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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.

View whole slide images

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

KEY CONSIDERATIONS

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.

 

Choosing the right tool to help answer your research question

THE FUNDAMENTALS

What is the difference between : number of colors, plex and multiplexing

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 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

Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI
(The protocol was carried out on the BOND RX stainer from Leica Biosystems fully automated stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system)

Fluorescence Multiplexing

Rat Neuron, 6-plex
DAPI
Spectrum Green
Spectrum Orange
Texas Red
CY5
CY7
Aqua
Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI
(The protocol was carried out on the BOND RX fully automated research stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system)

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

Normal human tonsil stained with CyclinD1 red nuclear stain, CD20 brown membranous stain, and hematoxylin counterstain
Cyclin D1 red nuclear stain
CD20 brown membranous stain
Hematoxylin Counterstain

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

CHROMOGENIC MULTIPLEXING

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.

(This is the battle field) Human tonsil, 4-plex assay
DAPI (Counterstain)
Spectrum Gold
CY7
CY5
Spectrum Green
2-plex FISH assay: green (CEP17) and orange (HER2 Gene)

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).

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

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.

 

MULTIPLEX USE CASES

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