The Vysis D7S486/CEP 7 FISH Probe Kit is a device intended for specimen characterization, and detects the LSI D7S486 probe target on chromosome 7q31 and the CEP 7 probe target on chromosome 7p11.1-q11.1 in bone marrow and peripheral blood specimens from patients with acute myeloid leukemia or myelodysplastic syndrome. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. This device is not intended for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening. The use of this device for diagnosis, prognosis, monitoring, or risk assessment has not been established.
Device Story
The Vysis D7S486/CEP 7 FISH Probe Kit is an in vitro diagnostic tool for detecting chromosomal abnormalities in bone marrow and peripheral blood. It utilizes fluorescence in situ hybridization (FISH) to target the LSI D7S486 locus (7q31) and the CEP 7 centromeric region (7p11.1-q11.1). The kit includes DNA probes labeled with SpectrumOrange and SpectrumGreen fluorophores, hybridization buffer, and counterstain. In the laboratory, specimens are prepared on slides, denatured, and hybridized with the probes. After washing, a pathologist or cytogeneticist uses a fluorescence microscope to visualize signal patterns. A typical cell shows two orange and two green signals (2R2G). A 1R1G pattern indicates monosomy 7; a 1R2G pattern indicates 7q deletion. The device provides objective data on chromosomal status to assist in specimen characterization; it is not intended for clinical diagnosis, prognosis, or therapeutic decision-making.
Clinical Evidence
No clinical trials were conducted for this submission. Evidence consists of analytical performance data (specificity, sensitivity, reproducibility) and literature review. Analytical specificity was 100% (200/200 loci). Analytical sensitivity was 98.1% for bone marrow and 98.5% for peripheral blood. Reproducibility studies across three sites and three lots showed high agreement with known status. Literature references (Vance et al., Cherry et al., Tefferi et al.) support the detection of 7q- and monosomy 7 in AML, MDS, and myelofibrosis patients.
Technological Characteristics
The device uses FISH technology with fluorophore-labeled DNA probes (SpectrumOrange/SpectrumGreen). Components include LSI D7S486/CEP 7 probes, hybridization buffer, DAPI II counterstain, NP-40, and 20X SSC. It requires a fluorescence microscope for visualization. The LSI D7S486 probe is ~308 kb. The CEP 7 probe targets the D7Z1 alpha satellite sequence. The assay is manual, performed on microscope slides using standard cytogenetic procedures.
Indications for Use
Indicated for specimen characterization in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) using bone marrow or peripheral blood specimens. Not for high-risk uses including therapy selection, therapeutic response prediction, or disease screening.
Regulatory Classification
Identification
An early growth response 1 (EGR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization is a device intended to detect the EGR1 probe target on chromosome 5q in bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The assay results are intended to be interpreted only by a qualified pathologist or cytogeneticist. These devices do not include automated systems that directly report results without review and interpretation by a qualified pathologist or cytogeneticist. These devices also do not include any device intended for use to select patient therapy, predict patient response to therapy, or to screen for disease as well as any device with a claim for a particular diagnosis, prognosis, monitoring, or risk assessment.
Special Controls
EGR1 Gene FISH test system for specimen characterization must comply with the following special controls: 1) Premarket notification submissions must include the following information: i) A detailed description of all probes included in the kit ii) Purpose of each probe iii) Probe molecular specificity iv) Probe specificity v) Probe limits vi) Probe sensitivity vii) Specification of required ancillary reagents, instrumentation and equipment viii) Specification of the specimen collection, processing, storage and slide preparation methods ix) Specification of the assay procedure x) Specification of control elements that are incorporated into the recommended testing procedures xi) Specification of risk mitigation elements: description of all additional procedures. methods, and practices incorporated into the directions for use that mitigate risks associated with testing xii) Specification of the criteria for test result interpretation and reporting xiii) Device analytical sensitivity data xiv) Device analytical specificity data xv) Device reference limit data xvi) Device precision/reproducibility data xvii) Device stability data to include: A) Real-time Stability B) Freeze-Thaw Stability C) Transport and Temperature Stability D) Post-Hybridization Signal Stability E) Photostability of Probe xviii) Documentation that demonstrates the clinical validity of the device. The documentation must include data from clinical studies, a minimum of two peer-reviewed published literature references using the specific device seeking marketing clearance, or both. Documentation for peer-reviewed published literature references cited must include the following elements: A) Documentation that the sponsor's probe was used in the literature reference B) Number & type of specimens C) Target population studied. Target population must include the intended use population D) Upper reference limit F) Range of positive probe results 2) 21 CFR 809.10(b)(12) compliant labeling must include a statement summarizing the data identified in subparagraphs (1)(xiii)-(xviii) and a description of the studies supporting the information, including the pre-specified acceptance criteria for these performance studies, justification for the prespecified acceptance criteria, and whether the prespecified acceptance criteria were met. 3) 21 CFR 809.10 compliant labeling must include: i) A warning that reads "The assay results are intended to be interpreted only by a qualified pathologist or cytogeneticist." ii) A warning that reads "This device is not for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening." iii) A warning that reads "The use of this device for diagnosis, monitoring or risk assessment has not been established."
*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must also include the following information:
(i) A detailed description of all probes included in the kit;
(ii) Purpose of each probe;
(iii) Probe molecular specificity;
(iv) Probe specificity;
(v) Probe limits;
(vi) Probe sensitivity;
(vii) Specification of required ancillary reagents, instrumentation, and equipment;
(viii) Specification of the specimen collection, processing, storage and slide preparation methods;
(ix) Specification of the assay procedure;
(x) Specification of control elements that are incorporated into the recommended testing procedures;
(xi) Specification of risk mitigation elements: Description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing;
(xii) Specification of the criteria for test result interpretation and reporting;
(xiii) Device analytical sensitivity data;
(xiv) Device analytical specificity data;
(xv) Device reference limit data;
(xvi) Device precision/reproducibility data;
(xvii) Device stability data to include:
(A) Real-time stability,
(B) Freeze-thaw stability,
(C) Transport and temperature stability,
(D) Post-hybridization signal stability,
(E) Photostability of probe, and
(xviii) Documentation that demonstrates the clinical validity of the device. The documentation must include data from clinical studies, a minimum of two peer-reviewed published literature references using the specific device seeking marketing clearance, or both. Documentation for the clinical studies and peer-reviewed published literature references cited must include the following elements:
(A) Documentation that the sponsor's probe was used in the literature reference,
(B) Number and type of specimens,
(C) Target population studied,
(D) Upper reference limit, and
(E) Range of positive probe results.
(2) Your § 809.10(b)(12) of this chapter compliant labeling must include a statement summarizing the data identified in paragraphs (b)(1)(xiii) through (xviii) of this section and a description of the studies supporting the information, including the pre-specified acceptance criteria for these performance studies, justification for the pre-specified acceptance criteria, and whether the pre-specified acceptance criteria were met.
(3) Your § 809.10 of this chapter compliant labeling must include:
(i) A warning that reads “The assay results are intended to be interpreted only by a qualified pathologist or cytogeneticist.”
(ii) A warning that reads “This device is not for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening.”
(iii) A warning that reads “The use of this device for diagnosis, monitoring or risk assessment has not been established.”
DEN170070 — MLL (KMT2A) Breakapart FISH Probe Kit; AML1 (RUNX1) Breakapart FISH Probe Kit, P53 (TP53) Deletion FISH Probe Kit; EVI1 (MECOM) Breakapart FISH Probe Kit, Del(20q) Deletion FISH Probe Kit; AML1/ETO (RUNX1/RUNXIT1)) Translocation, Dual Fusi, CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit,Del(5q)Deletion FISH Probe Kit; Del(7q)Deletion FISH Probe Kit · Cytocell, Ltd. · Dec 21, 2018
K091960 — VYSIS EGR1 FISH PROBE KIT (VYSIS LSI EGR1 SPECTRUM/ORANGE/D5S23, D5S721, SPECTRUM GREEN PROBE), MODEL 4N37-020 · Abbott Molecular, Inc. · Aug 29, 2011
DEN130010 — VYSIS EGR1 FISH PROBE KIT - SC (SPECIMEN CHARACTERIZATION) · Abbott Molecular, Inc. · Jul 29, 2013
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k131508
B. Purpose for Submission:
New device
C. Measurand:
LSI D7S486 probe target on chromosome 7q31 and the CEP 7 probe target on chromosome 7p11.1-q11.1
D. Type of Test:
Fluorescence in-situ hybridization (FISH)
E. Applicant:
Abbott Molecular, Inc.
F. Proprietary and Established Names:
Vysis D7S486/CEP 7 FISH Probe Kit
G. Regulatory Information:
1. Regulation section:
21 CFR 864.1870, Early growth response 1 (EGFR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization.
2. Classification:
II
3. Product code:
PFG - DNA FISH probe kit for specimen characterization, human chromosome, hematological disorders
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4. Panel:
Hematology and Pathology Devices Panel
H. Intended Use:
1. Intended use(s):
The Vysis D7S486/CEP 7 FISH Probe Kit is a device intended for specimen characterization, and detects the LSI D7S486 probe target on chromosome 7q31 and the CEP 7 probe target on chromosome 7p11.1-q11.1 in bone marrow and peripheral blood specimens from patients with acute myeloid leukemia or myelodysplastic syndrome. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. This device is not intended for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening. The use of this device for diagnosis, prognosis, monitoring, or risk assessment has not been established.
2. Indication(s) for use:
Same as intended use
3. Special conditions for use statement(s):
For prescription use only, with the following labeling warning:
- The assay results are intended to be interpreted only by a qualified pathologist or cytogeneticist.
- The Vysis D7S486/CEP7 FISH Probe Kit – is not for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening.
- The use of this product for diagnosis, prognosis, monitoring, or risk assessment has not been established.
- Caution: Federal law restricts this device to sale by or on the order of a physician or other practitioner licensed by the law of the State in which he practices, to use or order the use of the device.
4. Special instrument requirements:
Fluorescence microscope equipped with appropriate excitation and emission filters.
I. Device Description:
The Vysis D7S486/CEP 7 FISH Probe Kit is a device intended for specimen characterization. The device uses DNA FISH probe technology to detect the LSI D7S486 probe target on chromosome 7q31 and the CEP 7 probe target on chromosome 7p11.1-q11.1 in bone marrow and peripheral blood specimens. The Vysis D7S486/CEP 7 FISH Probe Kit consists of a mixture of two DNA FISH probes (item 1 below) and four general purpose reagents (items 2 through 5 below) which are sufficient to process 20 assays:
1. Vysis LSI D7S486 SpectrumOrange and CEP 7 SpectrumGreen Probes:
a. The SpectrumOrange labeled LSI D7S486 probe is approximately 308 kb in
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length (chr7:115983468-115675366; February 2009 Assembly UCSC Human Genome Browser).
b. The SpectrumGreen labeled CEP 7 probe targets the D7Z1 alpha satellite sequence at the centromere of chromosome 7.
2. Vysis LSI/WCP Hybridization Buffer
3. DAPI II Counterstain
4. NP-40
5. 20X SSC
# J. Substantial Equivalence Information:
1. Predicate device name(s): Vysis EGR1 FISH probe Kit- SC (Specimen Characterization)
2. Predicate 510(k) number(s): K123951
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Indications for Use | For specimen characterization from patients with acute myeloid leukemia and myeloplastic syndrome | Same |
| Technology | Fluorescence In Situ Hybridization | Same |
| Kit components | DNA fluorophore labeled Probes, Vysis LSI/WCP Hybridization buffer, DAPI II, NP-40 and 20XSSC | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection of LSI D7S486 probe target on chromosome 7q31 and the CEP 7 probe target on chromosome 7p11.1-q11.1 | Detection of LSI EGR1 probe target on chromosome 5q. |
| Upper reference limit | 4.5% 1 Red, 1 Green (1R1G) 6.5% 1 Red, 2 Green (1R2G) | 6% |
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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Specimen Type | Bone marrow and peripheral blood | Bone marrow |
K. Standard/Guidance Document Referenced (if applicable):
Not applicable
L. Test Principle:
FISH is a technique that allows visualization of specific nucleic acid sequences within a cellular preparation. Specifically, FISH involves precise annealing of a single-stranded, fluorophore-labeled DNA probe to a complementary target sequence. Hybridization of the probe with the cellular DNA is visible using fluorescence microscopy.
Bone marrow and peripheral blood cell specimens from patients are attached to microscope slides using standard cytogenetic procedures. The resulting DNA specimen is denatured to single-stranded form and subsequently allowed to hybridize with the probes of the Vysis D7S486/CEP 7 FISH Probe Kit. Following hybridization, the unbound probe is removed by a series of washes, and the nuclei are counterstained with DAPI II, a DNA-specific stain that fluoresces blue. Hybridization of the Vysis LSI D7S486 SpectrumOrange/CEP 7 SpectrumGreen Probes is viewed using a fluorescence microscope equipped with appropriate excitation and emission filters, allowing visualization of the orange and green fluorescent signals.
In a cell with normal copy numbers of the Vysis LSI D7S486 SpectrumOrange/CEP 7 SpectrumGreen probe targets, two orange (2R) signals (D7S486) and two green (2G) signals (CEP 7) will be expected. In a cell having only one copy of chromosome 7, 1 orange, 1 green (1R1G) pattern is expected, and in a cell with loss of 7q, 1 orange, 2 green (1R2G) pattern is expected.
FISH results are interpreted by utilizing appropriate controls and analytical techniques as well as taking into consideration other clinical and diagnostic test data.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Repeatability and reproducibility of the Vysis D7S486/CEP 7 FISH Probe Kit were tested as shown in the table below:
| Study | Study Protocol | Conclusion |
| --- | --- | --- |
| Precision and Reproducibility: Intra-Day, Inter-Day and Inter-Site | Testing Panel
• Number of bone marrow specimens: 6 panel members
– 2 high positive panel members (High Positive 1 | Acceptance criteria met |
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| | and 2 with >45% positivity) - 2 low positive panel members (Low Positive 1 and 2) - 2 negative panel members (Negative 1 and 2) • Number of peripheral blood specimens: 6 panel members - 2 high positive panel members (High Positive 1 and 2 with >45% positivity) - 2 low positive panel members (Low Positive 1 and 2) - 2 negative panel members (Negative 1 and 2) Study Design • Number of test sites: 3 • Number of testing days: 5 non-consecutive • Number of replicates: 2 • Number of lots tested: 1 • Signal Patterns - 1 Red, 1 Green (1R1G) - 1 Red, 2 Green (1R2G) • Number of technologists: a minimum of 2 and a maximum of 3 (if third reader needed per package insert) • Number of nuclei evaluated per panel by each technologist: 100 nuclei Pre-specified acceptance criteria: • High Positive - ≥95% for the high positive specimen category for each site • Negative - ≥90% for the negative specimen category across all sites with no more than 3 discordant results occurring at one site | |
| --- | --- | --- |
| Lot-to-Lot Reproducibility | Testing panel same as above • Number of lots tested: 3 • Number of replicates: 4 • Number of sites: 1 • Signal Patterns - 1 Red, 1 Green (1R1G) - 1 Red, 2 Green (1R2G) • Number of technologists: a minimum of 2 and a maximum of 3 (if third reader needed per package insert) • Number of nuclei evaluated per panel by each technologist: 100 nuclei | Acceptance criteria met |
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Pre-specified acceptance criteria:
- High Positive - ≥ 95% for the high positive specimen category across all lots
- Negative - ≥ 87% for the negative specimen category across all lots with no more than 2 discordant results occurring for 1 lot
## Overall Agreement Site-to-Site for Bone Marrow Specimen
| | | Number | | | |
| --- | --- | --- | --- | --- | --- |
| Category | Signal | Agree^{a} | Disagree^{b} | Total | Percent Agreement |
| Negative | 1R2G | 60 | 0 | 60 | 100% |
| Low Positive | 1R2G | 53 | 7 | 60 | 88% |
| High Positive | 1R2G | 60 | 0 | 60 | 100% |
| Negative | 1R1G | 60 | 0 | 60 | 100% |
| Low Positive | 1R1G | 58 | 2 | 60 | 97% |
| High Positive | 1R1G | 60 | 0 | 60 | 100% |
a Agree is number of concordant slides.
b Disagree is number of discordant slides.
## Overall Agreement Site-to-Site for Peripheral Blood Specimen
| | | Number | | | |
| --- | --- | --- | --- | --- | --- |
| Category | Signal | Agree^{a} | Disagree^{b} | Total | Percent Agreement |
| Negative | 1R2G | 60 | 0 | 60 | 100% |
| Low Positive | 1R2G | 57 | 3 | 60 | 95% |
| High Positive | 1R2G | 60 | 0 | 60 | 100% |
| Negative | 1R1G | 60 | 0 | 60 | 100% |
| Low Positive | 1R1G | 56 | 4 | 60 | 93% |
| High Positive | 1R1G | 60 | 0 | 60 | 100% |
a Agree is number of concordant slides.
b Disagree is number of discordant slides.
## Site-to-Site Analysis of Variance Components for Bone Marrow Specimen
| Sample | Signal | N | mean | Within Day (Component) | Between Day (Component) | Between Site (Component) | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | SD | SD | SD |
| Negative 1 | 1R2G | 30 | 0.5 | 0.27 | 0.32 | 0.06 | 0.43 |
| Negative 2 | 1R2G | 30 | 0.4 | 0.38 | 0.14 | 0.24 | 0.47 |
| Low Positive 1 | 1R2G | 30 | 10.5 | 2.11 | 1.41 | 1.14 | 2.78 |
| Low Positive 2 | 1R2G | 30 | 10.0 | 3.19 | 0.00 | 2.33 | 3.95 |
| High Positive 1 | 1R2G | 30 | 43.9 | 6.76 | 0.00 | 7.63 | 10.19 |
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| High Positive 2 | 1R2G | 30 | 42.0 | 4.61 | 0.0 | 5.44 | 7.13 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Negative 1 | 1R1G | 30 | 0.5 | 0.67 | 0.14 | 0.49 | 0.84 |
| Negative 2 | 1R1G | 30 | 0.3 | 0.37 | 0.00 | 0.25 | 0.44 |
| Low Positive1 | 1R1G | 30 | 8.9 | 2.61 | 0.00 | 1.19 | 2.87 |
| Low Positive2 | 1R1G | 30 | 9.3 | 2.43 | 0.00 | 0.00 | 2.43 |
| High Positive 1 | 1R1G | 30 | 48.3 | 6.20 | 1.30 | 8.06 | 10.25 |
| High Positive 2 | 1R1G | 30 | 43.9 | 3.78 | 3.97 | 4.19 | 6.90 |
## Site-to-Site Analysis of Variance Components for Peripheral Blood Specimen
| Sample | Signal | N | mean | Within Day (Component) | Between Day (Component) | Between Site (Component) | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | SD | SD | SD |
| Negative 1 | 1R2G | 30 | 0.4 | 0.53 | 0.27 | 0.38 | 0.71 |
| Negative 2 | 1R2G | 30 | 0.4 | 0.42 | 0.25 | 0.00 | 0.49 |
| Low Positive 1 | 1R2G | 30 | 10.4 | 2.46 | 0.00 | 2.03 | 3.19 |
| Low Positive 2 | 1R2G | 30 | 12.5 | 2.57 | 0.00 | 0.38 | 2.60 |
| High Positive 1 | 1R2G | 30 | 42.1 | 3.26 | 2.05 | 5.82 | 6.98 |
| High Positive 2 | 1R2G | 30 | 52.8 | 4.07 | 1.82 | 2.15 | 4.95 |
| Negative 1 | 1R1G | 30 | 0.3 | 0.65 | 0.00 | 0.32 | 0.72 |
| Negative 2 | 1R1G | 30 | 0.4 | 0.47 | 0.20 | 0.25 | 0.57 |
| Low Positive1 | 1R1G | 30 | 9.1 | 2.61 | 0.00 | 0.55 | 2.67 |
| low Positive2 | 1R1G | 30 | 6.9 | 2.35 | 0.52 | 0.27 | 2.42 |
| High Positive 1 | 1R1G | 30 | 44.8 | 3.77 | 0.00 | 5.35 | 6.55 |
| High Positive 2 | 1R1G | 30 | 38.8 | 3.90 | 3.17 | 1.63 | 5.29 |
## Lot-to-Lot Overall Agreement for Bone Marrow Specimen
| Sample | Signal | Number | | | Percent Agreement |
| --- | --- | --- | --- | --- | --- |
| | | Agree | Disagree | Total | |
| Negative | 1R2G | 24 | 0 | 24 | 100% |
| Low Positive | 1R2G | 21 | 3 | 24 | 88% |
| High Positive | 1R2G | 24 | 0 | 24 | 100% |
| Negative | 1R1G | 24 | 0 | 24 | 100% |
| Low Positive | 1R1G | 22 | 2 | 24 | 92% |
| High Positive | 1R1G | 24 | 0 | 24 | 100% |
## Lot-to-Lot Overall Agreement for Peripheral Blood Specimen
| Sample | Signal | Number | | | Percent Agreement |
| --- | --- | --- | --- | --- | --- |
| | | Agree | Disagree | Total | |
| Negative | 1R2G | 24 | 0 | 24 | 100% |
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b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Kit stability
The kit stability was assessed as detailed in the table below:
| Study | Study Protocol | Conclusion |
| --- | --- | --- |
| Real-Time Stability | Attributes evaluated: signal intensity, specificity, background, and cross-hybridization • Number of lots of the test device: 3 • Number of specimens: 1 (Normal lymphocyte slides) • Number of replicates: 3 • Components of the kit: - Vysis LSI D7S496/CEP 7 Probe - LSI/WCP Hybridization Buffer - 20X SSC - DAPI II - NP-40 Pre-specified acceptance criteria: • Acceptable quality of all evaluated attributes, for all samples tested | Acceptance criteria met for 12 month stability |
| Transport and Temperature Extreme (TTE) and | • Device components were removed from -20°C and placed at 30°C (±2°C) for 4 hours ± 15 minutes and 3 cycles of 24-48 hours on dry ice and then at 30°C (±2°C) for 4 hours ±15 minutes • 1 lot of the device • 3 specimens • Attributes evaluated: Same as above • Pre-specified acceptance criteria: Same as above | Acceptance criteria met |
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| In-Use Freeze-Thaw Stability | • A series of 20 freeze-thaw cycles was performed on the probes, hybridization buffer and DAPI II counterstain.
• 1 lot of the device
• 3 specimens
• Attributes evaluated: Same as above
• Pre-specified acceptance criteria: Same as above | Acceptance criteria met throughout and at the end of 20 freeze-thaw cycles |
| --- | --- | --- |
| Post-hybridization Signal Stability | • A single slide from each of 3 bone marrow and 3 peripheral blood specimens was tested at baseline and then stored at -20°C ± 10°C protected from light
• The samples were tested at the following time points: Day 1, Day 8, Day 21, and Day 28
• 1 lot of the device
• Attributes evaluated: Same as above
• Pre-specified acceptance criteria: Same as above | Post-hybridization signal stability met acceptance criteria at all time points up to Day 28 which support 3 weeks post-hybridized slides stability. |
| Probe Photostability | • Procedure: The Vysis D7S486 SpectrumOrange/CEP 7 SpectrumGreen probes were continuously exposed to white fluorescent light (to mimic standard laboratory conditions) at 15-30°C for difference lengths of time.
• Time intervals: 0 hour, 24 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks
• Number of lots of the test device: 1
• Attributes evaluated: Same as above
• Pre-specified acceptance criteria: Same as above | Acceptance criteria met for Photo-stability of 8 weeks. |
Controls:
There are no external calibrators or controls associated with this device. For any FISH probe, the best control to assure the correct hybridization occurs is signal production at the intended target sequence.
d. Detection limit:
The analytical sensitivity of the Vysis D7S486/CEP 7 Probes was established using interphase nuclei from 25 bone marrow and 25 peripheral blood specimens from
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either karyotypically normal individuals or patients with a 1R1G signal pattern and 1R2G signal pattern. The orange and green signal patterns of 200 nuclei for each specimen type were evaluated by 2 technologists each scoring 100 nuclei per specimen for a total of 200 nuclei per specimen. A total of 5000 nuclei were scored from normal specimens. The Vysis D7S486/CEP 7 FISH Probe Kit has an analytical sensitivity of $98.1\%$ (4903/5000) (95% CI 97.6-98.4%) for bone marrow and $98.5\%$ (4923/5000) (95% CI 98.1-98.8%) for peripheral blood.
| Probe | Specimen | Nuclei with Expected Signal Pattern | Total Number of Nuclei Scored | Analytical Sensitivity (95% Confidence Interval) |
| --- | --- | --- | --- | --- |
| LSI D7S486 | Bone marrow | 4903 | 5000 | 98.1 (97.6, 98.40) |
| LSI D7S486 | Peripheral blood | 4923 | 5000 | 98.5 (98.1, 98.88) |
# e. Analytical specificity:
Analytical specificity is defined as the percentage of signals that hybridize to the correct locus and no other location. The analytical specificity of the Vysis LSI D7S486 SpectrumOrange/CEP 7 SpectrumGreen Probes for their respective chromosome target loci (7q31 and 7p11.1-q11.1, respectively) was established using metaphase chromosomes prepared from peripheral blood cultures of 4 male and 1 female karyotypically normal specimens. The hybridization location of each FISH signal on chromosomes of 100 consecutive metaphase nuclei was evaluated by 1 technologist for a total of 200 target loci per probe.
For each probe and sample, the number of metaphase chromosome FISH signals hybridized to the correct locus and the number of metaphase chromosome FISH signals hybridized to the incorrect locus were enumerated. The analytical specificity of each probe was calculated as the number of metaphase chromosome FISH signals hybridized to the correct locus divided by the total number of metaphase chromosome FISH signals hybridized and multiplied by 100 to give a percentage.
The analytical specificity of the Vysis D7S486/CEP 7 FISH Probe Kit was considered acceptable if greater than or equal to $99\%$ and the D7S486/CEP 7 probes was $100\%$ (200/200) (95% CI 98-100%).
| Probe | Correct Target Locus | Number of Metaphase Chromosome Signals | | Analytical Specificity (95% Confidence Interval ) |
| --- | --- | --- | --- | --- |
| | | Hybridized to the Correct Target Locus | Total Hybridized Signals | |
| LSI D7S486 | 7q31 | 200 | 200 | 100 % (98, 100) |
| CEP 7 | 7p11.1-q11.1 | 200 | 200 | 100 % (98, 100) |
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f. Assay cut-off:
Same as reference range (see #5 below)
# 2. Comparison studies:
a. Method comparison with predicate device:
Not applicable
b. Matrix comparison:
Not applicable
# 3. Clinical studies:
a. Clinical Sensitivity:
Not applicable
b. Clinical specificity:
Not applicable
c. Other clinical supportive data (when a. and b. are not applicable):
The sponsor provided two peer-reviewed published papers to support the clinical validity of the device in characterizing bone marrow and peripheral blood specimens from patients with AML or MDS. Sponsor provided additional a third paper that used specimen from patients with MMM. The studies described in the three papers used the Abbott Vysis D7S486/CEP 7 FISH Probe Kit. This information is listed in the table below:
| Conditions | Data Source 1 Vance et al1 | Data Source 2 Cherry et al2 | Data Source 3 Tefferi et al3 |
| --- | --- | --- | --- |
| Was the specific device (probe) under review in this submission used in the study? | Yes | Yes | Yes |
| Was the specimen type in the study representative of the claimed specimen type(s) | Yes | Yes | Yes |
| Target population (disease status) | AML | MDS | MMM |
| Was the sample size (number of samples) used? | 10 | 10 | 10 |
| Was the sample size (number of patients) used in the analysis? | 10 | 10 | 10 |
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| Upper reference limit (percentage and per 200 nuclei) | (1R1G signal pattern) | | 4.5% 9/200 nuclei | 4.5% 9/200 nuclei | Not Applicable |
| --- | --- | --- | --- | --- | --- |
| | (1R2G signal pattern) | | 6.5% 13/200 nuclei | 6.5% 13/200 nuclei | 6.5% 13/200 nuclei |
| Total Number of specimens tested for each claimed type | Bone Marrow | | 179 | 48 | 42 |
| | Peripheral Blood | | 47 | Not Applicable | 42 |
| Number of specimens with a positive probe result | Bone Marrow | 1R1G | 5 | 2 | Not Applicable |
| | | 1R2G | 4 | 3 | 2 |
| | Peripheral Blood | 1R1G | 1 | Not Applicable | Not Applicable |
| | | 1R2G | 3 | | 1 |
| Range of positive probe results | Bone Marrow | 1R1G | 70-96% | 23 & 87.5% | Not Applicable |
| | | 1R2G | 58-93% | 22.5-44% | 9 and 16% |
| | Peripheral Blood | 1R1G | 11% | Not Applicable | Not Applicable |
| | | 1R2G | 37-96% | | 27% |
AML= acute myeloid leukemia; MDS= myelodysplastic syndrome; MMM= myelofibrosis with myeloid metaplasia.
# REFERENCES
1. Vance GH, Kim H, Hicks GA, et al. Utility of interphase FISH to stratify patients into cytogenetic risk categories at diagnosis of AML in an Eastern Cooperative Oncology Group (ECOG) clinical trial (E1900) Leuk Res 2007; 31:605-09.
2. Cherry A, Brockman S, Paternoster, S et al. Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study. Leukemia Research 2003; 27: 1085-1090.
3. Tefferi A, Meyer R, Wyatt W, et al. Comparison of peripheral blood interphase cytogenetics with bone marrow karyotype analysis in myelofibrosis with myeloid metaplasia. British J Hematology 2001; 115: 316-319.
# 4. Clinical cut-off:
Not applicable
# 5. Expected values/Reference range:
The upper reference limit is defined as the maximum quantity of scoreable interphase nuclei with an altered signal pattern from karyotypically normal specimens. The upper reference limit is expressed in terms of a percentage, or the actual number of a specific atypical nuclear FISH signal pattern per the standard number of nuclei tested.
{12}
The upper reference limit for 4.5% or nine 1R1G patterns per 200 scoreable interphase nuclei, and the upper reference limit is 6.5% or thirteen 1R2G patterns per 200 scoreable interphase nuclei. The 4.5% and 6.5% upper reference limits were adopted from the publication of Vance et al, who utilized the Vysis D7S486/CEP 7 FISH Probe.1
In order to validate the 4.5% and 6.5% upper reference limits, the Vysis D7S486/CEP 7 FISH assay was performed on interphase nuclei from 25 bone marrow and 25 peripheral blood specimens from either karyotypically normal individuals or patients with signal patterns of 1R1G or 1R2G. The orange and green signal patterns of 200 nuclei for each specimen type were evaluated by each of two technologists scoring 100 nuclei per specimen.
Among the 25 karyotypically normal specimens for both peripheral blood and bone marrow, none produced 1R1G and 1R2G signals above the 4.5% and 6.5% upper reference limits, respectively.
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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