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Laboratory Procedure Manual
Analyte: Thyroid Peroxidase Antibodies
Matrix: Serum
Method: Access 2 (Beckman Coulter)
Method No :
Revised:
as performed by:
University of Washington Medical Center
Department of Laboratory Medicine
Immunology Division
Director: Mark Wener M.D.
Supervisor: Kathleen Hutchinson M.S., M.T. (ASCP)
Authors: Michael Walsh, MT (ASCP), September 2006
contact: Mark Wener M.D.
Important Information for Users
University of Washington periodically refines 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.
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Public Release Data Set Information
This document details the Lab Protocol for testing the items listed in the following table:
File Name
Variable
Name
SAS Label
THYROD_E LBXTPO Thyroid Peroxidase Antibodies
(IU/mL)
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3. COMPUTERIZATION; DATA SYSTEM MANAGEMENT
A. Each shipment of specimens received from the NHANES mobile unit arrives with a corresponding transmittal sheet and an electronic version of the shipping/resulting file. The file structure is determined by NHANES and is described in the National Health and Nutrition Examination Survey (NHANES) Contract Laboratory Manual.
B. After the testing is completed results from the Access 2 are transferred to the laboratory server system, which is backed up daily. This instrument file contains the following information for each sample, control and calibrator tested. Patient ID Sample ID Rack Verify Test Name Interpretation Result Units Comp. Time Flags LIS Instrument RLU Pipettor Sample Type Sample Priority Test ID Reagent Pack Lot # Reagent Pack Serial # Dilution Calibrator level Comments Load Date/Time
C. QC results are transferred to an Excel file using laboratory-developed software. This file calculates the QC statistics, plots Levey-Jennings charts, displays relevant instrument flags, tracks reagent lots and recent calibrations. QC results are reviewed prior to resulting samples.
D. Sample results are transferred to an Excel file using laboratory-developed software that enters results after matching sample identifiers from the instrument file with those provided in the NHANES shipping/resulting file. This Excel file is formatted to match the NHANES shipping/resulting file and the program uses the conventions outlined in the NHANES Contract Laboratory Manual.
E. Data entry is checked for errors.
F. After the TPOAb testing has also been completed, resulted, and checked, the result
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file is transmitted electronically to NHANES WESTAT. Electronic and hard copies of the files are kept in the laboratory.
G. Technical support for this system is provided by Westat, Rockville, MD (1-301-294-
4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES; CRITERIA
FOR SPECIMEN REJECTION
A. No special instructions such as fasting or special diets are required.
B. Serum is the preferred specimen type. Heparin plasma is acceptable. If testing is to be done within 48 hours, samples can be refrigerated at 2 to 8°C. Freeze at -20°C or colder for longer storage.
C. Blood should be collected aseptically and the serum separated by standard laboratory techniques. Specimens may be collected by using regular or serum-separator Vacutainers. Serum should be separated from the cells within 2 hours of collection.
D. The requested sample volume for the assay is 1.0 mL, and the minimum sample volume is 0.3 mL.
E. Specimens may be stored in glass or plastic vials, as long as the vials are tightly sealed to prevent desiccation of the sample.
F. Turbid samples or those with particulate matter should be centrifuged prior to assay.
G. More than three freeze-thaw cycles is not recommended.
5. PROCEDURES FOR MICROSCOPIC EXAMINATIONS; CRITERIA FOR REJECTION
OF INADEQUATELY PREPARED SLIDES
Not applicable for this procedure.
6. PREPARATION OF REAGENTS, CALIBRATORS (STANDARDS), CONTROLS, AND
ALL OTHER MATERIALS; EQUIPMENT AND INSTRUMENTATION
A. Instrumentation
- Beckman Access or Access II Immunoassay System (Beckman Coulter, Fullerton, CA.)
The Beckman Access is a fully automated, random access, instrument that features on-board storage of reagent packs in a refrigerated compartment; an ultrasonic probe tip for level sense detection, sample and reagent delivery, mixing, and probe cleaning to minimize carryover; barcode identification of specimens and reagent packs; temperature controlled reaction reactions; and measurement and analysis of the light signal generated by the chemiluminescent reaction (RLU) using a weighted four parameter logistic curve math model.
The Thyroid Peroxidase Antibody assay parameter settings for the instrument are as follows:
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- Access Substrate Cat. No. 81906: 4 x 130 ml
Lumi-Phos530 (buffered solution containing dioxetane), Lumigen PPD, fluorescer, and surfactant. Bring to room temperature (15 – 30 ºC) at least 18 hours before use. Stable for 14 days at room temperature or after bottle has been opened.
- Access Wash Buffer II: Cat # A Provided ready to use. Store at room temperature (15 – 30 ºC), stable until expiration date on label.
D. Standards/Calibration Preparation
Access Thyroid Peroxidase Antibody Calibrators Cat. No. A18227: 2.5 ml/vial
Provided ready to use. Store at 2 to 10°C. Mix contents by gently inverting before use. Avoid bubble formation. Stable until the expiration date stated on the vial labels when stored at 2 to 10°C. Control values out of range are a sign of possible deterioration.
- Human thyroperoxidase antibody in human serum at levels of approx. 5, 20, 75, 300 and 1000 IU/mL. Zero calibrator is human serum with <0.1%sodium azide. S0 Std contains 0.0 IU/mL TPO antibody. Refer to calibration card for exact concentrations.
- Calibration Card (with actual calibrator concentration information)
E. Preparation of Quality Control Materials
Three different levels of serum controls are run with each run. The controls are purchased from BioRad Laboratories (Hercules, CA) or prepared in-house. Commercial controls are stored and used according to the manufacturer’s recommendations. In house controls are stored frozen (-20ºC or colder). Once thawed, the controls are stored at 2-8 ºC. All controls are used within their stated expiration dates.
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7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES
A. Calibration Curve
Example TPOAb Calibration:
TPOAb concentrations are calculated by using a calibration curve. This method utilizes a weighted four-parameter logistic curve with a direct relationship of measured light produced (RLU) to concentration of TPOAb in the serum sample. Serum results are expressed as IU/ mL.
Calibrators are standardized to the WHO 66/387 international standard.
An active calibration curve is required for all tests. For the Access TPOAb assay, calibration is required every 56 days or whenever new lot numbers of reagents are placed into use. Refer to the Operator’s Guide and Reference Manual for complete instructions on calibration procedures.
B. Verification
- Three levels of control are run for each test series. If, within a testing series, these controls do not conform to specifications as defined in the quality control manual, the entire series is invalidated.
- New lot numbers of calibrator are verified by running 100 or more samples tested on the previous lot number. The correlation is analyzed using one or more linear regression formulas.
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- Daily Maintenance: Start-up: Inspect fluidics module. Check system supplies and replace as needed. Clean exterior of substrate, dispense, and aspirate probes. Prime pipettor – 1X and substrate - 4X. Verify temperature. Shut-down: Check waste containers, empty if needed Perform clean
- Weekly Maintenance: Change probes and clean them Clean exterior of the analyzer Clean upper portion of the main pipettor with alcohol wipe Inspect waste filter bottle for fluid Run system check
- Periodic Maintenance to be performed by the manufacturer's service engineer.
E. Calculations
Patient test results are determined automatically by the system software. The amount of analyte in a sample is determined from the measured light production by means of a stored nonlinear calibration curve. Patient test results can be reviewed using the Sample Results screen. Refer to the Operator’s Guide for complete instructions on reviewing results.
9. REPORTABLE RANGE OF TEST RESULTS
Results are reported to the nearest tenth (0.1). The lowest reportable TPOAb result is 0. IU/mL. Results above the top standard (generally near 1000 IU/mL) are reported as “greater than” (e.g. >1000 IU/mL). Estimates of imprecision can be generated from long- term quality control pool results.
10. QUALITY CONTROL (QC) PROCEDURES
A. Bench quality controls are used in this analytical method. Bench quality control specimens are tested with each analytical run (a set of consecutive assays performed without interruption) so that judgements may be made on the day of analysis. The data from these materials are then used in estimating methodological imprecision and in assessing the magnitude of any time-associated trends.
B. The bench controls are purchased in sufficient quantity to provide serum samples for all the assays for approximately 1 year. Ranges are established after 20 parallel runs with previously established controls. The quality control pools comprise three levels of concentration spanning the low, borderline and high ranges for TPOAb.
C. Bench quality controls are placed at the beginning of each analytical run. After analysis, the long-term quality control charts (Levey-Jennings) for each control material are consulted to determine if the system is “in control.” The Levey Jennings
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chart plots the quality control material observations on the y-axis and the date of the observation on the x-axis. Quality control material observations are compared with the 95% and 99% confidence limits as well as with the center line (the overall mean of the characterization runs) prior to reporting any results. The system is out of control if any of the following events occur for any one of the quality control materials:
The observation from a single pool falls outside the 99% confidence limits. The observations from two pools fall either both above or both below the 95% confidence limits. The observations from eight successive runs for one pool fall either all above or all below the center-line and the current result is above or below the 95% confidence limits.
11. REMEDIAL ACTION IF CALIBRATION OR QC SYSTEMS FAIL TO MEET
ACCEPTABLE CRITERIA
If the run is declared "out of control", the system (instrument, calibration standards, etc.) is investigated to determine the root of the problem before any results are released. Consult with the supervisor for appropriate actions.
12. LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND CONDITIONS
A. The upper reportable value is approximately 1000 IU/mL, and is dependent upon the assigned value of the current lot of calibrator.
B. The lowest reportable value is approximately 0.3 IU/mL. According to the manufacturer, this is the lowest detectable level of TPOAb distinguishable from zero with 95% confidence.
C. The TPOAb results should be interpreted in light of the total clinical presentation of the patient, including: symptoms, clinical history, data from additional tests, and other appropriate information.
D. This assay does not demonstrate "high dose hook" below 10,000 IU/mL.
E. According to the manufacturer the following substances do not interfere with the assay: Hemoglobin up to 500 mg/dL Bilirubin up to 40 mg/dL Triglycerides up to 3000 mg/dl Human albumin levels of 6 g/dL
F. For assay employing antibodies, the possibility exists for interference by heterophile antibodies in the patient sample. Patients that have been regularly exposed to animals or have received immunotherapy or diagnostics procedures utilizing immunoglobulins or immunoglobulin fragments may produce antibodies, e.g. HAMA, that interfere with immunoassays. Additionally, other heterophile antibodies such as human anti-goat antibodies may also be present in patient samples. Such interfering antibodies may cause erroneous results. Carefully evaluate the results of patients suspected of having these antibodies.
G. WARNING: The concentrations of TPOAb in a given specimen determined with assays from different manufacturers can vary due to differences in assay methods
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19. Summary Statistics and QC Graphs
Summary Statistics for Thyroid Peroxidase Antibodies by Lot
Lot N Start Date End Date Mean
Standard Deviation
Coefficient of Variation
40671 72 2/5/2007 9/23/2007 9.835 0.625 6.
40673 72 2/5/2007 9/23/2007 26.992 1.795 6. 41542 205 9/13/2007 1/29/2009 99.327 6.959 7.
40701 199 12/4/2007 1/29/2009 13.561 1.035 7.
40703 199 12/4/2007 1/29/2009 24.125 1.514 6.
2007-2008 Thyroid Peroxidase Antibodies Quality Control
0
20
40
60
80
100
120
140
2/5/2007 5/16/2007 8/24/2007 12/2/2007 3/11/2008 6/19/2008 9/27/2008 1/5/
41542
40671 40701
40673 40703
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REFERENCES
- Manufacturer Information: Beckman Access Immunoassay System Operator’s Guide and Reference Manual Thyroid peroxidase antibody kit inserts, Beckman Coulter, Inc. 2006 Beckman Coulter
- National Health and Nutrition Examination Survey (NHANES) Contract Laboratory Manual. September 2006
- DeGroot LJ, Niepomniszcze H,. Biosynthesis of thyroid hormone: basic and clinical aspects. Metabolism 1977;26:665-
- Mariotti S, Caturegli P, Piccolo P, Barsesino G and Pinchera A. Antithyroid peroxidase antibodies in thyroid disease. J Clin Endocrinol Metab 1990;71:661-
- Demers LM, Specer CA. Laboratory medicine practice guidelines – Laboratory support for the diagnosis and monitoring of thyroid disease. The National Academy of Clinical Biochemistry.
- Feldt-Rasmussen, U. Analytical and clinical performance goals for testing autoantibodies to thyroperoxidase, thyroglobulin and thyrotropin receptor. Clinical Chemistry; 42:1 pp. 160- (1996)
