19‐color, 21‐Antigen Single Tube for Efficient Evaluation of B‐ and T‐cell Neoplasms

Abstract Non‐Hodgkin lymphoma (NHL) is a heterogeneous disease, encompassing a wide variety of individually distinct neoplastic entities of mature B‐, T‐, and NK‐cells. While they constitute a broad category, they are the most common hematologic malignancies in the world. The distinction between different neoplastic entities requires a multi‐modal approach, such as flow cytometric immunophenotyping, which can exclude a neoplastic proliferation and help narrow the differential diagnosis. This article describes a flow cytometric test developed at Memorial Sloan Kettering Cancer Center to assess B‐, T‐, and NK‐cells in a single tube, 21‐antibody, 19‐color assay. The assay can identify most B‐ and T‐cell NHLs with high specificity and sensitivity and significantly narrow the differential when a specific diagnosis cannot be made. The basic protocol provides a detailed operational procedure for sample processing, staining, and cytometric acquisition. The support protocol provides typical steps and caveats for data analysis in lymphoproliferative disorders and in discriminating a variety of specific disease entities from each other and normal lymphoid populations. © 2023 Wiley Periodicals LLC. Basic Protocol : Processing, staining, and cytometric analysis of samples for B‐ and T‐cell assessment Support Protocol : Analysis and interpretation of the B‐ and T‐cell lymphocyte assay