List Number. For In Vitro Diagnostic Use. Store at C

Transcription

1 E HBsAg (V2) 7A40-22 B7P /R10 HBsAg (V2) This package insert must be read carefully prior to use. Package insert instructions must be carefully followed. Reliability of assay results cannot be guaranteed if there are any deviations from the instructions in this package insert. Note Changes Highlighted 1998, 2005 Abbott / August 2005 Key to symbols used List Number Lot Number For In Vitro Diagnostic Use Expiration Date Store at 2-8 C Reagent Pack Store at C Reaction Vessels CAUTION: Handle human sourced materials as potentially infectious. Consult instructions for use. (Infection Risk) Matrix Cells Sample Cups Legal Manufacturer Consult instructions for use See REAGENTS section for a full explanation of symbols used in reagent component naming. ABBOTT Diagnostics Division 1

2 NAME HBsAg - Hepatitis B Surface Antigen INTENDED USE AxSYM HBsAg (V2) is a third generation microparticle enzyme immunoassay for the qualitative detection of Hepatitis B Surface Antigen (HBsAg) in human serum or plasma. SUMMARY AND EXPLANATION OF THE TEST Enzyme immunoassays for the detection of HBsAg were first described by Engvall and Perlmann 1-3 and VanWeemen and Schuurs 4 in In 1976 and 1977, solid phase "sandwich" enzyme immunoassays were developed in which HBsAg was captured on a solid phase coated with polyclonal antibodies against HBsAg (anti-hbs) and then detected with anti-hbs conjugated to an enzyme. 5-7 In the early 1980's, monoclonal anti-hbs based assays were developed for the detection of HBsAg is an enzyme immunoassay which uses microparticles coated with monoclonal anti-hbs for the detection of HBsAg. Assays for HBsAg are used to screen blood and blood products for the presence of HBsAg to prevent transmission of hepatitis B virus (HBV) to recipients of these products. HBsAg assays are also routinely used to diagnose suspected HBV infection and to monitor the status of infected individuals; i.e., whether the patient's infection has resolved or the patient has become a chronic carrier of the virus. 14 In addition, these assays are used to evaluate the efficacy of anti-viral drugs by monitoring the levels of HBsAg in patient sera or plasma. 15 The prenatal screening of all pregnant women has been recommended by the U.S. Centers for Disease Control so that newborns from HBV carrier mothers may obtain prophylactic treatment. 16,17 Samples nonreactive by are considered negative for HBsAg and need not be tested further. A reactive sample should be retested by to determine whether it is repeatedly reactive. A sample which is found to be repeatedly reactive should be confirmed by neutralization procedures utilizing human anti-hbs, such as the AxSYM HBsAg Confirmatory assay. If the sample is neutralized in a confirmatory assay, the sample is considered positive for HBsAg and does not need to be tested further. BIOLOGICAL PRINCIPLES OF THE PROCEDURE is based on the Microparticle Enzyme Immunoassay (MEIA) technology. Sample and all reagents required for one test are pipetted by the Sampling Probe into various wells of a reaction vessel (RV) in the Sampling Center. The RV is immediately transferred into the Processing Center. Further pipetting is done in the Processing Center by the Processing Probe. The reactions occur in the following sequence: Sample, Anti-HBs Coated Microparticles and Biotinylated Anti-HBs are combined in one RV well. When HBsAg is present in the sample, it binds to the Anti-HBs Coated Microparticles and Biotinylated Anti-HBs, forming an antibody-antigenantibody complex in the reaction mixture. A portion of the reaction mixture is transferred to the matrix cell. The microparticles bind irreversibly to the glass fiber matrix. The Anti-Biotin:Alkaline Phosphatase Conjugate is dispensed onto the matrix cell and binds with any microparticle-bound antibody-antigenantibody complex. The matrix cell is washed to remove materials not bound to the microparticles. The substrate, 4-Methylumbelliferyl Phosphate, is added. The alkaline phosphatase-labeled conjugate catalyzes the removal of a phosphate group from the substrate, yielding the fluorescent product, 4-Methylumbelliferone. This fluorescent product is measured by the MEIA optical assembly. The presence or absence of HBsAg in the sample is determined by comparing the rate of formation of fluorescent product to a cutoff rate determined from a previous Index Calibration. If the rate of formation of fluorescent product in the test sample is greater than or equal to the cutoff rate, the sample is considered reactive for HBsAg. For further information regarding MEIA technology, refer to the AxSYM System Operations Manual, Section 3. REAGENTS REAGENT PACK, 100 TESTS Reagent Pack (7A40-22) 1 Bottle (5.1 ml) Anti-HBs (Mouse, Monoclonal, IgM) Coated Microparticles in Phosphate buffer with protein stabilizers. Minimum concentration: 0.15%. Preservative: Sodium Azide. (Reagent Bottle 2) 1 Bottle (15.5 ml) Anti-Biotin (Rabbit):Alkaline Phosphatase Conjugate in TRIS buffer with (Rabbit) IgG. Minimum concentration: 0.03 µg/ml. Preservative: Sodium Azide. (Reagent Bottle 1) 1 Bottle (17.6 ml) Biotinylated Anti-HBs (Goat, IgG) in TRIS buffer containing animal sera (Goat, Calf, Rabbit, Mouse). Minimum concentration: 0.25 µg/ml. Preservatives: Sodium Azide, Nipasept, Quinolone. (Reagent Bottle 3) 1 Bottle (6 ml) Index Calibrator. Recalcified human plasma nonreactive for HBsAg, HIV RNA or HIV-1 Ag, anti-hbs, anti-hcv and anti-hiv-1/hiv-2. Preservative: Sodium Azide. Dye: Green (Acid Yellow No. 23 and Acid Blue No. 9). Nipasept is a registered trademark of Nipa Laboratories, Wilmington, Delaware. CONTROLS AxSYM HBsAg Controls (7A40-10) 2 Bottles (8 ml each) of AxSYM HBsAg Controls are prepared with recalcified human plasma. The Negative Control is nonreactive for HBsAg, HIV RNA or HIV-1 Ag, anti-hbs, anti-hcv and anti-hiv-1/hiv-2. The Positive Control is reactive for HBsAg and nonreactive for HIV RNA or HIV-1 Ag, anti-hcv, anti-hbs and anti-hiv-1/hiv-2. Preservative: Sodium Azide. The AxSYM HBsAg Controls have the following ranges: HBsAg Control Control Concen- Range Range Control Color tration S/N S/CO Natural 0 ng/ml Blue* 4-15 ng/ml * Dye: Bromophenol Blue. The reporting unit is factory set to S/N. An alternate unit, S/CO may be selected for reporting results (Assay Parameter 45). OTHER REAGENTS AxSYM Probe Cleaning Solution (9A35-04/9A35-05) 4 Bottles (110 ml each)/2 Bottles (220 ml each) AxSYM Probe Cleaning Solution containing 2% Tetraethylammoniumhydroxide (TEAH). Solution 1 (MUP) (8A47-04) 4 Bottles (230 ml each) Solution 1 (MUP) containing 4-Methylumbelliferyl Phosphate, 1.2 mm, in buffer. Preservative: Sodium Azide. Solution 3 (Matrix Cell Wash) (8A81-04) 4 Bottles (1000 ml each) Solution 3 (Matrix Cell Wash) containing 0.3 M Sodium Chloride in TRIS buffer. Preservatives: Sodium Azide and Antimicrobial Agents. Solution 4 (Line Diluent) (8A46) 1 Bottle (10 L) Solution 4 (Line Diluent) containing 0.1 M Phosphate buffer. Preservatives: Sodium Azide and Antimicrobial Agent. WARNINGS AND PRECAUTIONS For In Vitro Diagnostic Use. CAUTION: This product contains human sourced and/or potentially infectious components. For a specific listing, refer to the Reagents section of this package insert. Components sourced from human blood present in the Positive Control have been tested and found to be reactive for HBsAg and nonreactive for HIV RNA or HIV-1 Ag and antibodies to HCV, HBs and HIV-1/HIV-2. Components sourced from human blood present in other kit components have been tested and found to be nonreactive for HBsAg and HIV RNA or HIV-1 Ag and for antibodies to HBs, HIV-1/HIV-2 and HCV. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Therefore, all human sourced materials should be considered potentially infectious. It is recommended that these reagents and human specimens be handled in accordance with the OSHA Standard on Bloodborne Pathogens. 18 Biosafety Level 2 19 or other appropriate biosafety practices 20,21 should be used for materials that contain or are suspected of containing infectious agents. These precautions include, but are not limited to the following: 1. Wear gloves when handling specimens or reagents. 2. Do not pipette by mouth. 3. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in areas where these materials are handled. 4. Clean and disinfect all spills of specimens or reagents using a tuberculocidal disinfectant such as 0.5% sodium hypochlorite, or other suitable disinfectant. 22,23 2

3 5. Decontaminate and dispose of all specimens, reagents and other potentially contaminated materials in accordance with local, state and federal regulation. 24 SAFETY PRECAUTIONS The AxSYM Probe Cleaning Solution (2% TEAH) may cause mild eye irritation. If this solution comes in contact with eyes, rinse immediately with water. If irritation persists, seek medical attention. All components of this product contain Sodium Azide. For a specific listing refer to the Reagents section of this package insert. The components containing Sodium Azide are classified per the applicable European Community (EC) Directives as: Harmful (Xn). The following are the appropriate risk (R) and safety (S) phrases. R22 Harmful if swallowed. R32 Contact with acids liberates very toxic gas. S35 This material and its container must be disposed of in a safe way. S36 Wear suitable protective clothing. S46 If swallowed, seek medical advice immediately and show this container or label. For product not classified as dangerous per European Directive 1999/45/EC - Safety data sheet available for professional user on request. HANDLING PRECAUTIONS reagents are susceptible to splashes, air bubbles, foaming and require inspection and removal of bubbles before loading. If bubbles are present, refer to the AxSYM System Operations Manual, Section 9: Service and Maintenance, Subsection: Daily Maintenance. Do not use Solution 1 (MUP) beyond the expiration date or a maximum of fourteen days on-board the AxSYM System. When loading new Solution 1 (MUP), it is important to immediately tighten the instrument cap for MUP to minimize exposure to air. Prolonged exposure to air may compromise performance. Do not use kits beyond the expiration date or a maximum of 112 cumulative hours on-board the AxSYM System. Do not mix reagents from different reagent packs. Do not mix reagent packs and index calibrators from different lots. Avoid microbial contamination of samples and reagents. Use of disposable pipettes or pipette tips is recommended. Avoid chemical contamination of reagents and equipment. Ensure that sufficient sample volume is present. If sample volume is insufficient, the AxSYM System will give an error code and no result will be reported. For a description of the System error codes, refer to the AxSYM System Operations Manual, Section 10. Inadequate adherence to package insert instructions may result in erroneous results. Use accurately calibrated equipment. Use caution in handling patient samples to prevent cross contamination. Transfer of as little as 1 µl from one HBsAg positive sample may contaminate an adjacent negative sample and result in a falsely reactive result. Additional safety and handling precautions and limitations for the reagent packs, index calibrators, controls, patient samples and other reagents are described in the AxSYM System Operations Manual, Sections 7 and 8. STORAGE INSTRUCTIONS The Reagent Pack, Index Calibrator and AxSYM HBsAg Controls must be stored at 2-8 C. The Reagent Pack, Index Calibrator and Controls may be used immediately after removal from the refrigerator. Do not freeze the reagents. The Reagent Pack may be on-board the AxSYM System for a maximum of 112 cumulative hours. After 112 hours the reagent pack and its associated index calibrator must be discarded. Refer to the AxSYM System Operations Manual, Appendices, for further information on tracking on-board time. Solution 1 (MUP) must be stored at 2-8 C. It may be on-board the AxSYM System for a maximum of fourteen days. After fourteen days it must be discarded. The AxSYM Probe Cleaning Solution, Solution 3 (Matrix Cell Wash) and Solution 4 (Line Diluent) must be stored at C. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS When an AxSYM HBsAg Positive or Negative Control value is out of the expected range, it may indicate deterioration of the reagents or errors in technique. Associated test results are invalid and must be retested. Assay recalibration may be necessary. INSTRUMENT PROCEDURE Assay File Installation The AxSYM HBsAg Assay File must be installed on the AxSYM System from the software disk, 9A16-03, or higher, prior to performing the HBsAg (V2) assay. Refer to the AxSYM System Operations Manual, Section 2, for proper installation procedures. Assay Parameters The default assay parameters for the assay are listed below. These parameters can be printed, displayed and edited according to the procedure in the AxSYM System Operations Manual, Section 2. To print the assay parameters, press PRINT. Assay parameters that can be edited contain a (>) symbol. In order to obtain values for the parameters with an asterisk (*), review the specific Assay Parameter screen. Assay Parameters 1 Long Assay Name (English): HBsAg 6 Abbrev Assay Name (English): HBsAg 11 Assay Number: Assay Version: 2 13 Calibration Version: Assay File Revision: * 15 Assay Enabled > ON 17 Assay Type: MEIA 18 Standard Cal Reps > 5 19 Master Cal Reps: 0 20 Cal Adjust Reps: 0 21 Cal A Concentration: Cal B Concentration: Cal C Concentration: Cal D Concentration: Cal E Concentration: Cal F Concentration: Master Calibrator 1 Concentration: Master Calibrator 2 Concentration: Cal Adjust Concentration: Default Dilution Protocol > UNDILUTED 44 Default Calibration Method > Index Cal 45 Selected Result Concentration Units > S/N 46 Selected Result Decimal Places > 2 62 Blank I-Max background intensity: Min Tracer-Min net intensity: Max Intercept-Max MUP intercept : Min Intercept-Min MUP intercept : Upper limit for NRMSE for low rates : Upper limit for NRMSE for high rates : Max Rate-Max rate used to check Min MUP Intercept : Min Rate-Rate cutoff for NRMSE and Corr. Coef. : Min correlation coefficient for low rates : Min correlation coefficient for high rates : MUP T Delay-Time delay following MUP: Low Limit-Normal/Therapeutic Range lower limit > High Limit-Normal/Therapeutic Range upper limit > Low Extreme Value > High Extreme Value > Lo Norm-% Uptake Normal Range Low > Hi Norm-% Uptake Normal Range High > Interpretation Option to use > 1 84 Hold results with POS interpretation > ON 85 Hold results with NEG interpretation > OFF 86 Hold results with GRY interpretation > ON 91 Low Range Neat: High Range Neat: Low Range Dil1: High Range Dil1: Low Range Dil2: High Range Dil2: Low Range Dil3: High Range Dil3: Negative Interpretation Cutoff > 2.00 NOTE: Parameters 18, 43, 44 cannot be edited to another value. NOTE: Parameter 45 can be edited to the alternate Result Unit S/CO. Refer to the AxSYM System Operations Manual for a detailed description of Instrument Procedures. NOTE: A grayzone feature is available. To utilize the grayzone option, parameter 117, Negative Interpretation Cutoff, must be edited to desired value within the range of 1.60 to 1.99 S/N or 0.80 to 0.99 S/CO. Specimens with S/N values equal to the selected value and less than 2.00 or with S/CO values equal to the selected value and less than 1.00 are indicated as "GZ-NEGATIVE" or "GZ-NONREACTIVE" in the interpretation field. The report options are selected in parameter 80, Interpretation Option to use. 3

4 The values available for parameter #80 (Interpretation Option to use) are: POS Interp NEG Interp GRY Interp 1. REACTIVE NEGATIVE GZ-NEGATIVE 2. REACTIVE NONREACTIVE GZ-NONREACTIVE 3. REACTIVE BLANK BLANK NOTE: Parameter 46 must be set to 3 in order to edit the alternate Result Unit S/CO grayzone value to SAMPLE COLLECTION AND PREPARATION FOR ANALYSIS Serum (including serum collected in separator tubes) or plasma (collected in sodium heparin, sodium citrate, ACD, CPDA-1 or EDTA) may be used in the Assay. Use the ratio of anticoagulant (quantity) to sample (volume) that is recommended by the manufacturer. The AxSYM System does not have the capability to verify the sample type. It is the responsibility of the operator to verify the correct sample type(s) are used in the assay. Samples containing particulate matter or red blood cells may give inconsistent or erroneous results. Inspect all samples for splashing, air bubbles and foaming. If necessary remove bubbles prior to analysis using a new applicator stick for each sample. Refer to the AxSYM System Operations Manual, Section 7. Specimens with obvious microbial contamination should not be used. All patient samples to be tested in Primary Tubes must be centrifuged to remove red cells or particulate matter. Each sample that requires repeat or confirmatory testing, or that has been frozen and thawed, must be transferred to a centrifuge tube and centrifuged at an RCF* of at least 10,000 x g for 10 minutes. Transfer clarified sample to a sample cup for testing. WARNING: AxSYM System Software Version 3.0 and higher offers an Auto Retest/Auto Dilution feature. Due to requirements discussed above, this feature must not be used. Samples which contain fibrin may cause erroneous results. Serum samples must be completely clotted and centrifuged prior to testing to generate consistent results. Centrifuged specimens with a lipid layer on top of the liquid must be transferred to a sample cup or secondary tube. Care must be taken to transfer only the clarified specimen and not the lipemic material. Samples from heparinized patients may be partially coagulated and erroneous results could occur due to the presence of fibrin. To prevent this phenomenon, draw the sample prior to heparin therapy. Samples may be stored on the clot or red blood cells for up to 14 days at 2-8 C prior to being tested. If the assay will be performed after 14 days, the sample must be removed from the clot or red blood cells and stored frozen (-10 C or colder). 15 nonreactive and 15 reactive samples showed no qualitative performance differences when subjected to 6 freeze-thaw cycles; however, multiple freeze-thaw cycles should be avoided. Samples must be mixed thoroughly after thawing and centrifuged prior to use, as described in this section, to remove particulate matter and to ensure consistency in the results. When shipped, samples must be packaged and labeled in compliance with applicable federal and international regulations covering the transport of clinical samples and etiologic agents. Samples may be shipped under ambient conditions, refrigerated on wet ice (2-8 C) or frozen on dry ice (-10 C or colder). All samples (patient samples, controls and calibrators) should be tested within 3 hours of being placed on-board the AxSYM System. Refer to the AxSYM System Operations Manual, Section 5, for more detailed discussion of on-board sample storage constraints. Do not use heat-inactivated samples. Performance has not been established for cadaver samples or body fluids such as urine, saliva, semen or amniotic fluid. No qualitative performance differences were observed when HBsAg Negative and Positive Controls were tested with elevated levels of bilirubin (0-20 mg/dl), hemoglobin (0-250 mg/dl) or lipids ( mg/dl). The assay was designed and validated for use with human serum or plasma from individual patient and donor specimens. Pooled specimens should not be used since the accuracy of their test results has not been validated. SAMPLE VOLUME The sample volume required to perform a single HBsAg (V2) test on the AxSYM System varies according to the different sample containers. For sample cups, the minimum sample volume required is 190 µl. For every additional HBsAg (V2) test performed from the same sample container, an additional 140 µl of sample will be required. The sample cup minimum volume will be calculated by the AxSYM System. It will be displayed on the Order screen at the time the test(s) is(are) ordered and printed in the Orderlist Report. To obtain the recommended volume requirements for the Index Calibrator and AxSYM HBsAg Controls, hold the bottles vertically and dispense 15 drops of Index Calibrator or 5 drops of Positive or Negative Control into each respective sample cup. For sample volume requirements in primary or aliquot tubes, and control volume requirements for multiple reagent lots, refer to the AxSYM System Operations Manual, Section 5. PROCEDURE Materials Required 7A40-22 Reagent Kit, containing: A40-10 AxSYM HBsAg Controls 8A A A46 9A35-04/ AxSYM 9A A76-01 Materials Required But Not Provided Pipettes or pipette tips CAUTION: When manually dispensing samples, verify that dispensing equipment does not introduce cross contamination and that it delivers the specified sample volume. Use a separate disposable pipette or pipette tip for each sample. For optimal performance, it is important to follow the routine maintenance procedures defined in the AxSYM System Operations Manual, Section 9. If your laboratory requires more frequent maintenance, follow those procedures. Assay Procedure CAUTION: The System status must be WARMING, PAUSED, READY or STOPPED before adding or removing sample segments, reagent packs or reaction vessels. 1. Check for sufficient on-board inventory of matrix cells and bulk solutions, and for sample segment availability. 2. Check for sufficient waste collection capacity. CAUTION: Do not open the Interior Waste Door or the AxSYM Processing Center Cover while any test is in progress. If opened, all processing will stop. All tests will be terminated and must be repeated. 3. Order the HBsAg (V2) Index Calibrator, HBsAg Controls, and/or patient samples as required. Assign or modify sample segment position (S/P) for each sample, as necessary. Refer to the Quality Control Procedures section in this package insert for calibration and control requirements. Index Calibration: Perform calibration by testing 5 replicates of the Index Calibrator. Dispense 15 drops of the Index Calibrator into a sample cup. Do not simultaneously calibrate more than one reagent lot. Controls: Perform quality control by testing the Positive and the Negative Control (one test each). Dispense 5* drops each of Positive and Negative Controls into individual sample cups. * When more than one reagent lot is on-board the AxSYM System, multiply control volume by the number of lots. * RCF = Relative Centrifugal Force 4

5 Patient Samples: Ensure that sufficient volume is present in the sample cups or tubes. The sample cup minimum volume is 190 µl for the first HBsAg (V2) test plus 140 µl for each additional HBsAg (V2) test. For volume requirements in primary or aliquot tubes, refer to the AxSYM System Operations Manual, Section 5. NOTE: The operator may obtain an Orderlist Report by pressing PRINT. The printout contains sample placement information and minimum sample cup volume requirements for all tests ordered. When using Host Order Query, the Order screen information and the Orderlist Report is not available. Refer to the AxSYM System Operations Manual, Section 5: Ordering Patient Samples, for a description of the Host Query Option. 4. Place sample segments containing the ordered samples into the Sample Carousel. 5. Place the HBsAg (V2) Reagent Pack into the Reagent Pack Carousel. 6. Ensure that reaction vessels (RVs) are present on the RV Carousel. Additional RVs may be added as needed. 7. Press RUN. All entries on the Orderlist screen are transferred to the Order Status screen for sample processing. 8. Review the results to determine whether retesting is required. 9. When testing is completed, remove the samples and the AxSYM HBsAg (V2) Reagent Pack from the Sampling Center. Store at 2-8 C. Refer to Sections 5 and 6 of the AxSYM System Operations Manual for a detailed explanation of performing assay calibration and sample testing procedure. QUALITY CONTROL PROCEDURES CALIBRATION Five replicates of the HBsAg (V2) Index Calibrator must be tested for an Calibration. A single sample of both the Positive and Negative Controls must be tested as a means of evaluating the assay calibration. Once an calibration is accepted and stored, all subsequent samples may be tested without further calibration unless: A reagent pack with a new lot number is used for an assay. Either of the AxSYM HBsAg Control values is out of its specified range. Refer to the AxSYM System Operations Manual, Section 6, for additional information. The operator must verify that the AxSYM HBsAg Control values are within the acceptable ranges specified in this package insert (see Controls section). The AxSYM System verifies that the results of an assay calibration meet the specifications assigned to selected validity parameters. An error message occurs when the calibration fails to meet a specification. Refer to the AxSYM System Operations Manual, Section 10, for an explanation of the corrective actions for the error code. Refer to the AxSYM System Operations Manual, Appendix E, for an explanation of the calibration validity parameters that may be used by the AxSYM System. QUALITY CONTROL The minimum control requirement for an assay is a single sample of each of the Positive and Negative Controls tested once every 24 hours, each day of use. Controls may be placed in any position in the Sample Carousel. The AxSYM HBsAg Control values must be within the acceptable ranges specified in this package insert (see Controls section). If a control value is out of its specified range, the associated test results are invalid and must be retested. Recalibration may be indicated. If the quality control procedures in your laboratory require more frequent use of controls, follow those procedures. Fluorescence Background Acceptance Criteria Quality Control with regards to the MUP substrate blank is automatically determined by the instrument and checked under Assay Parameter 64 Max Intercept-Max MUP intercept each time a test result is calculated. If the MUP intercept value is greater than the maximum allowable value, the result is invalid. The test request will be moved to the Exceptions List where it will appear with the message "1064 Invalid test result, intercept too high" and the calculated intercept value. Refer to the AxSYM System Operations Manual, Section 10, when this error message is obtained. Refer to the AxSYM System Operations Manual, Section 2, for further information on parameter files. RESULTS The software evaluates performance of the assay by the following assay specific criteria: Maximum acceptable calibrator %CV: Minimum acceptable Index Calibrator (Rate): 3.00 Maximum acceptable Index Calibrator (Rate): CALCULATIONS The AxSYM System calculates the Cutoff rate from the mean rate of five Index Calibrator replicates and stores the result. The assay has two selectable Result Units. One reports the result as a ratio S/N and the other reports the result as a ratio S/CO. These units can be selected in the assay parameters according to the procedure in the AxSYM System Operations Manual, Section 2. The Cutoff rate is determined by multiplying the Index Calibrator mean rate by 2. Cutoff Rate (CO) = Index Calibrator mean rate x 2 S/N The assay calculates a result based on the ratio of the sample rate to the stored Index Calibrator mean rate for each sample and control. Sample Rate S/N = Index Calibrator Mean Rate S/CO The assay calculates a result based on the ratio of the sample rate to the Cutoff rate (CO) for each sample and control. Sample Rate S/CO = Cutoff Rate (CO) FLAGS Some results may contain information in the Flags field. For a description of the flags that may appear in this field, refer to the AxSYM System Operations Manual, Section 1 and 2. INTERPRETATION OF RESULTS Samples with S/N less than 2.00 are negative by the assay and need not be tested further. Samples with S/N values greater than or equal to 2.00 are considered reactive. Samples with S/CO values less than 1.00 are negative by the AxSYM HBsAg (V2) assay and need not be tested further. Samples with S/CO values greater than or equal to 1.00 are considered reactive. All samples that are reactive on initial testing should be retested in duplicate using the assay. If neither of the retests are reactive, the sample must be considered negative for HBsAg. If the sample is reactive in either of the repeated replicates, the sample must be considered repeatedly reactive. Repeatedly reactive samples should be tested by a neutralizing confirmatory test, such as the AxSYM HBsAg Confirmatory assay. Samples which are confirmed by neutralization with human anti-hbs must be considered positive for HBsAg. False reactive results may be obtained with any diagnostic test. Two types of false reactive results may occur with, nonrepeatable reactives and nonspecific reactives. Nonrepeatable Reactives: Some samples which are reactive in the AxSYM HBsAg (V2) assay may not be reactive on retesting. The most common sources of nonrepeatable reactives are: particulate matter in the patient sample, particularly fibrin clots and cellular material. contamination of nonreactive samples caused by transfer of a high titer antigen sample. 25 Nonspecific Reactives: All highly sensitive immunoassay systems have a potential for nonspecific reactions. The specificity of a repeatedly reactive sample should be confirmed by a neutralizing confirmatory test, such as the AxSYM HBsAg Confirmatory assay. A nonspecific reactive sample will be repeatedly reactive but will not be confirmed by neutralization. Therefore, it is recommended that a specific antibody neutralization be performed prior to informing the donor/patient of their HBsAg carrier status. For additional information on neutralization testing, refer to the AxSYM HBsAg Confirmatory assay package insert. LIMITATIONS OF THE PROCEDURE For diagnostic purposes and in order to differentiate acute HBV infection from chronic HBV infection, the detection of HBsAg must be correlated with patient symptoms and other hepatitis B viral serological markers. It is recognized that presently available methods for the detection of hepatitis B surface antigen may not detect all potentially infectious units of blood or infected individuals. A nonreactive test result does not exclude the possibility of exposure to or infection with hepatitis B. Nonreactive test results in individuals with prior exposure to hepatitis B may be due to antigen levels below the detection limit of this assay or lack of antigen reactivity to the antibodies used in this assay. 5

6 This assay was designed and validated for use with human plasma or serum from individual patient and donor specimens. Insufficient data are available to interpret tests performed on pooled blood or processed plasma and products made from such pools. Testing of these specimens is not recommended. Performance has not been established using cadaver samples or body fluids such as urine, saliva, semen or amniotic fluid. Do not use heat-inactivated samples. Frozen samples and those containing particulate matter or red blood cells must be centrifuged prior to running the assay. Samples which contain fibrin may cause erroneous results. Serum samples must be completely clotted to generate consistent results. Samples from heparinized patients may be partially coagulated. Erroneous results could occur due to the presence of fibrin. To prevent this phenomenon, draw the sample prior to heparin therapy. EXPECTED VALUES In random blood donor populations, the number of samples found repeatedly reactive for HBsAg by has typically been less than 0.20%. In a population reactive for HBsAg, detected HBsAg in 402/402 (100%) specimens. SPECIFIC PERFORMANCE CHARACTERISTICS PRECISION Assay reproducibility was determined during the clinical evaluation of AxSYM HBsAg. The six member panel was run in replicates of three, four times per day, over two days on each of three clinical lots. Calculations were made with a variance components analysis, using a nested analysis of variance model. 26 The results from these three clinical lots are summarized in Table 1.* TABLE 1 Reproducibility of AxSYM HBsAg Panel Mean Intra-Run Inter-Run Total Member N S/N SD %CV SD %CV SD %CV Index NC PC Index = Index Calibrator NC = Negative Control PC = Positive Control DETECTABILITY The ability of to detect HBsAg in specimens from blood donors, hospital patients (samples submitted to hospital laboratories for diagnostic testing) and hepatitis B patients is shown in Table 2.* The data includes a total of 4940 serum and plasma specimens. TABLE 2 Detection of HBsAg in Serum and Plasma Specimens from Blood Donors, Hospital Patients and Hepatitis B Patients Population Number Confirmed Group Tested Number (%) Positive (%) Blood Donors (0) 0 (0) Hospital Patients (0) 0 (0) Hepatitis B Patients (100) 402 (100) SPECIFICITY The percentage of samples found to be initially reactive with and the percentage of these samples found to be repeatedly reactive were determined by testing 4538 samples (plasma and sera) obtained from two blood banks and a hospital laboratory in a clinical evaluation. The presence of HBsAg in the repeatedly reactive specimens was confirmed by neutralization with anti-hbs using AxSYM HBsAg Confirmatory assay. The results of these tests are shown in Table 3.* TABLE 3 Number (%) of Reactive Samples Detected by and Confirmed as Positive for HBsAg Number of Initially Repeatedly samples reactive reactive Confirmed Group tested (%) (%) Positivea Specificity Blood Donors (0.10) 1 (0.05) % Blood Donors (0.05) 1 (0.05) % (including 401 first donors) Hospital Patients (0.40) 2 (0.40) % Medical Conditions 50 2 (4.00) 0 (0) % and Potentially 57 1 (1.75) 1 (1.75) % Interfering Substances (both unrelated to HBV Infection)b a A confirmed positive result in these studies was defined as neutralization by AxSYM HBsAg Confirmatory Assay. b Medical Conditions Unrelated to HBV Infection and Potentially Interfering Substances included the following categories of previously frozen specimens: high titre Immunoglobulin (IgG Myeloma, IgM Myeloma, Pregnant Females 1st, 2nd and 3rd trimester, Flu Vaccine Recipients), antibodies to E. coli, anti-cmv positive, anti-ebv positive, anti-hsv positive, rubella antibody positive, yeast infection, anti-nuclear antibody positive, syphilis positive, rheumatoid factor and toxoplasmosis antibody positive. The specificity of was 99.95% (4033/4035) in volunteer blood donors based on an assumed zero prevalence of HBsAg. In this calculation no repeatedly reactive volunteer blood donor sample was excluded due to confirmation by neutralization with anti-hbs. The specificity of was 99.60% (501/503) in hospital patients. Two of 503 specimens obtained from hospital patients were repeatedly reactive. In this calculation no repeatedly reactive sample was excluded due to confirmation by neutralization with anti-hbs. In 107 specimens from indiviuals with medical conditions unrelated to HBV infection and specimens containing potentially interfering substances, one specimen was repeatedly reactive and confirmed positive for HBsAg. SENSITIVITY The sensitivity of was evaluated using a six member proficiency panel of purified HBsAg (ad and ay) prepared at Abbott Laboratories. Each panel member was assayed in replicates of 3 on each of two instruments. Typical results are summarized in Tables 4, 5 and 6.* TABLE 4 Detection of Purified HBsAg/ad Concentration (ng/ml) S/N Value Result TABLE 5 Detection of Purified HBsAg/ay Concentration (ng/ml) S/N Value Result TABLE 6 Sensitivity Calculated by Linear Regression Analysis HBsAg Sensitivity Subtype (ng/ml) ad 0.19 ay

7 Typically sensitivity results from clinical trials and studies done at Abbott Laboratories have ranged from 0.10 to 0.60 ng/ml. In studies performed at Abbott Laboratories using HBsAg ad reference serum from Paul Ehrlich Institute (PEI), the assay calculated sensitivity was PEI Units/mL.* * These are representative performance data. Results obtained at individual BIBLIOGRAPHY 1. Engvall E and Perlmann P. Enzyme-Linked Immunosorbent Assay (ELISA) Quantitative Assay of Immunoglobulin G. Immunochem. 8: , Engvall E and Perlmann P. Enzyme-Linked Immunosorbent Assay (ELISA). In: Peeters H, editor. Protides of the Biological Fluids. Proceedings of the Nineteenth Colloquium, Burgge. Oxford: Pergamon Press, , Engvall E, Jonsson K and Perlmann P. Enzyme-Linked Immunosorbent Assay II. Quantitative Assay of Protein Antigen, Immunoglobulin G, By Means of Enzyme-Labelled Antigen and Antibody-Coated Tubes. Biochem. Biophys. Acta. 251: , VanWeemen BK and Schuurs AHWM. Immunoassay Using Antigen- Enzyme Conjugates. FEBS Letters 15: , Wisdom GB. Enzyme-Immunoassay. Clin. Chem. 22: , Wolters G, Kuijpers L, Kacaki J and Schuurs A. Solid-Phase Enzyme- Immunoassay for Detection of Hepatitis B Surface Antigen. J. Clin. Pathol. 29: , Wei R, Knight GJ, Zimmerman DH and Bond HE. Solid-Phase Enzyme Immunoassay for Hepatitis B Surface Antigen. Clin. Chem. 23: , David GS, Present W, Martinis J, Wang R, Bartholomew R, Desmond W and Sevier, ED. Monoclonal Antibodies in the Detection of Hepatitis Infection. Med. Lab. Sci. 38: , Drouet J, Courouce AM, Kalil J, Ropars C and Fellous M. Monoclonal Antibodies to HBsAg Produced by Murine Hybridomas. In: Szmuness W, Alter HJ and Maynard JE, editors. Viral Hepatitis. Philadelphia: Franklin Institute Press, , Goodall AH, Miescher G, Meek FM, Janossy G and Thomas HC. Monoclonal Antibodies in a Solid-Phase Radiometric Assay for HBsAg. Med. Lab. Sci. 38: , Kennedy RC, Ionescu-Matiu I, Alder-Storthz K, Henkel RD, Sanchez Y and Dreesman GR. Characterization of Anti-Hepatitis B Surface Antigen Monoclonal Antibodies. Intervirology 19: , Shih JW-K, Cote PJ, Dapolito GM and Gerin JL. Production of Monoclonal Antibodies Against Hepatitis B Surface Antigen (HBsAg) by Somatic Cell Hybrids. J. Virol. Methods 1: , Wands JR and Zurawski VR. High Affinity Monoclonal Antibodies to Hepatitis B Surface Antigen (HBsAg) Produced by Somatic Cell Hybrids. Gastroenterology 80: , Perrillo RP and Aach RD. The Clinical Course and Chronic Sequelae of Hepatitis B Virus Infection. Seminars in Liver Disease 1:15-25, Dusheiko G, Dibisceglie A, Bowyer S, Sachs E, Ritchie M, Schoub B and Kew M. Recombinant Leukocyte Interferon Treatment of Chronic Hepatitis B. Hepatology 5: , CDC. Prevention of Perinatal Transmission of Hepatitis B Virus: Prenatal Screening of all Pregnant Women for Hepatitis B Surface Antigen: Recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR, 37: , CDC. Hepatitis B Virus: A Comprehensive Strategy for Eliminating Transmission in the United States Through Universal Childhood Vaccination, Recommendations of the Immunization Practices Advisory Committee (ACIP), MMWR, 40:1-19, U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR part , Occupational Exposure to Bloodborne Pathogens; Final Rule. Federal Register, 235: , U.S. Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. HHS Publication No. (CDC) Washington, DC: U.S. Government Printing Office, May World Health Organization. Laboratory Biosafety Manual. Geneva: World Health Organization, National Committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Occupationally Acquired Infections: Approved Guideline - Second Edition. NCCLS Document M29-A2. Wayne, PA: NCCLS, CDC, Guidelines for the prevention of transmission of Human Immunodeficiency Virus and Hepatitis B Virus to health-care and publicsafety workers. MMWR 1989,38, (S-6); 16S. 23. Sehulster LM, Hollinger FB, Dreesman GR, and Melnick JL. Immunological and Biophysical Alteration of Hepatitis B Virus Antigens by Sodium Hypochlorite Disinfection. Applied and Environmental Microbiology. 42:762-7, Centers for Disease Control and Prevention Revised guidelines for the performance of CD4+T-cell determinations in persons with human immunodeficiency virus (HIV) infections. MMWR 1994, 43 (No. RR-3). 25. CDC. Epidemiologic Notes and Reports, Hepatitis B Contamination in a Clinical Laboratory - Colorado. MMWR 29: , Statistics for Experiments: An Introduction to Design, Data Analysis, and Model Building. Box GEP, Hunter WG and Hunter JS. Wiley J and Sons, Inc., , AxSYM and IMx are registered trademarks of Abbott Laboratories, Abbott Park, IL USA. For additional product information, please contact your local customer service organization. 7