(254 days)
GenetiSure Dx Postnatal Assay is a qualitative assay intended for the postnatal detection of copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) in genomic DNA obtained from peripheral whole blood in patients referred for chromosomal testing based on clinical presentation. GenetiSure Dx Postnatal Assay is intended for the detection of CNVs and cnLOH 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 only by healthcare professionals, board certified in clinical cytogenetics. The assay is intended to be used on the SureScan Dx Microarray Scanner System and analyzed by CytoDx Software.
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.
The GenetiSure Dx Postnatal Assay is a clinical laboratory in vitro diagnostic assay for performing molecular karyotyping based on array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) analysis from blood samples of post-natal patients who are suspected of having a genomic abnormality. This molecular karyotyping is a modified in situ hybridization technique that allows detection and mapping of DNA sequence copy difference(s) between two genomes in a single experiment. In molecular karyotyping analysis, two differentially labeled genomic DNAs (subject/test sample and a reference sample) are co-hybridized to complementary nucleic acid sequences synthesized in situ on a microarrav slide.
Locations of copy number variation (CNVs) and copy-neutral loss of heterozygosity (cnLOH) in the DNA segments of the subject sample genome are revealed by variable fluorescence intensity on the microarray.
The assay compares the patient sample against a sex-matched reference sample. Genomic DNA (gDNA) is extracted from the patient's whole blood and then is fluorescently labeled in parallel with the reference sample using two different fluorescent dyes. The two labeled samples are hybridized to complementary sequences (probes) that are printed on a CGH+SNP microarray.
After hybridization, the microarrays are washed and then scanned. The data from the microarray images are converted to numeric data. The relative abundance of the target sequences is computed based on the relative intensities of the fluorophores in the patient and reference samples hybridized to each of the probe sequences.
The numeric data is then processed using software specifically designed to report CNVs by chromosomal location. The reported CNVs are interpreted by a Board Certified Cytogeneticist, Molecular Geneticist, Molecular Pathologist, or similarly qualified clinician who has been trained to identify the clinically relevant CNVs, determine clinical significance, and report out these findings. cnLOH in patient samples is also reported to the clinician.
The provided text describes the GenetiSure Dx Postnatal Assay, a qualitative assay for detecting copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) in genomic DNA from peripheral whole blood.
Here's an analysis of the acceptance criteria and the study that proves the device meets them:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria for this device are not explicitly stated in a single, consolidated table with pass/fail values. Instead, they are described within the context of each analytical performance study (Reproducibility, Precision, Accuracy, Stability, Limit of Detection, Analytical Specificity). The "reported device performance" is essentially the results presented in various tables and text summaries throughout Section K, "Performance Characteristics."
Below is a synthesized table based on the provided text, outlining the implied acceptance criteria (where quantifiable and explicitly stated in relation to a target metric) and the reported performance:
| Category | Acceptance Criteria (Implied/Stated) | Reported Device Performance |
|---|---|---|
| Reproducibility (Overall Pairwise Replicate Agreement - 50% Overlap) | Not explicitly stated as a single numeric criteria for overall, but implied high agreement for various categories. | Overall CN gains and losses: 85.0% (50% overlap), 82.3% (80% overlap) |
| Small CNVs (5-20 probes): N/A (grouped by kb/probe count) | CN Gain: 85.7% (50% overlap), 84.4% (80% overlap) | |
| Large CNVs (>20 probes): N/A | CN Loss: 84.6% (50% overlap), 81.3% (80% overlap) | |
| cnLOH regions: N/A | cnLOH: 89.1% (50% overlap), 87.9% (80% overlap) | |
| Reproducibility (Positive Percent Agreement - 50% Overlap) | N/A | All CNVs: 89.3% |
| cnLOH: 92.7% | ||
| Precision (Between-Lot Reagent and Scanner - Pairwise Replicate Agreement - 50% Overlap) | Not explicitly stated as a single numeric criteria for overall, but implied high agreement for various categories. | Small CNVs (5-20 probes): 83.33% |
| Large CNVs (>20 probes): 98.39% | Large CNVs (>20 probes): 98.39% | |
| cnLOH regions: 80.80% | cnLOH regions: 80.80% | |
| Precision (DNA Extraction - Pairwise Replicate Agreement - 50% Overlap) | Small CNVs (5-20 probes): 70% | Small CNVs (5-20 probes): 82.17% |
| Large CNVs (>20 probes): 80% | Large CNVs (>20 probes): 98.47% | |
| cnLOH regions: N/A | cnLOH regions: 81.15% | |
| Accuracy (Confirmation Rate - 50% Overlap, Excluding Indeterminate CNVs) | N/A (grouped by probe count and kb) | All CNVs: 92.6% (Scheme b) |
| cnLOH: 85.8% (Scheme b) | ||
| Accuracy (Endpoint Agreement - ≤2 probes for CNVs, ≤50 probes for cnLOH) | N/A | All CNVs (combined start/stop): 87.4% |
| cnLOH (combined start/stop): 91.6% | ||
| Whole Blood Stability (Confirmation Rate - 50% Overlap) | Small CNVs (5-20 probes): 75% | Small CNVs (5-20 probes): Met criteria |
| Large CNVs (>20 probes): 90% | Large CNVs (>20 probes): Met criteria | |
| Interfering Substances (Confirmation Rate - 50% Overlap) | Small CNVs (5-20 probes): 75% | Small CNVs (5-20 probes): Met criteria |
| Large CNVs (>20 probes): 90% | Large CNVs (>20 probes): Met criteria | |
| Cross-Contamination | No suspected cross-contamination detected, supporting appropriateness of workflow. | No suspected cross-contamination detected. |
| Limit of Detection (DNA Input) | Data supports conservative LLOD at 375 ng and common ULOD at 1000 ng for CN and cnLOH. LLOD could be 250ng for CNVs only. | Performance does not decline down to 375 ng; robust at 500 ng. |
| Limit of Detection (Mosaicism) | Reliable detection of large CNV (>100 probes) down to 50% level. | Large CNV (>100 probes) reliably detected at 50% or greater admixture. |
| Clinical Validity (PPA for CNVs only) | N/A. The comparator here is the collection site's own interpretation. | 76.8% (95%CI=68.2%-83.6%) |
| Clinical Validity (NPA for CNVs only) | N/A. The comparator here is the collection site's own interpretation. | 95.5% (95%CI=93.7%-96.8%) |
| Clinical Validity (PPA for all aberrations) | N/A. The comparator here is the collection site's own interpretation. | 76.7% (95%CI=68.3%-83.5%) |
| Clinical Validity (NPA for all aberrations) | N/A. The comparator here is the collection site's own interpretation. | 89.8% (95%CI=87.3%-91.8%) |
2. Sample Size Used for the Test Set and the Data Provenance
- Reproducibility:
- Sample Size: 48 test samples.
- Data Provenance: Cell-lines with known aberrations ("common syndromes," "analytically challenging regions," "claimed minimal resolution," "varying aberration size ranges," and "genomic coverage of aberrations"). Origin not explicitly stated, but cell lines are generally laboratory-derived. It's a prospective experimental study.
- Precision (Between-Lot Reagent and Scanner):
- Sample Size: 48 test samples.
- Data Provenance: Cell-lines with known aberrations (copy number gains, losses, and cnLOH). Origin not explicitly stated. It's a prospective experimental study.
- Precision (DNA Extraction):
- Sample Size: 24 samples.
- Data Provenance: Cell-lines (implied, as they were tested by each operator for DNA extraction and then run in the assay). Origin not explicitly stated. It's a prospective experimental study.
- Accuracy:
- Sample Size: 556 eligible samples total, comprising:
- 451 aberrant genomic DNA (gDNA) samples from established commercial cell lines.
- 76 archived clinical gDNA samples from whole blood specimens of anonymized patients.
- 5 globally recognized syndrome reference panel gDNA samples.
- 24 fresh blood-derived gDNA samples from phenotypically normal subjects.
- Data Provenance:
- Commercial cell lines, globally recognized reference panels (implies international or broadly recognized sources).
- Archived clinical gDNA samples are retrospective, anonymized from whole blood specimens; country of origin not specified.
- Fresh blood samples from phenotypically normal subjects are likely prospective, implying a controlled collection process, but specific origin not detailed.
- Sample Size: 556 eligible samples total, comprising:
- Stability (Whole Blood):
- Sample Size: 24 whole blood specimens (12 male, 12 female).
- Data Provenance: Obtained from a blood bank; implies clinical samples. Origin not explicitly stated. It's a prospective experimental study.
- Limit of Detection (DNA Input):
- Sample Size: 24 gDNA samples.
- Data Provenance: Obtained from Coriell Institute for Medical Research (a well-known biobank).
- Limit of Detection (Mosaicism):
- Sample Size: Aberrant cell line DNAs mixed with reference background DNA. Specific number of samples/admixtures not explicitly quantified but described as "admixtures."
- Data Provenance: Cell-line DNAs. Origin not explicitly stated.
- Analytical Specificity (Interfering Substances):
- Sample Size: 24 whole blood samples (12 male, 12 female).
- Data Provenance: Drawn from phenotypically normal subjects. Origin not explicitly stated.
- Analytical Specificity (Cross Contamination):
- Sample Size: 2 male and 2 female Coriell DNA samples.
- Data Provenance: Coriell DNA samples.
- Clinical Validity:
- Sample Size: 800 samples from patients suspected of pathogenic aberrations (SPA samples) + 100 samples from phenotypically normal individuals = Total 900 samples.
- Data Provenance:
- SPA samples from three (3) regionally distinct clinical institutions (implies clinical, retrospective or newly collected for study, "collected from" rather than "archived"). Country of origin not explicitly stated.
- Phenotypically normal samples used to assess aberrations in a normal population. Origin not explicitly stated for these.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts
- Accuracy Study (Comparator step for Agilent aberrations): Ground truth was established by independent (non-Agilent) commercially available microarray-based assays, analytically validated for copy number detection, and if discrepancies arose, by qPCR. This suggests comparative technical methods rather than direct expert interpretation as the primary ground truth for individual aberration confirmation.
- Clinical Validity Study:
- Ground Truth Establishment: The aberrations detected by the GenetiSure Dx Postnatal Assay for all 900 samples were interpreted by one of four cytogeneticists.
- Qualifications of Experts: The experts were cytogeneticists. No specific experience level (e.g., "10 years of experience") is provided, but it is implicitly understood that a "cytogeneticist" is a qualified professional in this field.
4. Adjudication Method for the Test Set
- Reproducibility, Precision (Between-Lot Reagent and Scanner), DNA Extraction Precision, Whole Blood Stability, Interfering Substances, Cross-Contamination: The primary adjudication method for individual aberration calls was Pairwise Replicate Analysis (PRA), requiring at least a 50% overlap of chromosomal coordinates for confirmation between replicates. An 80% overlap criterion was also used for some analyses, described as "more stringent." For DNA extraction, results were based on 18 replicates per sample.
- Accuracy Study:
- Initial confirmation of Agilent aberrations was against microarray-based comparator platforms (requiring 50% overlap).
- Discrepancy Resolution/Adjudication: If an Agilent CNV aberration could not be confirmed by the microarray comparator, an analytically validated method (qPCR) was employed. Additionally, 5% randomly selected "confirmed" CNV aberrations were also subjected to qPCR, and CNVs near the limit of resolution (from normal whole blood samples) were also confirmed by the third method. This represents a 2+1 (or 1+1+1) type adjudication involving multiple technical methods.
- Clinical Validity Study:
- Initial Interpretation: Interpretations (Benign, Likely Benign, VOUS, Likely Pathogenic, Pathogenic) were made by one of four cytogeneticists.
- Confirmation: All reported Pathogenic and Likely Pathogenic CNVs were subject to confirmation by alternative methods:
- Review of prior confirmation data from the sample collection site (e.g., qPCR, FISH, karyotype).
- If no prior data, qPCR assays were performed for confirmation.
- For cnLOH, no analytical confirmation was done, as clinical practice involves follow-up studies, which were beyond the study scope. This implies that for clinical validity related to cnLOH, the cytogeneticist's interpretation was the primary "ground truth" within the study's scope without further technical confirmation for the study itself.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance?
No MRMC study was performed, and this device (GenetiSure Dx Postnatal Assay) is not an AI-assisted diagnostic tool. It is a molecular diagnostic assay using microarray technology, and its software (CytoDx) performs feature extraction and aberration identification, but there is no mention of AI or machine learning algorithms, nor human reader improvement with AI assistance. The interpretation step is performed by human experts (cytogeneticists).
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done?
Yes, the analytical performance studies (Reproducibility, Precision, Accuracy, Limit of Detection, Analytical Specificity) essentially evaluate the standalone performance of the assay and its associated software (CytoDx Software) in detecting and identifying aberrations before a human cytogeneticist's interpretation. The "Confirmation Rate" and "Pairwise Replicate Agreement" metrics in these sections reflect the algorithmic performance in agreement with reference methods or across replicates.
The clinical validity study also evaluates the device's ability to identify aberrations that are subsequently interpreted by experts, but the core analytical performance measures the direct output of the assay system.
7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)
- Analytical Studies (Reproducibility, Precision, LOD, Analytical Specificity):
- Known aberrations in cell lines/reference DNA: Many studies used samples with "known aberrations" from commercial cell lines or reference panels (e.g., Coriell Institute). The nature of "known" implies a pre-established characterization, likely through previous molecular testing, expert characterization, or consensus characterization of these widely used reference materials.
- Microarray-based comparators and qPCR: For the Accuracy study, the ground truth for establishing confirmation rates was based on agreement with independent, commercially available, analytically validated microarray-based assays, and/or qPCR for discrepancy resolution.
- Clinical Validity Study:
- Clinical site historical array data: For comparison, the device's results were compared to "historical array data from the respective collection site," which were generated using internal laboratory methods.
- Expert cytogeneticist interpretation (with confirmation): The final interpretation of pathogenicity (and thus the "ground truth" for classifying samples as pathogenic/non-pathogenic within the study) was established by one of four cytogeneticists, with Pathogenic and Likely Pathogenic CNVs subject to confirmation by alternative methods (prior site data or qPCR). For cnLOH, the cytogeneticist's interpretation served as the ground truth without further technical confirmation in the study.
8. The Sample Size for the Training Set
The provided documentation does not explicitly detail a "training set" size. This is a 510(k) submission for a diagnostic assay, and the studies described are primarily analytical validation and clinical validation studies, focused on demonstrating performance metrics (accuracy, precision, reproducibility) against defined standards or comparator methods. For this type of device, the "training" (development) of the assay and its algorithms would have occurred prior to these validation studies. Information about the data used during the development phase of the CytoDx software or the assay itself (which could be considered training data for algorithm development) is not included in this document. The samples described in the performance characteristics sections are test/validation sets.
9. How the Ground Truth for the Training Set Was Established
As no specific "training set" is described for this 510(k) submission, information on how its ground truth was established is not provided. It is presumed that any internal development or training data used to optimize the assay or software algorithms would have utilized similar methods for ground truth establishment as outlined in the analytical and clinical validation sections (e.g., reference materials, comparator methods).
§ 866.5920 Postnatal chromosomal copy number variation detection system.
(a)
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.(b)
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.