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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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The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9,22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9:22). This test is not intended for the diagnosis of CML.

The QuantideX qPCR BCR-ABL IS Kit reagents are adapted for use on the ABI 7500 Fast Dx Real-Time PCR Instrument. The assay includes reagents sufficient for 60 reactions. A description of the reagents provided is described below in Table 1.

Here's a summary of the acceptance criteria and study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document describes both analytical and clinical performance acceptance criteria.

Analytical Performance - Precision/Reproducibility

Analytical Performance - Linearity

Analytical Performance - Limit of Quantitation (LoQ)

Analytical Performance - Interfering Substances

Analytical Performance - Primer Specificity

Analytical Performance - RNA Input

Analytical Performance - Traceability

Analytical Performance - Specimen Stability (Whole Blood)

Clinical Performance

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

The primary test set for clinical performance involved a retrospective study with:

• Sample Size: 137 evaluable samples from 96 subjects (out of 139 samples from 98 patients initially collected).
• Data Provenance: Retrospective, collected at 2 clinical sites in the US (OHSU and Hospital of the University of Pennsylvania).

For analytical performance studies, various sample compositions and quantities were used:

• Precision (within-lab & site-to-site): 25 samples formulated from 5 human RNA specimens positive for BCR-ABL1 diluted into RNAs from CML-negative blood. The site-to-site reproducibility study involved 1200 measurements from this 25-sample pool.
• Linearity/Reportable Range: 2 separate RNA specimens (one e13a2, one e14a2) diluted into RNAs from CML-negative whole blood (ranging from MR 0.1 to MR 4.8).
• Limit of Blank (LoB): 30 non-leukemic human RNA specimens.
• Limit of Detection (LoD): 4 separate human RNA specimens positive for BCR-ABL1, serially diluted to 28 levels.
• Limit of Quantitation (LoQ): 6 specimens derived from 6 human RNA positive for BCR-ABL (diluted to target MR 4.7).
• Interfering Substances: Residual CML-positive blood diluted to approximately MR 4.0 into RNA from CML-negative whole blood (tested in 9 replicates).
• Primer Specificity: 11 leukemic specimens (CML, AML, ALL) and 2 non-leukemic RNA specimens.
• Specimen Carryover Contamination: Serially diluted total RNA from residual clinical CML-positive blood into RNA from CML-negative whole blood in a checkerboard pattern (25 high positive, 25 negative).
• RNA Isolation: CML-positive white blood cells serially diluted across 4 levels (MR 1-4) into CML-negative human anti-coagulated whole blood.
• RNA Input: 30 samples derived from 2 primary RNA samples diluted into non-leukemic human RNA (total 216 evaluable observations).
• Traceability: WHO Reference Panel (4 panel members tested in duplicate).
• Reagent Stability: 5 samples diluted to approximately MR 1, 2, 3, 4, and

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The RNARetain® device is a single-use, prefilled container intended for the collection, storage, and transportation of fresh breast tissue specimens for subsequent RNA isolation and further molecular diagnostic testing. For Professional Use Only.

The RNARetain® device consists of an aqueous, hypertonic tissue preservation solution that is provided in a single-use, non-sterile vial intended to serve as the container for the collection, storage, and transport of breast tissue specimens. The ability of the solution formulation to preserve fresh tissue and nucleic acids within the tissue is due to its rapid permeation of tissue to protect cellular nucleic acids from nucleases that would otherwise rapidly degrade the nucleic acids within the specimen. The demonstrated inhibitory effect of the RNARetain® solution on the growth of bacteria, yeast and mold protects the specimen from inadvertent microbial contamination during storage or usage of the device.

The RNARetain® device is a single-use, prefilled container intended for the collection, storage, and transportation of fresh breast tissue specimens for subsequent RNA isolation and further molecular diagnostic testing. The device's performance was evaluated through several non-clinical analytical studies, including precision/reproducibility, stability of MammaPrint® outcomes, comparison with a predicate device, and internal analytical performance by Asuragen.

1. Table of Acceptance Criteria and Reported Device Performance:

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

• Reproducibility (K070675): 5 tumor samples, each isolated in duplicate (total 10 isolations). Origin: Not explicitly stated, but part of Agendia's MammaPrint® submission. Retrospective.
• Stability of MammaPrint® outcomes in RNARetain® (K070675): One tumor, with sections preserved in RNARetain® and immediately snap-frozen. Both sections were labeled 5 times. Origin: Not explicitly stated, but part of Agendia's MammaPrint® submission. Retrospective.
• Comparison Studies (K070675):
  • RNARetain® vs. snap-frozen: 33 breast tumor samples. One part of each sample stored in RNARetain®, another part snap-frozen. Origin: Collected in 2003 as a pilot study for the Dutch Raster clinical trial. Retrospective.
  • Frozen vs. frozen: 18 tumors, with two parts of each immediately snap-frozen. Origin: Collected in the same time period as the 33 breast tumor samples. Retrospective.
• Repeatability and Reproducibility (Asuragen): 10 adjacent pairs of breast tissue from 3 subjects (total 30 tissue samples). Origin: Asuragen, within the US. Prospective (implied, as it was specifically collected for this study).
• Sample Stability (Asuragen for storage conditions): Human breast cancer cell line MCF-7. Quantitative sample size for each condition not explicitly stated (e.g., how many replicates per condition), but various time points and temperatures were tested. Origin: Asuragen labs.
• RNARetain® Reagent Stability: 3 lots of the 8mL vial (6 mL volume); unspecified number of lots for 6 mL vial (5 mL volume) and 2 mL vial (1 mL volume). Origin: Asuragen.

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

• Comparison Studies (K070675): "HE stained sections were re-examined by a pathologist to confirm invasive ductal carcinoma and sufficient tumor cell content." Specific number and qualifications of pathologists are not given beyond "a pathologist".

4. Adjudication Method for the Test Set:

• Not applicable as the reported studies primarily involve analytical performance metrics (RNA purity, integrity, yield, MammaPrint® index correlation) rather than subjective assessments by multiple readers.

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 MRMC comparative effectiveness study was done. This device is a pre-analytical system for tissue preservation, not an AI diagnostic tool that assists human readers.

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

• Yes, the performance studies described are essentially standalone analytical performance evaluations of the RNARetain® device's ability to preserve RNA, mimicking the "algorithm only" concept in the context of a pre-analytical device. The device's performance is measured directly through RNA metrics and comparison to a gold standard (flash-frozen tissue) or established assay (MammaPrint®).

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.):

• MammaPrint® Index: The MammaPrint® assay itself serves as a reference, with the "original index" used for reproducibility studies (Table 3), implying an established ground truth for those samples.
• Pathology: For tissue samples in the comparison studies, "HE stained sections were re-examined by a pathologist to confirm invasive ductal carcinoma and sufficient tumor cell content." This indicates pathology review was used to establish the suitability of the tissue samples.
• Reference Methods: The "gold standard" for tissue preservation (flash-frozen tissue) was used as a comparator/ground truth for RNA yield, purity, and integrity measurements. Fresh Frozen cells were used as a comparator for sample stability studies.

8. The Sample Size for the Training Set:

• Information on a separate "training set" for the RNARetain® device, in the context of an algorithm or learnable system, is not applicable. RNARetain® is a chemical preservation solution and does not involve AI or machine learning that would require a distinct training set. The studies described are validation studies of its chemical and physical performance.

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

• Not applicable, as there is no training set in the context of an AI/ML algorithm for this device.

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