Reiner Siebert

Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)

In this chapter, the FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms) technique will be detailed. This method has been described in 1992 by Weber-Mat-thiesen et al. as the first technique to combine immunophenotyping and FISH. In contrast to previous methods like the morphology-antibody-chromosome (MAC) technique (Teerenhovi et al. 1984; Schlegelberger et al. 1992), the FICTION technique can be applied on interphase cells, which is of great advantage especially for tumor cytogenetics. To briefly introduce the technique: In general, conventional unstained peripheral blood or bone marrow smears, imprints and cytospin slides from fresh tissue and cryostat sections are applicable for FICTION. In order to preserve cellular antigens, preparing high quality slides is one of the critical steps for successfully performing FICTION. Freshly prepared slides should be air dried overnight at room temperature. Thereafter, slides can be processed immediately or stored airtight at -80°C for years. For the FICTION procedure, slides are fixed in acetone and then immunophenotyped by a direct or indirect fluorescence detection system. Immunophenotyping is followed by a fixation step to preserve the fluorescent immunostaining during the harsh hybridization procedure. After fixation, standard direct or indirect fluorescence in situ hybridization (FISH) is performed using appropriately modified DNA probes. Using appropriate filter sets, the immunophenotype and the hybridization Signals can be evaluated simultaneously under a fluorescence microscope (for review see Siebert and Weber-Matthiesen 1997; Siebert and Schlegelberger 1997).