“Immuno-flowFISH”: Diagnostic assessment of structural chromosomal abnormalities in phenotyped chronic lymphocytic leukaemia cells with del(17p) by imaging flow cytometry


Immuno-flowFISH is a novel technique that combines the functionality of flow cytometry with high-resolution digital images that can capture fluorescent signals and quantifiable numerical data. This high-throughput method integrates the well-established diagnostic techniques of fluorescence in situ hybridisation (FISH) with immunophenotyping on cells in suspension. Previous studies within the group have demonstrated the efficacy of this technique for use with prognostically significant numerical chromosomal abnormalities in CLL i.e. trisomy 12 (1). This particular study, following an international exchange with the University of Western Australia, aimed to improve “spot” detection sensitivity with the locus-specific del(17p12) probe. Normal blood (n = 15) and diagnostic/follow-up CLL cases (n = 20) were assessed. CLL peripheral blood mononuclear cells were identified using a CD5+/CD19+/CD3- phenotype. Subsequently, cells were fixed, permeabilised and DNA was denatured before incubation with chromosome 17 enumeration (CEP17) and 17p12 locus-specific FISH probes. SYTOX AADvanced was used as a nuclear marker. Greater than 10,000 cells were analysed for each sample using the Amnis ImageStreamX Mark II imaging flow cytometer. Digital images (x40/x60 magnification with extended depth of field) and quantitative data were used to assess morphology and FISH probe spot counts in cells, using the IDEAS software. Majority two spots were detected on all normal and non-del(17p) cases, whilst loss of one 17p12 probe spot was identified in eight cases. T cells within each of the cases were used as an internal control. All CLL cases were independently verified by conventional FISH. This new method enables FISH probe signals from phenotypically-identified cells to be analysed rapidly, providing accurate analysis of chromosomal abnormalities.