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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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