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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
DAPI (counterstain)
DAPI (counterstain)
Cy7
Cy7
Spectrum Green
Spectrum Green
Spectrum Orange
Spectrum Orange
  • Plex: the number of targets to be analyzed, excluding counterstain
2-plex Red and Brown IHC with crystal light green counterstain
2-plex Red and Brown IHC with crystal light green counterstain
2-plex Red and Green RNA ISH with hematoxylin counterstain
2-plex Red and Green RNA ISH with hematoxylin 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

Rat Neuron, 6-plex
Rat Neuron, 6-plex
DAPI
DAPI
Spectrum Green
Spectrum Green
Spectrum Orange
Spectrum Orange
Texas Red
Texas Red
CY5
CY5
CY7
CY7
Aqua
Aqua
Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI
(The protocol was carried out on the Leica Biosystems BOND RX fully automated stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system)
Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI (The protocol was carried out on the Leica Biosystems BOND RX fully automated stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system)
  • 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.
Normal Human Tonsil stained with CyclinD1 Red Nuclear Stain CD20 Brown Membranous Stain and Hematoxylin Counterstain
Normal Human Tonsil stained with CyclinD1 Red Nuclear Stain CD20 Brown Membranous Stain and Hematoxylin Counterstain
Cyclin D1 red nuclear stain
Cyclin D1 red nuclear stain
CD20 brown membranous stain
CD20 brown membranous stain
CyclinD1 CD20 hematoxylin
CyclinD1 CD20 hematoxylin

Multiplex use cases

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.

(This is the battle field) Human tonsil, 4-plex assay (IHC to separate out the different immune cells)
(This is the battle field) Human tonsil, 4-plex assay (IHC to separate out the different immune cells)
DAPI (Counterstain)
DAPI (Counterstain)
CY7
CY7
CY5
CY5
Spectrum Gold
Spectrum Gold
Spectrum Green
Spectrum Green

Fluorescence In Situ Hybridization (FISH) assay

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

2 plex FISH assay, Green (CEP17) and orange (HER2 Gene)
2 plex FISH assay, Green (CEP17) and orange (HER2 Gene)

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).

UroVysion Bladder cancer FISH kit (4 different chromosomes have been assayed: Gold (9p21), Aqua(CEP17), Red(CEP3) & Green(CEP7)
UroVysion Bladder cancer FISH kit (4 different chromosomes have been assayed: Gold (9p21), Aqua(CEP17), Red(CEP3) & Green(CEP7)
UroVysion Bladder cancer FISH kit analyzed with Aperio FISH A/D algorithm provides color visualization of the scoring classes of the FISH assay
UroVysion Bladder cancer FISH kit analyzed with Aperio FISH A/D algorithm provides color visualization of the scoring classes of the FISH assay

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

Breast panel original image
Breast panel original image
Breast panel stroma analysis mask - Cell IF algorithm
Breast panel stroma analysis mask - Cell IF algorithm
Breast panel tumor analysis mask - Cell IF algorithm
Breast panel tumor analysis mask - Cell IF algorithm
Breast panel GENIE classifier mask
Breast panel GENIE classifier mask

For Research Use Only. Not for use in diagnostic procedures