OMIP 072: A 15‐color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia

Abstract

This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

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

“…Similar to our study, Petersen et al have demonstrated variant subpopulations of LSCs based on differential expression of LSC markers CD123, CD45RA, TIM3, CD25, CD93 and IL1RAP 39 . Intra‐patient heterogeneous subpopulations of LSCs were observed in 4.6% of AML samples in our study by MFC.…”

Imaging Flow Cytometry and Convolutional Neural Network-Based Classification Enable Discrimination of Hematopoietic and Leukemic Stem Cells in Acute Myeloid Leukemia

“…However, multiple other LSC markers have been described, including CD123, CD7, and CD19 [ 15 , 23 , 63 ]. Due to the considerable heterogeneity between and within AML samples [ 30 ], we refrained from following a one-tube approach as performed by Zeiljemaker et al [ 22 , 63 ], where multiple LSC markers are placed in a single channel. While some LSC markers have a clear boundary between positive and negative populations (e.g., CD7, CLEC12A, and CD45RA), others may show continuous expression levels and/or overexpression compared with healthy cells (e.g., CD33, CD47, and CD96) [ 5 ].…”

“…Importantly, considerable immunophenotypic heterogeneity exists, and no single marker identifies all LSCs across AML samples [ 5 , 28 ]. For example, Petersen et al detected 50 LSC subsets showing 41 different immunophenotypic profiles within BM samples from 38 pediatric AML patients [ 29 , 30 ]. Also, the absence of an LSC marker on AML stem cells does not necessarily equal absence of disease, as shown by Bill et al demonstrating disease-related aberrancies within CLEC12A − stem cell subsets from AML cases [ 31 ].…”

Immunophenotypically defined stem cell subsets in paediatric AML are highly heterogeneous and demonstrate differences in BCL‐2 expression by cytogenetic subgroups

“…Cryopreserved BM MNCs were thawed and stained as previously described (Data S2) (Table SI). 47 Data were acquired on an LSRFortessa flow cytometer equipped with four lasers (violet, 405 nm; blue, 488 nm; yellow/green, 561 nm; red, 640 nm) (BD Biosciences). As compensation controls, single stained beads (UltraComp eBeads™; ThermoFisher) and cells were used for monoclonal antibodies (MoAbs) and the viability marker, respectively.…”