Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy |
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Authors: | G Lizard M C Chignol P Roignot C Souchier Y Chardonnet D Schmitt |
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Institution: | (1) INSERM CJF 93/10, Laboratoire de Biochimie Medicale, CHRU/Hopital du Bocage, BP 1542, 21034 Dijon Cedex, France;(2) INSERM U 346, Hopital E. Herriot, 69437 Lyon Cedex 03, France;(3) Centre de Pathologie/Institut de Recherche Medicale de Bourgogne, 21000 Dijon, France;(4) Cytologie Analytique, Universite C. Bernard Lyon I, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France |
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Abstract: | Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in
uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on
cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red
TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as
by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate
that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching
when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence
and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions,
of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively.
The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization
signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in
such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a
suitable method for identifying and characterizing HPV DNA in cells and tissue sections
This revised version was published online in November 2006 with corrections to the Cover Date. |
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