Implementation of Multiplex Staining, Imaging and Analysis as a Standardized Service for Researchers
| Autor: | Selvaraj S, Valeria Mezzano, Dabovic B, Loomis C |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | Scopus-Elsevier Europe PubMed Central |
| Popis: | Several modalities of multiplex immunofluorescence histology currently available require significant time and resources to implement. Many research laboratories develop questions benefiting from multiplex staining and analysis but do not have the human resources and/or equipment to perform the assay. Furthermore, pilot studies use similar metrics to evaluate multiplex histology data making them ideal for a core laboratory setup. The objective of this study was to establish a semi-automated workflow for multiplex immunofluorescence staining and initial quantification of cell populations in whole slide microscopy scans. The requirements for the workflow included: A. minimal transfer of decision making from the researcher to core personnel (Semi-Automation), B. modifiable in terms of antigen targets and tissue types with minimal disruption to the process and C. reproducible across samples submitted at different time periods (eg patient samples). Methods: Leica Bond RX auto stainer was used for staining. Akoya Biosciences Phenoptics platform was used for multiplex staining, imaging was performed with the Vectra3 multispectral imaging system followed by InForm quantification. Further analysis was conducted with the statistical analysis package R. Cross platform imaging was tested with a Zeiss 880 confocal microscope. Results: Five investigators are using the workflow for their projects that range from clinical pilot studies to basic science with animal models. Our workflow for one slide involves 24 hours for staining, one hour for scanning setup, 10 minutes for whole slide scanning, up to 2 hours for automated sampling, and initial analysis setup 2 hours. Conclusions: the key steps for establishing a robust workflow include antibody optimization using a combination of chromogenic (for determination of specificity/sensitivity) and fluorescent testing in multiplex panels (to determine appropriate dilution according to corresponding fluorophore). Explicit statement from the researchers regarding what to quantify (populations of interest, areas of interest) optimizes analysis output. |
| Databáze: | OpenAIRE |
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