Probe design, labeling and digital imaging

Based on the data obtained from conventional double-color interphase FISH assays the most reliable probes for the detection of recurrent translocations are either probes spanning both breakpoints or flanking a promiscuous locus. Thus, in order to allow a comprehensive analysis of multiple recurrent alterations characteristic for a certain disease a considerable number of differently labeled probes has to be applied. A sufficient number of such probes can be achieved either by using multiple fluorescence dyes or by combinatorial labelling of probes with a restricted set of dyes. The combinatorial labelling proved to work reliably on metaphase spreads. However, its applicability to routine interphase FISH is limited due to frequent random overlapping of probes caused by the limited space of the nucleus which hampers unequivocal signal identification. Considering that the detection of structural chromosomal abnormalities relies on a fusion or split of differently labeled probes the combinatorial labelling might result in high false positive or negative rates due to random overlapping. This holds particularly true if the number of probes applied to a single nucleus is increased in order to detect multiple aberrations. Our M-FICTION assays, thus, applied FISH probes labeled with a single dye rather than using combinatorial labelling. The fluorochromes used for the DNA labelling were diethylaminocoumarin (DEAC), SpectrumGreen™ (SG), SpectrumOrange™ (SO), Texas Red® (TR) and Cyanine 5 (Cy™5). Detection of the immunophenotype was performed with aminomethylcoumarin (AMCA). Capturing several signals in a two-dimensional picture poses the problem, that signals are distributed within a three-dimensional structure (the cell nucleus) and some signals might be in different focus planes. Therefore, a critical issue to establish the M-FICTION technique was the multicolor imaging system. Among the two widely accepted multicolor imaging systems, spectral and filter-based technologies, the latter is superior for M-FICTION because it allows multiple focal planes to be reliably captured and integrated. Moreover, it is suitable for scanning using the multicolor spot counting software MetaCyte (MetaSystems, Germany). Thus, for evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes from each color channel using a Z-axis motorized microscope (Axioplan 2, Carl Zeiss, Germany) and MetaSystems’ Isis-mFISH imaging system. Subsequently, an integrated feature of Isis- mFISH calculates an extended focus image from the Z-stack images. The pixel intensity from the different acquired focal planes is automatically compared for each fluorochrome and those with higher intensity were selected to provide a two-dimensional picture integrating three-dimensional information. In order to obtain a multicolor picture all color channels were merged using an false color display. The M-FICTION experimental procedure modified from the previously published conventional FICTION protocol [3] was recently described in detail [4].

Results and Conclusion

By applying B- and T-cell lymphoma specific M-FICTION assays, the characteristic chromosomal aberrations associated to these diseases were confined to the B-cell (CD20 positive) or T-cell (CD3 positive) compartment. In anaplastic large cell lymphomas, chromosomal aberrations appeared only in cells expressing the ALK oncoprotein (Figure 1). In all cases, the immunophenotypically negative cells displayed unaltered signal constellations [4; Gesk et al., in preparation]. Here we present an improved FICTION technique, termed M-FICTION, which as well as assessment of morphology and fluorescence immunophenotyping allows the investigation of multiple chromosomal aberrations. Analyses of different tumors of the lymphatic system with newly developed M-FICTION assays confirmed the performance and validity of the technique and established its diagnostic power. Nevertheless, the application of the technique is certainly not limited to hematological neoplasms. Fields of future applications are the detection of micrometastasis of solid tumors and disseminated tumor cells in bone marrow or peripheral blood. The presented technique will also have its use in the early detection of cancer and the detection of contaminating tumor cells in stem cell transplants and minimal residual disease after treatment. Detection of these rare events can be carried out using MetaSystems’ automatic scanning system for rare cell detection Metafer-RCDetect, which scans for immunofluorescently labeled cells. Subsequent automatic relocation and imaging of the cells including multicolor spot counting will be possible using Metafer-MetaCyte.

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