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OMIP-072

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

Marianne A. Petersen, Marie Bill, Carina A. Rosenberg Cytometry PART A, Volume 99, Issue 4, 382-387 (2021)

PURPOSE: 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.

CELL TYPE: Leukemic Stem Cells (LSCs) in bone marrow (BM) Samples from pediatric patients diagnosed with acute myeloid leukemia (AML)

MACHINE: BD LSR Fortessa

Marker Target Clone Fluorochrome Purpose
Bcl-2 Human bcl-2/100 V450
CD45 Human HI30 Brilliant Violet 510
CD117 Human 104D2 Brilliant Violet 650
TIM-3 Human 7D3 Brilliant Violet 711
CD38 Human HIT2 Brilliant Violet 786
IL-1 RAcP Human REA558 VioBright FITC
CD34 Human 581 PerCP-Cy5.5
CD45RA Human HI100 PE-CF594
Dead Cell All Species Zombie Red viability
CD3 Human SK7 PE-Cy5.5
CD123 Human 7G3 PE-Cy7
CD93 Human VIMD2 APC
CD25 Human 2A3 APC-R700
CD99 Human 3B2/TA8 APC-Vio770
CLEC12A Human HB3 PE