Comprehensive leucocyte subsets assay in healthy adults using multiparametric flowcytometry

Background and objectives Comprehensive immune-cell subset characterisation in healthy adults is the prerequisite for enabling accurate diagnosis of primary immunodeficiencies and informed therapeutic decision-making in targeted immunotherapies. Herein, we aim to establish biological reference intervals (BRIs) for leukocyte subsets for interpreting immunophenotypic data and identifying deviations in different pathological settings. Methods This prospective study was conducted at a tertiary care hospital and research centre in northern India over a period of six months using standardised multiparametric flow-cytometric immunophenotyping (FCMI). Peripheral blood from 120 healthy adult donors was analysed by 13-color, 15-parameter flow cytometry (Beckman Coulter Dx Flex). Thirty-seven leucocyte subsets were identified based on expression of surface markers including lymphoid (B, T and their subsets), myeloid (granulocytes, dendritic cells, monocytes and its subsets) and NK cell subsets. Frequencies and absolute counts were compared across gender and age groups by Mann–Whitney U and Kruskal–Wallis tests ( P ≤0.05). Results Males showed higher median total lymphocytes, absolute T-cells, cytotoxic T and memory T-regs, while females exhibited higher % of T-lymphocytes, helper T cells, and early terminally differentiated effector memory re-activating cells (TEMRA ) fractions. B-cell analysis revealed higher mature, transitional, switched-memory, and circulating plasma-cell counts in males. Naïve B cells remained the predominant subset across adulthood, showing biphasic variation with age, while memory B cells peaked in early adulthood, followed by a decline. Interpretation and conclusions When compared with Euro-Flow data, Indian adults displayed overall concordance with global reference ranges, though with broader variability in T and B subsets. These baseline values will refine immune monitoring, improve diagnostic precision in immunodeficiency disorders and guide therapeutic decisions in immune-modifying therapies.