Modern Multiplex Solutions for the Research Lab

Multiplexing addresses the need for researchers to assess multiple biomarkers (protein and/or nucleic acid markers) at specific locations within a tissue sample. The information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity are now understood to be critical to developing effective therapeutic strategies.

The latest technology encompasses multiplex IHC as well as multiplex ISH and FISH.

Key considerations for choosing to Multiplex:

• The need to extract the maximum amount of data from a limited sample, multiplex technology enables the user to detect many biomarkers in a single tissue section.
• Multiplexing can help determine which targets are important, by starting with a large range of potential markers and using the resulting spatial data to refine to the critical few.
• Multiplex staining on tissue allows for cell-specific context that molecular techniques and PCR can’t provide.
• Tissue multiplexing allows the visualization of both protein and nucleic acid targets in the same tissue section.

The fundamentals

What is the difference between:

• Number of colors: every single stain on the slide including counterstains

• Plex: the number of targets to be analyzed, excluding counterstain

• Multiplexing: the ability to simultaneously detect three or more markers on a single slide (eg CD3, CD4, CD8 & counterstain). The nuclear counterstains most frequently used: Hematoxylin for brightfield and DAPI for fluorescence.
  • Fluorescence Multiplexing: fluorescence IHC and FISH

• Chromogenic Multiplexing (multiplex IHC and ISH) provides the ability to look at three or more markers on the same slide using brightfield microscopes. Many chromogens provide a permanent stain.

Immuno-Oncology

Get the full picture for your tumor microenvironment research

Immuno-oncology has been one of the primary drivers of the current multiplex technology development.

Multiplexed immunohistochemical (IHC) analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples allows researchers to study the spatial relationships between different cell phenotypes in situ.

Tonsil is often the first step to check that the antibodies are identifying the correct immune cells. After that the actual tumor microenvironment can be employed with confidence that the staining of immune cells is real. This phased process of optimization is long but ensures fidelity of results.

Nucleic Acids in Cancer research

FISH is a sensitive, accurate, and reliable technique widely applied in cancer research. The genetic defects uncovered by FISH represent early genetic triggers or events responsible for cancer at stem-cell level.

FISH provides cell-based context for specific genomic aberrations and plays an important role in detecting specific biomarkers in solid and hematologic neoplasms

Quantitative Multicolor QM-FISH

Pairs of probes have been conventionally used to detect a single genetic event like deletion or amplification of a locus or chromosomal translocation. However, with the discovery of multigenic diseases including cancer, simultaneous detection of such genes by using multiple probes on a single slide aids understanding of disease progression (quantitative multicolor FISH).

Tissue heterogeneity

It is easy to think of heterogeneity between tumor and non-tumor, but it is also well known that there is heterogeneity within the tumor itself. Not every cell will stain the same and not every marker will be present in the same cells. Multiplex IHC and ISH uncovers heterogeneity and gives it context.

Example of breast cancer image using Aperio IA SW

Characterizing biomarkers within the tumor or the stroma