A context-aware interpretive framework for lymphocyte immunophenotyping by flow cytometry.

Background: Flow cytometry-based immunophenotyping is a powerful tool in clinical and translational immunology; however, the interpretation of lymphocyte immunophenotype results remains incomplete, despite extensive methodological standardization. This challenge is particularly evident when phenotypically normal lymphocytes are quantified under non-physiological conditions, where numerical results are often interpreted using implicit and heterogeneous assumptions. Hypothesis: It is hypothesized that harmonization in flow cytometry-based lymphocyte immunophenotyping must extend beyond technical standardization and incorporate explicit, structured frameworks for biological and clinical interpretation for enumeration of phenotypically normal lymphocyte subsets. Implications: Five foundational guidelines are proposed for interpreting lymphocyte immunophenotypic enumeration: the non-equivalence of relative and absolute metrics, context-dependent relevance of immunophenotypic values, compartment-specific determination of meaning, analytical constraints imposed by assay architecture, and the impact of analytical variability on interpretative stability. By articulating principles that are already implicitly applied in expert practice, this framework complements existing technical guidelines and supports a more consistent, context-aware interpretation of lymphocyte immunophenotypes across diverse clinical and biological settings. Conclusion: By explicitly articulating principles that are typically implicit in expert practice, this framework complements existing technical guidelines and bridges the gap between technical and interpretative reproducibility. It provides a coherent structure for context-aware immunophenotypic interpretation and contributes to a shared conceptual language for the study of immunology and its clinical application.