(300 days)
The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia uses qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva collected from individuals 18 years and older with the AncestryDNA Saliva Collection Kit for the purpose of reporting and interpreting Genetic Health Risks (GHR).
The AncestryDNA Factor V Leiden Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent.
A user's saliva is self-collected using the AncestryDNA Saliva Collection Kit, which consists of a sealable collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to one of two Clinical Laboratory Improvement Amendments (CLIA) certified laboratories for processing.
DNA is isolated from the saliva, quantified, and tested in a multiplex assay using a customized genotyping chip and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the indication proposed herein.
The raw data is generated using Illumina GenomeStudio software, and then sent to Ancestry Genomics (the Manufacturer). The data are analyzed using the Manufacturer's proprietary GHR software, and a genotype is determined for each tested variant. The results for the Factor V Leiden variant are used to generate personalized reports for users that provide information about the disease associated with the detected variant.
Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which variant(s) has/have been detected in their sample and provide information on the risk of disease associated with the variant(s). If no variant was detected, that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results.
Here is a summary of the acceptance criteria and the study that proves the device meets the acceptance criteria, based on the provided text:
Acceptance Criteria and Reported Device Performance
| Criteria | Acceptance Criterion | Reported Device Performance |
|---|---|---|
| Reproducibility/Precision (Overall) | >99% point estimate Overall Percent Agreement (OPA) | 100.00% (99.84 - 100.00) for all operator teams combined; 100.00% (99.84 - 100.00) for all instrument combinations combined; 100.00% (99.84 - 100.00) for all reagent lot combinations combined. |
| Reproducibility/Precision (Per Genotype) | ≥99% agreement for each genotype | 100.00% (99.53 - 100.00) for GG; 100.00% (99.53 - 100.00) for GA; 100.00% (99.53 - 100.00) for AA. |
| Analytical Sensitivity (LoD) | Lowest DNA concentration at which ≥95% of samples yield correct call | LoD = 1.53 ng/uL at which all genotyping attempts on samples with call rates ≥ 98% produced concordant genotypes. |
| Analytical Sensitivity (LoB) | Limit of Blank | LoB = 1.004 ng/uL |
| Analytical Sensitivity (Upper Limit) | Maximum DNA concentration for valid results | 50 ng/uL |
| Interfering Substances (Endogenous) | ≥95% agreement with true variant status for all controls and endogenous substances | 100% agreement for PBS (control), Salivary α-amylase, Hemoglobin, IgA, and Total Protein (30/30 replicates for each). |
| Interfering Substances (Exogenous) | ≥95% agreement with true variant status for all samples passing QC | 100% agreement for all tested exogenous substances (Chicken, Alcohol, Mouthwash, Beef, Gum, Smoking) at T0 and T30 time points. |
| Interfering Substances (Microbial) | >95% agreement with true variant status | 100% agreement for all five microbial agents (S. epidermis, S. mutans, L. casei, A. odontolyticus, C. albicans) at both low/normal and high concentrations (36/36 replicates for each). |
| Accuracy / Method Comparison | ≥99% agreement with true variant determination overall and per genotype | 100% overall agreement (198/198) between AncestryDNA Factor V Leiden GHR Test and bi-directional sequencing. 100% agreement for GG (69/69), GA (65/65), and AA (64/64). |
| User Comprehension (Study 1) | ≥90% comprehension score for each domain and overall | Overall comprehension rate was 93.2%. Individual domain comprehension rates ranged from 83.7% to 100%. (e.g., Appropriate Follow-Up Action: 97.4%, Ethnic Relevance: 95.5%, Other Risk Factors: 92.1%, Limitations of Test: 91.5%, Purpose of Test: 92.7%, Results of Test: 90.7%). Note that "Results of Test" had one category (1 Variant) at 83.7% which is below 90% individually but the overall for that domain was 90.7%. |
| User Comprehension (Study 2) | ≥90% comprehension score for each domain and overall | Overall comprehension rate was 96%. Individual domain comprehension rates ranged from 88.8% to 99.1%. (e.g., Appropriate Follow-Up Action: 96.5%, Ethnic Relevance: 97.7%, Other Risk Factors: 95.8%, Limitations of Test: 96.2%, Purpose of Test: 99.1%, Results of Test: 90.9%). Note that "Results of Test" had one category (1 Variant) at 88.8% which is below 90% individually but the overall for that domain was 90.9%. |
Study Details
2. Sample Size and Data Provenance (Test Set):
- Reproducibility/Precision:
- Total replicates: 2,340 genotyping events (combining all replicates from Lab 1, Lab 2, inter-laboratory, by site/operator team, by site/instrument, and by site/reagent lot).
- Donors: 9 donors with known Factor V Leiden genotypes (3 homozygous common, 3 heterozygous, 3 homozygous rare).
- Provenance: Samples were collected using AncestryDNA Saliva Collection Kits (SCKs) and tested at two CLIA-certified laboratories (Lab 1 and Lab 2). The study was prospective in nature for data collection.
- Analytical Sensitivity:
- Saliva Study: 15 donors (5 homozygous common, 5 heterozygous, 5 homozygous rare). 450 replicates tested.
- Cell Line Study: 4 cell lines. 216 data points per cell line for dilutions, and 855 blank replicates.
- Provenance: Saliva samples collected using Oragene® Dx Collection Device and AncestryDNA SCK. Cell lines. Data collected prospectively for the study.
- Interfering Substances (Endogenous):
- Donors: 10 saliva donors. 450 initial genotyping attempts (3 replicates for each of 10 donors per interferent + 30 control replicates).
- Provenance: Saliva samples collected using Oragene® Dx Collection Device. Data collected prospectively for the study.
- Interfering Substances (Exogenous):
- Donors: 10 non-smokers and 10 smokers. 594 data points.
- Provenance: Saliva samples collected using AncestryDNA SCKs. Data collected prospectively for the study.
- Interfering Substances (Microbial):
- Cell Lines: 6 human cell lines (4 homozygous common, 1 heterozygous, 1 homozygous rare). 432 genotyping results.
- Provenance: Human cell lines. Data collected prospectively for the study.
- Accuracy / Method Comparison:
- Donors: 209 donors with known Factor V Leiden genotypes (73 homozygous common, 69 heterozygous, 67 homozygous rare). 198 samples passed QC.
- Provenance: Saliva samples collected using Oragene Dx Ogd-500.001 (OGD) and AncestryDNA SCK. Data collected prospectively for comparison.
- User Comprehension (Study 1):
- Participants: 378 individuals (N=96, N=86, N=106, N=90 for each of four study arms).
- Provenance: Participants were recruited from the U.S. matching demographics for education, age, sex/gender, and race/ethnicity, and geographic diversity across four U.S. Census regions. Performed in-person. Prospective study.
- User Comprehension (Study 2):
- Participants: 213 individuals (N=103 for "1 Variant", N=110 for "Result Not Determined").
- Provenance: Participants were recruited from the U.S., matching demographics and geographic diversity as in Study 1. Performed via live televideo interviews. Prospective study.
3. Number of Experts and Qualifications for Ground Truth (Test Set):
For analytical studies (Reproducibility, Analytical Sensitivity, Interfering Substances, Accuracy/Method Comparison), the ground truth for Factor V Leiden genotype was primarily established using bi-directional sequencing analysis at a third-party laboratory. The specific qualifications of the experts performing the bi-directional sequencing were not detailed in the provided text.
For User Comprehension Studies, the materials were reviewed by a Certified Genetic Counselor to confirm that the materials met specific criteria for explaining test concepts.
4. Adjudication Method (Test Set):
The text does not explicitly describe an adjudication method like 2+1 or 3+1 for the analytical studies. Instead, direct comparison was made between the device's genotype calls and the ground truth established by bi-directional sequencing. Any discrepancies would presumably be investigated, though the method is not detailed. For User Comprehension studies, a questionnaire was administered by a trained interviewer/moderator to assess comprehension.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not performed. This device is a genetic health risk test (an in vitro molecular diagnostic system), not an imaging device typically associated with MRMC studies in the context of human reader performance improvements with AI.
6. Standalone Performance:
Yes, standalone performance (algorithm only) was done. The studies described (reproducibility, analytical sensitivity, interfering substances, and accuracy/method comparison) evaluate the performance of the device itself (AncestryDNA Factor V Leiden GHR Test) in determining genotype status, independent of human interpretation of raw data. The "AncestryDNA GHR software" processes data from the instrumentation and generates the final analytical genotype information for each sample, which is then used to generate personalized reports.
7. Type of Ground Truth Used (Test Set):
The primary ground truth used for determining the Factor V Leiden genotype in analytical studies was bi-directional sequencing analysis.
8. Sample Size for the Training Set:
The document does not explicitly specify a "training set" sample size for the AncestryDNA Factor V Leiden Genetic Health Risk Test in the context of its 510(k) submission. For molecular diagnostic systems like this, the "training" aspect often refers to the development and optimization of the assay and software algorithms. The document instead focuses on analytical validation studies (test sets) for device performance. If the AncestryDNA GHR software performs certain control checks and analyses (as described in section "P. SYSTEM DESCRIPTION" - "Software"), the underlying algorithms would have been developed and potentially "trained" or optimized during the device's development phase, but specific training set sizes are not provided.
9. How the Ground Truth for the Training Set was Established:
Since a specific training set sample size is not explicitly mentioned for the reported studies, the method for establishing ground truth for a training set is also not detailed. However, for any developmental work or optimization, it is highly probable that the ground truth would have been established using bi-directional sequencing or other highly accurate, established reference methods, similar to how the ground truth for the test sets was established.
§ 866.5950 Genetic health risk assessment system.
(a)
Identification. A genetic health risk assessment system is a qualitative in vitro molecular diagnostic system used for detecting variants in genomic deoxyribonucleic acid (DNA) isolated from human specimens that will provide information to users about their genetic risk of developing a disease to inform lifestyle choices and/or conversations with a health care professional. This assessment system is for over-the-counter use. This device does not determine the person's overall risk of developing a disease.(b)
Classification. Class II (special controls). The genetic health risk assessment system device, when it has previously received a first-time FDA marketing authorization (e.g., 510(k) clearance) for the genetic health risk assessment system (a “one-time FDA reviewed genetic health risk assessment system”), is exempt from the premarket notification procedures in part 807, subpart E, of this chapter subject to the limitations in § 866.9. The device must comply with the following special controls:(1) The 21 CFR 809.10 compliant labeling and any prepurchase page and test report generated, unless otherwise specified, must include:
(i) A section addressed to users with the following information:
(A) The limiting statement explaining that this test provides genetic risk information based on assessment of specific genetic variants but does not report on a user's entire genetic profile. This test [does not/may not, as appropriate] detect all genetic variants related to a given disease, and the absence of a variant tested does not rule out the presence of other genetic variants that may be related to the disease.
(B) The limiting statement explaining that other companies offering a genetic risk test may be detecting different genetic variants for the same disease, so the user may get different results using a test from a different company.
(C) The limiting statement explaining that other factors such as environmental and lifestyle risk factors may affect the risk of developing a given disease.
(D) The limiting statement explaining that some people may feel anxious about getting genetic test health results. This is normal. If the potential user feels very anxious, such user should speak to his or her doctor or other health care professional prior to collection of a sample for testing. This test is not a substitute for visits to a doctor or other health care professional. Users should consult with their doctor or other health care professional if they have any questions or concerns about the results of their test or their current state of health.
(E) Information about how to obtain access to a genetic counselor, board-certified clinical molecular geneticist, or equivalent health care professional about the results of a user's test.
(F) The limiting statement explaining that this test is not intended to diagnose a disease, tell you anything about your current state of health, or be used to make medical decisions, including whether or not you should take a medication or how much of a medication you should take.
(G) A limiting statement explaining that the laboratory may not be able to process a sample, and a description of the next steps to be taken by the manufacturer and/or the customer, as applicable.
(ii) A section in your 21 CFR 809.10 labeling and any test report generated that is for health care professionals who may receive the test results from their patients with the following information:
(A) The limiting statement explaining that this test is not intended to diagnose a disease, determine medical treatment, or tell the user anything about their current state of health.
(B) The limiting statement explaining that this test is intended to provide users with their genetic information to inform lifestyle decisions and conversations with their doctor or other health care professional.
(C) The limiting statement explaining that any diagnostic or treatment decisions should be based on testing and/or other information that you determine to be appropriate for your patient.
(2) The genetic test must use a sample collection device that is FDA-cleared, -approved, or -classified as 510(k) exempt, with an indication for in vitro diagnostic use in over-the-counter DNA testing.
(3) The device's labeling must include a hyperlink to the manufacturer's public Web site where the manufacturer shall make the information identified in paragraph (b)(3) of this section publicly available. The manufacturer's home page, as well as the primary part of the manufacturer's Web site that discusses the device, must provide a hyperlink to the Web page containing this information and must allow unrestricted viewing access. If the device can be purchased from the Web site or testing using the device can be ordered from the Web site, the same information must be found on the Web page for ordering the device or provided in a publicly accessible hyperlink on the Web page for ordering the device. Any changes to the device that could significantly affect safety or effectiveness would require new data or information in support of such changes, which would also have to be posted on the manufacturer's Web site. The information must include:
(i) An index of the material being provided to meet the requirements in paragraph (b)(3) of this section and its location.
(ii) A section that highlights summary information that allows the user to understand how the test works and how to interpret the results of the test. This section must, at a minimum, be written in plain language understandable to a lay user and include:
(A) Consistent explanations of the risk of disease associated with all variants included in the test. If there are different categories of risk, the manufacturer must provide literature references that support the different risk categories. If there will be multiple test reports and multiple variants, the risk categories must be defined similarly among them. For example, “increased risk” must be defined similarly between different test reports and different variant combinations.
(B) Clear context for the user to understand the context in which the cited clinical performance data support the risk reported. This includes, but is not limited to, any risks that are influenced by ethnicity, age, gender, environment, and lifestyle choices.
(C) Materials that explain the main concepts and terminology used in the test that include:
(
1 )Definitions: Scientific terms that are used in the test reports.(
2 )Prepurchase page: This page must contain information that informs the user about what information the test will provide. This includes, but is not limited to, variant information, the condition or disease associated with the variant(s), professional guideline recommendations for general genetic risk testing, the limitations associated with the test (e.g., test does not detect all variants related to the disease) and any precautionary information about the test the user should be aware of before purchase. When the test reports the risk of a life-threatening or irreversibly debilitating disease or condition for which there are few or no options to prevent, treat, or cure the disease, a user opt-in section must be provided. This opt-in page must be provided for each disease that falls into this category and must provide specific information relevant to each test result. The opt-in page must include:(
i ) An option to accept or decline to receive this specific test result;(
ii ) Specification of the risk involved if the user is found to have the specific genetic test result;(
iii ) Professional guidelines that recommend when genetic testing for the associated target condition is or is not recommended; and(
iv ) A recommendation to speak with a health care professional, genetic counselor, or equivalent professional before getting the results of the test.(
3 )Frequently asked questions (FAQ) page: This page must provide information that is specific for each variant/disease pair that is reported. Information provided in this section must be scientifically valid and supported by corresponding publications. The FAQ page must explain the health condition/disease being tested, the purpose of the test, the information the test will and will not provide, the relevance of race and ethnicity to the test results, information about the population to which the variants in the test is most applicable, the meaning of the result(s), other risk factors that contribute to disease, appropriate followup procedures, how the results of the test may affect the user's family, including children, and links to resources that provide additional information.(iii) A technical information section containing the following information:
(A) Gene(s) and variant(s) the test detects using standardized nomenclature, Human Genome Organization nomenclature and coordinates as well as Single Nucleotide Polymorphism Database (dbSNP) reference SNP numbers (rs#).
(B) Scientifically established disease-risk association of each variant detected and reported by the test. This risk association information must include:
(
1 ) Genotype-phenotype information for the reported variants.(
2 ) Table of expected frequency and risks of developing the disease in relevant ethnic populations and the general population.(
3 ) A statement about the current professional guidelines for testing these specific gene(s) and variant(s).(
i ) If professional guidelines are available, provide the recommendations in the professional guideline for the gene, variant, and disease, for when genetic testing should or should not be performed, and cautionary information that should be communicated when a particular gene and variant is detected.(
ii ) If professional guidelines are not available, provide a statement that the professional guidelines are not available for these specific gene(s) and variant(s).(C) The specimen type (
e.g., saliva, capillary whole blood).(D) Assay steps and technology used.
(E) Specification of required ancillary reagents, instrumentation, and equipment.
(F) Specification of the specimen collection, processing, storage, and preparation methods.
(G) Specification of risk mitigation elements and description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
(H) Information pertaining to the probability of test failure (
i.e., percentage of tests that failed quality control) based on data from clinical samples, a description of scenarios in which a test can fail (i.e., low sample volume, low DNA concentration, etc.), how users will be notified of a test failure, and the nature of followup actions on a failed test to be taken by the user and the manufacturer.(I) Specification of the criteria for test result interpretation and reporting.
(J) Information that demonstrates the performance characteristics of the test, including:
(
1 ) Accuracy of study results for each claimed specimen type.(
i ) Accuracy of the test shall be evaluated with fresh clinical specimens collected and processed in a manner consistent with the test's instructions for use. If this is impractical, fresh clinical samples may be substituted or supplemented with archived clinical samples. Archived samples shall have been collected previously in accordance with the instructions for use, stored appropriately, and randomly selected. In some limited circumstances, use of contrived samples or human cell line samples may also be appropriate and used as an acceptable alternative. The contrived or human cell line samples shall mimic clinical specimens as much as is feasible and provide an unbiased evaluation of the device accuracy.(
ii ) Accuracy must be evaluated by comparison to bidirectional Sanger sequencing or other methods identified as appropriate by FDA. Performance criteria for both the comparator method and the device must be predefined and appropriate to the device's intended use. Detailed study protocols must be provided.(
iii ) Test specimens must include all genotypes that will be included in the tests and reports. The number of samples tested in the accuracy study for each variant reported must be based on the variant frequency using either the minimum numbers of samples identified in this paragraph or, when determined appropriate and identified by FDA, a minimum number of samples determined using an alternative method. When appropriate, the same samples may be used in testing to demonstrate the accuracy of testing for multiple genotypes by generating sequence information at multiple relevant genetic locations. At least 20 unique samples representing the wild-type genotype must be tested. To test samples that are heterozygous for the reported variant(s), common variants (>0.1 percent variant frequency in the relevant population) must be tested with at least 20 unique samples. Rare variants (≤0.1 percent variant frequency in the relevant population) must be tested with at least three unique samples. To test samples that are homozygous for the reported variant(s), variants with ≥2 percent variant frequency in a relevant population must be tested with at least 20 unique samples. Variants with a frequency in the relevant population