144
ERICH GEBHART
Fluorescence in situ hybridization
The recovered slides, which have been pretreated in the way described
above, are now ready for being subjected to FISH. In particular, interphase
FISH is a readily applicable technique, while chromosome painting proved
to be difficult, although not impossible, on archival slides.
Results
The efficacy ofthe described procedure in our hands was sufficient to obtain
I-FISH data not onlyfrom 32 outof43 breast tumors (74%), from 29 out of! 00
colorectal tumors (29%), from 20 out of35 cancerous effusions (57%), from 5
out of 5 non-cancerous fibroblast controls (100%), and from 12 out of 65
leukemia samples (18.5%), but also from all archival slides of normal bone
marrow and of peripherallyrnphocytes (100%). In the meantime, with increased experience, however, we could also increase the success rates in
leukemia slides considerably. The attained quality of signals in interphase
nuclei from archival material in a breast tumor case is shown in Fig. 1.
I-FISH results of our first examinations using the described method are
summarized in Table 1. They document that cell clones could be detected.
These results helped to characterize the genomic DNA changes as specific
gains and losses. Using a conservative estimate of the percentage of nuclei
showing either gains or losses of signals allowed guidelines to be established for determining the presence of significant clonality (Table 1). The
percentage of nuclei used to define clonality will probably decline as the
growth of the data base is increased, as, for instance shown for hematopoietic neoplasms (Gebhart et al 1995).
Troubleshooting
The percentage of slides accessible to interphase FISH, from our experience, depends on the tissue type, but not on the age of the slides. The most
critical step of the procedure is the removal of the embedding medium
without destroying too many of the nuclei. If the coverslip and embedding
medium can be removed totally, 100% of the slides will show reliable FISH
signals.
In colorectal tissue, the interphase nuclei proved to be extremely sensitive to washing in xylene: During this procedure, most of the nuclei expanded and were lost to subsequent analysis.
ERICH GEBHART
Fluorescence in situ hybridization
The recovered slides, which have been pretreated in the way described
above, are now ready for being subjected to FISH. In particular, interphase
FISH is a readily applicable technique, while chromosome painting proved
to be difficult, although not impossible, on archival slides.
Results
The efficacy ofthe described procedure in our hands was sufficient to obtain
I-FISH data not onlyfrom 32 outof43 breast tumors (74%), from 29 out of! 00
colorectal tumors (29%), from 20 out of35 cancerous effusions (57%), from 5
out of 5 non-cancerous fibroblast controls (100%), and from 12 out of 65
leukemia samples (18.5%), but also from all archival slides of normal bone
marrow and of peripherallyrnphocytes (100%). In the meantime, with increased experience, however, we could also increase the success rates in
leukemia slides considerably. The attained quality of signals in interphase
nuclei from archival material in a breast tumor case is shown in Fig. 1.
I-FISH results of our first examinations using the described method are
summarized in Table 1. They document that cell clones could be detected.
These results helped to characterize the genomic DNA changes as specific
gains and losses. Using a conservative estimate of the percentage of nuclei
showing either gains or losses of signals allowed guidelines to be established for determining the presence of significant clonality (Table 1). The
percentage of nuclei used to define clonality will probably decline as the
growth of the data base is increased, as, for instance shown for hematopoietic neoplasms (Gebhart et al 1995).
Troubleshooting
The percentage of slides accessible to interphase FISH, from our experience, depends on the tissue type, but not on the age of the slides. The most
critical step of the procedure is the removal of the embedding medium
without destroying too many of the nuclei. If the coverslip and embedding
medium can be removed totally, 100% of the slides will show reliable FISH
signals.
In colorectal tissue, the interphase nuclei proved to be extremely sensitive to washing in xylene: During this procedure, most of the nuclei expanded and were lost to subsequent analysis.