Intended for Use in the United States Lot 500 and Above

INSTRUCTIONS FOR USE

VITROS Immunodiagnostic Products CA 15-3TM Reagent Pack

CA 153
CA 15-3

Intended Use
For in vitro diagnostic use only. The VITROS CA 15-3 Reagent Pack quantitatively measures DF3 defined antigen concentration in human serum and plasma of patients previously treated for stage II or stage III breast cancer. Serial test results obtained with the VITROS CA 15-3 assay, in patients who are clinically free of disease, should be used in conjunction with all relevant information derived from diagnostic tests, physical examination and full medical history in accordance with appropriate patient management procedures used for early detection of recurrence. The test is also intended for use as an aid in the management of breast cancer patients with metastatic disease by monitoring progression or response to treatment.

Summary and Explanation of the Assay

Breast cancer is the most common malignancy among women. Approximately, one out of every nine women will develop breast cancer and approximately 30% of women who have this malignancy will die of the disease. Metastatic disease may be present at the time of initial diagnosis and can occur at any time following primary therapy. Up to 70% of patients with metastases will respond to systemic treatment with cytotoxic drugs or 1 endocrine therapy; therefore, early detection of recurrence is important to patient management. The median survival time following diagnosis of recurrent disease is approximately two years, but may range from a few 2, 3 months to decades. The VITROS CA 15-3 assay utilizes two monoclonal antibodies (115D8 as capture and DF3 as conjugate) which react with DF3 defined antigen, expressed by human breast carcinoma cells. The 115D8 antibody was raised 4, 5, 6, 7 against antigens of human milkfat globule membranes. The DF3 antibody was prepared against a 8, 9 The results of studies indicate that the membrane-enriched fraction of a human breast carcinoma. concentration of the DF3 defined antigen is frequently elevated in the serum of patients with breast cancer as 10, 11, 12, 13 well as with other malignancies, such as lung cancer, and in some non-malignant disorders. CA 15-3 assay concentrations were not elevated in the sera of the majority of normal individuals. In patients previously treated for stage II or stage III disease, early detection of recurrence cannot be readily accomplished by routine clinical or diagnostic studies alone. The use of a circulating serum marker assay, such as VITROS CA 15-3, can be useful in the identification of these patients.
10, 11, 12, 13

Principles of the Procedure

The VITROS CA 15-3 assay is performed using the VITROS CA 15-3 Reagent Pack and VITROS Immunodiagnostic Products CA 15-3 Calibrators on the VITROS ECi Immunodiagnostic System with Intellicheck. An immunometric technique is used. DF3 defined antigen present in the sample reacts with a biotinylated antibody (115D8 mouse monoclonal anti-DF3 defined antigen). The antigen-antibody complex is captured by streptavidin on the wells, and unbound materials are removed by washing. In a second incubation, a horseradish peroxidase (HRP)-labeled antibody conjugate (DF3 mouse monoclonal anti-DF3 defined antigen) binds to the immobilized DF3 defined antigen. Unbound conjugate is removed by washing. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer 14 agent (a substituted acetanilide) is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent increases the level and duration of light produced. The light signals are read by the VITROS ECi System. The amount of HRP conjugate bound is directly proportional to the concentration of DF3 defined antigen present in the sample.
Version 2.1 Pub. No. J03795 1

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

Assay Time and Temperature Incubation time: 32 minutes Time to first result: 42 minutes Temperature: 37C

Assay Type Immunometric assay

Reaction Scheme

Warnings and Precautions

Take care when handling material of human origin. Consider all clinical specimens and calibrators as potentially 15 infectious. Handle specimens and assay components in accordance with NCCLS or other published biohazard safety guidelines or regulations. No test method can offer complete assurance that infectious agents are absent.

Reagents
Reagent Pack Contents
One VITROS CA 15-3 Reagent Pack (CAT No. 680 1758) contains: 100 coated wells (streptavidin, binds 3 ng biotin/well). 20.2 mL* conjugate reagent (HRP-labeled DF3 mouse monoclonal anti-DF3 defined antigen, binds 36.2 U DF3 defined antigen/mL) in buffer with antimicrobial agent, bovine serum albumin and bovine gamma globulin. 15.8 mL** biotinylated antibody reagent (biotinylated 115D8 mouse monoclonal anti-DF3 defined antigen, binds 41.7 U DF3 defined antigen/mL) in buffer with antimicrobial agent, bovine serum albumin and bovine gamma globulin. * Lots below Lot 600 contain 24.1 mL
** Lots below Lot 600 contain 19.8 mL

Reagent Pack Handling

The reagent pack is supplied ready for use. Reagent packs do not need mixing. Avoid agitation, which may cause foaming or the formation of bubbles.

Reagent Pack Stability

When stored and handled as specified in the package labeling, the VITROS CA 15-3 Reagent Pack is suitable for use until the expiration date printed on the outside of the carton.
2 Pub. No. J03795 Version 2.1

INSTRUCTIONS FOR USE

Reagent Pack Storage and Preparation

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

Store the unopened reagent pack refrigerated at 2 8C (36 46F). Do not freeze. Load reagent packs directly from refrigerated storage to minimize condensation. Use opened reagent packs within 8 weeks. Store opened reagent packs in the VITROS ECi System reagent supply, or refrigerated at 2 8C (36 46F) in a sealed reagent pack storage box that contains dry desiccant.

Specimen Collection and Preparation

Patient Preparation
No special patient preparation is necessary.

Recommended Specimen Types

Serum, EDTA or heparin plasma.

Specimens Not Recommended

Turbidity in samples may affect assay results.

Special Precautions
Certain specimen collection devices have been reported to affect other analytes and assays. collection devices are compatible with this assay.
16

Confirm that your

Specimen Collection and Preparation

Collect specimens using standard procedures. The VITROS CA 15-3 assay uses 10 L of sample for each determination. For details on minimum fill volume, refer to the VITROS ECi Immunodiagnostic System Operators Guide. Mix samples, calibrators and controls by inversion and bring to 1530C (5986F) before use.
17, 18

Handling and Storage Conditions

Handle specimens in stoppered containers to avoid contamination and evaporation. Specimen storage recommendations: Refrigerate at 28C (3646F) for up to 7 days.

For long term storage, freeze at -20C (-4F) or below. Avoid repeated freeze and thaw cycles.

Version 2.1

Pub. No. J03795

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

Assay Procedure
Materials Required But Not Provided
The following items are required to perform the assay for CA 15-3: VITROS CA 15-3 Calibrators VITROS Immunodiagnostic Products Signal Reagent VITROS Immunodiagnostic Products Universal Wash Reagent Quality control materials, such as VITROS Immunodiagnostic Products Oncology Controls VITROS Immunodiagnostic Products High Sample Diluent B. VITROS Immunodiagnostic Products Reagent Pack Storage Box (optional) with desiccant

Operating Instructions
Refer to the VITROS ECi System Operators Guide for complete instructions on the operation of your VITROS ECi System.

Sample Dilution
Serum or plasma samples with concentrations greater than the reportable range may be diluted up to 5-fold (1 part sample with 4 parts diluent) with VITROS High Sample Diluent B prior to assay. Refer to the High Sample Diluent B package insert sheet.

Calibration
Required Calibrators
VITROS CA 15-3 Calibrators

Calibrator Preparation, Handling, and Storage

Refer to the calibrator package insert for information on the use of VITROS CA 15-3 Calibrators.

Calibration Procedure
Refer to the VITROS ECi System Operators Guide for detailed instructions on how to calibrate.

When to Calibrate
Calibrate when the assay reagent lot changes Calibrate every 28 days

The VITROS CA 15-3 assay may also need to be calibrated: After specified service procedures have been performed (see the VITROS ECi System Operators Guide) If quality control results are consistently outside acceptable limits For additional information on when to calibrate, refer to the VITROS ECi System Operators Guide.

Traceability
Calibration of the VITROS CA 15-3 assay is traceable to Centocor CA 15-3 RIA.

Pub. No. J03795

Version 2.1

INSTRUCTIONS FOR USE

Calibration Model
A modified four-parameter logistic curve fit.

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

Reportable Range (Dynamic Range)

0500 U/mL

Quality Control
Procedure Recommendations
Choose control levels that check the clinically relevant concentrations. To verify system performance, analyze control materials: After calibration

At least once every 24 hours After specified service procedures are performed (see the VITROS ECi System Operators Guide)
Analyze quality control materials in the same manner as patient specimens. If control results fall outside your acceptable range, investigate the cause before deciding whether to report patient results. For more detailed information on quality control procedures, refer to the VITROS ECi System Operators Guide. Refer to Internal Quality Control Testing: Principles and Definitions or other published guidelines for general 19 quality control recommendations.

Quality Control Material Selection

VITROS Oncology Controls are recommended for use with the VITROS ECi System. The performance of commercial control fluids should be evaluated for compatibility with this assay before they are used for quality control. Control materials may show a difference when compared with other CA 15-3 methods if they contain high concentrations of preservatives, stabilizers, or other nonphysiological additives, or otherwise depart from a true human serum/plasma matrix.

Quality Control Material Preparation and Storage

Refer to the manufacturers product literature for preparation, storage, and stability information.

Version 2.1

Pub. No. J03795

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

Expected Values and Reporting Units

Distribution of Results by Clinical Category
Category Normal Females Non-Malignant Conditions Breast Liver Malignant Disease Breast Stage I/II Stage III Stage IV Others* * 35 41 18 143 27 20 9 102 8 21 9 41 50 50 42 49 8 1 No. of Subjects 198

35 U/mL
194

> 35 U/mL 4

Ovarian, Gastric and Colorectal.

98% of the VITROS CA 15-3 assay concentrations in a study of 198 samples from normal females were found to be 35 U/mL or less. Of 94 samples from patients with breast cancer, 38 samples had VITROS CA 15-3 assay concentrations above 35 U/mL. Each laboratory should confirm the validity of these intervals for the population it serves.

Reporting Units
VITROS CA 15-3 assay concentrations are reported in U/mL.

Limitations of the Procedure

Known Interfering Substances
Turbidity may affect assay results. Heterophilic antibodies in the serum or plasma of certain individuals are known to cause interference with 20 immunoassays. These antibodies may be present in blood samples from individuals regularly exposed to animals or who have been treated with animal serum products.
22

The VITROS CA 15-3 assay was evaluated for interference as recommended by NCCLS Protocol EP7. Commonly encountered substances were tested on two lots of reagent. Of the compounds tested, none were found to cause a bias greater than 10%. Refer to Specificity for a list of compounds tested that did not show interference.

Pub. No. J03795

Version 2.1

INSTRUCTIONS FOR USE

Other Limitations

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

The VITROS CA 15-3 has no high dose hook effect up to 47,000 U/mL. 21 For changes in tumor marker concentrations during therapy: Progressive disease is defined by an increase of at least 25%. Sampling should be repeated within two to four weeks for additional evidence.

Partial remission is defined as a decrease of at least 50% in the tumor marker concentration.
Different assay methods cannot be used interchangeably. DF3 defined antigen in a given patient sample determined with different assays and from different manufacturers can vary due to differences in assay methods and reagent specificity. A change to the assay used during serial monitoring of a patient should be accompanied by additional sequential testing to confirm baseline concentrations. The results reported by the laboratory to the physician must include the identity of the CA 15-3 assay used. The results of studies indicate that the concentration of the DF3 defined antigen is frequently elevated in the serum of patients with breast cancer as well as with other malignancies, such as lung cancer, and in some 10, 11, 12, 13 non-malignant disorders. CA 15-3 assay concentrations were not elevated in the sera of the majority 10, 11, 12, 13 of normal individuals. Serial test results obtained with the VITROS CA 15-3 assay, in patients who are clinically free of disease, should be used in conjunction with all relevant information derived from diagnostic tests, physical examination, and full medical history in accordance with appropriate patient management procedures used for early detection of recurrence.

Performance Characteristics
Analytical Sensitivity
The analytical sensitivity of this assay is 0.5 U/mL. It is defined as the lowest concentration of analyte that can be differentiated from a sample that contains no analyte. It was determined as the two standard deviation limit of 20 measurements of a sample containing no analyte.

Precision
Precision was evaluated following NCCLS Protocol EP5. Two replicates each of three pools of patient serum were assayed in two separate runs per day on 20 different days. The evaluation was performed on two different VITROS ECi Systems using a different lot of reagents on each. The data presented are a representation of assay performance and are provided as a guideline. Variables such as sample handling and storage, reagent handling and storage, laboratory environment, and system maintenance can affect the reproducibility of assay results.
23

Version 2.1

Pub. No. J03795

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

Units = U/mL

Mean CA 15-3 Conc. System 1 32.5 129 210 System 2 35.7 130 215 *

Within-run* SD 0.870 2.32 4.29 0.839 2.89 4.14 CV (%) 2.7 1.8 2.0 2.4 2.2 1.9

Within-calibration** SD 2.15 5.27 9.12 2.20 6.32 11.3 CV (%) 6.6 4.1 4.3 6.2 4.9 5.3

Within-lab*** SD 2.12 5.22 9.13 2.71 5.85 10.9 CV (%) 6.5 4.0 4.3 7.6 4.5 5.1 No. Observ. 80 80 80 80 80 80 No. Days 20 20 20 20 20 20

Within-run. Within-run precision was determined using duplicate determinations.

** Within-calibration. Total within-calibration precision was determined using a single lot of reagents over a single calibration interval. *** Within-lab. Total within-lab precision was estimated using a single reagent lot calibrated weekly.

Accuracy
Accuracy was evaluated following NCCLS Protocol EP9. The plot shows the results of a method comparison study using patient samples analyzed on the VITROS ECi System with those analyzed using the Centocor RIA 25 assay. The relationship between the two methods was determined by Passing-Bablok regression.
24

Pub. No. J03795

Version 2.1

INSTRUCTIONS FOR USE

Specificity
Substances that do not Interfere

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

Specificity of the VITROS CA 15-3 assay was evaluated by testing the following substances as recommended by 22 NCCLS Protocol EP7. They were found not to interfere (bias <10%) with the assay at a CA 15-3 concentration of 16.935 U/mL.
Compound Acetaminophen Acetylsalicylic Acid Ascorbic Acid Bilirubin Biotin Cyclophosphamide Doxorubicin 5-Fluorouracil Hemoglobin* Ibuprofen Leucovorin Methotrexate Mitomycin Navelbine Novantrone Paclitaxel Tamoxifen Thioplex Triolein Vinblastine * Compound Concentration 1323 mol/L 2.78 mmol/L 0.17 mmol/L 0.342 mmol/L 40.9 nmol/L 0.32 mmol/L 1.30 mol/L 0.75 mmol/L 0.31 mmol/L 1.94 mmol/L 0.29 mmol/L 22 mol/L 8.97 mol/L 9 nmol/L 2.9 mol/L 13 mol/L 13 mol/L 7.93 mol/L 22.6 mmol/L 13 nmol/L 200 g/mL 50 mg/dL 3 mg/dL 20 mg/dL 1 g/dL 8.38 mg/dL 75 g/dL 9.76 mg/dL 500 mg/dL 40 mg/dL 15 mg/dL 1 mg/dL 300 g/dL 1.0 g/dL 150 g/dL 1.11 mg/dL 750 g/dL 150 g/dL 2000 mg/dL 1.2 g/dL

Hemolysate was added to a series of serum specimens with VITROS CA 15-3 assay concentrations of 36.666.5 U/mL.

Version 2.1

Pub. No. J03795

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Chittoor SR & Swain SM. Adjuvant Therapy in Early Breast Cancer. Am Fam Physician. 44:453462; 1991. Patterson AGH et al. Impact of chemotherapy on survival in breast cancer. Lancet. 2:312;1980. Fey MF et al. Prognostic factors in metastatic breast cancer. Cancer Clin Trials. 4:237247; 1981. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes. Prot Biol Fluids. 29:813 816; 1981. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes in carcinoma research. Prot Biol Fluids. 31:10131016; 1984. Hilkens J et al. MAM-6 antigen, a new serum marker for breast cancer monitoring. Cancer Res. 46:2582 2587; 1986. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer. 34:197206; 1984. Kufe D et al. Differential reactivity of a novel monoclonal antobody (DF3) with human malignant versus benign breast tumor. Hybridoma. 3:223232; 1984. Sekine H et al. Purification and characterization of a high molecular weight glycoprotein detectable human milk and breast carcinomas. J Immunol. 135:36103615; 1985. Fujino N et al. Clinical evaluation of an immunoradiometric assay for CA 15-3 antigen associated with human mammary carcinomas: Comparison with carcinoembryonic antigen. Jpn J Clin Oncol. 16:335346; 1986. Colomer R et al. Early results of a new breast cancer marker. Anticancer Res. 6:683684; 1986. Hayes DF et al. Comparison of circulating carcinoembryonic antigen levels in patients with breast cancer. J Clin Oncol. 4:15421550; 1986. Pons-Anticet DFM et al. Value of CA 15-3 in the follow-up of breast cancer patients. Brit J Cancer. 55:567 569; 1987. Summers M et al. Luminogenic Reagent Using 3-Chloro 4-Hydroxy Acetanilide to Enhance Peroxidase/Luminol Chemiluminescence. Clin Chem. 41:S73; 1995. NCCLS. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue; Approved Guideline. NCCLS document M29-A (ISBN 1-56238-339-6). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1997. Calam RR. Specimen Processing Separator Gels: An Update. J Clin Immunoassay. 11:8690; 1988. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture Third Edition; Approved Standard. NCCLS document H3-A3 (ISBN 1-56238-108-3). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1991. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Skin Puncture Third Edition; Approved Standard. NCCLS document H4-A3 (ISBN 1-56238-111-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1991. NCCLS. Internal Quality Control: Principles and Definitions; Approved Guideline. NCCLS document C24-A (ISBN 1-56238-112-1). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1991. Levinson SS. The Nature of Heterophilic Antibodies and Their Role in Immunoassay Interference. J Clin Immunoassay. 15:108115; 1992. Bonfrer JMG, Working Group on Tumor Marker Criteria (WGTMC). Tumor Biol.11:287-288;1990. NCCLS. Interference Testing in Clinical Chemistry; Proposed Guideline. NCCLS document EP7-P (ISBN 156238-020-6). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1986. NCCLS. Evaluation of Precision Performance of Clinical Chemistry Devices Second Edition; Tentative Guideline. NCCLS document EP5-T2 (ISBN 1-56238-145-8). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1992. NCCLS. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline. NCCLS document EP9-A (ISBN 1-56238-283-7). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1995. Passing H, Bablok W. A New Biometrical Procedure for Testing the Equality of Measurements from Two Different Analytical Methods. J Clin Chem Clin Biochem. 21: 709-720 (1983).

25.

10

Pub. No. J03795

Version 2.1

INSTRUCTIONS FOR USE

Revision History
Date of Revision: 2008-04-08 2002AUG21 Version: 2.1 2.0 Description:

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

Updated Fjuirebio information Reagent Pack Contents revised CAT. No. revised reagent fill volumes New format. Update to 2001AUG22 Revised trademark, back page.

2002FEB28

1.0

When this Instructions For Use is replaced, sign and date below and retain as specified by local regulations or laboratory policies, as appropriate.

_________________________________
Signature

_____________
Obsolete Date

Version 2.1

Pub. No. J03795

11

Intended for Use in the United States Lot 500 and Above

CA 153
CA 15-3

INSTRUCTIONS FOR USE

Supplied by Fujirebio Diagnostics, Inc., Malvern, PA, USA.

Distributed in the US by Ortho-Clinical Diagnostics, Inc. 100 Indigo Creek Drive Rochester, NY 14626-5101

VITROS is a trademark of Ortho-Clinical Diagnostics, Inc. CA 15-3 is a trademark of Fujirebio Diagnostics, Inc. Ortho-Clinical Diagnostics, Inc., 2002. All rights reserved. Printed in USA.

12

Pub. No. J03795

Version 2.1