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Multiplex chemiluminescence microscope imaging of P16INK4A and HPV DNA as biomarker of cervical neoplasia

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Abstract

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16INK4A (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.

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Acknowledgements

This work was supported by the University of Bologna (“Strategic University Projects" and "RFO - Focused fundamental research projects”) and the Italian Ministry for Education, University and Research (“PRIN 2007” projects).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, Bologna, 40126, Italy

    M. Mirasoli, M. Guardigli & A. Roda

  2. National Institute of Biostructure and Biosystems, I.N.B.B., Interuniversity Consortium, Rome, Italy

    M. Mirasoli, M. Guardigli, P. Simoni & A. Roda

  3. Department of Clinical Medicine, University of Bologna, via Massarenti 9, Bologna, 40138, Italy

    P. Simoni

  4. Division of Microbiology, University of Bologna, via Massarenti 9, Bologna, 40138, Italy

    S. Venturoli, S. Ambretti & M. Musiani

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  1. M. Mirasoli
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  2. M. Guardigli
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  3. P. Simoni
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  4. S. Venturoli
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  5. S. Ambretti
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  6. M. Musiani
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  7. A. Roda
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Correspondence to A. Roda.

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Mirasoli, M., Guardigli, M., Simoni, P. et al. Multiplex chemiluminescence microscope imaging of P16INK4A and HPV DNA as biomarker of cervical neoplasia. Anal Bioanal Chem 394, 981–987 (2009). https://doi.org/10.1007/s00216-009-2707-y

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  • DOI: https://doi.org/10.1007/s00216-009-2707-y

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