LIPASE Examination Procedure

1.Purpose of examination: Lipase estimation from serum or plasma by Quinone Dye 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 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 Cholic acid 5.34 mmol/L R1A 1,2-Diglyceride 1.1 mmol/L Monoglyceride lipase 0.88 U/mL Glycerol kinase < 1.34 U/mL Glycerol-3-phosphate oxidase < 40.0 U/mL Peroxidase < 1.34 U/mL Colipase < 40.0 U/mL TOOS 0.068% ATP 0.66 mmol/L R2 Deoxycholic acid 36.0 mmol/L 4-Aminoantipyrine 0.12%

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 2-8°C until expiration date.
• On board System temperature reagent is stable for 28 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: Lipase Calibrators- conc Frequency:

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

9.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

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

10.Principle of the procedure used for examinations: Lipase acts on a natural substrate, 1,2-diglyceride, to liberate 2‐monoglyceride. This is hydrolyzed by monoglyceride lipase into glycerol and free fatty acid. Glycerol kinase acts on glycerol to form glycerol‐3‐phosphate which is in turn acted on by glycerol-3-phosphate oxidase to generate hydrogen peroxide. Peroxidase converts the hydrogen peroxide, 4‐aminoantipyrine, N-ethyl-N-(2-hydroxy3-sulfopropyl)-m-toluidine (TOOS) into a quinone dye. The rate of formation of the dye, measured as an increase in absorbance at 548 nm, is proportional to the lipase concentration 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.

12.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

13.Performance Characteristics:

• Linearity: linear up to 1,200 U/L

If values exceed this linearity limit 15.00 mg/dl, dilute the sample by Manual Dilution Procedure, or the Automatic Dilution Protocol provided in the assay parameters. Automated Dilution Protocol: When using the Automated Dilution Protocol, the system performs a dilution of the specimen and automatically corrects the concentration by multiplying the result by the appropriate 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): 1.6 U/L.
• The limit of quantification(LOQ): 3.1 U/L.
• Unit: U/L

Normal and critical ranges:

14.Laboratory Clinical interpretation: The common causes of High LIPASE Level are as follows:Acute/chronic pancreatic disease.

15.Interference and cross reaction: Interference studies were conducted using CLSI protocol NCCLS EP7‐P.10 Interference effects were assessed by Dose Response and Paired Difference methods, at the medical decision level of the analyte. Interfering Substance

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 Hemoglobin 1000 mg/dL- 2000 mg/dL
• No interference from Bilirubin 15 mg/dL-30 mg/dL
• No interference fromIntralipid 750 mg/dL-1,000 mg/dL

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

17.Recording of observation: Software backup Machine raw data

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

Environmental & Safety control: Reagent R1 contains sufamic acid that Causes severe skin burns and eye Damage .

Precautions:

• Wear protective gloves / protective clothing / eye protection
• Do not breathe mist / vapors / spray
• Wash hands thoroughly after handling
• Keep only in original container

Response

• IF SWALLOWED: Rinse mouth. Do NOT induce vomiting.
• IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
• IF IN EYES: Rinse cautiously with water for several minutes.
• Remove contact lenses, if present and easy to do. Continue rinsing.
• Absorb spillage to prevent material damage.

19.References:

1. US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030, Bloodborne Pathogens.
2. US Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories, 5th ed. Washington,DC: US Government Printing Office, December 2009.
3. World Health Organization. Laboratory Biosafety Manual, 3rd ed. Geneva: World Health Organization, 2004.
4. Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory Workers From Occupationally Acquired Infections;Approved Guideline-Fourth Edition. CLSI Document M29-A4. Wayne,PA: CLSI: 2014
5. Guder WG, da Fonseca-Wollheim F, Hail W, et al. The Quality of Diagnostic Samples. Darmstadt, Germany: GIT Verlag: 2001:36-7