Alinity m HBV

P200013 · Abbott Molecular, Inc. · MKT · Aug 29, 2020 · Microbiology

Device Facts

• Record ID: P200013
• Device Name: Alinity m HBV
• Applicant: Abbott Molecular, Inc.
• Product Code: MKT · Microbiology
• Decision Date: Aug 29, 2020
• Decision: APRL
• Regulation: 21 CFR 866.3174
• Device Class: Class 2

Indications for Use

The Alinity m HBV assay is an in vitro polymerase chain reaction (PCR) assay for use with the automated Alinity m System to quantitate Hepatitis B Virus (HBV) DNA in human plasma or serum. The Alinity m HBV assay is intended for use as an aid in the management of patients with chronic HBV infection undergoing anti-viral therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing response to treatment. The results from the Alinity m HBV assay must be interpreted within the context of all relevant clinical and laboratory findings. This assay is not intended to be used for screening donors of blood, blood products, or cell, tissue, and cellular and tissue-based products (HCT/Ps) or as a diagnostic test to confirm the presence of HBV infection.

Device Story

The Alinity m HBV assay is a fully automated, in vitro, real-time PCR assay performed on the Alinity m System. It targets a conserved sequence in the overlap of the HBV Surface and Polymerase regions. The system performs automated sample preparation (lysis, magnetic microparticle-based nucleic acid capture, wash, elution), PCR assembly, amplification, and real-time fluorescence detection. It uses an internal control (hydroxypyruvate reductase gene from Cucurbita pepo) to validate processing. The system is a random-access analyzer used in clinical laboratories. Output is a quantitative HBV DNA concentration (IU/mL) calculated from a stored calibration curve. Clinicians use these results to monitor viral load changes during anti-viral therapy, aiding in the assessment of treatment response (e.g., histologic, biochemical, serological). Benefits include objective monitoring of therapy efficacy, potentially reducing morbidity associated with chronic HBV sequelae.

Clinical Evidence

Clinical performance evaluated in 412 subjects (HBeAg-positive and HBeAg-negative) with chronic HBV undergoing anti-viral therapy. 1,821 specimens analyzed across 3 sites. Study assessed association between viral load response (HBV DNA <2000 IU/mL or ≥2 Log decrease) and clinical responses (histologic, biochemical, HBeAg loss, seroconversion) at Week 48. Results demonstrated that viral responses measured by the assay were informative for determining clinical responses, with statistically significant odds ratios (lower bound of 95% CI >1.00) for various clinical response combinations.

Technological Characteristics

Real-time PCR assay; targets conserved HBV Surface/Polymerase region. Uses magnetic microparticle technology for nucleic acid extraction. Reagents include lyophilized unit-dose amplification/detection reagents and liquid activation reagents. Includes Uracil-DNA Glycosylase (UDG) for contamination control. System is a fully automated, random-access molecular diagnostics analyzer. Connectivity: Networked system. Software: Automated data reduction and validity evaluation via Application Specification File (JSON format).

Indications for Use

Indicated for patients with chronic HBV infection undergoing anti-viral therapy to quantitate HBV DNA in plasma or serum at baseline and during treatment to assess response.

Regulatory Classification

Identification

A nucleic acid-based hepatitis B virus (HBV) assay is identified as an in vitro diagnostic device intended for prescription use in the detection of HBV nucleic acid in specimens from individuals with antibody evidence of HBV infection. In these devices, the detection of HBV nucleic acid is used as an aid in the management of HBV-infected individuals. The assay is intended for use with human serum or plasma (and other matrices as applicable) from individuals with HBV. The assay is not intended for use as a donor screening assay for the presence of HBV nucleic acids in blood, blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the assay is not intended for use as a screening assay for the presence of HBV DNA in blood or blood products, plasma, cells, or tissue donors, or as a diagnostic assay to confirm the presence of HBV infection. (ii) A detailed explanation of the principles of operation and procedures for performing the assay. (iii) A detailed explanation of the interpretation of results. (iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and/or management. These limitations must include statements that indicate: (A) Management of patients undergoing HBV treatment should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures, *e.g.,* HBV serologic testing, liver function assays, liver elastography, etc.(B) The specimen types for which the device has been cleared, and that use of this assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results. (C) The results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay. (2) Design verification and validation must include the following: (i) Detailed device description, including the device components, ancillary reagents required but not provided, and an explanation of the device methodology. Additional information appropriate to the technology must be included such as design of primers and probes, rationale for the selected gene targets, specifications for amplicon size, and degree of nucleic acid sequence conservation. (ii) For devices with assay calibrators, the design and composition of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a standardized reference material that FDA has determined is appropriate ( *e.g.,* a recognized consensus standard). In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance or approval, or when there is a transition to a new calibration standard.(iii) Documentation and characterization ( *e.g.,* determination of the identity, supplier, purity, and stability) of all critical reagents (including nucleic acid sequences for primers and probes) and protocols for maintaining product integrity.(iv) Risk analysis and management strategies demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance. (v) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specification will meet the identified analytical and clinical performance characteristics as well as stability. (vi) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range. (vii) All stability protocols, including acceptance criteria. (viii) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including limit of detection (LoD), linearity, precision, endogenous and exogenous interferences, cross-reactivity, carryover, matrix equivalency, sample and reagents stability, and as applicable, upper and lower limits of quantitation (ULoQ and LLoQ, respectively). Samples selected for use must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HBV nucleic acid negative subjects with other viral or non-viral causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis C virus, primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each identified nucleic-acid isolation and purification procedure on detection must be evaluated. (ix) Analytical sensitivity of the assay that is the same or better than that of other cleared or approved assays. (x) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review. (xi) Detailed documentation of performance from a clinical study with a design and number of clinical samples (appropriately statistically powered) that is appropriate for the intended use of the device as well as conducted in the appropriate settings by the intended users. The samples must include prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas.

Submission Summary (Full Text)

{0}

# SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

## I. GENERAL INFORMATION

Device Generic Name: Hepatitis Viral B DNA Detection

Device Trade Name: Alinity m HBV

Device Product Code: MKT

Applicants Name and Address: Abbott Molecular Inc.
1300 E. Touhy Ave
Des Plaines, IL60018

Date of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P200013

Date of FDA Notice of Approval: August 29, 2020

## II. INDICATIONS FOR USE

The Alinity m HBV assay is an in vitro polymerase chain reaction (PCR) assay for use with the automated Alinity m System to quantitate Hepatitis B Virus (HBV) DNA in human plasma or serum. The Alinity m HBV assay is intended for use as an aid in the management of patients with chronic HBV infection undergoing anti-viral therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing response to treatment. The results from the Alinity m HBV assay must be interpreted within the context of all relevant clinical and laboratory findings.

This assay is not intended to be used for screening donors of blood, blood products, or cell, tissue, and cellular and tissue-based products (HCT/Ps) or as a diagnostic test to confirm the presence of HBV infection.

## III. CONTRAINDICATIONS

There are no known contraindications.

## IV. WARNINGS AND PRECAUTIONS

The warning and precautions can be found in the Alinity m HBV assay package insert and Alinity m System Operations Manual

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 1 of 50

{1}

V. DEVICE DESCRIPTION

The Alinity m HBV assay utilizes real-time polymerase chain reaction (PCR) to amplify and detect HBV DNA genomic sequences that have been extracted from human plasma or serum specimens. The steps of the Alinity m HBV assay consist of sample preparation, PCR assembly, amplification/detection, and result calculation and reporting.

The Alinity m HBV assay is designed to target a highly conserved sequence within the overlap of the Surface and the Polymerase region of the HBV genome. Additionally, HBV primers and two probes ensure assay robustness against new and emerging HBV variants. All steps of the Alinity m HBV assay procedure are executed automatically by the Alinity m System. Manual dilutions may be performed for low-volume specimens to meet the minimum volume requirement, and for high-titer specimens above the upper limit of quantitation (ULoQ).

The Alinity m System is designed to be a random-access analyzer that can perform the Alinity m HBV assay in parallel with other Alinity m assays on the same instrument. HBV DNA from human plasma or serum is extracted using the Alinity m Sample Prep Kit 2, proteinase K, 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 acid is then combined with liquid unit-dose Alinity m HBV assay activation reagent and lyophilized unit-dose Alinity m HBV assay 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 HBV.

At the beginning of the Alinity m HBV assay sample preparation process, a lyophilized unit- dose Internal Control (IC) and proteinase K on the AMP TRAY 1 is rehydrated by the Alinity m System and delivered into each sample preparation reaction. IC is unrelated to HBV target sequence and is derived from the hydroxypyruvate reductase gene from the pumpkin plant, Cucurbita pepo. IC is unrelated to HBV target sequence and is derived from the hydroxypyruvate reductase gene from the pumpkin plant, Cucurbita pepo. The IC is then processed through the entire sample preparation and PCR procedure along with the specimens, calibrators, and controls to demonstrate proper sample processing and validity.

The Alinity m HBV assay amplification/detection reagents consist of enzyme, primers, probes, and activation reagents that enable polymerization and detection. The Alinity m HBV assay amplification/detection reagent also contains Uracil-DNA Glycosylase (UDG) as a contamination control for amplicons containing uracil, which may be present in molecular laboratories.

An HBV calibration curve is required for the determination of HBV DNA concentration. Two levels of calibrators are processed through sample preparation and PCR to generate the calibration curve. The concentration of HBV 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

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 2 of 50

{2}

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.

Alinity m HBV is intended for use with the Alinity m System, a fully automated, self-contained system.

## A. Components of the Alinity m HBV Assay

### A.1. Alinity m HBV AMP Kit:

The Alinity m HBV AMP Kit is comprised of 2 types of multi-well trays:

- Alinity m HBV AMP TRAY 1 (4 trays × 48 tests): Alinity m HBV AMP TRAY 1 contains 48 unit-dose lyophilized amplification reagent wells and 48 unit-dose lyophilized internal control and proteinase K (PK) reagent wells.
- Alinity m HBV ACT TRAY 2 (4 trays × 48 tests): Alinity m HBV ACT TRAY 2 contains 48 unit-dose liquid activation reagent wells.

Each Alinity m HBV Tray 1 and Tray 2 supports testing of up to 192 samples (patient specimens, assay controls, or calibrators). Both trays contain 48 unit-dose reagent wells (with reagents as listed above) of which one well of each reagent is used per test (48 tests per tray).

## Additional materials required but purchased separately

### B.1. Alinity m HBV CAL Kit:

The Alinity m HBV assay calibrators are for calibration of the Alinity m HBV assay on the automated Alinity m System when used for the quantitative determination of HBV DNA. The Alinity m HBV CAL Kit is composed of the following reagents:

- Alinity m HBV CAL A (4 tubes × 1.65mL)
- Alinity m HBV CAL B (4 tubes × 1.65mL)

The Alinity m HBV CAL A and Alinity m HBV CAL B tubes are intended for single-use only. The Alinity m System will process 3 replicates from each calibrator tube. The calibrators are assigned lot-specific HBV DNA concentrations based on the results of testing against the Primary Calibrators.

### C.1. Alinity m HBV CTRL Kit:

The Alinity m HBV assay controls are for the validity determination of the quantitative Alinity m HBV assay on the automated Alinity m System. The Alinity m HBV CTRL Kit is composed of the following reagents:

- Alinity m HBV Negative CTRL (12 tubes × 1.15mL)
- Alinity m HBV Low Positive CTRL (12 tubes × 0.65mL)
- Alinity m HBV High Positive CTRL (12 tubes × 0.65mL)

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{3}

The Alinity m HBV Negative CTRL, Alinity m HBV Low Positive CTRL, and Alinity m HBV High Positive CTRL tubes are intended for single-use only. Controls are recommended to be tested at or above the minimum frequency of once every 24 hours.

## D.1. Alinity m HBV Application Specification File:

The application specification file is a data file that contains a set of parameters in a software-industry-standard JSON (JavaScript Object Notation) file format. The parameters determine how the software controls the instrument components to execute the selected assay.

To run an assay on an Alinity m System, an Application Specification File is required. The Alinity m System software interprets the assay information provided in the specific Application Specification File (including definitions for sample extraction, reagent addition, amplification/detection, dilution function, data analysis, and validity evaluation protocols), along with system information, to control the system hardware, run validity checks and identify the appropriate algorithms for data generation.

## E.1. Alinity m Sample Prep Kit 2:

The Alinity m Sample Prep Kit 2 is provided in a liquid, multi-dose format and is shared with other Alinity m assays. It consists of 2 reagents:

- Alinity m Elution Buffer 2 (4 bottles × 22mL)
- Alinity m Microparticles 2 (4 bottles × 24mL)

The Alinity m Sample Prep Kit 2 is used in conjunction with Alinity m System Solutions as part of the sample preparation protocol. The Alinity m Sample Prep Kit 2 is used on the Alinity m System to extract and concentrate target molecules from biological samples for subsequent Polymerase Chain Reaction (PCR) amplification, and to remove potential inhibitors from the resulting extract.

The sample preparation procedure consists of lysis/binding, washes, and elution. The sample preparation is performed within a disposable multi-well Integrated Reaction Unit that is loaded onto an Assay Processing Unit (APU) on the Alinity m System.

## F.1. Alinity m Specimen Dilution Kit I:

The Alinity m Specimen Dilution Kit I is intended to allow dilution of specimens for testing on the automated Alinity m System for the measurement of nucleic acid. The Alinity m Specimen Dilution Kit I consist of an Alinity m Specimen Diluent Tube with a pierceable cap (24 tubes × 2.45mL). Each Alinity m Specimen Dilution Kit I supports dilution of up to 24 samples (patient specimens). The Alinity m Specimen Diluent Tube is a transport tube with a pierceable cap containing Abbott Molecular Transport Buffer. The buffer contains guanidine thiocyanate (GITC) in Tris Buffer.

## G.1. Alinity m Tubes and Caps:

- Alinity m LRV Tube: Low Residual Volume (LRV) Tubes closed with caps (12 capped tubes per kit)

PMA P200013: FDA Summary of Safety and Effectiveness Data
Page 4 of 50

{4}

- Alinity m Transport Tube Pierceable Capped: transport tubes closed with pierceable caps (1500 capped transport tubes per case, 10 boxes of 150 capped tubes)
- Alinity m Transport Tube: 1600 transport tubes per kit
- Alinity m Pierceable Cap: 2000 pierceable caps per kit. The pierceable cap can be used to recap a transport tube
- Alinity m Aliquot Tube: 1600 aliquot tubes per kit

## H.1. Alinity m System Solution:

The Alinity m System Solutions described below are used as a part of the sample preparation protocol to extract nucleic acid and concentrate target molecules from biological samples for subsequent PCR amplification and to remove potential inhibitors from the resulting extract. Alinity m System solution consists of

- Alinity m Lysis Solution: 1 bottle × 975mL
- Alinity m Diluent Solution: 4 bottles × 975mL
- Alinity m Vapor Barrier Solution: 1 bottle × 975mL

## I.1. Instrumentation and Software:

The Alinity m System is a fully integrated and automated molecular diagnostics analyzer that utilizes real-time PCR technology in clinical laboratories. It provides a sample-to-result uninterrupted processing workflow. The Alinity m System enables continuous and random-access sample processing by using multiple sample processors and PCR thermal cycler/reader modules in parallel. Each sample occupies either one sample process lane or PCR Amplification and Detection (Amp-Detect) lane. Parallel lanes are provided to enable 300 tests in approximately 8 hours.

Each Alinity m System utilizes four independent Assay Processing Units (APUs) to achieve the throughput and random-access requirements. Each APU consists of one extraction unit and one Amp-Detect unit, which automate the steps for nucleic acid purification/extraction and real-time PCR, respectively. This results in the ability to process up to twenty-four different assay types simultaneously (i.e., up to 12 different assay types for purification/extraction and up to 12 different assay types for amplification and detection).

The Alinity m System software is the set of computer instructions that interprets system and assay information, calculates results, and provides the interface for controlling the system hardware. The Alinity m System software interprets the assay information provided in the specific Application Specification File, along with system information, to control the system hardware and identify the appropriate algorithms for data reduction.

Using application specifications, customers create orders for calibrators, controls, and specimens. Customers load racks of calibrators, controls, and specimens in the sample input to begin processing. Once the samples are processed, the results are reviewed and released through the software user interface.

Additional details can be found in the Alinity m Operator's Manual.

PMA P200013: FDA Summary of Safety and Effectiveness Data
Page 5 of 50

{5}

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are currently several FDA approved Class III in vitro diagnostic tests for the quantitation of HBV DNA. The patient’s medical history and thorough clinical examination, in addition to serology, PCR or nucleic acid testing (NAT), determination of liver enzyme levels, and biopsy of the liver will provide further information on the status of an HBV infection. Each alternative has its advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

VII. MARKETING HISTORY

The CE certified Alinity m HBV AMP Kit (List No. 08N47-090), Alinity m HBV CAL Kit (List No. 08N47-070), and Alinity m HBV CTRL Kit (List No. 08N47-080) are identical in formulation to the US kits except for kit labeling and were introduced to markets outside of the United States as listed in Table 1. Additionally, 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) and Alinity m System Solutions (List No. 09N20) received CE certification and are available in markets outside of the United States as listed in Table 1. The devices have not been withdrawn from marketing for any reason related to safety and effectiveness.

B. Table 1. Alinity m HBV assay in Foreign Markets Outside of the United States

| Australia | Greece | Poland |
| --- | --- | --- |
| Austria | Hungary | Portugal |
| Belgium | Iceland | Romania |
| Brazil | Ireland | Saudi Arabia |
| Bulgaria | Italy | Slovakia |
| Croatia | Latvia | Slovenia |
| Cyprus | Liechtenstein | South Korea |
| Czech Republic | Lithuania | Spain |
| Denmark | Luxembourg | Sweden |
| Estonia | Malta | Switzerland |
| Finland | Netherlands | Turkey |
| France | Norway | UK |
| Germany | Peru | |

VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

When used according to the instructions in the package insert, there are no known direct adverse effects of this device on the health of the user. No specific adverse effects occurred during the conduct of clinical studies.

An erroneous test result—too low or too high—may occur with the Alinity m HBV assay. An erroneously low result may lead a clinician to believe that the current therapy is effective when it is not. Consequently, the clinician could fail to implement more appropriate therapy. An

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 6 of 50

{6}

erroneously high result may lead a clinician to believe that the current therapy is not effective. Consequently, the clinician could implement an inappropriate change in therapy.

There were no specific adverse events that occurred in clinical studies.

## IX. SUMMARY OF NON-CLINICAL STUDIES

## C. Laboratory Studies

### 1. Limit of Detection (LoD)

The LoD was determined by testing dilutions of the 3rd World Health Organization (WHO) International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques (NIBSC code: 10/264; genotype A) prepared in HBV negative human plasma and serum. A total of 7 panel members were tested for each specimen matrix (plasma and serum) in 3 testing runs with each of the 4 Alinity m HBV AMP Kit lots performed across 3 days including a minimum of 8 replicates per day (i.e., 4 AMP Kit lots × 8 replicates/day × 3 days = minimum 96 total replicates per panel member tested). The analytical sensitivity of Alinity m HBV assay in plasma and serum are included in Table 3.

Probit analysis of the data determined that the concentration of HBV DNA in plasma detected with 95% probability (LoD by Probit) was 4.29 IU/mL (95% CI: 3.61 to 5.34 IU/mL).

Probit analysis of the data determined that the concentration of HBV DNA in serum detected with 95% probability (LoD by Probit) was 6.85 IU/mL (95% CI 5.44 IU/mL to 9.19 IU/mL).

The claimed LoD for Alinity m HBV assay is 10 IU/mL (1.00 Log IU/mL) in plasma and serum for HBV genotype A.

Table 3. Alinity m HBV assay Limit of Detection (LoD) in Plasma and Serum (Genotype A)

| Matrix | HBV DNA (IU/mL) | Number of Valid Replicates | Number of detected Replicates | Detection Rate (%) | LoD by Probit [95% CI] |
| --- | --- | --- | --- | --- | --- |
| Plasma | 15.00 | 111 | 111 | 100.0 | 4.29 IU/mL [3.61 – 5.34 IU/mL] |
| | 10.00 | 112 | 112 | 100.0 | |
| | 7.00 | 110 | 110 | 100.0 | |
| | 4.00 | 109 | 101 | 92.7 | |
| | 2.00 | 109 | 83 | 76.1 | |
| | 1.00 | 112 | 47 | 42.0 | |
| | 0.50 | 111 | 25 | 22.5 | |
| Serum | 15.00 | 113 | 112 | 99.1 | 6.85 IU/mL [5.44 – 9.19 IU/mL] |
| | 10.00 | 114 | 112 | 98.2 | |
| | 7.00 | 112 | 108 | 96.4 | |
| | 4.00 | 113 | 101 | 89.4 | |
| | 2.00 | 108 | 74 | 68.5 | |
| | 1.00 | 113 | 64 | 56.6 | |
| | 0.50 | 112 | 46 | 41.1 | |

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 7 of 50

{7}

# 2. Limit of Detection for Genotypes

The LoD of the Alinity m HBV assay was verified for HBV Genotypes B, C, D, E, F, G, H, and I in plasma and serum. For each HBV Genotype, 3 panel members were prepared by diluting a clinical specimen into HBV negative human serum and plasma. Panel quantitation values were established using internal reference standards that are traceable to 3rd WHO International Standard for Hepatitis B Virus (NIBSC code: 10/264). Testing was performed across 3 days including 8 replicates per day for a total of 24 replicates per panel member. This study design ensures a minimum of 20 valid replicates per panel member according to CLSI EP17-A2. The analytical sensitivity of Alinity m HBV assay for genotypes B, C, D, E, F, G, H, and I are summarized in Table 4 and Table 5. Alinity m HBV assay detected $&gt;95\%$ HBV replicates at and above $10~\mathrm{IU / mL}$ (1.00 Log IU/mL) in plasma and serum. The study demonstrates that the Alinity m HBV assay detects genotypes B, C, D, E, F, G, H, and I at the claimed LoD.

Table 4. Alinity m HBV assay Genotype Limit of Detection (LoD) in Plasma

| Genotype | HBV DNA (IU/mL) | No. of Valid Replicates | No. of Detected Replicates | Detection Rate (%) |
| --- | --- | --- | --- | --- |
| B | 20 | 20 | 20 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| C | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| D | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 23 | 21 | 91.3 |
| E | 20 | 24 | 24 | 100.0 |
| | 10 | 23 | 23 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| F | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| G | 20 | 23 | 23 | 100.0 |
| | 10 | 24 | 24 | 100.0 |

PMA P200013: FDA Summary of Safety and Effectiveness Data

{8}

Table 5. Alinity m HBV assay Genotype Limit of Detection (LoD) in Serum

| Genotype | HBV DNA (IU/mL) | No. of Valid Replicates | No. of Detected Replicates | Detection Rate (%) |
| --- | --- | --- | --- | --- |
| B | 20 | 22 | 22 | 100.0 |
| | 10 | 23 | 23 | 100.0 |
| | 7 | 23 | 23 | 100.0 |
| C | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| D | 20 | 24 | 24 | 100.0 |
| | 10 | 22 | 22 | 100.0 |
| | 7 | 24 | 24 | 100.0 |
| E | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 23 | 95.8 |
| F | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 23 | 23 | 100.0 |
| G | 20 | 24 | 24 | 100.0 |
| | 10 | 24 | 24 | 100.0 |
| | 7 | 24 | 23 | 95.8 |
| H | 20 | 23 | 22 | 95.7 |
| | 10 | 24 | 24 | 100.0 |

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 9 of 50

{9}

| | 5 | 24 | 18 | 75.0 |
| --- | --- | --- | --- | --- |
| I | 20 | 22 | 22 | 100.0 |
| | 10 | 23 | 23 | 100.0 |
| | 7 | 22 | 20 | 90.9 |

# 3. Linear Range

The linearity of Alinity m HBV assay was assessed by testing a dilution series of HBV genotype A in negative human plasma and serum, each consisting of 9 panel levels spanning from 7 to 2,000,000,000 IU/mL (0.85 to 9.30 Log IU/mL). Two kinds of panel members were created: panel members with concentrations from 7 to 100,000 IU/mL (0.85 to 5.00 Log IU/mL) were prepared using an HBV positive clinical sample, while panel members with concentrations 100 to 2,000,000,000 IU/mL (2.00 to 9.30 Log IU/mL) were prepared using synthetic DNA. Panel quantitation values were traceable to the $3^{\text{rd}}$ WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques, (NIBSC code: 10/264).

There was no significant difference in the linearity between the clinical sample and the synthetic DNA based panel members. Similarly, there was no significant difference in the linearity between the plasma and serum (Figure 1). The difference in the predicted concentration between the fitted nonlinear model and the linear model was less than or equal to $0.50\mathrm{LogIU / mL}$ for each panel member. The Alinity m HBV assay demonstrated linearity in plasma and serum across the range tested 7 to 2,000,000,000 IU/mL (0.85 to 9.3 Log IU/mL) for HBV Genotype A. The results support plasma and serum linearity claim of 10 IU/mL (LLoQ) to 1,000,000,000 IU/mL (ULoQ) for Alinity m HBV assay.

Figure 1: Linearity of Plasma and Serum. The markers in the plot represent the mean (in Log IU/mL) Alinity m HBV assay concentration for each panel member.


PMA P200013 : FDA Summary of Safety and Effectiveness Data

{10}

# 4. Linearity Across HBV Genotypes

The Linearity of Alinity m HBV assay was assessed for HBV genotypes A, B, C, D, E, F, G, H, and I in plasma and serum. For each HBV genotype and each matrix (plasma and serum), linearity was evaluated by testing 9 panel members that spanned the intended dynamic range of the assay: levels spanning from 10 to 1,000,000,000 IU/mL (1.00 to 9.00 Log IU/mL). Two kinds of panels were created: a panel with levels with lower concentrations (10 IU/mL to 20,000 IU/mL) were prepared using either an HBV positive clinical sample or synthetic DNA, and a panel with levels with higher concentrations (10 IU/mL to 1,000,000,000 IU/mL) were prepared using synthetic DNA. Least squares linear regression analysis was performed for Genotypes separately.

The Alinity m HBV assay was linear in plasma and serum across the range of HBV DNA concentrations from 10 IU/mL (LLoQ) to 1,000,000,000 IU/mL (ULoQ) for genotypes A, B, C, D, E, F, G, H, and I. The results are shown in Figure 2 and Table 6.

Figure 2. Linearity Across HBV Genotypes in Plasma and Serum. The markers in the plot represent the mean (in Log IU/mL) Alinity m HBV assay concentration for each panel member


PMA P200013 : FDA Summary of Safety and Effectiveness Data

{11}

Table 6. Linear Fit Equations across Genotypes

| Genotype | Plasma | | Serum | |
| --- | --- | --- | --- | --- |
| | Linear Equation | Maximum Non-linearity (log IU/mL) | Linear Equation | Maximum Non-linearity (log IU/mL) |
| A | Y = 0.96X + 0.22 | 0.04 | Y = 0.95X + 0.35 | 0.05 |
| B | Y = 1.01X + 0.00 | 0.03 | Y = 1.03X + 0.07 | NA* |
| C | Y = 1.00X + 0.19 | NA* | Y = 0.96X + 0.20 | 0.08 |
| D | Y = 0.90X + 0.41 | 0.08 | Y = 0.91X + 0.23 | NA* |
| E | Y = 0.95X + 0.14 | NA* | Y = 0.97X + 0.14 | 0.05 |
| F | Y = 0.93X + 0.36 | 0.20 | Y = 0.89X + 0.23 | 0.18 |
| G | Y = 1.00X + 0.29 | NA* | Y = 0.97X + 0.14 | 0.07 |
| H | Y = 0.93X + 0.05 | 0.08 | Y = 0.94X + 0.08 | 0.08 |
| I | Y = 0.99X + 0.02 | NA* | Y = 0.99X + 0.37 | 0.04 |

*No 2nd/3rd order polynomial fit is statistically better than a linear fit at the significance level.

# 5. Lower Limit of Quantitation

The lower limit of quantitation (LLoQ) is defined as the lowest concentration at which HBV DNA is reliably quantitated within a total error. Total error was estimated by 2 methods: Total Analytical Error (TAE) = |bias| + 2 × SD, and Total Error (TE) = SQRT (2) × 2 × SD.

TAE and TE of Alinity m HBV assay for genotypes A, B, C, D, E, F, G, H, and I in plasma and serum were calculated for panel members with observed concentrations at or near 10 IU/mL (1.00 Log IU/mL) tested in multiple non-clinical studies, as shown in Table 7 and Table 8. The concentrations of the panel members were traceable to the 3rd WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques (NIBSC code: 10/264).

The results of these analyses demonstrated that Alinity m HBV assay can determine the concentration of HBV DNA for genotypes A, B, C, D, E, F, G, H, and I in plasma and serum at 10 IU/mL (1.00 Log IU/mL) with an acceptable level of accuracy and precision, i.e., TAE and TE less than or equal to 1.00 Log IU/mL. The LLoQ of the Alinity m HBV is 10 IU/ml.

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{12}

Table 7. Total Error for Plasma

| Study | Genotype | Target Conc. (Log IU/mL) | Mean Conc. (Log IU/mL) | Bias^{a} (Log IU/mL) | SD (Log IU/mL) | TAE (Log IU/mL) | TE (Log IU/mL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Limit of Detection | A | 1.00 | 1.07 | 0.07 | 0.25 | 0.57 | 0.71 |
| Genotype Limit of Detection | B | 1.00 | 1.18 | 0.18 | 0.20 | 0.58 | 0.57 |
| | C | 1.00 | 1.46 | 0.46 | 0.17 | 0.81 | 0.49 |
| | D | 1.00 | 1.24 | 0.24 | 0.24 | 0.72 | 0.67 |
| | E | 1.00 | 1.25 | 0.25 | 0.17 | 0.59 | 0.48 |
| | F | 1.00 | 1.63 | 0.63 | 0.14 | 0.91 | 0.40 |
| | G | 1.00 | 1.35 | 0.35 | 0.13 | 0.61 | 0.37 |
| | H | 1.00 | 0.98 | -0.02 | 0.29 | 0.59 | 0.82 |
| | I | 1.00 | 0.94 | -0.06 | 0.21 | 0.49 | 0.61 |
| Linearity | A | 1.00 | 1.19 | 0.19 | 0.23 | 0.65 | 0.65 |
| Genotype Linearity | B | 1.00 | 0.97 | -0.03 | 0.22 | 0.46 | 0.61 |
| | C | 1.00 | 1.18 | 0.18 | 0.31 | 0.79 | 0.87 |
| | D | 1.00 | 1.21 | 0.21 | 0.32 | 0.85 | 0.91 |
| | E | 1.00 | 1.11 | 0.11 | 0.18 | 0.47 | 0.50 |
| | F | 1.00 | 1.28 | 0.28 | 0.32 | 0.93 | 0.91 |
| | G | 1.00 | 1.29 | 0.29 | 0.12 | 0.53 | 0.35 |
| | H | 1.00 | 0.99 | -0.01 | 0.32 | 0.66 | 0.92 |
| | I | 1.00 | 1.00 | -0.00 | 0.28 | 0.57 | 0.80 |
| Precision | C | 1.00 | 1.23 | 0.23 | 0.27 | 0.76 | 0.75 |

$^a$ Bias = Mean Concentration – Target Concentration.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 13 of 50

{13}

Table 8. Total Error for Serum

| Study | Genotype | Target Conc. (Log IU/mL) | Mean Conc. (Log IU/mL) | \( Bias^a \) (Log IU/mL) | SD (Log IU/mL) | TAE (Log IU/mL) | TE (Log IU/mL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Limit of Detection | A | 1.00 | 0.96 | -0.04 | 0.26 | 0.56 | 0.72 |
| Genotype Limit of Detection | B | 1.00 | 1.19 | 0.19 | 0.20 | 0.59 | 0.57 |
| | C | 1.00 | 1.26 | 0.26 | 0.22 | 0.70 | 0.63 |
| | D | 1.00 | 1.19 | 0.19 | 0.19 | 0.57 | 0.54 |
| | E | 1.00 | 1.21 | 0.21 | 0.23 | 0.67 | 0.65 |
| | F | 1.00 | 1.48 | 0.48 | 0.20 | 0.87 | 0.56 |
| | G | 1.00 | 1.67 | 0.67 | 0.13 | 0.93 | 0.36 |
| | H | 1.00 | 1.14 | 0.14 | 0.20 | 0.55 | 0.58 |
| | I | 1.00 | 1.16 | 0.16 | 0.20 | 0.56 | 0.56 |
| Linearity | A | 1.00 | 1.30 | 0.30 | 0.17 | 0.65 | 0.49 |
| Genotype Linearity | B | 1.00 | 1.12 | 0.12 | 0.20 | 0.52 | 0.57 |
| | C | 1.00 | 1.24 | 0.24 | 0.22 | 0.69 | 0.63 |
| | D | 1.00 | 1.23 | 0.23 | 0.27 | 0.76 | 0.76 |
| | E | 1.00 | 0.99 | -0.01 | 0.34 | 0.68 | 0.95 |
| | F | 1.00 | 1.10 | 0.10 | 0.26 | 0.63 | 0.75 |
| | G | 1.00 | 1.09 | 0.09 | 0.21 | 0.52 | 0.60 |
| | H | 1.00 | 1.13 | 0.13 | 0.25 | 0.63 | 0.70 |
| | I | 1.00 | 1.34 | 0.34 | 0.18 | 0.69 | 0.50 |
| Precision | C | 1.00 | 1.19 | 0.19 | 0.28 | 0.76 | 0.80 |

${}^{a}$ Bias $=$ Mean Concentration- Target Concentration.

# 6. Traceability to the WHO Standard

Primary calibrators and assay calibrators with known concentrations were used throughout product development and product manufacturing to establish traceability to the 3rd World Health Organization (WHO) International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques (NIBSC code: 10/264; genotype A). The concentrations tested for the WHO standard were 1.93, 2.93, and 3.93 Log IU/mL. The concentrations tested for the primary calibrators ranged from 2.53 to 6.42 Log IU/mL. The Alinity m HBV assay calibrators and controls were also tested along with the primary calibrators and the WHO standard. All of the panels had

PMA P200013: FDA Summary of Safety and Effectiveness Data

{14}

observed HBV concentrations similar to the target concentrations and were linear across the assay's quantitation range. The results are shown in Figure 3.

Figure 3. Traceability to the WHO Standard

# 7. Precision

The Alinity m HBV assay variability was assessed in an internal precision study (serum and plasma) and the external reproducibility study using plasma on the Alinity m system at 3 external sites with three reagent lots.

# a. Internal (within laboratory) Precision Study

The precision of the Alinity m HBV assay was determined by analyzing an 8-member plasma panel and an 8-member serum panel. Panel members spanning a range from 1 to 5 Log IU/mL (10 to 100,000 IU/mL) were prepared using an HBV positive genotype C sample, while panel members with concentrations greater than 5 Log IU/mL (100,000 IU/mL) were prepared using synthetic DNA for HBV genotype C. Each panel member was tested in 4 replicates, twice each day for 12 days, on 3 Alinity m Systems with 1 Alinity m AMP Kit operated by 3 operators (one operator per instrument), for a total of 288 replicates per panel member.

The study results for Alinity m HBV assay precision in plasma and serum are shown in Table 9 and Table 10. The analyses demonstrated that the Alinity m HBV assay has a within-laboratory standard deviation (SD) was less than or equal to $0.25\mathrm{LogIU / mL}$ for HBV DNA from 2 to 9 Log

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{15}

IU/mL (100 to 1,000,000,000 IU/mL), and less than or equal to 0.35 Log IU/mL near the LLoQ (1.00 to 1.48 Log IU/mL or 10 to 30 IU/mL).

Table 9. Precision in Plasma

| Panel Member | Na | Mean Conc (Log IU/mL) | Within-Run Component | | Between-Run Component | | Between-Day Component | | Within-Laboratoryb | | Between-Instrument/Operator Component | | Totalc | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 08 | 284 | 8.87 | 0.09 | 1.0 | 0.04 | 0.5 | 0.04 | 0.4 | 0.10 | 1.2 | 0.09 | 1.0 | 0.14 | 1.5 |
| 07 | 282 | 7.66 | 0.06 | 0.8 | 0.02 | 0.2 | 0.03 | 0.4 | 0.07 | 0.9 | 0.06 | 0.8 | 0.10 | 1.3 |
| 06 | 283 | 6.53 | 0.07 | 1.1 | 0.02 | 0.3 | 0.04 | 0.5 | 0.08 | 1.3 | 0.05 | 0.8 | 0.10 | 1.5 |
| 05 | 283 | 5.03 | 0.07 | 1.4 | 0.02 | 0.5 | 0.03 | 0.7 | 0.08 | 1.7 | 0.02 | 0.4 | 0.09 | 1.7 |
| 04 | 285 | 3.38 | 0.08 | 2.4 | 0.04 | 1.1 | 0.05 | 1.6 | 0.10 | 3.1 | 0.04 | 1.3 | 0.11 | 3.3 |
| 03 | 277 | 2.26 | 0.12 | 5.3 | 0.03 | 1.2 | 0.08 | 3.5 | 0.15 | 6.5 | 0.10 | 4.3 | 0.18 | 7.8 |
| 02 | 271 | 1.76 | 0.16 | 8.8 | 0.00 | 0.0 | 0.09 | 5.0 | 0.18 | 10.2 | 0.11 | 6.3 | 0.21 | 11.9 |
| 01 | 278 | 1.23 | 0.23 | 18.6 | 0.09 | 6.9 | 0.11 | 8.6 | 0.27 | 21.6 | 0.13 | 10.3 | 0.29 | 24.0 |

${}^{a}$ Number of valid replicates.
b Within-Laboratory includes Within-Run, Between-Run, and Between-Day components.
Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Operator components.

Table 10. Precision in Serum

| Panel Member | Na | Mean Conc (Log IU/mL) | Within-Run Component | | Between-Run Component | | Between-Day Component | | Within-Laboratoryb | | Between-Instrument/Operator Component | | Totalc | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 08 | 284 | 8.82 | 0.09 | 1.0 | 0.02 | 0.2 | 0.05 | 0.6 | 0.10 | 1.2 | 0.13 | 1.5 | 0.17 | 1.9 |
| 07 | 284 | 7.73 | 0.08 | 1.0 | 0.03 | 0.4 | 0.00 | 0.0 | 0.08 | 1.1 | 0.08 | 1.0 | 0.12 | 1.5 |
| 06 | 285 | 6.49 | 0.08 | 1.2 | 0.00 | 0.0 | 0.00 | 0.0 | 0.08 | 1.2 | 0.06 | 0.9 | 0.10 | 1.5 |
| 05 | 287 | 4.97 | 0.08 | 1.6 | 0.02 | 0.3 | 0.01 | 0.3 | 0.08 | 1.7 | 0.05 | 1.1 | 0.10 | 2.0 |
| 04 | 283 | 3.32 | 0.09 | 2.6 | 0.06 | 1.8 | 0.07 | 2.2 | 0.13 | 3.8 | 0.08 | 2.4 | 0.15 | 4.5 |
| 03 | 279 | 2.19 | 0.12 | 5.5 | 0.00 | 0.0 | 0.09 | 3.9 | 0.15 | 6.8 | 0.07 | 3.3 | 0.17 | 7.5 |
| 02 | 282 | 1.71 | 0.16 | 9.3 | 0.06 | 3.8 | 0.07 | 4.2 | 0.19 | 10.9 | 0.11 | 6.5 | 0.22 | 12.7 |
| 01 | 286 | 1.19 | 0.25 | 20.9 | 0.04 | 3.4 | 0.13 | 10.6 | 0.28 | 23.7 | 0.10 | 8.3 | 0.30 | 25.1 |

${}^{a}$ Number of valid replicates.
b Within-Laboratory includes Within-Run, Between-Run, and Between-Day components.
Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Operator components.

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{16}

b. Multi-site (external) Reproducibility Study

The reproducibility study was performed at three external U.S sites. Nine unique panel members for Genotype A and nine unique panel members for Genotype C covering the quantitation range of the assay (1 to 8.5 Log IU/mL) were formulated in plasma to make an 18-member panel. Each panel member was tested 5 times per test run. The Alinity m System is a random-access analyzer, therefore a run is defined as testing a batch of 5 replicates of each of the 18 panel members consecutively on the system within a day using the same Alinity m HBV assay reagent lots. A total of 3 Alinity m HBV AMP Kit lots were used. Each of the 3 clinical sites tested 2 Alinity m HBV AMP Kit lots, on 5 non-consecutive days for each lot. Each of the 3 clinical sites used a unique lot of Alinity m HBV CAL Kit, Alinity m HBV CTRL Kit, and Alinity m Sample Prep Kit 2. The study design (3 sites x 5 replicates in one-panel x 1 run/day x 5 days x 2 lots/site) accounts for a total of 150 replicates per panel member. The analyses demonstrated that the Alinity m HBV assay standard deviation (SD) was less than or equal to 0.25 Log IU/mL for HBV DNA from 2.29 to 7.98 Log IU/mL, and for near LLoQ to 3x LLoQ SD was less than or equal to 0.35 Log IU/mL. The precision/reproducibility results are acceptable, and the results are summarized in Table 11.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 17 of 50

{17}

Table 11. Reproducibility of Alinity m HBV assay

| Geno-type | Na | Mean Conc.(Log IU/mL) | Within-Run/Day Componenta | | Between-Run/Day Componenta | | Within-Laboratoryb | | Between-Lot Component | | Between-Site Component | | Totalc | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SDd | %CV | SDd | %CV | SDd | %CV | SDd | %CV | SDd | %CV | SDd | %CV |
| A | 150 | 7.98 | 0.11 | 1.4 | 0.02 | 0.2 | 0.11 | 1.4 | 0.05 | 0.7 | 0.04 | 0.5 | 0.13 | 1.6 |
| | 150 | 6.44 | 0.10 | 1.6 | 0.05 | 0.7 | 0.11 | 1.8 | 0.00 | 0.0 | 0.04 | 0.7 | 0.12 | 1.9 |
| | 150 | 5.11 | 0.13 | 2.6 | 0.08 | 1.6 | 0.16 | 3.1 | 0.06 | 1.2 | 0.21 | 4.0 | 0.27 | 5.2 |
| | 150 | 4.15 | 0.12 | 2.8 | 0.08 | 2.0 | 0.14 | 3.5 | 0.04 | 0.9 | 0.12 | 2.9 | 0.19 | 4.6 |
| | 150 | 3.17 | 0.14 | 4.4 | 0.08 | 2.4 | 0.16 | 5.0 | 0.04 | 1.3 | 0.15 | 4.6 | 0.22 | 6.9 |
| | 150 | 2.29 | 0.15 | 6.5 | 0.08 | 3.3 | 0.17 | 7.3 | 0.06 | 2.6 | 0.16 | 7.1 | 0.24 | 10.5 |
| | 150 | 1.74 | 0.22 | 12.8 | 0.09 | 5.0 | 0.24 | 13.7 | 0.00 | 0.0 | 0.14 | 7.8 | 0.28 | 15.8 |
| | 146 | 1.05 | 0.35 | 33.7 | 0.00 | 0.0 | 0.35 | 33.7 | 0.06 | 5.9 | 0.20 | 19.1 | 0.41 | 39.1 |
| | 146 | 1.09 | 0.27 | 24.8 | 0.00 | 0.0 | 0.27 | 24.8 | 0.09 | 8.5 | 0.12 | 10.6 | 0.31 | 28.3 |
| B | 150 | 7.96 | 0.11 | 1.4 | 0.02 | 0.2 | 0.11 | 1.4 | 0.04 | 0.5 | 0.11 | 1.4 | 0.16 | 2.0 |
| | 150 | 6.36 | 0.12 | 1.9 | 0.00 | 0.0 | 0.12 | 1.9 | 0.04 | 0.7 | 0.05 | 0.9 | 0.14 | 2.2 |
| | 150 | 5.03 | 0.13 | 2.7 | 0.06 | 1.3 | 0.15 | 3.0 | 0.00 | 0.0 | 0.06 | 1.1 | 0.16 | 3.2 |
| | 150 | 4.12 | 0.12 | 2.9 | 0.04 | 1.0 | 0.12 | 3.0 | 0.00 | 0.0 | 0.04 | 0.9 | 0.13 | 3.2 |
| | 150 | 3.13 | 0.11 | 3.5 | 0.03 | 1.0 | 0.11 | 3.6 | 0.00 | 0.0 | 0.07 | 2.1 | 0.13 | 4.2 |
| | 150 | 2.27 | 0.12 | 5.3 | 0.01 | 0.6 | 0.12 | 5.3 | 0.04 | 1.6 | 0.10 | 4.2 | 0.16 | 6.9 |
| | 150 | 1.82 | 0.16 | 8.5 | 0.06 | 3.5 | 0.17 | 9.2 | 0.00 | 0.0 | 0.12 | 6.6 | 0.21 | 11.3 |
| | 150 | 1.17 | 0.19 | 16.1 | 0.04 | 3.8 | 0.19 | 16.5 | 0.00 | 0.0 | 0.17 | 14.5 | 0.26 | 22.0 |
| | 141 | 0.85 | 0.30 | 35.7 | 0.08 | 9.3 | 0.31 | 36.9 | 0.00 | 0.0 | 0.20 | 23.0 | 0.37 | 43.5 |

${}^{a}$ Number of valid replicates.
b Within-Laboratory includes Within-Run, and Between-Run Components.
Total includes Within-Run, Between-Run, Between-Lot, and Between-Site Variance Components.
${}^{d}SD =$ Standard deviations in $\operatorname{Log}{IU}/{mL}$

# 8. Performance with HBV-Negative Specimens

The analytical specificity of Alinity m HBV assay was determined by testing 250 HBV-negative plasma and 250 HBV-negative serum specimens (as determined by an FDA approved HBV Ag/Ab screening, and HBsAg Confirmatory assay) from individual donors. HBV DNA was not detected in any of the 500 specimens tested (specificity $100.0\%$ ; $95\%$ CI: 99.2 to $100.0\%$ ).

# 9. Cross-Reactivity of the Alinity m HBV assay with Other Microorganisms

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{18}

The impact of potential cross-reactivity and/or interference of pathogens in the Alinity m HBV assay was evaluated. The study was designed according to CLSI EP7-A2. HBV-negative or positive plasma specimens were spiked with microorganisms or purified nucleic acid from microorganisms to achieve a final titer of $10^{5}$ units/mL for viruses, protozoans, and yeast or $10^{6}$ CFU/mL for bacteria (Table 12). Cross-reactivity was analyzed using an HBV negative sample and microbial interference was analyzed using an HBV-positive sample at 30 IU/mL (3x LLoQ) and at 2000 IU/mL. Three replicates for each cross reactant were tested.

No cross-reactivity or microbial interference was observed in the presence of the tested microorganisms.

Table 12. Microorganisms

| Viruses | Viruses |
| --- | --- |
| Adenovirus Type 5 (AV5) | Japanese Encephalitis |
| BK Polyomavirus | Murray Valley Encephalitis Virus |
| Dengue Virus 1 (DENV 1) | Parvo Virus B19 |
| Dengue Virus 2 (DENV 2) | Rubella Virus |
| Dengue Virus 3 (DENV 3) | St. Louis Encephalitis |
| Dengue Virus 4 (DENV 4) | Vaccinia Virus (VACV) |
| FSME Virus | Varicella-Zoster Virus (VZV) |
| GB Virus C (GBV-C: Hepatitis G Virus, HGV) | West Nile Virus (WNV) |
| Hepatitis A Virus (HAV) | Yellow Fever Virus |
| Hepatitis C Virus (HCV) | Zika Virus |
| Hepatitis D Virus (HDV) | |
| Human Herpesvirus 1 (HHV-1)/ | Bacteria |
| Herpes Simplex Virus 1 (HSV-1) | Chlamydia trachomatis |
| Human Herpesvirus 2 (HHV-2)/ | Corynebacterium diphtheriae |
| Herpes Simplex Virus 2 (HSV-2) | Mycobacterium gordonae |
| Human Herpesvirus 5 (HHV-5)/ | Mycobacterium smegmatis |
| Human Cytomegalovirus (CMV) | Neisseria gonorrhoeae |
| Human Herpesvirus 4 (HHV-4)/ | Propionibacterium acnes |
| Epstein Barr Virus (EBV) | Staphylococcus aureus |
| Human Herpesvirus 6B (HHV-6B) | Staphylococcus epidermidis |
| Human Herpesvirus 8 (HHV8)/ | Streptococcus pneumoniae |
| Kaposi Sarcoma Virus | Protozoan |
| Human Immunodeficiency Virus 2 (HIV-2) | Trichomonas vaginalis |
| Human Immunodeficiency Virus 1 (HIV-1) | |
| Human Papilloma Virus 16 (HPV-16) | Yeast |
| Human Papilloma Virus 18 (HPV-18) | |
| Human T-Lymphotropic Virus 1 (HTLV-1) | Candida albicans |
| Human T-Lymphotropic Virus 2 (HTLV-2) | |
| Influenza A | |

# a. Potentially Interfering Substances (Endogenous)

The impact of potentially interfering endogenous substances, the presence of autoimmune disorders, and markers of other diseases on the analytical specificity and detection/quantitation of the Alinity m HBV assay was evaluated by testing spiked samples as well as patient samples with

PMA P200013: FDA Summary of Safety and Effectiveness Data

{19}

naturally elevated levels of endogenous substances. Ten donor samples were tested for each interfering substance with one replicate each. As a control, one replicate of each donor sample was also tested without the addition of any potentially interfering endogenous substance. Potential interference was assessed by testing 10 HBV negative samples, and 10 positive samples containing 30 or 2,000 IU/mL HBV DNA, except hepatocellular carcinoma (HCC), for which 4 samples were tested at each HBV concentration.

No interference was observed in the presence of albumin (60 mg/mL), hemoglobin (2 mg/mL), triglycerides (37 mM), conjugated bilirubin (0.342 mM), unconjugated bilirubin (0.342 mM) or human genomic DNA (2 mg/L) that were introduced in the sample. In addition, no interference was observed in specimens collected from individual donors containing the naturally elevated interfering substances, albumin (&gt;5.1 g/dL), bilirubin (&gt;2 mg/dL), hemoglobin (&gt;2 g/L) or triglycerides (&gt;325 mg/dL).

No interference was observed for specimens collected from patients with the following disease states: systemic lupus erythematosus (SLE), anti-nuclear antibodies (ANA), rheumatoid factor (RF), alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis, auto-immune hepatitis, and hepatocellular carcinoma (HCC). In addition, no interference was observed for specimens collected from patients that have received Influenza and Hepatitis B vaccines.

## b. Potentially Interfering Substances (Exogenous)

The impact of potentially interfering drugs commonly prescribed for the treatment of HBV and other related disease states on the performance of Alinity m HBV assay was evaluated. Ten donor samples were tested for each interfering drug pool or single drug with one replicate each. As a control, one replicate of each donor sample was also tested without the addition of any potentially interfering drug compounds. Potential interference was assessed by testing 10 HBV-negative samples, and 10 positive samples containing 30 (3x LLoQ) or 2,000 IU/mL HBV DNA. The drug compounds listed in Table 13 were tested at three times the reported maximum concentration (Cmax) evaluated with and without HBV viral targets.

No interference was observed in the presence of drug compounds tested in pools at a concentration of 3 times the reported Cmax or higher.

PMA P200013: FDA Summary of Safety and Effectiveness Data
Page 20 of 50

{20}

Table 13. Drug Compounds

| Pools Tested | Drug Compounds |
| --- | --- |
| 1 | Abacavir sulfate, Acetaminophen, Acyclovir, Adefovir, Amitriptyline, Amlodipine, Aspirin, Atazanavir, Atenolol, Atorvastatin, Azithromycin, Celecoxib, Cidofovir, Clarithromycin, Clopidogrel |
| 2 | Didanosine, Efavirenz, Entecavir, Fluconazole, Fluoxetine, Ibuprofen, Indinavir, Kaletra (Lopinavir and Ritonavir), Lamivudine, Levofloxacin, Maraviroc, Nelfinavir, Nevirapine, Paroxetine |
| 3 | Prednisone, Raltegravir, Ribavirin, Rifamate (Rifampin and Isoniazid), Saquinavir, Sertraline, Stavudine, Stribild (Elvitegravir, Cobicistat, Emtricitabine, and Tenofovir), Bactrim (Sulfamethoxazole and Trimethoprim) |
| 4 | Darunavir, Ethambutol, Etravirine, Flucytosine, Fluticasone propionate / Salmeterol xinafoate, Furosemide, Hydrochlorothiazide, Levothyroxine, Rifabutin, Rilpivirine, Simeprevir, Sofosbuvir, Telaprevir, Tenofovir alafenamide, Trazodone, Warfarin, Zalcitabine |
| 5 | Fosamprenavir, Keflex (Cephalexin), Metformin, Naproxen, Pyrazinamide |
| 6 | Tipranavir |
| 7 | Ceftriaxone, Ciprofloxacin, Foscarnet, Lisinopril, Peginterferon alfa-2a, Enfuvirtide, Imipramine |
| 8 | Cyclosporine, Telbivudine, Valacyclovir, Valganciclovir, Zidovudine, Amphotericin B, Ganciclovir |
| 9 | Acetaminophen, Hydrocodone |
| 10 | Biotin |

10. Carryover

The carryover rate for Alinity m HBV assay was determined by analyzing 360 valid replicates of HBV negative samples processed from alternating positions with 360 valid replicates of high concentrated HBV-positive samples at 100,000,000 IU/mL, across a total of 17 runs. HBV DNA was not detected in any of the HBV- negative samples, resulting in an overall carryover rate of 0.0% (95% CI: 0.0 to 1.1%).

11. Matrix Equivalency

a. Serum/Plasma Equivalency Across the Linear Range

Serum/Plasma matrix equivalency in the Alinity m HBV assay was evaluated by analyzing 30 negative plasma and serum pairs and 53 positive plasma and serum pairs. Negative plasma and serum samples in each pair were collected from the same normal donor (individuals that report no history of HIV or liver disease, such as hepatitis). The positive pairs were prepared by spiking high titer virus (for &lt;5 Log IU/mL samples) or synthetic DNA (for &gt;5 Log IU/mL samples) in serum and plasma matched pairs collected from normal donors. The HBV DNA concentrations for the HBV positive serum/plasma pairs were distributed across the quantitation range of the assay, with the lowest concentration at 1.40 Log IU/mL and the highest concentration at 9.14 Log IU/mL. HBV genotypes A, B, C, D, E, F, G, H, and I were included in the positive samples.

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{21}

Figure 4: Least Squares Regression Plot with Genotype (Outliers included)

All HBV negative plasma and serum samples were not detected, and all HBV positive plasma and serum samples were detected, resulting in an overall percent agreement between plasma and serum samples of $100.0\%$ (95% CI: 95.6 to $100.0\%$ ).

The least-squares regression analysis of samples including genotypes between serum and plasma demonstrated that the Alinity m HBV Assay has an equivalent performance in reporting quantitative results with serum and plasma since the regression demonstrated a slope of 1.0, intercept of 0.1, $r = 1.0$ , and mean bias of 0.03 Log IU/mL when compared to the results in serum (Figure 4).

# b. Specimen and Collection Tube Type Equivalency

To demonstrate equivalent performance between plasma and serum collection tube types, 25 matched sets of HBV negative and positive samples were obtained in each of the following sample collection tubes and evaluated using the Alinity m HBV Assay. For testing, plasma samples were collected in either Di-Potassium Ethylenediaminetetraacetic Acid (K2 EDTA) tubes, Tri-Potassium EDTA (K3 EDTA) tubes, Acid Citrate Dextrose (ACD) tubes, Plasma Preparation Tubes (PPT), and serum samples were collected in serum tubes, Serum Separator Tubes (SST), and serum rapid-clot tubes (both Z-clot and thrombin-clot). For each tube type, an HBV-positive clinical sample was prepared at a concentration of $30\mathrm{IU / mL}$ (3x LLoQ) or 2000 $\mathrm{IU / mL}$ . The paired difference in concentration between the test condition (sample collection tubes except for K2 EDTA tube) and control condition (K2 EDTA tube) was calculated for each donor. The mean and SD of the paired difference were calculated for each test condition.

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{22}

Plasma (K2 EDTA, K3 EDTA, ACD, and PPT) and serum (serum and SST) collection tubes demonstrated a $100\%$ Not Detected interpretation for HBV-negative samples for each tube type. $100\%$ of the Positive samples were detected at 30 IU/mL (3x LLoQ) and at 2000 IU/mL. For the 30 IU/mL samples' quantification, the mean difference between the test condition and the K2 EDTA control condition across tube types ranged from -0.08 to 0.16 Log IU/mL. For the 2000 IU/mL samples, the $95\%$ confidence interval of the mean difference between each test condition and the K2 EDTA control condition was within [-0.50, 0.50] Log IU/mL with a maximum standard deviation of 0.18. The largest mean difference between the test condition and the control serum tube across all tube types was 0.22 Log IU/mL. The results are summarized in Table 14 below. The matrix and sample collection tube equivalence studies demonstrated acceptable performance.

Table 14: HBV Positive samples tube equivalency summary

| Panel | Collection Tube Typea | N | Mean Differenceb (Log IU/mL) | SD | 95% CI of the Mean Difference |
| --- | --- | --- | --- | --- | --- |
| Low 30 IU/mL | K3 EDTA | 25 | -0.08 | 0.236 | N/P |
| | ACD Plasma Tube | 25 | 0.12 | 0.267 | N/P |
| | Plasma Preparation Tube (PPT) | 25 | -0.03 | 0.183 | N/P |
| | Serum Tube | 25 | 0.16 | 0.216 | N/P |
| | Serum Separator Tube (SST) | 25 | 0.09 | 0.229 | N/P |
| | Serum Rapid-clot Tube (Z-clot) | 25 | 0.10 | 0.209 | N/P |
| | Serum Rapid-clot tube (thrombin-clot) | 25 | 0.10 | 0.184 | N/P |
| High 2000 IU/mL | K3 EDTA | 25 | -0.00 | 0.135 | (-0.06, 0.05) |
| | ACD Plasma Tube | 25 | 0.06 | 0.144 | (-0.00, 0.12) |
| | Plasma Preparation Tube (PPT) | 25 | -0.00 | 0.108 | (-0.05, 0.04) |
| | Serum Tube | 25 | 0.04 | 0.160 | (-0.03, 0.10) |
| | Serum Separator Tube (SST) | 25 | 0.03 | 0.136 | (-0.03, 0.08) |
| | Serum Rapid-clot Tube (Z-clot) | 25 | 0.04 | 0.181 | (-0.04, 0.11) |
| | Serum Rapid-clot tube (thrombin-clot) | 25 | 0.02 | 0.131 | (-0.04, 0.07) |

${}^{a}$ Twenty-five samples from K2 EDTA tubes were used as Control
${}^{b}$ Test condition - control condition, $N/P$ : Not provided

# 12. Alinity m HBV assay Testing Using Dilution Procedure

# a. Quantitation of Manually Diluted Specimens

The Alinity m HBV assay design provides optional manual dilution procedures for low volume or high viral load specimens (upper limit of quantification). To verify that the Alinity m HBV assay provides accurate quantitation, dilution procedures were evaluated by comparing quantitation of neat specimens and specimens tested using the Alinity m HBV assay dilution procedure of 1:2.5

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{23}

and 1:50. Specimens were diluted using Alinity m Specimen Dilution Kit I. Ten plasma panel members and 10 serum panel members, consisting of HBV concentrations ranging from 75 to 2,000,000,000 IU/mL. Panel members were prepared by spiking unique HBV negative plasma and serum specimens with a high titer HBV clinical specimen or synthetic HBV DNA stock, to a final target concentration ranging from 1.88 Log IU/mL (75 IU/mL) to 9.30 Log IU/mL (2,000,000,000 IU/mL). Synthetic HBV DNA stock was only used for panel members with high concentrations ( $\geq$ 5 Log IU/mL). Panel members were then manually diluted in the Specimen Diluent provided in the Alinity m Specimen Dilution Kit. Dilutions of 1:2.5 and 1:50 were performed according to the instructions for use. For each HBV panel member, 5 undiluted replicates and 5 replicates diluted in Specimen Diluent were tested and compared. Results are summarized in Table 15A and Table 15B.

For the 10 plasma panel members, the differences between the mean of the diluted samples and the mean of the neat samples ranged from -0.15 to 0.09 Log IU/mL for the 1:2.5 dilution, and from -0.12 to 0.14 Log IU/mL for the 1:50 dilution. For the 10 serum panel members, the differences between the mean of the diluted samples and the mean of the neat samples ranged from -0.04 to 0.27 Log IU/mL for the 1:2.5 dilution, and from -0.18 to 0.13 Log IU/mL for the 1:50 dilution. The observed differences in viral load quantitation between the replicates of the diluted and undiluted samples are negligible (&lt;0.3 Log IU/mL) and clinically insignificant. This study demonstrates that the Alinity m HBV assay can accurately quantify specimens diluted 1:2.5 and 1:50 as recommended in the package insert.

Table 15A: Quantitation of Manual Diluted Plasma Samples

| Specimen Type | Dilution Factor | Panel | Test Condition (Diluted) | | | Control Condition (Undiluted) | | | Mean Difference (Test - Control) | 95% CI of the Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | N | Mean (Log IU/mL) | SD | N | Mean (Log IU/mL) | SD | | |
| Plasma | 2.5 | 01 | 5 | 2.07 | 0.133 | 5 | 2.22 | 0.256 | -0.15 | (-0.45, 0.15) |
| | | 02 | 5 | 2.25 | 0.070 | 5 | 2.39 | 0.090 | -0.14 | (-0.26, -0.02) |
| | | 03 | 5 | 3.51 | 0.145 | 4 | 3.50 | 0.066 | 0.02 | (-0.17, 0.20) |
| | | 04 | 5 | 4.30 | 0.037 | 5 | 4.22 | 0.049 | 0.09 | (0.02, 0.15) |
| | | 05 | 5 | 4.56 | 0.072 | 5 | 4.52 | 0.077 | 0.04 | (-0.07, 0.15) |
| | | 06 | 5 | 5.33 | 0.049 | 5 | 5.40 | 0.069 | -0.07 | (-0.16, 0.02) |
| | | 07 | 5 | 6.37 | 0.084 | 4 | 6.33 | 0.037 | 0.04 | (-0.07, 0.15) |
| | | 08 | 5 | 7.36 | 0.059 | 5 | 7.30 | 0.049 | 0.06 | (-0.02, 0.14) |
| | | 09 | 5 | 8.38 | 0.038 | 5 | 8.29 | 0.030 | 0.09 | (0.04, 0.14) |
| | | 10 | 4 | 8.62 | 0.070 | 5 | 8.63 | 0.038 | -0.01 | (-0.10, 0.08) |

PMA P200013: FDA Summary of Safety and Effectiveness Data
Page 24 of 50

{24}

Table 15B: Quantitation of Manual Diluted Serum Samples

| Specimen Type | Dilution Factor | Panel | Test Condition (Diluted) | | | Control Condition (Undiluted) | | | Mean Difference (Test - Control) | 95% CI of the Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | N | Mean (Log IU/mL) | SD | N | Mean (Log IU/mL) | SD | | |
| Plasma | 50 | 03 | 5 | 3.42 | 0.124 | 4 | 3.50 | 0.066 | -0.07 | (-0.23, 0.09) |
| | | 04 | 5 | 4.15 | 0.058 | 5 | 4.22 | 0.049 | -0.07 | (-0.14, 0.01) |
| | | 05 | 5 | 4.40 | 0.044 | 5 | 4.52 | 0.077 | -0.12 | (-0.21, -0.03) |
| | | 06 | 5 | 5.53 | 0.080 | 5 | 5.40 | 0.069 | 0.13 | (0.02, 0.23) |
| | | 07 | 5 | 6.33 | 0.079 | 4 | 6.33 | 0.037 | 0.01 | (-0.09, 0.11) |
| | | 08 | 5 | 7.45 | 0.074 | 5 | 7.30 | 0.049 | 0.14 | (0.05, 0.23) |
| | | 09 | 5 | 8.37 | 0.054 | 5 | 8.29 | 0.030 | 0.08 | (0.02, 0.15) |
| | | 10 | 5 | 8.76 | 0.073 | 5 | 8.63 | 0.038 | 0.13 | (0.04, 0.22) |
| | | 11 | 5 | 9.42 | 0.090 | 5 | 9.46 | 0.064 | -0.04 | (-0.15, 0.08) |
| | | 12 | 5 | 9.67 | 0.011 | 5 | 9.77 | 0.077 | -0.10 | (-0.20, -0.01) |

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{25}

| Specimen Type | Dilution Factor | Panel | Test Condition (Diluted) | | | Control Condition (Undiluted) | | | Mean Difference(Test - Control) | 95% CI of the Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | N | Mean (Log IU/mL) | SD | N | Mean (Log IU/mL) | SD | | |
| Serum | 50 | 03 | 5 | 3.34 | 0.182 | 5 | 3.34 | 0.078 | -0.01 | (-0.21, 0.20) |
| | | 04 | 5 | 4.12 | 0.075 | 5 | 4.30 | 0.078 | -0.18 | (-0.29, -0.07) |
| | | 05 | 5 | 4.53 | 0.089 | 5 | 4.64 | 0.108 | -0.11 | (-0.26, 0.03) |
| | | 06 | 5 | 5.30 | 0.063 | 5 | 5.27 | 0.109 | 0.02 | (-0.11, 0.15) |
| | | 07 | 5 | 6.22 | 0.056 | 5 | 6.25 | 0.075 | -0.02 | (-0.12, 0.07) |
| | | 08 | 5 | 7.34 | 0.026 | 4 | 7.35 | 0.059 | -0.00 | (-0.07, 0.06) |
| | | 09 | 5 | 8.35 | 0.054 | 5 | 8.22 | 0.091 | 0.13 | (0.02, 0.23) |
| | | 10 | 5 | 8.58 | 0.076 | 4 | 8.51 | 0.063 | 0.07 | (-0.04, 0.18) |
| | | 11 | 5 | 9.32 | 0.064 | 5 | 9.45 | 0.039 | -0.13 | (-0.21, -0.05) |
| | | 12 | 5 | 9.51 | 0.101 | 5 | 9.66 | 0.104 | -0.15 | (-0.30, 0.00) |

# b. On-Board Stability of Diluted Specimens

The onboard/off-board stability of the diluted sample was evaluated in both serum and plasma. For each dilution factor, 12 aliquots of plasma and serum panel members ( $\sim$ 20,000 IU/mL) were prepared and stored frozen. The design of this study is as follows. Two test conditions were performed. For Test condition (X1) 4 aliquots were thawed and placed onboard the Alinity m System for a minimum of 4 hours, then diluted in Specimen Diluent, placed off-board (i.e., at $15^{\circ}\mathrm{C}$ to $30^{\circ}\mathrm{C}$ ) for a minimum of 2 hours, and placed onboard for a minimum of another 4 hours before testing. For Test condition (X2), 4 aliquots per panel member were thawed and placed onboard for a minimum of 4 hours, then diluted in Specimen Diluent and placed onboard for a minimum of another 4 hours before testing. For the undiluted control condition (X3), 4 aliquots were thawed once the X1 aliquots had already been incubating for a minimum of 6 hours and X3 was placed onboard for a minimum of 4 hours before testing. All conditions for each dilution factor were tested in the same run. The results from both test conditions X1 and X2 were compared to the results from the control condition X3.

The difference between the mean quantitation results from testing diluted specimens with onboard/off-board storage (test conditions X1 and X2) and the mean quantitation results from testing undiluted specimens (X3 control condition) across all panel members, dilution factors and matrices ranged from -0.10 Log IU/mL to +0.03 Log IU/mL. The data demonstrated that serum and plasma specimens diluted in specimen diluent may be stored onboard the Alinity m System for 4 hours before testing with the Alinity m HBV assay. The data also demonstrated that diluted

PMA P200013: FDA Summary of Safety and Effectiveness Data

{26}

serum and plasma specimens may be stored in specimen diluent for 2 hours at $15^{\circ}\mathrm{C}$ to $30^{\circ}\mathrm{C}$ and an additional 4 hours onboard the instrument before testing with the Alinity m HBV assay.

# c. Precision of Diluted Specimens

The precision of Alinity m HBV assay, using the dilution procedures, was determined by analyzing 3 panel members prepared by spiking HBV clinical specimen (panel member 1) or synthetic DNA (panel members 2 and 3) in HBV negative human plasma. The panel members were prepared with HBV concentrations, such that when diluted in Specimen Diluent, the concentrations (target concentration in Specimen Diluent) were within the linear range of the Alinity m HBV assay. Each panel member was tested in 5 replicates, twice each day for 12 days, on 3 Alinity m Systems with 3 Specimen Diluent lots by 3 operators (1 Specimen Diluent lot and 1 operator per instrument), for a total of 360 replicates.

The analyses demonstrated that the Alinity m HBV assay has a within-laboratory SD of 0.25 Log IU/mL or less for plasma samples tested using dilution procedures. These results further support the use of dilution procedure as recommended for low sample volume or high viral load HBV samples. The results are summarized in Table 16

Table 16. Precision of Alinity m HBV assay using Dilution Procedures

| Panel Member | Dilution Factor | Na | Mean Conc. (Log Copies/mL) | Within-Run Component | | Between-Run Component | | Between-Day Component | | Within-Laboratoryb | | Between-Instrument/Operator/Diluent Lot Component | | Totalc | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 1:2.5 | 355 | 2.96 | 0.10 | 3.2 | 0.07 | 2.2 | 0.07 | 2.5 | 0.14 | 4.7 | 0.08 | 2.8 | 0.16 | 5.4 |
| 2 | 1:50 | 359 | 8.43 | 0.09 | 1.0 | 0.01 | 0.2 | 0.01 | 0.2 | 0.09 | 1.0 | 0.07 | 0.8 | 0.11 | 1.3 |
| 3 | 1:50 | 358 | 6.04 | 0.09 | 1.4 | 0.02 | 0.3 | 0.04 | 0.7 | 0.10 | 1.6 | 0.07 | 1.1 | 0.12 | 2.0 |

a Number of valid replicates with detectable viral load.
b Within-Laboratory includes Within-Run, Between-Run, and Between-Day components
Total includes Within-Run, Between-Run, Between-Day, and Between Instrument/Operator/ Diluent Lot components.

# d. Confirmation of the LLoQ in Diluted Specimens

The LLoQ for the Alinity m HBV assay using dilution procedures was confirmed in serum and plasma by testing 2 panel members for each dilution factor, 1:2.5 and 1:50. The HBV concentrations in the panel members were targeted at 10 and $15\mathrm{IU / mL}$ (1.00 and 1.18 Log $\mathrm{IU / mL}$ ) after dilution in Specimen Diluent and were traceable to the $3^{\mathrm{rd}}$ WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques (NIBSC code: 10/264). A minimum of 14 replicates of each panel member were tested using the dilution procedure in 3 runs across 3 days (one run per day). The study was performed using 1 Alinity m HBV AMP Kit lot, 1 Specimen Diluent lot, and 1 Alinity m System. For each specimen type and each panel member, the detection rate, the mean, standard deviation (SD), bias, Error in Difference of two measurements (TE), and Total Analytical Error (TAE) were calculated. The

PMA P200013: FDA Summary of Safety and Effectiveness Data

{27}

accuracy and precision at 10 and 15 IU/mL were confirmed for Alinity m HBV assay testing of serum and plasma using 1:2.5 and 1:50 dilution procedures.

The detection rate for the plasma and serum panel members with the target concentration in Specimen Diluent at expected LLoQ (10 IU/mL) and above the assay's LLoQ (15 IU/mL) was 100% for both dilution factors 2.5 and 50. The TE and TAE for the plasma and serum panel members was &lt;1 Log IU/mL which is acceptable for an HBV viral load at or near the LLoQ. The results are summarized in Table 17.

Table 17. LLoQ Verification for Specimens in Specimen Diluent

| Specimen Type | Panel Member | Dilution Factor | Target Conc.in Specimen Diluent (Log IU/mL) | Target Conc. Neat (Log IU/mL) | Tested (N) | Detected (N) | Detection Rate (%) | Mean Conc.ª (Log IU/mL) | Biasᵇ (Log IU/mL) | SD (Log IU/mL) | TAE (Log IU/mL) | TE (Log IU/mL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Plasma | 1 | 2.5 | 1.00 | 1.40 | 48 | 48 | 100 | 1.44 | 0.04 | 0.29 | 0.62 | 0.83 |
| | 2 | 2.5 | 1.18 | 1.57 | 47 | 47 | 100 | 1.55 | -0.02 | 0.20 | 0.42 | 0.57 |
| | 3 | 50 | 1.00 | 2.70 | 48 | 48 | 100 | 2.67 | -0.03 | 0.24 | 0.50 | 0.67 |
| | 4 | 50 | 1.18 | 2.88 | 48 | 48 | 100 | 2.69 | -0.19 | 0.18 | 0.55 | 0.51 |
| Serum | 1 | 2.5 | 1.00 | 1.40 | 46 | 46 | 100 | 1.48 | 0.08 | 0.31 | 0.71 | 0.89 |
| | 2 | 2.5 | 1.18 | 1.57 | 48 | 48 | 100 | 1.55 | -0.02 | 0.27 | 0.56 | 0.77 |
| | 3 | 50 | 1.00 | 2.70 | 47 | 47 | 100 | 2.74 | 0.04 | 0.21 | 0.47 | 0.61 |
| | 4 | 50 | 1.18 | 2.88 | 48 | 48 | 100 | 2.91 | 0.03 | 0.14 | 0.31 | 0.40 |

ª Reported concentration for undiluted samples.
ᵇ Bias = Mean concentration - Target concentration neat.

## 13. Stability Studies

### a. Specimen Stability

Specimen stability studies demonstrated that, for the Alinity m HBV Assay, specimens should be stored as indicated in Table 18 and can be stored in a primary or secondary tube.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 28 of 50

{28}

Table 18: Final Sample Storage Claims

| Specimen | Temperature | Max. Storage Time | Special Instructions |
| --- | --- | --- | --- |
| Whole Blood | 2 to 8°C | 0-72 hours | Whole blood may be stored between draw and plasma/serum separation.
Whole blood storage plus separated plasma/serum storage at 2 to 8°C must not exceed a combined total of 72 hours. |
| | 15 to 30°C | 24 hours | |
| Plasma/Serum | 2 to 8°C | 0-72 hours | Plasma/Serum may be stored in primary tubes (with or without gel) or secondary tubes after separation from blood cells (plasma) or clot (serum).
Plasma/Serum may further stay onboard Alinity m System for up to 4 hours prior to processing. |
| | 15 to 30°C | 20 hours | |
| | -20°C | 60 days | Plasma/Serum may be stored frozen in primary gel tubes or secondary tubes after separation from blood cells (plasma) or clot (serum).
Plasma can be subjected to at most 2 freeze-thaw cycles. The serum can be subjected to at most 3 freeze-thaw cycles. Defrosted samples may be stored at 2 to 8°C for up to 6 hours prior to loading on Alinity m System.
Plasma/Serum may further stay onboard Alinity m System for up to 4 hours prior to processing. |
| | -70°C | Long term | Plasma/Serum may be stored frozen in primary gel tubes or secondary tubes after separation from blood cells (plasma) or clot (serum).
Plasma can be subjected to at most 2 freeze-thaw cycles. The serum can be subjected to at most 3 freeze-thaw cycles. Defrosted samples may be stored at 2 to 8°C for up to 6 hours prior to loading on Alinity m System.
Plasma/Serum may further stay onboard Alinity m System for up to 4 hours prior to processing. |

b. Real-time Reagent Stability (Shelf-life)

Real-time stability studies were performed to establish the shelf-life for the Alinity m HBV assay. Three lots of the reagent kits were stored at the intended storage temperature indicated in Table 19 and then tested at various time points throughout the study. The performance was assessed against clinically relevant acceptance criteria (change in viral load within 0.5 or 0.7 Log IU/mL) using controls and calibrators and an internal stability panel consisting of four panel members between ~3x LLoQ of the Alinity m HBV assay and ~4.5 Log IU/mL.

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{29}

The Shelf-life study included the assessment of an inverted condition as well as a condition that simulated fluctuating (hot/cold) temperature extremes during shipping.

Study results demonstrate that reagents are stable at their intended storage condition and continue to meet acceptance criteria fifteen months after the date of manufacture including when shipped upon exposure to fluctuating temperature extremes. Shelf-life conditions are summarized in Table 19.

## On-Board Storage

The effect of the On-Board Storage (OBS) on reagent performance was assessed by testing one lot of reagents at the maximum on-board (34 days) temperature/humidity conditions allowed by the Alinity m Instrument System (i.e., 30°C [± 2 °C], 65% [± 10%] relative humidity [RH]) for the intended OBS of the reagents. Results of the OBS conditions were compared to the results when the reagents were stored at their intended storage condition.

Study results demonstrate that reagents are stable on-board the Alinity m Instrument and continue to meet acceptance criteria for the intended on-board storage time summarized in Table 19.

Table 19: Reagent Shelf Life and On-Board Stability for the Alinity m HBV and Alinity m Accessory Kits

| Kit/Accessory | Intended Storage Condition (Shelf Life) | On-Board Storage |
| --- | --- | --- |
| HBV AMP Kit | 15 months
2°C to 8°C | 30 days |
| HBV CTRL Kit | 15 months
-15°C to -25°C | 4 hours |
| HBV CAL Kit | 15 months
-15°C to -25°C | 4 hours |
| Sample Prep Kit 2
(Elution Buffer 2, Microparticles 2) | 15 months
15°C to 30°C | 10 days |
| System Solutions
(Lysis Solution, Diluent Solution, Vapor Barrier Solution) | 15 months
15°C to 30°C | Lysis Solution: 30 days
Diluent Solution: 30 days
Vapor Barrier Solution: until expiration |
| Specimen Dilution Kit I | 15 months | Not Applicable |

Expiration dating for the Alinity m HBV assay has been established and approved at 15 months when reagents are stored at the intended storage conditions.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 30 of 50

{30}

# 14. Antimicrobial effectiveness testing

The purpose of the Antimicrobial Effectiveness Testing (AET) is to determine the antimicrobial effectiveness of a preservative system and establish the level of antimicrobial protection provided by the preservative system at 3 months, 12 months, and 25 months. For the Alinity m HBV assay AET is only required for reagents containing preservatives (i.e., Proclin 950). The following reagents containing preservatives were evaluated in this study;

- Alinity m HBV-1 ACT TRAY 2
- Controls and Calibrators
- Alinity m Diluent Solution
- Alinity m Elution Buffer 2

Aliquots of the reagents were inoculated at bioburden concentration levels of 10⁵ to 10⁶ CFU/mL of 7 bacterial, 2 fungal microbial organisms, and one uninoculated control. Effectiveness of the preservative for each tested organism was classified as cidal, static or neither cidal nor static depending on the microbial count at the testing time point.

This study demonstrated the effectiveness of the preservatives in the preservative containing reagents as cidal (i.e., no growth) at the intended testing duration.

# X. SUMMARY OF PRIMARY CLINICAL STUDIES

## A. Study Design

The applicant performed a clinical study to establish a reasonable assurance of safety and effectiveness of the Alinity m HBV assay for use with the automated Alinity m System in the US. A summary of the clinical study is presented below.

The clinical performance was evaluated by assessing the antiviral therapy response in chronic HBV-infected subjects. The de-identified, remnant specimens tested in this study were previously collected from subjects treated with adefovir dipivoxil or placebo (collected during GS-98-437 and GS-98-438 studies). The relationship between treatment and HBV DNA viral loads at various treatment time points as measured by Alinity m HBV results and clinical responses (histological, bio-chemical and/or serological) was determined in both HBeAg-positive subjects with compensated liver function (GS-98-437) and HBeAg-negative subjects with compensated liver function (GS-98-438). The results were used to determine whether a viral response is informative for determining the response to treatment in HBeAg-positive and HBeAg-negative subjects with chronic hepatitis B infection.

Testing with the Alinity m HBV assay was performed across 3 Alinity m Systems and 3 sites in US, using a total of 4 Alinity m HBV AMP Kit lots. Bar-coded aliquots were provided to each site for testing. Sites were provided with a minimum of one aliquot of each specimen for testing. A total of 412 subjects were included in the study.

## B. Study population and baseline demographics

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 31 of 50

{31}

The demographics and baseline clinical characteristics of the test subjects were summarized in Table 20. A majority of the HBeAg-positive subjects in this study were Asian and HBV Genotypes A and C, while a majority of the HBeAg-negative subjects in this study were White and HBV Genotype D.

Table 20. Summary of Subject Demographics and Baseline Characteristics

| Characteristic | Category | Summary Statistics | HBeAg Positive | HBeAg Negative | Total |
| --- | --- | --- | --- | --- | --- |
| Number of Subjects | | n | 228 | 184 | 412 |
| Treatment Arm | Antiviral | n (%) | 168 (73.7%) | 123 (66.8%) | 291 (70.6%) |
| | Placebo | n (%) | 60 (26.3%) | 61 (33.2%) | 121 (29.4%) |
| Total Number of Subjects with Demographic Information | | n | 219 | 183 | 402 |
| Age (Yr) | | Median (Min, Max) | 34 (16, 65) | 46 (18, 65) | 40 (16, 65) |
| Weight (kg) | | Median (Min, Max) | 71 (43, 118) | 75 (46, 135) | 73 (43, 135) |
| Sex | Male | n (%) | 164 (74.9%) | 151 (82.5%) | 315 (78.4%) |
| | Female | n (%) | 55 (25.1%) | 32 (17.5%) | 87 (21.6%) |
| Race | Asian | n (%) | 128 (58.4%) | 56 (30.6%) | 184 (45.8%) |
| | White | n (%) | 80 (36.5%) | 122 (66.7%) | 202 (50.2%) |
| | Other | n (%) | 11 (5.0%) | 5 (2.7%) | 16 (4.0%) |
| Genotype | A | n (%) | 64 (29.2%) | 11 (6.0%) | 75 (18.7%) |
| | B | n (%) | 40 (18.3%) | 31 (16.9%) | 71 (17.7%) |
| | C | n (%) | 82 (37.4%) | 24 (13.1%) | 106 (26.4%) |
| | D | n (%) | 27 (12.3%) | 114 (62.3%) | 141 (35.1%) |
| | Other | n (%) | 6 (2.7%) | 3 (1.6%) | 9 (2.2%) |
| Number of Subjects with Knodell Score | | n | 198 | 163 | 361 |
| Total Knodell Score | | Mean (SD) | 9.38 (3.29) | 9.26 (3.30) | 9.33 (3.29) |
| Necro-inflammatory Score | | Mean (SD) | 7.70 (2.73) | 7.42 (2.71) | 7.57 (2.72) |
| Fibrosis Score | | Mean (SD) | 1.68 (1.09) | 1.85 (1.15) | 1.75 (1.12) |

From these subjects, a total of 1830 specimens (from each subject at multiple time points) were tested, of which 1821 specimens were included in analysis. A Summary of the treatment arm subjects and associated specimens is presented in Table 21.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 32 of 50

{32}

Table 21. Summary of Subjects and Specimens.

| Population | Total Subjects | Subjects-Placebo | Subjects-Antiviral | Total Specimensa |
| --- | --- | --- | --- | --- |
| HBeAg-Positive | 228 | 60 | 168 | 949 |
| HBeAg-Negative | 184 | 61 | 123 | 872 |
| Overall | 412 | 121 | 291 | 1821 |

${}^{a}$ Number of specimens included in the analysis of clinical performance.

# Within-Subject Variability in Absence of Treatment

The objective of this study was to assess the change in viral load (in Log IU/mL) between two successive time points (baseline and Week 12) in placebo subjects as assessed by the Alinity m HBV assay. In the placebo arm, there were fifty-one HBeAg-negative and fifty-one HBeAg-positive subjects that had available results for both weeks 0 and 12. These results were used to estimate within-subject variability. As shown in Table 22 within-subject variability in the absence of treatment is $&lt; 2$ Log IU/mL.

Table 22. Within Subject Variability

| Study Population | N | Within-Subject Variability (Log IU/mL) | Median difference (Week 12 - Week 0) | Percent with <2 Log Changea |
| --- | --- | --- | --- | --- |
| HBeAg Positive | 51 | 0.72 | -0.01 | 90.2% |
| HBeAg Negative | 51 | 0.74 | -0.17 | 88.2% |

${}^{a}$ Percentage of subjects with the viral load change (absolute difference) of less than 2 Log IU/mL between Week 12 and Week 0.

# C. Safety and Effectiveness Results

# Safety results

The analysis of safety was based on the tested cohort of 412 patients in the Patient Management Study available for evaluation. There were no adverse effects during the study

# Effectiveness results

The analysis of effectiveness was based on the tested cohort of 412 patients in the Patient Management Study. Key effectiveness outcomes are presented below.

# Determination of Response to Antiviral Treatment (Clinical Utility)

PMA P200013: FDA Summary of Safety and Effectiveness Data

{33}

Alinity m HBV assay testing was performed using specimens collected at baseline and at weeks 12, 24, and 48 during treatment. The HBV viral load results were evaluated against histologic, biochemical, and serological responses at Week 48. Data from HBeAg-positive and HBeAg-negative subjects were analyzed separately.

## Clinical Response Definitions:

### Viral Response:
- HBV DNA &lt;2000 IU/mL at weeks 12, 24 and 48

### Alternate Response:
- HBV DNA ≥2 Log IU/mL decrease from baseline at weeks 12, 24 and 48

## Clinical Response at week 48:
- Histologic response: Improvement of histologic status by at least 2 units of the Knodell necro-inflammatory score without deterioration of the fibrosis score compared to the histologic status at baseline
- Biochemical response: Normalization of ALT test result compared to the biochemical status at baseline
- HBeAg Loss: HBeAg undetectable
- Anti-HBe Gain: Antibody against HBeAg detected
- Seroconversion: HBeAg undetectable and antibody against HBeAg detected

## Measures of association, Predictive Value and Odds Ratios:
- Positive Predictive Value (PPV) = True Positive/(True Positive + False Positive) or the probability of clinical response at various time points given the presence of viral response.
- Negative Predictive Value (NPV) = True Negative/(False Negative + True Negative) or the probability of absence of clinical response at various time points given the absence of viral response.
- Odds Ratio (OR) = (True Positive × True Negative)/(False Positive × False Negative)

## Statistical Analysis:

Viral load response was defined as either an HBV DNA viral load less than 2000 IU/mL or greater than or equal to 2 Log IU/mL decrease from baseline. Statistical analysis (PPV) was performed to evaluate the association between clinical responses at Weeks 48 and a viral load response at Weeks 12, 24, or 48. Statistical analysis (NPV) was performed to evaluate whether there is an association between the clinical non-responses at Weeks 48 and a viral load non-response at Weeks 12, 24, or 48.

PMA P200013 : FDA Summary of Safety and Effectiveness Data
Page 34 of 50

{34}

Acceptance criteria:

HBeAg-positive subjects: Odds Ratio from at least one of the following analyses shall be statistically significant (lower bound of two-sided 95% CI &gt;1): Association between the viral response (&lt;2000 IU/mL and/or ≥2 Log decrease) at week 12, 24 or 48 and at least one of clinical responses at week 48 should be demonstrated.

HBeAg-Positive Subjects:

Table 23 summarizes the associations of the baseline covariates (Race, Sex, Age and Genotype) with clinical responses to treatment (histological, biochemical, HBeAg Loss, Anti-HBe Gain, and Seroconversion) at Week 48.

Table 23. Association Between Baseline Covariates and Clinical Responses to treatment at Week 48 for HBeAg-Positive Subjects.

| Clinical Response to Treatment | Covariate | Category | N | No. of Subjects with Response | Proportion (%) of Subjects with Response | Unadjusted Odds Ratio (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Histologic | Race | Asian | 66 | 43 | 65.2% | 1.59 (0.70 ,3.61) |
| | | Other | 50 | 27 | 54.0% | |
| | Sex | Male | 92 | 56 | 60.9% | 1.11 (0.40 ,3.03) |
| | | Female | 24 | 14 | 58.3% | |
| | Age | <= 30 | 45 | 29 | 64.4% | 1.33 (0.58 ,3.10) |
| | | > 30 | 71 | 41 | 57.7% | |
| | Genotype | B or C | 64 | 42 | 65.6% | 1.64 (0.72 ,3.71) |
| | | Other | 52 | 28 | 53.8% | |
| Biochemical | Race | Asian | 72 | 42 | 58.3% | 1.72 (0.78 ,3.82) |
| | | Other | 49 | 22 | 44.9% | |
| | Sex | Male | 95 | 50 | 52.6% | 0.95 (0.36 ,2.48) |
| | | Female | 26 | 14 | 53.8% | |
| | Age | <= 30 | 50 | 30 | 60.0% | 1.63 (0.74 ,3.63) |
| | | > 30 | 71 | 34 | 47.9% | |
| | Genotype | B or C | 70 | 41 | 58.6% | 1.72 (0.78 ,3.80) |
| | | Other | 51 | 23 | 45.1% | |
| HBeAg Loss | Race | Asian | 74 | 19 | 25.7% | 0.83 (0.35 ,2.00) |
| | | Other | 51 | 15 | 29.4% | |
| | Sex | Male | 97 | 25 | 25.8% | 0.73 (0.27 ,2.09) |
| | | Female | 28 | 9 | 32.1% | |
| | Age | <= 30 | 52 | 12 | 23.1% | 0.70 (0.28 ,1.68) |
| | | > 30 | 73 | 22 | 30.1% | |
| | Genotype | B or C | 71 | 17 | 23.9% | 0.69 (0.29 ,1.64) |
| | | Other | 54 | 17 | 31.5% | |
| Anti-HBe Gain | Race | Asian | 74 | 7 | 9.5% | 0.43 (0.13 ,1.37) |
| | | Other | 51 | 10 | 19.6% | |
| | Sex | Male | 97 | 14 | 14.4% | 1.41 (0.35 ,8.21) |
| | | Female | 28 | 3 | 10.7% | |
| | Age | <= 30 | 52 | 6 | 11.5% | 0.74 |

PMA P200013 : FDA Summary of Safety and Effectiveness Data

{35}

| Clinical Response to Treatment | Covariate | Categ…