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The TruDiagnosis® System is an in vitro diagnostic device intended for processing and genotyping multiple genetic variants in a DNA sample utilizing on-slide PCR gel-drop microarray technology. The TruDiagnosis® System consists of the TruDx® 2000 Imager, the TruArray® Warfarin Sensitivity Test Kit, and the ProFlex™ PCR System using the ProFlex™ 2x Flat Sample Block.

The TruDx® 2000 Imager is an instrument intended for processing and genotyping multiple genetic variants in a DNA sample utilizing on-slide PCR gel-drop microarray technology.

The TruArray® Warfarin Sensitivity Test Kit is an in vitro diagnostic test for the detection and genotyping of the 2C92, 2C93 alleles of the cytochrome P450 (CYP450) 2C9 gene locus and Vitamin K epoxide reductase CL, VKORCI, gene promoter polymorphism (-1639) from genomic DNA of human saliva samples collected using the Oragene® Dx Device (OGD-500) as an aid in the identification of patients at risk for increased warfarin sensitivity. The TruArray® Warfarin Sensitivity Test Kit is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.

The TruDiagnosis® System is an in vitro diagnostic device intended for processing and genotyping multiple genes in a DNA sample utilizing on-slide Polymerase Chain Reaction (PCR) gel-drop microarray technology. The TruDiagnosis® System consists of the:

• Hardware: TruDx® 2000 Imager ●
• Software: TruSpot™ Software ●
• . Test Kit: TruArray® Warfarin Sensitivity Test Kit
  • TruArray® Test Slide o
  • o TruPlex™ reagents
• Thermal Cycler: ProFlex™ PCR System using the ProFlex™ 2x Flat Sample Block ●

Hardware: Akonni's microarray imager (TruDx®2000) is an instrument that consists of a highintensity green light emitting diode (LED), custom optics, and a digital grayscale camera.

• . The purpose of the TruDx® 2000 Imager is to capture a fluorescence image of the microarray after completing the test.
• The user inserts the TruArray® Test Slide into the TruDx® 2000 Imager and follows . the on-screen prompts. The resulting microarray image is automatically analyzed and reported with the TruSpot™ Software.

Software: Akonni's TruSpot™ Software, integrated within the imager, locates and segments each fluorescently labeled microarray Gel-Element and reports signal-to-noise ratios (SNR). Assay results interpreted by TruSpot™ Software program are assigned a genotype and presented to the end user in a report format.

Test Kit: The TruArray® Warfarin Sensitivity Test Kit includes consumables and reagents necessary to perform multiplex on-slide PCR amplification, and fluorogenic target-specific microarray-based hybridization. Specifically:

• The TruArray® Test Slide (loaded with target DNA) undergoes thermal cycling via . asymmetric amplification to enrich single stranded fluorescently labeled complimentary strands to capture probes printed on the microarray (asymmetric PCR and allele specific hybridization occur in the same chamber). After hybridization, arrays are washed and dried, and the user then inserts the microarray into the TruDx® 2000 Imager and follows the on-screen prompts.

The warfarin assay performed with the test kit is an in vitro diagnostic test for the detection and genotyping of the 2C92, 2C93 alleles of the cytochrome P450 (CYP450) 2C9 gene locus and Vitamin K epoxied reductase C1, VKORC1 3673, gene promotor polymorphism (-1639) mutations from genomic DNA of human saliva samples collected using the Oragene® Dx OGD-500 Device (K110701).

Thermal Cycler: The ProFlex™ PCR System with the ProFlex™ 2x Flat Sample Block, a component of the TruDiagnosis System, is an end-point thermal cycler, specifically designed for the amplification of nucleic acids using the Polymerase Chain Reaction (PCR) process. The user interface includes a touchscreen with a graphical display that shows the time, status, and temperature for each run. A touchscreen keypad allows you to enter information into fields on the display screen.

Here's an analysis of the acceptance criteria and the studies that prove TruDiagnosis® System meets them:

1. Table of Acceptance Criteria and Reported Device Performance

Based on the provided document, the acceptance criteria are not explicitly stated as distinct pass/fail thresholds in simple terms. Instead, the studies demonstrate the device's performance, aiming for high "Correct Call Rate" and agreement with Sanger sequencing, especially after addressing "No Calls". A reasonable interpretation of the implicit acceptance criteria, based on the studies, would be a high percentage of accurate genotype calls and 100% concordance with the reference method (Sanger sequencing) upon resolution of initial "No Calls".

2. Sample Sizes and Data Provenance for Test Set

• Limit of Detection Study:

  • Sample Size: 3 genomic DNA samples (Donors 1, 2, 3), tested at various concentrations with 40 replicates each (total 3 * 4 * 40 = 480 replicates for initial runs, but 362 samples were ultimately tested, implying some initial runs were repeated due to control failures, and some individual test samples were re-run).
  • Data Provenance: Genotypes for these 3 donors are provided (*1/*1, *1/*3, AA; *1/*2, *1/*3, AG; *2/*2, *1/*1, GG). The origin of these DNA samples (e.g., country) is not specified. It is a retrospective study since the DNA samples were presumably pre-collected and genotyped by Sanger sequencing.

• Method Comparison Study:

  • Sample Size: 303 unique human genomic DNA samples.
  • Data Provenance: Origin of the data (e.g., country) is not specified, but the samples were isolated from human saliva specimens. It is a retrospective study as the samples were pre-collected using the Oragene® Dx OGD-500 device, extracted, and a reference genotype established.

• Interference Studies (Endogenous and Exogenous):

  • Endogenous: 5 donors, each providing saliva samples, divided into 5 aliquots (spiked with interferents or un-spiked control). Three extractions performed on each spiked/un-spiked sample. (Total 5 donors * 5 conditions * 3 extractions = 75 samples).
  • Exogenous: 5 activity groups (Eating, Drinking, Chewing Gum, Mouth Wash, Smoking) with 5 donors each. Each donor provided 2 saliva samples (baseline and 30 min post-activity), tested in triplicate. (Total 5 groups * 5 donors * 2 timepoints * 3 replicates = 150 samples).
  • Data Provenance: Saliva samples from human donors. Origin not specified. Retrospective.

• Reproducibility Study:

  • Sample Size: Six specific saliva samples (genotypes provided in the table). Each sample was processed in triplicate by 4 operators over 5 non-consecutive days (15 tests per operator per genotype). 6 genotypes * 4 operators * 15 tests = 360 samples.
  • Data Provenance: Saliva samples from human donors. Origin not specified. Retrospective for samples, but prospective for the testing process across different sites/operators.

3. Number of Experts and Qualifications for Ground Truth

The document explicitly states that the ground truth for all performance studies (Limit of Detection, Method Comparison, Interference, and Reproducibility) was established by bi-directional Sanger sequencing. It does not mention the use of human experts or their qualifications for establishing this ground truth. Sanger sequencing is a widely accepted gold standard for DNA sequencing and thus acts as the independent reference for genotyping.

4. Adjudication Method for the Test Set

The document describes an adjudication method for "No Calls" found during the initial runs. For "No Calls," the samples were repeated (re-run). Incorrect calls, however, were generally not repeated ("Incorrect Calls were not repeated" in the LoD study footnote, and for the Method Comparison, an incorrect call was attributed to a sample mix-up and "was not re-run"). This indicates an adjudication process where "no-calls" lead to re-testing, but "incorrect calls" (when a result was given but it was wrong) are investigated for root cause and not necessarily re-run for a different result in the study context.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was mentioned. The device, the TruDiagnosis® System, is an in vitro diagnostic device for genotyping, which typically involves laboratory testing rather than interpretation by multiple human readers (like radiology images). Therefore, a multi-reader study is not applicable in this context.

6. Standalone Performance

Yes, a standalone performance study was done. The entire performance evaluation (Limit of Detection, Method Comparison, Interference, Reproducibility) assesses the TruDiagnosis® System (which includes the TruArray® Warfarin Sensitivity Test Kit, TruDx® 2000 Imager, and TruSpot™ Software acting together as an algorithm/system) against an independent reference method (bi-directional Sanger sequencing). This is a direct measure of the algorithm's diagnostic accuracy without human intervention in the genotype interpretation after the system generates a result.

7. Type of Ground Truth Used

The ground truth used for all studies was bi-directional Sanger sequencing.

8. Sample Size for the Training Set

The document does not provide information regarding a training set or its sample size. This is typical for a 510(k) submission where the focus is on the validation of the finished device and its performance characteristics against a predicate, rather than the development and training of the underlying algorithm. The "TruSpot™ Software" is mentioned as integrated within the imager and interpreting assay results, implying it's an algorithmic component, but details on its development or training are not included in this summary.

9. How Ground Truth for the Training Set Was Established

Since no information on a training set is provided, there is no detail on how its ground truth was established.

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The eSensor® Warfarin Sensitivity Saliva Test is an in vitro diagnostic for the detection and genotyping of the *2 and *3 alleles of the cytochrome P450 (CYP450) 2C9 gene locus and the Vitamin K epoxide reductase C1 (VKORC1) gene promoter polymorphism (-1639G>A) from genomic DNA extracted from human saliva samples collected using the Oragene® Dx and ORAcollect® Dx devices, as an aid in the identification of patients at risk for increased warfarin sensitivity.

The eSensor® XT-8 instrument is an in vitro diagnostic device intended for genotyping multiple mutations or polymorphisms in an amplified DNA sample utilizing electrochemical detection technology.

The kit consists of the eSensor® Warfarin Sensitivity Saliva Test cartridge, the eSensor® Warfarin Sensitivity Saliva Test amplification reagents (including PCR mix and DNA polymerase), the eSensor® Warfarin Sensitivity Saliva Test detection reagents (including exonuclease, probes and hybridization buffer ingredients) and the eSensor® XT-8 System. One eSensor® Warfarin Sensitivity Saliva Test Kit has sufficient materials for 24 tests.

The provided document, a 510(k) summary for the eSensor® Warfarin Sensitivity Saliva Test, details performance data primarily focused on a new specimen collection kit (ORAcollect®·Dx Device) and its impact on the existing device's performance. The study aims to demonstrate that incorporating this new collection kit does not compromise the accuracy or reliability of the Warfarin Sensitivity Saliva Test.

Here's an analysis of the acceptance criteria and study findings:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state 'acceptance criteria' in terms of specific numerical thresholds for accuracy, reproducibility, or call rates. However, the studies consistently aim for 100% agreement with the ground truth (bi-directional sequencing), and 100% correct calls with 0% incorrect calls. The "Method Comparison" section indicates an aspiration for high concordance and call rate.

2. Sample Sizes and Data Provenance

• Reproducibility (Sample-to-Sample, Lot-to-Lot, Day-to-Day, Operator-to-Operator):

  • Test set sample size: 10 donors, with 3 samples each (total 30 samples collected). These were processed by 3 operators, with genotyping data evaluated for each operator for each of the 3 SNPs. This implies 20 tests per SNP per operator (based on the "20" under "Samples Tested" for each SNP for each operator), totaling 60 tests per SNP across all operators, or 120 calls per SNP when considering all samples and operators for the "Summary of Results by Sample and Genotype" table.
  • Data Provenance: Not explicitly stated, but likely prospective and from a controlled lab environment.

• Multi-center Reproducibility:

  • Test set sample size: 30 donors. Multiple saliva samples collected from each.
  • Data Provenance: Saliva samples were collected from 3 sites. Two sites were described as "professional setting" with "supervised collections," and the third site had "unsupervised collections." One sample from each donor was transported to three independent sites for extraction. All eSensor Warfarin Sensitivity Saliva testing was conducted at Site 1. This suggests a prospective data collection design, with samples from multiple (unspecified) locations. No country of origin is mentioned.

• Method Comparison:

  • Test set sample size: 156 saliva samples.
  • Data Provenance: Not explicitly stated, but implies a prospective or retrospectively collected set of human saliva samples. No country of origin is mentioned.

• Interfering Substances (Endogenous):

  • Test set sample size: 14 donors, each providing 4 saliva samples.
  • Data Provenance: Not explicitly stated, but likely prospective, controlled lab study.

• Interfering Substances (Exogenous):

  • Test set sample size: Varied per activity, ranging from 5 to 9 donors per activity group. Each donor provided samples at two time-points (immediate and 30 minutes post-activity).
  • Data Provenance: Not explicitly stated, but likely prospective, controlled lab study.

3. Number of Experts and Qualifications for Ground Truth

The ground truth for all studies was established by bi-directional DNA sequencing. This is a laboratory-based method, not typically requiring "experts" in the sense of clinical specialists. The interpretation of sequencing results is a standard molecular biology technique. No specific number of experts or their qualifications (e.g., geneticists, molecular biologists) are mentioned for establishing the ground truth, as it's assumed to be a direct, objective measurement.

4. Adjudication Method for the Test Set

The studies used bi-directional DNA sequencing as the gold standard, not a human expert adjudication process. The agreement was calculated directly between the device's genotypes and the sequencing results. If there were discrepancies in the initial device results (e.g., initial miscalls or no-calls in the method comparison), retests were performed. For instance, in the method comparison, two miscalls were attributed to "operator error (sample mix-up) occurring during the first XT8 testing" and one remaining no-call to "an operator error at the purification step." This suggests an internal investigation and re-testing process rather than an external adjudication panel.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No Multi-Reader Multi-Case (MRMC) comparative effectiveness study was done. This device is a diagnostic test for genotyping, not an imaging device or a system requiring human interpretation with or without AI assistance. Therefore, the concept of "readers improving with AI vs. without AI" is not applicable here. The device itself performs the genotyping.

6. Standalone Performance Study

Yes, a standalone performance study was done. All the performance data described (reproducibility across operators/sites, method comparison with sequencing, and interfering substances studies) directly assesses the performance of the eSensor® Warfarin Sensitivity Saliva Test (algorithm/device only, without human-in-the-loop performance influencing the genotyping call itself, though human operators are involved in sample preparation and running the test). The percentage agreement and call rates reflect the direct output of the device compared to the ground truth.

7. Type of Ground Truth Used

The primary ground truth used for all studies was bi-directional DNA sequencing. This is a highly accurate molecular method for determining genetic sequences and polymorphisms (genotypes).

8. Sample Size for the Training Set

The document is a 510(k) summary for a labeling modification (addition of a new specimen collection kit) to an already cleared device. It primarily presents validation data for the continued performance of the device with the new collection kit. It does not mention a "training set" for the eSensor® Warfarin Sensitivity Saliva Test itself, as this device likely relies on established molecular biology principles and probes designed against known genetic targets, rather than machine learning algorithms that require extensive training data. It's a deterministic diagnostic test, not an AI model that learns from data in the way a computer vision algorithm would.

9. How Ground Truth for the Training Set Was Established

As noted above, the concept of a "training set" doesn't directly apply in the context of this device. The device's design is based on known genetic sequences and electrochemical detection rather than a machine learning model. The validation studies verify its performance against the established ground truth of bi-directional DNA sequencing.

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The eQ-PCR™ LC Warfarin Genotyping kit is an in vitro diagnostic test for the detection and genotyping of two single nucleotide polymorphisms (SNP) in the cytochrome P450 enzyme gene CYP2C9 known as CYP2C92 (C430T) and CYP2C93 (A1075C), and a SNP in the vitamin K epoxide reductase complex 1 gene VKORC1, known as VKORC1 (-1639G>A) obtained from human peripheral blood samples. The eQ-PCR LC Warfarin Genotyping kit is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.

The eO-PCR™ LC Warfarin Genotyping kit is indicated for use as an aid in identifying patients who may be at risk of warfarin sensitivity.

The eQ-PCR 1M LC Warfarin Genotyping Kit assay requires extracted DNA obtained by using any commercially available DNA extraction kit. The extracted DNA sample, within a range of 50-200ng of total DNA, is mixed with a PCR Mix, and an eQ-PCR "M specific Probe Mix reagent containing specific primers and fluorescent labeled probes for the CYP2C9 and/or VKORC1 gene polymorphisms. Amplification and detection are then performed in the Roche Diagnostics LightCycler® Real-Time PCR System instrument model 1.2 using conditions defined in the specific eQ-PCRTM LC Warfarin Genotyping Kit Product Insert. After the PCR reaction is completed, the Roche Diagnostics LightCycler® Real-Time PCR System instrument automatically proceeds to the melting curve-based detection method. This real time PCR test is a closed test system and does not require post PCR operations. It reduces human errors and eliminates post-PCR handling contamination.

The instrument's standard melting curve analysis software algorithm of peak patterns and melting temperatures (Tm) determine the genotype (wild type, mutant, heterozygous) for each of the three specified polymorphisms.

This document describes the TrimGen eQ-PCR™ LC Warfarin Genotyping Kit, an in vitro diagnostic test for genotyping SNPs associated with warfarin sensitivity. The study aims to demonstrate substantial equivalence to a predicate device through clinical performance and reproducibility testing.

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state pre-defined quantitative acceptance criteria in a dedicated section. However, the performance is reported as "overall 100% clinical sensitivity" for the assay compared to bi-directional sequencing after re-sequencing of discrepant samples. The table below presents the detailed clinical sensitivity for each SNP and genotype, which can be inferred as the performance measures.

For reproducibility, the criteria were implicitly met by "no discordance among observed results or between observed and expected results across sites, days or operators" and all results matching bi-directional sequencing.

2. Sample Size Used for the Test Set and Data Provenance:

• Sample Size: 159 donors provided whole blood samples for the clinical performance study. For the reproducibility study, a panel of 31 samples (whole blood) was used at each of the three sites across five days.
• Data Provenance: The whole blood samples were collected from adult volunteers under IRB approval and with Informed Consent. The country of origin is not explicitly stated, but the submission is to the US FDA, implying testing within the US or for the US market. The clinical performance study is a prospective collection for testing.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:

• The ground truth for the clinical performance study was established by bi-directional sequencing performed at a "reference laboratory." The number of experts and their specific qualifications (e.g., geneticists, molecular biologists) are not specified within the provided text.

4. Adjudication Method for the Test Set:

• The clinical performance study mentions that "Only 0.4% (2/450) of the results was discordant and resolved in agreement with the eQ-PCR LC Warfarin Genotyping Kit result when bi-directional sequencing was repeated." This indicates an adjudication method where initial discrepancies were re-evaluated by re-sequencing using the ground truth method (bi-directional sequencing). This could be considered a form of "tie-breaker" or confirmatory testing for discordant results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done, If So, What was the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance:

• No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic (IVD) kit for genotyping, not an AI-assisted diagnostic imaging or interpretation tool. Therefore, the concept of human readers improving with AI assistance is not applicable here.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was Done:

• Yes, the clinical performance study is essentially a standalone performance evaluation of the device (the eQ-PCR™ LC Warfarin Genotyping Kit) against the established ground truth (bi-directional sequencing). The device's algorithm for determining genotype from melting curve analysis is used independently.

7. The Type of Ground Truth Used:

• The ground truth used was bi-directional sequencing. This is a highly accurate method for determining DNA sequences and is considered a gold standard for genotyping in many contexts.

8. The Sample Size for the Training Set:

• The document describes a post-market clearance study demonstrating assay performance. It does not mention a separate training set for the device's algorithm. The algorithm for interpreting melting curves is likely pre-defined based on known principles of real-time PCR and melting curve analysis, rather than a machine learning model requiring a specific training dataset in the context presented here. The "training" of such a system would typically involve establishing robust assay conditions and expected melting curve profiles for different genotypes.

9. How the Ground Truth for the Training Set Was Established:

• As a dedicated training set is not explicitly mentioned for an algorithm that learns from data, the establishment of ground truth for a training set is not applicable as per the provided text. The device relies on a pre-established methodology (real-time PCR and melting curve analysis) for genotype determination, where the "ground truth" for defining peak patterns and melting temperatures for different genotypes would be based on well-characterized samples and molecular biology principles.

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The eSensor® Warfarin Sensitivity Test is an in vitro diagnostic for the detection and genotyping of the *2 and * 3 alleles of the cytochrome P450 (CYP450) 2C9 gene locus and the Vitamin K epoxide reductase C1 (VKORC1) gene promoter polymorphism (-1639G>A) from genomic DNA extracted from fresh whole blood samples preserved with EDTA, as an aid in the identification of patients at risk for increased warfarin sensitivity. The eSensor® Warfarin Sensitivity Test is for Rx only professional use within the confines of a licensed laboratory, as defined by the Clinical Laboratory Improvement Amendments (CLIA) of 1988.
The eSensor® XT-8 Instrument is an in vitro diagnostic device intended for genotyping multiple mutations or polymorphisms in an amplified DNA sample utilizing electrochemical detection technology.

The eSensor® XT-8 System is an in vitro diagnostic device for performing hybridization and genotyping of multiple mutations and/or polymorphisms in an amplified DNA sample. The XT-8 Instrument is configured with one to three processing towers which perform up to 8 simultaneous tests per tower. The XT-8 System uses a single-use, disposable test cartridge to perform hybridization and genotyping in approximately 30 minutes per sample. The cartridge contains an EEPROM chip which transmits the cartridge lot number, expiration date and protocol identity to the instrument.
The analysis process for each sample consists of three steps: 1) Genomic DNA isolated from whole blood obtained using EDTA as anti-coagulant is combined with PCR Mix and Taq polymerase enzyme and is subjected to amplification of target sequences by PCR using a thermal cycler. 2) Amplified DNA is treated with exonuclease enzyme to generate single-stranded target DNA. 3) Single-stranded, amplified target DNA is mixed with hybridization and genotyping reagents and transferred to an eSensor® Warfarin Sensitivity Test cartridge, and the cartridge is inserted in the eSensor® XT-8 Instrument. The instrument controls the circulation of the sample inside the cartridge containing to allow hybridization at a controlled temperature, and then detects and genotypes the sample by voltammetry.
Genotyping of the test panel polymorphisms is achieved by a sandwich assay principle: 1) Each pair of electrodes contains a different synthetic oligonucleotide capture probe which is complementary to one of the target DNA fragments. 2) The hybridization reagents contain pairs of ferrocene-labeled synthetic oligonucleotide signal probes; one member of each pair is complementary to the major allele sequence of the target polymorphism, while the second member of the pair is complementary to the minor allele sequence. Each member of the probe pair has a ferrocene label with a different oxidation potential for each allele. 3) Single-stranded, amplified target DNA hybridizes to its specific capture probe, and in turn hybridizes to the allele-specific, ferrocene-labeled signal probe. 4) Each electrode of the array is analyzed by voltammetry; the target polymorphism is determined by the location of the electrode containing the capture probe, and the genotype is identified by the ratio of signals from the allele-specific ferrocene labels. The array also includes positive and negative controls to confirm the hybridization reaction and detect non-specific signals.
Upon completion of the test, the EEPROM chip on the cartridge contains information that prevents its re-use with a new sample. The instrument analyzes the results and provides a report of the test results. The operator removes the used cartridge from the slot of the XT-8 Instrument, and that slot is ready to accept a new test.

Here's a breakdown of the acceptance criteria and the study details for the eSensor® Warfarin Sensitivity Test and XT-8 System, based on the provided 510(k) summary:

Acceptance Criteria and Device Performance

Study Details

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

   • Reproducibility Study Test Set:

     • Samples: 5 genomic DNA samples covering all possible genotypes for the three alleles (CYP2C92, CYP2C93, VKORC1).
     • Total Tests: 200 tests for each allele (CYP2C92, CYP2C93, VKORC1), for a grand total of 600 allele tests in the final analysis (5 samples * 4 operators/sites * 5 days * 2 runs per day = 200 tests per allele for an operator who performed 2 runs a day).
     • Data Provenance: Three sites were used: one internal (likely Osmetech Molecular Diagnostics) and two external. The country of origin is not specified, but given the submission is to the FDA, it is likely US-based or recognized for regulatory purposes. The study appears prospective, as it involves controlled testing of specific samples under varied conditions.

   • Genomic DNA Extraction Reproducibility Test Set:

     • Samples: 7 whole blood samples of different genotypes.
     • Total Tests: 21 tests for each allele per site (7 samples * 3 replicates). With 3 sites, this totals 63 tests per allele, and 189 allele tests overall.
     • Data Provenance: Three different sites, using different commercially available extraction methods. Similar to the above, country of origin is not specified but likely US-based, and the study is prospective.

   • Method Comparison Test Set:

     • Samples: 157 samples.
     • Total Tests: 157 samples tested on the eSensor device; 157 samples tested by DNA sequencing. On a per-SNP basis, this represents 471 data points (157 samples * 3 SNPs).
     • Data Provenance: Not explicitly stated, but implies collected samples for method comparison. The nature (retrospective/prospective) isn't directly stated, but typically, method comparison studies utilize a representative set of existing or collected samples in a controlled manner, making them essentially prospective for the purpose of the comparison.

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

   • The ground truth for all studies (reproducibility and method comparison) was established by DNA sequencing. The document does not specify the number of experts or their qualifications for interpreting the DNA sequencing results. DNA sequencing is generally considered a highly accurate gold standard for genotyping, and its interpretation often involves trained molecular biologists or geneticists, but no specific details are provided here.

3. Adjudication method for the test set:

   • No formal adjudication method (like 2+1, 3+1 consensus) is explicitly mentioned for the test set.
   • For the reproducibility study, "An additional run using the same kit lot and sample as for the first-pass test were performed for test that gave a no-call result." This implies a re-testing/re-run strategy for initial failures rather than human expert adjudication of output discrepancies. All no-calls were resolved to 100% agreement after additional runs.
   • For the method comparison and extraction reproducibility, there were no initial no-calls or incorrect calls, so no adjudication or re-testing was necessary beyond the initial DNA sequencing ground truth.

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

   • No MRMC comparative effectiveness study was performed or described. This device is an in vitro diagnostic for genotyping, meaning it produces a direct genetic result, and there is no "human reader" analogue in the typical sense of interpreting imaging or complex clinical data where AI assistance would be measured. The output is a genotype, which is then used by medical professionals.

5. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done:

   • Yes, the performance studies (reproducibility, extraction reproducibility, and method comparison) represent the standalone performance of the eSensor® Warfarin Sensitivity Test and XT-8 System. The device analyzes DNA samples and outputs a genotype. While human operators perform PCR and load samples, the critical genotyping step and result interpretation are performed by the instrument's software ("Assay signal results are interpreted by a software program and are assigned a genotype that is presented to the end-user in a report format"). The 100% agreement with DNA sequencing demonstrates this algorithmic performance.

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

   • The ground truth used for all performance studies was bi-directional DNA sequencing. This is considered a highly reliable molecular diagnostic method.

7. The sample size for the training set:

   • The document does not specify a separate "training set" or its size. As an in vitro diagnostic device, particularly for genetic testing, the development process generally involves analytical validation (like the studies described) rather than a machine learning training phase analogous to image analysis AI. The device's underlying "algorithm" is based on biochemical reactions and electrochemical detection, not a learned model from a dataset.

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

   • Since no training set is explicitly mentioned in the context of machine learning, there's no ground truth establishment for such a set described. The "knowledge" or parameters for the device's operation would have been developed through biochemical and engineering principles, with validation done against known standards (DNA sequencing) as detailed in the performance studies.

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The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is an in vitro diagnostic test for the detection and genotyping of the *2 and *3 CYP4502C9 genetic variants and the VKORC1 3673 (-1639) intronic variant in genomic deoxyribonucleic acid (DNA) obtained from EDTA-anticoagulated whole blood samples. The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.
The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is indicated for use to identify individuals at risk for sensitivity to warfarin.

The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is an in vitro diagnostic device which utilizes proprietary film-based microarray technology combined with process automation, reagent management, and software technology for the detection and genotyping of the 2C92, 2C93, and VKORC1 3673 (-1639) mutations from EDTA-anticoagulated whole blood samples.

The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is comprised of the BioFilmChip™ Microarray, the Intellipac Reagent Module and the PCR Amplification Mix. The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin should be run using the AutoGenomics INFINITI Analyzer.

The BioFilmChip Microarray consists of a polyester film coated with proprietary multi-layer components designed for DNA analysis. The layers have been designed to provide a versatile surface to enhance test performance. There can be up to 240 spots per microarray with each spot representing a different allele. The microarrays are designed to be assay specific.

The Intellipac Reagent Module contains up to eight reservoirs that house the test reagents and has an integrated memory chip. Information on the reagent such as lot number, expiration date and volume usage, are archived in the memory.

The PCR Amplification Mix consists of the reagents needed for the PCR amplification step of the assay.

The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is based on the following processes:
(a) DNA extraction
(b) PCR amplification of purified DNA from human genomic DNA
(c) Labeling of the amplified product (allele specific primer extension)
(d) Hybridization of the labeled amplified product to a microarray by signature Tag/Capture probe hybridization under isothermal conditions.
(e) Scanning of the microarray
(f) Signal detection and analysis [determination of the 2C92, 2C93 and VKORC1 3673 (-1639) genotypes]
Steps (c) through (f) are automated by the INFINITI Analyzer.

The INFINITI Analyzer automates the 2C9 and VKORC1 assays and integrates all the discrete processes of sample (PCR amplicon) handling, reagent management, hybridization, detection, and results analysis. The assays are processed automatically and read by the built-in confocal microscope. Results are analyzed and presented as genotype calls.

Here's an analysis of the acceptance criteria and study details for the INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin, based on the provided text:

Acceptance Criteria and Device Performance

The document describes the performance characteristics without explicitly stating pre-defined "acceptance criteria" as pass/fail thresholds. Instead, it presents the "reported device performance" and implies that these results demonstrate the device's suitability. For the purpose of this analysis, I will synthesize the reported performance values as the de facto "acceptance criteria" that the device met.

1. Table of Acceptance Criteria and Reported Device Performance

2. Sample Sizes Used for the Test Set and Data Provenance

Sample Size for Test Set:

• Agreement with Bi-directional Sequencing:
  • 150 patient samples tested in the initial comparison (first run data for 2C9*2, 2C9*3, VKORC1).
  • Table 2b "by Sample Type" also shows 150 samples, implying these are the same samples analyzed by genotype.
• Inter-Laboratory Reproducibility:
  • 7 genomic DNA samples and 5 whole blood samples (total 12 unique samples).
  • Each unique sample was run in duplicate per day/operator for six days at each of 3 sites.
  • Total tests: 12 samples * 2 replicates/day * 6 days * 3 sites = 432 tests per site, for a total of 1296 tests across all sites for the "first time run" and "final result".

Data Provenance:

• Agreement with Bi-directional Sequencing:
  • Origin: Not explicitly stated, but "patient samples" were used. Given the FDA submission context, it's highly likely these were de-identified samples from the United States.
  • Nature: The description "Each site tested its own patient samples" and that they were "from patients using or have used warfarin" suggests these were retrospective samples, collected from a patient population relevant to the intended use.
• Inter-Laboratory Reproducibility:
  • Origin: Not explicitly stated, but likely from the United States given the submission.
  • Nature: Controlled study using "seven genomic DNA samples and five whole blood samples." These would be prospective in the sense that they were specifically prepared and distributed for this reproducibility study, though the original source of the genetic material might have been retrospective.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications

The document does not mention the use of experts to establish ground truth.

For the Agreement with Bi-directional Sequencing study, the ground truth was established by:

• Bi-directional DNA sequencing. This is a recognized laboratory method for determining genetic sequences and is considered the gold standard for many genetic variants. Thus, the ground truth was established by laboratory method, not expert consensus.
• The document implies that the sequencing results were accepted as the definitive truth without the need for expert adjudication or review.

For the Inter-Laboratory Reproducibility study, the ground truth for the 7 genomic DNA samples and 5 whole blood samples was their known genotypes, which would have been previously determined, presumably also by a method like bi-directional sequencing.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method involving multiple human reviewers for the test set.

• In the "Agreement with Bi-directional Sequencing" study, "bi-directional sequencing" served as the comparator/ground truth. The device results were directly compared to these sequencing results.
• For the inter-laboratory reproducibility study, device results from different sites and operators were compared to the known genotype of the samples.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was done. This study assesses the improvement of human readers with AI assistance versus without AI assistance. The INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin is an in vitro diagnostic device for genotype detection, not an imaging or diagnostic support tool that assists human readers. Its output is a genotype call, not an interpretation that radiologists or other human experts would then refine or improve upon.

6. Standalone Performance Study (Algorithm Only)

Yes, a standalone performance study was done. The entire submission details the performance of the "INFINITI 2C9 & VKORC1 Multiplex Assay for Warfarin" as a standalone device. The device automates key steps and provides genotype calls:

• "Steps (c) through (f) are automated by the INFINITI Analyzer."
• "The INFINITI Analyzer automates the 2C9 and VKORC1 assays and integrates all the discrete processes of sample (PCR amplicon) handling, reagent management, hybridization, detection, and results analysis. The assays are processed automatically and read by the built-in confocal microscope. Results are analyzed and presented as genotype calls."
• The performance metrics (agreement with sequencing, reproducibility, LOD, specificity, etc.) are all measures of the algorithm's direct output.

There is no human-in-the-loop component described for its routine operation or performance evaluation.

7. Type of Ground Truth Used

The primary ground truth used for the performance studies was established laboratory method results, specifically bi-directional DNA sequencing. This method is considered highly accurate for determining DNA sequences and genetic variants.

8. Sample Size for the Training Set

The document does not specify a sample size for a training set. This is typical for in vitro diagnostic devices based on established molecular biology principles (PCR, hybridization, genotyping microarrays) where the design is more mechanistic and less dependent on machine learning models requiring extensive "training data" in the conventional sense. The "development" of the assay mentioned for analytical specificity would involve testing various known samples, but these are generally considered part of the assay development and validation rather than a distinct "training set" for an algorithm in the AI context.

9. How the Ground Truth for the Training Set Was Established

Since no explicit training set is detailed, the method for establishing its ground truth is also not described. If one were to consider the samples used during "assay development" as a form of training/optimization, their ground truth would also have been established by known genomic samples (e.g., cell lines with characterized genotypes, or samples sequenced by a gold-standard method).

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The Verigene Warfarin Metabolism Nucleic Acid Test is an in vitro diagnostic for the detection and genotyping of the *2 and *3 alleles of the CYP2C9 gene and a single-point polymorphism (C to T at position 1173) of the VKORC1 gene, from EDTA-anticoagulated whole blood samples, as an aid in the identification of patients at risk for increased warfarin sensitivity. The test is intended to be used on the Verigene System.
The Verigene System is an in vitro diagnostic device intended for processing and genotyping multiple genes in a DNA sample utilizing gold nanoparticle probe technology. The Verigene System consists of the Verigene Processor and the Verigene Reader, each with its own onboard proprietary software.

The Verigene System is an in vitro diagnostic device for processing and genotyping multiple genes in a DNA sample. The Verigene System consists of two instruments, the Verigene Processor and the Verigene Reader, and utilizes single-use, disposable Test Cartridges to process and genotype multiple genes in a DNA sample in approximately 1½ hours.
The analysis sequence is the same for each of the three tests (i.e., CYP2C9*2 and *3 and VKORC1). After extracted and purified DNA, mixed with hybridization buffer, is loaded into the sample well of the Test Cartridge, it is ready for processing and is inserted into the Verigene Processor. An internal barcode reader reads the cartridge ID and sends the information to the Verigene Reader. From this information, the Verigene Reader establishes the hybridization parameters and starts the hybridization process.
The genotyping process occurs with a hybridization of the target analyte to a synthetic gene-specific oligonucleotide capture strand on the Test Cartridge's substrate. A synthetic mediator target-specific oligonucleotide is included with the test-specific sample buffer to form a hybridization "sandwich" with the gene sequence of interest. Washing steps following the target hybridization remove the unbound DNA from the hybridization chamber. A probe, composed of a gold nanoparticle with covalently bound oligonucleotides complementary to a sequence on the intermediate oligonucleotide, is introduced after the target wash. After the probe hybridization is completed, a series of washing steps remove the unbound probe from the hybridization chamber. A two-part signal enhancement reagent is added to the hybridization chamber and reacts with the gold nanoparticle to amplify the signal for the Verigene Reader scanning and analysis.
Upon completion of the genotyping process, the user removes the Test Cartridge from the Verigene Processor which is now ready for the next test.
Once the reagent portion of the Test Cartridge is removed by the user, the substrate is inserted into the Verigene Reader. The Verigene Reader illuminates the signal-enhanced nanoparticles specifically bound to either the wild type or mutant captures for the gene. A photosensor reads the relative brightness of each spot and the Verigene Reader outputs a result based on relative levels of brightness of the wild type to mutant signals.

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Acceptance Criteria and Device Performance

The document doesn't explicitly state "acceptance criteria" in a separate section with pass/fail metrics. Instead, it presents performance characteristics (reproducibility and accuracy) as evidence of the device's suitability. Based on the data provided, the implied acceptance criteria would be high percent agreement (call rate) for genotyping across different sites and conditions, and no incorrect calls (mis-calls).

Here's a table summarizing the reported device performance, interpreted as meeting implied acceptance for accuracy and call rate:

Note on "Acceptance Criteria": The document focuses on demonstrating performance rather than explicitly stating pre-defined thresholds the device must meet. However, the data presented, especially the 0% incorrect calls and generally high correct call rates, suggests these were the implicit criteria for acceptable performance. The "No Calls" indicate samples where a definitive genotype could not be assigned, which is different from an incorrect call.

Study Details:

1. Sample size used for the test set and the data provenance:

   • Accuracy Study (Test Set): 238 samples.
   • Reproducibility (Initial Study): 5 genomic DNA samples, tested in triplicate daily for 3 days at each of 3 sites (total 90 test runs per locus at site 1, 45 test runs per locus at sites 2 & 3).
   • Reproducibility (Extraction Method Study): Panel of 23 blood specimens. At each of the 3 sites, each specimen was extracted and run once, with a re-test if there was a "no call."
   • Limit of Detection Study: 12 cartridges per DNA concentration level.
   • Data Provenance: The document does not explicitly state the country of origin for the samples or specify if the studies were retrospective or prospective. It states "All purified DNA samples were from whole blood collected using EDTA as the anticoagulant" for the accuracy study, and "aliquots of a panel of 23 blood specimens were utilized" for the extraction method reproducibility. This suggests they were clinical samples, but further detail is not provided.

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

   • The ground truth for the accuracy study was established by bi-directional sequencing analysis at an independent reference laboratory. The document does not specify the number of experts or their qualifications, but the method itself (bi-directional sequencing) is a gold standard for genetic genotyping.
   • For the second reproducibility study (extraction method), the "genotypes of the DNA samples were confirmed by bidirectional sequencing." Again, the number and qualifications of experts are not specified, but the ground truth method is clear.

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

   • The document does not describe any human adjudication method for the test set results. The comparison is made directly between the device's genotype calls and the results from bi-directional sequencing.

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

   • No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is a molecular diagnostic test for genotyping, not an imaging device that requires human interpretation or assistance from AI. The output of the device is a genotype call.

5. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done:

   • Yes, the performance studies described (reproducibility, accuracy, limit of detection) represent standalone performance of the Verigene System and Verigene Warfarin Metabolism Nucleic Acid Test. The device provides a direct genotype output, and its performance is evaluated against a ground truth without human intervention in the interpretation of the primary result. The summary states, "The Verigene Reader outputs a result based on relative levels of brightness of the wild type to mutant signals."

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

   • The primary ground truth used was bi-directional DNA sequencing, which is a highly accurate molecular method for determining DNA sequences and identifying genetic variants.

7. The sample size for the training set:

   • The document does not provide details on a separate "training set" or its sample size. The studies described are performance validation studies (accuracy, reproducibility, LOD) rather than studies focused on the development and training of a machine learning algorithm. The Verigene System utilizes "gold nanoparticle probe technology" and "onboard proprietary software" but does not explicitly mention a machine learning or AI component requiring a traditional training set in the context of this 510(k) summary.

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

   • As no explicit "training set" or description of its characterization is provided in the document, there's no information on how its ground truth was established.

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