Towards a better and more robust assessment of dyserythropoiesis in myelodysplastic syndrome (MDS) clinical scientists at Aarhus University Hospital have built on the community's development of multicolour flow cytometry in recent years. They have utilised imaging flow cytometry to gain both immunophenotypic and morphological information on a cell-by-cell basis.
Frozen bone marrow MNCs were thawed and treated with DNase. For exclusion of dead cells Zombie Violet™ Fixable Viability dye (Biolegend) was then added. Following a wash, an antibody panel targeted against CD235a, CD105 PE-CF594, CD71, CD117 and CD45 was added to the cells. Finally, for the morphological analysis, in addition to bright field (BF), DRAQ5™ (for DNA; 1.25 µM) and Thiazole Orange (TO; for RNA; 0.25 µg/ml) were added to the cells and analysis undertaken. The latter reagents are a well-tested combination from early work to unpick the normal course of erythropoiesis (by K. McGrath and colleagues, Rochester, NY).
To better perform the sample analysis on the ImagestreamX Imaging Flow Cytometer, novel machine learning tools were applied to enable the separation of true binucleated events from doublets, aided by a combination of DRAQ5, BF and CD235a.
The authors show proof-of-concept for the detailed analysis of dysplasia in erythroid differentiation towards more definitive diagnosis and monitoring of MDS, most importantly perhaps reducing the intra-operator variability and alleviating the issues related with cytopenic samples.
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