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The Papanicolaou (Pap) test, based solely on the morphologic examination of exfoliated cells from the cervix, has reduced deaths due to cervical cancer by 74% in the United States during the past 40 years. During that time, the molecular mechanisms of cervical cancer have largely been elucidated. Taken together, these observations have identified a need for a high-throughput cervical cancer screening assay. We report the development of a high-throughput assay consisting of simultaneous immunophenotyping and ultrasensitive in situ hybridization for human papillomavirus (HPV) E6 and E7 messenger RNA (mRNA). This assay can be performed in less than 3 hours directly from liquid-based cervical cytology specimens. Overall, HPV fluorescence in situ hybridization (FISH) for E6 and E7 mRNA demonstrated 83.3% sensitivity and 91.3% specificity for high-grade squamous intraepithelial lesions compared with the Pap test in 231 liquid-based cytology samples from 2 cohorts. In a subset of these samples, HPV FISH demonstrated higher sensitivity and specificity than Hybrid Capture (Digene, Gaithersburg, MD) for high-risk genotypes.

High-Throughput Cervical Cancer Screening Using Intracellular Human PapillomavirusE6andE7mRNA Quantification by Flow Cytometry