Abstract 3965: Sequential multiplex immunofluorescence technology (SMIFT): A new staining strategy for immunotherapy
Authors: Vargas Joseph , Tacha David , Masabanda Julio , Schollmeier Sarah , Douglas Cristin , Yang George 
Published: August 2018

Abstract
Introduction: Tumor tissues exhibit a multitude of different cell types that have extravasated from other regions in the body. These cells are likely to represent heterogeneous populations of immune and effector cells. Assessing immunotherapy markers in tissues could be of significant value for patient care. We therefore have developed an innovative method that will enable the application of three or more sequential mouse monoclonal antibody (MMA) applications that can detect these immune cells on a single tissue. Previous methods using a sequential staining technique of MMA required a manual application and time-consuming heating and stripping between steps. The assessment of multiple targets can now be determined in approximately eight hours using full automation without heating or stripping procedures. This method may be valuable when the quantification of specific immunotherapy markers is required.
Materials/Methods: Two assays were developed using Sequential Multiplex Immunofluorescence Technology (SMIFT): FOXP3, CD163, PD-1, CD8 as well as p40, Desmoglein-3, TTF-1, CD8. Tissues were fixed in 10% NBF for 24 hours and processed into paraffin. Tissue sections were cut at 5 microns and adhered to glass slides. Sections were then heated in a citrate-based buffer at pH 6.0 within a pressure cooker at 95°C for 40 minutes. All antibodies were diluted in diluent and applied to tissues to determine optimal IF (and IHC) titers. Detection of each antibody was accomplished with commercially available anti-mouse/anti-rabbit HRP polymer followed by fluorophores 594, 488, 546 and 480 for IF (or by DAB for IHC comparison). A blocking reagent was incorporated into the protocol to prevent cross-reactivity of multiple detection fluorophores. Both assays were comprised of two sequential MMA followed by a third application of a mouse/rabbit monoclonal antibody cocktail to complete the procedure. Sections were then counterstained in DAPI (or hematoxylin).
Results: Multiplexed staining intensities were compared to individual antibody formats for both assays. Staining of each target was clearly identified even when multiplexed. Fluorescence intensities were scored on a subjective scale from 1-3+ with 3 representing the highest intensity. Both assays exhibited 3-3+ staining intensities for each target fluorophore as well as in multiplexed formats. Individual IHC staining results were also comparable to individual and multiplexed IF targets with identical staining patterns.
Conclusion: Multiplexing can be achieved using SMIFT, a sequential and multiplex approach for assays tested on a single tissue section. Three sequential applications of four antibodies can be performed in less than eight hours with full automation. This is in contrast to previous overnight methods. Most importantly, this approach could be used to assess immunotherapy markers in tissues and may provide prognostic or predictive value.
Citation Format: Joseph Vargas, David Tacha, Julio Masabanda, Sarah Schollmeier, Cristin Douglas, George Yang. Sequential multiplex immunofluorescence technology (SMIFT): A new staining strategy for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3965.