water bath of 80 °C for 10 minutes and are immediately added to the metaphase slide preparation. This reaction is then covered with a coverslip and left for two to four days in a humid chamber at 40 °C. The coverslip is then removed and 5 minute washes are applied, three using 2xSSC at room temperature, one at 45 °C with 0.1xSSC and one using TNT at room temperature. The reaction is then preincubated for 10 minutes then followed by a 60-minute, 37 °C incubation, three more 5 minute washes with TNT then one with 2xSSC at room temperature. The slide is then dried using an ethanol series of 70%/96%/100% before counterstaining with DAPI (0.35 μg/ml), for chromosome identification, and sealing with a coverslip. Fluorescence visualisation and imaging A fluorescence microscope with the appropriate filters for the DAPI stain as well as the two fluorophores utilised is required for visualisation, and these filters should also minimise the crosstalk between the fluorophores, such as narrow band pass filters. The microscope must provide uniform illumination without chromatic variation, be appropriately aligned and have a "plan" type of objective which is apochromatic and give a magnification of x63 or x100. The image should be recorded using a camera with spatial resolution at least 0.1 μm at the specimen level and give an image of at least 600x600 pixels. The camera must also be able to integrate the image for at least 5 to 10 seconds, with a minimum photometric resolution of 8 bit. Dedicated CGH software is commercially available for the image processing step, and is required to subtract background noise, remove and segment materials not of chromosomal origin, normalize the fluorescence ratio, carry out interactive karyotyping and chromosome scaling to standard length. A "relative copy number karyotype" which presents chromosomal areas of deletions or amplifications is generated by averaging the ratios of a number of high quality metaphases and plotting them along an ideogram, a diagram identifying chromosomes based on banding patterns. Interpretation of the ratio profiles is conducted either using fixed or statistical thresholds (confidence intervals). When using confidence intervals, gains or losses are identified when 95% of the fluorescence ratio does not contain 1.0. Extra notes Extreme care must be taken to avoid contamination of any step involving DNA, especially with the test DNA as contamination of the sample with normal DNA will skew results closer to 1.0, thus abnormalities