hdl_cholesterol [Laboratory Service Sir T Hospital , Bhavnagar ]

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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:**

^Parameter^Desirable / Optimal^Near/ Above optimal^Borderline^High^Very High^
|HDL Cholesterol|>60 mg/dL|-|< 40 mg/dL|-|

**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:** 
  - Turn around time (TAT):
  - Routine: 6.0 hours
  - 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

|**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**: HDL Cholesterol 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^