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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A011
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.
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