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

Background: 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.

Methods: 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).

Results: Guided by the imagery available in IFC, we identified distinct TdT and TdT subpopulations with apparent differences in internal complexity. As TdT 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 blasts were comparable to the TdT cells and without morphometric apoptotic hallmarks, supporting that the TdT subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdT FSC cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdT FSC and TdT FSC subsets.

Conclusion: 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.