IMAGE-ANALYSIS IN COMPARATIVE GENOMIC HYBRIDIZATION

Comparative genomic hybridization (CGH) is a new technique by which ge nomic imbalances can be detected by combining in situ suppression hybr idization of whole genomic DNA and image analysis. We have developed s oftware for rapid, quantitative CGH image analysis by a modification a nd extension of the standard software used for routine karyotyping of G-banded metaphase spreads in the Magiscan chromosome analysis system. The DAPI-counterstained metaphase spread is karyotyped interactively. Corrections for image shifts between the DAPI, FITC, and TRITC images are done manually by moving the three images relative to each other. The fluorescence background is subtracted. A mean filter is applied to smooth the FITC and TRITC images before the fluorescence ratio betwee n the individual FITC- and TRITC-stained chromosomes is computed pixel by pixel inside the area of the chromosomes determined by the DAPI bo undaries. Fluorescence intensity ratio profiles are generated, and pea ks and valleys indicating possible gains and losses of test DNA are ma rked if they exceed ratios below 0.75 and above 1.25. By combining the analysis of several metaphase spreads, consistent findings of gains a nd losses in all or almost all spreads indicate chromosomal imbalance. Chromosomal imbalances are detected either by visual inspection of fl uorescence ratio (FR) profiles or by a statistical approach that compa res FR measurements of the individual case with measurements of normal chromosomes. The complete analysis of one metaphase can be carried ou t in approximately 10 minutes. (C) 1995 Wiley-Liss, Inc.