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
.
For Research Use Only. Not for use in diagnostic procedures.
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.

What is the difference between : number of colors, plex and multiplexing
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 2 or more markers on a single slide (eg CD3, CD4, CD8 & counterstain). The nuclear counterstains most frequently used are hematoxylin for brightfield and DAPI for fluorescent microscopy

Fluorescence Multiplexing









Chromogenic Multiplexing (multiplex IHC and ISH)
Chromogenic multiplexing provides the ability to look at 3 or more markers on the same slide using brightfield microscopy. Chromogens provide a more stable and permanent result compared to their fluorescent counterparts.

Normal tonsil tissue stained with:
Marker 1 = PD-L1 = LBS Red
Marker 2 = CD68 = LBS DAB
Marker 3 = CD8 = LBS Blue
Marker 4 = Pan-CK – LBS Green
Counterstain = Hematoxylin





Tissue – Skin- Melanoma stained with:
Marker 1 - PD-L1 - LBS Red
Marker 2 - CD68 - LBS DAB
Marker 3 - CD8 - LBS Blue
Marker 4 - Pan-CK - LBS Green


In human bladder stained with:
Marker 1 - CK20 - Red
Marker 2 - p53 - Brown
Marker 3 - CD44 - Green
Counterstain = Hematoxylin

Adenocarcinoma in human colon stained with:
Marker 1 - CDX2 - LBS Red
Marker 2 - CD3 - LBS Green
Counterstain = Hematoxylin
For Research Use Only. Not for use in diagnostic procedures.
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. Multiplex 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 hematologic samples, staining often determines cell lineage/origin as well. This phased process of optimization is long but ensures fidelity of results.







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
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 uncover this multi-faceted approach to tissue heterogeneity and provides context.
Example of breast cancer image using Aperio IA SW
Characterizing biomarkers within the tumor or the stroma" shall be placed below the images on the right side.



