Development of a 29-Color Single-Tube Full Spectrum Flow Cytometry Assay for the Detection of Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Multiparametric flow cytometry (MFC) is widely used to detect measurable residual disease (MRD) in acute myeloid leukemia (AML). However, conventional flow assays require multiple tubes, with an additional tube for leukemia stem cell (LSC) analysis and lack hemodilution evaluation. Spectral flow cytometry (SFC) can overcome the limitation of flow channels and has the potential for multifunctional design using a single tube. We developed a 29-color single-tube assay that adheres to the recommendations of the European Leukemia Network Flow-MRD Working Party and incorporates the simultaneous evaluation of MRD, LSC, and hemodilution. The Complexity Index of the assay was calculated at 9.08. Through limit dilution experiments using the KG-1α AML cell line, we determined the limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) for four leukemia-associated immunophenotypes (LAIP). The assay easily achieved the minimum sensitivity requirement for MRD detection ≤ 0.1% with minimal intra- and interassay variations. Background signals for 24 LAIPs and 10 LSC immunophenotypes were evaluated in eight healthy bone marrow (BM) samples. The single-tube SFC assay was compared with the five-tube conventional assay by analyzing 20 AML BM samples, demonstrating high concordance. To assess hemodilution, markers to detect established parameters, including immature granulocytes, neutrophils, mast cells, and plasma cells, were included. In summary, we provide a versatile single-tube 29-color SFC-based MRD assay that minimizes cell requirements, integrates LSC evaluation, and assesses hemodilution. This assay has the potential to improve the reliability and simplicity of MRD detection.