Chapter 11
Specimen Preparation Techniques for Cytogenetic
Analysis by Fluorescence in situ Hybridization
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
lntroduction
Analysis of chromosomal DNA by fluorescence in situ hybridization
(FISH) is used in many areas of biological research as well as in clinical
cytogenetics. A major advantage of this method for the clinical applications
is that chromosomal changes can be diagnosed not only on metaphase
chromosomes but also in interphase nudei (see Figure 1 and Chap. 12).
Thus, the analysis is not dependent on the preparation of metaphase chromosomes, which are often difficult or impossible to obtain. Furthermore,
preparation of metaphase chromosomes often requires stimulation of cells
by mitogens. Such mitogenic Stimulation frequently results in selective
growth of a subset of cells in the sample. This may lead to the diagnosis
of chromosomal aberrations that do not reflect the composition of the tumor in vivo.
Interphase analysis permits the investigation of a wide range of different
tissues by means of molecular cytogenetics. The following descriptions provide techniques for preparation of clinical materials commonly used for
analysis by FISH. For further information on this topic the reader is referred
to the literature (see for example Manuelidis 1985; Burns et al. 1986; Hopman et al. 1988; Emmerich et al. 1989a,b; Waltet al. 1989; Hopman et al.
1990; V an Dekken et al. 1990; Hopman et al. 1991; Arnoldus et al. 1991; Hopman et al. 1992; Schertban and Cremer 1993; Lichteret al. 1996). Recent
technical developments of high diagnostic relevance include comparative
Stefan J oos, Deutsches Krebsforschungszentrum, Im N euenheimer Feld 280, Heidelberg,
69120, Germany (phone ), Martin Bentz, Universität Heidelberg, Medizinische Klinik
and Polyklinik V, Heidelberg, 69120, Germany, Anton H. N. Hopman, University ofLimburg, Dept. Molecular Cell Biology & Genetics, Postbus 616, Maastricht, 6200, The N etherlands, Correspondence to Peter Lichter, Deutsches Krebsforschungszentrum, Abt. Organisation komplexer Genome, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany
(phone +49-6221-424-609;fax +49-6221-424-639; e-mail P.Lichter @dkfz-heidelberg.de)
Specimen Preparation Techniques for Cytogenetic
Analysis by Fluorescence in situ Hybridization
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
lntroduction
Analysis of chromosomal DNA by fluorescence in situ hybridization
(FISH) is used in many areas of biological research as well as in clinical
cytogenetics. A major advantage of this method for the clinical applications
is that chromosomal changes can be diagnosed not only on metaphase
chromosomes but also in interphase nudei (see Figure 1 and Chap. 12).
Thus, the analysis is not dependent on the preparation of metaphase chromosomes, which are often difficult or impossible to obtain. Furthermore,
preparation of metaphase chromosomes often requires stimulation of cells
by mitogens. Such mitogenic Stimulation frequently results in selective
growth of a subset of cells in the sample. This may lead to the diagnosis
of chromosomal aberrations that do not reflect the composition of the tumor in vivo.
Interphase analysis permits the investigation of a wide range of different
tissues by means of molecular cytogenetics. The following descriptions provide techniques for preparation of clinical materials commonly used for
analysis by FISH. For further information on this topic the reader is referred
to the literature (see for example Manuelidis 1985; Burns et al. 1986; Hopman et al. 1988; Emmerich et al. 1989a,b; Waltet al. 1989; Hopman et al.
1990; V an Dekken et al. 1990; Hopman et al. 1991; Arnoldus et al. 1991; Hopman et al. 1992; Schertban and Cremer 1993; Lichteret al. 1996). Recent
technical developments of high diagnostic relevance include comparative
Stefan J oos, Deutsches Krebsforschungszentrum, Im N euenheimer Feld 280, Heidelberg,
69120, Germany (phone ), Martin Bentz, Universität Heidelberg, Medizinische Klinik
and Polyklinik V, Heidelberg, 69120, Germany, Anton H. N. Hopman, University ofLimburg, Dept. Molecular Cell Biology & Genetics, Postbus 616, Maastricht, 6200, The N etherlands, Correspondence to Peter Lichter, Deutsches Krebsforschungszentrum, Abt. Organisation komplexer Genome, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany
(phone +49-6221-424-609;fax +49-6221-424-639; e-mail P.Lichter @dkfz-heidelberg.de)