Chapter 5
PROTOCOL
FISH on Blood or Bone Marrow Smears
THOMAS LIEHR
Introduction
Cytogenetic diagnostics of leukemia subtypes are nowadays done routinely by conventional banding analysis as well as by fluorescence in situ
hybridization (FISH). A lack of metaphase spreads in cytogenetic preparations in many cases can be compensated, using interphase cytogenetics
with specific probes like commercially available satellite probes for the
centromeric regions of the human chromosomes, cosmids, YACs,
BACs or PI-clones (for summary see e.g. Sawyer et al., 1992; Wolfe
and Herrington, 1997). Special approaches, i.e. the control ofbone marrow
transplants, can be performed on interphase nuclei without the necessity
of time consuming cytogenetic preparation at all. Moreover, for blood or
bone marrow samples ofleukemia, it has to be remembered, that the 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 uncultured bone marrow aspirate gives the clinician more valid
information 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transplantation (Liehr and Gebhart, unpublished data).
Interphase nuclei from peripheral blood or bone marrow can be prepared directly according to Liehr et al. (1995) (see Chapter 4) or - more
quickly - by making a blood or bone marrow smear on a slide (Gebhart et
al., 1995). Furthermore, the latter technique has the advantage that the
cellular structure can be maintained and tumor cells can be distinguished
from nontumor cells (e.g. Haferlach et al., 1998). In the protocol reported
in the following, this can be achieved when replacing a slide pretreatment
step with a 10-min incubation of the slides in 2x SSC at room temperature.
~ Thomas Liehr, Institut flir Humangenetik, Kollegiengasse 10, lena, 07743, Germany
(phone 49-3641-935533; fax +49-3641-935502; e-mail i8Iith@mti-n.mti.uni-jena.de)
PROTOCOL
FISH on Blood or Bone Marrow Smears
THOMAS LIEHR
Introduction
Cytogenetic diagnostics of leukemia subtypes are nowadays done routinely by conventional banding analysis as well as by fluorescence in situ
hybridization (FISH). A lack of metaphase spreads in cytogenetic preparations in many cases can be compensated, using interphase cytogenetics
with specific probes like commercially available satellite probes for the
centromeric regions of the human chromosomes, cosmids, YACs,
BACs or PI-clones (for summary see e.g. Sawyer et al., 1992; Wolfe
and Herrington, 1997). Special approaches, i.e. the control ofbone marrow
transplants, can be performed on interphase nuclei without the necessity
of time consuming cytogenetic preparation at all. Moreover, for blood or
bone marrow samples ofleukemia, it has to be remembered, that the 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 uncultured bone marrow aspirate gives the clinician more valid
information 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).
Interphase nuclei from peripheral blood or bone marrow can be prepared directly according to Liehr et al. (1995) (see Chapter 4) or - more
quickly - by making a blood or bone marrow smear on a slide (Gebhart et
al., 1995). Furthermore, the latter technique has the advantage that the
cellular structure can be maintained and tumor cells can be distinguished
from nontumor cells (e.g. Haferlach et al., 1998). In the protocol reported
in the following, this can be achieved when replacing a slide pretreatment
step with a 10-min incubation of the slides in 2x SSC at room temperature.
~ Thomas Liehr, Institut flir Humangenetik, Kollegiengasse 10, lena, 07743, Germany
(phone 49-3641-935533; fax +49-3641-935502; e-mail i8Iith@mti-n.mti.uni-jena.de)