Development of a robust immuno-S-FISH protocol using imaging flow cytometry.

Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe "spots." Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and "spot" detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and "spot" counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry.