4 FISH on Uncultured Blood or Bone Marrow
83
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 duplication can be detected exclusively due to locus-specific probes in interphase
nuclei and never in metaphase spreads, neither by GTG banding nor by
FISH analysis. Thus, the time-consuming and cost-intensive procedure of
short-term culture and metaphase preparation could be relinquished in
such cases (Liehr et al., 1995, 1997; for a summary of Charcot-Marie-Tooth
disease, see Patel and Lupski, 1994).
Moreover, for bone marrow samples of leukemia, it has to be remembered, that the detected percentage of subpopulations even after a 24-h
short-term culture does not necessarily represent the in vivo situation.
Thus, the interphase cell analysis of directly prepared bone marrow aspirate gives more valid information to the clinician in repeated analysis of
bone marrow aspirates during the course of a leukemic process or in the
control of reverse sex bone marrow transplantation (Liehr and Gebhart,
unpublished data).
83
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 duplication can be detected exclusively due to locus-specific probes in interphase
nuclei and never in metaphase spreads, neither by GTG banding nor by
FISH analysis. Thus, the time-consuming and cost-intensive procedure of
short-term culture and metaphase preparation could be relinquished in
such cases (Liehr et al., 1995, 1997; for a summary of Charcot-Marie-Tooth
disease, see Patel and Lupski, 1994).
Moreover, for bone marrow samples of leukemia, it has to be remembered, that the detected percentage of subpopulations even after a 24-h
short-term culture does not necessarily represent the in vivo situation.
Thus, the interphase cell analysis of directly prepared bone marrow aspirate gives more valid information to the clinician in repeated analysis of
bone marrow aspirates during the course of a leukemic process or in the
control of reverse sex bone marrow transplantation (Liehr and Gebhart,
unpublished data).