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Automatic quantification of gene amplification in clinical samples by IQ-FISH
1CCRI, Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria.
Cytometry. Part B, Clinical Cytometry
|December 30, 2003
Summary
Interphase quantitative FISH (IQ-FISH) enables accurate automatic quantification of gene amplifications by measuring fluorescence intensities. This method reliably distinguishes amplified from non-amplified cells, crucial for clinical applications.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Accurate detection and quantification of gene amplifications are vital in clinical settings.
- Fluorescence in situ hybridization (FISH) is a reliable method, but automatic quantification of gene amplification based on fluorescence intensities has been a challenge.
- Current spot-counting methods are limited to low amplification rates, necessitating new approaches.
Purpose of the Study:
- To establish and validate interphase quantitative FISH (IQ-FISH) for automatic quantification of gene amplifications using fluorescence intensity.
- To assess the reliability of IQ-FISH in distinguishing between amplified and non-amplified cells in various sample types.
Main Methods:
- Quantified fluorescence intensities of MYCN and D2Z FISH probes in neuroblastoma cell lines, peripheral blood, and patient samples using the Metafer4 system.
- Calculated the ratio between MYCN and D2Z fluorescence intensities to determine MYCN copy number per cell.
- Analyzed HER-2/neu amplification status using specific FISH probes and the automatic device.
Main Results:
- IQ-FISH demonstrated distinct fluorescence intensity peaks for amplified and non-amplified cells, with minimal overlap.
- MYCN amplification showed ratio values significantly exceeding those of normal cells (1000-25,000 vs. 200-800).
- The method reliably detected even single amplified neuroblastoma cells among 1000 normal cells and accurately quantified HER-2/neu amplification in breast cancer samples.
Conclusions:
- Automatic measurement of fluorescence signal intensities via IQ-FISH allows reliable discrimination and exact quantification of amplified gene sequences.
- This technique serves as a prerequisite for applications including detection of amplified cells in bone marrow, monitoring treatment response, and identifying focal gene amplifications.