Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry

Rudolph Park, Prabha Chandrasekaran, Heather Hernandez, Ines Lakhal-Naouar, Kristina K. Peachman, Holly R. Hack, Dante Coleman, Jason Ouellette, Janice M. Darden, Oussama M’hamdi, Victor A. Sugiharto, Hua Wei Chen, Megan A. Schilling, Mark P. Simons, Natalie D. Collins, Yuliya S. Johnson, Linda L. Jagodzinski, Sheila A. Peel*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multiparametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic’s Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92–2.41) and 10:1 (CT increase of 3.03–3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer’s setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic’s SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

Original languageEnglish
Article numbere0276729
JournalPLoS ONE
Issue number11 November
StatePublished - Nov 2022
Externally publishedYes


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