Imaging flow cytometry reveals a subset of TdT negative T-ALL blasts with very low forward scatter on conventional...

Studies in T-cell acute lymphoblastic leukemia (T-ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow-up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T-ALL blasts could correspond to differences in morphometric features.

We investigated diagnostic bone marrow samples from six individual T-ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC).

Guided by the imagery available in IFC, we identified distinct TdT^neg and TdT^pos subpopulations with apparent differences in internal complexity. As TdT^neg blasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC-based morphometric analyses demonstrated that cell size and shape of TdT^neg blasts were comparable to the TdT^pos cells and without morphometric apoptotic hallmarks, supporting that the TdT^neg subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdT^neg FSC^very-low cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdT^neg FSC^very-low and TdT^pos FSC^int subsets.

We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.