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Intended for Use in the United States

Lot 500 and Above

INSTRUCTIONS FOR USE CA 153VITROS Immunodiagnostic Products CA 15-3TMReagent

Pack

CA 15-3

Version 2.1 Pub. No. J03795 1

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

endocrine therapy; therefore, early detection of recurrence is important to patient management.1 The median

survival time following diagnosis of recurrent disease is approximately two years, but may range from a few

months to decades.2, 3

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

against antigens of human milkfat globule membranes.4, , ,5 6 7

The DF3 antibody was prepared against a

membrane-enriched fraction of a human breast carcinoma.

8,9

The results of studies indicate that theconcentration 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 non-malignant disorders.10, , ,11 12 13

CA 15-3 assay concentrations were not elevated in the sera of the majority of normal individuals. , , ,10 11 12 13

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.

Principles of the Procedure

The VITROS CA 15-3 assay is performed using theVITROS 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 biotinylatedantibody (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

agent (a substituted acetanilide) is added to the wells.14 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.

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Intended for Use in the United StatesLot 500 and Above

CA 153 INSTRUCTIONS FOR USECA 15-3

2 Pub. No. J03795 Version 2.1

Assay Type Assay Time and Temperature

Immunometric assay Incubation time: 32 minutes

Time to first result: 42 minutes

Temperature: 37C

Reaction Scheme

Warnings and Precautions

Take care when handling material of human origin. Consider all clinical specimens and calibrators as potentially

infectious. Handle specimens and assay components in accordance with NCCLS15

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.

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Lot 500 and Above

INSTRUCTIONS FOR USE CA 153CA 15-3


Reagent Pack Storage and Preparation

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 VITROSECi 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.16

Confirm that your

collection devices are compatible with this assay.

Specimen Collection and Preparation

Collect specimens using standard procedures.17, 18

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.

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.

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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 VITROSECi 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 VITROSECi 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 VITROSCA 15-3 assay may also need to be calibrated: After specified service procedures have been performed (see the VITROSECiSystem 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 CentocorCA 15-3 RIA.

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Lot 500 and Above



Calibration Model

A modified four-parameter logistic curve fit.

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 guidelinesfor general

quality control recommendations.19

Quality Control Material Selection

VITROS Oncology Controls are recommended for use with the VITROS ECi System. The performance ofcommercial 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.

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Expected Values and Reporting Units

Distribution of Results by Clinical Category

CategoryNo. of

Subjects 35 U/mL > 35 U/mL

Normal Females 198 194 4

Non-Malignant

Conditions

Breast 50 42 8

Liver 50 49 1

Malignant Disease

Breast

Stage I/II 35 27 8

Stage III 41 20 21

Stage IV 18 9 9

Others* 143 102 41

* 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 UnitsVITROS 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

immunoassays. 20

These antibodies may be present in blood samples from individuals regularly exposed to

animals or who have been treated with animal serum products.

The VITROSCA 15-3 assay was evaluated for interference as recommended by NCCLS Protocol EP7.22

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.

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Lot 500 and Above



Other Limitations

The VITROS CA 15-3 has no high dose hook effect up to 47,000 U/mL. For changes in tumor marker concentrations during therapy:

21

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

non-malignant disorders.10, , ,11 12 13

CA 15-3 assay concentrations were not elevated in the sera of the majority

of normal individuals. , , ,10 11 12 13

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. 23

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.

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Units = U/mL

Within-run* Within-calibration** Within-lab***MeanCA 15-3Conc. SD CV (%) SD CV (%) SD CV (%)

No.

Observ.

No.

Days

System 1 32.5 0.870 2.7 2.15 6.6 2.12 6.5 80 20

129 2.32 1.8 5.27 4.1 5.22 4.0 80 20

210 4.29 2.0 9.12 4.3 9.13 4.3 80 20

System 2 35.7 0.839 2.4 2.20 6.2 2.71 7.6 80 20

130 2.89 2.2 6.32 4.9 5.85 4.5 80 20

215 4.14 1.9 11.3 5.3 10.9 5.1 80 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 EP9.24

The plot shows the results of a method comparison

study using patient samples analyzed on the VITROS ECi System with those analyzed using the CentocorRIA

assay. The relationship between the two methods was determined by Passing-Bablok regression.25

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Lot 500 and Above



Specificity

Substances that do not Interfere

Specificity of the VITROS CA 15-3 assay was evaluated by testing the following substances as recommended by

NCCLS Protocol EP7.22

They were found not to interfere (bias

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References

1. Chittoor SR & Swain SM. Adjuvant Therapy in Early Breast Cancer.Am Fam Physician. 44:453462; 1991.2. Patterson AGH et al. Impact of chemotherapy on survival in breast cancer. Lancet. 2:312;1980.

3. Fey MF et al. Prognostic factors in metastatic breast cancer. Cancer Clin Trials. 4:237247; 1981.

4. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes. Prot Biol Fluids. 29:813816; 1981.

5. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes in carcinoma research.Prot Biol Fluids. 31:10131016; 1984.

6. Hilkens J et al. MAM-6 antigen, a new serum marker for breast cancer monitoring. Cancer Res.46:25822587; 1986.

7. Hilkens J et al. Monoclonal antibodies against human milkfat globule membranes detecting differentiationantigens of the mammary gland and its tumors. Int J Cancer. 34:197206; 1984.

8. Kufe D et al. Differential reactivity of a novel monoclonal antobody (DF3) with human malignant versusbenign breast tumor. Hybridoma. 3:223232; 1984.

9. Sekine H et al. Purification and characterization of a high molecular weight glycoprotein detectable human

milk and breast carcinomas. J Immunol. 135:36103615; 1985.10. Fujino N et al. Clinical evaluation of an immunoradiometric assay for CA 15-3 antigen associated with humanmammary carcinomas: Comparison with carcinoembryonic antigen. Jpn J Clin Oncol. 16:335346; 1986.

11. Colomer R et al. Early results of a new breast cancer marker.Anticancer Res. 6:683684; 1986.

12. Hayes DF et al. Comparison of circulating carcinoembryonic antigen levels in patients with breast cancer. JClin Oncol. 4:15421550; 1986.

13. Pons-Anticet DFM et al. Value of CA 15-3 in the follow-up of breast cancer patients. Brit J Cancer. 55:567569; 1987.

14. Summers M et al. Luminogenic Reagent Using 3-Chloro 4-Hydroxy Acetanilide to EnhancePeroxidase/Luminol Chemiluminescence. Clin Chem. 41:S73; 1995.

15. NCCLS. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmittedby 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.

16. Calam RR. Specimen Processing Separator Gels: An Update. J Clin Immunoassay. 11:8690; 1988.

17. 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, Suite1400, Wayne, Pennsylvania 19087, 1991.

18. 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, Suite1400, Wayne, Pennsylvania 19087, 1991.

19. 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.

20. Levinson SS. The Nature of Heterophilic Antibodies and Their Role in Immunoassay Interference. J ClinImmunoassay. 15:108115; 1992.

21. Bonfrer JMG, Working Group on Tumor Marker Criteria (WGTMC). Tumor Biol.11:287-288;1990.

22. NCCLS. Interference Testing in Clinical Chemistry; Proposed Guideline. NCCLS document EP7-P (ISBN 1-56238-020-6). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1986.

23. 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.

24. NCCLS. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline. NCCLSdocument EP9-A (ISBN 1-56238-283-7). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania19087, 1995.

25. 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).

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Lot 500 and Above



Revision HistoryDate of Revision: Version: Description:

2008-04-08 2.1 Updated Fjuirebio information

2002AUG21 2.0 Reagent Pack Contents

revised CAT. No.

revised reagent fill volumes

2002FEB28 1.0 New format.

Update to 2001AUG22 Revised trademark, back page.

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

_________________________________ _____________Signature Obsolete Date

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