Differences

This shows you the differences between two versions of the page.

Link to this comparison view

Next revision		 Previous revision
total_cholesterol [2025/01/27 17:31] – created admintotal_cholesterol [2025/01/28 05:01] (current) – admin
Line 1: Line 1:
 +|[[clinical_biochemistry_section|Home]]|[[clinical_biochemistry|]]|[[examination_procedures|]]|
  
 +                      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.
  
-Purpose of examination:  +**2.Principle of the procedure used for examinations: **  
-●Triglceride estimation from serum or plasma by Glycerol Phosphate Oxidase.+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.
  
-Responsibility and Authority:  +**3.Performance:** 
-● Calibration: Technician  +Linearity: upto 705mg/dl 
-● Quality Control: Technician  +Limit of Detection (LOD):Serum : 5.0 mg/dl 
-● Routine operation: Technician  +Limit of Quantitation (LOQ),:Serum :6.2 mg/dl 
-● Overall Monitoring: Quality Manager+The LOQ is the analyte concentration at which the CV = 20%.
  
- Sample Details:  +**4.Type of Primary Sample:** 
-● Type of Sample: Serum,  Plasma +Serum or Plasma [[sample_collection_manual|]]
-● 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:  +**5.Patient Preparation:** 
-●Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer+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..
  
-Required reagents: +**6.Type of Container and additives :** 
-       R1 84ml Active ingredients: ATP (2.5 mmol/L), Mg2+ (2.5 mmol/L), +Plain Vaccutainer with no additives
-       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).+
  
- Reagent Handling +**7.Required Equipment and reagents:** 
- ● 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 +Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, Fully Auto Chemistry Analyzer
-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.+
  
 +**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
  
-Calibration Procedure: +**10.Calibration Procedure:** 
-● Consolidated Chemistry Calibrator+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
  
-● Frequency: +**11.Procedural Steps:** 
-Reagent lot change +  * Verify that all necessary reagents present  
-QC out of range +  * Take100µl Sample. 
-After service or maintenance +  * Put on sample loader ,select & Order Test 
-Replacement in any parts of Instrument +  * Press run.
-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: +**12.Quality Control Procedure:** 
-Name: Bio Rad Level 1 & 2 +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. 
-Frequency: As per Quality Control Procedure +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.
-●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.+
  
 +**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.
  
-●Procedure to run IQC +**17.Manual Dilution Procedure:** 
-Press Control order  +  * Dilute the specimen with saline (0.85% to 0.90% NaCl). 
-Select Assay /Panel, to be run. +  * Enter the dilution factor in the Patient or Control order screen
-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: +**18.Stability of sample** 
-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.+Serum or plasma-At Room temperature18°–28°C (64°–82°F) stability ≤ 24 hours
  
- Sample Preparation: +**19.Storage of sample** 
- ● Required SampleVolume: 150 µl of the sample  + 2-8° C for 1 days (Retention period for re-examination and/or additional tests is 24 hrs.) 
- ● Temperature: 37°  +  
-Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot.  +**20.Reagent Storage and stability**  
-●Procedure to run Patient sample +Reagent is stored at 2-8 °c.Reagent stability is 30 days if the reagent is uncapped and on board.
-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:  +**21.Turn around time (TAT)** 
-● Linearity: up to 1420 mg/dL +6.0 hours after collection,in case of emergency 2.0 hours
-● The limit of detection (LOD): 0.62 mg/dL +
-● The limit of quantification(LOQ): 6.2 mg/dL +
-● Unit:  mg/dL+
  
- Normal and critical ranges:+**22.Critical Value:**NA
  
-        Triglyceride +**23.Laboratory Interpretation:** 
-Desirable +The common causes of high cholesterol level are as follows :hyperlipoproteinemias,stress,hormonal imbalance etc
-Borderline High +
-High +
-<200 +
-200-399 +
-400 and above+
  
 +**24.Potential source of variation:**
 +  - Expiry of Kit 
 +  - Instrumental Error
 +  - Presence of interfering source 
 +  - Instrumental Error
 +  - Reagent contamination
 +  - Storage condition not proper
  
- Laboratory Clinical interpretation: +**25.Reagent Handling** 
-The common causes of hyperlipidemia is nephrosis,DM,Endocrine disturbances.+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
  
- Interference and cross reaction: +**26.REFERENCE:** 
-InterferingSubstance +  - US department of labor,Occupational safety and health administration. 
-No interference from Bilirubin up to7.5 mg/dL  +  - US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories. 
-No interference from Hemoglobin up to750 mg/dL +  - World health organization. Biosafety manual,3rd edition. 
-No interference from Ascorbate up to 1.5 mg/dl  +  - World health organization. Biosafety manual,3rd edition. 
-No interference from Acetamenophen up to200mg/dl.  +  - Clinical and laboratory std institute . Protection of Laboratory workers from oocupationally acquired infection; Approved guideline -4th edition. 
-No interference from Dipyrone up to100mg/dl.  +  - Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry 
-No interference from N acetyl L cystenine up to800 mg/dl+  - ARCHITECT  Total cholesterol 7D62-21307192/R03 
 +  - NABL 112 effective from 01/06/2019
  
-Potential source of variation:  +|**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use|
-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 labor,Occupational safety and health administration. +^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^ 
-US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories. +^Document No.1^**Document Name**: Total Cholesterol Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^ 
-World health organization. Biosafety manual,3rd edition. +^Issue No. : 01^Issue Date :30/04/2024^Page No.^^ 
-World health organization. Biosafety manual,3rd edition. +^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^
-Clinical and laboratory std institute . Protection of Laboratory workers from oocupationally acquired infection; Approved guideline -4th edition. +
-Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry+