Multiplex IHC

Multiplex IHC allow the flexibility to simultaneously quantify up to five chromogenic stains localised to any subcellular compartment (nucleus, cytoplasm or membrane). This approach is particularly well suited for applications in immuno-oncology whereby multiple chromogenic markers are required to identify distinct populations of immune and tumor cells within the tissue.

Analysis Approach

We develop a customised algorithm tailored to the specific markers in your study. This involves a color deconvolution process within the Indica Labs HALO platform to separate the different chromogens plus counterstain. Cell objects are formed by applying weighted optical density values for the individual chromogens. Each positive cell type is then identified using defined size, shape and subcellular compartment staining parameters. A classifier is developed and integrated into the algorithm to automatically segment the tissue region of interest (ROI) for analysis. The completed algorithm is applied to all tissue section images in the study in an automated and objective manner to generate detailed cell-by-cell data. Example analysis and data is shown below.

A. Multiplex stained IHC whole tissue section (left) and classified regions of interest (ROI) overlay (tumor – orange; necrosis – dark blue; stroma – light blue; white space – white). B. Multiplex stained IHC tissue section (left) and detection of PanCK (yellow), PD-L1 (brown) and CD8 (purple) positive cells within the viable tumor ROI (right). Histology performed by Asterand Bioscience.

Quantitative Readouts

  • Area of specific tissue regions of interest (ROI) including Tumor, Stroma & Necrotic content across whole section
  • Quantification of cells positive for multiple chromogenic markers across a whole tissue section or within a defined ROI
  • Quantification of cells in which multiple chromogenic markers colocalise

Example Multiplex IHC Data


  • Receive highly detailed data quantifying multiple single markers or colocalised markers on a cell-by-cell basis across a whole tissue section or within a defined ROI.
  • Our automated batch analysis service objectively analyses each section using the same algorithm to reduce variability and improve data quality and interpretation.
  • Spatial relationships of the different cell populations identified may be further investigated using our Spatial Analysis services.