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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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cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BK virus (BKV) DNA in human EDTA plasma on the cobas® 6800/8800 Systems.

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

The results from cobas® BKV are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings. Test results must not be the sole basis for patient management decisions.

cobas® BKV is not intended for use as a screening test for blood products or human cells, tissues, and cellular and tissue-based products (HCT/Ps).

cobas® BKV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas® 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas® 6800/8800 software which assigns test results for all tests as either target not detected, BKV DNA detected < LLoQ (lower limit of quantitation), BKV DNA detected > ULoQ (upper limit of quantitation), or a value in the linear range LLoQ < x < ULoQ. Results can be reviewed directly on the system screen, exported, or printed as a report.

Nucleic acid from patient samples and added lambda DNA-QS molecules is simultaneously extracted. In summary, viral nucleic acid is released by addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris and potential PCR inhibitors are removed with subsequent wash reagent steps and purified nucleic acid is eluted from the glass particles with elution buffer at elevated temperature.

Selective amplification of target nucleic acid from the sample is achieved by the use of a dual target virus specific approach from highly-conserved regions of the BKV located in the BKV small t-antigen region and the BKV VP2 region. Selective amplification of DNA-QS is achieved by the use of sequence-specific forward and reverse primers which are selected to have no homology with the BKV genome. A thermostable DNA polymerase enzyme is used for amplification. The target and DNA-QS sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme, which is included in the PCR mix, when heated in the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

The cobas® BKV master mix contains two detection probes specific for BKV target sequences and one for the DNA-QS. The probes are labeled with target-specific fluorescent reporter dyes allowing simultaneous detection of BKV target and DNA-QS in two different target channels. The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the specific single-stranded DNA templates results in cleavage by the 5'-to-3' nuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye is concomitantly increased. Real-time detection and discrimination of PCR products are accomplished by measuring the fluorescence of the released reporter dyes for the viral targets and DNA-QS.

The provided document is a 510(k) Summary for the cobas® BKV test for use on the cobas® 6800/8800 Systems. This document focuses on demonstrating substantial equivalence to a predicate device through non-clinical performance evaluation, rather than an AI/ML device requiring a comparative effectiveness study with human readers. Therefore, several of the requested sections (e.g., sample size for test set, number of experts, adjudication method, MRMC study, standalone performance, ground truth for test set, training set details) are not directly applicable or explicitly stated in the context of this In Vitro Diagnostic (IVD) device submission for a quantitative nucleic acid amplification test.

However, I will extract relevant information to address the applicable criteria based on the provided text.

Acceptance Criteria and Device Performance for cobas® BKV

The acceptance criteria for this diagnostic device are primarily defined by various performance characteristics required for quantitative viral nucleic acid tests. The study proves the device meets these criteria through a series of non-clinical performance evaluations.

Table of Acceptance Criteria and Reported Device Performance

Study Details:

1. Sample Size and Data Provenance:

   • Test Set (Clinical Concordance): 550 valid samples (217 neat clinical, 303 diluted clinical derived from 129 transplant subjects, and 30 contrived samples).
   • Provenance: Clinical samples were from BKV infected and non-infected patients. Although not explicitly stated, the context of an FDA submission for an IVD test likely implies data collected under controlled conditions at testing sites, potentially from multiple geographical locations if a multi-site study was conducted (the Reproducibility study specified 3 testing sites). The samples included both retrospective (stored clinical samples) and prospective (contrived samples) elements.
   • Non-Clinical Studies: Sample sizes vary by test as detailed in the table (e.g., 63 replicates per level for LoD, 36 replicates per panel member for linear range, 72 replicates per level for precision, 240/225 replicates for cross-contamination, 270 valid tests per concentration for reproducibility).

2. Number of Experts and Qualifications: Not applicable. This is an IVD device for quantitative viral DNA measurement, not an AI/ML device for image interpretation or diagnosis by experts. Ground truth is established analytically (e.g., known concentrations of viral standards) or through comparison to a well-established laboratory method.

3. Adjudication Method: Not applicable. For IVD tests like this, objective analytical measurements are compared rather than subjective interpretations needing adjudication.

4. MRMC Comparative Effectiveness Study: No. This is an In Vitro Diagnostic (IVD) quantitative nucleic acid amplification test, not an AI-assisted diagnostic imaging device that involves human readers. The standard of comparison is against a well-established laboratory method (LDT) and analytical performance targets, not human reader improvement.

5. Standalone Performance: Yes, the entire document describes the standalone performance of the cobas® BKV assay. The device measures BKV DNA concentration directly without human-in-the-loop assistance for the measurement itself, though a qualified healthcare professional interprets the results in a clinical context.

6. Type of Ground Truth Used:

   • Analytical/Reference Standards: For LoD, linearity, traceability, precision, and specificity, the ground truth was established using known concentrations of the 1st WHO International Standard for BK Virus DNA (NIBSC 14/212), armored DNA for specific subgroups/subtypes, and well-characterized high titer BKV DNA (subgroup Ib).
   • Comparative Method: For clinical concordance, the ground truth was established by comparing results from the cobas® BKV test to those from a well-established laboratory developed nucleic acid test (LDT) from Indiana University (referred to as "comparator BKV LDT"). This LDT results served as the reference for comparison and agreement.

7. Sample Size for Training Set: Not applicable. This is a molecular diagnostic assay characterized through analytical and clinical performance studies, not an AI/ML model that undergoes a distinct training phase with a dedicated training set. The "training" of the assay involves its development and optimization based on scientific principles of nucleic acid amplification and detection, and then validated through the studies described.

8. How Ground Truth for Training Set was Established: Not applicable, as there isn't a "training set" in the AI/ML sense. The development of the assay (analogous to training) would rely on molecular biology knowledge, optimization experiments, and characterization against internally developed standards and reference materials, ultimately ensuring performance against defined analytical targets and external standards (like the WHO standard).

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