hcg 01 English - Ref.: 904 Ref.:904 Insert Intended use. System for quantitative determination of Human Chorionic Gonadotropin - hcg in serum.

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1 hcg Insert Ref.:904 Intended use. System for quantitative determination of Human Chorionic Gonadotropin - hcg in serum. [Only for in vitro diagnostic use.] Introduction 1. The hcg molecule is comprised of two sub-units: beta sub-unit with 145 amino acids and alpha sub-unit, with 92 amino acids. The substances are bound in a non-covalent form. Alpha sub-unit of the hcg has structural role in the molecule and is similar to the alpha sub-unit of the luteinizing hormones (LH), follicle-stimulating hormones (FSH) and thyroid-stimulating hormones (TSH). Opposite to the alpha, the beta subunit is functional and exclusive of the hcg molecule, distinguishing from the remaining hormones. Presence of the hcg in the urine or serum may be detected 10 days after ovulation, in other words, before setting of the amenorrhea or, by measuring the pregnancy time, in the 4th week. Its fast concentration increase renders it an excellent indicator for detection and confirmation of pregnancy. Principle. The hcg present in the sample is specifically bound to immobilized anti-hcg monoclonal antibodies in the immunotube surface. After washing, polyclonal antibodies marked with biotin and streptavidin-peroxidase conjugate are added, thus originating formation of a quaternary complex. After removal of excess reagents, the chemiluminescent reaction is unleashed by addition of peroxide and luminol. The amount of light issued, measured in relative light units (RLU), is associated with the hcg concentration present in the sample, which value is obtained through the calibration curve. System features. The hcg - 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 the chemiluminescent signal in a few seconds. 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 hemoglobin,, bilirubin, triglyceride and LH, FSH and TSH hormone concentrations, and it does not have the hook effect (decreased response by excess antigen). Stability of the reagents reduces frequency of the calibrations, making possible for adaptation to small routines, upon accomplishment of individual assays without cost increase. Methodology. Chemiluminescence in tubes. Reagents 1. - Sample Diluent. Store at 2-8 º C. Ready for use. It contains buffer 100 mmol/l ph 7,4; bromonitro dioxane 0.02%, surfactant and stabilizers. 2.-Biotinylated. Store at 2-8 º C. Ready for use. It contains biotinylated hcg anti-alpha polyclonal antibody 0.5 g/ml in buffer 100 mmol/l ph 7.4; bromonitro dioxane 0.02%; surfactant and stabilizers. 3.-Concentrated Conjugate 50X. Store at 2-8 º C. It contains streptavidin-peroxidase conjugate 50 g/ml, bromonitro dioxane 0.02%, surfactant and stabilizers. See item Procedure - Preparing Reagents. 4.-Conjugate Diluent. Store at 2-8 º C.. It contains buffer 100 mmol/l ph 7.4; bromonitro dioxane 0.02%, surfactant and stabilizers. 5 - Low Calibrator. Store at 2-8 º C. Human chorionic gonadotropin (hcg) freeze-dried preparation containing buffer 100 mmol/l ph 8.1; bromonitro dioxane 0.02% and stabilizers. See in the flask label the value assigned for the hcg concentration. See item Procedure - Preparing Reagents. After reconstitution, the calibrators are stable for 30 days when stored in the conditions pointed out. See item Reconstitution of calibrators.. High 6 - Calibrator. Store at 2-8 º C. Human chorionic gonadotropin (hcg) freeze-dried preparation containing buffer 100 mmol/l ph 8,1; bromonitro dioxane 0.02% and stabilizers. See in the flask label the value assigned for the hcg concentration. See item Procedure - Preparing Reagents. After reconstitution, the calibrators are stable for 30 days when stored in the conditions pointed out. See item 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 º C. Ready for use. Polystyrene tubes sensitized with hcg anti-beta monoclonal antibodies. The plastic packaging containing the tubes should be opened only when they are at the room temperature (15-30 º C), 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 with desiccator in the well-sealed plastic packaging. The calibrator concentration, which is showed in the calibrator label, must be added in test protocol. 01 English - Ref.: 904

2 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 Customary safety care should be applied when handling the product, which shall not be pipetted with the mouth. Do not mix reagents from different batches and do not use reagents out of the validity term. Do not allow for cross contamination between the components. Use only disposable tips to pipette each sample and each reagent. Appropriate cleaning and drying of the material used are core factors for stability of the reagents and obtainment of correct results. Soap, detergent or oxidizing agent wastes in the flasks used for preparation of the use Conjugate may interfere the reaction. Preferably use disposable plastic material. If glass flasks are used, it is advisable that they are washed with sulfur or hydrochloric acid 0.5 mol/l, exhaustively rinsed with distilled or deionized water and dried previously to use. The plastic packaging containing the immunotubes should be opened only when they are at room temperature (15-30 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 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. Flasks to prepare dilutions. 4. Chronometer. 5. Washing Solution Concentrated (Ref. 908). 6. Developing Solution (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 3 days between 2 - º 8 C and for up to 8 months at temperature lower than negative 20 º C when stored in an appropriate freezing flask. Ensure that the samples are defrost and homogenized before their use. Particles under suspension should be eliminated by centrifugation. Do not use samples with contamination signals or samples frozen or defrost 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 mg/dl, bilirubin up to 30 mg/dl, LH up to m IU/mL, FSH up to m IU/mL and TSH up to IU/mL do not produce significant interferences. System limitations 1. The system was developed for determination of the intact hcg molecule, not being able to detect specific beta sub-units. Therefore, it should not be used as a test for tumor markers. 2. The Labtest hcg system has anti-mice antibody inhibitors (HAMA). However, samples containing increased amounts of HAMA may produce falsely reduced results. 3. As in the general immunoassays, non-described interferences of 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 (15-30 º C), so as to avoid condensation of water steam in the tubes. Non-compliance with these guidelines shall lead to decreased reagent stability. The liquid reagents should be slightly homogenized previously to use. Preparing the reagents Use conjugate. Transfer the total volume of the Concentrated Conjugate to the flask containing the Conjugate Diluent. Slightly homogenize by inversion. Identify the flask and note down the expiration date. Optionally, 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. Once prepared, the Use Conjugate is stable for 4 months when stored between 2-8 º C. Identify the flask and note down the expiration date. 02 English - Ref.: 904

3 In order to preserve its performance, the reagent 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 reagent quality water volume pointed out in the label. Replace the rubber cap, leave resting during 15 minutes and slightly homogenize by inversion. Before using, slightly homogenize and remove the amount required for use. Immediately seal and store between 2-8 º C. After reconstitution, the calibrators remains stable for 30 days if stored under 2-8 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. For storage for a period greater than 30 days, we suggest, after reconstitution, separation of the calibrator in portions and storage at temperature below 20 º C negative for up to 6 months 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 calibrators are traceable to the Reference Material NIBSC code 75/589 (WHO International Standard - 4th IS Chorionic Gonadotropin, Human). The system calibration is performed through the usage of a Master Curve of 6 points (concentration of calibrators and respective RLUs) and adjusted 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 hcg 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 30 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; 3. Each batch change of the Developing Solution (Ref. 909). 4. 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 use more than 20 immunotubes in each run. 1. Separate the number of immunotubes required for the tests, identify them and position them in the shelf for immunotubes. 2. Add 0.20 ml of Sample Diluent to the bottom of each tube. 3. Add 0.05 ml of the sample (serum, controls or calibrators) directly to the contents of each tube. Homogenize. 4. Incubate 30 minutes in water bath at 37 º C. 5. Remove the tubes 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 º C. 11. Remove the tubes from water bath. 12. Repeat for six consecutive times stages 6 and English - Ref.: 904

4 13. In order to obtain the results, proceed as follows: 1. Effect the equipment prime by using the Developing Solution (check the instructions for use 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. Interpretation of the results 3. The hcg is a glycoprotein hormone mainly produced by the trophoblastic tissue during pregnancy. Therefore, the levels of such hormone in healthy women, not pregnant, are very low or non-detectable, being located below 5 m IU/mL. The hcg concentration rapidly increases in the initial stages of pregnancy. Levels between 5 and 50 m IU/mL may indicate initial pregnancy. For samples with values in such interval, it is suggested to request another collection after 48 hours for accomplishment of a new test. The hcg levels usually reach a peak in the first quarter and are slowly reduced until the end of pregnancy, as described in the table below: Weeks after last menstrual period 3 to4 weeks 4 to 5 weeks 5 to 6 weeks 6 to 7 weeks 7 to 12 weeks 12 to 16 weeks 16 to 29 weeks 29 to 41 weeks hcg Range (miu/ml) 9, The pregnancy diagnosis should not be only based on the result of the laboratorial exam, but on correlation of the result obtained with the clinical signals and symptoms, in addition to the results of other tests. Despite being less frequent, the hcg levels may be affected due to situations such as presence of neoplasias, heterophile antibodies or substances structurally similar to hcg. Such interferents may falsely increase or reduce the hcg levels in the entire dynamic range of the test. Too increased levels of hcg are frequently related to trophoblastic neoplasms and not trophoblastic, such as hydatidiform mole and chorionic carcinoma. These pathological conditions shall be considered previously to confirmation of pregnancy. Operating interval. The test operating interval is 1.0 m IU/mL up to 500 m IU /ml. Samples having results lower than the analytical sensitivity limit should be reported as <1.0 m IU/mL. Samples with concentrations greater than 500 m IU/mL may be diluted with saline solution 0.85%. After 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 to use the preparations of the Labtest Qualitrol Q Line (Ref. 907) for internal quality control in chemiluminescence assays. Performance features 4 Effects of the matrix dilution. Two samples with values equal to 19 and 582 m IU/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 16 average recovery of 103.2% was found. Method comparison. The method proposed was compared to similar method, with the following results being obtained: Number of samples Concentration range (miu/l) Estimation mean (miu/l) Regression equation Correlation coefficient Comparative Method By using the regression equation, the total estimated systematic bias was equal to -12.1% for a decision level equal to 50 m IU/mL and - 5.1% for a decision level equal to 500 m IU/mL. Accuracy studies. The accuracy studies were carried out by using two samples Labtest Method Labtest Method = x Comparative Method In male individuals, high levels of hcg may be detected in cases of testicular neoplasia development. As a consequence of high test sensitivity, positive samples in the first days of pregnancy may later become negative due to natural interruption of pregnancy, which fact takes place in around 22% of pregnancies. Imprecision - Within Run Sample 1 Sample 2 N Mean (miu/ml) DP (%) CV Low hcg concentrations may occur in healthy, non-pregnant women. Since during pregnancy the hcg levels double every 48 hours, samples containing low levels of such analyte should be recollected after 48 hours for accomplishment of a new test. Imprecision - Run-to-run Sample 1 Sample 2 N Mean (miu/ml) DP (%) CV English - Ref.: 904

5 Total imprecision found corresponds to 9.2 % in a sample of 50 m IU/mL and 13.2 % in a sample of 250 m IU/mL, which variation is acceptable according to the total variation specifications acceptable by RILIBAK 5 (German Guidelines for Quality) published by CLIA. Methodological or analytical sensitivity. Detection limit: 1.0 m IU/mL. It is equal to the hcg concentration that is away from the standard deviations of the low calibrator (0 m IU/mL) and represents the lower concentration of hcg that may be differentiated from zero. 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. Reference 1. Cole LA. (2009) New discoveries on the biology and detection of human chorionic gonadotropin. Reproductive Biology and Endocrinology. 7:8. 2. Spencer K, Macri JN, Carpenter P, Anderson R. (1993) Stability of Intact Chorionic Gonadotropin (hcg) in Serum, Liquid Whole Blood, and Dried Whole-Blood Filter-Paper Spots: Impact on Screening for Down Syndrome Clin. Chem. 39:6, Braunstein GD, Rasor J, Adler D, Danzer H, Wade ME. (1976) Serum Human Chorionic Gonadotropin levels throughout normal pregnancy. Am. J. Obstet. Gynecol. 126, Labtest: Data on file. 5. RiliBÄK (Richtlinien der Bundesärztekammer), publicado em 2009 no site do CLIA ( (accessed on 09/2011). Presentation Product Reference Contents X 10 ml 1 X 7.5 ml 1 X 0.1 ml hcg X 5 ml 1 X 1.0 ml 1 X 1.0 ml 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 sac@labtest.com.br X 25 un Edition: May, 2012 Ref.: Copyright by Labtest Diagnóstica S.A. Reproduction under previous autorization 05 English - Ref.: 904

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