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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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