Early experience in design, operation, and validation of a 21-marker panel on the Lunaphore COMET™

Multiplex immunofluorescence platforms capable of staining 40+ markers on a single 4 mm tissue are an incredible tool for interrogating the pathology of a variety of disease processes, in particular immune responses to cancers. While these platforms are capable of providing deep and high-resolution multimarker and spatial data from a single sample, there are specific risks to guard against. These include artifacts related to sensitivity, dynamic range, spectral bleed, and signal carryover from staining cycles. Of course, any significant observation made in multiplex IF must be corroborated using orthogonal measures, but due to the expense and effort required to perform the experiments, the potential impact of the results, and the precious nature of the samples, a properly validated assay is required. There is currently no standard validation approach for multiplex IF. Here we review our first experience with a beta unit of the Lunaphore COMET, inclusive of operation, performance, panel development, as well as a prototype validation:

• COMET technology walkthrough + mIF landscape;
• What a multiplex validation should cover;
• Comet validation plan;
• What worked & lessons learned.