A 20‐color 21‐antigen flow cytometric assay for disease monitoring of T‐cell lymphoblastic leukemia

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of immature T cells. Flow cytometry plays a critical role in the diagnosis and management of the disease. It is used to establish the abnormal immature T-cell phenotype and to distinguish the early T-cell precursor (ETP)-ALL from more mature types at diagnosis. The evaluation of mediastinal disease is often complicated by the difficulty of the phenotypic distinction between the normal thymic precursors and the abnormal T lymphoblasts. Follow-up measurements of minimal/measurable residual disease (MRD) are critical for therapy decision-making and prognostication. In the MRD setting, flow cytometry requires a high degree of analytical expertise and assessment of numerous antigens. To address the diagnostic and monitoring challenges, we developed a single-tube 21-antigen assessment with simplified analysis. The assay distinguishes between normal thymic precursors and T lymphoblasts in tissue samples, enables evaluation of ETP versus non-ETP phenotypes, and allows for MRD assessment below 0.01% robust to antigenic changes.