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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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