To facilitate precision medicine, we require a detailed understanding of the biological pathways that are affected. The Canberra Clinical Phenomics Service will deliver cellular phenotyping and sophisticated analysis of lymphocytes in patient blood samples. 

 

We aimed to create a flow cytometry panel for the purpose of deep phenotyping human lymphocyte subsets. To expand flow cytometry tests beyond what is already available, we utilised a spectral flow cytometer, which permits more parameters to be investigated at once compared to conventional flow cytometry.

 

Ficoll-separated PBMCs were stained with fluorescent antibodies and analysed on a Cytek Northern Lights 3-laser spectral cytometer.

 

Through careful panel design, panel testing, titration and method optimisation, we generated a 30-colour lymphocyte panel. We can consistently detect populations including T cells (alpha-beta and gamma-delta); CD4+ and CD8+ naïve, TEMRA, central and effector memory; cTfh cells, Tregs and Tfr cells; CD57+ exhausted T cells and B cells; naïve, T1 and T2 transitional; switched and unswitched memory; IgG+ and IgA+ memory; Ig-kappa and Ig-lambda chain expression, Bregs, atypical CD11c+ B cells; activated B cells, plasmablasts and plasma cells.

 

We have developed a panel of 30 markers that can be simultaneously analysed, allowing the characterisation of 62 T cell and B cell subpopulations in a single sample. 

 

Offering such a broad panel maximises both the amount of data generated and the number of research and diagnostic areas to which it can be applied. Our long-term aim is for this panel to be accredited by the TGA and NATA as a diagnostic test.


Ainsley R Davies1, Kristy Kwong1, Ann-Maree Hatch1, Philip Wu1, Minnal Abbasi1, Koula Diamand1, Fei-Ju Li1, Stuart Read1, Harpreet Vohra2, Michael Devoy2, Euan McNaughton1, Katrina Randall1,3
 
 

1.     Canberra Clinical Phenomics Service, John Curtin School of Medical Research, The Australian National University, Acton, ACT, 2000

2.     Flow Cytometry Facility, John Curtin School of Medical Research, The Australian National University, Acton, ACT, 2000

3.     Department of Clinical Immunology, Canberra Health Services, Garran, ACT, 2605