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The Xpert® FII & FV test is a qualitative in vitro diagnostic genotyping test for the detection of Factor V alleles from sodium citrate or EDTA anticoagulated whole blood. The test is performed on the GeneXpert® Instrument Systems. This test is intended to provide results for Factor II (G20210A) and Factor V Leiden (G1691A) mutations as an aid in the diagnosis in individuals with suspected thrombophilia.

Xpert FII & FV is an automated genotyping test for detecting Factor II and Factor V normal and mutant alleles directly from sodium citrate or EDTA anticoagulated whole blood specimens. Blood specimens are drawn into either sodium citrate or EDTA anticoagulant tubes. Following brief mixing of the sample. 50 uL of the blood sample is transferred to the bottom wall of the Sample opening of the Xpert FII & FV test cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert Instrument System.

The Xpert FII & FV test includes reagents for the detection of Factor II and Factor V normal and mutant alleles. The primers and probes in the Xpert FII & FV test determine the genotype of the Factor II gene (at position 20210) and/or the Factor V gene (at position 1691). The test includes a Sample Processing Control (SPC) to confirm adequate processing and to monitor the presence of inhibitor(s) in the PCR assay. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The GeneXpert Instrument Systems family is comprised of GeneXpert Dx System, GeneXpert Infinity System, and GeneXpert System with Touchscreen. The GeneXpert Instrument Systems automate and integrate sample processing, nucleic acid amplification and detection of the target sequences in simple or complex samples using real-time polymerase chain reaction (PCR). The systems consist of an instrument, computer or touchscreen, and preloaded software for running the tests and viewing the results. The GeneXpert Instrument Systems require the use of singleuse disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is minimized.

The GeneXpert Instrument Systems have 1 to 80 modules (depending upon the instrument) that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.

The Xpert FII & FV test performed on the GeneXpert Instrument Systems provides results in approximately 30 minutes.

This document is a 510(k) summary for the Xpert FII & FV diagnostic test, seeking to remove a limitation statement and make minor branding/catalog number changes. It does not contain a study explicitly detailing acceptance criteria and performance against those criteria for the current submission. Instead, it refers to prior clearance (K082118) for the clinical validation supporting the removal of the limitation statement, and asserts that the current changes do not impact performance.

Therefore, the following information is extracted or inferred based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance:

The document explicitly states: "No new performance data were provided in this submission." The basis for substantial equivalence relies on the device being identical to its predicate (K223046) in all technological characteristics relevant to performance. The one significant change (removal of the pediatric patient limitation) is supported by prior clinical validation data (K082118). Since no new performance data or acceptance criteria are presented for this specific submission, this table cannot be fully completed from the provided text.

However, based on the nature of a genetic mutation detection system, typical acceptance criteria would involve analytical sensitivity, analytical specificity, and clinical performance (e.g., concordance with a gold standard). Without the original K082118 submission, specific numerical criteria are not available.

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

• Sample Size: Not explicitly stated in this document. The document refers to "clinical validation data that was submitted and reviewed as part of the original device clearance (K082118)" as the basis for removing the pediatric limitation. The sample size for that original study is not provided here.
• Data Provenance: Not explicitly stated in this document. Given it's a 510(k) for a US market device, it is likely the original clinical validation (K082118) included US data, but this is not confirmed. It refers to "clinical validation data," which typically implies prospective collection of patient samples.

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

• Not explicitly stated in this document. This level of detail would typically be found in the original clinical validation report (K082118). For genotyping, ground truth is usually established by orthogonal molecular methods, not primarily by expert consensus in the same way as imaging or pathology interpretation.

4. Adjudication Method for the Test Set:

• Not applicable/Not mentioned. For genetic tests where the ground truth is often established through well-defined molecular techniques (e.g., Sanger sequencing or a validated reference method), adjudication by multiple experts in the traditional sense (like for imaging reads) is generally not performed. The ground truth is objective.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done:

• No, an MRMC comparative effectiveness study was not done. This type of study is relevant for interpretive tasks (e.g., radiologists reading images) where human performance is being evaluated and compared with and without AI assistance. The Xpert FII & FV is an automated genotyping test; it does not involve human readers interpreting results in the same manner.

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

• Yes, the performance characteristics mentioned (e.g., analytical sensitivity, specificity) for a molecular diagnostic test like the Xpert FII & FV are inherently "standalone" in nature. The device itself performs the assay and provides a qualitative genotype call. The results are automated and interpreted by the diagnostic software.

7. The Type of Ground Truth Used:

• Not explicitly stated in this document, but for genetic tests, the ground truth is typically established by orthogonal molecular methods, such as Sanger sequencing or another highly accurate, validated reference genotyping method. It is not based on expert consensus, pathology, or outcomes data in the way these terms are typically used for imaging or disease diagnosis.

8. The Sample Size for the Training Set:

• Not explicitly stated in this document. The device uses real-time PCR for detection, and while there might be algorithm tuning within the software, the foundational "training" often refers to the design and optimization of primers and probes, and establishment of cut-offs, rather than machine learning on a large training set of clinical samples. The document refers to "clinical validation data" for performance, but not specifically to a training set size for an algorithm.

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

• Not explicitly stated in this document. This would be part of the original developmental studies for the device (predating K082118). For a PCR-based test, ground truth for developing and optimizing the assay would involve samples with known genotypes confirmed through reference methods.

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The Xpert® FII & FV test is a qualitative in vitro diagnostic genotyping test for the detection of Factor II and Factor V alleles from sodium citrate or EDTA anticoagulated whole blood. The test is performed on the Cepheid GeneXpert® Instrument Systems. This test is intended to provide results for Factor II (G20210A) and Factor V Leiden (G1691A) mutations as an aid in the diagnosis in individuals with suspected thrombophilia.

The Cepheid Xpert® FII & FV is a rapid, automated DNA test for detecting FII and FV normal and mutant alleles directly from sodium citrate or EDTA anticoagulated whole blood specimens. Blood specimens are drawn into either sodium citrate or EDTA anticoagulant tubes. Following brief mixing of the sample, 50 uL of the blood sample is transferred to the bottom wall of the Sample opening of the Xpert FII & FV test cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert® Instrument system instrument platform (comprised of the GeneXpert Dx Systems and GeneXpert Infinity Systems), which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of DNA. In the GeneXpert Instrument Systems platform, sample preparation, amplification, and real-time detection are all fully-automated and completely integrated.

The GeneXpert Instrument Systems have 1 to 80 randomly accessible modules, depending upon the instrument, that are each capable of performing separate sample preparation and real- time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.

The Xpert FII & FV test includes reagents for the detection of Factor II and Factor V normal and mutant alleles. The primers and probes in the Xpert FII & FV test determine the genotype of the Factor II gene (at position 20210) and the Factor V gene (at position 1691). The test includes a Sample Processing Control to confirm adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR assay. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The test is performed on the Cepheid GeneXpert Instrument Systems, which automate and integrate sample purification, nucleic acid amplification of the target sequences in simple or complex samples using real-time PCR. The systems consist of an instrument, personal computer, and preloaded software for running the tests and viewing the results. The GeneXpert Instrument Systems require the use of single-use disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, crosscontamination between samples is minimized. In this platform, additional sample preparation, amplification, and real-time detection are all fully- automated and completely integrated. The Xpert FII & FV test performed on the GeneXpert Instrument Systems provides results in approximately 30 minutes.

The FDA 510(k) summary for the Cepheid Xpert® FII & FV device outlines its performance and validation, primarily focusing on demonstrating substantial equivalence to a predicate device when used with new instrument systems (GeneXpert Infinity Systems).

Here's an analysis of the provided information against your requested criteria:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not provide a formal table of quantitative acceptance criteria with corresponding performance metrics like sensitivity, specificity, accuracy, etc. This is typical for a Special 510(k) submission where the primary objective is to demonstrate that a change (new instrument compatibility) does not negatively impact the existing, previously cleared performance.

Instead, the "Performance Data" section states:

• Acceptance Criteria (Implicit): That the performance claims of the Xpert FII & FV test were not impacted by the use of the GeneXpert Infinity Systems.
• Reported Device Performance: This was assessed through verification studies, including a "Cartridge Hold Time study" and a "Functional Testing study." The document explicitly states that the results "determined that the performance claims of the Xpert FII & FV test were not impacted."

Therefore, the performance is essentially stated as "comparable to the predicate device and original claims," rather than providing new specific rates (e.g., "99% sensitivity," "98% specificity").

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

The document does not explicitly state the sample sizes for the "Cartridge Hold Time study" and "Functional Testing study." It only mentions that these were "verification studies."

• Data Provenance: Not specified in terms of country of origin.
• Retrospective or Prospective: Not explicitly stated, but typically, verification studies like these for in vitro diagnostic devices would involve prospective testing of samples under controlled conditions to assess functionality and stability on the new instrument.

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

This type of information is not applicable to this device and study. The Xpert® FII & FV test is a qualitative in vitro diagnostic genotyping test for detecting specific DNA mutations (Factor II and Factor V alleles). The "ground truth" for such a device is established by the known genetic status of the samples used in the studies (e.g., confirmed Factor II or Factor V mutation presence/absence via reference methods or defined synthetic constructs), not by expert human interpretation of images or other subjective data. No human experts are used to establish ground truth for this kind of molecular diagnostic test performance.

4. Adjudication Method for the Test Set

Not applicable. As explained above, the "ground truth" for a genotyping test is based on direct molecular confirmation, not on subjective interpretations requiring adjudication.

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

Not applicable. The Xpert® FII & FV is an automated diagnostic test that provides a definitive genetic result. It does not involve human readers interpreting output that would be improved with AI assistance, nor is it a diagnostic imaging device where MRMC studies are common.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

This is the primary mode of operation for this device. The Xpert® FII & FV test is an automated system as described: "perform[s] hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of DNA." The results ("Result," "Not Detected," or "Invalid") are generated by the instrument's internal algorithms based on PCR amplification signals. The human interaction is limited to sample loading and initiating the test. Therefore, the "Functional Testing study" and "Cartridge Hold Time study" effectively assess the standalone performance of the algorithm and instrument system.

7. The Type of Ground Truth Used

The ground truth for a genotyping test is based on the presence or absence of specific DNA sequences/mutations. While not explicitly stated how this truth was established for the specific "verification studies," for molecular diagnostic tests, ground truth is typically confirmed using:

• Reference molecular methods: Such as Sanger sequencing or highly validated in-house PCR assays.
• Characterized samples: Samples with known genetic status (e.g., from biobanks, synthetic constructs, or previously validated patient samples).
• Clinical diagnosis/outcomes data: In some cases, to correlate the genetic findings with the clinical presentation, although for genotyping tests, the primary ground truth is molecular.

Given the nature of this device, it would be based on the confirmed genetic status of the samples.

8. The Sample Size for the Training Set

This information is not provided. As this is a Special 510(k) for an instrument change, the focus is on verification rather than a full re-validation involving new training sets. The underlying algorithms and core test technology (primers, probes, PCR) remain the same as the predicate device. Training sets would have been used in the original development and validation of the Xpert® FII & FV test (K082118).

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

Not provided in this document as it pertains to the original development of the predicate device (K082118), not this Special 510(k). For the original development, ground truth would have been established as described in point 7 (reference molecular methods, characterized samples).

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The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time PCR for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation and the Factor V Leiden G1691A mutation in genomic DNA obtained from K2EDTA whole blood specimens as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and the cobas z 480 analyzer are used together for automated amplification and detection.

The cobas® Factor II and Factor V Test is a real-time polymerase chain reaction (PCR) test for the qualitative detection and genotyping of a single point mutation in the human Factor V gene (G to A at position 1691, referred to as the Factor V Leiden mutation) and a single point mutation in the human Factor II gene (G to A at position 20210), in genomic DNA isolated from peripheral whole blood, as an aid in diagnosis of patients with suspected thrombophilia.

DNA is extracted offline from whole blood specimens collected in K2EDTA tubes. The userselected DNA extraction method must provide DNA of sufficient concentration. Automated realtime PCR is then performed on the cobas z 480 analyzer. The Factor II and Factor V mutations are detected simultaneously in the same real-time PCR reaction. Depending upon the test order, results for one or both mutations are reported for each DNA sample.

The assay consists of one reagent kit and a system platform. The reagent kit provides the necessary reagents and controls to perform automated real-time PCR amplification and detection. The system platform consists of a real-time PCR thermal cycler (cobas z 480 analyzer) and a control unit (cobas® 4800 System CU). The amplification reactions generate a Factor II specific amplicon and a Factor V specific amplicon in all samples, and utilize four fluorescent-dye labeled oligonucleotide probes for detection of the Factor II and Factor V genotypes.

Acceptance Criteria and Study for cobas® Factor II and Factor V Test

This document outlines the acceptance criteria and the studies performed to demonstrate that the cobas® Factor II and Factor V Test meets these criteria.

1. Table of Acceptance Criteria and Reported Device Performance

Factor II (Prothrombin) G20210A Mutation Detection:

Factor V Leiden G1691A Mutation Detection:

Analytical Sensitivity (Lower Limit of Detection):

Analytical Sensitivity (Upper Limit of Detection):

DNA Extraction Method Study:

Lot-to-Lot Repeatability:

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

Method Comparison Study:

• Sample Size: 300 specimens.
• Data Provenance: The specimens were obtained from patients whose routine medical care called for Factor II and/or Factor V measurements, representing an intended use population. The commercial vendors provided these samples. The type of data (retrospective/prospective) is not explicitly stated, but the description of samples being "obtained to represent the intended use population" and collected for "routine medical care" suggests a retrospective collection or at least samples collected for clinical indications. The provenance is likely a mix of clinical labs or commercial biobanks.

Clinical Reproducibility Study:

• Sample Size: A 9-member panel was used, consisting of 4 unique K2EDTA blood samples, 3 contrived blood samples, and 2 extracted genomic DNA samples. Each panel member was tested multiple times across different sites, operators, instruments, runs, and days. A total of 540 tests were performed (with 539 valid results and 1 invalid).
• Data Provenance: The samples included K2EDTA whole blood samples and extracted genomic DNA samples. The "contrived blood samples" suggest laboratory-prepared samples. The "unique K2EDTA blood samples" were likely sourced from similar vendors as the method comparison study. The study was conducted at three test sites (2 external and 1 internal), implying a multi-center study on potentially diverse datasets.

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

The ground truth for both the Method Comparison Study and the Clinical Reproducibility Study was established by bi-directional Sanger sequencing. The document does not specify the number of experts or their qualifications for performing or interpreting the Sanger sequencing results. However, Sanger sequencing is a widely accepted gold standard for genetic mutation detection, and it's typically performed by trained laboratory professionals.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method for the test set where discrepancies between results from the cobas® test and Sanger sequencing were adjudicated by experts. The performance metrics are reported as direct agreements or disagreements with the Sanger sequencing results. In the DNA Extraction Method Study, for instance, one inconsistent triplicate result with a method was re-tested, and all results were correct upon re-testing, implying an internal review for outlier data.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, an MRMC comparative effectiveness study was not done. The cobas® Factor II and Factor V Test is an in vitro diagnostic device for automated amplification and detection of genetic mutations. It is an algorithm-only device; therefore, human readers are not directly involved in its performance or interpretation in a way that would necessitate an MRMC study.

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

Yes, the studies presented are standalone performance evaluations. The cobas® Factor II and Factor V Test is described as an "in vitro diagnostic device that uses real-time PCR for the detection and genotyping" and used with the "cobas z 480 analyzer for automated amplification and detection." This indicates an automated system where the algorithm performs the detection and genotyping without direct human intervention in the result generation process. Human interaction is limited to sample preparation and loading, and interpreting the final automated result.

7. The Type of Ground Truth Used

The type of ground truth used for both the Method Comparison Study and the Clinical Reproducibility Study was expert consensus through bi-directional Sanger sequencing. Sanger sequencing is generally considered the "gold standard" for confirming DNA sequences, and thus, genotypes.

8. The Sample Size for the Training Set

The document does not provide information regarding a separate training set or its sample size. The studies described are performance evaluations (method comparison, reproducibility, analytical sensitivity, etc.) which typically use independent test data to assess the device's accuracy and reliability, assuming the algorithm was developed and optimized prior to these evaluation studies. As this is not an AI/ML device in the traditional sense, but a PCR-based diagnostic, the concept of a "training set" for an algorithm's learning phase is not directly applicable in the same way as it would be for an image recognition AI, for example. The "training" for such a system would involve optimizing primers, probes, and reaction conditions during product development.

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

As mentioned above, the document does not discuss a separate training set. For the validation studies, ground truth was established by bi-directional Sanger sequencing.

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The IMPACT Dx Factor V Leiden and Factor II Genotyping Test is a qualitative in vitro diagnostic device intended for use in the detection and genotyping of a single point mutation (G1691A, referred to as the Factor V Leiden mutation or FVL) of the Factor V gene, located on Chromosome 1q23, and a single point mutation (G20210A) of the prothrombin gene (referred to as Factor II or FII), located on Chromosome 11p11-q12, from genomic DNA isolated from EDTA anti- coagulated human whole blood samples. The test is to be performed on the IMPACT Dx System and is indicated for use as an aid in the diagnosis of patients with suspected thrombophilia.

The IMPACT Dx Factor V Leiden and Factor II Genotyping Test is a qualitative, multiplexed genetic testing device for parallel detection and genotyping of the point mutations G1691A of the Factor V gene and G20210A of the Factor II gene from genomic DNA isolated from EDTA anti- coagulated human whole blood samples. The test is to be performed on the IMPACT Dx System.

The IMPACT Dx Factor V Leiden and Factor II Genotyping Test is performed using the IMPACT Dx System, which includes the IMPACT Dx NANO and the IMPACT Dx MA, a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer. The test involves Factor V and Factor II region-specific polymerase chain reaction (PCR) amplification of genomic DNA purified from human whole blood in a multiplexed reaction, followed by allele-specific single base primer extension reactions. The reaction products are desalted, dispensed onto a SpectroCHIP® Array using the IMPACT Dx NANO, and the genotyping products are resolved on the basis of mass using the IMPACT Dx MA.

The IMPACT Dx Factor V Leiden and Factor II Genotyping Test provides reagents for multiplex PCR, deoxynucleotide triphosphate dephosphorylation, and single base extension. The IMPACT Dx Factor V Leiden and Factor II Genotyping Test is comprised of the following components:

• IMPACT Dx Factor V Leiden and Factor II Primer Set
• IMPACT Dx PCR Reagent Set
• IMPACT Dx Extend Reagent Set

The IMPACT Dx Factor V Leiden and Factor II Genotyping Test utilizes a biochemistry process (Sequenom Biochemistry) that involves target-specific PCR amplification and single-base extension reactions with the subsequent analysis of the reaction products of the target nucleic acids by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS),

The IMPACT Dx System (System) is a platform for highly accurate and sensitive genomic analysis and is designed for use with FDA cleared or approved assays citing its use. The IMPACT Dx System is comprised of the following instruments, software and consumables;

• IMPACT Dx NANO (NANO)
• IMPACT Dx MA (MA)
• TYPER Dx Software (TYPER Dx)
• System Consumables
• SpectroCHIP® Arrays (Chip)
• Clean Resin
• 3-Point Calibrant (Calibrant)

The System is intended to be used by trained operators in a professional laboratory to perform the following key tasks:

• De-salt (using Clean Resin) amplified nucleic acid samples, upon completion of polymerase chain reaction (PCR) and single-base extension reactions following the instructions provided in the Sequenom test-specific package insert;
• Transfer (using the IMPACT Dx NANO) de-salted nucleic acid samples from a microtiter plate onto a disposable 96-pad sample Chip;
• Obtain mass spectra (using the IMPACT Dx MA) from samples and 3-Point Calibrant on a Chip; and
• Analyze (using the TYPER Dx software) the mass spectra of the samples for genotyping results.

The IMPACT Dx System accomplishes genomic analysis and genotyping testing by coupling a biochemistry process (Sequenom biochemistry) that involves target-specific PCR amplification and single-base extension reactions with the subsequent analysis of the reaction products of the target nucleic acids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This biochemistry process is homogeneous and does not require purification of the PCR products or the extension products and thus is very amenable to high-throughput genotyping testing.

The IMPACT Dx NANO is a self-contained, enclosed instrument that uses computer-controlled robotics to transfer nanoliter volumes of analyte from a 96-well microtiter plate onto a Chip, which is subsequently processed by means of MALDI-TOF MS analysis on the IMPACT Dx MA. This instrument includes an integrated computer pre-loaded with the Nanodispenser software and provides a simple touch-screen interface for users.

The IMPACT Dx MA is a bench top mass spectrometer that processes analyte-loaded Chips by means of MALDI-TOF MS analysis. This instrument includes an integrated computer pre-loaded with the TYPER Dx software, a monitor, and a firewall for secure communication with the IMPACT Dx NANO. The main function of the IMPACT Dx MA is to acquire mass spectra from analytes that have been transferred onto a Chip, which has a chemical matrix on each pad. The mass spectra are captured and further analyzed by the TYPER Dx software.

The TYPER Dx software (TYPER Dx) manages the processing of Sequenom genotyping tests. It is deployed on the computer embedded within the IMPACT Dx MA.

The TYPER Dx software provides the following key functions:

• Allows users to create and manage panel runs;
• Monitors analyte transfer activities on the IMPACT Dx NANO;
• Controls user-initiated, automated mass spectrum acquisition runs on the IMPACT Dx MA;
• Analyzes the mass spectra acquired by the IMPACT Dx MA and makes genotype calls per a test-specific algorithm;
• Enables users to view and export results; and
• Allows an administrator to manage users to ensure secure access to the IMPACT Dx MA and panel run data.

Here's a summary of the acceptance criteria and study detailed in the provided document:

Acceptance Criteria and Device Performance

The IMPACT Dx Factor V Leiden and Factor II Genotyping Test aims for high agreement with a reference method for genotyping Factor II and Factor V genes.

Study Details

2. Sample Size and Data Provenance

• Test Set Sample Size:

  • Clinical Data (Method Comparison Study): 860 clinical samples
  • Analytical Specificity: 13 clinical genomic DNA from patient samples
  • Analytical Sensitivity: Not explicitly stated, but clinical genomic DNA from patient samples were used with a series dilution.
  • Carry-over Contamination: A panel of 3 clinical genomic DNA samples and a no template control sample.
  • Interferences: Leukocyte-depleted whole blood specimens spiked with cell lines.
  • Reproducibility: 12 human genomic DNA samples.

• Data Provenance: The document states "clinical genomic DNA obtained from patient samples" and "fresh EDTA anti-coagulated whole blood samples collected from normal human subjects." This implies the data is derived from human clinical samples, likely from retrospective collections for the method comparison and analytical studies, and potentially prospective collection for the fresh blood study. No specific country of origin is mentioned, but the submission is to the FDA (USA).

3. Number of Experts and Qualifications for Ground Truth (Test Set)

Relevant for the method comparison study:

• Number of Experts: Not explicitly stated as "experts." The ground truth was established by a bi-directional DNA sequencing reference method. While sequencing itself is a technical process, the interpretation of sequencing results could involve expert review. However, the document doesn't detail this.
• Qualifications of Experts: Not provided, as the ground truth relied on a reference method (bi-directional sequencing) rather than human expert consensus on image interpretation, for example.

4. Adjudication Method (Test Set)

Not applicable in the context of this document. The study compares the device's results directly against a bi-directional DNA sequencing reference method. Discrepancies would likely be investigated through re-testing or re-sequencing, as implied by the "Number of FII/FVL Calls After Repeat Testing" in the tables, but formal "adjudication" by multiple human readers is not described.

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

No, a MRMC comparative effectiveness study was not explicitly done or reported in this document. The study focuses on the analytical and clinical performance of the device itself, comparing it to a reference sequencing method, not on human reader performance with or without AI assistance.

6. Standalone Performance Study

Yes, a standalone (algorithm only without human-in-the-loop performance) study was performed. The entire context of the "Non-clinical Bench Data" and "Clinical Data" sections describes the performance of the IMPACT Dx Factor V Leiden and Factor II Genotyping Test as a diagnostic device, which processes samples and generates genotype calls automatically or semi-automatically via the IMPACT Dx System's TYPER Dx software. The results (e.g., percent agreement with sequencing) reflect the algorithm's performance.

7. Type of Ground Truth Used

The ground truth used for the clinical data (method comparison study) was bi-directional DNA sequencing. This is considered a highly accurate and established reference method for genotyping.

8. Sample Size for the Training Set

The document does not explicitly mention a separate "training set" or its sample size. The reported studies primarily describe testing the device's performance (analytical and clinical validation) against known samples or a reference method. For in-vitro diagnostic devices, particularly those based on established molecular biology principles, the development might involve iterative testing and refinement, but a formal "training set" in the machine learning sense is not typically documented as a distinct phase with a specified sample size in regulatory submissions like this unless the device employs novel AI/ML components requiring such.

9. How Ground Truth for the Training Set Was Established

As a distinct "training set" is not explicitly mentioned, the method for establishing its ground truth is also not described. If development involved internal studies, it's highly probable that ground truth would have been established using methods similar to the validation, such as DNA sequencing or other orthogonal molecular techniques.

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The eSensor® Warfarin Sensitivity Saliva Test is an in vitro diagnostic test 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 of human saliva samples collected using the Oragene® Dx Device, as an aid in the identification of patients at risk for increased warfarin sensitivity.

The eSensor® Warfarin Sensitivity Saliva Test is a multiplex microarray-based genotyping test system. It is based on the principles of competitive DNA hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to sequence-specific solution-phase signal probe and solid-phase electrode-bound capture probe. The test employs polymerase chain reaction amplification, exonuclease digestion and hybridization of target DNA. In the process, the double stranded PCR amplicons are digested with exonuclease to generate single stranded DNA suitable for hybridization. Hybridization occurs in the eSensor XT-8 Cartridge (described below) where the singlestranded target DNA is mixed with a hybridization solution containing labeled signal probes.

During hybridization, the target DNA binds to a complementary, single-stranded capture probe immobilized on the working electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to the target adjacent to the capture probe. When inserted into the eSensor XT-8 instrument (described below), simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each pair of working electrodes on the array contains a different capture probe, and sequential analysis of each electrode allows genotyping of multiple mutations or polymorphisms.

The Assay Cartridge (eSensor XT-8 Cartridge): The eSensor XT-8 cartridge device consists of a printed circuit board (PCB) with a multi-layer laminate and a plastic cover that forms a hybridization chamber has a volume of approximately 140 µl. The cartridge consists of a diaphragm pump and check valves (microfluidic components) that circulate the hybridization solution in the hybridization chamber when inserted into the eSensor XT-8 instrument. The PCB chip consists of an array of 72 gold-plated working electrodes, a silver/silver chloride reference electrode, and two gold-plated auxiliary electrodes. Each working electrode has a connector contact pad on the opposite side of the chip for electrical connection to the eSensor XT-8 instrument. Each electrode is modified with a multicomponent, self-assembled monolayer that includes presvnthesized oligonucleotide capture probes specific for each polymorphic site on the test panel and insulator molecules. The cartridge also contains an electrically erasable programmable read-only memory component (EEPROM) that stores information related to the cartridge (e.g., assay identifier, cartridge lot number, and expiration date).

The eSensor XT-8 Instrument (Same as cleared under K073720): The eSensor XT-8 is a clinical multiplex instrument that has a modular design consisting of a base module and one, two, or three cartridge-processing towers containing 8, 16, or 24 cartridge slots, respectively. The cartridge slots operate independently of each other. Any number of cartridges can be loaded at one time, and the remaining slots are available for use while the instrument is running. The base module controls each processing tower, provides power, and stores and analyzes data. The base module includes the user interface, and a 15-in. portrait-orientation display and touch panel. The instrument is designed to be operated solely with the touch screen interface. Entering patient accession numbers and reagent lot codes can be performed by the bar code scanner, the touch screen, or uploading a text file from a USB memory stick. Each processing tower consists of eight cartridge modules, each containing a cartridge connector, a precision-controlled heater, an air pump, and electronics. The air pumps drive the diaphragm pump and valve system in the cartridge, eliminating fluid contact between the instrument and the cartridge. The pneumatic pumping enables recirculation of the hybridization solution allowing the target DNA and the signal probes to hybridize with the complementary capture probes on the electrodes. The diaphragm pump in the cartridge is connected to a pneumatic source from the eSensor XT-8 instrument and provides unidirectional pumping of the hybridization mixture through the microfluidic channel during hybridization. Using microfluidic technology to circulate the hybridization solution minimizes the unstirred boundary layer at the electrode surface and continuously replenishes the volume above the electrode that has been depleted of complementary targets and signal probes. The XT-8 instrument provides electrochemical detection of bound signal probes by ACV and subsequent data analysis and test report generating functions. All hybridization, ACV scanning and analysis parameters are defined by a scanning protocol loaded into the XT-8 Software, and then specified for use by the EEPROM on each cartridge.

The Assay Kit: The Warfarin Sensitivity Saliva Test consists of the test cartridge and the following components: 1) PCR REAGENTS consisting of: PCR Mix [PCR buffer containing primers and dNTP mixture (dCTP, dGTP, dATP, and dUTP)], MgCl2 thermostable DNA polymerase (Taq Polymerase ); and 2 GENOTYPING REAGENTS consisting of: lambda exonuclease, signal probes and hybridization buffer ingredients (Buffer-1 and Buffer-2).

Here's a breakdown of the acceptance criteria and the study details for the eSensor® Warfarin Sensitivity Saliva Test, based on the provided document:

Acceptance Criteria and Device Performance

The acceptance criteria are implied by the comparison to DNA sequencing, which is considered the gold standard for genotyping. The device performance is reported as agreement percentages with DNA sequencing.

Note: The document implies acceptance criteria by comparing the device's performance to DNA sequencing, which is implicitly considered the standard for accuracy. Specific numeric thresholds for "acceptance" are not explicitly stated, but the high agreement percentages (mostly 100%) and the lower bounds of the 95% confidence intervals (LCB) suggest a requirement for high concordance.

Study Information

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

• Sample Size: A total of 316 gDNA samples were extracted from saliva specimens for the method comparison study.
• Data Provenance: The document does not explicitly state the country of origin or if the data was retrospective or prospective. It uses "saliva specimen" without further context on donor recruitment or source.

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

• Experts: Not applicable. The "ground truth" for the genotyping results was established by DNA sequencing, not by human experts.
• Qualifications: Not applicable.

4. Adjudication Method for the Test Set:

• Adjudication Method: Not applicable in the traditional sense of human consensus. The reference method (ground truth) was DNA sequencing. The study reports "No-Calls" and "Miscalls" by the eSensor® device when compared to DNA sequencing. The "After Retest" results suggest that initial "No-Calls" were re-evaluated and resolved, leading to higher agreement.

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:

• MRMC Study: No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in-vitro diagnostic (IVD) for genotyping, which is an automated process, not an imaging device requiring human interpretation alongside AI.

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

• Standalone Performance: Yes, the performance reported is essentially a standalone (algorithm only) performance. The eSensor® Warfarin Sensitivity Saliva Test is an automated system that generates results which are then compared to DNA sequencing, without human-in-the-loop interpretation being part of the primary performance evaluation.

7. The Type of Ground Truth Used:

• Ground Truth Type: DNA sequencing (referred to as "bidirectional DNA sequencing" or "DNA Sequencing Result"). This is considered a gold standard for genetic polymorphism detection.

8. The Sample Size for the Training Set:

• Training Set Sample Size: The document does not specify a separate training set. For IVD devices, especially those based on established molecular biology principles, the "training" (development) often involves optimizing reagents and protocols, rather than machine learning algorithm training with distinct datasets. The method comparison study appears to be the primary validation of the device's performance against a reference method.

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

• Training Set Ground Truth: As no explicit training set is mentioned in the context of machine learning, this question is not directly applicable. For the performance validation, as stated above, DNA sequencing was used as the ground truth.

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The Invader® Factor II test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

The Invader Factor II test consists of the following components: Factor II Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor II Wild Type Control, Factor II Heterozygous Control, Factor II Mutant Control, Invader Call Reporter™ Software, Invader® Factor II Software.

This document describes the Invader® Factor II test, an in vitro diagnostic test for detecting a specific mutation (G20210A) in the human Factor II gene, associated with thrombophilia.

Here's an analysis of the acceptance criteria and supporting studies:

1. Table of Acceptance Criteria and Reported Device Performance

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

• External Reproducibility (Study #1):
  • Sample Size: 9 unique leukocyte-depleted whole blood samples (3 WT, 3 HET, 3 MUT), tested in duplicate over 5 days by 2 operators at 3 sites. Total tests: 9 samples * 2 duplicates * 5 days * 2 operators * 3 sites = 540 initial tests.
  • Data Provenance: Not explicitly stated, but implied to be from internal and external validation sites. Likely retrospective as they were "spiked cell lines."
• Lot-to-Lot Reproducibility (Study #9):
  • Sample Size: 4 genomic DNA samples (3 WT, 1 HET), tested in quadruplicate using 3 different kit lots. Total tests: 4 samples * 4 replicates * 3 lots = 48 tests.
  • Data Provenance: Not explicitly stated. Likely retrospective as they are "genomic DNA samples."
• Real-Time Stability Study (Study #5):
  • Sample Size: 3 controls (WT, HET, MUT) and 4 gDNA samples (WT, WT, HET, MUT), tested in quadruplicate at each time point (T0, T4, T7) for 3 lots. Total tests are not explicitly summed, but 7 sample types * 4 replicates * 3 time points * 3 lots = 252 tests.
  • Data Provenance: Not explicitly stated. Likely retrospective (genomic DNA and controls).
• Reagent Freeze-Thaw Stability Study (Study #6):
  • Sample Size: 3 Controls (WT, HET, MUT) and 3 gDNA samples (WT, HET, MUT) with varying numbers of replicates tested at each of 15 freeze/thaw cycles. Total tests: 255.
  • Data Provenance: Not explicitly stated. Likely retrospective (genomic DNA and controls from cell lines).
• Analytical Sensitivity and Normal Range (Study #3):
  • Sample Size: 2 genomic DNA samples (1 HET, 1 WT), each diluted to 8 concentrations and tested in 40 replicates. Total tests: 2 samples * 8 concentrations * 40 replicates = 640 tests.
  • Data Provenance: Whole blood collected in potassium EDTA, then extracted and diluted. Retrospective for the samples, but the dilution and testing process is controlled.
• Analytical Specificity (Interfering Substances) (Study #4):
  • Sample Size: 4 whole blood samples differing genotype (3 WT, 1 HET), each with 9 different interfering substances/conditions. Total tests: 4 samples * 2 (with/without substance) * number of replicates not specified, but results given as "8 of 8" for percent agreement, suggesting at least 8 replicates per condition/sample. Total presented data points: 8 conditions * 8 replicates = 64 tests.
  • Data Provenance: Whole blood samples. Retrospective.
• Pre-Analytical Equivalency Study (Study #7):
  • Sample Size: 30 human whole blood samples and 10 leukocyte-depleted whole blood samples (total 40 samples), extracted using 4 different methods. Each resulting DNA analyzed in singlicate. Total tests: 40 samples * 4 extraction methods = 160 tests.
  • Data Provenance: Human whole blood samples. Implied retrospective for the samples themselves.
• Instrument Equivalency (Study #8):
  • Sample Size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples (total 39 samples), extracted (using 2 methods). These extracts tested with the device on 3 thermal cyclers and raw data acquired on 3 fluorometers. Total presented for agreement: 78 tests per thermal cycler/fluorometer combination (likely 39 samples tested in duplicate, or 39 samples * 2 extraction methods = 78 sample preparations). Total 78 across all combinations * 3 thermal cyclers * 3 fluorometers = 702 measurements.
  • Data Provenance: Human whole blood samples. Implied retrospective.
• Secondary Polymorphism Impact (Study #10):
  • Sample Size: 6 samples (1 homozygous normal, 1 heterozygous, 4 homozygous normal each with a known secondary polymorphism). 40 replicates for each sample. Total tests: 6 samples * 40 replicates = 240 tests.
  • Data Provenance: Samples with known genotypes and secondary polymorphisms. Likely retrospective.
• Method Comparison (Study #2):
  • Sample Size: 336 human whole blood samples.
  • Data Provenance: Human whole blood samples. Retrospective.

3. Number of Experts and Qualifications for Ground Truth

• General Ground Truth Method: The primary and most frequently cited ground truth method across all studies is bi-directional DNA sequencing.
• Number of Experts: The document does not specify the number of experts used for establishing ground truth via bi-directional DNA sequencing. Sequencing is typically a highly automated process with results interpreted by trained molecular biologists or laboratory staff, rather than a consensus of "experts" in the clinical sense (like radiologists).
• Qualifications of Experts: Not specified. It's assumed that standard molecular biology laboratory practices for sequencing and interpretation were followed.

4. Adjudication Method for the Test Set

• Not Applicable in the traditional sense. For diagnostic tests like this, adjudication by multiple human experts (e.g., radiologists) is not a typical part of ground truth establishment.
• The ground truth is established by a definitive molecular method (bi-directional DNA sequencing).
• In the external reproducibility study (Study #1), two initial "No Call" results were resolved by retesting, which then agreed with the sequencing results. This implies a retesting/re-evaluation process for indeterminate results, rather than expert adjudication of the initial "No Call."

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

• No, an MRMC comparative effectiveness study was not done.
• This device is an in vitro diagnostic test for genotyping, where the output is a molecular result (genotype). It does not involve human readers interpreting images or data where AI assistance would directly improve human performance in the same way as, for example, a radiology AI.
• The "Invader Call Reporter™ Software" mentioned is for data analysis and conversion of raw fluorescence data into genotype calls, not for assisting human interpretation that would typically be evaluated in an MRMC study.

6. Standalone Performance Study

• Yes, a standalone performance study was done.
• The entire set of analytical performance studies (Reproducibility, Stability, Analytical Sensitivity, Analytical Specificity, Extraction Equivalency, Instrument Equivalency, Secondary Polymorphism Impact) and the method comparison study against bi-directional sequencing demonstrate the standalone performance of the algorithm/device system.
• The device takes raw fluorescence data and, through its software ("Invader Call Reporter™ Software" and "Invader® Factor II Software"), outputs a genotype call. The studies rigorously validate that these automated calls are accurate and reproducible compared to the gold standard of sequencing. The system is designed to provide automated calls, not to serve as an aid to a human reader's interpretation in a separate step.

7. Type of Ground Truth Used

• The primary ground truth used across all key performance studies is bi-directional DNA sequencing. This is considered a gold standard molecular method for determining specific genetic mutations.
• For controls, "expected genotype" based on the composition of the control material (e.g., cell lines) is used.

8. Sample Size for the Training Set

• The document does not explicitly describe a separate "training set" for the device's algorithm.
• For molecular diagnostic assays like this, the algorithm (e.g., for converting fluorescence data to genotype calls) is typically developed based on known statistical models and pre-defined thresholds related to the chemistry, rather than being "trained" on a large dataset of classified samples in the machine learning sense.
• The various controls (No DNA, WT, HET, MUT) are used within each run to validate performance and inform signal-to-noise calculations, serving as internal calibration rather than an external training set for an AI model.

9. How Ground Truth for the Training Set Was Established

• As a formal "training set" is not explicitly mentioned or implied in the context of supervised machine learning, the process of establishing ground truth for such a set is not described.
• However, the underlying principles for the chemistry and software's interpretive rules would have been developed using well-characterized samples with ground truth established by methods like DNA sequencing during the research and development phase before formal validation studies.

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The Invader® Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

The Invader Factor V test consists of the following components: Factor V Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor V Wild Type Control, Factor V Heterozygous Control, Factor V Mutant Control, Invader Call Reporter™ Software, Invader® Factor V Software.

Here's an analysis of the acceptance criteria and supporting study details for the Invader® Factor V test, based on the provided text:

Acceptance Criteria and Device Performance for Invader® Factor V Test

1. Table of Acceptance Criteria and Reported Device Performance

The submission primarily focuses on analytical performance criteria, demonstrating the assay's reliability and accuracy in detecting the Factor V G1691A mutation.

2. Sample Size for Test Set and Data Provenance

• External Reproducibility (Study #1):
  • Sample Size: 9 unique leukocyte-depleted whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant) spiked cell lines.
  • Provenance: "Spiked cell lines" implies controlled, laboratory-prepared samples. The text does not specify the country of origin, but the study was conducted at "3 different sites (2 external sites and 1 internal site)", suggesting multi-center testing, likely within the US given the FDA submission. This was a prospective study where samples were tested newly.
• Lot-to-Lot Reproducibility (Study #9):
  • Sample Size: 5 genomic DNA samples (3 wild type, 2 heterozygous).
  • Provenance: Not explicitly stated, likely laboratory-prepared or acquired genomic DNA.
• Real-Time Stability (Study #5):
  • Sample Size: Samples representing all three genotypes (WT, HET, MUT) (number not explicitly stated, but tested in quadruplicate at each time point). Also mentions three product lots.
  • Provenance: Likely laboratory-prepared/controlled samples.
• Reagent Freeze-Thaw Stability (Study #6):
  • Sample Size: Controls (WT, HET, MUT) and gDNA (WT, HET, MUT). Total of 255 tests.
  • Provenance: Genomic DNA isolate from cell lines.
• Detection limit/Analytical Sensitivity and Normal Range (Study #3):
  • Sample Size: 2 genomic DNA samples (1 WT, 1 HET), each diluted to 8 concentrations and tested in 40 replicates (total 2 * 8 * 40 = 640 tests).
  • Provenance: Genomic DNA extracted from whole blood collected in potassium EDTA.
• Analytical specificity (Study #4):
  • Sample Size: 4 whole blood samples (3 WT, 1 HET). Each substance tested on 8 of these samples.
  • Provenance: Human whole blood samples.
• Pre-Analytical Equivalency Study (Study #7):
  • Sample Size: 30 human whole blood samples and 10 leukocyte-depleted whole blood spike cell lines (total 40 unique samples). These were processed by 4 different extraction methods, leading to 160 extracted DNAs.
  • Provenance: Human whole blood samples and cell lines.
• Instrument Equivalency (Study #8):
  • Sample Size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 unique samples). These were tested across various instrument combinations, leading to 78 samples per combination (likely some samples were tested multiple times or a subset of the 39 samples were used to create 78 data points.
  • Provenance: Human whole blood samples and cell lines.
• Secondary Polymorphism Impact (Study #10):
  • Sample Size: 5 samples (one homozygous normal, one heterozygous, and three homozygous normal with different secondary polymorphisms). Tested in 40 replicates each (total 5 * 40 = 200 tests).
  • Provenance: Known Factor V genotypes with or without secondary polymorphisms.
• Method comparison (Bi-directional Sequencing) (Study #2):
  • Sample Size: 352 human whole blood samples (289 Homozygous Wild Type (GG), 45 Heterozygous (GA), 18 Homozygous Mutant (AA)).
  • Provenance: Human whole blood samples. The text does not specify the country of origin, but it is implied to be a retrospective collection based on the phrasing "underwent DNA extraction and subsequent bi-directional DNA sequence analysis" then "The same DNA samples were then analyzed using the Invader® Factor V test."

3. Number of Experts and Qualifications for Ground Truth

The primary method for establishing ground truth across most studies is bi-directional DNA sequencing. The text does not specify the number of experts or their specific qualifications (e.g., molecular geneticists) used to interpret these sequencing results. However, bi-directional sequencing is a standard and highly accurate method for genotyping, and its interpretation would typically be performed by trained molecular diagnosticians or geneticists.

4. Adjudication Method for the Test Set

The text does not explicitly describe an adjudication method (e.g., 2+1, 3+1). For discrepancies, the sequencing results are considered the gold standard to which the device's results are compared. In cases like the "No Call" results in Study #1, troubleshooting and retraining of the operator were performed, and retesting was done against sequencing as the reference.

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

No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done.

This device (Invader® Factor V test) is an in vitro diagnostic assay for genotyping, not an AI-assisted diagnostic imaging or clinical decision support system that would typically involve human readers. Its output is a genotype call (WT, HET, MUT), which is then interpreted by a healthcare professional. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" does not apply to this type of device.

6. Standalone (Algorithm Only) Performance

Yes, the standalone performance (algorithm only without human-in-the-loop performance) was done and is the primary focus of the performance studies.

The Invader® Factor V test is an automated in vitro diagnostic system. The "Invader® Factor V Software, in combination with Invader Call Reporter™ software," processes raw fluorescence data to determine genotype calls (WT, HET, MUT). All the performance studies described (Precision, Stability, Sensitivity, Specificity, Method Comparison, etc.) evaluate the accuracy of these software-generated genotype calls against a recognized gold standard (bi-directional sequencing). There is no "human-in-the-loop" step described for data interpretation that would alter the genotype call generated by the software. The human operator's role is primarily experimental execution and loading/reviewing results from the software.

7. Type of Ground Truth Used

The primary type of ground truth used across all studies is bi-directional DNA sequencing. Where applicable, expected genotypes from known control materials (cell lines) also served as ground truth. This is a highly accurate and widely accepted method for determining genetic mutations.

8. Sample Size for the Training Set

The document does not explicitly mention a separate "training set" for the device's algorithm. For in vitro diagnostic kits like the Invader® Factor V test, the "training" (development and optimization) phase would typically involve a proprietary set of samples used by the manufacturer to establish the assay parameters, cutoff values for fluorescence, and the logic within the software for genotype calling. These samples are not typically disclosed or enumerated as a formal "training set" in regulatory submissions but are part of the overall development process. The studies described are validation studies (test sets) demonstrating the finalized device's performance.

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

Since a formal "training set" is not explicitly defined in the provided document, the method for establishing ground truth for any internal development samples would likely be the same as for the validation studies: bi-directional DNA sequencing, or comparison to previously characterized genomic DNA samples.

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The Invader® MTHFR 677 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5.10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood Potassium EDTA samples from patients with suspected thrombophilia.

The Invader MTHFR 677 test consists of the following components:
MTHFR 677 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 677 Wild Type Control
MTHFR 677 Heterozygous Control
MTHFR 677 Mutant Control
Invader Call Reporter™ Software

The Invader® MTHFR 677 test is an in vitro diagnostic test for detecting and genotyping the C to T mutation at position 677 of the MTHFR gene.

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state "acceptance criteria" but demonstrates performance through several studies of 100% agreement with the ground truth for most tests. The primary performance metric is agreement with bi-directional sequencing.

2. Sample Sizes and Data Provenance:

• Inter-laboratory Reproducibility (Study #1):
  • Test set sample size: 9 whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant). Tested in duplicate over 5 non-consecutive days by 6 operators (2 from each of 3 sites), resulting in 90 samples tested per operator, totaling 540 data points.
  • Data provenance: Not explicitly stated, but includes 2 external sites and 1 internal site, suggesting geographically diverse testing within a controlled study. Prospective based on the study design.
• Lot-to-Lot Reproducibility (Study #9):
  • Test set sample size: 9 genomic DNA samples (3 wild type, 3 heterozygous, 3 mutant). Tested in quadruplicate using 3 different kit lots, totaling 108 data points.
  • Data provenance: Not explicitly stated, but part of analytical performance studies, likely within a controlled laboratory setting.
• Real-Time Stability Study (Study #5):
  • Test set sample size: Samples representing all 3 genotypes (including 3 controls and 4 gDNA samples) tested in quadruplicate at each time point.
  • Data provenance: Not explicitly stated, likely internal laboratory testing.
• Reagent Freeze-Thaw Stability Study (Study #6):
  • Test set sample size: Genomic DNA isolated from cell lines, representing all possible genotypes.
  • Data provenance: Not explicitly stated, likely internal laboratory testing.
• Analytical Sensitivity (Study #3):
  • Test set sample size: 3 genomic DNA samples (WT, HET, MUT). Each sample diluted to 8 different concentrations and tested in replicates of 40, totaling 960 data points.
  • Data provenance: Whole blood collected in potassium EDTA, likely laboratory-controlled study.
• Analytical Specificity (Interfering Substances) (Study #4):
  • Test set sample size: 9 whole blood samples of different genotypes (3 WT, 3 HET, 3 MUT).
  • Data provenance: Not explicitly stated, likely laboratory-controlled study.
• Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7):
  • Test set sample size: 30 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 40 samples). These were analyzed with 4 different extraction methods, leading to 160 extracted DNA samples tested.
  • Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
• Instrument Equivalency (Study #8):
  • Test set sample size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 samples). Tested across 3 thermal cyclers and 3 fluorometers (39*3 = 117 tests per fluorometer in Table 9, but recorded as 78 of 78 = 100% for each cell, implying 78 distinct samples or tests per fluorometer/thermal cycler combination).
  • Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
• Method Comparison (Bi-directional Sequencing) (Study #2):
  • Test set sample size: 361 human whole blood samples.
  • Data provenance: Human whole blood samples. Not specified if retrospective or prospective or country of origin, but generally such studies involve controlled collection.

3. Number of Experts and Qualifications for Ground Truth:

The document does not explicitly mention the number or qualifications of experts used to establish the ground truth. However, the ground truth for all performance studies is established by bi-directional DNA sequencing, which is a widely accepted gold standard for determining genetic variants. This method is inherently objective and does not rely on expert interpretation in the same way as, for example, image-based diagnostics.

4. Adjudication Method:

Given that the ground truth is established by bi-directional DNA sequencing, which is a definitive molecular method, there is no mention of an adjudication method in the context of human interpretation (e.g., 2+1, 3+1). The testing process involves comparing the device's output to the objective sequencing results. In cases of discrepancies or invalid results (e.g., in Study #2), these are noted and factored into the agreement calculations.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The device is an in vitro diagnostic test for genetic analysis, not an imaging device or one requiring human interpretation of complex visual data. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.

6. Standalone Performance:

Yes, a standalone performance study (algorithm only without human-in-the-loop performance) was done. All the analytical performance studies (precision, stability, sensitivity, specificity, equivalency, and method comparison against sequencing) represent the standalone performance of the Invader® MTHFR 677 test, as it directly outputs genotype calls from fluorescence data without human interpretation influencing the result. The Invader Call Reporter™ software performs the data analysis and determines results autonomously following the import of fluorescence data.

7. Type of Ground Truth Used:

The primary type of ground truth used across all studies is bi-directional DNA sequencing. This is explicitly stated for the method comparison study and implied as the reference method for verifying all genotype calls throughout the analytical performance sections (e.g., "percent agreement between Invader® MTHFR 677 test and sequencing").

8. Sample Size for the Training Set:

The document does not specify the sample size used for a "training set." This device is based on a well-established molecular biology technique (Invader Plus® chemistry, PCR, FRET) and software for data interpretation, rather than a machine learning or AI model that typically requires a distinct training phase with large datasets. The controls included with the kit (Wild Type, Heterozygous, Mutant, No DNA Control) serve to ensure proper functioning and calibration for each run, effectively acting as internal references for the test.

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

Since there is no explicit mention of a "training set" in the context of machine learning, the question of how its ground truth was established is not directly applicable. The performance is validated against the gold standard of bi-directional DNA sequencing, which inherently provides the "ground truth" for the MTHFR 677 genotype. The development and optimization of the assay itself would have involved molecular biology techniques to ensure accurate detection of the specific SNP.

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The Invader® MTHFR 1298 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (A to C at position 1298) of the human 5,10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

The Invader MTHFR 1298 test consists of the following components:
MTHFR 1298 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 1298 Wild Type Control
MTHFR 1298 Heterozygous Control
MTHFR 1298 Mutant Control
Invader Call Reporter™ Software
Invader® MTHFR 1298 Software

Here's an analysis of the Invader® MTHFR 1298 device based on the provided text, addressing your specific questions.

1. Table of Acceptance Criteria and Reported Device Performance

(Note: The document does not explicitly state "acceptance criteria" in a separate section with pass/fail thresholds for each study. Instead, it presents study results and concludes that the information "supports a substantial equivalence decision." The table below infers the implicit acceptance criterion as 100% agreement/concordance where achieved, and notes the precise percentage reported for other metrics.)

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

• Inter-laboratory Reproducibility (Study #1):
  • Test Set Sample Size: 9 (panel of genomic DNA samples representing 3 genotypes: wild type, heterozygous, homozygous mutant).
  • Total Tests Performed: 540 (9 samples x 2 operators/site x 3 sites x 2 duplicates/day x 5 days of testing).
  • Data Provenance: Not specified, but involved 2 external sites and 1 internal site. The samples were "whole blood samples specific for each of the three possible genotypes." Implied retrospective as specific genotypes were selected.
• Lot-to-Lot Reproducibility (Study #9):
  • Test Set Sample Size: 9 (genomic DNA samples: 3 wild type, 3 heterozygous, 3 mutants).
  • Total Tests Performed: 108 (9 samples x 4 replicates x 3 lots).
  • Data Provenance: Not specified, but samples were "genomic DNA samples." Implied retrospective.
• Real-time Stability (Study #5):
  • Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
  • Total Tests Performed: Performed in quadruplicate at each time point (initial, 4 months, 7 months) for 3 lots. Total: (3 gDNA + 3 controls) x 4 replicates x 3 lots x 3 time points = 216 tests.
  • Data Provenance: Not specified.
• Freeze-Thaw Stability (Study #6):
  • Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
  • Total Tests Performed: The table indicates varying numbers of tests per sample per freeze/thaw cycle. Total for gDNA: 36 (WT), 48 (HET), 36 (MUT). Total for controls: 45 each. Total overall is 255.
  • Data Provenance: Genomic DNA isolated from cell lines.
• Analytical Sensitivity (Detection Limit) (Study #3):
  • Test Set Sample Size: 3 genomic DNA samples (1 MUT, 1 HET, 1 WT).
  • Total Tests Performed: 960 (3 samples x 8 concentrations x 40 replicates).
  • Data Provenance: "Genomic DNA samples with different genotypes (i.e. WT, HET, MUT) were extracted from whole blood collected in potassium EDTA." Implied retrospective.
• Analytical Specificity (Interfering Substances) (Study #4):
  • Test Set Sample Size: 9 whole blood samples (3 WT, 3 HET, 3 MUT).
  • Total Tests Performed: 18 tests per substance (9 samples x 2 replicates). 7 substances + 1 control = 8 conditions. Total: 18 x 8 = 144 tests.
  • Data Provenance: "human whole blood samples." Implied retrospective.
• Pre-Analytical Equivalency (Genomic DNA Extraction Reproducibility) (Study #7):
  • Test Set Sample Size: 30 human whole blood samples + 10 leukocyte depleted whole blood spiked with cell lines = 40 unique samples.
  • Total Tests Performed: 160 (40 samples x 4 extraction methods, analyzed in singlicate).
  • Data Provenance: "human whole blood samples and leukocyte depleted whole blood spiked with cell lines." Implied retrospective.
• Instrument Equivalency (Study #8):
  • Test Set Sample Size: 29 human whole blood samples + 10 leukocyte depleted whole blood samples = 39 unique samples.
  • Total Tests Performed: 78 tests were reported per combination of fluorometer/thermal cycler. Overall: 39 samples x 2 extraction methods x 3 thermal cyclers x 3 fluorometers (but results are combined for each fluorometer/cycler pairing). The table shows 78 total tests for each of the 9 combinations.
  • Data Provenance: "human whole blood samples and leukocyte depleted whole blood samples spiked with cell lines." Implied retrospective.
• Method Comparison (Bi-directional Sequencing) (Study #2):
  • Test Set Sample Size: 348 human whole blood samples.
  • Data Provenance: "Human whole blood samples (n = 348)." Implied retrospective.

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

The document does not explicitly state the number or qualifications of experts used to establish ground truth.

For ground truth established by bi-directional sequencing, it's a laboratory method that typically involves trained molecular biologists or laboratory technicians. The results are considered highly accurate.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test sets. The ground truth was primarily established by bi-directional sequencing, which is considered a definitive method, minimizing the need for adjudication in the traditional sense (e.g., of discrepant expert reads).

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

This section is not applicable. The Invader® MTHFR 1298 is an in vitro diagnostic molecular test for genotyping a specific gene mutation. It is not an imaging or interpretive AI device that involves human "readers" or assesses "AI assistance." The device outputs a direct genotype call (WT, HET, MUT).

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

Yes, the device's performance studies were conducted in a standalone manner. The Invader® MTHFR 1298 test is an automated system where the Invader Call Reporter™ Software processes raw fluorescence data to determine genotypes. Human input is in sample preparation and loading, and reviewing the generated report, but the genotyping call itself is algorithm-driven without real-time human interpretation embedded in the performance measurement. The various studies (reproducibility, sensitivity, specificity, method comparison) directly evaluate this standalone algorithmic performance against a known ground truth (sequencing).

7. The Type of Ground Truth Used

The primary ground truth used for all performance studies was bi-directional DNA sequencing. This is considered the gold standard for confirming specific genetic variations. For some studies, expected genotypes based on controls or known cell lines were also used, which would have initially been characterized by sequencing.

8. The Sample Size for the Training Set

The document primarily describes validation studies and does not explicitly mention a "training set" in the context of device development. For IVD devices, a training set generally refers to the data used to initially develop and optimize the assay parameters and interpretation algorithms. Such details are typically proprietary and not extensively disclosed in 510(k) summaries, which focus on performance validation. The studies described are validation and verification studies using independent "test sets."

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

As no specific "training set" details are provided, the method for establishing its ground truth is also not described. However, it can be inferred that any samples used for initial assay development and algorithm optimization would also have had their MTHFR 1298 genotype established using a highly accurate method, most likely bi-directional DNA sequencing, similar to the validation studies.

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The VeraCode Genotyping Test for Factor V and Factor II is an in vitro diagnostic device for the detection and genotyping of Factor V Leiden G1691A and Factor II (Prothrombin) G20210A point mutations in DNA obtained from EDTA-anticoagulated human blood samples. The test is intended for use on the BeadXpress System. The VeraCode Genotyping Test for Factor V and Factor II on the BeadXpress System is indicated for use as an aid to diagnosis in the evaluation of patients with suspected thrombophilia.

The VeraCode Genotyping Test for Factor V and Factor II assay consists of reagents sufficient for 96 tests, consisting of two boxes containing pre-PCR and post-PCR reagents. The pre-PCR box contains the following reagents: MTR1 (1 x 1.2 mL). AB1 (1 x 4 mL), AOP1 (1 x 4.8 mL), ELM (1 x 4.8 mL), FSB (1 x 4.8mL), UB3 buffer (2 x 4.8 mL) and AE1 reagent (2 x 4.8 mL). The post-PCR box contains MSS reagent (1 x 4.8 mL) and Fast Start Taq DNA Polymerase (1 x 60 µL), VW2 buffer (1 x 60 mL), a VeraCode FV/FII Bead Plate with holographically inscribed glass microbeads aliquoted in strip-well plates, a test-specific kit manifest file and sample sheet files (containing test specific outcome specifications and sample plate layout files used to interpret and report genotype results). A magnet plate is also required but sold separately.

Here's a breakdown of the acceptance criteria and the study details for the Illumina VeraCode® Genotyping Test for Factor V and Factor II, based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The device's performance is primarily evaluated through reproducibility (precision) and accuracy (method comparison).

Acceptance Criteria for Reproducibility (Precision):
While explicit numerical acceptance criteria for reproducibility are not stated as "acceptance criteria," the study aimed to demonstrate high agreement for correct calls across different operators, sites, and days. The results presented in the summary show very high percentage agreements and 95% lower confidence bounds (LCB) nearing 100% after retesting, indicating that the implicit acceptance criterion was near-perfect agreement for calling genotypes correctly.

Note: The 95% LCB for homozygous Factor V (70.33%) and Factor II (65.18%) in the accuracy study, while lower than other categories, likely reflect the smaller sample size for these less common genotypes.

2. Sample Size and Data Provenance for the Test Set

• Test Set Sample Size:
  • Reproducibility (Precision) Study: A panel of 15 genomic DNA samples (3 for each of the 5 possible genotypes including Wild Type). Each sample was tested in duplicate once a day for 5 non-consecutive days at each of three sites by two operators. This results in a significant number of replicates (e.g., for Factor V Wild Type, 3 samples * 2 replicates * 5 days * 3 sites * 2 operators = 180 total instances, with slight variations in the tables indicating initial no-calls).
  • Accuracy (Method Comparison) Study: 92 patient samples were accrued at each of the three sites, yielding a total of 276 patient samples. Additionally, each site received two archived DNA samples as positive controls.
• Data Provenance: The report does not explicitly state the country of origin. However, the samples for the accuracy study were "prospectively drawn blood samples" (for interference testing) and "pre-selected or unscreened patients undergoing Factor V testing" (for method comparison), suggesting a combination of prospective and retrospective (archived) samples for the accuracy study. For reproducibility, it uses "genomic DNA samples isolated from blood" and "commercially available cultured cells."

3. Number of Experts and Qualifications for Ground Truth

• Number of Experts: For the accuracy study (method comparison), the ground truth was established by "bi-directional DNA sequencing analysis performed at an independent reference laboratory." The number of individuals/experts involved in this independent sequencing and interpretation is not specified, nor are their specific qualifications (e.g., number of years of experience). However, "bi-directional DNA sequencing" is a well-established and highly accurate method for genotyping, implying expert-level analysis.

4. Adjudication Method for the Test Set

• There appears to be no explicit adjudication method described for resolving discrepancies between the device's results and the ground truth (bi-directional sequencing). The summary reports the raw agreement.
• For the device's own internal "no calls" (samples generating an invalid result), the "no calls" were retested once. This retesting resolved most, but not all, initial "no calls," as seen in the tables (e.g., one FVL Homozygous sample in the accuracy study remained a "no call" after retesting).

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

• No MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) genetic test, which typically has a standalone performance assessment against a gold standard (like sequencing) rather than evaluating human reader performance with or without AI assistance.

6. Standalone Performance Study (Algorithm Only)

• Yes, a standalone performance study was done. The entire analytical performance section (Precision/Reproducibility and Method Comparison) describes the performance of the device (VeraCode Genotyping Test for Factor V and Factor II on the BeadXpress System, with VeraScan software) as a standalone system. The results of the device were directly compared against the established ground truth (bi-directional sequencing), without human intervention in the interpretation process of the device's output. The software interprets and reports the genotype.

7. Type of Ground Truth Used

• The primary ground truth used for both the reproducibility and accuracy studies was bi-directional DNA sequencing. This is considered a highly reliable and definitive method for determining gene mutations.

8. Sample Size for the Training Set

• The document does not provide information on the sample size used for the training set. As a 510(k) submission for a diagnostic test kit based on established molecular biology principles (PCR, allele-specific primer extension, hybridization), the development of the "algorithm" (the VeraScan software and kit manifest file) would typically rely on well-characterized samples and biochemical parameters rather than a large machine learning training set in the modern sense. The "assay-specific kit manifest file" contains the parameters and cutoffs used to interpret and report genotype results, implying these were established during development.

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

• Similar to the training set sample size, the document does not explicitly detail how ground truth for the training set (development of the kit manifest and software interpretation rules) was established. It's inferable that the development team used well-characterized reference samples (likely with known genotypes confirmed by sequencing or other gold-standard methods) to define the assay parameters, thresholds, and interpretation rules encoded in the kit manifest file that guides the VeraScan software. The "Preliminary passing criteria where all replicates of all genotypes produce the correct result when compared to bi-directional sequencing" mentioned in the detection limit section suggests an iterative process referencing sequencing during development.

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