C. Loss of heterozygosity (LOH) (see theallele difference in this figure)
29. If the debubbling does not work, manually fi ll the array with Array Holding Buffer. Stick a p200 pipette tip into the top
Acknowledgment
We would like to thank Dr. Charles Lee and other colleagues for their kind help and support.
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Chapter 16
Multicolor Karyotyping and Fluorescence In Situ Hybridization-Banding (MCB/mBAND)
Thomas Liehr , Moneeb A. K. Othman , and Katharina Rittscher
Abstract
Multicolor fl uorescence in situ hybridization (mFISH) approaches are routine applications in tumor as well as clinical cytogenetics nowadays. The fi rst approach when thinking about mFISH is multicolor karyo- typing using human whole chromosome paints as probes; this can be achieved by narrow-band fi lter-based multiplex-FISH (M-FISH) or interferometer/spectroscopy-based spectral karyotyping (SKY). Besides, various FISH-based banding approaches were reported in the literature, including multicolor banding (MCB/mBAND) the latter being evaluated by narrow-band fi lters, and using specifi c software. Here, we describe the combined application of multicolor karyotyping and MCB/mBAND for the characterization of simple and complex acquired chromosomal changes in cancer cytogenetics.
Key words Multicolor karyotyping , Multicolor fl uorescence in situ hybridization (mFISH) , Multiplex-FISH (M-FISH) , Spectral karyotyping (SKY) , Multicolor banding (MCB/mBAND)
1 Introduction
In cancer, more specifi cally in leukemia and lymphoma diagnostics, karyotyping is still one of the most crucial approaches to be done for correct disease assessment, treatment decisions, and follow-up [ 1 ]. However, as chromosome morphology and black and white banding pattern are the only two evaluated parameters in banding cytogenetics [ 2 ], origin of additional material in a structurally altered acquired tumor-chromosome often remained unresolved.
This kind of limitations was overcome in major parts by the intro- duction of fl uorescence in situ hybridization (FISH) approaches in the 1980s [ 3 ] and more recently by multicolor FISH ( mFISH ) techniques. mFISH is “the simultaneous use of at least three dif- ferent ligands or fl uorochromes for the specifi c labeling of DNA, excluding the counterstain ” [ 4 ]. Following this defi nition fi rst suc- cessful mFISH experiments were done not before the end of the 1990s [ 5 ] and fi rst mFISH probe sets were established by the mid- 1990s [ 6 , 7 ] enabling multicolor karyotyping. The latter describes
the simultaneous staining of each of the 24 human chromosomes in different colors using whole chromosome painting (wcp) probes (Fig. 1A -1). Interestingly, multicolor karyotyping was reported at least eight times in the literature as M-FISH (= multiplex- FISH ) [ 6 ], SKY (= spectral karyotyping ) [ 7 ], multicolor FISH [ 8 , 9 ], COBRA -FISH (= COmbined Binary RAtio labeling FISH) [ 10 ], 24-color FISH [ 11 ], as well as IPM-FISH (= IRS-PCR multiplex FISH) [ 12 ]. While SKY and COBRA-FISH are interferometer/
spectroscopy-based, all other approaches have evaluation systems that are narrow-band fi lter-based. All mentioned multicolor karyo- typing possibilities are based on four to seven different
Fig. 1 Examples for multicolor karyotyping and FISH-banding (MCB/mBAND) ( A ) In case of an acute lymphatic leukemia [ 1 ] a complex karyotype was found in GTG-banding (result not shown). M- FISH showed that indeed the three following chromosomes were involved in the rearrangement: 10, 11, and 14 ( arrows in A-1). The rearrangements of all chromosomes were further characterized by chromosome-specifi c homemade MCB probes. In A-2 only the result for chromosome 14 is depicted. MCB revealed that indeed no normal chromo- some 14 was present; one chromosome 14 had a paracentric inversion, the second was involved in a translo- cation with chromosome 11, and a derivative der(10)t(10;14) was also stained by MCB-14-probe set. Overall, a fi nal karyotype was determined as: 46,XX,der(10)(10pter->10p12.31::11q23.3->11q23.3::10p12.31-
>10q11.23::14q24.2->14qter),der(11)(10qter->10q11.23::11p15.3->11q23.3::10p12.31-
>10p12.31::11q23.3->11qter),der(14)t(11;14)(q15.3;q24.2),inv(14)(q11q23) using additional probes and approaches (see also [ 1 ]). [Abbreviations: Cy5 cyanine 5, DAPI 4,6-diamidino-2-phenylindole.2HCl, DEAC diethylaminocoumarine, inv . inverted, spect . spectrum] ( B ) A simple insertion translocation between chromo- somes 10 and 11 was suggested in this acute myeloid leukemia case according to banding cytogenetics (result not shown). No M- FISH was done, but fi rst a whole chromosome paint ( wcp ) probe for chromosome 11 was used together with inverted DAPI banding (B-1) to characterize the breakpoint in chromosome 10 to subband p12.31 (B-2). In B-3 the result of MCB probe set for chromosome 11 is shown, characterizing the breakpoints in 11q13.1 and 11q23.3. This part is inserted directly in the derivative chromosome 10. The rear- rangement after MCB could be reported as ins(10;11)(p12.31;q13.1q23.3)
fl uorescence dyes. However, those that are routinely used by the majority of laboratories are M-FISH and SKY, which work with fi ve fl uorochromes used for combinatorial labeling and one coun- terstain (DAPI = 4,6-diamidino-2-phenylindoleã 2HCl ) [ 13 ].
As multicolor karyotyping reaches its limits when an exact localization of a chromosomal breakpoint is required, or when intrachromosomal aberrations need to characterized, different so- called FISH banding approaches were developed to overcome this kind of limitations . As defi ned in 2002, FISH-banding methods are any kind of FISH technique, which provide the possibility of characterizing simultaneously several chromosomal subregions smaller than a chromosome arm (excluding the short arms of the acrocentric chromosomes). FISH banding methods fi tting that defi nition may have quite different characteristics, but share the ability to produce a DNA-specifi c chromosomal banding [ 14 ]. For humans the following FISH banding probe sets were established yet: IPM-FISH [ 12 ], cross-species color banding (Rx-FISH) [ 15 ], locus-specifi c probe -based [ 16 – 19 ] and somatic cell hybrid-based chromosome bar coding [ 20 ], multicolor banding (MCB) [ 21 – 23 ], multitude multicolor banding ( mMCB ) [ 24 ], spectral color banding ( SCAN ) [ 25 , 26 ], and M- FISH using chromosome- region- specifi c probes ( CRP ) [ 27 ]. To the best of our knowledge, the only commercially available FISH-banding probe set is MCB/ mBAND .
The applications of M- FISH and SKY or MCB/mBAND cover the whole spectrum of human cytogenetics including tumor cytogenetics; while in leukemia and lymphoma these probe sets may be used in routine diagnostics, solid tumors (including cell lines) are most often studied by these approaches mainly under research aspects [ 13 ].
The protocol how to do M- FISH for characterization of chro- mosomes involved in a complex rearrangement and MCB/ mBAND to resolve the breakpoints in more detail are provided here. In case of more simple rearrangements those may be resolved by directly starting with chromosome-specifi c MCB/mBAND probe sets.
Examples for a complex rearrangement resolved by both M-FISH and MCB and only by MCB are given in Fig. 1 . For research- associated screening of cancer cytogenetic cases appearing unal- tered according to banding cytogenetics it might be better fi rst to do M-FISH or SKY followed by mMCB [ 1 ]. Also, it is necessary to keep in mind that M- FISH and MCB/mBAND have resolution limitations of 3–10 megabase pairs; thus in cases higher resolution of breakpoints needs to be achieved locus-specifi c probes and/or MLPA and array-comparative genomic hybridization need to be applied (the latter two approaches for sure can only resolve unbal- anced rearrangements) [ 1 ].
2 Materials
To perform M- FISH and/or MCB/ mBAND a fully equipped molecular cytogenetic laboratory is the prerequisite.
1. 20 × Saline-sodium citrate (SSC) buffer.