Multicolor-FICTION: expanding the possibilities of combined morphologic, immunophenotypic, and genetic single cell analyses.

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkin's lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases.