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Abstract
Methods to detect immunolabeled molecules at increasingly higher resolutions, even when present at low levels, are revolutionizing immunohistochemistry (IHC). These technologies can be valuable for the management and examination of rare patient tissue specimens, and for improved accuracy of early disease detection. The purpose of this article is to highlight recent multiplexing methods that are candidates for more prevalent use in clinical research and potential translation to the clinic. Multiplex IHC methods, which permit identification of at least 3 and up to 30 discrete antigens, have been divided into whole-section staining and spatially-patterned staining categories. Associated signal enhancement technologies that can enhance performance and throughput of multiplex IHC assays are also discussed. Each multiplex IHC technique, detailed herein, is associated with several advantages as well as tradeoffs that must be taken into consideration for proper evaluation and use of the methods.
Financial & competing interests disclosure
S Takayama and J White own stock in PHASIQ, Inc., a company that is commercializing technology related to ATPS. S Takayama is supported by grants from the National Institutes of Health (CA170198) and the Coulter Foundation Grant to the University of Michigan. A R Dixon is supported by the National Institutes of Health sponsored, University of Michigan Hearing, Balance, and Chemical Senses Program (T32 DC00011). A R Dixon is supported by the University of Michigan Hearing, Balance, and Chemical Senses Program, sponsored by the National Institutes of Health (T32 DC00011). C Bathany is supported by the Basic Science Research Program (2013R1A1A2064729) of the National Research Foundation of Korea through the Ministry of Education, Science and Technology. K F Barald is supported by a grant from the National Science Foundation (IOS1146132). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No single biomarker or protein can specifically and sensitively reveal all relevant histological correlates to pathological states.
Multiplex immunohistochemistry allows visualization of more than one antigen at a time, but can suffer from antibody cross-talk, signal overlap and procedural complexities.
Multiplex immunohistochemistry can reduce inter-experiment error compared to performing several singleplex assays.
Use of multiplexing methods is critical when available tissue samples are minimal in number or in tissue area.
Increasing levels of multiplexing demand higher skills and experience from users or development of new automated approaches.
With colorimetric and fluorescent multiplexing techniques, methods to minimize autofluorescence and deconvolute spectral overlap become critical.
With spatially-patterned multiplexing techniques, it is difficult to know whether different antigens are co-localized or not, and there is a larger chance that rare markers may be missed.
User-friendly and cost-effective multiplexing systems that improve upon signal sensitivity and specificity while reducing batch-to-batch and user-to-user variability are needed.