Recent developments in multiplexing techniques for immunohistochemistry - PubMed


Angela R Dixon 1 ,

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Recent developments in multiplexing techniques for immunohistochemistry

Angela R Dixon et al. Expert Rev Mol Diagn. 2015.

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.

Keywords: colorimetric multiplexing; immunohistochemistry; multiplex; signal enhancement; spatially-patterned multiplexing.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1

High-level multiplexing IHC systems. Listed are whole-section and spatially-patterned multiplexing immunohistochemistry systems and the key procedural challenges they address. Whole-section and spatially-patterned images reproduced with permission [30], [8].

Figure 2
Figure 2

Whole section multiplex staining systems for IHC. A) Multi-epitope ligand cartography (MELC) technology consists in automated cycles of fluorescent staining, imaging, and photobleaching. Each cycle can target a different protein to produce a set of thresholded images mapping the different proteins’ distribution. The resulting image represents a combinatorial molecular phenotype for any desired protein. B) The indirect layer peptide array (iLPA) is one variant of the layer expression system that positions a stack of antigen-coated membranes atop the tissue sample to capture antibodies detached from the tissue, and each free antibody binds to a complementary antigen-specific membrane. The original x-y position of each antibody is maintained. C) Schematic of a protocol combining LA-ICP-MS with metal tagged antibody capture using CyTOF capabilities and IHC/ICC techniques for subcellular resolution screening of biomarkers in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. IHC: Immunohistochemistry; ICC: Immunocytochemistry; MELC: Multi-epitope ligand cartography; iLPA: Indirect layer peptide array; LA-ICP-MS: Laser ablation inductively coupled plasma mass spectrometry. Figure 2A. Reproduced with permission from Nature Publishing Group [85]. Figure 2B. Reproduced with permission from Clinica Chimica Acta, 376(1–2), Gannot G, Tangrea MA, Richardson AM et al., Layered expression scanning: Multiplex molecular analysis of diverse life science platforms, 9–16, Copyright 2007, with permission from Elsevier [17]. Figure 2C. Reproduced with permission from Nature Publishing Group [28].

Figure 3
Figure 3

Spatially-patterned multiplexing systems for IHC. A) Tissue microarray manufacturing and use. Tissue microarrays are constructed by transfer of tissue cores from a ”donor” paraffin block (a) to a pre-punctured “recipient” paraffin block (b) using a tissue microarrayer. A microtome can be used to cut slices from tissue microarrays. An adhesive-tape transfer system can be applied to the tissue microarray to keep tissue intact during transfer to and while mounted on the slide (c & d). B) 1. Microfluidic platform for multiplex screening of 10 biomarkers using 10 independently run microfluidic channels to deliver reagent-loaded solutions on a cancer breast tissue sample. 2. Multiplex-immunostain chip (MI-chip) uses flipping function to deliver antibodies to discrete regions of tissue. C) Microfluidic microprobe is a vertical probe actuated across a breast cancer tissue to deliver reagents for multiplex screening of cancer biomarkers. D) Multiplex primary antibody patterning of a cell monolayer using ATPS of Dextran microdroplets submerged in a bulk solution of polyethylene glycol (PEG) (left). ATPS technique was applied to a dorsal root ganglion (DRG) explant of a chick embryo, where Dextran droplets containing tetramethylrhodamine (TRITC) were localized to DRG axons (top right) or fluorescein isothiocyanate (FITC)-wheat germ agglutinin delivered to non-labeled DRG axons (bottom right). Reproduced with permission [60] Copyright 2015. Figure 3A. Reproduced with permission from Nature Publishing Group [30]. Figure 3B1. Reproduced with permission from [47], Biomaterials, 32(5), Kim M, Kwon S, Kim T, Lee E, Park J-K, Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry and immunocytochemistry, 1396–1403, Copyright 2011, with permission from Elsevier [47]. Figure 3B2. Reproduced with permission from Furuya, et al. [51]. Figure 3C. Reproduced with permission from the Royal Society of Chemistry [50]. Figure 3D. Reproduced with permission from John Wiley and Sons [60].

Figure 4
Figure 4

Signal Enhancements for Immunohistochemistry (A) Quantum dots are conjugated to secondary antibodies to enhance fluorescent signal. (B) In silver enhancement, nucleation of silver (Ag) around a gold (Au) core enhances visibility of the gold particles. (C) In tyramide signal amplification, horseradish peroxidase catalyzes the deposition of biotinylated tyramine in proximity to the tagged antigen (D) ImmunoRCA, enables easy visualization of antigens via surface-anchored DNA replication. OLN = oligonucleotide. Ab = antibody. Detection with 1:25,600 antibody dilution (E) Polymer-Based signal amplification involves binding a polymer backbone that is conjugated with multiple antibodies and enzymes for higher contrast visualization. Permissions were obtained for microscopy images of stained cells and tissues (enclosed in shaded boxes) [86], [72], [87],[74], [88]. Microscopy images of stained cells and tissues (enclosed in shaded boxes) are reproduced with kind permission from Nature Publishing Group (A) [86], Springer Science and Business Media (B) [87], Methods, 18(4) Soontornniyomkij V, Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy, 459–464, Copyright 1999, from Elsevier (C) [72], the American Journal of Pathology, 159(1), Gusev Y, Sparkowski J, Raghunathan A et al. Rolling Circle Amplification: A New Approach to Increase Sensitivity for Immunohistochemistry and Flow Cytometry, 63–69, Copyright 2001, with permission from Elsevier (D) [74], and Wolters Kluwer Health (E) [88].

Figure 5
Figure 5

Comparison of whole specimen multiplexing methods for immunohistochemistry. All figure permissions have been obtained as follows: mass spectroscopy, reproduced with permission from John Wiley and Sons [24], Clinica Chimica Acta, 376(1–2); SIMPLE, reprinted by Permission of SAGE Publications [8]; MELC, reproduced with permission from Nature Publishing Group [4].

Figure 6
Figure 6

Comparison of spatially-patterned multiplexing methods for immunohistochemistry. dia = diameter; w = width; l = length All figures reproduced with permissions as follows: permission obtained from Nature Publishing Group [30], PLOS One [46], Biomaterials,32(5), Kim M, Kwon S, Kim T, Lee E, Park J-K. Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry (IHC) and immunocytochemistry, 1396–1403, Copyright 2011, with permission from Elsevier [47]. Reprinted with Permission of SAGE Publications [50,51], and Reprinted with permission from John Wiley and Sons [60].

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