Alinity m HR HPV

P230003 · Abbott Molecular, Inc. · MAQ · Nov 1, 2023 · Microbiology

Device Facts

• Record ID: P230003
• Device Name: Alinity m HR HPV
• Applicant: Abbott Molecular, Inc.
• Product Code: MAQ · Microbiology
• Decision Date: Nov 1, 2023
• Decision: APPR
• Device Class: Class 3

Intended Use

Alinity m HR HPV is a qualitative in vitro test for the detection of Human Papillomavirus DNA in cervical specimens collected by a health care professional using an endocervical brush/spatula placed in ThinPrep PreservCyt Solution or an endocervical broom placed in SurePath Preservative Fluid. This test identifies high-risk (HR) HPV types 16, 18, 45, while reporting the concurrent detection of the other HR genotypes (31/33/52/58) and (35/39/51/56/59/66/68). Alinity m HR HPV is indicated for use in routine cervical cancer screening as per professional medical guidelines, including triage of ASC-US cytology, co-testing (adjunctive screening) with cytology, and HPV primary screening of women to assess the risk for cervical pre-cancer and cancer. Patients should be followed-up in accordance with professional medical guidelines, results from prior screening, medical history, and other risk factors.

Device Story

Alinity m HR HPV is an in vitro diagnostic assay for detecting 14 high-risk HPV genotypes in cervical specimens (ThinPrep or SurePath). Used on the Alinity m System, the device automates sample preparation (nucleic acid extraction via magnetic microparticles) and real-time PCR amplification/detection. The system processes samples in parallel using independent Assay Processing Units. Output includes qualitative detection of HPV16, 18, 45, and two groups of other HR genotypes (A: 31/33/52/58; B: 35/39/51/56/59/66/68), plus an endogenous human beta globin control. Operated by laboratory technicians in clinical settings, the system provides sample-to-result workflow. Healthcare providers use results alongside cytology and clinical history to guide patient management, such as colposcopy referral, potentially benefiting patients through early detection of cervical pre-cancer/cancer.

Clinical Evidence

Prospective, multi-center study of 14,935 women (≥25 years) in Screening and Enriched populations. Performance evaluated against CPRP-adjudicated histologic diagnosis (≥CIN2, ≥CIN3). Clinical sensitivity and false positivity rate (FPR) ratios compared to an FDA-approved HPV test. In Screening population, sensitivity ratio for ≥CIN3 was 1.00 (ThinPrep/SurePath). Genotype agreement with NGS ranged from 84% to 100%. No adverse events attributed to the assay.

Technological Characteristics

Real-time PCR assay; detects 14 HR HPV genotypes and human beta globin. Uses magnetic microparticle-based nucleic acid extraction. Reagents: Alinity m HR HPV AMP Kit, CTRL Kit, Sample Prep Kit 1. System: Alinity m System (automated molecular analyzer). Connectivity: Networked/integrated. Software: Application Specification File (JSON format) controls instrument parameters and data reduction algorithms.

Indications for Use

Indicated for routine cervical cancer screening in women ≥ 25 years, including ASC-US cytology triage, co-testing with cytology, and HPV primary screening to assess cervical pre-cancer/cancer risk. No known contraindications.

Reference Devices

• Roche cobas HPV Test
• Aptima HPV Assay
• Qiagen Hybrid Capture 2 High-Risk HPV DNA Test
• BD Onclarity HPV Assay

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: In vitro polymerase chain reaction (PCR)-based assay for detection of Human Papillomavirus (HPV) DNA.

Device Trade Name: Alinity m HR HPV (used on Alinity m System)

Device Procode: MAQ

Applicant’s Name and Address: Abbott Molecular Inc.
1300 E. Touhy Ave
Des Plaines, IL 60018

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P230003

Date of FDA Notice of Approval: November 1, 2023

II. INDICATIONS FOR USE

Alinity m HR HPV is a qualitative in vitro test for the detection of Human Papillomavirus DNA in cervical specimens collected by a health care professional using an endocervical brush/spatula placed in ThinPrep PreservCyt Solution or an endocervical broom placed in SurePath Preservative Fluid. This test identifies high-risk (HR) HPV types 16, 18, 45, while reporting the concurrent detection of the other HR genotypes (31/33/52/58) and (35/39/51/56/59/66/68).

Alinity m HR HPV is indicated for use in routine cervical cancer screening as per professional medical guidelines, including triage of ASC-US cytology, co-testing (adjunctive screening) with cytology, and HPV primary screening of women to assess the risk for cervical pre-cancer and cancer. Patients should be followed-up in accordance with professional medical guidelines, results from prior screening, medical history, and other risk factors.

III. CONTRAINDICATIONS

There are no known contraindications.

IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the Alinity m HR HPV labeling.

V. DEVICE DESCRIPTION

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The Alinity m HR HPV assay utilizes real-time PCR to amplify and detect DNA from 14 HR HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and human genomic DNA sequences that have been extracted from cervical specimens. Cervical specimens collected in ThinPrep PreservCyt Solution or SurePath Preservative Fluid can be used with Alinity m HR HPV assay.

The Alinity m HR HPV assay consists of two assay-specific components:

- Alinity m HR HPV AMP Kit (two trays, AMP TRAY 1 and 2)
- Alinity m HR HPV CTRL Kit

Additional necessary components include:

- Alinity m Sample Prep Kit 1
- Alinity m Tubes and Caps
- Alinity m System Solutions
- Alinity m System

The steps of the Alinity m HR HPV assay consist of sample preparation, PCR assembly, amplification/detection, and result calculation and reporting. All steps of the Alinity m HR HPV assay procedure are executed automatically by the Alinity m System. No intermediate processing or transfer steps are performed by the user.

## Principles of Procedure

1. Sample Preparation (Nucleic acid extraction and purification)

Nucleic acids from specimens are extracted using the Alinity m Sample Prep Kit 1 and Alinity m System Solutions. Nucleic acid extraction (lysis) disrupts the biological matrix to release the nucleic acid materials and allows for nucleic acids to adhere to the surface of magnetic microparticles. The magnetic microparticle technology facilitates nucleic acid capture, wash and elution. Nucleic acids bound to the surface of the magnetic microparticles are washed multiple times to remove any substances from the sample (i.e., lipids, proteins, therapeutic drugs) and from earlier sample preparation (i.e., ethanol, proteinase) that may potentially interfere with the later amplification and detection processes. Purified nucleic acids are released from the magnetic microparticle surface by mixing microparticles with an elution buffer. The resulting purified nucleic acids are then combined with the liquid unit-dose activator reagent (AMP TRAY 1), lyophilized unit-dose Alinity m HR HPV amplification/ detection reagents (AMP TRAY 2) and transferred into a reaction vessel.

2. Nucleic acid amplification

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The lyophilized, unit-dose Alinity m HR HPV amplification/detection reagents are reconstituted following mixing with liquid unit-dose activator reagent, mixed with purified nucleic acids from the tested specimens. 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 HR HPV. Purified specimen nucleic acids are amplified by real-time polymerase chain reaction (PCR) using a mix composed of thermostable DNA polymerase, dNTPs, MgCl₂, and short oligonucleotide primers targeting the 14 HR-HPV targets and an endogenous human beta globin (BG) sequence. Amplification and detection of the BG sequence is a sample validity control for cell adequacy, sample extraction and amplification efficiency. The Alinity m HR HPV amplification/detection reagent also contains Uracil-DNA Glycosylase (UDG) as a control for contamination from amplicons containing uracil, which may be present in molecular laboratories.

## 3. Nucleic acid detection

Alinity m HR HPV assay primers allow for amplification of the 14 HR HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Within a single PCR well, the Alinity m HR HPV probes are labeled with different fluorophores that allow for genotype specific detection of HPV16, 18, and 45 while the remaining 11 HR HPV genotypes are detected as Other HR HPV A (HPV31/33/52/58 genotypes) or Other HR HPV B (HPV35/39/51/56/59/66/68). Amplification of BG is detected also reported separately using a uniquely labeled fluorescent probe. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence for the HPV targets and BG, respectively.

## 4. Assay Controls

Assay controls are tested at or above an established minimum frequency to help ensure that instrument and reagent performance remain satisfactory. During each control event, a negative control and a positive control are processed through sample preparation and real-time PCR procedures that are identical to those used for specimens.

## Instrumentation and Software

### 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.

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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 individual 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 eight 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 (24) 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).

## Alinity m Software

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, results are reviewed and released through the software user interface.

## Alinity m HR HPV 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 and is needed to run an Alinity m assay on the Alinity m System. The parameters determine how the software controls the instrument components to execute the selected assay. 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.

## Interpretation of Alinity m HR HPV test results

HPV16, 18, and 45 are detected using unique fluorophores and can be individually detected and reported in a specimen sample. The remaining 11 HR HPV genomes are amplified using unique oligonucleotide primers, but are detected and reported as two different groups, Other HR HPV A (HPV31/33/52/58) and Other HR HPV B (HPV35/39/51/56/59/66/68). Table 1 below summarizes reported result and interpretation by the Alinity m HR HPV assay on the Alinity m System.

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Table 1: Summary of possible Alinity genotype results and specimen interpretation

| Alinity m HR HPV Result | Interpretation |
| --- | --- |
| Negative | Negative |
| Negative | Negative |
| HPV16 | HR HPV Positive (16 Positive) |
| HPV18 | HR HPV Positive (18 Positive) |
| HPV45 | HR HPV Positive (45 Positive) |
| Other HR HPV A | HR HPV Positive (A Positive) |
| Other HR HPV B | HR HPV Positive (B Positive) |
| HPV16; HPV18 | HR HPV Positive (16;18 Positive) |
| HPV16; HPV45 | HR HPV Positive (16;45 Positive) |
| HPV16; Other HR HPV A | HR HPV Positive (16;A Positive) |
| HPV16; Other HR HPV B | HR HPV Positive (16;B Positive) |
| HPV18; HPV 45 | HR HPV Positive (18;45 Positive) |
| HPV18; Other HR HPV A | HR HPV Positive (18;A Positive) |
| HPV18; Other HR HPV B | HR HPV Positive (18;B Positive) |
| HPV45; Other HR HPV A | HR HPV Positive (45;A; Positive) |
| HPV45; Other HR HPV B | HR HPV Positive (45;B Positive) |
| Other HR HPV A; Other HR HPV B | HR HPV Positive (A;B Positive) |
| HPV16; HPV18; HPV45 | HR HPV Positive (16;18;45 Positive) |
| HPV16; HPV18; Other HR HPV A | HR HPV Positive (16;18;A Positive) |
| HPV16; HPV18; Other HR HPV B | HR HPV Positive (16;18;B Positive) |
| HPV16; HPV45; Other HR HPV A | HR HPV Positive (16;45;A; Positive) |
| HPV16; HPV45; Other HR HPV B | HR HPV Positive (16;45;B Positive) |
| HPV16; Other HR HPV A; Other HR HPV B | HR HPV Positive (16;A;B Positive) |
| HPV18; HPV45; Other HR HPV A | HR HPV Positive (18;45;A Positive) |
| HPV18; HPV45; Other HR HPV B | HR HPV Positive (18;45;B Positive) |
| HPV18; Other HR HPV A; Other HR HPV B | HR HPV Positive (18;A;B Positive) |
| HPV45; Other HR HPV A; Other HR HPV B | HR HPV Positive (45;A;B Positive) |

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are several other alternatives for the correction of cervical cancer precursors, including testing by cytology alone, co-testing with HPV alongside or as a follow-up to cytology, or HPV testing as a first line screening test for cervical cancer. There are also multiple FDA approved in vitro nucleic acid amplification tests for the qualitative measurement of HPV DNA in human specimens (i.e., Roche cobas HPV Test and cobas HPV assay, Aptima HPV Assay, Qiagen Hybrid Capture 2 High-Risk HPV DNA Test, BD Onclarity HPV Assay). Each alternative has its own advantages and disadvantages. A patient should fully

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discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

The woman's age, medical history and thorough physical examination will provide further information on the risk of cervical disease, as well as the need for referral to colposcopy. The Alinity m HR HPV assay should only be used in conjunction with this clinical information in accordance with appropriate clinical patient management guidelines.

## VII. MARKETING HISTORY

The product is currently distributed/marketed in 34 countries. The product has not been withdrawn to date from the market in any country for reasons related to the safety or effectiveness of the device. The Alinity m HR HPV AMP Kit and Alinity m HR HPV CTRL Kit are identical in formulation and use similar application specification files to the US kits and are marketed outside the US as listed in Table 2.

Table 2. Current distribution of the Alinity m HR HPV assay

| Australia | France | Portugal |
| --- | --- | --- |
| Austria | Germany | Romania |
| Belgium | Italy | Slovenia |
| Brazil | Latvia | South Africa |
| Bulgaria | Malaysia | Spain |
| Chile | Mexico | Sweden |
| Colombia | Montenegro | Switzerland |
| Croatia | Netherlands | Taiwan |
| Czech Republic | New Zealand | Thailand |
| El Salvador | Norway | United Kingdom |
| Estonia | Poland | Vietnam |
| Finland | | |

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

Below is a list of the potential adverse effects (e.g., complications) associated with the use of the device.

As with any in vitro diagnostic test, the potential adverse effects are associated with incorrect test results or result interpretations. Failure of this device to perform as expected or failure to correctly interpret results may lead to incorrect HPV test results and subsequently, improper patient management decisions in cervical cancer screening. False negative results may lead to delays in the timely diagnosis of cervical cancer, allowing an undetected condition to worsen and

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potentially increasing morbidity and mortality. False positive results could lead many women to unnecessarily undergo more frequent screening and potentially invasive procedures such as colposcopy and biopsy.

For the specific adverse events that occurred in the clinical study, please see Section X below.

## IX. SUMMARY OF NONCLINICAL STUDIES

### A. Laboratory Studies

#### 1. Clinical cutoff determination

The Alinity m HR HPV assay cutoffs were established using clinical specimens from subjects undergoing routine cervical cancer screening (Screening population) and subjects referred to colposcopy follow-up based on previous cervical screening that included HR HPV testing (Enriched population). The methods used for cutoff selection were chosen to achieve the maximum sensitivity for detecting ≥ CIN2 (Cervical Intraepithelial Neoplasia 2) disease while maintaining a clinically acceptable level of specificity. Based on these criteria, the clinical cutoff was set at a cycle number (CN) of 33 for HPV 16 and a CN of 32 for the remaining 13 HR HPV genotypes detected by the Alinity m HR HPV assay.

#### 2. Analytical Sensitivity

##### a. Limit of Detection at the Clinical Cutoff

The limit of detection (LoD) at the clinical cutoff of the Alinity m HR HPV assay was determined by testing plasmids for 14 HR HPV genotype sequences (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and HPV positive cell lines (HPV16 SiHa and HPV18 HeLa) in pooled HR HPV negative clinical specimens collected in ThinPrep PreservCyt Solution (ThinPrep clinical matrix) and in pooled HR HPV negative clinical specimens collected in SurePath Preservative Fluid (SurePath clinical matrix). Each plasmid and cell line were diluted to concentrations at, below, and above the expected LoD level for each matrix. HPV 16, 18, 45, 58 (Other HR HPV A), and 39 (Other HR HPV B) plasmids and SiHa and HeLa cell lines were tested with three amplification reagent lots; the remaining HR HPV plasmids (HPV 31, 33, 35, 51, 52, 56, 59, 66, and 68) were tested with two amplification reagent lots. For each lot, a minimum of 20 replicates were tested across three days for each plasmid and cell line target concentration in each matrix.

The LoD was defined as the lowest target concentration having a ≥ 95% detection rate with all higher concentrations having a > 95% detection rate. Table 3 and Table 4 list result from the reagent lot producing the most conservative (highest) LoD at the clinical cutoff in the analysis for 14 HR HPV genotypes in ThinPrep and SurePath, respectively. These results are reported at a

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concentration of cells/assay for the HPV positive cell lines and copies/assay for the 14 HR HPV plasmids. Displayed are the positivity rates and the 95% CI (confidence interval).

Table 3. Limit of Detection for HR HPVs in ThinPrep

| HPV Target | Concentration | Positivity Rate% | 95% CI |
| --- | --- | --- | --- |
| Cell line | Cells/assay | | |
| SiHa (HPV16) | 40 | 95.7 | (79.0%, 99.2%) |
| HeLa (HPV18) | 9.2 | 95.8 | 79.8%, 99.3%) |
| Plasmid | Copies/assay | | |
| HPV16 | 480 | 100.0 | 86.2%, 100.0%) |
| HPV18 | 120 | 100.0 | 86.2%, 100.0%) |
| HPV31 | 300 | 95.8 | 79.8%, 99.3%) |
| HPV33 | 600 | 100.0 | 86.2%, 100.0%) |
| HPV35 | 300 | 95.8 | 79.8%, 99.3%) |
| HPV39 | 9600 | 100.0 | 86.2%, 100.0%) |
| HPV45 | 120 | 100.0 | 86.2%, 100.0%) |
| HPV51 | 600 | 95.5 | 78.2%, 99.2%) |
| HPV52 | 2500 | 100.0 | 86.2%, 100.0%) |
| HPV56 | 300 | 100.0 | 86.2%, 100.0%) |
| HPV58 | 1200 | 95.8 | 79.8%, 99.3%) |
| HPV59 | 150 | 100.0 | 83.9%, 100.0%) |
| HPV66 | 150 | 100.0 | 83.9%, 100.0%) |
| HPV68 | 300 | 100.0 | 86.2%, 100.0%) |

Table 4. Limit of Detection for HR HPVs in SurePath

| HPV Target | Concentration | Positivity Rate% | 95% CI |
| --- | --- | --- | --- |
| Cell line | Cells/assay | | |
| SiHa (HPV16) | 80 | 100.0 | (86.2%, 100.0%) |
| HeLa (HPV18) | 4.8 | 95.7 | (79.0%, 99.2%) |
| Plasmid | Copies/assay | | |
| HPV16 | 120 | 95.8 | 79.8%, 99.3%) |
| HPV18 | 120 | 100.0 | 86.2%, 100.0%) |
| HPV31 | 300 | 100.0 | 86.2%, 100.0%) |
| HPV33 | 600 | 95.7 | 79.0%, 99.2%) |
| HPV35 | 600 | 100.0 | 86.2%, 100.0%) |
| HPV39 | 1200 | 100.0 | 86.2%, 100.0%) |
| HPV45 | 120 | 100.0 | 86.2%, 100.0%) |
| HPV51 | 600 | 95.7 | 79.0%, 99.2%) |
| HPV52 | 1250 | 100.0 | 86.2%, 100.0%) |
| HPV56 | 300 | 95.8 | 79.8%, 99.3%) |

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Table 4. Limit of Detection for HR HPVs in SurePath

| HPV Target | Concentration | Positivity Rate% | 95% CI |
| --- | --- | --- | --- |
| HPV58 | 300 | 100.0 | 83.9%, 100.0%) |
| HPV59 | 1200 | 100.0 | 85.7%, 100.0%) |
| HPV66 | 2400 | 100.0 | 85.1%, 100.0%) |
| HPV68 | 4800 | 100.0 | 83.9%, 100.0%) |

# 3. Analytical Specificity

# a. Cross-reactivity

The analytical specificity of the Alinity m HR HPV assay was evaluated with a panel of microorganisms that included low-risk HPV, bacteria, viruses, protozoan, and yeast as well as human cellular DNA (Table 5). Microorganisms were tested at $10^{5}$ Units/mL (viruses and eukaryotes) or $10^{6}$ Units/ml (bacteria), where the unit of measure is specific to each microorganism Human cellular DNA was tested at $10^{6}$ Copies/ml. Each potential cross reactant was tested with HR HPV negative samples in both ThinPrep and SurePath matrices. No cross reactivity or interference in Alinity m HR HPV assay performance was observed in the presence of the tested potential cross reactants in either sample type.

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Table 5. Microorganisms Tested

| Virus | | | | Bacteria continued | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Adenovirus | | | | Klebsiella pneumoniae ss ozaenae | | | |
| Cytomegalovirus (CMV) | | | | Lactobacillus acidophilus | | | |
| Epstein Barr Virus (EBV) | | | | Mycoplasma genitalium | | | |
| Hepatitis B virus (HBV) | | | | Mycoplasma hominis | | | |
| Herpes simplex virus I | | | | Neisseria gonorrhoeae | | | |
| Herpes simplex virus II | | | | Neisseria meningitidis Serogroup A | | | |
| Human herpes virus 3 | | | | Peptostreptococcus anaerobius | | | |
| Hepatitis C virus (HCV) | | | | Proteus mirabilis | | | |
| Human immunodeficiency virus (HIV-1) | | | | Pseudomonas aeruginosa | | | |
| Bacteria | | | | Staphylococcus aureus | | | |
| Bacteroides fragilis | | | | Staphylococcus epidermidis | | | |
| Bacteroides ureolyticus | | | | Streptococcus agalactiae | | | |
| Bifidobacterium adolescentis | | | | Streptococcus faecalis | | | |
| Chlamydia trachomatis | | | | Streptococcus pneumoniae | | | |
| Clostridium perfringens | | | | Streptococcus pyogenes | | | |
| Corynebacterium genitalium | | | | Treponema pallidum | | | |
| Enterococcus faecalis | | | | Ureaplasma urealyticum | | | |
| Enterobacter cloacae | | | | Yeast | | | |
| Escherichia coli | | | | Candida albicans | | | |
| Fusobacterium necrophorum | | | | Other | | | |
| Gardnerella vaginalis | | | | Human Cellular DNA | | | |
| Haemophilus ducreyi | | | | Trichomonas vaginalis | | | |
| Low-risk (LR) HPV genomes | | | | | | | |
| HPV6 | HPV26 | HPV34 | HPV43 | HPV54 | HPV61 | HPV70 | HPV85 |
| HPV11 | HPV30 | HPV40 | HPV44 | HPV55B | HPV67 | HPV73 | |
| HPV13 | HPV32 | HPV42 | HPV53 | HPV57 | HPV69 | HPV82 | |

## b. Interference

The effect of potentially interfering endogenous and exogenous substances that may be present in cervical specimens was assessed for the Alinity m HR HPV assay. Each substance was tested with HR HPV negative samples (above the corresponding cutoff) and HR HPV positive samples containing SiHa (HPV 16) and HeLa (HPV 18) cell lines at approximately 3X LoD in both ThinPrep and SurePath clinical matrices.

For both ThinPrep and SurePath, no interference was observed for any substance at the concentrations listed in Table 6. Assay interference was observed for Monistat-1 at concentrations higher than 0.75% in ThinPrep. Based on the reported CN values, detection of the 14 HR HPV genotypes for cervical samples stored in SurePath may be impacted by the presence of mucus at a concentration of 5% w/v.

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Table 6. Endogenous and Exogenous Substances Evaluated

| Endogenous Substance | Test Level |
| --- | --- |
| Whole Blood | 10.00% v/v |
| Mucus | 5.00% w/v^{b} |
| PBMC (leukocytes) | 10^{6} Cells/mL |
| Exogenous Substance | Test Level |
| Clotrimazole Vaginal Cream | 1.00% w/v |
| Terconazole Vaginal Cream 0.8% | 1.00% w/v |
| Monistat-1 | 0.75% w/v^{a} |
| KY Jelly Personal Lubricant | 1.00% w/v |
| KY Warming Liquid | 1.00% w/v |
| Zovirax (Acyclovir) | 1.00% w/v |
| Summer’s Eve Douche | 1.00% w/v |
| Norforms Deodorant Suppositories | 0.50% w/v |
| Metrogel-Vaginal | 0.50% w/v |
| Vaginal Contraceptive Foam (VCF)
Contraceptive Gel | 0.50% w/v |
| Options Gyncol II Vaginal Contraceptive Gel | 0.50% w/v |
| Hydrocortisone | 0.50% w/v |
| Sigma Acetic Acid | 0.50% w/v |
| Vagisil Dry Wash | 0.50% w/v |

a 1.00% w/v produced false negative results in ThinPrep clinical matrix.
b 5.00% w/v mucus may impact performance in SurePath clinical matrix

c. Competitive Inhibition

A study was performed to test whether high concentrations of HR HPV DNA could interfere with the genotyping capability of the Alinity m HR HPV assay to detect HPV types 16, 18, 45, Other HR HPV A (represented by HPV58), and Other HR HPV B (represented by HPV39) in ThinPrep and SurePath matrices. Each plasmid (at low concentration) was tested for detection in the presence of the other four competing, high concentration plasmids (at a concentration of 10⁵ copies/ml). Testing was done in ThinPrep and SurePath matrices containing C33A cells.

No competitive inhibition was detected in ThinPrep matrix.

In SurePath, detection of Other HR HPV A genotypes (represented by HPV58 during testing) was inhibited by the presence of HPV18 at 10⁵ copies/ml. Other HR HPV B genotypes (represented by HPV39 during testing) was inhibited by the presence of HPV16, 18, and 45 at a concentration of 10⁵ copies/ml. During retesting, Group A and Group B were detected when the competing targeting analyte was present at a concentration of 10⁴ copies/ml in SurePath.

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# 4. Precision

# Study 1

Within-laboratory precision of the Alinity m HR HPV assay was evaluated with a 22-member panel of samples in ThinPrep PreservCyt Solution and a 13-member panel of samples in SurePath Preservative Fluid. The ThinPrep panel included six pools made from clinical specimens collected in ThinPrep PreservCyt Solution and 16 contrived samples prepared with SiHa (HPV 16), HeLa (HPV 18), HPV 45, HPV 39, or HPV 58 plasmids in ThinPrep PreservCyt Solution containing HR HPV negative C33A cells. The SurePath panel included six pools made from clinical specimens collected in SurePath Preservative Fluid and seven contrived samples prepared with SiHa or HeLa in SurePath Preservative Fluid containing HR HPV negative C33A cells. For each sample type, one negative clinical pool and five moderate positive clinical pools representative of each genotype category (HPV 16, 18, 45, Other HR HPV A, and Other HR HPV B) were prepared. ThinPrep contrived panels consisted of high negative, low positive, and moderate positive target levels for each genotype category, in addition to a negative panel member. SurePath contrived panels consisted of high negative, low positive, and moderate positive target levels for SiHa and HeLa cell lines, in addition to a negative panel member. Each panel member was tested with two replicates, twice a day, for 12 days on three Alinity m Systems with three reagent lots.

A summary of the panel members and agreement rate relative to expected result (positive agreement for positive samples and negative agreement for negative samples), and analysis of CN variability are presented in Table 7 and Table 8 for the ThinPrep panel and Table 9 and Table 10 for the SurePath panel. For ThinPrep panel members, the total % CV (coefficient of variation) for target CN ranged from $1.4\%$ to $6.5\%$ across all genotypes at low and moderate positive levels (Table 8). For SurePath panel members, the total % CV for target CN ranged from $1.4\%$ to $5.7\%$ across all genotypes at low and moderate positive levels (Table 10).

Table 7. Summary of Within-laboratory Precision Study 1 Panel Members, Median CN, and Agreement Rates: ThinPrep

| Panel Composition | Panel Description | No. Valida | Median (CN)b | %Positive (n/N)c | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| Pooled negative clinical sample | Negative | 144 | NA | 0.0 (0/144) | 0.0 (0/144) | 100.0 (144/144) |
| HPV negative cell line | Negative | 144 | NA | 0.0 (0/144) | 0.0 (0/144) | 100.0 (144/144) |
| HPV16 positive SiHa cell line | HPV16 high negative | 144 | 32.86 | 56.9 (82/144) | 24.3 (35/144) | 18.8 (27/144) |
| HPV18 positive HeLa cell line | HPV18 high negative | 144 | 36.00 | 10.4 (15/144) | 2.8 (4/144) | 86.8 (125/144) |
| HPV45 plasmid | HPV45 high negative | 144 | 32.20 | 37.5 (54/144) | 61.1 (88/144) | 1.4 (2/144) |

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Table 7. Summary of Within-laboratory Precision Study 1 Panel Members, Median CN, and Agreement Rates: ThinPrep

| Panel Composition | Panel Description | No. \(Valid^a\) | Median (CN)\(^b\) | %Positive (n/N)\(^c\) | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| Other HR HPV Plasmid A\(^d\) | Other HR HPV A high negative | 144 | 36.00 | 17.4 (25/144) | 11.1 (16/144) | 71.5 (103/144) |
| Other HR HPV Plasmid B\(^e\) | Other HR HPV B high negative | 144 | 36.00 | 33.3 (48/144) | 9.0 (13/144) | 57.6 (83/144) |
| Pooled clinical sample | HPV 16 moderate positive | 144 | 27.67 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 18 moderate positive | 144 | 28.01 | 98.6 (142/144) | 0.0 (0/144) | 1.4 (2/144) |
| | HPV 45 moderate positive | 144 | 28.37 | 98.6 (142/144) | 0.0 (0/144) | 1.4 (2/144) |
| | Other HR HPV A moderate positive | 144 | 27.72 | 99.3 (143/144) | 0.0 (0/144) | 0.7 (1/144) |
| | Other HR HPV B moderate positive | 144 | 27.00 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| HPV 16 positive SiHa cell line | HPV 16 low positive | 144 | 28.89 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 16 moderate positive | 144 | 27.45 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| HPV 18 positive HeLa cell line | HPV 18 low positive | 144 | 28.29 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 18 moderate positive | 144 | 27.15 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| HPV 45 plasmid | HPV 45 low positive | 144 | 30.50 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 45 moderate positive | 144 | 29.95 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| Other HR HPV A Plasmid | Other HR HPV A low positive | 144 | 30.18 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | Other HR HPV A moderate positive | 144 | 29.96 | 99.3 (143/144) | 0.7 (1/144) | 0.0 (0/144) |
| Other HR HPV B Plasmid | Other HR HPV B low positive | 144 | 30.73 | 97.2 (140/144) | 0.0 (0/144) | 2.8 (4/144) |
| | Other HR HPV B moderate positive | 144 | 28.75 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |

a N is the number of valid replicates.
b The median CN value reported is calculated for all replicates measured in the negative and positive panel members, above and below the clinical cutoff. Negative replicates with no virus detected were assigned the last cycle of PCR (36.00). Due to $0\%$ virus detection, median CN was not reported for negative panels (NA)
c The percent agreement is based on the number of valid replicates with a CN value below the clinical cutoff.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

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Table 8. Variance Component Analysis Results for Within-Laboratory Precision Study 1: ThinPrep

| | | | | | Within-Run | | Between-Run | | Between-Day | | Between Instrument/Lot | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Panel Description | Nb | nc | Mean (CN) | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Pooled Clinical Sample | HPV16 moderate positive | 144 | 144 | 27.52 | 1.330 | 4.8 | 0.747 | 2.7 | 0.000 | 0.0 | 0.000 | 0.0 | 1.525 | 5.5 |
| | HPV 18 moderate positive | 144 | 142 | 27.67 | 1.425 | 5.1 | 0.344 | 1.2 | 0.362 | 1.3 | 0.213 | 0.8 | 1.525 | 5.5 |
| | HPV 45 moderate positive | 144 | 142 | 28.08 | 1.084 | 3.9 | 0.000 | 0.0 | 0.192 | 0.7 | 0.080 | 0.3 | 1.104 | 3.9 |
| | Other HR HPV A moderate positive | 144 | 143 | 27.17 | 1.510 | 5.6 | 0.852 | 3.1 | 0.000 | 0.0 | 0.310 | 1.1 | 1.762 | 6.5 |
| | Other HR HPV B moderate positive | 144 | 144 | 27.01 | 0.771 | 2.9 | 0.441 | 1.6 | 0.000 | 0.0 | 0.000 | 0.0 | 0.889 | 3.3 |
| HPV16 positive SiHa cell line | HPV 16 low positive | 144 | 144 | 28.89 | 0.378 | 1.3 | 0.087 | 0.3 | 0.175 | 0.6 | 0.000 | 0.0 | 0.425 | 1.5 |
| | HPV 16 moderate positive | 144 | 144 | 27.45 | 0.382 | 1.4 | 0.000 | 0.0 | 0.030 | 0.1 | 0.101 | 0.4 | 0.396 | 1.4 |
| HPV18 positive HeLa cell line | HPV 18 low positive | 144 | 144 | 28.30 | 0.752 | 2.7 | 0.000 | 0.0 | 0.205 | 0.7 | 0.204 | 0.7 | 0.806 | 2.8 |
| | HPV 18 moderate positive | 144 | 144 | 27.22 | 0.570 | 2.1 | 0.261 | 1.0 | 0.000 | 0.0 | 0.213 | 0.8 | 0.662 | 2.4 |
| HPV45 plasmid | HPV 45 low positive | 144 | 144 | 30.52 | 0.373 | 1.2 | 0.025 | 0.1 | 0.123 | 0.4 | 0.196 | 0.6 | 0.439 | 1.4 |
| | HPV 45 moderate positive | 144 | 144 | 29.96 | 0.373 | 1.2 | 0.000 | 0.0 | 0.172 | 0.6 | 0.090 | 0.3 | 0.421 | 1.4 |
| Other HR HPV A plasmidd | Other HR HPV A low positive | 144 | 144 | 30.23 | 0.386 | 1.3 | 0.000 | 0.0 | 0.133 | 0.4 | 0.000 | 0.0 | 0.409 | 1.4 |
| | Other HR HPV A moderate positive | 144 | 143 | 29.91 | 0.319 | 1.1 | 0.000 | 0.0 | 0.193 | 0.6 | 0.173 | 0.6 | 0.411 | 1.4 |
| Other HR HPV B plasmid e | Other HR HPV B low positive | 144 | 140 | 30.75 | 0.369 | 1.2 | 0.000 | 0.0 | 0.227 | 0.7 | 0.205 | 0.7 | 0.479 | 1.6 |
| | Other HR HPV B moderate positive | 144 | 144 | 28.83 | 0.327 | 1.1 | 0.000 | 0.0 | 0.146 | 0.5 | 0.167 | 0.6 | 0.395 | 1.4 |

Because only positive, detected test results with CN values below the cutoff were included, estimates of SD (and % CV may be underestimated.
a N is the number of valid replicates.
b n is the number of valid replicates with a CN value below the clinical cutoff
c Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot Components
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

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Table 9. Summary of Within-laboratory Precision Study 1 Panel Members, Median CN, and Agreement Rates: SurePath

| Panel Composition | Panel Description | No. \(Valid^a\) | Median (CN)\(^b\) | %Positive (n/N)\(^c\) | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| Pooled negative clinical sample | Negative | 144 | NA | 0.0 (0/144) | 0.0 (0/144) | 100.0 (144/144) |
| HPV negative cell line | Negative | 144 | NA | 0.0 (0/144) | 0.0 (0/144) | 100.0 (144/144) |
| HPV16 positive SiHa cell line | HPV16 high negative | 144 | 36.00 | 25.0 (36/144) | 0.7 (1/144) | 74.3 (107/144) |
| HPV18 positive HeLa cell line | HPV18 high negative | 144 | 36.00 | 14.6 (21/144) | 0.0 (0/144) | 85.4 (123/144) |
| Pooled clinical sample | HPV 16 moderate positive | 144 | 28.39 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 18 moderate positive | 144 | 27.14 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 45 moderate positive | 144 | 28.24 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | Other HR HPV A moderate \(positive^d\) | 144 | 27.39 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | Other HR HPV B moderate \(positive^e\) | 144 | 28.22 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| HPV 16 positive SiHa cell line | HPV 16 low positive | 144 | 29.35 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| | HPV 16 moderate positive | 144 | 27.91 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |
| HPV 18 positive HeLa cell line | HPV 18 low positive | 144 | 28.07 | 97.2 (140/144) | 0.0 (0/144) | 2.8 (4/144) |
| | HPV 18 moderate positive | 144 | 26.77 | 100.0 (144/144) | 0.0 (0/144) | 0.0 (0/144) |

a N is the number of valid replicates.
b The median CN value reported is calculated for all replicates measured in the negative and positive panel members, above and below the clinical cutoff. Negative replicates with no virus detected were assigned the last cycle of PCR (36.00). Due to $0\%$ virus detection, median CN was not reported for negative panels (NA)
c The percent agreement is based on the number of valid replicates with a CN value below the clinical cutoff.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

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Table 10. Variance Component Analysis Results for Within-Laboratory Precision Study 1: SurePath

| | | | | | Within-Run | | Between-Run | | Between-Day | | Between Instrument/Lot | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Panel Description | Nb | nc | Mean (CN) | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Pooled Clinical Sample | HPV16 moderate positive | 144 | 144 | 28.23 | 0.743 | 2.6 | 0.449 | 1.6 | 0.167 | 0.6 | 0.140 | 0.5 | 0.896 | 3.2 |
| | HPV 18 moderate positive | 144 | 144 | 27.21 | 1.271 | 4.7 | 0.000 | 0.0 | 0.347 | 1.3 | 0.000 | 0.0 | 1.318 | 4.8 |
| | HPV 45 moderate positive | 144 | 144 | 28.18 | 0.441 | 1.6 | 0.222 | 0.8 | 0.158 | 0.6 | 0.029 | 0.1 | 0.520 | 1.8 |
| | Other HR HPV A moderate positive d | 144 | 144 | 26.93 | 1.493 | 5.5 | 0.000 | 0.0 | 0.330 | 1.2 | 0.132 | 0.5 | 1.535 | 5.7 |
| | Other HR HPV B moderate positive e | 144 | 144 | 28.19 | 0.600 | 2.1 | 0.304 | 1.1 | 0.212 | 0.8 | 0.000 | 0.0 | 0.705 | 2.5 |
| HPV16 positive SiHa cell line | HPV 16 low positive | 144 | 144 | 29.38 | 0.370 | 1.3 | 0.092 | 0.3 | 0.171 | 0.6 | 0.081 | 0.3 | 0.425 | 1.4 |
| | HPV 16 moderate positive | 144 | 144 | 27.93 | 0.309 | 1.1 | 0.117 | 0.4 | 0.160 | 0.6 | 0.110 | 0.4 | 0.383 | 1.4 |
| HPV18 positive HeLa cell line | HPV 18 low positive | 144 | 140 | 28.26 | 0.834 | 3.0 | 0.000 | 0.0 | 0.311 | 1.1 | 0.000 | 0.0 | 0.890 | 3.1 |
| | HPV 18 moderate positive | 144 | 144 | 26.71 | 0.483 | 1.8 | 0.192 | 0.7 | 0.000 | 0.0 | 0.000 | 0.0 | 0.520 | 1.9 |

Because only positive, detected test results with CN values below the cutoff were included, estimates of SD (and % CV may be underestimated.
a N is the number of valid replicates.
b n is the number of valid replicates with a CN value below the clinical cutoff
c Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot Components
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

## Study 2

An additional within-laboratory precision study was conducted with a six-member panel of contrived samples in SurePath Preservative Fluid. Contrived panel members were prepared with HPV 45, HPV 39, or HPV 58 plasmids in SurePath Preservative Fluid containing HR HPV negative C33A cells. Each HPV genotype was targeted to low positive and moderate positive target levels. Each panel member was tested with a minimum of two replicates twice each day for 12 days on each of three reagent lots on each of three instruments. A description of the panels, agreement rate relative to expected result (percent positive results), and analysis of CN variability are presented in Table 11 and 12 for the SurePath panel. The total % CV for CN ranged from 1.0% to 1.2% across all HPV genotypes targeted at low positive or higher (Table 12).

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Table 11. Summary of Precision Study 2 Panel Members, Median CN, and Agreement Rates: SurePath

| Panel Composition | Panel Description | No. \(Valid^a\) | Median (CN)\(^b\) | %Positive (n/N)\(^c\) | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| HPV45 Plasmid | HPV 45 low positive | 643 | 29.06 | 99.8 (642/363) | 0.2 (1/643) | 0.0 (0/643) |
| | HPV 45 moderate positive | 646 | 28.90 | 100.0 (646/646) | 0.0 (0/646) | 0.0 (0/646) |
| Other HR HPV A Plasmid \(^d\) | Other HR HPV A low positive | 647 | 30.76 | 97.4 (630/647) | 0.3 (2/647) | 2.3 (15/647) |
| | Other HR HPV A moderate positive | 645 | 29.77 | 99.5 (642/645) | 0.2 (1/645) | 0.3 (2/645) |
| Other HR HPV A Plasmid \(^e\) | Other HR HPV A low positive | 594 | 31.40 | 86.4 (513/594) | 5.4 (32/594) | 8.2 (49/594) |
| | Other HR HPV A moderate positive | 647 | 30.39 | 99.8 (646/647) | 0.0 (0/647) | 0.2 (1/647) |

a N is the number of valid replicates.
b The median CN value reported is calculated for all replicates measured in the negative and positive panel members, above and below the clinical cutoff. Negative replicates with no virus detected were assigned the last cycle of PCR (36.00).
c The percent agreement is based on the number of valid replicates with a CN value below the clinical cutoff.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

Table 12. Variance Component Analysis Results for Precision Study 2: SurePath

| | | | | | Within Run | | Between Run | | Between Day | | Between Lot | | Between Instrument | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Panel Description | Nb | nc | Mean (CN) | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| HPV45 Plasmid | HPV 45 low positive | 643 | 642 | 29.06 | 0.238 | 0.8 | 0.121 | 0.4 | 0.000 | 0.0 | 0.000 | 0.0 | 0.111 | 0.4 | 0.289 | 1.0 |
| | HPV 45 moderate positive | 646 | 646 | 28.90 | 0.247 | 0.9 | 0.067 | 0.2 | 0.061 | 0.2 | 0.044 | 0.2 | 0.170 | 0.6 | 0.316 | 1.1 |
| Other HR HPV A Plasmid \(^d\) | Other HR HPV A low positive | 647 | 630 | 30.76 | 0.314 | 1.0 | 0.084 | 0.3 | 0.000 | 0.0 | 0.065 | 0.2 | 0.057 | 0.2 | 0.336 | 1.1 |
| | Other HR HPV A moderate positive | 645 | 642 | 29.78 | 0.332 | 1.1 | 0.000 | 0.0 | 0.074 | 0.2 | 0.026 | 0.1 | 0.107 | 0.4 | 0.358 | 1.2 |
| Other HR HPV B Plasmid \(^e\) | Other HR HPV A low positive | 594 | 513 | 31.33 | 0.282 | 0.9 | 0.137 | 0.4 | 0.000 | 0.0 | 0.000 | 0.0 | 0.062 | 0.2 | 0.320 | 1.0 |
| | Other HR HPV A moderate positive | 647 | 646 | 30.39 | 0.306 | 1.0 | 0.046 | 0.2 | 0.075 | 0.2 | 0.098 | 0.3 | 0.093 | 0.3 | 0.346 | 1.1 |

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Because only positive, detected test results with CN values below the cutoff were included, estimates of SD (and % CV may be underestimated.

a N is the number of valid replicates.
b n is the number of valid replicates with a CN value below the clinical cutoff
c Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot Components
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

# 5. Lot-to-Lot Variability

The variability of Alinity m HR HPV reagent lots was assessed with HPV positive panels in ThinPrep and SurePath matrices containing HPV negative C33A cells. For both matrices, HPV positive panels were prepared with HPV 16, HPV 18, HPV 45, HPV 58, or HPV 39 plasmids at a low positive and moderate positive level. Each positive panel member was tested with three different reagent lots on one Alinity m System. A minimum of 20 replicates were tested per panel member per lot. For ThinPrep panel members, the total $\% \mathrm{CV}$ for HPV CN ranged from $1.1\%$ to $2.1\%$ across all genotypes (Table 13). For SurePath panel members, the total $\% \mathrm{CV}$ for HPV CN ranged from $1.0\%$ to $2.0\%$ across all genotypes (Table 14).

Table 13. Lot-to-Lot Precision Study: ThinPrep

| | | | | | | Within-Lot Component | | Between-Lot Component | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Target Level | Nb | nc | Detection Rate | Mean (CN) | SD | %CV | SD | %CV | SD | %CV |
| HPV 16 plasmid | Low positive | 72 | 72 | 100.0% | 29.94 | 0.314 | 1.0 | 0.097 | 0.3 | 0.329 | 1.1 |
| | Moderate positive | 72 | 72 | 100.0% | 28.90 | 0.301 | 1.0 | 0.109 | 0.4 | 0.321 | 1.1 |
| HPV 18 plasmid | Low positive | 72 | 72 | 100.0% | 28.87 | 0.347 | 1.2 | 0.190 | 0.7 | 0.395 | 1.4 |
| | Moderate positive | 72 | 72 | 100.0% | 27.99 | 0.310 | 1.1 | 0.109 | 0.4 | 0.328 | 1.2 |
| HPV 45 plasmid | Low positive | 72 | 72 | 100.0% | 30.08 | 0.315 | 1.0 | 0.108 | 0.4 | 0.333 | 1.1 |
| | Moderate positive | 72 | 72 | 100.0% | 29.15 | 0.300 | 1.0 | 0.132 | 0.5 | 0.328 | 1.1 |
| Other HR HPV A plasmidd | Low positive | 60 | 60 | 100.0% | 30.71 | 0.344 | 1.1 | 0.368 | 1.2 | 0.504 | 1.6 |
| | Moderate positive | 60 | 60 | 100.0% | 29.11 | 0.274 | 0.9 | 0.220 | 0.8 | 0.351 | 1.2 |
| Other HR HPV B plasmide | Low positive | 60 | 60 | 100.0% | 30.20 | 0.278 | 0.9 | 0.282 | 0.9 | 0.396 | 1.3 |
| | Moderate positive | 60 | 60 | 100.0% | 29.39 | 0.297 | 1.0 | 0.548 | 1.9 | 0.623 | 2.1 |

a Total includes Within-Lot and Between-Lot Components.
b N is the number of valid replicates.
c n is the number of positive tests, which contribute CN values to the variance component analysis.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

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Table 14. Lot-to-Lot Precision Study: SurePath

| | | | | | | Within-Lot Component | | Between-Lot Component | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Target Level | Nb | nc | Detectio n Rate | Mean (CN) | SD | %CV | SD | %CV | SD | %CV |
| HPV 16 plasmid | Low positive | 60 | 60 | 100.0% | 30.50 | 0.413 | 1.4 | 0.437 | 1.4 | 0.601 | 2.0 |
| | Moderate positive | 60 | 60 | 100.0% | 29.58 | 0.294 | 1.0 | 0.471 | 1.6 | 0.555 | 1.9 |
| HPV 18 plasmid | Low positive | 72 | 71 | 98.6% | 30.06 | 0.426 | 1.4 | 0.000 | 0.0 | 0.426 | 1.4* |
| | Moderate positive | 71 | 71 | 100.0% | 29.39 | 0.355 | 1.2 | 0.246 | 0.8 | 0.432 | 1.5 |
| HPV 45 plasmid | Low positive | 60 | 60 | 100.0% | 30.32 | 0.345 | 1.1 | 0.000 | 0.0 | 0.345 | 1.1 |
| | Moderate positive | 60 | 60 | 100.0% | 29.47 | 0.281 | 1.0 | 0.109 | 0.4 | 0.302 | 1.0 |
| Other HR HPV A plasmidd | Low positive | 60 | 59 | 98.3% | 30.84 | 0.351 | 1.1 | 0.285 | 0.9 | 0.452 | 1.5* |
| | Moderate positive | 60 | 60 | 100.0% | 30.00 | 0.265 | 0.9 | 0.248 | 0.8 | 0.363 | 1.2 |
| Other HR HPV B plasmide | Low positive | 60 | 58 | 96.7% | 31.06 | 0.366 | 1.2 | 0.167 | 0.5 | 0.402 | 1.3* |
| | Moderate positive | 60 | 59 | 98.3% | 30.44 | 0.323 | 1.1 | 0.161 | 0.5 | 0.361 | 1.2* |

a Total includes Within-Lot and Between-Lot Components.
b N is the number of valid replicates.
c n is the number of positive tests, which contribute CN values to the variance component analysis.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.
*Calculated SD and CV (%) may be underestimated.

# 6. Reproducibility

Site-to-site reproducibility performance of the Alinity m HR HPV assay was evaluated with a 22-member panel of samples in ThinPrep PreservCyt Solution and a 13-member panel of samples in SurePath Preservative Fluid. The ThinPrep panel included six pools made from clinical specimens collected in ThinPrep PreservCyt Solution and 16 contrived samples prepared with SiHa (HPV 16), HeLa (HPV 18), HPV 45, HPV 39, or HPV 58 plasmids in ThinPrep PreservCyt Solution containing HR HPV negative C33A cells. The SurePath panel included six pools made from clinical specimens collected in SurePath Preservative Fluid and seven contrived samples prepared with SiHa or HeLa in SurePath Preservative Fluid containing HR HPV negative C33A cells. For each sample type, one negative clinical pool and five moderate positive clinical pools representative of each genotype category (HPV 16, 18, 45, Other HR HPV A, and Other HR HPV B) were prepared. ThinPrep contrived panels consist of high negative, low positive, and moderate positive target levels for each genotype category, in addition to a negative panel member. SurePath contrived panels consist of high negative, low positive, and moderate positive target levels for SiHa and HeLa cell lines, in addition to a negative panel member. Each panel member was tested with three lots of Alinity m HR HPV AMP Kit across three clinical sites, with one unique lot per site on five non-consecutive days. Each site

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tested two runs per day and four replicates of each panel member per run on each of the five days. Each of the three clinical sites used different lots of Alinity m HR HPV CTRL Kits and Alinity m Sample Prep Kit 1. A summary of the reproducibility panel members and agreement rates, and analysis of cycle number (CN) variability are presented in Table 15 and Table 16 for the ThinPrep panel and Table 17 and Table 18 for the SurePath panel.

Table 15. Summary of Reproducibility Study Panel Members, Median CN, and Agreement Rates: ThinPrep

| Panel Composition | Panel Description | No. \(Valid^a\) | Median (CN)\(^b\) | %Positive (n/N)\(^c\) | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| Pooled negative clinical sample | Negative | 119 | NA | 0.0 (0/119) | 0.0 (0/119) | 100.0 (119/119) |
| HPV negative cell line | Negative | 119 | NA | 0.0 (0/119) | 0.0 (0/119) | 100.0 (119/119) |
| HPV16 positive SiHa cell line | HPV16 high negative | 118 | 33.03 | 50.0 (59/118) | 18.6 (22/118) | 31.4 (37/118) |
| HPV18 positive HeLa cell line | HPV18 high negative | 117 | 36.00 | 10.3 (12/117) | 1.7 (2/117) | 88.0 (103/117) |
| HPV45 plasmid | HPV45 high negative | 120 | 32.17 | 36.7 (44/120) | 59.2 (71/120) | 4.2 (5/120) |
| Other HR HPV Plasmid A\(^d\) | Other HR HPV A high negative | 119 | 36.00 | 18.5 (22/119) | 22.7 (27/119) | 58.8 (70/119) |
| Other HR HPV Plasmid B\(^e\) | Other HR HPV B high negative | 111 | 32.66 | 24.3 (27/111) | 36.9 (41/111) | 38.7 (43/111) |
| Pooled clinical sample | HPV 16 moderate positive | 119 | 27.72 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |
| | HPV 18 moderate positive | 119 | 28.28 | 98.3 (117/119) | 0.0 (0/119) | 1.7 (2/119) |
| | HPV 45 moderate positive | 120 | 28.40 | 98.3 (118/120) | 0.0 (0/120) | 1.7 (2/120) |
| | Other HR HPV A moderate positive | 119 | 28.02 | 98.3 (117/119) | 0.0 (0/119) | 1.7 (2/119) |
| | Other HR HPV B moderate positive | 118 | 26.95 | 100.0 (118/118) | 0.0 (0/118) | 0.0 (0/118) |
| HPV 16 positive SiHa cell line | HPV 16 low positive | 118 | 29.41 | 100.0 (118/118) | 0.0 (0/118) | 0.0 (0/118) |
| | HPV 16 moderate positive | 117 | 28.06 | 100.0 (117/117) | 0.0 (0/117) | 0.0 (0/117) |
| HPV 18 positive HeLa cell line | HPV 18 low positive | 120 | 28.16 | 96.7 (116/120) | 0.8 (1/120) | 2.5 (3/120) |
| | HPV 18 moderate positive | 119 | 27.28 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |
| HPV 45 plasmid | HPV 45 low positive | 120 | 30.45 | 99.2 (119/120) | 0.8 (1/120) | 0.0 (0/120) |
| | HPV 45 moderate positive | 116 | 29.89 | 100.0 (116/116) | 0.0 (0/116) | 0.0 (0/116) |
| Other HR HPV A Plasmid | Other HR HPV A low positive | 119 | 30.31 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |
| | Other HR HPV A moderate positive | 118 | 29.75 | 100.0 (118/118) | 0.0 (0/118) | 0.0 (0/118) |
| Other HR HPV B Plasmid | Other HR HPV B low positive | 120 | 30.47 | 97.5 (117/120) | 0.0 (0/120) | 2.5 (3/120) |
| | Other HR HPV B moderate positive | 119 | 28.73 | 99.2 (118/119) | 0.0 (0/119) | 0.8 (1/119) |

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a N is the number of valid replicates.
b The median CN value reported is calculated for all replicates measured in the negative and positive panel members, above and below the clinical cutoff. Negative replicates with no virus detected were assigned the last cycle of PCR (36.00). Due to $0\%$ virus detection, median CN was not reported for negative panels (NA)
c The percent agreement is based on the number of valid replicates with a CN value below the clinical cutoff.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

Table 16. Variance Component Analysis Results for Reproducibility Study: ThinPrep

| | | | | | Within-Run | | Between-Run | | Between-Day | | Between Instrument/Lot | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Panel Description | Nb | nc | Mean (CN) | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Pooled Clinical Sample | HPV16 | 119 | 119 | 27.47 | 1.391 | 5.1 | 0.000 | 0.0 | 0.000 | 0.0 | 0.441 | 1.6 | 1.459 | 5.3 |
| | HPV 18 moderate positive | 119 | 117 | 27.82 | 1.464 | 5.3 | 0.624 | 2.2 | 0.184 | 0.7 | 0.000 | 0.0 | 1.602 | 5.8 |
| | HPV 45 moderate positive | 120 | 118 | 28.12 | 1.355 | 4.8 | 0.000 | 0.0 | 0.319 | 1.1 | 0.189 | 0.7 | 1.405 | 5.0 |
| | Other HR HPV A moderate positive | 119 | 117 | 27.42 | 1.506 | 5.5 | 0.000 | 0.0 | 0.569 | 2.1 | 0.115 | 0.4 | 1.614 | 5.9 |
| | Other HR HPV B moderate positive | 118 | 118 | 26.94 | 0.550 | 2.0 | 0.285 | 1.1 | 0.147 | 0.5 | 0.035 | 0.1 | 0.638 | 2.4 |
| HPV16 positive SiHa cell line | HPV 16 low positive | 118 | 118 | 29.43 | 0.525 | 1.8 | 0.155 | 0.5 | 0.177 | 0.6 | 0.210 | 0.7 | 0.612 | 2.1 |
| | HPV 16 moderate positive | 117 | 117 | 28.18 | 0.525 | 1.9 | 0.134 | 0.5 | 0.387 | 1.4 | 0.148 | 0.5 | 0.682 | 2.4 |
| HPV18 positive HeLa cell line | HPV 18 low positive | 120 | 116 | 28.24 | 0.892 | 3.2 | 0.000 | 0.0 | 0.000 | 0.0 | 0.308 | 1.1 | 0.944 | 3.3 |
| | HPV 18 moderate positive | 119 | 119 | 27.25 | 0.667 | 2.4 | 0.000 | 0.0 | 0.000 | 0.0 | 0.000 | 0.0 | 0.667 | 2.4 |
| HPV45 plasmid | HPV 45 low positive | 120 | 119 | 30.51 | 0.435 | 1.4 | 0.221 | 0.7 | 0.000 | 0.0 | 0.054 | 0.2 | 0.491 | 1.6 |
| | HPV 45 moderate positive | 116 | 116 | 29.90 | 0.357 | 1.2 | 0.065 | 0.2 | 0.311 | 1.0 | 0.000 | 0.0 | 0.478 | 1.6 |
| Other HR HPV A plasmidd | Other HR HPV A low positive | 119 | 119 | 30.35 | 0.380 | 1.3 | 0.194 | 0.6 | 0.124 | 0.4 | 0.000 | 0.0 | 0.444 | 1.5 |
| | Other HR HPV A moderate positive | 118 | 118 | 29.85 | 0.383 | 1.3 | 0.200 | 0.7 | 0.324 | 1.1 | 0.039 | 0.1 | 0.542 | 1.8 |
| Other HR HPV B plasmid e | Other HR HPV B low positive | 120 | 117 | 30.46 | 0.391 | 1.3 | 0.122 | 0.4 | 0.184 | 0.6 | 0.102 | 0.3 | 0.461 | 1.5 |
| | Other HR HPV B moderate positive | 119 | 118 | 28.77 | 0.376 | 1.3 | 0.215 | 0.7 | 0.184 | 0.6 | 0.149 | 0.5 | 0.494 | 1.7 |

Because only positive, detected test results with CN values below the cutoff were included, estimates of SD (and $\%$ CV may be underestimated.
a N is the number of valid replicates.
b n is the number of valid replicates with a CN value below the clinical cutoff

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c Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot Components
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

Table 17. Summary of Reproducibility Study Panel Members, Median CN, and Agreement Rates: SurePath

| Panel Composition | Panel Description | No. \(Valid^a\) | Median (CN)\(^b\) | %Positive (n/N)\(^c\) | % Negative, HPV amplified (n/N) | % Negative, HPV not amplified (n/N) |
| --- | --- | --- | --- | --- | --- | --- |
| Pooled negative clinical sample | Negative | 119 | NA | 0.0 (0/119) | 0.0 (0/119) | 100.0 (119/119) |
| HPV negative cell line | Negative | 119 | NA | 0.0 (0/119) | 0.0 (0/119) | 100.0 (119/119) |
| HPV16 positive SiHa cell line | HPV16 high negative | 119 | 36.00 | 20.2 (24/119) | 0.8 (1/119) | 79.0 (94/119) |
| HPV18 positive HeLa cell line | HPV18 high negative | 118 | 36.00 | 25.4 (30/118) | 0.0 (0/118) | 74.6 (88/119) |
| Pooled clinical sample | HPV 16 moderate positive | 119 | 28.52 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |
| | HPV 18 moderate positive | 117 | 27.20 | 99.1 (116/117) | 0.9 (1/117) | 0.0 (0/117) |
| | HPV 45 moderate positive | 119 | 28.11 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |
| | Other HR HPV A moderate \(positive^d\) | 118 | 27.46 | 99.2 (117/118) | 0.0 (0/118) | 0.8 (1/118) |
| | Other HR HPV B moderate \(positive^e\) | 118 | 28.26 | 99.2 (117/118) | 0.0 (0/118) | 0.8 (1/118) |
| HPV 16 positive SiHa cell line | HPV 16 low positive | 120 | 29.57 | 100.0 (120/120) | 0.0 (0/120) | 0.0 (0/120) |
| | HPV 16 moderate positive | 118 | 27.98 | 100.0 (118/118) | 0.0 (0/118) | 0.0 (0/118) |
| HPV 18 positive HeLa cell line | HPV 18 low positive | 118 | 27.91 | 98.3 (116/118) | 0.0 (0/118) | 1.7 (2/118) |
| | HPV 18 moderate positive | 119 | 26.54 | 100.0 (119/119) | 0.0 (0/119) | 0.0 (0/119) |

a N is the number of valid replicates.
b The median CN value reported is calculated for all replicates measured in the negative and positive panel members, above and below the clinical cutoff.
c The percent agreement is based on the number of valid replicates with a CN value below the clinical cutoff.
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

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Table 18. Variance Component Analysis Results for Reproducibility Study: SurePath

| | | | | | Within-Run | | Between-Run | | Between-Day | | Between Instrument/Lot | | Totala | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel | Panel Description | Nb | nc | Mean (CN) | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Pooled Clinical Sample | HPV16 | 119 | 119 | 28.44 | 0.889 | 3.1 | 0.000 | 0.0 | 0.054 | 0.2 | 0.189 | 0.7 | 0.911 | 3.2 |
| | HPV 18 moderate positive | 117 | 116 | 27.49 | 1.265 | 4.6 | 0.000 | 0.0 | 0.256 | 0.9 | 0.303 | 1.1 | 1.326 | 4.8 |
| | HPV 45 moderate positive | 119 | 119 | 28.05 | 0.785 | 2.8 | 0.265 | 0.9 | 0.000 | 0.0 | 0.181 | 0.6 | 0.848 | 3.0 |
| | Other HR HPV A moderate positive d | 118 | 117 | 27.28 | 1.110 | 4.1 | 0.287 | 1.1 | 0.000 | 0.0 | 0.045 | 0.2 | 1.148 | 4.2 |
| | Other HR HPV B moderate positive e | 118 | 117 | 28.14 | 1.102 | 3.9 | 0.000 | 0.0 | 0.371 | 1.3 | 0.542 | 1.9 | 1.283 | 4.6 |
| HPV16 positive SiHa cell line | HPV 16 low positive | 120 | 120 | 29.62 | 0.592 | 2.0 | 0.000 | 0.0 | 0.077 | 0.3 | 0.156 | 0.5 | 0.617 | 2.1 |
| | HPV 16 moderate positive | 118 | 118 | 28.02 | 0.439 | 1.6 | 0.025 | 0.1 | 0.121 | 0.4 | 0.051 | 0.2 | 0.458 | 1.6 |
| HPV18 positive HeLa cell line | HPV 18 low positive | 118 | 116 | 28.06 | 0.888 | 3.2 | 0.000 | 0.0 | 0.059 | 0.2 | 0.321 | 1.1 | 0.946 | 3.4 |
| | HPV 18 moderate positive | 119 | 119 | 26.57 | 0.636 | 2.4 | 0.000 | 0.0 | 0.000 | 0.0 | 0.066 | 0.2 | 0.640 | 2.4 |

Because only positive, detected test results with CN values below the cutoff were included, estimates of SD (and % CV may be underestimated.
a N is the number of valid replicates.
b n is the number of valid replicates with a CN value below the clinical cutoff
c Total includes Within-Run, Between-Run, Between-Day, and Between-Instrument/Lot Components
d HPV 58 was used for the Other HR HPV A panel.
e HPV 39 was used for the Other HR HPV B panel.

## 7. Alinity m System Carryover

Carryover studies were conducted to stress two areas of potential carryover on the Alinity m System: Sample Preparation and the AMP TRAY 1. Carryover rates were determined for each by testing alternating replicates of HR HPV high positive samples and HR HPV negative samples across multiple runs. The HR HPV high positive sample was prepared with HPV 16 at 6.9 x107 copies/ml.

The highest potential for carryover was determined to be at the Sample Preparation stage. Of 480 negative samples tested adjacent to high positive samples during sample preparation, 4 samples were detected, resulting in an overall carryover rate of 0.83% (4/480) and 95% confidence interval (CI) ranging from 0.32% to 2.12%.

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## 8. Upstream Cytology Processor Carryover

The potential for sample carryover from upstream cytology processing systems when testing residual cytology samples with the Alinity m HR HPV assay was evaluated by processing alternating replicates of HPV high-positive samples and HPV negative samples on each of 3 systems: the ThinPrep 2000, ThinPrep 5000, and the BD PrepMate, followed by testing of the negative HPV samples on the Alinity m System. For each of the systems, the carryover rate was 0.0% (0 detected out of 120 valid HR HPV negative samples) with an upper bound of the two-sided 95% CI of 3.1%.

## 9. Reagent Stability

Expiration dating for the Alinity m HR HPV reagents has been established at 15 months for the Alinity m HR HPV AMP Kit stored at 2°C to 8°C and for the Alinity m HR HPV CTRL Kit stored at -25°C to -15°C. The shelf lives of the Alinity m HR HPV reagents were established in a real-time stability study.

## 10. Specimen Stability

Specimen stability studies demonstrated that when testing with Alinity m HR HPV, cervical specimens can be stored in ThinPrep at -20°C, 2-8 °C, and 15-30°C for up 6 months from the date of collection. Cervical specimens stored in SurePath can be stored at -20°C and 2-8°C for up to 3 months and at 15-30°C for up to 14 days from the date of collection. The observed changes in CN value between baseline and the different storage conditions did not change any reported results

## B. Animal Studies

N/A

## C. Additional Studies

N/A

## X. SUMMARY OF PRIMARY CLINICAL STUDY(IES)

The applicant performed a clinical study to establish a reasonable assurance of safety and effectiveness of HR HPV testing with Alinity m HR HPV for detection of HPV DNA in cervical specimens collected by a health care professional using an endocervical brush/spatula placed in ThinPrep PreservCyt Solution (ThinPrep) or an endocervical broom placed in SurePath Preservative Fluid (SurePath) in the US. Data from this clinical study were the basis for the PMA approval decision. A summary of the clinical study is presented below.

### A. Study Design

A prospective, multi-center clinical study was conducted and recruited 14,935 women ≥ 25 years across 96 collection sites throughout the US. The study included patients undergoing routine cervical cancer screening (Screening

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population) and was supplemented with individuals referred for colposcopy follow-up based on previous cervical cancer screening that included HR HPV testing (Enriched population). A total of 14,702 (11,636 Screening and 3,066 Enriched) women were eligible to participate in the study. Eligible patients were treated between February 16, 2021, and June 16, 2022. Alinity m HR HPV testing was performed at four clinical testing sites within these dates.

A detailed description of the two populations (Screening and Enriched), the inclusion/exclusion criteria used to determine patient eligibility, and an account of the specimens collected and tested for either population is detailed below. Patients with valid HR HPV testing (Alinity m HR HPV and FDA approved tests) and final disease diagnosis results, used to assess the clinical performance of Alinity m HR HPV in either population, is further detailed in Subsection B. below entitled, "Accountability of PMA Cohort."

## Screening Population

The collection sites served populations at low to high risks for HPV infection and included but were not limited to health clinics, OB/GYN practices, health integrated site networks/health systems, private practice and research centers, and universities and academic centers. All women in the Screening population had cervical specimens collected using an endocervical brush/spatula placed in ThinPrep PreservCyt Solution (ThinPrep). Approximately 80% of the women also had cervical specimens collected using a cervical broom-like collection device placed in SurePath Preservative Fluid (SurePath). When both cervical specimens were collected, the collection order was randomized to ensure that approximately half of the women had ThinPrep collected first, and the other half had SurePath collected first.

In the Screening population, specimens collected as the first (or only) specimen were used for cytology testing also. Cytology slides generated using ThinPrep cytology processors (ThinPrep 2000 (TP 2000), ThinPrep 5000 (TP 5000)), and SurePath cytology processors (SurePath PrepMate/PrepStain Systems) were diagnosed using the 2014 Bethesda System¹.

All specimens collected were tested for HPV infection by Alinity m HR HPV and an FDA approved HPV test performed according to manufacturer's instructions. Cytology results and up to four HPV results (from both the Alinity m HR HPV and the FDA approved HPV testing performed on both ThinPrep and SurePath specimens) were utilized to inform referral to colposcopy (see Follow-up Schedule). Women in the Screening population with ≥ ASC-US cytology, regardless of HPV test results, or with any cytology result and a positive HPV status (in at least one of four test results) were referred to colposcopy. Those with a NILM (Negative for Intraepithelial Lesion Malignancy) cytology and negative or undetermined HPV, or negative HPV and UNSAT (Unsatisfactory) or unavailable cytology, were not referred to colposcopy. All cytology and specimen testing with an FDA approved HPV test were performed at a reference laboratory and used for routine clinical management. A randomly selected subset of

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specimens was sent for NGS at a reference laboratory to support Alinity m HR HPV genotyping performance.

## Enriched population

Women referred for colposcopy follow-up based on previous cervical cancer screening that included HR HPV testing represented the Enriched population. All women in the Enriched population had both ThinPrep and SurePath specimens collected and were tested by both the Alinity m HR HPV assay and the FDA approved HPV test using non-cytology aliquots. All women in the Enriched population underwent colposcopy procedures (see Follow-up Schedule). All cytology and HPV testing by another FDA approved HPV test were performed at a reference laboratory and used for routine clinical management.

### 1. Clinical Inclusion and Exclusion Criteria

Enrollment in the Alinity m HR HPV study was limited to patients who met the following inclusion criteria for the Screening and Enriched populations, respectively.

#### Screening population inclusion criteria:

Individuals were eligible if she met all the following criteria:

- Is 25 years of age or older
- Is attending a participating clinic for routine cervical cancer screening following screening guidelines
- Has an intact cervix
- Is willing and able to provide documented informed consent
- Is willing and able to undergo colposcopy, biopsy, and endocervical curettage (ECC) within 12 weeks (≤ 84 days) from the baseline visit (if required)
- Is willing and able to allow collection of two cervical cytology specimens

#### Enriched population inclusion criteria:

Individuals were eligible if she met all the following criteria:

- Is willing and able to provide documented informed consent (or her legal representative is willing and able)
- Is 25 years of age or older
- Has had a colposcopy referral in which routine cervical cancer screening has included HR HPV testing (HPV primary screening, co-testing, or ASC-US cytology triage) performed within 12 weeks preceding the colposcopy visit
- Has an intact cervix
- Is willing and able to undergo colposcopy, biopsy, and endocervical curettage (ECC)
- Is willing and able to allow collection of two cervical cytology specimens

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Patients were not permitted to enroll in the Alinity m HR HPV study if they met any of the following exclusion criteria as it pertained to the Screening and Enriched populations:

**Screening population exclusion criteria:**

An individual was ineligible for the study if she met any of the following criteria:

- Is pregnant at the time of visit or plans to become pregnant within the following 12 weeks
- Has any known medical condition that, in the opinion of the investigator, would result in increased risk of bleeding at biopsy
- Has a known history of excisional or ablative therapy (e.g., LEEP [Loop Electrosurgical Excision Procedure], cone biopsy, cervical laser surgery, or cryotherapy) to the cervix in the last 12 months prior to the baseline visit
- Had a cervical cytology specimen collected within the last 4 months
- Is currently participating in any diagnostic trial for cervical cancer
- Had a complete or partial hysterectomy, either supra cervical or involving removal of the cervix
- Is currently participating or planning to participate in any clinical trial for HPV treatment (for the duration of this study)
- Previous participation in this study (in the Screening/Enriched population)

**Enriched population exclusion criteria:**

An individual was ineligible for the study if she met any of the following criteria:

- Is pregnant when presenting for colposcopy
- Has any known medical condition that, in the opinion of the investigator, would result in increased risk of bleeding at biopsy
- Has a known history of excisional or ablative therapy (e.g., LEEP, cone biopsy, cervical laser surgery, or cryotherapy) to the cervix in the last 12 months prior to the colposcopy visit
- Is currently participating in any diagnostic trial for cervical cancer
- Had a complete or partial hysterectomy, either supracervical or involving removal of the cervix
- Is currently participating or planning to participate in any clinical trial for HPV treatment (for the duration of this study)
- Is referred to colposcopy based on a cytology-only screening program
- HR HPV referral test is known to be the same FDA approved test used to assess clinical performance
- Previous participation in this study.

2. **Follow-up Schedule**

Colposcopy procedures were conducted in the Screening and Enriched populations according to a standardized protocol: 1) an endocervical curettage was performed for all participants; 2) biopsies were obtained on all visible lesions, and 3) a single random biopsy of the visualized or partially visualized

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squamocolumnar junction was obtained if no lesion was visible. In addition, during the colposcopy visit, loop electrosurgical excision procedure (LEEP) was performed when required by the standard of care. To avoid potential bias, study participants and colposcopists were blinded to HPV test results until colposcopy was completed. Cytology results were made available to colposcopists prior to colposcopy.

All tissue biopsies were examined by a Central Pathology Review Panel (CPRP) consisting of three expert pathologists. Discordant results were adjudicated according to a pre-defined standardized protocol. The CPRP was masked to patient information, including cytology, HPV results, and previous diagnoses. The slides prepared from the tissue biopsies were stained using conventional hematoxylin and eosin (H&E) staining, and with p16 IHC (immunohistochemistry) staining where applicable. For all tissue biopsies, the expert pathologists evaluated H&E slides to establish an H&E histologic diagnosis. When a tissue biopsy case met LAST (Lower Anogenital Squamous Terminology) Workshop guidelines, the expert pathologists evaluated both p16 and H&E slides for that tissue biopsy to establish a p16/H&E histologic diagnosis. The final CPRP histologic results for the biopsies meeting the LAST guidelines were defined by the p16/H&E histologic diagnosis. The final CPRP histologic results for the biopsies not meeting the LAST guidelines were defined by the H&E histologic diagnosis. After the slides from all collected tissues were reviewed for a woman, the disease status for the woman was determined based on the most severe histologic result across all tissues collected (≥ cervical intraepithelial neoplasia 3 [CIN3], CIN2, or ≤ CIN1). Adverse events and complications were recorded at all visits.

## 3. Clinical Endpoints

With regards to safety, as an in vitro diagnostic test, the Alinity m HR HPV assay is performed on cervical cells collected during routine pelvic exam (i.e., cervical cytology) using an endocervical brush/spatula or broom. The test, therefore, does not present any more safety hazard to an individual being tested than other tests where cervical cells are sampled in this manner (i.e., cervical cytology).

With regards to effectiveness, the clinical performance of the Alinity m HR HPV assay was evaluated against CPRP determined histologic diagnosis, with ≥CIN2 and ≥CIN3 as the disease endpoints in both the Screening and Enriched populations.

Clinical performance of the Alinity m HR HPV device in this study was evaluated by comparing the ratio of clinical sensitivity and the false positivity rate (FPR) between the Alinity test and a FDA approved test. These ratios were evaluated in both specimen matrices (ThinPrep and SurePath) and in both populations (Screening and Enriched) against the disease endpoints listed above.

## B. Accountability of PMA Cohort

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A total of 14,935 patients were recruited of which 14,702 (11,636 from the Screening population and 3,066 from the Enriched population) were eligible and enrolled in the PMA study. In the Screening population, 11,532 patients had valid HR HPV (Alinity and FDA approved) test and cytology results to determine colposcopy referral. In the Enriched population, 3,059 patients had patients had valid HR HPV (Alinity and FDA approved) test and cytology results.

Patient accountability, including the final number of subjects with a valid HR HPV test results (for Alinity and FDA approved tests) and CPRP diagnosis of the collected biopsy, for either matrix (ThinPrep and SurePath) and study population (Screening and Enriched), are presented in Figures 1 through 4 below. The final number of subjects used for the primary analysis (i.e., clinical sensitivity and FPR) are also indicated in the figures below.

## Screening Population Accountability

In the Screening population, 11,532 subjects had a valid HR HPV testing and cytology result to determine a colposcopy referral. A total of 11,521 valid ThinPrep results and 9,466 valid SurePath results were generated for these 11,532 women. Of 11,532 subjects, a total of 1,922 (16.7%) were referred to colposcopy based on cytology and/or HR-HPV status of their ThinPrep or SurePath specimen, while the remaining 9,610 subjects exited the study. Among the population who completed colposcopy and had successful biopsy diagnosis, 1,508 subjects had a valid ThinPrep result (35 ≥ CIN3, 80 CIN2, and 1,393 ≤ CIN1) and, separately, 1,269 subjects had a valid SurePath result (27 ≥ CIN3, 66 CIN2, and 1,176 ≤ CIN1). The Alinity clinical sensitivity and FPR (primary analysis) in the Screening population was determined using 10,956 patient ThinPrep and 8,455 patient SurePath results.

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Figure 1. Subject and result accountability for ThinPrep Screening population samples
10,956 TP samples used in primary analysis for clinical sensitivity

a1 subject in the Screening population had two cervixes; Four cervical swabs were collected (two stored in ThinPrep and two stored in SurePath). TP=ThinPrep and SP=SurePath, UND= Undetermined; UNSAT= Unsatisfactory

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Figure 2. Subject and result accountability for SurePath Screening population samples

a 1 subject in the Screening population had two cervixes; Four cervical swabs were collected (two stored in ThinPrep and two stored in SurePath). TP=ThinPrep, SP=SurePath, UND= Undetermined; UNSAT= Unsatisfactory

# Enriched Population Accountability

In the Enriched population, 3,059 subjects had a valid HR HPV testing (3,055 valid ThinPrep results and, separately, 3,013 SurePath results) and cytology result. Among the population who completed colposcopy and had successful biopsy diagnosis, 2,990 subjects had a valid ThinPrep result $(184\geq \mathrm{CIN}3$ 403 CIN2, and $2,403\leq \mathrm{CIN}1)$ and, separately, 2,951 subjects had a valid SurePath result $(183\geq \mathrm{CIN}3$ 403 CIN2, and $2,361\leq \mathrm{CIN}1)$ . Alinity clinical sensitivity (primary analysis) in the Enriched population was determined in 2,981 patient ThinPrep and 2,931 patient SurePath results.

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Figure 3. Subject and result accountability for ThinPrep Enriched population

TP = ThinPrep, SP= SurePath

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Figure 4. Subject and result accountability for SurePath Enriched population
TP = ThinPrep, SP= SurePath

# C. Study Population Demographics and Baseline Parameters

The demographics of the study population are typical for a clinical study performed in the US for a HPV molecular test.

In the Screening population, the median age of the women was 41 years, with $13.4\%$ in age group 25-29 years, $30.9\%$ in age group 30-39 years, and $55.7\%$ in age group $\geq$ 40 years. In the Enriched population, the median age of the women was 37 years, with $18.9\%$ in age group 25-29 years, $39.5\%$ in age group 30-39 years, and $41.5\%$ in age group $\geq 40$ years.

The demographics of the study population (age, age group, HPV vaccination status, ethnicity, and race) for Screening and Enriched populations, is shown in Table 19 and Table 20.

| Table 19. Demographic Characteristics Statistics | |
| --- | --- |
| Screening Population | |
| Age (years) | (N=11532) |
| Table 20. Demographic Characteristics Statistics | |
| --- | --- |
| Enriched Population | |
| Age (years) | (N=3059) |

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| Table 19. Demographic Characteristics Statistics | |
| --- | --- |
| n | 11532 |
| Mean | 43 |
| Median | 41 |
| Minimum | 25 |
| Maximum | 85 |
| Age Group | n (%) |
| 25-29 | 1548 (13.4) |
| 30-39 | 3562 (30.9) |
| >=40 | 6422 (55.7) |
| Vaccination Status | n (%) |
| Has been vaccinated | 1541 (13.4) |
| Never been vaccinated | 9135 (79.2) |
| Unknown | 856 (7.4) |
| Ethnicity | n (%) |
| Hispanic or Latino | 1901 (16.5) |
| Not Hispanic or Latino | 9190 (79.7) |
| Not Reported | 441 (3.8) |
| Race | n (%) |
| White | 8537 (74.0) |
| Black or African American | 2369 (20.5) |
| Asian | 233 (2.0) |
| American Indian or Alaska Native | 35 (0.3) |
| Native Hawaiian or Other Pacific Islander | 28 (0.2) |
| Other | 72 (0.6) |
| Not Reported | 258 (2.2) |
| Table 20. Demographic Characteristics Statistics | |
| --- | --- |
| n | 3059 |
| Mean | 39 |
| Median | 37 |
| Minimum | 25 |
| Maximum | 81 |
| Age Group | n (%) |
| 25-29 | 579 (18.9) |
| 30-39 | 1209 (39.5) |
| >=40 | 1271 (41.5) |
| Vaccination Status | n (%) |
| Has been vaccinated | 606 (19.8) |
| Never been vaccinated | 2238 (73.2) |
| Unknown | 215 (7.0) |
| Ethnicity | n (%) |
| Hispanic or Latino | 827 (27.0) |
| Not Hispanic or Latino | 2161 (70.6) |
| Not Reported | 71 (2.3) |
| Race | n (%) |
| White | 2326 (76.0) |
| Black or African American | 503 (16.4) |
| Asian | 77 (2.5) |
| American Indian or Alaska Native | 13 (0.4) |
| Native Hawaiian or Other Pacific Islander | 6 (0.2) |
| Other | 26 (0.8) |
| Not Reported | 108 (3.5) |

Table 21 shows the HPV positivity rate for the Alinity m HR HPV assay by specimen type and testing site in the Screening population. In the screening population…