AncestryDNA Factor V Leiden Genetic Health Risk Test

K192944 · Ancestry Genomics, Inc. · PTA · Aug 13, 2020 · Immunology

Device Facts

• Record ID: K192944
• Device Name: AncestryDNA Factor V Leiden Genetic Health Risk Test
• Applicant: Ancestry Genomics, Inc.
• Product Code: PTA · Immunology
• Decision Date: Aug 13, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5950
• Device Class: Class 2

Indications for Use

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 Test 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.

Device Story

Device uses saliva samples collected via AncestryDNA Saliva Collection Kit; shipped to CLIA-certified labs. DNA isolated, quantified, and tested via multiplex genotyping assay on Illumina Infinium platform (Tecan Evo, Illumina iScan). GenomeStudio software generates raw genotype data; Ancestry Genomics' proprietary GHR software performs quality control and determines genotype for Factor V Leiden variant. Results provided to users via personalized reports explaining disease risk associated with detected variant. Used by consumers (OTC) to understand genetic health risks; intended for informational purposes, not medical decision-making. Benefits include providing users with genetic risk information for hereditary thrombophilia, encouraging consultation with healthcare providers.

Clinical Evidence

Clinical performance supported by meta-analysis (Simone et al., 2013) establishing odds ratios for Factor V Leiden variants and post-test risk calculations. User comprehension studies (n=378 and n=213) demonstrated high comprehension rates (90.7%–99.1%) for test purpose, limitations, and follow-up actions. Analytical accuracy study (n=209) showed 100% agreement with bi-directional sequencing for all genotypes. Precision studies across two labs, multiple operators, and instruments showed >99% reproducibility.

Technological Characteristics

Qualitative in vitro molecular diagnostic system. Uses Illumina Infinium HumanOmniExpress-24 BeadChip assay. DNA isolated from saliva; quantified; fragmented; hybridized to variant-specific primers; extended with hapten-labeled nucleotides; detected via fluorescently labeled antibodies. Instrumentation: Tecan Evo (extraction/processing), Illumina iScan (scanning/quantification). Software: GenomeStudio (genotype calling), proprietary GHR software (data analysis).

Indications for Use

Indicated for individuals 18+ years old to detect Factor V Leiden variant in F5 gene. Relevant for European descent. Not a diagnostic for specific health conditions; not a substitute for healthcare provider visits. Does not detect all hereditary thrombophilia variants.

Regulatory Classification

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.

Special Controls

*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 <2 percent and ≥0.5 percent must be tested with at least 10 unique samples. Variants with a frequency in the relevant population <0.5 percent must be tested with at least three unique samples. If variants with a frequency of <0.5 percent are not found within the relevant population and homozygous samples are not tested, then the test results for this homozygous rare variant must not be reported to the user.( *iv* ) Information about the accuracy study shall include the number and type of samples that were compared to bidirectional Sanger sequencing or other methods identified as appropriate by FDA. This information must either be reported in tabular format and arranged by clinically relevant variants or reported using another method identified as appropriate by FDA. As an example, for samples with different genotypes DD, Dd, and dd, the following table represents data from the accuracy study presented in tabular format: ( *v* ) The accuracy represents the degrees of agreement between the device results and the comparator results. The accuracy must be evaluated by measuring different percent agreements (PA) of device results with the comparator results and percent of 'no calls' or 'invalid calls.' Calculate the rate of 'no calls' and 'invalid calls' for each comparator output as %Inv(DD) = A4 /NDD, %Inv(Dd) = B4 /NDd , %Inv(dd) = C4 /Ndd . If 'no calls' or 'invalid calls' are required to be retested according to the device instructions for use, the percent of final 'no calls' or 'invalid calls' must be provided. In the table presenting the results of the accuracy study, use only the final results (*i.e.,* after retesting the initial 'no calls' or 'invalid calls', if required according to the instructions for use). Samples that resulted in a 'no call' or 'invalid call' after retesting must not be included in the final calculations of agreement. If the percentages of 'no calls' or 'invalid calls' for each comparator output are similar, combine these estimates as (A4 + B4 + C4 )/(NDD + NDd + Ndd ) and provide a 95 percent two-sided confidence interval. The percent of final 'no calls' or 'invalid calls' must be clinically acceptable.( *vi* ) Point estimates of percent agreement for each genotype must be calculated as the number of correct calls for that genotype divided by the number of samples known to contain that genotype excluding 'no calls' or 'invalid calls'. The calculations must be performed as follows: ( *vii* ) For percent agreements for DD, Dd and dd (PA(DD|DD), PA(Dd|Dd) and PA(dd|dd)) as described in paragraph (b)(3)(iii)(J)(*1* )(*vi* ) of this section, the 95 percent two-sided confidence intervals must be provided. The accuracy point estimates for percent agreements for DD, Dd and dd must be ≥99 percent per reported variant and overall. Any variants that have a point estimate for either PA(DD|DD), PA(Dd|Dd), or PA(dd|dd) of <99 percent compared to bidirectional sequencing or other methods identified as appropriate by FDA must not be incorporated into test claims and reports. Accuracy results generated from clinical specimens versus contrived samples or cell lines must be presented separately. Results must be summarized and presented in tabular format by sample type and by genotype or must be reported using another method identified as appropriate by FDA (see paragraph (b)(3)(iii)(J)(*1* )(*iv* ) of this section).( *viii* ) Information must be reported on the Technical Positive Predictive Value (TPPV) related to the analytical (technical) performance of the device for genotypes in each relevant subpopulation (*e.g.,* ethnicity, gender, age, geographical location, etc.). TPPV is the percentage of individuals with the genotype truly present among individuals whose test reports indicate that this genotype is present. The TPPV depends on the accuracy measures of percent agreements and on the frequency of the genotypes in the subpopulation being studied. The f(DD) is the frequency of DD and f(Dd) is the frequency of Dd in the subpopulation being studied; TPPV must be calculated as described in paragraphs (b)(3)(iii)(J)(*1* )(*ix* ) through (*xi* ) of this section.( *ix* ) For variants where the point estimates of PA(DD|DD), PA(Dd|Dd) and PA(dd|dd) are less than 100 percent, use these point estimates in TPPV calculations.( *x* ) Point estimates of 100 percent in the accuracy study may have high uncertainty about performance of the test in the population. If these variants are measured using highly multiplexed technology, calculate the random error rate for the overall device. The accuracy study described in paragraph (b)(3)(iii)(J) of this section in those cases is more to determine that there is no systematic error in such devices. In those cases, incorporate that rate in the estimation of the percent agreements as calculated in paragraph (b)(3)(iii)(J)(*1* )(*vi* ) of this section and include it in TPPV calculations.( *xi* ) The TPPV for subpopulations with genotype frequencies of f(dd), f(Dd) and f(DD) = 1−f(dd)−f(Dd) in the subpopulation is calculated as: ( *2* ) Precision and reproducibility data must be provided using multiple instruments and multiple operators, on multiple non-consecutive days, and using multiple reagent lots. The sample panel must either include specimens from the claimed sample type (*e.g.,* saliva) representing all genotypes for each variant (*e.g.,* wild type, heterozygous, and homozygous) or, if an alternative panel composition of specimens is identified by FDA as appropriate, a panel composed of those specimens FDA identified as appropriate. A detailed study protocol must be created in advance of the study and must include predetermined acceptance criteria for performance results. The percentage of samples that failed quality control must be indicated (*i.e.,* the total number of sample replicates for which a sequence variant cannot be called (no calls) or that fail sequencing quality control criteria divided by the total number of replicates tested). It must be clearly documented whether results were generated from clinical specimens, contrived samples, or cell lines. The study results shall report the variants tested in the study and the number of replicates for each variant, and what conditions were tested (*i.e.,* number of runs, days, instruments, reagent lots, operators, specimens/type, etc.). Results must be evaluated and presented in tabular format and stratified by study parameter (*e.g.,* by site, instrument(s), reagent lot, operator, and sample variant). The study must include all extraction steps from the claimed specimen type or matrix, unless a separate extraction reproducibility study for the claimed sample type is performed. If the device is to be used at more than one laboratory, different laboratories must be included in the reproducibility study and reproducibility across sites must be evaluated. Any no calls or invalid calls in the study must be listed as a part of the precision and reproducibility study results.( *3* )*Analytical specificity data:* Data must be provided that evaluates the effect of potential endogenous and exogenous interferents on test performance, including specimen extraction and variant detection. Interferents tested must include those reasonably likely to be potentially relevant to the sample type used for the device.( *4* )*Interfering variant data:* Nucleotide mutations that can interfere with the technology must be cited and evaluated. Data must be provided to demonstrate the effect of the interfering variant(s) on the performance of the correct calls. Alternatively, for each suspected interfering mutation for which data is not provided demonstrating the effect of the interfering variant, the manufacturer must identify the suspected interfering variants in the labeling and indicate that the impact that the interfering variants may have on the assay's performance has not been studied by providing a statement that reads “It is possible that the presence of [insert clearly identifying information for the suspected interfering variant] in a sample may interfere with the performance of this test. However, its effect on the performance of this test has not been studied.”( *5* )*Analytical sensitivity data:* Data must be provided demonstrating the minimum amount of DNA that will enable the test to perform correctly in 95 percent of runs.( *6* )*Reagent stability:* The manufacturer must evaluate reagent stability using wild-type, heterozygous, and homozygous samples. Reagent stability data must demonstrate that the reagents maintain the claimed accuracy and reproducibility. Data supporting such claims must be provided.( *7* )*Specimen type and matrix comparison data:* Specimen type and matrix comparison data must be generated if more than one specimen type can be tested with this device, including failure rates for the different specimens.(K) Clinical performance summary. ( *1* ) Information to support the clinical performance of each variant reported by the test must be provided.( *2* ) Manufacturers must organize information by the specific variant combination as appropriate (*e.g.,* wild type, heterozygous, homozygous, compound heterozygous, hemizygous genotypes). For each variant combination, information must be provided in the clinical performance section to support clinical performance for the risk category (*e.g.,* not at risk, increased risk). For each variant combination, a summary of key results must be provided in tabular format or using another method identified as appropriate by FDA to include the appropriate information regarding variant type, data source, definition of the target condition (*e.g.,* disease), clinical criteria for determining whether the target disease is present or absent, description of subjects with the target disease present and target disease absent (exclusion or inclusion criteria), and technical method for genotyping. When available, information on the effect of the variant on risk must be provided as the risk of a disease (lifetime risk or lifetime incidences) for an individual compared with the general population risk.( *i* ) If odds ratios are available, using information about the genotype distribution either among individuals with the target disease absent, or in the general population, or information about the risk variant frequency and odds ratios, the likelihood ratios for the corresponding device results along with 95 percent confidence intervals must be calculated. Using information about pretest risk (π), an estimate of likelihood ratio (LR), and a relationship between post-test risk R as R/(1−R) = LR·π/(1−π), the post-test risk R must be calculated.( *ii* ) When available, likelihood ratios (LR) for different test results must be presented in a tabular format along with references to the source data or using another method identified as appropriate by FDA as stated in paragraph (b)(3)(iii)(K)(2) of this section. When these values are not directly available in published literature, likelihood ratios can be separately calculated along with the 95 percent confidence interval with references to the source data. Note that a minimum requirement for the presence of the variant's effect on the risk is that a corresponding LR is statistically higher than 1 (a lower bound of 95 percent two-sided confidence interval is larger than 1). It means that the post-test risk is statistically higher than the pretest risk (an observed value of the difference between the post-test and pretest risks).(L) Materials that explain the main concepts and terminology used in the test that includes, but is not limited to: ( *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 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 on the test results, information about the population to which the variants in the test is most applicable, the meaning of the result(s), other risks 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.(M) User comprehension study: Information on a study that assesses comprehension of the test process and results by potential users of the test must be provided. ( *1* ) The test manufacturer must provide a genetic risk education module to naïve user comprehension study participants prior to their participation in the user comprehension study. The module must define terms that are used in the test reports and explain the significance of genetic risk reports.( *2* ) The test manufacturer must perform pre- and post-test user comprehension studies. The comprehension test questions must include directly evaluating a representative sample of the material being presented to the user as described in paragraph (b)(3)(ii) of this section.( *3* ) The manufacturer must provide a justification from a physician and/or genetic counselor that identifies the appropriate general and variant-specific concepts contained within the material being tested in the user comprehension study to ensure that all relevant concepts are incorporated in the study.( *4* ) The user study must meet the following criteria:( *i* ) The study participants must comprise a statistically sufficient sample size and demographically diverse population (determined using methods such as quota-based sampling) that is representative of the intended user population. Furthermore, the study participants must comprise a diverse range of age and educational levels and have no prior experience with the test or its manufacturer. These factors shall be well defined in the inclusion and exclusion criteria.( *ii* ) All sources of bias must be predefined and accounted for in the study results with regard to both responders and non-responders.( *iii* ) The testing must follow a format where users have limited time to complete the studies (such as an onsite survey format and a one-time visit with a cap on the maximum amount of time that a participant has to complete the tests).( *iv* ) Users must be randomly assigned to study arms. Test reports in the user comprehension study given to users must define the target condition being tested and related symptoms, explain the intended use and limitations of the test, explain the relevant ethnicities in regard to the variant tested, explain genetic health risks and relevance to the user's ethnicity, and assess participants' ability to understand the following comprehension concepts: The test's limitations, purpose, appropriate action, test results, and other factors that may have an impact on the test results.( *v* ) Study participants must be untrained, be naïve to the test subject of the study, and be provided the labeling prior to the start of the user comprehension study.( *vi* ) The user comprehension study must meet the predefined primary endpoint criteria, including a minimum of a 90 percent or greater overall comprehension rate (*i.e.,* selection of the correct answer) for each comprehension concept. Other acceptance criteria may be acceptable depending on the concept being tested. Meeting or exceeding this overall comprehension rate demonstrates that the materials presented to the user are adequate for over-the-counter use.( *vii* ) The analysis of the user comprehension results must include results regarding reports that are provided for each gene/variant/ethnicity tested, statistical methods used to analyze all data sets, and completion rate, non-responder rate, and reasons for nonresponse/data exclusion. A summary table of comprehension rates regarding comprehension concepts (*e.g.,* purpose of test, test results, test limitations, ethnicity relevance for the test results, etc.) for each study report must be included.(4) The intended use of the device must not include the following indications for use: (i) Prenatal testing; (ii) Determining predisposition for cancer where the result of the test may lead to prophylactic screening, confirmatory procedures, or treatments that may incur morbidity or mortality to the patient; (iii) Assessing the presence of genetic variants that impact the metabolism, exposure, response, risk of adverse events, dosing, or mechanisms of prescription or over-the-counter medications; or (iv) Assessing the presence of deterministic autosomal dominant variants.

Predicate Devices

• 23andMe Personal Genome Service Test for Hereditary Thrombophilia (DEN160026)

Submission Summary (Full Text)

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FDA

U.S. FOOD &amp; DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I Background Information:

A 510(k) Number

K192944

B Applicant

Ancestry Genomics, Inc.

C Proprietary and Established Names

AncestryDNA Factor V Leiden Genetic Health Risk Test

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PTA | Class II | 21 CFR 866.5950 - Genetic Health Risk Assessment System | IM - Immunology |

## II Submission/Device Overview:

A Purpose for Submission:

New Device

B Measurand:

Factor V Leiden c.1601G&gt;A variant in the F5 gene from a human saliva sample

C Type of Test:

The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia determines and interprets if a person has variants associated with a higher risk of developing harmful blood clots. The report is based on a qualitative genetic test for a single nucleotide polymorphism detection of Factor V Leiden variant in the F5 gene (rs6025).

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

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 Test 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.

C Special Conditions for Use Statement(s):

OTC - Over the Counter

This test is not a substitute for visits to a healthcare provider. It is recommended that you consult with a healthcare provider if you have any questions or concerns about your results or your current state of health.

The AncestryDNA Factor V Leiden Genetic Health Risk Test does not detect all genetic variants associated with Hereditary Thrombophilia. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related.

The test is intended for users ≥ 18 years old.

The test does not diagnose any specific health conditions. Results should not be used to make medical decisions.

The laboratory may not be able to process a user's sample. The probability that the laboratory cannot process a sample can be up to 3%.

A user's race, ethnicity, age, and other life style factors may affect how the genetic test results are interpreted.

Subject to meeting the limitations contained in the special controls under regulation 21 CFR 866.5950.

D Special Instrument Requirements:

K192944 - Page 2 of 17

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The AncestryDNA Factor V Leiden Genetic Health Risk Test is to be performed using the Tecan Evo and Illumina iScan instruments.

GenomeStudio Software is a modular software application that is used to view and analyze genotypic data obtained from the Illumina iScan System and based on the cluster definition file. AncestryDNA Factor V Leiden Genetic Health Risk Test software conducts a variety of control checks on the file, resulting in a final genotype profile for each sample. These data are used to generate test reports on a user's genotype and associated risk of disease.

## IV Device/System Characteristics:

### A Device Description:

The AncestryDNA Genetic Health Risk (GHR) Test for Factor V Leiden is intended for the detection of single nucleotide polymorphism (SNP) in the F5 gene associated with Hereditary Thrombophilia from human saliva collected in the AncestryDNA Saliva Collection Kit (SCK).

The user's saliva is self-collected using the AncestryDNA SCK, which consists of a single-use 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, testing and analysis. DNA is extracted, fragmented and tested with the AncestryDNA Factor V Leiden GHR Test, 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 the Illumina GenomeStudio software and then delivered to Ancestry Genomics for analysis using the proprietary AncestryDNA GHR Software. A genotype is determined for each tested variant. The results for the Factor V Leiden variant are used to generate personalized reports for users which 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 scientific 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.

### B Principle of Operation:

The AncestryDNA Factor V Leiden GHR Test is performed by CLIA-certified laboratories using the BeadChip v10 assay (Illumina Infinium HumanOmniExpress-24 format chip) on the Illumina Infinium platform. Samples collected using the AncestryDNA Saliva Collection Kit are delivered to laboratories for testing and analysis. DNA from saliva is extracted and quantified. Samples

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with DNA concentrations in the range of 1.53 ng/μL – 50 ng/μL are eligible for further processing. These samples are fragmented and captured on a bead array by hybridization to immobilized variant-specific primers, followed by extension with hapten-labeled nucleotides. The primers hybridize adjacent to the variants and are extended with a single nucleotide corresponding to the variant allele. The incorporated hapten-modified nucleotides are detected by adding fluorescently labeled antibodies in several steps to amplify the signals. The Tecan Evo and Illumina iScan instruments are used for extraction and processing of the DNA, and the BeadChip for scanning and quantification of the results. Genotypes are determined using the GenomeStudio software package and delivered to Ancestry Genomics for analysis using the AncestryDNA GHR software.

C Instrument Description Information:

| Modes of Operation | Yes | No |
| --- | --- | --- |
| Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device? | ☑ | ☐ |
| Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission? | ☑ | ☐ |
| Software | | |
| FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types. | ☑ | ☐ |

1. Instrument Name:

Tecan Evo, Illumina iScan BeadChip scanner with GenomeStudio software

2. Specimen Identification:

Consumers must register their saliva collection kit, linking their saliva sample to a secure online account with a valid email address through a unique activation code, in order to use the test. The activation code is matched to records of kits shipped to consumers to ensure it is a valid kit. A timestamp of the user completing the entries to activate the kit is recorded.

3. Specimen Sampling and Handling:

Samples are collected using the AncestryDNA Saliva Collection Kit and delivered to one of two CLIA-certified laboratories for testing and analysis. The recommended volume of saliva is 1 mL. Saliva is collected directly by the user spitting into the provided saliva collection tube via the pre-installed funnel. After providing saliva, the user is instructed to remove the funnel and screw on tightly the provided cap. Affixing the cap by screwing on releases the stabilization solution. The saliva sample can be immediately processed, transported, or stored for future use. Device and sample integrity are preserved during typical ambient transport and storage conditions for up to 12 months.

4. Calibration:

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Calibration and calibration verification procedures are established to demonstrate continued accuracy of the test systems.

## 5. Quality Control:

The AncestryDNA Factor V Leiden Genetic Health Risk Test uses one control material, which serves as both the sample processing control and the reproducibility control. The control material is genotyped on the Illumina BeadChip according to routine standard procedures at the laboratory. Each new lot of the control is tested by comparison with reference BeadChip genotype results.

The sample processing control is run on every sample genotyping plate and the reproducibility control is run approximately once per week.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

23andMe Personal Genome Service Test for Hereditary Thrombophilia

### B Predicate 510(k) Number(s):

DEN160026

### C Comparison with Predicate(s):

| Device & Predicate Device(s): | K192944 | DEN160026 |
| --- | --- | --- |
| Device Trade Name | AncestryDNA Factor V Leiden Genetic Health Risk Test | 23andMe Personal Genome Service (PGS) Test |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | 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 23andMe Personal Genome Service (PGS) Test uses qualitative genotyping to detect the following clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥ 18 years with the Oragene Dx model OGD-500.001 for the purpose of reporting and interpreting Genetic Health Risks (GHR):
The 23andMe PGS Genetic |

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| | The AncestryDNA Factor V Leiden Genetic Health Risk Test 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. | Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F gene, and the Prothrombin G20210A variant in the F2 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood blots, 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. |
| --- | --- | --- |
| Special Instrument Requirements | The AncestryDNA Factor V Leiden Genetic Health Risk Test is to be performed using the Tecan Evo and Illumina iScan instruments.
GenomeStudio Software is a modular software application that is used to view and analyze genotypic data obtained from the Illumina iScan System and based on the cluster definition file. AncestryDNA GHR software conducts a variety of control checks on the file, resulting in a final genotype profile for each sample. These data are used to generate test reports on a user's genotype and associated risk of disease. | The 23andMe PGS Genetic Health Risk Tests for Hereditary Thrombophilia is to be performed using the Tecan Evo and Illumina iScan instruments.
GenomeStudio is a modular software application that is used to view and analyze genotypic data obtained from the iScan. Coregen software conducts a variety of control checks on the file, resulting in a final genotype profile for each sample. These data are used to generate test reports on a user's genotype and associated risk of disease. |
| Classification | Class II | Same |
| Measurand | Factor V Leiden c.1601G>A variant in the F5 gene | Factor V Leiden variant (rs6025, c.1601G>A) in the F5 gene and Prothrombin G20210A |
| Type of Test | Qualitative in vitro molecular diagnostic system | Same |
| Sample Preparation | DNA extraction from human saliva | Same |

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| Calibration | Calibration and calibration verification procedures are established to demonstrate continued accuracy of the test systems. | Same |
| --- | --- | --- |
| Results | The test report describes if a person has variants associated with a higher risk of developing harmful blood clots. The report does not describe a person’s overall risk of developing harmful blood clots. | The report provides results of which variant(s) has/have been detected and provided information on the risk of disease associated with the variant(s). |
| General Device Characteristic Differences | | |
| Sample Collection Device | AncestryDNA Saliva Collection Kit (K192947) | Oragene®·Dx Collection Device, model OGD-500.001 (K141410) |
| Analytical Sensitivity | The performance requirement for the AncestryDNA GHR Test has been set at a minimum of 1.53 ng/μL DNA and maximum of 50 ng/μL DNA. | The performance requirement for the PGS Test has been set at a minimum of 15 ng/μL DNA and maximum of 50 ng/μL DNA. |
| Interfering Mutations | The potential interfering mutations include rs770011773, rs773367113 and rs143663052. The impact of these mutations on the performance of the assay has not been evaluated. | For Factor V Leiden the potential interfering mutations include rs760488939 and rs763859650. The impact of these mutations on the performance of the assay has not been evaluated. |

VI Standards/Guidance Documents Referenced:

- Special Controls for Genetic Health Risk Assessment System as detailed in 21 CFR 866.5950
- CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition
- CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline – Second Edition
- CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition
- CLSI EP37-A1, Supplement Tables for Interference Testing in Clinical Laboratory; Approved Guideline – First Edition
- CLSI EP25-A, Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline

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VII Performance Characteristics (if/when applicable):

# A Analytical Performance:

# 1. Precision/Reproducibility:

The purpose of the precision studies was to determine the imprecision of the AncestryDNA Factor V Leiden GHR Test under the following changed conditions: assay run, critical reagent lot, instrument, operator, day and site. Saliva samples from nine donors, three for each genotype (homogenous common - GG, heterozygous - GA and homozygous rare - AA), were collected with three lots of the Ancestry DNA Saliva Collection Kit. Genotypes of these samples were confirmed through bi-directional sequencing.

The precision studies were performed at two independent CLIA-certified testing laboratories (Lab 1 and Lab 2). Saliva samples were genotyped by the AncestryDNA Factor V Leiden GHR Test using three lots of critical reagents by six different operator teams (three per laboratory) on eight instrument combinations (four per laboratory) over multiple days. The precision study yielded $100\%$ correct genotype calls for all samples with a valid call across multiple days, operator teams, instruments and reagent lot at both laboratory sites.

Information regarding samples that failed quality control (FQC) was also evaluated. Sample replicates that did not pass sample call rate (SCR) QC in the first run underwent second, and when eligible, third genotyping run per laboratory standard operating procedures (SOPs). The overall precision exceeded $99\%$ point estimate. The results of the study are demonstrated in the tables below.

Summary of the within-run repeatability results at Lab 1

| Genotype | Total Number of Replicates | Number of Concordant Calls | Number of “No-Calls” | Number of Call Rate QC Failures | FQC (%) |
| --- | --- | --- | --- | --- | --- |
| GG | 15 | 15 | 0 | 0 | 0.00 |
| GA | 15 | 15 | 0 | 0 | 0.00 |
| AA | 15 | 15 | 0 | 0 | 0.00 |
| Total | 45 | 45 | 0 | 0 | 0.00 |

Summary of the within-laboratory testing results at Lab 1

| Genotype | Total Number of Replicates | Number of Concordant Calls | Number of “No-Calls” | Number of Call Rate QC Failures | FQC (%) |
| --- | --- | --- | --- | --- | --- |
| GG | 540 | 540 | 0 | 0 | 0.00 |
| GA | 540 | 537 | 0 | 3 | 0.56 |
| AA | 540 | 540 | 0 | 0 | 0.00 |
| Total | 1620 | 1617 | 0 | 3 | 0.19 |

Summary of the within-laboratory testing results at Lab 2

| Genotype | Total Number of Replicates | Number of Concordant Calls | Number of “No-Calls” | Number of Call Rate QC Failures | FQC (%) |
| --- | --- | --- | --- | --- | --- |
| GG | 180 | 180 | 0 | 0 | 0.00 |

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| GA | 180 | 180 | 0 | 0 | 0.00 |
| --- | --- | --- | --- | --- | --- |
| AA | 180 | 180 | 0 | 0 | 0.00 |
| Total | 540 | 540 | 0 | 0 | 0.00 |

Summary of the inter-laboratory reproducibility

| Genotype | Total Number of Replicates | | Number of Concordant Calls | | Number of “No-Calls” | | Number of Call Rate QC Failures | | FQC (%) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 |
| GG | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| GA | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| AA | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| Total | 81 | 81 | 81 | 81 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |

2. Linearity:

Not applicable

3. Analytical Specificity/Interference:

The study was to establish the analytical specificity of the AncestryDNA Factor V Leiden GHR Test from samples collected using the AncestryDNA Saliva Collection Kit (SCK) by evaluating the impact of potential endogenous interferents on assay performance. A series of studies were conducted to assess the effects of endogenous substances, exogenous substances, microbial substances and mutation on the AncestryDNA Factor V Leiden GHR Test.

i. Endogenous Interference

Saliva samples from ten donors were collected using the AncestryDNA SCK and their genotypes were determined with bi-directional sequencing. The endogenous substances were individually spiked into saliva prior to DNA extraction and genotyping. Saliva spiked with PBS served as the control. The results indicated that the performance of the AncestryDNA Factor V Leiden GHR Test is not affected by salivary $\alpha$ -amylase (395 U/mL), hemoglobin (20 mg/mL), IgA (0.44 mg/mL) and total protein (including 0.185 mg/mL salivary $\alpha$ -amylase, 0.44 mg/mL IgA and 2.05 mg/mL human serum albumin).

ii. Exogenous Interference

The AncestryDNA Factor V Leiden GHR Test requires the use of an FDA cleared saliva collection kit (K192947). The saliva collection kit includes Instructions for Use (IFU) instructing the user not to eat, drink, smoke, or chew gum for 30 minutes prior to collecting their saliva, thus minimizing the presence of interferents in the sample. The saliva collection IFU was tested for user comprehension and a paper version of the IFU is included in every collection kit. Should an interfering substance be present after DNA has been extracted and an insufficient concentration or quality of DNA is available, then the sample is managed per standard operating procedures that are pre-determined by the manufacturer.

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iii. Microbial Interference

Microbial DNA from Staphylococcus epidermis, Streptococcus mutans, Lactobacillus casei, Actinomyces odontolyticus and Candida albicans were tested to determine their impact on the performance of the AncestryDNA Factor V Leiden GHR Test. DNA from each of the six human cell lines was spiked with two concentrations (low/normal: 2.8 ng/μL; high: 12.5 ng/μL) of the five different species of microbial DNA. The results indicate that there is no significant impact of common microbial interferents on the performance of the AncestryDNA Factor V Leiden GHR test in either low/normal or high concentrations.

iv. Mutational Interference

An in silico analyses was performed to identify potential interfering variants within the 50 nucleotide probe-binding region downstream of the variant being detected. The genome aggregation database (gnomAD database) was utilized to search for mutations in this region. Only mutations that have been observed in more than one individual were evaluated. Three potential interfering mutations (rs770011773, rs773367113 and rs143663052) were identified for the AncestryDNA Factor V Leiden GHR Test. The impact of these mutations on the performance of the assay has not been evaluated.

4. Assay Reportable Range:

Not applicable

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

The AncestryDNA Factor V Leiden GHR Test uses one control material, which serves as both the sample processing control and the reproducibility control. The control material is genotyped on the Illumina BeadChip according to the standard of procedures at the laboratory. Each new lot of the control is tested by comparison with reference BeadChip genotype results.

The stability studies are ongoing and current data support a shelf-life stability claim of one month and an in-use stability claim of 30 days when the control material is stored at 2–8°C.

6. Detection Limit:

i. Limit of Blank

The Limit of Blank (LoB) study was performed to incorporate measurement variability due to the noisy nature of UV-Vis spectroscopy and potentially erroneous fluorescence signal in the assay. A plate of 95 blanks containing 1 mL of molecular grade water and the standard volume of DNA stabilizing solution from the AncestryDNA Saliva Collection Kit was extracted, quantified and genotyped using three lots of critical reagents. LoB calculation was performed using the non-parametric rank method in accordance with CLSI EP17-A2. No blanks returned a sample call rate above 98%. The LoB was determined to be 1.004 ng/μL.

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ii. Limit of Detection

The Limit of Detection (LoD) study was performed to establish the lowest concentration of DNA obtained from the AncestryDNA Saliva Collection Kit that is necessary when tested with the AncestryDNA Factor V Leiden GHR Test to successfully assign the correct variant. The study used both saliva samples from 15 donors (five for each genotype) and DNA from four cell lines. DNA from each sample was diluted to multiple concentrations and genotyped by the AncestryDNA Factor V Leiden GHR Test using three lots of critical reagents. To confirm the genotype call, each sample was also sequenced by bi-directional sequencing. The LoD was defined as the lowest DNA concentration at which at least 95% of samples yielded the correct call and statistically different from LoB. The LoD was determined to be 1.53 ng/μL. The manufacturer has also claimed the upper limit of DNA concentration of 50 ng/μL.

7. Assay Cut-Off:

Not applicable

8. Accuracy (Instrument):

Refer to Section B.1.i.

9. Carry-Over:

Not applicable

B Comparison Studies:

1. Comparison with Sanger Bi-directional Sequencing

i. Accuracy

The accuracy study was performed at one laboratory site to calculate the agreement of the genetic variant determination between the AncestryDNA Factor V Leiden GHR Test and results from bi-directional sequencing. Saliva samples were collected from 209 donors with known Factor V Leiden genotypes: 73 homozygous common (GG), 69 heterozygous (GA) and 67 homozygous rare (AA). Of the 200 samples initially genotyped, 11 samples failed the sample call rate (SCR) quality control (QC) and nine alternate samples were added to the same cohort, all of which passed SCR QC on the first run. All samples were genotyped using bi-directional sequencing to determine their true variant status. Genotyping results were compared between the GHR test and bi-directional sequencing to calculate percent agreement with the sequencing results considered to be "truth". The percent agreement for each genotype, the overall percent agreement and confidence intervals were calculated based on a binomial distribution. Data analysis results did not include FQC samples. The results of the study are summarized in the tables below.

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Distribution of the 11 FQC samples by genotypes

| Genotype | Total Number of Samples | Number of FQC | FQC (%) | 95% Confidence Interval |
| --- | --- | --- | --- | --- |
| GG | 73 | 4 | 5.5 | 1.5% to 13.4% |
| GA | 67 | 3 | 4.5 | 0.9% to 12.5% |
| AA | 69 | 4 | 5.8 | 1.6% to 14.2% |

Comparison of the genotyping results between AncestryDNA Factor V Leiden GHR Test and bi-directional sequencing

| | Bi-directional Sequencing Genotypes | | | Total | |
| --- | --- | --- | --- | --- | --- |
| | | GG | GA | AA | |
| AncestryDNA Factor V Leiden GHR Test | GG | 69 | 0 | 0 | 69 |
| | GA | 0 | 65 | 0 | 65 |
| | AA | 0 | 0 | 64 | 64 |
| | No call or invalid | 0 | 0 | 0 | 0 |
| | FQC | 4 | 4 | 3 | 11 |
| Total | | 73 | 69 | 67 | 209 |

Percent agreement and confidence intervals for AncestryDNA Factor V Leiden GHR Test genotypes

| | Correct/Incorrect | No Call | FQC | Percent Agreement (PA) | 95% Confidence Interval |
| --- | --- | --- | --- | --- | --- |
| GG | 69/0 | 0 | 4 | 100% | 94.8% to 100% |
| GA | 65/0 | 0 | 4 | 100% | 94.5% to 100% |
| AA | 64/0 | 0 | 3 | 100% | 94.4% to 100% |
| All Genotypes | 198/0 | 0 | 11 | 100% | 98.2% to 100% |
| No call | | 0/198 | | 0% | 0% to 1.8% |

As reported in published literature, factor V Leiden heterozygosity is present in 5.1%, 2.0% and 1.2% of Caucasians, Hispanics and African Americans, respectively. The frequencies of homozygosity for the above populations are 65, 10 and 4 per 100,000 individuals correspondingly. Technical (analytical) Positive Predictive Value (TPPV) of the AncestryDNA Factor V Leiden GHR Test results are 100% for both GA and AA.

2. Matrix Comparison:

Not applicable

C Clinical Studies:

1. Clinical Summary

Clinical performance for this test was assessed using published data and studies to demonstrate user comprehension of the labeling and test results.

The AncestryDNA Factor V Leiden GHR Test for Hereditary Thrombophilia is indicated for the detection of the Factor V Leiden variant (c.1601G&gt;A) in the F5 gene. This variant is associated with higher risk for developing harmful blood clots or venous thromboembolism

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(VTE; includes deep vein thrombosis and pulmonary embolism (Simone et al., 2013) $^{1}$ . Odds ratios are available in the meta-analysis performed by Simone et al. (2013). Odds ratios adjusted for age and sex using logistic regression and $95\%$ confidence intervals for each variant status are given below:

| Factor V Leiden variant status | Odds ratio (95% confidence interval) |
| --- | --- |
| One copy, heterozygote | 4.22 (3.35 to 5.32) |
| Two copies, homozygote | 5.45 (6.79 to 19.29) |

To support test results, a meta-analysis study conducted by Simone et al. (2013) was used to calculate likelihood ratios (LR, an estimate of how the test result affects the chances of a condition). Post-test risk $(\mathrm{R}_{\mathrm{post}})$ was calculated from the LR and using a pre-test risk $(\mathrm{R}_{\mathrm{pre}})$ of $11\%$ from the 2018 ACMG venous thromboembolism laboratory testing standard (Zhang, 2018) $^{2}$ . LR and $95\%$ confidence intervals calculated using a normal approximation are listed in the table below along with the post-test risk calculated using the relationship $\mathrm{R}_{\mathrm{post}} / (1 - \mathrm{R}_{\mathrm{post}}) = \mathrm{LR}[\mathrm{R}_{\mathrm{pre}} / (1 - \mathrm{R}_{\mathrm{pre}})]$ .

| FVL variant count (genotype) | Case/control distribution | LR | 95% confidence interval | Post-test risk |
| --- | --- | --- | --- | --- |
| 0 variant (homozygous common / wild type) | 1758 cases and 1201 controls among 2959 Factor V Leiden carriers. 7323 cases and 16312 controls among 23635 wild type individuals. | 0.87 | 0.86 to 0.88 | 10% |
| 1 variant (heterozygous) | 1758 cases and 1201 controls among 2959 Factor V Leiden carriers. 7323 cases and 16312 controls among 23635 wild type individuals. | 2.82 | 2.64 to 3.02 | 26% |
| 2 variants (homozygous rare) | 92 cases and 24 controls among 116 Factor V Leiden homozygous individuals. 4524 cases and 11643 controls among 16167 wild type individuals. | 9.69 | 6.19 to 15.16 | 54% |

# 2. Other Clinical Supportive Data

# a. User Comprehension Study

Two user comprehension studies (Study 1 and Study 2) were conducted to assess user comprehension of the AncestryDNA Factor V Leiden GHR Test process and reports. The user comprehension studies were performed using a sampling of individuals that were demographically diverse in a controlled environment. Quota-based sampling was used to

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recruit study participants representative of the U.S. population according to education, age, sex/gender and race/ethnicity.

In Study 1, geographic diversity was addressed through participant recruitment and comprehension testing was conducted from each of the four U.S. census regions: South, Midwest, Northeast and West. Approximately 120 participants were tested at each location. These subjects were randomly assigned to each of the four study arms, testing comprehension of four GHR Test reports:

- 0 Variants Identified
- 1 Variant Identified
- 2 Variants Identified
- Result Not Determined

The study was conducted in person and each interview session was administered by a trained interviewer/moderator using a series of pre-defined questions. Participants were given a time limit from start of report review to completion of the comprehension study. After excluding several participants according to pre-defined exclusion criteria, the final 378 participants completed the survey talk for the four AncestryDNA Factor V Leiden GHR Test reports and were included in the endpoint analysis. The manufacturer determined comprehension accuracy rates for multiple core comprehension concepts. The comprehension assessment addressed the overall comprehension and the following core comprehension concepts: purpose of test, limitations of test, ethnic relevance, results of test, other risk factors and appropriate follow-up action.

In Study 2, geographically diverse participants were randomly assigned to each of two study arms, representing the two most challenging reports:

- 1 Variant Identified
- Result Not Determined

The study was conducted via live tele-video interview and each interview session was administered online by a trained interviewer/moderator using a series of pre-defined questions. After excluding participants per pre-defined exclusion criteria, a total of 213 individuals completed the study and were included in the endpoint analysis.

Both studies were performed on the different types of the GHR Test reports developed using representative samples of the materials below (supplemental materials):

- Education Module including definition of terms
- Pre-purchase page
- Frequently Asked Questions, and
- Technical Details

The representative samples were developed per FDA guidance for medical device patient labeling and designed for readability no higher than a defined reading level using a Flesch-Kincaid Readability test. The representative samples were reviewed by a Certified Genetic Counselor to confirm that the materials tested accomplished the following:

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- Defined the target condition being tested and related symptoms
- Explained the intended use and limitations of the test
- Explained the relevant ethnicities in regard to the variant tested
- Explained genetic health risks and relevance to the user's ethnicity, and
- Assessed participants' ability to understand the following comprehension concepts: the test's limitations, purpose, appropriate follow-up action, test results, ethnic relevance and other risk factors that may have an impact on the test results.

For both studies, the completion rate was 100% (378/378 for Study 1 and 213/213 for Study 2) for all subjects who appeared for their interviews in the study after passing the pre-defined exclusion criteria. The average comprehension rates per core comprehension concept were 90.7% to 97.4% for Study 1 and 90.9% to 99.1% for Study 2. The overall comprehension score across all GHR reports was 93.2% for Study 1 and 96% for Study 2.

Overall user comprehension rates for Factor V Leiden GHR Test reports in Study 1

| Core Concept | Comprehension Rates by GHR Report Type (%) | | | | Overall Comprehension Rates (%) |
| --- | --- | --- | --- | --- | --- |
| | 0 Variant | 1 Variant | 2 Variants | Result Not Determined | |
| Appropriate Follow-Up Action | 95.8 | 97.7 | 100 | 95.6 | 97.4 |
| Ethnicity Relevance | 98.9 | 89.5 | 97.2 | N/A | 95.5 |
| Other Risk Factors | 89.6 | 94.2 | 92.5 | 92.2 | 92.1 |
| Limitations of Test | 88.5 | 89.5 | 98.1 | 88.9 | 91.5 |
| Purpose of Test | 89.0 | 95.3 | 93.4 | 93.3 | 92.7 |
| Results of Test | 93.8 | 83.7 | 93.4 | 91.1 | 90.7 |
| Total Number of Reports | 96 | 86 | 106 | 90 | 378 |

Overall user comprehension rates for Factor V Leiden GHR Test reports in Study 2

| Core Concept | Comprehension Rates by GHR Report Type (%) | | Overall Comprehension Rates (%) |
| --- | --- | --- | --- |
| | 1 Variant | Result Not Determined | |
| Appropriate Follow-Up Action | 94.2 | 98.6 | 96.5 |
| Ethnic Relevance | 98.1 | 97.3 | 97.7 |
| Other Risk Factors | 98.1 | 93.6 | 95.8 |
| Limitations of Test | 93.7 | 98.6 | 96.2 |
| Purpose of Test | 98.5 | 99.6 | 99.1 |
| Results of Test | 88.5 | 92.7 | 90.9 |
| Total Number of Reports | 103 | 110 | 213 |

Comparison of pre- and post-test by core concept in Study 2

| Core Concept | Pre-test (%) | Post-test (%) | % Improvement | p-value |
| --- | --- | --- | --- | --- |
| Purpose | 89.0 | 99.1 | 10.1 | <0.001 |

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| Other Risk Factors | 91.1 | 95.8 | 4.7 | 0.002 |
| --- | --- | --- | --- | --- |
| Ethnic Relevance | 95.8 | 97.7 | 1.9 | 0.117 |
| Limitations | 87.7 | 96.2 | 8.5 | <0.001 |

b. Frequently Asked Questions Material

The manufacturer has developed a Frequently Asked Question (FAQ) section for each Genetic Health Risk Test Report. The FAQ section was created to provide users information to adequately understand the purpose, limitations and the meaning of the results of the test. The FAQ section was developed using methodology consistent with the Manufacturer's labeling design, identification of primary communication messages and label comprehension. The concepts covered in the FAQ section include: the test results, the purpose of the test, limitations of the test, relevance of race and ethnicity on test results, the meaning of the result, other risk factors that may affect the user's family and children, and links to resources that provide additional information. Additionally, the FAQ section provide definitions for terminology found in Genetic Health Risk Reports that is used to describe risks associated with detected variants. The questions included in the FAQ section for the test include, but are not limit to:

- What is the purpose of this test?
- What is a blood clot?
- What is thrombophilia?
- What can this test tell me?
- What can this test not tell me?
- What does it mean if I have an increased risk?
- What does it mean if I don't have an increased risk?
- Can other things increase my risk for an abnormal blood clot?
- Is factor V Leiden thrombophilia more common in certain ethnic groups?
- What should I do with my results?
- Where can I get more information?

Each Genetic Health Risk Test Report has answers to FAQ that are specific to the variant(s) and disease being reported, where applicable. The FAQ section is included in the Genetic Health Risk Test Report.

D Clinical Cut-Off:

Not applicable

E Expected Values/Reference Range:

Not applicable

F Other Supportive Instrument Performance Characteristics Data:

i. Reagent Shelf-Life Stability

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The purpose of the real-time stability study is to establish the shelf-life stability for the five critical reagents used in the AncestryDNA Factor V Leiden GHR Test. Three lots of critical reagents were assessed with DNA from three commercially available cell lines (one cell line for each genotype). The study is ongoing and current results support a shelf-life stability claim of 2 months when the critical reagents are stored under the following conditions:

| Critical Reagent | Storage Temperature |
| --- | --- |
| MSM | -15°C to -25°C |
| FMS | -15°C to -25°C |
| BeadChip | 2°C to 8°C |
| X-Stain Plate | -15°C to -25°C |
| EML | -15°C to -25°C |

ii. Specimen Stability

Saliva samples for testing are collected with the AncestryDNA Saliva Collection Kit. Refer to K192947 for sample stability information as well as a description of the device and performance characteristics.

VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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