6th ESACP Congress, Heidelberg, April 7-11, 1999

A055
A RAPID WASH-LESS PROCEDURE FOR KI-67 (MIB 1) EVALUATION IN SOLID TUMOR SAMPLES BY FLOW CYTOMETRY
Ferrari C, Dionigi P, Guizetti M, Mazzini G

Dept. Surgery, Univ. Pavia, CNR Histochemistry Study Center, Dept. Biology, Univ.Pavia, Italy

Proliferation assessment and ploidy status are important findings in solid tumors study. Flow cytometry is playing an important role in this field. DNA analysis is routinely employed and DNA indexes are acquired in a quite reproducible and standard approach. Estimation of the proliferation potential is, on the contrary, still under experimental phase, while many proliferation "markers" as well as different methodology can be applied. Among others, Ki-67 seems to be the most interesting one, in clinical pathology of solid tumors. The aim of our study had been the critical review of the available methodology. Different fixation procedures have been tested on human cell colture lines. Best results were acquired using cold 3:1 methanol-acetone. In case of small bioptic samples the limited amount of the material may be a problem for conventional methodology. A rapid single step procedure had been tested on frozen unfixed tissue samples simply stored in a normal -20°C freezer without conservative medium like DMSO or glycerol. After thawing samples are mechanically disaggregated and all the required reagents are sequentially (MIB 1 Immunotech 0505 dil 1:10 3h + FITC IgG Sigma F 2012 dil 1:50 1h + PI 5 mg/ml 1h) added to the original cell suspension without interstep washings and centrifugations. Good results have been obtained in terms of intensity of fluorescence labelling. Experiments are in progress on the possibility to apply the above described methodology on the dual labelling Ki-67 and CK+ aimed to assess proliferation on the restricted epithelial cell subpopulation.