KATH CLINICAL BIOCHEMISTRY LABORATORY

Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

Title:
ASPARTATE AMINOTRANSFERASE (AST) TEST SOP
Approved by: Date Approved:
ERIC OSEKRE ADJEI 10th May, 2022

Prepared by: Effective Supersedes Document:

Date:
Kenneth K. desBordes 1st June, 2022
Reviewed By
Alex Agyei

Date Reviewed: Date Revised: Comments Initials:

Distribution:
Clinical Biochemistry A&E laboratory

Date of
Discontinuation:

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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

1. PURPOSE
. AST is one of the major liver enzymes used to identify hepatocellular damage as part
of a liver function test. This SoP is to guide the user on how to run the AST test using
the Vitros 5600 analyzer.

2. PRINCIPLE
The Vitros AST 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. In the assay for aspartate aminotransferase,
the amino group of L-aspartate is transferred to α-ketoglutarate in the presence of
pyridoxal-5-phosphate (P-5-P) to produce glutamate and oxaloacetate. The
oxaloacetate formed in the deamination of the L-aspartate is converted to pyruvate
and carbon dioxide by oxaloacetate decarboxylase. Pyruvate is oxidized to
acetylphosphate and hydrogen peroxide by pyruvate oxidase.

The final reaction step involves the peroxidase-catalyzed oxidation of a leuco dye to
produce a colored dye. The rate of oxidation of the leuco dye is monitored by
reflectance spectrophotometry. The rate of change in reflectance density is
proportional to enzyme activity in the sample. The low wavelength cutoff filter on
the slide support minimizes the blank rate effects of incident light during the dye
development.

Test Type and Conditions

Approximate
Wavelength Reaction Sample
Test Type Incubation Temperature
Volume
Time

Multi-point rate 5 minutes 37 °C (98.6 °F) 670nm 7 μL

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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

Reaction Scheme

3. SCOPE OF THIS SOP

This SOP will describe the standard way of performing the AST test using the Vitros
5600 analyzer.

4. DEFINITION OF TERMS
ABBREVIATION FULL NAME
CLSI Clinical & Laboratory Standards Institute
LHIMS Lightwave Hospital Information Management System
AST Aspartate AminoTransferase

5. PERFORMANCE CHARACTERISTICS
See Manufacturer SOP attached

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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

6. REAGENTS

6.1 Slide Ingredients

Reactive Ingredients per cm2

Sodium aspartate 0.27 mg; sodium α-

ketoglutarate 0.13 mg; sodium
pyridoxal-5-phosphate 11 µg; sodium
phosphate 42 µg; 2-(3,5-dimethoxy- 4-
hydroxyphenyl)-4,5-bis(4-
dimethylaminophenyl) imidazole
(leuco dye) 30 µg; pyruvate oxidase
(Aerococcus sp.) 0.20 U; peroxidase
(horseradish root) 0.50 U and
oxaloacetate decarboxylase
(Pseudomonas sp.) 0.30 U.

Other Ingredients

Enzyme cofactors, pigment,

binders, buffer, surfactants,
stabilizer, scavenger, dye
solubilizer, filter dyes and cross-
linking agent.

6.2 Reagent 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.

6.3 Reagent Preparation

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

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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

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).

6.4 Reagent Storage and Stability

VITROS AST Slides are stable until the expiration date on the carton
when they are stored and handled as specified. Do not use beyond the
expiration date.

Reagent Storage Condition Stability

Unopened Refrigerated 2–8 °C (36–46 °F) ≤ 3 months
Frozen ≤-18 °C (≤0 °F) Until expiration date
Opened On-analyzer System turned on ≤ 2 weeks
On-analyzer System turned off ≤ 2 hours

7. EQUIPMENT
a. VITROS 5600 analyzer
b. Heraeus Megafuge 8 Centrifuge

8. SPECIMEN COLLECTION, PREPARATION AND STORAGE

8.1 Specimens Recommended
• Serum
• Plasma: Heparin

8.2 Specimens Not Recommended

 Plasma:
– EDTA
– Citrate
– Fluoride oxalate
 Do not use hemolyzed specimens because of high levels of AST activity in erythrocytes
Page 5 of 12

BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

8.3 Serum and Plasma Specimen Collection and Preparation

 Collect specimens using standard laboratory procedures.
 Due to the very low density of platelets, it is important to centrifuge plasma
specimens at a minimum of 1000 x g for a minimum of ten minutes in order to avoid
contamination of plasma with AST derived from platelets.

8.4 Patient Preparation

No special patient preparation is necessary

8.5 Specimen Handling and Storage

• 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.

8.6 Specimen Storage and Stability: Serum a n d Plasma

Storage Temperature Stability
Room temperature 18–28 °C (64–82 °F) ≤ 3 days
Refrigerated 2–8 °C (36–46 °F) ≤ 7 days
Frozen ≤-18 °C (≤0 °F) ≤ 3 months

8.8 Sample Dilution

If aspartate aminotransferase activities exceed the system’s measuring (reportable
or dynamic) range:
Refer to the operating instructions for more information on the On-Analyzer
Dilution Procedure. Use VITROS Chemistry Products FS Diluent Pack 2 for the
dilution.

Note: A DP or TR flag indicates substrate depletion. This may

indicate a high pyruvate concentration. Refer to “Limitations of
the Procedure.”

Page 6 of 12

BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

On-Analyzer Sample Dilution

Use VITROS Chemistry Products FS Diluent Pack 2 for the dilution.

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 aspartate aminotransferase activity.

9. SAFETY PRECAUTIONS
 Lab coat and gloves should be worn during the procedure.
 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 CLSI Guideline M29 or other
published biohazard safety guidelines

10. PROCEDURE
Materials required
 VITROS Chemistry Products AST Slides
 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)

Sample processing and analysis

i. Serum/Plasma samples collected in well-labelled serum separator tubes must be
centrifuged at 4000rpm for four minutes.
ii. Samples are placed on the sample tray and each sample’s position recorded in the
workbook
iii. On the analyzer, the sodium test is selected and the sample type indicated as well as
entering of patient’s demographics.
iv. The sample trays are placed in the machine

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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

v. The start icon is clicked on if machine is in a resting mode for the tests to begin.
vi. Completed results are verified and transmitted and reported using the appropriate
means (Either printing out or uploading onto the LHIMS).

11. RESULTS
Reporting Units - The VITROS Chemistry System may be programmed to report
AST results in conventional or SI units.

Conventional and SI Units Alternate Unit

U/L
µkat/L (U/L x 0.0167)

12. NOTES
 Bring all fluids and samples to room temperature, 18–28 °C (64–82 °F), prior to
analysis.
 Verify performance of reagents 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.
 Check reagent inventories at least daily to ensure that quantities are sufficient for the
planned workload.

13. RESULT INTERPRETATION

Aspartate aminotransferase is present in high activity in heart, skeletal muscle, and
liver. Increased serum AST activity commonly follows myocardial infarction,
pulmonary emboli, skeletal muscle trauma, alcoholic cirrhosis, viral hepatitis, and
drug-induced hepatitis.

Page 8 of 12

BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

14. PRECAUTIONS
a. Do not draw specimen from an arm receiving an intravenous transfusion.
b. Fibrin clots may cause incomplete sampling of the specimen.
 Allow specimens to clot completely in order to prevent fibrin clots.
 Inspect plasma specimens for fibrin clots.
c. Centrifuge specimens and remove the serum or plasma from the cellular
material within 2 hours of collection
d. Avoid agitation or mixing of plasma samples after centrifugation. Re-
suspension of platelets into previously centrifuged plasma may lead to
artificially elevated AST results because of high AST activity in platelets.
e. Centrifuge specimens and remove the serum or plasma from the cellular
material within 3 days of collection.

15. LIMITATIONS OF PROCEDURE

Known Interferences

Serum and Plasma

The VITROS AST Slide method was screened for interfering substances following
NCCLS Protocol EP7. The substances listed in the table, when tested at the
concentrations indicated, caused the bias shown.

* It is possible that other interfering substances may be encountered. These results are
representative; however, your results may differ somewhat due to test-to-test variation.
The degree of interference at concentrations other than those listed might not be
predictable.
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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

Other Interferences
 High levels of pyruvate will trigger either TR or DP flags. Such samples need to be
repeated following dilution.
 Certain drugs and clinical conditions are known to alter aspartate aminotransferase
activity in vivo. For additional information, refer to one of the published summaries.

16. EXPECTED VALUES

17. CRITICAL VALUES

See hand book

Page 10 of 12

BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

18. REFERENCES

 Tietz NW (ed). Fundamentals of Clinical Chemistry. ed. 3. Philadelphia: WB Saunders;

369–371; 1987.

 CLSI. Protection of Laboratory Workers from Occupationally Acquired Infections;

Approved Guideline – Fourth Edition. CLSI document M29-A4. Wayne, PA: Clinical and
Laboratory Standards Institute; 2014.

 Doumas BT, et al. Differences Between Values for Plasma and Serum in Tests Performed
in the Ektachem 700 XR Analyzer, and Evaluation of “Plasma Separator Tubes (PST).”
Clin. Chem. 35:151–153; 1989.

 Calam RR. Specimen Processing Separator Gels: An Update. J Clin Immunoassay.

11:86–90; 1988.

 Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests. ed. 2.

Washington D.C.: AACC Press; 3-69, 3-70; 1997.

 CLSI. Collection of Diagnostic Venous Blood Specimens. 7th ed. CLSI standard GP41.
Wayne, PA: Clinical and Laboratory Standards Institute; 2017.

 NCCLS. Procedures and Devices for the Collection of Diagnostic Capillary Blood
Specimens; Approved Standard—Fifth Edition. NCCLS document H4-A5 [ISBN 1-56238-
538-0]. CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA; 2004.

 Clinical Laboratory Handbook for Patient Preparation and Specimen Handling. Fascicle
VI: Chemistry/Clinical Microscopy. Northfield, IL: College of American Pathologists;
1992.

 Bergmeyer H U, Horder M, Rej R. Approved Recommendation on IFCC Methods for the

Measurement of Catalytic Concentration of Enzymes. Part 2. IFCC Method for Aspartate
Aminotransferase. J. Clin. Chem. Clin. Biochem. 24:497–510; 1986.

 CLSI. Statistical Quality Control for Quantitative Measurements: Principles and

Definitions; Approved Guideline – Third Edition. CLSI document C24-A3 (ISBN 1-
56238-613-1). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA;
2006.

 NCCLS. Interference Testing in Clinical Chemistry. NCCLS Document EP7. CLSI, 940
West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA; 1986.
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BCHM GEN 009

 KATH CLINICAL BIOCHEMISTRY LABORATORY
Prepared by: KENNETH K. desBORDES Effective Date: 01/06/2022

Approved by: ERIC OSEKRE ADJEI Revision Date:

TITLE: ASPARTATE AMINOTRANSFERASE (AST) TEST SOP

APPENDIX
USER LOG SHEET

SOP NUMBER DATE INITIALS SIGNATURE

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BCHM GEN 009