Poster Presentation 32nd Lorne Cancer 2020

The clinical utility of an automated, ultra-fast and highly sensitive “Immuno-flowFISH” method (#350)

Jason Stanley 1 , Henry Hui 1 , Kathy A Fuller 1 2 , James McQuillan 1 , Venkat R Doddi 1 , Carly George 1 3 , Hun Chuah 1 4 , Teng Fong Ng 1 4 , Wendy N Erber 1 2 4 5
  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, Perth Children's Hospital, Nedlands, Western Australia, Australia
  4. Department of Haematology, Royal Perth Hospital, Perth, Western Australia, Australia
  5. Department of Haematology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia

Aim: Fluorescence in situ Hybridisation (FISH) is a commonly utilised cytogenetic assay however, this technology is insensitive, laborious and slow with little innovation over the past decade. The Immuno-flowFISH (IFF) is a next generation flowFISH (FISH in suspension) that combines immuno-phenotyping with flowFISH to enable rapid, automated and highly sensitive FISH analysis. This study examined the clinical utility of IFF for the analysis of haematological malignancies.

Method: Mononuclear cells were isolated from fresh or cryopreserved leukemic patient and healthy donor peripheral blood samples or patient bone marrow aspirate. Cells were immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies prior to fixation. DNA denatured and hybridised with chromosome 12 or 17 and 17p12 locus‐specific FISH probes. Samples were analysed on the Amnis ImageStream®X Mark II to determine the cytogenetic of the leukemic cells using an automated analysis software. To determine clinical utility, a subset of samples are stored post fixation for up to five days before hybridisation to simulate batch processing.

Result: A total of 10,000 – 100,000 cells were analysed within 48 hours with 100% concordance to diagnostic FISH. The protocol enabled the hybridisation of multiple FISH probes with >90% efficiency. IFF hybridisation of batched samples was concordant with samples processed immediately, with similar stain index and cell population recovered across the five days. The IFF method was able to detect leukemic cells with a sensitivity of at least 0.13% and was also able to detect the presence of subclonal heterogeneity in leukemic samples.

Conclusion: The flexibility of sample types, processing time and multi-probe analysis provided by IFF would allow implementation in a diagnostic pipeline. In addition, the fast turnaround time for results coupled with high sensitivity and ability to detect clonality within a sample would enable FISH to be used for regular MRD detection. IFF could provide rapid and highly sensitive automated flowFISH analysis which could improve the utility of FISH in a diagnostic and research laboratory.