TUMOR MARKERS CA 15-3

TUMOR MARKERS CA 15-3 E Tumor Markers CA 15-3 7K56-20 34-8878/R4 B7K560 Read Highlighted Changes Revised January, 2008 Customer Service For additional product information, please contact your local customer service organization. This package insert must be read carefully prior to product 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. Key to symbols used List Number In Vitro Diagnostic Medical Device Lot Number Expiration Date Store at 2-8 C Assay Number Standard Calibrator (A-F) Control Low, High (L, H) Reagent Pack Reaction Vessels Matrix Cells Store at 15-30 C CAUTION: Consult accompanying documents Manufacturer Sample Cups Checksum Consult instructions for use See REAGENTS section for a full explanation of symbols used in reagent component naming. ABBOTT Diagnostics Division 1

WARNING: CA 15-3 assay values obtained with different assay methods cannot be used interchangeably due to differences in assay methods and reagent specificity. The results reported by the laboratory to the physician must include the identity of the CA 15-3 assay used. If, in the course of monitoring a patient, the assay method used for determining serial CA 15-3 levels is changed, additional sequential testing should be carried out. Prior to changing assays, the laboratory MUST confirm baseline values for patients being serially monitored. The Abbott AxSYM CA 15-3 assay is based on the 115D8 and DF3 antibodies which are available exclusively through Fujirebio, Inc., its distributors and licensees. Specimens from patients who have received preparations of mouse monoclonal antibodies for diagnosis or therapy may contain human anti-mouse antibodies (HAMA). Such specimens may show either falsely elevated or depressed values when tested with assay kits which employ mouse monoclonal antibodies. These specimens should not be assayed with the AxSYM CA 15-3 assay. Refer to the LIMITATIONS OF THE PROCEDURE section in this assay package insert. NAME AxSYM CA 15-3: Cancer Antigen 15-3 INTENDED USE The AxSYM CA 15-3 assay is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative measurement of CA 15-3 assay values in human serum and plasma (EDTA) to aid in the management of Stage II and III breast cancer patients. Serial testing for patient CA 15-3 assay values should be used in conjunction with other clinical methods for monitoring breast cancer. SUMMARY AND EXPLANATION OF THE TEST CA 15-3 assay values are defined by using the 115D8 and DF3 monoclonal antibodies. 1,2 Monoclonal antibody 115D8, raised against human milk-fat globule membranes, and monoclonal antibody DF3, raised against a membrane enriched fraction of metastatic human breast carcinoma, react with epitopes expressed by a family of high molecular weight glycoproteins designated as polymorphic epithelial mucins (PEMs). 3-6 Research studies have indicated that CA 15-3 assay values are frequently elevated in patients with breast cancer. 7-17 These studies have suggested that the CA 15-3 assay may be of clinical value for monitoring the response of patients undergoing therapy because increasing and decreasing values correlated with disease progression and regression, respectively. 1,7,10,15-18 Additional published studies have suggested that increasing CA 15-3 assay values in patients at risk for breast cancer recurrence after primary therapy may be indicative of recurrent disease before it can be detected clinically. 10,15,16,19 Elevations of CA 15-3 assay values have been reported in individuals with nonmalignant conditions such as cirrhosis, hepatitis, autoimmune disorders, and benign diseases of the ovary and breast. 7,8 Non-mammary malignancies in which elevated CA 15-3 assay values have been reported include lung, colon, pancreatic, primary liver, ovarian, cervical, and endometrial. 7,20 CA 15-3 assay values are not elevated in most normal individuals. 7 The CA 15-3 assay is not recommended as a screening procedure to detect cancer in the general population; however, use of the CA 15-3 assay as an aid in the management of breast cancer patients has been reported. 7-19 BIOLOGICAL PRINCIPLES OF THE PROCEDURE The AxSYM CA 15-3 assay is based on Microparticle Enzyme Immunoassay (MEIA) technology. MEIA technology uses a solution of suspended, submicron sized latex particles to measure analytes. The particles are coated with a capture molecule specific for the analyte being measured. The effective surface area of microparticles increases assay kinetics and decreases assay incubation time. Reactants and sample for one assay are transferred to a reaction vessel (RV). In the RV, the reagents and sample are combined and then incubated to allow the reactants to come to reaction temperature. The reaction mixture is transferred to an inert glass fiber matrix. Irreversible binding of the microparticles causes the immune complex to be retained by the glass fibers while the reaction mixture flows rapidly through the large pores in the matrix. An Alkaline Phosphatase-labeled conjugate is added to the glass fiber matrix prior to the addition of 4-Methylumbelliferyl Phosphate (MUP). The conjugate catalyzes the hydrolysis of MUP to Methylumbelliferone (MU). Measurement of the fluorescent MU as it is generated on the matrix is proportional to the concentration of the analyte in the test sample. This assay is unique in that the Calibrators are supplied prediluted: The AxSYM System dilutes all Controls and specimens by the same final dilution factor as the prediluted Calibrators during the course of the assay. The AxSYM CA 15-3 reagents and sample are pipetted in the following sequence: SAMPLING CENTER Prediluted Calibrator, 115D8 Antibody Coated Microparticles, and Specimen Diluent are combined in a well of the reaction vessel (RV), forming an antigen-antibody complex. OR Specimen or Control is pipetted to one well of the reaction vessel where it is diluted by the same final dilution factor as the prediluted Calibrator with Specimen Diluent. The diluted specimen or Control, 115D8 Antibody Coated Microparticles, and Specimen Diluent are combined in a different well of the reaction vessel (RV), forming an antigen-antibody complex. The RV is immediately transferred into the Processing Center. Further pipetting is done in the Processing Center by the Processing Probe. PROCESSING CENTER The reaction mixture is incubated. 115D8 reactive determinants in the sample bind to the 115D8 Antibody Coated Microparticles. An aliquot of the reaction mixture is transferred to the matrix cell. The microparticles bind irreversibly to the glass fiber matrix. The matrix cell is washed to remove unbound materials. The DF3 Antibody: Alkaline Phosphatase Conjugate is dispensed onto the matrix cell and binds to the antigen-antibody complex. The matrix cell is washed to remove unbound materials. The substrate, 4-Methylumbelliferyl Phosphate, is added to the matrix cell and the rate of fluorescent product formation is measured by the MEIA optical assembly. The concentration of 115D8:DF3 reactive determinants in the sample is determined using a previously generated calibration curve. For further information, refer to the AxSYM System Operations Manual, Section 3. REAGENTS REAGENT PACK, 100 TESTS AxSYM CA 15-3 Reagent Pack (7K56-20)* 1 Bottle (9.4 ml) 115D8 Antibody (Mouse, Monoclonal) Coated Microparticles in TRIS buffer. Preservative: Sodium Azide. (Reagent Bottle 1) 1 Bottle (12.1 ml) DF3 Antibody (Mouse, Monoclonal): Alkaline Phosphatase Conjugate in TRIS buffer with protein (bovine) stabilizers. Minimum Concentration: 0.1 μg/ml. Preservatives: Sodium Azide and Antimicrobial Agents. (Reagent Bottle 2) 1 Bottle (45.2 ml) Specimen Diluent, TRIS buffer with protein (bovine and murine) stabilizers. Preservatives: Sodium Azide and Antimicrobial Agents. (Reagent Bottle 4) * 7K56-20 includes an AxSYM CA 15-3 Reagent Pack (100 tests), reaction vessels (100 each) and matrix cells (100 each). CALIBRATORS AxSYM CA 15-3 Standard Calibrators (7K91-01) 6 Bottles (4 ml each) of AxSYM CA 15-3 Standard Calibrators with 115D8: DF3 reactive determinants (human) prediluted in TRIS buffer with protein (bovine) stabilizers to yield the following assay values: Bottle CA 15-3 Assay Value (U/mL)* 0 15 60 120 180 250 Preservatives: Sodium Azide and Antimicrobial Agents. * CA 15-3 assay values are expressed as U/mL. A unit is a value related to an Abbott maintained reference preparation. The calibrators for the CA 15-3 assay are manufactured either gravimetrically or volumetrically and are referenced to this standard prepared by Abbott Laboratories. There is no internationally recognized standard available at this time. 2

CONTROLS AxSYM CA 15-3 Controls (7K91-10) 2 Bottles (8 ml each) of AxSYM CA 15-3 Controls with 115D8:DF3 reactive determinants (human) prepared in TRIS buffer with protein (bovine) stabilizers to yield the following assay value ranges: Bottle CA 15-3 Assay Value (U/mL) Range (U/mL) 35 26.3-43.8 150 112.5-187.5 Preservatives: Sodium Azide and Antimicrobial Agents. SPECIMEN DILUENT AxSYM CA 15-3 Specimen Diluent (7K56-50) 1 Bottle (100 ml) AxSYM CA 15-3 Specimen Diluent, TRIS buffer with protein (bovine and murine) stabilizers. Preservatives: Sodium Azide and Antimicrobial Agents. OTHER REAGENTS Solution 1 (MUP) (8A47-04) 4 Bottles (230 ml each) Solution 1 (MUP) containing 4-Methylumbelliferyl Phosphate, 1.2 mm in AMP 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. AxSYM Probe Cleaning Solution (9A35-05) 4 Bottles (110 ml each)/2 Bottles (220 ml each) AxSYM Probe Cleaning Solution containing 2% Tetraethylammoniumhydroxide (TEAH). WARNINGS AND PRECAUTIONS For In Vitro Diagnostic Use. SAFETY PRECAUTIONS CAUTION: This product contains human sourced infectious and/or potentially infectious components. Refer to the REAGENTS section of this package insert. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Therefore, it is recommended that all human sourced materials be considered potentially infectious and handled with appropriate biosafety practices. This product contains sodium azide; for a specific listing, refer to the REAGENTS section. Contact with acids liberates very toxic gas. This material and its container must be disposed of in a safe way. For product not classified as dangerous per European Directive 1999/45/EC as amended - Safety data sheet available for professional user on request. HANDLING PRECAUTIONS Do not use Solution 1 (MUP) beyond the expiration date or a maximum of 14 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 of MUP 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. Refer to the AxSYM System Operations Manual, Sections 7 and 8 for a more detailed discussion of the safety and handling precautions during system operation. STORAGE INSTRUCTIONS The AxSYM CA 15-3 Reagent Pack, Calibrators, Controls, and Specimen Diluent must be stored at 2-8 C and may be used immediately after removing them from the refrigerator. Do not freeze the AxSYM CA 15-3 Reagents, Calibrators, Controls, or Specimen Diluent. Calibrators, Controls, and Specimen Diluent should be returned to 2-8 C storage immediately after use. Reagents are stable until the expiration date when stored and handled as directed. The AxSYM CA 15-3 Reagent Pack may be on-board the AxSYM System for a maximum of 112 cumulative hours; for example, 14 eight hour shifts. Refer to the AxSYM System Operations Manual, Sections 2, 5, and Appendices, for further information on tracking on-board time. Solution 1 (MUP) must be stored at 2-8 C (do not freeze). It may be on-board the AxSYM System for a maximum of 14 days. After 14 days, it must be discarded. It may be used immediately after removing it from the refrigerator. The AxSYM Probe Cleaning Solution, Solution 3 (Matrix Cell Wash), and Solution 4 (Line Diluent) must be stored at 15-30 C. INSTRUMENT PROCEDURE Assay File Installation The AxSYM CA 15-3 Assay File must be installed on the AxSYM System from the software disk, 03D50-02, or higher, prior to performing CA 15-3 assays. Refer to the AxSYM System Operations Manual, Section 2, for proper installation procedures. AxSYM CA 15-3 Assay Parameters The default values for the assay parameters used for the AxSYM CA 15-3 assay are listed below. Assay parameters that can be edited contain a (>) symbol. These parameters can be displayed and edited according to the procedure in the AxSYM System Operations Manual, Section 2. In order to obtain values for the parameters with an asterisk (*), review the specific Assay Parameter screen. Press PRINT to print the assay parameters. Assay Parameters 1 Long Assay Name (English): CA_15-3 6 Abbrev Assay Name (English): CA_15-3 11 Assay Number: 496 12 Assay Version: * 13 Calibration Version: * 14 Assay File Revision: * 15 Assay Enabled > ON 17 Assay Type: MEIA 18 Standard Cal Reps > 2 21 Cal A Concentration: 0 22 Cal B Concentration: 15.0 23 Cal C Concentration: 60.0 24 Cal D Concentration: 120.0 25 Cal E Concentration: 180.0 26 Cal F Concentration: 250.0 43 Default Dilution Protocol > SMPL/CTRL 44 Default Calibration Method > Standard Calibration 45 Selected Result Concentration Units > U/mL 46 Selected Result Decimal Places > 1 75 Low Extreme Value > 0.50 76 High Extreme Value > 2500.00 92 High Range Undiluted: * Note: Assay parameter #43, Default Dilution Protocol option SMPL/CTRL is an undiluted protocol. Note: Values associated with the Low and High Extreme flags, Assay Parameter #75 and #76, are assay specific and should not be edited. 3

It is recommended that the General Configuration Parameter, Release Mode, be set to the Manual or Hold release mode to ensure that all flagged results are reviewed prior to reporting assay results. Refer to the AxSYM System Operations Manual, Section 2, for a detailed description of Instrument Procedures. If General Configuration parameter, Release Mode, is configured to the Automatic release mode, ensure that all flagged results are reviewed prior to reporting assay results. SPECIMEN COLLECTION AND PREPARATION FOR ANALYSIS Human serum or plasma (EDTA) may be used with the AxSYM CA 15-3 assay. When serial specimens are being evaluated, the same type of specimen should be used throughout the study. The AxSYM System does not provide the capability to verify specimen type. It is the responsibility of the operator to verify the correct specimen type(s) is(are) used in the AxSYM CA 15-3 assay. Do not test grossly hemolyzed specimens. Ensure that complete clot formation has taken place prior to centrifugation. Some specimens, especially those from patients receiving anticoagulant or thrombolytic therapy, may exhibit increased clotting time. If a serum specimen is centrifuged before a complete clot forms, the presence of fibrin may cause erroneous results. For optimal results, specimens should be free of fibrin, red blood cells, or other particulate matter. Specimens showing particulate matter, erythrocytes, or turbidity must be clarified by centrifugation before testing. Specimens may be stored for up to 24 hours at 2-8 C prior to being tested. If testing will be delayed more than 24 hours, the specimen should be stored at -20 C or colder. Specimens that have been stored at -20 C or colder for 12 months have shown no performance differences. Multiple freeze/thaw cycles should be avoided. Mix specimens thoroughly after thawing, by low speed vortexing or gently inverting, to ensure consistency in the results. To minimize the effects of evaporation, all samples (patient specimens, 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 a more detailed discussion of on-board sample storage constraints. When shipped, samples must be packaged and labeled in compliance with applicable state, federal, and international regulations covering the transport of clinical samples and infectious substances. Inspect all samples for bubbles. Remove all bubbles prior to analysis. SAMPLE VOLUME The sample volume required to perform a single CA 15-3 test on the AxSYM System varies according to the different sample containers. For sample cups, a ROUTINE test requires 150 μl and a STAT test requires 105 μl. For every additional CA 15-3 test performed (ROUTINE or STAT) from the same container, an additional 55 μl of sample is required. The sample cup minimum volumes for both STAT and ROUTINE tests are calculated by the AxSYM System. They are displayed on the Order screen at the time the test(s) is(are) ordered, and printed in the Orderlist Report. When using Host Order Query, the Order screen information and the Orderlist Report are not available. Refer to the AxSYM System Operations Manual, Section 5: Ordering Patient Samples, for a description of the Host Order Query option. If the assay is configured for auto retest/auto dilution, the additional sample volume needed for the retest/dilution will not be displayed on the Order screen at the time the test(s) is(are) ordered. Therefore, the total sample volume should include an additional 55 μl of sample. To obtain the recommended volume requirements for the AxSYM CA 15-3 Calibrators and Controls, invert, hold the bottles vertically and dispense 5 drops of each Calibrator and 4 drops of each Control into each respective sample cup. Refer to the AxSYM System Operations Manual, Section 5 for sample volume requirements in primary or aliquot tubes and Calibrator/Control requirements for multiple reagent lots. AxSYM CA 15-3 PROCEDURE Materials Required 7K56-20 AxSYM CA 15-3 Reagent Kit, containing: AxSYM CA 15-3 100 100 Materials Required but not Provided AxSYM System 7K91-10 AxSYM CA 15-3 Controls 7K91-01 AxSYM CA 15-3 Standard Calibrators 7K56-50 AxSYM CA 15-3 8A47-04 8A81-04 8A46 9A35-05 AxSYM 8A76-01 CAUTION: Use a separate Pipette tip for each sample. Use accurately calibrated equipment. When manually dispensing sample into sample cups, verify that dispensing equipment does not introduce cross-contamination and delivers specified sample volume. 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 Sections 5 and 6 of the AxSYM System Operations Manual can easily be removed for use at the instrument. They contain detailed steps for performing assay calibration and sample testing procedures. Prior to ordering tests, confirm that the system inventory of matrix cells, bulk solutions, and waste levels are acceptable. Note: The AxSYM CA 15-3 Calibrators are supplied prediluted, a D (dilution) appears in the Flags field of the Order List and Order Status reports for Calibrators. The Orderlist Report contains sample placement information and sample cup volume requirements for all ordered tests. It is recommended that this report be referenced when loading samples into sample segments. CAUTION: When operating the AxSYM System, always observe the following: The System status must be WARMING, PAUSED, READY, or STOPPED before adding or removing sample segments, reagent packs, or reaction vessels. Do not open the Interior Waste Door or the AxSYM Processing Center Cover while any test is in process. If opened, all processing will stop. Any tests will be terminated and must be repeated. The cap for reagent bottle 4 must be manually opened prior to running an AxSYM CA 15-3 Assay. Upon completion of the run, close the reagent bottle 4 cap securely. When testing is completed, it is recommended that samples and the AxSYM CA 15-3 Reagent Pack be removed from the Sampling Center to maximize the on-board reagent pack use. Store at 2-8 C. SAMPLE DILUTION PROCEDURES Patient specimens with a CA 15-3 assay value exceeding 250 U/mL (HIGH RANGE, assay parameter 92), are flagged with the code >250. To quantitate the concentration of these specimens, perform either the Automated Dilution Protocol or the Manual Dilution Protocol. Automated Dilution Protocol The Automated Dilution Protocol is provided to assist in quantitating test results greater than 250 U/mL up to 2,500 U/mL. The AxSYM System performs a 1:10 dilution of the unknown specimens using one reaction vessel. The AxSYM System automatically calculates the concentration of the diluted specimen and reports the results. If the assay is configured for auto dilution, the additional 55 μl of specimen volume needed for the dilution should be included in the sample container when ordering tests. Refer to the AxSYM System Operations Manual, Section 5, for additional information on ordering sample dilutions. 4

Manual Dilution Protocol A manual dilution can be performed by making a dilution of the specimen with the AxSYM CA 15-3 Specimen Diluent (7K56-50) before pipetting the specimen into the sample cup. It is desirable to perform the dilution so that the diluted specimen reads above 15 U/mL on the calibration curve. Example: A ten-fold dilution is prepared by adding 100 μl of specimen to 900 μl of AxSYM CA 15-3 Specimen Diluent. Mix thoroughly before assaying. To determine the final CA 15-3 assay value of the specimen, multiply the assay value of the diluted specimen by the dilution factor. QUALITY CONTROL PROCEDURES CALIBRATION The AxSYM CA 15-3 assay must be calibrated using a Standard Calibration (6-point) procedure. Standard Calibration To perform an AxSYM CA 15-3 Standard Calibration, test CA 15-3 Standard Calibrators A, B, C, D, E, and F in duplicate. A single sample of the CA 15-3 Low and High Controls must be tested as a means of evaluating the assay calibration. Once the AxSYM CA 15-3 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. Controls are out of range. Refer to the AxSYM System Operations Manual, Section 6, for: Setting up an assay calibration When recalibration may be necessary Calibration verification 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 an error code. Refer to the AxSYM System Operations Manual, Appendices, for an explanation of the calibration validity parameters that may be used by the AxSYM System. QUALITY CONTROL The recommended control requirement for an AxSYM CA 15-3 Assay is a single sample of the CA 15-3 Low and High Controls tested once every 24 hours. Controls may be placed in any Segment position in the Sample Carousel. If the quality control procedures in your laboratory require more frequent use of controls to verify test results, follow those procedures. Ensure that assay control values are within assay value ranges specified in the package insert. Refer to the REAGENTS, CONTROLS section of this package insert for AxSYM CA 15-3 Control ranges. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS When a CA 15-3 control value is out of its specified range, it may indicate deterioration of the reagents or errors in technique. Associated test results may be invalid and require retesting. Assay recalibration may be indicated. Refer to the AxSYM System Operations Manual, Section 10, Subsection: Observed Problems, for further troubleshooting information. The AxSYM System has a capability to generate a Levey-Jennings plot of each assay s quality control performance. Refer to the AxSYM System Operations Manual, Section 5. At the discretion of the laboratory, selected quality control rules may be applied to the quality control data. Fluorescence Background Acceptance Criteria Quality control of the MUP substrate blank is automatically determined by the instrument and checked under Assay Parameter 64 (Max Intercept minus 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 this parameter. RESULTS AxSYM CA 15-3 utilizes a four parameter logistic curve fit (4PLC Analysis) to generate a calibration curve. Refer to the AxSYM System Operations Manual, Appendix F for further information. FLAGS Some results may contain information in the Flags field. Samples flagged as low extreme values (LL), assay parameter #75, must be reviewed prior to reporting assay results. Results at or near the assay sensitivity should be verified prior to reporting CA 15-3 assay values. For a description of the flags that may appear in this field, refer to AxSYM System Operations Manual, Sections 1 and 2. LIMITATIONS OF THE PROCEDURE Specimens from patients who have received preparations of mouse monoclonal antibodies for diagnosis or therapy may contain human anti-mouse antibodies (HAMA). Such specimens may show either falsely elevated or depressed values when tested with assay kits which employ mouse monoclonal antibodies. 21-23 These specimens should not be assayed with the AxSYM CA 15-3 assay. Patients with confirmed breast carcinoma frequently have CA 15-3 assay values in the same range as healthy individuals. Elevations in circulating 115D8:DF3 reactive determinants may be observed in patients with nonmalignant disease. For these reasons, a CA 15-3 assay value, regardless of level, should not be interpreted as absolute evidence for the presence or absence of malignant disease. The CA 15-3 assay value should be used in conjunction with information available from clinical evaluation and other diagnostic procedures. THE AxSYM CA 15-3 ASSAY SHOULD NOT BE USED AS A CANCER SCREENING TEST. The AxSYM CA 15-3 Calibrators are supplied prediluted. A specialized protocol dilutes all controls and specimens by the same final dilution factor as the prediluted Calibrators. Refer to the SPECIMEN COLLECTION AND PREPARATION FOR ANALYSIS section in this package insert. EXPECTED VALUES Changes observed in serial CA 15-3 assay values when monitoring Stage II or Stage III breast cancer patients should be used in conjunction with other clinical methods used for monitoring breast cancer patients. The distribution of CA 15-3 assay values determined in 2,380 specimens is shown in the table below. (Values greater than 31.3 U/mL were considered elevated) Distribution of AxSYM CA 15-3 Assay Values Number of Subjects 0-31.3 U/mL Percent (%) 31.4-60 U/mL 60.1-120 U/mL >120.1 U/mL HEALTHY FEMALES < 50 years 391 99.7 0.3 0.0 0.0 50 years 307 98.4 1.6 0.0 0.0 Total 698 99.1 0.9 0.0 0.0 MALIGNANT DISEASES Breast Stage I 132 90.9 9.1 0.0 0.0 Stage II 307 77.5 11.4 8.5 2.6 Stage III 129 61.2 11.6 3.1 24.0 Stage IV 175 46.9 11.4 8.6 33.1 Colorectal 110 90.0 6.4 3.6 0.0 Lung 115 87.8 7.8 2.6 1.7 Liver 26 69.2 23.1 0.0 7.7 Ovarian 130 61.5 15.4 11.5 11.5 Pancreas 27 77.8 22.2 0.0 0.0 Uterine/Cervical 122 86.1 10.7 3.3 0.0 NONMALIGNANT DISEASES Breast 130 98.5 1.5 0.0 0.0 Lung 50 84.0 4.0 4.0 8.0 Kidney 50 90.0 6.0 2.0 2.0 Liver 67 92.5 6.0 1.5 0.0 Other 112 100.0 0.0 0.0 0.0 5

In this study, 97.4% of the healthy female subjects had CA 15-3 assay values at or below 28.0 U/mL, and 99.1% of the healthy female subjects had CA 15-3 assay values at or below 31.3 U/mL (mean=14.1, SD=6.0). The Malignant Diseases portion of the distribution table above is derived primarily from monitored carcinoma patients representing both active (clinical evidence of disease progression) and inactive (no clinical evidence of disease progression) disease states. It is recommended that each laboratory establish its own reference value for the population of interest. Clinical Performance in Breast Cancer Patients Seventy-nine (79) Stage II and III breast cancer patients, who were clinically free of disease following surgery were identified from previously conducted prospective trials of adjuvant therapies. The group included patients who subsequently experienced disease recurrence and patients who experienced no disease recurrence during follow-up. Serial plasma samples from seventy-seven (77) patients who met pre-established eligibility criteria were selected and analyzed in a blinded study using the Abbott AxSYM CA 15-3 assay and the TRUQUANT BR RIA. All specimens (n=359) from the 77 evaluable patients were analyzed to determine the concordance (agreement) between the two assays using their respective reference values. Ninety-one percent (91%) of the samples tested using both assays were in agreement. AxSYM CA 15-3 Assay Value TRUQUANT BR RIA 31.3 U/mL > 31.3 U/mL Total 37.7 U/mL 304 29 333 > 37.7 U/mL 4 22 26 Total 308 51 359 Concordance = 91% (326/359) Using the last value prior to relapse (0.1-46.5 months prior to relapse) or the last value prior to discontinuation (for patients who did not relapse), the clinical sensitivity of both assays was 35% (95% Confidence Interval = 16-57), and the specificities were 94% (95% CI = 85-99) and 91% (95% CI = 80-97) for the AxSYM CA 15-3 assay and TRUQUANT BR RIA, respectively. The sensitivity of both assays improved when the assay data for relapsed patients was restricted to values obtained within 6 months of relapse as shown in the following tables: Diagnosis No. AxSYM CA 15-3 TRUQUANT BR RIA Patients Assay Value 31.3 > 31.3 37.7 > 37.7 U/mL U/mL U/mL U/mL Recurrence 13 6 7 5 8 No Recurrence 54 51 3 49 5 Total 67 57 10 54 13 Assay Sensitivity (%) 95% CI Specificity (%) 95% CI AxSYM CA 15-3 54 25-81 94 85-99 TRUQUANT BR RIA 62 32-86 91 80-97 SPECIFIC PERFORMANCE CHARACTERISTICS PRECISION Reproducibility was determined as described in National Committee for Clinical Laboratory Standards (NCCLS) protocol EP5-T2. 24 A five member panel was assayed, at 3 laboratories, in replicates of 2 at two separate times per day for twenty days (n=80 for each sample at each site) using a single lot of reagents and a single calibration. Panel members 1 and 2 are buffered protein based, and panel members 3, 4, and 5 are human serum based. Data from this study are summarized below. Panel Reproducibility of AxSYM CA 15-3 Assay Mean Within Between Between Value Run Run Day Lab (U/mL) (%CV) (% CV) (% CV) Total (% CV) 1 1 33.5 3.5 1.8 2.3 4.5 2 33.9 5.4 5.2 4.2 8.6 3 38.1 3.6 3.0 2.1 5.1 2 1 129.7 5.1 3.2 0.0 6.0 2 137.5 5.7 0.0 4.1 7.0 3 158.5 6.1 0.0 3.6 7.1 3 1 12.9 3.5 1.0 1.1 3.8 2 13.3 3.3 2.0 3.5 5.2 3 14.5 2.8 2.3 2.0 4.1 4 1 87.2 3.9 4.0 2.3 6.0 2 94.2 3.8 2.6 3.1 5.6 3 104.8 3.3 3.2 3.0 5.4 5 1 130.5 5.5 4.8 0.0 7.2 2 136.6 5.1 3.3 3.3 6.9 3 160.8 4.7 3.9 3.3 6.9 The standard deviation may be calculated by multiplying the mean CA 15-3 assay value by the percent CV and dividing by 100. SD = Mean (U/mL) x %CV 100 MEASUREMENT RANGE The measurement (reportable) range of the AxSYM CA 15-3 assay is 0.3 U/mL to 250 U/mL, as defined by the analytical sensitivity lower limit and the upper limit of the calibration range. For patient specimens with a CA 15-3 assay value exceeding 250 U/mL refer to the sample dilution procedures section of this package insert. RECOVERY Varying amounts of a sample with a known CA 15-3 assay value were added to human serum or plasma containing an endogenous level of 115D8:DF3 reactive determinants. The CA 15-3 assay value was determined using the AxSYM CA 15-3 assay and the resulting percent recovery was calculated. Recovery Serum Sample Endogenous CA 15-3 Assay Value (U/mL) 115D8:DF3 Reactive Determinants Added (U/mL) CA 15-3 Assay Value Obtained (U/mL) Percent Recovery* (%) A 14.5 20.9 36.3 103 75.3 88.8 99 142.9 146.5 93 B 11.3 20.9 32.6 101 75.3 87.2 101 142.9 158.7 103 C 17.5 20.9 39.5 103 75.3 99.9 108 142.9 169.9 106 * % Recovery = U/mL Assay Value Obtained x 100 U/mL Endogenous Assay Value + 115D8:DF3 Reactive Determinants Added 6

ANALYTICAL SENSITIVITY The sensitivity of the AxSYM CA 15-3 assay was calculated to be better than 0.3 U/mL (n = 36 runs in replicates of 10). This sensitivity is defined as the concentration two standard deviations greater than the AxSYM CA 15-3 Calibrator A (0 U/mL) and represents the lowest concentration of 115D8:DF3 reactive determinants that can be distinguished from zero. ANALYTICAL SPECIFICITY The specificity of the AxSYM CA 15-3 assay was analyzed by performing recovery studies on sera containing the following compounds. Recoveries were determined to have no clinically significant effect on AxSYM CA 15-3 assay values at the levels indicated. INTERFERING SUBSTANCES Test Compound Bilirubin Hemoglobin IgG Total Protein Triglyceride CHEMOTHERAPEUTIC DRUGS Test Compound Cisplatin Cyclophosphamide Doxorubicin β-estradiol Estrone 3-Sulfate 5-Fluorouracil Megestrol Acetate Methotrexate Mitomycin C Paclitaxel Tamoxifen Testosterone Vinblastine THERAPEUTIC DRUGS Test Compound Acetaminophen Coumadin Heparin Multi-Vitamin Salicylate Vitamin B Vitamin C Vitamin E Test Concentration 20 mg/dl 600 mg/dl 250-2900 mg/dl 3-12 gm/dl 3000 mg/dl Test Concentration 66.7 μg/ml 330.0 μg/ml 6.6 μg/ml 6.7 μg/ml 6.7 μg/ml 280.0 μg/ml 39.6 μg/ml 13.2 μg/ml 12.7 μg/ml 3.5 ng/ml 5.0 μg/ml 33.0 μg/ml 1.3 μg/ml Test Concentration 747 μg/ml 1.4 mg/ml 7 U/mL 200% RDA 3.02 mg/dl 200% RDA 200% RDA 200% RDA METHOD COMPARISON The AxSYM CA 15-3 assay was compared to the TRUQUANT BR RIA on 560 specimens with AxSYM CA 15-3 assay values ranging from 4.0 U/mL to 248.7 U/mL. Linear regression analysis yielded the following results: (AxSYM) = 0.67 (TRUQUANT) + 4.2 U/mL r = 0.888 BIBLIOGRAPHY 1. Hayes DF, Zurawski V, Kufe D. Comparison of Circulating CA15-3 and Carcinoembryogenic Antigen Levels in Patients with Breast Cancer. J Clin Onc 1986;4:1542-1550. 2. Tobias R, Rothwell C, Wagner J, et al. Development and Evaluation of a Radioimmunoassay for the Detection of a Monoclonal Antibody Defined Breast Tumor Associated Antigen 115D8/DF3. Clin Chem 1985;31:986. 3. Hilkens J, Buijs F, Hilgers J, et al. Monoclonal Antibodies Against Human Milk-Fat Globule Membranes Detecting Differentiation Antigens of the Mammary Gland and its Tumors. Int J Cancer 1984;34:197-206. 4. Hilkens J, Hilgers J, Buijs F, et al. Monoclonal Antibodies Against Human Milk-Fat Globule Membranes Useful in Carcinoma Research. Prot Biol Fluids 1984;31:1013-1016. 5. Kufe D, Inghirami G, Abe M, et al. 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Relative Operating Characteristic Analysis and Group Modeling for Tumor Markers: Comparison of CA 15-3, Carcinoembryonic Antigen, and Mucin-like Carcinoma-associated Antigen in Breast Carcinoma. Cancer Research 1991;51:1904-1909. 17. Tondini C, Hayes DF, Gelman R, et al. Comparison of CA 15-3 and Carcinoembryonic Antigen in Monitoring the Clinical Course of Patients with Metastatic Breast Cancer. Cancer Research 1988;48:4107-4112. 18. Robertson JFR, Pearson D, Price MR, et al. Assessment of Four Monoclonal Antibodies as Serum Markers in Breast Cancer. Eur J Cancer 1990;26:1127-1132. 19. Geraghty JG, Coveney MB, Sherry F, et al. CA 15-3 in Patients with Locoregional and Metastatic Breast Carcinoma. Cancer 1992;70:2831-2834. 20. Colomer R, Ruibal A, Genollà J, et al. Circulating CA 15-3 Antigen Levels in Non-mammary Malignancies. Br J Cancer 1989;59:283-286. 21. Primus FJ, Kelley EA, Hansen HJ, et al. Sandwich -Type Immunoassay of Carcinoembryonic Antigen in Patients Receiving Murine Monoclonal Antibodies for Diagnosis and Therapy. Clin Chem 1988;34:261-264. 22. Hansen HJ, Solving the Problem of Antibody Interference in Commercial Sandwich -Type Immunoassays of Carcinoembryonic Antigen. Clin Chem 1989;35:146-151. 23. Schroff RW, Foon KA, Beatty SM, et al. Human Anti-Murine Immunoglobulin Responses in Patients Receiving Monoclonal Antibody Therapy. Cancer Res 1985;45:879-885. 7

24. National Committee for Clinical Laboratory Standards. Evaluation of Precision Performance of Clinical Chemistry Devices; Second Edition: Tentative Guideline. NCCLS Document EP5-T2. Villanova, PA:NCCLS, 1992. All trademarks are properties of their respective owners. Abbott Ireland Diagnostics Division Finisklin Business Park Sligo Ireland + 353-71-9171712 January 2008 2005, 2008 Abbott Laboratories 8