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                    HDL Cholesterol Examination Procedure

1.Purpose of examination: HDL Cholesterol estimation from serum or plasma by Accelerator Selective Detergent 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: Cholesterol oxidase (E. coli) (< 1000 U/L), Peroxidase (Horseradish) (< 1300 ppg U/L), N, N-bis (4-sulfobutyl)-m-toluidine-disodium (DSBmT) (< 1.0 mmol/L), Accelerator (< 1.0 mmol/L), Ascorbic oxidase (Cucurbita sp.) (< 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 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.

7.Calibration Procedure:

• Consolidated Chemistry Calibrator
• 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 calibrator solution in separate aliquots.
• 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: 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

• 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.

9.Principle of the procedure used for examinations:

• This method is based on accelerating the reaction of cholesterol oxidase (CO) with non-HDL

unesterified cholesterol and dissolving HDL cholesterol selectively using a specific detergent. In the first reagent, non-HDL unesterified cholesterol is subject to an enzyme reaction and the peroxide generated is consumed by a peroxidase reaction with DSBmT yielding a colorless product. The second reagent consists of a detergent (capable of solubilizing HDL cholesterol), cholesterol esterase (CE), and chromagenic coupler to develop color for the quantitative determination of HDL cholesterol.

10.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

• 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

11.Performance Characteristics:

• Linearity: up to 180 mg/dL
• The limit of detection (LOD): 5.0 mg/dL
• The limit of quantification(LOQ): 2.5 mg/dL
• Unit: mg/dL

Normal and critical ranges:

12.Laboratory Clinical interpretation: The principle role of HDL cholesterol in lipid metabolism is the uptake and transport of cholesterol from peripheral tissues to the liver through a process known as reverse cholesterol transport proposed cardioprotective mechanism).Low HDL cholesterol levels are strongly associated with an increased risk of coronary heart disease.

13.Interference and cross reaction:

• No interference from Bilirubin up to32.6mg/dL
• No interference from Hemoglobin up to1000mg/dL
• No interference from Intralipid up to1000 mg/dL
• No interference from Ascorbic acid up to 2.9mg/dl

14.Potential source of variation:

1. Turn around time (TAT):
2. Routine: 6.0 hours
3. Urgent: 2.0 hours

15.Recording of observation: Software backup Machine raw data

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

17.Environmental & Safety control: Reagent R1 contains sodium azide.contact with acids liberates very toxic gas

18.Precautions:

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

19.References: US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories. World health organization. Biosafety manual,3rd edition. Gotto AM. Lipoprotein metabolism and etiology of hyperlipidemia.Hosp pract 1988;23