The MLL (KMT2A) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the MLL (KMT2A) region on chromosome 11 at location 11g23.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The P53 (TP53) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion of the P53 (TP53) region on chromosome 7 at location 17p13 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The Del(20q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion within the long arm of chromosome 20 at locations 20g12 and 20q13.1, in fixed bone marrow specimens from patients with myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The CBFB (CBFB)/MYH11 Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the chromosome 16 causing the CBFB-MYH11 (CBFB-MYH11) fusion in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The Del(5q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 5 at location 5g31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The Del(7q) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletions within the long arm of chromosome 7 at locations 7g22 and 7q31.2 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the AML1 (RUNX1) region on chromosome 21 at location 21g22.1 and the ETO (RUNXITI) region on chromosome 8 at location 8q21.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

The EVI1 (MECOM) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement involving the EVII (MECOM) region on chromosome 3 at location 3g26.2, in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.
For prescription use. For in vitro diagnostic use

Device Description

Each Cytocell FISH Probe Kit device consists of one vial containing specific probes as described below. The probes are provided premixed in hybridization solution (formamide: dextran sulfate; saline-sodium citrate (SSC)) and are ready to use. The Kit also includes one vial of 4,6-diamidino-2-phenylindole (DAPI) counterstain. The kits are available in a 10-test format.

The MLL (KMT2A) Breakapart FISH Probe Kit consists of an 87kb probe, labeled in Texas red, covering a region telemetric to the MLL (KMT2A) gene including the marker SHGC-111513 and a FITC green probe covering a 170kb region centromeric to the MLL (KMT2A) gene spanning the CD3G and UBE4A genes.

The P53 (TP53) Deletion FISH Probe Kit consists of a 161kb probe, labeled in Texas red, covering the whole P53 (TP53) gene, extending 74kb telomeric to the gene and covering a region centromeric to the gene, to just beyond the marker D17S655; and a probe, labelled in FITC green, covering the chromosome 17 centromere (D17Z1) region.

The Del(20q) Deletion FISH Probe Kit consists of a 331kb probe, labeled in Texas red, covering a region within the PTPRT gene and including the D20S108 marker; and two (141kb and 174kb) probes labeled in FITC green covering the MYBL2 gene and including the D20S150 marker.

The CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit consists of a 617kb probe, labeled in Texas red, covering a region, within 16q22 including the CBFB gene; and a 621kb probe, labeled in FITC green, covering a region within 16p13.1 including the MYH11 gene.

The Del(5q) Deletion FISH Probe Kit consists of a 186kb probe, labeled in Texas red, covering a region within 5q31.2, including the D5S500 marker; and a 376kb probe, labeled in FITC green, within 5p15.3, including the D5S630 marker.

The Del(7q) Deletion FISH Probe Kit consists of a 396kb probe, labeled in Texas red, covering a region within 7q22 including the telomeric end of the RELN gene and extending beyond the D7S658 marker; and a 203kb probe, labeled in FITC green, covering a region within 7q31.2 including the TES gene.

The AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit consists of a 156kb probe labeled in Texas red, centromeric to the AMLI (RUNXI) gene, including the CLIC6 gene; a 169kb probe labelled in Texas red, telomeric to AMLI (RUNXI) gene, extending beyond the marker D21S1921; and two (151kb and 194kb) probes, labeled inn FITC green, on either side of the ETO (RUNXIT) gene.

The EVI1 (MECOM) Breakapart FISH Probe Kit consists of a 158kb probe, labeled in Texas red, telomeric to the D3S4415 marker and including the LRRC34 gene, a FITC green probe covering a 181kb region. including the entire EVI1 (MECOM) gene and flanking regions and a PF-415 blue probe, which covers a 563kb region centromeric to the EVI I (MECOM) gene, including the D3S1614 marker.

AI/ML Overview

Acceptance Criteria and Study for Cytocell FISH Probe Kits

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for these devices are defined by adherence to various analytical performance metrics and comparison to established literature prevalence ranges. The performance is reported across multiple tables within the document. Below is a summary, with specific numerical criteria inferred from the successful outcome of the studies.

Category	Acceptance Criteria (Inferred from successful study outcome)	Reported Device Performance
Analytical Performance
Precision/Reproducibility (Intra-day, Inter-day, Between-site SD)	Acceptable precision in terms of standard deviation (SD) for negative, near cut-off/low positive, and high positive specimens. Agreement of replicates should be high, particularly for negative and high positive samples.	Tables 2-10 show standard deviations across intra-day, inter-day, and between-site measurements. For example, for MLL (KMT2A) Breakapart: Negative 1 (Total SD: 0.00-0.09), Near cut-off 1 (Total SD: 1.98), Positive 1 (Total SD: 6.60). Agreement replicates for negative and positive samples are generally 100%, with near cut-off samples showing lower but acceptable agreement (e.g., 63-90% for MLL, 27-43% for CBFβ/MYH11). Additional studies for near cut-off samples of CBFβ/MYH11 and EVI1 (MECOM) Breakapart Inversion also demonstrated acceptable precision. Each probe kit demonstrated acceptable precision.
Between-Lot Reproducibility	Acceptable standard deviation (SD) for intra-lot, inter-lot, and total SD using one normal, one low positive, and one high positive specimens across 3 different reagent lots. High agreement of replicates.	Tables 11-19 show acceptable total SDs for negative, near cut-off, and positive specimens. Agreement of replicates for negative and positive samples is generally 100%, with near cut-off samples showing lower but acceptable agreement (e.g., 67-100%). Each probe kit demonstrated acceptable lot-to-lot reproducibility.
Real-time Kit Stability	Probes should maintain performance (accuracy) for the claimed shelf-life (e.g., 24 months at -20°C).	Supported a claimed shelf life of 24 months when stored at -20°C.
Transport Stability	No significant change in device performance under defined stress conditions (e.g., 2 weeks at +40°C, then -20°C).	No change in device performance under the stress conditions.
Freeze/Thaw Stability	Probes should maintain performance after a specified number of freeze/thaw cycles (e.g., 10 cycles).	Ten freeze-thaw cycles were found to be acceptable.
Post Hybridization Stability	Hybridized slides should remain stable for a specified duration (e.g., 1 month).	Post-hybridization signal was determined to be stable to one month.
Photostability	Probes should maintain performance after a specified exposure to light (e.g., 24-36 hours).	Photo-stability of slides was supported to 24-36 hours. Prolonged exposure of slides to light should be avoided.
Analytical Sensitivity	Analytical sensitivity of greater than 95% (proportion of interphase nuclei showing the expected normal signal pattern from karyotypically normal samples).	All nine probes evaluated had an analytical sensitivity of greater than 95%, meeting the required acceptance criteria (Table 21). Ranges from 97.98% to 99.4% with 95% Confidence Intervals.
Analytical Specificity	100% specificity (percentage of signals hybridizing to the correct locus and no other location) from normal male peripheral blood samples.	All probes met the required acceptance criteria for analytical specificity with all components having a specificity of 100% (Table 22).
Assay Cut-off (Upper Reference Limit)	Normal cut-offs confirmed by analytical sensitivity data; an analytical result above the cut-off is positive, below is negative.	Clinical cut-offs were either generated by a central clinical laboratory using a large patient dataset or derived from analytical sensitivity data of 25 karyotypically normal bone marrow samples. The normal cut-offs were confirmed as none of the 25 normal samples showed an abnormal signal pattern at or above the established cut-offs (Table 23 shows the cut-offs for each probe).
Clinical Validation
Incidence Rate Comparison	Device-measured incidence rates (and 95% CI) from clinical specimens should fall within the expected prevalence range from peer-reviewed literature for the target population (AML/MDS patients).	For each probe, incidence rates from two data sources (GOP and YAL) using the Cytocell FISH probe kits were compared to expected ranges from 3-6 literature sources. In most cases, the observed incidence rates, including their 95% CIs, fell within or aligned closely with the expected literature ranges (Tables 25-32). For example, for MLL (KMT2A) Breakapart, GOP (2%) and YAL (1.45%) were within the literature range of 0.2%-4.5%.
Concordance with Karyotyping (for specific probes)	High percentage agreement between the FISH probe kit results and G-band karyotyping, especially for normal and confirmed abnormal specimens. Any discordance should be justifiable (e.g., near cut-off).	For AML1/ETO (RUNX1/RUNX1T1) and CBFβ (CBFB)/MYH11 probes, 100% agreement was observed for both data sets with G-band karyotyping, except for one specimen that fell into the re-test zone or was near the cut-off (Tables 33-34). This demonstrates acceptable clinical concordance.

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

Analytical Performance Test Set:
- Precision/Reproducibility: For each probe kit, 6 specimens were used (2 normal, 2 near cut-off/low positive, 2 high positive). These were tested over 5 non-consecutive days in duplicates at 3 sites (n=30 per specimen). In some cases, additional specimens were re-assessed for "near cut-off" performance.
- Between-Lot Reproducibility: 3 specimens (1 normal, 1 low positive, 1 high positive) were tested with 3 replicates per lot across 3 reagent lots.
- Analytical Sensitivity: 25 karyotypically normal bone marrow samples.
- Analytical Specificity: 5 normal male peripheral blood samples.
Clinical Performance Test Set:
- Incidence Rate Comparison:
  - GOP data set: 100 known or suspected AML and MDS specimens in total.
  - YAL data set: Re-analyzed specimen data ranging from 266 to 746 AML or MDS specimens depending on the probe.
- G-band Concordance: Additional clinical data sets (sample size not explicitly stated for each, but tables 33 and 34 imply sufficient numbers to show concordance for AML1/ETO and CBFβ/MYH11).
Data Provenance: The document does not explicitly state the country of origin for the clinical data (GOP and YAL data sets). The analytical validation studies appear to have been conducted by the applicant, Cytocell, Ltd. (a UK-based company). The literature references used for prevalence rates are international. The clinical data sets (GOP and YAL) are implied to be from clinical laboratories. The studies mentioned (clinical and analytical) were prospective studies for the current submission.

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

Analytical Studies (Analytical Sensitivity & Specificity): 2 independent analysts analyzed the interphase nuclei for analytical sensitivity. The qualifications of these analysts are not explicitly stated, but it is implied they are trained cytogeneticists or laboratory personnel performing FISH analysis.
Clinical Performance Studies (Incidence Rate & Concordance): The ground truth for the clinical specimens used in the incidence rate studies (GOP and YAL data sets) and the G-band concordance studies was established by established alternative cytogenetic methods (competitor probe or G-banding) prior to distribution for analytical studies, or by standard clinical diagnostic procedures. Final assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The number of experts establishing the initial "known" status of the clinical samples is not specified, but the references to "established alternative cytogenetic method (competitor probe or G-banding)" imply experienced cytogenetic laboratories. For the concordance studies, the G-band result serves as the established ground truth.

4. Adjudication Method (e.g., 2+1, 3+1, none) for the Test Set

For the analytical studies (e.g., Analytical Sensitivity), the document states: "Each analyst scores independently 100 nuclei for each sample. In some cases, depending on the number of abnormal nuclei each analyst has seen, a third reader may be required." This suggests an adjudication method of 2+1 (two initial independent reads, with a third if there is disagreement or uncertainty regarding the number of abnormal nuclei for a final count near a threshold).

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size

No, a traditional MRMC comparative effectiveness study comparing AI-assisted vs. unassisted human readers was not performed or described in this document.
This document describes the validation of FISH probe kits, which are reagents for a laboratory test, not an AI-powered diagnostic device. The "device" in question refers to these biological reagents for in-situ hybridization.
Therefore, the concept of "how much human readers improve with AI vs. without AI assistance" is not applicable to this submission.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

No, this is not applicable. The Cytocell FISH Probe Kits are biological reagents, and the process of FISH analysis inherently requires human interpretation (qualified pathologist or cytogeneticist) of the hybridized signals under a microscope. This is not an algorithmic or AI-based device intended for standalone performance.

7. The Type of Ground Truth Used

Analytical Studies:
- Analytical Sensitivity: Karyotypically normal bone marrow samples were used. The ground truth was "normal" based on karyotyping.
- Analytical Specificity: Normal male peripheral blood samples were used. The ground truth was "normal" for the target chromosome locations.
Clinical Studies:
- Incidence Rate Comparison: "Known or suspected AML or MDS specimens" were used. The ground truth for identifying the presence/absence of specific chromosomal rearrangements was established by "established alternative cytogenetic method (competitor probe or G-banding)" or by "clinical thresholds defined in the literature." The literature itself acts as a form of "expert consensus" on prevalence.
- Concordance with Karyotyping: G-band karyotyping served as the ground truth for confirming the presence or absence of specific rearrangements (e.g., t(8;21) or inv(16)/t(16;16)).

8. The Sample Size for the Training Set

The document describes the analytical and clinical validation of the FISH probe kits. It does not describe an AI/algorithm training set as this is not an AI-based device.
However, for the establishment of cut-off values, the following "datasets" were used, which could be considered analogous to "training data" for establishing thresholds:
- For seven of the nine probes, a "large patient dataset exceeding the ACMG minimum sample size guidelines" was used by a central clinical laboratory to generate the clinical cut-off. The exact number is not explicitly stated.
- For the other two probes (AML1 and EVI1), "≥ 25 karyotypically normal bone marrow samples" were enumerated for establishing normal cut-offs.

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

As per point 8, this is not an AI device with a traditional training set.
For the establishment of cut-off values:
- The "large patient dataset" used by the central clinical laboratory likely had ground truth established through comprehensive clinical and cytogenetic diagnoses (including techniques like G-banding and potentially other FISH probes) based on WHO guidelines.
- For the "≥ 25 karyotypically normal bone marrow samples," the ground truth was "karyotypically normal" established through standard karyotyping procedures (e.g., G-banding).
- These established cut-off values represent a consensus or standard derived from expert interpretation of a larger dataset over time.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Cytocell FISH Probe Kits for AML and MDS DECISION SUMMARY

A. DEN Number:

DEN170070

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the following Cytocell FISH Probe Kits for AML (Acute Myeloid Leukemia) and/or MDS (Myelodysplastic Syndromes) (refer to the list of devices in Section C below).

C. Measurands:

Chromosomal rearrangements as listed below:

Device	Chromosomal Rearrangement
MLL (KMT2A) Breakapart FISHProbe Kit	Rearrangement of MLL (KMT2A) region onchromosome 11 at location 11q23.3
P53 (TP53) Deletion FISH Probe Kit	Deletions of the P53 (TP53) region on chromosome17, at location 17p13
Del(20q) Deletion FISH Probe Kit	Deletions within the long arm of chromosome 20 atlocations 20q12 and 20q13.1
CBFβ (CBFB) /MYH11Translocation, Dual Fusion FISHProbe Kit	Rearrangements of chromosome 16 causing theCBFβ-MYH11 (CBFB-MYH11) fusion
Del(5q) Deletion FISH Probe Kit	Deletions within the long arm of chromosome 5 atlocation 5q31.2
Del(7q) Deletion FISH Probe Kit	Deletions within the long arm of chromosome 7 atlocations 7q22 and 7q31.2
AML1/ETO (RUNX1/RUNX1T1))Translocation, Dual Fusion FISHProbe Kit	Rearrangements involving the AML1 (RUNX1)region on chromosome 21 at location 21q22.1 and theETO (RUNX1T1) region on chromosome 8 at location8q21.3
EVI1 (MECOM) Breakapart FISHProbe Kit	Rearrangements involving the EVI1 (MECOM) regionon chromosome 3 at location 3q26.2

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D. Type of Test:

Fluorescent in situ hybridization (FISH).

E. Applicant:

Cytocell, Ltd

F. Proprietary and Established Names:

MLL (KMT2A) Breakapart FISH Probe Kit
P53 (TP53) Deletion FISH Probe Kit
Del(20q) Deletion FISH Probe Kit
CBFß (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit
Del(5q) Deletion FISH Probe Kit
Del(7q) Deletion FISH Probe Kit
AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion FISH Probe Kit ●
EVI1 (MECOM) Breakapart FISH Probe Kit

G. Regulatory Information:

1. Regulation section:
  21 CFR 864.1880
1. Classification:
  Class II
1. Product code(s):
  QDI
1. Panel:
  Pathology

H. Indications for use:

1. Indications for use:
  The MLL (KMT2A) Breakapart FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect rearrangement of the MLL (KMT2A) region on chromosome 11 at location 11g23.3 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated for characterization of patient specimens consistent with World Health

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Organization (WHO) guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th Edition) and in conjunction with other clinicopathological criteria. The assay results are intended to be interpreted by a qualified pathologist or cytogeneticist. The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

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intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.

1. Special conditions for use statement(s) For prescription use. For in vitro diagnostic use

I. Device Description:

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vial of 4,6-diamidino-2-phenylindole (DAPI) counterstain. The kits are available in a 10-test format.

K. Standard/Guidance Document Referenced:

Guidance for Industry and FDA Staff - Content and Format for Abbreviated 510(k)s for Early Growth Response 1 (EGR1) Gene Fluorescence In-Situ Hybridization (FISH) Test

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System for Specimen Characterization Devices

L. Test Principle:

Fluorescence in situ hybridization (FISH) is a technique that allows chromosomal aberrations to be detected on metaphase chromosomes or in interphase nuclei from fixed cytogenetic samples.

Bone marrow cells from patients are attached to microscope slides using standard cytogenetic procedures. After fixation and denaturation of cellular DNA to single-stranded form, target DNA is available for annealing to a similarly denatured, fluorescently labelled DNA probe, which has a complementary sequence. Following hybridization, unbound and nonspecifically bound DNA probe is removed by a series of washes, and the DNA is counterstained for visualization with DAPI, a DNA-specific stain that fluoresces blue. Fluorescence microscopy equipped with appropriate excitation and emission filters then allows the visualization of the hybridized probe on the target material. Two analysts should analyze each sample. Each analyst scores independently 100 nuclei for each sample. In some cases, depending on the number of abnormal nuclei each analyst has seen, a third reader may be required.

The expected signal pattern is dependent on the number of copies of each probe. Enumeration of the signals provide a mechanism for determining absolute copy number of the probe targets and the presence of chromosomal aberrations of interest. Refer to Table 1 -Supported Signal Patterns & Cut-Offs for the expected normal signal pattern and expected abnormal signal pattern for each probe kit (R = red, G = Green, B = blue).

Catalogue	Name	NegativeSignal
USA-LPH013	MLL (KMT2A) Breakapart	(b)(4)
USA-LPH017	P53 (TP53) Deletion
USA-LPH020	Del (20q) Deletion
USA-LPH022	CBFβ (CBFB) /MYH11Translocation, Dual Fusion
USA-LPH024	Del (5q) Deletion
USA-LPH025	Del (7q) Deletion
USA-LPH026	AML1/ETO(RUNX1/RUNX1T1)Translocation, Dual Fusion
USA-LPH036	EVI1 (MECOM) Breakapart InversionSignalEVI1 (MECOM) BreakapartTranslocation Signal

Table 1 - Supported Signal Patterns & Cut-Offs

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M. Performance Characteristics:

Analytical validation was provided in support of each probe kit:

1. Analytical performance:

a. Precision/Reproducibility

Repeatability and reproducibility of each probe kit was assessed using ""bone marrow specimens to represent the range of results [two normal (negative), two near cut-off or low positive, and two high positive]. Specimens were analyzed according to protocol and tested over 5 non-consecutive days in duplicates at " sites to assess precision (n =30 per specimen).

In some cases, to ensure there was ample volume to allow for consistent testing amongst all sites one or more normal samples were combined (pooled) and distributed to testing sites. The majority of the near cut-off and low positive samples were contrived by spiking known normal samples with known positive specimens. All samples were confirmed to be normal by an established alternative cytogenetic method (competitor probe or G-banding) prior to distribution.

Data was analyzed for intra-day, between-day, between-site and total SD.

Two probe kits [CBFB (CBFB) /MYH11 Translocation, Dual Fusion FISH Probe Kit (LPH 022) and EVI1 (MECOM) Breakapart FISH Probe Kit (LPH 036))) had specimens too close the cut-off to assess precision and therefore additional specimens were re-assessed at "" and "" | the cut-off at one site with 2 operators and 5 days in duplicate to confirm acceptable precision at the low end of the enumeration range (n (6)(4) ). The data with the clinical thresholds used in the study are shown in Tables 2 through 10 demonstrate that all probe kits had acceptable precision.

Table 2. Precision of the MLL (KMT2A) Breakapart (cut-off: 3.8%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-DaySD	Inter-DaySD	Between-site SD	TotalSD
Negative 1	(b)(4)	100	0.00	0.00	0.00	0.00
Negative 1		100	0.00	0.00	0.00	0.09
Near cut-off 1		63	0.00	0.00	1.17	1.98
Near cut-off 2		90	0.00	1.69	0.87	3.12
Positive 1		100	0.00	0.77	4.57	6.60
Positive 2		100	0.81	0.35	3.06	4.14

N/A = not applicable

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Specimen	Mean of abnormal percentage	Agreement (%)	Inter-Day SD	Between-site SD	Total SD
Negative 1	(b)(4)	97	0.00	1.11	2.09
Negative 1		93	0.00	1.65	2.50
Near cut-off 1		83	0.00	1.71	3.62
Near cut-off 2		100	2.18	1.34	4.46
Positive 1		100	0.00	0.00	5.84
Positive 2		100	0.92	4.18	6.04

Table 3. Precision of the P53 (TP53) Deletion (cut-off: 6.8%)

Table 4. Precision of the Del (20q) Deletion (cut-off: 5.7%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Inter-DaySD	Between-site SD	TotalSD
Negative 1	(b)(4)	100	0.48	0.00	1.41
Negative 1		100	0.33	0.67	1.01
Near cut-off 1		80	0.00	0.00	4.45
Near cut-off 2		100	2.01	2.79	5.31
Positive 1		100	0.00	0.00	6.93
Positive 2		100	0.00	0.48	4.96

Table 5. Precision of the CBFβ (CBFB) /MYH11 Translocation, Dual Fusion (cut-off: 2.3%)

Specimen	Mean of abnormal percentage	Agreement (%)	Intra-Day SD	Inter-Day SD	Between-site SD	Total SD
Negative 1	(b)(4)	100	0.00	0.00	0.00	0.00
Negative 1		100	0.00	0.00	0.00	0.09
Near cut-off 1		27	0.00	0.51	0.00	1.30
Near cut-off 2		43	0.75	0.00	0.10	2.47
Positive 1		100	0.00	0.84	0.64	4.55
Positive 2		100	0.00	1.76	3.20	7.50

N/A = not applicable

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Specimen	Mean ofabnormalpercentage	Range	Agreementreplicates	Agreement(%)	Intra-DaySD	Inter-DaySD	TotalSD
----------	-----------------------------------	-------	-------------------------	------------------	---------------------	---------------------	-------------

100

0.56

0.00

0.25

0.00

1.34

2.42

Table 5.a. Additional supporting date for precision of the CBFβ (CBFB) /MYH11
Translocation, Dual Fusion

Table 6. Precision of the Del (5q) Deletion (cut-off: 6.3%)

(b)(4)

Near cut-off 1 Near cut-off 2

Specimen	Mean ofabnormal	Agreement(%)	Intra-DaySD	Inter-DaySD	Between-site SD	TotalSD
	(b)(4)
Negative 1		100	0.32	0.58	0.73	1.25
Negative 1		100	0.00	0.06	1.30	1.59
Near cut-off 1		80	0.00	2.47	3.67	5.85
Near cut-off 2		97	2.06	0.00	1.08	4.49
Positive 1		100	1.93	3.27	0.89	7.04
Positive 2		100	0.00	1.21	3.18	5.09

Table 7. Precision of the Del (7q) Deletion (cut-off: 7.4%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-DaySD	Inter-DaySD	Between-site SD	TotalSD
Negative 1	(b)(4)	100	0.00	0.11	0.87	1.12
Negative 1		100	0.00	0.00	0.98	1.67
Near cut-off 1		100	0.00	0.30	2.54	4.86
Near cut-off 2		100	0.39	0.00	2.28	4.21
Positive 1		100	0.00	0.00	3.00	5.09
Positive 2		100	0.00	0.31	2.16	3.79

Table 8. Precision of the AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion (cutoff: 2.3%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-DaySD	Inter-DaySD	Between-site SD	TotalSD
Negative 1	(b)(4)	100	0.00	0.00	0.00	0.00
Negative 1		100	0.00	0.00	0.00	0.00
Near cut-off 1		80	0.23	0.00	0.12	1.72
Near cut-off 2		97	1.57	0.00	0.82	2.40
Positive 1		100	0.00	1.06	0.00	8.30
Positive 2		100	0.25	0.00	0.00	6.25

N/A = not applicable

{9}------------------------------------------------

Table 9. Precision of the EVI1 (MECOM) Breakapart_Inversion (cut-off: 4.0%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-DaySD	Inter-DaySD	Between-site SD	TotalSD
Negative 1	(b)(4)	100	0.24	0.40	0.54	1.02
Negative 1		100	0.52	0.00	0.26	1.24
Near cut-off 1		53	0.00	1.20	0.00	3.49
Near cut-off 2		73	0.00	1.25	1.06	4.13
Positive 1		100	0.68	3.01	7.05	11.93
Positive 2		100	2.42	0.00	5.56	13.26

these two specimens are too close to cut-of to allow assessment of precision An additional onesite precision study with low positive specimens is provided below.

Table 9.a. Additional supporting date for precision of the EVI1 (MECOM) Breakapart Inversion

Specimen	Mean ofabnormalpercentage	Range	Agreementreplicates	Agreement(%)	Intra-DaySD	Inter-DaySD	TotalSD
Near cut-off 1	(b)(4)			100	0.26	1.78	2.75
Near cut-off 2				100	0.00	0.00	2.11

Table 10. Precision of the EVI1 (MECOM) Breakapart Translocation (cut-off: 4.0%)

Specimen	Mean of	Agreement	Agreement	Intra-	Inter-	Between-	Total
	abnormal	replicates	%)	Day	Day	site SD	SD
	percentage			SD	SD
Negative 1	(b)(4)		100	0.00	0.00	0.95	1.02
Negative 1			100	0.00	0.32	0.84	1.09
Near cut-off 1			97	0.00	1.47	2.87	4.27
Near cut-off 2			100	0.00	0.00	1.27	3.38
Positive 1			100	0.00	0.00	3.34	6.79
Positive 2			100	1.11	0.37	0.00	6.78

Between- Lot reproducibility:

Three reagent lots were evaluated using one normal, one low positive and one high positive specimens were tested with " replicates per lot to assess lot to lot variation (total of [" replicates evaluated). Intra-lot, inter-lot and total SD for each lot are shown in Tables 11-19 below.

{10}------------------------------------------------

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	100	0.0	0.00	0.00
Near cut-off		100	0.9	1.43	2.09
Positive		100	0.0	1.46	3.97
N/A = not app

Table 11. Lot to lot reproducibility of the MLL (KMT2A) Breakapart (cut-off: 3.8%)

Table 12. Lot to lot reproducibility of the P53 (TP53) Deletion (cut-off: 6.8%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	TotalSD
Negative	(b)(4)	100	0.00	1.20
Near cut-off		100	0.00	4.85
Positive		100	2.25	2.76

Table 13. Lot to lot reproducibility of the Del (20q) Deletion (cut-off: 5.7%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	92	0.00	0.00	2.17
Near cut-off		67	1.41	0.00	3.25
Positive		100	0.00	2.62	4.95

Table 14. Lot to lot reproducibility of the CBFβ (CBFB) /MYH11 Translocation, Dual Fusion (cut-off: 2.3%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	TotalSD
Negative	(b)(4)	100	0.00	0.00
Near cut-off		33	1.12	2.06
Positive		100	0.00	7.69

Table 15. Lot to lot reproducibility of the Del (5q) Deletion (cut-off: 6.3%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	83	0.98	0.49	2.09
Near cut-off		92	0.00	0.00	4.06
Positive		100	0.00	1.40	3.66

{11}------------------------------------------------

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	100	0.00	0.08	0.94
Near cut-off		100	0.00	0.00	5.78
Positive		100	1.82	0.00	4.03

Table 16. Lot to lot reproducibility of the Del (7q) Deletion (cut-off: 7.4%)

Table 17. Lot to lot reproducibility of the AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion (cut-off: 2.3%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	100	0.00	0.00	0.14
Near cut-off		100	0.87	0.58	1.84
Positive		100	0.80	1.18	4.85

Table 18. Lot to lot reproducibility of the EVI1 (MECOM) Breakapart Inversion (1RG/1B/1RGB) (cut-off: 4.0%)

Specimen	Mean ofabnormalpercentage	' Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	92	0.00	0.00	2.52
Near cut-off		67	1.63	2.82	6.29
Positive		100	1.80	3.01	6.99

Table 19. Lot to lot reproducibility of the EVI1 (MECOM) Breakapart Translocation (1R/1GB/1RGB) (cut-off: 4.0%)

Specimen	Mean ofabnormalpercentage	Agreement(%)	Intra-lot SD	Inter-lot SD	TotalSD
Negative	(b)(4)	100	0.25	0.00	0.70
Near cut-off		100	0.96	1.80	3.55
Positive		100	4.67	3.74	7.62

b. Linearity/assay Reportable Range:

Not applicable

{12}------------------------------------------------

c. Traceability, Stability, Expected Values (controls, calibrators, or methods):
Several stability studies were conducted in support of the probe kits. The specifics of each study are described below and summarized in Table 20 below.

Real-time Kit stability study:

The real-time stability of the probes was assessed using an isochronal study whereby probe lots of differing ages were assessed at the same time on identical samples. One normal, one low positive, and one high positive specimens were each tested in triplicate on each lot. Three lots of probes with minimum age of 25 months were tested in support of a 24-month shelf-life claim. Probe lots of intermediate ages between 0 and 25 months were also included to provide interim data. The result supported the claimed shelf life of 24 months when stored at -20 C.

Transport Stability

The transport study was conducted by stressing probes under potential extremes of transport conditions. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. There was no change in device performance under the stress conditions.

Freeze/Thaw Stability

The freeze/thaw stability was assessed by testing the probes at 0, 5, and 11 freeze/thaw cycles to support the "" cycles claim. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. Ten freeze-thaw cycles were found to be acceptable for this kit.

Post Hybridization Stability

The post hybridization stability was assessed by re-analyzing hybridized slides at '0)(4) weeks after hybridization to support the 1month claim. One normal, one low and" positive, and one high positive specimens were each tested in triplicate using one lot of probe. The post-hybridization signal was determined to be stable to one month.

Photostabilitv

The photostability of the probes was assessed by exposing probes to light for 0, 24, and "" hours to support the limited exposure to light claim. One normal, one low positive, and one high positive specimens were each tested in triplicate using one lot of probe. The photo-stability of slides was supported to "" hours. Prolonged exposure of slides to light should be avoided.

Table 20. Summary of stability studies

{13}------------------------------------------------

Test	Samples	Storage	Intervals
Real TimeStability	(b)(4)	-20 °C	(b)(4)
TransportStability		2 weeks at+40 °C, then-20 °C
Freeze/ThawStability		-20 °C
PostHybridizatioStability		Refrigeratortemperature(in the dark)
Photostabilit		RoomTemperature(fluorescentlight)

BM - bone marrowN = negative; LP = low positive; HP = high positive; F/T = freeze thaw; t = time point

d. Detection Limit:

The analytical sensitivity of the probes was established by analyzing interphase nuclei from 25 karyotypically normal bone marrow samples. Each sample was analyzed by 2 independent analysts and the signal pattern of each interphase was recorded. Each analyst analyzed 100 nuclei per sample, for a total of 200 nuclei per sample, resulting in 5000 scorable nuclei per probe evaluated. All nine probes evaluated in this study have an analytical sensitivity of greater than 95%, meeting the required acceptance criteria for analytical sensitivity (Table 21).

Table 21. Analytical sensitivity of the probes
------------------------------------------------	--	--	--

Probe name	Number ofinterphase nucleiwith the expectednormal signalpattern	Total numberof interphasenucleianalyzed	Analyticalsensitivity(%)	95%Confidenceinterval (%)
MLL (KMT2A)Breakapart probe	(b)(4)
P53 (TP53) DeletionProbe

{14}------------------------------------------------

Del(20q) DeletionProbe	4924	5000	98.48	98.1-98.78
CBFB(CBFB)/MYH11Translocation, DualFusion Probe	(b)(4)
Del(5q) Deletion Probe
Del(7q) Deletion Probe
AML1/ETO(RUNX1/RUNX1T1)Translocation, DualFusion Probe
EVI1 (MECOM)Breakapart Probe

e. Analytical specificity:
Analytical specificity is defined as the percentage of signals that hybridize to the correct locus and no other location. To establish the probe analytical specificity, each probe was assessed to verify that it hybridizes to the specific target chromosome location. Five(b) normal male peripheral blood samples ("" metaphase spreads in
each sample, of " metaphase spreads if the probe is located on a sex chromosome giving(0)(9) unique loci evaluated for each analysis). The analytical specificity of each product 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. All probes met the required acceptance criteria for analytical specificity with all components having a specificity of 100%. (Table 22).

Table 22. Analytical specificity of the probes

Probe	Target	Number ofmetaphasechromosomeshybridized	Number ofcorrecthybridizedloci	AnalyticalSpecificity	95%ConfidenceInterval
MLL (KMT2A) Breakapart FISH Probe Kit	(b)(4)

{15}------------------------------------------------

Probe	Target	Number ofmetaphasechromosomeshybridized	Number ofcorrecthybridizedloci	AnalyticalSpecificity	95%ConfidenceInterval
(b)(4)
EVI1 (MECOM) Breakapart FISH Probe Kit
EVI1, Red	3q26.2	200	200	100%	98.12% - 100%
EVI1, Green	3q26.2	200	200	100%	98.12% - 100%
EVI1, Blue	3q26.2	200	200	100%	98.12% - 100%

Assay Cut-off (Upper Reference Limit): f.

The assay cut off was assigned as follows: For seven of the nine probes, a central clinical laboratory generated the clinical cut-off using a large patient dataset exceeding the ACMG minimum sample size guidelines. In addition, analytical sensitivity data from 25 karyotypically normal bone marrow samples was used to confirm the normal cut-offs. None of the 25 normal samples showed an abnormal signal pattern at or above the normal cut-offs confirming the clinical cut-off established.

For two of the nine probes included in this study, AML1 (RUNX1) Breakapart FISH Probe Kit and EVI1 (MECOM) Breakapart FISH Probe Kit, the results generated by the analytical sensitivity study were utilized (25 karyotypically normal bone marrow samples). In this case: ≥ 25 karyotypically normal bone marrow samples were enumerated and the normal cut-offs were calculated using the ß inverse function (BETAINV) (Table 23).

{16}------------------------------------------------

An analytical result above the normal cut-off (upper reference limit) is deemed to be positive. Conversely, an analytical result below the cut-off is deemed to be negative.

Name	NegativeSignal	Positive Signal	Cut-off
MLL (KMT2A) Breakapart	(b)(4)	1F/1R/1G	3.8%
P53 (TP53) Deletion		1R/2G	6.8%
Del (20q) Deletion		1R/1G	5.7%
CBFβ (CBFB) /MYH11Translocation, Dual Fusion		2F/1R/1G	2.3%
Del (5q) Deletion		1R/2G	6.3%
Del (7q) Deletion		1R/1G	7.4%
AML1/ETO(RUNX1/RUNX1T1)Translocation, Dual Fusion		2F/1R/1G	2.3%
EVI1 (MECOM) BreakapartInversion Signal		1RG/1B/1RGB	4.0%
EVI1 (MECOM) BreakapartTranslocation Signal		1R/1GB/1RGB	4.0%

Table 23: Supported Signal Patterns & Cut-Offs

g. Probe limit
Not applicable. The probes are intended to be used only at the concentration provided and are not intended to be diluted.

Comparison Studies: 2.

a. Method comparison with predicated device:
Not applicable
b. Matrix Comparison
Not applicable
1. Clinical Studies:
- a. Clinical sensitivity Not applicable
- b. Clinical specificity Not applicable

{17}------------------------------------------------

C. Other clinical supportive data

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS)

AML and MDS are neoplastic hematological disorders that arise from myeloid progenitor cells in the bone marrow. AML is characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow or other tissues, while MDS is characterized by the simultaneous proliferation and apoptosis of hematopoietic cells.

Refer to the WHO Guidelines for the most up-to-date use of the probes.

Incident rate

Reference to the clinical significance in WHO guidelines and peer-reviewed published papers (3 for AML and 3 for MDS) were provided to support the clinical validity of the device in characterizing bone marrow specimens from patients with AML and/or MDS. Prevalence rates based on clinical thresholds defined in the literature were reviewed and summarized. Clinical specimens were tested in using the Cytocell AML FISH probe sets and assigned cut-offs and the incidence rates were compared to literature. Laboratory 1 (GOP) analyzed 100 known and suspected AML and MDS specimens in total. Laboratory 2 (YAL) re-analyzed specimen data (266-742 AML or MDS specimens depending on the probe) tested in their laboratory based on the assigned cut-off. See Table 24 for the summary of results and Tables 25-33 for individual probe results.

Probes/Rearrangement	ExpectedPrevalenceRate fromLiterature	GOP data setPrevalence(95% CI)	YAL data setPrevalence (95% CI)
MLL (KMT2A)Breakapart	(b)(4)
P53 (TP53) deletion
Del(20q) deletion
CBFB/MYH11translocation, dualfusion
Del(5q) deletion

{18}------------------------------------------------

	(b)(4)
Del(7q) deletion
AML/ETO(RUNX1/RUNX1T1)dual fusion,translocation
EVI1 (MECOM)Breakapart

N/A = not applicable as data was not obtained from this site for this probe.

Table 25. MLL (KMT2A) breakapart probe incidence rate
-------------------------------------------------------	--

Condition	LiteratureSource 1Schanz etal.	LiteratureSource 2Zhao et al.	LiteratureSource 3Wang et al.	LiteratureSource 4Papaemmanuilet al.	LiteratureSource 5Grimwadeet al.	LiteratureSource 6Bacher etal.	DataSource 1GOP	DataSource 2YAL
Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	No	No	No	Yes	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS orAML	Known orsuspectedMDS orAML
Upperreferencelimit - 'Cut-off value'(percentageand per 200nuclei)	N/A	N/A	N/A	N/A	N/A	N/A	(b)(4)1R1G1Fpatternsper 200scoreableinterphasenuclei	1R1G1Fpatternsper 200scoreableinterphasenuclei

{19}------------------------------------------------

Total Numberof specimenstested foreach claimedtype	2902	2404	435	1540	5876	2235	100	413
Number ofspecimenswith apositive proberesult	N/A	N/A	N/A	N/A	N/A	N/A	2	6
Range ofpositive proberesults	N/A	N/A	N/A	N/A	N/A	N/A	(b)(4)
Sourceincidence rateforrearrangement(95% CI)	0.2%	1.24%	1.9%	2.86%	4.5%	2.72%
			*'11qabnormalities'		Expected range fromliterature:

Table 26. P53(TP53) deletion probe incidence rate

Condition	LiteratureSource 1Schanz et al.	LiteratureSource 2Bernasconi et al.	LiteratureSource 3Wang et al.	LiteratureSource 4Papaemmanuil et al.	LiteratureSource 5Grimwade et al.	LiteratureSource 6Lazarevic et al.	DataSource 1GOP
Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	No	No	No	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Yes	Yes

{20}------------------------------------------------

Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS orAML
Upperreference limit- 'Cut-offvalue'(percentageand per 200nuclei)	N/A	N/A	N/A	N/A	N/A	N/A	(b)(4)r14 1R2Gpatternsper 200scoreableinterphasenuclei
Total Numberof specimenstested for eachclaimed type	2902	331	435	1540	5876	3251	100
Number ofspecimenswith a positiveprobe result	N/A	N/A	N/A	N/A	N/A	N/A	5
Range ofpositive proberesults	N/A	N/A	N/A	N/A	N/A	N/A	(b)(4)
Sourceincidence rateforrearrangement(95% CI)	0.6%	1.8%	1.9%	4%	4%	8.8%	Expected range fromliterature:

Table 27. Del(20q) deletion probe incidence rate

Condition	LiteratureSource 1Schanz etal.	LiteratureSource 2Haas et al.	LiteratureSource 3Wang etal.	Data Source1GOP	Data Source2YAL
Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	Yes	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes
Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	Known orsuspected	Known orsuspected
Upperreferencelimit - 'Cut-off value'(percentageand per 200nuclei)	N/A	N/A	N/A	(b)(4)
Total Numberof specimenstested foreach claimedtype	2902	2072	435	100	742
Number ofspecimenswith apositive proberesult	N/A	N/A	N/A	5	9
Range ofpositive proberesults	N/A	N/A	N/A	(b)(4)
Sourceincidence rateforrearrangement(95% CI)	1.7%	3.6%	6.6%
Condition	LiteratureSource 1Papaemmanuilet al.	LiteratureSource 2Grimwadeet al.	LiteratureSource 3Dores etal.	Data Source 1GOP	Data Source 2YAL
Was thespecific deviceunder reviewin thesubmissionused in thestudy?	No	No	No	Yes	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes
Targetpopulation(disease status)	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspected MDSor AML	Known orsuspectedMDS or AML
Upperreference limit- 'Cut-offvalue'(percentageand per 200nuclei)	N/A	N/A	N/A	2.3% or 51R1G2Fpatterns per200 scoreableinterphasenuclei	2.3% or 51R1G2Fpatterns per200 scoreableinterphasenuclei
Total Numberof specimenstested for eachclaimed type	(b)(4)
Number ofspecimenswith a positiveprobe result	N/A	N/A	N/A	2	7
Range ofpositive proberesults	N/A	N/A	N/A	83.0%-93.0%	14%-99.5%
Sourceincidence rateforrearrangement%(95% CI)	5.26%	5%	1.04%	2%(0.24% to7.04%)	2.63%(1.06% to5.35%)
		Expected range fromliterature:		1.04% - 5.26%

{21}------------------------------------------------

{22}------------------------------------------------

Table 28. CBFB/MYH11 translocation, dual fusion probe incidence rate

{23}------------------------------------------------

Table 29. Del(5q) deletion probe incidence rate

Condition	LiteratureSource 1Schanz etal.	LiteratureSource 2Haase etal.	LiteratureSource 3Bernasconiet al.	LiteratureSource 4Papaemmanuilet al.	LiteratureSource 5Grimwadeet al.	LiteratureSource 6Sandersonet al.	DataSource 1GOP	DataSource 2YAL
Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	No	No	No	Yes	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS orAML	Known orsuspectedMDS orAML
Upperreferencelimit - 'Cut-off value'(percentageand per 200nuclei)	N/A	N/A	N/A	N/A	N/A	N/A	6.3% or 131R2Gpatternsper 200scoreableinterphasenuclei	6.3% or 131R2Gpatternsper 200scoreableinterphasenuclei
Total Numberof specimenstested foreach claimed	2902	2072	331	1540	5876	1709	100	723

{24}------------------------------------------------

type
Number ofspecimenswith apositive proberesult	N/A	N/A	N/A	N/A	N/A	N/A	9	73
Range ofpositive proberesults	N/A	N/A	N/A	N/A	N/A	N/A	8%-98%	8%-99.5%
Sourceincidence rateforrearrangement(95% CI)	8%	15.1%	16%	6%	5%	11%	9%(4.20% to6.40%)	10.9%(8% to12.53%)
							Expected range fromliterature:5% - 16%

Table 30. Del(7q) deletion probe incidence rate

Condition	LiteratureSource 1Schanz etal.	LiteratureSource 2Haase etal.	LiteratureSource 3Bernasconiet al.	LiteratureSource 4Papaemmanuilet al.	LiteratureSource 5Grimwadeet al.	LiteratureSource 6Sandersonet al.	Data Source1GOP	Data Source2YAL
Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	No	No	No	Yes	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS orAML	Known orsuspectedMDS orAML

{25}------------------------------------------------

Upperreferencelimit - 'Cut-off value'(percentageand per 200nuclei)	N/A	N/A	N/A	N/A	N/A	N/A	7.4% or 151R1Gpatterns per200scoreableinterphasenuclei	7.4% or 151R1Gpatterns per200scoreableinterphasenuclei
Total Numberof specimenstested foreach claimedtype	2902	2072	331	1540	5876	1709	100	746
Number ofspecimenswith apositive proberesult	N/A	N/A	N/A	N/A	N/A	N/A	4	48
Range ofpositive proberesults	N/A	N/A	N/A	N/A	N/A	N/A	21.5%-96%	9%-98%
Sourceincidence rateforrearrangement(95% CI)	3.6%	11.1%	7.8%	5.7%	8%	9%	4%(1.10% to9.93%)	6.43%(4.78% to8.44%)
					Expected range fromliterature		3.6% - 11.1%

Table 31. AML1/ETO (RUNX1/RUNX1T1) dual fusion translocation probe incidence rate

Condition	LiteratureSource 1Papaemmanuilet al.	LiteratureSource 2Grimwadeet al.	LiteratureSource 3Dores et al.	Data Source1GOP	Data Source2YAL	Condition	LiteratureSource 1Schanz etal	LiteratureSource 2Haase	LiteratureSource 3Pozdnyakovaet al	LiteratureSource 4Papaemmanuilet al	LiteratureSource 5Grimwadeet al	LiteratureSource 6Byrd et al	Data Source 1GOP
Was thespecific deviceunder reviewin thesubmissionused in thestudy?	No	No	No	Yes	Yes	Was thespecificdevice underreview in thesubmissionused in thestudy?	No	No	No	No	No	No	Yes
Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Was thespecimen typein the studyrepresentativeof the claimedspecimentype(s)	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Targetpopulation(disease status)	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS orAML	Known orsuspectedMDS orAML	Targetpopulation(diseasestatus)	ConfirmedMDS	ConfirmedMDS	ConfirmedMDS	ConfirmedAML	ConfirmedAML	ConfirmedAML	Known orsuspectedMDS or AML
Upperreference limit- 'Cut-offvalue'(percentageand per 200nuclei)	N/A	N/A	N/A	2.3% or 51R1G2Fpatterns per200scoreableinterphasenuclei	2.3% or 51R1G2Fpatterns per200 scoreableinterphasenuclei	Upperreferencelimit - 'Cut-off value'(percentageand per 200nuclei)	N/A	N/A	N/A	N/A	N/A	N/A	4% or 81RG/1B/1RGBor1R/1GB/1RGBpatterns per200 scoreableinterphasenuclei
Total Numberof specimenstested for eachclaimed type	1540	5876	19497	100	414	Total Numberof specimenstested foreach claimedtype	2902	2072	1029	1540	5876	1213	100
Number ofspecimens witha positiveprobe result	N/A	N/A	N/A	0	6	Number ofspecimenswith apositive proberesult	N/A	N/A	N/A	N/A	N/A	N/A	4
Range ofpositive proberesults	N/A	N/A	N/A	N/A	69.5%-98.5%
Sourceincidence rateforrearrangement%(95% CI)	3.8%	7%	1.6%	0%(0.00% to3.62%)	1.45%(0.53% to3.13%)
		Expected range fromliterature			1.6% - 7.0%

{26}------------------------------------------------

{27}------------------------------------------------

Table 32. EVI1 Breakapart Probe incidence rate

{28}------------------------------------------------

Range ofpositive proberesults	N/A	N/A	N/A	N/A	N/A	N/A	11%-90.5%
Sourceincidence rateforrearrangement(95% CI)	0.4%	2%	0.3%	1.3%	1.5%	1%Expected range fromliterature	4%(1.10% to9.93%)0.3% - 2.0%

Additional supporting data

To further support the performance of AML1/ETO (RUNX1/RUNX1T1) and CBFB (CBFB) /MYH11 probes, additional clinical data sets covering the full range of the signal distribution were provided to demonstrate concordance between FISH and G-band. 100% agreement were observed for both data sets expect for one specimen that falls into re-test zone. Tables 33 and 34 demonstrate acceptable clinical concordance.

Table 33. Concordance between the AML1/ETO (RUNX1/RUNX1T1) Probe kit and Karyotyping

AML1/ETO (RUNX1/RUNX1T1) Translocation, DualFusion FISH probe kit% agreement (n)
G-band result	Normal	Abnormal
No t(8;21) rearrangement	(b)(4)
Confirmed t(8;21) rearrangement

Table 34. Concordance between the CBFβ (CBFB) /MYH11 Probe kit and Karyotyping

	CBFβ (CBFB) /MYH11 Translocation, Dual Fusion FISHProbe Kit % agreement (n)
G-band result	Normal	Abnormal
No inv(16)/t(16;16)rearrangement	(b)(4)
Confirmed inv(16)/t(16;16)rearrangement

The discordance was determined to be a sample with results very near the cut-off.

References:

1. Bacher U, Kern W, Schnittger S, Hiddemann W, Schoch C, Haferlach T. Further correlations of morphology according to FAB and WHO classification to cytogenetics in de novo acute myeloid leukemia: A study on 2,235 patients. Ann Hematol. 2005;84(12):785–91.

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1. Bernasconi P, Klersy C, Boni M, Cavigliano PM, Calatroni S, Giardini I, et al. Incidence and prognostic significance of karyotype abnormalities in de novo primary myelodysplastic syndromes: a study on 331 patients from a single institution. Leukemia. 2005:19(8):1424-31.
1. Byrd JC. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002 Dec 15:100(13):4325-36.
1. Dores, G.M. et al., 2011. Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood, 119(1), pp.34-43.
1. Grimwade, D. et al., 2010. Refinement of cytogenetic classification in acute myeloid leukemia: Determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood, 116(3), pp.354-365.
1. Haase D. Cytogenetic features in myelodysplastic syndromes. Ann Hematol. 2008 Jul:87(7):515-26.
1. Lazarevic V, Hörstedt A-S, Johansson B, Antunovic P, Billström R, Derolf Å, et al. Incidence and prognostic significance of karyotypic subgroups in older patients with acute myeloid leukemia: the Swedish population-based experience. Blood Cancer J. 2014 Feb 28:4(2):e188.
1. Papaemmanuil, E. et al., 2016. Genomic Classification and Prognosis in Acute Myeloid Leukemia. New England Journal of Medicine, 374(23), pp.2209-2221.
1. Pozdnyakova O. Miron PM. Tang G. Walter O. Raza A. Woda B. et al. Cytogenetic abnormalities in a series of 1,029 patients with primary myelodysplastic syndromes: a report from the US with a focus on some undefined single chromosomal abnormalities. Cancer. 2008 Dec 15:113(12):3331-40.
1. Sanderson RN, Johnson PRE, Moorman a V, Roman E, Willett E, Taylor PR, et al. Population-based demographic study of karyotypes in 1709 patients with adult acute myeloid leukemia. Leukemia. 2006 Mar 19:20(3):444-50.
1. Schanz, J. et al., 2012. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. Journal of Clinical Oncology, 30(8), pp.820-829.
1. Wang XQ, Ryder J, Gross SA, Lin G, Irons RD. Prospective analysis of clinical and cytogenetic features of 435 cases of MDS diagnosed using the WHO (2001) classification: a prognostic scoring system for predicting survival in RCMD. Int J Hematol. 2009 Oct:90(3):361-9.
1. Zhao X, Li S, Li N, Fan R, Lin G, Wang X. 11q23 abnormalities in adult Chinese patients with hematological malignancies. Med Oncol. 2014 Aug;31(8):115
1. Clinical cut-off

Not applicable

న. Expected values/Reference range: Same as Assay Cut-off (Upper Reference Limit) (section 1.f) above

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N. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

The labeling supports the decision to grant the De Novo request for this device

O. Patient Perspective

This submission did not include specific information on patient perspectives for this device.

Identified Risks to Health	Mitigation Measures
Incorrect test results	Special controls (1), (2), (3), and (4)
Incorrect interpretation of test results	Special controls (1), (2), (3), and (4)

P. Identified Risks to Health and Mitigation Measures:

O. Benefit/Risk Summary:

Clinical reviewer memo will contain details for benefit risk determination.

Summary of theBenefits	Summary of theRisks	Summary of OtherFactors	Conclusions
AML and MDSpatients may benefit byuse of the device onbone marrowspecimens to obtainresults that can be usedin accordance with theWorld HealthOrganization criteriafor assessment of thesepatient specimenswhen assay results areinterpreted by aqualified pathologist orcytogeneticist.	Erroneous deviceperformance can yieldfalse negative or falsepositive results orincorrect interpretationof test results by theuser which mayadversely influencemanagement of AMLor MDS patients.	Risks are mitigated byanalytical and clinicalvalidation studies usingthe device probes, alongwith labeling andsupports the intendeduse. The device userequires a qualifiedpathologist orcytogeneticist in thecontext ofhistopathologicalevaluation (e.g.,immunohistochemistry).	Yes. Based on thesupporting clinicalstudies for thediagnostic devicealong with review ofthe analyticalperformance andlabeling, the probablebenefits outweigh theprobable risks in lightof the mitigationsprovided by the specialcontrols, in addition tothe general controls.

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 864.1880. FDA believes that the stated special controls, in combination with the general controls, provide a reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code: QDI

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Device Type:	Fluorescence in-situ hybridization based detection of chromosomalabnormalities from patients with hematologic malignancies
Class:	II (Special Controls)
Regulation:	21 CFR 864.1880

(a) IDENTIFICATION: Fluorescence in-situ hybridization based detection of chromosomal abnormalities from patients with hematologic malignancies is used to detect chromosomal abnormalities in human specimens from patients with hematologic malignancies. The test is indicated for the clinical management of patients consistent with internationally accepted guidelines (e.g. World Health Organization guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues) and in conjunction with other clinical and clinicopathological criteria. The results are to be interpreted by a pathologist or equivalent professional.
(b) CLASSIFICATION: Class II (special controls). The special controls for this device are:
- (1) Design verification and validation (b)(4) -must include:
  - (i) A detailed description of all probes included in the kit;
- Purpose of each probe; (ii)
- (iii) Probe molecular specificity;
- (iv) Probe specificity:
- Probe limits: (v)
- Probe sensitivity; (vi)
- Specification of required ancillary reagents, instrumentation, and equipment; (vii)
- Specification of the specimen collection, processing, storage and slide (viii) preparation methods;
- Specification of the assay procedure: (ix)
- Specification of control elements that are incorporated into the recommended (x) testing procedures;
- Specification of the criteria for test result interpretation and reporting; (xi)
- (xii) Documentation demonstrating analytical validation that includes:
  - (A)Device analytical sensitivity data with a minimum of 25 specimens from karyotypically normal males.
  - (B)Device analytical specificity data with a minimum of 5 specimens from karyotypically normal males.

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(C) Description of how the clinical threshold was assigned and verification of the assigned clinical threshold.
(D) Device precision/reproducibility data with a minimum of 6 clinical specimens including 2 negative specimens, 2 positive specimens near the clinical decision threshold (cut-off) and 2 positive specimens. The data must include results obtained from 3 sites (as applicable), with 2 operators at each site, with the assay run for a minimum of 3-5 non-consecutive days and each specimen run in duplicate for a minimum of 30 replicates.
(E) Between-reagent lot reproducibility using 3 reagent lots and 3 clinical specimens representing negative, near cut-off /low positive, and positive.
(F) Device stability data to include:
- (1) Real-time Stability,
- (2) Freeze-Thaw Stability,
- (3) Transport and Temperature Stability, as applicable,
- (4) Post-Hybridization Signal Stability, and
- (5) Photostability of Probe.
(xiii) Documentation demonstrating the clinical validity of the device that includes:
- (A) A summary of the prevalence and clinical thresholds reported in 3 peerreviewed published literature references for the intended use population of the device and device performance data demonstrating conformance with the published prevalence as reported in peer-reviewed published literature references based on testing clinical specimens, selected without bias (e.g., consecutively selected) from the intended use population using the specific device seeking marketing clearance. A minimum number of clinical specimens must be tested to ensure sufficient positives are evaluated by the device, or alternatively, in the absence of a sufficient number of positives, an additional comparison of results obtained with the device to clinical truth (e.g., confirmed clinical diagnosis and/or G-banded karyotyping) with an independent specimen set must be conducted.
- (B) Documentation for peer-reviewed published literature references must include the following elements:
  - (1) Whether the specific device was used in the literature reference;
  - (2) Number and type of specimens;
  - (3) Target population studied;
  - (4) Upper reference limit; and

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(5) Prevalence range estimated based on the number of positive probe results.
(C) In the absence of clinical data obtained from paragraphs (b)(1)(xiii)(A) and (b)(1)(xiii)(B) of this section, clinical data obtained from a method comparison to the predicate with positives and negative clinical specimens.
(2) The intended use required on the label under 21 CFR 809.10(a)(4) and on the labeling under 21 CFR 809.10(b)(5)(ii), must include a statement that

"The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic."

(3) The 21 CFR 809.10(b) labeling must include information that demonstrates the performance characteristics of the test, including a detailed summary of the performance studies conducted and their results, as described in paragraphs (b)(1)(iv) through (b)(1)(xiii) of this section. The 21 CFR 809.10(b) labeling must include the pre-specified acceptance criteria for these performance studies, justification for the pre-specified acceptance criteria, and whether the prespecified acceptance criteria were met.
(4) The 21 CFR 809.10(b) labeling must include the following limiting statements:
"Reporting and interpretation of FISH results should be consistent with (i) professional standards of practice and should take into consideration other clinical and diagnostic information. This kit is intended as an adjunct to other diagnostic laboratory tests and therapeutic action should not be initiated on the basis of the FISH result alone. Failure to adhere to the protocol may affect the performance and lead to false results."
(ii) "Each lab is responsible for establishing their own cut-off values. Each laboratory should test sufficiently large number of samples to establish normal population distribution of the signal levels and to assign a cut-off value. The product is for professional use only and is intended to be interpreted by a qualified Pathologist or Cytogeneticist."

Regulation Number and Section

§ 864.1880 Fluorescence in situ hybridization (FISH)-based detection of chromosomal abnormalities from patients with hematologic malignancies.

(a)
Identification. A fluorescence in situ hybridization (FISH)-based detection of chromosomal abnormalities from patients with hematologic malignancies is used to detect chromosomal abnormalities in human specimens from patients with hematologic malignancies. The test is indicated for the clinical management of patients consistent with internationally accepted guidelines (e.g., World Health Organization guidelines for Classification of Tumours of Haematopoietic and Lymphoid Tissues) and in conjunction with other clinical and clinicopathological criteria. The results are to be interpreted by a pathologist or equivalent professional.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include:
(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 the criteria for test result interpretation and reporting;
(xii) Documentation demonstrating analytical validation that includes:
(A) Device analytical sensitivity data with a minimum of 25 specimens from karyotypically normal males.
(B) Device analytical specificity data with a minimum of five specimens from karyotypically normal males.
(C) Description of how the clinical threshold was assigned and verification of the assigned clinical threshold.
(D) Device precision/reproducibility data with a minimum of six clinical specimens including two negative specimens, two positive specimens near the clinical decision threshold (cut-off) and two positive specimens. The data must include results obtained from three sites (as applicable), with two operators at each site, with the assay run for a minimum of 3-5 non-consecutive days and each specimen run in duplicate for a minimum of 30 replicates.
(E) Between-reagent lot reproducibility using three reagent lots and three clinical specimens representing negative, near cut-off/low positive, and positive.
(F) Device stability data to include:
(
1 ) Real-time stability,(
2 ) Freeze-thaw stability,(
3 ) Transport and temperature stability, as applicable,(
4 ) Post-hybridization signal stability, and(
5 ) Photostability of probe.(xiii) Documentation demonstrating the clinical validity of the device that includes:
(A) A summary of the prevalence and clinical thresholds reported in three peer-reviewed published literature references for the intended use population of the device and device performance data demonstrating conformance with the published prevalence as reported in peer-reviewed published literature references based on testing clinical specimens, selected without bias (
e.g., consecutively selected) from the intended use population using the specific device seeking marketing clearance. A minimum number of clinical specimens must be tested to ensure sufficient positives are evaluated by the device, or alternatively, in the absence of a sufficient number of positives, an additional comparison of results obtained with the device to clinical truth (e.g., confirmed clinical diagnosis and/or G-banded karyotyping) with an independent specimen set must be conducted.(B) Documentation for peer-reviewed published literature references must include the following elements:
(
1 ) Whether the specific device was used in the literature reference;(
2 ) Number and type of specimens;(
3 ) Target population studied;(
4 ) Upper reference limit; and(
5 ) Prevalence range estimated based on the number of positive probe results.(C) In the absence of clinical data obtained from paragraphs (b)(1)(xiii)(A) and (B) of this section, clinical data obtained from a method comparison to the predicate with positives and negative clinical specimens.
(2) The intended use required on the label under § 809.10(a)(4) of this chapter and on the labeling required under § 809.10(b)(5)(ii) of this chapter must include a statement that “The test is not intended for use as a stand-alone diagnostic, disease screening, or as a companion diagnostic.”
(3) The labeling required under § 809.10(b) of this chapter must include information that demonstrates the performance characteristics of the test, including a detailed summary of the performance studies conducted and their results, as described in paragraphs (b)(1)(iv) through (xiii) of this section. The labeling required under § 809.10(b) of this chapter must include 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.
(4) The labeling required under § 809.10(b) of this chapter must include the following limiting statements:
(i) “Reporting and interpretation of FISH results should be consistent with professional standards of practice and should take into consideration other clinical and diagnostic information. This kit is intended as an adjunct to other diagnostic laboratory tests and therapeutic action should not be initiated on the basis of the FISH result alone. Failure to adhere to the protocol may affect the performance and lead to false results.”
(ii) “Each lab is responsible for establishing their own cut-off values. Each laboratory should test sufficiently large number of samples to establish normal population distribution of the signal levels and to assign a cut-off value. The product is for professional use only and is intended to be interpreted by a qualified pathologist or cytogeneticist.”