c Two important preanalytical protocols performed on liquid-based cytological specimens, namely, automated cytology processing and glacial acetic acid (GAA) treatment, may occur prior to the arrival of specimens in a molecular diagnostics laboratory. Ninety-two ThinPrep vials previously positive for high-risk human papillomavirus (HPV) via the Cervista HPV HR test were preselected and alternated with 92 previously negative ThinPrep vials. The specimen set was processed in a consecutive fashion by an automated cytology processor without fastidious decontamination precautions. Carryover potential was subsequently assessed by performance of the Aptima HPV assay on aliquots from reprocessed ThinPrep vials. All previously negative ThinPrep vials yielded a negative result following routine automated cytology processing, despite close proximity to known-positive ThinPrep vials. In separate experiments, aliquots from 236 ThinPrep vials were forwarded for tandem analysis with and without GAA treatment. Data from GAA-and mock-treated specimens generated by Aptima HPV were compared to correlate data generated by Cervista. A 99.2% concordance of Aptima HPV results from GAA-treated and mock-treated specimens was noted. This result differed from the concordance result derived from Cervista (91.5%; P < 0.0002). Of the initially positive Cervista results, 21.9% reverted to negative following GAA treatment; the correlate value was 2.7% for Aptima HPV (P ‫؍‬ 0.01). While deleterious effects of GAA treatment on genomic DNA were noted with Cervista (P ‫؍‬ 0.0015), GAA treatment had no significant effects on Aptima HPV specimen signal/cutoff ratios or amplification of internal control RNA (P > 0.07). The validity of an Aptima HPV result is independent of GAA treatment and routine automated cytology processing. D etection of high-risk human papillomavirus (HPV) E6/E7transcripts is a recent diagnostic advancement in the context of cervical cancer triage. Transcription-mediated amplification (TMA) of these targets (Aptima HPV; Hologic/Gen-Probe, San Diego, CA) performs in an equivalent fashion to high-risk HPV DNA hybridization assays, such as the Digene HC2 high-risk HPV DNA test (Qiagen, Gaithersburg, MD) (1-4) and the Cervista HPV HR (Cervista; Hologic, Madison, WI) (5), for the detection of indolent cervical intraepithelial neoplasia (CIN) 2ϩ. The aforementioned studies also document improved specificity of Aptima HPV over that of DNA hybridization in the context of atypical squamous cells of undetermined significance (ASC-US) or nonsignificant cytology diagnoses and in cases of non-CIN 2ϩ biopsy results.Aptima HPV is an FDA-cleared assay for the analysis of liquidbased cytology collections (ThinPrep; Hologic, Marlborough, MA) on high-throughput instrumentation such as the Tigris DTS system (6). Past package insert guidelines have referred to meticulous and costly cross-contamination mitigation steps required when the ThinPrep vials are subjected to automated cytology processing prior to HPV detection (7). In addition, specimen car...