Comparison of Multiplexed Immunofluorescence Imaging to Chromogenic Immunohistochemistry of Skin Biomarkers in Response to Monkeypox Virus Infection.

Over the last 15 years, advances in immunofluorescence-imaging based cycling methods, antibody conjugation methods, and automated image processing have facilitated the development of a high-resolution, multiplexed tissue immunofluorescence (MxIF) method with single cell-level quantitation termed Cell DIVETM. Originally developed for fixed oncology samples, here it was evaluated in highly fixed (up to 30 days), archived monkeypox virus-induced inflammatory skin lesions from a retrospective study in 11 rhesus monkeys to determine whether MxIF was comparable to manual H-scoring of chromogenic stains. Six protein markers related to immune and cellular response (CD68, CD3, Hsp70, Hsp90, ERK1/2, ERK1/2 pT202_pY204) were manually quantified (H-scores) by a pathologist from chromogenic IHC double stains on serial sections and compared to MxIF automated single cell quantification of the same markers that were multiplexed on a single tissue section. Overall, there was directional consistency between the H-score and the MxIF results for all markers except phosphorylated ERK1/2 (ERK1/2 pT202_pY204), which showed a decrease in the lesion compared to the adjacent non-lesioned skin by MxIF vs an increase via H-score. Improvements to automated segmentation using machine learning and adding additional cell markers for cell viability are future options for improvement. This method could be useful in infectious disease research as it conserves tissue, provides marker colocalization data on thousands of cells, allowing further cell level data mining as well as a reduction in user bias.