Multiple myeloma is an incurable haematological malignancy of plasma cells, with some of the highest global incidence rates reported in Australia and New Zealand. The pathogenesis of myeloma is complex, and patient prognosis is strongly influenced by both cytogenetic abnormalities and the depth and duration of remission. Consequently, comprehensive diagnosis, sensitive disease monitoring, and accurate identification of prognostically relevant mutations are essential for managing myeloma. Current diagnostic strategies rely on a combination of histopathology, flow cytometry, and fluorescence in situ hybridisation (FISH) on bone marrow samples. However, these approaches are invasive, costly, and logistically demanding, particularly when repeat sampling is required for longitudinal monitoring. Immuno-flowFISH is a novel diagnostic and monitoring technique that utilises imaging cytometry to integrate morphological, immunophenotypic, and cytogenetic analyses into a single, highly sensitive blood-based test. While immuno-flowFISH shows significant promise for clinical translation, the tolerance of the protocol to processing delays has not been assessed, which is critical for real-world implementation.This project aimed to determine whether pause points could be introduced into the immuno-flowFISH workflow without compromising data quality, enabling sample storage and transport prior to imaging. The workflow involves isolation of peripheral blood mononuclear cells from whole blood, antibody staining, fixation, FISH probe hybridisation, and imaging. Three potential pause points suitable for sample transport were identified and evaluated: following blood collection, after fixation, and before imaging. Healthy donor blood was used for this preliminary investigation, with flow cytometry and confocal microscopy employed to approximate the capabilities of an imaging cytometer. Samples were assessed for antibody-positive population resolution and fluorescence signal intensity following storage. Whole blood could be stored for up to three days before processing while maintaining clear CD19+ and CD45+ populations and strong fluorescent signals, with no added benefit observed from the use of BD OMICS-Guard preservation buffer. Processing could be delayed for up to three days following fixation, after which FISH probe photobleaching became apparent. A delay of up to nine days prior to imaging was also tolerated, with minimal loss of probe fluorescence and preservation of distinct CD19+ and CD45+ populations. Collectively, these findings preliminarily demonstrate that immuno-flowFISH can accommodate processing delays while maintaining data quality, providing a foundation for future studies using imaging cytometry and myeloma patient samples. Incorporation of delay resilience into the immuno-flowFISH workflow could support clinical implementation across a range of settings, expanding patient access to sensitive, minimally invasive myeloma diagnostics and monitoring.
Lia Alyssa Chalmers (Fri,) studied this question.