The xTAG® Cystic Fibrosis 60 Kit v2 is a device used to simultaneously detect and identify a panel of mutations and variants in the Cystic Fibrosis transment conductance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG), plus some of the world's most common and North American-prevalent mutations. The xTAG Cystic Fibrosis 60 Kit v2 is a qualitative genotyping test that provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

Not Found

The provided text is a 510(k) premarket notification letter for the Luminex Molecular Diagnostics xTAG® Cystic Fibrosis 60 Kit v2. This document is a regulatory approval letter and does not contain the detailed study information typically found in a clinical trial report or a scientific publication. Therefore, I cannot provide all the requested information.

However, based on the nature of the device (a molecular diagnostic kit for detecting genetic mutations), I can infer some aspects and explicitly state what is not present in the document.

Here's a breakdown of what can and cannot be answered:

1. A table of acceptance criteria and the reported device performance

This information is not available in the provided document. A 510(k) clearance letter acknowledges substantial equivalence to a predicate device but does not typically detail the specific performance metrics (like sensitivity, specificity, accuracy) or the pre-defined acceptance criteria from the validation studies.

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

This information is not available in the provided document. The letter does not describe the specific studies conducted, including sample sizes or data provenance.

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)

This information is not available in the provided document. For genetic testing, ground truth would typically be established by Sanger sequencing or another highly accurate molecular method, not by human experts in the way that imaging studies use radiologists.

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

This information is not available in the provided document. Adjudication methods are typically relevant for subjective assessments, which is not the primary mode of operation for a genetic diagnostic kit.

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 information is not applicable and not available in the provided document. This device is a genetic testing kit, not an AI-assisted diagnostic tool that involves human readers interpreting images or other data.

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

This information is partially applicable but not detailed in the provided document. The xTAG® Cystic Fibrosis 60 Kit v2 is a qualitative genotyping test. This implies it operates in a "standalone" fashion in terms of detecting mutations, producing results without human interpretive input beyond running the assay and reading the output. However, the letter does not provide details of such a "standalone performance" study or its results.

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

For a genetic testing kit, the ground truth would typically be established by a gold standard molecular method, such as:

• Sanger Sequencing: Considered the gold standard for definitive mutation identification.
• Next-Generation Sequencing (NGS): Can also be used to confirm mutations.
• Reference materials with known genotypes: Commercially available or laboratory-validated samples with confirmed mutation status.

However, the specific method used for establishing ground truth for the xTAG® Cystic Fibrosis 60 Kit v2 is not detailed in this document.

8. The sample size for the training set

This information is not available in the provided document. This type of device does not typically have a "training set" in the machine learning sense. Instead, assay development involves extensive analytical validation and clinical validation studies.

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

This information is not applicable and not available in the provided document. As mentioned, this is not an AI/machine learning device with a training set.

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The xTAG® Cystic Fibrosis 39 Kit v2 is a device used to simultaneously detect and identify a panel of mutations and variants in the Cystic Fibrosis transmembrance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG), plus some of the world's most common and North American-prevalent mutations. The xTAG Cystic Fibrosis 39 Kit v2 is a qualitative genotyping test that provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

Not Found

The provided text is a cover letter from the FDA to Luminex Molecular Diagnostics, Inc. regarding their xTAG™ Cystic Fibrosis 39 Kit v2. While it confirms the device's substantial equivalence and lists its indications for use, it does not contain the detailed study information required to answer your questions about acceptance criteria, device performance, sample sizes, ground truth establishment, or expert involvement.

The document states: "We have reviewed your Section 510(k) premarket notification... and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices..." This implies that the study data was submitted to the FDA as part of the 510(k) premarket notification, but this specific document does not provide the study details.

Therefore, I cannot provide the requested information based on the text provided. To answer your questions, details from the actual 510(k) submission or subsequent study publications would be needed.

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The Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay is a qualitative in vitro diagnostic system used to simultaneously detect 139 clinically relevant cystic fibrosis disease-causing mutations and variants of the cystic fibrosis transmembrance regulator (CFTR) gene in genomic DNA isolated from human peripheral whole blood specimens. The variants include those recommended in 2004 by the American College of Medical Genetics (ACMG) and in 2011 by the American College of Obstetricians and Gynecologists (ACOG). The test is intended for carrier screening in adults of reproductive age, in confirmatory diagnostic testing of newborns and children, and as an initial test to aid in the diagnosis of individuals with suspected cystic fibrosis. The results of this test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available laboratory and clinical information. This test is not indicated for use for newborn screening, fetal diagnostic testing, pre-implantation testing, or for stand-alone diagnostic purposes.

The test is intended to be used on the Illumina MiSeqDxTM Instrument.

The Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay consists of library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software. Testing begins with genomic DNA from a peripheral whole blood sample. The genomic DNA is processed through the library preparation steps, which specifically amplifies the intended genomic regions of each sample while also adding the indexes for sample identification. Flow cell capture sequences are also added to the amplified products. The resulting sample libraries are then transferred into a MiSeqDx reagent cartridge which contains all of the reagents required for cluster generation and sequencing (Sequencing By Synthesis). The MiSeqDx Cartridge, MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into the MiSeqDx instrument, which performs cluster generation, sequencing and data analysis.

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

Acceptance Criteria and Device Performance

Note regarding "Acceptance Criteria": The document K124006 primarily presents the results of validation studies rather than explicitly stating pre-defined "acceptance criteria" in a separate section. The "acceptance criteria" implied above are derived from the consistently high performance reported across various studies and the overall conclusion of the device being substantially equivalent.

Study Details

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

• Accuracy Study (Primary Source):
  • Test Set Size: 500 samples in total, comprised of:
    • 366 samples from a clinical accuracy study (primary source).
    • 68 cell line samples from the reproducibility study (supplementary).
    • 14 clinical samples from the extraction method evaluation analytical study (supplementary).
    • 52 synthetic plasmid samples (supplementary).
  • Data Provenance:
    • Clinical Samples: Majority (n=355) were archived, anonymized clinical gDNA specimens isolated from human blood. (No specific country of origin mentioned, but implied to be human clinical samples).
    • Cell Line Samples: 11 samples were commercially available cell line specimens.
    • Synthetic Plasmid Samples: Designed to include genomic context of rare variants, linearized, diluted, and blended with human genomic DNA (lab-generated, not from patients).
    • Retrospective/Prospective: The 355 clinical gDNA specimens were "archived, anonymized clinical gDNA specimens," suggesting a retrospective approach for these samples. The cell line and synthetic samples were laboratory-generated.
• Reproducibility Study:
  • Test Set Size: 46 samples (in each of two panels, total 92 unique samples) were tested. Each sample was tested for a total of 810 calls per site.
  • Data Provenance: Genomic DNA from lymphoblastoid cell lines with known mutations and leukocyte-depleted blood spiked with lymphoblastoid cell lines with known mutations. This suggests laboratory-controlled samples, not directly from clinical patients in a prospective manner.
• DNA Extraction Study:
  • Test Set Size: 14 unique blood samples per extraction method, with 2 operators performing 3 runs each, and each run having 2 replicates, totaling 168 (14 x 2 x 3 x 2) for each of the three extraction methods.
  • Data Provenance: K2EDTA anti-coagulated whole blood samples, including wild type and three mutant genotypes (samples with F508del, I506V, and D110H).
• DNA Input Study:
  • Test Set Size: 14 representative DNA samples (with 16 unique CF variants) tested in duplicate at 9 DNA input levels. Additional testing involved 4 representative samples with 20 replicates each (n=80), and 14 samples with 20 replicates each (n=280).
  • Data Provenance: Representative DNA samples containing CF variants.
• Interfering Substances Study:
  • Test Set Size: 8 whole blood specimens (including 3 CF positive samples with unique genotypes). 16 replicates each for specific interferents.
  • Data Provenance: Whole blood specimens.

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

The document specifies the reference methods used to establish ground truth, but does not explicitly state the number or qualifications of experts involved in reviewing these reference results or establishing consensus.

• For 137 SNA/small InDel sites and PolyTG/Poly T region: Sanger bi-directional sequence analysis was the reference method.
• For two large deletions: A PCR-based assay was the reference method, confirmed for accuracy using Sanger Sequencing.

While Sanger sequencing and PCR assays are considered robust reference methods, the process of interpreting and confirming these results as "ground truth" and addressing any discrepancies would typically involve qualified molecular geneticists or lab personnel. However, this level of detail is not provided.

4. Adjudication Method for the Test Set

The document does not describe a formal adjudication method (e.g., 2+1, 3+1, none) for the test set. Ground truth was established by reference methods (Sanger bi-directional sequencing and a PCR-based assay). Any "miscalls" (one in the accuracy study and a few in the reproducibility study) or "no calls" are noted and sometimes explained (e.g., insufficient coverage, sample handling issues), implying internal review rather than a multi-expert adjudication process for primary ground truth establishment.

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

No Multi-Reader Multi-Case (MRMC) comparative effectiveness study was done. This device is an in vitro diagnostic system for detecting genetic mutations, not an imaging device or a diagnostic aid meant to directly assist human readers in interpreting complex visual data. Therefore, the concept of "human readers improve with AI vs. without AI assistance" is not applicable in this context.

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

Yes, a standalone performance assessment was conducted. The accuracy and reproducibility studies directly evaluated the device's ability (including its data analysis software) to detect mutations and variants in genomic DNA. The "results of this test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available laboratory and clinical information," indicating that while human interpretation is part of the overall clinical process, the performance metrics (PA, NA, OA) are for the device (algorithm + instrument) alone.

7. Type of Ground Truth Used

The ground truth used was primarily based on:

• Sanger bi-directional sequence analysis: For 137 SNA/small InDel sites and the PolyTG/Poly T region. This is considered a gold standard for sequence verification.
• PCR-based assay: For two large deletions, confirmed by Sanger Sequencing.

This represents established molecular diagnostic laboratory methods, widely accepted as reliable for genetic variant detection.

8. Sample Size for the Training Set

The document does not report the sample size for a training set. This is typical for in vitro diagnostic devices based on established sequencing technology and a defined set of targets, where the "training" may involve internal development and optimization using design verification samples rather than a formally described, distinct "training set" in the context of machine learning model development. The focus is on validation against known samples to determine performance characteristics.

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

As no explicit "training set" is described, how its ground truth might have been established is not mentioned in the document. Development and optimization would have relied on samples with known CFTR genotypes, likely confirmed by methods similar to those used for the test set (Sanger sequencing, PCR).

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The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system that re-sequences the protein coding regions and intrones of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in genomic DNA isolated from human periphers collected in K2EDTA. The test detects single nucleotide variants, and small InDels within the region sequenced, and additionally reports on two deep intronic mutations and two large deletions. The test is intended to be used on the Illumina MiSeqDx Instrument.

The test is intended to be used as an aid in the diagnosis of individuals with suspected cystic fibrosis (CF). The test is most appropriate when the patient has an atypical or non-classic presentation of CF or when other mutation panels have failed to identify both causative mutations. The results of the test are intented by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available information including clinical symptoms, other diagnostic tess, and family history. This test is not indicated for use for stand-alone diagnostic testing, for pre-implantation testing, carrier screening, newborn screening, or population screening.

The Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay consists of library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software. Testing begins with genomic DNA from a peripheral whole blood sample. The genomic DNA is processed through the library preparation steps, which specifically amplifies the intended genomic regions of each sample while also adding the indexes for sample identification. Flow cell capture sequences are also added to the amplified products. The resulting sample libraries are then transferred into a MiSeqDx reagent cartridge which contains all of the reagents required for cluster generation and sequencing (Sequencing By Synthesis). The MiSeqDx Cartridge, MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into the MiSeqDx instrument, which performs cluster generation, sequencing and data analysis.

Here's a summary of the acceptance criteria and study details for the Illumina MiSeqDx™ Cystic Fibrosis Clinical Sequencing Assay based on the provided text:

Acceptance Criteria and Device Performance

Study Details

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

• Accuracy Study Test Set:

  • Total Samples: 500
  • Clinical Samples: 355 (archived, anonymized gDNA from human blood) - Country of origin not specified, but the review is by the US FDA.
  • Commercial Cell Line Samples: 11 (from clinical accuracy study) + 68 (from reproducibility study) = 79
  • Clinical Samples (from extraction method evaluation): 14
  • Synthetic Plasmid Samples: 52
  • Data Provenance: Retrospective for archived clinical samples, prospective for cell line and synthetic samples generated for the study.

• Reproducibility Study Test Set:

  • Total Samples: 46 distinct samples (implied from the HGVS tables, with many tested in multiple replicates). The tables show analysis for 68 samples, each tested across 3 sites, with 2 operators each doing 3 runs per site, and with some samples having multiple replicates within those runs. This results in a large number of individual tests. The text explicitly states 68 cell line samples were evaluated in the reproducibility study for accuracy data.
  • Data Provenance: Prospective (generated specifically for the study). The operator details suggest this was done as part of the validation study.

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

• The ground truth reference methods were Sanger bi-directional sequencing and validated PCR based assays.
• The text does not specify the number of experts used to establish the ground truth for Sanger sequencing or the PCR assays, nor their specific qualifications. It only states these were "reference methods."

4. Adjudication Method for the Test Set

• The text mentions that "All results are based on initial testing. No repeat testing was done for this study" for the accuracy study. For reproducibility, some samples had "no calls" or "miscalls" initially, and further investigation was done (e.g., misidentified variant, low level contamination, improperly prepared synthetic specimen, switched samples). However, a formal adjudication method (e.g., 2+1, 3+1) involving a specific number of experts to resolve discrepancies against the reference is not explicitly described. The determination of miscalls and no calls seems to stem from comparison with the reference methods.

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

• No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. This study focuses on the standalone analytical performance (accuracy, reproducibility) of the device (an in vitro diagnostic system for genetic sequencing), not on its impact on human reader performance.

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

• Yes, a standalone performance study was done. The entire accuracy and reproducibility analysis describes the performance of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay (which includes the instrument and data analysis software) in detecting genetic variants. The results (PA, NA, OA, call rates) are indicative of the algorithm's performance compared to established reference methods (Sanger sequencing and PCR assays). The "human-in-the-loop" aspect for interpretation is mentioned in the intended use, where results are to be interpreted by a board-certified clinical molecular geneticist, but the performance metrics provided are for the device's ability to 'call' the variants.

7. The Type of Ground Truth Used

• Expert Consensus (implied via Gold Standard Methods): The primary ground truth for SNVs and small InDels was Sanger bi-directional sequencing. For large deletions, two validated PCR-based assays were used, with accuracy confirmed by Sanger Sequencing. These are considered gold-standard methods in molecular diagnostics, implying an expert-established and validated ground truth.

8. The Sample Size for the Training Set

• The document does not specify a training set size. As this is a performance validation document for a sequencing assay, it describes the testing of the device rather than the development or training of an AI model in the conventional sense. The "MiSeqDx instrument and data analysis software" are components, but details on their development/training are not provided in this regulatory summary.

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

• Since a training set is not explicitly mentioned or detailed for the device's software/algorithm development, the method for establishing its ground truth is not provided in this document. The focus is on validating the final product's performance against reference standards.

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The xTAG® Cystic Fibrosis 60 kit v2 is a device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG) plus some of the world's most common and North American prevalent mutations. The xTAG Cystic Fibrosis 60 kit v2 is a qualitative genotyping test which provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

Indication(s) for use: same as intended use.

The xTAG CFTR 60 kit v2 includes the following components:

• xTAG PCR Primer Mix v2
• . xTAG ASPE Mix A v2
• xTAG ASPE Mix B v2 .
• xTAG Bead Mix A v2 .
• . xTAG Bead Mix B.v2
• xTAG 10X Buffer .
• . Platinum® TFI Exo(-) DNA Polymerase
• Platinum® TFI Reaction Buffer, 5x .
• . TFI 50mM MgCl2
• xTAG Shrimp Alkaline Phosphatase .
• xTAG Exonuclease I .
• xTAG Strepavidin-Phycoerythrin Conjugate .

Here is an analysis of the provided text regarding the xTAG® Cystic Fibrosis 60 kit v2's acceptance criteria and studies:

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on analytical performance characteristics rather than formally defined acceptance criteria with numerical targets. However, based on the results presented, "100% accuracy" and "precision of >99.99%" appear to be the overarching goals the device achieved.

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

• Accuracy Study (Clinical Performance Characteristics):

  • Sample Size: 396 clinical samples. These samples contained a total of 488 mutant alleles. Some samples were compound heterozygotes or homozygous mutants.
  • Data Provenance: The majority of samples consisted of "left-over, anonymized, banked whole-blood specimens." These were supplemented with "genomic DNAs from EBV-transformed lymphoid cell lines" and "several custom-designed plasmids engineered to contain 1-2 CFTR mutations each." "Archived clinical genomic DNA samples were obtained from a variety of sources." The origin (e.g., country) of these banked specimens is not explicitly stated. The study appears to be retrospective as it used archived and banked samples.

• Precision/Reproducibility Study:

  • Sample Size: Not explicitly stated as a single number, but involves multiple replicates. Each assay point was run in duplicate within a run.
    • For the overall precision study of all 60 mutations and wild-type: "Each set of samples representing all mutations and variants probed by the xTAG Cystic Fibrosis 60 kit v2."
    • For dF508mut reproducibility: 36 replicates of a sample.
    • For 2307insA allele reproducibility: 36 test points.
  • Data Provenance: Purified genomic DNAs extracted from clinical (whole blood) samples, purified genomic DNA extracted from lymphoid cell lines, and/or plasmids. The study was "multi-centre" (3 independent sites) and "multi-operator" (2 operators per site). The origin of these specific samples is not detailed, but it's reasonable to infer they are similar to the accuracy study samples. The study design implies a prospective collection and testing for the purpose of the study, even if the underlying samples were banked.

• Detection Limit and Range of Assay Study:

  • Sample Size: "Genomic DNA samples representing a subset of mutations in the CFTR 60 kit v2 test" (number not specified). Each sample was run in duplicate. For LoD determination: 22 replicates of each of 10 genomic DNAs, plus 8 negative controls.
  • Data Provenance: Genomic DNA samples used at various concentrations, plus wild-type positive control and negative controls.

• Interference Study:

  • Sample Size: Eight whole blood samples.
  • Data Provenance: Whole blood samples.

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 that experts were used to establish ground truth for the test samples in the typical sense of a diagnostic interpretation.

• For the accuracy study, the FDA-cleared xTAG Cystic Fibrosis Kit (predicate device) was used as the comparator for Panel A mutations, and dideoxy-sequencing was used for Panel B mutations. This implies that the ground truth for these samples was established by these reference methods, which are considered highly reliable for genetic variant detection.

• For the reproducibility and other analytical studies, the ground truth for the samples (e.g., presence or absence of specific mutations) would have been known from their characterization by reference methods or their design (e.g., custom-designed plasmids).

Therefore, there's no mention of a "number of experts" or their "qualifications" involved directly in adjudicating the ground truth in this context, as the ground truth was established by established genetic testing methodologies.

4. Adjudication Method for the Test Set

As the ground truth was established by reference methods (predicate device and dideoxy-sequencing), an "adjudication method" involving multiple human readers or experts to determine the ground truth for each case is not described or applicable to this type of genetic assay. The device's results were compared directly against the results from the reference methods.

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

No. The xTAG® Cystic Fibrosis 60 kit v2 is a qualitative genotyping test. It does not involve human readers interpreting images or data in a way that an MRMC study (typically used for imaging diagnostics or complex diagnostic interpretations by humans) would be relevant. The "reader" in this context is the Luminex instrument and the xTAG Data Analysis Software (TDAS CFTR), which generate a definitive genotype output.

6. Standalone Performance Study

Yes. The entire performance evaluation reported (accuracy, precision, LoD, interference) describes the standalone performance of the xTAG® Cystic Fibrosis 60 kit v2. The kit's output, analyzed by its proprietary software, provides a final qualitative genotype. While the intended use notes it "is not indicated for stand-alone diagnostic purposes" in the clinical context (meaning clinicians integrate the result with other information), the technical performance studies presented are for the algorithm/device itself operating independently.

7. Type of Ground Truth Used

• For Panel A mutations (Accuracy Study): Results from the FDA cleared xTAG Cystic Fibrosis Kit (K043011 and K060627).
• For Panel B mutations (Accuracy Study): Dideoxy-sequencing.
• For other analytical studies (Precision, LoD, Interference): Pre-characterized genomic DNAs from clinical samples, lymphoid cell lines, and custom-designed plasmids where the presence/absence of mutations was known with high certainty through established genetic analysis methods. This effectively falls under a form of expert consensus on the genetic makeup of the samples, derived from gold-standard molecular techniques.

8. Sample Size for the Training Set

The document does not provide information on a separate "training set" or its sample size. This type of genetic assay, based on PCR and allele-specific primer extension, is designed based on known genetic sequences and variants. While there would be an inherent development and optimization phase (analogous to training for an AI model), the detailed data for such a phase is not presented in this 510(k) summary. The described studies are primarily for validation/verification of the final product.

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

As no explicit "training set" and its size are mentioned, the method for establishing its ground truth is also not described. For such a molecular diagnostic, the "ground truth" for developing the assays would derive from established knowledge of CFTR gene mutations and their sequences, along with empirical testing and optimization using characterized samples (similar to those used in the validation, but within a developmental context).

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The xTAG® Cystic Fibrosis 39 kit v2 is a device used to simultaneously detect and identify a panel of mutations and variants in the cystic fibrosis transmembrance regulator (CFTR) gene in human blood specimens. The panel includes mutations and variants currently recommended by the American College of Medical Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG), plus some of the worlds most common and North Americanprevalent mutations. The xTAG Cystic Fibrosis 39 kit v2 is a qualitative genotyping test which provides information intended to be used for carrier testing in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

The kit is not indicated for use in fetal diagnostic or pre-implantation testing. This kit is also not indicated for stand-alone diagnostic purposes.

The xTAG CFTR 39 kit v2 includes the following components:

• PCR Primer Mix v2 including dNTPs designed to simultaneously produce 23 amplimers of the CFTR gene (24 in the presence of CFTR del 2, 3).
• ASPE Mix A v2 including dNTPs contains primers designed to hybridize to either wild-type or mutant alleles ◆ with proprietary sequences at their 5' ends designed to specifically hybridize to complementary sequences coupled to a given bead population in Bead Mix A.
• Bead Mix A v2 contains spectrally distinguishable populations of polystyrene beads internally dyed with red and . infrared fluorochromes coupled to proprietary DNA sequences designed to specifically hybridize to complementary sequences on the ASPE primers in ASPE Mix A v2.
• 10X Buffer
• Platinum® TFI DNA Polymerase .
• Platinum® TFI Reaction Buffer .
• TFI MgCl2 .
• Shrimp Alkaline Phosphatase .
• Exonuclease I .
• Strepavidin-Phycoerythrin Conjugate .
• xTAG Data Analysis Software (TDAS) CFTR .

Here's a breakdown of the acceptance criteria and study details for the xTAG® Cystic Fibrosis 39 kit v2, based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the "overall accuracy" and "agreement with comparator" percentages, which were consistently 100% or very close to it across various sample types and individual alleles, especially after any allowed reruns.

2. Sample Sizes Used for Test Set and Data Provenance

• Test Set Sample Size:
  • Clinical Samples: 319 independent clinical samples.
  • Cell Lines: 8 cell lines.
  • Plasmids: Number of plasmids tested is not explicitly stated for the accuracy study calculation, but they were used to supplement samples.
  • Per Allele Reproducibility: Varied per allele, ranging from 6 to 468 total calls across all sites (e.g., 36 calls for G85E, 468 for dF508).
• Data Provenance: The majority of samples consisted of left-over, anonymized, banked whole-blood specimens. These were supplemented with genomic DNAs from EBV-transformed lymphoid cell lines and custom-designed plasmids. Archived clinical genomic DNA samples were obtained from a variety of sources. The document does not specify the country of origin, but study sites included Hartford Hospital, Connecticut, USA; Luminex Molecular Diagnostics, Toronto, Canada; and Hospital for Sick Children, Toronto, Canada, suggesting a North American provenance. The nature of the samples (archived clinical genomic DNA, banked specimens) indicates a retrospective study design.

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

The document does not explicitly mention the use of "experts" to establish ground truth in the traditional sense of medical image interpretation or clinical diagnosis. Instead, the ground truth was established by comparison with a predicate device. The "FDA cleared xTAG Cystic Fibrosis Kit (K043011 and K060627)" was used as the comparator for all clinical specimens. No human experts are described as part of ground truth establishment for the test set.

4. Adjudication Method for the Test Set

Not applicable. The ground truth was established by comparison with a predicate device, not by human expert consensus requiring adjudication. The study mentions "allowable re-runs" for device performance, but this is an internal process, not an adjudication of a human expert ground truth.

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

No. This type of study is typically performed for AI systems that aid human readers in diagnostic tasks, such as medical image interpretation. The xTAG® Cystic Fibrosis 39 kit v2 is a laboratory diagnostic test for gene mutation detection, which does not involve human readers interpreting images with or without AI assistance.

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

Yes, this was a standalone study of the device. The device, which includes "xTAG Data Analysis Software (TDAS) CFTR," processes MFI signals from the Luminex instrument to "provide a final qualitative genotype for the sample." The performance evaluation focuses on the accuracy and reproducibility of this automated process against a comparator device, without human intervention in the interpretation phase for the reported performance metrics.

7. Type of Ground Truth Used

The ground truth was established by comparison with a predicate device ("FDA cleared xTAG Cystic Fibrosis Kit (K043011 and K060627)"). This indicates that the established genotypes of the samples (both positive and negative for various mutations/variants) were determined using another FDA-cleared CFTR mutation detection system.

8. Sample Size for the Training Set

The document does not provide details on a separate "training set" or its size. This is common for traditional in vitro diagnostic (IVD) kits, where performance is typically validated through studies demonstrating accuracy, precision, and reproducibility, rather than through machine learning model training. The device uses "proprietary software," but the development and validation process is likely based on established molecular diagnostics principles and analytical validation rather than a machine learning training/validation split.

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

Not applicable, as a distinct training set (in the machine learning sense) and its ground truth establishment are not discussed in the provided document. The device's performance evaluation focuses on its accuracy against a predicate device and its internal reproducibility.

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The Verigene CFTR and Verigene®CFTR PolyT Nucleic Acid Tests are qualitative in vitro diagnostic devices used to genotype a panel of mutations and variants in vitro transmembrance regulator (CFTA) consisted institute and variants in the cystic fibrosis transmission is see about to the of micrations and variants in the cystic fibrosis transmembrance regulator (CFFT) gene in genomic DNA isolated from human peripheral whole blood (CF7 / ) gene includes mutations and variations and variations and virus and virus and variations and variations and variations and Obstericians and Gyneologists (ACOG). The Verigene CFTA Nucleic Acid Test provides information intended to be used for carrier testing in adults of reproductive age and in confirmatory diagnostic testing of newborns and children. These tests are not indicated for use in fetal diagnostic or pre-implantation testing and not indicated for stand-alone is in retal dagnostic of pre-implantation testing and not indicated for stand-alone diagnostic purposes. The results should be used in conjunction w other available alono diagnostic purposes. The results should be used in conjunction with other available laboratory and clinical information. Both tests are intended to be u

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This document is an FDA 510(k) clearance letter for the Verigene® CFTR and Verigene® CFTR PolyT Nucleic Acid Tests. It primarily focuses on the regulatory aspects and does not contain detailed information about the acceptance criteria or a specific study proving the device meets those criteria.

Therefore, I cannot provide the requested information based solely on the provided text. The document states that the FDA reviewed the premarket notification and determined the device is "substantially equivalent" to legally marketed predicate devices. This determination is based on information provided in the 510(k) submission itself, which would include performance data, but that data is not present in this clearance letter.

To answer your request, I would need access to the actual 510(k) premarket notification submission (document K083294) from Nanosphere, Inc., which would contain the performance study details.

However, based on the general nature of such submissions, I can infer what typical elements would be included, but I cannot provide specific data from this document.

If you can provide the 510(k) submission document (K083294), I would be able to extract the specific information you are looking for.

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The eSensor® CF Genotyping 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 eSensor® CF Genotyping 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.

The eSensor® CF Genotyping Test is intended for use on the eSensor® XT-8 System.

The eSensor® CF Genotyping Test on the eSensor® XT-8 System is an in vitro diagnostic device for performing hybridization and genotyping of multiple mutations and/or polymorphisms in an amplified DNA sample. A single-use, disposable test cartridge is used to perform hybridization and genotyping. The cartridge contains an EEPROM chip which transmits the cartridge lot number, expiration date and protocol identity to the XT-8 instrument.

The analysis process for each sample consists of three steps: 1) Genomic DNA isolated from whole blood obtained using EDTA as anti-coagulant is combined with PCR Mix and Taq polymerase enzyme and is subjected to amplification of target sequences by PCR using a thermal cycler. 2) Amplified DNA is treated with exomuclease enzyme to generate single-stranded target DNA. 3) Single-stranded, amplified target DNA is mixed with hybridization and genotyping reagents and transferred to an eSensor® CF Genotyping Tet cartinge, and the cartridge is inserted in the eSensor® XT-8 Instrument controls the circulation of the sample inside the cartridge to allow hybridization at a controlled temperature and then detects and genotypes the sample by voltammetry.

Genotyping of the test panel polymorphisms is achieved by a sandwich assay principle: 1) Each pair of electrodes contains a different synthetic oligonucleotide capture probe which is complementary to one of the target DNA fragments. 2) The hybridization reagents contain pairs of ferrocenc-labeled synthetic oligonucleotide signal probes; one member of each pair is complementary to the major allele sequence of the target polymorphism, while the second member of the pair is complementary to the minor allele sequence. Each member of the probe pair has a ferrocene label with a different oxidation potential for each allele. 3) Single-stranded, amplificd target DNA hybridizes to its specific capture probe, and in turn hybridizes to the allele-specific, ferrocenc-labeled signal probe. 4) Each electrode of the array is analyzed by voltammetry; the target polymorphism is determined by the location of the electrode containing the capture probe, and the genotype is identified by the ratio of signals from the allclespecific ferrocene labels. The array also includes positive and negative confirm the hybridization reaction and detect non-specific signals.

Upon completion of the test, the EEPROM chip on the cartridge contains information that prevents its re-use with a new sample. The eSensor® XT-8 instrument analyzes the results and provides a report of the test results.

The eSensor® CF Genotyping Test on the XT-8 System is an in vitro diagnostic device intended for genotyping multiple mutations or polymorphisms in an amplified DNA sample utilizing electrochemical detection technology.

Here's an analysis of the acceptance criteria and the study that proves the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied through the successful demonstration of 100% agreement with DNA sequencing after repeat testing for initial no-calls across various studies. The primary performance metric is the agreement with the gold standard (DNA sequencing).

2. Sample Sizes and Data Provenance

• Test Set Sample Sizes:
  • Reproducibility Study: 22 gDNA samples containing positive calls for all ACOG/ACMG panel mutations and the 5/7/9T polymorphism. Each sample was run in duplicate, generating 1320 total replicates across multiple sites and operators.
  • Lot-to-Lot Reproducibility: 21 genomic DNA samples covering all possible genotypes.
  • Genomic DNA Extraction Reproducibility: 20 whole blood samples of different genotypes.
  • Method Comparison: 112 gDNA samples extracted from whole blood.
• Data Provenance: The document does not explicitly state the country of origin for the data or whether it was retrospective or prospective. Given the nature of a 510(k) submission for a diagnostic test, it is highly likely that samples were collected prospectively or obtained from biobanks with appropriate ethical approvals, and the studies were conducted in a controlled, prospective manner to validate device performance. The reproducibility study involved an "Internal Site" and "External Sites," suggesting multi-center data collection, likely within the USA where the manufacturer is located.

3. Number of Experts and Qualifications for Ground Truth Establishment

The document does not explicitly state the number of experts or their qualifications for establishing the ground truth. However, the ground truth was established using DNA sequencing, which is a widely accepted gold standard in genetic testing. The interpretation of DNA sequencing results for CFTR mutations is a specialized task typically performed by molecular geneticists or clinical laboratory directors with expertise in sequencing analysis.

4. Adjudication Method for the Test Set

The document does not describe a formal adjudication method (e.g., 2+1, 3+1). Instead, discrepancies (initial "no-calls") in the device's output were resolved by re-running the tests, leading to "Final Correct Calls." Miscalls were not observed.

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

No multi-reader multi-case (MRMC) comparative effectiveness study was done. This device is an automated genotyping test, not an imaging device that would typically involve human readers for interpretation. The comparison is between the automated device's output and the established DNA sequencing results, and not aimed at quantifying human reader improvement with AI assistance.

6. Standalone Performance

Yes, standalone performance (algorithm only without human-in-the-loop) was performed. The eSensor® CF Genotyping Test is an automated system that provides results via a software program on the eSensor® XT-8 instrument. The performance data presented (reproducibility, method comparison, limit of detection, interfering substances) all reflect the standalone analytical performance of the device without explicit human intervention in the result interpretation process (beyond standard laboratory procedures for running the test and reviewing the final report).

7. Type of Ground Truth Used

The type of ground truth used was DNA sequencing. This is explicitly stated as the reference method for comparison throughout the performance characteristics section (e.g., "All samples gave 100% correct calls when compared with DNA sequencing," and "All samples gave 100% agreement with DNA sequencing").

8. Sample Size for the Training Set

The document does not specify a separate training set size for the device's development or algorithm. This is common for predicate-based 510(k) submissions of in vitro diagnostic devices where the focus is on analytical and clinical validation of the final product, rather than the development process of a machine learning algorithm that typically requires a distinct training phase. The device uses established electrochemical detection technology and a predefined panel of mutations, not a learning algorithm that would necessitate a large training dataset as seen in modern AI/ML submissions.

9. How Ground Truth for the Training Set Was Established

As no separate training set is explicitly mentioned or seems applicable in the context of this device's technology, the method for establishing ground truth for a training set is not provided. The development of the device likely relied on established scientific knowledge of CFTR mutations and electrochemical detection principles, rather than an iterative machine learning training process. The validation studies (using DNA sequencing as ground truth) confirm the performance of the final device.

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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 Cystic Fibrosis Genotyping Assay is a qualitative in vitro diagnostic device used to genotype a panel of mutations and variants in the cystic fibrosis transmembrane conductance requlator (CFTR) gene in genomic DNA isolated from human whole blood specimens. The panel includes mutations and variants recommended by the American College of Medical Genetics (ACMG, 2004) and the American College of Obstetricians and Gynecologists (ACOG, 2005) plus additional multiethnic mutations. The Cystic Fibrosis Genotyping Assay provides information intended to be used for carrier screening in adults of reproductive age, as an aid in newborn screening, and in confirmatory diagnostic testing in newborns and children.

This test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes.

The Cystic Fibrosis Genotyping Assay is designed to genotype the normal and mutant alleles at 30 loci of the CFTR gene using purified human genomic DNA. Genotype coverage includes the panel of 23 mutations recommended by the American College of Medical Genetics (ACMG) 2004 guidelines for use in CF population carrier screening. Coverage also includes 9 additional mutations as part of an expanded core panel to support genetic diversity of multiethnic populations that may be underserved by the ACMG panel alone (e.g. Hispanic, African American). In addition to core panel coverage, the assay is designed to detect polythymidine variants (5/7/9T) within intron 8 of the CFTR gene and polymorphisms (1506V, I507V, and F508C) within Exon 10 of the CFTR gene, in accordance with ACMG guidelines.

Purified genomic human DNA is prepared by standard purification methods. A multiplex polymerase chain reaction (PCR) is then performed to amplify the genomic DNA sample with 16 pairs of PCR primers and DNA polymerase. Next, the oligonucleotide ligation assay (OLA) is performed on the CFTR amplicons. Allele-specific OLA probes hybridize to the respective normal, mutant, and variant alleles and become ligated with fluorescent-labeled common probes by the ligase enzyme. The OLA probes are varied in length due to the addition of inert mobility modifiers. The ligated, fluorescent-labeled DNA fragments are separated on the Celera CEGA-16™ Instrument System by electrophoresis. Detection is based on size and fluorescent label. The ligation products are then identified and genotyped by analysis with the CEGA-16 software and assay-specific configuration disk.

The CF GT Assay also contains Reflex OLA reagents for the detection of the polythymidine 5/7/9T variants in intron 8 of the CFTR gene and for the detection of the I506V, I507V, and F508C polymorphisms in Exon 10 of the CFTR gene. The CFTR R117H mutation, along with the 5T variant of the 5/7/9T polymorphism in intron 8 on the same chromosome (cis), can cause classical CF if another CF mutation is present on the other chromosome. As a result, reflex testing for the 5/7/9T variant with the CF 5/7/9T Reflex OLA assay is recommended when the R117H mutation is detected. The CF Exon 10 Reflex OLA assay is used to verify a homozygous deletion of the F508 or 1507 codon and to exclude a potential false-positive result due to interference by certain non-CF causing variants at codons 506, 507, and 508. The CF Exon 10 Reflex OLA assay will distinguish between a true homozygous F508del or I507del from a sample containing one F508del or I507del allele plus the benign variants of I506V, I507V or F508C, respectively. The same software contained on the configuration disk is used to report reflex testing genotyping information.

1. Acceptance Criteria and Reported Device Performance

2. Sample Size and Data Provenance

• Test Set Sample Size:
  • Accuracy Study: 163 unique samples, which generated 201 samples after creating 38 additional independent aliquots. These 201 samples were tested twice, resulting in 402 replicates.
  • Reproducibility Study: 48 unique genomic DNA samples (25 from frozen whole blood, 23 from frozen cell line pellets). Each unique sample was replicated 3 times, for a total of 144 reproducibility study samples. Each sample was tested 3 times at each of 3 sites, leading to 1296 results (48 unique samples x 27 replicates).
• Data Provenance: Clinical trial samples were "genomic DNA obtained from frozen whole blood and frozen pellets from commercially available cell lines". The document does not specify the country of origin of the data. The studies were retrospective, as samples were collected prior to testing.

3. Number of Experts and Qualifications

The document does not explicitly state the number of experts used to establish the ground truth for the test set or their qualifications. However, it explicitly states that "Bi-directional dideoxy DNA sequencing was performed on all clinical trial samples by an independent supplier operating under applicable good laboratory practices (GLPs) and current good manufacturing practices (cGMP) according to 21 CFR Parts 58, 211 and 820, respectively." This implies that the ground truth was established by a qualified and accredited laboratory.

4. Adjudication Method

The document describes using bi-directional dideoxy DNA sequencing as the "gold standard" for assessing accuracy. There is no mention of a traditional expert adjudication method (e.g., 2+1, 3+1) for discordant results. Instead, any discrepancies were directly compared against the sequencing results.

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

No. This study is for a genetic genotyping assay, not a medical imaging device that typically involves human readers interpreting images. Therefore, an MRMC study comparing human readers with and without AI assistance is not applicable.

6. Standalone Performance

Yes, the study describes the standalone performance of the Celera Cystic Fibrosis Genotyping Assay (algorithm only). The accuracy and reproducibility results are directly from the device's performance against the sequencing gold standard. The device's "Intended Use" states: "This test is also not indicated for stand-alone diagnostic purposes," which refers to the broader clinical context of the test, not the analytical performance being standalone from human interpretation in this specific validation study.

7. Type of Ground Truth Used

The ground truth used was bi-directional dideoxy DNA sequencing, which is considered highly accurate for genetic variant identification and served as the "gold standard" for the study.

8. Sample Size for the Training Set

The document does not specify a separate training set or its sample size. The description of the assay suggests it relies on established molecular biology principles (PCR, OLA, electrophoresis) rather than a machine learning model that typically requires a distinct training phase.

9. How Ground Truth for Training Set Was Established

As no separate training set is explicitly mentioned for a machine learning model, this question is not directly applicable. The assay's "knowledge" is embedded in its design to detect specific CFTR gene mutations and variants based on human genomic DNA and established scientific understanding, with sequencing serving as the gold standard for validation.

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