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1 BIO-FLASH anti-hbs tests The BIO-FLASH anti-hbs is a fully automated chemiluminescent simultaneous immunoassay for quantitative measurement of antibodies to Hepatitis B surface antigen (anti-hbs) in human serum or plasma on the BIO-FLASH instrument. Summary Hepatitis B is a liver infection produced by the Hepatitis B Virus (HBV) that represents a major global health concern. It causes a number of liver diseases, ranging from acute and chronic hepatitis to cirrhosis and primary liver cancer. Diseases caused by HBV have a global distribution. It is estimated that approximately one-third of the world population has been infected with HBV. Of these, approximately 350 million individuals are chronically infected and at risk of serious illness and death, mainly from liver cirrhosis and hepatocellular carcinoma. HBV infection varies markedly in different geographic areas, and chronic HBV prevalence can range from 0.2 to 15%. The main modes of HBV transmission are perinatal exposure to an infected mother, horizontal, parenteral and sexual, and the relative rates of these vary throughout the world. Parenteral and sexual transmission predominates in industrialized countries, whereas horizontal and perinatal transmission predominates in developing countries. 1,2 During the viral infection many serological markers appear. One of these markers is the anti-hbs. The majority (> 80%) of patients infected with HBV presents an acute episode followed by convalescence and recovery. In these patients, antibodies against hepatitis B surface antigen (HBsAg) generally appear within a few weeks after HBsAg is cleared from circulation and indicate clinical recovery and subsequent immunity to HBV. Therefore the anti-hbs determination is the best marker of recovery and immunity. 3 The usefulness of anti-hbs determination has increased by the use of hepatitis B vaccines, as a tool in pre-immunization "screening" (individuals should be negative for HBsAg, anti-hbc and anti-hbs) and in monitoring seroconversion after immunization. 3-7 Principle The BIO-FLASH anti-hbs paramagnetic microparticles and tracer are mixed and incubated simultaneously with the sample. If present in the sample, the specific anti-hbs antibodies will bind to the HBsAg (subtypes ad and ay) coated on the microparticles. The tracer, consisting of an isoluminol labeled HBsAg (subtypes ad and ay), may bind to the anti-hbs captured by the microparticles. A magnetic separation followed by a wash step is done to remove residual sample and tracer. Immediately after, reagents that trigger the chemiluminescent reaction are added. The emitted light is measured as relative light units (RLU) by the BIO-FLASH luminometer. The RLUs are directly proportional to the anti-hbs concentration in the sample. The BIO-FLASH utilizes a 4 Parameter Logistic Curve fit data reduction method (4PLC) to generate a Master Calibration Curve (MCC). The MCC is predefined and lot dependent and it is stored in the instrument through the cartridge barcode. With the measurement of calibrators (supplied in a separate kit), the predefined MCC is transformed to a new, instrument specific Working Calibration Curve (WCC). The concentration values of the calibrators are included in the calibrator tube barcodes. Reaction Scheme: READ HIGHLIGHTED CHANGES Triggers RLU Magnetic particle Sample Tracer Light emission

2 Components Reagent cartridge The BIO-FLASH anti-hbs kit contains a reagent cartridge for 100 determinations (REF ). Note: Cartridge design is protected under patent (US D565,741 / EC Design ) Reagent cartridge composition: Cartridge has 3 different vials with the following contents: A. 1 cylindrical vial of microparticles suspension coated with HBsAg in a phosphate buffer. Contains <0.1% Sodium azide. B. Empty position. C. 1 opaque vial of tracer consisting of HBsAg labeled with isoluminol. Contains <0.1% Sodium azide. D. 1 empty vial. Preparation See the figure below. Cartridge: Microparticles settle during shipment and storage and require mixing to resuspend. The first time that the cartridge is used, gently invert the cartridge 30 times avoiding foam formation. Bubbles may interfere with the instrument liquid sensors. Check for the complete resuspension of the microparticles. If the microparticles are not totally resuspended continue to invert the cartridge until the microparticles have been completely resuspended. If the microparticles do not resuspend or it is detected that the reagent seal is broken, DO NOT USE THE CARTRIDGE. Once the microparticles have been resuspended, place the cartridge on a solid surface and gently remove the Red Secure Shipping Tab from the cartridge. With the cartridge still on a solid surface, press the two tabs placed on the sides of the piercing cap (grey part) and apply pressure to the top portion of the cartridge until it snaps into a lock position. Once in the locked position the tabs should not be visible. Do not invert the open cartridge. Let the cartridge stand for a period of 5 minutes before loading it onto the instrument. Once the cartridge is placed on the instrument additional periodic mixing is automatically performed on board.

3 Precautions The BIO-FLASH reagents are intended for IN VITRO diagnostic use. For professional use only. Sodium azide may react with lead or copper pipes and plumbing creating highly explosive metal azides. Flush drains with water thoroughly after disposing of the remains of reagents. WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL. All human source material used in the preparation of this product was found to be negative for the presence of HIV-1/2 and HCV antibodies using a method approved by the Food and Drug Administration (USA). The HBsAg used in this reagent has been inactivated at 60 C for 10 hours. Because no test method can offer complete assurance of the absence of infectious agents, this product should be handled with caution. Dispose of all used materials in a suitable biohazardous waste container. Please do not reuse nor reintroduce any reagent in the cartridges or vials. Sample collection and storage Use fresh serum (including serum separator tube) or plasma (EDTA, Li-Heparin, Na-Heparin, Na-Citrate and ACD). Other anticoagulants should be evaluated before use. Liquid anticoagulants as Na-Citrate have a dilution effect resulting in lower concentrations for individual patient specimens. Samples should not be heat inactivated. Samples showing visible particles should be clarified by centrifugation. Refer to the CLSI H18-A3 and H21-A5 guidelines for further information on handling, transport, processing and storage of samples. Serum Serum samples can be stored at 2-8 C for 8 days. For longer periods, sera should be frozen at -20 C or colder. Samples can be freeze/thawed up to 3 times. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Plasma Plasma samples can be stored at 2-8 C for 8 days. For longer periods, plasma samples should be frozen at -20 C or colder. Thaw plasma at 37 C. Thaw samples only once. Mix thoroughly after thawing. Inspect all samples for bubbles and remove all of them prior to analysis. Sample volume The sample volume required to perform a single BIO-FLASH anti-hbs test varies depending on the type of sample container used. A test requires at least 100 µl plus dead volume, which is 200 µl for the recommended sample cup (REF ). Additional materials The following materials are not supplied with the reagent cartridge and must be purchased separately. REF REF BIO-FLASH anti-hbs Calibrators BIO-FLASH anti-hbs Controls Read carefully their corresponding inserts for more information. Do not use other calibrators. The information required by the BIO-FLASH instrument to calibrate the BIO-FLASH anti-hbs assay is included in the barcoded vials. Using controls from other manufacturers may lead to unexpected results. Ensure that the BIO-FLASH instrument has enough quantity of the following consumables on board before running samples, calibrators or controls: REF BIO-FLASH Cuvettes Note: Cuvette design is protected under patent (US D560,816 / EC Design ) REF REF REF BIO-FLASH Triggers BIO-FLASH System Rinse BIO-FLASH Sample Diluent (if re-run option is enabled) Instrument / test procedure Refer to the BIO-FLASH Operator s Manual for the complete assay procedure instructions.

4 Calibration Each cartridge barcode contains the Master Calibration Curve (MCC) valid for the reagent lot; however a Working Calibration Curve (WCC) is required for all tests. The WCC is lot specific and valid throughout the shelf life of the lot. A new WCC is indicated when controls report outside the acceptable range or when adjustments are made to the instrument. Read carefully BIO-FLASH Operator s Manual for configuring a WCC. Quality control Three levels of control are recommended for a complete quality control program. BIO-FLASH anti-hbs Controls Negative, Positive and High Positive are designed for this program. Controls should be analyzed at least once every 24 hours each day of use. Ensure that control results are within the acceptable ranges. A control result not falling within the acceptable range may indicate invalid test results and corrective measures should be taken by the user. Examine all tests results generated since obtaining the last acceptable quality control check for this analyte. Recalibration may be indicated. Refer to the instrument s Operator s Manual for additional information. Refer to Westgard et al. for identification and resolution of out-of-control situations. 8 Storage and stability Unopened reagents are stable until the expiration date shown on the cartridge label when stored at 2-8 C in the upright position. Once open the cartridge stability on board the BIO-FLASH instrument or stored at 2-8 C is 12 weeks. Traceability of calibrators and controls The values are reported in miu/ml and were determined over multiple runs on BIO-FLASH instruments using specific lots of reagents and against internal standards referenced to the WHO First International Reference Preparation for Hepatitis B Immunoglobulin (Netherlands Red Cross code: W1042). Interpretation of results The amount of analyte in every sample is determined from the emitted light (RLU) by interpolation in the stored Working Calibration Curve. BIO-FLASH anti-hbs results are reported in miu/ml. The quantitative measurement of anti-hbs, using the BIO-FLASH anti-hbs assay, helps to determine the patient immune status: samples with a concentration < 10.0 miu/ml are considered non reactive (negative). samples with a concentration 10.0 miu/ml are considered reactive (positive). It is widely accepted that a sustained level of an anti-hbs concentration equal or higher than 10.0 miu/ml is indicative of recovery of a past infection or protection in vaccinated people. 3-7 Limitations This assay does not differentiate between a vaccine-induced immune response and an immune response induced by natural infection with HBV. To determine if the anti-hbs response is due to vaccine or HBV infection, anti-hbc assay should be performed. For diagnostic purposes, the BIO-FLASH anti-hbs results should be used in conjunction with other data; e.g., symptoms, clinical history, and other hepatitis markers for diagnosis of acute, chronic or recovered infection. Although quantitative results show a good correlation with other assays for the majority of samples, there is some degree of variation, and even important variations for some individual samples. This is due to several factors such as the variability of HBsAg, which has many different antigenic subtypes, the characteristics of anti-hbs contained in the sample (specificity and affinity to different epitopes) and others related to the assay (manufacture method, calculation of results, etc.). Expected results The majority of patients with HBV infection clears the virus, develops anti-hbs, and recovers completely. The prevalence of anti-hbs positive samples due to HBV past infection can range from 4 to 95% depending on the geographical area. 2 The rate of positive samples due to HBV vaccination will vary in different countries according to the extension of immunization programs. Since 1982, when first hepatitis B vaccine was introduced, millions of hepatitis B vaccines have been used worldwide. For this reason, the prevalence of HBsAg carriers is falling and anti-hbs due to vaccination is increasing. Performance characteristics Note: The following data are representative; results in individual experiments may vary from these data.

5 Method comparison BIO-FLASH anti-hbs was evaluated in comparative studies with other commercial assays. Evaluation A panel of 717 samples from different sources, including positive and negative sera for anti-hbs, was tested with BIO-FLASH anti-hbs in comparison with a commercially available EIA anti-hbs method. Reference method BIO-FLASH anti-hbs POS NEG Total POS NEG Total The following results were obtained for relative sensitivity, specificity and overall agreement: Relative Sensitivity Relative Specificity Overall Agreement N Value 95% CI Value 95% CI Value 95% CI % 95.7% to 99.7% 100.0% 99.3% to 100.0% 99.6% 98.8% to 99.9% Three hundred and fifty (350) samples from the previous panel were also tested with a commercially available chemiluminescent anti-hbs assay (CLIA). A consensus result was established considering the agreement between at least two of the assays, including the BIO-FLASH anti-hbs. One sample for which a consensus could not be achieved was not taken into account for the calculations. The summary of results of BIO-FLASH against the consensus result is shown in the following table: Consensus BIO-FLASH anti-hbs POS NEG Total POS NEG Total The following results were obtained for consensus relative sensitivity, specificity and overall agreement: Consensus Sensitivity Consensus Specificity Consensus Overall Agreement N Value 95% CI Value 95% CI Value 95% CI % 96.4% to 99.9% 100.0% 97.6% to 100.0% 99.4% 97.9% to 99.9% Seroconversion follow-ups Additional sensitivity studies were carried out by testing 12 vaccination follow-ups. Samples were collected from individuals before they were vaccinated for Hepatitis B and one month after vaccination. Anti-HBs results obtained were compared to a commercially available chemiluminescent anti-hbs assay (CLIA). In all cases follow-up samples showed evidence of seroconversion. Precision Within run and total (run to run and day to day) precision were assessed (following CLSI EP05-A2 Guidelines) over multiple runs. Results are summarized in the following table: Mean CV% (Within run) CV% (Total) Negative Control* 778 RLU Positive Control 44.7 miu/ml High Positive Control miu/ml Around Cut-Off Level 9.4 miu/ml * The negative control is close to the limit of quantitation and for this reason the mean RLU was calculated instead of the mean concentration.

6 Reproducibility Repeatability between replicates, between lots and between instruments was assessed using Passing & Bablok. Results are shown in the following table: Slope Intercept R N Value 95% CI Value 95% CI Value 95% CI Replicate 2 vs. Replicate to to to 1.00 Lot 2 vs. Lot to to to 1.00 Instrument 2 vs. Instrument to to to 1.00 Interferences As assessed by the CLSI EP7-A2 Guidelines, BIO-FLASH anti-hbs results are not affected by the following potentially interfering substances: Potentially Interfering substance Concentration % Interference Hemoglobin 500 mg/dl 10 Free bilirubin 18 mg/dl 10 Complexed bilirubin 18 mg/dl 10 Triglycerides 1300 mg/dl 10 HAMA 1 µg/ml 10 Cross reactivity A total of 113 positive specimens for a range of potential cross-reactants and samples from pregnant women were analyzed with BIO-FLASH anti-hbs. It is considered that a potential cross-reactant causes cross-reactivity when the diagnostic changes from negative to positive or vice versa. The samples were compared to commercially available anti-hbs assays. Results are summarized in the following table: Potential Cross-Reaction Agreement ANA (Anti-Nuclear Antibodies) 5/5 anti-cmv (Cytomegalovirus) 10/11 anti-ebv (Epstein-Barr Virus) 5/5 anti-hav IgG (Hepatitis A Virus) 6/6 anti-hav IgM (Hepatitis A Virus) 5/5 anti-hdv IgG (Hepatitis delta Virus) 2/2 anti-hev (Hepatitis E Virus) 2/2 anti-hiv (Human Immunodeficiency Virus) 4/4 anti-hsv (Herpes Simplex Virus) 2/2 anti-rubella (Rubella Virus) 11/11 anti-toxoplasma (Toxoplasma gondii) 5/5 anti-vzv (Varicela Zoster Virus) 4/4 Elevated IgG 4/4 Elevated IgM 4/4 Mononucleosis 4/4 Pregnant (multiparous) 5/5 Pregnant 10/10 RF (Rheumatoid Factor)* 15/15 RPR (Rapid Plasma Reagin) 9/9 *RF concentration in the samples tested varied from 24 to 805 IU/mL. Limit of Detection Assessed by the CLSI EP17-A Guidelines, the Limit of Detection (LoD) is set at 2.6 miu/ml. Limit of Quantitation Assessed by the CLSI EP17-A Guidelines, the Limit of Quantitation (LoQ) is set at 3.6 miu/ml.

7 Linearity The BIO-FLASH anti-hbs assay is linear between 3.6 and 1900 miu/ml as assessed following the CLSI EP6-A Guidelines. However, the linearity can be extended by dilution up to miu/ml by enabling the automatic re-run option. When the re-run option is not enabled, samples that contain more than 1900 miu/ml will be reported as higher than 1900 miu/ml. When the re-run option is enabled, the instrument makes an automatic dilution and corrects the final result for the dilution factor (20x), thereby expanding the test range up to miu/ml. Refer to BIO-FLASH Operator s Manual for enabling the automatic re-run option. Prozone effect Experimentally, no prozone effect was observed with a sample as high as miu/ml.