Triglyceride Examination Procedure

1.Purpose of examination: Triglyceride estimation from serum or plasma by Glycerol Phosphate Oxidase.

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 84ml Active ingredients: ATP (2.5 mmol/L), Mg2+ (2.5 mmol/L),
• 4-aminoantipyrine (0.4 mmol/L), 4-chlorophenol (2 mmol/L),
• Peroxidase (horseradish) (> 2000 U/L), GK (microbial) (> 600 U/L),
• GPO (microbial) (> 6000 U/L), Lipoprotein lipase (microbial) (> 3000 U/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:

• Consolidated Chemistry Calibrator
• Frequency:
• Reagent lot change
• QC out of range
• After service or maintenance
• Replacement in any parts of Instrument

8.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 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

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: Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3-phosphate and adenosine diphosphate (ADP). Glycerol-3-phosphate is oxidized to dihydroxyacetone phosphate (DAP) by glycerophosphate oxidase (GPO) producing hydrogen peroxide (H2O2). In a color reaction catalyzed by peroxidase, the H2O2 reacts with4-aminoantipyrine (4-AAP) and 4-chlorophenol (4-CP) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglyceride present in the sample. This analytical methodology is based on the reaction sequence described by Fossati et al.4 and by McGowan et al.5 In this reagent,4-chlorophenol is used rather than 2-hydroxy-3,5-dichloro benzene sulfonate, used in the Fossati and McGowan studies.

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:

1. Linearity: up to 1420 mg/dL
2. The limit of detection (LOD): 0.62 mg/dL
3. The limit of quantification(LOQ): 6.2 mg/dL
4. Unit: mg/dL

Normal and critical ranges:

Triglyceride

13.Laboratory Clinical interpretation: The common causes of hyperlipidemia is nephrosis,DM,Endocrine disturbances.

14.Interference and cross reaction:

InterferingSubstance

• No interference from Bilirubin up to7.5 mg/dL
• No interference from Hemoglobin up to750 mg/dL
• No interference from Ascorbate up to 1.5 mg/dl
• No interference from Acetamenophen up to200mg/dl.
• No interference from Dipyrone up to100mg/dl.
• No interference from N acetyl L cystenine up to800 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.torage & Disposal of waste: Follow storage & discard procedure

18.Environmental & Safety control:

Reagent R1 contains sodium azide.contact with acids liberates very toxic gas

19.Precautions:

1. Wear protective gloves / protective clothing / eye protection
2. Do not breathe mist / vapors / spray
3. Wash hands thoroughly after handling
4. Keep only in original container

. 20.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. World health organization. Biosafety manual,3rd edition.
5. Clinical and laboratory std institute . Protection of Laboratory workers from oocupationally acquired infection; Approved guideline -4th edition.
6. Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry