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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 EnLite™ Neonatal TREC Kit is an in vitro diagnostic device intended for the semiquantitative determination of TREC (T-cell receptor excision circle) DNA in blood specimens dried on filter paper. The test is for use on the VICTOR™ EnLite instrument. The test is indicated for use as an aid in screening newborns for severe combined immunodeficiency disorder (SCID).

This test is not intended for use as a diagnostic test or for screening of SCID-like Syndromes, such as DiGeorge Syndrome, or Omenn Syndrome. It is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID.

The EnLite™ Neonatal TREC Kit is comprised of the EnLite™ Neonatal TREC Kit, the VICTOR™ EnLite instrument and the EnLite™ workstation software. The EnLite™ Neonatal TREC Kit contains reagents sufficient for 384 reactions or 1152 reactions, and multi-level, dried blood spot (DBS) calibrators and controls. The DBS calibrators and DBS controls have been prepared from porcine whole blood with a hematocrit value of 48-55%, and contain purified salmon-sperm, TREC, and beta-actin DNA.

The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device for semi-quantitative determination of T-cell receptor excision circles (TRECs) in dried blood specimens, used as an aid in screening newborns for severe combined immunodeficiency disorder (SCID).

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for the EnLite™ Neonatal TREC Kit are outlined in the regulatory information, specifically within the "Special Controls" section (Section T, point 1(iii)). These criteria detail the required analytical and clinical performance characteristics for the device. The reported device performance is presented throughout the "Performance Characteristics" section (Section M).

Here's a table summarizing key acceptance criteria and reported performance, focusing on the clinical validation study as that directly addresses the intended use of screening:

Table of Acceptance Criteria and Reported Device Performance

A minimum of 10-15 confirmed positive specimens from more than one site, with relevant annotation, and SCID diagnosis by flow cytometry or clinically meaningful information regarding subject status at one year or beyond.

Additional specimens characterized by other disorders with low/absent TREC (e.g., other T-cell lymphopenic disorders) to supplement the range of results.

Pre-specified clinical decision point (cut-off) before studies.

Results summarized in tabular format comparing interpretation to reference method.

Point estimates and 95% CIs for PPA, NPA, and OPA.

Data must include retest rate, false positive rate before retest, final false positive rate, and false negative rate. | The primary clinical study objective was to demonstrate the EnLite™ Neonatal TREC Kit's screening performance in the intended use population and its ability to discriminate between normal and SCID cases. The study was conducted retrospectively.

SCID Positive Specimens: 17 archived confirmed SCID positive DBS specimens were obtained from newborn screening laboratories in the US. All 17 were confirmed for SCID by flow cytometry. These enriched the study due to the low incidence of SCID.

Other Low TREC Specimens: An additional 9 DBS specimens from babies with low TREC values (0 to 20 TREC Copies/uL) were included.

Comparator: For routine clinical study specimens, the comparator was the clinical assessment from medical records at one year of age or older (365 days), confirming the newborn was not identified with SCID, was not deceased from SCID-related complications, and was apparently healthy. For confirmed SCID cases, the comparator was the reference tests results for SCID confirmation.

Pre-specified Cut-off: The cut-off for TREC was pre-determined to be 36 copies/uL and for beta-actin as 56 copies/uL, based on the 2.5th percentile of normal distribution data from a separate cut-off confirmation study using 2846 archived, retrospective newborn specimens from the Danish Newborn Screening Biobank.

Retest Rate: The retest rate was 1.9%.

False Positive Rate: The false positive rate using the cut-off of 36 in the first round of testing was 1.5%. After repeat testing on follow-up cases, the final false positive rate was 0.5%.

False Negative Rate: The clinical data indicates 0 false negative results among the 16 confirmed SCID positives classified after the final testing round (Table 14).

Performance (from Table 14, excluding invalid results):
- Overall Percent Agreement (OPA): 99.7% (95% CI: 99.4% to 99.8%)
- Positive Percent Agreement (PPA): 100% (95% CI: 79.4% to 100%)
- Negative Percent Agreement (NPA): 99.7% (95% CI: 99.4% to 99.8%)

Note: One SCID positive specimen in the clinical study was classified as an invalid result, leading to 16 confirmed SCID positives being used for final agreement calculations. |

2. Sample Sizes and Data Provenance

• Test Set Sample Size:

  • Clinical Study: A total of 6,471 neonatal specimens were run, with 6,373 included in the final analysis. This included 6,389 routine Danish newborn screening biobanked newborn routine DBS samples and 82 enrichment samples (17 confirmed SCID positive samples, 9 confirmed low-level TREC specimens, and 56 samples used for blinding purposes). For the final agreement calculations, 5,454 specimens (after some exclusions and loss-to-follow-up) were used, specifically 5,442 after removing invalid results (16 confirmed SCID positives and 5,426 normal/presumptive normal).
  • Cut-off Establishment Study: 3,243 archived, retrospective newborn specimens initially, with 2,846 included in the analysis after exclusions.
  • Analytical Performance Studies (Examples):
    • Reproducibility (Site-to-Site): 90 measurements per sample (6 unique TREC levels, 10 runs x 3 laboratories x 3 replicates/sample).
    • Precision: 27 runs performed over 20 days. For TREC precision, 10 samples were assessed with 4 replicates/sample. For beta-actin, 7 samples were used.
    • LoB/LoD/LoQ: 5 samples for LoB (60 results per sample); 5 samples for LoD/LoQ (108 results per sample).

• Data Provenance:

  • Clinical Study & Cut-off Establishment:
    • Country of Origin: Denmark (samples from the Danish Newborn Screening Biobank, comprising the Danish population).
    • Retrospective/Prospective: All samples were archived, retrospective.
  • SCID Enrichment Samples: 17 confirmed SCID positive DBS specimens were obtained from newborn screening laboratories in the US (retrospective).

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of experts used to establish the ground truth for the test set, nor their specific qualifications (e.g., "radiologist with 10 years of experience").

• However, the ground truth for the 17 confirmed SCID positive specimens was established by flow cytometry, which is a specialized laboratory test requiring expert interpretation, presumably by qualified clinical immunologists or pathologists.
• For the routine newborn specimens, the comparator for ground truth was the clinical assessment of the study subjects obtained from their medical records at one year of age or older (365 days), confirming they were not identified with SCID, were not deceased from SCID-related complications, and were apparently healthy. This clinical assessment would implicitly involve input from various medical professionals (pediatricians, specialists).
• The "expert" component primarily comes from the reference method (flow cytometry) for SCID diagnosis and the subsequent clinical follow-up for the larger cohort.

4. Adjudication Method for the Test Set

The adjudication method for the test set was not explicitly described as a multi-expert consensus process like "2+1" or "3+1" that is common in medical imaging studies. Instead, the ground truth for SCID confirmation was primarily based on:

• Laboratory Confirmation: Flow cytometry for the 17 confirmed SCID cases.
• Clinical Outcomes: Medical record review at one year of age or older for the large cohort of routine newborns to determine the absence of SCID.

The device itself has an internal retesting algorithm (Section P.4 and P.4, Figure 8). Initial results below the cut-off are "presumptive positive" and are retested in duplicate. This internal retesting acts as a form of "internal adjudication" for the device's own classification, but it's not external expert adjudication of the ground truth.

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

No MRMC comparative effectiveness study was mentioned. This device is a laboratory diagnostic kit and not an AI-assisted diagnostic tool for human readers (like a CAD system for radiologists). Therefore, a study to measure how much human readers improve with AI vs. without AI assistance is not applicable to this type of device.

6. Standalone Performance

The study primarily assessed the standalone performance of the device/kit (EnLite™ Neonatal TREC Kit, VICTOR™ EnLite instrument, and EnLite™ workstation software) in classifying samples as "presumptive positive" or "normal" based on its quantitative TREC and beta-actin measurements and the predefined cut-offs. The results (PPA, NPA, OPA) reflect the performance of the integrated system in a laboratory setting, without direct human cognitive interpretation of raw data for diagnosis. The output from the device is a quantitative TREC value, which is then used with a hard cut-off.

7. Type of Ground Truth Used

The ground truth used was a combination of:

• Laboratory Test Confirmation: For the known SCID positive cases, flow cytometry was used to confirm SCID.
• Clinical Outcomes Data: For the large cohort of routine newborn screening samples, the absence of SCID was determined through medical record review (vaccination records, national patient registry, civil registration system) at one year of age or older, looking for signs of SCID or SCID-related complications/death.

8. Sample Size for the Training Set

The document describes the evaluation of an already developed device/kit, not a machine learning model. Therefore, there is no explicit "training set" in the context of machine learning model development. The data used for establishing the clinical cut-off (2,846 samples from the Danish Newborn Screening Biobank) could be considered analogous to a "development" or "calibration" dataset, which informed the final cut-off value used in the pivotal study (test set).

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

As noted above, there isn't a "training set" for a machine learning model. For the dataset used to establish the clinical cut-off (2,846 samples):

• These were archived, retrospective newborn specimens from the Danish Newborn Screening Biobank.
• The ground truth in this context was based on the distribution of TREC and beta-actin values in this "normal newborn population". The 2.5 percentile of this distribution was then chosen as the clinical cut-off for TREC (36 copies/uL) and beta-actin (56 copies/uL). This is a statistical approach to defining "normal" for screening purposes, rather than a direct disease diagnosis for each individual sample.

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