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                    LDL Cholesterol Examination Procedure

1.Purpose of examination: LDL cholesterol estimation from serum or plasma by Measured, Liquid Selective Detergent method.

2.Responsibility and Authority:

  • Calibration: Technician
  • Quality Control: Technician
  • Routine operation: Technician
  • Overall Monitoring: Quality Manager

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

Required Equipment: ●Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

Required reagents: R1: MES buffer (pH 6.3) 

      Detergent 1 < 1.0%
     Cholesterol esterase (Microorganism) < 1,500 U/L
     Cholesterol oxidase (Microorganism) < 1,500 U/L
     Peroxidase (Horseradish) < 1,300 ppg U/L
     4-aminoantipyrine < 0.01%
     Ascorbic acid oxidase (Cucurbita sp.) < 3,000 U/L

R2:MES buffer (pH 6.3) 

    Detergent 2 < 1.0%
    N,N-bis(4-sulfobutyl)-m-toluidine, disodium (DSBmT) < 1.0 mmol/L

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.

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.

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.

Principle of the procedure used for examinations: The method is in a two-reagent format and depends on the properties of a unique detergent. This detergent, , solubilizes only the non-LDL particles. The cholesterol released is consumed by cholesterol esterase and cholesterol oxidase in a non-color-forming reaction. A second detergent, , solubilizes the remaining LDL particles and a chromogenic coupler allows for color formation. The enzyme reaction with LDL in the presence of the coupler produces color that is proportional to the amount of LDL cholesterol present in the sample.

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

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:

Parameter Desirable / Optimal Near/ Above optimal Borderline High Very High

HDL Cholesterol

60 mg/dL

- < 40 mg/dL -

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).3 Low HDL cholesterol levels are strongly associated with an increased risk of coronary heart disease.

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

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

Recording of observation: Software backup Machine raw data Storage & Disposal of waste: Follow storage & discard procedure Environmental & Safety control: Reagent R1 contains sodium azide.contact with acids liberates very toxic gas Precautions: Wear protective gloves / protective clothing / eye protection Do not breathe mist / vapors / spray Wash hands thoroughly after handling Keep only in original container . 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