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A Clinically Applicable Unmixing Approach for Spectral MRD Detection in AML
Julian-Philipp Waclawski1,2, Isabell Arnhardt1, Hendrik Fokken1,2
1Department of Hematology, Hemostasis, Oncology, and Cell Therapy, Hannover Medical School, 30625 Hannover, Germany.
Cancers
|July 28, 2026
Summary
A new 22-color spectral flow cytometry assay for acute myeloid leukemia (AML) measurable residual disease (MRD) uses peripheral blood leukocytes (PBLs) for unmixing. This simplifies MRD diagnostics, making it more clinically applicable and standardizable.
Area of Science:
- Immunology
- Hematology
- Analytical Chemistry
Background:
- Spectral flow cytometry allows single-tube assessment of acute myeloid leukemia (AML) measurable residual disease (MRD).
- Clinical adoption is hindered by the need for specific single-stained controls for spectral unmixing.
- Developing a panel using universally available peripheral blood leukocytes (PBLs) for unmixing is key for clinical implementation.
Purpose of the Study:
- To develop a multifunctional, single-tube 22-color spectral flow cytometry assay for AML MRD.
- To establish a robust unmixing strategy using only PBLs, simplifying clinical workflow.
- To incorporate viability, hemodilution, and leukemic stem cell (LSC) markers into the AML MRD panel.
Main Methods:
- Reconfigured a 19-color spectral MRD assay to a 22-color panel, reassigning primitive markers to non-tandem fluorochromes.
- Utilized fluorochromes as CD14 surrogate controls and for unmixing with single-stained PBLs.
- Validated the assay using spike-in experiments, KG-1 dilution experiments with normal bone marrow (nBM), and patient samples compared to a 5-tube reference assay.
Main Results:
- PBL-based controls achieved accurate spectral unmixing and reproducible assessment of hemodilution and cell viability.
- The assay demonstrated linear leukemia-associated immunophenotype (LAIP) detection, sensitivity, and precision in dilution experiments.
- The 22-color assay showed high concordance with the reference assay in patient samples, identifying novel aberrant marker combinations.
Conclusions:
- A single-tube 22-color spectral flow cytometry assay with a PBL-based unmixing strategy is feasible for AML MRD diagnostics.
- This approach offers a more clinically applicable and potentially standardizable method for AML MRD assessment.
- The assay facilitates comprehensive immunophenotypic analysis, including LSCs, in a single tube.
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