Poster Presentation Indian Ocean Rim Laboratory Haematology Congress 2019

Improving the clinical utility of “Immuno-flowFISH”: Analysis of antibody fluorophores performance for expansion of the diagnostic immunophenotyping panel. (#113)

Jason Stanley 1 , Henry Hui 1 , Kathy Fuller 1 2 , Venkat Doddi 1 , James McQuillan 1 , Kathyrn Clarke 3 , Carly George 1 4 , Wendy Erber 1 2
  1. Translational Cancer Pathology Laboratory, The University of Western Australia, Crawley, WA, Australia
  2. PathWest Laboratory Medicine, PathWest, Nedland, WA, Australia
  3. Department of Haematology, Haemato-Oncology Diagnostic Service, Addenbrooke's Hospital, Cambridge, UK
  4. Department of Haematology, Princess Margaret Hospital, Perth, WA, Australia

Immuno-flowFISH (IFF) is a novel diagnostic technique that combines immunophenotyping with fluorescence in situ hybridisation (FISH) enabling the assessment of genetic abnormalities in immunophenotyped neoplastic cells. The IFF methodology utilises an imaging cytometer which has the capacity of flow cytometry and imaging to enable FISH probe signals to be viewed and counted within the nucleus of the immunophenotyped cells. However the combination of acid denaturation and high temperature incubation for DNA hybridisation are known to limit the choices of fluorophores. To date IFF panels have been optimised for diagnosis of Chronic Lymphocytic Leukaemia. In order to expand the IFF panel to allow for diagnosis of other diseases i.e. Multiple Myeloma and Acute Lymphoblastic Leukaemia, it is important to assess the performance of commonly used antibodies with a range of fluorophores.

Method: Red blood cells are lysed and stained with fluorescent conjugated monoclonal antibodies such as CD3, CD4, CD5, CD19 and CD45. Samples were fixed, permeabilised and treated with an acid solution prior to overnight FISH hybridisation. Aliquots of the samples were taken after each stage and acquired on the AMNIS ImagestreamX mkII imaging flow cytometer. Data was analysed to determine percent of cells positive for each immunophenotype at each stage of processing and the stain index for each fluorophore.

Results: Synthetic polymer-based (BV and BB) fluorophores were more stable throughout IFF analysis. All protein based (APC, PE, FITC) fluorophores and their tandem conjugates lost fluorescence after either acid denaturation or hybridisation.

Conclusion: This study found that synthetic polymer fluorophores were more stable than protein-based during IFF analysis. This experiment has improved our understanding of the fluorophore chemistry and expanded the range of fluorophores that we can access for IFF. Validating the use of these fluorophores in the IFF protocol, will provide greater flexibility when designing immunophenotyping panels for other applications.