An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures.

Introduction: Peripheral blood leukocyte immunophenotyping offers a minimally invasive insight into an individual's immune system and is an attractive biomarker for detecting and monitoring disease. Advances in high-parameter spectral flow cytometry now resolve leukocyte subsets in unprecedented detail, but this depth adds complexity to sample preparation, acquisition and analysis, introducing technical variability that can obscure the biological signatures required for longitudinal monitoring. Methods: We built and validated an optimized, high-parameter spectral flow cytometry pipeline for repeated blood leukocyte profiling and asked whether the resulting signatures are stable and distinct enough to identify individuals over time. Results: Using rainbow beads and monoclonal-antibody spectra, we confirmed that instrument performance and spectral unmixing remained stable across more than two years, and that this dual measure localized faults to either the instrument or a reagent. Against a stabilized biological-invariant control, gating built on the dimensionality-reduction method PaCMAP was substantially more reproducible between operators than traditional two-dimensional gating and produced cleaner populations. Chemical fixation and one-week antibody pre-storage preserved leukocyte composition, whereas delayed processing and, more severely, cryopreservation altered it, the latter through near-complete granulocyte loss. Of the count-normalization schemes tested, normalization to the percentage of CD45 Conclusion: Together these results define a technically sound, operator-reproducible and handling-robust pipeline whose normalized signatures capture a temporally stable individual immune fingerprint, providing a practical foundation for blood leukocyte profiling as a longitudinal biomarker of immune state in cancer and other diseases.