Contents of the Kit. Instruction Manual B R A H M S TRAK human LIA. B R A H M S Service

Transcription

1 B R A H M S GmbH Neuendorfstr. 25 D Hennigsdorf Luminescence receptor assay (LRA) for the quantitative determination of TSH receptor autoantibodies (TRAb) in human serum (Coated Tube System) Article number: (50 determinations), (100 determinations) respectively B R A H M S is a registered trademark of B R A H M S GmbH. Other product names in this document are used for identification purposes; they may be trademarks and/or registered trademarks of their respective companies. Contents of the Kit Reagent Quantity for 50 (100) det. Contents A 1 (2) x 11 ml vial(s) Tracer, luminescence-labelled (acridine derivative) b-tsh (bovine), lyophilized, blue coloured, before use to be reconstituted with 11 ml buffer B B 1 (2) x 11 ml vial(s) Buffer for the reconstitution of tracer A, ready for use C 1 (2) x 50 tubes Coated tubes, coated with TSH-receptor (human, recombinant), ready for use D 1 (2) x 11 ml vial(s) Buffer for the incubation of samples, ready for use W 2 x 11 ml vials B R A H M S Washing solution universal, concentrate, 11 ml S0 S5 6 x 0.5 ml vials TRAK standards (human serum), ready for use, concentrations: 0; 1; 2; 4; 16; 40 IU/L K1, K2 2 x 0.5 ml vials TRAK controls 1 and 2 (human serum), ready for use, concentrations see leaflet enclosed Instruction Manual B R A H M S TRAK human LIA Date: (This version supersedes all earlier instruction manuals.) Protected by following patents: US , US ; EP975970; JP Content changes versus previous version Insert the address of ALPCO B R A H M S Service Address B R A H M S GmbH Neuendorfstr. 25 D Hennigsdorf Switchboard Tel.: +49 (0)3302/883 0 Fax: +49 (0)3302/ info@brahms.de USA Thermo Fisher Scientific B R A H M S LLC 8365 Valley Pike, Middletown, Virginia Phone: Fax: techservice.mgc@thermofisher.com Internet Xi: R36/38 S22-24 Offered in the US by ALPCO 26G Keewaydin Drive, Salem, NH Ph: (800) REF REF see label for expiry date 2 8 C B R A H M S TRAK human LIA Instruction manual (Version R07us) Page 1 of 5

2 Introduction Intended use(s): B R A H M S TRAK human LIA is a luminescence receptor assay (LRA) for the quantitative determination of antibodies to the human thyrotropin (TSH) receptor. The measurement of TSH receptor autoantibodies is used in the assessment of patients with suspect Graves disease (autoimmune hyperthyroidism). Clinical use TSH receptor antibodies (TRAb) are the cause of hyperthyroidism in Graves disease (= autoimmune hyperthyroidism). The main indications for TRAb determination are therefore the detection or exclusion of Graves disease and differentiation from disseminated autonomy of the thyroid gland. By contrast to the systems most commonly used to date, which are based on TSH receptors isolated from the pig thyroid gland (= porcine systems) and bovine TSH as the standard material, a significant improvement in clinical sensitivity, with identical specificity, is obtained with the use of the human TSH receptor and human standard material in the B R A H M S TRAK human LIA. More recent data suggest that TRAb determination during the course of Graves disease allows a prognostic statement, thereby Important Notes This kit contains materials of human origin (e.g. human serum). These materials have been screened for HBsAg, HIV I/II antibodies, and HCV antibodies; all tests were negative. However, the reagents and patient samples should be handled with care, as all materials of human origin are potentially hazardous. The following kit reagents contain the preservative sodium azide at concentrations of < 0.1 percent by weight: buffer B, standards and controls. These reagents should not be swallowed or allowed to come in contact with the skin or mucous membranes. The tracer contains Tris (Xi irritant: R36/38 S22-24). Irritating to eyes. Irritating to skin. Do not breathe dust. Avoid contact with skin. Our Customer Service Department, on +49 (0)3302/ , will gladly send the reagent-specific EU Safety Data Sheets in accordance to regulation 1907/2006-EC upon request. Standardisation and Reference Ranges One international unit (IU) of B R A H M S TRAK human LIA is equivalent to one IU of TSAb WHO standard 90/672. In the clinical trial in 86 patients with untreated Graves disease and 282 healthy individuals, the research version of B R A H M S TRAK human LIA, with an evaluation limit of 1 IU/L, showed a diagnostic sensitivity of 98.8 % with a specificity of 99.6 % (ref. 1). TRAK values < 1 IU/L are regarded as negative, values > 2.0 IU/L are considered positive. Values between 1 and 2.0 IU/L are regarded as indeterminant (grey-zone). It is recommended that each laboratory establish its own reference ranges based on representative patient populations in order to test the validity of the data provided with commercial test kits. This not only makes it possible to identify regional features and variations in the prevalence of certain thyroid diseases and dysfunctions, but also serves as a test of the methodological quality of the individual laboratories and test kits. Therefore, the data given should be treated as orientational guidelines. Quality control: National quality assurance guidelines for quantitative tests in the medical laboratory (current version) must be complied with. For instance, test accuracy and precision can be monitored by means of laboratory in-house and/or Stability and Storage Conditions Store all reagents and coated tubes at 2 to 8 C in their original shipping containers until directly prior to use. Observe the expiry dates specified on the main container and the vial labels. Reconstituted tracer (Tracer A reconstituted in Buffer B) may be used for up to 2 weeks if stored at 2 8 C. constituting an important decision-making aid in the management of the treatment. Furthermore, in pregnant women with Graves disease, TRAb determination provides a method of assessing the risk of the onset of hyperthyroidism in the foetus. Assay principle The B R A H M S TRAK human LIA is a test kit for the quantitative determination of antibodies against the human thyrotropin (TSH) receptor. Detection is based on the ability of TRAb to prevent the binding of labelled TSH to the TSH receptor. In the first step of the assay, the patient sample with an unknown TRAb concentration and the standard samples with a known TRAb concentration are incubated in test tubes coated with the human TSH receptor. After an initial wash, labelled bovine TSH, which binds to the still free TSH receptors on the test tube wall is added. In a second wash, unbound labelled TSH is removed and the remaining bound TSH is measured in the measuring instrument. The level of the measuring signal is therefore inversely proportional to the quantity of TRAb in the tested sample. The TRAb concentration in the patients sample can be calculated from a standard curve of samples with a known TRAb concentration. In the event that glass vials are included in the reagent kit, we explicitly point out that there will be a breakage hazard, and consequently a risk of injury. The reagents as well as waste originated by the test must be disposed of in accordance with the specifications of local authorities. The results, obtained from this assay should always be assessed in combination with the clinical examination, patients medical history, and other findings before momentous actions will be prefaced. commercially available control materials. If unacceptable control values are obtained, proceed as outlined in standard laboratory diagnostic procedures to determine the cause and implement corrective measures. Bibliography 1. Second generation assay for TSH-receptor antibodies has superior diagnostic sensitivity for Graves disease. S. Costagliola, N. G. Morgenthaler, R. Hoermann, K. Badenhoop, J. Struck, D. Freitag, S. Poertl, W. Weglöhner, J. M. Hollidt, B. Quadbeck, J. E. Dumont, P.-M. Schumm-Draeger, A. Bergmann, K. Mann, G. Vassart and K.-H. Usadel. J Clin Endocrinol Metab 84 (1); (1999) 2. The prognostic value of thyrotropin receptor antibody measurement in the early stages of the treatment of Graves disease with antithyroid drugs. V. Michelangeli, C. Poon, J. Taft, H. Newnham, D. Topliss and P. Colman. Thyroid 8 (2); (1998) 3. Guidelines for TSH-receptor antibody measurements in pregnancy: results of an evidence-based symposium organized by the European Thyroid Association. P. Laurberg, B. Nygaard, D. Glinoer, M. Grussendorf and J. Orgiazzi. Eur J Endocrinol 139 (6); (1998) 4. The thyroid stimulating hormone receptor in human disease. L. C. Harrison and P. J. Leedman. Clin Biochem 23 (1); (1990) Diluted washing solution may be used for up to 4 weeks if stored at 2 8 C. Contaminated washing solution must not be used. This is the case either if the liquid is clouded or the ph value is < 6. B R A H M S TRAK human LIA Instruction manual (Version R07us) Page 2 of 5

3 Test Procedure Incubation Scheme 1. Number Test tubes (a, b) 0 5 I/II P 1 P n 2. Pipette Buffer D µl Standards µl 100 Controls µl 100 Pat. sera µl Incubate at least 3 h at RT (17 27 C) while shaking at 300 rpm: cf. note in Test Procedure. 4. Pipette Wash. solution ml Decant Repeat washing step 4 one time and turn the tubes upside down for max. 5 min on blotting paper. 5. Pipette Tracer µl Incubate 1 h +/- 5 min at RT (17 27 C) while shaking at 300 rpm. 7. Pipette Wash. solution ml Decant Repeat washing step 7 three times and turn the tubes upside down for 5 15 min on blotting paper. 8. Measure Place all coated tubes in a luminometer. Measurement by automatic injection of Basiskit reagents 1 and 2. Measurement time: 1 sec. Specimen Handling It is recommended to use serum. If plasma is used, separate reference values should be created. If serum samples are not analyzed immediately after preparation, they can be stored for 3 days at 2 8 C and for longer periods of time at 20 C. Repeated freezing and thawing should be avoided. Notes on Test Execution Do not use any reagents that have exceeded the expiration date printed on the label. The individual components of the kit are perfectly attuned to each other. If components from different batches are exchanged or mixed, B R A H M S GmbH can assume no liability for the accuracy of results. In large test series, reagents of the same batch designation are pooled. The indicated sequence of steps must be followed. Patient samples with a concentration above the measuring range are to be rated as > highest standard. The result must not be extrapolated. The patient sample in question should be diluted and retested. Further information can be obtained from B R A H M S GmbH customer service. The quantity of preparation must be scaled in such a way that it can all be pipetted within minutes, in order to avoid shift effects. Test Procedure 1. Preparations Allow all kit components and patient samples to warm up to room temperature. Reconstitute tracer (buffer B in vial A). Agitate all liquid reagents including patient sera gently before use (avoid foam formation). Number the coated tubes (preferentially using a, b for duplicates). Prepare washing solution: dilute 11 ml concentrate with distilled water to yield 550 ml. We strongly recommend to contact the manufacturer or distributor before using other washing solutions. Prepare the luminometer for use. 2. Into each tube pipette 200 µl buffer D. Into the tubes 0 a, b... 5 a, b pipette 100 µl TRAK standards with increasing concentrations. Into the tubes I a, b and II a, b pipette 100 µl TRAK controls. Into the tubes P 1... P n a, b pipette 100 µl patient sera. A fresh plastic micropipette tip should be used for each sample to avoid carry-over from one sample to the next. The whole batch must be mixed thoroughly. 3. Incubate the tubes for 3 hours at room temperature (17 27 C) while shaking (300 rpm). 4. Wash the coated tubes with 2 ml of diluted washing solution, which is decanted afterwards. Note The washing solution should always be added in such a way that even the top of the inner surface of the tubes is washed completely. Only by carrying out the washing procedure in this manner the complete removal of the non-specific bound tracer from all parts of the tube, including the upper areas of the inner surface, can be guaranteed. Note This can be carried out by using a wash comb, washing 5 tubes simultaneously (available from B R A H M S GmbH). Repeat washing step 4 one time and turn the tubes upside down for max. 5 min on blotting paper. 5. Into each tube pipette 200 µl tracer. The whole batch must be mixed thoroughly. 6. Incubate the tubes for 1 hour +/- 5 minutes at room temperature (17 27 C) while shaking (300 rpm). The test tubes should be protected from light during incubation. 7. Wash the coated tubes with 1 ml washing solution, which is decanted afterwards. For washing, please heed the notes under 4. Appropriate care should be taken to avoid any carryover of tracer from tube to tube during the washing procedure. Repeat washing step 7 three times and turn the tubes upside down for 5 15 minutes on blotting paper. Afterwards, remove any residual liquid by tapping the tubes on the blotting paper. B R A H M S TRAK human LIA Instruction manual (Version R07us) Page 3 of 5

4 8. Place all test tubes in the luminometer in the correct order as defined by the measuring protocol. Start luminescence measurement by automatic injection of 300 µl B R A H M S Basiskit LIA reagents 1 and 2. Recommended measurement time: 1 second per tube. Follow the manufacturer s instructions carefully. Improper handling of the reagents may falsify the test results. B R A H M S GmbH accepts no liability for faulty test results arising from improper storage, use or handling. Additionally available Zero serum for dilution of patient samples (10 ml) (This zero serum can also be used in B R A H M S TRAK human RIA, B R A H M S anti-tpo n RIA, B R A H M S anti-tpo n LIA, B R A H M S anti-tg n RIA, and B R A H M S anti-tg n LIA.) Additionally required B R A H M S Basiskit LIA (available from B R A H M S GmbH) mm tubes for priming the luminometer and for measurement of the reagent blank (RLW/NSB) Micropipettes (100, 200 µl) Sample mixer Horizontal rotator Dispenser (5 ml) for the B R A H M S Washing solution universal, concentrate, 11 ml Distilled water Luminometer with two injectors Washing equipment for the washing/decanting procedure Assay Characteristics Intra-assay Precision The patient values mentioned below have been determined in an assay-run on 10-fold determination. Patient Mean [IU/L] CV (%) Inter-assay Precision The patient values specified below were determined in 10 assayruns in duplicate. Time of 1 st incubation: 3 hours. Patient Mean [IU/L] CV (%) Sensitivity The analytical sensitivity read at the optimal curve is 0,4 IU/L. It was based on 10-fold determination of the zero standard as follows: x 2 s deviation in. The functional assay sensitivity (20 % inter-assay variation coefficient) is IU/L (values below IU/L should be declared as < IU/L) Method comparison with predicate device: A correlation study was performed between the predicate KRONUS TRAb assay and the B R A H M S TRAK human LIA assay. 52 serum samples abtained from either confirmed Graves disease patients or patients with non-graves thyroid disease were tested in parallel. Overall agreement between the two assays was 75%. Cross Reactivity Substance Maximum permissible concentration without danger of interference human TSH 1,000 mu/l human LH 9,000 U/L human FSH 15,000 U/L Anti-TPO antibodies (1) 3,000 U/mL Anti-Tg antibodies (2) 2,000 U/mL (1) Measured with LUMItest anti-tpon (2) Measured with LUMItest anti-tgn Interferants : Hemoglobin: Two control samples were diluted in three steps with plasma plus red blood cells and plasma neat as reference. Recovery ranged from %. Bilirubin: Concentrations of bilirubin (0.625, 1.25, 2.5, 5, 10 and 20 mg/dl) were added to two different control samples. Recovery ranged from %. Lipids: A TRAK positive control sample was diluted serially with lipemic serum (634 mg/dl triglycerides). Serial dilutions of the sample with a non-lipemic serum served as referee. Recovery ranged from %. Dilution Patient Dilution Observed Value [IU/L] 1 undiluted 1:2 1:4 1:8 1:16 2 undiluted 1:2 1:4 1: Mean: Mean: 6.6 Expected Value [IU/L] Recovery (%) TRAK-positive samples can be diluted with TRAK zero standard or TRAK-negative patient serum. Because of the heterogeneity of autoantibodies, for some patient samples a nonlinear dilution is possible. B R A H M S TRAK human LIA Instruction manual (Version R07us) Page 4 of 5

5 Calculation of Results For computer-aided evaluation, an evaluation programme (spline/unsmoothed) must be selected that suits the specific combination of processor and measuring equipment used. When calculating the results without the assistance of a computer, the mean count rate of each sample (B) should be related to the mean count rate of the zero standard (B 0 ). The results should be expressed as B/B 0 (%). In case of the zero standard B/B 0 = 100 %. Using lin-log graph paper the mean percent value of each standard (ordinate, linear) is plotted against the corresponding TRAb concentration (abscissa, logarithmic), in order to obtain a standard curve. The mean percentage values B/B 0 of the serum samples are then used to determine the corresponding TRAb concentrations in International Units per Liter (IU/L). For technical support, please contact the customer service of B R A H M S GmbH or the appropriate distribution partner / sales representative. Calculation example Standard curve B/B (%) Test tubes (a) (b) (mean) B/B 0 (%) Zero standard Standard 1 (1 IU/L) Standard 2 (2 IU/L) Standard 3 (4 IU/L) Standard 4 (16 IU/L) Standard 5 (40 IU/L) Serum sample P The luminescence values specified here can differ depending on the individual type luminometer used Concentration IU/l Symbols Symbol Usage Symbol Usage Symbol Usage Manufacturer CE Conformity Marking According to Directive 98/79/EC on In Vitro Diagnostic Medical Devices 50 Contains sufficient for (Number of) tests, e.g. 50 Use by Temperature Limitation REF Article Number/ Catalogue Number Green Dot according to German Law Buffer Tracer / Enzyme conjugate Lyophilized, freeze dried Consult Instruction for use Batch code Reconstitute with 11 ml Irritant B R A H M S TRAK human LIA Instruction manual (Version R07us) Page 5 of 5