The 3 Biggest Challenges When Designing a Spatial Proteomic Panel
By Tad George, PhD · Senior Vice President, Biology R&D · RareCyte
Designing an effective spatial proteomic panel can be a complex balancing act. From selecting the right biomarkers to validating performance, each step is critical in ensuring you get the most meaningful, reproducible multiplex immunofluorescence results. This blog explores the top three challenges in panel design and how Orion™'s validated reagents, combined with high-plex spatial protein profiling, simplify workflows to deliver accurate, reproducible results.
Selecting biomarkers to include in your panel
Selecting biomarkers that best capture your study's microenvironment is essential — but so is balancing practical considerations such as reagent availability, cost, throughput, and data analysis complexity. For these considerations, finding the optimal plex is key:
- Too few markers may restrict your ability to draw meaningful conclusions
- Too many markers can inflate costs, slow workflows, and introduce unnecessary complexity
The goal: design a panel that maximizes biological insight without compromising efficiency.
Validating clone performance in single-plex
Not all antibody clones are created equal, and performance can vary significantly with tissue type and antigen retrieval conditions. With current workflows, it's critical to validate each marker in single-plex under the exact conditions you'll use in your study. This step helps identify false positives, weak signals, or incompatibilities before they impact larger experiments. Skipping this step can lead to data quality issues that are costly and time-consuming to fix later.
Using validated antibodies for your platform helps with this step, as each marker has already been proven to perform under the required antigen retrieval conditions. Orion maintains a comprehensive list of validated biomarkers to streamline this process.
Verifying multiplex panel performance
Reagents that work well alone don't always behave the same in a multiplex environment. This is particularly true for cyclic staining approaches, where you'll need to account for:
- Antigen expression or accessibility changes between staining rounds, making it essential to determine the optimal cycle for each biomarker
- Incomplete antibody stripping after each round, which can introduce unwanted background noise
- Epitope masking ("Umbrella Effect") caused by Tyramide amplification, potentially obscuring target detection
Whenever possible, staining all markers simultaneously minimizes these risks and preserves assay performance.
Careful biomarker selection, thorough single-plex validation, and diligent panel verification set the foundation for more accurate, biologically relevant spatial proteomics data.
How Orion Simplifies Panel Design
Orion was designed to help researchers overcome these very challenges. It offers:
A comprehensive catalog of validated reagents and verified biomarker panels — reducing time spent on validation and providing you with reliable results from the start.
Single-round staining and imaging of up to 18 biomarkers simultaneously — minimizing cyclic staining rounds and reducing risks like antigen expression changes, background noise, and epitope masking.
With Orion, researchers can focus on generating spatial insights, not troubleshooting panel performance.
Tad has over 15 years of startup experience dedicated to creating scientific markets for novel instrumentation platforms that span basic research, drug discovery and clinical applications. Prior to joining RareCyte, Tad has held similar positions at Biodesy, Inc. and DVS Sciences, and was Director of Biology at Amnis Corporation. Tad completed his B.A. in Biochemistry from the University of Texas at Austin, Ph.D. in Immunology from UT Southwestern Medical Center at Dallas, and post-doctoral training at Immunex Corp. in Seattle.
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Tad has over 15 years of startup experience dedicated to creating scientific markets for novel instrumentation platforms that span basic research, drug discovery and clinical applications. Prior to joining RareCyte, Tad has held similar positions at Biodesy, Inc. and DVS Sciences, and was Director of Biology at Amnis Corporation. Tad completed his B.A. in Biochemistry from the University of Texas at Austin, Ph.D. in Immunology from UT Southwestern Medical Center at Dallas, and post-doctoral training at Immunex Corp. in Seattle.
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