Automated Multi-plex Immunofluorescence with TSA for CD4, CD8, FOXP3, CD21, PD1 and CD68 in Follicular Lymphoma v1

Observing immune cell infiltration and spatial organisaion in the tumour microenvironment of follicular lymphoma is important for the development of novel biomarkers for prognosis and treatment selection. However the identification of multiple immune cell sub-populations is limited in routine single-plex immunohistochemical staining. Our goal was to develop and validate an automated protocol for multiplex immunofluoresence using tyramide signal amplification, the Opal™ 7 color Kit (Akoya Biosciences) and the Ultra Discovery (Roche) autostainer, which would enable concurrent visualisation on the same tissue sections of: T follicular helper cells (CD4+) cytotoxic T cells (CD8+) Tregs (CD4+FOXP3+) Macrophages (CD68+) PD1+ lymphocytes DAPI nuclear counterstain We aimed in addition to observe the spatial arrangement of these cells in reference to the follicle areas, therefore the CD21 marker, which is expressed in mature B cells and follicular dendtritic cells located in the follicles was added in the panel. We validated quantitatively the agreement between single-plex and multiplex assays by comparing stained area in sequential sections of a follicular lymphoma FFPE tissue micro-array (TMA). The TMA was constructed from a follicular lymphoma cohort of 44 patients collected retrospectively from the archives of the Christie NHS Foundation Trust (Manchester), with ethical permission granted by the Central Manchester Multi-centre Research Ethical Committee (03/08/016). The protocol was automated with the DISCOVERY ULTRA IHC/ISH Roche autostainer and stained areas were quantified using the Indica Labs’ Area Quantification Fluorescence module (HALO software). Statistically significant linear correlations were seen between single-plex and multiplex assays (R² >0.72, p<7.1× 10-14, see attached) for all markers.Therefore, this multiplex immunofluorescence (mIF) protocol can be used to conserve tissue and observe immune cell spatial organisation in follicular lymphoma.