A Clinically Applicable Unmixing Approach for Spectral MRD Detection in AML.

Background/Objectives: Spectral flow cytometry enables the single-tube integration of all markers required for the assessment of measurable residual disease (MRD) in AML. The routine clinical use of spectral flow cytometry is challenged by the need for adequate single-stained unmixing controls. To pave the way for the clinical implementation of spectral flow cytometry, we opted to develop a multifunctional AML MRD panel, whose unmixing routine solely depends on ubiquitously available peripheral blood leukocytes (PBLs). Methods: We reconfigured our previously developed 19-color single-tube spectral MRD assay to incorporate viability, hemodilution, and leukemic stem cell (LSC) markers. In parallel, primitive markers were reassigned to non-tandem fluorochromes, resulting in a finalized 22-color panel. These fluorochromes were additionally leveraged as CD14 surrogate controls, whereas all other markers retained their dedicated fluorochromes for unmixing based on single-stained PBLs. Results: PBL-based controls enabled accurate spectral unmixing and reproducible assessment of CD16-based hemodilution and cell viability in spike-in experiments. Linear leukemia-associated immunophenotype (LAIP) detection, sensitivity, as well as intra- and inter-assay precision within predefined acceptance criteria were demonstrated in KG-1 dilution experiments using normal bone marrow (nBM). Furthermore, the 22-color assay resolved LAIPs and LSCs in patient samples with strong concordance to the 5-tube reference assay and revealed aberrant marker combinations that could not be assessed using the multi-tube design. Conclusions: This single-tube 22-color spectral flow cytometry assay may support a more clinically applicable and potentially standardizable approach to AML MRD diagnostics through a universal PBL-based unmixing strategy.