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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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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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cobas® BKV is an in vitro nucleic acid amplification test for the quantitation of BKV) DNA in human EDTA plasma and urine stabilized in cobas® PCR media on the cobas® 6800/8800 Systems.

In EDTA plasma, cobas® BKV is intended for use as an aid in the 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.

In urine stabilized in cobas® PCR Media, cobas® BKV is intended for use as an aid in the management of BKV in transplant patients.

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 (Figure 1) 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-OS 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).13 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.45 The fluorescent signal of the intact probes is suppressed by the quencher dye. During the PCR amplification step, hybridization of the probe to 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-OS.

This document describes the acceptance criteria and supporting studies for the cobas® BKV device for the quantitation of BK virus (BKV) DNA in human urine. The information is extracted from a 510(k) summary.

1. Table of Acceptance Criteria and Reported Device Performance

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

The studies focused on analytical performance (Limit of Detection, Linearity, Precision, Specificity, Interference, Cross-contamination) and clinical performance (Reproducibility, Clinical Concordance).

• Limit of Detection (LoD):
  • WHO International Standard: 63 replicates per concentration level (total 7 levels, x3 lots = 1323 replicates).
  • Subgroups/Subtypes Verification: 63 replicates per concentration level for each genotype (total 5 genotypes, 3 levels each, x3 lots = 2835 replicates).
• Linear Range:
  • Main linearity: 36 replicates per panel member (10 panel members, x3 lots = 1080 replicates).
  • Genotype linearity: 12 replicates per level for each genotype (8 panel members each, 5 genotypes, x3 lots = 1440 replicates).
• Lower Limit of Quantitation (LLoQ): Data from the Linearity study at 100, 200, and 300 IU/mL concentrations.
• Precision (Within-Laboratory): 72 replicates for each of 5 dilution levels (x3 lots = 1080 replicates).
• Analytical Specificity: 3 replicates for each of the microorganisms listed in Table 10, both in BKV-negative and BKV-positive urine (number of microorganisms not explicitly totalled, but substantial).
• Interfering Substances: Replicates for each substance in presence and absence of BKV DNA (number of replicates not explicitly stated, but implies multiple for each substance in Table 11).
• Cross Contamination: 240 replicates of BKV-negative matrix samples, 225 replicates of high titer BKV DNA urine samples.
• Clinical Reproducibility: 270 tests per concentration (5 concentrations, total 1350 tests, not including controls).
• Clinical Performance / Concordance: 308 neat urine samples from 84 transplant subjects (for concordance analysis). 61 negative samples for NPA. 153 BKV positive urine samples from 55 transplant subjects (for correlation analysis).

Data Provenance: The document implies that the non-clinical studies were conducted internally or at authorized labs. The clinical performance evaluation was conducted at 3 testing sites, suggesting multi-site prospective data collection. The data samples were human EDTA plasma and urine. The origin of the samples (country) is not explicitly stated in the provided text. The clinical study used a retrospective cohort of samples from transplant patients.

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

The ground truth for the analytical studies was established based on known concentrations of BKV international standards or armored DNA. For clinical performance, the comparator was a "well-established laboratory developed nucleic acid test (LDT)".

The document does not mention the use of experts to establish ground truth for the test sets in the typical sense of human readers for image-based diagnostics. The "ground truth" for this diagnostic device study is based on the highly controlled properties of the spiked samples (known concentrations, genotypes) for analytical performance, and the results from a comparator LDT for clinical concordance.

4. Adjudication Method for the Test Set

Not applicable in the context of this in vitro diagnostic device, as the "ground truth" is based on quantitative measurements and known concentrations, not subjective expert assessment requiring adjudication.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is an in vitro nucleic acid amplification test (NAAT) for quantitative measurement of BKV DNA. It is not an AI-assisted diagnostic device requiring human reader input or interpretation in the way an imaging diagnostic device would. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not relevant to this submission.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the studies described are for the standalone performance of the cobas® BKV system, which is an automated molecular diagnostic assay. The system performs "fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection." The results are "assigned... by the cobas® 6800/8800 software." While "results are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings," the primary performance metrics are based on the direct output of the automated system.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

• Analytical Studies: The ground truth for analytical studies (LoD, Linearity, Precision, Specificity, Interference) was established using known concentrations of BKV DNA, including the WHO International Standard (NIBSC 14/212), BKV armored DNA, and clinical specimens diluted to specific concentrations. Samples were spiked into BKV-negative urine.
• Clinical Studies: For clinical concordance, the ground truth was based on the results from a "well-established laboratory developed nucleic acid test (LDT) (comparator BKV LDT)" on actual clinical urine samples. DNA sequencing was also used in some cases to confirm BKV presence in discordant results.

8. The Sample Size for the Training Set

The document describes performance evaluation studies (validation and verification) rather than a machine learning model development process that typically involves distinct training and test sets.
Therefore, a separate "training set" sample size for a machine learning algorithm is not applicable in the context of this in vitro diagnostic device, which is based on established molecular biological techniques (PCR).

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

As this is not an AI/ML-based device with a "training set," this question is not applicable. The ground truth for the evaluation of the device was established through known concentrations of viral standards and comparison to a comparator LDT, as described in point 7.

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cobas EBV is an in vitro nucleic acid amplification test for the quantitation of Epstein-Barr virus (EBV) DNA in human EDTA plasma on the cobas 6800/8800 Systems.

cobas 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 response to treatment.

The results from cobas EBV are intended to be read and analyzed by a qualified licensed healthcare professional in conjunction with clinical signs and symptoms and relevant laboratory findings. Negative test results do not preclude EBV infection or EBV disease. Test results must not be the sole basis for patient management decisions.

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

cobas EBV is a quantitative test performed on the cobas 6800 System and cobas 8800 System. cobas EBV enables the detection of EBV DNA in plasma specimens. The cobas EBV assay is a dual target assay, with both targets using the same dye. The DNA Internal Control, used to monitor the entire sample preparation and PCR amplification process, is introduced into each specimen during sample processing. cobas EBV enables the detection and quantitation of EBV DNA in EDTA plasma from solid organ transplant patients (SOT) and from hematopoietic stem cell transplant (HSCT) patients. The test is intended for use as an aid in the management of SOT patients and HSCT patients.

The cobas EBV consists of:

• Proteinase Solution ●
• DNA Quantitation Standard (DNA QS) ●
• Elution Buffer ●
• Master Mix Reagent 1
• . EBV Master Mix Reagent 2

The EBV viral load is quantified against a non-EBV DNA quantitation standard (DNA-OS), which is introduced into each specimen during sample preparation. The DNA-QS also functions as an internal control for sample preparation and the PCR amplification process.

In addition, the test utilizes the following separately packed and sold control materials:

• 1. cobas EBV Positive Control Kit:
  • . EBV Low Positive Control (EBV L(+)C)
  • EBV High Positive Control (EBV H(+)C) ●

The positive control contains phage packaged EBV DNA in normal human plasma and serves as a control for the cobas EBV test.

• 2. cobas Negative Control Kit:
  • cobas Buffer Negative Control (BUF (-) C) ●

Testing with the cobas EBV test requires the following materials that are not provided:

• cobas OMNI Reagents: Including the following reagents used for specimen ● processing, PCR and detection:
• cobas EBV Assay Specific Analysis Package (ASAP) software .

The cobas EBV test uses sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection, all steps are fully automated by the cobas 6800/8800 platform.

Here's an analysis of the acceptance criteria and the studies that prove the device meets these criteria, based on the provided text:

Acceptance Criteria and Device Performance

Study Details

2. Sample Size and Data Provenance

• Test Set (Clinical Study):
  • Sample Size: 464 samples (439 neat or diluted clinical samples from 72 transplant subjects, and 25 contrived samples).
  • Data Provenance: Clinical samples from a major transplant center in the United States (implied prospective or retrospective collection for comparison, but not explicitly stated as either for the clinical samples themselves). Contrived samples were prepared in the lab.

3. Number of Experts and Qualifications (for Ground Truth)

• The document does not specify the number of experts or their qualifications for establishing the ground truth for the clinical test set. The comparator EBV test is described as a "validated well-established comparator nucleic acid test currently used in clinical practice at a major transplant center in the United States." This implies expert-driven clinical practice but no explicit panel for ground truth review is mentioned.

4. Adjudication Method (for Test Set)

• None specified for the concordance study. The primary method was direct comparison to a "validated well-established comparator nucleic acid test." Discordant results were defined and analyzed based on a quantitative difference (more than one box away from the diagonal in the concordance table). DNA sequencing was used on representative samples for sequence mismatches in large discrepancies, which is a form of expert adjudication for cause but not for initial ground truth determination.

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

• No, a MRMC comparative effectiveness study was not done. The study focused on the analytical and clinical concordance of the automated device with an existing laboratory-developed test (LDT), not on human reader performance with and without AI assistance.

6. Standalone Performance Study (Algorithm Only)

• Yes, a standalone study was done. The entire analytical performance section (Precision, Reproducibility, Linearity, Traceability, Stability, LoD, LLoQ, Analytical Specificity, Cross-Contamination) evaluates the cobas EBV test (algorithm and instrument) in a standalone capacity, without human intervention in the result generation. The clinical concordance study also compares the device's output to a comparator, effectively assessing its standalone performance.

7. Type of Ground Truth Used

• For Analytical Studies: Spiked biological samples (e.g., EBV phagemid, EBV cell culture supernatant, WHO EBV Standard) with known concentrations, or EBV-negative plasma samples, were used to establish ground truth for parameters like LoD, Linearity, and Specificity.
• For Clinical Concordance Study: The ground truth was established by a "validated well-established comparator nucleic acid test" currently used in clinical practice at a major transplant center. This is a form of reference method/comparator assay ground truth.

8. Sample Size for Training Set

• The document does not explicitly state the sample size used specifically for the training set. It describes the reagents and the test principle, but not the specific data used for initial algorithm development or 'training' in the machine learning sense. The performance studies use various panels and clinical samples for validation of the finalized assay.

9. How Ground Truth for Training Set Was Established

• As the document does not specify a distinct 'training set' or 'algorithm training', it also does not explain how ground truth was established for it. For a PCR-based quantitative assay, the 'training' often involves optimizing primer/probe design and assay conditions against well-characterized reference materials (like the WHO standard or cultured virus) to achieve desired analytical performance parameters before formal validation studies.

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