TSH. 01 English - Ref.: 903. Ref.:903. Insert. Intended use. System for quantitative determination of the thyroid stimulating hormone (TSH) in serum.

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1 TSH Insert Intended use. System for quantitative determination of the thyroid stimulating hormone (TSH) in serum. [Only for in vitro diagnostic use.] Test principle. The TSH present in the sample is specifically bound to anti-tsh monoclonal antibodies immobilized in the surface of immunotubes (IT). After washing, monoclonal antibody marked with biotin and streptavidin-peroxidase conjugate are added, originating formation of a quaternary complex. After removal of excess reagents the chemiluminescent reaction is initiated by adding peroxide and luminol. The amount of light issued, measured in relative light units (RLU), is proportional to the TSH concentration in the sample. S ummary. The TSH - Labtest is a semi-automatic system developed with an innovative approach that makes possible for using the chemiluminescent substrates luminol and peroxide in fast kinetics, with obtainment of results in a few seconds of reaction. The sensitivity levels are equivalent to those of other chemiluminescent substrates that use alkaline phosphatase as enzyme. The studies carried out show that the assay does not suffer significant interference caused by increased concentrations of hemoglobin, bilirubin, triglycerides and the LH, FSH and hcg hormones, in addition to having no hook effect. Increased stability of the reagents reduces frequency of the calibrations, making possible for adaptation to small routines, upon accomplishment of individual tests without increase of costs. Methodology. Chemiluminescence in tubes. Reagents 1. - Sample Diluent - Store at 2. - Biotinylated - Store at 2-8 ºC. 2-8 ºC. It contains buffer 100 mmol/l ph 7.4, preservative, surfactant and stabilizers. Ready for use Concentrated It contains anti-tsh monoclonal antibody biotinylated in buffer 100 mmol/l ph 7.4, preservative, surfactant and stabilizers. Ready for use Conjugate Conjugate 50X - Store at 2-8 ºC. It contains streptavidin-peroxidase, preservative, surfactant and stabilizers. See item Procedure - Preparing Reagents. Diluent - Store at 2-8 ºC. It contains buffer 100 mmol/l ph 7.4, preservative, surfactant and stabilizers. See item Procedure - Preparing Reagents Low Calibrator - Store at 2-8 ºC. Thyroid stimulating hormone (TSH) freeze-dried preparation containing buffer 100 mmol/l ph 8.1; preservative and stabilizers. See in the flask label the value assigned for the TSH concentration. See item Procedure - Preparing Reagents - Reconstitution of calibrators. The. calibrator concentration, which is showed in the calibrator label, must be added in test protocol. 6 - High Calibrator - Store at 2-8 ºC. Thyroid stimulating hormone (TSH) freeze-dried preparation containing buffer 100 mmol/l ph 8,1; preservative and stabilizers. See in the flask label the value assigned for the TSH concentration. See item Procedure - Preparing Reagents - Reconstitution of calibrators. The. calibrator concentration, which is showed in the calibrator label, must be added in test protocol. 7 - Immunotubes - Store at 2-8 ºC. Polystyrene tubes sensitized with anti-tsh monoclonal antibodies. Ready for use. The plastic packaging containing the tubes should be opened only when they are at the room temperature ( Temperature below 30 ºC), to avoid condensation of water steam in the tubes. Non-compliance with these guidelines shall lead to decrease of reagent stability. After opening the packaging, the tubes are stable for 6 months, when stored from 2 ºC to 8 ºC and with desiccator in the well-sealed plastic packaging. The non-open reagents, when stored in the conditions pointed out, are stable until the expiration date printed on the label. After reopened, the reagents should be handled according to the good laboratorial practices so as to avoid chemical and microbial contaminations, that may cause stability reduction. Precautions and warnings Ref.:903 The calibrator contains human blood derivatives and was tested for the presence of HBsAg and anti-hcv and anti-hiv antibodies with approved tests, yielding negative results for all of them. However, no known test method can offer complete assurance that human blood samples will not transmit infectious diseases. Therefore, the usual security cares should be applied to the reagent handling which must not be pipetted by mouth aspiration. All blood derivatives should be considered potentially infectious. Do not mix reagents from different batches and do not use expired reagents. Do not allow for cross contamination between the components. Use only disposable tips to pipette each sample and each reagent. The suitable cleaning and drying of the material used are fundamental factors for stability of the reagents and obtainment of correct results. 01 English - Ref.: 903

2 Soap, detergent or oxidizing agents residues in the containers used for preparation of the use Conjugated may interfere the reaction. Preferably use disposable plastic material. The plastic packaging containing the immunotubes (IT) should be opened only when they are at room temperature ( Temperature below 30 º C), so as to avoid water steam condensation in the tubes. Non-compliance with these guidelines shall lead to decrease of the reagent stability. After opening the packaging, the immunotubes are stable for 6 months, when stored with the desiccator in the well-sealed plastic packaging. The immunotubes (IT) shall be disposed of after use. Do not reuse for other purposes. In order to dispose of the reagents and the biological material, we suggest application of the local, state or federal environmental protection standards. Material required not provided 1. Disposable micropipettes and tips. 2. Water bath (37 1 º C). 3. Containers to prepare dilutions. 4. Chronometer. 5. Washing Solution Concentrated (Ref. 908). 6. Developing Solutions (Ref. 909). 7. Shelf for tubes 12 x 75 mm with fixation system. 8. Computer. 9. Lumiquest luminometer or similar device equipped with two injectors and capacity to store 12 x 75 mm tubes. 10. Sanitizing solution (Ref. 604). Sample 4 Use only serum. The analyte is stable 5 days from 2 to 8 ºC and for up to 1 month at temperature below negative 20 ºC when stored in a container suitable for freezing. Ensure that the samples are defrosted and homogenized before their use. Particles under suspension should be eliminated by centrifugation. Do not use samples with contamination signals or samples frozen or defrosted more than once. A Standard Operating Procedure (SOP) should be drawn-up for collection, preparation and storage of the sample. We emphasize that the bias due to the sample may be much larger than the bias occurred in the analytical procedure. Since no known test may ensure that blood samples do not transmit infections, all of them should be considered as potentially infectious. Therefore, when handling them, the biosecurity standards should be followed. Interferences Concentrations of hemoglobin up to 750 mg/dl, bilirubin up to 20 mg/dl, triglycerides up to 1000 mg/dl, hcg up to mui/ml, LH up to mui/ml, and FSH up to mui/ml do not produce significant interferences. System limitations 1. The TSH Labtest system has anti-mice antibody inhibitors (HAMA). However, samples containing increased amounts of HAMA may produce falsely reduced results. 2. As in general immunoassays, interferences not described from medications or other endogenous substances may affect the results. Procedure Special care. The packaging containing the immunotubes should be opened only when they are at room temperature (Temperature below 30 ºC), so as to avoid condensation of water steam in the tubes. Noncompliance with these guidelines shall lead to decreased reagent stability. The liquid reagents should be slightly homogenized previously to use. Preparing the Reagents Use Conjugate. In order to prepare the Use Conjugate, pipette the entire contents of the Concentrated Conjugate, transfer to the flask containing the Conjugate Diluent and homogenize. If necessary, centrifuge the material to ensure that the entire contents of the Concentrated Conjugate flask is at the bottom of the tube. After transferring the entire volume, rinse the Concentrated Conjugate flask with part of the mix of the Concentrated Conjugate and Conjugate Diluent. Slightly homogenize by inversion. Once prepared, the Use Conjugate is stable for 4 months when stored from 2 to 8 ºC. Identify the flask and note down the expiration date. As required, lower volumes of the Use Conjugate may be prepared by transferring to a clean and dry flask one (1) volume of the Concentrated Conjugate and 50 volumes of the Conjugate Diluent. Identify the flask and note down the expiration date. In order to preserve its performance, the Use Conjugate should remain out of the fridge only for the time required for use. Reconstitution of the calibrators See item 1 - Notes. Remove the aluminum seal and carefully take out the rubber cap. By using a volumetric pipette, add to the calibrator flask the deionized water volume pointed out in the label. Replace the rubber cap, leave resting during 10 minutes and slightly homogenize by inversion. Before using, slightly homogenize and remove the amount required for use. Immediately seal and store from 2 to 8 º C. After reconstitution, the calibrators remains stable for 21 days if stored from 2 to 8 ºC, well-sealed and protected from light. In order to preserve performance, keep the calibrator out of the storage temperature only for the minimum time required to obtain the volume to be used. 02 English - Ref.: 903

3 For storage for a period greater than 21 days, we suggest, after reconstitution, separation of the calibrator in portions and storage at temperature below 15 º C negative for up to 90 days in a hermeticallysealed flask, protected from light. In order to avoid evaporation of the material during the storage period, it is essential to use appropriate flasks for freezing (cryotubes). The portions of the calibrator should be defrosted only once. Calibrators should be handled in accordance with the good laboratorial practices to avoid chemical and microbial contaminations that may cause stability reduction. Calibration. The system calibration is performed through the usage of a Master Curve of 6 points (concentration of calibrators and respective RLUs) and adjusted it when necessary using both of the calibrators (Low Calibrator - Ref e High Calibrator - Ref ). The Master Curve is provided in a document attached jointly with the TSH product and is specific for each reagent batch. When inserting the Master Curves data during the creation of the protocol, it must be also insert the adjustment calibrators concentrations indicated on the calibrator's labels. (Low Calibrator - Ref e High Calibrator - Ref ). When a batch of the product is used for the first time, the data of Master Curve must be inserted in the luminometer program and then must be performed an adjustment of the calibration curve, assaying in double both calibrators provided within the kit (Low Calibrator - Ref e High Calibrator - Ref ). In order to define the device setting and correctly insert the Master Curve data, check the equipment manual. When the reagents are kept in the conditions indicated, the calibration remains stable for 90 days. The system calibration shall be adjusted in the following conditions: 1. When a product batch is used for the first time; 2. Each new preparation of the Use Conjugate, as the case may be; 3. When the quality control determines so. Test Procedure Important 1. The reading parameters of the tests as well as the Master curve data should be inserted into the luminometer previously to accomplishment of the tests. See item Calibration and check the equipment manual for details. 2. Do not test more than 20 immunotubes per round. 1. Separate the number of immunotubes (IT) required for the tests, identify them and position them in the shelf for immunotubes. 2. Add 0.15 ml of Sample Diluent to the bottom of each tube. 3. Add 0.10 ml of the sample (serum, controls or calibrators) directly to the contents of each immunotube. Homogenize. 4. Incubate 30 minutes in water bath at 37 ºC. 5. Remove the immunotubes from water bath. 6. Add 1.0 ml of Washing Solution Concentrated (Ref. 908) ready for use. 7. Dispose of the contents of the tubes by inversion and with the shelf still inverted, remove excess liquid, leaning the top edge of the tubes against an absorbing surface (2-3 layers of towel paper). 8. Add 0.15 ml of Biotinylated to the bottom of each tube. 9. Add 0.10 ml of Use Conjugate directly to the contents of each tube. Homogenize. 10. Incubate 30 minutes in water bath at 37 ºC. 11. Remove the tubes from water bath. 12. Repeat for six consecutive times stages 6 and In order to obtain the results, proceed as follows: 1. Effect the equipment prime by using the Developing Solutions (check the insert of the product Developing Solution (Ref. 909) and the equipment operating manual); 2. Select the test protocol having the Master Curve data; 3. Measure the adjustment calibrators (as required), samples and controls. Operating range. The operational range for measuring is UI/mL up to 25 UI/mL. The upper limit of the range is defined by concentration of the last calibrator of the calibration curve. The concentration of the last calibrator may undergo slightly variations from batch to batch. Specimen that present results below the limit of analytical sensitivity must be reported as <0. 05 UI/mL. Specimen with concentrations above the value measured in the last calibrator may be diluted with the buffered solution 0. 85%. After the dilution, multiply the value found by the dilution factor used. Internal quality control. The laboratory should keep an internal quality control program clearly defining the applicable regulations, goals, procedures, standards, criteria for quality specifications and tolerance limits, corrective measures and activity record. Use of the controls in all sets of test is essential to monitor inaccuracy of the measurement and calibration deviations. It is suggested that the specifications for the variation coefficient and total bias are based on the components of 1,2,3 biological variation (VB). It is suggested to use the preparations of the Labtest Qualitrol Q Line (Ref. 907) for internal quality control in chemiluminescence assays. 03 English - Ref.: 903

4 Reference interval 4 The interval should be used only as guideline. It is recommended that each laboratory establishes its own reference interval in the population served. Age newborn up to 4 day TSH ( Ul/mL) to 20 weeks weeks up to 20 years to 54 years to 87 years Pregnancy 1st Quarter Performance features 5 Effects of the matrix dilution. Two samples with values equal to 13.9 and 20.7 UI/mL were used to assess the system response in matrix dilutions with NaCl 150 mmol/l (0.85%). By using dilution factors that ranged from 2 to 8 recoveries between 98.6 and 99.4% were found. Method comparison. The method proposed was compared to a product available in the market, which uses a similar methodology, and the following results were obtained: By using the regression equation, the total estimated systematic bias was equal to 2.74% for a decision level equal to 4.49 UI/mL and 0.58% for a decision level equal to UI/mL. Such bias are lower than the analytical systematic bias of the specification based on biological variation which is 7.8 2%. Accuracy studies. The accuracy studies were carried out by using native samples, with concentrations equal to UI/mL. Imprecision - Within Run Sample 1 Sample 2 2nd Quarter 3rd Quarter Number of samples Concentration range ( UI/mL) Estimation mean ( UI/mL) Regression equation Correlation coefficient N Comparative Method Mean ( UI/mL) SD Labtest Method Labtest Method = x Comparative Method UI/mL and (%) CV Imprecision - Run-to-run Sample 1 Sample 2 N The imprecision found meets the specifications for total imprecision based on the VB components which is 9. 65%. Total estimated bias (random bias + systematic bias) is equal to 12.3% for the decision level 4.49 UI/L and 12.4% for the decision level UI/L. Results point out that the method meets the specification for total bias ( %) based on the VB components. Methodological or analytical sensitivity. Detection limit: 0.05 UI/mL The analytical sensitivity corresponds to the lower detection limit. It represents the lower concentration of TSH possible to be distinguished from zero. It is calculated as the concentration of TSH located two standard deviations above the zero calibrator. 4 Clinical meaning. Mean ( UI/mL) TSH is a glycoprotein with molecular weight equal to 30 KDa and comprised of two polypeptide sub-units, both essential for its metabolic action. The sub-unit β is specific for the TSH and the sub-unit is shared with other hormones such as LH, FSH and hcg. By acting through the cyclical AMP mechanism, the TSH acts by stimulating production of the T3 (triiodothyronine) and T4 (tyrosine) hormones by the thyroid gland. Biochemical integration of the anterior hypophysis, TSH producer and thyroid, target gland, is made through the Thyrotropin Releasing Hormone (TRH), which acts directly to the hypophysis stimulating production of the TSH. Thus, increased levels of T4 inhibit hypothalamic production of TRH, thus reducing stimulation for production of the hypophysis TSH. On the other hand, reduction of the T4 plasmatic levels has as a consequence an increase in TSH production through reduction of the hypothalamus control. Understanding of the basic mechanisms above directly leads to the clinical interpretations of the laboratorial findings found in the several pathologies. Thus, in primary hypothyroidism, resulting from attack of the thyroid gland, the T3 and T4 levels are reduced, which leads to an increase of the TSH. Even in discrete hypothyroidism or its initial stage, when the thyroidal hormones may still be in the reference interval, the TSH levels are already high. In secondary hypothyroidism, the metabolic defect is in the central level and occurs due to a disturbance in the hypothalamic-hypophyseal axis, resulting from injuries in the hypothalamus, in hypophysis or both. In such case, the T3 and T4 levels are reduced, but the TSH levels are reduced or in the reference interval, i.e., the TSH levels are below the expectations for the T4 levels. SD (%) CV English - Ref.: 903

5 In primary hyperthyroidism, as that which occurs in the Graves Disease, thyroidal nodules or thyroid adenoma, the TSH levels are almost always reduced or even undetectable, except for some rare metabolic situations. In most cases the T4 (mainly specific T4) will be high. Notes 1. The clinical laboratory is aimed at providing accurate and precise results. Use of inappropriate quality water is a potencial cause of analytical bias. The water used in the laboratory should have the appropriate quality for each application. Thus, to prepare reagents, use in the measures and for use in the final rinsing of the flasks, the water should have resistivity 1 megaohm.cm or conductivity 1 microsiemens/cm and silicate concentration <0.1 mg/l. When the deionizing column is with its capacity saturated, occurs release of several ions, silicates and substances with large oxidation or reduction power that deteriorate the reagents in a few days or even hours, thus changing the results unpredictably. Consequently, it is essential to establish a quality control program for the water. 2. It is suggested to consult Young DS. Effects of Drugs on Clinical Laboratory Tests, 3rd edition, Washington: AACC Press, in order to review physiopathological source and drugs interference in results and methodology. Reference 1. Sociedad Española de Bioquímica Clínica y Patología Molecular. Available in: < 7C_Bases_de_datos_y_documentos_del_Comite_de_Garantia_de_l a_cali...> ( accessed on 04/2014). 2. Basques JC. Especificações da Qualidade Analítica. Labtest Diagnóstica Burtis CA, Ashwood ER, Bruns DE: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4.ed. St. Louis: Sauders/Elsevier, p Labtest: Data on file. Presentation Product Reference Contents X 7.5 ml 1 X 7.5 ml 1 X 0.1 ml TSH X 5 ml 1 X 1 ml 1 X 1 ml 2 X 25 un Consumer information [Warranty conditions] Labtest Diagnóstica warrants the performance of this product under the specifications until the expiration date shown in the label provided that the procedures and storage conditions indicated on the label and in this insert have been followed correctly. Labtest Diagnóstica S.A. CNPJ: / Av. Paulo Ferreira da Costa, Vista Alegre - CEP Lagoa Santa. Minas Gerais Brasil - Consumer Service 1 sac@labtest.com.br 3. Westgard JO, Barry PL, Hunt MR, Groth T. Clin Chem 1981;27: Edition: May, 2012 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 05 English - Ref.: 903

6 06 English - Ref.: 903