PRINCIPLE REF ANNUAL REVIEW Reviewed by. Date. Date INTENDED USE
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1 SYNCHRON System(s) Chemistry Information Sheet Copyright 2013 Beckman Coulter, Inc. T-Uptake REF For In Vitro Diagnostic Use ANNUAL REVIEW Reviewed by Date Reviewed by Date PRINCIPLE INTENDED USE reagent, when used in conjunction with UniCel DxC 600/800 System(s) and T-Uptake Calibrator, is intended for the quantitative determination of thyroxine binding capacity in human serum. CLINICAL SIGNIFICANCE Changes in the concentration of thyroid-binding proteins affect free hormone levels, triggering the depression or stimulation of hormone production until a new equilibrium is reached. The clinical usefulness of total thyroid hormone measurement is compromised if there are marked changes in the binding capacity or concentration of thyroxine-binding proteins. 1 METHODOLOGY reagent is used to measure unoccupied thyroxine-binding sites. This reagent used recombinant DNA technology. The enzyme β-galactosidase (β-gal) is split into two inactive components, an enzyme-donor (ED) and an enzyme-acceptor (EA), which, when mixed together, spontaneously form active enzyme. Enzyme-donor-thyroxine conjugate binds directly to the unoccupied thyroxine-binding protein (TBP) sites in serum, preventing spontaneous reassociation of the two enzyme fragments to form active enzyme. 2 Thyroid-binding proteins regulate the amount of active β-galactosidase formed. The unoccupied thyroxine-binding sites in the sample are inversely proportional to the amount of β-galactosidase formed from the reassembly of the remaining ED and EA as monitored by the hydrolysis of the substrate o-nitrophenyl-β-d-galactopyranoside (ONPG). The SYNCHRON System(s) automatically proportions the appropriate sample and reagent volumes into a cuvette. The ratio used is one part sample to 17 parts reagent. The System monitors the ONPG hydrolysis by measuring the change in absorbance at 410 nanometers. This change in absorbance is inversely proportional to the unoccupied thyroxine-binding sites in the sample and is used by the System to calculate and express T-Uptake as a percentage based upon a two-point calibration curve. FEBRUARY 2014 Page 1 of 11
2 The free thyroxine index (FTI) is a means of normalizing the effect of thyroxine-binding protein concentrations on total T4 levels. The FTI yields an index which is related to the biologically active free T4 concentration. The FTI calculation, as recommended by the American Thyroid Association is: The mean % referred to in the above calculation refers to the mean % T-Uptake of the normal range distribution which should be established by each laboratory. CHEMICAL REACTION SCHEME SPECIMEN TYPE OF SPECIMEN Biological fluid samples should be collected in the same manner routinely used for any laboratory test. 3 Freshly drawn serum is the preferred specimen. Whole blood, plasma or urine are not recommended for use as a sample. SPECIMEN STORAGE AND STABILITY 1. Tubes of blood are to be kept closed at all times and in a vertical position. It is recommended that the serum be physically separated from contact with cells within two hours from the time of collection Separated serum should not remain at room temperature longer than 8 hours. If assays are not completed within 8 hours, serum should be stored at +2 C to +8 C. If assays are not completed within 48 hours, or the separated sample is to be stored beyond 48 hours, samples should be frozen at -15 C to -20 C. Frozen samples should be thawed only once. Analyte deterioration may occur in samples that are repeatedly frozen and thawed. 4 Additional specimen storage and stability conditions as designated by this laboratory: SAMPLE VOLUME The optimum volume, when using a 0.5 ml sample cup, is 0.3 ml of sample. For optimum primary sample tube volumes and minimum volumes, refer to the Primary Tube Sample Template for your system. CRITERIA FOR UNACCEPTABLE SPECIMENS Refer to the PROCEDURAL NOTES section of this chemistry information sheet for information on unacceptable specimens. Page 2 of 11 FEBRUARY 2014
3 Criteria for sample rejection as designated by this laboratory: PATIENT PREPARATION Special instructions for patient preparation as designated by this laboratory: SPECIMEN HANDLING Special instructions for specimen handling as designated by this laboratory: REAGENTS CONTENTS Each kit contains the following items: One Empty Reagent Cartridge (250 tests when filled as described) One Calibrator Diskette One EA Reagent (lyophilized) One ED Reagent (lyophilized) One EA Reconstitution Buffer One ED Reconstitution Buffer One 4 ml Low Calibrator/Cal 1 (lyophilized) One 4 ml High Calibrator/Cal 2 (lyophilized) One EA Adapter One ED adapter VOLUMES PER TEST Sample Volume 15 µl Total Reagent Volume 252 µl Cartridge Volumes A 209 µl FEBRUARY 2014 Page 3 of 11
4 B 43 µl C REACTIVE INGREDIENTS REAGENT CONSTIENTS T-Uptake Enzyme-Donor (ED) Reagent: Enzyme-Donor conjugated to thyroxine o-nitrophenyl-β-d-galactopyranoside T-Uptake Enzyme-Acceptor (EA) Reagent: Enzyme-Acceptor Also non-reactive chemicals necessary for optimal system performance. 14 ml (after reconstitution with ED buffer) 62 ml (after reconstitution with EA buffer) CAUTION Sodium azide preservative may form explosive compounds in metal drain lines. See National Institute for Occupational Safety and Health Bulletin: Explosive Azide Hazards (8/16/76). MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT At least two levels of control material REAGENT PREPARATION NOTICE To ensure solubility of combined reagents, reconstitute immediately upon removal from refrigerator. Reconstitute in the following order: 1. Enzyme-Donor Reagent (ED) A. Open cold ED Reconstitution Buffer bottle and snap adapter on tightly. B. Connect the bottle of ED Reagent to the bottle of ED Reconstitution Buffer. C. Mix by gentle inversion to ensure all the ED Reagent is transferred into the ED Buffer bottle. Avoid foaming. Allow to stand at room temperature for 5 minutes. Mix again. D. Detach the ED Reagent bottle and adapter from the ED Buffer bottle and discard. E. Transfer the entire contents of the bottle to compartment B (middle) of the reagent cartridge. F. Record the reconstitution date on the cartridge label. 2. Enzyme-Acceptor Reagent (EA) A. Open cold EA Reconstitution Buffer bottle and snap adapter on tightly. B. Connect the bottle of EA Reagent to the bottle of EA Reconstitution Buffer. C. Mix by gentle inversion to ensure all the EA Reagent is transferred into the EA Buffer bottle. Avoid foaming. Allow to stand at room temperature for 5 minutes. Mix again. D. Detach the EA Reagent bottle and adapter from the EA Buffer bottle and discard. Page 4 of 11 FEBRUARY 2014
5 E. Transfer the entire contents of the bottle to compartment A (large) of the reagent cartridge. Cross contamination of reagents must be avoided. The enzyme-donor solution should appear colorless to very pale yellow. A deep yellow or yellow-orange color indicates that the reagent has been contaminated and must be discarded. The enzyme-donor and enzyme-acceptor reagents are matched sets. Do not mix bottles from different kit lot numbers in the same cartridge. Always add freshly reconstituted reagents to an unused cartridge. ACCEPTABLE REAGENT PERFORMANCE The acceptability of a reagent is determined by successful calibration and by ensuring that quality control results are within your facility s acceptance criteria. REAGENT STORAGE AND STABILITY T-Uptake reagent when stored lyophilized and unopened at +2 C to +8 C will remain stable until the expiration date printed on the kit label. Once reconstituted, the reagent is stable for 21 days at +2 C to +8 C unless the expiration date is exceeded. DO NOT FREEZE. Do not expose T-Uptake Reagent to room temperature for more than 24 hours, either in single or cumulative exposure. Reagent storage location: CALIBRATION CALIBRATOR REQUIRED T-Uptake Calibrator Levels 1 and 2 (included in the T-Uptake Reagent Kit) CALIBRATOR PREPARATION 1. Reconstitute each bottle with 4.0 ml of deionized water using a volumetric pipette. 2. Let stand 45 minutes to dissolve and equilibrate. Mix by gentle inversion (avoid foaming). CALIBRATOR STORAGE AND STABILITY If unopened, T-Uptake Calibrators should be stored at +2 C to +8 C until the expiration date printed on the calibrator bottle. Prepared calibrators that are resealed and stored at +2 C to +8 C are stable for 30 days unless the expiration date is exceeded. NOTICE Prior to the first calibration of a specific lot number of T-Uptake Reagent, the lot specific calibrator diskette that accompanies the kit must have been loaded onto the system. FEBRUARY 2014 Page 5 of 11
6 Calibrator storage location: CAUTION Because this product is of human origin, it should be handled as though capable of transmitting infectious diseases. Each serum or plasma donor unit used in the preparation of this material was tested by United States Food and Drug Administration (FDA) approved methods and found to be negative for antibodies to HIV and HCV and nonreactive for HbsAg. Because no test method can offer complete assurance that HIV, hepatitis B virus, and hepatitis C virus or other infectious agents are absent, this material should be handled as though capable of transmitting infectious diseases. This product may also contain other human source material for which there is no approved test. The FDA recommends such samples to be handled as specified in Centers for Disease Control s Biosafety Level 2 guidelines. 5 CALIBRATION INFORMATION 1. The system must have valid calibration factors in memory before controls or patient samples can be run. 2. Under typical operating conditions the reagent cartridge must be calibrated every 24 hours and also with certain parts replacement or maintenance procedures, as defined in the UniCel DxC 600/800 System Instructions For Use (IFU) manual. 3. For detailed calibration instructions, refer to the UniCel DxC 600/800 System Instructions For Use (IFU) manual. 4. The system will automatically perform checks on the calibration and produce data at the end of calibration. In the event of a failed calibration, the data will be printed with error codes and the system will alert the operator of the failure. For information on error codes, refer to the UniCel DxC 600/800 System Instructions For Use (IFU) manual. TRACEABILITY T-Uptake measurand (analyte) in this calibrator is traceable to Manufacturer s Working Calibrator. process is based on pren ISO QUALITY CONTROL The traceability At least two levels of control material should be analyzed daily. In addition, these controls should be run with each new calibration, with each new reagent cartridge, and after specific maintenance or troubleshooting procedures as detailed in the appropriate system manual. More frequent use of controls or the use of additional controls is left to the discretion of the user based on good laboratory practices or laboratory accreditation requirements and applicable laws. The following controls should be prepared and used in accordance with the package inserts. Discrepant quality control results should be evaluated by your facility. NOTICE Quality control material containing ethylene glycol is not recommended for use with this assay. Page 6 of 11 FEBRUARY 2014
7 Table 1.0 Quality Control Material CONTROL NAME STORAGE TESTING PROCEDURE(S) 1. If necessary prepare reagent as defined in the Reagent Preparation section of this chemistry information sheet and load the reagent onto the system. 2. After reagent load is completed, calibration may be required. 3. Program samples and controls for analysis. 4. After loading samples and controls onto the system, follow the protocols for system operations. For detailed testing procedures, refer to the UniCel DxC 600/800 System Instructions For Use (IFU) manual. CALCULATIONS The SYNCHRON System(s) performs all calculations internally to produce the final reported result. The system will calculate the final result for sample dilutions made by the operator when the dilution factor is entered into the system during sample programming. REPORTING RESULTS Equivalency between the SYNCHRON LX and UniCel DxC 600/800 Systems has been established. Chemistry results between these systems are in agreement and data from representative systems may be shown. REFERENCE INTERVALS Each laboratory should establish its own reference intervals based upon its patient population. The reference intervals listed below were taken from literature and a study performed on SYNCHRON Systems. 6 Table 2.0 Reference intervals a Literature SYNCHRON INTERVALS Serum (Male) Serum (Female) Serum (Male) Serum (Female) CONVENTIONAL UNITS 30.6 to 40.4% uptake 25.3 to 40.4% uptake 27.9 to 39.5% uptake 21.6 to 34.1% uptake Laboratory INTERVALS CONVENTIONAL UNITS FEBRUARY 2014 Page 7 of 11
8 Refer to References (7,8,9) for guidelines on establishing laboratory-specific reference intervals. Additional reporting information as designated by this laboratory: PROCEDURAL NOTES ANTICOAGULANT TEST RESULTS Serum is the specimen of choice. The following anticoagulant data was derived from a study of plasma: Table 3.0 Incompatible Anticoagulants a ANTICOAGULANT LEVEL TESTED FOR IN VITRO INTERFERENCE AVERAGE PLASMA-SERUM BIAS (%) b Lithium Heparin 14 Units/mL +2.3 Sodium Heparin 14 Units/mL +2.6 LIMITATIONS 1. Specimens containing particulate matter should be clarified by centrifugation. 2. Specimens quantifying outside of the assay range should be repeated for validity, but not diluted. They should be reported greater than the Hyperthyroid-assigned (Calibrator 2) or less than the Hypothyroid-assigned (Calibrator 1) values. 3. Ethylene glycol-based controls are not recommended for use with this product. INTERFERENCES 1. The following substances were tested for interference with this methodology: Table 4.0 Interferences a SUBSTANCE SOURCE LEVEL OBSERVED EFFECT Bilirubin Bovine 24 mg/dl NSI b Hemoglobin RBC hemolysate 500 mg/dl NSI Lipemia Intralipid c 300 mg/dl NSI a Data shown was collected using SYNCHRON CX Systems. Equivalency between SYNCHRON LX Systems has been established by Deming regression analysis to SYNCHRON CX Systems. b NSI = No Significant Interference (within ± 1.3% uptake or 5%). c Intralipid is a registered trademark of KabiVitrum, Inc., Clayton, NC Refer to References (10,11,12) for other interferences caused by drugs, disease and preanalytical variables. PERFORMANCE CHARACTERISTICS ANALYTIC RANGE The analytical range is defined by the % uptake values printed on the low and high calibrator bottles. Values outside this range should not be diluted but reported out as less than the low calibrator value or higher than the high calibrator value. Page 8 of 11 FEBRUARY 2014
9 REPORTABLE RANGE (AS DETERMINED ON SITE): Table 5.0 Reportable Range CONVENTIONAL UNITS S.I. UNITS SENSITIVITY Sensitivity for the thyroxine binding capacity is defined by the % uptake values printed on the low calibrator bottle. EQUIVALENCY Equivalency was assessed by Deming regression analysis of patient samples to accepted clinical methods. Serum (in the range of 18.1 to 48.3% uptake): Y (SYNCHRON Systems) = 0.993X N = 77 MEAN (SYNCHRON Systems) = 34.6 MEAN (SYNCHRON CX7 DELTA) = 34.5 CORRELATION COEFFICIENT (r) = Refer to References (13) for guidelines on performing equivalency testing. PRECISION A properly operating SYNCHRON System(s) should exhibit precision values less than or equal to the following: Table 6.0 Precision Values TYPE OF PRECISION 1 SD % uptake CHANGEOVER VALUE a % uptake Within-run Serum Total Serum % CV Comparative performance data for a SYNCHRON LX System evaluated using the NCCLS Proposed Guideline EP5-T2 appears in the table below. 14 Each laboratory should characterize their own instrument performance for comparison purposes. Table 7.0 NCCLS EP5-T2 Precision Estimate Method TYPE OF IMPRECISION Within-run No. Systems No. Data Points a Test Mean Value (% uptake) EP5-T2 Calculated Point Estimates SD %CV Serum Control Serum Control Serum Control FEBRUARY 2014 Page 9 of 11
10 Table 7.0 NCCLS EP5-T2 Precision Estimate Method, Continued a TYPE OF IMPRECISION Total No. Systems No. Data Points a Test Mean Value (% uptake) EP5-T2 Calculated Point Estimates SD %CV Serum Control Serum Control Serum Control The point estimate is based on the pooled data from one system, run for twenty days, two runs per day, two observations per run on an instrument operated and maintained according to the manufacturer s instructions. NOTICE These degrees of precision and equivalency were obtained in typical testing procedures on a SYNCHRON LX System and are not intended to represent the performance specifications for this reagent. ADDITIONAL INFORMATION For more detailed information on UniCel DxC Systems, refer to the appropriate system manual. Beckman Coulter, the Beckman Coulter Logo, Synchron, UniCel and DxC are trademarks of Beckman Coulter, Inc and are registered in the USPTO. SHIPPING DAMAGE If damaged product is received, notify your Beckman Coulter Clinical Support Center. REVISION HISTORY Revision AE Revised Quality Control section, and removed the sodium azide warning. Revision AF Updated corporate address; removed insert reference from content description. Revision AG Updated kit contents, and the Calibrator Storage and Stability section. Revision AH Added Revision History. Revision AJ Revised Traceability section. Revision AK Added new language requirement: Czech, and Korean. Revision AL Removed references to CX and LX systems as they are discontinued effective 12/2013. Added Beckman Coulter trademark statement and disclaimer. Page 10 of 11 FEBRUARY 2014
11 REFERENCES 1. Henry, J. B., Kreig, A. F., "Endocrine Measurements", Clinical Diagnosis by Laboratory Methods, W. B. Saunders Company, Philadelphia, PA (1974). 2. Henderson, D. R., Friedman, S. B., Harris, J. D., Manning, W. B., Zoccoli, M. A., "CEDIA, A New Homogeneous Immunoassay System", Clin. Chem., 32(9): (1986). 3. Tietz, N. W., "Specimen Collection and Processing; Sources of Biological Variation", Textbook of Clinical Chemistry, 5th Edition, W. B. Saunders, Philadelphia, PA (2005). 4. National Committee for Clinical Laboratory Standards, Procedures for the Handling and Processing of Blood Specimens Approved Guideline, NCCLS publication H18-A, Villanova, PA (1990). 5. CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, (Washington, D.C.: U.S. Government Printing Office, 2009). (CDC ) 6. Tietz, N. W., Clinical Guide to Laboratory Tests, 3rd Edition, W. B. Saunders Company, Philadelphia, PA (1995). 7. National Committee for Clinical Laboratory Standards, How to Define, Determine, and Utilize Reference Intervals in the Clinical Laboratory Approved Guideline, NCCLS publication C28-A, Villanova, PA (1995). 8. Tietz, N. W., ed., Fundamentals of Clinical Chemistry, 6th Edition, W. B. Saunders, Philadelphia, PA (2007). 9. Henry, J. B., Clinical Diagnosis and Management by Laboratory Methods, 22nd Edition, W. B. Saunders Company, Philadelphia, PA (2006). 10. Young, D. S., Effects of Drugs on Clinical Laboratory Tests, 5th Edition, AACC Press, Washington, D. C. (2000). 11. Friedman, R. B., Young, D. S.,Effects of Disease on Clinical Laboratory Tests, 4th Edition, AACC Press, Washington, D.C. (2001). 12. Young, D. S., Effects of Preanalytical Variables on Clinical Laboratory Tests, 3rd Edition, AACC Press, Washington, D. C. (2007). 13. National Committee for Clinical Laboratory Standards, Method Comparison and Bias Estimation Using Patient Samples Approved Guideline, NCCLS publication EP9-A, Villanova, PA (1995). 14. National Committee for Clinical Laboratory Standards, Precision Performance of Clinical Chemistry Devices Tentative Guideline, 2nd Edition, NCCLS publication EP5-T2, Villanova, PA (1992). Beckman Coulter Ireland, Inc., Mervue Business Park, Mervue, Galway, Ireland Beckman Coulter, Inc., 250 S. Kraemer Blvd., Brea, CA U.S.A. FEBRUARY 2014 Page 11 of 11
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