Alinity m HCV
Device Facts
| Record ID | P190025 |
|---|---|
| Device Name | Alinity m HCV |
| Applicant | Abbott Molecular, Inc. |
| Product Code | MZP · Microbiology |
| Decision Date | Mar 23, 2020 |
| Decision | APRL |
| Regulation | 21 CFR 866.3170 |
| Device Class | Class 2 |
Intended Use
The Alinity m HCV assay is an in vitro reverse transcription-polymerase chain reaction (RT-PCR) assay for both the detection and quantitation of hepatitis C virus (HCV) RNA, in human plasma (EDTA, Acid Citrate Dextrose) or serum, from HCV antibody positive individuals. The assay is intended for use as an aid in the diagnosis of active HCV infection in individuals with antibody evidence of HCV infection, and to aid in the management of patients with known active HCV infection, including SVR determination. The results from the Alinity m HCV assay must be interpreted within the context of all relevant clinical and laboratory findings. The Alinity m HCV assay is not intended to be used in screening blood, plasma, serum, tissue or tissue donors for HCV.
Device Story
Alinity m HCV is an automated, random-access, in vitro diagnostic assay for detection and quantitation of HCV RNA in human plasma or serum. It uses real-time RT-PCR technology. The Alinity m System performs all steps: sample preparation (magnetic microparticle technology for nucleic acid capture/wash/elution), RT-PCR assembly, amplification/detection, and result calculation. The system uses an Application Specification File (JSON) to control hardware and algorithms. It is used in clinical laboratories by trained personnel. The output is a quantitative HCV RNA result (IU/mL) or qualitative detection status. Clinicians use these results to confirm active infection, monitor treatment response (SVR), and guide antiviral therapy decisions. Benefits include accurate diagnosis and management of HCV, potentially reducing liver disease complications and transmission.
Clinical Evidence
Clinical performance evaluated in two studies: a Diagnostic Utility Study (n=511) and a Patient Management Study (n=232). Diagnostic study showed 100% positive agreement and 94.7% negative agreement versus FDA-approved NAAT. Patient management study demonstrated effectiveness in determining SVR across genotypes 1-6 in patients undergoing DAA therapy. Analytical studies confirmed LoD of 12 IU/mL, linearity up to 8.3 Log IU/mL, and precision (SD ≤0.25 Log IU/mL). No cross-reactivity or interference observed with common endogenous substances, drugs, or other viral/bacterial pathogens.
Technological Characteristics
Real-time RT-PCR assay. Utilizes magnetic microparticle technology for nucleic acid extraction. Reagents include enzymes, primers, probes, and UDG for contamination control. Form factor: unit-dose lyophilized reagents in multiwell trays. Connectivity: integrated, automated system. Software: Application Specification File (JSON) controls instrument parameters. Sterilization: not applicable (reagents).
Indications for Use
Indicated for HCV antibody positive individuals to diagnose active HCV infection and manage patients with known active HCV infection, including SVR determination. Not for screening blood, plasma, serum, or tissue donors.
Regulatory Classification
Identification
A nucleic acid-based hepatitis C virus (HCV) ribonucleic acid (RNA) test is identified as an in vitro diagnostic device intended for prescription use as an aid in the diagnosis of HCV infection in specified populations, and/or as an aid in the management of HCV-infected patients including guiding the selection of genotype-specific treatment in individuals with chronic HCV infection. The test is intended for use with human serum or plasma. The test is not intended for use as a donor screening test for the presence of HCV antibodies in blood, blood products, or tissue donors.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) For all nucleic acid-based HCV RNA tests, the labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the test is not intended for use as a donor screening test for the presence of HCV RNA from human cells, tissues, and cellular and tissue-based products. (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 management. These limitations must include, but are not limited to, statements that indicate: (A) The specimen types for which the device has been cleared and that use of this test kit with specimen types other than those specifically cleared for this device may result in inaccurate test results. (B) When applicable, that assay performance characteristics have not been established in populations of immunocompromised or immunosuppressed patients or, other populations where test performance may be affected. (C) Test results are to be interpreted by qualified licensed healthcare professionals in conjunction with the individual's clinical presentation, history, and other laboratory results. (2) For all nucleic acid-based HCV RNA tests, the design verification and validation must include: (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 nature 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) Detailed documentation of analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including, but not limited to, limit of detection (LoD), upper and lower limits of quantitation (ULoQ and LLoQ, respectively), linearity, precision, endogenous and exogenous interferences, cross reactivity, carryover, matrix equivalency, and sample and reagent stability. Samples selected for use in analytical studies or used to prepare samples for use in analytical studies must be from subjects with clinically relevant circulating genotypes in the United States. Cross-reactivity studies must include samples from HCV RNA negative subjects with other causes of liver disease, including autoimmune hepatitis, alcoholic liver disease, chronic hepatitis B virus, primary biliary cirrhosis, and nonalcoholic steatohepatitis, when applicable. The effect of each claimed nucleic-acid isolation and purification procedure on detection must be evaluated. (v) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on test performance. (vi) Final release criteria to be used for manufactured test lots with appropriate evidence 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) Multisite reproducibility study that includes the testing of three independent production lots. (viii) All stability protocols, including acceptance criteria. (ix) Final release test results for each lot used in clinical studies. (x) Analytical sensitivity and specificity of the test must be the same or better than that of other cleared or approved tests. (xi) Lot-to-lot precision studies, as appropriate. (3) For devices intended for the qualitative detection of HCV RNA, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, the design verification and validation must include detailed documentation of performance from a multisite clinical study. Performance must be analyzed relative to an FDA cleared or approved qualitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must be conducted using appropriate patient samples, with appropriate numbers of HCV positive and negative samples in applicable risk categories. Additional genotypes must be validated using appropriate numbers and types of samples. The samples may be a combination of fresh and repository samples, sourced from within and outside the United States, as appropriate. The study designs, including number of samples tested, must be sufficient to meet the following criteria: (i) Clinical sensitivity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 95 percent. (ii) Clinical specificity of the test must have a lower bound of the 95 percent confidence interval of greater than or equal to 96 percent. (4) For devices intended for the quantitative detection of HCV RNA, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply: (i) Labeling required under § 809.10(b) of this chapter must include a prominent statement that the test is not intended as a diagnostic test to confirm the presence of active HCV infection, when applicable. (ii) Design verification and validation must include the following: (A) Detailed documentation of the following analytical performance studies conducted as appropriate to the technology, specimen types tested, and intended use of the device, including but not limited to: LoD, ULoQ and LLoQ. LoD, LLoQ, and linearity studies must demonstrate acceptable device performance with all HCV genotypes detected by the device. (B) Detailed documentation of clinical performance testing from either: ( *1* ) A multisite clinical study with an appropriate number of clinical samples from chronically HCV infected patients in which the results are compared to an FDA-cleared or approved quantitative HCV RNA test, or a comparator that FDA has determined is appropriate. This study must include a sufficient number of HCV positive samples containing an analyte concentration near the LLoQ to describe performance at this level. Clinical samples must cover the full range of the device output and must be consistent with the distribution of these genotypes in the U.S. population. Clinical samples may be supplemented with diluted clinical samples for those viral load concentrations that are not sufficiently covered by natural clinical specimens, or( *2* ) A clinical study with prospectively collected samples demonstrating clinical validity of the device.(C) Detailed documentation of a qualitative analysis near the lower end of the measuring range demonstrating acceptable performance when used as an aid in diagnosis. (5) For devices intended for HCV RNA genotyping, in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, design verification and validation must include the following: (i) Detailed documentation of an analytical performance study demonstrating the LoD for all HCV genotypes detected by the device. (ii) Detailed documentation, including results, of a multisite clinical study that assesses genotyping accuracy ( *i.e.,* the proportion of interpretable results that match with the reference method result) and the genotyping rate (*i.e.,* the proportion of results that were interpretable).(6) For any nucleic acid-based HCV RNA test intended for Point of Care (PoC) use, the following special controls, in addition to those listed in paragraphs (b)(1) and (2) of this section, apply: (i) Clinical studies must be conducted at PoC sites. (ii) Additional labeling must include a brief summary of the instructions for use that are appropriate for use in a PoC environment.
Related Devices
- P100017 — ABBOTT REALTIME HCV, ABBOTT REALTIME HCV AMPLIFICATION REAGENT KIT, ABBOTT REALTIME HVC CONTROL KIT, ABBOTT REALTIME HCV · Abbott Molecular, Inc. · May 17, 2011
- K252102 — Alinity m HCV · Abbott Molecular, Inc. · Sep 25, 2025
- K221007 — cobas HCV · Roche Molecular Systems, Inc. · Nov 4, 2022
- P160023 — APTIMA HCV QUANT DX ASSAY · Hologic, Inc. · Feb 13, 2017
Submission Summary (Full Text)
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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)
I. GENERAL INFORMATION
Device Generic Name: In vitro reverse transcription-polymerase chain reaction (PCR)-based assay for detection of HCV RNA.
Device Trade Name: Alinity m HCV
Device Procode: MZP
Applicant’s Name and Address: Abbott Molecular, Inc. 1300 E. Touhy Avenue Des Plaines, IL 60018
Date(s) of Panel Recommendation: None
Premarket Approval Application (PMA) Number: P190025
Date of FDA Notice of Approval: March 23, 2020
II. INDICATIONS FOR USE
The Alinity m HCV assay is an in vitro reverse transcription-polymerase chain reaction (RT-PCR) assay for both the detection and quantitation of hepatitis C virus (HCV) RNA, in human plasma (EDTA, Acid Citrate Dextrose) or serum, from HCV antibody positive individuals. The assay is intended for use as an aid in the diagnosis of active HCV infection in individuals with antibody evidence of HCV infection, and to aid in the management of patients with known active HCV infection, including SVR determination. The results from the Alinity m HCV assay must be interpreted within the context of all relevant clinical and laboratory findings.
The Alinity m HCV assay is not intended to be used in screening blood, plasma, serum, tissue or tissue donors for HCV.
III. CONTRAINDICATIONS
The Alinity m HCV assay is not intended to be used in screening blood, plasma, serum, tissue or tissue donors for HCV.
IV. WARNINGS AND PRECAUTIONS
The warnings and precautions can be found in the Alinity m HCV labeling.
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V. DEVICE DESCRIPTION
The Alinity m HCV assay utilizes real-time reverse transcriptase polymerase chain reaction (RT-PCR) to amplify and detect HCV RNA genomic sequences that have been extracted from human plasma or serum specimens. The steps of the Alinity m HCV assay consist of sample preparation, reverse transcriptase RT-PCR assembly, amplification/detection, and result calculation and reporting. All steps of the Alinity m HCV 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 HCV assay in parallel with other Alinity m assays on the same instrument.
The Alinity m HCV assay requires the following three assay-specific kits in addition to other, non-assay specific accessory reagents and consumables:
- Alinity m HCV AMP Kit
- Alinity m HCV CTRL Kit
- Alinity m HCV CAL Kit
HCV RNA from human plasma or serum is extracted 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 RNA is then combined with liquid unit-dose Alinity m HCV activation reagent and lyophilized unit-dose Alinity m HCV 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 reverse transcription, PCR amplification, and real-time fluorescence detection of HCV.
At the beginning of the Alinity m HCV 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 RT-PCR procedure along with the specimens, calibrators, and controls to demonstrate proper sample processing and assay validity.
The Alinity m HCV amplification/detection reagents consist of enzymes, primers, probes, and activation reagents that enable reverse transcription, polymerization, and detection. The Alinity m HCV 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 HCV calibration curve is required for determining the HCV RNA concentration. Two levels of calibrators are processed through sample preparation
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and RT-PCR to generate the calibration curve. The concentration of HCV RNA in specimens and controls is then calculated from the stored calibration curve.
Assay controls are tested at or above an established minimum frequency to help ensure that instrument and reagent performance remains satisfactory. During each control event, a negative control, a low-positive control, and a high-positive control are processed through sample preparation and RT-PCR procedures that are identical to those used for specimens.
## 1. Alinity m HCV AMP Kit
The Alinity m HCV AMP Kit consists of 2 types of multiwell trays:
- Alinity m HCV AMP TRAY 1 (4 trays × 48 tests): The Alinity m HCV AMP TRAY 1 contains separate wells of lyophilized, unit-dose RT-PCR amplification/detection reagents and lyophilized, unit-dose internal control.
- Alinity m HCV ACT TRAY 2 (4 trays × 48 tests): Alinity m HCV ACT TRAY 2 contains liquid activation reagent.
Each Alinity m HCV AMP TRAY 1 and Alinity m HCV ACT TRAY 2 is provided in a sealed foil pouch (4 pouches of each tray type per Alinity m HCV AMP Kit for up to 192 samples [patient specimens and/or 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).
## 2. Alinity m HCV CAL Kit
The Alinity m HCV CAL Kit is composed of the following reagents:
- Alinity m HCV CAL A (4 tubes × 1.95mL)
- Alinity m HCV CAL B (4 tubes × 1.95mL)
The Alinity m HCV CAL A and Alinity m HCV 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 HCV RNA concentrations based on the results of testing against the Primary Calibrators.
## 3. Alinity m HCV CTRL Kit
The Alinity m HCV CTRL Kit is composed of the following reagents:
- Alinity m HCV Negative CTRL (12 tubes × 1.15mL)
- Alinity m HCV Low Positive CTRL (12 tubes × 0.75mL)
- Alinity m HCV High Positive CTRL (12 tubes × 0.75mL)
The Alinity m HCV Negative CTRL, Alinity m HCV Low Positive CTRL, and Alinity m HCV 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.
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## 4. Alinity m HCV Application Specification File
The application specification file is a data file that contains a set of parameters in a software-industry-standard JSON (Java Script 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.
## 5. 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 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 APU on the Alinity m System.
## 6. Alinity m Specimen Dilution Kit I
The Alinity m Specimen Dilution Kit I is intended to allow dilution of specimens for measurement of nucleic acid. It consists of Alinity m Specimen Diluent Tubes with a pierceable cap (24 tubes × 2.45mL). Each Specimen Dilution Kit I supports dilution of up to 24 samples (patient specimens); each tube may only be used once and must not be reused. The Alinity m Specimen Diluent Tubes contain Abbott Molecular Transport Buffer which contains guanidine thiocyanate (GITC) in Tris Buffer.
## 7. Alinity m Tubes and Caps
- Alinity m LRV Tube: capped Low Residual Volume (LRV) Tubes (12 per kit)
- Alinity m Transport Tube Pierceable Capped: transport tubes closed with pierceable caps (1500 capped tubes per case, 10 boxes of 150 capped tubes)
- Alinity m Transport Tube: 1600 tubes per kit
- Alinity m Pierceable Cap: 2000 caps per kit
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- Alinity m Aliquot Tube: 1600 tubes per kit
## 8. Alinity m System Solutions
The Alinity m System Solutions (below) are used as part of the sample preparation protocol to extract and concentrate target molecules from biological samples for subsequent PCR amplification and to remove potential inhibitors from the resulting extract.
- The Alinity m Lysis Solution: consists of 1 bottle × 975 mL.
- The Alinity m Diluent Solution: consists of 4 bottles × 975 mL
- The Alinity m Vapor Barrier Solution: consists of 1 bottle × 975 mL
## 9. Alinity m System
The Alinity m System is a fully integrated and automated molecular diagnostics analyzer which utilizes real-time PCR technology in clinical laboratories. It is an integrated system for performing sample preparation and performing fluorescence-based real-time PCR to provide quantitative and qualitative detection of nucleic acid sequences. It provides sample-to-result uninterrupted processing workflow.
The Alinity m System enables continuous and random-access sample processing by utilizing four (4) independent Assay Processing Units (APUs), each of which has a sample processor and a PCR thermal cycler/reader modules that operate in parallel. The system can process up to 300 tests in approximately 8 hours.
APU sample preparation lanes use multi well disposables and provide functionality for heating wells, mixing the contents of wells, capturing magnetic particles, and moving the magnetic particles from one well to another (extraction unit). Once the sample is purified, the remaining eluate is mixed with amplification reagent and dispensed into a reaction vessel (1 reaction vessel per sample) where it undergoes thermal cycling and amplification/detection (Amp-Detect unit).
The Alinity m System software interprets system and assay specific (provided in the Application Specification File) information, calculates results, and provides the interface for controlling the system hardware.
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, results are reviewed and released through the software user interface.
Additional details can be found in the Alinity m Operator's Manual.
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VI. ALTERNATIVE PRACTICES AND PROCEDURES
There are several other FDA approved in vitro diagnostic tests for the quantitation of HCV RNA. 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 noninvasive liver elasticity measurements or biopsy of the liver, will provide further information on the status of an HCV infection. Each alternative has its own advantages and disadvantages.
VII. MARKETING HISTORY
The Alinity m HCV AMP Kit (List No. 08N50 095), Alinity m HCV CTRL Kit (List No. 08N50 085), and Alinity m HCV CAL Kit (List No. 08N50 075) are intended to be marketed in the United States.
At this time, the Alinity m HCV assay (List No. 08N50-095) has not been introduced or distributed for sale, and there are no tests sold to date. However, the CE certified Alinity m HCV AMP Kit (List No. 08N50-090/091), Alinity m HCV CTRL Kit (List No. 08N50-080), and Alinity m HCV CAL Kit (List No. 08N50-070) are identical in formulation to the US kits, except for kit labeling, and were introduced to foreign markets outside of the United States as listed in Table 1.
Table 1: Alinity m HCV in Foreign Markets Outside of the United States
| Australia^{a} | Greece | Peru |
| --- | --- | --- |
| Austria | Hungary | Poland |
| Belgium | Iceland | Portugal |
| Brazil | Ireland | Romania |
| Bulgaria | Italy | Saudi Arabia |
| Croatia | Kenya | Slovakia |
| Cyprus | Latvia | Slovenia |
| Czech Republic | Liechtenstein | Spain |
| Denmark | Lithuania | Sweden |
| Estonia | Luxembourg | Switzerland |
| Finland | Malta | Turkey |
| France | Netherlands | UK |
| Germany | Norway | |
a 08N-50-091 only
The Alinity m System (List No. 08N53-002) is intended to be marketed in the United States. At this time, the Alinity m System (List No. 08N53-002) has not been introduced for sale into the United States. However, The system was self-certified for commercialization in the European Union, and the European Free Trade Association (EFTA) since December 2017 and is marketed in the countries listed in Table 2 below:
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Table 2: Alinity m System - Registered for Sale in Following Countries
| Australia | Greece | Netherlands |
| --- | --- | --- |
| Austria | Hungary | Norway |
| Belgium | Iceland | Poland |
| Brazil | Ireland | Portugal |
| Bulgaria | Israel | Romania |
| Colombia | Italy | Saudi Arabia |
| Croatia | Japan | Slovakia |
| Cyprus | Kenya | Slovenia |
| Czech Republic | Latvia | Spain |
| Denmark | Liechtenstein | Sweden |
| Estonia | Lithuania | Switzerland |
| Finland | Luxembourg | Turkey |
| France | Malta | UK |
| Germany | | |
The Alinity m HCV and the Alinity m System have not been withdrawn from marketing for any reason related to its safety or effectiveness.
## 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 either the device operator or the patient who is tested. No adverse effects occurred during conduct of the clinical studies.
An erroneous "Not Detected" (false negative) result may lead to an HCV infected patient not being identified and not admitted into care. A false negative result is considered a public health concern because of the potential for HCV transmission from an unidentified HCV infected patient; however, this is partially mitigated by current testing guidelines that recommend repeat HCV RNA testing within a certain timeframe for patients with symptoms indicative of an HCV infection, and for patients at high risk of HCV infection. For HCV infected patients undergoing direct acting antiviral (DAA) therapy, a false negative result is not significant because current treatment regimens are all of fixed duration.
An erroneous "HCV Detected" (false positive) result is not considered to be a public health concern because HCV RNA testing is used to confirm serology positive results. In addition, repeat HCV RNA testing and consideration of other clinical evidence of an active HCV infection is recommended prior to initiating HCV DAA therapy. However, a false positive test result could lead to unnecessary exposure to HCV therapy with the potential for adverse effects.
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# IX. SUMMARY OF NONCLINICAL STUDIES
## A. Laboratory Studies
### 1. Limit of Detection (LoD)
The limit of detection (LoD) was determined by testing dilutions of the 4th World Health Organization (WHO) International Standard for Hepatitis C Virus for Nucleic Acid Amplification Techniques (NIBSC code: 06/102; genotype 1) prepared in HCV negative human plasma and serum. Testing for each HCV RNA concentration was performed with 4 lots of amplification reagents across multiple days. The results, representative of the analytical sensitivity performance of Alinity m HCV assay in plasma and serum are included in Table 3.
Probit analysis of the data determined that the concentration of HCV RNA in plasma detected with 95% probability (LoD by Probit) was 8.50 IU/mL (95% CI: 6.20 to 14.53 IU/mL).
Probit analysis of the data determined that the concentration of HCV RNA in serum detected with 95% probability (LoD by Probit) was 7.96 IU/mL (95% CI:5.06 to 25.52 IU/mL).
The LOD of Alinity m HCV is 12 IU/mL (1.08 Log IU/mL) in plasma and serum.
Table 3: LOD with HCV 4th WHO International Standard (NIBSC 06/102) in EDTA Plasma and Serum Across all Lots
| Matrix | Nominal Concentration (IU/mL) | Number of Valid Replicates | Number of detected* Replicates | Hit Rate [%] | LOD by Probit [95% CI] |
| --- | --- | --- | --- | --- | --- |
| Plasma | 1 | 95 | 11 | 11.6 | 8.50 IU/mL
[6.20 – 14.53 IU/mL] |
| | 2 | 93 | 25 | 26.9 | |
| | 3 | 93 | 66 | 71.0 | |
| | 6 | 96 | 84 | 87.5 | |
| | 9 | 95 | 90 | 94.7 | |
| | 12 | 96 | 94 | 97.9 | |
| | 15 | 95 | 95 | 100.0 | |
| Serum | 1 | 94 | 14 | 14.9 | 7.96 IU/mL
[5.06 – 25.52 IU/mL] |
| | 2 | 93 | 24 | 25.8 | |
| | 3 | 95 | 67 | 70.5 | |
| | 6 | 93 | 84 | 90.3 | |
| | 9 | 95 | 91 | 95.8 | |
| | 12 | 95 | 92 | 96.8 | |
| | 15 | 96 | 96 | 100.0 | |
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# 2. Genotype Specific LoD
The LoD of the Alinity m HCV assay was verified for HCV Genotypes 2, 3, 4, 5, and 6 in plasma and serum. For each HCV Genotype, 3 panel members were prepared by diluting a clinical specimen into HCV negative human serum and plasma. Testing was performed across three days. Alinity m HCV detected $>95\%$ of HCV replicates at and above 9 IU/mL in plasma and serum. As such, the claimed LOD of the Alinity m HCV was confirmed for all genotypes.
# 3. Linearity
Linearity of Alinity m HCV was assessed by testing a dilution series of HCV genotype 1 in negative human plasma and serum, each consisting of 9 panel members spanning from 10 to 200,000,000 IU/mL (1.00 to 8.30 Log IU/mL). Two kind of panel members were created: panel members with concentrations from 10 to 10,000 IU/mL (1.00 to 4.00 Log IU/mL) were prepared using an HCV positive clinical specimen, panel members with concentrations 100 to 200,000,000 IU/mL (2.00 to 8.30 Log IU/mL) were prepared using Armored RNA.
There was no significant difference in the linearity between the clinical sample and the armored RNA based panel members. As such they were analyzed in a combined regression (Figure 1). Alinity m HCV demonstrated linearity in plasma and serum across the range tested with an upper limit of quantitation (ULoQ) of 8.3 Log IU/mL.
Plasma
Serum
Figure 1: Alinity m HCV Linearity in Serum and Plasma for Genotype 1 - Outliers Included
Linearity was also performed with genotypes 2, 3, 4, 5, and 6. The following equations were obtained for the different genotypes using a least square regression model across the linear range of the Alinity m HCV (outliers were included). The Alinity m HCV is linear across the measuring range for all tested genotypes.
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Table 4: Least Square Regression Equations
| Genotype | Plasma | | Serum | |
| --- | --- | --- | --- | --- |
| | Linear Equation | Maximum Non-linearity (log IU/mL) | Linear Equation | Maximum Non-linearity (log IU/mL) |
| 1 | y=1.05x - 0.04 | 0.06 | y=1.05x + 0 | -0.12 |
| 2 | y=0.99x + 0.17 | 0.07 | y=1.06x = 0.12 | -0.13 |
| 3 | y=1.02x - 0.02 | 0.03 | y=1.02x + 0.05 | 0.05 |
| 4 | y=1.04x - 0.07 | 0.16 | y=0.99x + 0.09 | 0.15 |
| 5 | y=1.01x - 0.04 | 0.15 | y=1.00x + 0.04 | 0.26 |
| 6 | y=1.03x - 0.02 | 0.08 | y=1.05x - 0.11 | -0.08 |
## 4. Lower Limit of Quantitation (LLoQ)
The lower limit of quantitation (LLOQ) is defined as the lowest concentration at which HCV RNA is reliably quantitated within a total error. Total error was estimated by two methods: Total Analytical Error (TAE) = |bias| + 2 × SD, and Total Error (TE) = √2 × 2 × SD.
TAE and TE of Alinity m HCV for genotypes 1, 2, 3, 4, 5 and 6 in plasma and serum were calculated for panel members with observed concentrations at or near 12 IU/mL (1.08 Log IU/mL) tested in multiple non-clinical studies (LOD/Genotype specific LOD, Linearity, Precision [see below]) as shown in Table 5. The panel members were either generated directly from the 4th WHO International Standard for HCV (NIBSC code: 06/102) or their concentrations were traceable to the 4th WHO International Standard for HCV.
The results of these analyses demonstrated that Alinity m HCV can determine the concentration of HCV RNA for genotypes 1, 2, 3, 4, 5, and 6 in plasma and serum at 12 IU/mL (1.08 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 HCV is 12 IU/mL.
Table 5: LLoQ of the Alinity m HCV (TAE and TE for all Genotypes based on various studies as indicated)
| Genotype | Study | Target (Log IU/mL) | Mean (Log IU/mL) | Bias (Log IU/mL) | SD | TAE | TE |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PLASMA | | | | | | | |
| 1 | Precision | 1.08 | 1.07 | -0.01 | 0.19 | 0.40 | 0.55 |
| | LOD | 1.08 | 0.82 | -0.26 | 0.24 | 0.75 | 0.69 |
| | Linearity | 1.08 | 1.01 | -0.07 | 0.20 | 0.47 | 0.57 |
| 2 | Genotype LOD | 1.08 | 0.95 | -0.13 | 0.24 | 0.61 | 0.68 |
| | Genotype Linearity | 1.08 | 1.04 | -0.04 | 0.18 | 0.39 | 0.50 |
| 3 | Genotype LOD | 1.08 | 1.06 | -0.02 | 0.20 | 0.41 | 0.56 |
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| Genotype | Study | Target (Log IU/mL) | Mean (Log IU/mL) | Bias (Log IU/mL) | SD | TAE | TE |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PLASMA | | | | | | | |
| | Genotype Linearity | 1.08 | 1.05 | -0.03 | 0.23 | 0.49 | 0.65 |
| 4 | Genotype LOD | 1.08 | 1.10 | 0.02 | 0.23 | 0.48 | 0.64 |
| | Genotype Linearity | 1.08 | 1.30 | 0.22 | 0.20 | 0.63 | 0.58 |
| 5 | Genotype LOD | 1.08 | 1.10 | 0.02 | 0.25 | 0.51 | 0.70 |
| | Genotype Linearity | 1.08 | 1.15 | 0.07 | 0.23 | 0.53 | 0.64 |
| 6 | Genotype LOD | 1.08 | 0.97 | -0.11 | 0.17 | 0.45 | 0.49 |
| | Genotype Linearity | 1.08 | 1.11 | 0.03 | 0.28 | 0.59 | 0.80 |
| SERUM | | | | | | | |
| 1 | Precision | 1.08 | 0.96 | -0.12 | 0.23 | 0.57 | 0.64 |
| | LOD | 1.08 | 0.85 | -0.23 | 0.22 | 0.67 | 0.62 |
| | Linearity | 1.08 | 1.23 | 0.15 | 0.20 | 0.55 | 0.56 |
| 2 | Genotype LOD | 1.08 | 1.07 | -0.01 | 0.14 | 0.30 | 0.40 |
| | Genotype Linearity | 1.08 | 0.96 | -0.12 | 0.15 | 0.42 | 0.41 |
| 3 | Genotype LOD | 1.08 | 1.10 | 0.02 | 0.21 | 0.44 | 0.60 |
| | Genotype Linearity | 1.08 | 1.13 | 0.05 | 0.26 | 0.56 | 0.72 |
| 4 | Genotype LOD | 1.08 | 1.14 | 0.05 | 0.20 | 0.45 | 0.57 |
| | Genotype Linearity | 1.08 | 1.27 | 0.19 | 0.17 | 0.54 | 0.49 |
| 5 | Genotype LOD | 1.08 | 1.27 | 0.19 | 0.15 | 0.48 | 0.41 |
| | Genotype Linearity | 1.08 | 1.16 | 0.08 | 0.16 | 0.40 | 0.46 |
| 6 | Genotype LOD | 1.08 | 1.06 | -0.02 | 0.15 | 0.33 | 0.44 |
| | Genotype Linearity | 1.08 | 1.15 | 0.07 | 0.29 | 0.65 | 0.82 |
# 5. 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 $4^{\text{th}}$ WHO International Standard for Hepatitis C Virus for Nucleic Acid Amplification Techniques (NIBSC code: 06/102). The concentrations tested for the WHO standard were 3.00 and 2.00 Log IU/mL. The concentrations tested for the primary calibrators ranged from 3.00 to 7.00 Log IU/mL. The Alinity m HCV calibrators and controls were also tested along with the primary calibrators and the WHO standard. All of the panels had observed HCV concentrations similar to the target concentrations, and were linear across the assay's quantitation range, as presented in Figure 2.
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Figure 2: Traceability to the 4th WHO International Standard for HCV (NIBSC code: 06/102)
# 6. Precision
These studies evaluated the lot-to-lot variability in an internal precision study and the reproducibility of the Alinity m HCV test in plasma on the Alinity m system at 3 external sites with four reagent lots.
# a. Internal 12-Day Precision Study
Precision of Alinity m HCV was determined by analyzing a 9-member plasma panel and a 9-member serum panel. Panels span the range of $12\mathrm{IU / mL}$ (the assay's LLOQ) to 200,000,000 IU/mL. Panel members 1, 2, 3, 5, 7, 8 and 9 were prepared by diluting HCV genotype 1 into HCV negative human plasma and serum, whereas panel members 4 and 6 were prepared by diluting HCV genotype 2 into HCV negative human plasma and serum. Panel members with concentrations from 2 to 5 Log IU/mL (100 to 100,000 IU/mL) were prepared using HCV positive clinical specimens, while panel members with concentrations greater than 5 Log IU/mL (100,00 IU/mL) were prepared using Armored RNA. 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 lot by 3 operators (one per instrument) for a total of 288 replicates.
The results, representative of the precision of Alinity m HCV in plasma and serum, (Table 6), demonstrated that the within-laboratory standard deviation (SD) is less than or equal to $0.25\mathrm{Log}$ IU/mL of HCV RNA from 2 to 8 Log IU/mL (100 to 100,000,000 IU/mL), and less than or equal to $0.35\mathrm{Log}$ IU/mL from 1 to 3 times the LLOQ (1.08 to 1.56 Log IU/mL or 12 to 36 IU/mL).
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Table 6: Precision Analysis in Plasma and Serum
| Panel | Mean Concentration | Within-Run Component | | Between-Run Component | | Between-Day Component | | Within-Laboratorya | | Between-Instrument/Operator Component | | Totalb | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| PLASMA | | | | | | | | | | | | | |
| 01 | 285 | 1.07 | 0.18 | 16.9 | 0.07 | 6.4 | 0.00 | 0.0 | 0.19 | 18.1 | 0.08 | 7.9 | 0.21 |
| 02 | 286 | 1.42 | 0.15 | 10.8 | 0.03 | 2.4 | 0.00 | 0.0 | 0.16 | 11.0 | 0.08 | 6.0 | 0.18 |
| 03 | 284 | 1.91 | 0.13 | 6.9 | 0.02 | 1.0 | 0.00 | 0.0 | 0.13 | 6.9 | 0.10 | 5.2 | 0.17 |
| 04 | 281 | 2.88 | 0.12 | 4.2 | 0.00 | 0.0 | 0.04 | 1.5 | 0.13 | 4.5 | 0.11 | 3.7 | 0.17 |
| 05 | 285 | 3.94 | 0.12 | 3.0 | 0.00 | 0.0 | 0.02 | 0.5 | 0.12 | 3.1 | 0.07 | 1.8 | 0.14 |
| 06 | 285 | 5.15 | 0.10 | 2.0 | 0.01 | 0.2 | 0.02 | 0.5 | 0.11 | 2.1 | 0.07 | 1.4 | 0.13 |
| 07 | 282 | 6.14 | 0.07 | 1.2 | 0.01 | 0.1 | 0.00 | 0.0 | 0.07 | 1.2 | 0.09 | 1.4 | 0.11 |
| 08 | 285 | 7.11 | 0.12 | 1.7 | 0.03 | 0.4 | 0.00 | 0.0 | 0.13 | 1.8 | 0.10 | 1.5 | 0.16 |
| 09 | 285 | 8.55 | 0.06 | 0.7 | 0.03 | 0.4 | 0.00 | 0.0 | 0.06 | 0.8 | 0.15 | 1.7 | 0.16 |
| SERUM | | | | | | | | | | | | | |
| 01 | 284 | 0.96 | 0.22 | 23.0 | 0.00 | 0.0 | 0.04 | 4.1 | 0.23 | 23.4 | 0.07 | 7.0 | 0.24 |
| 02 | 283 | 1.34 | 0.16 | 12.1 | 0.05 | 3.5 | 0.03 | 2.3 | 0.17 | 12.8 | 0.08 | 6.3 | 0.19 |
| 03 | 286 | 1.81 | 0.12 | 6.4 | 0.05 | 2.6 | 0.03 | 1.8 | 0.13 | 7.2 | 0.06 | 3.5 | 0.14 |
| 04 | 286 | 2.78 | 0.09 | 3.4 | 0.05 | 1.9 | 0.04 | 1.6 | 0.12 | 4.2 | 0.15 | 5.2 | 0.19 |
| 05 | 286 | 3.82 | 0.09 | 2.5 | 0.06 | 1.7 | 0.01 | 0.3 | 0.11 | 3.0 | 0.05 | 1.3 | 0.12 |
| 06 | 286 | 5.10 | 0.09 | 1.7 | 0.00 | 0.0 | 0.00 | 0.0 | 0.09 | 1.7 | 0.08 | 1.7 | 0.12 |
| 07 | 282 | 6.08 | 0.08 | 1.3 | 0.03 | 0.5 | 0.00 | 0.0 | 0.09 | 1.4 | 0.07 | 1.1 | 0.11 |
| 08 | 286 | 6.85 | 0.08 | 1.2 | 0.06 | 0.9 | 0.05 | 0.7 | 0.11 | 1.7 | 0.01 | 0.2 | 0.12 |
| 09 | 288 | 8.60 | 0.06 | 0.7 | 0.02 | 0.2 | 0.00 | 0.0 | 0.06 | 0.7 | 0.13 | 1.5 | 0.15 |
a Within-Laboratory includes Within-Run, Between-Run and Between-Day Components.
b Total includes Within-Run, Between-Run, Between-Day and Between-Instrument Components.
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b. External 5-Day Reproducibility Study
A multi-site reproducibility study was performed at three external U.S. based sites. Six unique panel members for Genotype 1a and five unique panel members for Genotype 2 covering the measuring range of the assay were formulated in plasma to make an 11 member panel (see Table 7 below). Each panel member was tested with 5 replicates per run. Because the Alinity is a random-access analyzer a run is defined as testing a batch of 5 replicates of each of the 11 panel members tested consecutively on the system within 8 hours (i.e., one run per work day) using the same Alinity m HCV reagent lot. Testing was performed on 5 non-consecutive days. Three different Alinity m HCV Amplification Reagent Kit lots were tested (2 per site). Each site used unique lots of the Calibrator Kit, Control Kit, Sample Prep Kit 2 and Systems Solutions. A minimum of three operators at each site were part of the testing. The 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. Results are summarized in Table 8.
Table 7: Genotypes and Concentration of Reproducibility Panel Members
| Panel Member | Genotype | HCV RNA Target Concentration [Log IU/mL] | HCV RNA Target Concentration [IU/mL] |
| --- | --- | --- | --- |
| 1 | 1a | 8 | 100,000,000 |
| 2 | 1a | 6 | 1,000,000 |
| 3 | 1a | 4 | 10,000 |
| 4 | 1a | 2 | 100 |
| 5 | 1a | 1.18 | 15 |
| 6 | 2 | 7 | 10,000,000 |
| 7 | 2 | 5 | 100,000 |
| 8 | 2 | 3 | 1,000 |
| 9 | 2 | 1.7 | 50 |
| 10 | 2 | 1.18 | 15 |
| 11 | 1a | 1.0 | 12 |
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Table 8: Summary of Reproducibility (All Sites)
| Genotype | Na | Mean Concentration (Log IU/mL) | Within-Run | | Between-Run | | Within-Laboratoryc | | Between-Lot | | Between- Site | | Totald | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SDb | %CV | SDb | % CV | SDb | % CV | SDb | % CV | SDb | % CV | SDb | % CV |
| 1 | 150 | 8.50 | 0.08 | 0.9 | 0.05 | 0.6 | 0.09 | 1.1 | 0.12 | 1.4 | 0.00 | 0.0 | 0.15 | 1.7 |
| | 150 | 6.15 | 0.15 | 2.5 | 0.03 | 0.5 | 0.15 | 2.5 | 0.03 | 0.6 | 0.01 | 0.1 | 0.16 | 2.6 |
| | 150 | 3.89 | 0.16 | 4.2 | 0.04 | 1.1 | 0.17 | 4.3 | 0.04 | 1.0 | 0.03 | 0.7 | 0.18 | 4.5 |
| | 150 | 1.95 | 0.21 | 10.6 | 0.00 | 0.0 | 0.21 | 10.6 | 0.06 | 3.1 | 0.00 | 0.0 | 0.22 | 11.0 |
| | 149 | 1.06 | 0.30 | 28.0 | 0.09 | 8.6 | 0.31 | 29.3 | 0.16 | 15.2 | 0.00 | 0.0 | 0.35 | 33.0 |
| | 128 | 0.65 | 0.34 | 53.0 | 0.07 | 10.6 | 0.35 | 54.0 | 0.11 | 17.5 | 0.00 | 0.0 | 0.37 | 56.8 |
| 2 | 150 | 7.30 | 0.09 | 1.3 | 0.01 | 0.2 | 0.09 | 1.3 | 0.02 | 0.3 | 0.05 | 0.6 | 0.11 | 1.5 |
| | 150 | 5.27 | 0.12 | 2.3 | 0.00 | 0.0 | 0.12 | 2.3 | 0.04 | 0.8 | 0.05 | 0.9 | 0.14 | 2.6 |
| | 150 | 3.06 | 0.20 | 6.5 | 0.00 | 0.0 | 0.20 | 6.5 | 0.06 | 1.9 | 0.00 | 0.0 | 0.21 | 6.7 |
| | 150 | 1.44 | 0.21 | 14.7 | 0.08 | 5.3 | 0.22 | 15.6 | 0.10 | 6.7 | 0.00 | 0.0 | 0.24 | 17.0 |
| | 149 | 0.88 | 0.28 | 31.4 | 0.09 | 10.2 | 0.29 | 33.0 | 0.08 | 8.8 | 0.00 | 0.0 | 0.30 | 34.2 |
a Number of valid replicates with detectable viral load
b SD = Standard deviations in Log IU/mL
c Within-Laboratory includes Within-Run, Between-Run and Between-Day component
Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument components
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# 7. Cross Reactivity of the Alinity m HCV with Other Microorganisms
A comprehensive list of organisms containing viral, fungal and bacterial analytes as indicated in Table 9 was evaluated for cross-reactivity with the Alinity m HCV test. The study was designed according to CLSI EP7-A2. Organisms were tested by adding either the microorganism or its purified nucleic acid to HCV negative and HCV positive (36 IU/mL and 10,000 IU/mL) plasma at a final concentration of 10^5 U/mL for viruses, protozoans and yeast and 10^6 CFU/mL for bacteria. Cross-reactivity was analyzed from the HCV negative sample, microbial interference was analyzed from the 3x LLoQ (i.e., 36 IU/mL) sample (i.e., detectability) and from the 10,000 IU/mL sample (i.e., quantitation). Three replicates for each cross reactant were tested.
No cross reactivity or microbial interference was observed for any of the tested organisms.
Table 9: Potential Cross-Reactants
| Viruses | Bacteria |
| --- | --- |
| Adenovirus Type 5 | Chlamydia trachomatis |
| BK polyomavirus | Corynebacterium diphtheriae |
| Dengue virus 1 | Mycobacterium gordonae |
| Dengue virus 2 | Mycobacterium smegmatis |
| Dengue virus 3 | Neisseria gonorrhoeae |
| Dengue virus 4 | Propionibacterium acnes |
| FSME virus | Staphylococcus aureus |
| GB virus C | Staphylococcus epidermidis |
| Hepatitis A Virus | Streptococcus pneumoniae |
| Hepatitis B Virus | Yeast/Protozoan |
| Hepatitis D Virus | Candida albicans |
| HSV-1 | Trichomonas vaginalis |
| HHV-2 | |
| HHV-5 (CMV) | |
| HHV-4 (EBV) | |
| HHV-6B | |
| HHV-8 (Kaposi Sarcoma Virus) | |
| HIV-2 | |
| HIV-1 | |
| HPV-16 | |
| HPV-18 | |
| HTLV-1 | |
| HTLV-2 | |
| Influenza A | |
| Japanese encephalitis virus | |
| Murray Valley encephalitis virus | |
| Parvovirus B19 | |
| Rubella virus | |
| St. Louis encephalitis | |
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Vaccinia virus (VACV)
VZV
West Nile virus
Yellow Fever virus
Zika virus
# 8. Interfering Substances
## a. Interference with Endogenous Substances
The impact of potentially interfering endogenous substances and disease states on the analytical specificity (detection and quantitation) of the Alinity m HCV assay was evaluated by testing spiked samples as well as patient samples with naturally elevated levels of these substances. Hemoglobin (2g/L), triglycerides (37mM), conjugated and unconjugated bilirubin (342μM), albumin (60g/L) and human DNA (2μg/mL) were spiked into HCV negative and HCV positive plasma. 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. The HCV positive samples were prepared at two HCV levels by adding HCV from a positive patient specimen to HCV negative plasma at a final concentration of 3 × the assay's LLOQ and at 10,000 IU/mL.
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. No interference was observed in specimens collected from individual donors containing the naturally elevated interfering substances, i.e. albumin (>5.1 g/dL), bilirubin (>2 mg/dL), hemoglobin (>2 g/L) or triglycerides (>325 mg/dL).
## b. Interference with Autoimmune Disorders, Serological Markers and non-viral Hepatitis
This study tests the interference of non-viral hepatitis and other conditions (autoimmune) with the detection and quantitation of the Alinity m HCV in the presence of 3× LLOQ HCV. HCV negative clinical specimens from 10 individual donors from each of the following disease states were tested in the presence and absence of HCV at a concentration of 3 × LLOQ: systemic lupus erythematosus (SLE), anti-nuclear antibodies (ANA), rheumatoid factor (RF), alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), cirrhosis, auto-immune hepatitis, and hepatocellular carcinoma.
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 or hepatocellular carcinoma (HCC). Please also refer to the Diagnostic Study in the clinical study section.
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c. Interference with Potentially Interfering Drugs
Interference was tested in the presence of drug compounds tested in pools that are listed in Table 12, at a concentration of 3 times the reported Cmax or higher. Not interference was observed.
Table 10: Potentially Interfering Drugs
| Pool Tested | Drug Compounds in Pool |
| --- | --- |
| 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 (Trimethoprim/Sulfamethoxazole) |
| 4 | Darunavir, Ethambutol, Etravirine, Flucytosine, Fluticasone propionate, Furosemide, Hydrochlorothiazide, Levothyroxine, Rifabutin, Rilpivirine, Salmeterol xinafoate, 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 | Hydrocodone |
| 10 | Biotin |
| 11 | Acetaminophen/Hydrocodone) |
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# 9. Carryover
The carryover rate for Alinity m HCV was determined by analyzing 362 replicates of HCV negative plasma samples processed from alternating positions with high concentration HCV armored RNA positive plasma samples at 10,000,000 IU/mL, across a total of 16 runs. HCV RNA was not detected in any of the HCV negative samples, resulting in an overall carryover rate of $0.0\%$ (95% CI: 0.0 to $1.1\%$ ).
# 10. Matrix Equivalency Study
# a. Serum/Plasma Equivalency Across the Linear Range
Serum/plasma matrix equivalency in Alinity m HCV results for plasma and serum was evaluated by analyzing 27 negative plasma and serum pairs and 58 positive plasma and serum pairs. Ten positive pairs were collected from HCV positive patients. Forty-eight positive pairs were prepared by spiking patient specimens or Armored RNA in paired serum and plasma specimens collected from individuals without a history of HCV. The HCV RNA concentrations for the HCV positive serum/plasma pairs were distributed across the quantitative range of the assay, with the lowest concentration at 1.23 Log IU/mL and the highest concentration at 8.21 LogIU/mL. HCV genotypes 1, 2, 3, 4, 5 and 6 were represented in the positive samples.
No detection was observed for any of HCV negative plasma and serum samples; all HCV positive plasma and serum samples were detected, resulting in an overall percent agreement between plasma and serum of $100.0\%$ (95% CI: 95.7 to $100.0\%$ ). The least square regression analysis in Figure 3 between serum and plasma demonstrated that the Alinity m HCV Assay has equivalent performance in reporting quantitative results with serum and plasma since the regression demonstrated a slope of 1.00, intercept of $-0.06$ , $r = 0.994$ , and mean bias of $-0.06$ Log IU/mL (plasma minus serum). Accordingly, performance of the test in serum and plasma can be considered equivalent.
Figure 3: Matrix Equivalence Study (Outliers included) - Least Squares Regression Plot (equation: $\mathbf{Y} = 1.0 \times \mathbf{X} + (-0.1)$ ; 95% CI Slope: 1.0, 1.0; 95% CI Intercept: -0.2,0.1).
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# b. Specimen and Collection Tube Type Equivalency
To demonstrate equivalent performance between serum and plasma collection tube types, 25 matched sets of HCV- negative and positive samples were obtained in each of the following sixprimary collection tubes and tested using the Alinity m HCV Assay: plasma preparation tubes (PPT), K2 EDTA, K3 EDTA, ACD, serum tubes, and serum separation tubes (SST). To generate the positive samples aliquots of each of the 25 sets were spiked with a high positive clinical sample at a concentration of (a) $3\mathrm{x}$ LLoQ and (b) $4\log \mathrm{IU / mL}$ HCV.
Serum (serum and SST) and plasma collection tubes (K2 EDTA, K3 EDTA, ACD and PPT) demonstrated $100\%$ positive and negative agreement both at 3x LLoQ and at 4 log IU/mL. For the 3x LLoQ samples the mean difference between the test condition and the control serum tube across all tube types ranged from -0.04 to 0.05 log IU/mL. For HCV positive samples targeted to 10,000 IU/mL the largest $95\%$ confidence interval of the mean difference between each test condition and the control condition was -0.17, 0.11 Log IU/mL with a maximum standard deviation of 0.34 log IU/mL. The largest mean difference between the test condition and the control serum tube across all tube types was 0.08 log IU/mL. The results are summarized in Table 11 below.
Table 11: Matrix Equivalency Study - Summary
| Panel Member | Collection Tube | Test Condition | | | Mean Difference (Test-Control) | SD of Mean Difference | 95% CI of Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | N | Mean | SD | | | |
| Low Positive | Serum (Control Plasma) | 25 | 1.53 | 0.165 | N/A | N/A | N/A |
| | Serum (Control Serum) | 25 | 1.52 | 0.119 | N/A | N/A | N/A |
| | ACD-A | 25 | 1.57 | 0.102 | 0.05 | 0.156 | N/P |
| | K3 EDTA | 25 | 1.56 | 0.146 | 0.04 | 0.191 | N/P |
| | K2 EDTA | 25 | 1.53 | 0.160 | 0.01 | 0.157 | N/P |
| | PPT | 25 | 1.49 | 0.131 | -0.03 | 0.129 | N/P |
| | Serum Rapid-Clot Tube | 25 | 1.56 | 0.167 | 0.03 | 0.163 | N/P |
| | SST | 25* | 1.49 | 0.163 | -0.04 | 0.232 | N/P |
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| Panel Member | Collection Tube | Test Condition | | | Mean Difference (Test-Control) | SD of Mean Difference | 95% CI of Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | N | Mean | SD | | | |
| High Positive | Serum (Control Plasma) | 25 | 3.94 | 0.186 | N/A | N/A | N/A |
| | Serum (Control Serum) | 25 | 3.89 | 0.088 | N/A | N/A | N/A |
| | ACD-A | 25 | 3.97 | 0.065 | 0.08 | 0.108 | (0.03, 0.12) |
| | K3 EDTA | 25 | 3.89 | 0.161 | 0.00 | 0.179 | (-0.07, 0.08) |
| | K2 EDTA | 25 | 3.93 | 0.108 | 0.04 | 0.115 | (-0.00, 0.09) |
| | PPT | 25 | 3.95 | 0.096 | 0.05 | 0.140 | (-0.00, 0.11) |
| | Serum Rapid-Clot Tube | 25 | 3.91 | 0.228 | -0.03 | 0.340 | (-0.17, 0.11) |
| | SST | 25 | 3.98 | 0.065 | 0.04 | 0.172 | (-0.03, -0.11) |
N/P = Not provided; N/A – Not Applicable.
* one replicate was detected but below the LLoQ, however, the software returned a value that was used in calculation of the mean and SD (such a result in the final software would not be reported with a value)
## 11. Validation of Dilution Procedure
### a. Confirmation of the LLoQ in Diluted Specimens
The LLOQ for the Alinity m HCV assay in diluted samples was confirmed in serum and plasma by testing 2 panel members for each dilution factor targeting HCV concentrations at the assay's LLOQ and a level above LLOQ. HCV positive clinical specimen(s) were used to prepare panel members in HCV negative serum and plasma. Each panel member was diluted using the Specimen Diluent Kit per the instructions for use. 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. Results are summarized in Table 12.
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Table 12: LLoQ Verification for Specimens in Specimen Diluent
| Matrix | Panel Member | Target Concentration Undiluted (Log IU/mL) | Dilution Factor | Target Concentration Diluted (Log IU/mL) | Tested | Detected | Mean Concentration (Log IU/mL) | Bias (Log IU/mL) | SD | TAE | TE |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Plasma | 1 | 1.48 | 2.5 | 1.08 | 44 | 44* | 1.55 | 0.07 | 0.19 | 0.45 | 0.53 |
| Plasma | 2 | 1.57 | 2.5 | 1.18 | 45 | 45 | 1.91 | 0.34 | 0.15 | 0.64 | 0.42 |
| Plasma | 3 | 2.78 | 50 | 1.08 | 45 | 45 | 2.71 | -0.07 | 0.2 | 0.48 | 0.57 |
| Plasma | 4 | 2.88 | 50 | 1.18 | 43 | 43 | 2.84 | -0.04 | 0.22 | 0.48 | 0.62 |
| Serum | 1 | 1.48 | 2.5 | 1.08 | 45 | 45 | 1.55 | 0.07 | 0.16 | 0.4 | 0.46 |
| Serum | 2 | 1.57 | 2.5 | 1.18 | 45 | 45 | 1.64 | 0.07 | 0.16 | 0.39 | 0.46 |
| Serum | 3 | 2.78 | 50 | 1.08 | 45 | 45 | 2.72 | -0.06 | 0.2 | 0.47 | 0.58 |
| Serum | 4 | 2.88 | 50 | 1.18 | 45 | 45 | 2.9 | 0.02 | 0.22 | 0.45 | 0.62 |
* Without considering the dilution factor one replicate was detected but not quantifiable per the assays established LLOQ (i.e., < 1.08 log IU/mL). All other replicates were above 1.08 log IU/mL though some replicates were below a concentration of 1.48 log IU/mL – which is equivalent to the final concentration of a sample that returns a result equal to the LLoQ when diluted by factor 2.5 (i.e., 1.08 log IU/mL x 2.5 = 1.48 log IU/mL or 30 IU/mL)
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b. Quantitation of Manually Diluted Specimens
This study was designed to demonstrate that the Alinity m HCV assay provides accurate quantitation of specimens when tested using the manual dilution procedures of 1:2.5 and 1:50 as recommended as an option in the package insert.
Ten plasma panel members and 10 serum panel members, ranging from 2.0 to 8.3 Log IU/mL, were tested for each of the dilution procedures. Panel members were prepared by spiking unique HCV negative plasma/serum with a high titer HCV patient specimen or HCV Armored RNA to a final concentration ranging from 2.0 Log IU/mL to 8.3 Log IU/mL. HCV Armored RNA was only used for panel members with high concentrations (≥4.5 Log IU/mL). Panel members were then manually diluted in the Specimen Diluent buffer provided in the Alinity m Specimen Dilution Kit. Dilutions of 1:2.5 and/or 1:50 were performed according to the instructions for use. For each HCV panel member, 5 undiluted replicates and 5 replicates diluted in Specimen Diluent were tested and compared.
Note that not all panel members were tested with both dilutions.
Correct quantitation was assessed by calculating the difference between the mean quantitation results from the diluted and the undiluted replicates, and the two-sided 95% confidence interval (CI) of the difference in means. Results are summarized in Table 13.
The standard deviation of the panel members in both matrices was consistent with the precision of the Alinity m HCV (i.e., was <0.2 log IU/mL) with the exception of the 3.0 log IU/mL panel member in plasma that showed a slightly larger difference of +0.38 log IU/mL and a standard deviation of 0.25 log IU/mL (slightly above the SD of the precision panel member with the lowest precision). However, considering the relatively small number of replicates in this study (versus the number of replicates in the precision study) and the concentration of panel member 3, the observed differences are clinically insignificant. This study demonstrates that the Alinity m HCV can accurately quantify specimens diluted 1:2.5 and 1:50 as recommended in the package insert (i.e., the obtained viral load of diluted samples is comparable to the viral load as determined in the neat, undiluted specimen).
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Table 13: Quantitation of Manual Diluted Plasma and Serum Samples
| Dilution Factor | Panel | Diluted | | | Undiluted (Neat) | | | Mean Difference (Test - Control) | 95% CI of Mean Difference |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | N | Mean (Log IU/mL) | SD | N | Mean (Log IU/mL) | SD | | |
| PLASMA | | | | | | | | | |
| 2.5 | 0 | 5 | 2.06 | 0.123 | 5 | 1.98 | 0.077 | 0.08 | (-0.07, 0.23) |
| | 1 | 5 | 2.51 | 0.068 | 5 | 2.42 | 0.155 | 0.09 | (-0.08, 0.26) |
| | 2 | 5 | 3.05 | 0.084 | 5 | 3.00 | 0.045 | 0.05 | (-0.05, 0.15) |
| | 4 | 5 | 3.59 | 0.090 | 5 | 3.52 | 0.066 | 0.07 | (-0.04, 0.18) |
| | 5 | 5 | 4.05 | 0.077 | 5 | 4.15 | 0.058 | -0.09 | (-0.19, 0.01) |
| | 6 | 5 | 4.56 | 0.053 | 5 | 4.72 | 0.027 | -0.17 | (-0.23, -0.11) |
| | 7 | 5 | 5.07 | 0.057 | 4a | 5.24 | 0.044 | -0.17 | (-0.25, -0.09) |
| | 8 | 5 | 5.59 | 0.057 | 5 | 5.71 | 0.048 | -0.11 | (-0.19, -0.04) |
| | 9 | 5 | 6.61 | 0.083 | 5 | 6.77 | 0.041 | -0.16 | (-0.26, -0.07) |
| | 10 | 5 | 7.45 | 0.055 | 5 | 7.42 | 0.038 | 0.03 | (-0.04, 0.10) |
| 50 | 2 | 5 | 2.83 | 0.268 | 5 | 3.00 | 0.045 | -0.17 | (-0.50, 0.16) |
| | 3 | 5 | 3.31 | 0.173 | 5 | 3.33 | 0.101 | -0.02 | (-0.23, 0.18) |
| | 4 | 5 | 3.42 | 0.206 | 5 | 3.52 | 0.066 | -0.1 | (-0.36, 0.15) |
| | 5 | 5 | 3.96 | 0.121 | 5 | 4.15 | 0.058 | -0.18 | (-0.32, -0.04) |
| | 6 | 5 | 4.4 | 0.079 | 5 | 4.72 | 0.027 | -0.33 | (-0.41, -0.24) |
| | 7 | 5 | 4.9 | 0.062 | 4a | 5.24 | 0.044 | -0.34 | (-0.43, -0.25) |
| | 8 | 5 | 5.44 | 0.061 | 5 | 5.71 | 0.048 | -0.26 | (-0.34, -0.18) |
| | 9 | 5 | 6.46 | 0.086 | 5 | 6.77 | 0.041 | -0.32 | (-0.41, -0.22) |
| | 10 | 5 | 7.23 | 0.087 | 5 | 7.42 | 0.038 | -0.19 | (-0.29, -0.10) |
| | 11 | 5 | 8.31 | 0.08 | 5 | 8.67 | 0.015 | -0.35 | (-0.45, -0.25) |
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| SERUM | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 1 | 5 | 2.13 | 0.048 | 4^{a} | 1.86 | 0.099 | 0.26 | (0.15, 0.38) |
| | 2 | 5 | 2.53 | 0.041 | 5 | 2.39 | 0.079 | 0.14 | (0.05, 0.23) |
| | 3 | 5 | 3.04 | 0.063 | 5 | 2.66 | 0.252 | 0.38 | (0.07, 0.68) |
| | 4 | 5 | 3.51 | 0.144 | 5 | 3.52 | 0.075 | -0.02 | (-0.19, 0.15) |
| | 5 | 5 | 4.05 | 0.068 | 5 | 3.95 | 0.023 | 0.1 | (0.03, 0.17) |
| | 6 | 5 | 4.61 | 0.076 | 5 | 4.61 | 0.175 | 0 | (-0.20, 0.20) |
| | 7 | 5 | 5.09 | 0.033 | 5 | 5.14 | 0.033 | -0.06 | (-0.10, -0.01) |
| | 8 | 4^{a} | 6.44 | 0.026 | 5 | 6.63 | 0.065 | -0.19 | (-0.27, -0.11) |
| | 9 | 5 | 7.45 | 0.039 | 5 | 7.38 | 0.089 | 0.07 | (-0.03, 0.17) |
| | 7b | 5 | 5.56 | 0.068 | 5 | 5.65 | 0.043 | -0.09 | (-0.17, -0.01) |
| 50 | 3 | 4^{a} | 3.04 | 0.029 | 5 | 2.66 | 0.252 | 0.38 | (0.07, 0.69) |
| | 4 | 5 | 3.53 | 0.151 | 5 | 3.52 | 0.075 | 0 | (-0.17, 0.18) |
| | 5 | 5 | 3.92 | 0.078 | 5 | 3.95 | 0.023 | -0.04 | (-0.13, 0.06) |
| | 6 | 5 | 4.48 | 0.093 | 5 | 4.61 | 0.175 | -0.13 | (-0.34, 0.07) |
| | 7 | 5 | 4.94 | 0.064 | 5 | 5.14 | 0.033 | -0.21 | (-0.28, -0.13) |
| | 8 | 5 | 6.51 | 0.084 | 5 | 6.63 | 0.065 | -0.13 | (-0.24, -0.02) |
| | 9 | 5 | 7.26 | 0.057 | 5 | 7.38 | 0.089 | -0.12 | (-0.23, -0.01) |
| | 10 | 5 | 8.33 | 0.034 | 5 | 8.59 | 0.135 | -0.27 | (-0.43, -0.10) |
| | 3b | 5 | 3.15 | 0.147 | 5 | 3.24 | 0.054 | -0.09 | (-0.26, 0.07) |
| | 7b | 5 | 5.42 | 0.07 | 5 | 5.65 | 0.043 | -0.23 | (-0.32, -0.15) |
CI = Confidence Interval
<sup>a</sup> The missing replicate was invalid and was not retested
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c. Precision of Diluted Specimens
Precision was evaluated in both plasma and serum by testing 3 panel members (see Table 14), that were prepared with HCV concentrations, such that when diluted in Specimen Diluent, the concentrations (target concentration in Specimen Diluent) were within the linear range of the Alinity m HCV assay. Plasma and serum panel members were prepared by diluting a high titer HCV genotype 1 patient specimen (panel member 1) or HCV genotype 1 Armored RNA stock (panel members 2 and 3) into HCV negative human plasma and serum. Testing was performed with 3 Diluent lots (1 lot per Instrument/Operator). Results are summarized in Table 15.
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Table 14: Panel Target Concentrations and Testing Plan for Diluted Specimen Precision
| Panel Member | Concentration (Log IU/mL) | Dilution Factor Tested | Concentration after Specimen Dilution (Log IU/ mL) | Minimum Replicates per Run | Minimum Replicates per Day | Minimum Days | Minimum Replicates per Specimen Diluent Lot | Total Replicates |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2.9 | 1:2.5 | 2.5 | 3 | 2 | 12 | 72 | 216 |
| 2 | 7.2 | 1:50 | 5.5 | 3 | 2 | 12 | 72 | 216 |
| 3 | 5.7 | 1:50 | 4.0 | 3 | 2 | 12 | 72 | 216 |
Table 15: Precision of Diluted Plasma and Serum Specimens
| Matrix | Panel | N | Mean Concentration (Log IU/mL) | Within-Run Component | | Between-Run Component | | Between-Day Component | | Within-Laboratory^{a} | | Between-Instrument Component | | Total^{b} | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Plasma | 01 | 276 | 2.95 | 0.09 | 3.1 | 0.04 | 1.3 | 0.23 | 7.7 | 0.25 | 8.5 | 0.06 | 2.0 | 0.26 | 8.7 |
| | 02 | 284 | 7.20 | 0.08 | 1.1 | 0.01 | 0.1 | 0.02 | 0.3 | 0.08 | 1.1 | 0.06 | 0.9 | 0.10 | 1.4 |
| | 03 | 283 | 5.65 | 0.12 | 2.1 | 0.00 | 0.0 | 0.01 | 0.2 | 0.12 | 2.1 | 0.07 | 1.3 | 0.14 | 2.5 |
| Serum | 01 | 283 | 2.97 | 0.12 | 3.9 | 0.05 | 1.7 | 0.04 | 1.2 | 0.13 | 4.5 | 0.05 | 1.7 | 0.14 | 4.8 |
| | 02 | 282 | 7.13 | 0.12 | 1.6 | 0.02 | 0.2 | 0.03 | 0.4 | 0.12 | 1.7 | 0.09 | 1.2 | 0.15 | 2.1 |
| | 03 | 280 | 5.57 | 0.09 | 1.7 | 0.02 | 0.4 | 0.02 | 0.3 | 0.10 | 1.8 | 0.08 | 1.5 | 0.13 | 2.3 |
<a>^{a}</a> Within-Laboratory includes Within-Run, Between-Run and Between-Day Components.
<a>^{b}</a> Total includes Within-Run, Between-Run, Between-Day and Between-Instrument Components.
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d. On-Board Stability of Diluted Specimens
Onboard/off-board stability of the diluted sample was evaluated in both serum and plasma using some of the panel members described in Table 13 (above). The panel members were tested undiluted onboard the Alinity m System for a minimum of 4 hours prior to testing (control) and diluted and stored as follows: For each dilution factor, the undiluted panel member was placed onboard for a minimum of 4 hours, then diluted in Specimen Diluent (using factors 2.5 and 50), placed off-board (i.e., at 15°C to 30°C) for a minimum of 2 hours, and then placed onboard for a minimum of another 4 hours prior to testing.
The difference between the mean quantitation results from testing diluted specimens with onboard/off-board storage (test conditions 1 and 2) and the mean quantitation results from testing undiluted specimens (control condition) across all panel members, dilution factors and matrices ranged from -0.09 Log IU/mL to +0.26 Log IU/mL with standard deviations that were consistent with the precision of the test. The study confirmed on-board stability for diluted serum and plasma specimens.
12. Specimen Stability
Specimen stability studies demonstrated that, for the Alinity m HCV Assay, specimens should be stored as listed in Table 16 and can be stored in primary or secondary tubes.
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Table 16: 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 | 4 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. 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. 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. |
## 13. Reagent Stability
### a. Shelflife
Realtime stability studies were performed to establish the shelf-life for the Alinity m HCV assay. Three (3) lots of the following reagent kits were stored at the intended storage temperature indicated in Table 17 and then tested at various time points throughout the study. Performance was assessed against clinically relevant acceptance criteria using controls and calibrators and an internal stability panel consisting of three panel members between 3xLLOQ of the Alinity m HCV and 5 log IU/mL.
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The Shelflife 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 (15) months after the date of manufacture including when shipped upon exposure to fluctuating temperature extremes. Shelflife conditions are summarized in Table 17.
## b. 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 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. On-board storage conditions are summarized in Table 17
Table 17: Reagent Shelf Life and On-Board Stability for the Alinity m HCV and Alinity m Accessory Kits
| Kit/Accessory | Intended Storage Condition (Shelf Life) | On-Board Storage |
| --- | --- | --- |
| HCV AMP Kit | 15 months
2°C to 8°C | 30 days |
| HCV CTRL Kit | 15 months
-15°C to -25°C | 4 hours |
| HCV CAL Kit | 15 months
-15°C to -25°C | 4 hours |
| Sample Prep Kit 2
(Elution Buffer 2, Microparticles 2) | 12 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 | 18 months | None
(single unit packaged) |
Expiration dating for the Alinity m HCVassay has been established and approved at 15 months when reagents are stored at the intended storage conditions.
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# 14. Antimicrobial Effectiveness (AET)
Testing was performed for the Alinity m HCV assay for all reagents that contained preservatives. 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. Nine different bacteria and yeast were tested. Results of the study demonstrated the effectiveness of the preservatives in the preservative containing reagents.
# B. Animal Studies
None
# C. Additional Studies
Table 18: Electromagnetic Compatibility (EMC) Testing
| Test | Acceptance Criteria | Results |
| --- | --- | --- |
| Radiated Emissions Testing | Met Class A, Group 1 limits | Passed |
| AC Mains conducted emissions | Met Class A, Group 1 limits | Passed |
| Electrostatic Discharge | Contact: at ±4 kV, both polarities | Passed |
| | Contact: at ±8 kV, both polarities | Passed |
| | Air: up to 15 kV, both polarities | Passed |
| Radiated Radio-Frequency, Electromagnetic Immunity | Frequencies up to 2700 MHz | Passed |
| Conducted Disturbances, RF Electromagnetic Immunity | up to 6 Vrms in the ISM bands. | Passed |
# X. SUMMARY OF PRIMARY CLINICAL STUDIES
The applicant performed the following two clinical studies to establish a reasonable assurance of safety and effectiveness for HCV testing with the Alinity m HCV assay when used as an aid in the diagnosis of active HCV infection in individuals with antibody evidence of HCV infection, and to aid in the management of patients with known active HCV infection, including SVR determination:
- Diagnostic Utility Study:
Evaluation of the ability of the assay to correctly diagnose anti-HCV positive subjects with active HCV infection (Section 2, below)
- Patient Management Study:
Evaluation of the ability of the assay to predict clinical outcome in patients undergoing treatment (Section 1, below)
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Data from these clinical studies were the basis for the PMA approval decision. A summary of the clinical studies is presented below.
## DIAGNOTIC UTILITY STUDY
### A. Study Design
Patient samples were tested between June 2019 and August 2019. The database for this PMA reflected data collected through August 19, 2019, and included a total of 522 patients. There were three investigational sites.
The study was designed to evaluate the ability of the assay to correctly diagnose HCV antibody positive subjects with active HCV infection. The study consisted of two parts:
1. Testing HCV antibody positive subjects of unknown HCV RNA status (Diagnostic Population) to determine whether they have active HCV infection, and
2. Testing subjects with non-HCV related liver disease to determine specificity of the Alinity m HCV for HCV related liver disease.
### a. Testing of HCV Antibody Positive Subjects of Unknown HCV RNA Status
Plasma or serum specimens were collected from 307 subjects who were at risk for HCV and tested positive for HCV antibodies with an FDA approved HCV antibody test (i.e. individuals with antibody evidence of HCV with evidence of liver disease, individuals suspected to be actively infected with HCV antibody evidence, individuals at risk for HCV infection with antibodies to HCV). 5 subjects were excluded. All specimens were tested with a FDA approved HCV nucleic acid test to establish the patient infected status (PIS). All specimens were then tested across four Alinity m Systems and three clinical sites.
According to AASLD guidelines¹, an FDA-approved NAAT with a detection level of 25 IU/mL or lower should be used to confirm a positive HCV infection status following a reactive HCV antibody test result. The patient infected status (PIS) for the tested cohort was defined as “Active HCV Infection” if the HCV antibody was positive and the FDA approved HCV nucleic acid test result was ≥25 IU/mL. The PIS was defined as “Resolved HCV Infection” if the HCV antibody was positive and the FDA approved HCV nucleic acid test result was <25 IU/mL. Specimens with Alinity m HCV result ≥25 IU/mL were considered positive. Specimens with Alinity m HCV <25 IU/mL were considered negative.
### b. Testing of Subjects with Non-HCV Related Liver Diseases
The cross-reactivity of Alinity m HCV was evaluated with 215 plasma and serum specimens from subjects with hepatitis not caused by HCV. Specimens were retrospectively collected by specimen vendors using vendor IRB approved consent forms and collection protocols or were de-identified remnant specimens not requiring
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consent. All the specimens were tested across 4 Alinity m Systems and 3 clinical sites. Six (6) samples were excluded.
# Clinical Endpoints
With regard to safety, as an in vitro diagnostic test, the Alinity m HCV test involves taking a sample of plasma or serum from a patient. The test, therefore, presents no more safety hazard to an individual being tested than other tests where blood samples are drawn. Safety issues regarding false positive and negative test results are discussed in section VIII.
With regard to effectiveness, the clinical performance of the Alinity m HCV in this study was evaluated versus the HCV infected status of patients actively infected with HCV. Patient infected status was determined by FDA approved tests for antibodies to HCV and HCV viral load.
# B. Accountability of PMA Cohort
The following Figure accounts for subjects in the Diagnostic Study (Dx).
Figure 4: Accountability Diagnostic Study Cohort
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a. Testing of HCV Antibody Positive Subjects of Unknown HCV RNA Status At the time of database lock, of 307 patients enrolled in the PMA study, $98\%$ (302) patients were available for analysis at the completion of the study.
# b. Testing of Subjects with Non-HCV Related Liver Diseases
At the time of database lock 215 subjects were enrolled in the PMA study. Four subjects did not meet the inclusion criteria (i.e., being negative for HCV) and were excluded. A total of 211 Anti-HCV – /HCV RNA – subjects were available for testing with the Alinity m HCV. Of these 211 subjects 209 subjects produced a valid result with the Alinity m HCV test and were included. Therefore, $97.2\%$ (209) of subjects were available for analysis at the completion of the study.
# C. Study Population Demographics and Baseline Parameters
The demographics of the study population are typical for a HCV Diagnostic Study performed in the US. Demographics and baseline characteristics of the Diagnostic Study subjects are presented in Table 19 for the Diagnostic population and Table 20 for the subjects with non-HCV related liver disease.
Table 19: Demographics of HCV Antibody Positive Subjects (Diagnostic Population)
| Characteristics | Category | N (%) |
| --- | --- | --- |
| Age (years) | Mean | 50.2 |
| | Median | 52.0 |
| | SD | 11.42 |
| | <21 | 3 (1.0) |
| | 21 - 49 | 118 (38.4) |
| | 50 - 70 | 183 (59.6) |
| | >70 | 3 (1.0) |
| Gender | Female | 80 (26.1) |
| | Male | 227 (73.9) |
| Race | Asian | 1 (0.3) |
| | Black | 159 (51.8) |
| | White | 62 (20.2) |
| | Mixed | 2 (0.7) |
| | Other | 83 (27.0) |
| Ethnicity | Hispanic/Latino | 83 (27.0) |
| | Non-Hispanic/Latino | 224 (73.0) |
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| Characteristics | Category | N (%) |
| --- | --- | --- |
| Risk Factor | Baby Boomers (Born: 1945 - 1965) | 28 (9.1) |
| | IV Drug Users | 116 (37.8) |
| | Baby Boomers and IV Drug Users | 153 (49.8) |
| | Other* | 10 (3.3) |
| HCV Infection Status | HCV RNA positive | 249 (81.1) |
| | HCV RNA negative | 58 (18.9) |
| Matrix | Plasma | 163 |
| | Serum | 144 |
| Total | | 307 |
| * neither Baby Boomers nor IV Drug Users | | |
Table 20: Demographics of Subjects with Non-HCV Related Liver Diseases
| Characteristics | Category | N (%) |
| --- | --- | --- |
| Age (years) | Mean | 56.0 |
| | Median | 58.0 |
| | SD | 13.52 |
| | 21 - 49 | 63 (29.9) |
| | 50 - 70 | 117 (55.5) |
| | > 70 | 30 (14.2) |
| | Not Available | 1 (0.5) |
| Gender | Female | 125 (59.2) |
| | Male | 86 (40.8) |
| Race | Asian | 42 (19.9) |
| | Black | 19 (9.0) |
| | White | 149 (70.6) |
| | Mixed | 1 (0.5) |
| Ethnicity | Hispanic/Latino | 13 (6.2) |
| | Non-Hispanic/Latino | 194 (91.9) |
| | Not Available | 4 (1.9) |
| Baby Boomers (Born: 1945 - 1965) | Yes | 105 (49.8) |
| | No | 70 (33.2) |
| | Not Available | 36 (17.1) |
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| Characteristics | Category | N (%) |
| --- | --- | --- |
| Hepatitis Conditions not Caused by HCV | Autoimmune hepatitis | 30 (14.2) |
| | Alcoholic liver disease | 49 (23.2) |
| | HBV | 44 (20.9) |
| | Primary biliary cirrhosis | 29 (13.7) |
| | Nonalcoholic steatohepatitis (NASH) | 30 (14.2) |
| | Fatty liver disease | 29 (13.7) |
| Total | | 211 |
## D. Safety and Effectiveness Results
### 1. Safety Results
The analysis of safety was based on the tested cohort of 511 analyzable patients in the Diagnostic Utility Study available for evaluation. There were no adverse effects during the study. As an in vitro diagnostic test, the Alinity m HCV test involves taking a sample of plasma or serum from a patient. The test, therefore, presents no more safety hazard to an individual being tested than other tests where blood samples are drawn.
### 2. Effectiveness Results
The analysis of effectiveness was based on the cohort of 511 patients in the Diagnostic Utility Study available for evaluation. Key effectiveness outcomes are presented in Table 21 and Table 22. The analysis of effectiveness of the Alinity m HCV assay assessed whether the Alinity m HCV can accurately diagnose active HCV infection in RNA positive and negative plasma and serum specimens from patients that are HCV antibody positive. The testing cohort included two sub-cohorts for which the analysis is shown below.
#### a. Testing of HCV Antibody Positive Subjects of Unknown HCV RNA Status
Results of the Testing of HCV antibody positive subjects for whom the RNA status (i.e., the status of an acute infection) is unknown is summarized in Table 21 below.
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Table 21: Agreement between Alinity m HCV and Patient Infected Status for HCV Antibody Positive Subjects Using the AASLD Recommended Threshold of 25* IU/mL
| | Patient Infected Status | | Total | |
| --- | --- | --- | --- | --- |
| | | Active HCV Infection | | Resolved HCV Infection |
| Alinity m HCV | Positive* | 226 | 4** | 230 |
| | Negative* | 0 | 72 | 72 |
| Total | | 226 | 76 | 302 |
| % Positive Agreement (95% CI) | | 100.0% (98.3%, 100.0%) | | |
| % Negative Agreement (95% CI) | | 94.7% (87.2%, 97.9%) | | |
CI: Confidence Interval (Score Method)
* "Negative" if HCV RNA was detected but < 25 IU/mL (i.e., 1.40 log10 IU/mL); otherwise, assay result interpretation was defined as "Positive"
** All 4 samples were detected below 25 IU/mL by the FDA approved HCV viral load test but were detected above 25 IU/mL with the Alinity m HCV (between 1.72 and 2.2 log IU/mL).
This study demonstrates the clinical utility of the Alinity m HCV assay to correctly diagnose subjects with ongoing active HCV RNA infections and to distinguish them from subjects with inactive infections in a population with prior exposure to HCV (HCV antibody-positive serology).
## b. Testing of Subjects with Non-HCV Related Liver Diseases
Table 22 shows the Alinity m HCV assay specificity by liver disease and the distribution of test results across viral load categories. Using the AASLD recommended threshold of 25 IU/mL, the specificity was 100% in subjects with chronic hepatitis B virus (HBV), primary biliary cirrhosis, nonalcoholic steatohepatitis (NASH), and multiple liver diseases reported. HCV RNA was detected at very low levels (<LLoQ) in 1 subject with alcoholic liver disease and in 1 subject with chronic hepatitis B virus infection.
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Table 22: Distribution of Results in Subjects with Non-HCV Related Liver Diseases
| Liver Disease | % Specificity (95% CI^{a}) | Not Detected | <12 IU/mL (LLoQ) | 12 to <25 IU/mL | 25 IU/mL to 8 log10 IU/mL | >8 log10 IU/mL | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Autoimmune hepatitis | 100 (88.3-100) | 30 (100) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 30 |
| Alcoholic liver disease | 98 (89.5-99.7) | 49 (98) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 49 |
| Chronic HBV | 97.7 (88.2-99.6) | 43 (100) | 1 (2.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 44 |
| Primary biliary cirrhosis | 100 (88.3-100) | 29 (100) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 29 |
| NASH | 100 (88.7-100) | 30 (100) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 30 |
| Fatty Liver Disease | 100 (87.6-100) | 27 (100) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 27 |
| Total | 99.0 (96.6-99.7) | 207 (99.0) | 2 (1.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 209* |
* Specimens from two subjects did not produce a valid Alnity HCV result and were excluded
3. **Subgroup Analyses**
The study design enabled an assessment of assay performance by subgroup as depicted in Table 22 above.
4. **Pediatric Extrapolation**
In this premarket application, existing clinical data was not leveraged to support approval of a pediatric patient population.
E. **Financial Disclosure**
The Financial Disclosure by Clinical Investigators regulation (21 CFR 54) requires applicants who submit a marketing application to include certain information concerning the compensation to, and financial interests and arrangement of, any clinical investigator conducting clinical studies covered by the regulation. The pivotal clinical study included three investigators. None of the clinical investigators had disclosable financial interests/arrangements as defined in sections 54.2(a), (b), (c), and (f). The information provided does not raise any questions about the reliability of the data.
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# PATIENT MANAGEMENT STUDY
# A. Study Design
Clinical specimen testing was conducted between April 15, 2019 and August 20, 2019. The database for this PMA reflected testing data collected through August 20, 2019 and included serum and plasma specimens from 232 patients (61 patients with plasma samples; 171 patients with serum samples). There were 3 external investigational sites in the U.S. that collected and tested samples.
The study was a prospectively archived, multi-center clinical study. The study subjects had been treated with 8-week or 12-week therapeutic regimens containing Sofosbuvir or Mavyret. For each subject, specimens were collected from the following timepoints: Baseline, Week 4 On Treatment, End of Treatment, and End of Follow-up. Depending on the therapeutic regimen, the End of Follow-up timepoint was 12 or 24 weeks after the completion of the treatment. Specimens were tested across 4 Alinity m Systems and 3 clinical sites.
The distribution of subjects in different treatment regimens and with different genotypes is presented in Table 23; the table excludes four subjects whose genotype (GT) could not be verified (i.e., two GT 1 subjects, one GT 3 subject and one GT 4 subject were excluded per accountability section below).
Table 23: Patient Management Study - Genotype/Treatment Distribution
| Treatment* | GT 1 | GT2 | GT3 | GT4 | GT5 | Total |
| --- | --- | --- | --- | --- | --- | --- |
| Sofosbuvir Containing | 100 | 31 | 43 | 1 | 4 | 179 |
| - Harvoni ± Ribavirin | 100 | - | - | 1 | - | 101 |
| - Epclusa ± Ribavirin | - | 25** | 40 | - | - | 65** |
| - Sovaldi | - | 6 | 3 | - | - | 9 |
| - Daklinza | - | - | - | - | 4 | 4 |
| Mavyret | - | 20** | 33** | - | - | 53** |
| Total | 100 | 51 | 76 | 1 | 4 | 232 |
* Harvoni (Sofosbuvir + Ledipasvir); Epclusa (Sofosbuvir + Velpatasvir); Sovaldi (Sofosbuvir + Ribavirin); Daklinza (Sofosbuvir + daclatasvir); Mavyret (Glecaprevir + Pibrentasvir)
** One Epclusa treated subjects and 3 Mavyret treated subjects (one Gt 2 and two Gt 3) were excluded from SVR analysis in
Table 25 below because they were missing the EOF sample needed to determine SVR.
# Clinical Endpoints
With regard to safety, as an in vitro diagnostic test, the Alinity m HCV test involves taking a sample of plasma or serum from a patient. The test, therefore, presents no more safety hazard to an individual being tested than other tests where blood samples are
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drawn. Safety issues regarding false positive and negative test results are discussed in section VIII.
With regard to effectiveness, the clinical performance of the Alinity m HCV in the Patient Management Study was evaluated by determining sustained virological response (SVR) of patients actively infected with HCV and undergoing antiviral treatment with Direct Acting Antiviral agents. Clinical truth (i.e., SVR or Non-SVR) was established by testing with an FDA approved HCV viral load test.
## B. Accountability of PMA Cohort
The following Figure accounts for all subjects in the Patient Management Study.
Figure 5: Accountability Patient Management Cohort
At the time of database lock, of 236 chronic HCV infected patients enrolled in the PMA study, four subjects were excluded because their genotype could not be verified. A total of 98.3% (232) of patients were available for testing and 96.6% (228) of the patients were available for SVR analysis at the completion of the study.
## C. Study Population Demographics and Baseline Parameters
The demographics of the study population are typical for a HCV Patient Management study performed in the US. Demographics and baseline characteristics of the patient management study subjects are presented in Table 24.
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Table 24: Demographics of Actively HCV Infected Subjects (Management Population)
| Characteristics | Category | N (%) |
| --- | --- | --- |
| Age | < 40 years | 44 (19.0) |
| | ≥ 40 years | 188 (81.0) |
| Gender | Female | 88 (37.9) |
| | Male | 144 (62.1) |
| Race | Asian | 9 (3.9) |
| | Black | 24 (10.3) |
| | White | 172…
