CODEX™: A Comprehensive Solution for Highly Multiplex Tissue Imaging
Characterizing the complexities of the tissue microenvironment is a critical element to understanding the molecular and cellular mechanisms driving the disease as well as therapeutic response. The role of spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity.
Akoya has developed the CODEX Technology to provide a comprehensive solution for spatially-resolved, highly multiplexed biomarker analysis.
Akoya’s CODEX Technology
The CODEX™ (CO-Detection by indEXing) technology uses an antibody conjugated to proprietary Akoya Barcodes as a unique label. The CODEX Assay targets specific barcodes with a dye-labeled Reporter for highly specific detection. Akoya’s CODEX Barcode technology enables an almost unlimited level of flexibility in the generation of highly multiplexed panels while maintaining the tissue integrity ensuring high quality data.
Antibody Panel Cocktails for Single-Staining Step
Individual Barcoded-Antibodies are combined into a cocktail, enabling a single staining step of the full tissue. Akoya provides inventoried, pre-validated antibodies targeting a broad range of tissues, immune cells, critical oncology biomarkers and stromal cell proteins. Alternative, user-validated antibodies can be incorporated into panel designs using Akoya’s proprietary conjugation method. Akoya-validated antibodies are designed for use with human fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE), as well as mouse fresh frozen samples.
The CODEX assay overcomes challenges of traditional multiplexed immune-fluorescence, by targeting subsets of the antibody panel through iterative imaging cycles. For each imaging cycle, three CODEX Reporters, each with a spectrally-distinct dye, are applied to the stained tissue to assay the corresponding Antibody Barcode. The tissue is imaged, revealing the fluorescence signal levels of the first three targets plus a nuclear stain. The CODEX instrument applies gentle wash steps to remove the Reporters without any chemical modifications, followed by the applying the next set of Reporters. In this manner, more than 50 parameters can be measured in a single tissue specimen.
Cycle 1: CD8 – CD19 – CD90
Cycle 2: CD21 – CD3 – CD54
Cycle 3: CD49f – CD63 – Ki67
Cycle 4: CD38 – CD5 – CD279
Human tonsil germinal centers imaged in four cycles with three markers per cycle.
Akoya’s CODEX System is a fluidics control instrument that interfaces with existing microscope hardware to facilitate the cyclic Reveal, Image, Wash steps. The CODEX Stage Insert is mounted within standard microscope stages for easy integration into standard inverted microscopes. The CODEX Driver software controls the coordination of cyclic imaging and the wash steps in a completely automated manner, converting a standard fluorescent microscope into a highly multiplexed imaging system.
Multiplex Image Analysis
The fully automated CODEX Analysis Pipeline processes the multi-channel images from each cycle and computationally combines them to provide signal quantification data for each marker. Akoya’s segmentation algorithms use the nuclear stain and protein marker data to identify individual cells and define the cellular boundaries to create a matrix table containing a cell identifier with the corresponding spatial location and signal values for each of the panels biomarkers. Segmentation data can be used for statistical methods for cell phenotyping, population comparisons and spatial analysis including defining cellular niches and neighborhood analysis.
Cell segmentation identifies individual cells to provide spatial and protein expression quantification data for further analysis.