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The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro diagnostic device that uses polymerase chain reaction (PCR) and capillary electrophoresis to detect and identify the number of cytosine-guanine (CGG) repeats in the fragile X mental retardation-1 (FMR1) gene using genomic DNA isolated from peripheral whole blood specimens. It is solely intended as an aid in the post-natal diagnosis of fragile X syndrome, and fragile Xassociated disorders [i.e., fragile X-associated tremor/ataxia syndrome (FXTAS) or fragile Xassociated primary ovarian insufficiency (FXPOI)], and for carrier testing in adults of reproductive age. Assay results are solely intended to be interpreted by healthcare professionals who are board certified in molecular genetics and to be used in conjunction. with other clinical and diagnostic findings, consistent with professional standards of practice. Reflex testing, clinical genetic evaluation, and genetic counseling should be offered as appropriate. The test is for use on the 3500Dx Series Genetic Analyzer.

This test is not indicated for use for fetal diagnostic testing, newborn screening or for standalone diagnostic purposes.

The AmplideX Fragile X Dx & Carrier Screen Kit (hereafter referred to as the AmplideX Kit) includes reagents sufficient for 100 reactions and are for use on the Applied Biosystems® 3500 Dx Series Genetic Analyzer (8 and 24 capillary) with AmplideX reporter software.

The AmplideX® Fragile X Dx & Carrier Screen Kit is an in vitro diagnostic device used to detect and identify the number of cytosine-guanine (CGG) repeats in the FMR1 gene, which is associated with fragile X syndrome and related disorders. The device uses PCR and capillary electrophoresis.

Here's a breakdown of the acceptance criteria and the studies proving the device's performance:

1. Acceptance Criteria and Reported Device Performance

The device's performance was evaluated against specific criteria for genotype categorization and allele size precision.

• 71-120 CGG repeats: ± 3
• 121-199 CGG repeats: ± 5%
• ≥ 200 CGG repeats: N/A (reported as >200) | Three-site Reproducibility:
• For 10 out of 11 samples, >97% genotype agreement was observed for categorical calls. Sample ASGN-018 (Full Mutation mosaic) showed 54.7% agreement due to the mosaic allele not being found in 81/179 replicates, prompting a limitation statement in IFU.
• Reproducibility of expected allele lengths (excluding mosaic alleles) showed >98% within target precision range.

Lot-to-Lot Reproducibility:

• 99% categorical genotype agreement was observed for all samples (excluding invalids). Samples ASGN-112 and ASGN-016 were not 100% due to third alleles in a few replicates.

• Reproducibility of all alleles (excluding mosaic alleles) showed 100% within target precision specifications.

Mosaicism Reproducibility/LoD:

• Greater than 95% hit rate demonstrated for claimed LoD for mosaic alleles (Table 9 shows various MAFs from 2.0% to 10.0% achieved 94.4% to 100% detection). |
  | Diagnostic Performance (Full Mutation) | PPA, NPA, and OPA ≥ 95% | Full Mutation vs. Southern Blot:
• PPA: 95.71% (95% CI: 88.1-98.5)
• NPA: 99.30% (95% CI: 96.0-99.9)
• OPA: 97.6% (95% CI: 94.5-99.0)
• One discordant sample (194-repeat AmplideX, >200 Southern Blot) was borderline. |
  | Diagnostic Performance (Premutation) | PPA, NPA, and OPA ≥ 95% | Premutation vs. Normal or Intermediate (Southern Blot):
• PPA: 100% (95% CI: 94.7-100.0)
• NPA: 97.10% (95% CI: 90.0-99.2)
• OPA: 98.6% (95% CI: 94.9-99.6) |
  | Carrier Screening Performance | Overall agreement ≥ 95% for Full Mutation/Premutation, Intermediate, or Normal genotypes. | AmplideX vs. FMR1 Dual-PCR Reference Method:
• Premutation/Full Mutation: 100% (95% CI: 94.7-100.0)
• Intermediate: 85.7% (95% CI: 75.7-92.1)
• Normal: 98.6% (95% CI: 92.2-99.7)
• Note: When considering assay precision, 8 borderline samples (54/55 CGG) were reclassified as concordant, increasing Intermediate agreement to 97.1%. |
  | LoD for mosaic alleles | > 95% hit rate for claimed LoD | - Intermediate on Normal: 2.0% MAF, 100.0% detected
• Premutation on Normal: 2.0% MAF, 94.4% detected
• Full Mutation on Normal: 6.1% MAF, 100.0% detected
• Normal on Premutation: 2.0% MAF, 100.0% detected
• Premutation on Premutation: 5.0% MAF, 97.1% (low), 100.0% (high) detected
• Full Mutation on Premutation: 7.0% MAF, 100.0% detected
• Normal on Full Mutation: 2.0% MAF, 100.0% detected
• Premutation on Full Mutation: 10.0% MAF, 100.0% detected |
  | DNA Input Range (20-80 ng) | 100% categorical genotype agreement for valid samples, all expected allele peaks within target precision range, and acceptable QC failure rates within the recommended range. | - 100% categorical genotype agreement and all expected allele peaks within target precision for all valid samples and all input levels.
• QC failure rates: 1 ng (6.25%), 10 ng (1.88%), 20 ng (0.6%), 40 ng (1.3%), 80 ng (0%), 160 ng (0.63%). These results support the recommended 20-80 ng range. |
  | DNA Extraction Methods | 100% categorical genotype agreement and 100% of all measured alleles within target precision range for common commercial DNA extraction methods. | - 100% categorical genotype agreement and 100% of all measured alleles within target precision range for the three common commercial DNA extraction methods tested (solution precipitation, manual silica spin column, and manual magnetic bead). |
  | Thermal Cycler Equivalence | 100% genotype category calls agreement and 100% of all identified independent alleles within their respective target precision ranges across all thermal cycler types. | - 100% genotype category calls agreement and 100% of all identified independent alleles within their respective target precision ranges across three separate thermal cyclers. |
  | Interfering Substances (Hemoglobin, EDTA) | For processable samples, >95% genotype category agreement and 100% of alleles within expected precision range. (Limitation statement regarding impact on assay failure rate for excess hemoglobin/EDTA is included in IFU). | - Effects on DNA concentration yield observed (hemoglobin reduced, EDTA enhanced).
• For samples that could be processed, genotype category agreement was >95% and 100% of alleles within expected precision range. (Limitation statement for IFU). |
  | Specimen Handling Stability | 100% genotype category agreement and all expected allele peaks within target precision for DNA extracted from whole human blood stored at 2-8°C for up to 2 weeks. | - 100% genotype category agreement and all expected allele peaks within target precision for all timepoints across all samples (up to 2 weeks storage at 2-8°C). |
  | DNA Freeze/Thaw Stability | 100% genotype category agreement and all expected allele peaks within target precision for extracted DNA across (b)(4) freeze/thaw cycles. | - 100% genotype category agreement and all expected allele peaks within target precision for up to (b)(4) freeze/thaw cycles. |
  | PCR & CE Reagent Product Stability | PCR Product: 100% categorical genotype agreement and >95% of measured alleles within target precision for PCR products stored up to 3 days at 2-8°C.
  CE Product: 100% categorical genotype agreement and 100% of measured alleles within target precision for samples prepared for CE analysis and stable at ambient temperature for up to 24 hours. | - PCR Product: 100% categorical genotype agreement for all timepoints across all samples and >95% of all measured alleles within target precision range (up to 3 days at 2-8°C).
• CE Product: 100% categorical genotype agreement for all timepoints across all samples and 100% of all measured alleles within target precision range (up to 24 hours at ambient temperature). |
  | Real Time Kit Stability | 100% categorical genotype agreement and 100% of measured alleles within target precision. (Supports a minimum of 1 year, with ongoing study for 24 months). | - 100% categorical genotype agreement for all timepoints across all samples and 100% of all measured alleles within target precision range. (Supports at least 1-year storage stability at present). |
  | Freeze/Thaw Kit Stability | 100% categorical genotype agreement and 100% of measured alleles within target precision for up to eight freeze/thaw cycles. | - 100% categorical genotype agreement for all timepoints across all samples and 100% of all measured alleles within target precision range (up to eight freeze/thaw cycles). |
  | Kit Shipping Stability | 100% of measured alleles within target precision range and 100% genotype category agreement across different shipping configurations. | - 100% of their measured alleles within the target precision range and 100% genotype category agreement across all different shipping configurations (based on ISTA 7D, 24-hour summer profile). |
  | WHO Standards Testing Accuracy | Correctly classifies and sizes international Fragile X reference standards. | - The test correctly classified and sized the 5 WHO International Standard Fragile X Syndrome Reference Panel samples (NIBSC code: 08/158) with 100% of measured values falling within the expected CGG range, even when reported as ">200" for full mutations. |

2. Sample Sizes and Data Provenance (Test Set)

• Precision (Reproducibility) Studies:
  • Three-site reproducibility: 11 samples (7 clinical whole blood specimens, 4 contrived from cell lines spiked into leukocyte-depleted whole human blood). This yielded 1980 total sample measurements (excluding controls).
  • Lot-to-lot reproducibility: 11 samples (same panel as above). This yielded 2376 total sample measurements (excluding controls).
  • Mosaicism reproducibility/LOD: Panel of mixed cell line DNA with known genotypes and a panel of 30 samples involving mixing DNA from clinical specimens. This yielded 36 replicate measurements per specimen.
• DNA Input Study: 8 samples (6 clinical, 2 contrived). This resulted in 960 sample measurements (20 replicates per dilution level per sample).
• Clinical Performance Studies:
  • Diagnostic Performance: 207 leftover clinical specimens (111 female, 96 male) obtained from patient samples submitted for routine FMR1 5'UTR mutation testing across multiple clinical sites.
  • Carrier Screening Performance: (b)(4) specimens from females ((b)(4) years of age) enrolled across (b)(4) clinical sites, resulting in 207 evaluable subjects.
  • Mosaic Allele Calling Accuracy: 49 specimens identified as mosaic by AmplideX across the two clinical studies (27 from diagnostic, 22 from carrier screening). An additional 40 randomly selected samples were further evaluated for mosaicism resolution after initial discordant cases were addressed.
  • WHO Standards Testing: 5 samples from the WHO International Standard Fragile X Syndrome Reference Panel (NIBSC code: 08/158). This yielded 135 sample measurements (excluding control).

Data Provenance: The data primarily appears to be retrospective for the clinical performance studies, using "leftover specimens" and "patient samples submitted for routine FMR1 5'UTR mutation testing" or "enrolled across clinical sites." The analytical performance studies used a mix of clinical specimens and contrived samples (cell lines spiked into blood). The document does not explicitly state the country of origin for the clinical data.

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

The document does not explicitly state the number of experts or their specific qualifications for establishing the ground truth for the clinical test sets (Southern Blot and FMR1 Dual-PCR Reference Method). However, it mentions that the interpretation of assay results is "solely intended to be interpreted by healthcare professionals who are board certified in molecular genetics."

4. Adjudication Method (Test Set)

The document does not describe a formal adjudication method for the test set.

• In the diagnostic performance study, results were compared directly against the reference methods (Southern Blot or FMR1 Dual-PCR). Discordant samples were noted, and in one case for full mutation, a borderline sample was identified.
• For mosaicism, there was a manual evaluation of orthogonal PCR method results for cases discordant with AmplideX.

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

There is no mention of a multi-reader multi-case (MRMC) comparative effectiveness study to assess the effect size of human readers improving with AI vs. without AI assistance. This device is a diagnostic kit for laboratory use, and its performance is evaluated against reference methods, not typically in a human-in-the-loop context.

6. Standalone Performance Study (Algorithm Only)

Yes, the studies described are for standalone performance (algorithm only, as applied to the output of the PCR and capillary electrophoresis). The AmplideX Fragile X Reporter Software performs automated analysis and categorization of FMR1 alleles based on size. The performance is assessed by comparing the device's output (categorical calls and CGG repeat lengths) directly against established reference methods (Southern Blot, FMR1 Dual-PCR) or known values (WHO standards, contrived samples).

7. Type of Ground Truth Used

• Clinical Performance (Diagnostic):
  • Southern Blot: For Full Mutation classification and Premutation vs. Normal or Intermediate assessment. Southern Blot is a molecular biology method considered a gold standard for detecting large FMR1 expansions.
• Clinical Performance (Carrier Screening):
  • FMR1 Dual-PCR Test: An alternate orthogonal independently validated PCR assay was used as the comparator for carrier screening, as Southern blot is noted to not have accurate sizing in the premutation range.
• Analytical Performance (Precision, LoD):
  • Clinical Samples: Whole blood specimens from patients with known FMR1 genotypes.
  • Contrived Samples: Genomic DNA extracted from cell lines with known gender, genotype, and expected allele sizes, sometimes spiked into leukocyte-depleted whole human blood.
  • Mixed Cell Line DNA: With known genotypes and mosaic allele frequencies for LoD studies.
• WHO Standards Testing:
  • WHO International Standard Fragile X Syndrome Reference Panel (NIBSC code: 08/158): These are internationally established reference materials with characterized mean repeat lengths and ranges.

8. Sample Size for the Training Set

The document does not provide a specific sample size for a "training set." This device is a diagnostic assay with software for interpretation, but the context here suggests a traditional verification and validation study rather than a machine learning model where a distinct training set (for model parameters) and test set (for performance evaluation) would be explicitly separated and reported. The analytical and clinical studies serve as the validation of the device's performance.

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

As mentioned above, there isn't an explicit "training set" in the context of a machine learning-based device. The ground truth for the reference materials and clinical samples used in the validation studies was established through:

• Reference Methods: Southern Blot analysis and an independently validated FMR1 Dual-PCR assay are considered the ground truth for clinical performance.
• Known Characteristics: For contrived samples and cell lines, the genotypes and allele sizes are "known" based on prior characterization.
• International Standards: The WHO/NIBSC standards have established and published characterizations from a study consortium.

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