Multiplex immunofluorescence IHC is a powerful technique to address the increasing research needs in immuno-oncology to better understand the tumor microenvironment.

In this webinar we present a rapid and automated high-plex fluorescent immunostaining procedure, using our microfluidic-based staining device LabSat® Research and TSA staining reagents. The 6-marker protocol could be performed in less than 4 hours and 30 minutes, resulting in high-quality marker detection, efficient removal of antibodies, and remarkable signal uniformity.

The RNAscope® assay, based on ACD’s technology for signal amplification and background suppression, was optimized on LabSat® to result in well-preserved morphology, uniform staining with excellent signal-to-noise ratio, and readily detectable punctate signal dots – comparable to the manual assay on mouse and human FFPE samples. Concomitantly, the assay turnaround time was shortened with respect to the manual and other automated CISH protocols. Overall, we have proven the capability of the microfluidic platform LabSat® to automate the RNAscope® 2.5 HD-BROWN CISH assay with reproducible staining results. The microfluidic platform brings the benefits of a shorter run time and reliability of a compact desktop system.

To simultaneously visualize multiple markers within the same sample enables a more detailed view of cellular phenotypes and how they orient in space. This in turn brings deeper understanding of the function and interplay of different cell types in situ, of value for research related to many biological conditions. For most labs, accessing and applying emerging spatial methods for highly multiplexed detection of various types of molecules is not straight forward. The Spatial and Single Cell Biology (SSCB) platform at SciLifeLab offers several spatial omics methods as a service, including advanced single cell sequencing, spatial transcriptomics, in situ sequencing, spatial mass spectrometry and spatial proteomics. In this talk, Dr Stadler will present the possibilities for spatial omics as a service at Scilifelab, with emphasis on the methods offered for spatial proteomics using highly multiplexed immunofluorescence. Her Spatial Proteomics team as part of the SSCB platform currently offers a service, to analyze up to 30 proteins in the same tissue section using DNA-barcoded antibodies. From next year, the COMET instrument from Lunaphore will be fully integrated in the SSCB platform and offered as a service. COMET performs sequential indirect immunofluorescence to allow for multiplexed protein detection in 4 samples in parallel. In this talk, Dr. Stadler will show results obtained with COMET on several tissue types.

Multiplex protein expression analysis in tissue using immunohistochemistry has rapidly evolved over the last decade from 2- and 3-plex assays to a field of new technologies enabling highly multiplexed assays of 40 or more targets. Knowing which of these spatial proteomic technologies would be most effective in your lab or group is not simple. In this presentation we will walk through the value proposition for spatial proteomics, focusing on antibody- and immunohistochemistry-based spatial proteomics platforms, as well as review technical vignettes describing our lab’s experience evaluating and selecting the most appropriate platforms for our lab. We hope that our experience can empower you on your own journey into spatial proteomics.

Live presentation & demo featuring COMET™: the first all-in-one, rapid hyperplex solution for spatial biology.

You will be able to virtually join our team in the lab, learn more about how it can support your research work, ask questions and watch them operating it live.

Purpose of this study: A small number of maternal SARS-CoV-2 infections are complicated by placentitis leading to obstetric complications including foetal growth restriction and/or intra-uterine death. The aim of this study is to understand the immunological processes that drive this potentially devastating condition.

Methods: FFPE tissue from 13 cases of placentitis from mothers with COVID-19, 6 diseased controls (chronic histiocytic villousitis (CHI) and villousitis of unknown origin (VUE)) and 5 normal placentas were examined by high-plex 32 marker immunohistochemistry (mIHC) on the Lunaphore COMET™ platform. Transcriptomic profiling was performed by RNAscope in-situ hybridisation (ISH), bulk gene expression sequencing (GES) and GeoMX digital spatial profiling (DSP).

Results: Direct SARS-CoV-2 infection of the trophoblast was demonstrated in 6/13 cases from SARS-CoV-2 positive mothers by mIHC (dual positivity for viral spike and nucleocapsid proteins) and confirmed by ISH and bulk GES. Virus was also detected within histiocytes consistent with virus phagocytosis. SARS-CoV-2+ cases of placentitis showed a predominantly histiocytic (CD68+) intervillous infiltrate with an associated smaller population of T-cells (CD3+) and B-cells (CD20+), distinguishing COVID-19 placentitis from diseased controls. GES showed upregulation of the CXCL10 and type I interferon pathways. CXCL10 expression within histiocytes was confirmed by mIHC and RNAscope.

Conclusion: SARS-CoV-2 can directly infect the placental villous trophoblast. SARS-CoV-2 placentitis is characterised by chronic histiocytic intervillousitis accompanied by high levels of CXCL10 production and activation of type I interferon signalling pathways. Overall, SARS-CoV-2 positive placentas showed a distinct immunopsthological phenotype compared to SARS-CoV-2 negative placentas from COVID-19 positive mothers and disease controls.

Immune checkpoint blockade (ICB) utilizing antibodies that block PD-1, PD-L1, and CTLA-4 has changed cancer patient care and started a new era, with long-term durable disease control in a subset of patients across multiple cancers. However, most patients do not respond to ICB therapy and many of those who initially respond eventually develop recurrent and progressive disease. Our laboratory seeks to understand why this approach is active in some patients but ineffective in others, why do some patients develop resistance to therapy, and how to overcome these barriers to ICB and generate effective systemic antitumor immunity. We will be presenting examples of rationally designed combination therapies based on the changes in the immune system, systemically in the tumor microenvironment. These studies have been paralleled both in murine models and clinical settings. We hope these studies provide a framework for future early pre-clinical and clinical studies.

Abstract and learning goals of Mike Surace: Validation of multiplex panels can mean a variety of different things. In this webinar, Michael Surace will review the factors which determine how stringent a validation should be, including the assay, the platform, the signal of interest, the reference signal, the degree of precision/ dimensions/ depth required in the analysis, the application, the purpose, the context, and the criteria for success. The standard development workflow for a 6-plex panel will be dissected and described in detail, and the critical components of the low plex validation, including development tasks, pressure tests, and the formal validation will be redeployed in a manner appropriate for a 40 plex iterative mIF assay, including caveats, potential artifacts, and criteria for success.