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Disease specific chromosome abnormalities that have originated from visible chromosome aberrations or have occurred at a molecular level (cryptic aberrations) can be identified by FISH (Fluorescence In-Situ Hybridisation).
FISH analysis is based on the main feature of the DNA molecule - the ability of identical sequences to form a double helix. Stretches of DNA of any length and known sequence can be labelled with fluorescent dye to produce a FISH probe. This probe can be used to study either dividing or quiescent cells from any tissue. The strands of sample DNA (ie the cell or chromosomes) form a target that couples with the probe and its attached fluorochrome to form a DNA double helix. The results are visualised under a fluorescence microscope and recorded with the help of sensitive camera, computer and dedicated software (see figure L1 & L2). A summary of the available FISH technologies used to study metaphase cells (lower left) and non-dividing cells (upper right) is illustrated in figure L3.
An important part of our laboratory's work is devoted to assisting with the management of patients undergoing sex mismatched bone marrow transplantation. If possible the bone marrow donor is chosen from the opposite sex, which would allow follow-up of the engraftment by identification of the cell's gender using FISH. Here we apply centromere probes specific for the X & Y chromosomes. The presence of a high level of host cells in post BMT samples indicates graft rejection, while the detection of aberrant host cells demonstrates disease relapse (figure L4).
Classic G-banding methods are augmented with a range of FISH tests. The list of assays is growing daily. At disease presentation G banding and FISH analysis are performed to identify the underlying genetic abnormalities and determine a suitable marker for post-treatment disease monitoring. For example, recently a young female patient was admitted in the department with suspected diagnosis of acute leukaemia. While G banding analysis of her bone marrow cells showed a normal karyotype (figure L5a), screening by FISH revealed a BCR/ABL fusion gene (figure L5b) resulting from a complex chromosome rearrangement as depicted in figure L5c. Similarly, a subtle translocation specifically associated with a subtype of AML, can be reliably detected by FISH while easily missed by conventional analysis (figure L6). Results such as these, support the clinical diagnosis and help to select an appropriate course of treatment. In another example, seminal work (Sinclair et al, Blood, 1999 & 2000) on CML patients used FISH to detect cryptic deletions in the flanking region of the t(9;22)(q34;q11) translocation leading to the formation of the BCR/ABL chimeric gene (figure L7). The deletions affect only the derivative 9 chromosome in some 15% CML patients but these are associated with a poor overall survival (figure L8). The detection of these hitherto unknown cryptic deletions in an otherwise balanced chromosome translocation has led to the discovery of numerous other examples that form the foundation of many modern cytogenetic tests.
Diagnostic Services are jointly supported by the Royal Free NHS Trust Hospital and the UCL Medical School, Royal Free Campus. Our research programme is jointly funded by the Kay Kendall Leukaemia Fund, the Leukaemia Research Fund, the NHS Trust and the UCL Medical School.
All text and Imagery copyright Dr EP Nacheva MD PhD FRCPath, Royal Free and University College School of Medicine
Figure L5 A/B/C - click to view larger image
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Figure L8 - click to view larger image