Interview with a scientist: multiplex IHC - past, present and future challenges

Multiplex immunohistochemistry is a powerful investigative tool that allows for the simultaneous visualization of multiple proteins on a single section by using a specific antibody for each protein of interest. Read our interview with Dr. Kristian Moller about the past, present, and future of multiplex immunohistochemistry.

Dr. Kristian Moller is a Principal Scientist at Atlas Antibodies. He holds a Ph.D. in Molecular Neurobiology from the Medical Faculty at Lund University, Sweden. Dr. Moller has a profound national and international R&D experience as a specialist in applied molecular histology from the private pharmaceutical, diagnostic, and immunotherapy sectors.

His work has emphasized research devoted to tumor diagnostic antibodies, T-cell mediated cancer immunotherapies and early drug discovery within dermatology. In addition to his general expertise in tissue biomarkers, he is strongly specialized in the technical aspects of immunohistochemistry and RNA in situ hybridization.


Photo: Dr. Kristian Moller, Principal Scientist at Atlas Antibodies.

Q1: Kristian, could you explain multiplex IHC in simple words?

To summarize, multiplexing allows for the simultaneous interrogation of two or more staining parameters within the same tissue section using similar or different affinity reagents consisting of different detection labels. Most often these are proteins but can also be coding and non-coding transcripts (mRNAs and non-coding RNAs). Until quite recently, multiplexing rested upon chromogenic or fluorochrome-labeled antibodies or probes which limited the number of targets that could be interrogated simultaneously due to spectral overlap.


Q2: What does it deliver in terms of biological insights?

In simple words, a multifunctional level of information within a tissue context. Multiplexed immunohistochemistry allows for the simultaneous spatial interrogation and identification of several proteins in one tissue section at sub-micrometer resolution.

This method is for example highly advantageous for investigating immune evasion mechanisms, for phenotyping the plethora of cell types in a particular brain region. Multiplexing is also an indispensable tool in the discovery of potential biomarkers for the prediction of any response to a given treatment. 


Q3: What is the translational application?

This translates amenably to applications within diagnostic pathology where it is required to elucidate whether, or not a patient will respond to a particular therapy Other highly relevant applications include drug discovery and development.


Q4: Share a turning point or defining moment in multiplex IHC history.

There are several milestones, one being the development of highly validated and target-specific antibodies raised in different host species enabling multiplexing.  Another milestone is the development of sensitive, stable, and spectrally diverse fluorochromes and chromogens.

The issue with spectral overlap between fluorochromes can now be overcome using in situ imaging mass spectrometry and the use of metal-labeled antibodies and third-party digital image reconstruction. Another relates to DNA-barcoding of antibodies and iterated staining cycling allowing for truly high-dimensional multiplexing.

I would also like to stress that nothing of this could be realized without the invention of intelligent, and user-friendly hard- and software solutions for molecular imaging of multispectral images.


Q5: How prevalent is the use of multiplex IHC today compared to similar techniques?

Within basic research, I would state that multiplexed IHC is frequent. This is mostly true for immunofluorescent staining, but less so for chromogenic IHC assays which are prevalent in diagnostic pathology. Hence for tumor diagnostics, single-staining still dominates for practical and throughput reasons.

This is about to change with the advent of novel advanced particular therapies which require more complex interrogations to elucidate whether a directed therapy is effective or not. This requires multiple analyses of the different molecular pathways at play within the same patient tissue section.


Q6: What do you see as the barriers to the wider adoption of multiplex IHC?

The main barrier is the lack of biological understanding and technical experience as well as possibly the complexity of the staining protocols. Back in the old days, one could always blame the lack of reagents and equipment.

The situation is nowadays different due to the many methodological breakthroughs within the field and wide availability of highly diverse and improved reagents, staining automation, and digital image analysis platforms.


Q7: Neuroscience and cancer research: how does multiplex IHC enhance understanding of diseases?

The molecular and cellular complexity of the brain necessitates high-level multiplexing. Here you may deal with diverse subclasses of neurons even within a very limited subregion of the brain wherein subtle differences in the expression of a specific neuropeptide or transcription factor determine their specific roles and wiring. In this case, you are at a loss if you cannot make use of multiplexing.

Cancer represents a diverse spectrum of conditions with a high degree of complexity. In oncological research and clinical oncology, it is necessary to investigate particular pathways being active in a malignant cell population and to capture their responsiveness to a particular therapeutic regimen and indeed the development of drug resistance. This can be accomplished by multiplexed staining methods. The phenotypic nature of the tumor microenvironment, which is key for tumor diagnosis, can only be inquired by multiplexing IHC or IHC/RNA-ISH. 

Reading Tip

Read our blog post Do you have your IHC controls under control?


Q8: What questions were you addressing when you started using multiplex IHC?

My first consideration was whether at-all multiplexing was necessary to address the biological question of relevance. Then I considered the choice of reagents, how to combine these in a clever way with the secondary reagents and which detection method would make the best sense and give the most unequivocal and crisp staining results.


Q9: What happens next in the process of multiplex IHC and discovery?

I observe a distinct clear trend in the research and diagnostic community towards a higher level of multiplexing. Reiterated immunostaining with DNA-barcoded antibodies in the same section represents a key methodological breakthrough now commercialized by Nanostring Inc.

Another major dividend is the establishment of the Hyperion/CyTOF system for in situ mass-spectrometry by Fluidigm which allows for the analysis of more than 100 different markers within the same tissue due to lack of spectral overlap.


Q10: If you could offer readers interested in multiplex IHC one key piece of advice, what would it be? 

Let biology decide and define the next steps. Set the conditions straight for each antibody in a singleplex staining format before applying the multiplexing protocol.

Thoroughly consider which detection system to use. Select one that provides an optimal signal-to-noise-ratio i.e. minimize the spectral overlap. If you are using an immunofluorescence method make sure that you have all the filter settings, you need in your fluorescence microscope.

If you are considering a chromogenic method be aware that the chromogens can be visually distinguished and make adjustments to the choice of counterstaining method.

For chromogenic multiplexing, spectral deconvolution methods are available to distinguish chromogenic stains within the same cellular sub-compartment provided they are of a different color.