CYTOSCAN(R) DX
Device Facts
| Record ID | DEN130018 |
|---|---|
| Device Name | CYTOSCAN(R) DX |
| Applicant | Affymetrix, Inc. |
| Product Code | PFX · Immunology |
| Decision Date | Jan 17, 2014 |
| Decision | DENG |
| Submission Type | Post-NSE |
| Regulation | 21 CFR 866.5920 |
| Device Class | Class 2 |
Intended Use
CytoScan® Dx Assay is a qualitative assay intended for the postnatal detection of copy number variations (CNV) in genomic DNA obtained from peripheral whole blood in patients referred for chromosomal testing based on clinical presentation. CytoScan® Dx Assay is intended for the detection of CNVs associated with developmental delay, intellectual disability, congenital anomalies, or dysmorphic features. Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counseling, as appropriate. Interpretation of assay results is intended to be performed only by healthcare professionals, board certified in clinical cytogenetics or molecular genetics. The assay is intended to be used on the GeneChip® System 3000Dx and analyzed by Chromosome Analysis Suite Dx Software (ChAS Dx Software). This device is not intended to be used for standalone diagnostic purposes, preimplantation or prenatal testing or screening, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
Device Story
The CytoScan Dx Assay is a chromosomal microarray used for genome-wide detection of chromosomal copy number variations (CNVs). It processes gDNA isolated from peripheral whole blood; gDNA is digested with Nsp1, ligated to adapters, PCR-amplified, purified, fragmented, and biotin-labeled. The resulting DNA is hybridized to microarrays containing ~2.7 million markers (non-polymorphic and SNP) using the GeneChip® Fluidics Station 450Dx v.2. Arrays are scanned via GeneChip® Scanner 3000Dx v.2. ChAS Dx Software analyzes signal intensity ratios (log2) against a reference set to determine copy number states (0, 1, 2, 3, 4) and visualize CNV regions, mosaicism, and loss of heterozygosity (LOH). The assay is performed in clinical laboratories by trained personnel; results are interpreted by board-certified clinical cytogeneticists or molecular geneticists. The output assists clinicians in diagnosing constitutional genomic disorders, providing diagnostic yield for patients with developmental delay or congenital anomalies. It is used in conjunction with other clinical findings and professional counseling.
Clinical Evidence
Clinical performance evaluated in a retrospective study of 960 samples from patients with developmental delay/intellectual disability. Diagnostic yield was 13.8% for CytoScan Dx vs 13.3% for routine patient care. Clinical PPA was 82.0% (95% CI: 74.5-87.7%) and NPA was 96.8% (95% CI: 95.3-97.8%) compared to routine patient care. Analytical accuracy was assessed against sequencing and composite methods, showing high agreement for CNVs. Reproducibility was established across sites, operators, and days.
Technological Characteristics
Microarray-based assay with ~2.7 million markers (25-mer probes). Includes 5 reagent modules for amplification, fragmentation, labeling, and hybridization. Uses GeneChip® System 3000Dx v.2 (Fluidics Station 450Dx v.2 and Scanner 3000Dx v.2). Software: ChAS Dx v1.0.0. Analyzes gDNA input (recommended 250ng). Detects CNVs and LOH. Not networked/cloud-dependent for core function.
Indications for Use
Indicated for postnatal detection of CNVs in genomic DNA from peripheral whole blood in patients referred for chromosomal testing due to developmental delay, intellectual disability, congenital anomalies, or dysmorphic features. Not for prenatal/preimplantation testing, population screening, or acquired/somatic genetic aberrations.
Regulatory Classification
Identification
A postnatal chromosomal copy number variation detection system is a qualitative assay intended for the detection of copy number variations (CNVs) in genomic DNA obtained from whole blood in patients referred for chromosomal testing based on clinical presentation. It is intended for the detection of CNVs associated with developmental delay, intellectual disability, congenital anomalies, or dysmorphic features. Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counseling, as appropriate. Interpretation of assay results is intended to be performed by qualified healthcare professionals such as clinical cytogeneticists or molecular geneticists. This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing or screening, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) Design verification and validation must include the following information: (i) A detailed description of all components in the test system that includes: (A) A description of the assay components, array composition and layout, all required reagents, instrumentation, and equipment, including illustrations or photographs of non-standard equipment or methods; (B) A description of the design of the array in terms of chromosomal coverage and probe density for different regions; (C) An identification of the number of probes and size of the CNVs reported at the lower range of the assay; (D) Detailed documentation of the device software, including standalone software applications and hardware-based devices that incorporate software; (E) Methodology and protocols for detecting copy number and visualizing results; (F) A description of the result outputs along with sample reports, and a description of any links to external databases provided by the device to the user or accessed by the device; (G) Specifications for the methods to be used in specimen collection, extraction (including DNA criteria for DNA quality and quantity to perform the assay), and storage; and (H) A description of appropriate internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure. (ii) Information that demonstrates the performance characteristics of the system, including: (A) Device reproducibility data generated, at a minimum, using three sites, with two operators at each site, for three non-consecutive days using at least three instruments. A well-characterized panel of samples that provide a wide range of CNVs ( *i.e.,* gains, losses, adequate size coverage across the range of sizes claimed by the device, adequate chromosomal coverage, challenging regions in the genome, CNVs reported at the lower range of the assay, interstitial, subtelomeric, and pericentromeric rearrangements, aneuploidy, unbalanced translocations, mosaicism, and known syndromic regions) must be used. The results must be itemized for all CNVs detected in each sample across all replicates and summarized in a tabular format stratified by size range and range of probe numbers for gains and losses separately and calculated for overall. The results must be analyzed using pairwise replicate agreement, and summarized as overall pairwise replicate agreement as well as pairwise replicate agreement conditional on replicates having a positive copy number state call (gains or losses), call rate, CNV size variation, and endpoint agreement;(B) Device accuracy data using cell lines and clinical samples representing a variety of CNVs and syndromes. In this analytical study, accuracy must be determined for every CNV detected in a particular sample. The accuracy data provided must include the copy number state determination and endpoint accuracy. The accuracy samples must cover different genomic variations across the genome ( *i.e.,* gains, losses, adequate CNV size coverage across the range of sizes claimed by the device, adequate chromosomal coverage, challenging regions in the genome, CNVs reported at the lower range of the assay, interstitial, subtelomeric, and pericentromeric rearrangements, aneuploidy, unbalanced translocations, mosaicism, and known syndromic regions). CNVs identified by the device must be compared to comparator method(s). Agreement between the CNVs detected by the array and the comparator must be summarized in a tabular format that includes the positive percent agreement and false positive rate stratified by size range and range of probe numbers for gains and losses separately and calculated for overall;(C) Assay performance data for CNVs reported at the lower range of the assay for both gains and losses; (D) Device analytical sensitivity data, including DNA input and limit of detection for mosaicism, if applicable; (E) Device analytical specificity data, including interference, carryover, and cross-contamination data; (F) Device stability data, including real-time stability under various storage times, temperatures, and freeze-thaw conditions; (G) Specimen matrix comparison data if more than one specimen type or anticoagulant can be tested with the device; (H) Data that demonstrates the clinical validity, including diagnostic yield, of the device using a minimum of 800 retrospective clinical samples that were collected prospectively and obtained from three or more clinical laboratories, with results interpretation equally divided between two or more qualified healthcare professionals ( *e.g.,* cytogeneticists). Patients must be representative of the intended use population and not limited to common syndromes. Diagnostic yield data must be summarized in tabular format and stratified by the comparison methodologies. Data must also be summarized comparing interpretation of results, with description of reasons for variability in calls between the device and the standard of care methods. Data to support the accuracy of calls for known syndromes must be included; and(I) Data that demonstrates device results when a minimum of 100 apparently healthy, phenotypically normal individuals are tested and interpreted by one or more cytogeneticists blinded to the patient status. (iii) Identification of risk mitigation elements used by the device, including a description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing. (2) The labeling required under § 809.10 of this chapter must include: (i) A warning statement that the device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing or screening, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations; (ii) Limitations regarding the assay's performance with respect to validated CNVs reported at the lower range of the assay, stratified by size range and range of probe numbers for gains and losses separately; and limitations regarding problematic (hypervariable) regions, loss of heterozygosity, mosaicism, and inability to detect balanced translocations, as appropriate; (iii) A warning statement that interpretation of assay results is intended to be performed by qualified healthcare professionals such as clinical cytogeneticists or molecular geneticists; and, (iv) A description of the performance studies performed in accordance with paragraph (b)(1)(ii) of this section and a summary of the results.
Reference Devices
- HapMap (Gibson et al.)
Related Devices
- K163367 — GenetiSure Dx Postnatal Assay · Agilent Technologies, Inc. · Aug 11, 2017
- K080995 — MODIFICATION TO AFFYMETRIX GENECHIP MICROARRAY INSTRUMENTATION SYSTEM · Affymetrix, Inc. · Jul 30, 2008
- DEN040012 — AFFYMETRIX GENECHIP MICROARRAY INSTRUMENTATION SYSTEM · Affymetrix, Inc. · Dec 23, 2004
Submission Summary (Full Text)
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# EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Affymetrix® CytoScan® Dx Assay DECISION SUMMARY
# A. 510(k) Number:
k130313
## B. Purpose for Submission:
De novo request for evaluation of automatic class III designation of the Affymetrix® CytoScan® Dx Assay
#### C. Measurand:
Genome-wide chromosomal copy number variations
## D. Type of Test:
Chromosomal Microarray
# E. Applicant:
Affymetrix, Inc.
## F. Proprietary and Established Names:
Affymetrix® CytoScan® Dx Assay
## G. Regulatory Information:
- 1. Regulation section:
21 CFR 866.5920
- 2. Classification:
Class II (special controls)
- 3. Product code:
PFX -- System, Microarray-based, genome-wide, postnatal chromosomal abnormality detection
- 4. Panel:
Immunology
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# H. Intended Use:
# 1. Intended use(s):
CytoScan® Dx Assay is a qualitative assay intended for the postnatal detection of copy number variations (CNV) in genomic DNA obtained from peripheral whole blood in patients referred for chromosomal testing based on clinical presentation. CytoScan® Dx Assay is intended for the detection of CNVs associated with developmental delay, intellectual disability, congenital anomalies, or dysmorphic features. Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counseling, as appropriate. Interpretation of assay results is intended to be performed only by healthcare professionals, board certified in clinical cytogenetics or molecular genetics. The assay is intended to be used on the GeneChip® System 3000Dx and analyzed by Chromosome Analysis Suite Dx Software (ChAS Dx Software).
This device is not intended to be used for standalone diagnostic purposes, preimplantation or prenatal testing or screening, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
- 2. Indication(s) for use:
Same as Intended use above.
- 3. Special conditions for use statement(s):
For prescription use.
- 4. Special instrument requirements: GeneChip® System 3000Dx v.2 and with Chromosome Analysis Suite Dx Software v.1.0 (ChAS Dx Software).
# I. Device Description:
The CytoScan Dx consists of five reagent modules, a microarray kit, and analysis software. The five reagent modules are:
- 1. MOD R L A, CytoScan® Dx Pre-PCR contains buffers, nucleotides, enzyme, and primers and adaptors for amplification;
- 2. MOD T E W, CytoScan® Dx Pre-PCR contains buffer and nuclease free water for amplification;
- 3. MOD F L H, CytoScan® Dx Post-PCR contains buffers, nucleotides, and enzyme for fragmentation, labeling and hybridization;
- MOD S AH W PB, CytoScan® Dx Post-PCR contains buffers, nuclease free water, ব and purification beads for stain and array hold;
- 5. MOD E PW CytoScan® Dx Post-PCR contains buffer for elution and purification wash.
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The CytoScan® Dx Post-PCR CytoScan® Dx Array kit, 6-pack is designed for 6 runs. The microarray contains approximately 2,696,550 functional markers, each of which is approximately 25 bases long.
ChAS Dx Analysis Software and Browser v1.0.0 analyzes CEL file microarray data.
# J. Substantial Equivalence Information:
- 1. Predicate device name(s) and 510(k) number(s):
Not applicable.
- 2. Comparison with predicate:
Not applicable.
# K. Standard/Guidance Document Referenced (if applicable):
Not applicable.
# L. Test Principle:
CytoScan Dx Assay provides genome-wide coverage for the detection of chromosomal imbalances. The CytoScan Dx array contains approximately 2.7 million markers which are representative of DNA sequences distributed throughout the genome with spacing, on average, approximately 880 bases apart in genic regions, and approximately 1700 bases apart in non-genic regions. The majority of the markers (1.9 million) are non-polymorphic markers, which provide overall genomic coverage of relevant cytogenetic regions and are used for assessing copy number. Approximately 750,000 SNP markers on the array are included to maximize genomic coverage and to enable detection of homozygosity. Both the SNP and non-polymorphic markers are approximately 25 bp long.
CytoScan Dx Assay consists of the following steps: (1) gDNA is isolated from peripheral blood and the isolated gDNA is digested with the restriction enzyme Nsp1 to reduce genomic complexity; (2) The digested gDNA is ligated to Nsp1 adapters and amplified in a multiplex PCR reaction to produce optimized amplicons in the 200 1100 bp size range; (3) The amplified PCR products are purified and then randomly fragmented using DNAse I to generate species of 25 125 bp, which are optimal for hybridization to 25-mer markers; (4) Reaction intermediates are visualized by gel electrophoresis after the PCR and fragmentation steps to confirm proper size distribution; (5) The final DNA product is end-labeled by the addition of a modified biotinylated base and hybridized to CytoScan Dx Arrays: (6) The arrays are sequentially washed and stained with a combination of a streptavidin-coupled dye and a biotinylated anti-streptavidin antibody in GeneChip® Fluidics Station 450Dx v.2; (7) The washed arrays are scanned using GeneChip® Scanner 3000Dx v.2 to acquire the signal intensity from each marker.
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Chromosome Analysis Suite (ChAS Dx) software is used to analyze and visualize microarray data. The signal intensity of the hybridized DNA from the patient sample is compared to a reference DNA, which is based on an average of over 400 samples. The ratio of patient sample to reference intensity is expressed as a log2 ratio, and represents the relative intensity for each marker. A discrete copy number value is computed from the relative intensity data, and is displayed as the marker copy number state. The noninteger copy number states are calculated and displayed as the smoothed signal track, which can used to support an interpretation of a mosaic gain or loss. The SNP marker A- and B-allele intensities are also visualized in the Allele Track, which can be used to confirm copy number variation regions. The allele tracks show 3 bands (AA, AB, BB) in normal diploid regions, 4 bands (AAA, AAB, ABB, BBB) in triploid regions, and 2 bands (A, B) in haploid regions. The SNP markers are also analyzed for long contiguous stretches of homozygosity, which are visualized in the loss of heterozygosity (LOH) track. The absence or loss of heterozygosity (AOH / LOH) is calculated as a region significantly devoid of heterozygous genotype calls.
CytoScan Dx reports the copy number state (loss, gain), copy number (i.e., 0, 1, 2, 3, or 4 or greater), and position/location of chromosomal segment copy number changes across the queried genome.
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
# a. Precision/Reproducibility:
Two reproducibility studies were conducted. The first was a site to site reproducibility study, and the second was a between-lot reproducibility study.
In each study, the reproducibility of CytoScan Dx results was evaluated for both copy number state determination (gain or loss), and localization of each CNV based on overlapping size of the CNVs or overlapping marker number, for two criteria of agreement: 50% overlap and 80% overlap. For every sample replicate tested, every CNV detected in each replicate was analyzed for reproducibility with the other replicates by pairwise agreement (i.e., CNV 1 in replicate 1 was compared to CNV 1 in replicate 2, then CNV 1 in replicate 1 was compared to CNV 1 in replicate 3, and so on). A pair of replicates was considered to agree if the two CNVs in each pair compared, overlapped by at least 50% or 80% of the CNV length at a given location on the chromosome, or marker number, provided the copy number state (gain/loss) was the same. Because pairwise analysis considers agreement between two CNVs for all combined pairs of replicates (e.g., a CNV detected in 9 replicates will have a total of 36 paired comparisons), in the scenario where two of 9 replicates had no CNV result, the 2 no calls were considered to be agreed in the pairwise replicate agreement calculation. Positive percent agreement (PPA) measures the pairwise agreement conditional on a replicate being gain or loss. Call rate is calculated as the average of the percent of replicates that call each CNV. Reproducibility was also assessed for agreement of copy number (0, 1, 2, 3, 4) without regard to size or marker number, and assessed for localization based on endpoint agreement between each CNV
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replicate. To determine the precision estimate for localizing a CNV, % coefficient of variance of CNV length, median % absolute endpoint deviation, and standard deviation of the endpoints (both left and right endpoint) were calculated. The results in the tables below are grouped by size range and marker number. Results are shown including, and excluding Affymetrix-defined hypervariable regions. A more detailed description of the statistical methods for assessing the reproducibility variables is shown below:
Description of variables:
- (1) Percent (%) overlap: For the criteria shown (50% overlap or 80% overlap between CNVs), the overlap data is the average of results for all pairwise replicates for the range of CNVs listed (row).
- (2) Pairwise replicate agreement was determined by examining all pairs of replicates for overlap at 50% or 80% CNV length, summarized (for 9 replicates) as follows:
$$\sum_{i=1}^{9} \sum_{j>i}^{9} Pr\{rap_i = rap_j\}/3.6$$
Two replicates are considered to be agreed (or equal) if the CNVs overlap at least 50% (or 80%) and the copy number states are identical. Two replicates of no calls are considered to be agreed.
- (3) Positive percent agreement (PPA) measures the pairwise agreement conditional on a replicate having the gain or loss, summarized (for 9 replicates) as follows:
$$\sum_{l=1}^{i} \sum_{\substack{l=1 \ l \neq i}}^{i} \Pr\{rap_l = rap_j | rap_l = gain \text{ or } l \text{as} \} / \ll \sum_{l=1}^{i} \Pr\{rap_l = gatin \text{ or } l \text{as} \}$$
Two replicates are considered to be agreed (or equal) if the CNVs overlap at least 50% (or 80%) and the copy number states are identical.
- (4) Call rate is calculated as the average of the percentage of replicates that call each CNV.
- (5) Length, % Coefficient of Variance (%CV): The %CV of each CNV, calculated for both size in terms of kilobases (kb) and number of markers. For clarity. CV is calculated as the standard deviation divided by the mean length across the replicates (%CV = SD/Mean). The mean, minimum, median, and maximum for all CNVs, or stratified groups of CNVs, are presented.
- (6) Median % Absolute Endpoint Deviation: For each CNV, the median left and right endpoint was determined. For each replicate CNV, the distance from the median endpoint was calculated for the left and right endpoints (DL and DR. respectively). The combined endpoint distance, DL+DR, was calculated. The fractional endpoint error is the combined endpoint distance divided by the CNV length, expressed as a percentage. For a CNV with replicate measurements, the CNV endpoints are er and er, the median left and right endpoints are defined as a and an respectively. For each replicate measurement, the absolute distance of the CNV endpoints from the medians, $D_L = |e_L - \tilde{e}_L|$ and $D_R = |e_R - \tilde{e}_R|$.
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$$\% \, Endpoint \, Error = \frac{\mathbf{e}_{\mathbf{k}} + \mathbf{e}_{\mathbf{k}}}{\mathbf{e}_{\mathbf{k}} - \mathbf{e}_{\mathbf{k}} + \mathbf{1}} \times \mathbf{100}$$
#### Median % Absolute endpoint Deviation = % Endpoint Error
- (7) Standard deviation of identifying each endpoint is the standard deviation of the endpoint, separately for left and right endpoint.
#### Study 1. Site-to-site reproducibility:
To assess the reproducibility metrics described above across multiple sites, a study was performed on 93 genomic DNA samples (48 purified from blood, 44 purified from cell lines obtained from Coriell and the World Health Organization, 1 control sample from American Tissue Culture Collection) representing different CNV sizes and gains and losses. These samples were run by 2 operators at each of 3 sites across 3 non-consecutive days. Samples contained either gains or losses which covered a total of 50.8% of the genome with at least 1 CNV region on every chromosome with 42.3% of the CNVs detected as gains and 20.5% detected as losses.
A total of 9 chromosomal regions were defined by the sponsor as hypervariable regions because they contain genetic components of the immune system or members of gene families that have been shown to be associated with extensive copy number variations and/or rearrangement (e.g., the olfactory receptor family genes at 1q44 and 11q11), segmental duplication (17q21), and nonfunctional pseudogenes (ADAM3A at 8p11.2). None of these regions overlap with the CNV target regions for constitutional genomic disorders identified by the International Standards for Cytogenomic Arrays Consortium (ISCA). These excluded regions add up to approximately 3.6 Mb in size, or approximately 0.1% of the entire human genome (~3×109 bp). Only those CNV regions that were contained completely within the boundaries of the regions defined in the table above were removed. CNV regions containing any marker outside the boundaries of defined regions were still included in the analysis. Performance of the device including and excluding these 9 hypervariable regions were both evaluated. These 9 hypervariable regions are described as the limitations of the device and listed below.
| Chromosomal Region | Boundaries | Size of Region (in bases) |
|--------------------|---------------------------|---------------------------|
| 1q44 | 248,681,754 – 248,835,053 | 153,300 bp |
| 5q35.3 | 180,376,952 – 180,432,918 | 55,967 bp |
| 7p14.1 | 38,273,345 - 38,419,181 | 145,837 bp |
| 8p11.22 | 39,226,075 - 39,390,890 | 164,816 bp |
| 11q11 | 55,347,529 - 55,481,854 | 134,326 bp |
| 14q11.2 | 22,329,745 - 23,005,312 | 675,568 bp |
| 14q32.33 | 106,035,612 - 107,297,169 | 1,261,558 bp |
| 17q21.31 | 44,107,114 - 44,854,730 | 747,617 bp |
| 22q11.22 | 22,992,312 - 23,260,235 | 267,924 bp |
Table 1. List of Affymetrix-defined Hypervariable Regions of the Human Genome (hg19 build).
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The reproducibility results demonstrated that when agreement across replicates was considered for CNVs that overlapped by 50% or more of the CNV length, the overall pairwise replicate agreement was 79.4% for all CNVs; range 65.7 to 100%. The agreement was 75.5% for all gains and 82.4% for all losses). For marker number, the overall pairwise replicate agreement is 82.2%; range 70.2% to 82.2%. The agreement was 79.8% for all gains and 83.9% for all losses. The results are shown in Tables 2B and 2A below, respectively.
The following tables 2A-2D show the results of the study to evaluate reproducibility of the device.
Table 2A. Reproducibility of CNVs Grouped by State (Gains and Losses) and Marker Number Based on Call Rate, Pairwise Agreement between Replicates and Positive Percent Agreement (PPA) for Two Criteria (50% and 80% Overlap) in All Regions in the Site-to-Site Study.
| | CNV Range<br>(Markers) | | | Pairwise Replicate<br>Agreement* | | PPA* | |
|-------|------------------------|--------|------------|----------------------------------|-------|-------|-------|
| State | | # CNVs | Call Rate* | 50% | 80% | 50% | 80% |
| Gain | 50-75 | 230 | 49.7 | 70.8 | 68.7 | 72.7 | 68.7 |
| | 75-100 | 150 | 68.3 | 70.2 | 54.7 | 77.4 | 54.3 |
| | 100-150 | 131 | 70.2 | 81.5 | 71.6 | 86.5 | 73.0 |
| | 150-200 | 68 | 82.5 | 87.8 | 81.7 | 91.0 | 83.8 |
| | 200-300 | 72 | 92.4 | 91.0 | 81.1 | 92.4 | 81.9 |
| | 300-400 | 8 | 100.0 | 100.0 | 98.9 | 100.0 | 98.9 |
| | 400-1000 | 22 | 100.0 | 97.5 | 95.9 | 97.5 | 95.9 |
| | 1000-3000 | 22 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 3000-5000 | 7 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 60 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | Total | 770 | 71.4 | 79.8 | 72.5 | 85.3 | 75.3 |
| Loss | 25-50 | 430 | 48.8 | 79.5 | 76.4 | 75.4 | 68.0 |
| | 50-75 | 204 | 74.8 | 77.7 | 69.0 | 84.2 | 72.9 |
| | 75-100 | 107 | 81.9 | 87.5 | 80.7 | 92.1 | 83.8 |
| | 100-150 | 58 | 82.4 | 85.2 | 78.8 | 87.6 | 79.9 |
| | 150-200 | 24 | 86.4 | 82.4 | 69.4 | 85.1 | 70.2 |
| | 200-300 | 26 | 96.1 | 92.5 | 85.4 | 93.9 | 86.7 |
| | 300-400 | 12 | 100.0 | 94.2 | 83.0 | 94.2 | 83.0 |
| | 400-1000 | 37 | 100.0 | 95.2 | 77.4 | 95.2 | 77.4 |
| | 1000-3000 | 26 | 100.0 | 100.0 | 99.9 | 100.0 | 99.9 |
| | 3000-5000 | 12 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 41 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | Total | 977 | 68.9 | 83.9 | 78.7 | 87.0 | 79.3 |
| All | Total | 1747 | 70.0 | 82.2 | 76.1 | 86.3 | 77.5 |
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| | | | | | Pairwise Replicate<br>Agreement* | | PPA* | |
|-------|----------------|--------|------------|--|----------------------------------|---------|-------|-------|
| | | | | | | Overlap | | |
| State | CNV Range (kb) | # CNVs | Call Rate* | | 50% | 80% | 50% | 80% |
| Gain | 50-75 | 99 | 50.4 | | 71.4 | 67.2 | 70.4 | 62.1 |
| | 75-100 | 98 | 63.7 | | 70.7 | 62.6 | 74.4 | 63.1 |
| | 100-150 | 149 | 64.9 | | 70.8 | 63.6 | 76.3 | 65.2 |
| | 150-200 | 101 | 62.3 | | 65.7 | 47.1 | 64.9 | 36.4 |
| | 200-300 | 100 | 78.2 | | 73.3 | 67.5 | 78.1 | 70.8 |
| | 300-400 | 27 | 68.6 | | 70.3 | 65.3 | 71.5 | 64.2 |
| | 400-1000 | 94 | 88.8 | | 86.8 | 71.3 | 89.5 | 70.7 |
| | 1000-3000 | 39 | 86.6 | | 87.4 | 78.5 | 90.5 | 80.2 |
| | 3000-5000 | 3 | 100.0 | | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 60 | 100.0 | | 99.1 | 98.4 | 99.1 | 98.4 |
| | Total | 770 | 71.4 | | 75.5 | 66.5 | 79.4 | 67.0 |
| Loss | 25-50 | 351 | 53.7 | | 78.8 | 73.9 | 78.5 | 68.8 |
| | 50-75 | 177 | 65.6 | | 77.9 | 70.1 | 76.7 | 63.2 |
| | 75-100 | 88 | 66.9 | | 77.4 | 68.5 | 72.4 | 57.1 |
| | 100-150 | 133 | 80.9 | | 82.4 | 76.3 | 88.0 | 80.7 |
| | 150-200 | 30 | 73.6 | | 82.5 | 80.1 | 85.5 | 82.2 |
| | 200-300 | 29 | 86.4 | | 81.8 | 62.9 | 84.8 | 63.0 |
| | 300-400 | 35 | 88.3 | | 89.8 | 69.5 | 91.0 | 68.1 |
| | 400-1000 | 43 | 86.9 | | 89.1 | 74.4 | 91.9 | 74.9 |
| | 1000-3000 | 35 | 98.5 | | 96.3 | 92.8 | 96.8 | 93.3 |
| | 3000-5000 | 16 | 60.0 | | 84.2 | 84.2 | 86.9 | 86.9 |
| | 5000+ | 40 | 100.0 | | 100.0 | 99.9 | 100.0 | 99.9 |
| | Total | 977 | 68.9 | | 82.4 | 75.9 | 84.7 | 75.1 |
| All | Total | 1747 | 70.0 | | 79.4 | 71.9 | 82.4 | 71.5 |
Table 2B. Reproducibility of CNVs Grouped by State (Gains and Losses) and Sizes (in kb) Based on Call Rate, Pairwise Agreement between Replicates and Positive Percent Agreement (PPA) for Two Criteria (50% and 80% Overlap) in All Regions in the Site-to-Site Study.
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Table 2C. Reproducibility of CNVs Grouped by State (Gains and Losses) and Marker Number Based on Call Rate, Pairwise Agreement between Replicates and Positive Percent Agreement (PPA) for Two Criteria (50% and 80% Overlap) in Regions Excluding Affymetrix-defined Hypervariable Regions in the Site-to-Site Study.
| | | | | Pairwise Replicate<br>Agreement* | | PPA* | |
|-------|------------------------|--------|------------|----------------------------------|-------|-------|-------|
| | CNV Range<br>(Markers) | # CNVs | Call Rate* | Overlap | | 50% | 80% |
| State | | | | 50% | 80% | | |
| Gain | 50-75 | 166 | 45.4 | 75.1 | 73.6 | 74.7 | 71.6 |
| | 75-100 | 34 | 57.0 | 77.9 | 73.7 | 75.5 | 66.5 |
| | 100-150 | 75 | 63.6 | 85.1 | 76.9 | 88.3 | 76.4 |
| | 150-200 | 48 | 78.7 | 90.8 | 88.9 | 94.0 | 91.8 |
| | 200-300 | 36 | 85.6 | 91.3 | 87.3 | 94.0 | 89.3 |
| | 300-400 | 8 | 100.0 | 100.0 | 98.9 | 100.0 | 98.9 |
| | 400-1000 | 22 | 100.0 | 97.5 | 95.9 | 97.5 | 95.9 |
| | 1000-3000 | 22 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 3000-5000 | 7 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 60 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | Total | 478 | 68.9 | 85.4 | 82.5 | 89.1 | 85.0 |
| Loss | 25-50 | 387 | 49.8 | 81.0 | 78.1 | 77.4 | 70.6 |
| | 50-75 | 74 | 71.5 | 84.8 | 82.8 | 89.0 | 86.4 |
| | 75-100 | 67 | 92.1 | 95.5 | 89.0 | 97.2 | 90.1 |
| | 100-150 | 42 | 82.7 | 87.0 | 82.8 | 88.5 | 83.4 |
| | 150-200 | 19 | 86.9 | 84.9 | 74.9 | 87.3 | 75.8 |
| | 200-300 | 16 | 99.5 | 99.4 | 96.7 | 99.6 | 96.9 |
| | 300-400 | 8 | 100.0 | 100.0 | 94.9 | 100.0 | 94.9 |
| | 400-1000 | 17 | 100.0 | 100.0 | 99.8 | 100.0 | 99.8 |
| | 1000-3000 | 26 | 100.0 | 100.0 | 99.9 | 100.0 | 99.9 |
| | 3000-5000 | 12 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 41 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | Total | 709 | 67.9 | 87.3 | 84.5 | 89.8 | 85.6 |
| All | Total | 1187 | 68.3 | 86.6 | 83.8 | 89.5 | 85.4 |
{9}------------------------------------------------
Table 2D. Reproducibility of CNVs Grouped by State (Gains and Losses) and Sizes (in kb) Based on Call Rate, Pairwise Agreement between Replicates and Positive Percent Agreement (PPA) for Two Criteria (50% and 80% Overlap) in Regions Excluding Affymetrix-defined Hypervariable Regions in the Site-to-Site Study.
| State | CNV Range (kb) | # CNVs | Call Rate* | Pairwise Replicate<br>Agreement*<br>50% | 80% | PPA*<br>50% | 80% |
|-------|----------------|--------|------------|-----------------------------------------|-------|-------------|-------|
| Gain | 50-75 | 79 | 49.5 | 75.4 | 73.4 | 74.2 | 70.1 |
| | 75-100 | 55 | 58.3 | 75.9 | 73.0 | 75.0 | 70.9 |
| | 100-150 | 89 | 66.8 | 79.8 | 74.0 | 84.2 | 75.7 |
| | 150-200 | 48 | 43.5 | 77.6 | 74.0 | 71.5 | 63.0 |
| | 200-300 | 56 | 80.3 | 90.9 | 89.4 | 93.9 | 92.1 |
| | 300-400 | 17 | 71.1 | 80.0 | 74.8 | 81.2 | 73.9 |
| | 400-1000 | 32 | 73.8 | 78.7 | 76.2 | 78.6 | 74.4 |
| | 1000-3000 | 39 | 86.6 | 87.4 | 78.5 | 90.5 | 80.2 |
| | 3000-5000 | 3 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
| | 5000+ | 60 | 100.0 | 99.1 | 98.4 | 99.1 | 98.4 |
| | Total | 478 | 68.9 | 82.6 | 79.2 | 85.1 | 80.4 |
| Loss | 25-50 | 286 | 54.3 | 81.0 | 77.2 | 80.9 | 73.4 |
| | 50-75 | 115 | 65.4 | 85.6 | 81.8 | 84.8 | 77.8 |
| | 75-100 | 54 | 59.6 | 81.2 | 77.2 | 74.0 | 65.8 |
| | 100-150 | 68 | 82.2 | 93.1 | 89.3 | 95.3 | 91.0 |
| | 150-200 | 26 | 75.7 | 85.3 | 84.2 | 87.8 | 86.4 |
| | 200-300 | 21 | 85.6 | 87.4 | 73.6 | 89.9 | 73.7 |
| | 300-400 | 17 | 75.8 | 83.5 | 76.4 | 84.4 | 75.1 |
| | 400-1000 | 31 | 82.0 | 87.6 | 78.8 | 91.3 | 80.5 |
| | 1000-3000 | 35 | 98.5 | 96.3 | 92.8 | 96.8 | 93.3 |
| | 3000-5000 | 16 | 60.0 | 84.2 | 84.2 | 86.9 | 86.9 |
| | 5000+ | 40 | 100.0 | 100.0 | 99.9 | 100.0 | 99.9 |
| | Total | 709 | 67.9 | 86.4 | 82.7 | 88.4 | 82.8 |
| All | Total | 1187 | 68.3 | 85.0 | 81.4 | 87.2 | 81.8 |
*refer to Description of variables section above for detail
CytoScan Dx Assay determines the copy number of each identified CNV as being 0, 1, 2, 3 or 4. For copy number agreement, reproducibility of the numerical copy number was evaluated by performing all pairwise comparisons for replicate measurements of each CNV. Each pairwise comparison with exactly matching copy number was scored as concordant. The summary table 2E presents the fraction of the pairwise comparisons scored as percent agreement, for each size range shown. Agreement = Number of Matching Pairwise Comparisons * 100 / Total Number of Pairwise comparisons, where matching is defined as having identical copy numbers for both of the pair. Copy number agreement was assessed without regard to location, size or marker number agreement. Note that this is a different calculation of agreement because it relies on identical copy number, not just copy number state.
{10}------------------------------------------------
| | | All Regions (Markers) | | All Regions (kb) | | Regions Excluding Affymetrix-Defined Hypervariable Regions (Markers) | | Regions Excluding Affymetrix-Defined Hypervariable Region (kb) | | Gain/Loss | CNV Range<br>(Markers) | N | % CV CNV Length<br>Mean (Min, Median, Max) | Average<br>%<br>Overlap | Median % Absolute Endpoint Deviation<br>Mean (Min, Median, Max) | SD Left Endpoint (M)<br>Mean (Min, Median, Max) | SD Right Endpoint (M)<br>Mean (Min, Median, Max) | | | % CV CNV length | Average | Median % Absolute Endpoint Deviation | SD Left Endpoint (kb) | SD Right Endpoint (kb) | |
|------|-----------|-----------------------|-------------|------------------|-------------|----------------------------------------------------------------------|-------------|----------------------------------------------------------------|-------------|-----------|------------------------|------|--------------------------------------------|-------------------------|-----------------------------------------------------------------|-------------------------------------------------|--------------------------------------------------|-----------|-------------------|-----------------|----------------------------|--------------------------------------|-------------------------|---------------------------|---------------------------|
| | | CNV (N) | % Agreement | CNV (N) | % Agreement | CNV (N) | % Agreement | CNV (N) | % Agreement | Gain | 50-75 | 157 | 5.1 (0.0, 4.1, 30.1) | 68.5 | 0.03 (0.00, 0.01, 0.22) | 1.8 (0.0, 1.2, 12.5) | 2.2 (0.0, 1.6, 11.3) | Gain/Loss | CNV<br>Range (kb) | N | Mean (Min, Median,<br>Max) | %<br>Overlap | Mean (Min, Median, Max) | | |
| Gain | 50-75 | 230 | 69.6% | 99 | 71.1% | 166 | 73.5% | 79 | 74.3% | | 75-100 | 136 | 11.6 (0.0, 11.4, 29.9) | 63.8 | 0.06 (0.00, 0.04, 0.30) | 8.1 (0.0, 8.4, 27.9) | 4.6 (0.0, 2.0, 21.6) | Gain | 50-75 | 67 | 9.1 (0.0, 3.9, 54.8) | 69.6 | 0.04 (0.00, 0.00, 0.37) | 5.1 (0.0, 0.9, 40.2) | 2.9 (0.0, 1.1, 14.0) |
| | 75-100 | 150 | 70.7% | 98 | 78.0% | 34 | 78.6% | 55 | 87.5% | | 100-150 | 111 | 11.7 (0.0, 7.0, 66.2) | 76.9 | 0.06 (0.00, 0.02, 0.63) | 10.1 (0.0, 3.0, 78.1) | 6.2 (0.0, 2.1, 48.8) | | 75-100 | 77 | 11.7 (0.0, 8.9, 55.6) | 68.6 | 0.06 (0.00, 0.02, 0.56) | 7.6 (0.0, 4.4, 46.8) | 7.3 (0.0, 5.7, 52.7) |
| | 100-150 | 131 | 81.9% | 149 | 71.2% | 75 | 86.3% | 89 | 78.7% | | 150-200 | 59 | 11.7 (0.3, 4.5, 54.2) | 85.0 | 0.05 (0.00, 0.01, 0.47) | 15.1 (0.0, 3.3, 107.5) | 9.8 (0.0, 2.8, 93.4) | | 100-150 | 130 | 13.8 (0.0, 6.4, 65.8) | 67.4 | 0.07 (0.00, 0.00, 0.59) | 13.6 (0.0, 5.5, 63.8) | 7.0 (0.0, 2.6, 61.4) |
| | 150-200 | 68 | 90.1% | 101 | 77.5% | 48 | 91.6% | 48 | 79.7% | | 200-300 | 68 | 9.7 (0.0, 5.9, 76.5) | 86.1 | 0.05 (0.00, 0.02, 0.77) | 12.5 (0.0, 5.4, 79.3) | 13.2 (0.0, 9.6, 166.9) | | 150-200 | 76 | 35.9 (0.0, 29.7, 137.4) | 62.6 | 0.13 (0.00, 0.09, 0.62) | 31.6 (0.0, 31.5, 91.7) | 37.2 (0.0, 16.6, 253.5) |
| | 200-300 | 72 | 93.5% | 100 | 80.4% | 36 | 90.5% | 56 | 90.5% | | 300-400 | 8 | 1.9 (0.0, 1.0, 7.9) | 98.1 | 0.01 (0.00, 0.01, 0.02) | 1.4 (0.0, 1.3, 3.1) | 5.7 (0.0, 2.6, 24.1) | | 200-300 | 89 | 25.5 (0.0, 6.4, 118.6) | 70.8 | 0.07 (0.00, 0.01, 0.68) | 22.8 (0.0, 9.1, 238.5) | 43.5 (0.0, 5.5, 269.0) |
| | 300-400 | 8 | 100.0% | 27 | 79.7% | 8 | 100.0% | 17 | 86.7% | | 400-1000 | 22 | 2.7 (0.0, 0.5, 38.9) | 95.8 | 0.02 (0.00, 0.00, 0.28) | 12.5 (0.0, 1.4, 210.1) | 10.3 (0.0, 1.9, 152.7) | | 300-400 | 23 | 41.9 (0.0, 14.1, 144.4) | 69.2 | 0.42 (0.00, 0.02, 2.23) | 24.8 (0.0, 5.1, 298.4) | 123.8 (0.0, 15.2, 488.7) |
| | 400-1000 | 22 | 98.1% | 94 | 88.3% | 22 | 98.1% | 32 | 82.0% | | 1000-3000 | 22 | 0.3 (0.0, 0.2, 1.4) | 99.3 | 0.00 (0.00, 0.00, 0.01) | 3.0 (0.0, 1.4, 15.6) | 3.2 (0.0, 1.5, 34.1) | | 400-1000 | 90 | 16.2 (0.0, 9.8, 100.6) | 79.3 | 0.08 (0.00, 0.04, 1.21) | 36.7 (0.0, 16.8, 358.2) | 71.8 (0.0, 44.7, 695.3) |
| | 1000-3000 | 22 | 97.4% | 39 | 89.7% | 22 | 97.4% | 39 | 89.7% | | 3000-5000 | 7 | 0.3 (0.0, 0.1, 1.1) | 99.5 | 0.00 (0.00, 0.00, 0.00) | 4.3 (0.0, 3.1, 11.3) | 7.7 (0.0, 0.5, 41.8) | | 1000-3000 | 35 | 12.1 (0.0, 1.4, 61.7) | 83.8 | 0.06 (0.00, 0.00, 0.44) | 106.2 (0.0, 4.5, 538.4) | 83.8 (0.0, 3.2, 1011.9) |
| | 3000-5000 | 7 | 100.0% | 3 | 100.0% | 7 | 100.0% | 3 | 100.0% | | 5000+ | 60 | 0.0 (0.0, 0.0, 0.5) | 99.7 | 0.00 (0.00, 0.00, 0.00) | 3.1 (0.0, 0.9, 55.0) | 2.0 (0.0, 0.6, 25.5) | | 3000-5000 | 3 | 0.8 (0.1, 0.2, 2.1) | 98.9 | 0.00 (0.00, 0.00, 0.00) | 20.1 ( 2.2, 6.9, 51.1) | 20.4 (0.0, 4.8, 56.5) |
| | 5000+ | 60 | 93.6% | 60 | 93.6% | 60 | 93.6% | 60 | 93.6% | | Total | 650 | 7.8 (0, 4.3, 76.5) | 76.1 | 0.04 (0.00, 0.01, 0.77) | 7.4 (0.0, 2.3, 210.1) | 5.6 (0.0, 2.0, 166.9) | | 5000+ | 60 | 3.0 (0.0, 0.0, 146.8) | 98.6 | 0.00 (0.00, 0.00, 0.03) | 183.1 (0.0, 0.3, 10583.8) | 352.8 (0.0, 0.0, 11297.1) |
| | All | 770 | 79.6% | 770 | 79.6% | 478 | 84.3% | 478 | 84.3% | Loss | 25-50 | 282 | 6.9 (0.0, 5.4, 63.7) | 77.3 | 0.04 (0.00, 0.02, 0.48) | 1.4 (0.0, 1.0, 18.4) | 1.8 (0.0, 0.9, 26.8) | | Total | 650 | 17.4 (0.0, 7.2, 146.8) | 72.4 | 0.08 (0.00, 0.01, 2.23) | 39.6 (0.0, 5.4, 10583.8) | 64.4 (0.0, 4.7, 11297.1) |
| Loss | 25-50 | 430 | 79.3% | 351 | 78.8% | 387 | 81.0% | 286 | 81.1% | | 50-75 | 186 | 13.1 (0.0, 8.2, 73.1) | 73.3 | 0.08 (0.00, 0.03, 0.71) | 3.5 (0.0, 1.7, 22.4) | 6.2 (0.0, 3.5, 42.4) | Loss | 25-50 | 252 | 9.5 (0.0, 6.6, 60.3) | 76.3 | 0.04 (0.00, 0.00, 0.66) | 2.0 (0.0, 0.1, 22.9) | 2.8 (0.0, 1.3, 24.3) |
| | 50-75 | 204 | 79.5% | 177 | 82.7% | 74 | 86.3% | 115 | 87.7% | | 75-100 | 101 | 8.2 (0.0, 5.2, 33.1) | 82.9 | 0.04 (0.00, 0.01, 0.30) | 3.3 (0.0, 1.7, 22.4) | 4.7 (0.0, 2.8, 22.6) | | 50-75 | 145 | 20.3 (0.0, 12.7, 71.4) | 75.4 | 0.08 (0.00, 0.02, 0.68) | 8.4 (0.0, 4.0, 43.4) | 6.1 (0.0, 3.6, 33.8) |
| | 75-100 | 107 | 88.0% | 88 | 87.0% | 67 | 96.2% | 54 | 87.4% | | 100-150 | 51 | 10.8 (0.0, 1.9, 66.0) | 82.9 | 0.06 (0.00, 0.01, 0.53) | 8.6 (0.0, 1.4, 71.6) | 6.5 (0.0, 1.6, 70.5) | | 75-100 | 69 | 25.7 (0.0, 12.5, 115.9) | 76.2 | 0.12 (0.00, 0.03, 0.50) | 18.2 (0.0, 8.2, 116.5) | 7.2 (0.0, 3.3, 47.0) |
| | 100-150 | 58 | 90.7% | 133 | 85.2% | 42 | 93.0% | 68 | 94.2% | | 150-200 | 22 | 14.2 (0.0, 5.4, 57.7) | 78.6 | 0.04 (0.00, 0.01, 0.23) | 17.3 (0.0, 3.0, 109.7) | 10.5 (0.0, 3.5, 78.9) | | 100-150 | 121 | 11.7 (0.0, 8.1, 57.3) | 79.5 | 0.03 (0.00, 0.00, 0.38) | 8.0 (0.0, 1.6, 78.5) | 8.0 (0.0, 3.8, 40.3) |
| | 150-200 | 24 | 90.5% | 30 | 86.3% | 19 | 91.8% | 26 | 89.0% | | 200-300 | 26 | 14.7 (0.0, 6.8, 68.1) | 90.5 | 0.07 (0.00, 0.02, 0.33) | 27.8 (0.0, 5.8, 136.0) | 8.4 (0.0, 1.7, 46.9) | | 150-200 | 24 | 10.6 (0.2, 2.8, 104.1) | 80.6 | 0.03 (0.00, 0.00, 0.36) | 14.6 (0.0, 3.5, 163.9) | 3.7 (0.0, 1.8, 43.9) |
| | 200-300 | 26 | 97.0% | 29 | 89.2% | 16 | 99.6% | 21 | 92.2% | | 300-400 | 12 | 8.3 (0.3, 1.1, 62.8) | 90.0 | 0.04 (0.00, 0.00, 0.41) | 19.0 (0.0, 1.4, 204.9) | 10.7 (0.0, 1.8, 63.9) | | 200-300 | 27 | 16.2 (0.0, 14.3, 53.4) | 76.2 | 0.07 (0.00, 0.02, 0.42) | 29.4 (0.0, 13.1, 128.7) | 15.0 (0.0, 3.5, 79.3) |
| | 300-400 | 12 | 100.0% | 35 | 95.5% | 8 | 100.0% | 17 | 90.5% | | 400-1000 | 37 | 8.2 (0.0, 3.2, 50.0) | 87.6 | 0.04 (0.00, 0.02, 0.26) | 41.5 (0.0, 12.2, 216.5) | 6.5 (0.0, 5.2, 21.4) | | 300-400 | 32 | 12.9 (0.0, 7.7, 94.8) | 82.2 | 0.03 (0.00, 0.02, 0.17) | 35.3 (0.0, 22.4, 293.4) | 13.0 (0.0, 3.9, 184.3) |
| | 400-1000 | 37 | 100.0% | 43 | 92.4% | 17 | 100.0% | 31 | 89.5% | | 1000-3000 | 26 | 0.5 (0.0, 0.2, 6.1) | 99.1 | 0.00 (0.00, 0.00, 0.01) | 5.1 (0.0, 0.9, 42.5) | 5.1 (0.0, 2.0, 65.9) | | 400-1000 | 40 | 17.2 (0.0, 5.8, 167.4) | 82.4 | 0.10 (0.00, 0.01, 1.26) | 55.2 (0.0, 8.9, 466.3) | 54.7 (0.0, 8.3, 1442.1) |
| | 1000-3000 | 26 | 100.0% | 35 | 98.9% | 26 | 100.0% | 35 | 98.9% | | 3000-5000 | 12 | 0.1 (0.0, 0.0, 0.1) | 99.3 | 0.00 (0.00, 0.00, 0.00) | 1.1 (0.0, 0.7, 3.2) | 1.4 (0.0, 1.1, 5.3) | | 1000-3000 | 35 | 3.2 (0.0, 0.4, 33.4) | 94.5 | 0.01 (0.00, 0.00, 0.10) | 21.3 (0.0, 1.1, 342.6) | 28.5 (0.0, 4.7, 202.4) |
| | 3000-5000 | 12 | 100.0% | 16 | 84.2% | 12 | 100.0% | 16 | 84.2% | | 5000+ | 41 | 0.0 (0.0, 0.0, 0.3) | 99.8 | 0.00 (0.00, 0.00, 0.00) | 1.5 (0.0, 1.1, 6.2) | 1.8 (0.0, 0.5, 21.9) | | 3000-5000 | 11 | 0.3 (0.0, 0.0, 2.6) | 83.9 | 0.00 (0.00, 0.00, 0.00) | 11.4 (0.0, 0.0, 117.9) | 4.0 (0.0, 0.0, 24.6) |
| | 5000+ | 41 | 100.0% | 40 | 100.0% | 41 | 100.0% | 40 | 100.0% | | Total | 796 | 8.6 (0.0, 4.8, 73.1) | 81.1 | 0.05 (0.00, 0.01, 0.71) | 6.2 (0.0, 1.4, 216.5) | 4.4 (0.0, 1.5, 78.9) | | 5000+ | 40 | 0.5 (0.0, 0.0, 9.4) | 99.6 | 0.00 (0.00, 0.00, 0.06) | 8.1 (0.0, 0.6, 164.7) | 50.9 (0.0, 0.1, 1484.5) |
| | All | 977 | 85.1% | 977 | 85.1% | 709 | 87.9% | 709 | 87.9% | All | Total | 1446 | 8.3 (0.0, 4.6, 76.5) | 79.0 | 0.04 (0.00, 0.01, 0.77) | 6.7 (0.0, 1.7, 216.5) | 4.9 (0.0, 1.8, 166.9) | | Total | 796 | 13.1 (0.0, 7.0, 167.4) | 79.8 | 0.05 (0.00, 0.00, 1.26) | 12.1 (0.0, 1.6, 466.3) | 11.6 (0.0, 2.3, 1484.5) |
| All | | 1747 | 82.7% | 1747 | 82.7% | 1187 | 86.6% | 1187 | 86.6% | All | Total | 1446 | 15.1 (0.0, 7.1, 167.4) | 76.6 | 0.07 (0.00, 0.00, 2.23) | 24.5 (0.0, 2.9, 10583.8) | 35.3 (0.0, 3.0, 11297.1) | | | | | | | | |
Table 2E. Copy Number Reproducibility in the Site-to-Site Reproducibility Study.
* Agreement = Number of Matching Pairwise Comparisons * 100 / Total Number of Pairwise comparisons, where matching is defined as having identical copy numbers for both of the pair. Copy number agreement was assessed without regard to location, size or marker number agreement.
For endpoint analysis, only those CNVs detected were assessed for endpoint agreement (i.e.,no calls in replicates could not be included). The CNVs need to have same copy number state (gain, loss) in order to be included in the endpoint agreement calculation. Endpoint agreement is assessed by median % absolute endpoint deviation, standard deviation of left endpoint, and standard deviation of right endpoint, as indicated in the Description of variables section. The results are shown in Tables 2F-2I.
{11}------------------------------------------------
Table 2F. Reproducibility of CNV Length and Endpoints (in Markers) in All Regions in the Site-to-Site Study *
{12}------------------------------------------------
Table 2G. Reproducibility of CNV Length and Endpoints (in kb) in All Regions in the Site-to-Site Study.*
{13}------------------------------------------------
| | | % CV CNV Length | Average | Median % Absolute<br>Endpoint Deviation | SD Left Endpoint (M) | | SD Right Endpoint (M) | |
|------------|---------------------------|-----------------|----------------------------|-----------------------------------------|-------------------------|---------------------------|---------------------------|--|
| Gain/ Loss | CNV<br>Range<br>(Markers) | N | Mean (Min, Median,<br>Max) | %<br>Overlap | Mean (Min, Median, Max) | | Mean (Min, Median, Max) | |
| Gain | 50-75 | 97 | 4.3 (0.0, 2.6, 30.1) | 73.1 | 0.03 (0.00, 0.02, 0.22) | 1.9 (0.0, 1.4, 12.5) | 1.5 (0.0, 1.0, 11.3) | |
| | 75-100 | 23 | 7.5 (0.0, 5.4, 29.7) | 75.6 | 0.05 (0.00, 0.02, 0.26) | 3.9 (0.0, 2.0, 13.5) | 4.2 (0.0, 2.1, 21.6) | |
| | 100-150 | 56 | 11.7 (0.0, 3.4, 66.2) | 81.3 | 0.08 (0.00, 0.02, 0.63) | 10.8 (0.0, 2.4, 78.1) | 6.8 (0.0, 2.6, 48.8) | |
| | 150-200 | 39 | 6.9 (0.3, 1.7, 54.2) | 89.0 | 0.03 (0.00, 0.01, 0.23) | 8.4 (0.0, 2.0, 65.1) | 5.6 (0.0, 1.5, 93.4) | |
| | 200-300 | 32 | 7.9 (0.0, 2.4, 76.5) | 88.1 | 0.05 (0.00, 0.01, 0.77) | 9.4 (0.0, 2.0, 79.3) | 11.4 (0.0, 2.4, 166.9) | |
| | 300-400 | 8 | 1.9 (0.0, 1.0, 7.9) | 98.1 | 0.01 (0.00, 0.01, 0.02) | 1.4 (0.0, 1.3, 3.1) | 5.7 (0.0, 2.6, 24.1) | |
| | 400-1000 | 22 | 2.7 (0.0, 0.5, 38.9) | 95.8 | 0.02 (0.00, 0.00, 0.28) | 12.5 (0.0, 1.4, 210.1) | 10.3 (0.0, 1.9, 152.7) | |
| | 1000-3000 | 22 | 0.3 (0.0, 0.2, 1.4) | 99.3 | 0.00 (0.00, 0.00, 0.01) | 3.0 (0.0, 1.4, 15.6) | 3.2 (0.0, 1.5, 34.1) | |
| | 3000-5000 | 7 | 0.3 (0.0, 0.1, 1.1) | 99.5 | 0.00 (0.00, 0.00, 0.00) | 4.3 (0.0, 3.1, 11.3) | 7.7 (0.0, 0.5, 41.8) | |
| | 5000+ | 60 | 0.0 (0.0, 0.0, 0.5) | 99.7 | 0.00 (0.00, 0.00, 0.00) | 3.1 (0.0, 0.9, 55.0) | 2.0 (0.0, 0.6, 25.5) | |
| | Total | 366 | 5.1 (0.0, 1.6, 76.5) | 83.2 | 0.03 (0.00, 0.00, 0.77) | 5.7 (0.0, 1.7, 210.1) | 4.7 (0.0, 1.4, 166.9) | |
| Loss | 25-50 | 250 | 6.3 (0.0, 4.9, 63.7) | 78.8 | 0.03 (0.00, 0.02, 0.48) | 1.5 (0.0, 1.1, 18.4) | 1.5 (0.0, 0.8, 26.8) | |
| | 50-75 | 61 | 7.5 (0.0, 2.9, 73.1) | 83.0 | 0.05 (0.00, 0.02, 0.71) | 1.5 (0.0, 0.7, 11.2) | 3.7 (0.0, 1.4, 42.4) | |
| | 75-100 | 64 | 6.4 (0.0, 3.1, 33.1) | 91.1 | 0.02 (0.00, 0.00, 0.30) | 2.7 (0.0, 1.3, 22.4) | 3.6 (0.0, 1.5, 20.9) | |
| | 100-150 | 36 | 8.3 (0.0, 1.6, 55.1) | 85.6 | 0.04 (0.00, 0.00, 0.46) | 4.5 (0.0, 1.3, 65.1) | 6.9 (0.0, 0.9, 70.5) | |
| | 150-200 | 17 | 9.5 (0.0, 2.1, 50.7) | 81.7 | 0.02 (0.00, 0.01, 0.12) | 7.7 (0.0, 1.9, 40.7) | 10.7 (0.0, 1.7, 78.9) | |
| | 200-300 | 16 | 4.6 (0.0, 1.0, 19.2) | 97.7 | 0.03 (0.00, 0.01, 0.21) | 3.9 (0.0, 0.8, 19.7) | 7.2 (0.0, 1.4, 43.5) | |
| | 300-400 | 8 | 3.0 (0.3, 0.5, 19.1) | 96.5 | 0.01 (0.00, 0.00, 0.09) | 1.1 (0.0, 0.7, 4.1) | 9.5 (0.9, 1.8, 63.9) | |
| | 400-1000 | 17 | 0.7 (0.0, 0.3, 2.9) | 97.8 | 0.00 (0.00, 0.00, 0.02) | 2.0 (0.0, 0.0, 11.9) | 2.5 (0.0, 1.4, 21.4) | |
| | 1000-3000 | 26 | 0.5 (0.0, 0.2, 6.1) | 99.1 | 0.00 (0.00, 0.00, 0.01) | 5.1 (0.0, 0.9, 42.5) | 5.1 (0.0, 2.0, 65.9) | |
| | 3000-5000 | 12 | 0.1 (0.0, 0.0, 0.1) | 99.3 | 0.00 (0.00, 0.00, 0.00) | 1.1 (0.0, 0.7, 3.2) | 1.4 (0.0, 1.1, 5.3) | |
| | 5000+ | 41 | 0.0 (0.0, 0.0, 0.3) | 99.8 | 0.00 (0.00, 0.00, 0.00) | 1.5 (0.0, 1.1, 6.2) | 1.8 (0.0, 0.5, 21.9) | |
| | Total | 548 | 5.5 (0.0, 2.7, 73.1) | 85.2 | 0.03 (0.00, 0.00, 0.71) | 2.3 (0.0, 1.1, 65.1) | 3.2 (0.0, 1.1, 78.9) | |
| All | Total | 914 | 5.3 (0.0, 2.2, 76.5) | 84.5 | 0.03 (0.00, 0.00, 0.77) | 3.6 (0.0, 1.3, 210.1) | 3.8 (0.0, 1.3, 166.9) | |
| | | % CV CNV Length | Average<br>% | Median % Absolute<br>Endpoint Deviation | SD Left Endpoint (kb) | | SD Right Endpoint (kb) | |
| Gain/Loss | CNV<br>Range (kb) | N | Mean (Min, Median,<br>Max) | %<br>Overlap | Mean (Min, Median, Max) | | Mean (Min, Median, Max) | |
| Gain | 50-75 | 48 | 6.2 (0.0, 2.0, 54.8) | 74.2 | 0.01 (0.00, 0.00, 0.18) | 3.2 (0.0, 0.4, 40.2) | 1.6 (0.0, 0.0, 8.4) | |
| | 75-100 | 36 | 10.8 (0.0, 6.2, 55.6) | 76.9 | 0.06 (0.00, 0.00, 0.51) | 7.4 (0.0, 3.1, 46.8) | 6.8 (0.0, 3.0, 52.7) | |
| | 100-150 | 72 | 11.8 (0.0, 5.5, 55.1) | 76.3 | 0.05 (0.00, 0.00, 0.46) | 12.5 (0.0, 4.0, 63.8) | 4.3 (0.0, 1.6, 56.4) | |
| | 150-200 | 24 | 10.5 (0.0, 3.2, 61.7) | 76.2 | 0.05 (0.00, 0.00, 0.36) | 10.9 (0.0, 4.2, 51.8) | 9.1 (0.0, 0.7, 114.7) | |
| | 200-300 | 47 | 8.0 (0.0, 2.9, 89.3) | 88.5 | 0.02 (0.00, 0.00, 0.40) | 15.0 (0.0, 3.7, 238.5) | 7.6 (0.0, 4.1, 99.3) | |
| | 300-400 | 13 | 24.1 (0.0, 5.5, 144.4) | 78.9 | 0.05 (0.00, 0.01, 0.40) | 34.8 (0.0, 8.0, 298.4) | 55.2 (0.0, 3.2, 488.7) | |
| | 400-1000 | 28 | 16.1 (0.0, 1.8, 100.6) | 77.8 | 0.08 (0.00, 0.01, 1.21) | 41.2 (0.0, 3.6, 358.2) | 78.0 (0.0, 5.4, 695.3) | |
| | 1000-3000 | 35 | 12.1 (0.0, 1.4, 61.7) | 83.8 | 0.06 (0.00, 0.00, 0.44) | 106.2 (0.0, 4.5, 538.4) | 83.8 (0.0, 3.2, 1011.9) | |
| | 3000-5000 | 3 | 0.8 (0.1, 0.2, 2.1) | 98.9 | 0.00 (0.00, 0.00, 0.00) | 20.1 (2.2, 6.9, 51.1) | 20.4 (0.0, 4.8, 56.5) | |
| | 5000+ | 60 | 3.0 (0.0, 0.0, 146.8) | 98.6 | 0.00 (0.00, 0.00, 0.03) | 183.1 (0.0, 0.3, 10583.8) | 352.8 (0.0, 0.0, 11297.1) | |
| | Total | 366 | 9.7 (0.0, 2.5, 146.8) | 81.1 | 0.04 (0.00, 0.00, 1.21) | 51.0 (0.0, 2.7, 10583.8) | 77.2 (0.0, 1.6, 11297.1) | |
| Loss | 25-50 | 201 | 7.3 (0.0, 5.0, 60.3) | 78.7 | 0.03 (0.00, 0.00, 0.66) | 1.6 (0.0, 0.5, 22.6) | 1.8 (0.0, 0.4, 23.9) | |
| | 50-75 | 84 | 11.3 (0.0, 6.5, 71.4) | 83.5 | 0.04 (0.00, 0.00, 0.68) | 4.0 (0.0, 2.2, 43.4) | 4.0 (0.0, 0.6, 33.8) | |
| | 75-100 | 38 | 22.5 (0.0, 6.3, 115.9) | 80.5 | 0.09 (0.00, 0.01, 0.50) | 15.6 (0.0, 2.3, 116.5) | 6.5 (0.0, 2.4, 47.0) | |
| | 100-150 | 57 | 6.8 (0.0, 3.8, 41.5) | 91.0 | 0.01 (0.00, 0.00, 0.20) | 6.5 (0.0, 2.0, 55.6) | 2.9 (0.0, 1.5, 16.4) | |
| | 150-200 | 21 | 8.5 (0.2, 2.1, 104.1) | 83.8 | 0.02 (0.00, 0.00, 0.17) | 11.0 (0.0, 3.0, 163.9) | 3.6 (0.0, 0.6, 43.9) | |
| | 200-300 | 19 | 11.2 (0.4, 7.1, 43.4) | 83.2 | 0.04 (0.00, 0.00, 0.42) | 18.0 (0.0, 4.4, 121.4) | 12.3 (0.0, 1.6, 79.3) | |
| | 300-400 | 14 | 15.4 (0.0, 0.9, 94.8) | 82.1 | 0.01 (0.00, 0.00, 0.05) | 35.5 (0.0, 0.3, 293.4) | 21.8 (0.0, 2.4, 184.3) | |
| | 400-1000 | 28 | 19.7 (0.0, 4.6, 167.4) | 82.9 | 0.12 (0.00, 0.01, 1.26) | 57.9 (0.0, 5.3, 466.3) | 75.1 (0.0, 12.0, 1442.1) | |
| | 1000-3000 | 35 | 3.2 (0.0, 0.4, 33.4) | 94.5 | 0.01 (0.00, 0.00, 0.10) | 21.3 (0.0, 1.1, 342.6) | 28.5 (0.0, 4.7, 202.4) | |
| | 3000-5000 | 11 | 0.3 (0.0, 0.0, 2.6) | 83.9 | 0.00 (0.00, 0.00, 0.00) | 11.4 (0.0, 0.0, 117.9) | 4.0 (0.0, 0.0, 24.6) | |
| | 5000+ | 40 | 0.5 (0.0, 0.0, 9.4) | 99.6 | 0.00 (0.00, 0.00, 0.06) | 8.1 (0.0, 0.6, 164.7) | 50.9 (0.0, 0.1, 1484.5) | |
| | Total | 548 | 9.0 (0.0, 3.7, 167.4) | 84.4 | 0.03 (0.00, 0.00, 1.26) | 10.1 (0.0, 1.2, 466.3) | 12.6 (0.0, 1.3, 1484.5) | |
| All | Total | 914 | 9.3 (0.0, 3.2, 167.4) | 83.2 | 0.03 (0.00, 0.00, 1.26) | 26.4 (0.0, 1.5, 10583.8) | 38.5 (0.0, 1.4, 11297.1) | |
Table 2H. Reproducibility of CNV Length and Endpoints (in Markers) in Regions Excluding Affymetrix-defined Hypervariable Regions in the Site-to-Site Study.*
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Table 21. Reproducibility of CNV Length and Endpoints (in kb) in Regions Excluding Affymetrix-defined Hypervariable Regions in the Site-to-Site Study.
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Reproducibility of Loss of Heterozygosity (LOH) regions:
Reproducibility of LOH calls was calculated in the site-to-site study and is shown in Table 3.
| | | | | Pairwise Replicate Agreement | PPA | |
|----------------------------------------|-----|------|---------|------------------------------|-----------|--|
| | | | Overlap | | | |
| Size Range (Mb)# LOH regions Call Rate | | | 50% | 80% | 50% 80% | |
| 3-4 | 228 | 84.6 | 90.0 | 82.4 | 93.5 83.9 | |
| 4-2 | 38 | 97.9 | 95.4 | 81.0 | 96.3 81.6 | |
| 2-10 | 68 | 96.7 | 91.5 | 84.9 | 93.4 86.6 | |
| >10 | 147 | 99.7 | 96.0 | 93.4 | 96.3 93.6 | |
| Total | 481 | 91.9 | 92.4 | 86.0 | 94.6 87.4 | |
Table 3. Reproducibility of LOH calls in the site-to-site study.
# Study 2. Lot-to-Lot reproducibility:
A between-lot reproducibility study was conducted to test the impact of array lot, reagent lot, and operator on the reproducibility of the CytoScan Dx Assay. Fortyseven (47) genomic DNA (gDNA) samples (including 1 control) were randomized across 2 plates with each plate including 12 blood gDNA, 11 cell-line gDNA, and 1 cell-line control gDNA. Samples in each replicate plate were independently randomized for plate location. All 47 gDNA samples were evaluated with the 3 lots of arrays, 3 lots of reagents, and 6 operators at 1 investigative site - Affymetrix, Santa Clara, CA.
The samples were selected to maximize the variation across the genome with consideration to gain and loss segments, chromosomal representation, CNV regions in genic and non-genic regions, and in telomeric and centromeric regions. The samples had aberrations that collectively covered 31.3% of the genome. Each sample was run 10 times across the study, except for one control sample which was run in every batch and therefore run 24 times. Data analysis methods are same as those used in the site-to-site reproducibility study. Reproducibility results observed in the lot-tolot reproducibility study were similar to the results in the site-to-site study.
- b. Linearity/assay reportable range:
N/A
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- Traceability, Stability, Expected values (controls, calibrators, or methods): c.
# Stability:
Stability studies were performed which included packaging/shipping stability, shelf life, and freeze/thaw and inversion stability. The stability of gDNA samples under expected storage conditions was also evaluated. In process OC standard: visual inspection for discoloration, leakage, volume loss or precipitation of reagents; and array QC metrics were assessed.
Reagent packaging/shipping stability was evaluated under ambient, refrigerated and frozen shipping conditions, and array stability was evaluated under ambient conditions (summer or winter) over 120 hour periods in a kit box of 6 arrays. One gDNA sample was used for both shelf life testing and freeze/thaw and inversion stability studies. CytoScan Dx microarrays and reagents were each randomly selected from 3 design validation lots and 12 replicates of the gDNA samples were tested with CytoScan Dx reagents and array at storage time = 3 months. A variety of storage. freeze/thaw, in use and inversion conditions were tested.
Real-time stability studies currently support a three month shelf life. CytoScan® Dx reagents can undergo a total of 10 freeze/thaw cycles (-20°C/Wet Ice Freeze/Thaw) total, including 6 in-use cycles and 4 cycles prior to use in the assay. Enzymes can withstand 5 dry ice/-20°C freeze/thaw cycles. The recommended number of freeze/thaw cycles for components stored at -15 and -25°C are 4 in-use cycles within 30 days, 3 cycles prior to use in the assay, and 4 dry ice/-20℃ freeze/thaw cycles for the enzymes. Purified gDNA samples are stable when stored for up to 6 weeks following DNA extraction, at either 15°C to 30°C (ambient), 2°C to 8°C (refrigerated), or -15°C to -25°C (frozen, with up to 4 freeze/cycles).
## Assay controls:
The package insert recommends including a positive control in every batch of samples processed.
- d. Detection limit:
# DNA input:
The amount of genomic DNA recommended for testing per sample with the CytoScan Dx is 250 ng. To determine the performance of the CytoScan Dx across a range of genomic DNA input concentrations, the amount of genomic DNA used in the initial fragmentation step of the assay was tested at levels ranging from 1 ng to 500 ng. Eight genomic DNA samples containing 31 CNV regions (representing 317 kb- 83.1 Mb of sizes of CNV region gains and losses) were tested in the study. These include 7 cell line derived samples from Coriell or ATCC that have known chromosomal aberrations and 1 blood derived sample from a healthy donor. A set of input DNA
{17}------------------------------------------------
levels serially diluted were tested at concentrations of 1, 2, 10, 50, 250, 300, 375, and 500 ng. The parameters measuring CytoScan performance, copy number state within copy number variation region (PMC), the endpoints of the copy number region (PMCB), and the number of non-copy number 2 segments (AASC) were compared with predefined acceptance criteria. The Limit of Detection is defined as the DNA input level where 100% of CNV markers can be detected accurately 95% of the time. DNA input of 250 ng was set as the control level. The results demonstrated that 10 ng of genomic DNA is a conservative lower limit for the CytoScan Dx assay based on the evaluated criteria. Array OC metrics including median absolute pair-wise difference (MAPD) and single nucleotide polymorphism quality control (SNPQC) also showed concordant results. supporting that array data meeting prespecified acceptance criteria were achievable for input DNA levels ≥ 10 ng. The assay functions appropriately with genomic DNA input of up to 500 ng.
## Mosaicism:
To determine the level of mosaicism reliably detected by the CytoScan Dx, a total of 8 gDNA samples purified from Coriell cell lines were tested in the study. Each sample contained 1 gain and 1 loss on autosomal chromosomes, for a total of 16 CNV regions. The study was designed to simulate different percentages of mosaicism by mixing different proportions of 2 different gDNA samples. Four pairs of samples were mixed at 12 different mixture levels (0, 5, 10, 20, 30, 40, 60, 70, 80, 90, 95, and 100%) to generate samples representative of specified levels of mosaicism. This design generated 48 different DNA mixtures that were analyzed on 48 CytoScan Dx microarrays.
Receiver operating characteristics (ROC) curve analysis was used as a metric of how well the mosaic copy number markers are separated from the adjacent normal copy number markers. An ROC curve was generated for each mosaic region in each sample mixture. The mosaic region was scored as detected if the area under the curve (AUC) of the ROC curve met or exceeded the predefined threshold of 0.90. An AUC of 0.90 means that each marker considered has a 90% probability of detecting a mosaic segment correctly. Results with the 8 gDNA samples showed that the CytoScan Dx can detect mosaic segments in samples with greater than 15% mosaicism.
Another 2 gDNA samples at 8 different mixture levels were run as 9 replicates on the CytoScan Dx system to establish repeatability of mosaicism detection, which generated data from 72 CytoScan Dx microarrays. Samples NA13330 and NA07216 were mixed in 11 different proportions scored for the ability to distinguish a mosaic segment in 5 genomic regions (chromosomes 1, 3, 4, 7 and X). Each condition was run on 9 replicate tests in CytoScan Dx to assess repeatability. Mosaic regions were scored by calculating the AUC for the markers in the region and an equal number of adjacent markers. The results show that at a 15% mosaicism level, the mean AUC was 0.96 with 50% of the data falling within the inter quartile range of ± 1.9%. The CV at the 15% mosaicism level was 1.6%. Overall, the claimed detection level is that the CytoScan Dx reliably detects mosaicism greater than 20%, and that mosaicism less than 20% may not be reliably detected.
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- e. Analytical specificity:
#### Interfering Substances:
To assess the impact of interfering substances on the CytoScan Dx Assay, simulated aberrant or normal blood samples were either spiked with conjugated and unconjugated bilirubin (60 mg/dL), triglycerides (triolein, 3000 mg/dL), and hemoglobin (>0.5g/dL). Twenty-four (24) normal blood samples with no known chromosomal abnormalities and 9 spike-in cell lines with 19 known aberrant regions were utilized in the study. Each cell line was cultured, harvested, counted and resuspended in leukocyte-depleted blood to a final concentration of 8x106 cells/mL. DNA was extracted from each sample to be tested using CytoScan assay and the assay performance was measured against critical limits of in-process QC, array-based QC and performance metrics. For each interferent test condition, array QC and assay performance results met the sponsor's acceptance criteria and no interference was observed with any of the tested substances.
#### Carryover:
To determine the effect of potential gDNA carry-over from 1 array to the next when processing multiple arrays on the same Fluidics workstation 450Dx v.2, 12 gDNA samples that represent a variety of copy number gains and losses on over 15 chromosomes were tested in the study. For a given fluidic station module, a sequence of 1 normal sample followed by 3 successive aberrant samples (with the same aberrations) then followed by another run of the same normal sample was used to determine any potential carry-over effect due to genomic DNA. These 5 sequential runs were performed in a predefined order on 12 modules of 3 fluidics stations. Prespecified criteria for establishing the absence of carryover was to compare the array performance metric values and copy number state determination on a set of predefined aberrations between the first run and the fifth run. There was no significant difference in copy number state determination (Wilcoxon signed rank statistic (S) = 0.5, p-value = 1.00) or array QC metrics (Wilcoxon signed rank statistic, p > 0.05) between the first run and the fifth run, suggesting no carry-over from the aberrant gDNA samples by potential sources such as the fluidic station.
#### Cross-Contamination:
Sensitivity to cross-contamination was evaluated under simulated crosscontaminating conditions using 8 gDNA samples, mixed into 4 pairs at 12 different ratios before the hybridization step. A total of 48 arrays were analyzed for the assay performance and OC metrics. Prespecified acceptance criteria were that the singlenucleotide polymorphism quality control (SNPQC) should not fall below the standard acceptance limit (SNPQC ≥12) at approximately 20% contamination. SNPQC values ≥12 indicate the absence of substantial cross-contamination.
- f. Assay cut-off:
N/A
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#### 2. Comparison studies:
200-300
ર્સ્વ
#### a. Accuracy:
Accuracy of the CytoScan Dx Assay results was assessed by comparing the CNVs identified by CytoScan Dx Assay to the results obtained using alternative methods. A total of 1515 CNVs were identified by the CytoScan Dx Assay in a total of 137 gDNA samples (48 purified from blood, 86 from Coriell Cell Repository cell lines, 3 from ATCC cell lines). The samples were selected to maximize the variation across the genome with consideration to gain and loss segments of various sizes/number of probes, chromosomal representation, CNV regions in genic and non-genic regions, and in telomeric and centromeric regions. Of the 137 samples in the sequencing study, 5 samples were excluded for a total of 132 evaluable samples (3 of the 5 omitted samples were incorrectly annotated samples and 2 samples were hypersegmented i.e., >40 segments, indicating poor quality DNA). These 132 samples had 1280 eligible CNVs for inclusion in the analysis. The CNVs covered 63.5% of the genome and were more prevalent in non-telomeric/non-centromeric regions than in telomeric/centromeric (62.3% vs 37.7%) and in genic (79.1%) than non-genic regions. A total of 28.91% of the CNVs had high (>45%) GC content.
The 132 samples were analyzed by a validated high throughput method to confirm the accuracy of the CytoScan Dx CNV results. The criterion for accuracy was agreement between the CNVs identified by the CytoScan Dx Assay and sequencing method based on a >50% overlap in markers or size, and the same copy number state (gain or loss) between the two methods. The results are summarized and stratified by copy number state, size or marker range, and genomic region in Tables 4A-E. Due to the number of CNVs that did not show gain or loss (i.e., were copy number neutral state) using high throughput sequencing, another analytically validated molecular method was performed on a statistically appropriate number of CNVs, and CytoScan Dx Assay results were then compared to this composite analytical method (Tables 5A-C). This proportion analysis demonstrated a modest improvement in results from 78.8% (95%CI, 76.4-80.9%) to 88.7% (95% CI, 84.2-92.2%). The results in Tables 4A-E and 5A-C summarize the accuracy (% agreement between CytoScan Dx Assay and comparison method) and the false positive rate (FPR) (i.e., the alternate method(s) did not confirm the CNV detected by the Cytoscan Dx; 1-agreement; see footnote to tables for explanation).
| (kb) when Compared to Sequencing Method. | | | | | |
|------------------------------------------|----------------|-----------------|-------|----------------------------------------------|----------------------------------------|
| Gain/Loss | CNV Range (kb) | Sample Size (N) | Agree | % Agreement (95% CI)*<br>Based on Sequencing | FPR** (95% CI)*<br>Based on Sequencing |
| Gain | 50-75 | 49 | 39 | 79.6% (66.4%, 88.5%) | 20.4% (11.5%, 33.6%) |
| | 75-100 | 48 | 21 | 43.8% (30.7%, 57.7%) | 56.3% (42.3%, 69.3%) |
| | 100-150 | 133 | 95 | 71.4% (63.2%, 78.4%) | 28.6% (21.6%, 36.8%) |
| | 150-200 | 39 | 20 | 51.3% (36.2%, 66.1%) | 48.7% (33.9%, 63.8%) |
67.9% (54.8%, 78.6%)
38
Table 4A. CytoScan Dx Assay Accuracy for All CNV Regions Stratified by Gain/Loss and Size
32.1% (21.4%, 45.2%)
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| Gain/Loss | CNV Range (kb) | Sample Size<br>(N) | Agree | % Agreement (95% CI)*<br>Based on Sequencing | FPR** (95% CI)*<br>Based on Sequencing |
|-----------|----------------|--------------------|-------|----------------------------------------------|----------------------------------------|
| | 300-400 | 42 | 31 | 73.8% (58.9%, 84.7%) | 26.2% (15.3%, 41.1%) |
| | 400-1000 | 123 | 67 | 54.5% (45.7%, 63.0%) | 45.5% (37.0%, 54.3%) |
| | 1000+ | 83 | 82 | 98.8% (93.5%, 99.8%) | 1.2% (0.2%, 6.5%) |
| | Total | 573 | 393 | 68.6% (64.7%, 72.3%) | 31.4% (27.7%, 35.3%)…
