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K Number
K212778
Device Name
Alinity m EBV AMP Kit (List No. 09N43-095), Alinity m EBV CTRL Kit (List No. 09N43-085), Alinity m EBV CAL Kit (List No. 09N43-075)
Manufacturer
Abbott Molecular, Inc.
Date Cleared
2022-07-15

(317 days)

Product Code
QLX
Regulation Number
866.3183
Type
Traditional
Panel
Microbiology
Reference & Predicate Devices
DEN200015
Predicate For
K243489
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

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.

Device Description

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
AI/ML Overview

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.

Performance MetricAcceptance Criteria (Implicit from Results)Reported Device Performance
Limit of Detection (LoD) for EBV Type 1Detect with 95% probability19.56 IU/mL (LoD by Probit) with 95% CI (13.09 IU/mL, 39.39 IU/mL) for least sensitive lot. Claimed LoD: 20 IU/mL.
LoD for EBV Type 2Detect 95% or greater of EBV samples95.7% at 20 IU/mL and 95.8% at 15 IU/mL.
Linearity RangeLinear across the quantitation range50 IU/mL to 200,000,000 IU/mL (1.70 Log IU/mL to 8.30 Log IU/mL) for both EBV types 1 & 2. Correlation coefficient r = 0.999.
Precision (Within-Laboratory SD)≤ 0.25 Log IU/mL for 2.70-8.30 Log IU/mL≤ 0.25 Log IU/mL.
≤ 0.50 Log IU/mL for 1.30-<2.70 Log IU/mL≤ 0.50 Log IU/mL.
Lower Limit of Quantitation (LLoQ)Acceptable accuracy and precision (TAE and TE ≤ 1.00 Log IU/mL)50 IU/mL (1.70 Log IU/mL) confirmed. TAE ≤ 0.59 Log IU/mL; TE ≤ 0.78 Log IU/mL at LLoQ.
Analytical Specificity (Cross-reactivity)No cross-reactivity or interferenceNo cross-reactivity observed with panel of microorganisms.
Analytical Specificity (Interfering Substances)No interferenceNo interference observed from tested endogenous substances or therapeutic drugs.
Carryover (Detectable concentration ≥ LoD)Low (specific threshold not stated, but 0.3% is good)0.3% (95% CI: 0.1% to 1.1%).
Carryover (EBV detection)Low (specific threshold not stated, but 1.2% is good)1.2% (95% CI: 0.6% to 2.4%).
Clinical ReproducibilityAcceptable precision across sites/lotsTotal SD (Log IU/mL) ranged from 0.10 to 0.30 for positive panels. Negative Rate of 97.8%.
Agreement with Comparator (HSCT & SOT combined)High agreementColumn Agreement ranges from 97.6% to 100.0%.
Systematic Difference (Alinity m EBV - Comparator)Low bias0.09 Log IU/mL (95% CI: 0.06, 0.12).

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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July 15, 2022

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health and Human Services seal on the left and the FDA acronym and name on the right. The seal is a stylized depiction of an eagle and a caduceus, while the FDA acronym and name are written in blue, with the word "Administration" appearing below the acronym and name.

Abbott Molecular, Inc. Gina Sammarco Senior Specialist Regulatory Affairs 1300 E. Touhy Ave Des Plaines, Illinois 60018

Re: K212778

Trade/Device Name: Alinity m EBV AMP Kit (List No. 09N43-095). Alinity m EBV CTRL Kit (List No. 09N43-085), Alinity m EBV CAL Kit (List No. 09N43-075) Regulation Number: 21 CFR 866.3183 Regulation Name: Quantitative Viral Nucleic Acid Test For Transplant Patient Management Regulatory Class: Class II Product Code: QLX Dated: August 31, 2021 Received: September 1, 2021

Dear Gina Sammarco:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Maria Garcia, Ph.D. Assistant Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K212778

Device Name Alinity m EBV (09N43)

Indications for Use (Describe)

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.

Type of Use (Select one or both, as applicable)
☑ Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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Section 5: 510(k) Summary

Table of Contents

5.0510(k) SummaryPage 2
5.1Submitter2
5.2Device Information3
5.3Predicate Device3
5.4Device Description4
5.5Intended Use6
5.6Similarities and Differences to Predicate Devices6
5.7Performance Data10
5.7.1 Specific Performance Characteristics10
5.7.2 Clinical Reproducibility26
5.7.3 Clinical Performance29
5.8Conclusions Drawn from the Studies35

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5.0 510(k) Summary

This 510(k) summary of safety and effectiveness information is being submitted in accordance with the requirement of 21 CFR Section 807.92(c).

5.1 Submitter

Applicants Name and Address:Abbott Molecular Inc.1300 E. Touhy AveDes Plaines, IL 60018
Contact Person:Gina SammarcoManager Regulatory AffairsAbbott Molecular, Inc.1300 E. Touhy AvenueDes Plaines, IL 60018Phone: 224-361-7627Fax: 224-361-7646
Date Prepared:August 31, 2021

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Trade NameRegulation NameProductCodeRegulationNumberClass
Alinity m EBVQuantitative viral nucleic acid testfor transplant patient managementQLX866.3183II

Common Name: Nucleic acid test for quantitation of EBV DNA

5.3 Predicate Device

Device NamePredicate DeviceDe NovoCleared
Alinity m EBVcobas® EBVDEN200015July 30, 2020

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5.4 Device Description

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

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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

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  • . 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

ર્સ્ટ Intended Use

Alinity m EBV AMP Kit

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.

5.6 Similarities and Differences to Predicate Devices

The primary functional components of the Alinity m EBV assay are substantially equivalent to other legally marketed nucleic acid amplification tests (NAAT) intended for the quantitative detection of EBV DNA.

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The Alinity m EBV assay has the same general intended use as the predicate device. Although there are some technological differences between the Alinity m EBV assay and the predicate device, these differences do not raise new types of safety or effectiveness questions.

These devices are similar in that they are designed to prepare nucleic acids for amplification, amplify specific EBV DNA sequences, detect the amplified products, and report quantitative results.

The primary similarities and differences between the Alinity EBV assay and the predicate device are shown in Table 1.

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Table 1. Similarities and Differences Between Alinity m EBV and Predicate Device
Device &PredicateDevice(s):Alinity m EBVK212778cobas EBV testDEN200015
General DeviceCharacteristicSimilarities
Assay TypeQuantitativeQuantitative
Specimen TypesEDTA PlasmaEDTA Plasma
SamplePreparationProcedureAutomated liquid handling and robotic manipulation platform.Automated liquid handling and robotic manipulation platform.
AmplificationTechnologyReal-time polymerase chain reaction (PCR)PCR
Assay ControlsNegative Control Low Positive Control High Positive Control Internal ControlNegative Control Low Positive Control High Positive Control DNA Quantitation Standard (DNA-QS)
General DeviceCharacteristicDifferences
Intended UseAlinity m EBV is an in vitro polymerase chain reaction (PCR)assay for the quantitation of Epstein-Barr Virus (EBV) DNA inhuman EDTA plasma on the automated Alinity m System.cobas EBV is an in vitro nucleic acid amplification test for thequantitation of Epstein-Barr virus (EBV) DNA in human EDTAplasma on the cobas 6800/8800 Systems.
Alinity m EBV is intended for use as an aid in the management ofEBV in transplant patients. In patients undergoing monitoring ofEBV, serial DNA measurements can be used to indicate the needfor potential treatment changes and to assess viral response totreatment.The results from Alinity m EBV must be interpreted within thecontext of all relevant clinical and laboratory findings. Alinity mEBV 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.cobas EBV is intended for use as an aid in the management of EBVin transplant patients. In patients undergoing monitoring of EBV,serial DNA measurements can be used to indicate the need forpotential treatment changes and to assess response to treatment.The results from cobas EBV are intended to be read and analyzedby a qualified licensed healthcare professional in conjunction withclinical signs and symptoms and relevant laboratory findings.Negative test results do not preclude EBV infection or EBVdisease. Test results must not be the sole basis for patientmanagement decisions. cobas EBV is not intended for use as ascreening test for donors of blood or blood products or humancells, tissues, and cellular and tissue-based products (HCT/Ps).
Assay Targets2 highly conserved regions of the EBV genomeEBV EBNA-1 geneEBV BMRF gene

CONFIDENTIAL

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5.7 Performance Data

The following performance data were provided in support of the safety, effectiveness and substantial equivalence determination of the device.

5.7.1 Specific Performance Characteristics

5.7.1.1 Traceability to the WHO Standard

Primary calibrators and assay product calibrators with known concentrations were used throughout product development and product manufacturing to establish traceability to the 1st World Health Organization (WHO) International Standard for Epstein-Barr virus for Nucleic Acid Amplification Techniques (NIBSC code: 09/260). The concentrations tested for the WHO standard were 3.00 Log IU/mL and 5.00 Log IU/mL. The target concentrations tested for the primary calibrators were 3.00 Log IU/mL and 7.00 Log IU/mL. The Alinity m EBV product calibrators and controls were also tested along with the primary calibrators and the WHO standard. All of the tested material had observed EBV concentrations similar to the target concentrations, and were linear across the assay's quantitation range, as presented in Figure 1.

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Image /page/13/Figure/1 description: This image is a scatter plot showing the relationship between the target concentration and the Alinity m EBV observed mean. The x-axis represents the target concentration in Log IU/mL, and the y-axis represents the Alinity m EBV observed mean in Log IU/mL. The plot includes data points for WHO, Primary Calibrator, Product Calibrator, and Control, and a dashed line representing the equation y = 1.00x - 0.02 with an R-squared value of 0.999.

Limit of Detection 5.7.1.2

The limit of detection (LoD) was determined for EBV type 1 by testing dilutions of the 1 st World Health Organization (WHO) International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (NIBSC code: 09/260) prepared in EBV negative human plasma. Testing for each EBV DNA concentration was performed with 4 lots of amplification reagents across multiple days. The results, representative of the analytical sensitivity performance of Alinity m EBV, are summarized in Table 2 (Lot 1), Table 3, (Lot 2), Table 4 (Lot 3), and Table 5 (Lot 4) .

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Probit analysis of the data using the least sensitive lot (Lot 3) determined that the concentration of EBV DNA in plasma detected with 95% probability (LoD by Probit) was 19.56 IU/mL with a 95% confidence interval (CI) of (13.09 IU/mL, 39.39 IU/mL) (Table 4).

Table 2. Alinity m EBV Limit of Detection (LoD) - Lot 1
EBV DNA(IU/mL)No. of ValidReplicatesNo. of DetectedReplicatesDetectionRate (%)
100.002424100.0
50.002424100.0
20.002424100.0
15.002424100.0
12.50242395.8
10.00242291.7
7.50242291.7
5.00241458.3
2.5024625.0
1.2524937.5
Table 3. Alinity m EBV Limit of Detection (LoD) - Lot 2
EBV DNA(IU/mL)No. of ValidReplicatesNo. of DetectedReplicatesDetectionRate (%)
100.002424100.0
50.002424100.0
20.002423તે જેવી જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલ
15.002424100.0
12.502424100.0
10.002423તે જેવી જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલ
7.50242187.5
5.00241770.8
2.50241354.2
1.2524937.5

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Table 4. Alinity m EBV Limit of Detection (LoD) - Lot 3 (Least Sensitive Lot)
EBV DNA(IU/mL)No. of ValidReplicatesNo. of DetectedReplicatesDetectionRate (%)
100.002424100.0
50.002424100.0
20.002424100.0
15.00232295.7
12.50232087.0
10.00242187.5
7.50241979.2
5.00241666.7
2.5024937.5
1.2524729.2
Table 5. Alinity m EBV Limit of Detection (LoD) - Lot 4
EBV DNA(IU/mL)No. of ValidReplicatesNo. of DetectedReplicatesDetectionRate (%)
100.002424100.0
50.002424100.0
20.002424100.0
15.00242291.7
12.50242395.8
10.00242291.7
7.50242083.3
5.00241979.2
2.50241354.2
1.2523521.7

The claimed LoD of Alinity m EBV is 20 IU/mL (1.30 Log IU/mL) in plasma.

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5.7.1.3 Limit of Detection for EBV Type 2

Cultured virus for EBV type 2 was diluted to 3 different concentrations in EBV negative human plasma. Testing was performed using one lot of amplification reagents across multiple days. The results, representative of the analytical sensitivity performance of Alinity m EBV for EBV type 2, are summarized in Table 6.

Alinity m EBV detected 95% or greater of EBV samples at and above 15 IU/mL (1.18 Log IU/mL) in plasma. These results demonstrate the ability of Alinity m EBV to detect EBV type 2 at the claimed LoD.

Table 6. Alinity m EBV Type 2 Limit of Detection (LoD)
EBV DNA(IU/mL)No. of ValidReplicatesNo. of DetectedReplicatesDetectionRate (%)
50.002424100.0
20.00232295.7
15.00242395.8

5.7.1.4 Linear Range

The quantitation range of Alinity m EBV is from the lower limit of quantitation (LLQ) of 50 IU/mL (1.70 Log IU/mL) to the upper limit of quantitation (ULoQ) of 200,000,000 IU/mL (8.30 Log IU/mL).

Linearity of Alinity m EBV was assessed by testing a dilution series of EBV type 1 in negative human plasma, consisting of 16 panel levels targeted in the range of 10 IU/mL to 400,000,000 IU/mL (1.00 Log IU/mL to 8.60 Log IU/mL). Panel levels with concentrations from 10 IU/mL to 1,500 IU/mL (1.00 Log IU/mL to 3.18 Log IU/mL) were prepared using clinical specimen, while panel levels with concentrations from 15 IU/mL to 400,000,000 IU/mL (1.18 Log IU/mL to 8.60 Log IU/mL) were prepared using synthetic DNA. Panel quantitation values were traceable to the 1st World Health Organization (WHO) International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (NIBSC code: 09/260).

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Alinity m EBV was linear across the quantitation range from 50 IU/mL to 200.000.000 IU/mL (1.70 Log IU/mL to 8.30 Log IU/mL). Representative results for Alinity m EBV linearity performance are shown in Figure 2.

Image /page/17/Figure/1 description: The figure is a scatter plot that shows the correlation between Alinity m EBV (Log IU/mL) and Expected Concentration (Log IU/mL). The x-axis represents the Expected Concentration (Log IU/mL), and the y-axis represents the Alinity m EBV (Log IU/mL). The data points are clustered around a straight line, indicating a strong positive correlation. The correlation coefficient, r, is 0.999, which further confirms the strong positive correlation between the two variables.

Figure 2. Linearity a

3 The markers in the plot represent the mean Alinity m EBV concentration (in Log IU/mL) for each panel level.

Linearity of EBV Type 2 5.7.1.5

Linearity of Alinity m EBV for EBV type 2 was confirmed by testing a dilution series in negative human plasma, consisting of 16 panel levels targeted in the range of 10 IU/mL to 400,000,000 IU/mL (1.00 Log IU/mL to 8.60 Log IU/mL). Panel levels with concentrations from 10 IU/mL to 1,500 IU/mL (1.00 Log IU/mL to 3.18 Log IU/mL) were prepared using a cultured virus, while panel levels with concentrations from

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15 IU/mL to 400,000,000 IU/mL (1.18 Log IU/mL to 8.60 Log IU/mL) were prepared using synthetic DNA. Panel quantitation values were traceable to the 1st World Health Organization (WHO) International Standard for Epstein-Barr Virus for Nucleic Acid Amplification Techniques (NIBSC code: 09/260).

Alinity m EBV was linear across the quantitation range from 50 IU/mL to 200,000,000 IU/mL (1.70 Log IU/mL to 8.30 Log IU/mL) for EBV type 2.

Representative results for Alinity m EBV linearity performance for type 2 and for type 1 (Section 5.7.1.4) are shown in Figure 3.

Image /page/18/Figure/3 description: The image shows the title of a figure. The title is "Figure 3. Linearity for EBV Type 1 and Type 2 ª". The title is written in a clear, sans-serif font and is centered on the page. The figure number is 3.

Image /page/18/Figure/4 description: The image is a scatter plot comparing expected concentration to Alinity m EBV. The x-axis is labeled 'Expected Concentration (Log IU/mL)' and ranges from 0 to 9. The y-axis is labeled 'Alinity m EBV (Log IU/mL)' and ranges from 0 to 9. There are two lines plotted on the graph, one for Type 1 and one for Type 2, both with r=0.999.

a The markers in the plot represent the mean Alinity m EBV concentration (in Log IU/mL) for each panel level.

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Alinity m EBV was demonstrated to be linear across the quantitation range for EBV type 1 and type 2 from 50 IU/mL to 200.000.000 IU/mL (1.70 Log IU/mL to 8.30 Log IU/mL).

5.7.1.6 Precision

Precision of Alinity m EBV was determined by analyzing a 9-member plasma panel. Panel members with concentrations targeted to 1.30 Log IU/mL and 2.00 Log IU/mL (20 IU/mL and 100 IU/mL) were prepared with positive clinical sample, panel members targeted in the range of 2.70 Log IU/mL to 5.00 Log IU/mL (500 IU/mL to 100,000 IU/mL) were prepared using cultured virus, and panel members with targeted concentrations greater than 5.00 Log IU/mL were prepared using synthetic DNA. Each panel member was tested in 4 replicates, twice each day for 12 days, on 3 Alinity m Systems operated by 3 operators (1 operator per instrument), using 3 AMP kit lots (1 lot per instrument), for a total of 288 replicates per panel member.

The representative precision study results in Table 8 demonstrated that Alinity m EBV within-laboratory standard deviation (SD) was less than or equal to 0.25 Log IU/mL for EBV DNA panels targeted in the range of 2.70 Log IU/mL to 8.30 Log IU/mL (500 IU/mL to 200.000.000 IU/mL), and less than or equal to 0.50 Log IU/mL for EBV DNA panels targeted in the range of 1.30 Log IU/mL to less than 2.70 Log IU/mL (20 IU/mL to less than 500 IU/mL).

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Table 7. Precision
MeanConcentration(Log IU/mL)Within-RunComponentBetween-RunComponentBetween-DayComponentWithin-LaboratorycBetween-InstrumentComponentdTotale
PanelNaSDb% CVSD% CVSD% CVSD% CVSD% CVSD% CV
92878.220.040.50.040.50.000.00.060.70.050.60.080.9
82878.040.050.60.040.40.000.00.060.70.060.80.091.1
72887.050.050.70.020.30.000.00.050.70.020.40.060.8
62846.090.050.80.030.40.000.00.060.90.040.70.071.1
52875.050.050.90.030.60.010.10.051.10.050.90.071.4
42874.060.041.10.030.70.020.40.051.30.061.40.081.9
32882.770.072.70.072.70.000.00.113.80.083.00.134.9
22862.200.135.70.136.10.000.00.188.30.073.10.208.9
12831.430.2517.20.075.20.010.60.2618.00.053.70.2618.4

ª Number of valid replicates with detectable viral load

b Standard deviations (SD) are in Log IU/mL.

· Within-Laboratory includes Within-Run, Between-Run, and Between-Day components.

d Alinity m System, AMP Kit lot, and Operator are confounding effect is represented by Instrument.

e Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument Components.

Alinity m EBV 510(k) Section 5 May 2022 K212778 510k summary FINAL.docx CONFIDENTIAL

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Table 8. Precision
CV(%)c
PanelNaMeanConcentrationb(IU/mL)Within-RunComponentBetween-RunComponentBetween-DayComponentBetween-InstrumentComponentdTotale
9287169,046,88310.09.50.010.917.7
8287111,475,84110.78.20.014.920.2
728811,275,29110.84.30.05.713.0
62841,231,68511.46.10.09.416.0
5287112,52310.76.61.210.916.8
428711,59610.06.33.513.318.2
328862017.417.20.019.531.8
228617629.531.50.016.047.5
12833261.317.22.012.366.2

Number of valid replicates with detectable viral load

b Titer data are considered to be log-normally distributed and the mean values for titer data are calculated as exp(mean * In(10) + [SD * In(10) (22).

& Titer data are considered to be log-normally distributed and %CV (%) = sqrt(10"[SD^2 * In(10)] = 1) * 100.

d Alinity m System, AMP Kit lot, and Operator are confounding effect is represented by Instrument.

e Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument Components.

Alinity m EBV 510(k) Section 5 May 2022 K212778 510k summary FINAL.docx CONFIDENTIAL

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5.7.1.7 Lower Limit of Quantitation

The lower limit of quantitation (LLoQ) is defined as the lowest concentration at which EBV DNA is reliably quantitated within an acceptable total error. Total error was estimated for detected samples from the LoD study by 2 methods:

  • Total Analytical Error (TAE) = |bias| + 2 × SD, and .
  • . Total Error (TE) = SORT(2) × 2 × SD.

The results of the calculations are shown in Table 9.

Panel members were dilutions of the 1st World Health Organization (WHO) International Standard for Epstein Barr Virus for Nucleic Acid Amplification Techniques (NIBSC code: 09/260) prepared in EBV negative plasma.

The results of these analyses support a claimed LLoQ of 50.00 IU/mL (1.70 Log IU/mL) for Alinity m EBV, with an acceptable level of accuracy and precision, ie, TAE and TE less than or equal to 1.00 Log IU/mL.

Table 9. Total Error
TargetConcentration(Log IU/mL)MeanConcentration(Log IU/mL)Biasa(Log IU/mL)SD(Log IU/mL)TAE(Log IU/mL)TE(Log IU/mL)
1.301.340.040.270.590.78
1.701.770.070.220.510.62
2.002.120.120.140.400.40

ª Mean concentration - target concentration

Analytical Specificity – Potential Cross-Reactants 5.7.1.8

The analytical specificity of Alinity m EBV was evaluated with a panel of microorganisms (see Table 10) in EBV negative plasma, positive plasma targeted to 60 IU/mL EBV DNA, and positive plasma targeted to 10,000 IU/mL EBV DNA. Microorganisms were tested at a final concentration of 105 Units/mL for viruses and fungi or 106 Units/mL for bacteria. No cross-reactivity or interference in the performance of the Alinity m EBV assay was observed in the presence of the tested microorganisms.

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Table 10. Potential Cross-Reactants
VirusesBacteriaFungi
Adenovirus 2Actinomyces israeliiAspergillus niger
BK polyomavirusClostridium perfringensCandida albicans (CA)
Cytomegalovirus (CMV)Enterococcus faecalisCryptococcus neoformans
Enterovirus Type 71Escherichia coli
Hepatitis A Virus (HAV)Klebsiella pneumoniae
Hepatitis B Virus (HBV)Listeria monocytogenes
Hepatitis C Virus (HCV)Morganella morganii
Herpesvirus 6AMycobacterium smegmatis
Herpesvirus 6BMycoplasma pneumoniae
Herpesvirus 7Pseudomonas aeruginosa
Herpesvirus 8(Kaposi's sarcoma associated virus)Salmonella enterica
Human immunodeficiency virus 1 (HIV-1)Staphylococcus aureus (SA)
Human immunodeficiency virus 2 (HIV-2)Staphylococcus epidermidis
Human papilloma virus 16(HPV-16)Streptococcus pneumoniae
Human papilloma virus 18(HPV-18)
Herpes Simplex Virus-1 (HSV-1)
Human T-lymphotropic virus type 1(HTLV-1)
Mumps orthorubulavirus
Parvo virus B19
Simian Virus 40
Vaccinia virus (VACV)
Varicella-Zoster virus (VZV)

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5.7.1.9 Analytical Specificity - Potentially Interfering Substances

The effects of endogenous substances and the presence of high levels of therapeutic drugs commonly prescribed in transplant patients were evaluated. Potential interference on Alinity m EBV performance in plasma was assessed by testing 8 negative samples, 8 positive samples targeted to 60 IU/mL and 8 positive samples targeted to 10,000 IU/mL EBV DNA.

No interference was observed in the presence of albumin (60 g/L), hemoglobin (10 g/L), triglycerides (16.94 mmol/L), conjugated bilirubin (475 µmol/L), unconjugated bilirubin (684 umol/L), or human genomic DNA (2 µg/mL) that were introduced in the sample.

No interference was observed in the presence of drug compounds tested in pools or individually that are listed in Table 11, at a concentration of 3 times the reported Cmax or higher.

Table 11. Drug Compounds
Pools TestedDrug Compounds
1Mycophenolic acid
2AmoxicillinClavulanateFoscarnetPiperacillinTazobactam sodiumVancomycin
3AcyclovirAmlodipine besylateAtenololAzathioprineCefotetanCyclosporineEverolimusFamotidineFluconazoleLisinoprilMycophenolate mofetilPrednisoneRabeprazoleSirolimusSulfamethoxazoleTacrolimusTrimethoprimValacyclovirValsartan

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5.7.1.10 Carryover

The carryover rate for Alinity m EBV was determined by analyzing 648 valid replicates of EBV negative samples processed from alternating positions with 647 valid replicates of high concentrated EBV positive samples greater than or equal to 20,000,000 IU/mL, across a minimum of 27 runs. The carryover resulting in a detectable concentration greater than or equal to LoD was 0.3% (95% CI: 0.1% to 1.1%). The carryover resulting in EBV detection was 1.2% (95% CI: 0.6% to 2.4%).

5.7.1.11 Alinity m EBV Testing Using Dilution Procedure

The 1:2.5 dilution procedure was evaluated by comparing quantitation of neat samples and samples tested using the Alinity m EBV dilution procedure. Five plasma panel members with EBV levels targeted in the range of 150 IU/mL to 100,000,000 IU/mL (2.18 Log IU/mL to 8.00 Log IU/mL) were tested. Each panel member was tested, neat or using the dilution procedures, in multiple replicates. For the 5 panel members, the differences in mean, ie, diluted minus neat, ranged from -0.01 Log IU/mL to 0.23 Log IU/mL.

5.7.1.12 Precision of Alinity m EBV Using Dilution Procedure

Precision of Alinity m EBV, using the dilution procedure, was determined by analyzing 3 plasma panel members. Panel members 1 and 2 were prepared by spiking cultured virus in EBV negative sample, and panel member 3 was prepared by spiking synthetic DNA in EBV negative sample. Each panel member was tested in at least 3 replicates, twice each day for 12 days, on 3 Alinity m Systems with 3 Alinity m Specimen Dilution Kit I lots and 3 Alinity m EBV AMP Kit lots by 3 operators (1 Specimen Diluent lot, 1 AMP kit lot and 1 operator per instrument), for a total of at least 216 replicates.

The results, representative of the precision of Alinity m EBV using dilution procedures, are summarized in Table 12 and Table 13.

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MeanConcentrationWithin-RunComponentBetween-RunComponentBetween-DayComponentWithin-LaboratorycBetween-InstrumentComponentdTotale
PanelNa(Log IU/mL)SDb% CVSD% CVSD% CVSD% CVSD% CVSD% CV
12743.500.072.10.061.60.061.70.113.20.041.20.123.4
22734.890.051.10.091.90.000.00.112.20.061.10.122.5
32747.690.060.80.091.20.000.00.111.40.040.50.121.5

ª Number of valid replicates with detectable viral load.

b Standard deviations (SD) are in Log IU/mL.

& Within-Laboratory includes Within-Run, Between-Run and Between-Day Components.

d Alinity m System, AMP Kit lot, Specimen Diluent lot, and Operator are confounding effect is represented by Instrument.

e Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument Components.

Table 13. Precision Using Dilution Procedure
PanelNaMeanConcentrationb(IU/mL)CV(%)c
Within-RunComponentBetween-RunComponentBetween-DayComponentBetween-InstrumentComponentdTotale
12743,26317.013.313.79.727.7
227381,25312.621.70.012.928.5
327451,057,72914.421.20.09.627.6

ª Number of valid replicates with detectable viral load

b Titer data are considered to be log-normally distributed and the mean values for titer data are calculated as exp(mean * In(10) + [SD * In(10) (22).

& Titer data are considered to be log-normally distributed and %CV (%)= sqrt(10°[SD^2 * In(10)]= 1) * 100.

d Alinity m System, AMP Kit lot, and Operator are confounding effect is represented by Instrument.

Page 24 of 35

e Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument Components.

CONFIDENTIAL

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5.7.1.13 Confirmation of the LLoQ Using Dilution Procedure

Confirmation testing for Alinity m EBV LLoQ using the dilution procedure was performed by testing 2 panel members at 50 IU/mL (LLoQ) and 60 IU/mL (near LLoQ) with a dilution factor of 1:2.5. The EBV concentrations in the panel members were targeted at 20 IU/mL and 24 IU/mL (1.30 Log IU/mL) after dilution in Specimen Diluent. Panel members were dilutions of cultured virus spiked into EBV negative plasma.

PanelTargetConcentrationUndiluted(Log IU/mL)DilutionFactorTargetConcentrationin SpecimenDiluent(Log IU/mL)MeanConcentration a(Log IU/mL)Bias b(Log IU/mL)SD(Log IU/mL)TAE(Log IU/mL)TE(Log IU/mL)
11.702.51.301.710.010.170.350.48
21.782.51.381.74-0.040.230.500.65

ª Reported concentration for undiluted samples.

b Mean concentration - target concentration for undiluted samples

A minimum of 14 replicates per day of each panel level were tested using the dilution procedure in 3 runs across 3 days (one run per day). The study was performed using 1 Alinity m EBV AMP Kit lot, 1 Specimen Diluent lot, and 1 Alinity m System. Total error was estimated by TAE and TE, as shown in Table 14. The accuracy and precision at 20 IU/mL were confirmed for Alinity m EBV testing using the 1:2.5 dilution procedure.

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5.7.2 Clinical Reproducibility

Reproducibility performance of Alinity m EBV was evaluated by testing a 9-member reproducibility panel, including 8 positive panel members and 1 negative panel member. The positive panel members were prepared using an EBV positive clinical specimen, cultured virus, or plasmid DNA diluted in human EDTA plasma. The concentration levels targeted for the reproducibility panels spanned the quantitation range of the assay. A total of 3 Alinity m EBV AMP Kit lots, 3 Alinity m EBV CAL Kit lots, 3 Alinity m EBV CTRL Kit lots and 3 Alinity m Sample Prep Kit 2 lots were used. Three clinical sites each tested 2 unique reagent lot combinations (consisting of 2 Alinity m EBV AMP Kit lots along with 1 lot of each Alinity m EBV CAL Kit, Alinity m EBV CTRL Kit and Alinity m Sample Prep Kit 2) on 5 non-consecutive days for each lot combination. Six replicates of each panel member were tested on each of 5 days to ensure a minimum of 5 valid replicates for analysis. The reproducibility results are summarized in Table 15 and Table 16 (for the positive panel members) and Table 17 (for the negative panel member).

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Table 15. Reproducibility for Positive Panel Members
PanelNaTargetConcentration(Log IU/mL)MeanConcentration(Log IU/mL)Within-Run/DayComponentBetween-Run/DayComponentWithin-LaboratorycBetween-LotComponentBetween-Site/InstrumentComponentTotald
SDb% CVSD% CVSD% CVSD% CVSD% CVSD% CV
11788.308.400.050.50.030.30.050.60.060.70.182.10.192.3
21777.007.040.040.60.000.10.040.60.040.60.121.80.142.0
31796.005.760.050.80.020.30.050.90.040.70.081.30.101.8
41795.005.040.061.10.040.90.071.40.040.70.091.80.122.4
51794.004.010.051.20.030.90.061.50.041.00.051.30.092.2
61723.003.070.072.20.031.00.072.40.020.70.041.30.092.8
71791.781.640.1911.40.021.00.1911.50.042.40.137.60.2314.0
81741.301.200.2823.50.054.20.2923.80.000.00.108.00.3025.1

4 Number of valid replicates with detectable viral load

6 Standard deviations (SD) are in Log IU/mL.

? Within-Laboratory includes Within-Run/Day and Between-Run/Day components.

Total includes Within-Run/Day, Between-Run/Day, Between-Lot, and Between-Site/Instrument Components.

CONFIDENTIAL

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CV(%)c
PanelNaMeanConcentrationb(IU/mL)Within-Run/DayComponentBetween-Run/DayComponentBetween-LotComponentBetween-Site/InstrumentComponentTotal
1178279,192,22010.66.413.042.847.1
217711,407,3419.51.19.329.332.5
3179586,12611.33.99.418.023.7
4179112,77213.110.18.221.428.6
517910,57211.68.09.711.720.8
61721,18715.36.94.99.319.9
71795145.43.99.029.456.8
81742072.111.50.022.378.6

CONFIDENTIAL

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Table 17. Reproducibility for Negative Panel Member
Expected EBV DNA ConcentrationNo. of ReplicatesNegative Rate (%)95% Confidence Interval
ValidNegative
Negative18017697.8 (176/180)(94.4, 99.1)

5.7.3 Clinical Performance

Alinity m EBV results were compared to those of an FDA-cleared EBV nucleic acid test in a representative study. A total of 558 EDTA plasma samples were tested (neat or diluted), including 542 clinical specimens from hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) subjects, and 16 contrived samples prepared by spiking inactivated EBV virus into the individual clinical specimens. The Alinity m EBV assay testing was performed at 3 clinical testing sites with 3 Alinity m EBV reagent kit lots.

Of the 558 clinical samples, 550 produced valid results with Alinity m EBV and the comparator assay, including 388 samples detected by Alinity m EBV and 162 samples not detected by Alinity m EBV. Out of 550 valid samples, 168 were from HSCT subjects, 379 were from SOT subjects, and 3 were from dual-transplant (HSCT/SOT) subjects. The agreement between Alinity m EBV and comparator results is shown in Table 18 (HSCT samples), Table 19 (SOT samples), and Table 20 (HSCT and SOT samples combined).

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Table 18. HSCT Samples – Agreement Between Alinity m EBV and Comparatora
Comparator EBV (Log IU/mL)
Alinity m EBV(Log IU/mL)TargetNotDetected<LLoQbLLoQbto <2.702.70to <3.003.00to <3.703.70to <4.00≥4.00Total
Target NotDetected4400000044
<LLoQb4206000030
LLoQb to <2.700624100031
2.70 to < 3.0000441009
3.00 to < 3.700005132020
3.70 to <4.0000002428
≥4.000000012829
Total4826341016730171
ColumnAgreement (%)(48/48)100.0%(26/26)100.0%(34/34)100.0%(10/10)100.0%(16/16)100.0%(7/7)100.0%(30/30)100.0%
95%Score CI(92.6%,100.0%)(87.1%,100.0%)(89.8%,100.0%)(72.2%,100.0%)(80.6%,100.0%)64.6%,100.0%)(88.6%,100.0%)

ª Three dual-transplant specimens were included in both HSCT and SOT agreement analyses.

b The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

Table 19. SOT Samples – Agreement Between Alinity m EBV and Comparator a
Comparator EBV (Log IU/mL)
Alinity m EBV(Log IU/mL)TargetNotDetected<LLoQbLLoQbto <2.702.70to <3.003.00to <3.703.70to <4.00≥4.00Total
Target NotDetected110710000118
<LLoQb28618000097
LLoQb to <2.7011661410083
2.70 to <3.00005900014
3.00 to <3.700006200026
3.70 to <4.0000004228
≥4.000000043236
Total13984751925634382
ColumnAgreement (%)(138/139)99.3%(84/84)100.0%(74/75)98.7%(19/19)100.0%(24/25)96.0%(6/6)100.0%(34/34)100.0%
95%Score CI(96.0%,99.9%)(95.6%,100.0%)(92.8%,99.8%)(83.2%,100.0%)(80.5%,99.3%)61.0%,100.0%)(89.8%,100.0%)

a Three dual-transplant specimens were included in both HSCT and SOT agreement analyses.

b The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

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Table 20. HSCT and SOT Samples Combined – Agreement Between Alinity m EBV and Comparator
Comparator EBV (Log IU/mL)
Alinity m EBV(Log IU/mL)TargetNotDetected<LLOQ aLLOQ ato <2.702.70to <3.003.00to <3.703.70to <4.00≥4.00Total
Target NotDetected154710000162
<LLOQa3280140000126
LLOQa to <2.70122835100112
2.70 to <3.000091310023
3.00 to <3.7000011332046
3.70 to <4.00000066416
≥4.000000056065
Total18710910729411364550
ColumnAgreement (%)(186/187)99.5%(109/109)100.0%(106/107)99.1%(29/29)100.0%(40/41)97.6%(13/13)100.0%(64/64)100.0%
95%Score CI(97.0%,99.9%)(96.6%,100.0%)(94.9%,99.8%)(88.3%,100.0%)(87.4%,99.6%)(77.2%,100.0%)(94.3%,100.0%)

a The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

Discordant results were defined as those that are more than one box away from the diagonal (indicated by shading). For Target Not Detected (TND) by Comparator Column Agreement, the Alinity m EBV Target Not Detected and < LLoQ cells were combined. The rationale for adding the adjacent <LLoQ and TND cells for the TND column was that the difference between a TND and <LLoQ were not clinically meaningful and that these were analytically at the lower end of the quantitation range, which may be impacted by random error.

Of the 550 samples, 44 were collected for the estimation of Negative Percent Agreement (NPA) and were confirmed as EBV DNA negative. For this subset of confirmed EBV DNA negative clinical specimens, the NPA with the Comparator assay was 100.0% (44/44) with a 95% confidence interval of (92.0%, 100.0%).

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Agreement between Alinity m EBV assay and the comparator assay was also evaluated using different clinical thresholds and is shown in Table 21 (HSCT samples), Table 22 (SOT samples), and Table 23 (HSCT and SOT samples combined).

Table 21. HSCT Samples – Agreement between Alinity m EBV and Comparator EBV usingDifferent Thresholds
ThresholdPercent Agreement < Threshold (%)95% Score CI (n/N)Percent Agreement ≥ Threshold (%)95% Score CI (n/N)
Not Detected100.0 (92.6, 100.0)(48/48)100.0 (97.0, 100.0)(123/123)
< LLoQ a91.9 (83.4, 96.2)(68/74)93.8 (87.2, 97.1)(91/97)
< 3.00 Log IU/mL95.8 (90.5, 98.2)(113/118)98.1 (90.1, 99.7)(52/53)
< 4.00 Log IU/mL99.3 (96.1, 99.9)(140/141)93.3 (78.7, 98.2)(28/30)

ª The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

Table 22. SOT Samples – Agreement between Alinity m EBV and Comparator EBV usingDifferent Thresholds
ThresholdPercent Agreement < Threshold (%)95% Score CI (n/N)Percent Agreement ≥ Threshold (%)95% Score CI (n/N)
Not Detected99.3 (96.0, 99.9)(138/139)96.7 (93.6, 98.3)(235/243)
< LLoQ a92.4 (88.1, 95.2)(206/223)94.3 (89.6, 97.0)(150/159)
< 3.00 Log IU/mL98.1 (95.9, 99.1)(311/317)98.5 (91.8, 99.7)(64/65)
< 4.00 Log IU/mL98.9 (97.1, 99.6)(344/348)94.1 (80.9, 98.4)(32/34)

ª The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

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Table 23. HSCT and SOT Samples Combined – Agreement between Alinity m EBV andComparator EBV using Different Thresholds
ThresholdPercent Agreement < Threshold (%)95% Score CI (n/N)Percent Agreement ≥ Threshold (%)95% Score CI (n/N)
Not Detected99.5 (97.0, 99.9)(186/187)97.8 (95.7, 98.9)(355/363)
< LLoQ a92.2 (88.6, 94.8)(273/296)94.1 (90.5, 96.4)(239/254)
< 3.00 Log IU/mL97.5 (95.5, 98.6)(421/432)98.3 (94.0, 99.5)(116/118)
< 4.00 Log IU/mL99.0 (97.6, 99.6)(481/486)93.8 (85.0, 97.5)(60/64)

ª The LLoQ used here is the higher LLoQ between Alinity m EBV and comparator.

Regression and bias analysis included a total of 239 samples with results that were within the common quantitation range of both Alinity m EBV and the comparator assay.

Figure 4 shows the results of the Deming regression analysis with a slope of 1.01, intercept of 0.05, and correlation coefficient of 0.974. The mean bias between Alinity m EBV and the comparator (Alinity m EBV minus comparator) was 0.09 Log IU/mL with a 95% CI of (0.06, 0.12).

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Image /page/36/Figure/0 description: The image is a scatter plot titled "Figure 4. Deming Regression Analysis". The x-axis is labeled "Comparator EBV Quantitation (Log IU/mL)" and ranges from 1 to 8. The y-axis is labeled "Alinity m EBV Quantitation (Log IU/mL)" and ranges from 1 to 8. The plot shows a strong positive correlation between the two variables, with the equation of the regression line being y = 1.01x + 0.05, r = 0.974, and n = 239.

Systematic difference between Alinity m EBV and the comparator at 4 selected viral load levels is shown in Table 24.

Table 24. Systematic Difference at Selected Viral Load Levels
Target Viral Load Level(based on comparator)Systematic Difference
LLoQ0.07 Log IU/mL
3.00 Log IU/mL0.09 Log IU/mL
4.00 Log IU/mL0.10 Log IU/mL
5.00 Log IU/mL0.11 Log IU/mL

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5.8 Conclusions Drawn from the Studies

The analytical and clinical study results demonstrate that the Alinity m EBV assay for use on the Alinity m System is safe, effective, and performs comparably to the predicate device. The results support a substantial equivalence decision for Alinity m EBV.

§ 866.3183 Quantitative viral nucleic acid test for transplant patient management.

(a)
Identification. A quantitative viral nucleic acid test for transplant patient management is identified as a device intended for prescription use in the detection of viral pathogens by measurement of viral DNA or RNA using specified specimen processing, amplification, and detection instrumentation. The test is intended for use as an aid in the management of transplant patients with active viral infection or at risk for developing viral infections. The test results are intended to be interpreted by qualified healthcare professionals in conjunction with other relevant clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include:
(i) A prominent statement that the device is not intended for use as a donor screening test for the presence of viral nucleic acid in blood or blood products.
(ii) Limitations which must be updated to reflect current clinical practice. These limitations must include, but are not limited to, statements that indicate:
(A) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with clinical signs and symptoms and other relevant laboratory results; and
(B) Negative test results do not preclude viral infection or tissue invasive viral disease and that test results must not be the sole basis for patient management decisions.
(iii) A detailed explanation of the interpretation of results and acceptance criteria must be provided and include specific warnings regarding the potential for variability in viral load measurement when samples are measured by different devices. Warnings must include the following statement, where applicable: “Due to the potential for variability in [analyte] measurements across different [analyte] assays, it is recommended that the same device be used for the quantitation of [analyte] when managing individual patients.”
(iv) A detailed explanation of the principles of operation and procedures for assay performance.
(2) Design verification and validation must include the following:
(i) Detailed documentation of the device description, including all parts that make up the device, ancillary reagents required for use with the assay but not provided, an explanation of the methodology, design of the primer/probe sequences, rationale for the selected gene target, and specifications for amplicon size, guanine-cytosine content, and degree of nucleic acid sequence conservation. The design and nature of all primary, secondary and tertiary quantitation standards used for calibration must also be described.
(ii) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's functions;
(iii) Documentation and characterization (
e.g., determination of the identity, supplier, purity, and stability) of all critical reagents and protocols for maintaining product integrity throughout its labeled shelf-life.(iv) Stability data for reagents provided with the device and indicated specimen types, in addition to the basis for the stability acceptance criteria at all time points chosen across the spectrum of the device's indicated life cycle, which must include a time point at the end of shelf life.
(v) All stability protocols, including acceptance criteria.
(vi) Final lot release criteria along with documentation of an appropriate justification that lots released at the extremes of the specifications will meet the claimed analytical and clinical performance characteristics as well as the stability claims.
(vii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Mode Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel viral stains (
e.g., regular review of published literature and annual in silico analysis of target sequences to detect possible primer or probe mismatches). All results of this protocol, including any findings, must be documented.(viii) Analytical performance testing that includes:
(A) Detailed documentation of the following analytical performance studies: limit of detection, upper and lower limits of quantitation, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, quality control, specimen stability studies, and additional studies as applicable to specimen type and intended use for the device;
(B) Identification of the viral strains selected for use in analytical studies, which must be representative of clinically relevant circulating strains;
(C) Inclusivity study results obtained with a variety of viral genotypes as applicable to the specific assay target and supplemented by in silico analysis;
(D) Reproducibility studies that include the testing of three independent production lots;
(E) Documentation of calibration to a reference standard that FDA has determined is appropriate for the quantification of viral DNA or RNA (
e.g., a recognized consensus standard); and(F) Documentation of traceability performed each time a new lot of the standardized reference material to which the device is traceable is released, or when the field transitions to a new standardized reference material.
(ix) Clinical performance testing that includes:
(A) Detailed documentation from either a method comparison study with a comparator that FDA has determined is appropriate, or results from a prospective clinical study demonstrating clinical validity of the device;
(B) Data from patient samples, with an acceptable number of the virus-positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision points. If an acceptable number of virus-positive samples containing an analyte concentration near the lower limit of quantitation and any clinically relevant decision cannot be obtained, contrived samples may be used to supplement sample numbers when appropriate, as determined by FDA;
(C) The method comparison study must include predefined maximum acceptable differences between the test and comparator method across all primary outcome measures in the clinical study protocol; and
(D) The final release test results for each lot used in the clinical study.