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Journal of Histochemistry and Cytochemistry, Vol. 48, 709-718, May 2000, Copyright © 2000, The Histochemical Society, Inc.

ARTICLE

AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix

Harold M.J. Kerstensa, Johanna C.M. Robbena, Pino J. Poddighea, Willem J.G. Melchersb, Henk Boonstrac, Petrus C.M. de Wildea, Merryn V.E. Macvillea, and Antonius G.J.M. Hanselaara
a Departments of Pathology, University Hospital Nijmegen, Nijmegen, The Netherlands
b Medical Microbiology, University Hospital Nijmegen, Nijmegen, The Netherlands
c Obstetrics & Gynecology, University Hospital Nijmegen, Nijmegen, The Netherlands

Correspondence to: Harold M.J. Kerstens, Dept. of Pathology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: h.kerstens@pathol.azn.nl

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer. (J Histochem Cytochem 48:709–718, 2000)

Key Words: cell block processing, cytology, molecular diagnosis, uterine cervix, immunocytochemistry, in situ hybridization, PCR, image cytometry


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