Developing your own multiplex immunohistochemistry panels can be a challenging, time-consuming process. In our latest webinar, Victoria Duckworth, Product Manager for Opal & TSA Reagents at Akoya Biosciences, introduced our new MOTiF™ PD-1/PD-L1 Panel Kits, which are part of a fully optimized workflow from staining to analysis for lung cancer and melanoma samples.
Using Opal™ with autostainers
Before discussing the new panel kits, Victoria went over the process of staining with Opal™ fluorophores for multiplex immunohistochemistry (mIHC). Opal™ is an iterative IHC assay, she explained. The fluorophores have been optimized for spectral unmixing and data quantification. Opal™ enables you to capture up to 8 markers on a single tissue section, supporting high-throughput biomarker discovery without tissue ablation.
“In your lab, if you’re able to implement single stain DAB, you can just as easily implement Opal IHC – because it’s the same workflow,” said Victoria.
The Opal™ workflow can be performed manually or on an autostainer of your choice. After normal preparation for IHC, the next step is to add the primary antibody and then the secondary antibody, which is species-specific and labeled with HRP. Once the Opal™ fluorophore is added, Victoria explained, it interacts with HRP to create a free radical which covalently binds to any tyramide residue within 10 nm of the target on the tissue.
With the signal bound to the antigen, heat stripping is performed to remove the previously bound primary and secondary antibodies. A new primary antibody of the same species can then be used without the concern of interspecies crosstalk. The markers can then be visualized using the Vectra® Polaris™. Traditional narrow-band fluorescent scanners use filters to visualize individual markers. However, some fluorophores excite in multiband channels.
The Vectra® Polaris™ overcomes this problem with spectral unmixing, isolating every signal appropriate for the marker of interest. This ensures that signal intensity from the marker is only being received from the channel in which you are interrogating it. Polaris™ also isolates autofluorescence spectra and removes it from the sample.
MOTiF™: Whole Slide Multispectral Imaging
The new MOTiF™ PD-1/PD-L1 Panel Kits
We’ve released two MOTiF™ PD-1/PD-L1 Panel Kits, validated for melanoma and lung carcinoma. These kits are robust and reproducible, easy-to-use with pre-selected, clinically relevant antibodies, and provide quick turn-around with same day imaging and analysis, making them the ideal plug-and-play solution for immuno-oncology researchers.
Victoria emphasized that these kits are not standalone products. Instead, they fit into the Phenoptics™ workflow to provide a complete turn-key solution for immuno-oncology translational research. Staining is performed, either manually or with an autostainer, using reagents included in the kits. Pre-optimized acquisition protocols are available for imaging on the Vectra® Polaris™. These protocols are already adjusted for the expected level of intensity from each of the markers in the panels.
Analysis is performed using inForm® , machine learning software used for cell segmentation, tissue segmentation, and phenotyping. We’ve developed algorithms for inForm® that are specifically pre-configured for these panels. They include Opal™ and antibody pairing and the order in which they’re applied and incorporate co-positivity and exclusivity rules. Data outputs from inForm® can then be moved into R, or whichever large-scale analytical software you prefer.
MOTiF™ Panel Kit Workflow
Victoria shared some pre-analytical considerations for successful staining, not only with Opal™, but for IHC in general. She focused on three key factors: tissue handling, tissue fixation, and section storage.
When handling tissue, it’s important to be conscious of re-section and ischemic times, as certain antigens can be sensitive to these timings. For tissue fixation, she noted that formalin penetrates tissue sections during the process. This means larger sections can be fixed longer than smaller sections. A general rule of thumb is 24 hours of fixation time. When storing sections, the key is to keep them dry, as water causes oxidation and destroys antigenicity. It’s generally recommended to store a cut slide for no more than two months.
Customization and optimization
A key feature of Opal™ is its flexibility. With open detection, you can choose to use any primary antibody. In the MoTIF™ panel kits, the most clinically relevant antibodies have been pre-selected. However, it is still possible to adjust the kits to suit your needs.
There are a couple things to keep in mind when customizing the MOTiF™ panels, Victoria noted. One of these is the antibody you choose. She presented two CD8 clones run on serial sections, with approximately equal concentrations. Clone A had significantly more robust staining compared to clone B, highlighting the importance of antibody selection.
Titrations are another factor – an antibody concentration that much higher than required for epitope saturation could lead to problems during stripping, while one that is too low will not visualize properly on the slide.
It’s also necessary to consider how to best optimize antigen retrieval in order to determine the best way to visualize your target. She suggested paying close attention to the parameters which produce the best results for your antibody. This may mean testing recommendations from antibody vendors – in an example, Victoria showed how an KI67 staining was improved when using a different buffer than the recommended type.
When deciding on your substrate-antibody pairings and staining order, said Victoria, it’s important to keep in mind the colocalization of your markers, their expression patterns, and your epitope retrieval conditions. For example, if you have multiple antibodies that may be present in the same cell compartment, we suggest not using consecutive Opal™ dyes in order to avoid cross-talk.
This webinar is now available on-demand – click here to watch.