Design, optimisation and standardisation of a high-dimensional spectral flow cytometry workflow assessing T-cell immunophenotype in patients with melanoma

Jack M. Edwards, Miles C. Andrews, Hayley Burridge, Robin Smith, Carole Owens, Mark Edinger, Katherine Pilkington, Juliette Desfrancois, Mark Shackleton, Sashendra Senthi, Menno C. van Zelm*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

8 Downloads (Pure)



Despite the success of immune checkpoint blockade, most metastatic melanoma patients fail to respond to therapy or experience severe toxicity. Assessment of biomarkers and immunophenotypes before or early into treatment will help to understand favourable responses and improve therapeutic outcomes. 


We present a high-dimensional approach for blood T-cell profiling using three multi-parameter cytometry panels: (1) a TruCount panel for absolute cell counts, (2) a 27-colour spectral panel assessing T-cell markers and (3) a 20-colour spectral panel evaluating intracellular cytokine expression. Pre-treatment blood mononuclear cells from patients and healthy controls were cryopreserved before staining across 11 batches. Batch effects were tracked using a single-donor control and the suitability of normalisation was assessed. The data were analysed using manual gating and high-dimensional strategies. 


Batch-to-batch variation was minimal, as demonstrated by the dimensionality reduction of batch-control samples, and normalisation did not improve manual or high-dimensional analysis. Application of the workflow demonstrated the capacity of the panels and showed that patients had fewer lymphocytes than controls (P = 0.0027), due to lower naive CD4+ (P = 0.015) and CD8+ (P = 0.011) T cells and follicular helper T cells (P = 0.00076). Patients showed trends for higher proportions of Ki67 and IL-2-expressing cells within CD4+ and CD8+ memory subsets, and increased CD57 and EOMES expression within TCRγδ+ T cells.



Our optimised high-parameter spectral cytometry approach provided in-depth profiling of blood T cells and found differences in patient immunophenotype at baseline. The robustness of our workflow, as demonstrated by minimal batch effects, makes this approach highly suitable for the longitudinal evaluation of immunotherapy effects.

Original languageEnglish
Article numbere1466
JournalClinical and Translational Immunology
Issue number9
Publication statusE-pub ahead of print - 7 Sept 2023
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.


Dive into the research topics of 'Design, optimisation and standardisation of a high-dimensional spectral flow cytometry workflow assessing T-cell immunophenotype in patients with melanoma'. Together they form a unique fingerprint.

Cite this