AgarCyto: A novel cell-processing method for multiple molecular diagnosticanalyses of the uterine cervix

In diagnostic cytology, it has been advocated that molecular techniques wil l improve cytopathological diagnosis and may predict clinical course. Ancil lary molecular techniques, however, can be applied only if a sufficient num ber of preparations are made from a single cell sample. We have developed t he AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol inclu des 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 pro duced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for K i-67 and p53, and in situ hybridization for the centromere of human chromos ome 1 and human papilloma virus Type 16. In addition, single AgarCyto secti ons yielded sufficient input DNA for specific HPV detection and typing by L iPA-PCR, and the protocol includes an option for DNA image cytometry. The A garCyto cell block protocol is an excellent tool for inventory studies of d iagnostic and potentially prognostic molecular markers of cervical cancer.