Flow cytometric detection of leukemic stem cells in Acute Myeloid Leukemia: current status and future directions

The assessment of measurable residual disease (MRD) plays a critical role in acute myeloid leukemia (AML) treatment response evaluation and prognosis. However, current AML MRD detection by flow cytometry (FC) is limited in sensitivity due to immunophenotypic variability, similarities to normal hematopoietic stem/progenitor cells, and the lack of stable leukemia-associated immunophenotypes. A significant proportion of AML patients classified as MRD-negative by FC eventually relapse, likely due to the persistence of therapy-resistant leukemic stem cells (LSCs) that are not sensitively detected by routine clinical flow panels. Flow cytometry panels designed to detect LSC antigens, while promising, face challenges like immunophenotypic heterogeneity across AML subtypes, lack of standardized marker panels across laboratories, and limited validation. Here, we summarize the current state of FC-based LSC detection in AML, discussing commonly used markers, immunophenotypic variability, assay setup challenges, and we review recent clinical studies on LSC assessment, outlining their main findings and implications for prognosis and MRD integration. We also consider advances in spectral flow cytometry for improved LSC detection.