This assay is designed to provide a reliable method for the simultaneous detection and enumeration of X and Y chromosomes in interphase nuclei and metaphase spreads in bone marrow by fluorescence in situ hybridization (FISH).

Device Description

The CEP X/Y probe is a combination of CEP X SpectrumOrange and CEP Y SpectrumGreen continuous DNA probes for the centromeric region of chromosome X and the satellite III DNA at the Yq12 region of chromosome Y.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the CEP X SpectrumOrange/Y SpectrumGreen DNA Probe Kit, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

Acceptance Criteria	Reported Device Performance
Hybridization Efficiency: <2% cells with only one hybridization signal	Average percentage of cells with only one hybridization signal was 0.012% (S.D.=0.015%) on 143 bone marrow specimens.
Analytical Sensitivity (Limit of Detection): 1.0%	1% confidence limit for the 1% specimen was 0.31% for XY and 1.28% for XX. Estimated limit of detection for CEP X/Y is 1.0%.Reproduction study results: 0% XX specimen: 0.00% (s.d.=0.00%) XX nuclei and 0.95% (s.d. 34%) XY nuclei. 1% XY specimen: 0.94% (s.d.=0.32%) XY nuclei and 0.00% (s.d.=0.00%) XX nuclei.
Analytical Specificity (Cross-hybridization): No cross-hybridization to other chromosome loci	No cross-hybridization to other chromosome loci was observed in any of the 65 metaphase spreads examined; hybridization was limited to the centromere of chromosome X and the Yq12 region of chromosome Y.
Reproducibility (Inter-day, Inter-observer): (Implicit, based on presented data)	The study assessed inter-day and inter-observer reproducibility. Detailed mean, standard deviation, and coefficient of variation (CV) for different specimen levels (0%, 1%, 5%, 95%, 99%, 100% XY/XX) are provided in Table 1. Examples from Table 1: - 1% XY/99% XX: XY Mean=0.88%, SD=0.48%, CV=54.8% - 5% XY/95% XX: XY Mean=4.90%, SD=0.99%, CV=20.2%
Methods Comparison (Clinical Specimens): Relative sensitivity for donor cell detection in opposite-sex BMTs	Interphase FISH analysis identified 141/141* specimens as positive for donor cells (100% relative sensitivity).
Methods Comparison (Clinical Specimens): Relative specificity for absence of donor cells in like-sex BMTs (Interphase FISH)	Interphase FISH designated 149/153 (97.4%) as negative. 4 false positive cases.
Methods Comparison (Clinical Specimens): Relative specificity for absence of donor cells in like-sex BMTs (Metaphase FISH)	FISH metaphase analyses designated 151/153 (98.7%) as negative. Both false positive metaphase cases were the same patients as two of the false positives from interphase.

Note: The document implicitly defines acceptance criteria through stated performance goals (e.g., "<2% cells with only one signal is a realistic standard of acceptance") or by reporting outcomes that confirm no issues (e.g., no cross-hybridization).

2. Sample Sizes Used for the Test Set and Data Provenance

Hybridization Efficiency: 143 bone marrow specimens.
Analytical Specificity: 65 metaphase spreads.
Reproducibility: Mixtures with various XY/XX percentages (0%/100%, 1%/99%, 5%/95%, 95%/5%, 99%/1%, 100%/0%). Specific n values for each mixture are in Table 1 (ranging from 10 to 24). Additionally, four bone marrow specimens with approximately 0%/100%, 1%/99%, 5%/95%, and 95%/5% were prepared and analyzed at one site.
Methods Comparison (Clinical Specimens):
- Opposite-sex BMT: 143 patients (72 males and 71 females were recipients of opposite-sex BMTs). 143 specimens collected, but only 141 had metaphase spreads for FISH analysis.
- Like-sex BMT: 153 patients.
Data Provenance: The document does not explicitly state the country of origin. It mentions a "multi-center" study and "three sites" for the clinical specimen comparison. The study is described as a "pivotal study," and the data appears to be retrospective as it involves "bone marrow specimens, which were previously evaluated by standard cytogenetic analysis."

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

The document does not explicitly state the number of experts or their qualifications for establishing ground truth. However:

For analytical specificity, the 65 metaphase spreads were "examined sequentially by G-banding to identify chromosomes X and Y, followed by FISH." This G-banding analysis would have been performed by a cytogeneticist.
For the Methods Comparison (Clinical Specimens), standard cytogenetic analysis was used as the comparator for previously collected bone marrow specimens. This implies that the ground truth for these clinical specimens was established by cytogeneticists through traditional karyotyping.

4. Adjudication Method

The document does not mention an explicit adjudication method (e.g., 2+1, 3+1). It describes "inter-observer" assessments for reproducibility, implying multiple observers, but not a formal adjudication process to resolve discrepancies for ground truth. For the clinical studies, standard cytogenetic analysis served as the established ground truth for comparison.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a multi-reader multi-case (MRMC) comparative effectiveness study comparing human readers with AI assistance versus human readers without AI assistance was not done. This study is evaluating a standalone diagnostic kit (a DNA probe kit), not an AI-assisted diagnostic tool.

6. Standalone Performance Study

Yes, the studies described are standalone (algorithm/device only) performance studies. The CEP X/Y probe kit is the device, and its performance characteristics (hybridization efficiency, sensitivity, specificity, reproducibility) and agreement with standard cytogenetics are being evaluated. It is not an AI algorithm with a human-in-the-loop.

7. Type of Ground Truth Used

Hybridization Efficiency, Analytical Sensitivity, Reproducibility: These were assessed against known compositions (e.g., 0% XX, 1% XY specimen, various XY/XX mixtures). The "ground truth" here is the expected percentage or presence/absence of signals based on the prepared specimens.
Analytical Specificity: Ground truth was established by sequential G-banding to identify chromosomes X and Y.
Methods Comparison (Clinical Specimens): The ground truth was established by standard cytogenetic analysis on bone marrow specimens from BMT recipients. This is a well-established diagnostic method.

8. Sample Size for the Training Set

The document does not mention a training set in the context of machine learning or AI. This is a DNA probe kit, not an AI model.

9. How the Ground Truth for the Training Set Was Established

Not applicable, as there is no training set for an AI model.

Summary

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K954214

JAN 2 1 1997

SUMMARY: SAFETY AND EFFECTIVENESS INFORMATION FOR SAFETY AND ERBICTAVERSE Y SpectrumGreen DNA Probe Kit

The CEP X/X probe is a combination of CEP X SpectrumOrange and CRP Y The CEP 7/1 probes is continuous DNA probes for the sights of characters of charmers. region of chromosome X and the sutellite III DNA at the Yq12 region of chromosome Y. This assay is designed to provide a reliable method for the simulancous detection and I mis assay is clesigned to provise and 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 Sundato Cytigenets andysis coose and promosomes with a dye in cultured tissue cells.

Safety and effectiveness issues relevant to FISH assays such as the CEP XY assay may Salely and effectivelies issues resitivity, poor specificity, or poor reproducibility.

Analytical Sensitivity and Specificity

Hybridization Rifficiency

In a pivotal study, the average percentage of cells with only one hybridization signal was 0.012% (S.D.o.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 0,00% (s.d.m0.00%) 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 0 % XX specimen, 0.95% (s.d. 34%). There was little overlap between the 0% the 1% & speciment; 0.9% (1% confidence limit for the 1% specimen was 0.31% and 1.28% 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 crosshybridization to other chromosome loci was observed in any of the 65 cells examined; hybridization was limited to the centromer of chromosome X and the Yq12 region of chromosome Y.

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Reproducibility

To assess the reproducibility of the CBP X/Y interplase analysis for the percentage To assess the reproductions of the case of immentow speciments with approximately 0%/100%, 1%/99%, 5%/95%, 95%/5%, 95%/5%, 99%/1%, and 100%/0%, 100%/100%, 17%/100%, 11%/100%, 11%/100%, 11%/100%, 11%/100%, 11%/100%, 11%/100%, 11%/100%, 11%/99%, 11%/99%, 11% 0%/100%, 1%/99%, 5%/95%, 5%/3% 3% inter-day, and inter-observer productions.
were assessed in a pivotal study, with two of ther-observer mixtures
were assessed in a pivotal s (approximately 99%/1% and 100%/0% XY/XX, and two mixtures of hemstologically decived human cells with approximately 09/100% and 19699%
hemstologically decived human cells with XX stock y gignals were evaluated hemstologically decived number cour with XX and XY signals were evaluated
XY/XX. The percentage of cells with humansism in the peckage insert XY/XX. The percentage of cells will A.A. and of eighten in the peckes e insect. Using according to the instructions in signal canner and observer variations were observed, reflecting the subjectivity of the visual enumeration process. In addition to the pivotal study, four bone marrow specimens with appersymately 0%/100%, to the pivotal study, Tour obternet CV of the was prepared and analyzed at one alte. 15/99%, 5%/95% and 95%/5% and percent CV of the observed percentage of XX The mean, standard teviation, and percein of ender are shown in Table 1.

		Mean(%)		Standard Deviation(%)		Coefficient ofVariation (%)
SpecimenLevel of XY/XX:	n	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	-

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

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Methods Comparison: Clinical Specimens

A multi-center, blinded, controlled, controlled, comparative study was conducted to A multi-center, blinded, controlled, controlled arobe arobe arobe areastic analysis, in characterize the performatics of the care to standerd cynogenetic analysis, in recipients of opposite-sex bone marrow transplants (BMT). And lived bone marrow specimens, which were previously evaluated by standard cytorenetic analysis, were
specimens, which were previously evaluated by standard who were the specimens, which were proviously of any and and 71 females were the selected from a total of 14.5 patients 72 mass and 12 remansvere selected and recipients of opposites sites in 1 ... Collected ve specificans; aite 2, 52
evaluated at three sites; site 1 provided and analyzed from pa evaluated a three since, Sile I provided and andyied to specialize, were derived from patients with one of the following diagnoses.

1. Chronic myelogenous leukemia (CML): 69 specimens
I . Chronic myelogenous (AMI.) or Acute nonlymphocytic leukemia (ANLL): 30 specimens
1. Myelodysplastic syndrome (MDS): 7 specimens
1. Acute lymphoid leukemia (ALL): 21 speciment
1. Hematological disorder not otherwise specified, but in which cytogenetics Hematorogical enocreted (HDNOS): 16 specimens

All sites utilized unstimulated, cultured specimens for both standard cytogenctic and FISH analyses. Each site followed its own in-house protoxool for standard cytogenetic analysis; FISH analyses were performed according to the instructions in cytogenetic analysis; HISH analyses were perioding of donor and recipied.
the CEP XY DNA probe kit package insert. The number of donor and recipied cells.

As expected for specimens with presumed sex chromosome chimserism after opposite-sex BMT, donor cells were detected in each of the 143 specimens by
opposite-sex BMT, donor cells were detected in cash of desimated 143/143 standard cytogenetic analysis. Interphase FISH andysis designatied 143/143 standard cytogenetic analysis. Interprise of donor cells (100% generalive sensitivity). specimens as positive for the presence of conor cells in 141/141* specimens (100% relative sensitivity).

In addition to assessing the performance of FISH in the target population of patients In addition to assessing the poility of interphase and metaphase FISH to correctly with opposite-sex BMT, the ability of merestive was assessed in 153 patients designate speciments with IIE-sex Blvi i as negative the societies was similar to
with like-sex BMTs; the distribution of cliagnoses for the somethy designated with like-sex BMTs. He distribution of the four career or using of the segment in may 149/153 (97.4%) as negative. All of the four false positive cases occurred in male 149/153 (97.4%) as negative. An of the 100 16.30 . T. X karyotype, which led to
recipients of like-sex BMT. One case had a sharts of the other recipients of like-sex BMT. One case nation in the PISH results of the other three cases showed low levels of XX cells (4.6%, 1.6%, and 0.8%).
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 metaphase analyses designated 1377153 (1) director and MSH interphase analysis. cases were the same patients as unose which had to a FISH result of 20% of One case had a 46,X 1,- 1,+X Karyolype, which issues showed 7.1% XX cells.

# Two specimens had no metaphase spreads for FISH analysis, thus the total number was 141, instead of 143.

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The misclassification of a like-sex BMT recipient with an abnormal sequired The misclassincessor of a interportance of performing per-BMT cytogenetic karyotype demonstrates the importance of personing peoples a see by HISH had low levels of XX cells; both recipient and doner cells showed a 40,XX karyotype. low levels of XX couls; bodifferent and conce were misclassified by FISE, low
All levels of donor/recipient colls by FISH should be interpressed with caution. All levels of doner/recupent could by I its a microstion with standard cycles of FISH results should be merpreted in conjunt clinical information.

Conclusions

The porformance of CER XY is suppored by the Vysis Quality Control Procedures and is
demonstrated in clinical studies. When the CEP X SpectumScranger / CRP Y
. Box Can BMA B demonstrated in clinked stoules. Which the OLA 11 Special States statements describe its performance.

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Image /page/4/Picture/1 description: The image is a black and white logo for the U.S. Department of Health & Human Services. The logo features the department's name in a circular arrangement around an emblem. The emblem is a stylized image of three human profiles facing right, with flowing lines suggesting movement or connection.

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

AUG 26 2011

Re: K954214 Trade/Device Name: CEP X Spectrum Orange/Y SpectrumGreen 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 (301) 796-5450. 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 tollfree number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Mana Eden

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

Regulation Number and Section

§ 866.6010 Tumor-associated antigen immunological test system.

(a)
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.(b)
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.