Technical Note: CODEX® Commercial Antibody Validation Background: This technical note describes the validation process for commercial CODEX® antibodies. The CODEX technology allows tissues to be stained with an entire antibody panel in a single step, thus decreasing experiment time and preserving sample integrity. In this technical note we show how the CODEX antibodies perform similarly to other immunofluorescence (IF) and immunohistochemistry (IHC) grade dye-conjugated antibodies. We also describe the quantitative and qualitative validation process to ensure the right specificity is achieved while optimizing sensitivity. And finally, we also demonstrate an assay for measuring potential steric hindrance from CODEX antibody panels and show that there are minimal effects of this from the demonstrated example.
Poster Title: Highly multiplexed single-cell spatial analysis of FFPE tumor tissues using CODEX® Background: In this poster, 28-plex analysis of human FFPE tissues was performed with CODEX® to understand spatial interactions in the tumor microenvironment. The CODEX® workflow was adjusted to incorporate TSA-mediated dyes for the amplification of low-expressing markers, including FOXP3, PD-L1, and PD-1. Antibodies for these markers were run and imaged with both the standard CODEX® workflow and the new amplification workflow. Signal intensity for the amplified markers was significantly increased when compared to the standard CODEX® run. We plan to integrate these TSA-mediated dyes into the standard CODEX® workflow to enable researchers to study key low-expressing markers.
Poster Title: A novel platform for highly multiplexed, single-cell imaging of cell suspensions Background: This poster, a collaboration between Akoya and researchers at Stanford University, was a preliminary study into the compatibility of the CODEX® system with cell suspensions. An in-depth immune profile of PBMC samples was generated using CODEX®, with PBMCs fixed and spread on a coverslip and stained using 26 different markers. CODEX® performed well, with most markers displaying good signal/noise separation. Staining patterns were qualitatively assessed: mutually exclusive markers showed no overlap in staining, and co-expression was observed in similar cell types. Signal intensity also remained relatively constant over 9 cycles. With validation and optimization of markers for cell suspensions, staining performance could improve further. The results from this initial study indicate that CODEX® can be used as a faster, simpler, and more affordable alternative for cellular biomarker analysis.
Poster Title: Highly multiplexed single-cell spatial analysis of tissue specimens using CODEX® Background: In this poster we showcase examples of profiling FFPE and fresh frozen tumor samples with highly multiplexed CODEX panels that include relevant tumor and immune markers. Tissue types analyzed are renal cell carcinoma (FFPE), breast cancer (FFPE) and metastatic breast cancer lymph node (Fresh Frozen). Using the metastatic breast cancer lymph node tissue as an example, we've also demonstrated how complex the tissue architecture and detailed cellular phenotypes can be uncovered using a highly multiplexed panel of markers.
Poster Title: Spatially-resolved deep biomarker profiling of single cells in FFPE tissue samples through CODEX® Background: In this poster we describe the analysis of FFPE samples using the CODEX platform. Spatially resolved, deep biomarker profiling of tissue specimens is crucial for investigating the architecture and cell diversity in complex matrices, such as the tumor microenvironment (TME). CODEX assays can be performed on a variety of tissue sections, ranging from FFPE to fresh frozen samples. CODEX assays are also non-destructive: tissues can be cleared and stored after one or more CODEX runs. In the poster we show representative images of CODEX multicycles on FFPE human tonsil, melanoma and breast cancer tissues. CODEX antibody staining is also shown in tissue biopsy cores from tissue microarrays (TMAs) as proof of concept.
Poster Title: Multiparametric Proteomic Profiling Via Imaging Dozens of Biomarkers Simultaneously Background: In this poster we show the chemistry behind CODEX technology – single step staining with the multiplexed antibody panel followed by iterative cycles of labeling, imaging and removing Reporters using the fully automated CODEX fluidics unit, until all biomarkers of interest are imaged. We demonstrate the compatibility of the system with different tissue types – Spleen, Lymph Note and Tonsil and FFPE melanoma and the ability of the platform to multiplex 40+ markers in a single panel. And finally, this poster also shows examples of how to analyze highly multiplexed tissue images to uncover complex cellular interactions and phenotypes.