SGPT (ALT) Examination Procedure

1.Purpose of examination:

• ALT estimation from serum or plasma by NADH (without Pyridoxal Phosphate) Method.

2.Responsibility and Authority:

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

3.Sample Details:

• Type of Sample: Serum, Plasma
• Type of container and additives: Plain without any additives
• Patient Preparation: As per Primary Sample Collection Manualsample_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-alanine (66.820 g/L) β-NADH (0.305 g/L) lactate dehydrogenase (5.000 KU/L) * R2. L-alanine (89.090 g/L) α-ketoglutaric acid (13.150 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:

1. Consolidated Chemistry Calibrator
2. Frequency:
3. Reagent lot change
4. QC out of range
5. After service or maintenance
6. Replacement in any parts of Instrument

Procedure:

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

8.Quality control Procedure:

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

Procedure for Reconstitution of IQC

1. Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
2. Leave to stand for 30 min in the dark place.
3. Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
4. 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.
5. Prepare aliquots of 150 µl from the reconstituted QC material.
6. Store these aliquots at -15° C to -20° C.
7. 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: ALT present in the sample catalyzes the transfer of the amino group from L-alanine to α-ketoglutarate, forming pyruvate and L-glutamate. Pyruvate in the presence of NADH and lactate dehydrogenase is reduced to L-lactate. 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.

11.Procedure to run Patient sample

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

12.Performance Characteristics:

• Linearity: 7 to 3271 U/L

Automated Dilution Protocol:The system performs a 1:5 dilution of the sample and automatically calculates the concentration by multiplying the result by the dilution factor. Manual Dilution Procedure: Dilute the specimen with saline (0.85% to 0.90% NaCl). Enter the dilution factor in the Patient or Control order screen

• The limit of detection (LOD): 4 U/L
• The limit of quantification(LOQ):7 U/L
• Unit: U/L

Normal and critical ranges:

13.Laboratory Clinical interpretation:

• ALT rises in disease states that cause hepatocellular injury. The cause of hepatocellular injury may result in varying magnitudes of elevation in ALT and AST. Borderline ALT elevation is defined as <2 times the upper limit of normal (ULN), mild ALT elevation is defined as 2 to 5 times the ULN, moderate ALT elevation is defined as 5 to 15 times the ULN, and severe ALT elevation is defined as >15 times the ULN.

14.Interference and cross reaction: The Following analytes were tested up to the levels indicated at Direct Bilirubin concentrations of 0.14mg/dl and 5.03 mg/dl, and found not to interfere:

• No interference from Bilirubin up to 30 mg/dL
• No interference from Hemoglobin up to 750 mg/dL
• No interference from Intralipid up to 550 mg/dL
• No interference from Sulfapyrdine up to 60 mg/dl
• No interference from Sufasalazine up to 20 mg/dl
• No interference from Temozolomyde up to 20 mg/dl

15.Potential source of variation: Turn around time (TAT):

• Routine: 6.0 hours
• Urgent: 2.0 hours

16.Recording of observation:

• Software backup
• Machine raw data

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

18.Environmental & Safety control:

• For in vitro diagnostic use
• Do not use component before the expiration date
• Do not mix material from different kit lot number.

19.References:

1. US department of labor, Occupational safety and health administration.
2. US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.
3. World health organization. Biosafety manual,3rd edition.
4. Clinical and laboratory std institute . Protection of Laboratory workers from oocupationally acquired infection; Approved guideline -4th edition.