The CEP 12 SpectrumOrange Direct Labeled Chromosome Enumeration DNA Probe Kit is designed to be an adjunct to standard cytogenetic analysis for in vitro diagnostic use in identifying and enumerating chromosome 12 via fluorescence in situ hybridization (FISH) in interphase nuclei of cells obtained from peripheral blood lymphocytes in patients with chronic lymphocytic leukemia (CLL). Results from the CEP 12 SpectrumOrange Direct Labeled Chromosome Enumeration DNA Probe Kit are intended for use in conjunction with standard cytogenetics and for further assessing the trisomy 12 status in normal and abnormal tissue specimens characterized by standard cytogenetics, in patients with and/or without clinical symptoms of CLL. It is not intended to be a stand alone assay for test reporting.

Device Description

The CEP 12 SpectumOrange DNA Probe is a SpectumOrange fluorescent labeled DNA probe specific for the centromeric region of chronosome 12. This assey is designed IDN provide a rapid and reliable method for the desertion and quantification of chromosome 12 In interphase nuclei by fluorescence in situ hybridization (FISH).

AI/ML Overview

Here's a breakdown of the acceptance criteria and study information for the CEP 12 SpectrumOrange DNA Probe Kit, based on the provided text:

Acceptance Criteria and Reported Device Performance

Acceptance Criteria Category	Specific Metric	Acceptance Standard	Reported Device Performance
Hybridization Efficiency	Percentage of cells with no hybridization signal	≤ 2% (realistic standard)	Average of 0.43% (S.D.=1.48%) on 402 peripheral blood specimens.
Analytical Sensitivity	Limit of Detection (LOD)	4.0% (estimated)	0% specimen estimated with mean 1.72%. Slight overlap with 5% specimen (upper 95% CI for 0% was 3.86%, lower 95% CI for 5% was 2.93%).
Analytical Specificity	Cross-hybridization to other loci	No cross-hybridization to other chromosomes	No cross-hybridization observed in any of the 56 metaphase spreads examined. Hybridization was limited to chromosome 12 centromere.
Reproducibility	Intra-assay variation (CV) for 0% specimen	Not explicitly stated as a numerical acceptance criterion, but implicitly expected low CV.	CV of 63.1% for 0% specimen (Mean=1.72%, SD=1.09%, N=22).
	Intra-assay variation (CV) for 5% specimen	Not explicitly stated as a numerical acceptance criterion.	CV of 20.4% for 5% specimen (Mean=4.87%, SD=0.99%, N=23).
	Intra-assay variation (CV) for 10% specimen	Not explicitly stated as a numerical acceptance criterion.	CV of 19.4% for 10% specimen (Mean=9.19%, SD=1.78%, N=23).
	Intra-assay variation (CV) for 13% specimen	Not explicitly stated as a numerical acceptance criterion.	CV of 13.3% for 13% specimen (Mean=12.07%, SD=1.61%, N=24).
	Inter-site/inter-observer variation	Expected to be acceptable reflecting visual enumeration	"significant site-to-site and observer-to-observer variations were observed, reflecting the subjectivity of the visual enumeration process."
Clinical Sensitivity	Relative Sensitivity	Not explicitly stated as a numerical acceptance criterion, but expected high.	100% (95% CI 91.2% to 100%) compared to standard cytogenetics.
Clinical Specificity	Relative Specificity	Not explicitly stated as a numerical acceptance criterion, but expected high.	91.47% (95% CI 86.66% to 96.22%) compared to standard cytogenetics.

Study Details

2. Sample Size Used for the Test Set and Data Provenance:

Pivotal Study (Hybridization Efficiency): 402 peripheral blood specimens.
Analytical Specificity: 56 metaphase spreads.
Reproducibility Study:
- 0% specimen: 22 replicates
- 5% specimen: 23 replicates
- 10% specimen: 23 replicates
- 13% specimen: 24 replicates
- Provenance: Hematologically derived human cells (mixtures with known percentages of trisomy 12).
Clinical Specimens (Methods Comparison):
- Total 402 peripheral blood specimens from patients with B-cell chronic lymphocytic leukemia (B-CLL).
- Provenance: Multi-center study; specimens from three sites (Site 1: 97 specimens, Site 2: 205 specimens, Site 3: 100 specimens). One site (from the United Kingdom) provided 157 specimens.
- Retrospective/Prospective: Not explicitly stated, but the collection of "peripheral blood specimens... from a total of 402 patients" suggests they were likely retrospective, selected for the study.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:

Analytical Specificity: Examination "sequentially by G-banding" followed by FISH. This implies cytogeneticists, but the number and specific qualifications are not detailed.
Reproducibility Study: "Known percentages of trisomy 12" in the cell mixtures suggests a predefined truth, likely established by advanced cytogenetic techniques, but no "experts" are explicitly described for this part of ground truth establishment.
Clinical Specimens (Methods Comparison): Standard cytogenetic analysis was used as the comparator (ground truth). Each site followed "its own in-house protocol for standard cytogenetic analysis." This implies trained cytogeneticists performed the standard cytogenetic analysis and interpretation due to the nature of G-banding and karyotyping. The number of experts per site is not specified, nor are their detailed qualifications (e.g., years of experience).

4. Adjudication Method for the Test Set:
The document does not explicitly describe a formal adjudication method (e.g., 2+1, 3+1) for the interpretation of either the FISH results or the standard cytogenetic results. For clinical specimens, it notes that "inter-site and observer-to-observer variations were observed" in the reproducibility study, suggesting independent assessments without a specific adjudication process mentioned for discrepancies in the broader clinical study.

5. Multi Reader Multi Case (MRMC) Comparative Effectiveness Study:
No, an MRMC comparative effectiveness study, in the sense of evaluating how much human readers improve with AI vs. without AI assistance, was not performed. This study compares a device (FISH probe) to a standard clinical method (cytogenetics), which are both human-interpreted methods, not an AI system.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study:
Yes, the analytical sensitivity, specificity, and reproducibility studies for the CEP 12 SpectrumOrange DNA Probe Kit essentially represent standalone performance where the probe kit itself is being evaluated. The reading of the FISH signals is a human-in-the-loop step, but the performance metrics (hybridization efficiency, LOD, cross-hybridization) are inherent to the probe and its interaction with the biological sample.

7. Type of Ground Truth Used:

Analytical Specificity: G-banding (a traditional cytogenetic technique for chromosome identification).
Reproducibility: "Known percentages of trisomy 12" in cell mixtures, implying a highly characterized true state.
Clinical Specimens (Methods Comparison): Standard cytogenetic analysis (G-banded karyotyping).

8. Sample Size for the Training Set:
The document does not mention a training set or machine learning/AI algorithms that would require one. This submission is for a DNA probe kit, not an AI-powered diagnostic device.

9. How the Ground Truth for the Training Set Was Established:
Not applicable, as there is no training set mentioned.

Summary

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K962873

SUMMARY: SAFETY AND EFFECTIVENESS INFORMATION FOR CEP 12 SpectrumOrange DNA Probe Kit

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Standard cytogenetic analysis detects cytogenetic abnormalities such as trisomy 12 by keyotyping metaphase spreads after staining the chromosomes with a dye in cultured tissue cells.

Safety and effectiveness issues relevant to FISH assays such as the CEP 12 assay may include cross-reactivity, poor sensitivity, poor specificity, or poor reproducibility.

Analytical Sensitivity and Specificity

Hybridization Efficiency

In a pivotal study, the average percentage of cells with no hybridization signal was 0.43% (S.D.=1.48%) on 402 peripheral blood specimens. Thus, 2% cells was signal is a realistic standard of acceptance.

Analytical Sensitivity

The analytical sensitivity of the CEP 12 probe was tested in the reproducibility study described below. In that study, the 0% specimen was estimated with a mean of 1.72% There was elight overlean between the ST specimen, 4.87% (S.D.= 0.99%) There was slight overlap between the Off and 5% speciment; the upper 95% confidence limit for the 0% specime was 3.86% and the lower 95% confidence limit for the 5% specimen was 2.93%. Thus, the limit of detection for CHP 12 is estimated to be 4.0%,

Analytical Specificity

Locus specificity studies were performed with metaphase spreads according to standard
Vysis QC protocols. A total of 56 metaphase speeds were examined sequentially by G-banding to identify chromosome 12, followed by FISH. No cross-hybridization to other chromosome loci was observed in any of the 56 cells examined; hybridization was limited to the centromere region of chromosome 12.

Reproducibility

To assess the reproducibility of the CEP 12 assay, CEP 12 analyses for the percentage of tri-signaled cells were assessed for inter-site, inter-lov, inter-lov, and inter-love, and inter-observer reproducibility. Four mixtures of hematologically derived haman cells with known percentages of trisomy 12 (approximately 0%, 5%, 10%, and 13%) were evaluated for the percentage of tri-signated cells according to the instructions for signal enumeration in the package insert. For intra assay variation, the N, mean, SD, and percent CV of the observed percentage of tri-signaled nuclei were 22, 1.72%, 1.09%, and 63.1%, respectively, for the 0% specimen; 23, 4.87%, 0.99%, and 20.4%, respectively, for the 5% specimen; 23, 9.19%, 1.78%, and 19.4%, respectively, for the 10% specimen; and 24, 12.07%, 1.61%, and 13.3%, respectively, for the 13% specimen. For inter varietions wom changed significally significant site-to-site and observer-10-observer-10-observer variations were observed, reflecting the subjectivity of the visual enumeration process.

Safety and Effectiveness Statement

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Methods Comparison; Clinical Specimens

A multi-center, blinded, controlled, comparative study was conducted to further define the performance of the CBP 12 Spectrum Crange DNA probe kit relative to runner bel cytogenetic analysis. Peripheral blood specimens were obtained from a total of 402 patients with B-cell chronic lymphocytic levitemia (B-CLL) for standard cyngeneeric and FISH analysis. Specimens were evaluated at three sites; 97 specimens were analyzed at site 1, 205 at site 2, and 100 at site 3. All sites willized cultured specificas for standard cytogenetic and FISH analyses except site 1; it utilized direct preparations for FISH only, but the same patient specimen was utilized for both methods. Each site followed its own in-house protocol for standard cytogenetic analysis; FISH analyses were performed according to the instructions in the CEP 12 SpectrumOrange INA probe kit package insert".

A total of 177 specimens had a sufficient number of metaphase cells (≥20, or at least two metaphases with trisomy 12) for standard cytogenetic analysis. In addition, 157 speciments from one site had insufficient metaphases for complete cytogenetic analysis, but were evaluated by FISH.

Of those specimens with sufficient metaphases for analysis, 41 were classified as positive for trisomy 12; 132 negative; and 4 ambiguous (one trisomy 12 cell per 30 metaphases), by standard cytogenetic analysis. By interphase FTSH analysis, 33 specimens were classified as positive for trisomy 12; 119 were negative; and 5 were uninformative (with less than 500 evaluable interphase nuclei).

When results between interphase FISH and standard cytogenetics were compared utilizing only specimens with sufficient members for malysis, the CTP 12 DN12
probe kit showed a relaxy sensitivity of 100% (95% CT 91.2% to 100%), and a relative specificity of 91.47% (95% CI 86.66% to 96.22%). FISH interphase analysis designated 54 specimens as positive; 13 more than were positive by standard cytogenetic analysis.

One study site (from the United Kingdom) evaluated a significant number of specimens (157) with less than the minimum number (20) of metaphases required by the International System for Human Cytogenetic Nomenclature (ISCN) standards for standard cytogenetic analysis. At this site, a minimum of 10 metaphase colls vere examined and specimens were designated as negative or ambiguous if 0 or 1 cells, respectively, showed trisomy 12. If >2 cells were positive for trisomy 12, the specimen was reported as positive. FISH analysis designated 25 of these 157 specimens as positive for trisomy 12; they were reported as negative or ambiguous at this site by standard cytogenetic analysis.

Although the true status of specimens designated as trisomy 12 by FISH and negative or ambiguous by standard cytogenetic analysis has not been established, hese discrepancies may, in part, be a reflection of the difference in the reported analytic sensitivity between the two methods.

Conclusions

Performance of CEP 12 is supported by the Vysis Quality Control Procedures and is demonstrated in the clinical studies. When the CEP 12 Spectrum Crange DNA Probe is used as instructed in the package insert, the above statements describe its porformance.

Safery and Effectiveness Statement

* Site 2 used a slight modification to the recommended time and temperature for the FISH bybridization step.

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Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

AUG 26 2011

Vysis c/o Dr. Russel K. Enns Vice President, Regulatory Affairs 3100 Woodcreek Dr. Downers Grove, IL 60515

Re: K962873

Trade/Device Name: CEP 12 SpectrumOrange Direct Labeled Chromosome Enumeration DNA Probe Regulation Number: 21 CFR §866.6040 Regulation Name: Gene expression profiling test system for breast cancer prognosis. Regulatory Class: Class II Product Code: OVQ, KIR Dated: October 29, 1996 Received: October 30, 1996

Dear Dr. Enns:

This letter corrects our substantially equivalent letter of January 13, 1997.

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807);

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Page 2 - Dr. Russel K. Enns

labeling (21 CFR Parts 801 and 809); medical device reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours.

Marie M. Chan

Maria M. Chan. Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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2000 2

Page 1 of 1

510(k) Number (if known): K962873

Device Name: CEP 12 SpectrumOrange Direct Labeled

Chromosome Enumeration DNA Probe Kit

Indications For Use:

Aitu E. Macin
(Division Sign-Off)

vision of Clinical Laboratory De STOlki Number

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use (Per 21 CFR 801.109)

Over-The-Counter Use

(Optional Format 1-2-96)

Regulation Number and Section

§ 866.6040 Gene expression profiling test system for breast cancer prognosis.

(a)
Identification. A gene expression profiling test system for breast cancer prognosis is a device that measures the ribonucleic acid (RNA) expression level of multiple genes and combines this information to yield a signature (pattern or classifier or index) to aid in prognosis of previously diagnosed breast cancer.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Gene Expression Profiling Test System for Breast Cancer Prognosis.” See § 866.1(e) for the availability of this guidance document.