The Vysis EGR1 FISH Probe Kit - SC detects the LSI EGR1 probe target on chromosome 5q in bone marrow specimens. The Vysis EGR1 FISH Probe Kit -SC assay results characterize bone marrow 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 product for diagnosis, monitoring or risk assessment has not been established.

Device Description

The Vysis EGR1 FISH Probe Kit - Specimen Characterization uses fluorescence in situ hybridization (FISH) DNA probe technology to detect probe target LSI EGR1 (containing early growth response 1 gene; location chromosome 5g31). The LSI D5S23, D5S721 probe (location chromosome 5p15.2) serves as a control.

The Vysis EGR1 FISH Probe Kit – SC (List No. 04N37-001) consists of the following components which are sufficient to process 20 assays:

Vysis LSI EGR1 SpectrumOrange/D5S23, D5S721 Spectrum Green Probes
Vysis LSI/WCP Hybridization Buffer
DAPI II Counterstain
NP-40
20X SSC Salt

Items 2 through 5 above are general purposes reagents.

DNA Probes: Vysis LSI EGR1 SpectrumOrange/ D5S23, D5S721 SpectrumGreen.

a. The SpectrumOrange-labeled LSI EGR1 probe, approximately 209 kb in length (chr5:137654208-137862738; February 2009 Assembly; UCSC Human Genome Browser) is located at 5g31 and contains the complete EGR1 gene.
b. The SpectrumGreen-labeled LSI D5S23, D5S721 probe, approximately 561 kb in length (chr5:9397109-9958407; February 2009 Assembly; UCSC Human Genome Browser) is located at 5p15.2.

AI/ML Overview

Here's a summary of the acceptance criteria and the studies performed for the Vysis EGR1 FISH Probe Kit - SC, based on the provided text:

Acceptance Criteria and Device Performance for Vysis EGR1 FISH Probe Kit - SC

1. Table of Acceptance Criteria and Reported Device Performance:

Study Category	Study Type	Acceptance Criteria	Reported Device Performance
Analytical Performance
Precision/Reproducibility	Intra-Day and Inter-Day	Agreement with the expected result of greater than or equal to 90% for the high positive specimen category for each site and 90% for the negative specimen category across all sites with no more than 3 discordant results occurring at one site.	Acceptance criteria met
	Reproducibility: Inter-Site	Same as above.	Acceptance criteria met
	Lot to Lot Reproducibility	Same as above.	Acceptance criteria met
Stability	Real-Time Stability	Acceptable quality of all attributes (signal intensity, target background, cross-hybridization, specificity, overall readability) for all samples tested.	Acceptance criteria met for 12-month stability
	In-Use Freeze-Thaw Stability	Same as above.	Acceptance criteria met throughout and at the end of 20 freeze-thaw cycles
	Transport and Temperature Extreme Stability	Same as above.	Acceptance criteria met
	Post-Hybridization Signal Stability	Same as above.	Acceptance criteria met for Post-hybridization stability of 3 weeks
	Probe Photostability	Same as above.	Acceptance criteria met for Photo-stability of 48 hours
Detection Limit	Analytical Sensitivity	Not explicitly stated as a pre-specified acceptance criterion but implied to be high for "expected 2 red/2 green signal pattern".	99.6% (95% CI: 99.4, 99.7) of nuclei showed expected signal pattern
Analytical Specificity	Analytical Specificity	Not explicitly stated as a pre-specified acceptance criterion but implied to be high for correct locus hybridization.	100% (95% CI: 98, 100) for both D5S23, D5S721 and EGR1 probes
Reference Range	Upper Reference Limit	The assay identifies 1R2G patterns at or below 6% or 12 1R2G patterns per 200 scoreable interphase nuclei in karyotypically normal or deletion-free specimens.	None of the 25 normal specimens produced 1R2G signals at or above the 6% upper reference limit.

2. Sample sizes used for the test set and the data provenance:

Precision/Reproducibility (Intra-Day, Inter-Day, Inter-Site, Lot-to-Lot):
- Test Set Sample Size: 6 bone marrow specimens (2 high positive, 2 low positive, 2 negative).
- Nuclei evaluated: 200 nuclei per panel member (100 by each of 2 technologists) for a total of 1200 nuclei per study type (e.g., Intra-day, Inter-day, etc.).
- Data Provenance: Not explicitly stated (e.g., country of origin). Appears to be prospective analytical testing conducted by the manufacturer.
Analytical Sensitivity:
- Test Set Sample Size: 25 bone marrow specimens (karyotypically normal or 5p15 and 5q31 deletion-free).
- Nuclei evaluated: 200 nuclei per specimen (100 by each of 2 technologists) for a total of 5000 scoreable nuclei.
- Data Provenance: Not explicitly stated. Appears to be prospective analytical testing.
Analytical Specificity:
- Test Set Sample Size: Metaphase chromosomes prepared from peripheral blood cultures of five karyotypically normal males (pooled).
- Nuclei evaluated: 100 consecutive metaphase nuclei by one technologist, for a total of 200 target loci (100 for each probe).
- Data Provenance: Not explicitly stated. Appears to be prospective analytical testing.
Reference Range Validation:
- Test Set Sample Size: 25 bone marrow specimens (karyotypically normal or 5p15.2 and 5q31 deletion-free).
- Nuclei evaluated: 200 nuclei per specimen (100 by each of 2 technologists).
- Data Provenance: Not explicitly stated. Appears to be prospective analytical testing.
Clinical Supportive Data (Clinical Validity):
- Test Set Sample Size:
  - Data Source 1 (Sun et al.): 320 bone marrow specimens
  - Data Source 2 (Galvan et al.): 28 bone marrow specimens
  - Data Source 3 (Vance et al.): 181 bone marrow specimens
- Data Provenance: Retrospective, derived from peer-reviewed published literature (implicitly based on patient data from the respective study locations, not specified).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

Analytical Performance Studies (Precision, Sensitivity, Specificity, Reference Range):
- "Two technologists" or "one technologist" were involved in evaluating nuclei. Their specific qualifications (e.g., "cytogeneticist with X years of experience") are not explicitly stated, beyond being referred to as "technologists."
- The overall interpretation is intended for a "qualified pathologist or cytogeneticist."
Clinical Supportive Data:
- Ground truth for the clinical studies would have been established by the methods described in those published papers, likely involving consensus diagnostics by pathologists and/or cytogeneticists based on conventional cytogenetics and clinical information. The number and qualifications of these experts are not detailed in this summary document.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set:

For the analytical performance studies where two technologists evaluated slides (e.g., Precision, Analytical Sensitivity, Reference Range Validation), the text states "each technologist evaluated 100 nuclei per panel member" or "100 nuclei per specimen." It does not explicitly describe an adjudication method (e.g., 2+1, 3+1) if their readings disagreed. It simply reports aggregated results.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

No, an MRMC comparative effectiveness study was not done.
This device is described as an assay (FISH probe kit) intended to be interpreted by a qualified pathologist or cytogeneticist, not an automated system or AI-assisted diagnostic tool. The text explicitly states: "These devices do not include automated systems that directly report results without review and interpretation by a qualified pathologist or cytogeneticist." Therefore, the concept of human readers improving with 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, a standalone (algorithm only) performance study was not done.
As noted above, this is a FISH probe kit, which is a laboratory assay requiring manual interpretation by human experts. It is not an algorithm that performs standalone analysis.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

Analytical Performance Studies:
- Expected signal pattern: This serves as the ground truth for parameters like precision and sensitivity. It's based on the known genetic characteristics of the specimens (e.g., high positive, low positive, negative, normal karyotype).
- Known chromosomal location: For analytical specificity, the ground truth is the established chromosomal location of the gene targets.
Clinical Supportive Data:
- The ground truth for the clinical studies (validation of clinical utility) was likely established through a combination of pathology reports, conventional cytogenetics, and clinical diagnoses of AML or MDS as carried out in the respective peer-reviewed studies. The "percentage of cells with 1R2G signal pattern" would have been correlated with these clinical states.

8. The sample size for the training set:

Not applicable. The Vysis EGR1 FISH Probe Kit is a diagnostic assay (chemical reagents and probes), not a machine learning algorithm. Therefore, there is no "training set" in the context of AI/ML. All samples discussed are for analytical or clinical validation/support.

9. How the ground truth for the training set was established:

Not applicable, as there is no "training set" for this type of device.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR VYSIS EGR1 FISH PROBE KIT - SC (SPECIMEN CHARACTERIZATION)

Decision Summary

A. 510(k) Number	K123951
B. Purpose of the Submission:	Clearance of new assay
C. Measurand:	LSI EGR1 probe target on chromosome 5q inbone marrow specimens
D. Type of Test:	Flourescence in-situ hybridization (FISH)
E. Applicant:	Abbott Molecular Inc.
F. Device Name:	Vysis EGR1 FISH Probe Kit - SC (SpecimenCharacterization)

G. Regulatory Information:

FDA identifies this type of device as:

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.

1. New Regulation Number:	21 CFR 864.1870
2. Classification:	Class II (special controls)
3. Product code:	PDO
4. Panel:	Hematology and Pathology Devices Panel

H. Intended Use:

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1. Intended use(s):

2. Indication(s) for use:

Same as intended use

1. Special conditions for use statement(s):
  Warnings included in the labeling are as follows:
The assay results are intended to be interpreted only by a qualified pathologist or cytogeneticist.
· The Vysis EGR1 FISH Probe Kit SC is not for high-risk uses such as selecting therapy, predicting therapeutic response or disease screening.
· The use of this product for diagnosis, 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.
1. Special instrument requirements:

Fluorescence microscope equipped with appropriate excitation and emission filters

I. Device Description:

The Vysis EGR1 FISH Probe Kit – SC (List No. 04N37-001) consists of the following components which are sufficient to process 20 assays:

1. Vysis LSI EGR1 SpectrumOrange/D5S23, D5S721 Spectrum Green Probes
1. Vysis LSI/WCP Hybridization Buffer
1. DAPI II Counterstain
1. NP-40

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5. 20X SSC Salt

Items 2 through 5 above are general purposes reagents.

DNA Probes: Vysis LSI EGR1 SpectrumOrange/ D5S23, D5S721 SpectrumGreen.

The SpectrumOrange-labeled LSI EGR1 probe, approximately 209 kb in a. length (chr5:137654208-137862738; February 2009 Assembly; UCSC Human Genome Browser) is located at 5g31 and contains the complete EGR1 gene.
b. The SpectrumGreen-labeled LSI D5S23, D5S721 probe, approximately 561 kb in length (chr5:9397109-9958407; February 2009 Assembly; UCSC Human Genome Browser) is located at 5p15.2.

J. Substantial Equivalence Information:

1. Predicate device name(s):
  No predicate device exists for this intended use
1. Predicate 510(k) number(s):
  Not applicable
1. Comparison with predicate:
  Not Applicable

K. Standard/Guidance Document Referenced (if applicable):

Not Applicable

L. Test Principle:

Alterations in chromosome 5 are common aberrations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Chromosome band 5q31 contains the early growth response 1 gene (EGR1). The Vysis locus specific identifier (LSI) EGR1 SpectrumOrange Probe and D5S23, D5S721 SpectrumGreen Probe are components of the Vysis EGR1 FISH Probe Kit, and are used to detect the target EGR 1 using FISH DNA probe technology. The results of this assay are used to characterize bone marrow specimens.

Bone marrow cells from AML patients attached to microscope slides using standard cytogenetic procedures are used for this assay. The resulting cellular DNA is denatured to single-stranded form and subsequently allowed to hybridize with the LSI EGR1 and LSI D5S23, D5S721 probes. Following hybridization, the unbound probe is removed by a series of washes, and the nuclei are counterstained with DAPI, a DNA-specific stain that fluoresces blue. Hybridization of the LSI EGR1 and LSI D5S23, D5S721 probes is viewed using a fluorescence microscope equipped with

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appropriate excitation and emission filters, allowing visualization of the orange and green fluorescent signals.

The expected signal pattern in a cell with two copies of the EGR1 gene covered by the probes using the Vysis EGR1 FISH Probe Kit is 2 orange and 2 green signals (2R2G). In a cell with the 5q alteration, one orange signal (LSI EGR1) and two green signals (LSI D5S23, D5S721) will be expected. Enumeration of the orange LSI EGR 1 and green LSI D5S23, D5S721 signals provide a mechanism for determining absolute copy number of the probe targets and the presence of chromosomal alterations of interest.

M. Performance Characteristics (if/when applicable):

The sponsor provided the following information to support the analytical performance of the device:

1. Analytical performance:
- a. Precision/Reproducibility: Repeatability and reproducibility of the Vysis EGR1 FISH Probe Kit were tested as shown in the table below:

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Study	Study Protocol	Conclusion
Precision:Intra-Day andInter-Day	• 6 Bone marrow specimens- 2 high positive (HP1 & 2)(69.3%, 49.9% positivity)- 2 low positive (LP1 & 2)(12% positivity)- 2 negative (Neg1 & 2)• 3 test sites• 5 different testing days• 1 lot of probe• Red and green signalpatterns of 200 nucleievaluated by 2technologists (eachtechnologist evaluated 100nuclei per panel member)• Pre-specified acceptance criteria:Agreement with the expected resultof greater than or equal to 90% forthe high positive specimencategory for each site and 90% forthe negative specimen categoryacross all sites with no more than 3discordant results occurring at onesite	Acceptance criteria met

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Study	Study Protocol	Conclusion
Reproducibility:Inter-Site	• Testing configuration sameas above• In addition, 2 replicates ofeach panel member weretested• Pre-specified acceptancecriteria: Same as above	Acceptance criteria met
Lot to LotReproducibility	• 6 Bone marrow specimens- 2 high positive (HP1 & 2)(69.3%, 49.9% positivity)- 2 low positive (LP1 & 2)(12% positivity)- 2 negative (Neg1 & 2)• 3 lots of probe• 4 replicates• 1 site• 2 operators per site• Pre-specified acceptancecriteria: Same as above	Acceptance criteria met

Between Site Analysis of Variance

Sample Type	N	Mean %a	SDb
High Positive 1	30	70.0	5.44
High Positive 2	30	47.6	0.74
Low Positive 1	30	18.1	1.03
Low Positive 2	30	14.9	0.00
Negative 1	30	0.7	0.99
Negative 2	30	0.9	1.5

ª Percentage of cells with 1R2G signal pattern

bSD = standard deviation

Sample Type	N	Mean %a	Within-DayComponentSDb	Between-DayComponentSD
High Positive 1	30	70.0	3.28	4.01
High Positive 2	30	47.6	5.56	0.00
Low Positive 1	30	18.1	3.00	3.82
Low Positive 2	30	14.9	3.25	1.54
Negative 1	30	0.7	0.71	0.00
Negative 2	30	0.9	0.66	1.42

Within-day/Between-day Analysis of Variance

ª Percentage of cells with 1R2G signal pattern

bSD = standard deviation

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Sample Type	N	Mean %a	SDb
High Positive 1	12	66.2	0.00
High Positive 2	12	47.4	3.04
Low Positive 1	12	12.7	0.00
Low Positive 2	12	12.3	1.12
Negative 1	12	0.0	0.00
Negative 2	12	0.1	0.20

Lot-to-Lot Analysis of Variance

ª Percentage of cells with 1R2G signal pattern

bSD = standard deviation

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	StudyProtocol	Conclusion
Real-TimeStability	• Attributes evaluated: signalintensity, targetbackground, cross-hybridization,specificity, overall readability• 3 lots of the device• 3 specimens• The Vysis EGRI FISH Probe Kitdating is determined by thecomponent with the shortestexpiration dating.• Pre-specified acceptance criteria:Acceptable quality of all attributesfor all samples tested	Acceptance criteria met for12 month stability
In-Use Freeze-Thaw Stability	• A series of 20 freeze-thaw cycleswas performed on the probes,hybridization buffer and DAPI IIcounterstain.• 1 lot of the device• 3 specimens• Attributes evaluated: Sameas above• Pre-specified acceptance criteria:Same as above	Acceptance criteria metthroughout and at the endof 20 freeze-thaw cycles
Transport andTemperatureExtreme	• Device components were removedfrom -20°C and cycled for 48 hourson dry ice, 72 hours at 25°C, 72	Acceptance criteria met

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Stability	hours at 40°C, and -20°C for 24hours prior to testing• 1 lot of the device• 3 specimens• Attributes evaluated: Same asabove• Pre-specified acceptance criteria:Same as above
Post-hybridizationSignal Stability	• A single slide from each of 3 bonemarrow specimens was tested atbaseline and then stored at -20°C ±10°C protected from light• The samples were tested at threetime points -Day 7, Day 14, andDay 25• 1 lot of the device• Attributes evaluated: Sameas above• Pre-specified acceptance criteria:Same as above	Acceptance criteria met forPost-hybridization stabilityof 3 weeks
ProbePhotostability	• The device was exposedcontinuously under whitefluorescent light (to mimicstandard laboratory conditions) at15-30°C for different time lengths(0 hours, 3 hours, 8 hours, 24 hoursand 48 hours)• 1 lot of the device• Attributes evaluated: Same asabove• Pre-specified acceptance criteria:Same as above	Acceptance criteria met forPhoto-stability of 48 hours

Controls:

The Vysis EGR1 FISH Probe Kit also contains the LSI D5S23, D5S721 probe (location chromosome 5p15.2) which serves as a control. Observation of at least 1R signal and 1G signal, assures that the assay conditions are adequate to allow both the orange and green labeled probes to bind properly to their respective targets.

d. Detection limit:
The analytical sensitivity of the Vysis LSI EGRI SpectrumOrange/D5S23, D5S721SpectrumGreen probes was established using interphase nuclei prepared from 25 bone marrow specimens that were either karyotypically normal or 5pl5 and 5q31 deletion-free. The orange and green signal patterns of nuclei for these 25 specimens were evaluated by two technologists. Each

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technologist evaluated 100 nuclei per specimen for a total of 200 nuclei per specimen and 5000 scoreable nuclei from normal specimens. The analytical sensitivity was calculated as the percentage of scoreable interphase nuclei with the expected 2 red/2 green signal pattern.

Probe	Total Numberof NucleiScored	Number of NucleiWith ExpectedSignal Pattern	Analytical Sensitivity(95% Confidence Interval)
VysisEGR1/D5S23,D5S721	5000	4979	99.6% (99.4, 99.7)

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 EGR1 SpectrumOrange/D5S23, D5S721 SpectrumGreen probes for their respective chromosome target loci was established using metaphase chromosomes prepared from peripheral blood cultures of five karyotypically normal males that were pooled prior to dropping on microscope slides. The hybridization location of each FISH signal on chromosomes of 100 consecutive metaphase nuclei was evaluated by one technologist for a total of 200 target loci.

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 LSI EGR1 SpectrumOrange/D5S23, D5S721 SpectrumGreen Probes was 100%.

Probe	Target	Total No. ofMetaphaseChromosomeHybridized	No. ofMetaphaseChromosomeHybridized toCorrect Locus	Analytical Specificity(95% ConfidenceIntervals)
D5S23, D5S721	5p15.2	200	200	100%(98, 100)
EGR 1	5q31	200	200	100%(98, 100)

f. Assay cut-off:

Same as reference range (see #5 below)

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2. Comparison studies:

a. Method comparison with predicate device:
Not applicable
b. Matrix comparison:
Not applicable
1. 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 three peer-reviewed published papers to support the clinical validity of the device in characterizing bone marrow specimens from patients with AML. All three papers used the Abbott Vysis EGR1 FISH Probe Kit. The information is listed in the table below:

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Conditions	Data Source 1Sun et. al	Data Source 2Galvan et. al	Data Source 3Vance et. al
Was the specificdevice underreview in thesubmission used inthe study?	Yes	Yes	Yes
Was the specimentype in the studyrepresentative ofthe claimedspecimen type(s)	Yes	Yes	Yes
Target population(disease status)	Known orsuspected del(5q)having MDS orAML	MDS, therapy-related MDS (t-MDS) and AMLpatients	AML
Upper referencelimit (percentageand per 200 nuclei)	6% or 12 1R2Gpatterns per 200scoreableinterphase nuclei	6% or 12 1R2Gpatterns per 200scoreableinterphase nuclei	6% or 12 1R2Gpatterns per 200scoreableinterphase nuclei
Total Number ofspecimens testedfor each claimedtype	320 bone marrowspecimens	28 bone marrowspecimens	181 bone marrowspecimens*
Number ofspecimens with apositive proberesult [5q- (1R2G)]	66	23MDS - 6t-MDS - 3t-MDS&MM – 1AML - 13	8AML - 8
Range of positiveprobe results	Not available	MDS: 35 - 81.5%t-MDS: 25 - 75%t-MDS&MM: 76%AML: 20 - 99%	45 - 91 %

MDS – Myelodysplastic syndrome

AML – Acute myeloid leukemia

t-MDS – Therapy related MDS

MM – Multiple Myeloma

Line data unpublished, but this data was provided in the 510(k) submission

Sun Y, Cook JR. Fluorescence in situ hybridization for del(5q) in myelodysplasia/acute myeloid lukemia: Comparison of EGR1 vs. CSF1R probes and diagnostic yield over metaphase cytogenetics alone. Leuk Res 2010; 34: 340-343.

Galvan AB, Mallo M, Arenillas L, et al. Does monosomy 5 really exist in myelodysplastic syndromes and acute myeloid leukemia? Leuk Res 2010; 34: 1242– 1245.

Vance GH, Haesook K, Hicks GA, et al. Utility of interphase FISH to stratify patients into cytogenetic

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risk categories at diagnosis of AML in an Eastern Cooperative Oncology Group (ECOG) clinical trial (E1900). Leuk Res 2007; 31:605-609.

1. Clinical cut-off:
  Not applicable
1. Expected values/Reference range:
  The upper reference limit is defined as the maximum quantity of scoreable interphase nuclei with an altered signal pattern from either karyotypically normal specimens or 5p15.2 and 5g31 deletion-free specimens. The upper reference limit is expressed in terms of a percentage or the actual number of atypical nuclear FISH signal pattern per the standard number of nuclei tested.

The upper reference limit for this assay is 6% or 12 1R2G patterns per 200 scoreable interphase nuclei. Published literature was used to establish the reference range for this assay.

In order to validate the 6% upper reference limit of the Vysis EGR 1 FISH Probe Kit, the assay was performed on interphase nuclei from 25 bone marrow specimens from either karyotypically normal specimens or 5p15.2 and 5q31 deletion-free specimens. The signal patterns of 200 nuclei were evaluated by counting the number of orange and green signals. Each of two technologists evaluated 100 nuclei per specimen. Among the 25 normal specimens, none produced 1R2G signals at or above the 6% upper reference limit.

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

None

O. Proposed Labeling:

Labeling satisfies the requirements of 21 CFR 809.10, 21 CFR 801.109, including an appropriate prescription statement as required by 21 CFR 801.109(b), and the special controls for this type of device.

Identified Potential Risk	Required Mitigations
False negative result	1) Premarket notification submissions mustalso include the following information:i) A detailed description of all probesincluded in the kitii) Purpose of each probeiii) Probe molecular specificityiv) Probe specificityv) Probe limits
vi)	Probe sensitivity
vii)	Specification of required ancillaryreagents, instrumentation andequipment
viii)	Specification of the specimencollection, processing, storage andslide preparation methods
ix)	Specification of the assay procedure
x)	Specification of control elementsthat are incorporated into therecommended testing procedures
xi)	Specification of risk mitigationelements: description of alladditional procedures, methods, andpractices incorporated into thedirections for use that mitigate risksassociated with testing
xii)	Specification of the criteria for testresult interpretation and reporting
xiii)	Device analytical sensitivity data
xiv)	Device analytical specificity data
xv)	Device reference limit data
xvi)	Device precision/reproducibilitydata
xvii)	Device stability data to include:A) Real-time StabilityB) Freeze-Thaw StabilityC) Transport and TemperatureStabilityD)Post-Hybridization SignalStabilityE) Photostability of Probe
xviii)	Documentation that demonstrates theclinical validity of the device. Thedocumentation must include datafrom clinical studies, a minimum oftwo peer-reviewed publishedliterature references using thespecific device seeking marketingclearance, or both. Documentationfor the clinical studies and peer-reviewed published literaturereferences cited must include thefollowing elements:A) Documentation that the
	literature referenceB) Number & type of specimensC) Target population studiedD) Upper reference limitE) Range of positive probe results
	2) Your 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 pre-specified acceptance criteria, and whether the pre-specified acceptance criteria were met.
	3) Your 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.”
False Positive Result	1) Premarket notification submissions must also include the following information:i) A detailed description of all probes included in the kitii) Purpose of each probeiii) Probe molecular specificityiv) Probe specificityv) Probe limitsvi) Probe sensitivityvii) Specification of required ancillary reagents, instrumentation and equipmentviii) 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 StabilityB) Freeze-Thaw StabilityC) Transport and Temperature StabilityD) Post-Hybridization Signal StabilityE) 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 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 referenceB) Number & type of specimensC) Target population studiedD) Upper reference limitE) Range of positive probe results
2)	Your 21 CFR 809.10(b)(12) compliant labeling must include a statement summarizing the data identified in
	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 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.”

P Risks to Health and Mitigation Measures:

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Q. Benefit/Risk Analysis:

Summary of theBenefits	Summary ofthe Risks	Summary of OtherFactors	Conclusions
AML and MDSpatients maypotentiallybenefit in theintended usepopulation by useof the device tocharacterize bonemarrowspecimens withassay results	Erroneousdevice resultscould adverselyinfluenceexpectation ofmore or lessfavorableclinical courseof AML orMDS patientsdue to false	In addition to citedclinical studies usingthe device probes,analytical evaluationand labeling supportsthe intended use. Denovo regulatoryapproach leveragesdevice use by aqualified pathologist orcytogeneticist in the	Yes. Based onthe supportingclinical studiesfor thediagnosticdevice alongwith review ofthe analyticalperformance andlabeling, theprobable
interpreted by aqualifiedpathologist orcytogeneticist.	negative orfalse positiveresults.	context ofhistopathologicalevaluation (e.g.,immunohistochemistry).	benefitsoutweigh theprobable risks.

R. Conclusion:

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The request for Evaluation of Automatic Class III Designation for this device is granted. The device is classified as Class II under regulation 21 CFR 864.1870 with special controls. The device is classified under the following:

Product Code:	PDO
Device Type:	Early growth response 1 (EGR1) gene fluorescence in-situhybridization (FISH) test system for specimencharacterization
Class:	II (special controls)
Regulation:	21 CFR 864.1870
	(a) IDENTIFICATION: An early growth response 1(EGR1) gene fluorescence in-situ hybridization (FISH) testsystem for specimen characterization is a device intendedto detect the EGR1 probe target on chromosome 5q in bonemarrow specimens from patients with acute myeloidleukemia (AML) or myelodysplastic syndrome (MDS).The assay results are intended to be interpreted only by aqualified pathologist or cytogeneticist. These devices donot include automated systems that directly report resultswithout review and interpretation by a qualified pathologistor cytogeneticist. These devices also do not include anydevice intended for use to select patient therapy, predictpatient response to therapy or to screen for disease as wellas any device with a claim for a particular diagnosis,prognosis, monitoring or risk assessment.
	(b) CLASSIFICATION: Class II (special controls). EGR1Gene FISH test system for specimen characterization mustcomply with the following special controls:1) Premarket notification submissions must include thefollowing information:i) A detailed description of all probes included in the kitii) Purpose of each probeiii) Probe molecular specificityiv) Probe specificityv) Probe limitsvi) Probe sensitivityvii) Specification of required ancillary reagents,instrumentation and equipment

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viii) Specification of the specimen collection, processing, storage and slide preparation methods
- Specification of the assay procedure ix)
Specification of control elements that are x) 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
Device precision/reproducibility data xvi)
Device stability data to include: xvii)
- 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
1. 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

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the pre-specified acceptance criteria for these performance studies, justification for the prespecified acceptance criteria, and whether the prespecified acceptance criteria were met.

1. 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."

Regulation Number and Section

§ 864.1870 Early growth response 1 (EGR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization.

(a)
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.(b)
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.”