Alkaline Phosphatase(ALP) Examination Procedure

1.Purpose of examination: Alkaline Phosphatase estimation from serum or plasma by Para-nitrophenyl 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 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. 2-amino-2-methylpropanol
• R2. 4-nitrophenyl phosphate 30.430g/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 10 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:

• Consolidated Chemistry Calibrators(CONCC) - Level 1 & 2
• 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.

8.Quality control Procedure: Name: Biorad Level 1 & 2 Frequency: As per Quality Control Procedure

9.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.

10.Principle of the procedure used for examinations: ALP in sample catalyzes the hydrolysis of colorless Para Nitrophenyl Phosphate to give paranitrophenol and inorganic phosphate. In alkaline pH paranitrophenol is in the yellow phenoxide form.The rate of absorbance increase at 404 nm is a directly proportional to ALP activity in 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

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

13.Performance Characteristics:

• Linearity: 9.0 to 4522 U/L

If values exceed this linearity limit 4500 U/L, 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): 7.0 U/L
• The limit of quantification(LOQ): 9.0 U/L
• Unit: U/L

Normal and critical ranges:

14.Laboratory Clinical interpretation:

• A rise in ALP activity occurs with all forms of cholestasis, particularly with obstructive jaundice. ALP is also elevated in disorders of the skeletal system that involve osteoblast hyperactivity and bone remodeling, such as Paget disease, rickets, osteomalacia, fractures, and malignant tumors. Moderate elevation of ALP may be seen in other disorders such as Hodgkin disease, congestive heart failure, ulcerative colitis, regional enteritis, and intra-abdominal bacterial infections.

15.Interference and cross reaction:

• No interference from Conjugated Bilirubin 15 up to U/L
• No interference from Unconjugated Bilirubin 20 up to U/L
• No interference from Hemoglobin up to 250 U/L
• No interference from Intralipid up to1500 U/L

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

19.Environmental & Safety control:

20.Precautions:

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

21.Response

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

Reagent R2 contains methylisothiazolones that Causes severe skin burns and eye Damage .

• Do not breathe mist / vapors / spray
• Wear protective gloves / protective clothing / eye protection

22.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. ARCHITECT ALP 7D55 307217/R01
5. NABL 112 effective from 01/06/2019