An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures
- 1Irradiation Immunity Interaction Lab, Division of Genome Sciences and Cancer, John Curtin School of Medical Research, Australian National University
- 2Radiation Oncology Department, Canberra Hospital, Canberra Health Services
Abstract
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 ⁺ leukocytes best preserved individual signatures over time, whereas counting-bead normalization performed worst. Applying the pipeline to ten donors sampled repeatedly across a year, we found that leukocyte signatures were dominated by stable, individually distinct differences between people — so distinct that a held-out visit could be assigned back to its donor, both by a trained random forest and by simple proximity to that donor’s own mean.
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.
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BibTeX
@article{gosling2026optimized,
title = {{An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures}},
author = {Katharine Gosling and Jason Price and David A. Simon Davis and Farhan M. Syed and Benjamin J. C. Quah},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1959895},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1959895/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures
AU - Katharine Gosling
AU - Jason Price
AU - David A. Simon Davis
AU - Farhan M. Syed
AU - Benjamin J. C. Quah
PY - 2026
DA - 2026/10/05
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1959895
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1959895/full
AB - 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 ⁺ leukocytes best preserved individual signatures over time, whereas counting-bead normalization performed worst. Applying the pipeline to ten donors sampled repeatedly across a year, we found that leukocyte signatures were dominated by stable, individually distinct differences between people — so distinct that a held-out visit could be assigned back to its donor, both by a trained random forest and by simple proximity to that donor’s own mean. 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.
SN - 1664-3224
ER -