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 |[[clinical_biochemistry_section|Home]]|[[clinical_biochemistry|]]|[[examination_procedures|]]| |[[clinical_biochemistry_section|Home]]|[[clinical_biochemistry|]]|[[examination_procedures|]]|
  
-                      Triglyceride Examination Procedure +                      Total Cholesterol Examination Procedure                                                                                                                                                                                                                                                                                                                                                                                                                   
-**1.Purpose of examination:**  +**1.Purpose of Examination:**  
-Triglyceride estimation from serum or plasma by Glycerol Phosphate Oxidase.+To lay down standard operating procedure for serum Total Cholesterol estimation by CHOD-PAP Method in Biochemistry section of LSSTH, Bhavnagar.
  
-**2.Responsibility and Authority:**  +**2.Principle of the procedure used for examinations: **  
-  * Calibration: Technician  +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 
-  * Quality Control: Technician  +(quinoneimine dye) which is quantitated at 604 nm.
-  * Routine operation: Technician  +
-  * Overall Monitoring: Quality Manager+
  
-**3.Sample Details:**  +**3.Performance:** 
-  * Type of Sample: Serum,  Plasma +Linearity: upto 705mg/dl 
-  * Type of container and additives: Plain without any additives  +Limit of Detection (LOD):Serum : 5.0 mg/dl 
-  * Patient Preparation: As per Primary Sample Collection Manual  [[sample_collection_manual|]] +Limit of Quantitation (LOQ),:Serum :6.2 mg/dl 
-  * Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours +The LOQ is the analyte concentration at which the CV = 20%.
-  * Handling and transport: As per Primary Sample collection manual  +
-  * Storage: 24 hours at 2-8° C+
  
-**4.Required Equipment:**  +**4.Type of Primary Sample:** 
-Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer+Serum or Plasma [[sample_collection_manual|]]
  
-**5.Required reagents:** +**5.Patient Preparation:** 
-  * R1 84ml Active ingredients: ATP (2.5 mmol/L), Mg2+ (2.5 mmol/L), +Instruction should be given to the patient  
-  * 4-aminoantipyrine (0.4 mmol/L), 4-chlorophenol (2 mmol/L), +Verbal consent of patient should be taken before collecting blood sample. 
-  * Peroxidase (horseradish) (> 2000 U/L), GK (microbial) (> 600 U/L), +Patient should relax for about 5 minutes before the venepuncture..
-  * GPO (microbial) (> 6000 U/L), Lipoprotein lipase (microbial) (> 3000 U/L).+
  
-**6.Reagent Handling** +**6.Type of Container and additives :** 
-  * 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 +Plain Vaccutainer with no additives
-  * 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.+
  
 +**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
  
-**7.Calibration Procedure:** +**9.Precautions:** 
-  * Consolidated Chemistry Calibrator +Wear protective gloves / protective clothing / eye protection 
-  * Frequency: +Do not breathe mist / vapors / spray 
-  * Reagent lot change +Wash hands thoroughly after handling 
-  * QC out of range +Keep only in original container
-  * After service or maintenance +
-  * Replacement in any parts of Instrument+
  
-**8.Procedure:** +**10.Calibration Procedure:** 
-  - Start the equipment.WDI abbotte fully.docx +Give Calibration with Consolidated Chemistry  Calibrator ConCC (See calibrator chart for lot specific concentration) 
-  - Calibrators are ready to use. +**Manual calibration is done on following circumstances:**  
-  - Put calibrator 15-20 minutes at room temperature   +  * After instrument repair 
-  - Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation.  +  * After new reagent lot 
-  - Take a 150 µl calibrator solution in separate aliquots. +  * QC data not satisfactory - QC outlier 
-  - Go to the calibration and give the calibration order. +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
-  - Verify calibration with at least two levels of controls. +
-  - If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.+
  
-**9.Quality control Procedure:** +**11.Procedural Steps:** 
-  * ** Name:** Bio Rad Level 1 & 2 +  * Verify that all necessary reagents present  
-  * **Frequency:** As per Quality Control Procedure+  * Take100µl Sample. 
 +  * Put on sample loader ,select & Order Test 
 +  * Press run.
  
-**Procedure for Reconstitution of IQC**  +**12.Quality Control Procedure:** 
-  - Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette. +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. 
-  - Leave to stand for 30 min in the dark place. +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.
-  - 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** +**13.Interferences & Cross-Reactions:** 
-  - Press Control order  +InterferingSubstance 
-  - Select Assay /Panel, to be run. +  * No interference from Bilirubin up to7.5 mg/dL  
-  - Select the control/s and its level/s +  * No interference from Hemoglobin up to750 mg/dL  
-  - Give Carrier Number and Position number +  * No interference from Intralipid up to 1000 mg/dl  
-  - Press F3 / Add order +  * No interference from Ascorbate up to 1.5 mg/dl
-  - Put respected carrier in RSH rack +
-  - Check IQC results, in case outliers call residents.+
  
-**10.Principle of the procedure used for examinations:** +**14.Principle of Procedure for Calculating Results including where relevant, the measurement uncertainty of measured quantity values:** 
-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.+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
  
-**11.Sample Preparation:** +**15.Biological Reference Interval:** 
-  * Required SampleVolume: 150 µl of the sample  +Adult : <200mg/dl.
-  * 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** +**16.Reportable interval of patient examination results:** 
-  - Press Patient order  +Up to 500mg/dl
-  - Select Assay /Panel, to be run. +
-  - Give Carrier Number and Position number +
-  - Press F3 / Add order +
-  - Put respected carrier in RSH rack+
  
-**12.Performance Characteristics**:  +  * Instruction for determining Quantitative results when a results is not within the measurement 
-  - Linearity: up to 1420 mg/dL +  * 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 
-  - The limit of detection (LOD): 0.62 mg/dL +  * Automated Dilution Protocol 
-  - The limit of quantification(LOQ): 6.2 mg/dL +  * 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.
-  - Unit:  mg/dL+
  
-**Normal and critical ranges:**+**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
  
-**Triglyceride** +**18.Stability of sample** 
-^Desirable^ Borderline High^High^ +Serum or plasma-At Room temperature18°–28°C (64°–82°F) stability ≤ 24 hours
-|<200|200-399|400 and above|+
  
 +**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.
  
-**13.Laboratory Clinical interpretation:** +**21.Turn around time (TAT)** 
-The common causes of hyperlipidemia is nephrosis,DM,Endocrine disturbances.+6.0 hours after collection,in case of emergency 2.0 hours
  
-**14.Interference and cross reaction:**+**22.Critical Value:**NA
  
-InterferingSubstance +**23.Laboratory Interpretation:** 
-  * No interference from Bilirubin up to7.5 mg/dL  +The common causes of high cholesterol level are as follows :hyperlipoproteinemias,stress,hormonal imbalance etc
-  * No interference from Hemoglobin up to750 mg/dL +
-  * No interference from Ascorbate up to 1.5 mg/dl  +
-  * No interference from Acetamenophen up to200mg/dl.  +
-  * No interference from Dipyrone up to100mg/dl.  +
-  * No interference from N acetyl L cystenine up to800 mg/dl+
  
-**15.Potential source of variation:**  +**24.Potential source of variation:** 
-  * Turn around time (TAT): +  - Expiry of Kit  
-  * Routine: 6.0 hours +  - Instrumental Error 
-  * Urgent: 2.0 hours+  - Presence of interfering source  
 +  - Instrumental Error 
 +  - Reagent contamination 
 +  - Storage condition not proper
  
-**16.Recording of observation:** +**25.Reagent Handling** 
-  * Software backup  +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
-  * Machine raw data +
-**17.torage & Disposal of waste:** Follow storage & discard procedure+
  
-**18.Environmental & Safety control:** +**26.REFERENCE:**
- +
-**Reagent R1** contains sodium azide.contact with acids liberates very toxic gas +
- +
-**19.Precautions:** +
-  - Wear protective gloves / protective clothing / eye protection +
-  - Do not breathe mist / vapors / spray +
-  - Wash hands thoroughly after handling +
-  - Keep only in original container +
-. +
-**20.References:**+
   - US department of labor,Occupational safety and health administration.   - US department of labor,Occupational safety and health administration.
   - US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.   - US department of health and human services. Biosafety in MIcrobiological and biomedical laboratories.
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   - Clinical and laboratory std institute . Protection of Laboratory workers from oocupationally acquired infection; Approved guideline -4th edition.   - 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   - Burtis CA,Ashwood ER,editors,Tietz Textbook of clinical chemistry
 +  - ARCHITECT  Total cholesterol 7D62-21307192/R03 
 +  - NABL 112 effective from 01/06/2019
  
 |**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use| |**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 ^^^^ ^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
-^Document No.1^**Document Name**: Triglyceride Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^+^Document No.1^**Document Name**: Total Cholesterol Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^
 ^Issue No. : 01^Issue Date :30/04/2024^Page No.^^ ^Issue No. : 01^Issue Date :30/04/2024^Page No.^^
 ^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^ ^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^
 +