lipase [Laboratory Service Sir T Hospital , Bhavnagar ]

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                      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:
  - 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 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** 
  - 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**
  * 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:**

^Lipase^Adult^<38^

**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**
^Bilirubin^15 mg/dL-30 mg/dL^ 
^Hemoglobin^1,000 mg/dL- 2,000 mg/dL^
^Intralipid^750 mg/dL-1,000 mg/dL^

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:**
  - US Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910.1030, Bloodborne Pathogens.
  - US Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories, 5th ed. Washington,DC: US Government Printing Office, December 2009.
  - World Health Organization. Laboratory Biosafety Manual, 3rd ed. Geneva: World Health Organization, 2004.
  - 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
  - Guder WG, da Fonseca-Wollheim F, Hail W, et al. The Quality of Diagnostic Samples. Darmstadt, Germany: GIT Verlag: 2001:36-7

|**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use|

^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
^Document No.1^**Document Name**: Lipase Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^
^Issue No. : 01^Issue Date :30/04/2024^Page No.^^
^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^