Multiplexing Immunohistochemistry - A new method for integrating biomarker expression

Visualising multiple antigens in a single formalin-fixed paraffin embedded tissue section allows for a greater understanding of the complexities of protein expression in situ and their temporal and spatial relationships. The need to co-evaluate potential clinical biomarkers and/or immuno-oncology markers has placed growing demands on very small, and increasingly precious, biopsy samples.

Dual or triple labelling has been in use for many years but has its limitations:

Risk of species cross-reactivity. If the best primary antibodies for the targets of interest are raised in the same species (many of the best antibodies are either mouse or rabbit), cross-reactivity arising from detection with secondary regents is somewhat difficult to avoid and the compromise may be a second-choice primary antibody raised in a different species for at least one of the antigens.

The alternative of directly conjugated primary antibodies (antibodies carrying a covalently bonded detection molecule, such as a fluorophore) do not allow signal amplification and can have low sensitivity.

A new wave of multiplexing immunohistochemistry methodology has been developed that circumvents these issues by covalently bonding fluorophores to the site of the antigen. Fluorescently-conjugated tyramide catalysed by horse-radish peroxidase of the secondary antibody, binds to tyrosine residues near to the epitope. The primary and secondary antibodies used to detect the antigen are then no longer needed and are stripped off by heat, preventing cross-reactivity with subsequent antibodies. In this way sequential labelling of several antigens irrespective of the primary antibody species, can be achieved using different fluorophores.

This technology, like older methods, may still be limited by tissue autofluorescence. Other issues may be that there is a loss of antigen signal or tissue integrity following multiple heat cycles. Defining the most robust sequence of antigen labelling will therefore be needed. Image analysis may also be challenging due to overlapping fluorophores and blending where there is co-expression. However where successful, multiplexed biomarkers and diagnostics can provide a more accurate indication of the response of a tissue to a drug, or of disease stage, allowing further progress towards personalised medicines.

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