Automated Multiplex Immunofluorescence

Hello everyone and welcome to today's live webinar automated multiplex immunofluorescence presented by Dr. Bethany Remeniuk and Traci DeGeer. I'm Christy with Labroots and I'll be your moderator for today's event. Today's educational web seminar is presented by Labroots and brought to you by Leica Biosystems. For more information on our sponsor, please visit the sponsor tab at the left side of your screen and click on the logos to be directed to the sponsor website. 

Before we begin, I would like to remind everyone that this event is interactive and we encourage you to participate by submitting as many questions as you want at anytime you want during the presentation. To do so, simply type them into the ask a question box and click send. We will answer as many questions as we have time for at the end of the presentation. If you have trouble seeing or hearing the presentation, click on the support tab found at the top right of the presentation window. Or use the ask a question box to let us know you're experiencing a problem. 

This presentation is educational and thus offers continuing education credits. Please click on the continuing education credits tab located at the top right of your presentation window and follow the process to obtain your credits. I would now like to present today's speakers doctor Bethany Remeniuk, Global Application Scientist, Akoya Biosciences, and Tracy DeGeer, Director, Advanced Staining Innovation, Leica Biosystems. For complete biographies on our speakers, please visit the biography tab at the top of your screen. Bethany and Tracy, you may now begin your presentation.

Thank you, Christy for that introduction. Hello everyone. My name is Bethany Remeniuk and I am the Global Application Scientist at Akoya Biosciences. Today, we will be discussing the advantages of utilizing Akoya's opal multiplex immunohistochemistry, automated detection kits on the Leica Biosystems BOND RX auto stainer to meet your research needs. In addition, I will be highlighting some tips and recommendations to help design your multiplex panel and optimize your staining. However, before we can delve into this, we must first take a step back to understand how these two platforms combined help to facilitate a streamlined workflow and drive cancer research forward. 

From 1991 to 2015, cancer deaths in the United States alone fell by 26%. This is due in part to significant advances in the treatment of cancer patients, including the use of more personalized therapies for individuals as well as FDA-approved immunotherapy treatments such as Anti-PD-1 and Anti-PD-L1. However not all individuals who test positive for these markers will respond to targeted treatment. 

The Underlying Biology of the Tumor Microenvironment is Complex

To help understand why we may have responders and non-responders, we must first acknowledge that the biology and composition of the tumor microenvironment is very complex. There are several specialized cell types, including immune mediated cells such as macrophages and B&T cell lymphocytes that interact directly and indirectly with tumor cells, resulting in an ever-evolving relationship between immune cells trying to destroy the tumor cells while simultaneously the tumor cells attempting to evade and promote their own self growth. 

Because of this, there are several influencing factors that need to be taken into consideration when interrogating the tumor microenvironment, including cellular phenotypes that could influence the biology of the tumor, the functional status of the surrounding cell types, as well as other contributing factors such as inflammatory mediators and how these all interact within the spatial biology of the tumor microenvironment. Having a greater understanding of the relationships happening within the tumor microenvironment are key to improving diagnosis and designing new therapeutic regimens. 

Limitations of Current Analysis Platforms

Currently, clinical laboratory tests are automated and performed by calibrated machines to reduce human error. However, most anatomic pathology and disease diagnosis is still based upon physician interpretation of microscopic tissue characteristics such as cells, markers, and tissue architecture. In fact, this visual assessment using conventional immunohistochemistry remains the gold standard for cancer diagnosis. 

Despite this, it is recognized that visual assessment can be subjective and interpretation of results can differ between pathologists. Additionally, because immunohistochemistry such as DAB can only be conducted for a single marker on tissue samples, very little information can be obtained about the spatial context and interplay of other cellular phenotypes within the tumor microenvironment. 

On the other hand, genomics and proteomics studies including flow cytometry and DNA microarrays, provide significant quantitative information regarding different cellular phenotypes within the tumor. However, the spatial context is lost as analysis needs to be conducted using homogenized tissue and thus were at a crossroads. Where on one platform we are able to retain morphology but lose phenotypic information or conversely, have a wealth of phenotype data but lose spatial biology and interactions between them. 

Immunofluorescence Reveals Complex Biology

To advance research forward, we need to be able to retain the spatial biology of the tumor microenvironment while simultaneously identifying and labeling multiple markers of interest. This is where immunofluorescence is truly able to bridge the divide. Immunofluorescence overcomes the limitations of conventional immunohistochemistry, which are constrained by detecting two or three markers of interest, by allowing for multiplexing of several markers. By using this methodology, researchers can conserve their tissue samples, while identifying and characterizing cellular interactions. 

Immunofluorescence can then identify and reveal weak expressors that may be lost using conventional immunohistochemistry staining, and furthermore, the balancing of signals can allow researchers to detect multiple markers that colocalize on the same cell. Lastly, immunofluorescence can provide better signal for quantification and data analysis. 

Challenges with Conventional Immunofluorescence

While there are many benefits associated with immunofluorescence, there are challenges with conventional methodologies that can unfortunately limit your staining success. First, staining will need to be done using antibodies raised in different species, as secondary antibodies may crossreact and ruin your results. This unfortunately can be restricting as some rare antibodies may only be raised in one species. 

Next, some antigens may be sheltered from the primary antibody due to stearic hinderance. Furthermore, signals can be unbalanced or low if you have an overabundance of a marker or very little expression of another. Other factors that may impact your results include background staining, contributing tissue autofluorescence, which can be a result of the tissue itself as well as how the tissue was processed, and photobleaching, or quenching, of the fluorophores as they are exposed to light. 

Opal Fluorophores for Multiplexing

Thus, the Opal flurophores were created to overcome the limitations of conventional immunofluorescence. As part of the synoptics workflow, Opal dyes are accessible to anyone who has previously worked with standard immunohistochemistry. Opal allows for detection of multiple cellular phenotypes to be visualized and quantified simultaneously in the same formalin-fixed paraffin embedded tissue sections, enabling researchers to study the complex relationships and distribution of these cells while retaining spatial context within the tumor microenvironment. 

With Opal you can select any primary antibody you wish for immunohistochemistry detection based upon their performance, rather than being relegated to a species type. And lastly, Opal's multiplex assays and kits provide a practical workflow that allows for the simultaneous detection of upwards of eight biomarkers of interest post tapping nuclear counter stain within a single tissue section. 

Automated Staining with Opal on the BOND RX

Akoya offers Opal automated IHC detection kits for researchers to perform Opal multiplex staining on one of the leading research automated stating platforms, the BOND RX by Leica Biosystems. Automation provides you with the flexibility to support the dynamic demands of translational research. Open platform promotes a simplified workflow that frees up users’ time for other research endeavors. 

High-throughput Opal BOND protocols can perform upwards of seven color immunofluorescence staining on 30 slides, which can be performed overnight as opposed to typically running manually, which may take two to three days. And lastly, automation can achieve quality, consistency and reproducibility with every sample and supports the transition into clinical studies. 

How to Stain with Opal on the BOND RX

How does one go about staining with Opal using the BOND RX? As was previously mentioned, Opal dyes allow for the use of any standard unlabeled primary antibody, including multiple antibodies raised in the same species. Here we have a microscope slide that has been barcoded to recognize to be recognized by the BOND RX. After introduction of the primary antibody, the Opal polymer, HRP, is applied. 

The Opal system then uses tyramide signal amplification, or TSA, to amplify IHC detection by covalently depositing multiple fluorophores near the targeted antigen. After labeling is complete, antibodies are removed in a manner that does not disrupt the Opal fluorescence signal, allowing for the next target to be detected without antibody cross reactivity. These steps are repeated for all markers of interest. The Opal technology thus enables development of multiplex assays with balanced quantitative signal for both rare and abundant targets of interest. 

Opal Dyes are Stable Through ER Repetition in the BOND RX

Moving forward, we wanted to test the stability and reproducibility of Opal on the BOND RX. First, to confirm fluorescent signal stability of the Opal dyes on the BOND RX, we conducted a series of repeated stripping steps with each Opal fluorophore and determined that over the course of six repeated high intensity epitope retrievals that the Opal dye signal remains stable for all fluorophores, including our classic six fluorophores and our latest edition of fluorophores including the Opal 480. 

Detect Co-localized Markers with Opal

Next, to demonstrate Opal's ability to detect markers in the same cellular compartments without interference from TSA deposition, we stained tonsil with both CD3 and CD8, as they are often found in close proximity with one another on the same cell. On the left we have a merged overlay of the CD3 and CD8 channels where signal overlap appears in white. Then we have the individual channels appearing to the right. As you can see, TSA does not interfere with the detection of co-localization of markers of interest. 

BOND RX Produces Consistent Opal Staining: Monoplex

Next, we wanted to test the consistency and reproducibility of the BOND RX at staining Opal. Here we ran monoplexes staining for CD20 using Opal 520 on serial sections of tonsil for all 30 slide positions. We demonstrate that slide variability between all 30 slides for per cell expression of CD20 produced a coefficient of variation less than 5%, indicating that the BOND can produce consistent and reliable staining results. 

BOND RX Produces Consistent Opal Staining: Multiplex

For the next testing procedure, we assessed the BOND RX’s ability to reliably stain 15 tonsil serial sections for seven color multiplex panels. For each of the Opal fluorophores, we analyzed the mean counts of the top 20 brightest cells per slide and found that for each marker there was a calculated CV of 14% or less. Which, given the nature of serial sections, speaks to the consistency of both Opal staining as well as the functionality and reliability of the BOND RX in producing consistent results for all your staining needs.

Opal Workflow Overview on the BOND RX

Next, we're going to delve into some tips, tricks, and recommendations to help optimize your Opal staining. Here on the left we have an overview of how staining is run on the BOND RX using the newest Opal Polaris immunohistochemistry automated detection kit, which includes our newest fluorophores, the Opal Polaris 480, and the Opal Polaris 780. 

Opal protocols are auto populated on the BOND RX and can be used as they are for all of our other kits that we have. However, if you wish to do staining with the Opal Polaris kit, you will need to modify that protocol to include Opal Polaris 780 at the end of the staining run. At the end of this presentation as well, I will also share some additional staining resources to help support your staining endeavors. 

Considerations for Monoplex Development

First we have some recommendations from Akoya to help in your monoplex development. First, we recommend that you use tonsil to optimize all of your monoplexes. This is ideal because tonsil has several markers of interest that are highly expressed, making it ideal to help optimize your primary antibody dilution. 

We recommend going ahead with a series of three DAB titrations and then optimizing from there based on staining pattern and intensity. Once you feel you've optimized your primary antibody dilution using DAB, we recommend then converting it to immunofluorescence. Here then you would want to pick an ideal Opal pairing to go with the primary antibody and some factors to consider include co-expression. If you happen to be looking at multiple markers of interest on the same cellular compartment, you might want to choose an Opal pairing for that primary antibody that is perhaps a little bit far removed from the other marker of interest that you are looking to image. 

For example, you may want to choose Opal 520 paired to CD3 and then maybe Opal 570 or Opal 620 to look at CD8. Furthermore, you also want to take into consideration whether you are investigating rare versus abundant markers. If you are looking at markers that may not be highly expressed or weak expresses, you may want to pair them with an Opal that is very bright. In this case, Opal Polaris 480 or Opal 520 versus if you happen to have a blended marker of interest such as cytokeratin, you may want to pair that with some of the more lower bright Opal dyes, including Opal 690 and Opal Polaris 780. 

For Opal concentrations, we recommend starting at a 1 to 150 dilution and then titrating up or down as needed. We like you to remain within already predetermined primary antibody dilution. 

Next, you'll want to go ahead and assess your target brightness, and we recommend that target brightness counts fall within a range of 10 to 30, except for Opal Polaris 780 because it is near the infrared. We recommend that you target anywhere from a range of 1 to 10. This can be done using our one platform, which is free, it's Phenochart. You can go ahead and then look at the normalized counts and then you can adjust your Opal titration from there. 

Once you get done optimizing your tonsil monoplexes, we recommend transitioning those dilution factors onto project tissue samples and reoptimizing as needed. We recommend not really adjusting the primary and body dilution factor and trying to titrate out your Opal.

Considerations for Library Development