33 Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
433
Several such combined IC/ISH methods have been described with
either enzyme precipitation reactions (Porter et al. 1990; Losada et al.
1991), fluorochromes (Van den Berg et al. 1991; Weber-Matthiesen et
al. 1993; Steilen et al. 1994, Ermis et al. 1995), or a combination of
both (Strehl and Ambros 1993). The methods can be subdivided into
two groups, those which use fluorochromes for IC, and those which
use enzyme reactions. For the detection with fluorochromes FITC, TRITC
or AMCA are mainly used. Enzyme precipitation reactions have also been
used efficiently for combined IC/ISH staining of proteins in cell preparations and tissue sections. Frequently used enzyme precipitation products
that withstand the proteolytic digestion and denaturation steps in the ISH
procedure are:
- Stain precipitates (Fast Red, New Fuchsin and BCIP/NBT) formed by
alkaline phosphatase
- Diaminobenzidine precipitate formed by horseradish peroxidase
Non-random chromosomal abnormalities have been observed in many
types of solid human tumors (Sandberg, 1990). Most information on
the genomic changes of solid tumors is acquired by karyotype analysis
of cell cultures, but in recent years FISH procedure has been used for targeted analyses of numerical chromosome abnormalities within interphase
nuclei of malignancies. Since tumor samples usually consist of both nontumorous and tumor cells and may therefore contain resident cells with a
normal chromosome complement, the interpretation of FISH results can
be difficult. Moreover, tumor cells may lack chromosome alterations at the
regions detected by hybridization and, for this reason, may be indistinguishable from normal cells. Therefore, techniques that enable characterization of the tumor tissue e.g. by determining cell-surface antigens or
proliferation markers, in combination with interphase cytogenetics offer
several advantages. Preselection of cells that are positive or negative for a
certain antigen reduces the number of cells that need to be examined and
avoids the potential for misinterpretation due to the analysis of normal
cells. To combine the detection of antigen staining and visualization of
DNA probes in the same cell makes this procedure highly suitable for detection of tumor cell heterogeneity, detection of rare phenotypes, and in
cases where only a few cells can be obtained for analysis (e.g. in needle
biopsy material).
We conclude that the combination of immunocytochemistry and ISH
(FISH) is a valuable method of examining the correlation between genotype and phenotype characteristics.
433
Several such combined IC/ISH methods have been described with
either enzyme precipitation reactions (Porter et al. 1990; Losada et al.
1991), fluorochromes (Van den Berg et al. 1991; Weber-Matthiesen et
al. 1993; Steilen et al. 1994, Ermis et al. 1995), or a combination of
both (Strehl and Ambros 1993). The methods can be subdivided into
two groups, those which use fluorochromes for IC, and those which
use enzyme reactions. For the detection with fluorochromes FITC, TRITC
or AMCA are mainly used. Enzyme precipitation reactions have also been
used efficiently for combined IC/ISH staining of proteins in cell preparations and tissue sections. Frequently used enzyme precipitation products
that withstand the proteolytic digestion and denaturation steps in the ISH
procedure are:
- Stain precipitates (Fast Red, New Fuchsin and BCIP/NBT) formed by
alkaline phosphatase
- Diaminobenzidine precipitate formed by horseradish peroxidase
Non-random chromosomal abnormalities have been observed in many
types of solid human tumors (Sandberg, 1990). Most information on
the genomic changes of solid tumors is acquired by karyotype analysis
of cell cultures, but in recent years FISH procedure has been used for targeted analyses of numerical chromosome abnormalities within interphase
nuclei of malignancies. Since tumor samples usually consist of both nontumorous and tumor cells and may therefore contain resident cells with a
normal chromosome complement, the interpretation of FISH results can
be difficult. Moreover, tumor cells may lack chromosome alterations at the
regions detected by hybridization and, for this reason, may be indistinguishable from normal cells. Therefore, techniques that enable characterization of the tumor tissue e.g. by determining cell-surface antigens or
proliferation markers, in combination with interphase cytogenetics offer
several advantages. Preselection of cells that are positive or negative for a
certain antigen reduces the number of cells that need to be examined and
avoids the potential for misinterpretation due to the analysis of normal
cells. To combine the detection of antigen staining and visualization of
DNA probes in the same cell makes this procedure highly suitable for detection of tumor cell heterogeneity, detection of rare phenotypes, and in
cases where only a few cells can be obtained for analysis (e.g. in needle
biopsy material).
We conclude that the combination of immunocytochemistry and ISH
(FISH) is a valuable method of examining the correlation between genotype and phenotype characteristics.