# Advanced immunophenotyping of lymphocyte and monocyte subsets in healthy Australian adults using a novel spectral flow cytometry panel

**Authors:** Ainsley R. Davies, Kristy Kwong, Zhijia Yu, Koula E. M. Diamand, Fei-Ju Li, Laurensia Kannitha, Sidra A. Ali, Abolfazl Amjadipour, Ann-Maree Padarin, Michael Devoy, Harpreet Vohra, Bahar Miraghazadeh, Simon H. Jiang, Anne Brüstle, Nicolas Cherbuin, Christopher J. Nolan, Matthew C. Cook, Elizabeth E. Gardiner, Stuart Read, Euan McNaughton, Katrina L. Randall

PMC · DOI: 10.3389/fimmu.2025.1577206 · 2025-07-22

## TL;DR

This paper introduces a new 30-color flow cytometry panel to measure over 50 lymphocyte and monocyte populations in healthy adults, aiming to improve disease diagnosis.

## Contribution

A novel 30-color spectral flow cytometry panel for comprehensive immunophenotyping of lymphocyte and monocyte subsets.

## Key findings

- The panel measures over 50 distinct lymphocyte and monocyte populations from a single sample.
- Data was collected from 148 healthy Australian adults to establish baseline immunophenotyping profiles.

## Abstract

Lymphocytes play pivotal roles in disease pathogenesis and can be used as potential biomarkers for various immunological conditions. Yet, current flow cytometry methods used in clinical settings are often only capable of measuring between four to eight distinct lymphocyte populations. The purpose of our study was to measure many lymphocyte and monocyte populations from a single sample, with the long-term aim of validating our assay for diagnostic use in the Australian regulatory environment.

We designed and optimised a novel 30-colour lymphocyte immunophenotyping panel tailored for use on a 3-laser (V-B-R) spectral flow cytometer. This panel measures over 50 lymphocyte and monocyte populations.

In this report we present data derived from 148 healthy individuals.

This lays the groundwork for future clinical application of spectral flow cytometry tests and offers a more comprehensive approach to lymphocyte and monocyte analysis with future implications for disease diagnosis and monitoring.

## Full-text entities

- **Genes:** CD8A (CD8 subunit alpha) [NCBI Gene 925] {aka CD8, CD8alpha, IMD116, Leu2, p32}, KRT20 (keratin 20) [NCBI Gene 54474] {aka CD20, CK-20, CK20, K20, KRT21}, CD27 (CD27 molecule) [NCBI Gene 100520023], CR2 (complement C3d receptor 2) [NCBI Gene 1380] {aka C3DR, CD21, CR, CVID7, SLEB9}, LOC102723407 (immunoglobulin heavy variable 4-38-2-like) [NCBI Gene 102723407] {aka IGHV4, IGHV4-30, IGHV4-38-2, IGHV4-39, IGHV4-b, IGVH4-39}, CD3E (CD3 epsilon subunit of T-cell receptor complex) [NCBI Gene 397455] {aka CD3}, CXCR3 (C-X-C motif chemokine receptor 3) [NCBI Gene 100524123], CD38 (CD38 molecule) [NCBI Gene 952] {aka ADPRC 1, ADPRC1, cADPR1}, CD19 (CD19 molecule) [NCBI Gene 930] {aka B4, CVID3}, IL7R (interleukin 7 receptor) [NCBI Gene 3575] {aka CD127, CDW127, IL-7R-alpha, IL-7Ralpha, IL7RA, IL7Ralpha}, CCR7 (C-C motif chemokine receptor 7) [NCBI Gene 396663], CCR6 (C-C motif chemokine receptor 6) [NCBI Gene 1235] {aka BN-1, C-C CKR-6, CC-CKR-6, CCR-6, CD196, CKR-L3}, PTPRC (protein tyrosine phosphatase receptor type C) [NCBI Gene 5788] {aka B220, CD45, CD45R, GP180, IMD105, L-CA}, Ighd (immunoglobulin heavy constant delta) [NCBI Gene 380797] {aka IgD, Igh-5}, NCAM1 (neural cell adhesion molecule 1) [NCBI Gene 4684] {aka CD56, MSK39, NCAM}, CD14 [NCBI Gene 100620530], IL2RA (interleukin 2 receptor subunit alpha) [NCBI Gene 3559] {aka CD25, IDDM10, IL2R, IMD41, TCGFR, p55}, FOXP3 (forkhead box P3) [NCBI Gene 50943] {aka AIID, DIETER, IPEX, JM2, PIDX, XPID}, IGG (Immunoglobulin G level) [NCBI Gene 102658792], CCR6 [NCBI Gene 100037929], CD24 (CD24 molecule) [NCBI Gene 100133941] {aka CD24A}, ITGAX (integrin subunit alpha X) [NCBI Gene 3687] {aka CD11C, SLEB6}, APC (APC regulator of Wnt signaling pathway) [NCBI Gene 324] {aka BTPS2, DESMD, DP2, DP2.5, DP3, GS}, CD19 (CD19 molecule) [NCBI Gene 397669], CD4 (CD4 molecule) [NCBI Gene 920] {aka CD4mut, IMD79, Leu-3, OKT4D, T4}, IGHA (immunoglobulin alpha heavy chain constant region) [NCBI Gene 100568455] {aka IGA}, CD38 (CD38 molecule) [NCBI Gene 100511702], CXCR5 (C-X-C motif chemokine receptor 5) [NCBI Gene 100515679] {aka CD185}, Ccr7 (C-C motif chemokine receptor 7) [NCBI Gene 12775] {aka CC-CKR-7, CCR-7, CD197, Cdw197, Cmkbr7, EBI1}, CD4 (CD4 molecule) [NCBI Gene 404704], APP (amyloid beta precursor protein) [NCBI Gene 351] {aka AAA, ABETA, ABPP, AD1, APPI, CTFgamma}, IL7R (interleukin 7 receptor) [NCBI Gene 100271930] {aka CD127}, CD27 (CD27 molecule) [NCBI Gene 939] {aka S152, S152. LPFS2, T14, TNFRSF7, Tp55}, IL2RA (interleukin 2 receptor subunit alpha) [NCBI Gene 396814] {aka CD25, IL-2RA}
- **Diseases:** dermatomyositis (MESH:D003882), RA (MESH:D001172), autoimmune (MESH:D001327), rheumatic disorders (MESH:D012216), inclusion body myositis (MESH:D018979), MS (MESH:D009103), inborn errors of immunity (MESH:D007154), SS (MESH:D012859), anti-synthetase syndrome (MESH:D020159), acquired immune deficiency disease (MESH:D000163), systemic sclerosis (MESH:D012595), infectious illness (MESH:D003141), UMAP (MESH:C567162), inflammatory conditions (MESH:D007249), blood malignancies (MESH:D009369), SLE (MESH:D008180)
- **Chemicals:** ACD (MESH:C002113), sodium azide (MESH:D019810), Cy5.5 (MESH:C098793), paraformaldehyde (MESH:C003043), trypan blue (MESH:D014343), polymer (MESH:D011108), BD (MESH:C028491), Ficoll (MESH:D005362), dimethyl sulfoxide (MESH:D004121), AF660 (-)
- **Species:** Human immunodeficiency virus 1 (no rank) [taxon 11676], Homo sapiens (human, species) [taxon 9606], Mus musculus (house mouse, species) [taxon 10090]
- **Mutations:** S17015F

## Figures

6 figures with captions in the complete paper: https://tomesphere.com/paper/PMC12322900/full.md

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Source: https://tomesphere.com/paper/PMC12322900