Total Cholesterol Examination Procedure                                                                                                                                                                                                                                                                                                                                                                                                                  

1.Purpose of Examination: To lay down standard operating procedure for serum Total Cholesterol estimation by CHOD-PAP Method in Biochemistry section of LSSTH, Bhavnagar.

2.Principle of the procedure used for examinations: The Cholesterol2 assay is an automated clinical chemistry assay. Cholesterol esters are enzymatically hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-ene-3-one and hydrogen peroxide. The hydrogen peroxide oxidatively couples with N,N-Bis(4-sulfobutyl)-3-methylaniline (TODB) and 4-aminoantipyrine to form a chromophore (quinoneimine dye) which is quantitated at 604 nm.

3.Performance: Linearity: upto 705mg/dl Limit of Detection (LOD):Serum : 5.0 mg/dl Limit of Quantitation (LOQ),:Serum :6.2 mg/dl The LOQ is the analyte concentration at which the CV = 20%.

4.Type of Primary Sample: Serum or Plasma sample_collection_manual

5.Patient Preparation: Instruction should be given to the patient Verbal consent of patient should be taken before collecting blood sample. Patient should relax for about 5 minutes before the venepuncture..

6.Type of Container and additives : Plain Vaccutainer with no additives

7.Required Equipment and reagents: Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, Fully Auto Chemistry Analyzer

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

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

10.Calibration Procedure: Give Calibration with Consolidated Chemistry Calibrator ConCC (See calibrator chart for lot specific concentration) Manual calibration is done on following circumstances:

• After instrument repair
• After new reagent lot
• QC data not satisfactory - QC outlier

Note: Verify calibration with at least two levels of controls according to the established quality control requirements for your laboratory. If control results fall outside acceptable ranges, recalibration may be necessary

11.Procedural Steps:

• Verify that all necessary reagents present
• Take100µl Sample.
• Put on sample loader ,select & Order Test
• Press run.

12.Quality Control Procedure: The Laboratory runs Lypocheck Assayed Chemistry Control level 1 & level 2 Once a day. Monthly L-J charts are reviewed & for any QC outlier root cause analysis are done & corrective actions are taken according to the error. The Laboratory Participates in EQAS programme EQAS report is reviewed and if any result go outlier, then root cause analysis is done & corrective actions are taken according to the error.

13.Interferences & Cross-Reactions: InterferingSubstance

• No interference from Bilirubin up to7.5 mg/dL
• No interference from Hemoglobin up to750 mg/dL
• No interference from Intralipid up to 1000 mg/dl
• No interference from Ascorbate up to 1.5 mg/dl

14.Principle of Procedure for Calculating Results including where relevant, the measurement uncertainty of measured quantity values: Instrument measures the absorbance of a calibrator of known concentration. This point determines the slope of a straight line that relates absorbance to concentration. Instrument calculates a constant which is the inverse of the slope of the straight line. Sample Concentration: Cs = K x As Cs = Sample Concentration K = Constant As = Sample Absorbance Measurement uncertainty: Highest CV% of last 6 months ± 1.96 %CV Refer Annexure I

15.Biological Reference Interval: Adult : <200mg/dl.

16.Reportable interval of patient examination results: Up to 500mg/dl

• Instruction for determining Quantitative results when a results is not within the measurement
• Serum and plasma specimens with Cholesterol values exceeding the 705mg/dl, dilution need by following the 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.

17.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

18.Stability of sample Serum or plasma-At Room temperature18°–28°C (64°–82°F) stability ≤ 24 hours

19.Storage of sample 2-8° C for 1 days (Retention period for re-examination and/or additional tests is 24 hrs.)

20.Reagent Storage and stability Reagent is stored at 2-8 °c.Reagent stability is 30 days if the reagent is uncapped and on board.

21.Turn around time (TAT) 6.0 hours after collection,in case of emergency 2.0 hours

22.Critical Value:NA

23.Laboratory Interpretation: The common causes of high cholesterol level are as follows :hyperlipoproteinemias,stress,hormonal imbalance etc

24.Potential source of variation:

1. Expiry of Kit
2. Instrumental Error
3. Presence of interfering source
4. Instrumental Error
5. Reagent contamination
6. Storage condition not proper

25.Reagent Handling Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to sit at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles

26.REFERENCE:

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
7. ARCHITECT Total cholesterol 7D62-21307192/R03
8. NABL 112 effective from 01/06/2019