Aim: We have invented a world-first automated high-throughput imaging flow cytometry method called “Immuno-flowFISH” that integrates fluorescence in situ hybridisation (FISH) on immunophenotyped cells in suspension. We aimed to assess the utility of immuno-flowFISH for the detection of chromosomal abnormalities in chronic lymphocytic leukaemia (CLL), specifically trisomy 12 (+12) and del(17p).
Methods: Peripheral blood mononuclear cells were isolated from the blood of 55 CLL patients (at diagnosis and monitoring). Immunophenotyping was performed with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilised, double-stranded DNA denatured and hybridised with chromosome 12 or 17 enumeration (CEP 12, CEP17) and 17p12 locus-specific FISH probes. Cells were analysed on the Amnis ImageStreamX Mark II to assess the number and percent FISH-positive CD5/CD19-positive CLL cells and the ratio of FISH spot counts for CLL cells to CD3/CD5-positive T cells (FISH “mean spot ratio”).
Results: The mean number of cells analysed was 20,000 (range = 10,000 – 50,000). Trisomy 12 was detected in 15% of cases and del(17p) in 8% of CLL cases; there was 100% concordance with standard FISH testing. The number of +12 CLL cells ranged from 0.13 – 45% and del(17p) from 3.5 – 22.8%. The FISH “mean spot ratios” were 1.089 -1.362 for +12 and 0.86 – 0.96 for del(17p).
Conclusions: Immuno-flowFISH could detect both +12 and del(17p) in phenotypically identified CD5/CD19-positive CLL cells. Both imagery and quantitative data, including spot count ratio, were used in the analysis. Since a minimum of 10,000 cells were analysed, this was a 100-fold increase compared with standard FISH. Furthermore, the sensitivity and specificity of the FISH result was greater as only cells with the CLL phenotype were analysed. The limit of detection was 1 abnormal cell in 1,000 normal cells.