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Alinity m BKV is an in vitro nucleic acid amplification test for the quantitation of BK virus (BKV) DNA in human EDTA plasma (K2 EDTA, K3 EDTA, and PPT) and urine stabilized using the Alinity m Urine Transport Kit on the automated Alinity m System.

In EDTA plasma (K2 EDTA, K3 EDTA, and PPT) and urine stabilized using the Alinity m Urine Transport Kit, Alinity m BKV is intended for use as an aid in the diagnosis and management of BKV in transplant patients.

In patients undergoing monitoring of BKV in EDTA plasma, 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 BKV must be interpreted in conjunction with clinical signs and other relevant laboratory findings. Alinity m BKV is not cleared as a screening test for blood or blood products or human cells, tissues, and cellular and tissue-based products.

The Alinity m BKV assay utilizes real-time polymerase chain reaction (PCR) to amplify and detect BKV genomic DNA sequences that have been extracted from human EDTA plasma or urine specimens. The steps of the Alinity m BKV assay consist of sample preparation, real-time PCR assembly, amplification/detection, result calculation, and reporting. One transfer step of urine specimens into the Alinity m Urine Transport Kit by the user is required prior to placing urine specimens on the Alinity m System. Remaining steps of the Alinity m BKV assay procedure are executed automatically by the Alinity m System. Manual dilutions may be performed for low-volume plasma specimens to meet the minimum volume requirement. The Alinity m System is designed to be a randomaccess analyzer that can perform the Alinity m BKV assay in parallel with other Alinity m assays on the same instrument.

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

• . Alinity m BKV AMP Kit (List No. 09N85-095), consisting of 2 types of multi-well assay trays. The amplification tray (AMP TRAY 1) contains liquid, unit-dose PCR amplification/detection reagents and liquid, unit-dose Internal Control (IC) in separate wells; and the activation tray (ACT TRAY 2) contains liquid, unit-dose activation reagent. The intended storage condition for the Alinity m BKV AMP Kit is -25°C to -15°C.
• . Alinity m BKV CTRL Kit (List No. 09N85-085), 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 BKV CTRL Kit is -25°C to -15°C.
• Alinity m BKV CAL Kit (List No. 09N85-075), consisting of 2 calibrator levels, . each supplied as liquid in single-use tubes. The intended storage condition for the Alinity m BKV CAL Kit is -25°C to -15°C.

The Alinity m BKV assay requires a transport kit for testing all urine specimens:

• Alinity m Urine Transport Kit (List No. 09N85-001) consisting of a transport tube . and transfer pipette. The transport tube contains transport buffer. The intended storage condition for the Alinity m Urine Transport Kit is 15℃ to 30℃.
  BKV DNA from human plasma or urine is extracted automatically on board 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 DNA is then combined with liquid unit-dose Alinity m BKV activation reagent and liquid unit-dose Alinity m BKV 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 BKV DNA.

At the beginning of the Alinity m BKV sample preparation process, a liquid unit-dose IC on the AMP Tray is transferred 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 BKV amplification/detection reagents consist of enzymes, primers, probes, and activation reagents that enable amplification and detection of dual targets in the BKV genome. Amplification and detection of the two BKV targets ensures sensitive detection of the viral genome even at low levels. In addition to the BKV primers and probes, the assay utilizes an IC primer/probe set for amplification and detection of the IC target sequence, which is not related to BKV. The IC probe is labeled with a different fluorophore than the BKV probes. This allows for simultaneous detection and discrimination of both the BKV and IC amplified products within the same reaction vessel.

A BKV calibration curve is required for determination of BKV DNA concentration. Two levels of calibrators are processed through sample preparation and PCR to generate the calibration curve. The concentration of BKV 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 BKV assay also utilizes the following:

• Alinity m BKV Application Specification File (List No. 09N85-05A) .
• 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 System Solutions, (List No. 09N20):
  • o Alinity m Lysis Solution (List No. 09N20-001)
  • o Alinity m Diluent Solution (List No. 09N20-003)
  • o 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) o
  • o Alinity m Transport Tubes Pierceable Capped (List No. 09N49-010)
  • o Alinity m Transport Tube (List No. 09N49-011)
  • o Alinity m Pierceable Cap (List No. 09N49-012)
  • o Alinity m Aliquot Tube (List No. 09N49-013)

1. Acceptance Criteria and Reported Device Performance

2. Sample Size for Test Set and Data Provenance

• Plasma Clinical Test Set: 579 EDTA plasma clinical specimens (555 neat and 24 diluted clinical specimens) from 556 subjects.
• Urine Clinical Test Set: 380 urine specimens (1 specimen per subject).
• Provenance: Clinical specimens were collected from solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) subjects. The document does not specify the country of origin of the data or explicitly state if the studies were retrospective or prospective, but the nature of "clinical specimens" and "clinical sites" suggests they were likely collected in a clinical setting, potentially in a prospective or mixed fashion for evaluation. The "reproducibility" studies were performed at "3 clinical sites," indicating multi-center data collection.

3. Number of Experts and Qualifications for Ground Truth

• The document does not explicitly mention the use of experts to establish ground truth for the clinical test set. Instead, the device's performance is compared against an "FDA-cleared BKV nucleic acid test" (the predicate device, cobas® BKV (K203220)) to establish clinical agreement. This predicate device serves as the reference standard.

4. Adjudication Method for the Test Set

• As the ground truth for clinical performance was established by comparison to an FDA-cleared predicate device, "adjudication" in the sense of expert consensus on individual cases is not directly applicable. The FDA-cleared predicate device's results were considered the reference for comparison. Discordant results were analyzed but not adjudicated by an independent panel of experts. Instead, results more than one box away from the diagonal in the agreement tables were defined as "discordant."

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

• No MRMC comparative effectiveness study was done. This device is a quantitative viral nucleic acid test (an in vitro diagnostic medical device), not an imaging device or AI-assisted diagnostic tool that would typically involve human readers. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not relevant to this type of device.

6. Standalone Performance

• Yes, a standalone (algorithm only) performance study was conducted. The entire analytical and clinical performance evaluation of the Alinity m BKV assay, as described in the document, represents the standalone performance of the device without human-in-the-loop assistance for result generation. The system is automated, performing sample preparation, PCR, detection, calculation, and reporting.

7. Type of Ground Truth Used

• Analytical Performance Studies (LoD, Linearity, Precision, Specificity, Carryover):
  • External Standard: The 1st World Health Organization (WHO) International Standard for BK virus for Nucleic Acid Amplification Techniques (NIBSC code: 14/212) was used as the primary reference for quantitation and traceability.
  • Defined Samples: BKV-negative human EDTA plasma and BKV-negative stabilized urine were used as matrices, spiked with known concentrations of BKV (WHO standard, armored DNA, or clinical specimens).
• Clinical Performance Studies (Plasma and Urine):
  • Comparative Reference: An "FDA-cleared BKV nucleic acid test" (the predicate device) was used as the comparative reference for clinical agreement. This implies that the results from the predicate device served as the de-facto "ground truth" for the comparison of clinical performance.
  • Clinical Specimens: Actual patient samples from transplant subjects were used.

8. Sample Size for the Training Set

• The document describes performance studies, not the development of a machine learning model that would typically have a distinct "training set." Therefore, a specific sample size for a training set in that context is not provided. The development and optimization of the assay itself would involve internal studies, but these are not explicitly termed "training sets" in the context of AI/ML.

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

• As no specific "training set" for an AI/ML model is described, this question is not directly applicable. The development and validation of the BKV assay would have relied on a combination of:
  • Scientific principles of nucleic acid amplification and detection.
  • Characterization of BKV strains and their genomic sequences.
  • Internal R&D experimentation and optimization using known concentrations of BKV and control samples, often traceable to international standards (like the WHO standard mentioned).
  • Extensive analytical studies to define assay characteristics like sensitivity, specificity, and linearity before clinical validation.

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