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The Invitae Common Hereditary Cancers Panel is a qualitative high-throughput sequencing based in vitro diagnostic test system intended for analysis of germline human genomic DNA extracted from whole blood for detection of substitutions, small insertion and deletion alterations and copy number variants (CNV) in a panel of targeted genes.

This test system is intended to provide information for use by qualified health care professionals, in accordance with professional guidelines, for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with a diagnosed cancer.

The test is not intended for cancer screening or prenatal testing. Results are intended to be interpreted within the context of additional laboratory results, family history, and clinical findings.

The test is a single-site assay performed at Invitae Corporation.

The Invitae Common Hereditary Cancers Panel uses hybridization-based capture, nextgeneration sequencing (NGS), and a custom-built bioinformatics pipeline to compare all positions in targeted regions of 47 genes to a reference sequence and identify variants, including single nucleotide variants (SNVs), insertions and deletions (Indels), and copy number variants (CNVs). Sequence analysis covers clinically important regions of each gene, including coding exons and 10 to 20 base pairs of adjacent intronic sequence on either side of the coding exons in the transcript listed in Table 1. Genes of "high clinical significance" are defined as those for which the test result(s) may lead to prophylactic screening, confirmatory procedures, or treatment that may incur morbidity or mortality to the patient and are shown in bold text. In addition, the analysis covers the select non-coding variants specifically defined in the table. Any variants that fall outside these regions are not analyzed. Identified variants are assessed by clinical professionals using currently available literature and data from public genetic variant databases. Variants are assigned a score, calculated according to an algorithm that weights the available clinical evidence. Possible outcomes include the following, which are based on joint ACMG/AMP Committee guidelines: Benign (not reported), Likely benign (not reported), Likely pathogenic, Pathogenic, Variant of Uncertain Significance. Variants are reported using HGVS nomenclature and the human reference genome GRCh37.

The Invitae Common Hereditary Cancers Panel is a high-throughput sequencing-based in vitro diagnostic test system designed for detecting germline substitutions, small insertions and deletions, and copy number variants (CNVs) in 47 targeted genes. It is intended for use by qualified healthcare professionals for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with diagnosed cancer.

Here's a breakdown of the acceptance criteria and the study proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the Invitae Common Hereditary Cancers Panel are primarily based on the analytical performance metrics of Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Technical Positive Predictive Value (TPPV), and Technical Negative Predictive Value (TNPV).

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The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) 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) adult patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is a reverse transcription-quantitative PCR performed on the Sniper Digital PCR All-in-One System 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 BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is intended for use only on the Sniper Digital PCR All-in-One System.

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 BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is designed for detection of the BCR-ABL1 fusion gene (p210) and ABL1 gene, with specific primers and specific fluorescence probes. The test process includes three parts. The first part is to extract ribonucleic acid (RNA) from peripheral blood of CML patients. The second part is to detect BCR-ABL1 fusion gene (p210) and ABL1 internal reference gene in RNA samples by RT-dPCR (Reverse Transcription-Droplet PCR) reaction solution using the Sniper Digital PCR All-in-One System (DQ24-Dx). The third part is to analyze the results.

The Sniper Digital PCR All-in-One System consists of one instrument, which can be used together with it's supporting consumables and BCR-ABL1 (p210) %IS Kit (Digital PCR Method) to complete the detection of samples.

The Sniper Digital PCR All-in-One System divides the sample into about 20000 droplets and carries out PCR amplification, read the number of positive and negative droplets through fluorescent signals, and then calculate the concentration of nucleic acid quantitatively according to the volume of the droplets and the principle of Poisson Distribution.

DQ24-Dx-Sight Software (v1.0.2) is used to control the system and analyze test results. This software is embedded in the Sniper Digital PCR All-in-One System.

Here's a breakdown of the acceptance criteria and study detailed in the provided document:

Acceptance Criteria and Device Performance

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Bladder EpiCheck Kit is intended for the qualitative detection of DNA methylation patterns of 15 loci in human DNA that are associated with transitional cell carcinoma of the bladder. The test is performed on voided urine samples and run on the ABI® 7500 Fast Dx Real-Time PCR system.

Bladder EpiCheck Kit is indicated for use as a non-invasive method to monitor for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with Non-Muscle Invasive Bladder Cancer.

The Bladder EpiCheck Test is a real-time PCR-based in vitro diagnostic assay intended for the qualitative detection of DNA methylation patterns associated with transitional cell carcinoma of the bladder to monitor for tumor recurrence (in conjunction with cystoscopy) in patients previously diagnosed with non-muscle invasive bladder cancer (NMIBC).

The assay consists of a panel of 15 novel DNA methylation (covalent addition of methyl (CH3) groups to the C5 position of the pyrimidine ring of cytosines, typically in a CpG dinucleotide) biomarkers that were found to distinguish between patients with bladder cancer and patients without bladder cancer. The Bladder EpiCheck Test differentiates between methylated and non-methylated DNA, creating a unique platform for methylation profiling of urine specimens towards the detection of bladder cancer recurrence in patients previously diagnosed with the disease. The test is comprised of reagents for end-to-end (sample-to-answer) processing of urine samples (reagents for DNA extraction, DNA digestion, PCR amplification, and analysis software), and is performed using the Applied Biosystems® 7500 Fast Dx Real-Time PCR system.

A voided urine specimen is centrifuged, and the cells (both normal and cancerous if present) are separated from the urine supernatant. DNA is then extracted from the cell pellet using the Bladder EpiCheck Extraction kit (P/N NX899090-01C). The extracted DNA is digested using a methylation-sensitive restriction enzyme mix. which cleaves DNA at specific recognition sequences if they are unmethylated. Methylated DNA is protected from enzymatic digestion and therefore remains intact.

Here's a breakdown of the acceptance criteria and study details for the Bladder EpiCheck Kit, based on the provided FDA 510(k) summary:

Device: Bladder EpiCheck Kit
Intended Use: Qualitative detection of DNA methylation patterns of 15 loci in human DNA associated with transitional cell carcinoma of the bladder, used as a non-invasive method to monitor for tumor recurrence in conjunction with cystoscopy in patients previously diagnosed with Non-Muscle Invasive Bladder Cancer.

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in a dedicated table format. However, performance metrics are reported. Based on the "Method Comparison" section (9.2 Clinical), the de facto acceptance criteria appear to be tied to non-inferiority against the predicate device (UroVysion Bladder Cancer Kit) and sufficient performance against a Gold Standard.

2. Sample Size and Data Provenance for Test Set (Clinical Performance Study)

• Sample Size:
  • Against Gold Standard: 583 subjects (total voided urine specimens collected from 583 subjects). Valid Bladder EpiCheck and GS results were obtained from 449 subjects.
  • Against Predicate Device (Matched Cases): Valid Bladder EpiCheck, UroVysion, and GS results were obtained from 352 samples.
  • Specificity in Urology Patients without Bladder Cancer: 147 subjects.
  • Clinical Specificity - Cross Reactivity with Other Cancers: 147 urine samples.
• Data Provenance:
  • Country of Origin: U.S. and Canada (from 11 academic and urology specialty medical centers).
  • Retrospective or Prospective: The main clinical study (Method Comparison) was a multi-center, prospective, IRB-approved longitudinal study. The specificity study in urology patients without bladder cancer was also multi-center, prospective. The cross-reactivity study utilized banked remnant de-identified urine samples, which would generally be considered retrospective.

3. Number of Experts and their Qualifications for Establishing Ground Truth for the Test Set

The document does not specify the number of experts or their qualifications for establishing the ground truth. It states that positive cases were confirmed by "cystoscopy and pathology." This implies that the ground truth was established by clinical diagnoses and pathological examination of tissue, presumably performed by trained urologists and pathologists, which are standard practices. No "experts" are explicitly described as reviewing cases for the purpose of establishing a "ground truth" consensus for the study, beyond the routine clinical workflow.

4. Adjudication Method for the Test Set

The adjudication method is implicitly described for the Gold Standard (GS) definition:

• "a subject was considered 'positive' if the interpretation for either cytology or the combined cystoscopy/pathology results were positive"
• "and a subject was considered 'negative' if both cytology and the combined cystoscopy/pathology results were negative."

This indicates a hierarchical or "any positive result makes it positive" adjudication for the ground truth definition. There is no explicit mention of an adjudication panel (e.g., 2+1, 3+1) for cases of disagreement between cytology and pathology results, or for disagreements among multiple readers of the ground truth modalities.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not conducted. This device is a molecular diagnostic test (in-vitro diagnostic) and not an imaging AI device that assists human readers. Therefore, the concept of human readers improving with AI vs. without AI assistance does not apply in this context. The comparison was between the Bladder EpiCheck test result and clinical ground truth (cytology/pathology), and between Bladder EpiCheck test results and the predicate device's test results.

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

Yes, the device's performance, as reported in the "Method Comparison" section, is a standalone (algorithm only without human-in-the-loop) performance. The Bladder EpiCheck Kit provides a qualitative result (positive/negative) based on its algorithm (EpiScore), and this result is compared directly to the established Gold Standard.

7. Type of Ground Truth Used

The primary ground truth used for the clinical performance study consisted of:

• Combined Cystoscopy/Pathology data: This is the gold standard for definitive diagnosis of bladder cancer recurrence.
• Clinical Cytology: Urine cytology was also part of the Gold Standard definition.

Therefore, the ground truth is a combination of pathology (histopathological examination of biopsy/resection specimens) and outcomes data (clinical diagnosis via cystoscopy, supplemented by cytology).

8. Sample Size for the Training Set

The document refers to "Clinical Cutoff (Training and Feasibility Data)" in section 9.1.

• Total for software algorithm development: 178 samples.
• First set (for cut-off definition): 109 samples (40 control, 69 UCC positive).
• Second set (for cut-off validation): 67 samples (51 control, 16 UCC positive).

It's important to note that this "training" refers to the development and validation of the EpiScore algorithm's cutoff, not necessarily a machine learning training set in the AI sense.

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

For the "training" set (used for algorithm development and cutoff definition, section 9.1), the ground truth was established by:

• "urine samples collected from control patients with a history of bladder cancer and bladder cancer positive patients confirmed by cystoscopy and pathology."
• "Urothelial Cell Carcinoma (UCC) positive patients confirmed by pathology."

Similar to the test set, the ground truth for algorithm development was based on definitive clinical diagnosis and pathological confirmation.

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The Eonis™ SCID-SMA kit is intended for the qualitative detection of the SMN1 gene exon 7 as an aid in screening newborns for Spinal Muscular Atrophy (SMA). The test is intended for DNA from blood specimens dried on a filter paper and for use on the QuantStudio™ Dx Real-Time PCR instrument.

This test is only intended for use for screening of SMA that bear the homozygous deletion of SMN1 exon 7.

This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing.

The Eonis SCID-SMA kit contains reagents to detect three biomarkers: TREC, KREC and exon 7 in the SMN1 gene. Detection of TREC and KREC was cleared in K203035.

The newborn screening workflow for the Eonis SCID-SMA kit includes:

• Two liquid handling platforms (one for DNA extraction and one for PCR master mix . setup)
• QuantStudio Dx Real-Time PCR instrument .
• . Eonis Analysis Software

Each Eonis SCID-SMA kit contains reagents for up to 384 reactions or 1152 reactions including kit controls. The kit contents are listed in Table 1. Materials required but not provided include the Eonis DNA Extraction Kit, Eonis Analysis Software and consumables (Table 2).

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

• Precision (SMN1 presence call): Sample 11 (SMA positive) showed 100% (107/107) "Above Cut-off" (Presumptive Positive). Other normal/carrier samples (1-8, 10, 12-13) showed 100% "Below Cut-off" (Presumptive Normal), with one exception in Sample 9 (99.1% Below Cut-off, 1/106 incorrect call).
• Reproducibility (SMN1 presence call): All 13 samples showed 100% agreement (150/150 replicates) for qualitative calls across study sites, operators, and runs. This includes Sample 11 (SMA positive) consistently yielding "Above Cut-off" (Presumptive Positive) results, and other samples consistently yielding "Below Cut-off" (Presumptive Normal).
• Filter Paper Reproducibility: 100% qualitative agreement for all tested samples across different filter paper brands and lots.
• qPCR Method Equivalency: 100% qualitative agreement between 384-well and 96-well qPCR methods.
• DNA Extraction Equivalency: 100% concordance for qualitative calls among JANUS handler, a second commercial liquid handler, and manual extraction processes. |
  | False Positive Rate for SMN1 Detection (Desirable: Low) | Clinical Study: 0.0% false positive rate (0 historical SMA cases misclassified as normal out of 3018 normal newborns).
  Limit of Blank Study: 0.0% false positive rate (analytes-negative samples consistently yielded no Ct value) |
  | False Negative Rate for SMN1 Detection (Desirable: Low) | Clinical Study: 0.0% false negative rate (0 historical SMA cases misclassified as normal out of 51 confirmed SMA cases). |
  | Concordance with Genetic Testing (Accuracy) | Accuracy Study: 100% positive percentage agreement (51/51 confirmed SMA cases correctly identified) and 100% negative percentage agreement (55/55 confirmed negative samples correctly identified), resulting in 100% overall agreement. |
  | Specimen Stability for DBS samples | No differences in qualitative calls or SMN1 Ct values at day 28 compared to day 0 under varying temperature and humidity conditions. |
  | Eonis DNA Extraction Kit In-Use and On-Board Stability | Stable for 14 days at +19 - +25 °C after first opening. |
  | Eonis DNA Extraction Kit Real-Time and Transport Simulation Interim Stability | No difference in SMN1 Ct values up to 7 months. Can be shipped at room temperature. Supports a shelf life of 6 months. |
  | Eonis SCID-SMA Kit Interim In-Use and On-Board Stability | PCR Reagents 1 and 2 stable for 14 days at +2°C to +8°C after thawing. SCID-SMA Kit Controls stable for 14 days at -30°C to -16°C after first use. |
  | Eonis SCID-SMA Kit Real-Time and Transport Simulation Interim Stability | No change in SMN1 Ct values for assay controls or PCR Reagents 1 or 2 up to 10 months. Supports a shelf life of 180 days (6 months). |
  | Control of Contamination (Carry-Over) | Analytical Study: 4% false-negative rate observed in artificially high analyte-positive samples in a checkerboard configuration.
  Clinical Validation: 0% false negative rate; no clinically significant carry-over observed. |

Study Details

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

• Precision Study (Test Set 1):
  • Sample Size: 13 representative DBS samples (SMA positive, carrier, and normal), tested in 108 replicates (106 for some) per sample over 54 runs. Total measurements: 13 samples * 108 measurements = 1404 measurements.
  • Data Provenance: Analytical performance studies conducted using contrived samples (cord blood or adult whole blood with hematocrit adjusted to neonate levels). SMA positive sample created by spiking SMN1 negative Coriell cells into leukocyte-depleted blood.
• Reproducibility Study (Test Set 2):
  • Sample Size: 13 samples (same as precision study), tested in 150 replicates per sample across 3 study sites over 5 operating days. Total measurements: 13 samples * 150 measurements = 1950 measurements.
  • Data Provenance: Contrived samples (cord blood or adult whole blood with hematocrit adjusted to neonate levels).
• Filter Paper Reproducibility Study (Test Set 3):
  • Sample Size: 6 samples (from precision study set) prepared on 3 lots of 2 brands of filter paper each (total 36 conditions). 5 replicates per condition. Total 900 results.
  • Data Provenance: Contrived samples.
• Limit of Blank Study (Test Set 4):
  • Sample Size: 150 replicates of contrived analyte-negative samples per kit lot (total 300 replicates across 2 kit lots).
  • Data Provenance: Contrived samples (SMN1-negative cells from Coriell Institute into leukocyte-depleted human blood).
• Interference Study (Test Set 5):
  • Sample Size: 7 interfering substances, 2 interferent levels, 3 target DNA levels, 13 replicates per level. Total 544 sample results.
  • Data Provenance: Contrived samples (SMN1 presumptive normal).
• qPCR Method Equivalency Study (Test Set 6):
  • Sample Size: 13 samples (from precision study set), 5 replicates per sample, test for 2 PCR methods. Total 1560 results.
  • Data Provenance: Contrived samples.
• DNA Extraction Equivalency Study (Test Set 7):
  • Sample Size: 7 samples (from precision study set), 5 replicates per sample, test for 3 extraction/PCR methods. Total 1050 results.
  • Data Provenance: Contrived samples.
• Clinical Screening Study (Test Set 8):
  • Sample Size: 3069 DBS specimens. This included 51 retrospective archived DBS specimens from subjects confirmed positive for SMA and 3018 routine newborn screening specimens.
  • Data Provenance: Retrospective archived DBS specimens from the US and Denmark. Routine newborn screening specimens obtained from the Danish Newborn Screening Biobank (NBS-Biobank).
• Accuracy Study (Test Set 9):
  • Sample Size: 51 confirmed positive SMA samples and 55 presumed negative DBS samples. Total 106 samples.
  • Data Provenance: Confirmed positive SMA samples (molecular genetic testing result showing homozygous deletion of SMN1 exon 7) and presumed negative DBS samples matched by storage time.

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

The document mentions that the clinical status of the routine subjects in the Clinical Screening Study was determined through a "retrospective review by clinical experts." However, it does not specify the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience"). For the confirmed SMA cases, "confirmatory test results" (molecular genetic testing) were used as the comparator, which is a definitive method rather than expert consensus on imaging.

4. Adjudication Method for the Test Set

The document does not describe any specific adjudication method (like 2+1 or 3+1) for the interpretation of results in the test sets. For the Eonis SCID-SMA kit, the interpretation of results appears to be largely automated by the Eonis Analysis Software based on pre-set Ct cut-off values. For the clinical screening study, samples with values above the cut-off were re-tested in duplicate to obtain the final result.

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

No, an MRMC comparative effectiveness study was not performed. This device is a quantitative PCR-based assay with automated interpretation software, not an imaging-based AI system that assists human readers. Therefore, the concept of human readers improving with AI assistance is not applicable here.

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

Yes, the performance of the Eonis SCID-SMA Kit is essentially standalone. The Eonis Analysis Software "automatically flags quality control (QC) violations and interprets results according to the cut-offs," presenting results as "Presumptive Positive" or "Presumptive Normal." While human operators perform the lab procedures (DNA extraction, PCR setup), the final interpretation of the test result itself is automated by the algorithm based on the measured Ct values against a pre-set cut-off.

7. The Type of Ground Truth Used

• Analytical Studies (Precision, Reproducibility, Limit of Blank, Interference, Method/Extraction Equivalency, Carry-over): Ground truth was based on the contrived nature of the samples. For example, SMA positive samples were created by spiking specific cells, and analyte-negative samples were prepared to contain no target analyte.
• Clinical Screening Study:
  • For SMA positive cases (51 samples): Ground truth was established by "confirmatory test results" (molecular genetic testing showing homozygous deletion of SMN1 exon 7).
  • For routine newborn screening specimens (3018 samples): Ground truth was established by "retrospective review by clinical experts to confirm the routine subject cohort samples were from unaffected individuals." This suggests a form of clinical outcome/diagnosis as ground truth, likely based on further clinical evaluations, not just genetic testing for SMN1 deletion.
• Accuracy Study:
  • For confirmed SMA samples (51 samples): Ground truth was "molecular genetic testing result showing homogenous deletion of SMN1 gene exon 7."
  • For presumed negative samples (55 samples): Ground truth was confirmed by "molecular genetic testing for SMN1" using a CE-IVD labeled assay. This is molecular genetic testing/pathology ground truth.

8. The Sample Size for the Training Set

The document does not explicitly state a separate "training set" for the Eonis SCID-SMA Kit. As a PCR-based assay, its "learning" primarily involves setting the appropriate Ct cut-off value (31.24). This cut-off is pre-set in the Eonis Analysis Software. The document does not describe how this specific cut-off was initially determined (e.g., through a separate study for calibration or training). The studies described here are verification and validation studies to demonstrate the performance with that pre-set cut-off.

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

Since a distinct "training set" is not explicitly mentioned for algorithmic development in a machine learning sense, the establishment of ground truth for training is not detailed. The inherent "training" of such a system would involve optimizing the Ct cut-offs based on a set of known positive and negative samples to achieve desired diagnostic sensitivity and specificity. However, the provided text focuses on the validation of the device's performance given its pre-determined operational parameters (like the 31.24 Ct cut-off).

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The Eonis™ SCID-SMA kit is intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) as an aid in screening newborns for Severe Combined Immunodeficiency (SCID) and for the semi-quantitative determination of KREC (Kappa-deleting recombination excision circle) as an aid in screening newborns for X-linked agammaglobulinemia (XLA). The test is intended for DNA from blood specimens dried on a filter paper and for use on the QuantStudio™ Dx Real-Time PCR instrument.

This test is not intended for screening of SCID-like Syndromes, such as DiGeorge Syndrome, or Omenn Syndrome. lt is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID. The test is not indicated for screening B-cell deficiency disorders other than XLA, such as atypical XLA, or for screening of XLA carriers.

This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing.

The Eonis SCID-SMA kit is a multiplex real-time PCR-based assay. It uses target sequence-specific primers and TaqMan™ probes to amplify and detect three targets: TREC, and RPP30, in the DNA extracted from newborn dried blood spot (DBS) using Eonis DNA Extraction kit in a single PCR reaction.

Each Eonis SCID-SMA kit contains reagents for up to 384 reactions (for 3241-001U) or 1152 reactions (for 3242-001U) including kit controls.

The document describes the Eonis SCID-SMA kit, a real-time PCR-based assay for newborn screening of Severe Combined Immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA). The study provided demonstrates the device's analytical and screening performance to support its substantial equivalency to a predicate device.

Here's a breakdown of the requested information:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" as a separate table. However, it presents Sensitivity and Specificity for both TREC and KREC analytes, which serve as key performance metrics. These values are compared to the predicate device.

Reported Device Performance of Eonis SCID-SMA Kit:

Comparison to Predicate Device (PerkinElmer EnLite Neonatal TREC Kit) for TREC:

The reported performance clearly aims to meet or exceed the performance of the predicate device, demonstrating 100% sensitivity and high specificity for both analytes.

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

• Sample Size for Screening Performance Study (Test Set):
  • Total DBS specimens: 3090
  • Confirmed SCID positive: 17
  • Confirmed XLA positive: 6
  • Normal newborn screening specimens: 3018 (retrospective archived)
• Data Provenance: Retrospective archived dried blood spot specimens.
  • Country of Origin: US and Denmark.
  • Study conducted in Denmark.

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

• Number of Experts: Not explicitly stated as a specific number. The document mentions "clinical experts" were used.
• Qualifications of Experts: The document states "clinical experts" retrospectively reviewed the clinical status of routine subjects to confirm they were from unaffected individuals. Further specific qualifications (e.g., specific medical specialty, years of experience) are not provided in this document.

4. Adjudication Method for the Test Set

• Adjudication Method: The document describes a retesting protocol for initial "screen positive" results.
  • "The specimens having TREC and KREC levels below the cut-off values in the initial round of testing were re-tested in duplicate."
  • "The final results (presumptive positive, invalid result) were classified after the second round of testing."
  • This implies a form of internal re-adjudication based on duplicate retesting for samples near the cut-off. There is no mention of external expert adjudication for discordant results or a specific "X+Y" type of adjudication.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

• MRMC Study: No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an in-vitro diagnostic (IVD) kit for semi-quantitative determination of biomarkers, not an AI assisting human readers of medical images. Therefore, the concept of human readers improving with AI assistance is not applicable to this type of device.

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

• Standalone Performance: Yes, the entire performance data regarding sensitivity, specificity, reproducibility, precision, limit of detection, and linearity are based on the standalone performance of the Eonis SCID-SMA kit (the algorithm of the kit combined with the instrument) on dried blood spot samples. This device operates as an automated assay, therefore, its performance is inherently "standalone" in terms of its analytic and clinical validity.

7. The Type of Ground Truth Used

• Ground Truth Type:
  • Confirmatory testing: For SCID and XLA positive cases, "Confirmatory test results were used as the comparator." This implies clinical diagnosis or gold standard laboratory tests.
  • Clinical expert retrospective review: For normal newborn screening specimens, "The clinical status of the routine subjects was determined through a retrospective review by clinical experts to confirm the routine subject cohort samples were from unaffected individuals." This indicates clinical outcomes or medical records adjudicated by experts.

8. The Sample Size for the Training Set

• Training Set Sample Size: The document does not explicitly state the sample size of a separate "training set" for the assay. The study described is a clinical validation (test set). For assay development (which would include "training" for establishing parameters like cut-offs), the document mentions:
  • Cut-off values were established using "an independent dataset." The size of this independent dataset is not specified.
  • Reproducibility and precision studies used panels of dried blood spots at different TREC/KREC levels, but these are for analytical validation rather than establishing classification criteria.

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

• Training Set Ground Truth Establishment: As no specific "training set" is detailed, the method for establishing ground truth for any data used during the assay's development or cut-off determination (the "independent dataset" mentioned for cut-off study) is not explicitly described. However, it's reasonable to infer that similar methods to the test set ground truth (confirmatory testing for affected individuals and clinical review for unaffected individuals) would have been applied during the development phase.

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The Parsortix® PC1 system is an in vitro diagnostic device intended to enrich circulating tumor cells (CTCs) from peripheral blood collected in K>EDTA tubes from patients diagnosed with metastatic breast cancer. The system employs a microfluidic chamber (a Parsortix cell separation cassette) to capture cells of a certain size and deformability from the population of cells present in blood. The cells retained in the cassette are harvested by the Parsortix PC1 system for use in subsequent downstream assays. The end user is responsible for the validation of any downstream assay. The standalone device, as indicated, does not identify, enumerate or characterize CTCs and cannot be used to make any diagnostic/prognostic claims for CTCs, including monitoring indications or as an aid in any disease management and/or treatment decisions.

The Parsortix® PC1 system is a bench top laboratory instrument consisting of five main subsystem components:

• . Parsortix PC1 instrument incorporating a computer, keypad and display, pneumatic and hydraulic components including reservoir bottles and tubes, a separation cassette mounting clamp and other electronics to control the instrument hardware and behavior.
• Parsortix PC1 Software consisting of a Windows 7 Embedded operating system together . with dedicated Parsortix PC1 proprietary Windows application software (Software).
• A set of embedded and encrypted Protocol Files (Protocols) that are sequences of simple . instructions, interpreted by the Software and used to control the instrument fluidic and hydraulic components and circuits. The Protocols supplied embedded within the Software enable the four core instrument processes: Clean, Prime, Separate, and Harvest.
• Parsortix PC1 MBC-01 Metastatic Breast Cancer Kit which contains Separation Cassettes . (n = 10, 50 or 100), Cleaning Cassettes [(n = 1, 5, or 10), one Cleaning Cassette for every multiple of 10 x separation cassette], Encrypted Instrument protocol file distributed on a USB memory stick as required to perform the proposed intended use, Cassette labels and one package insert (per kit) containing instructions for use and expected performance data for the Parsortix PC1 instrument, when used in conjunction with the MBC-001 Metastatic Breast Cancer Kit.
• Parsortix PC1 ICT-01 Instrument Control Test Kit which contains Control tubes . containing a known, aliquoted cell suspension which is used to periodically confirm acceptable performance of the system, Separation Cassettes Polystyrene 12mL 16x100 mm tubes (n = 10 or 25) and one package insert (per kit) containing instructions for use for the ICT-001 Instrument Control Test Kit.

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

Acceptance Criteria and Device Performance

The provided document details various analytical performance studies demonstrating the device's capabilities. While explicit "acceptance criteria" are not presented in a single, clear list with pass/fail thresholds, the studies' objectives and reported results implicitly define what was considered acceptable performance for the device's de novo classification. The performance data is summarized below based on these implicit criteria.

Table of Implicit Acceptance Criteria and Reported Device Performance

• SKBR3: ~69% (CI: 65%-73%)
• MCF7: ~76% (CI: 73%-79%)
• Hs578T: ~76% (CI: 74%-79%) |
  | Comparison of live vs. fixed cell recovery | Fixed SKBR3 recovery: 88% (more efficient than live). Live SKBR3 recovery: 69%. |
  | Detection Limit | |
  | Minimum number of spiked tumor cells to recover at least one cell >95% of the time | - SKBR3: 3 cells
• Hs 578T: 4 cells
• MCF7: 5 cells |
  | Limit of Blank | 0 cells (no tumor cells detected in unspiked healthy donor blood). |
  | Blood Volume Impact | |
  | No significant impact on efficiency across 5mL, 7.5mL, 10mL volumes (direct harvest) | Mean % SKBR3 Harvest:
• 7.5mL: 71.1%
• 5mL: 62.3%
• 10mL: 66.3%
  (Avg Difference from 7.5mL: -8.8% for 5mL, -4.8% for 10mL; CIs indicate no significant differences across volumes) |
  | Impact of Cytospin™ slide deposition on recovery | Significant cell loss observed. Mean % SKBR3 Deposited:
• 7.5mL: 23.5%
• 5mL: 24.2%
• 10mL: 29.1% |
  | Blood Stability | |
  | No significant impact on recovery for samples stored at RT or 4°C for up to 72 hours | Mean % Harvest (control 71.2%):
• 24h RT: 81.6%
• 48h RT: 74.9%
• 72h RT: 71.1%
• 24h 4°C: 72.9%
• 48h 4°C: 72.5%
• 72h 4°C: 74.1%
  (CIs indicate no significant impact) |
  | Impact on processing time / residual nucleated cells | Storage at RT >4h or 4°C >48h increases residual nucleated cells. RT >24h may increase processing time. |
  | Cell Carryover | |
  | Absence of any cell carryover between samples | 0 of 220 PBS harvests showed fluorescently labeled cells. |
  | Cleaning Reagent Carryover | |
  | Residual cleaning detergent not interfering with cell recovery/morphology/molecular evaluation | No more than 0.01% residual cleaning detergent, which was demonstrated not to impact recovery, morphology, or RNA evaluation. |
  | Cassette Lot Performance | |
  | Consistent performance across multiple cassette lots | - Overall mean % harvest: 81.4% (SD 14.4%, %CV 17.7%) Range: 52.6% to 100%.
• Overall mean % capture: 84.0% (SD 13.2%, %CV 15.6%) Range: 57.6% to 100%. |
  | Interfering Substances | |
  | No significant interference from tested cancer drugs | No significant differences in captured/harvested SKBR3 cells. (Paclitaxel at 80ug/mL, however, showed potential for sample loss/quality reduction). |
  | No significant interference from high albumin or triglycerides | No impact on harvested cells or processing time. |
  | No significant interference from different hematocrit levels on cell capture/harvest | No interference for capture/harvest; high hematocrit increased processing time/residual WBCs, low hematocrit significantly increased residual WBCs. |
  | High WBC count not interfering with SKBR3 cell capture/harvest | No interference with capture/harvest (up to 16x10^9 cells/L). Elevated WBCs lead to increased residual nucleated cells (addressed by downstream assay compatibility). |
  | Compatibility of WBC background with downstream qPCR assay | No negative impact on qPCR performance for most genes (except ERBB2). |
  | Compatibility of WBC background with downstream cytology, FISH, and IF evaluation | No significant impact on quality of WBCs or SKBR3 cells observed in these evaluations. |
  | Reproducibility and Repeatability (Precision) | |
  | Acceptable %CVs for various precision studies (fixed/live cells, PBS/blood, single/multi-site) | - 10-day single site (fixed SKBR3, PBS): Overall avg harvest 81.3%, repeatability %CV 14.4%, within-laboratory %CV 14.5%.
• 20-day 3-site (fixed SKBR3, PBS): Overall avg harvest 75.3%, repeatability %CV 17.0%, reproducibility %CV 20.6%.
• 20-day single site (fixed SKBR3, blood): Overall avg harvest 89.4%, repeatability %CV 10.2%, within-laboratory %CV 10.3%.
• 20-day single site (live SKBR3, blood): Overall avg harvest 70.4%, repeatability %CV 21.1%, within-laboratory %CV 22.0%.
• Combined 20-day precision (fixed/live SKBR3, blood): Repeatability %CV 15.4%, reproducibility %CV 23.2%.
• 5-day single site (live SKBR3, MCF7, Hs578T, blood, various spike levels): Within-run repeatability %CVs ranged from 12.3% to 32.4%, within-laboratory %CVs ranged from 13.3% to 34.1%. Overall (5-50 cells): Repeatability and reproducibility %CV 26.3%. |
  | Clinical Performance (Enrichment of CTCs) | |
  | Comparison of CTC detection in MBC patients vs. healthy volunteers (IF staining) | - HV: 6.9% (5/72) had ≥1 CTC (DAPI+, CD45-, EpCAM+/CK+).
• MBC: 45.3% (34/75) had ≥1 CTC. **Significantly larger proportion in MBC patients (Fisher's exact p

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The 23andMe Personal Genome Service (PGS) uses qualitative genotyping to detect select clinically relevant variants in genomic DNA isolated from human saliva collected from individuals ≥18 years for the purpose of reporting and interpreting genetic health risks, including the 23andMe PGS Genetic Health Risk Report for Hereditary Prostate Cancer (HOXB13-Related). The 23andMe PGS Genetic Health Risk Report for Hereditary Prostate Cancer (HOXB13- Related) is indicated for reporting of the G84E variant in the HOXB13 gene. The report describes if a person has the G84E variant and if a male is at increased risk for prostate cancer. The variant included in this report is most common in people of European descent. The test report does not describe a person's overall risk of developing any type of cancer, and the absence of a variant tested does not rule out the presence of other variants that may be cancer-related. This test is not a substitute for visits to a healthcare provider for recommended screenings or appropriate follow-up and should not be used for diagnosis, to determine any treatments or medical interventions.

The 23andMe Personal Genome Service (PGS) is an over-the-counter (direct-to-consumer), DNA testing service that provides information and tools for consumers to learn about and explore their DNA.

The 23andMe Personal Genome Service (PGS) is a currently marketed, non-invasive genetic information service that combines qualitative genotyping data covering genetic ancestry, traits, and certain heritable health conditions from a single multiplex assay with descriptive information derived from peer reviewed, published genetic research studies. It is a home use, over-thecounter (direct-to-consumer) DNA testing service intended to provide information and tools for consumers to learn about and explore their DNA.

Customer saliva is self-collected using the Oragene-Dx® Device manufactured by DNA Genotek, Inc. (previously cleared for carrier screening indications under K141410, and the same collection kit used to generate performance data for DEN140044, DEN160026, DEN170046, K182784, DEN180028, and K193492, which consists of a sealable collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to one of our Clinical Laboratory Improvement Amendments (CLIA) certified laboratories for testing.

DNA is isolated from the saliva and tested in a multiplex assay using a customized genotyping beadchip, and off the shelf reagents and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the previously authorized indications, as well as for the indications proposed herein.

Raw data is generated using Illumina GenomeStudio software, and then sent to 23andMe. The data is then analyzed using 23andMe's proprietary Coregen software, where a genotype is determined for each tested SNP. The results for certain of these SNPs are used to generate personalized reports for the customer that provide information about the detected genotype.

Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which genetic health risk variant(s) have been detected in their sample and provide information about the disease associated with the variant(s). If no variant was detected, that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results.

The modified components of the Personal Genome Service included in this 510(k) submission are new labeling to include (a) one new variant to be reported, and (b) the qualitative reporting of one's Genetic Health Risk for Hereditary Prostate Cancer (HOXB13-Related).

Engineering drawings, schematics, etc. of Genetic Health Risk Report for Hereditary Prostate Cancer (HOXB13-Related) are not applicable to this device.

The provided document describes the acceptance criteria and study proving the device meets these criteria for the 23andMe PGS Genetic Risk Report for Hereditary Prostate Cancer (HOXB13-Related).

Here's the breakdown of the information requested:

1. Table of Acceptance Criteria and Reported Device Performance

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

• Accuracy/Method Comparison Study:
  • Sample Size: Not explicitly stated as a number, but "Saliva samples were selected from the 23andMe customer biobank, based on their predetermined genotype and minimum volume required for testing." This implies a varied sample size based on the availability of specific genotypes.
  • Data Provenance: From the "23andMe customer biobank" and "approved contract laboratory sites." The origin of the customers is not specified beyond "23andMe customer" which is a US-based company, suggesting primarily US data. The study was retrospective, using pre-existing samples from the biobank.
• Precision Study:
  • Sample Size: "DNA samples were selected based on their confirmed genotypes, and were obtained from the 23andMe biobank." Not an explicit number.
  • Data Provenance: From the "23andMe biobank." Implies primarily US data, retrospective.
• DNA Input Study:
  • Sample Size: "DNA samples were obtained from the 23andMe biobank based on their listed genotypes." Not an explicit number.
  • Data Provenance: From the "23andMe biobank." Implies primarily US data, retrospective.
• Interfering Substance Study (referenced from DEN140044):
  • Sample Size: Over 35,000 sample replicates.
  • Data Provenance: Not explicitly stated for this particular study, but given it's for a US regulatory submission by a US company, it's highly likely to be US data, retrospective.
• Labeling Comprehension Study (referenced from DEN160026):
  • Sample Size: Not explicitly stated.
  • Data Provenance: Not explicitly stated, but also likely US data.

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

• Ground Truth Method: For the analytical studies (Method Comparison, Precision, DNA Input), the ground truth for genotyping was established by bi-directional Sanger sequencing.
• Number/Qualifications of Experts: The document does not specify the number or qualifications of experts involved in performing or interpreting the Sanger sequencing results to establish the "truth." It only states that sequencing was performed "by an approved supplier" and that the sequencing results were "considered to be 'truth.'"

4. Adjudication Method for the Test Set (e.g., 2+1, 3+1, none)

• The document does not describe any human adjudication method for establishing the ground truth from Sanger sequencing. It implies that the sequencing results themselves were directly taken as ground truth without further expert consensus or adjudication.

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 or comparative effectiveness study involving human readers (e.g., radiologists) with or without AI assistance was performed or described. This device is a direct-to-consumer genetic test, not an imaging-based AI diagnostic tool.
• The closest concept is the "Labeling Comprehension" study, which assesses how well consumers understand the report. It indicates that the report and educational materials were effective in communicating relevant concepts for safe use. This is a measure of user comprehension, not human reader improvement with AI assistance.

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

• Yes, the performance studies (Accuracy, Precision, DNA Input, Interfering Substance) represent a standalone evaluation of the genotyping assay, which is essentially the "algorithm" or technical process of the device. The accuracy and precision figures are "algorithm only" performance metrics, as they compare the device's genotype calls directly against Sanger sequencing as the ground truth.

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

• For the analytical performance studies (Accuracy, Precision, DNA Input), the ground truth for specific genetic variants (genotype) was established by bi-directional Sanger sequencing.
• For the clinical performance, the document refers to "published studies of variant frequencies in various populations and the results of analytical studies" and "allele frequencies in the 23andMe customer database." This relies on established scientific literature and aggregated anonymized real-world data rather than individual outcomes or pathology reports.

8. The Sample Size for the Training Set

• The document primarily describes validation studies (test sets) for the analytical performance of the device. It does not provide information about a separate "training set" sample size for developing the genotyping assay or the underlying "Coregen software." The genotyping method described relies on physical beadchip arrays and established principles of DNA analysis, not on a machine learning model that would typically have a distinct training phase with a dedicated dataset.
• The "Customer biobank" is used for selecting samples for the performance studies, which may implicitly reflect data used in the development or refinement of their overall genotyping process, but it's not explicitly defined as a separate 'training set' for an AI model.

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

• As mentioned above, the document does not elaborate on a distinct "training set" with established ground truth in the context of an AI/ML model for this genetic test. The "Coregen software" analyzes raw data from the beadchip, and its accuracy is validated against Sanger sequencing. The development process of this proprietary software, and any data used to "train" it (if it involves statistical modeling beyond simple rule-based interpretation of genotyping signals), is not detailed in terms of ground truth establishment.

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The Helix Laboratory Platform is a qualitative in vitro diagnostic device intended for exome sequencing and detection of single nucleotide variants (SNVs) and small insertions and deletions (indels) in human genomic DNA extracted from saliva samples collected with Oragene® Dx OGD-610. The Helix Laboratory Platform is only intended for use with other devices that are germline assays authorized by FDA for use with this device. The device is performed at the Helix laboratory in San Diego, CA.

The Helix Laboratory Platform (HLP) is a high throughput DNA sequencing platform for targeted sequencing of an individual's whole exome. It is intended for targeted sequencing of an individual's whole exome for use with a genetic test application. Genetic test applications may be third party partner ("Partner") genetic test applications or a Helix genetic test application such as the Helix Genetic Health Risk App (HRA; K192073). The DNA sequence generated by this device is intended as input to clinical germline DNA assays intended for use with this device that have FDA marketing authorization. A brief overview of the commercialized workflow is shown in Figure 1 (refer to the section titled "Test Principle" for more specific information regarding the commercialized workflow.) HLP consists of a HiSeq sequencing instrument, cBot system, library preparation reagents, sequencing reagents, and data analysis software. The Helix Laboratory Platform also interacts with the Helix Laboratory Automation Systems and Content Mapping Systems which serve as repositories for the data and do not perform data analysis. The test detects single nucleotide variants (SNVs) insertions and deletions (indels) up to 20 base pairs (bp) and is limited to making high-confidence variant calls that meet prespecified quality metrics (i.e., the analytical range) within the reportable range. Sequencing is performed at the Helix clinical laboratory in San Diego. CA.

Acceptance Criteria and Device Performance Study

This document describes the acceptance criteria for the Helix Laboratory Platform (HLP) and the studies conducted to demonstrate that the device meets these criteria. The HLP is a qualitative in vitro diagnostic device intended for exome sequencing and detection of single nucleotide variants (SNVs) and small insertions and deletions (indels) in human genomic DNA extracted from saliva samples, for use with other FDA-authorized germline assays.

1. Table of Acceptance Criteria and Reported Device Performance

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

The major testing was performed across several studies:

• Precision (Cell lines): 6 unique reference cell line samples (NA12877, NA12878, NA24385, NA24149, NA24143, NA24631). Each was tested with 72 replicates, for a total of 432 replicates.
• Precision (Clinical Specimens): 18 unique saliva (clinical) samples. Originally intended for 72 replicates each, resulting in ~1296 replicates. However, due to QC failures, 118 replicates were not evaluable, leaving 1178 evaluable replicates across 17 samples (one sample had all replicates fail).
• Between-Lot Reproducibility: 24 samples (6 cell lines and 18 saliva-derived DNAs). Each sample produced 54 replicate sequences with combinatorial sets of reagents, totaling 1296 intended replicates. 1287 evaluable replicates passed QC.
• DNA Input: 20 unique samples with known variants. Tested at 35ng, 50ng, 70ng, and 100ng DNA input, each in triplicate (totaling 240 intended samples). 219 samples were evaluable.
• Index Swapping - Barcoding: 48 saliva samples with known variants. Run in triplicate, totaling 160 libraries. 157 were used in analysis.
• Interfering Substances (Endogenous): 60 donors, each providing 3 aliquots (no treatment, plus 2 different endogenous substances). 180 no-treatment libraries and 120 treatment libraries were generated. 299 out of 300 samples were evaluable.
• Interfering Substances (Exogenous): 22 donors (originally 20, 2 added for food group), each providing samples for various conditions (before, immediately after, 30 min after consumption of food, drink, gum, mouthwash). 198 intended samples. Number of evaluable samples varied by condition.
• Interfering Substances (Microbial): 6 cell line DNA samples across 5 bacterial content conditions (0%, 10%, 20%, 30%, 50%), each in triplicate (totaling 90 samples). 81 samples evaluable. Also, fresh saliva from 3 donors tested across 3 conditions (baseline, bacteria spiked-in, yeast spiked-in), each in triplicate (totaling 27 samples).
• Interfering Substances (Smoking): 5 donors, each providing samples for 3 conditions (before, immediately after, 30 min after smoking), each in triplicate (totaling 45 samples).
• Accuracy Study 1 (Reference Cell Lines): 6 well-characterized cell lines (same as Precision study).
• Accuracy Study 2 (Clinical Specimens): 1002 clinical samples and 96 unique cell line samples.

Data Provenance:
The reference cell line samples (NA12877, NA12878, NA24385, NA24149, NA24143, NA24631) are publicly available from the Genome in a Bottle (GIAB) consortium and Platinum Genomes project, primarily representing Northern European (Utah) and Ashkenazim Jewish ethnicities. One GIAB sample was Asian Chinese.
Clinical samples were saliva samples collected from donors within the Helix lab's specimen collection. These are therefore retrospective samples. The country of origin is not explicitly stated for the clinical samples but is assumed to be the USA, where the Helix lab is located.

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

The ground truth for the reference cell lines (NA12877, NA12878, NA24385, NA24149, NA24143, NA24631) was established by publicly available reference datasets from the Genome in a Bottle (GIAB) consortium and the Platinum Genomes project. These consortia involve multiple expert groups and diverse sequencing technologies to establish highly confident variant calls. No specific number of, or qualifications for, individual experts are listed, as the ground truth relies on these highly vetted, community-accepted reference standards.

For the clinical samples in Accuracy Study 2, a validated Sanger sequencing method was used as the comparator method to confirm the accuracy of specific variants. This implies expert interpretation of Sanger sequencing results, but the number and qualifications of these experts are not explicitly stated.

4. Adjudication Method for the Test Set

The ground truth for reference cell lines was based on publicly available, highly vetted datasets (GIAB, Platinum Genomes), which typically involve a consensus-based approach from multiple sequencing technologies and analyses rather than active, real-time adjudication by a small group of experts for this specific study.

For samples where a reference sequence was generated within a study (e.g., Precision, DNA Input, Endogenous/Exogenous Substances, Microbial Interference, Smoking studies), it was established by majority call comparison over multiple replicates of a sample within the study. This implies an internal consensus mechanism rather than external expert adjudication.

For Accuracy Study 2 (clinical samples), Sanger sequencing was used as the comparator. Discrepancies between HLP and Sanger sequencing would be reviewed, but a formal adjudication method (e.g., 2+1, 3+1) involving external experts for these specific discrepancies is not described.

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

No MRMC comparative effectiveness study was performed or described in this document. The evaluation focuses on the standalone analytical performance of the Helix Laboratory Platform and its concordance with established ground truth or comparator methods, not on how human readers' performance improves with or without AI assistance from this platform.

6. Standalone Performance Study

Yes, extensive standalone performance studies were conducted. The entire "Performance Characteristics" section (Section L) details the intrinsic analytical performance of the Helix Laboratory Platform (HLP) without human intervention for variant calling and quality assessment. The reported PPA, NPA, and TPPV values across various studies (Precision, Between-Lot Reproducibility, DNA Input, Index Swapping, Interfering Substances, Accuracy) demonstrate the algorithm's performance in detecting SNVs and indels when operating independently.

7. Type of Ground Truth Used

The primary types of ground truth used were:

• Expert Consensus / Community Standards: For reference cell lines, publicly available datasets from the Genome in a Bottle (GIAB) consortium and the Platinum Genomes project were used. These are highly confident, multi-platform consensus truth sets.
• Validated Comparator Method: For clinical samples where specific variants were assessed (e.g., Accuracy Study 2), a validated Sanger sequencing method served as the comparator ground truth.
• Internal Majority Call: For various precision and interference studies where multiple replicates of a sample were generated and analyzed, a "majority call" across these replicates was used to establish an internal reference sequence for performance comparison.

8. Sample Size for the Training Set

The document does not explicitly state the sample size used for training the Helix bioinformatics pipeline's algorithms. It mentions optimization processes in Section L.1, such as "Optimization of variant read depth, allele fraction and callability thresholds" and "Establishment of filter and QC threshold for variant calling." These optimizations were "based on historical reference sample runs" and "analyzed with [...redacted...] of the bioinformatics pipeline representing different conditions relative to the quality metric criteria." While this indicates that data was used for optimizing and establishing parameters, specific training set sizes are not provided.

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

Similar to point 8, the document does not explicitly detail how the ground truth for any training set was established. However, the optimization efforts heavily relied on "historical reference sample runs" and "reference samples." It is highly likely that these reference samples would have included publicly available, well-characterized control genomes like those from the GIAB and Platinum Genomes projects, for which the "truth" variants are established through extensive, multi-platform sequencing and expert consensus by those consortia. The process described for establishing QC thresholds (e.g., in Section L.1.b) implies a comparison against these known reference datasets to fine-tune filtering parameters and improve accuracy.

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The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/1gH (J) and BCL2/1gH (J) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL).

The clonoSEQ Assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.

The clonoSEQ Assay is a single-site assay performed at Adaptive Biotechnologies Corporation.

The clonoSEQ Assay is a next-generation sequencing (NGS) based assay that identifies rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences. The assay also includes primers that amplify specific genomic regions present as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content.

Testing begins with gDNA extracted from the specimen supplied (Figure 1). Extracted gDNA quality is assessed and rearranged immune receptors are amplified using a multiplex PCR. Reaction-specific index barcode sequences for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared from barcoded amplified DNA, which are then sequenced by synthesis using NGS. Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample's sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias. When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported LoD and LoO on the patient report. During clonoSEQ Tracking (MRD) assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The clonoSEQ Assay MRD assessment measures residual disease in a biologic sample.

Here's a breakdown of the acceptance criteria and study details for the Adaptive Biotechnologies clonoSEQ Assay, based on the provided FDA 510(k) summary:

This device, the Adaptive Biotechnologies clonoSEQ Assay, is an in vitro diagnostic (IVD) that identifies and quantifies rearranged immune receptor gene sequences (IgH, IgK, IgL) and translocated BCL1/IgH and BCL2/IgH sequences using multiplex PCR and Next-Generation Sequencing (NGS). It measures Minimal Residual Disease (MRD) in patients with B-cell acute lymphoblastic leukemia (ALL), multiple myeloma (MM), and chronic lymphocytic leukemia (CLL) to monitor disease burden. The current submission is an expansion of indications to include blood samples from CLL patients.

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

The provided document doesn't explicitly list "acceptance criteria" in a single table, but rather describes the performance characteristics that were measured and the outcomes for each. I will compile these for the CLL in Blood indication, as this is the focus of the 510(k) expansion.

Acceptance Criteria & Reported Device Performance for clonoSEQ Assay (CLL in Blood)

• At 500 ng DNA input: 21.9% - 54.9% CV
• At 2 µg DNA input: 20.8% - 51.6% CV
• At 20 µg DNA input: 18.7% - 49.2% CV
  (Comparable to BMA precision) |
  | Precision (Malignant Cells Detected) | %CV within acceptable clinical/analytical limits, primarily influenced by cell number. | Range: 19% CV (at 765.70 cells) to 53% CV (at 3.10 cells).
  Primarily due to residual variability; other factors (operator, instruments, reagents, day, run) contributed 0%-10% CV. |
  | Linearity | Maximum deviation from linearity (based on quadratic or cubic fit) less than 5%. | Met for all DNA inputs (20µg, 2µg, 500ng) across the entire tested MRD frequency range (0 to 1x10^-3 / 4x10^-3).
• Slopes: 0.989 - 0.997 (indicating strong linearity)
• Intercepts: -0.009 to -0.075 |
  | Accuracy (Concordance with mpFC) | High positive percent agreement (PPA) and understanding of negative percent agreement (NPA) reflecting greater sensitivity. | PPA: 98.9% (95% CI: 94.3%-100%)
  NPA: 47.5% (95% CI: 40.5%-54.6%)
  (NPA lower due to clonoSEQ's higher sensitivity detecting MRD where mpFC calls negative) |
  | Limit of Blank (LoB) | LoB should be zero or negligible. | LoB was confirmed as zero (95th percentile of trackable sequences in healthy blood was zero). |
  | Limit of Detection (LoD) / Limit of Quantitation (LoQ) | LoD/LoQ for blood should be comparable to or lower than previously determined values for bone marrow. | LoD and LoQ for CLL in blood were lower or within the 95% CI of bootstrapped prior BMA data, confirming comparability. |
  | Analytical Specificity (Interfering Substances) | Mean MRD frequency difference ± 30% when comparing with and without interferent substances. | All tested endogenous and exogenous substances met acceptance criteria.
• Endogenous: Bilirubin (conjugated & unconjugated), Hemoglobin, Cholesterol, Triglycerides.
• Exogenous: K2EDTA, K3EDTA, Heparin, Chloroform.
  (MRD results not substantially influenced). |
  | Cross-Contamination/Sample Carryover | No significant contamination events leading to false positive ID or MRD results. | - No false calibrations for run-to-run (0/44 BMA, 0/44 BMMC).
• One single well-to-well false calibration (1/44 BMA) at a very low template count (83 templates), not associated with cell lines.
• Blood: No contamination or disease clone-sharing events leading to false positive ID/MRD results.
• PCR/Library/Sequencing: No run-to-run contamination (0/36 tests). 8/712 well-to-well events (likely vendor-related primer barcode issue)

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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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