High-Throughput, Multiplex Immunofluorescence for the Computer-automated Immunophenotyping of Mycosis Fungoides

The diagnosis of Mycosis Fungoides (MF) is difficult and often delayed, exacerbated by the constraint of conventional immunohistochemistry (IHC) to analyze only one antigen per tissue section, often necessitating repeat biopsies and extensive workups. We sought to validate a high-throughput Multiplex Immunofluorescence (MIF) method, coupled with computer-automated image analysis, to generate comprehensive immunophenotyping data from a single formalin-fixed, paraffin-embedded (FFPE) biopsy. We applied an 11-biomarker MIF panel across 18 archived skin specimens (9 MF/TCR clonality positive and 9 control/TCR clonality negative). Initial validation confirmed that MIF antigen expression and spatial localization were concordant with sequential IHC-stained sections. Whole slide image stacks were analyzed using both computer-assisted and fully computer-automated pipelines. Both methods successfully delineated immunophenotypic differences. MF specimens showed a significant expansion of hematopoietic cells and proliferative T-lymphocytes compared to controls. Crucially, MF tissues also exhibited a significant increase in the percentage of atypical T-lymphocytes. Our results validate the potential of MIF to obtain comprehensive, high-dimensional diagnostic information from a single tissue section. Integration with computer-automated analysis offers a scalable, high-throughput platform that can significantly aid in the timely and accurate diagnosis of cutaneous lymphomas.