Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10‐colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity

Abstract Introduction Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single‐cell sequencing. Materials and Methods Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube‐10‐colour‐panel containing LSC‐associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL‐1, TIM‐3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single‐cell sequencing of the whole transcriptome was also done in a bone marrow sample. Results LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs ( p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ ( n = 184) and CD34‐ LSCs ( n = 41), no difference was observed between the groups. More than one sub‐population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. Conclusion A single tube‐10‐colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter‐ and intra‐sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.