High-throughput, multiplex immunofluorescence for the computer-automated immunophenotyping of Mycosis Fungoides

The diagnosis of mycosis fungoides is difficult and often delayed, exacerbated by the limitation of conventional immunohistochemistry to analyze only 1 antigen per tissue section, often necessitating repeat biopsies and extensive workups. We sought to validate a high-throughput multiplex immunofluorescence method, coupled with computer-automated image analysis, to generate comprehensive immunophenotyping data from a single formalin-fixed, paraffin-embedded biopsy. We applied an 11-biomarker multiplex immunofluorescence panel across 18 archived skin specimens (9 mycosis fungoides/TCR clonality positive and 9 control/TCR clonality negative). Initial validation confirmed that multiplex immunofluorescence antigen expression and spatial localization were concordant with those of sequential immunohistochemistry-stained sections. Whole-slide image stacks were analyzed using both computer-assisted and computer-automated pipelines. Both methods successfully delineated immunophenotypic differences. Mycosis fungoides specimens showed a significant expansion of hematopoietic cells, atypical T-lymphocytes, and proliferative T-lymphocytes when compared with controls. Our results validate the potential of multiplex immunofluorescence 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.