Multicenter Flow Cytometry For Acute Myeloid Leukemia Measurable Residual Disease With LAIP/DfN And CD34+CD38-...

Measurable residual disease (MRD) follow-up is recommended for treatment response evaluation in acute myeloid leukemia (AML) clinical trials according to ELN 2025 guidelines. The aim of this study was to implement a standardized follow-up of patients using a harmonized MRD flow approach across 30 French hematology laboratories participating in AML clinical trials. To obtain comparable results, the network established recommendations from wet-lab procedures to clinical reports. We designed a 3-tube panel with mandatory 8-color common markers per tube, according to ELN recommendations, to identify leukemia-associated immunophenotype/different-from-normal (LAIP/DfN) patterns in bulk cells and leukemic stem cell (LSC)-enriched populations in the CD34⁺CD38^- fraction. A backbone of CD34/CD38/CD45/CD117 was used, completed by lineage markers for the first tube, LSC-associated markers for the second tube, and monocytic and differentiation markers for the third tube. This panel can be used in 8-, 10-, and 12-color formats and implemented on multiple conventional flow cytometer platforms. We propose flow cytometer settings adapted to each platform. Harmonization of sensitivity between the four platforms was performed using 8-peak rainbow beads. Immunostaining was performed after bulk lysis. To detect bias between platforms, the staining index was tested using fresh healthy bone marrow samples in parallel on the four platforms. Regular bone marrow quality-control samples were shared among laboratories for wet external quality assessment (EQA) to verify all steps of the protocol. Finally, standardization of the data analysis strategy obtained in the centers was evaluated using dry EQA by sharing MRD FCS data files. The feasibility of this multicenter approach requires harmonization of instrument sensitivity and sample preparation, as well as training and systematic education of analytical operators.