Analytical Performance of the APTIMA HPV Assay on the PANTHER AnalyzerAndrew Worlock, Marilyn Vi, Jimmykim Pham, Melissa Hunsicker, Trisha LeNguyen, Lori Villegas, Jennifer Tidd and Craig Clark

Gen-Probe Incorporated, San Diego, CA

Objective: The PANTHER analyzer is a new fully automated molecular diagnostic instrument under development at Gen-Probe. The objective of the studies was to assess performance of the APTIMA HPV Assay on prototype PANTHER instruments. Methods: Six positive panels were made by spiking RNA transcript into Swab Transport Media (STM) at concentrations close to the limit of detection for HPV subtypes 16,18, 31, 51, 52 and 68. The negative panel member was unspiked STM. Panels were tested with the APTIMA HPV Assay on 4 prototype PANTHER instruments in replicates of at least 30/instrument. The total precision for signal output was calculated. Analytical sensitivity was assessed by spiking RNA transcript from the 6 HPV subtypes in STM at 6 different concentrations. A Probit analysis was used to calculate the analytical sensitivity for the assay as determined by 95% positivity. Results: Total precision for output signal was 12% or less for the positive panels and 8% for the internal calibrator in the negative panel. The analytical sensitivity estimates for the APTIMA HPV Assay run on the prototype PANTHER instruments were 16 copies/reactions for subtype 16, 13 copies/reaction for subtype 18, 13 copies/reaction for subtype 45, 37 copies/reaction for subtype 51, 273 copies/reaction for subtype 52 and 165 copies/reaction for subtype 68. Conclusions: The newly developed, fully automated PANTHER molecular diagnostic analyzer running the APTIMA HPV Assay generates precise results and has good analytical sensitivity. In addition to excellent performance, the PANTHER analyzer offers the optimum throughput and workflow that is ideal for the low-to-mid volume molecular testing laboratory.

Updated Abstract APTIMA HPV Assay Analytical Sensitivity (LOD)

Conclusions

The PANTHER Analyzer

Andy Worlock, PhDGen-Probe Incorporated10210 Genetic Center DriveSan Diego, CA 92121Phone: 858.410.8000andrewwo@gen-probe.com

• The newly developed, fully automated PANTHER analyzer provides precise results and comparable analytical sensitivity to the TIGRIS DTS System when running the APTIMA HPV Assay.

• The PANTHER instrument offers the optimum throughput, workflow and turn around time that is ideal for the low-to-mid volume molecular testing laboratory.

Figure 2. Probit analyses comparing the APTIMA HPV Assay sensitivity on PANTHER and TIGRIS instruments.

Methods

The APTIMA HPV Assay is a target amplification nucleic acid probe test for the in vitro qualitative detection and differentiation of E6/E7 viral messenger RNA (mRNA) from 14 high-risk human papillomavirus HPV types

Target Capture - Target sequences are captured with magnetic particles and complementary oligonucleotides.

Amplification - Isothermal exponential amplification is performed using Transcription-Mediated Amplification (TMA).

Hybridization / Detection - Acridinium ester labeled probes detect amplicon using the Hybridization Protection Assay (HPA). The assay incorporates an internal control to control for nucleic acid capture, amplification, and detection, as well as operator or instrument error.

Precision Study Panels Six positive panel members were made by spiking RNA transcript

into swab transport media (STM) at 100 copies/test for HPV subtypes 16, 18, 31, at 300 copies/reaction for subtypes 51 and 68 and at 1000 copies/reaction for subtype 52. An unspiked negative panel of STM was also tested. Panels were tested on 4 PANTHER instruments in 2 runs per instrument in a total of 8 runs. Panels were run in replicates of at least 15/run for a total n of at least 120 for each panel member. The total precision for signal out was calculated.

Analytical Sensitivity Panels Six positive panel members were made by spiking RNA transcript into STM for subtypes 16, 18, 45, 51, 52 and 68 at 6 concentrations. Ten or 15 replicates of each panel member were tested on two PANTHER instruments and two TIGRIS DTS Systems (Gen-Probe Incorporated), a fully-automated, high-throughput instrument. A Probit analysis was used to calculate the analytical sensitivity for the assay as determined by 95% positivity.

APTIMA HPV Assay Precision on the PANTHER Analyzer

Figure 1. Relative Light Unit (RLU) output for 6 HPV panel members run on 4 prototype PANTHER instruments. The figures show the mean for each subtype combining all run data 9 (horizontal line) and the means for each run (horizontal line within each box). The boxes depict the 25 th and 75th quartiles and the whiskers the upper or lower quartiles +/- 1.5 x the inter-quartile range. Individual points beyond the whiskers are potential outliers

Fully Automated and Integrated

Sample to Result

Positive Sample Identification

Inventory Management

Dual Format

Chemiluminescent (qualitative)

Real-Time (quantitative)

Improved Customer Workflow

Random access sample loading

Continuous access to reagents and consumables

260 specimen tubes in 8 hours; 500 specimen tubes in 12.5 hours

Bi-Directional host interface for sample processing and reporting

3.5 hours to first result

Primary tube sampling

Small Footprint

Floor standing

Self-contained modules (computer, pumps, reagents, etc. on-board)

The PANTHER analyzer is the second fully automated molecular diagnostic instrument from Gen-Probe. Designed for the low-to-mid volume laboratories, it is the first molecular diagnostic instrument to incorporate many of the features available on Chemistry and Immunoassay platforms.

APTIMA, PANTHER, TIGRIS and DTS are trademarks of Gen-Probe Incorporated.

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HPV Subtype 18 Precision HPV Subtype 31 Precision

HPV Subtype 51 Precision HPV Subtype 52 Precision

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HPV Subtype 68 Precision

Table 1. Total precision and agreement with the expected result for 6 HPV panel members run on 4 prototype PANTHER instruments

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Table 2. Analytical sensitivity as measured by Probit analysis of subtype dilutions shown in Figure 2

Subtype TIGRIS PANTHER16 15 (10 to 23) 16 (11 to 23)18 12 (9 to 16) 13 (10 to 17)45 5 (0 to 300) 13 (9 to 19)51 40 (26 to 63) 37 (24 to 58)52 221 (159 to 307) 273 (184 to 406)68 103 (60 to 176) 165 (94 to 290)

Copies/reaction giving 95% positivity (95% confidence limits)

Subtype n

Total RLU Precision

(%CV)%

Agreement16 120 3.7 10018 120 8.9 10031 120 3.5 10051 120 3.5 10052 120 8.4 10068 120 11.9 100

Negative (IC) 190 8.0 100