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Alinity m EBV is an in vitro polymerase chain reaction (PCR) assay for the quantitation of Epstein-Barr Virus (EBV) DNA in human EDTA plasma on the automated Alinity m System.

Alinity m EBV is intended for use as an aid in the management of EBV in transplant patients. In patients undergoing monitoring of EBV, serial DNA measurements can be used to indicate the need for potential treatment changes and to assess viral response to treatment.

The results from Alinity m EBV must be interpreted within the context of all relevant clinical and laboratory findings.

Alinity m EBV is not cleared for use as a screening test for donors of blood, blood products, or human cells, tissues, and cellular and tissue-based products (HCT/Ps) for EBV.

Alinity m EBV is an in vitro polymerase chain reaction (PCR) assay for the quantitation of EBV DNA in human plasma.

This device is similar to the predicate device originally cleared (K212778) with the exception that the subject device may use MomentaTaq DNA Polymerase as an alternative to KAPA2G DNA Polymerase in the reagent formulation of the assay. This formulation difference does not introduce any changes to sample processing, assay procedure, or data reduction.

Additional studies were initiated to support the formulation of the assay with MomentaTaq DNA Polymerase. Supplemental data from these studies were used with data previously obtained from the analytical and clinical testing studies submitted in K212778.

The steps of the Alinity m EBV consist of sample preparation, real-time PCR assembly, amplification/detection, result calculation, and reporting. All stages of the Alinity m EBV procedure are executed automatically by the Alinity m System. No intermediate processing or transfer steps are performed by the user. The Alinity m System is designed to be a random-access analyzer that can perform the Alinity m EBV assay in parallel with other Alinity m assays on the same instrument.

Alinity m EBV requires three separate assay specific kits as follows:

• Alinity m EBV AMP Kit (List No. 09N43-095), consisting of 2 types of multi-well assay trays. The amplification trays (AMP TRAY 1) contain lyophilized, unit-dose PCR amplification/detection reagents and lyophilized, unit-dose IC in separate wells, and the activation trays (ACT TRAY 2) contain liquid unit-dose activation reagent. The intended storage condition for the Alinity m EBV AMP Kit is 2°C to 8°C.

• Alinity m EBV CTRL Kit (List No. 09N43-085), consisting of negative controls, low positive controls, and high positive controls, each supplied as liquid in single-use tubes. The intended storage condition for the Alinity m EBV CTRL Kit is –25°C to –15°C.

• Alinity m EBV CAL Kit (List No. 09N43-075), consisting of 2 calibrator levels, each supplied as liquid in single-use tubes. The intended storage condition for the Alinity m EBV CAL Kit is –25°C to –15°C.

EBV DNA from human plasma is extracted automatically on-board in the Alinity m System using the Alinity m Sample Prep Kit 2, Alinity m Lysis Solution, and Alinity m Diluent Solution. The Alinity m System employs magnetic microparticle technology to facilitate nucleic acid capture, wash, and elution. The resulting purified nucleic acids are then combined with liquid unit-dose Alinity m EBV activation reagent and lyophilized unit-dose Alinity m EBV amplification/detection reagents and transferred into a reaction vessel. Alinity m Vapor Barrier Solution is then added to the reaction vessel which is then transferred to an amplification/detection unit for PCR amplification, and real-time fluorescence detection of EBV targets.

At the beginning of the Alinity m EBV sample preparation process, a lyophilized unit-dose IC on the AMP Tray is rehydrated by the Alinity m System and delivered into each sample preparation reaction. The IC is then processed through the entire sample preparation and real-time PCR procedure along with the specimens, calibrators, and controls to demonstrate proper sample processing and validity.

The Alinity m EBV amplification/detection reagents consist of enzymes, primers, probes, and activation reagents that enable polymerization and detection.

An EBV calibration curve is required for determination of EBV DNA concentration. Two levels of calibrators are processed through sample preparation and PCR to generate the calibration curve. The concentration of EBV DNA in specimens and controls is then calculated from the stored calibration curve.

Assay controls are tested at or above an established minimum frequency to help ensure that instrument and reagent performance remains satisfactory. During each control event, a negative control, a low-positive control, and a high-positive control are processed through sample preparation and PCR procedures that are identical to those used for specimens.

The Alinity m EBV assay also utilizes the following:

• Alinity m EBV Application Specification File, (List No. 09N43-05B)
• Alinity m System and System Software (List No. 08N53-002)
• Alinity m Sample Prep Kit 2 (List No. 09N12-001)
• Alinity m Specimen Dilution Kit I (List No. 09N50-001)
• Alinity m System Solutions, (List No. 09N20):
  • Alinity m Lysis Solution (List No. 09N20-001)
  • Alinity m Diluent Solution (List No. 09N20-003)
  • Alinity m Vapor Barrier Solution, (List No. 09N20-004)
• Alinity m Tubes and Caps (List No. 09N49):
  • Alinity m LRV Tube (List No. 09N49-001)
  • Alinity m Transport Tubes Pierceable Capped (List No. 09N49-010)
  • Alinity m Transport Tube (List No. 09N49-011)
  • Alinity m Pierceable Cap (List No. 09N49-012)
  • Alinity m Aliquot Tube (List No. 09N49-013)

This document, K243489, is a 510(k) clearance letter for the Alinity m EBV assay, specifically focusing on the use of MomentaTaq DNA Polymerase as an alternative to KAPA2G DNA Polymerase. The primary goal of the studies described is to demonstrate that the device formulated with MomentaTaq DNA Polymerase performs equivalently to the previously cleared device formulated with KAPA2G DNA Polymerase (K212778).

Here's an analysis of the acceptance criteria and study information provided:

1. Table of Acceptance Criteria and Reported Device Performance

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

The document describes several additional studies conducted specifically for the MomentaTaq formulation, referring back to the K212778 submission for other characteristics.

• Limit of Detection (LoD):
  • Sample Size: For EBV type 1, tested dilutions with 3 lots of amplification reagents across multiple days. For 20 IU/mL, there were 142 replicates (47-48 per lot). Total replicates across all concentrations and lots were higher (e.g., 143 for 15 IU/mL, 142 for 100 IU/mL).
  • Data Provenance: Prepared in EBV negative human plasma using the 1st World Health Organization (WHO) International Standard for Epstein-Barr Virus. This suggests controlled laboratory samples.
• Linearity:
  • Sample Size: 16 panel members spanning the intended quantitation range.
  • Data Provenance: Prepared from plasmid DNA, cultured virus, and clinical specimens. The specific origin country for clinical specimens is not provided, but the use of the WHO International Standard points to internationally recognized reference material.
• Precision:
  • Sample Size: 8 panel members, with 270 replicates per panel member (3 lots x 3 operators x 15 days x 2 runs/day x 3 replicates/run, then averaged).
  • Data Provenance: Panel members prepared by diluting EBV-positive clinical specimens, EBV cultured virus, or synthetic DNA in human plasma. Quantitation traceable to the 1st WHO International Standard. Specific origin country not provided.
• Lower Limit of Quantitation (LLoQ):
  • Sample Size: Calculated from detected samples in the LoD study.
  • Data Provenance: Prepared in EBV-negative plasma using the 1st WHO International Standard.
• Clinical Reproducibility:
  • Sample Size: 9-member reproducibility panel (8 positive, 1 negative). 1 lot of kits used. Testing performed at 3 clinical sites on 5 non-consecutive days with 2 runs per day. 4 replicates of each panel member tested, ensuring a minimum of 3 valid replicates. For the positive panels, N ranged from 113 to 120. For the negative panel, N = 119 for all sites combined.
  • Data Provenance: Positive panel members were prepared using EBV positive clinical specimen, cultured virus, or plasmid DNA diluted in human EDTA plasma. Specific origin country for clinical specimens not provided.
• Clinical Performance (Method Comparison):
  • Sample Size: 124 samples with results within the common quantitation range of both assays.
  • Data Provenance: Samples used for method comparison between the MomentaTaq formulation and the KAPA2G formulation. Implies ex vivo clinical samples. Specific origin country not provided.

The studies for the MomentaTaq formulation are analytical performance studies and a method comparison to an already cleared device, not direct clinical outcome studies. They appear to be retrospective as they involve testing banked or prepared samples. The reference to "3 clinical sites" for reproducibility suggests a multi-site testing environment, but it's an analytical reproducibility study, not a clinical trial on patient outcomes.

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

The provided document does not mention the number or qualifications of experts used to establish ground truth for the test sets. The ground truth for quantitation appears to be established by:

• Using the 1st World Health Organization (WHO) International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (NIBSC code 09/260) for LoD, Linearity, and LLoQ.
• Using EBV-positive clinical specimens, cultured virus, or plasmid DNA for precision and reproducibility, with quantitation traceable to the WHO standard.
• The "comparator" assay (on-market Alinity m EBV with KAPA2G DNA Polymerase) as a reference for clinical performance (method comparison).

For a quantitative viral load test, the ground truth is typically defined by reference materials (like WHO standards) or highly characterized samples, rather than by expert consensus on interpretation of results.

4. Adjudication Method for the Test Set

Given that the ground truth for these quantitative assays is based on reference standards and characterized samples, no expert adjudication method (like 2+1, 3+1, none) is described or would typically be applicable in this context. The output is a quantitative value, not a subjective interpretation.

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

No, an MRMC comparative effectiveness study was not done. This type of study is more common for imaging diagnostics where human readers interpret results with and without AI assistance. This document describes an in vitro diagnostic (IVD) quantitative PCR assay, which is an automated device producing numerical results, not something that human readers interpret in the conventional sense of an MRMC study.

6. If a Standalone Study (Algorithm Only Without Human-in-the-Loop Performance) was Done

Yes, effectively, all the studies described are standalone performance studies. The Alinity m EBV device is an automated system that performs sample preparation, PCR, detection, and result calculation without human intervention in the actual assay run. The performance metrics (LoD, linearity, precision, reproducibility, method comparison) directly evaluate the algorithm's ability to accurately quantify EBV DNA in samples. There is no "human-in-the-loop" component in the performance of the assay itself.

7. The Type of Ground Truth Used

The ground truth used in these studies is primarily based on:

• Reference Materials: The 1st World Health Organization (WHO) International Standard for Epstein-Barr Virus (NIBSC code 09/260) is extensively used for establishing and verifying the quantitative values of the panel members.
• Characterized Samples: EBV positive clinical specimens, cultured virus, and synthetic plasmid DNA where their concentrations are known or precisely determined relative to the WHO standard.
• Comparison to Predicate Device: For the method comparison study, the performance of the new MomentaTaq formulation is benchmarked against the FDA-cleared Alinity m EBV assay (K212778), which serves as a clinical reference.

This is a form of "expert consensus" on the value of reference materials and highly characterized samples, rather than pathology or outcomes data in the usual sense for IVDs.

8. The Sample Size for the Training Set

The document does not specify the sample size used for the training set for the Alinity m EBV assay. Regulatory submissions like 510(k) clearances typically focus on the validation (test set) rather than the development and training data of the algorithm itself, especially for established PCR technologies that do not heavily rely on machine learning requiring distinct training sets in the same manner. This assay is based on PCR technology, where the "training" would involve optimizing reagent concentrations, primer/probe design, and thermocycling conditions, rather than training a machine learning model on a "training set" of patient data.

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

As mentioned above, specific "training set" details are not provided. For PCR-based assays, the "ground truth" during development (analogous to training) would involve:

• Known Viral Copies: Using synthetic nucleic acids or viral stock solutions with precisely quantified viral load to optimize assay components and parameters.
• Spiked Samples: Spiking known amounts of virus or viral nucleic acid into negative matrix (e.g., human plasma) to simulate different concentrations and evaluate early performance.
• Clinical Samples with Confirmed Status: Using retrospectively collected clinical samples with well-established EBV positive/negative status and viral loads determined by highly accurate laboratory methods or other validated assays to refine and confirm assay performance.

The development process would aim to ensure the assay accurately detected and quantified EBV DNA, aligning with established reference standards and biological knowledge of the virus.

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