Feasibility study: phospho-specific flow cytometry enabling rapid functional analysis of bone marrow samples from...

Background: Multiple myeloma (MM) is an incurable cancer accounting for about 2% of cancer deaths. Its diagnosis is based on a combination of criteria, which are not always easily measurable. Flow cytometry now allows multiplex analysis of intracellular signaling at the single cell level. We investigated the feasibility of using intracellular protein phosphorylation analysis by flow cytometry on primary plasma cells from bone marrow and its usefulness in MM diagnosis. Methods: Cells from frozen bone marrow of five MM patients and four normal donors were stimulated with LPS, IL-6, IL-21, IFN? and TNF?. Cells were stained by fluorescent cell barcoding to allow multiplex analysis. Staining with antibodies against phosphorylated NFkB-p65, Stat1, Stat3 and p38 were used to identify cellular responses following stimulation. Results: Activation profile of MM and normal plasma cells have been established. MM cells showed heterogeneous response profiles while normal cells responses were homogeneous between donors. We also noticed that many MM samples seemed to show elevated basal level of Stat3 phosphorylation. These results suggest that different response profiles in primary MM cells might correspond to different subtypes of the disease. Thus, we provide an example of how these results may be used as a criterion for MM subtypes classification. Conclusions: We demonstrate that flow cytometry can be used to study signaling pathways in primary MM cells. The heterogeneity observed in MM cells from different patients can prove valuable for MM characterization and represents an interesting avenue for future research in MM diagnosis. © 2013 Clinical Cytometry Society.