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Laboratory Procedure Manual
Analyte: Chloride
Matrix: Serum
Method: Roche Cobas 6000 (c501 module)
Method No.: Revised:
As performed by: University of Minnesota Advanced Research and Diagnostic Laboratory (ARDL) 1200 Washington Ave S, Suite 175 Minneapolis, MN 55415
Contact: Anthony Killeen, MD, PhD, Laboratory Director Jennifer Peters, MT, ASCP, Laboratory Manager
Important Information for Users
The Advanced Research and Diagnostic Laboratory (ARDL) periodically refine these laboratory methods. It is the responsibility of the user to contact the person listed on the title page of each write-up before using the analytical method to find out whether any changes have been made and what revisions, if any, have been incorporated.
Public Release Data Set Information
This document details the Lab Protocol for testing the items listed in the following table:
Data File Name (^) Variable Name SAS Label BIOPRO_J LBXSCLSI Chloride (mmol/L)
Caution: This product is of human and animal origin. Handle as though capable of transmitting infectious disease. Wear appropriate PPE when handling equipment, reagents, and samples.
3. COMPUTERIZATION; DATA SYSTEM MANAGEMENT
ARDL utilizes a highly specialized Laboratory Information System (LIS) (STARLIMS, Abbott Informatics Corporation; Hollywood, FL, 33021-6755) for all lab functions. Major instrument platforms are interfaced directly to the LIS, allowing data to be electronically transferred directly to the main database. The system provides an extensive quality assurance package and data management tools. Numerous networked computer workstations are used in the laboratory for data management and transmission, and also include software for word and spreadsheet creation and manipulation, statistical analysis, report presentation, and electronic communication. All workstations are user password protected with job specific security access levels and have idle time out functionality. All systems are redundantly backed up on a real time basis.
4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES; CRITERIA FOR SPECIMEN REJECTION a. Specimen Type and Requirements: Serum, lithium heparin plasma, and urine are acceptable specimens. The NHANES Biochem study uses refrigerated serum. This test is analyzed from NHANES Vial 018. b. Specimen Volume: Optimum/Minimum volume: 100 μL in a sample cup or 2 mL microtube (9.7 μL serum or plasma for test; remainder for dead volume). c. Acceptable Specimens/Unacceptable Specimens: Serum or lithium heparin plasma is acceptable. Other anticoagulants are not acceptable. d. Specimen Stability and Storage: Separated serum or plasma should be removed from the cells within one hour of collection. Serum or plasma is stable for 7 days at 15- 25°C, 7 days at 2-8ºC, indefinitely at -20°C or colder. Specimens must be at room temperature prior to assay e. Interferences or limitations: Hemolysis does not interfere with the assay up to an H index of 1000 (approximately 1000 mg/dL hemoglobin). Intralipid does not interfere in
the tested concentration range up to 2000 mg/dL (L index of 2000). However, there is poor correlation between triglyderides concentration and L index. Gross lipemia can result in pseudohyponatremia as a result of fluid displacement Excessively lipemic specimens should be ultracentrifuged, and specimens with particulate matter should be cleared by centrifugation. Bilirubin does not interfere up to an I index of 60. f. Specimen Handling and Transport: Mix specimens well, allow clot to fully form (if serum), and centrifuge 10 minutes at 2000 x g before use. Aliquot a minimum of 0. mL. Store sample in refrigerator until shipment. Ship at refrigerated temperature.
5. PROCEDURES FOR MICROSCOPIC EXAMINATIONS; CRITERIA FOR REJECTION OF INADEQUATELY PREPARED SLIDES Not applicable for this procedure. 6. EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS (STANDARDS), AND CONTROLS a. Reagents and Supplies •ISE Diluent (ready for use), Roche Cat. No. 04522630190, 5x300 mL. HEPES buffer: 10 mmol/L; Triethanolamine: 7 mmol/L; Preservative. Store at 15-25°C. On board stability 14 days. •ISE Internal Standard (ready for use), Roche Cat. No. 04522320190, 5x600 mL. HEPES buffer: 10 mmol/L; Triethanolamine: 7 mmol/L; Sodium chloride: 3. mmol/L; Sodium acetate: 1.45 mmol/L; Potassium chloride: 0.16 mmol/L; Preservative. Store at 15-25°C. On board stability 14 days. •ISE Reference Electrolyte (ready for use), Roche Cat. No. 11360981216, 5x mL. Potassium chloride: 1 mol/L. Store at 15-25°C. On board stability 28 days. •Electrodes, Roche Cat. No. 03246353001 (Chloride) and Roche Cat. No. 03149501001 (Reference). Store electrodes at 7-40 °C. See label for expiration date. On board stability: chloride (2 months), reference (6 months). - ISE Cleaning Solution, Roche Cat. No. 11298500316, 5x100 mL. Sodium hydroxide solution (12%) with sodium hypochloride solution < 2 % active Cl.
7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES
Calibrators S1, S2 and S S1: ISE Standard Low, Roche Cat. No. 11183974216, 10x3 mL. 120 mmol/L Na+, 3 mmol/L K+, 80 mmol/L Cl- S2: ISE Standard High, Roche Cat. No. 11183982216, 10x3 mL 160 mmol/L Na+, 7 mmol/L K+, 120 mmol/L Cl- S3: ISE Standard High (compensated), Roche Cat. No. 11183982216, 10x3 mL 160 mmol/L Na+, 7 mmol/L K+, 120 mmol/L Cl Storage and stability Store S1, S2 and S3 at 15-25°C. See label for expiration date. On-board stability Calibrators S1, S2 and S3: to be used for one calibration only. Full calibration for Na+, K+ and Cl- requires the following 3 calibrator solutions: ISE Standard Low, ISE Standard High, and ISE Standard High (compensated). The slope of the calibration curve is calculated from Standards 1 and 2. ISE Standard High (compensated) is designed to reduce matrix effects; it only affects the intercept, not the slope. An internal standard is also measured during calibration and between samples to compensate for any system deviations. Refer to the Operator’s Manual of the analyzer for detailed calibration instructions. US Traceablity: This method has been standardized against primary calibrators prepared gravimetrically from purified salts. Calibration frequency: Perform a full calibration •every 24 hours •after ISE cleaning and maintenance
•after changing the reagent bottles •after replacing any electrode •as required following quality control procedures If calibration fails perform the following corrective action steps in sequence: •Check reagent and calibrator for appropriate lot numbers, expiration dates, preparation and storage conditions. •Repeat calibration with new calibrator. •Repeat calibration with new reagent and new calibrator. If successful calibration is not achieved, discontinue testing and notify the supervisor.
8. OPERATING PROCEDURE INSTRUCTIONS; CALCULATIONS; INTERPRETATION OF RESULTS a. Instrument Operation: The Roche/Hitachi Cobas 6000 analyzer series is a fully automated, random-access, software controlled system for immunoassay and photometric analyses intended for qualitative and quantitative in vitro determinations using a wide variety of tests. The Cobas 6000 analyzer series is optimized for workloads using a combination of photometric and ion-selective electrode (ISE) determinations (c501 module), and electrochemiluminescence (ECL) signal in the immunoassay analysis module (e601 module). The ISE system is used in the quantitation of sodium, potassium and chloride. The photometric system can measure colorimetric or immunoturbidimetric reactions utilizing end point or kinetic (rate) absorbance measurements. Test ordering end execution on the Cobas 6000 and data entry in the STARLIMS host computer system may be done manually or these tasks may be executed via a barcode- based bi-directional interface. The Cobas 6000 can utilize both of these two systems simultaneously. b. Professional Judgement: Check results for error flags and take appropriate corrective action. Investigate alert values and delta checks. c. Result Entry
Limit of Detection (standard 1 + 3 SD): Specimens exceeding the linear limits are not diluted by the Cobas 6000. If a dilution is desired, dilute the specimen in CLRW water, and multiply the result by the corresponding dilution factor. Maximum dilution is 1:2. Specimens reading above or below the linear range are essentially incompatible with life, and would most likely indicate an analytical problem or a contaminated or incorrect specimen (i.e. urine specimen labeled as a serum specimen).
10. QUALITY CONTROL (QC) PROCEDURE
Quality Control
- Normal pooled serum control (CQ). Stable at -80°C for up to 4 years, at refrigerated temperature for up to 1 day and at room temperature for up to 4 hours.
- Roche Precinorm U Plus Control (Roche Cat. No. #12149435160, 10x3 mL.). Roche Diagnostics 9115 Hague Road Indianapolis, IN 46250-0457). Stable until expiration date on package when unopened and stored at 2-8°C. To prepare, open bottle 1 and pipette in exactly 3.0 mL of diluent (bottle 2). Dissolve by gentle swirling for 30 minutes. Prepared control is stable for 12 hours at room temperature, 5 days at 2-8°C, and one month at -20°C (when frozen once).
- Both levels of quality control are analyzed at the start of the day and results are verified for acceptability prior to testing specimens. Quality control is also analyzed at the end of the shift, with change in reagent, after major maintenance, or as needed for troubleshooting.
- The analytical measurement range (AMR) must be validated every 6 months or after major maintenance or service procedures. The laboratory enrolls in the College of American Pathologist (CAP) linearity program. Chloride is included in the LN2 kit that is shipped twice per year. Follow kit instructions for preparation. Analyze samples in duplicate. Results are due within two to four weeks of receipt of kit. Results are submitted online to the CAP website by the lead or supervisor. The linearity report is available online at the CAP website shortly after the due
date. Confirm reported values are within acceptability limits. Place instrument printouts, worksheets and CAP results in the CAP three ring binder.
- New Lot Verification: Each new electrode lot must be verified for acceptability before being placed into use. Calibration, quality control, and comparison of at least 5 patient samples on the old and new lots must be performed and found to be within acceptable limits before a new lot can be placed into use.
- Quarterly Technical Progress Report: Progress reports for each quarter of the calendar year are submitted to NHANES. The report includes 1) a summary of the status of all specimens including date of arrival, deviations from protocol, handling, storage, and manipulation of vials and all laboratory procedures used, 2) a description of any major problems such as missing data, out of range values or inconsistent data and what was done to overcome them, internal and external quality control for runs containing NHANES specimens, instrument calibration and repairs, reagent lots and dates of use. 11. REMEDIAL ACTION IF CALIBRATION OR QC SYSTEMS FAIL TO MEET ACCEPTABLE CRITERIA
- If QC values are outside of specified ranges, do the following, in order, until QC is acceptable:
- Repeat the analysis using fresh QC material.
- Perform a calibration.
- Check for system problems.
- Contact Roche Technical Support for assistance and possible service dispatch. Phone: 1-800-428-2336; account number: 55042919 12. LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND CONDITIONS
a. Analytical Measurement Range: 60-140 mmol/L b. Hemolysis does not interfere with the assay up to an H index of 1000 (approximately 1000 mg/dL hemoglobin). Intralipid does not interfere in the tested
Shipments for NHANES generally arrive on Tuesdays and/or Wednesdays. These shipments are recorded on the Log of Quality Assurance located in a binder labeled NHANES Shipping Log in the receiving area. The specimen barcode numbers in the boxes are checked against the manifests. The receipt date is written on top of the boxes. The frozen samples (vial 11-Iron/UIBC & vial 13-CRP) are placed in the designated -70°C freezer and the refrigerated samples (vial 18-Biochem panel) are placed in the designated 2-8°C refrigerator until analysis. The manifests are filed in a binder labeled NHANES Shipping Manifests located in the receiving area. All labels are removed from the shipping box and the provided airbill is attached for return shipment.
Quality Assurance Log:
A Quality Assurance Specimen Receipt and Specimen Return Log is maintained by laboratory staff. The following parameters are tracked: NHANES shipper I.D., NHANES Container I.D., Vial #, Date Received, Specimen Receipt Conditions, Number of Specimens Received, 2.5% QC Repeats, Total Number of Specimens, 21 Day Due Date, Analysis Date, Date Results Sent, Number of Days For Result Return, Thaw Date (if applicable), Return To Freezer Date, Number of Days at Refrigerated Temperature, 1 Year Discard or Return Date, NHANES Quarterly Report Date
Specimen Ordering/Labeling:
Electronic files for all NHANES specimens are sent via email from Westat, Inc to the NHANES contact person shortly before they are to be received. These files include the Sample ID, Analyte Type, Slot No, Sample Collection Date, Sample Comment, Age Grouping, Astro ID, Receipt Date, Analysis Date, Run Number, Tech ID, Analyte Result, Result Comment, Adjusted Result, QC Repeat, LOD, Change Reason, and Change Reason Other. The first seven columns are protected and cannot be altered. The files are saved on the laboratory’s common S drive in the NHANES Biochem folder. After analysis, the contact person returns the completed files via their website to Westat, Inc.
The NHANES spreadsheets are used to set up pending batches for batch accession upload in the Laboratory Information system (STARLIMs). New labels are generated out of the Laboratory Information System (STARLIMs). The new bar-coded labels are attached to a carrier tube. The Cobas analyzer reads the bar-coded label for the sample ID and test information.
Specimen Storage:
The temperatures for all freezers and refrigerators are monitored 24 hours a day/ 7 days a week. If the temperature for any unit falls outside the allowable range, action is taken to resolve the problem. If the temperature cannot be corrected, the contents are moved to a different unit.
Summary Statistics and QC Chart for Chloride (mmol/L)
Lot N
Start Date
End Date Mean
Standard Deviation
Coefficient of Variation Q 122 18JAN17 22JAN19 101.0 0.8 0. 176407 52 18JAN17 15NOV17 85.4 1.0 1. 186350 32 22NOV17 30MAY18 87.0 1.2 1. 203067 29 04JUN18 13NOV18 88.9 0.9 1. 203068 9 20NOV18 22JAN19 89.2 1.0 1.
REFERENCES
- Cobas 6000 Analyzer Operator’s Manual. Roche Diagnostics Inc., 9115 Hague Road Indianapolis, IN 46250-0457. Version 5.0.
- Roche ISE reagent package insert. Version 2016-04, V13.0. Roche Diagnostics Inc., 9115 Hague Road Indianapolis, IN 46250-0457.
- Roche Precinorm U Plus package insert, Version 2015-03, V7.0. Roche Diagnostics Inc., 9115 Hague Road Indianapolis, IN 46250-0457.
