Optimisation and application of fluorescence in situ hybridization in selected leukaemias

The overall aim of this project was to optimise and then apply the technique of dual-colour fluorescence in situ hybridization (FISH). The first main aim of this study was optimisation of dual-colour FISH by using alpha-satellite centromeric probes, chromosome specific libraries and locus specific cosmid probes. The second main aim was the application of uni- and dual-colour FISH using these probes. At the beginning, to gain experience with the non-isotopic in situ hybridization, a highly repetitive probe GMGYIO (DYS59) was applied by using the alkaline phosphatase technique and then the same probe was used for the fluorescence in situ hybridization technique. The next step was optimisation of whole chromosome specific library probes (pBS-21 and pBS-18) for chromosome 21 and chromosome 18. Less than 600 ng (per hybridization area) of the pBS-21 probe was found to be insufficient to give complete coverage of the chromosome. With a higher concentration of pBS-21 (600-700 ng), a complete coverage was obtained. Use of this probe was discontinued since each hybridization required too much of the library probe DNA. The whole chromosome specific library probe for chromosome 18 (pBS-18) was optimised using 600 ng (per hybridization area) of pBS-18 probe concentration for uni-colour FISH. Using different concentrations of this probe, it was consistently found that the centromeric area of chromosome 2 visualised more strongly than chromosome 18. The study then progressed to the development of the dual-colour FISH technique. The alpha-satellite centromeric probes and the locus specific cosmid probes were individually labelled either with biotin by using nick translation. or with digoxigenin using random primed labelling or the nick translation system. The probes to be used in the study were the a-satellite centromeric probes for chromosome 3, 8, 12 and 22, the locus specific cosmid probes for abl and bcr, and library probes 6, 8, 9, and 22. The first step was to optimise the conditions for each probe for uni-colour FISH. The centromeric probes (for chromosomes 8 and 12) always gave sharp and specific signals. The hybridization signal intensity of the abl cosmid probe was very specific and bright. In contrast, as reported by others, the locus specific bcr cosmid probes did not give specific sharp signals due to the instability of the probe. However, by manipulating each of the steps in the uni-colour FISH procedure, successful results were obtained with bcr probes. Following the optimisation of uni-colour FISH using a-satellite repetitive sequence probes for chromosome 8, 12 and 22, and the locus specific cosmid probes for ab1 and bcr, these probes were optimised for dual-colour FISH. First, in order to overcome the technical difficulties incurred by the dualcolour FISH procedure, all steps were optimised by using a-satellite repetitive sequence probes for chromosomes 8 and 12. Dual-colour FISH results were successfully optimised with those centromeric probes. Using uni-colour FISH specific parameters for cosmid probes such as probe and competitor DNA concentration and hybridization condition, these probes unfortunately could not tolerate some parameters described for the centromeric probe, such as detection, counterstaining and visualisation of signals. This failure may be due to the size and quality of the probe. Therefore, these steps were refined for dual-colour FISH of the cosmid abl and bcr probes. After optimisation of uni- and dual-colour FISH by using the a-satellite probes for chromosomes 8 and 12 , and the locus specific probes for abl and bcr , the techniques were applied to selected leukaemias. Commercial probes were also used either individually or in combination with self-prepared probes. In this project, three different groups of leukaemias were studied. These are (1) Chronic Myeloid Leukaemia (CML), (2) Acute Promyelocytic Leukaemia (AML-M3) and (3) Chronic Lymphocytic Leukaemia (CLL). The first group investigated was the CML group. Fourteen cytogenetically Philadelphia (Ph) positive CML cases were evaluated for the presence of the bcr-abl fusion using uni-colour FISH on metaphase spreads. Despite the low number of metaphases obtained, the FISH metaphase results showed the translocation of abl signal from chromosome 9 to the derivative chromosome 22q. These results were found to be in agreement with the cytogenetic results. Moreover, twelve of Ph negative patients with a provisional diagnosis of CML were studied by using uni-colour FISH on metaphase spreads. Interestingly, one case proved to be bcr-abl fusion positive, while in the remaining cases cytogenetics is in agreement with FISH result. (Abstract shortened by ProQuest.).