Leukaemia and other haematological malignancies are characterised by features such as cell morphology, phenotype and genetic abnormalities. These features define the subtype of the malignancy and provide prognostic information. Immuno-flowFISH (IFF) is a new technique that combines immunophenotyping with fluorescence in situ hybridisation (FISH) and enables the assessment of genetic abnormalities in neoplastic cells that have been identified by phenotype. The IFF methodology utilises an imaging cytometer which has the capacity of high-throughput flow cytometry and imaging to enable FISH probe signals or “spots” to be viewed and counted within the stained nucleus of the immunophenotyped cells. Optimisation of IFF was conducted to achieve optimal staining, hybridisation and reproducibility. IFF involves acid denaturation and high temperature incubations for DNA hybridisation. These steps are important as they are known to affect fluorophore performance.
Aim: To evaluate the performance of commercially available antibodies with a range of fluorophores in IFF assessment of blood samples of healthy controls and leukaemia patients.
Method: Nucleated cells were stained with fluorescent conjugated monoclonal antibodies. Samples were fixed, permeabilised and treated with an acid solution to denature DNA prior to overnight hybridisation. Aliquots of the samples were taken after staining, fixation/permeabilisation, acid denaturation and hybridisation, prior to analysis on an imaging flow cytometer.
Results: Most synthetic (BB and BV) polymer-based were stable throughout IFF analysis. All protein based (APC, PE, FITC) fluorophores and their tandem conjugates lost fluorescence after either acid denaturation or hybridisation. Treatment with BS3 (an amine cross-linker) after staining was found to maintain several fluorophores during permeabilisation, acid treatment and high temperature FISH.
Conclusion: This study found that synthetic polymer fluorophores were more stable than protein based during IFF analysis. This experiment has improved our understanding of fluorophore chemistry and expanded the range of fluorophores that we can access with IFF. Validating the use of these fluorophores in the IFF protocol will provide greater flexibility when designing immunophenotyping panels for other haematological malignancies.