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

A011
IMPROVED FISH SIGNAL AMPLIFICATION METHODS BRIDGE THE GAP BETWEEN RESEARCH AND DAILY PRACTICE
Hopman AHN, Ramaekers FCS

Dept. Molecular Cell Biology and Genetics, University Maastricht, Netherlands

Fluorescence in situ hybridization has become a routine molecular cytogenetic method to detect genetic changes in metaphase chromosomes and interphase cells. In fresh biological material up to a few kb can be detected in multicolor assays. For routinely processed formalin fixed and paraffin embedded material the efficiency as well as the detection sensitivity is reduced. We improved the processing of formalin fixed material and incorporated multicolor catalyzed reporter deposition (CARD) detection procedures. The CARD methods were based on own synthesized haptenized conjugated tyramides (BIO, DIG) or red, green and blue fluorescent tyramides. The peroxidase substrates were used in indirect immunocytochemical- and direct-fluorescent detection methods respectively. The amplification techniques were applied to detect aneusomies, chromosomal imbalances and amplifications. As a result of these improvements: 1) A limited number of tissue sections are needed to complement data obtain by CGH and LOH. 2) Detection of numerical chromosome aberrations and amplifications is within the detection limits of ordinary microscopes and 3) The intensities of the FISH signal also allow automated image analyses measuring deletions, amplifications and the mapping of tumor heterogeneity. We used different repeat sequence, locus specific and viral probes. All probes were applied in multicolor CARD methods to map aneuploidy formation, chromosomal imbalances, chromosome arm imbalances and viral infections. Examples will be shown of the genetic analyzes of preneoplasia of the bladder, cervix and head & neck.