Extended routine lymphocyte immunophenotyping refines individual immune organization across a heterogeneous patient cohort

Abstract

Routine TBNK immunophenotyping detects the major lymphocyte compartments but may underestimate the biologically relevant variation contributed by less frequent but well‐defined minor lymphocyte subsets. This study evaluated whether extending routine TBNK immunophenotyping with minor lymphocyte subsets provides complementary, analytically retrievable information that improves patient‐level immune stratification beyond clinical diagnosis. In this retrospective cross‐sectional study, peripheral blood immunophenotypes from 159 individuals were analyzed using a standardized workflow of eight DuraClone TBNK markers with manual hierarchical selection and absolute counting on a single platform. Multivariate profiling at the patient level was performed using derived immunophenotypic variables rather than single‐cell event data. Canonical and extended panels were compared using nonparametric tests, PERMANOVA, principal component analysis, UMAP, k‐means clustering, inter/intra distance indices, feature importance analysis, and hierarchical heat maps. Canonical immunophenotyping showed the strongest diagnostic associations for the B cell and CD4+ T cell compartments, while the extended panel identified an additional discriminatory structure, with absolute T double negative counts as the most informative minor lymphocyte subsets. Both panels were significantly associated with diagnostic clustering, but the extended panel showed a broader distribution of variance than the diagnostic panel. Compared with the canonical panel, the extended panel increased the inter/intra distance index (0.369 vs. 0.302, p  = 0.0027) and substantially repositioned individuals within the immune space, with 59.1% shifting more than 10 percentiles. The clustering agreement between models was partial (ARI = 0.28; NMI = 0.42), and heat maps revealed continuous immunophenotypic axes with an internal substructure enriched in minor lymphocyte subsets. Extended routine immunophenotyping refines the multivariate representation of peripheral immune organization without requiring additional cytometry markers. Rather than replacing standard TBNK analysis, it complements it by revealing complementary, analytically retrievable information from minor lymphocyte subsets, thereby improving patient‐level stratification and facilitating a more accurate, context‐specific interpretation of immune status.