Effect of Preanalytical Processing of ThinPrep Specimens on Detection of High-Risk Human Papillomavirus by the Aptima HPV Assay

Abstract

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...

Determinants of Viral Oncogene E6-E7 mRNA Overexpression in a Population-Based Large Sample of Women Infected by High-Risk Human Papillomavirus Types

“…This phenomenon can be observed also within other studies (11) and when comparing different studies (e.g., references 11 and 12) and could result from lack of HPV mRNA positivity in the presence of HPV DNA test results that are either analytical false positives or true positives for very low viral loads (see discussion below). Indeed, variations in HPV DNA test reproducibility and specificity with different preanalytical procedures have been observed (13,14,31). Interestingly, we did not observe any effect of age on the HPV E6-E7 mRNA positivity rate in the range that we explored, suggesting that despite a higher probability of CIN2 and CIN3 regression at a younger age (15), the probability of an infection to initiate the transforming process is not age dependent.…”

Nucleic acid‐based assays for the detection of high‐risk human papillomavirus: A technical review

“…At least 1 study has documented that 21.9% of specimens with Cervista-positive results reverted to negative when treated with glacial acetic acid, 11 whereas no such effects have been reported for the hc2, cobas HPV, and APTIMA HPV tests. [11][12][13] In addition, performance characteristics of the assay were determined through data obtained during a multicenter prospective clinical study and crossreactivity was identified with HPV genotypes 67 and 70, which currently are of uncertain clinical risk. 10 A subset of specimens that represent "triple positives" can present a unique set of challenges with respect to this assay.…”

“…Limitations of the Cervista HPV HR test include impacts on assay performance of preprocessing treatment with glacial acetic acid, which is commonly used to lyse red blood cells in liquid‐based cytology specimens. At least 1 study has documented that 21.9% of specimens with Cervista‐positive results reverted to negative when treated with glacial acetic acid, whereas no such effects have been reported for the hc2, cobas HPV, and APTIMA HPV tests . In addition, performance characteristics of the assay were determined through data obtained during a multicenter prospective clinical study and cross‐reactivity was identified with HPV genotypes 67 and 70, which currently are of uncertain clinical risk .…”

Clinical sensitivity of HPV assays for the detection of high grade cervical disease in cervical samples treated with glacial acetic acid

“…Of the small number of studies where the impact of GAA has been assessed, the evidence would indicate that the COBAS 4800HPV Assay (Roche Molecular Systems, Pleasanton, CA, USA) and APTIMA HPV Test (Gen-Probe Inc., San Diego, CA) are unaffected by GAA treatment − as opposed to the Cervista Test (Hologic, Inc., Bedford, MA) where it has a deleterious influence [3][4][5][6]. Furthermore, in an earlier splitspecimen study of untreated vs treated specimens undertaken in our laboratory, while the impact of GAA on HPV detection was insignificant at the qualitative level, GAA treatment was associated with lower read-outs according to the semi-quantitative measures of the assays chosen: HC2 and rtHPV [7].…”