FISH on Uncultured Blood or Bone Marrow
Interphase cytogenetics (= assay to obtain some “cytogenetic information” from interphase nuclei) has been made possible since the advent of the fluorescence in situ hybridization (FISH) technique (Langer et al., 1981, Pinkel et al., 1986). It is an appro
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FISH on Uncultured Blood or Bone Marrow THOMAS LIEHR
Introduction Interphase cytogenetics (= assay to obtain some "cytogenetic information" from interphase nuclei) has been made possible since the advent of the fluorescence in situ hybridization (FISH) technique (Langer et al., 1981, Pinkel et al., 1986). It is an approach widely used in diagnostics since reliable and well estimable probes have been made available for this technique; i.e. commercially available satellite probes for the centromeric regions of the human chromosomes to study numerical chromosomal changes, and cosmids, YACs, BACs or P1-clones suitable for the detection of translocations, inversions, deletions, duplications or amplifications (for summary see e.g. Sawyer et al., 1992; Wolfe and Herrington, 1997). Peripheral blood or bone marrow aspirate are normally subjected to a short-term culture for 24 to 72 h to obtain metaphase spreads for standard cytogenetic banding analyses (according to Verma and Babu, 1989). However, in some exceptional cases, direct preparation of uncultured blood or bone marrow should be given priority (Liehr et al. 1995). In some cases clinicians send EDTA- or sodium acetate-treated instead of heparinized blood samples for cytogenetic analysis. It is a well-known fact that such blood or bone marrow samples cannot be successfully cultured in short-term culture. However, for some FISH analyses, interphase nuclei are sufficient, as in the detection of microdeletion or microduplication syndromes (i.e. CATCH 22, hereditary neuropathy with liability to pressure palsies = HNPP, Miller-Diecker syndrome, Cri du Chat syndrome, Williams-Beuren syndrome, Prader-Willi syndrome or CharcotMarie-Tooth disease Type 1A and others), numerical chromosome aberrations detectable by centromeric probes (see Figure 1), or in control of
Thomas Liehr, Institut rur Humangenetik, Kollegiengasse 10, Jena, 07743, Germany (phone +49-3641-935533; fax +49-3641-935502; e-mail [email protected])
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B. W. Rautenstrauss et al. (eds.), FISH Technology © Springer-Verlag Berlin Heidelberg 2002
4 FISH on Uncultured Blood or Bone Marrow
Fig. 1. Interphase nuclei prepared from EDTA-treated blood, prepared according to the protocol described in this chapter. Alphoid probes specific for chromosome 8 (red) and 17 (green) have been applied. Probes were derived from Oncor digoxigenated (D8Z2) and biotinylated (DIn!) and detected as described using rhodamine and FITC. Two normal nuclei - with respect to the number of centromeric signals of chromosomes 8 and 17 are shown. Images were captured on a Zeiss Axioplan microscope using the ISIS digital FISH imaging system (MetaSystems, Altlussheim, Germany) using an XC77 CCD camera with onchip integration (Sony)
the course of a leukemic process with a known aberration (e.g. bcr-abl rearrangement). Even heparinized blood samples could be prepared directly, for example in cases to be tested for Charcot-Marie-Tooth disease Type lA, as the disease causing duplication is only 1.5 Mbp in size. This kind of du
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