Multiplex tissue imaging: An introduction to the scope and challenges

Abstract

Prompted by the multiplex study of Calvani et al (page 942), this editorial discusses opportunities and practicalities of a nextgeneration pathology, in which convergent technologies deep-mine a tissue section to reveal unsuspected networks of spatial, social, and functional organization. Prompted by the multiplex study of Calvani et al (page 942), this editorial discusses opportunities and practicalities of a nextgeneration pathology, in which convergent technologies deep-mine a tissue section to reveal unsuspected networks of spatial, social, and functional organization. That allograft biopsies are still needed reflects that histopathology outperforms other modalities in difficult settings, because the underlying truth in a tissue sample cannot be recapitulated with other approaches. The unique advantage of tissue sections is the retention of spatial relationships to molecular level. This is exploited with clinical histopathology, where trained observers categorize anatomic patterns into clinically relevant interpretations of a tissue snapshot. That paradigm taps only a fraction of the data richness within a tissue sample, but the convergence of multiplexed biomarker labeling in formalin-fixed paraffin-embedded tissue (epitopes, RNA, spectroscopically reactive molecules) with imaging technology and computational platforms offers us a next-generation histopathology. In this issue, Calvani and colleagues use multiplex immunofluorescence to evaluate macrophages, NK and T cells in renal allograft rejection.1Calvani J Terada M Eloudzen M et al.In situ multiplex immunohistofluorescence analysis of the inflammatory burden in kidney allograft rejection: a new tool to characterise the alloimmune response.Am J Transplant. 2019; 20 (https://doi.org/10.1111/ajt.15699)Google Scholar They found unexplained variability between patients in relative proportions of graft-infiltrating interstitial T cells and macrophages, while peritubular capillaries (ptc) contained mainly CD3 T cells (>80%). They also show that antibody-mediated rejection (AMR) and T cell–mediated rejection (TCMR) have similar average ptc infiltrate density, despite the diagnostic association of ptc leucocytosis with AMR. Moreover, ptc inflammation density in AMR was outweighed by that in interstitium, despite case selection censoring for interstitial inflammation in an effort to present “pure” examples on Banff scores (glomerular inflammation in TCMR was also censored). The findings suggest that even polarized examples of AMR/TCMR overlap more in tissue compartment involvement than pathologist scoring identifies and redirect attention to infiltrate composition, pattern, and setting. Indeed, differential counts showed 2-3-fold more ptc NK cells and macrophages in AMR, consistent with disease-specific effector roles. The authors focused here on validating the multiplex process and their data contain richness not presented. For example, despite similar average ptc leukocytosis, how did the nature and content of individual ptc distribute between the diseases? Pathologists might predict NK- and macrophage-rich capillaritis “hot spots” in AMR only, co-localizing with ptc injury (dilation and endothelial cell swelling), providing quantified topologic support for effector roles and the Banff ptc score design. Such deeper questions are now addressable with multiplex staining. Indeed, new proposals for immune monitoring with multiplex cytometry in immunotherapy trials2Hartmann FJ Babdor J Gherardini PF et al.Comprehensive immune monitoring of clinical trials to advance human immunotherapy.Cell Rep. 2019; 28: 819-831Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar anticipate expectations for multiplex tissue-based monitoring, so it is timely to consider the potential of multiplexed epitope-targeted tissue imaging. At a prosaic level, multiplex tissue imaging identifies marked specific, experimentally labeled structures with spatial reference to differently marked oth