Phenotyping of the Tumor Microenvironment Using DNA Barcoded Multiplex Technology

Recorded On: 10/15/2020

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Multiplex immunohistochemistry and immunofluorescence offer powerful ways to understand target expression in the tumor microenvironment. However, there are challenges with the development of traditional multiplex methods due to the limitations of chromogenic dyes and secondary antibody conjugated fluorophores. DNA barcoded InSituPlex technology provides us with an innovative way to detect multiple markers simultaneously without new capital investment or the use of antibody stripping methods. Using this technology, we successfully combined UltiVue’s InSituPlex and DNA exchange in a custom 7 plex assay that was applied to sections of human NSCLC and bladder cancer. This method used two separate panels that were designed using barcoded primary antibodies against CD8/CD68/PD-L1 and Ki67/Granzyme B/CD3/PanCK. Following the application of the first round of staining, slides were temporarily coverslipped then imaged, then processed using the DNA exchange protocol and application of the second antibody panel then imaged again. For each section, two whole slide images were acquired, fused into one single image and analyzed using HALO image analysis. Using this technology we were able to detect 7 markers simultaneously using the same tissue section, which significantly expanded our capability to understand the expression and distribution of the immune cells in these tumors.

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David Krull, HT(ASCP)QIHC

Scientific Leader

GlaxoSmithKline

David Krull received his science degree in Zoology/Animal Biology from Connecticut college, and his certificate from Hartford Hospital School of Allied Health. He is also chair of the NSH VIR committee, a member of the NSH program team – which if you are not aware selflessly dedicates weeks to helping the NSH curate and execute an outstanding program the annual Symposium/Convention, and also worked with NSH to develop the Digital Pathology Certificate program.  

Chifei Sun, MSc

Senior Histotechnologist

GlaxoSmithKline

Chifei is a highly motivated histotechnologist with >9 years experience in pharma R&D. During  his time with GSK, his job scope spans from animal model characterization, target validation, to clinical biomarker assay development and validation. Despite rich knowledge in drug discovery and development, he also has fine technical skills in routine histology, special stain, immunohistochemistry, multiplex immunofluorescence, digital pathology and quantitative image analysis. He has special interest in using multiplex IHC to elucidate disease relevant molecular mechanism.

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Phenotyping of the Tumor Microenvironment Using DNA Barcoded Multiplex Technology
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Open to view video. Multiplex immunohistochemistry and immunofluorescence offer powerful ways to understand target expression in the tumor microenvironment. However, there are challenges with the development of traditional multiplex methods due to the limitations of chromogenic dyes and secondary antibody conjugated fluorophores. DNA barcoded InSituPlex technology provides us with an innovative way to detect multiple markers simultaneously without new capital investment or the use of antibody stripping methods. Using this technology, we successfully combined UltiVue’s InSituPlex and DNA exchange in a custom 7 plex assay that was applied to sections of human NSCLC and bladder cancer. This method used two separate panels that were designed using barcoded primary antibodies against CD8/CD68/PD-L1 and Ki67/Granzyme B/CD3/PanCK. Following the application of the first round of staining, slides were temporarily coverslipped then imaged, then processed using the DNA exchange protocol and application of the second antibody panel then imaged again. For each section, two whole slide images were acquired, fused into one single image and analyzed using HALO image analysis. Using this technology we were able to detect 7 markers simultaneously using the same tissue section, which significantly expanded our capability to understand the expression and distribution of the immune cells in these tumors.
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