Standardized Multicolor Flow Cytometry Protocol for Measurable Residual Disease Assessment in Acute Leukemia

Measurable residual disease (MRD) is an important prognostic indicator in the management of acute leukemia. However, routine multicolor flow cytometric (MFC) assessment remains vulnerable to pre-analytical and analytical variability, including hemodilution, inconsistent acquisition depth, and subjective interpretation. This article presents a standardized operational workflow for routine MRD evaluation in B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The protocol incorporates strict specimen acceptance criteria, with integrated hemodilution assessment, and uses a bulk red blood cell lysis method for bone marrow and peripheral blood specimens. Analytical standardization is achieved through disease-specific two-tube, eight-color antibody panels, harmonized acquisition targets requiring at least 500,000 CD45-positive events, and fixed sequential gating strategies. In addition, the workflow applies denominator-adjusted limits of detection (LOD) and quantification (LOQ) to support an objective three-tiered reporting system. Validation across 197 clinical specimens demonstrated strong repeatability, robust dilutional linearity, and high concordance with paired orthogonal molecular assays. Collectively, this protocol provides a practical framework for standardized MRD monitoring. for standardized MRD monitoring in routine clinical laboratories by integrating controlled pre-analytical handling and harmonized analytical parameters.