Amylase Examination Procedure

1.Purpose of examination: Amylase estimation from serum or plasma by Enzymatic/Colorimetric Method.

2.Responsibility and Authority:

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

3.Sample Details:

1. Type of Sample: Serum, Plasma
2. Type of container and additives: Plain without any additives
3. Patient Preparation: As per Primary Sample Collection Manual sample_collection_manual
4. Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
5. Handling and transport: As per Primary Sample collection manual
6. 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. α-glucosidase 16.000 KU/L. ● R2. Ethylidene-4-NP-G7 (EPS) 6.501 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

7.Reagent Storage and stability Unopened reagent stable at 15-30°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.

8.Calibration Procedure:

• Consolidated Chemistry Calibrators(ConCC)
• Frequency:

1. Reagent lot change
2. QC out of range
3. After service or maintenance
4. 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 aliquots.
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.

9.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.

10.Principle of the procedure used for examinations: At an alkaline pH, creatinine in the sample reacts with picrate to form a creatinine-picrate complex. The rate of increase in absorbance at 500 nm due to the formation of this complex is directly proportional to the concentration of creatinine in the sample.

11.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

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: 3 to 3300 U/L
• The limit of detection (LOD): 1 U/L
• The limit of quantification(LOQ): 3 U/L
• Unit: U/L

Normal and critical ranges:

13.Laboratory Clinical interpretation: Amylase is used primarily in the diagnosis of acute pancreatitis. Acute pancreatitis is a reversible inflammation due to enzymatic necrosis. The most common cause of acute pancreatitis is gallstones. The second most common cause is heavy alcohol consumption. Serum amylase elevation and concomitant urine amylase elevation is seen in patients with acute pancreatitis.

14.Interference and cross reaction: The Following analytes were tested up to the levels indicated at Creatinine concentrations of(approximately 50 U/L and 200 U/L). mg/dl, and found not to interfere: Avoid contaminents like saliva,cough , since these contain many units of amylase which might effect the reagent for color development.

15.Potential source of variation:

1. Turn around time (TAT):
2. Routine: 6.0 hours
3. 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: Contact with acids liberates very toxic gas. Dispose of contents / container in accordance with local regulations.

19.References:

1. Tietz NW, Huang WY, Rauh DF, et al. Laboratory tests in the differential diagnosis of hyperamylasemia. Clin Chem 1986 32(2):301-307
2. 6 Junge W. Troge B, Klein G, et al. Evaluation of a New Assay for Pancreatic Amylase: Performance Characteristics and Estimation of Reference Intervals Clin Biochem 1989,22 109-114
3. Stuttgart/New York Georg Thieme Verlag 1991 354-361. 3 Salt WB II, Schenker S. Amylase its clinical significance: a review ofthe literature [Review] Medicine 1976,55 269-281.
4. Steinberg WM, Goldstein SS, Davies ND, et al. Diagnostic assays in acute pancreatitis [Review]. Ann Intern Med 1985,102.576-580