Siemens Healthcare Diagnostics Inc.

Instructions for Use

2017-08

Alanine Aminotransferase, Concentrated Reagents (ALT_c)

System New Information
ADVIA® 1200 Updates: Limitations of the Procedure
ADVIA 1650/1800 Updates: Limitations of the Procedure
ADVIA 2400 Updates: Limitations of the Procedure

Note To run the concentrated Alanine Aminotransferase reagents, you must use a
special parameter set. Refer to the system- and software-specific ALT_c parameter
sheets.

Method Summary
Item Description
Method Principle Modified IFCC

Specimen Type Human serum and plasma (lithium heparin)

On-board Stability ADVIA 1200: 45 days
ADVIA 1650/ADVIA 1800: 45 days
ADVIA 2400: 45 days
Reagent Storage Temperature 2–8°C
Calibration Frequency 45 days

Reagent Blank (RBL) Frequency Daily

Reaction Type Rate (RRA)
Measurement Wavelength 340/410 nm
Standardization International Federation of Clinical Chemistry (IFCC)
reference method
Analytical Range Serum/Plasma: 8–1100 U/L
Expected Values 10–49 U/L
Reagent Code 74708
Calibrators ADVIA Chemistry Enzyme 2 Calibrator
(REF 10916058)

© 2009 Siemens Healthcare Diagnostics. All rights reserved.

Siemens Healthcare Diagnostics Inc., Tarrytown, NY 10591-5097 USA
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Intended Use
For in vitro diagnostic use in the quantitative determination of alanine aminotransferase
activity in human serum and plasma on ADVIA Chemistry systems. Such
measurements are used mainly in the diagnosis and treatment of liver disease and to
monitor the course of treatment for hepatitis and active post necrotic cirrhosis.

Summary and Explanation

The ADVIA Chemistry concentrated Alanine Aminotransferase (ALT_c) method is
based on work by Wroblewski and LaDue. The procedure was later modified by
H.U. Bergmeyer.1

Principles of the Procedure

The reaction is initiated by the addition of -ketoglutarate as a second reagent. The
concentration of reduced nicotinamide adenine dinucleotide (NADH) is measured by its
absorbance at 340/410 nm and the rate of absorbance decrease is proportional to the
alanine aminotransferase (ALT) activity.

Reaction Equations

L-Alanine + -Ketoglutarate Pyruvate + L-Glutamate

Pyruvate + NADH Lactate + NAD

Reagents
The reagents are provided in the package as listed below. Components of the package
are available as a kit only.

REF (PN) No. of

Container Size Symbol Contents Amount Tests

06860469 Alanine 6 x 850

Aminotransferase
Reagents

40-mL Reagent 1 6 x 38 mL

40-mL Reagent 2 6 x 27 mL

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Components and Concentrations

Reagent Component Concentration

Reagent 1 L-Alanine 1.22 mol/L
LD (pig heart)  2.4 KU/L
Sodium azide 0.09%
Reagent 2 Ketoglutarate 93 mmol/L
Reduced nicotinamide adenine 1.41 mmol/L
dinucleotide (NADH)
Sodium azide 0.09%

CAUTION! This device contains material of animal origin and should be handled as a
potential carrier and transmitter of disease.

Note Sodium azide can react with copper and lead plumbing to form explosive metal
azides. If disposal into a drain is in compliance with federal, state, and local
requirements, flush reagents with a large amount of water to prevent the buildup of
azides.

Safety data sheets (MSDS/SDS) available on siemens.com/healthcare.

For in vitro diagnostic use.

Reagent Preparation and Use

Reagents are ready to use. Before use, gently swirl the reagent to disrupt bubbles and
assure homogeneity. If bubbles still exist or foam is present, using a clean transfer
pipette, aspirate them from the reagent container prior to use.
ADVIA Chemistry systems automatically dilute concentrated ADVIA Chemistry
reagents on a per-test basis.

On-board Reagent Stability (OBS)

System Stability
ADVIA 1200 45 days
ADVIA 1650/ADVIA 1800 45 days
ADVIA 2400 45 days

For all systems, unopened reagents are stable until the expiration date printed on the
product label when stored at 2° to 8°C. Do not freeze reagents.

Sample Handling
Siemens Healthcare Diagnostics recommends using serum or plasma (lithium heparin)
for this method.

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Materials Required but not Provided

The following list contains the materials required, but not provided, to perform this
method:
• sample containers
• system solutions
• calibrator (refer to the Method Summary section for the REFs)
• control materials (refer to the Quality Control section)*
• 40-mL adapter (REF 08163594, PN 073-0788-01) for 70-mL slot
(ADVIA 1650/ADVIA 2400)
*For storage and stability information, refer to the package insert.

Calibration
To calibrate the ADVIA Chemistry ALT_c assay, use the ADVIA Chemistry Enzyme 2
Calibrator (REF 10916058).

Enter the lot-specific calibrator values that are provided with each lot of calibrator.
Perform the calibration as described in the calibrator instructions for use.

For setup and use instructions, refer to the Calibration Overview section of the
system-specific operator’s guide.

Calibration Frequency
Calibrate the assay every 45 days.

Calibrate the assay after the following events:

• When the reagent lot number changes
• When a reagent pack is replaced by a new reagent pack with the same lot number,
and the previous reagent pack was recalibrated during use
• When a reagent pack is replaced by a new reagent pack with the same lot number,
and an additional reagent blank was run on the previous reagent pack during use
• After replacing critical optical or hydraulic components
• When indicated by quality control procedures

Individual laboratory quality control programs and procedures may require more
frequent calibration.

Reagent Blank (RBL) Frequency

Siemens recommends measurement of the RBL daily.

Run an additional RBL when a reagent pack is replaced by a new reagent pack with the
same lot number and an additional reagent blank was run during use.

Note Use deionized water as the sample for the RBL in the ADVIA Chemistry ALT_c
assay.

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Quality Control
Follow government regulations or accreditation requirements for quality control
frequency.

Siemens recommends the use of commercially available quality control materials with
at least 2 levels (low and high). A satisfactory level of performance is achieved when
the analyte values obtained are within the Acceptable Control Range for the system or
within your range, as determined by an appropriate internal laboratory quality control
scheme.
The actual frequency of control in a laboratory is based on many factors, such as
workflow, system experience, and government regulation. Each laboratory should
evaluate the controls based on the frequency established by their laboratory guidelines.
When the method is performed, analyze at least 2 levels of controls daily.
Also, assay controls under the following conditions:
• whenever you use a new reagent lot
• following the performance of any system maintenance, cleaning, or troubleshooting
procedure
• after performing a new calibration

For more information, refer to the Quality Control Overview section of the
system-specific operator’s guide.

Limitations of the Procedure

A number of substances cause physiological changes in serum or plasma analyte
concentrations. A comprehensive discussion of these and other interfering substances,
their serum or plasma concentrations, and their possible physiological involvements is
beyond the scope of this document. Consult listed reference for specific details on
known potential interfering substances.2

As with any chemical reaction, you must be alert to the possible effect on results of
unknown interferences from medications or endogenous substances. The laboratory
and physician must evaluate all patient results in light of the total clinical status of the
patient.

Venipuncture should occur prior to sulfasalazine administration due to the potential for
falsely depressed results.

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Interferences
At the Analytical Parameters (Serum) window, you can set up the ADVIA Chemistry
system to flag different levels of lipemia (turbidity), hemolysis, and icterus for samples
run on the system.

Note Turbidity resulting from equivalent levels of Intralipid and naturally occurring
triglycerides in actual patient samples may not correlate.

Siemens tested the following potential interferents and found the results shown below:

ADVIA 1200
ALT Sample
Interferent Interferent Level Concentration Interference*

Bilirubin 30 mg/dL 33 U/L NSI

(conjugated/unconjugated) (513 µmol/L)

Hemolysis 1000 mg/dL 34 U/L NSI

(hemoglobin) (10.0 g/L)

Lipemia 188 mg/dL 33 U/L NSI

(from Intralipid) (2.1 mmol/L)**

375 mg/dL 33 U/L -15.2%

(4.2 mmol/L)**

* NSI = No Significant Interference. A percentage effect  10% is considered a significant

interference.
**as triolein

ADVIA 1650/ADVIA 1800

Interferent ALT Sample
Interferent Level Concentration Interference*

Bilirubin 30 mg/dL 34 U/L NSI

(conjugated/unconjugated) (513 µmol/L)

Hemolysis 1000 mg/dL 32 U/L NSI

(hemoglobin) (10.0 g/L)

Lipemia 188 mg/dL 33 U/L NSI

(from Intralipid) (2.1 mmol/L)**

375 mg/dL 33 U/L -10.6%

(4.2 mmol/L)**

* NSI = No Significant Interference. A percentage effect  10% is considered a significant

interference.
**as triolein

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ADVIA 2400
Interferent ALT Sample
Interferent Level Concentration Interference*

Bilirubin 30 mg/dL 34 U/L NSI

(conjugated/unconjugated) (513 µmol/L)

Hemolysis 500 mg/dL 32 U/L NSI

(hemoglobin) (5.0 g/L)

750 mg/dL 32 U/L 15.6%

(7.5 g/L)

Lipemia 500 mg/dL 31 U/L NSI

(from Intralipid) (5.7 mmol/L)**

* NSI = No Significant Interference. A percentage effect  10% is considered a significant

interference.
**as triolein

Performance Characteristics
Precision
Each sample was assayed 2 times per run, 2 runs per day, for at least 10 days.
Precision estimates were computed according to CLSI document EP5-A2, Evaluation
of Precision Performance of Quantitative Measurement Methods; Approved Guideline.3

Data contained in this section represents typical performance for

ADVIA Chemistry systems. Your laboratory data may differ from these values.

ADVIA 1200
Within-Run Total

Specimen Type Level (U/L) SD CV (%) SD CV (%)

Control 1 34 0.9 2.7 1.1 3.2

Control 2 133 1.4 1.0 1.7 1.3

ADVIA 1650/ADVIA 1800

Within-Run Total

Specimen Type Level (U/L) SD CV (%) SD CV (%)

Control 1 41 0.8 1.9 1.1 2.7

Control 2 107 1.2 1.1 1.3 1.2

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ADVIA 2400
Within-Run Total

Specimen Type Level (U/L) SD CV (%) SD CV (%)

Control 1 34 0.8 2.3 1.1 3.4

Control 2 94 0.7 0.7 1.1 1.2

Analytical Range
This method is linear from 8 to 1100 U/L for serum and plasma (lithium heparin).
Siemens has validated an automatic rerun condition for this method that extends the
reportable range for serum and plasma up to 3300 U/L on the ADVIA 1200 Chemistry
system, and up to 6600 U/L on the ADVIA 1650, 1800 and 2400 Chemistry systems.

Expected Values
The expected values for adults are 10 to 49 U/L.4 Siemens provides this information for
reference. Each laboratory should establish its own normal range. At the Analytical
Parameters (Chemistry) window, enter expected range values and abnormal range
values.

System Correlation
The performance of the ADVIA Chemistry ALT_c assay (y) was compared with the
performance of the comparison assay on the indicated system (x).

ADVIA 1200
Regression Sample
Specimen Type Comparison System (x) N Equation Sy.x r Range

Serum ADVIA 1200 (Serum) 70 y = 1.028x - 1.25 4.38 1.000 3–928 U/L
non-concentrated
reagent
Plasma* ADVIA 1200 (Serum) 30 y = 0.952x + 0.26 0.66 0.998 11–61 U/L
concentrated reagent
Serum** IFCC Reference Method 48 y = 0.990x - 3.00 37.00 0.980 9–646 U/L
* lithium heparin plasma (matrix comparison)
** Refer to Standardization

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ADVIA 1650/ADVIA 1800

Regression Sample
Specimen Type Comparison System (x) N Equation Sy.x r Range

Serum ADVIA 1650/ 69 y = 0.995x + 0.41 2.97 1.000 6–1093 U/L

ADVIA 1800 (Serum)
non-concentrated
reagent
Plasma* ADVIA 1650/ 30 y = 0.964x - 1.01 0.56 0.998 12–57 U/L
ADVIA 1800 (Serum)
concentrated reagent
Serum** IFCC Reference Method 49 y = 1.030x - 3.10 16.80 0.997 12–829 U/L
* lithium heparin plasma (matrix comparison)
** Refer to Standardization

ADVIA 2400
Regression Sample
Specimen Type Comparison System (x) N Equation Sy.x r Range

Serum ADVIA 2400 (Serum) 68 y = 1.012x - 1.04 3.78 1.000 3–1073 U/L
non-concentrated
reagent

Plasma* ADVIA 2400 (Serum) 45 y = 0.992x - 1.70 0.66 0.998 12–57 U/L
concentrated reagent

Serum** IFCC Reference Method 49 y = 1.010x - 5.40 14.70 0.997 12–829 U/L

* lithium heparin plasma (matrix comparison)

** Refer to Standardization

Standardization
The ADVIA Chemistry ALT_c method is traceable to the IFCC reference method via
patient sample correlation using the non-concentrated version of the assay which
provides identical levels of all reaction components. See the correlation data in the
System Correlation section for the relationship.

References
1. International Federation of Clinical Chemistry, Committee on Reference Systems
for Enzymes, Chem Clin Lab Med 2002; 40 (7):718–724.
2. Young DS, Effects of Drugs on Clinical Laboratory Tests. 5th ed. Washington,
AACC Press; 2000.
3. Clinical and Laboratory Standards Institute (formerly NCCLS). Evaluation of
Precision Performance of Quantitative Measurement Methods; Approved Guideline
- Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2004.
NCCLS Document EP5-A2.
4. Data on file. Siemens Healthcare Diagnostics.

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Technical Assistance
For customer support, contact your local technical support provider or distributor.

siemens.com/healthcare

Trademarks
ADVIA is a trademark of Siemens Healthcare Diagnostics.
Intralipid is a trademark of Fresenius Kabi AB.

Made in: UK

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Definition of Symbols
The following symbols may appear on the product labeling:

Symbol Definition Symbol Definition

In vitro diagnostic medical device Catalog number

Authorized Representative in the

Legal manufacturer
European Community

CE Mark with identification number

CE Mark
of notified body
0088

Consult instructions for use Biological risk

Keep away from sunlight and heat Temperature limitation

Lower limit of temperature Upper limit of temperature

Do not freeze (> 0°C) Up

Use by Contains sufficient for (n) tests

Recycle Printed with soy ink

Rev. Revision YYYY-MM-DD Date format (year-month-day)

Batch code

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