SGOT (AST) Examination Procedure

1.Purpose of examination: AST estimation from serum NADH (without P-5’-P) Method.

2.Responsibility and Authority:

• Calibration: Technician
• Quality Control: Technician
• Routine operation: Technician
• Overall Monitoring: Quality Manager

3.Sample Details:

• Type of Sample: Serum,
• Type of container and additives: Plain without any additives
• Patient Preparation: As per Primary Sample Collection Manual sample_collection_manual
• Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
• Handling and transport: As per Primary Sample collection manual
• Storage: 24 hours at 2-8° C

4.Required Equipment:

• Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

5.Required reagents: R1 L-aspartic acid (103.860 g/L), lactate dehydrogenase (4.000 KU/L), and malate dehydrogenase (2.000 KU/L). R2 α-ketoglutaric acid (6.570 g/L).

6.Reagent Handling

• Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to settle at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles
• Reagent Storage and stability
• Unopened reagent stable at 2-8°C until expiration date.
• On board System temperature reagent is stable for 30 days
• Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or
• contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

7.Calibration Procedure:

• SGOT Calibrators-WATER BLANK
• Frequency:
• Reagent lot change
• QC out of range
• After service or maintenance
• Replacement in any parts of Instrument

Procedure:

• Start the equipment.WDI abbotte fully.docx
• Calibrators are ready to use.
• Put calibrator 15-20 minutes at room temperature
• Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation.
• Take a 150 µl calibrator solution in to separate aliquot.
• Go to the calibration and give the calibration order.
• Verify calibration with at least two levels of controls
• If control results fall outside acceptable ranges, root cause analysis or recalibration may be necessary.

8.Quality control Procedure:

• Name: Bio Rad Level 1 & 2
• Frequency: As per Quality Control Procedure

Procedure for Reconstitution of IQC

• Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
• Leave to stand for 30 min in the dark place.
• Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
• Prior to use, mix the contents by inverting the vial. Do not shake the vial as the information of foam should be avoided. Ensure that no lyophilized material remains un-reconstituted.
• Prepare aliquots of 150 µl from the reconstituted QC material.
• Store these aliquots at -15° C to -20° C.
• Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

Procedure to run IQC

1. Press Control order
2. Select Assay /Panel, to be run.
3. Select the control/s and its level/s
4. Give Carrier Number and Position number
5. Press F3 / Add order
6. Put respected carrier in RSH rack
7. Check IQC results, in case outliers call residents.

9.Principle of the procedure used for examinations:

• AST present in the sample catalyzes the transfer of the amino group from L-aspartate to α-ketoglutarate, forming oxaloacetate and L-glutamate. Oxaloacetate in the presence of nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH) is reduced to L-malate. In this reaction, NADH is oxidized to NAD+. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH to NAD+.

10.Sample Preparation:

• Required SampleVolume: 150 µl of the sample
• Temperature: 37°
• Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot.

Procedure to run Patient sample

• Press Patient order
• Select Assay /Panel, to be run.
• Give Carrier Number and Position number
• Press F3 / Add order
• Put respected carrier in RSH rack

11.Performance Characteristics:

• Linearity: 5 to 4001 U/L
• The limit of detection (LOD): 4 U/L
• The limit of quantification(LOQ): 5 U/L
• Unit:U/L

Normal and critical ranges: Aspartate aminotransferase

12.Laboratory Clinical interpretation: AST is found to be elevated in alcohol-induced liver disease, viral hepatitis, cirrhosis and acute fulminant hepatic failure.

13.Interference and cross reaction: The Following analytes were tested up to the two levels indicated at SGOT concentrations of 30 U/L and 120 U/L, and found not to interfere:

1. No interference from Bilirubin up to 30 mg/dL
2. No interference from Hemoglobin up to 62 mg/dL
3. No interference from Intralipid up to 550 mg/dL
4. No interference from Sulfapyrdine up to 300 mg/dl
5. No interference from Sufasalazine up to 300 mg/dl
6. No interference from Temozolomyde up to20 mg/dl

14.Potential source of variation: Turn around time (TAT): Routine: 6.0 hours Urgent: 2.0 hours

15.Recording of observation:

• Software backup
• Machine raw data

16.Storage & Disposal of waste:Follow storage & discard procedure

17.Environmental & Safety control:

• For in vitro diagnostic use
• Do not use component before the expiration date
• Do not mix material from different kit lot numbers.
• Reagent 2 causes mild skin irritation.

18.References:

1. Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry.
2. World health organization. Biosafety manual,3rd edition.
3. Clinical and laboratory std institute . Protection of Laboratory workers from occupationally acquired infection; Approved guideline -4th edition.
4. Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry
5. Kaplan LA,Pesce AJ editors. Clinical Chemistry Theory, Analysis, and Correlation, 3rd ed.