BACKGROUND: The simultaneous detection of protein expression and gene copy 
number changes in patient samples, like paraffin-embedded tissue sections, is 
challenging since the procedures of immunohistochemistry (IHC) and Fluorescence 
in situ Hybridization (FISH) negatively influence each other which often results 
in suboptimal staining. Therefore, we developed a novel automated algorithm 
based on relocation which allows subsequent detection of protein content and 
gene copy number changes within the same cell.
METHODS: Paraffin-embedded tissue sections of colorectal cancers were stained 
for CD133 expression. IHC images were acquired and image coordinates recorded. 
Slides were subsequently hybridized with fluorescently labeled DNA probes. FISH 
images were taken at the previously recorded positions allowing for direct 
comparison of protein expression and gene copy number signals within the same 
cells/tissue areas. Relocation, acquisition of the IHC and FISH images, and 
enumeration of FISH signals in the immunophenotyped tumour areas were done in an 
automated fashion.
RESULTS: Automated FISH analysis was performed on 13 different colon cancer 
samples that had been stained for CD133; each sample was scored for MYC, ZNF217 
and Chromosome 6 in CD133 positive and negative glands. From the 13 cases four 
(31%) showed amplification for the MYC oncogene and seven of 13 (54%) cases were 
amplified for ZNF217. There was no significant difference between CD133 positive 
tumour and CD133 negative tumour cells.
CONCLUSION: The technique and algorithm presented here enables an easy and 
reproducible combination of IHC and FISH based on a novel automated algorithm 
using relocation and automated spot counting.