Introduction: Integrated immunophenotyping and fluorescence in situ hybridisation (immunoFISH) via imaging flow cytometry enables high-throughput analysis of morphology, phenotype and genotype of cells in suspension. This novel method may significantly enhance the detection and stratification of tumor clonal heterogeneity in hematological malignancies with high sensitivity. We aimed to develop a robust immunoFISH protocol on the Amnis ImageStreamX (ISX) MkII platform.
Methods: Peripheral blood mononuclear cells were stained with fluorescently conjugated antibodies to cell surface antigens, fixed and permeabilised. DNA was denatured followed by probe hybridisation, cells were then stained with Hoechst 33342 and analysed. Optimisation included preservation of fluorescent conjugates and morphology after fixation and denaturation, single cell suspension buffers and determining nuclear probe specificity.
Results: Cross-linking preserved antigen-antibody binding during DNA denaturation and hybridisation. Co-localisation with a nuclear marker (Hoechst 33342) deduced accurate probe hybridisation and spot counting. Increased serum and EDTA in buffers minimised cell clumping, maximising sample throughput.
Conclusion: ImmunoFISH enabled automated FISH analysis of large numbers of phenotypically identified cells, including low frequency subsets. Further optimisation may enable the detection of different chromosomal aberrations associated with cellular phenotypes. We present a novel and potentially powerful avenue for assessing haematological malignancies and minimal residual disease detection.