A practical difference from the normal approach to evaluate natural killer (NK) cells and NK cell neoplasms by flow cytometry

Abstract Natural killer (NK) cell neoplasms span indolent to highly aggressive entities, and flow cytometric immunophenotyping is central to their detection and classification. However, distinguishing reactive from neoplastic NK cell expansions can be challenging because NK cells lack somatically recombined antigen receptors, and reactive states may produce restricted phenotypes that mimic clonality. This best‐practices manuscript outlines a practical, flow cytometry‐based approach to NK cell analysis that integrates (i) robust lineage definition and gating, (ii) subset‐aware interpretation of CD16/CD56 patterns, (iii) inference of clonality using killer cell immunoglobulin‐like receptor (KIR; CD158) repertoire restriction and other NK cell receptors. We highlight key pitfalls—particularly the limited utility of KIR‐based clonality inference in CD56 bright CD16 − NK cells and the potential for adaptive NK cell expansions to appear KIR‐restricted—and propose an operational reflex algorithm triggered by NK cell expansion and/or defined immunophenotypic abnormalities in screening tubes. Case‐based examples illustrate common scenarios, including NK‐large granular lymphocytic leukemia, extranodal NK/T‐cell lymphoma with circulating CD56 bright CD16 − cells, reactive adaptive NK cell expansions, and concurrent neoplastic and reactive NK clones. This framework supports standardized, reproducible NK cell assessment and improves specificity of flow‐based clonality inference in routine clinical practice.