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Updated: Sep 3, 2025
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
Digital Image Analysis and Quantitative Bead Standards in Root Cause Analysis of Immunohistochemical Staining
Rebecca Rojansky1, Seshi R Sompuram2, Ellen Gomulia1
1Department of Pathology, Stanford University School of Medicine, Stanford, CA.
Abstract:
Assessment of automated immunohistochemical staining platform performance is largely limited to the visual evaluation of individual slides by trained personnel. Quantitative assessment of stain intensity is not typically performed. Here we describe our experience with 2 quantitative strategies that were instrumental in root cause investigations performed to identify the sources of suboptimal staining quality (decreased stain intensity and increased variability). In addition, these tools were utilized as adjuncts in validation of a new immunohistochemical staining instrument. The novel methods utilized in the investigation include quantitative assessment of whole slide images (WSI) and commercially available quantitative calibrators. Over the course of ~13 months, these methods helped to identify and verify correction of 2 sources of suboptimal staining. One root cause of suboptimal staining was insufficient/variable power delivery from our building's electrical circuit. This led us to use uninterruptible power managers for all automated immunostainer instruments, which restored expected stain intensity and consistency. Later, we encountered one instrument that, despite passing all vendor quality control checks and not showing error alerts was suspected of yielding suboptimal stain quality. WSI analysis and quantitative calibrators provided a clear evidence that proved critical in confirming the pathologists' visual impressions. This led to the replacement of the instrument, which was then validated using a combination of standard validation metrics supplemented by WSI analysis and quantitative calibrators. These root cause analyses document 2 variables that are critical in producing optimal immunohistochemical stain results and also provide real-world examples of how the application of quantitative tools to measure automated immunohistochemical stain output can provide a greater objectivity when assessing immunohistochemical stain quality.
