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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 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 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.

The steps of the Alinity m EBV assay 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 multi-well amplification trays (AMP Trays) containing lyophilized, unit-dose PCR amplification/detection reagents and multi-well activation trays (ACT Trays) containing liquid, unit-dose activation reagents (MgCl2, TMAC, KCl, and ProClin). 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 a negative control, a low positive control, and a high positive control, each supplied as liquid in single-use tubes. The intended storage condition for the Alinity m EBV CTRL Kit is —15°C to —25°C.
• . Alinity m EBV CAL Kit (List No. 09N43-075) consisting of two levels of calibrators (CAL A and CAL B), each supplied as liquid in single-use tubes. The intended storage condition for the Alinity m EBV CAL Kit is -15°C to -25°C.

EBV DNA from specimens 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 the 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.

An EBV calibration curve is required for the quantitation of EBV targets. Two levels of calibrators are processed through sample preparation and real-time 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 remain satisfactory. During each control event, a negative control, a low-positive control, and a high-positive control are processed through sample preparation and real-time PCR procedures that are identical to those used for specimens.

At the beginning of the Alinity m EBV sample preparation process, a lyophilized unit -dose of Internal Control on the AMP Tray is rehydrated by the Alinity m System and delivered into each sample preparation reaction. The Internal Control 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 assay validity.

The Alinity m EBV amplification and detection reagents include primers and probes that amplify and detect dual targets in the EBV genome. Amplification and detection of the two EBV targets ensures sensitive detection of the viral genome even at low levels.

The Alinity m EBV assay also utilizes the following accessories:

• . Alinity m EBV Application Specification File, List No. 09N43-05A
• . 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 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
• . 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

Here's a summary of the acceptance criteria and study details for the Alinity m EBV AMP Kit, extracted from the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document describes performance characteristics but doesn't explicitly state "acceptance criteria" for each in a table. Instead, it presents the validated performance values. I've constructed a table based on the key performance metrics and their demonstrated values.

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

• Limit of Detection (LoD) Study (Test Set):
  • Sample Size: 24 replicates per EBV DNA concentration level, tested with 4 lots of amplification reagents. This means 96 replicates for each concentration level shown in Tables 2-5 (24 * 4).
  • Data Provenance: Dilutions of the 1st World Health Organization (WHO) International Standard for Epstein-Barr virus (NIBSC code: 09/260) prepared in EBV negative human plasma. No country of origin is specified for the EBV negative human plasma, but the WHO standard is internationally recognized. This is retrospective/contrived data.
• Linearity Study (Test Set):
  • Sample Size: 16 panel levels (dilution series). Number of replicates per panel level not explicitly stated for individual tests but the figures show mean concentrations.
  • Data Provenance: Dilution series of EBV type 1 (prepared using clinical specimen for lower concentrations, synthetic DNA for higher) and EBV type 2 (prepared using cultured virus for lower concentrations, synthetic DNA for higher) in negative human plasma. Quantitation values traceable to WHO International Standard. This is retrospective/contrived data.
• Precision Study (Test Set):
  • Sample Size: 9-member plasma panel, each panel member tested in 4 replicates, twice each day for 12 days, on 3 systems by 3 operators using 3 AMP kit lots, totaling 288 replicates per panel member.
  • Data Provenance: Panel members prepared with positive clinical sample, cultured virus, or synthetic DNA spiked into EBV negative plasma. This is retrospective/contrived data.
• Analytical Specificity (Test Set):
  • Sample Size: Undisclosed number of individual microorganisms and drug compounds tested in EBV negative plasma and positive plasma (60 IU/mL and 10,000 IU/mL EBV DNA).
  • Data Provenance: Various microorganisms and drug compounds. This is retrospective/contrived data.
• Carryover Study (Test Set):
  • Sample Size: 648 valid replicates of EBV negative samples and 647 valid replicates of high concentrated EBV positive samples.
  • Data Provenance: Contrived EBV negative and high positive samples. This is retrospective/contrived data.
• Clinical Reproducibility Study (Test Set):
  • Sample Size: 9-member reproducibility panel (8 positive, 1 negative). 6 replicates of each panel member tested on each of 5 non-consecutive days for each of 2 reagent lot combinations per site, across 3 clinical sites. (6 * 5 * 2 * 3 = 180 replicates per panel member usually, but tables show 172-180 for positive, 180 for negative).
  • Data Provenance: Positive panel members prepared using EBV positive clinical specimen, cultured virus, or plasmid DNA diluted in human EDTA plasma. This is a mix of retrospective (clinical specimens) and contrived data.
• Clinical Performance Study (Test Set):
  • Sample Size: 558 EDTA plasma samples (542 clinical specimens, 16 contrived).
  • Data Provenance: Clinical specimens from hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) subjects. The 16 contrived samples were prepared by spiking inactivated EBV virus into individual clinical specimens. The text does not specify the country of origin for these clinical specimens, but it's implied they are from a clinical setting. This is a mix of retrospective clinical data and contrived data.

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

The provided text does not explicitly mention the use of human experts or their qualifications to establish ground truth for the test set.

For the analytical studies (LoD, linearity, precision, specificity, carryover), the ground truth was established by:

• Using internationally recognized standards (1st WHO International Standard for EBV).
• Precisely diluting known concentrations of virus or synthetic DNA.
• Using confirmed EBV negative plasma.

For the clinical performance study, the ground truth appears to be established by comparison to an "FDA-cleared EBV nucleic acid test" (the predicate device). The results of the comparator assay served as the reference for agreement analysis.

4. Adjudication Method for the Test Set

No adjudication method (e.g., 2+1, 3+1) is mentioned or implied for any of the studies described. The "ground truth" for the clinical performance study was primarily the results of the FDA-cleared comparator device.

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

No MRMC comparative effectiveness study was done. The device is an in vitro diagnostic (IVD) assay quantifying viral nucleic acid directly, not an imaging device requiring human interpretation, so the concept of human readers improving with AI assistance is not applicable here. The clinical performance section compares the device's quantitative results to those of a predicate IVD device.

6. Standalone Performance Done

Yes, the studies evaluate the standalone performance of the Alinity m EBV assay. The precision, analytical specificity, limit of detection, and linearity are all measures of the algorithm and system's performance without human intervention in the result generation or interpretation beyond operating the instrument and following its procedures. The "Clinical Performance" section compares the standalone device results to a predicate device.

7. Type of Ground Truth Used

• Analytical Studies (LoD, Linearity, Precision, LLoQ, Carryover): Ground truth was established by known, precisely prepared concentrations of EBV (WHO International Standard, cultured virus, synthetic DNA) in EBV negative plasma.
• Analytical Specificity (Cross-Reactants/Interfering Substances): Ground truth was based on the presence of specific microorganisms or substances at known concentrations, with the expectation of no interference.
• Clinical Reproducibility: Similar to precision, ground truth was based on known concentrations of EBV positive clinical specimens, cultured virus, or plasmid DNA in human plasma.
• Clinical Performance: Ground truth was primarily defined by the results of an FDA-cleared predicate EBV nucleic acid test. For a subset, "confirmed EBV DNA negative clinical specimens" were used.

8. Sample Size for the Training Set

The document does not provide information on the training set for the device. As an IVD assay, the development process differs from AI/ML-based algorithms that typically involve explicit training data sets for model development. The assays are developed based on established molecular biology principles (PCR) and optimized through internal R&D, rather than machine learning on a distinct "training set" of patient data.

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

Since no explicit "training set" is mentioned in the context of an AI/ML algorithm, this question is not directly applicable to the information provided for this IVD device. The development and optimization of such assays typically involve laboratory experiments using characterized viral strains, reference materials, and defined concentrations, which would implicitly form the basis for establishing assay parameters and performance.

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