Spot count variability in FFPE specimens

The establishment of cutoff values to set the threshold or false positive level is also influenced by other biological variables affecting both FISH signals and relative nuclear volume in tumor sections:

• The condensation of the DNA, the size of the nucleus,
• Thickness of FFPE sections,
• Assay design (the signal distance in 3 or 4 color FISH assay depends partially on the genomic distance of the probes),
• Ploidy status (higher likelihood of higher levels of ploidy convey larger volume nuclei).

In addition to these nuclear variables:

• The actual FISH signal itself (the "spot size") may occupy more three-dimensional space in decondensed nuclear chromatin (typical open DNA packing of expressed genes) in comparison to condensed regions of the genome (DNA of repressed genes for example).
• Individual FISH signals will often be hard to interpret if DNA replication has already taken place, since G2 nuclei exhibit "doublet" or paired spot counts, and scoring criteria must ensure that this effect does not distort DDP assays.
• Several of these variables such as ploidy, nuclear size, and chromatin condensation can dramatically vary between normal control samples and tumor specimens.

Therefore it must be taken into account that these factors cannot be properly modeled in negative controls used to establish cutoff levels for interphase FISH assays. This is of particular importance in samples with low-tumor cell content or additional subclonal genomic changes.

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