Measurable Residual Disease (MRD) in Haematological Malignancies: A Comparative Overview of Flow Cytometry, Molecular, and Next-Generation Sequencing Approaches

Measurable residual disease (MRD) has emerged as a cornerstone of disease evaluation in haematological malignancies, providing prognostic insight and informing treatment decisions beyond what is achievable through conventional morphological assessment. During morphological remission—defined by less than 5% bone marrow blasts—MRD testing detects persistent leukemic cells at sub-microscopic levels, enabling early identification of relapse risk and supporting individualized therapy planning. The principal platforms for MRD assessment include flow cytometry (FCM), molecular diagnostic techniques, and next-generation sequencing (NGS), each offering distinct advantages across different disease contexts. Technological progress has extended detection sensitivity to as low as 10−6—levels unattainable with conventional assays. Flow cytometry is the most universally applicable method, capable of interrogating leukaemia-specific surface marker profiles across virtually all patients. Instruments range from basic four-colour configurations to next-generation flow cytometry (NGF) platforms employing more than ten markers, permitting the analysis of millions of cells and approaching the sensitivity of molecular methods. However, FCM has notable limitations, including dependence on fresh specimens, significant operator expertise, and susceptibility to immunophenotypic drift that may generate false-positive or false-negative results. On the molecular side, methodologies have progressed from conventional quantitative PCR (qPCR) to sophisticated platforms such as droplet digital PCR (dPCR) and NGS. While NGS offers unparalleled sensitivity and the capacity to identify clonal evolution, its deployment remains confined to specialist centres because of cost and technical complexity. dPCR permits absolute target quantification and surpasses traditional PCR in sensitivity, though standardization remains challenging for genetically heterogeneous targets such as NPM1. MRD assessment is well-established in B-ALL, T-ALL, AML, MM, and CLL, with each malignancy requiring tailored immunophenotypic and molecular strategies. In CML, serial molecular monitoring by qRT-PCR forms the backbone of response assessment, enabling evaluation of deep molecular remission and informing decisions around treatment-free remission. Liquid biopsy approaches utilizing ctDNA have attracted growing interest in lymphoma, where conventional sampling is less informative. Despite these advances, key challenges persist: the absence of harmonized global standards, biological heterogeneity causing false results, clonal evolution undermining established targets, and restricted access to technologies such as NGS in resource-limited settings.