11 Formalin-Fixed and Paraffin-Embedded Tissue Sections
155
sing the whole depth of the section is therefore necessary. It has to be kept
in mind that part of the nuclei is truncated by the preceding tissue sectioning, leading to a loss of genetic material and reduced numbers of hybridization signals. Thus diagnostic results in tissue sections, although
based on information from a single cell, require the analysis oflarge numbers of nuclei and statistical evaluation.
Results
We performed this method on tissue sections of various thicknesses (2, 5,
10, 15 and 20 !Jm) of archived, formalin-fIxed and paraffIn-embedded tissue specimens derived from placenta, fetal organs, lymphomas and breast
cancers. Centromeric probes as well as single copy probes were successfully employed. Mainly centromeric probes used in a large variety of samples of different tissue origin were the basis for our statistical evaluation of
FISH results.
The hybridization effIciency was dependent on the tissue type, the
conservation of the tissues with respect to autolysis as well as necrosis,
and the period of formalin fIxation rather than of paraffm embedding.
Regarding centromeric probes, diagnostic results could be achieved in approximately 60% of placental tissues, 20-90% of fetal tissues (depending
on the different tissue types), 90% of lymphoma and 60-70% of breast
cancer samples.
Characteristic signal distribution patterns were found after successfully performed hybridization. They clearly differentiated between chromosome and/or gene copy numbers, but not between different levels of
ploidies. Exclusion of polyploidy required the application of several chromosome probes in single, double or multiple color FISH. The combined
use of differently labeled gene specifIc and centromeric probes of the same
chromosomes helped to distinguish between changes in the gene copy
number, due to complete chromosome gains or losses and due to structural chromosome rearrangements (Figs. I, 2). Double in situ hybridization using human DNA and viral RNA probes was performed for the identifIcation of Ebstein Barr virus positive tumor cells. By the combination of
using chromosome-specifIc FISH with immunhistochemistry to identify
tumor cell-specifIc antigens, a correlation of cytogenetic, immunological
and morphological criteria was possible.
The section thickness as well as the chromosome copy numbers directly influenced the FISH results with respect to the detection of the
true karyotype. The highest number of spots per nucleus present in at least
155
sing the whole depth of the section is therefore necessary. It has to be kept
in mind that part of the nuclei is truncated by the preceding tissue sectioning, leading to a loss of genetic material and reduced numbers of hybridization signals. Thus diagnostic results in tissue sections, although
based on information from a single cell, require the analysis oflarge numbers of nuclei and statistical evaluation.
Results
We performed this method on tissue sections of various thicknesses (2, 5,
10, 15 and 20 !Jm) of archived, formalin-fIxed and paraffIn-embedded tissue specimens derived from placenta, fetal organs, lymphomas and breast
cancers. Centromeric probes as well as single copy probes were successfully employed. Mainly centromeric probes used in a large variety of samples of different tissue origin were the basis for our statistical evaluation of
FISH results.
The hybridization effIciency was dependent on the tissue type, the
conservation of the tissues with respect to autolysis as well as necrosis,
and the period of formalin fIxation rather than of paraffm embedding.
Regarding centromeric probes, diagnostic results could be achieved in approximately 60% of placental tissues, 20-90% of fetal tissues (depending
on the different tissue types), 90% of lymphoma and 60-70% of breast
cancer samples.
Characteristic signal distribution patterns were found after successfully performed hybridization. They clearly differentiated between chromosome and/or gene copy numbers, but not between different levels of
ploidies. Exclusion of polyploidy required the application of several chromosome probes in single, double or multiple color FISH. The combined
use of differently labeled gene specifIc and centromeric probes of the same
chromosomes helped to distinguish between changes in the gene copy
number, due to complete chromosome gains or losses and due to structural chromosome rearrangements (Figs. I, 2). Double in situ hybridization using human DNA and viral RNA probes was performed for the identifIcation of Ebstein Barr virus positive tumor cells. By the combination of
using chromosome-specifIc FISH with immunhistochemistry to identify
tumor cell-specifIc antigens, a correlation of cytogenetic, immunological
and morphological criteria was possible.
The section thickness as well as the chromosome copy numbers directly influenced the FISH results with respect to the detection of the
true karyotype. The highest number of spots per nucleus present in at least