Multiplex Immunofluorescence (mIF) and Multiplexed IHC Consecutive Staining on a Single Slide (MICSSS)

Multiplex Immunofluorescence Biomarker Imaging

Multiplex immunofluorescence (mIF) biomarker imaging allows for multi-label analysis of formalin-fixed paraffin embedded (FFPE) tissue biopsy samples, enabling scientists to study complex biological questions. This assay allows scientists to precisely measure and quantify multiple fluorescent molecular markers simultaneously, even when co-localized within a single tissue section. Among the advantages of this quantitative pathology imaging system are the preservation of the morphological context down to the subcellular level, elimination of auto-fluorescence, capture of low signal levels, and excellent overall quantification of protein expression.

Each mIF biomarker panel includes six to seven antibodies used to stain a single slide. Currently, the CIMAC network offers four optimized mIF panels, with six additional panels in development:

Panel 1: PD-L1, CD68, PD-1, CD8, CD3, pan-cytokeratin, DAPI.

Panel 2: FOXP3, Granzyme B, CD45RO, CD8, CD3, pan-cytokeratin, DAPI.

Panel 3: pan-cytokeratin, CD8, PD-1, PD-L1, DAPI.

Panel 4: SOX10, CD8, PD-1, PD-L1, DAPI.

For each mIF panel, a report on marker co-expression has been prepared and will be made available to investigators on request.

Each antibody is labeled with a specific fluorophore, which is then covalently transferred to the target biomarker using a tyramide signal amplification kit (Opal™, PerkinElmer).  Images are scanned and captured with a multispectral microscope (Vectra™/Polaris™, PerkinElmer), and analyzed with a specialized image analysis software (InForm™, PerkinElmer).

The scoring of PD-L1 and similar markers is expressed as the percent positive cells within the cell population of interest: for example, PD-L1 positive macrophages (CD68+/PDL1+) and PD-L1 positive tumor cells (SOX10+/PD-L1+).  Immune cell subpopulations are defined by co-expression of specific markers, e.g., T-cytotoxic (CD3+/CD8+) and exhausted T-cytotoxic cells (CD8+/PD-1+), and are evaluated as cell density per tumor tissue (number of positive cells per square millimeter of tumor area). The staining procedure is performed by specialized technicians under pathologist supervision, and the scanning and analysis is done by senior scientists and pathologists trained in the use of Vectra and InForm.  Finally, the data is consolidated using Spotfire software (PerkinElmer) for the final report.

Multiplexed IHC Consecutive Staining on a Single Slide

Multiplexed IHC Consecutive Staining on a Single Slide (MICSSS) is an alternative method developed by Mount Sinai CIMAC to address the clinical need for high-dimensional tissue analysis for in situ characterization of immune infiltrates. MICSSS is performed on FFPE tissue using iterative cycles of chromogenic IHC staining, revelation, scanning, and bleaching of labile chromogenic substrate. Image analysis is then performed using QuPath software to segment cells and quantify each marker as a percentage of cells stained or as a density per square millimeter. This method overcomes a major limitation of high-dimensional analyses, namely tissue availability, by allowing for up to 10 markers to be analyzed from a single tissue slide. MICSSS is also independent of proprietary reagents and equipment, so it can be readily integrated into routine clinical pathology.

The MICSSS method can characterize many parameters on a single tissue section, including co-localization of markers on single cells, while preserving tissue antigenicity and architecture. This allows us to build a comprehensive spatiotemporal map of the local tissue microenvironment, including, for example, immunocytes, tumor associated markers, and drivers of immunosuppression. The goal is to offer a comprehensive phenotypic characterization, organization and functionality assessment of samples in relation to clinical parameters and treatment outcome. Currently available markers include CD2, CD3, CD8, CD20, CD66b, CD68, DC-LAMP, Foxp3, PD-L1, PD-1, HLA-DR, CD163, CD206, Langerin, CD1a, and Ki-67.

mIF assays are available at Dana-Farber and MD Anderson sites; MICSSS is available at Mount Sinai.