Multiple Myeloma is an incurable malignancy of bone-marrow plasma cells. It is characterized by unpredictable and highly variable therapeutic response and patient survival, largely attributed to the development of multidrug resistance (MDR) in response to chemotherapy. Presently, no clinical procedures allow for a continuous, non-invasive monitoring of MDR.
In our earlier work, we detected and analysed circulating extracellular vesicles (EVs) from myeloma patients by flow cytometry. We also showed that EV counts corresponded to disease burden and that EV biomarkers have potential for monitoring for the presence of MDR. The application of flow cytometry for the study of EVs is gaining momentum, however technical challenges exist in their analysis.
In the present study, we have commenced an analytical validation and standardization of our prototype test. We describe studies for the evaluation and optimization of scatter resolution, enumeration accuracy and instrument precision. In improving the detection sensitivity of EVs, we have identified the most common sources of background noise and applied strategies to minimize these into the workflow. We assessed the contribution of coincidence and defined the clinical operating range to avoid swarm detection, one of the most common artefacts in flow cytometric EV analysis.
The outcome of this study is a defined working protocol which supports the use of flow cytometry in a liquid biopsy application.
Funding
Research Funds supporting this project were provided by SPARK OCEANIA and through the UTS Innovation Commercialisation Seed Fund Scheme to Mary Bebawy.