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InPlex™ CF Molecular Test is an in vitro diagnostic device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic DNA samples isolated from human peripheral whole blood specimens. The panel includes mutations and variants recommended by the 2004 American College of Medical Genetics (ACMG). The InPlex™ CF Molecular Test is a qualitative genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the 2005 American College of Obstetricians and Gynecologists (ACOG) for adults of reproductive age, as an aid in newborn screening for cystic fibrosis, and in confirmatory diagnostic testing for cystic fibrosis in newborns and children.

The test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes and results should be used in conjunction with other available laboratory and clinical information.

InPlex™ CF Molecular Test amplifies specific regions of the CF7R gene in genomic DNA extracted from human whole peripheral blood. Each amplified DNA sample is subsequently mixed with Cleavase® enzyme and buffer then added to a loading port on an InPiex™ microfluidic card. An InPlex™ card contains eight sample-loading ports, each connected to 48 independent reaction chambers. Twenty-eight of these reaction chambers contain dried assay mixes specific for reporting the 23 ACMG/ACOG recommended CF7R mutations and variants. The remaining chambers consist of a "No Invader" Control", an Independent Quality Control. and several unused chambers.

After an InPlex™ card is loaded; the channels are mechanically sealed using a micro-fluidic card sealer, isolating each individual reaction chamber from all other chambers. The card is then incubated to allow individual Invader® reactions to occur. Following incubation, the card is read in a multi-well fluorometer and the raw signal data are imported into the InPlex™ CF Molecular Test Call Reporting Software for final result analysis.

The InPlex™ CF Molecular Test is a qualitative genotyping test designed to detect and identify a panel of mutations and variants in the CFTR gene for cystic fibrosis carrier screening, newborn screening, and confirmatory diagnostic testing. The acceptance criteria and performance are detailed across several analytical studies rather than a single overarching study with a unified set of criteria and results. The studies consistently aim for high percent agreement with known genotypes, typically emphasizing a 1-sided lower 95% Confidence Limit.

Here's a breakdown of the requested information based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance:

The document presents several analytical studies, each with its own performance and acceptance criterion (implicitly, high agreement with ground truth). The overall accuracy and reproducibility studies are the most central to demonstrating the device's main function.

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

• DNA Extraction Equivalency: 7 CF positive genomic DNA samples from human whole peripheral blood.
• Fluorometer Equivalency: 8 genomic DNA (gDNA) samples (some CFTR mutation positive, some normal).
• Incubator Equivalency: 8 gDNA samples.
• Interfering Substances: 8 CF positive genomic DNA samples from human whole peripheral blood.
• Limit of Detection: 8 gDNA samples.
• Lot-to-Lot Equivalency: 23 CFTR gDNA samples.
• Accuracy and Repeat Rate: 123 unique genomic DNA samples from peripheral whole blood and cell lines.
• Freeze-Thaw Tolerance: 23 CFTR gDNA samples.
• Real-Time Stability: A panel of seven CFTR gDNA samples and a panel of eight control samples.
• Reproducibility Study (Proficiency Phase): 8 pre-characterized gDNA samples.
• Reproducibility Study (Performance Phase): 23 samples containing mutations representing the ACMG recommended panel.

Data Provenance: The document does not explicitly state the country of origin for the samples or whether they were retrospective or prospective. It consistently refers to "genomic DNA samples isolated from human peripheral whole blood" and "cell lines," implying human biological samples. The use of "pre-characterized commercially available reference materials" in the Reproducibility Study suggests some samples might be from commercial sources. Without further information, the provenance is unknown.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

The ground truth for most studies was established by DNA sequencing (bidirectional DNA sequencing). In the context of genetic testing, DNA sequencing itself is the "gold standard" and is not typically "read" by human experts in the same way an image would be. The results from sequencing are analyzed by bioinformatics tools and specialists in molecular genetics. The document does not specify the number or qualifications of individuals interpreting the sequencing data; it's assumed to be standard laboratory practice for DNA sequencing result analysis.

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

There is no mention of an adjudication method involving multiple human readers for discrepancies. The comparison is directly between the InPlex™ CF Molecular Test results and the DNA sequencing results. The "root cause" investigation described after the initial miscalls in the "Accuracy and Repeat Rate" study implies a process of technical investigation rather than expert adjudication of conflicting interpretations.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not conducted. This device is a molecular diagnostic test, not an imaging device typically involving human readers for interpretation. The "reproducibility study" involved multiple technicians at different sites, but this was to assess the assay's consistency across operators and sites, not to compare human reader performance with and without AI (or this assay's) assistance.

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

Yes, the studies described are essentially standalone performance studies of the InPlex™ CF Molecular Test. The device, including its Call Reporting Software, provides final result analysis. The performance data (e.g., percent agreement with DNA sequencing) reflects the algorithm's ability to accurately detect mutations without human interpretation being part of the primary outcome measure for accuracy. Human involvement would be in operating the device and performing the laboratory procedures, but the "call" itself is algorithm-generated.

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

The primary ground truth used across all analytical studies is DNA sequencing (specifically, bidirectional DNA sequencing). This is considered the gold standard for genetic mutation detection. In some instances, samples were "pre-characterized gDNA genotypes," implying their mutation status was already known from previous (likely sequencing-based) analysis.

8. The sample size for the training set

The document does not explicitly mention a separate "training set" for the InPlex™ CF Molecular Test. As a molecular diagnostic test, such devices are typically developed and optimized using various samples during their R&D phase, but the rigorous performance studies presented (like accuracy, LoD, etc.) constitute the test set performance verification. The document describes analytical validation studies, not an AI model requiring distinct training and test sets in the conventional machine learning sense. The "samples" referred to throughout the document are for validation of the fully developed device.

9. How the ground truth for the training set was established

Since a distinct "training set" is not described, the method for establishing ground truth for development/optimization samples (if any) is not specified. However, given the nature of the device and the ground truth used for validation, it is highly probable that DNA sequencing would also have been the method used to establish ground truth for any samples used during the development and optimization phases.

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The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles of the UDP glucuronosyltransferase 1A1 (UGTIA1) gene in genomic DNA from whole peripheral blood as an aid in the identification of patients with greater risk for decreased UDP-glucunorosyltransferase activity.

The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test which utilizes sequence specific Invader DNA probes, a structure-specific cleavage enzyme and a fluorescent resonance energy transfer (FRET) system combined with universal interpretative software and third party microtiter plate reader instrumentation. Invader® is the term used to generically refer to the patented chemistry on which the Invader® UGT A 1 Molecular Assay is based. The assay is designed to identify specific nucleic acid sequences and query for the presence of known sequence polymorphisms through the structurespecific cleavage of a series of probes that are specifically complementary to TA repeat sequences in in the "TATA Box" of of the UGT1A1 promoter region.

In the Invader® UGT1A1 Molecular Assay, two oligonucleotides (a discriminatory Primary Probe and an Invader® Oligo) hybridize in tandem to the target DNA to form an overlapping structure. The 5'-end of the Primary Probe includes a 5'-flap that does not hybridize to the target DNA. The 3'-nucleotide of the bound Invader® Oligo overlaps the Primary Probe, but need not hybridize to the target DNA. The Cleavase® enzyme recognizes this overlapping structure and cleaves off the unpaired 5'-flap of the Primary Probe, releasing it as a target-specific product. The Primary Probe is designed to have a melting temperature close to the reaction temperature. Therefore, under the isothermal assay conditions, Primary Probes, which are provided in excess, cycle on the target DNA. This allows for multiple rounds of Primary Probe cleavage for each target DNA, and amplification of the number of released 5'-flaps.

In the secondary reaction, each released 5'-flap can serve as an Invader® Oligo on a fluorescence resonance energy transfer (FRET) Cassette to create another overlapping structure that is recognized and cleaved by the Cleavase® enzyme. When the FRET Cassette is cleaved, the fluorophore and quencher are separated, generating detectable fluorescence signal. Similar to the initial reaction, the released 5'-flap and the FRET Cassette cycle, resulting in amplified fluorescence signal. The initial and secondary reactions run concurrently in the same well.

The biplex format of the Invader® UGT1A1 Molecular Assay enables simultaneous detection of two DNA sequences, a non-varying segment of the human alpha actin (ACTAI) gene and the TA repeat in the TATA box of the human UGTIA1 gene, in a single well. The biplex format uses two different discriminatory Primary Probes, each with a unique 5'-flap, and two different FRET Cassettes, each with a spectrally distinct fluorophore. By design, the released 5'-flaps will bind only to their respective FRET Cassettes to generate a target-specific signal.

The Invader® UGT1A1 Molecular Assay utilizes four independent wells per sample (one well for each of the TA Oligo mix reactions), to make a single genotype call. Each well contains a TATA box specific probe and an alpha actin probe. The alpha actin probe serves as an internal control to confirm the validity of a given result when a particular TATA box polymorphism is absent.

Here's a summary of the acceptance criteria and study details for the Invader® UGT1A1 Molecular Assay, based on the provided text:

Acceptance Criteria and Reported Device Performance

Study Details:

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

   • Limit of Detection (Lower): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 50, 100, 150 ng DNA/µL. Total genotype calls for calculation: 120 (40 at each concentration for 3 genotypes).
   • Limit of Detection (Upper): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 80 ng DNA/µL. Total genotype calls: 120.
   • Genotype Detection: 285 blood samples.
   • Repeat Rate (Initial): 285 blood samples.
   • Repeat Rate (External Study): 20 samples, tested in triplicate at 3 sites on 5 separate days (900 possible genotype calls).
   • Stability (Freeze-Thaw): 20 genomic DNA samples.
   • Stability (Storage): 3 genomic DNA samples.
   • Lot-to-Lot Reproducibility: 40 whole blood samples, analyzed with 3 different lots of reagents (120 data points).
   • Sample Preparation Equivalency: 60 human genomic DNA samples.
   • Reproducibility (Multi-site): 20 blood samples, tested across 3 sites (900 sample points generated).
   • Interference Studies: 16 whole blood samples (for bilirubin, lipids, EDTA); 16 whole blood samples (for hemoglobin, AW2 buffer).

   The data provenance is not explicitly stated as retrospective or prospective, nor is the country of origin mentioned for the samples. It is implied to be clinical samples, likely from a patient population relevant to UGT1A1 genotyping.

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

   • The ground truth for all performance studies (Limit of Detection, Genotype Detection, Stability, Lot-to-Lot Reproducibility, Sample Preparation Equivalency, Interference, Reproducibility) was established using bi-directional DNA sequence analysis.
   • The document does not specify the number or qualifications of experts who performed or interpreted the bi-directional DNA sequencing. It is presented as a gold standard laboratory method.

3. Adjudication method for the test set:

   • The document does not describe an adjudication method involving multiple human readers for discrepancies. The genotype calls from the Invader assay were directly compared to the results of bi-directional DNA sequencing.
   • For the reproducibility study, where "misidentified" samples occurred at one site, it states "Discrepancy resolution by resequencing of the template in the original Invader assay confirmed that the genotypes present in the assay wells were consistent with the reported genotypes of the assay." This suggests a re-analysis by the measurement method itself rather than a formal expert adjudication panel.

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

   • No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic molecular assay, not an AI-powered diagnostic imaging tool that would typically involve human readers. The performance is evaluated against a gold standard molecular method (bi-directional DNA sequencing), not against human interpretation of images.

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

   • Yes, this entire submission focuses on the standalone performance of the Invader® UGT1A1 Molecular Assay, which is an algorithm-driven automated test. The "algorithm" here refers to the sequence-specific Invader DNA probes, cleavage enzyme, FRET system, and universal interpretative software that automates genotype calling. There is no human-in-the-loop performance described beyond standard laboratory handling and setup. The assay signal results are interpreted by a software program and assigned a genotype.

6. The type of ground truth used:

   • The primary ground truth used for all performance evaluations was bi-directional DNA sequence analysis. This is a highly accurate molecular method for determining DNA sequences.

7. The sample size for the training set:

   • The document does not mention a separate training set or training data used to develop the assay. The focus is on the performance data from non-clinical studies (validation studies) comparing the Invader assay results to bi-directional DNA sequencing. This suggests the assay's underlying chemistry and interpretive software were already developed and are being validated, rather than being developed using a specific training dataset in the context of machine learning.

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

   • As no training set is explicitly described, the method for establishing its ground truth is not applicable or detailed in this document. The assay's fundamental design relies on established molecular biology principles and specific probe-target recognition, rather than learning from a large, pre-labeled training dataset in the way a machine learning algorithm would.

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