← Product Code MAQ · P190028

# cobas HPV for use on the cobas 6800/8800 Systems (P190028)

_Roche Molecular Systems, Inc. · MAQ · Apr 3, 2020 · Microbiology · APPR_

**Canonical URL:** https://fda.innolitics.com/device/P190028

## Device Facts

- **Applicant:** Roche Molecular Systems, Inc.
- **Product Code:** MAQ
- **Decision Date:** Apr 3, 2020
- **Decision:** APPR
- **Device Class:** Class 3
- **Review Panel:** Microbiology

## Intended Use

cobas HPV for use on the cobas 6800/8800 Systems (cobas HPV) is a qualitative in vitro test for the detection of Human Papillomavirus in clinician-collected cervical specimens using an endocervical brush/spatula or broom and placed in the ThinPrep Pap Test PreservCyt Solution. This test detects the high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. cobas HPV is indicated for use for routine cervical cancer screening as per professional medical guidelines, including triage of ASC-US cytology, co-testing (or adjunctive screen) with cytology, and HPV primary screening of women to assess the risk for cervical precancer 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

Qualitative real-time PCR test; detects 14 high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in cervical specimens. Input: cervical samples in PreservCyt Solution. Process: automated nucleic acid extraction/purification; PCR amplification using thermostable DNA polymerase; real-time detection via fluorescent oligonucleotide probes. Output: positive/negative/invalid results for HR HPV and specific genotyping (HPV16, HPV18). Used in clinical labs; operated by technicians. Results reviewed by clinicians to guide cervical cancer screening, triage, and management decisions. Benefits: enables risk assessment for cervical precancer/cancer; supports clinical decision-making.

## Clinical Evidence

Multicenter, prospective IMPACT study (n=35,263). Evaluated performance for ASC-US triage, adjunctive screening, and primary screening against CPR-adjudicated histology (H&E+p16). Sensitivity for ≥CIN2 in ASC-US population: 86.29%; specificity: 68.99%. Primary screening algorithm showed significantly higher sensitivity and specificity for ≥CIN2/≥CIN3 compared to cytology alone.

## Technological Characteristics

Real-time PCR; detects 14 high-risk HPV genotypes via L1 region primers (~200 nt) and β-globin internal control (330 bp). Uses magnetic glass particles for extraction. Automated platform (cobas 6800/8800). Fluorescent dye detection (HPV16, HPV18, and 12 Other HR HPV types). Standalone system with RFID reagent tracking.

## Reference Devices

- ATHENA study (Addressing the Need for Advanced HPV Diagnostics study)

## Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Real-time PCR test

Device Trade Name: cobas HPV for use on the cobas 6800/8800 Systems

Device Procode: MAQ

Applicant’s Name and Address: Roche Molecular Systems, Inc.
4300 Hacienda Drive
Pleasanton, CA 94588

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P190028

Date of FDA Notice of Approval: April 3, 2020

II. INDICATIONS FOR USE

cobas HPV for use on the cobas 6800/8800 Systems (cobas HPV) is a qualitative in vitro test for the detection of Human Papillomavirus in clinician-collected cervical specimens using an endocervical brush/spatula or broom and placed in the ThinPrep Pap Test PreservCyt Solution. This test detects the high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

cobas HPV is indicated for use for routine cervical cancer screening as per professional medical guidelines, including triage of ASC-US cytology, co-testing (or adjunctive screen) with cytology, and HPV primary screening of women to assess the risk for cervical precancer 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 cobas HPV labeling.

V. DEVICE DESCRIPTION

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cobas HPV is a qualitative real-time PCR test that detects 14 high-risk HPV genotypes. Of 14 HPV genotypes, 13 HPV genotypes are classified as carcinogenic or high-risk (HR): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and an additional genotype, 66, that is classified as "possibly carcinogenic" based on its relatively low prevalence in invasive cervical carcinoma. cobas HPV uses primers to define a sequence of approximately 200 nucleotides within the polymorphic L1 region of the HPV genome. A pool of HPV primers present in the Master Mix is designed to amplify HPV DNA from 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The test includes a primer pair that amplifies the human  $\beta$ -globin gene as an internal control to monitor the entire sample preparation and PCR amplification process (330 base pair amplicon). Fluorescent oligonucleotide probes bind to polymorphic regions within the sequence defined by these primers. In addition, the test utilizes a low titer positive and a negative control.

cobas HPV consists of:

- cobas 6800/8800 Systems
- cobas HPV assay specific analysis package (ASAP) software
- cobas HPV reagents in cassettes
- cobas HPV Positive Control Kit
- cobas Buffer Negative Control Kit
- Specimen preparation reagents (cobas omni Reagents)

cobas HPV is based on fully automated sample preparation (nucleic acid extraction and purification) followed by PCR amplification and detection. The cobas 6800/8800 Systems consist of the sample supply module, the transfer module, the processing module, and the analytic module. Automated data management is performed by the cobas 6800/8800 software, which assigns test results for all tests as positive, negative or invalid. Results can be reviewed directly on the system screen, exported, or printed as a report.

## Principle of Procedure

### 1. Sample Preparation (Nucleic Acid Extraction and Purification)

Nucleic acid from a patient sample is released upon addition of proteinase and lysis reagent to the sample. The released nucleic acid binds to the silica surface of the added magnetic glass particles. Unbound substances and impurities, such as denatured protein, cellular debris, and potential PCR inhibitors are removed with subsequent wash steps, and purified nucleic acid is eluted from the magnetic glass particles with elution buffer at elevated temperature. External controls (positive and negative) are processed in the same way with each cobas HPV run.

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2. Nucleic Acid Amplification

A thermostable DNA polymerase enzyme is used for PCR amplification. The HPV and β-globin sequences are amplified simultaneously utilizing a universal PCR amplification profile with predefined temperature steps and number of cycles. The master mix includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), which is incorporated into the newly synthesized DNA (amplicon). Any contaminating amplicon from previous PCR runs are eliminated by the AmpErase enzyme, which is included in the PCR master mix, during the first thermal cycling step. However, newly formed amplicons are not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C.

3. Nucleic Acid Detection

The cobas HPV master mix contains detection probes specific for twelve High Risk HPV target sequences, one detection probe specific for the HPV16 target sequence, one detection probe specific for the HPV18 target sequence and one for β-globin. The amplified signal from twelve high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) is detected using the same fluorescent dye while HPV16, HPV18, and β-globin signals are each detected with their own dedicated fluorescent dye. When not bound to the target sequence, the fluorescent signal of the intact probes is suppressed by a quencher dye. During the PCR amplification step, hybridization of the probes to the specific single-stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye increases concomitantly. Real-time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes for the HPV targets and β-globin, respectively.

Instrumentation and Software

1. cobas 6800/8800 System Platform Overview

The cobas 6800/8800 System is a platform that allows users to perform multiple PCR-based in vitro nucleic acid amplification tests. The platform consists of two separate instruments, the cobas 6800 System and the cobas 8800 System, both of which provide automated sample preparation, nucleic acid extraction, and target amplification and detection.

2. cobas 6800/8800 Systems Software Overview

The cobas 6800/8800 Systems Software is the primary interface for operators to access, control, and manage the cobas 6800/8800 Systems. The cobas 6800/8800 Systems Software includes off the shelf software components as well as software tools that are used for diagnosis and maintenance of the system.

The main system functionality is provided by two software components; i) the cobas 6800/8800 System Software and ii) Assay Specific Analysis Package

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(ASAP) software. The cobas 6800/8800 System software provides basic functionality, such as a Graphical User Interface (GUI), instrument management, database functionality, report engines, and LIS interfaces. These basic functions do not change when a new ASAP is added onto the system. The ASAPs are built using a common software framework and provide the assay test run conditions (sample preparation and PCR parameters), result analysis functionality (result calculation and algorithms), and result report formatting.

## 3. cobas 6800/8800 Systems Workflow

A workflow defines how the system processes the samples designated for a specific test, including any required user interactions. The sample gets loaded by the user and then processed automatically without any further user interaction until results are generated. More than one test can be ordered for the same sample. The system identifies the orders, and manages the process automatically.

The workflow on the cobas 6800/8800 Systems is centered on the batch process and linked to the design of the Process plate and Amplification plate:

- The Process Plate has 48 wells
- Both the cobas 6800 and cobas 8800 Systems have two 48 channel process heads per process cell which can process 96 test orders in parallel, using two Process plates
- The Amplification Plate has 96 wells

Both systems are capable of processing up to 96 samples in one run batch. As a result, there is only one common sample workflow on both systems. This common workflow is used for parallel processing of 96 samples in two Process plate and one Amplification plate on the cobas 6800/8800 Systems. If 48 or fewer test orders have to be processed, then only one Process plate will be used on both systems.

The cobas 6800/8800 Systems use the same hardware, software, and workflow for processing the samples. As a result, samples run on either system will have identical processing. The Sample Transfer process depends on the number of samples and orders and volumes being transferred to the Process plate. The timing for the Sample Preparation process is independent of the number of samples being processed, since all process steps are controlled by a time box. The timing of the amplification and detection process is independent of the number of samples, since all samples in the Amplification Plate undergo PCR in parallel.

The cobas 6800/8800 Systems utilize a core set of common reagents, the cobas omni reagents, which are designed to be used with all assays that are run on the systems. Identification, validity described by the remaining number of determinations, onboard time and expiry date of all reagents are tracked by RFID.

In addition, the omni (common) reagents and consumables, such as the P-plates, racks, AD-plates, waste bags, pipette tips, and secondary tubes, can be used by any of

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the cobas 6800/8800 System assays, and on either the cobas 6800 or the cobas 8800 instrument. Figure 1 below, depicts the cobas 6800/8800 platform.

![img-0.jpeg](img-0.jpeg)
Figure 1: cobas 6800/8800 platform

Additional details can be found in the operator's manual of the device.

# 4. Interpretation of Test Results

Results and their corresponding interpretation for detecting overall HR HPV and HPV-Genotyping are shown in Table 1 and Table 2, respectively.

Table 1: The interpretations for the overall HR-HPV results

|  Target 1 | Target 2 | Target 3 | Interpretation  |
| --- | --- | --- | --- |
|  HR HPV Positive | <Blank> | <Blank> | Specimen is positive for the DNA of any one of, or combination of, the following high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.  |
|  HR HPV Negative |   |   | HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold.  |
|  Invalid |   |   | The result for HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 is invalid.  |

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Table 2: The interpretations for the HPV-genotyping results

|  Target 1 | Target 2 | Target 3 | Interpretation  |
| --- | --- | --- | --- |
|  Other HR HPV Positive |  |  | Specimen is positive for the DNA of any one of, or combination of the following high risk HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.  |
|  Other HR HPV Negative |   |   | HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were undetectable or below the pre-set threshold.  |
|  Invalid |   |   | The result for HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 is invalid.  |
|   | HPV 16 Positive |   | Specimen is positive for HPV type 16 DNA.  |
|   |  HPV 16 Negative |   | HPV type 16 DNA was undetectable or below the pre-set threshold.  |
|   |  Invalid |   | The result for HPV type 16 is invalid.  |
|   |   | HPV 18 Positive | Specimen is positive for HPV type 18 DNA.  |
|   |   |  HPV 18 Negative | HPV type 18 DNA was undetectable or below the pre-set threshold.  |
|   |   |  Invalid | The result for HPV type 18 is invalid.  |

# VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are several alternatives for the detection 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. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

The patient'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 cobas HPV test should only be used in conjunction with this clinical information in accordance with professional clinical patient management guidelines.

# VII. MARKETING HISTORY

The product is currently distributed/marketed in forty-one 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 forty-one countries where the product is distributed includes:

Australia

Germany

Mexico

Austria

Greece

Norway

Belgium

Hungary

Poland

Brazil

Iceland

Portugal

Bulgaria

Ireland

Romania

Colombia

Italy

Slovakia

Croatia

Japan

Slovenia

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Cyprus
Latvia
Spain
Czech Republic
Liechtenstein
Sweden
Denmark
Lithuania
Switzerland
El Salvador
Luxembourg
Turkey
Estonia
Malta
United Kingdom
Finland
Myanmar
Vietnam
France
Netherlands

# VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

As with any in vitro diagnostic test, the potential risks are associated with an incorrect test result or result interpretation. Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect HPV test results and subsequently, improper patient management decisions.

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 methods used for cutoff selection was chosen to achieve the maximum sensitivity for detecting ≥ CIN2 disease while maintaining a clinically acceptable level of specificity. Based on these criteria, the clinical cutoff was set at Ct of 38.5 for HPV 16, Ct of 38.0 for HPV 18 and Ct of 34.5 for all HR HPV genotypes other than 16 and 18.

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

The Limit of Detection (LoD) at the clinical cutoff for HPV16 and HPV18 was assessed using SiHa and HeLa cell lines in the background of pooled HPV negative patient specimens collected in PrervCyt Solution. Cell lines were diluted to concentrations below, above, and at the expected LoD at the Clinical cutoff levels. A minimum of 24 replicates were tested for each cell line level using three reagent lots with an equal number of runs performed on the cobas 6800 and the cobas 8800 Systems. The LoD at the clinical cutoff was defined as the level of HPV DNA in the sample that has positive test results at least 95% of the time. The LoD at the clinical cutoff for SiHa and HeLa was 16 cells/mL. Table 3 and Table 4 list results from the reagent lot producing the most conservative (highest) LoD at the clinical cutoff in the analysis for HPV16 and HPV18.

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Table 3: LoD at the clinical cutoff for HPV16 (SiHa cell line)

|  SiHa Concentration (cells/mL) | Number of Positive/Tested | % Positive | 95% Confidence Interval  |
| --- | --- | --- | --- |
|  32 | 24 / 24 | 100% | 86.2% - 100%  |
|  16 | 24 / 24 | 100% | 86.2% - 100%  |
|  8 | 22 / 24 | 91.7% | 74.1% - 97.7%  |

Table 4: LoD at the clinical cutoff for HPV18 (HeLa cell line)

|  HeLa Concentration (cells/mL) | Number of Positive/Tested | % Positive | 95% Confidence Interval  |
| --- | --- | --- | --- |
|  32 | 24 / 24 | 100% | 86.2% - 100%  |
|  16 | 24 / 24 | 100% | 86.2% - 100%  |
|  8 | 22 / 24 | 91.7% | 74.1% - 97.7%  |

## 3. Inclusivity

Plasmids for high risk genotypes 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 were tested close to the LoD at the clinical cutoff in the background of pooled HPV negative patient specimens. All 12 of the high risk genotypes tested were detected by the assay.

## 4. Analytical Specificity

A panel of bacteria, fungi and viruses, including those commonly found in the female urogenital tract, as well as several human papillomavirus types classified as low or undetermined risk were tested with cobas HPV to assess analytical specificity. The organisms listed in Table 5 were spiked at concentrations of approximately $1 \times 10^{6}$ Colony Forming Units (CFU)/mL for bacteria (except for *Chlamydia trachomatis* which was quantified as Inclusion Forming Units (IFU) and *Trichomonas vaginalis* which was quantified as cells/mL) and approximately $1 \times 10^{5}$ TCID$_{50}$/mL for viruses (except *Adenovirus Type 40* which was tested at $2.82 \times 10^{4}$ TCID$_{50}$/mL and Epstein Barr virus which was tested at $1 \times 10^{5}$ copies/mL) into pools of HPV negative cervical specimens in PreservCyt Solution. Testing was performed with each potential interfering organism alone as well as with each organism mixed with HPV31 plasmid, SiHa (HPV16) and HeLa (HPV18) cell lines at approximately $3 \times$ LoD at the clinical cutoff of cobas HPV. Results indicated that these organisms neither interfered with detection in the 12 Other High Risk HPV, HPV16 and HPV18 channels nor produced a false positive result in the HPV negative specimen.

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Table 5: Microorganisms tested for analytical specificity

|  Adenovirus Type 40 | Herpes Simplex Virus 2 | HPV84  |
| --- | --- | --- |
|  Bacteroides caccae | HPV6 | HPV85  |
|  Bacteroides ureolyticus | HPV11 | HPV89  |
|  Bifidobacterium adolescentis | HPV26 | Klebsiella oxytoca  |
|  Bifidobacterium breve | HPV30 | Klebsiella pneumoniae  |
|  Bifidobacterium longum | HPV34 | Lactobacillus acidophillus  |
|  Candida albicans | HPV40 | Neisseria gonorrhoeae  |
|  Chlamydia trachomatis | HPV42 | Peptostreptococcus anaerobius  |
|  Clostridioides difficile | HPV53 | Peptostreptococcus asaccharolyticus  |
|  Clostridium perfringens | HPV54 | Proteus mirabilis  |
|  Corynebacterium genitalium | HPV55 | Proteus penneri  |
|  Cytomegalovirus | HPV61 | Proteus vulgaris  |
|  Enterobacter aerogenes | HPV62 | Pseudomonas aeruginosa  |
|  Enterobacter cloacae | HPV64 | Pseudomonas fluorescens  |
|  Enterococcus avium | HPV67 | Pseudomonas putida  |
|  Enterococcus casseliflavus | HPV69 | Staphylococcus aureus  |
|  Enterococcus faecalis | HPV70 | Staphylococcus epidermidis  |
|  Enterococcus faecium | HPV71 | Streptococcus agalactiae  |
|  Epstein Barr Virus | HPV72 | Streptococcus pyogenes  |
|  Escherichia coli | HPV73 | Treponema pallidum  |
|  Finegodia magna* | HPV81 | Trichomonas vaginalis  |
|  Fusobacterium nucleatum | HPV82 |   |
|  Herpes Simplex Virus 1 | HPV83 |   |

*formerly Peptostreptococcus magnus

# 5. Interference

The effects of endogenous and exogenous substances on the performance of the cobas HPV were evaluated. HPV negative and positive specimens were tested in the presence or absence of each potential interferent that may be present in clinical cervical specimens. The concentrations of exogenous and endogenous substances tested in this study represent concentrations that could potentially occur during specimen collection. Specimens were prepared from pooled negative clinical matrix in PreservCyt Solution. All testing for interference was performed with each potential interfering substance alone as well as with the substance mixed with SiHa (HPV16) and HeLa (HPV18) cell lines at approximately 3x LoD at the clinical cutoff of cobas HPV in HPV negative samples. The study design was acceptable and the results of this study are described below.

Endogenous substances tested were cervical mucus, peripheral blood mononuclear cells and whole blood. Levels of endogenous substances tolerated by the assay are shown in Table 6. Exogenous substance testing included 18 over-the-counter (OTC)

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feminine hygiene and prescription products and glacial acetic acid that are listed in Table 7.

Of OTC feminine hygiene and prescription products tested, Metronidazole Gel, Replens, RepHresh Odor Eliminating Vaginal Gel and RepHresh Clean Balance Feminine Freshness Kit produced false negative results. An appropriate limitation has been included in the Package Insert.

Table 6: Endogenous substances tested for interference
|  Endogenous Substance | PreservCyt  |
| --- | --- |
|  Mucus | Presence*  |
|  Peripheral Blood Mononuclear Cells (PBMCs as cells/mL) | 1.00E+06  |
|  Whole Blood (% v/v) | 10%  |

*Presence refers to the amount of cervical mucus normally removed from the cervix prior to sampling.

Table 7: Exogenous substances tested for interference
|  Product Name | Concentration  |
| --- | --- |
|  Clindamycin Phosphate Vaginal Cream | 1.40 mg/mL  |
|  CVS Tioconazole 1 (Equate™ tioconazole 1) | 8.02 mg/mL  |
|  Equate™ Vagicaine Anti-Itch Cream | 5.87 mg/mL  |
|  Estrace® Cream | 4.38 mg/mL  |
|  K-Y® Ultra Gel | 6.59 mg/mL  |
|  Metronidazole Vaginal Gel§ | 0.20 mg/mL*  |
|  Monistat® 3 Vaginal Antifungal Combination Pack | 1.57 mg/mL  |
|  Monistat® Complete Care Itch Relief Cream | 4.76 mg/mL  |
|  Gyne-Lotrimin® 7 | 3.13 mg/mL  |
|  Norforms® Suppositories | 1.10 mg/mL  |
|  Premarin® Vaginal Cream | 3.65 mg/mL  |
|  Replens™ Long-Lasting Vaginal Moisturizer§ | 0.96 mg/mL†  |
|  RepHresh™ Odor Eliminating Vaginal Gel§ | ‡  |
|  RepHresh™ Clean Balance™ Feminine Freshness Kit§ | ‡  |
|  Summer's Eve® Feminine Deodorant Spray | 0.90 mg/mL  |
|  VCF® - Vaginal Contraceptive Foam | 1.42 mg/mL  |
|  Yeast Gard Advanced® | 3.04 mg/mL  |
|  ZOVIRAX® (acyclovir) Cream 5% | 10.37 mg/mL  |
|  Glacial acetic acid | 5% (v/v)  |
|  * Concentration of product that did not cause interference with test performance.
† Concentration of product that did not cause interference with test performance.
‡ Concentrations of product that did not interfere with test performance were not determined
§ Products containing carbomer(s) have been shown to cause interference  |   |

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# 6. Competitive Inhibition

A competitive inhibition study was performed to test whether high concentrations of high risk or low risk HPV DNA could interfere with the genotyping capability of cobas HPV to detect HPV 16 and HPV18. Competitive inhibition of HPV16 and HPV18 detection was assessed by testing samples containing low concentrations of HPV16 and HPV18 along with high concentration of non-targeted low risk HPV and targeted 12 Other high risk HPV types. The HPV16 and HPV18 were spiked to concentrations close to about 1x LoD at the clinical cutoff; each of the 25 low risk and 12 Other high risk HPV tested were at a concentration 1000-fold (3log10) higher than that of HPV16 and HPV18.

Results of this study showed that cobas HPV can detect low concentrations of HPV16 and HPV18 in the presence of 1000-fold higher concentration of any of the 25 non-targeted low risk and 12 Other high risk HPV types. No competitive interference was observed from any of the competing targets.

# 7. Within-Laboratory Precision

Within-laboratory precision was performed using a panel composed of either HPV cell lines or HPV positive clinical samples diluted into a pool of negative cervical specimen matrix. The precision panel was designed to include members with high negative, very low (&lt; LoD at the clinical cutoff), low (~LoD at the clinical cutoff) and moderate (~3x LoD at the clinical cutoff) concentrations of HPV as well as an HPV negative. Testing was performed with three lots of cobas HPV reagents, two instruments, and two users. There was an equal number of runs performed on the cobas 6800 and the cobas 8800 Systems over 12 days for a total of 24 runs for each panel member. The observed hit rates for each panel member are shown in Table 8. Table 9, Table 10, and Table 11 summarize the variance components analysis in positive panel members separated by 12 Other High Risk HPV, HPV16, and HPV18. The overall CV (%) ranged from  $4.32\%$  to  $6.19\%$  for 12 Other High Risk HPV,  $1.09\%$  to  $4.61\%$  for HPV16, and  $1.23\%$  to  $3.76\%$  for HPV18.

Table 8: Summary of within laboratory precision study

|  Panel Level | Expected Hit Rate | Target Source | HPV Concentration | Target Channel | N Tested | N Positive | Hit Rate | 95% CI  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |   |   |   |  LL | UL  |
|  Negative | 0% | N/A | N/A | 12 Other HR HPV | 72 | 0 | 0% | 0% | 5%  |
|  Negative | 0% | N/A |   | HPV16 | 72 | 0 | 0% | 0% | 5%  |
|  Negative | 0% | N/A |   | HPV18 | 72 | 0 | 0% | 0% | 5%  |
|  High Negative | ≤ 5% | Clinical sample | N/A | 12 Other HR HPV | 72 | 0 | 0% | 0% | 5%  |
|  High Negative | ≤ 5% | Clinical sample |   | HPV16 | 72 | 0 | 0% | 0% | 5%  |
|  High Negative | ≤ 5% | Clinical sample |   | HPV18 | 72 | 5 | 7% | 3% | 15%  |
|  < 1x LoD | < 95% | Clinical sample | N/A | 12 Other HR HPV | 72 | 30 | 42% | 31% | 53%  |
|  < 1x LoD | < 95% | Clinical sample | N/A | HPV16 | 71 | 33 | 47% | 35% | 58%  |
|  < 1x LoD | < 95% | Clinical sample | N/A | HPV18 | 72 | 49 | 68% | 57% | 78%  |

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|  Panel Level | Expected Hit Rate | Target Source | HPV Concentration | Target Channel | N Tested | N Positive | Hit Rate | 95% CI  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |   |   |   |   |  LL | UL  |
|  < 1x LoD | 20-80% | SiHa cell line | 4.8 cells/mL | HPV16 | 72 | 44 | 61% | 50% | 72%  |
|  < 1x LoD | 20-80% | HeLa cell line | 4.8 cells/mL | HPV18 | 72 | 49 | 68% | 57% | 78%  |
|  ~ 1x LoD | ≥ 95% | Clinical sample | N/A | 12 Other HR HPV | 72 | 72 | 100% | 95% | 100%  |
|  ~ 1x LoD | ≥ 95% | SiHa cell line | 16 cells/mL | HPV16 | 72 | 72 | 100% | 95% | 100%  |
|  ~ 1x LoD | ≥ 95% | HeLa cell line | 16 cells/mL | HPV18 | 72 | 72 | 100% | 95% | 100%  |
|  > 1x LoD | ≥ 99% | Clinical sample | N/A | 12 Other HR HPV | 72 | 72 | 100% | 95% | 100%  |
|  > 1x LoD | ≥ 99% | SiHa cell line | 48 cells/mL | HPV16 | 72 | 72 | 100% | 95% | 100%  |
|  > 1x LoD | ≥ 99% | HeLa cell line | 48 cells/mL | HPV18 | 72 | 72 | 100% | 95% | 100%  |

CI = Confidence interval, LL = Lower limit, UL = Upper limit

Table 9: Overall mean, standard deviations and coefficients of variation (%) for cycle threshold - 12 Other High Risk HPV

|   |   |   | Between-Day |   | Between-Instrument |   | Between-Operator |   | Between-Lot |   | Between-Run |   | Within-Run |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Level | Hit Rate | Mean Ct | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV  |
|  < LoD | 41.7% | 33.2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.47 | 1.43 | 1.72 | 5.18 | 1.78 | 5.37  |
|  ~ LoD | 100% | 32.4 | 0 | 0 | 0 | 0 | 0.49 | 1.50 | 0.16 | 0.51 | 0 | 0 | 1.94 | 5.98 | 2.01 | 6.19  |
|  > LoD | 100% | 30.7 | 0 | 0 | 0 | 0 | 0 | 0 | 0.27 | 0.88 | 0 | 0 | 1.30 | 4.23 | 1.33 | 4.32  |

Table 10: Overall mean, standard deviations and coefficients of variation (%) for cycle threshold - HPV16

|   |   |   | Between-Day |   | Between-Instrument |   | Between-Operator |   | Between-Lot |   | Between-Run |   | Within-Run |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Level | Hit Rate | Mean Ct | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV  |
|  < LoD | 46.5 % | 35.7 | 0.84 | 2.34 | 0.29 | 0.80 | 0.85 | 2.39 | 0 | 0 | 0 | 0 | 1.10 | 3.07 | 1.65 | 4.61  |
|  < LoD | 61.1 % | 36.1 | 0.44 | 0.67 | 0 | 0 | 0.16 | 0.45 | 0.21 | 0.57 | 0 | 0 | 0.49 | 1.36 | 0.61 | 1.68  |
|  ~ LoD | 100 % | 35.0 | 0 | 0 | 0.02 | 0.06 | 0.02 | 0.07 | 0.38 | 1.09 | 0 | 0 | 0.45 | 1.28 | 0.59 | 1.69  |
|  > LoD | 100 % | 34.0 | 0.03 | 0.09 | 0.04 | 0.12 | 0 | 0 | 0.27 | 0.78 | 0 | 0 | 0.25 | 0.74 | 0.37 | 1.09  |

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Table 11: Overall mean, standard deviations and coefficients of variation (%) for cycle threshold - HPV18

|   |   |   | Between-Day |   | Between-Instrument |   | Between-Operator |   | Between-Lot |   | Between-Run |   | Within-Run |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Level | Hit Rate | Mean Ct | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV  |
|  < LoD | 68.1% | 35.9 | 0 | 0 | 0.55 | 1.52 | 0 | 0 | 0.18 | 0.51 | 0.17 | 0.49 | 1.21 | 3.37 | 1.35 | 3.76  |
|  < LoD | 68.1% | 35.3 | 0.19 | 0.54 | 0 | 0 | 0.02 | 0.06 | 0 | 0 | 0 | 0 | 0.97 | 2.75 | 0.99 | 2.80  |
|  ~ LoD | 100% | 33.8 | 0 | 0 | 0 | 0 | 0 | 0 | 0.37 | 1.11 | 0 | 0 | 0.73 | 2.17 | 0.82 | 2.44  |
|  > LoD | 100% | 32.2 | 0 | 0 | 0 | 0 | 0 | 0 | 0.22 | 0.68 | 0.03 | 0.10 | 0.33 | 1.02 | 0.39 | 1.23  |

# 8. Lot-to-lot variability

Lot-to-lot variability was evaluated at one testing site, using three reagent lots for each of the two systems separately (cobas 6800 and cobas 8800). This study was performed along with the reproducibility study at testing site 3. Each panel member was tested over 15 days (5 days per lot) with three replicates per run on each of the two cobas systems. Two operators performed one run per day for 5 days for each reagent lot. The lot-to-lot variability study design was identical for both the cobas 6800 and the cobas 8800 Systems. A 13-member panel composed of pools made from clinical samples collected into PreservCyt Solution and from samples derived from SiHa and HeLa cell lines was tested.

Table 12 and Table 13 show results by reagent lot, operator/run, and day on the cobas 6800 System for the negative and positive panel members, respectively. All negative panel members were correctly identified as negative across reagent lot, operator/run and testing day. Analysis of variance of the Ct values from valid tests performed on positive panel members yielded total CV (%) ranging from  $0.9\%$  to  $5.0\%$  across all panel members. The CV(%) ranged from  $0.9\%$  to  $2.2\%$  for the cell line panel members and  $1.7\%$  to  $5.0\%$  for the pooled clinical panel members.

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Table 12: Agreement and variability of negative panel members by lot, operator/run, and day on the cobas 6800 System

|   |   |   | Number Negative / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Negative agreement (%) | Negative/ Valid | ID* | Negative agreement (%) | Negative/ Valid | ID | Negative agreement (%) | Negative/ Valid  |
|  Negative background cell line | n/a | n/a | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Negative pooled clinical samples | n/a | n/a | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

*Note: Operators 5 and 6 were at testing site 3.
Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation; n/a=not applicable.

Table 13: Agreement and variability of positive panel members by lot, operator/run, and day on the cobas 6800 System

|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive/ Valid | ID* | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive/ Valid  |
|  Positive Cell Line Panel Members: HPV16/18 Weak Positive (0.3x LOD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Weak Positive (0.3x LOD) | 0.75 | 2.0 | 1 | 46.7 | 14/30 | 5 | 57.8 | 26/45 | 1 | 61.1 | 11/18  |
|   |   |  | 2 | 46.7 | 14/30 | 6 | 53.3 | 24/45 | 2 | 44.4 | 8/18  |
|   |   |  | 3 | 73.3 | 22/30 |  |  |  | 3 | 38.9 | 7/18  |
|   |   |  |  |  |  |  |  |  | 4 | 77.8 | 14/18  |
|   |   |  |  |  |  |  |  |  | 5 | 55.6 | 10/18  |
|  HPV18 Weak Positive (0.3x LOD) | 0.77 | 2.2 | 1 | 60.0 | 18/30 | 5 | 62.2 | 28/45 | 1 | 66.7 | 12/18  |
|   |   |  | 2 | 60.0 | 18/30 | 6 | 66.7 | 30/45 | 2 | 66.7 | 12/18  |
|   |   |  | 3 | 73.3 | 22/30 |  |  |  | 3 | 66.7 | 12/18  |
|   |   |  |  |  |  |  |  |  | 4 | 66.7 | 12/18  |
|   |   |  |  |  |  |  |  |  | 5 | 55.6 | 10/18  |

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|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive/ Valid | ID* | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive/ Valid  |
|  Positive Cell Line Panel Members: HPV16/18 Low Positive (1x LOD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Low Positive (1x LOD) | 0.50 | 1.4 | 1 | 100.0 | 30/30 | 5 | 97.8 | 44/45 | 1 | 94.4 | 17/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 96.7 | 29/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  HPV18 Low Positive (1x LOD) | 0.67 | 2.0 | 1 | 96.7 | 29/30 | 5 | 97.8 | 44/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 94.4 | 17/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Positive Cell Line Panel Members: HPV16/18 Positive (3x LOD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Positive(3x LOD) | 0.31 | 0.9 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  HPV18 Positive(3x LOD) | 0.39 | 1.2 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LOD) | 1.13 | 3.4 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

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|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive/ Valid | ID* | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive/ Valid  |
|  Pooled HPV18 Low Positive (1x LOD) | 0.60 | 1.7 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Pooled HPV18 Positive (3x LOD) | 0.86 | 2.5 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Pooled HPV45 Low Positive (1x LOD) | 1.60 | 5.0 | 1 | 100.0 | 30/30 | 5 | 97.8 | 44/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 96.7 | 29/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 94.4 | 17/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Pooled HPV45 Positive (3x LOD) | 1.46 | 4.9 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Pooled HPV39 Low Positive (1x LOD) | 0.75 | 2.3 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Pooled HPV39 Positive (3x LOD) | 0.84 | 2.6 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

*Note: Operators 5 and 6 were at testig site 3.
Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation.

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Table 14 and Table 15 show results for the negative and positive panel member by reagent lot, operator/run, and day on the cobas 8800 System respectively. All negative panel members were correctly identified as negative across reagent lot, operator/run and testing day. Analysis of variance of the Ct values from valid tests performed on positive panel members yielded total CV (%) ranging from 1.1% to 7.4% across all panel members. The CV (%) ranged from 1.1% to 3.0% for the cell line panel members and 2.0% to 7.4% for the pooled clinical panel members.

Table 14: Agreement and variability of negative panel members by lot, operator/run, and day on the cobas 8800 System

|   |   |   | Number Negative / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Negative Agreement (%) | Negative/ Valid | ID* | Negative Agreement (%) | Negative/ Valid | ID | Negative Agreement t (%) | Negative/ Valid  |
|  Negative background cell line | n/a | n/a | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Negative pooled clinical samples | n/a | n/a | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

*Note: Operators 5 and 6 were at testig site 3.
Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation; n/a=not applicable.

Table 15: Agreement and variability of positive panel members by lot, operator/run, and day on the cobas 8800 System

|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive / Valid | ID¹ | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive / Valid  |
|  Positive Cell Line Panel Members: HPV16/18 Weak Positive (0.3x LOD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Weak Positive (0.3x LOD) | 0.67 | 1.8 | 1 | 60.0 | 18/30 | 5 | 57.8 | 26/45 | 1 | 66.7 | 12/18  |
|   |   |  | 2 | 63.3 | 19/30 | 6 | 68.9 | 31/45 | 2 | 61.1 | 11/18  |
|   |   |  | 3 | 66.7 | 20/30 |  |  |  | 3 | 72.2 | 13/18  |
|   |   |  |  |  |  |  |  |  | 4 | 66.7 | 12/18  |
|   |   |  |  |  |  |  |  |  | 5 | 50.0 | 9/18  |
|   | 1.07 | 3.0 | 1 | 70.0 | 21/30 | 5 | 73.3 | 33/45 | 1 | 77.8 | 14/18  |

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|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive / Valid | ID¹ | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive / Valid  |
|  HPV18 Weak Positive (0.3x LOD) |  |  | 2 | 70.0 | 21/30 | 6 | 64.4 | 29/45 | 2 | 72.2 | 13/18  |
|   |   |  | 3 | 66.7 | 20/30 |  |  |  | 3 | 72.2 | 13/18  |
|   |   |  |  |  |  |  |  |  | 4 | 72.2 | 13/18  |
|   |   |  |  |  |  |  |  |  | 5 | 50.0 | 9/18  |
|  Positive Cell Line Panel Members: HPV16/18 Low Positive (1x LOD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Low Positive (1x LOD) | 0.44 | 1.2 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 96.7 | 29/30 | 6 | 95.6 | 43/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 96.7 | 29/30 |  |  |  | 3 | 94.4 | 17/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 94.4 | 17/18  |
|  HPV18 Low Positive (1x LOD) | 0.74 | 2.2 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Positive Cell Line Panel Members: HPV16/18 Positive (3x LOD)²  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Positive (x LOD) | 0.38 | 1.1 | 1 | 100.0 | 29/29 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 44/44 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 17/17  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  HPV18 Positive (3x LOD) | 0.41 | 1.2 | 1 | 100.0 | 29/29 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 44/44 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 17/17  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LOD) | 0.91 | 2.7 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

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|  | Number of Positives / Total Number Valid Results |
| --- | --- |
|  | Between-Lot | Between-Operator | Between-Day |
| Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive / Valid | ID¹ | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive / Valid |
| Pooled HPV16 Positive (3x LOD) |  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |
| Pooled HPV18 Low Positive (1x LOD) | 0.70 | 2.0 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18 |
|  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |
| Pooled HPV18 Positive (3x LOD) | 1.02 | 3.0 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18 |
|  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |
| Pooled HPV45 Low Positive (1x LOD) | 2.32 | 7.4 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18 |
|  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |
| Pooled HPV45 Positive (3x LOD) | 1.74 | 5.9 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18 |
|  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |
| Pooled HPV39 Low Positive (1x LOD) | 1.06 | 3.2 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18 |
|  |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18 |
|  |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 4 | 100.0 | 18/18 |
|  |  |  |  |  |  |  |  | 5 | 100.0 | 18/18 |

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|   |   |   | Number of Positives / Total Number Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Lot |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV % | ID | Positive Agreement (%) | Positive / Valid | ID¹ | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive / Valid  |
|  Pooled HPV39 Positive (3x LOD) | 1.52 | 4.8 | 1 | 100.0 | 30/30 | 5 | 100.0 | 45/45 | 1 | 100.0 | 18/18  |
|   |   |  | 2 | 100.0 | 30/30 | 6 | 100.0 | 45/45 | 2 | 100.0 | 18/18  |
|   |   |  | 3 | 100.0 | 30/30 |  |  |  | 3 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 4 | 100.0 | 18/18  |
|   |   |  |  |  |  |  |  |  | 5 | 100.0 | 18/18  |

¹Note: Operators 5 and 6 were at testing site 3.
²One replicate failed due to processing error and excluded from analysis.
Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation.

Analyses were performed for between lot, between operator, between day, and within run variability for both the cobas 6800 and 8800 Systems separately as shown in Table 16 and Table 17, respectively.

Table 16: Overall mean, standard deviation, and coefficients of variation (%) for cycle threshold, estimated from positive panel members on cobas 6800 System

|   |   |   | Standard Deviation, Coefficient of Variation (%)  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | N | Mean Ct | Between-Lot | Between-Operator | Between-Day | Within-Run | Total CV  |
|  Positive Cell Line Panel Members  |   |   |   |   |   |   |   |
|  HPV16/18 Weak Positive (0.3x LOD)  |   |   |   |   |   |   |   |
|  HPV16 Weak Positive (0.3x LOD) | 52 | 36.5 | 0.07, (0.20%) | 0.00, (0.00%) | 0.28, (0.78%) | 0.69, (1.88%) | 2.0  |
|  HPV18 Weak Positive (0.3x LOD) | 58 | 35.4 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.77, (2.19%) | 2.2  |
|  HPV16/18 Low Positive (1x LOD)  |   |   |   |   |   |   |   |
|  HPV16 Low Positive (1x LOD) | 89 | 35.6 | 0.09, (0.25%) | 0.04, (0.12%) | 0.00, (0.00%) | 0.49, (1.37%) | 1.4  |
|  HPV18 Low Positive (1x LOD) | 89 | 34.1 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.67, (1.97%) | 2.0  |
|  HPV 16/18 Positive (3x LOD)  |   |   |   |   |   |   |   |
|  HPV16 Positive (3x LOD) | 90 | 34.6 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.31, (0.88%) | 0.9  |
|  HPV18 Positive (3x LOD) | 90 | 32.9 | 0.00, (0.00%) | 0.00, (0.00%) | 0.13, (0.41%) | 0.36, (1.10%) | 1.2  |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LOD) | 90 | 33.5 | 0.11, (0.31%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.12, (3.35%) | 3.4  |

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|   |   |   | Standard Deviation, Coefficient of Variation (%)  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | N | Mean Ct | Between-Lot | Between-Operator | Between-Day | Within - Run | Total CV  |
|  Pooled HPV16 Positive (3x LOD) | 90 | 33.1 | 0.11, (0.32%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.00, (3.01%) | 3.0  |
|  Pooled HPV18 Low Positive (1x LOD) | 90 | 35.1 | 0.14, (0.41%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.58, (1.67%) | 1.7  |
|  Pooled HPV18 Positive (3x LOD) | 90 | 33.7 | 0.00, (0.00%) | 0.26, (0.76%) | 0.14, (0.43%) | 0.81, (2.39%) | 2.5  |
|  Pooled HPV45 Low Positive (1x LOD) | 90 | 32.0 | 0.00, (0.00%) | 0.00, (0.00%) | 0.42, (1.31%) | 1.55, (4.84%) | 5.0  |
|  Pooled HPV45 Positive (3x LOD) | 90 | 29.7 | 0.18, (0.62%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.45, (4.89%) | 4.9  |
|  Pooled HPV39 Low Positive (1x LOD) | 90 | 33.3 | 0.00, (0.00%) | 0.00, (0.00%) | 0.23, (0.69%) | 0.71, (2.14%) | 2.3  |
|  Pooled HPV39 Positive (3x LOD) | 90 | 31.6 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.84, (2.65%) | 2.6  |

Table 17: Overall mean, standard deviation, and coefficients of variation (%) for cycle threshold, estimated from positive panel members on cobas 8800 System

|   |   |   | Standard Deviation, Coefficient of Variation (%)  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | N | Mean Ct | Between-Lot | Between-Operator | Between-Day | Within - Run | Total CV  |
|  Positive Cell Line Panel Members  |   |   |   |   |   |   |   |
|  HPV16/18 Weak Positive (0.3x LOD)  |   |   |   |   |   |   |   |
|  HPV 16 Weak Positive (0.3 x LOD) | 58 | 36.6 | 0.00, (0.00%) | 0.16, (0.45%) | 0.16, (0.44%) | 0.63, (1.72%) | 1.8  |
|  HPV 18 Weak Positive (0.3 x LOD) | 63 | 35.5 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.07, (3.01%) | 3.0  |
|  HPV16/18 Low Positive (1x LOD)  |   |   |   |   |   |   |   |
|  HPV 16 Low Positive (1 x LOD) | 88 | 35.6 | 0.00, (0.00%) | 0.00, (0.00%) | 0.14, (0.39%) | 0.42, (1.18%) | 1.2  |
|  HPV 18 Low Positive (1 x LOD) | 90 | 34.2 | 0.00, (0.00%) | 0.16, (0.46%) | 0.30, (0.86%) | 0.66, (1.94%) | 2.2  |
|  HPV16/18 Positive (3x LOD)  |   |   |   |   |   |   |   |
|  HPV 16 Positive (3 x LOD) | 89 | 34.6 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.38, (1.10%) | 1.1  |
|  HPV 18 Positive (3 x LOD) | 89 | 32.7 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.41, (1.24%) | 1.2  |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LOD) | 90 | 33.6 | 0.07, (0.21%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.91, (2.71%) | 2.7  |

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|   |   |   | Standard Deviation, Coefficient of Variation (%)  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | N | Mean Ct | Between-Lot | Between-Operator | Between-Day | Within - Run | Total CV  |
|  Pooled HPV16 Positive (3x LOD) | 90 | 32.9 | 0.00, (0.00%) | 0.00, (0.00%) | 0.13, (0.39%) | 0.87, (2.64%) | 2.7  |
|  Pooled HPV18 Low Positive (1x LOD) | 90 | 35.0 | 0.00, (0.00%) | 0.05, (0.15%) | 0.16, (0.47%) | 0.68, (1.94%) | 2.0  |
|  Pooled HPV18 Positive (3x LOD) | 90 | 33.6 | 0.24, (0.70%) | 0.25, (0.75%) | 0.18, (0.54%) | 0.94, (2.80%) | 3.0  |
|  Pooled HPV45 Low Positive (1x LOD) | 90 | 31.2 | 0.40, (1.27%) | 0.00, (0.00%) | 0.74, (2.37%) | 2.16, (6.93%) | 7.4  |
|  Pooled HPV45 Positive (3x LOD) | 90 | 29.5 | 0.00, (0.00%) | 0.41, (1.40%) | 0.59, (2.00%) | 1.59, (5.39%) | 5.9  |
|  Pooled HPV39 Low Positive (1x LOD) | 90 | 33.1 | 0.00, (0.00%) | 0.00, (0.00%) | 0.30, (0.92%) | 1.02, (3.07%) | 3.2  |
|  Pooled HPV39 Positive (3x LOD) | 90 | 31.4 | 0.00, (0.00%) | 0.00, (0.00%) | 0.59, (1.88%) | 1.40, (4.46%) | 4.8  |

# 9. Equivalence between cobas 6800 and 8800 Systems

The lot-to-lot variability study data was also analyzed to compare the results obtained between the cobas 6800 cobas 8800 Systems to assess systems equivalency. The two systems generated comparable hit rates as shown below in Table 18.

Table18: Comparison of hit rates between cobas 6800 and cobas 8800 Systems

|   | cobas 6800 System |   |   | cobas 8800 System  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | No. of Valid Tests | No. of Correct Results N | Percent of Correct Results % (95% CI) | No. of Valid Tests | No. of Correct Results N | Percent of Correct Results % (95% CI)  |
|  Negative background cell line | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  HPV 16/18 Weak Positive (0.3 x LoD)  |   |   |   |   |   |   |
|  HPV 16 Weak Positive (0.3x LoD) | 90 | 50 | 55.6 (44.7, 66.0) | 90 | 57 | 63.3 (52.5, 73.2)  |
|  HPV 18 Weak Positive (0.3x LoD) | 90 | 58 | 64.4 (53.7, 74.3) | 90 | 62 | 68.9 (58.3, 78.2)  |
|  HPV 16/18 Low Positive (1 x LOD)  |   |   |   |   |   |   |
|  HPV 16 Low Positive (1x LoD) | 90 | 89 | 98.9 (94.0, 100.0) | 90 | 88 | 97.8 (92.2, 99.7)  |
|  HPV 18 Low Positive (1x LoD) | 90 | 89 | 98.9 (94.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  HPV 16/18 Positive (3 x LoD)  |   |   |   |   |   |   |
|  HPV 16 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 89 | 89 | 100.0 (95.9, 100.0)  |

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|   | cobas 6800 System |   |   | cobas 8800 System  |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | No. of Valid Tests | No. of Correct Results N | Percent of Correct Results % (95% CI) | No. of Valid Tests | No. of Correct Results N | Percent of Correct Results % (95% CI)  |
|  HPV 18 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 89 | 89 | 100.0 (95.9, 100.0)  |
|  |   |   |   |   |   |   |
|  Negative pooled clinical samples | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 16 Low positive (1x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 16 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 18 Low Positive (1x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 18 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 45 Low Positive (1x LoD) | 90 | 89 | 98.9 (94.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 45 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 39 Low Positive (1x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |
|  Pooled HPV 39 Positive (3x LoD) | 90 | 90 | 100.0 (96.0, 100.0) | 90 | 90 | 100.0 (96.0, 100.0)  |

## 10. Reproducibility

A site-to-site reproducibility study was performed using the same panel as described in the lot-to-lot variability study. Testing was conducted at three testing sites using one reagent lot and four cobas systems (three cobas 6800 Systems at all three testing sites and one cobas 8800 System at one of those three sites). Each panel member was tested over five days with three replicates per run on the four systems. Two operators performed one run per day for five days for each system. Overall, 41 runs were performed, with 30 runs on the cobas 6800 and 11 on the cobas 8800 (with 1 failed run).

Testing of the negative panel members by site/instrument, operator/run, and day on the three cobas 6800 Systems and one cobas 8800 System are summarized in Table 19. All negative panel members were correctly identified as negative across site/instrument, operator/run and testing day. The study demonstrated that the cobas HPV for use on the cobas 6800/8800 Systems produced results that were reproducible across reagent lots, sites/systems, operators, days, and within- and between-runs.

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Table 19: Agreement and variability for negative panel member for site/instrument, operator/run, and day on the cobas 6800/8800 System

|   |   |   | Number of Negatives/Total Number of Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Site/Instrument |   |   | Between-Operator |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Negative Agreement (%) | Negative/Valid | ID¹ | Negative Agreement (%) | Negative/Valid | ID | Negative Agreement (%) | Negative/Valid  |
|  Negative background cell line | n/a | n/a | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Negative pooled clinical samples | n/a | n/a | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |

Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation; n/a=not applicable.
¹Operators 1 and 2 were at testing site 1; Operators 3 and 4 were at testing site 2; Operators 5 and 6 were at testing site 3.
Operators 5 and 6 from site 3 ran both the cobas 6800 and cobas 8800 Systems.

Percent of positive results for the positive panel members are presented in Table 20. Analysis of variance of the Ct values from tests performed on the positive panel members yielded total CV(%) ranging from 1.1% to 5.6% across all panel members. The CV(%) ranged from 1.1% to 2.7% for the cell line panel members and 2.1% to 5.6% for the pooled clinical panel members. The overall mean, standard deviation, and coefficient of variation were also calculated for positive panel members as shown in Table 21.

Table 20: Agreement and variability for positive panel members for site/instrument, operator/run, and day on the cobas 6800/8800 Systems

|   |   |   | Number of Positive Results / Total Number of Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Site/Instrument |   |   | Between-Operator/Run |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Positive Agreement (%) | Positive/Valid | ID¹ | Positive Agreement (%) | Positive/Valid | ID | Positive Agreement (%) | Positive/Valid  |
|  Positive Cell Line Panel Members HPV16/18 Weak Positive (0.3 x LoD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Weak Positive (0.3x LoD) | 0.76 | 2.1 | 11 | 66.7 | 20/30 | 1 | 60.0 | 9/15 | 1 | 58.3 | 14/24  |
|   |   |  | 21 | 76.7 | 23/30 | 2 | 73.3 | 11/15 | 2 | 54.2 | 13/24  |
|   |   |  | 31 | 46.7 | 14/30 | 3 | 93.3 | 14/15 | 3 | 62.5 | 15/24  |
|   |   |  | 32 | 60.0 | 18/30 | 4 | 60.0 | 9/15 | 4 | 83.3 | 20/24  |
|   |   |  |  |  |  | 5 | 53.3 | 16/30 | 5 | 54.2 | 13/24  |
|   |   |  |  |  |  | 6 | 53.3 | 16/30 |  |  |   |

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|   |   |   | Number of Positive Results / Total Number of Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Site/Instrument |   |   | Between-Day |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Positive Agreement (%) | Positive/ Valid | ID1 | Positive Agreement (%) | Positive/ Valid | ID | Positive Agreement (%) | Positive/ Valid  |
|  HPV18 Weak Positive (0.3x LoD) | 0.96 | 2.7 | 11 | 53.3 | 16/30 | 1 | 40.0 | 6/15 | 1 | 70.8 | 17/24  |
|   |   |  | 21 | 60.0 | 18/30 | 2 | 66.7 | 10/15 | 2 | 66.7 | 16/24  |
|   |   |  | 31 | 60.0 | 18/30 | 3 | 60.0 | 9/15 | 3 | 45.8 | 11/24  |
|   |   |  | 32 | 70.0 | 21/30 | 4 | 60.0 | 9/15 | 4 | 70.8 | 17/24  |
|   |   |  |  |  |  | 5 | 73.3 | 22/30 | 5 | 50.0 | 12/24  |
|   |   |  |  |  |  | 6 | 56.7 | 17/30 |  |  |   |
|  Positive Cell Line Panel Members HPV16/18 Low Positive (1 x LoD)  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Low Positive (1x LoD) | 0.47 | 1.3 | 11 | 96.7 | 29/30 | 1 | 100.0 | 15/15 | 1 | 95.8 | 23/24  |
|   |   |  | 21 | 96.7 | 29/30 | 2 | 93.3 | 14/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 93.3 | 14/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 95.8 | 23/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  HPV18 Low Positive (1x LoD) | 0.63 | 1.9 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 96.7 | 29/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 95.8 | 23/24  |
|   |   |  |  |  |  | 5 | 96.7 | 29/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Positive Cell Line Panel Members HPV16/18 Positive (3 x LoD)2  |   |   |   |   |   |   |   |   |   |   |   |
|  HPV16 Positive (3x LoD) | 0.37 | 1.1 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 29/29 | 4 | 100.0 | 15/15 | 4 | 100.0 | 23/23  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 29/29 |  |  |   |
|  HPV18 Positive (3x LoD) | 0.40 | 1.2 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 29/29 | 4 | 100.0 | 15/15 | 4 | 100.0 | 23/23  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 29/29 |  |  |   |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LoD) | 1.07 | 3.2 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV16 Positive (3x LoD) | 0.89 | 2.7 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV18 Low Positive (1x LoD) | 0.74 | 2.1 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 96.7 | 29/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |

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|   |   |   | Number of Positive Results / Total Number of Valid Results  |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   | Between-Site/Instrument |   |   | Between-Day |   |   | Between-Day  |   |   |
|  Panel Member | Ct SD | Ct CV% | ID | Positive Agreement (%) | Positive/Valid | ID¹ | Positive Agreement (%) | Positive/Valid | ID | Positive Agreement (%) | Positive/Valid  |
|   |  |  | 32 | 100.0 | 30/30 | 4 | 93.3 | 14/15 | 4 | 95.8 | 23/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV18 Positive (3x LoD) | 0.92 | 2.7 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV45 Low Positive (1x LoD) | 1.80 | 5.6 | 11 | 96.7 | 29/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 93.3 | 14/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 95.8 | 23/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV45 Positive (3x LoD) | 1.54 | 5.2 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV39 Low Positive (!x LoD) | 1.04 | 3.1 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |
|  Pooled HPV39 Positive (3x LoD) | 1.45 | 4.6 | 11 | 100.0 | 30/30 | 1 | 100.0 | 15/15 | 1 | 100.0 | 24/24  |
|   |   |  | 21 | 100.0 | 30/30 | 2 | 100.0 | 15/15 | 2 | 100.0 | 24/24  |
|   |   |  | 31 | 100.0 | 30/30 | 3 | 100.0 | 15/15 | 3 | 100.0 | 24/24  |
|   |   |  | 32 | 100.0 | 30/30 | 4 | 100.0 | 15/15 | 4 | 100.0 | 24/24  |
|   |   |  |  |  |  | 5 | 100.0 | 30/30 | 5 | 100.0 | 24/24  |
|   |   |  |  |  |  | 6 | 100.0 | 30/30 |  |  |   |

Notes: Ct=Cycle Threshold; SD=Standard Deviation; CV=Coefficient of Variation.
¹Operators 1 and 2 were at testing site 1; Operators 3 and 4 were at testing site 2; Operators 5 and 6 were at testing site 3.
²One replicate failed due to processing error and excluded from analysis.

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Table 21: Overall mean, standard deviation, and coefficients of variation (%) for cycle threshold, estimated from positive panel members

|   |   |   | Standard Deviation, Coefficient of Variation (%)  |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member | N | Mean Ct | Between-Site/Instru-ment | Between-Operator/Run | Between-Day | Within-Run | Total CV  |
|  Positive Cell Line Panel Members  |   |   |   |   |   |   |   |
|  HPV16/18 Weak Positive (0.3x LoD) |  |  |  |  |  |  |   |
|  HPV16 Weak Positive (0.3x LoD) | 77 | 36.6 | 0.00, (0.00%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.76, (2.08%) | 2.1  |
|  HPV18 Weak Positive (0.3x LoD) | 74 | 35.3 | 0.00, (0.00%) | 0.00, (0.00%) | 0.12, (0.34%) | 0.95, (2.69%) | 2.7  |
|  HPV16/18 Low Positive (1x LoD) |  |  |  |  |  |  |   |
|  HPV16 Low Positive (1x LoD) | 118 | 35.6 | 0.10, (0.27%) | 0.00, (0.00%) | 0.15, (0.43%) | 0.43, (1.22%) | 1.3  |
|  HPV18 Low Positive (1x LoD) | 119 | 34.1 | 0.00, (0.00%) | 0.09, (0.28%) | 0.00, (0.00%) | 0.63, (1.83%) | 1.9  |
|  HPV16/18 Positive (3x LoD) |  |  |  |  |  |  |   |
|  HPV16 Positive (3x LoD) | 119 | 34.7 | 0.05, (0.16%) | 0.00, (0.00%) | 0.11, (0.31%) | 0.35, (1.01%) | 1.1  |
|  HPV18 Positive (3x LoD) | 119 | 32.9 | 0.05, (0.16%) | 0.08, (0.25%) | 0.00, (0.00%) | 0.39, (1.19%) | 1.2  |
|  Positive Clinical Panel Members  |   |   |   |   |   |   |   |
|  Pooled HPV16 Low Positive (1x LoD) | 120 | 33.6 | 0.25, (0.73%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.05, (3.11%) | 3.2  |
|  Pooled HPV16 Positive (3x LoD) | 120 | 33.1 | 0.30, (0.90%) | 0.00, (0.00%) | 0.00, (0.00%) | 0.84, (2.53%) | 2.7  |
|  Pooled HPV18 Low Positive (1x LoD) | 119 | 35.1 | 0.00, (0.00%) | 0.00, (0.00%) | 0.11, (0.31%) | 0.74, (2.09%) | 2.1  |
|  Pooled HPV18 Positive (3x LoD) | 120 | 34.0 | 0.56, (1.64%) | 0.00, (0.00%) | 0.21, (0.62%) | 0.70, (2.06%) | 2.7  |
|  Pooled HPV45 Low Positive (1x LoD) | 120 | 31.9 | 0.56, (1.74%) | 0.00, (0.00%) | 0.00, (0.00%) | 1.71, (5.37%) | 5.6  |
|  Pooled HPV45 Positive (3x LoD) | 120 | 29.7 | 0.00, (0.00%) | 0.00, (0.00%) | 0.60, (2.04%) | 1.42, (4.79%) | 5.2  |
|  Pooled HPV39 Low Positive (1x LoD) | 120 | 33.4 | 0.20, (0.61%) | 0.00, (0.00%) | 0.33, (0.98%) | 0.97, (2.90%) | 3.1  |
|  Pooled HPV39 Positive (3x LoD) | 120 | 31.5 | 0.00, (0.00%) | 0.00, (0.00%) | 0.62, (1.95%) | 1.31, (4.15%) | 4.6  |

# 11. Cross Contamination

A study was performed to evaluate the risk of producing a false positive result in either the same run (within run carry-over) or in a subsequent run (between run carry-over) on the cobas 6800/8800 Systems. Three runs were performed using a checkerboard pattern of HPV positive and negative samples, followed by a full run of negative samples. Each run was arranged in an alternating checkerboard pattern, with HPV negative samples (consisting of an HPV negative cell line (HCT-15) at 10,000 cells/mL) placed in an alternating pattern with samples consisting of an HPV 16 positive cell line spiked at a concentration targeting a Ct value of  $\leq 20$ . This Ct value represents a signal that is stronger than  $95\%$  of the

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positive results in the intended use population based on the clinical study. Both the sample to sample and run to run cross-contamination rates were 0% (0/288, 95% CI: 0.00, 1.27 and 0/187, 95% CI: 0.00, 1.95, respectively).

## 12. Reagent Stability

Expiration dating for the cobas HPV reagents has been established and approved at 18 months for the cobas HPV and cobas HPV Positive Control Kit and at 24 months for the cobas Buffer Negative Control Kit when stored at 2-8°C. The shelf lives of the cobas HPV reagents were established in a real-time stability study.

## 13. Specimen Stability

Specimen stability studies demonstrated that for the cobas HPV, cervical specimens can be stored in PreservCyt Solution at 2-30 °C for up to 3 months from the date of collection. The observed changes in Ct value between baseline and the different storage conditions did not change any reported results.

## B. Animal Studies

Not Applicable

## C. Additional Studies

Not Applicable

## X. SUMMARY OF PRIMARY CLINICAL STUDIES

The applicant performed a clinical study to establish a reasonable assurance of safety and effectiveness of the cobas HPV for use on the 6800/8800 Systems in detecting high-risk HPV nucleic acid during routine cervical cancer screening 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

Subjects were enrolled between September 2017 to October 2018. The database for this PMA reflected specimen data collected through October 2018 and included 35,263 women. There were 32 clinical investigational sites in the United States.

A multicenter, prospective study (IMPACT study, IMproved Primary screening And Colposcopy Triage) was conducted to evaluate the performance of the cobas HPV on cobas 6800/8800 Systems (hereafter referred as cobas HPV) as a triage test to stratify women with ASC-US Pap cytology results for colposcopy, as an adjunctive test to cervical cytology to guide management decisions in women with NILM Pap cytology, and as a first-line primary test for cervical cancer screening.

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In total, 35,263 women were enrolled from September 2017 to October 2018 at 32 clinical sites in the United States. Following written informed consent, demographic information and gynecologic histories were obtained. Approximately half of the women had one cervical sample collected using a brush/spatula while the other half had one cervical sample collected using the broom-type device. Cervical samples were collected for HPV testing and ThinPrep Pap Test liquid based cytology (LBC). Specimens were tested with two HPV devices: an FDA-approved HPV test and the cobas HPV for use on the 6800/8800 systems. Both tests were performed according to manufacturer's instructions. HPV testing was performed on pre-aliquoted samples in secondary vials prior to cytology processing at four testing laboratories. LBC testing was conducted at the same four laboratories. Cytology samples were classified according to the criteria of the 2001 Bethesda System. Results from pap cytology, the FDA-approved HPV test, and the cobas HPV were used to inform referral to colposcopy as per the study protocol.

To determine the clinical study endpoint, a subset of non pregnant women identified at the enrollment visit was selected to undergo colposcopy, where a biopsy/endocervical curettage (ECC) was collected. The subset included women aged 25-65 years with ≥ ASC-US cytology and women 25-65 years with positive HPV Test results by the FDA-approved HPV Test and/or cobas HPV. In addition, 59 women with unsatisfactory Pap cytology and HPV-negative results by both the FDA-approved and cobas HPV tests, and a randomly selected subset of subjects with NILM cytology and HPV-negative results by both the FDA-approved and cobas HPV tests (approximately 1:50) were referred to colposcopy. In order to avoid bias, study participants and colposcopists were blinded to all HPV test and cytology results until after the colposcopy procedure was completed.

Colposcopy was conducted according to a standardized protocol following the principles recommended by the American Society for Colposcopy and Cervical Pathology (ASCCP), which is as follows: biopsies were obtained on all visible lesions; ECC was performed in all patients in whom the squamocolumnar junction was not visualized, and a single random cervical biopsy was obtained if no lesions were visible. All biopsies were examined by a Central Pathology Review (CPR) panel consisting of three expert pathologists, and discordant results adjudicated according to a pre-defined protocol. The slides that were prepared from the biopsies were stained using conventional hematoxylin and eosin (H&amp;E) staining and H&amp;E with p16 IHC assay (CINtec Histology, Ventana Medical Systems, Inc.). Clinical performance of the cobas HPV is presented using interpretation of H&amp;E-stained slides with adjunctive use of p16-stained slides in accordance with the 2012 Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions (LAST) excluding ASC-US/HPV16+ as a LAST criterion (CPRH&amp;E+p16 per LAST) at the clinical endpoints ≥CIN2 and ≥CIN3.

Subjects were recruited through general obstetrics and gynecology (OB/GYN) practices, and/or other healthcare facilities that routinely performed cervical cancer

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screening, where either the same facility or an affiliated facility frequently performed colposcopy and cervical biopsy (hereafter referred to as "collection sites").

## 1. Clinical Inclusion and Exclusion Criteria

Enrollment in the IMPACT study was limited to patients who met the following inclusion criteria:

- Female 25-65 years of age presenting for routine cervical cancer screening
- Intact cervix
- Willing and able to undergo colposcopy and biopsy (and possibly ECC) within 12 weeks from the date of the cervical sample collection
- Willing and able to provide written informed consent
- Willing and able to participate in the 1-year Follow-Up Phase, should it be required

Patients were not permitted to enroll in the IMPACT study if they met any of the following exclusion criteria:

- Known pregnancy at enrollment
- Current or planned participation in another cervical cancer screening study or in a cervical treatment or vaccine study
- Incomplete informed consent
- Any medical condition that, in the opinion of the Investigator, would have resulted in increased risk of bleeding at biopsy
- Known history of ablative or excisional therapy (e.g., loop electrosurgical excision procedure (LEEP), cone biopsy) within the past 12 months
- Known history of hysterectomy (including supracervical)

## 2. Clinical Endpoints

With regard to safety, as an in vitro diagnostic test, the cobas HPV 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 regard to effectiveness, the following clinical endpoints were used:

For ASC-US Triage (25-65 years): The clinical performance of the cobas HPV was evaluated against CPR determined histologic diagnosis, with ≥CIN2 and ≥CIN3 as the disease endpoints.

For Adjunctive screening (NILM 30-65 years): The clinical performance of the cobas HPV was evaluated against CPR determined histologic diagnosis, with ≥CIN2 and ≥CIN3 as the disease endpoints.

For HPV Primary screening (25-65 years): The clinical performance of the cobas HPV was evaluated ag…

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**Source:** [https://fda.innolitics.com/device/P190028](https://fda.innolitics.com/device/P190028)

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