
950 System

Precision
Precision was evaluated with quality control materials on VITROS 250, 750, 950, and 5,1 FS Systems following NCCLS
Protocol EP5. 13
The data presented are a representation of test performance and are provided as a guideline. Variables such as sample
handling and storage, reagent handling and storage, laboratory environment, and system maintenance can affect
reproducibility of test results.

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 INSTRUCTIONS FOR USE ALT
References Alanine Aminotransferase

Precision for ALT: Serum

Conventional and SI Units (U/L) Alternate Units (µkat/L) Within
Mean Within Within Mean Within Within Lab No. No.
System Activity Day SD* Lab SD** Activity Day SD* Lab SD** CV%** Observ. Days
VITROS 250 37 3.5 4.1 0.6 0.06 0.07 11.2 78 20
204 3.8 4.9 3.4 0.06 0.08 2.4 78 20
VITROS 750 34 2.0 2.7 0.6 0.03 0.05 8.1 90 23
189 2.3 3.7 3.2 0.04 0.06 2.0 92 23
VITROS 950 34 1.7 3.2 0.6 0.03 0.05 9.6 84 23
186 1.7 3.2 3.1 0.03 0.05 1.7 86 23
VITROS 5,1 FS 44 1.6 2.9 0.7 0.03 0.05 6.6 96 22
187 1.9 3.5 3.1 0.03 0.06 1.9 96 22
* Within Day precision was determined using two runs/day with two to three replications.
** Within Lab precision was determined using a single lot of slides and calibrating weekly.

Specificity
Substances That Do Not Interfere
The substances listed in the table were tested with VITROS ALT Slides following NCCLS Protocol EP7 14 and found not to
interfere, bias <5.5 U/L, at the concentration shown.

Substances That Do Not Interfere With ALT

Compound Concentration Compound Concentration
AST 200 U/L 200 U/L Glutathione 1 mg/dL 33 µmol/L
Ascorbic Acid 3 mg/dL 170 µmol/L Intralipid 800 mg/dL 8 g/L
Bilirubin 40 mg/dL 684 µmol/L Salicylic acid 35 mg/dL 2.5 mmol/L
Ethanol 300 mg/dL 65 mmol/L

References
1. Tietz NW (ed). Fundamentals of Clinical Chemistry. ed. 3. Philadelphia: WB Saunders; 369–371; 1987.
2. NCCLS. Protection of Laboratory Workers from Instrument Biohazards and Infectious Diseases Transmitted by Blood, Body Fluids and
Tissue; Approved Guideline. NCCLS Document M29 (ISBN 1-56238). NCCLS, Wayne, PA 19087; 1997.
3. Calam RR. Specimen Processing Separator Gels: An Update. J Clin Immunoassay. 11:86–90; 1988.
4. Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests. Washington D.C.: AACC Press; 3–7; 1993.
5. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture. NCCLS Document H3. Wayne, PA: NCCLS;
1991.
6. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Skin Puncture. NCCLS Document H4. Wayne, PA: NCCLS;
1991.
7. Clinical Laboratory Handbook for Patient Preparation and Specimen Handling. Fascicle VI: Chemistry/Clinical Microscopy. Northfield,
IL: College of American Pathologists; 1992.
8. Bergmeyer HU, Horder M, Rej R. Approved Recommendation (1985) on IFCC Methods for the Measurement of Catalytic Concentration
of Enzymes. Part 3. IFCC Method for Alanine Aminotransferase. J. Clin. Chem. Clin. Biochem. 24:481; 1986.
9. NCCLS. Statistical Quality Control for Quantitative Measurements: Principles and Definitions; Approved Guideline-Second Edition.
NCCLS Document C24. Wayne, PA: NCCLS; 1999.
10. Young DS. Effects of Drugs on Clinical Laboratory Tests. ed. 4. Washington D.C.: AACC Press; 1995.
11. Friedman RB, Young DS. Effects of Disease on Clinical Laboratory Tests. Washington, D.C.: AACC Press; 1990.
12. NCCLS. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline. NCCLS Document EP9. Wayne, PA:
NCCLS; 1995.
13. NCCLS. User Evaluation of Precision Performance with Clinical Chemistry Devices. NCCLS Document EP5. Wayne, PA: NCCLS;
1992.
14. NCCLS. Interference Testing in Clinical Chemistry. NCCLS Document EP7. Wayne, PA: NCCLS; 1986.

Version 3.0 Pub. No. MP2-36_EN 7

 ALT INSTRUCTIONS FOR USE
Alanine Aminotransferase Glossary of Symbols

Glossary of Symbols

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 INSTRUCTIONS FOR USE ALT
Revision History Alanine Aminotransferase

Revision History
Date of
Revision Version Description of Technical Changes*
2004-09-13 3.0 • Added VITROS 5,1 FS Chemistry System
• Specificity – added Intralipid; updated Bilirubin
• Glossary of Symbols – updated data
2003-07-28 2.0 • New organization and sections consistent with IVD Directive
• Reference Interval – adult: corrected values
• Limitations of the Procedure – removed statements regarding elevated total
protein
• Method Comparison – updated comparisons for 750 System and plots
• Precision – updated data for the 750 System
• References – added 2, 3, 12, 13, 14
2002APR19 1.0 – English only New format, technically equivalent to 11/96.
* The change bars indicate the position of a technical amendment to the text with respect to the previous version of the document.

When this Instructions For Use is replaced, sign and date below and retain as specified by local regulations or laboratory
policies, as appropriate.

_________________________________ _____________
Signature Obsolete Date

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 ALT INSTRUCTIONS FOR USE
Alanine Aminotransferase

Ortho-Clinical Diagnostics Brasil:

Johnson & Johnson Distribuidor:Johnson&Johnson Produtos Profissionais LTDA
50-100 Holmers Farm Way Rod. Presidente Dutra, Km 154, S.J. dos Campos – SP –
High Wycombe CEP: 12240-908 - Brasil
Buckinghamshire CNPJ:54.516.661/0002-84
HP12 4DP Farm.Resp.: Nancy M.R.B Lopes C.R.F.-SP N° 10965
United Kingdom SAC : 0800787865

Ortho-Clinical Diagnostics, Inc. VITROS is a trademark of Ortho-Clinical Diagnostics, Inc.

100 Indigo Creek Drive © Ortho-Clinical Diagnostics, Inc., 2003, 2004.
Rochester, NY 14626-5101

10 Pub. No. MP2-36_EN Version 3.0