![]() |
6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A055
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.
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