CEP X SPECTRUMORANGE\Y SPECTRUMGREEN DNA PROBE KIT

K954214 · Vysis · OXP · Jan 21, 1997 · Immunology

Device Facts

• Record ID: K954214
• Device Name: CEP X SPECTRUMORANGE\Y SPECTRUMGREEN DNA PROBE KIT
• Applicant: Vysis
• Product Code: OXP · Immunology
• Decision Date: Jan 21, 1997
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6010
• Device Class: Class 2

Intended Use

The CEP X/Y probe is a combination of CEP X SpectrumOrange and CEP Y SpectrumGreen fluorescently labeled DNA probes for the alpha DNA on the centromeric region of chromosome X and the satellite III DNA at the Yq12 region of chromosome Y. This assay is designed to provide a reliable method for the simultaneous detection and enumeration of chromosomes X and Y in both interphase nuclei and metaphase spreads in bone marrow by fluorescence in situ hybridization (FISH).

Device Story

Device uses fluorescently labeled DNA probes (CEP X SpectrumOrange, CEP Y SpectrumGreen) to target centromeric/satellite DNA regions. Input: bone marrow specimens (unstimulated, cultured). Process: Fluorescence in situ hybridization (FISH) allows visualization of X and Y chromosomes. Output: visual signal enumeration in interphase nuclei and metaphase spreads. Used in clinical laboratories by cytogeneticists/technicians. Healthcare providers use results to assess donor cell engraftment post-bone marrow transplant. Interpretation requires integration with standard cytogenetic analysis and clinical context. Benefits: provides reliable method for detecting sex chromosome chimerism.

Clinical Evidence

Multi-center, blinded, controlled study of 143 opposite-sex bone marrow transplant (BMT) patients (69 CML, 30 AML/ANLL, 7 MDS, 21 ALL, 16 HDNOS). FISH interphase analysis showed 100% relative sensitivity (143/143) for donor cell detection. Specificity assessed in 153 like-sex BMT patients; interphase FISH correctly identified 149/153 (97.4%) as negative. False positives occurred in male recipients, particularly those with abnormal karyotypes (e.g., 46,XY,-Y,+X).

Technological Characteristics

Fluorescently labeled DNA probes targeting chromosome X centromere and Yq12 region. Principle: Fluorescence in situ hybridization (FISH). Form factor: DNA probe kit. Analysis: Visual enumeration of signals in interphase nuclei and metaphase spreads. No software or automated algorithm described.

Indications for Use

Indicated for the detection and enumeration of X and Y chromosomes in bone marrow specimens via FISH, specifically for monitoring donor cell engraftment in patients who have undergone opposite-sex bone marrow transplantation.

Regulatory Classification

Identification

A tumor-associated antigen immunological test system is a device that consists of reagents used to qualitatively or quantitatively measure, by immunochemical techniques, tumor-associated antigens in serum, plasma, urine, or other body fluids. This device is intended as an aid in monitoring patients for disease progress or response to therapy or for the detection of recurrent or residual disease.

Special Controls

*Classification.* Class II (special controls). Tumor markers must comply with the following special controls: (1) A guidance document entitled “Guidance Document for the Submission of Tumor Associated Antigen Premarket Notifications (510(k)s) to FDA,” and (2) voluntary assay performance standards issued by the National Committee on Clinical Laboratory Standards.

Submission Summary (Full Text)

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K954214

JAN 21 1997

# SUMMARY: SAFETY AND EFFECTIVENESS INFORMATION FOR CEP X SpectrumOrange/CEP Y SpectrumGreen DNA Probe Kit

The CEP X/Y probe is a combination of CEP X SpectrumOrange and CEP Y SpectrumGreen fluorescently labeled DNA probes for the alpha DNA on the centromeric region of chromosome X and the satellite III DNA at the Yq12 region of chromosome Y. This assay is designed to provide a reliable method for the simultaneous detection and enumeration of chromosomes X and Y in both interphase nuclei and metaphase spreads in bone marrow by fluorescence *in situ* hybridization (FISH).

Standard cytogenetic analysis detects the presence of the X and Y chromosomes by karyotyping 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 X/Y 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 only one hybridization signal was 0.012% (S.D.=0.15%) on 143 bone marrow specimens. Thus, <2% cells with only one signal is a realistic standard of acceptance.

## Analytical Sensitivity

The analytical sensitivity of the CEP X/Y probe was tested in the reproducibility study described below. In that study, the 0% XY specimen was estimated with a mean of 0.00% (s.d.=0.00%) XY nuclei and the 1% XY specimen, 0.94% (s.d.=0.32%). The 0% XX specimen was estimated with a mean of 0.00% (s.d.=0.00%) XX nuclei and the 1% XX specimen, 0.95% (s.d.=0.34%). There was little overlap between the 0% and 1% specimens; the lower 95% confidence limit for the 1% specimen was 0.31% and 0.28% for XY and XX, respectively. Thus, the limit of detection for CEP X/Y is estimated to be 1.0%.

## Analytical Specificity

Locus specificity studies were performed with metaphase spreads according to standard Vysis QC protocols. A total of 65 metaphase spreads were examined sequentially by G-banding to identify chromosomes X and Y, followed by FISH. No cross-hybridization to other chromosome loci was observed in any of the 65 cells examined; hybridization was limited to the centromere of chromosome X and the Yq12 region of chromosome Y.

Safety and Effectiveness Statement

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SISAN 870212802

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# Reproducibility

To assess the reproducibility of the CEP X/Y interphase analysis for the percentage of cells with XX and XY signals, bone marrow specimens with approximately 0%/100%, 1%/99%, 5%/95%, 95%/5% XY/XX, 99%/1% and 100%/0% XY/XX were prepared. Inter-site, inter-lot, inter-day, and inter-observer reproducibility were assessed in a pivotal study with two of these bone marrow specimen mixtures (approximately 99%/1% and 100%/0% XY/XX,) and two mixtures of hematologically derived human cells with approximately 0%/100% and 1%/99% XY/XX. The percentage of cells with XX and XY signals were evaluated according to the instructions for signal enumeration in the package insert. Using ANOVA, significant, site-to-site and observer-to-observer variations were observed, reflecting the subjectivity of the visual enumeration process. In addition to the pivotal study, four bone marrow specimens with approximately 0%/100%, 1%/99%, 5%/95% and 95%/5% XY/XX were prepared and analyzed at one site. The mean, standard deviation, and percent CV of the observed percentage of XX and XY nuclei for the bone marrow specimens are shown in Table 1.

Table 1 Precision of the Observed % XY/XX Signaled Nuclei Detection

| Specimen Level of XY/XX: | n | Mean (%) | | Standard Deviation (%) | | Coefficient of Variation (%) | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | XY | XX | XY | XX | XY | XX | |
| 0% | 100% | 10 | 0.00 | 97.4 | 0.00 | 1.18 | — | 1.21 |
| 1% | 99% | 20 | 0.88 | 97.2 | 0.48 | 2.00 | 54.8 | 2.06 |
| 5% | 95% | 20 | 4.90 | 94.9 | 0.99 | 0.99 | 20.2 | 1.04 |
| 95% | 5% | 10 | 95.0 | 4.96 | 1.60 | 1.60 | 1.68 | 32.3 |
| 99% | 1% | 24 | 98.3 | 0.95 | 0.41 | 0.34 | 0.41 | 36.3 |
| 100% | 0% | 24 | 99.0 | 0.00 | 0.47 | 0.00 | 0.48 | — |

Safety and Effectiveness Statement

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20:01 26.91 N# 90d 22L

SISN 8p0212802

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# Methods Comparison: Clinical Specimens

A multi-center, blinded, controlled, comparative study was conducted to characterize the performance of the CEP X/Y DNA probe kit in identifying the proportion of XX and XY cells, relative to standard cytogenetic analysis, in recipients of opposite-sex bone marrow transplants (BMT). Archived bone marrow specimens, which were previously evaluated by standard cytogenetic analysis, were selected from a total of 143 patients 72 males and 71 females), who were the recipients of opposite-sex BMTs. Consecutive specimens were selected and evaluated at three sites; site 1 provided and analyzed 40 specimens; site 2, 52 specimens; and site 3, 51 specimens. These specimens were derived from patients with one of the following diagnoses.

1. Chronic myelogenous leukemia (CML): 69 specimens
2. Acute myeloid leukemia (AML) or Acute nonlymphocytic leukemia (ANLL): 30 specimens
3. Myelodysplastic syndrome (MDS): 7 specimens
4. Acute lymphoid leukemia (ALL): 21 specimens
5. Hematological disorder not otherwise specified, but in which cytogenetics are commonly requested (HDNOS): 16 specimens

All sites utilized unstimulated, cultured specimens for both standard cytogenetic and FISH analyses. Each site followed its own in-house protocol for standard cytogenetic analysis; FISH analyses were performed according to the instructions in the CEP X/Y DNA probe kit package insert. The number of donor and recipient cells were enumerated by FISH in a minimum of 20 metaphase and 500 interphase cells.

As expected for specimens with presumed sex chromosome chimerism after opposite-sex BMT, donor cells were detected in each of the 143 specimens by standard cytogenetic analysis. Interphase FISH analysis designated 143/143 specimens as positive for the presence of donor cells (100% relative sensitivity). FISH metaphase analysis detected donor cells in 141/141* specimens (100% relative sensitivity).

In addition to assessing the performance of FISH in the target population of patients with opposite-sex BMT, the ability of interphase and metaphase FISH to correctly designate specimens with like-sex BMT as negative was assessed in 153 patients with like-sex BMTs; the distribution of diagnoses for these patients was similar to those with opposite-sex BMTs. FISH interphase analysis correctly designated 149/153 (97.4%) as negative. All of the four false positive cases occurred in male recipients of like-sex BMT. One case had a 46,XY,-Y,+X karyotype, which led to a FISH result of 37.4% of cells with XX signals; the FISH results of the other three cases showed low levels of XX cells (4.6%, 1.6%, and 0.8%). FISH metaphase analyses designated 151/153 (98.7%) as negative. Both false positive cases were the same patients as those with discrepant FISH interphase analysis. One case had a 46,XY,-Y,+X karyotype, which led to a FISH result of 20% of cells with XX signals; the FISH results of the other case showed 7.1% XX cells.

Safety and Effectiveness Statement
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80:16.91 N51 20d 23L
SISAN 8p021L2802

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The misclassification of a like-sex BMT recipient with an abnormal acquired karyotype demonstrates the importance of performing pre-BMT cytogenetic analysis in conjunction with FISH. The other 3 false positive cases by FISH had low levels of XX cells; both recipient and donor cells showed a 46,XY karyotype. Although no females with like-sex BMT cases were misclassified by FISH, low levels of donor/recipient cells by FISH should be interpreted with caution. All FISH results should be interpreted in conjunction with standard cytogenetic analysis and within the context of other relevant clinical information.

## Conclusions

The performance of CEP X/Y is supported by the Vysis Quality Control Procedures and is demonstrated in clinical studies. When the CEP X SpectrumOrange / CEP Y SpectrumGreen DNA Probe is used as instructed in the package insert, the above statements describe its performance.

Safety and Effectiveness Statement

80:16,91 Nf 80d 2εL

SISN 8p0212802

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DEPARTMENT OF HEALTH & HUMAN SERVICES
Public Health Service
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993

Vysis
c/o Ms. Vicki Anastasi
Manager, Regulatory Affairs
3100 Woodcreek Dr.
Downers Grove, IL 60515

Re: k954214
Trade/Device Name: CEP X Spectrum Orange/Y Spectrum Green DNA Probe Kit
Regulation Number: 21 CFR §866.6010
Regulation Name: Tumor-associated antigen immunological test system
Regulatory Class: II
Product Code: OXP, KIR
Dated: November 19, 1996
Received: November 20, 1996

Dear Ms. Anastasi:

This letter corrects our substantially equivalent letter of January 21, 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 (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 either class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. 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); labeling (21 CFR Parts 801 and

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Page 2 – Ms. Vicki Anastasi

809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); 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 Part 801 and809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR 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 [phone] or [phone] or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

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