Multiplex Fluorescence Immunohistochemistry using the Ultivue InSituPlex Platform on the Leica Biosystems BOND RX

Hello everyone and welcome to today's live broadcast, “Multiplex Fluorescence Immunohistochemistry Using the Ultivue InSituPlex Platform on the Leica Biosystems BOND RX,” presented by Traci deGeer, Global Product and Innovation Manager BOND RX, Leica Biosystems and Alex Klimowicz, Principal Scientist, Boehringer Ingelheim Pharmaceuticals. I'm Alexis Krauss of Labroots, and I'll be your moderator for today's event. Today's educational web seminar is brought to you by Labroots and sponsored by Leica Biosystems. For more information on our sponsor, please visit leicabiosystems.com. 

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This presentation is educational and thus offers continuing education credits. Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits. I'd like to now introduce our presenters, Traci deGeer and Alex Klimowicz. For complete biographies on our speakers, please visit the Biography tab at the top of your screen. Traci, you may now begin your presentation.

Thank you so much Alexis. I'm an open innovation manager for the research platform here at Leica and one of the things that I do in my position is bring companies like Ultivue to Leica to form partnerships so that we can bring innovation to the researchers that we work with. One way that we do that is through the BOND RX system, which is our research platform. My presentation today is going to walk you through a little bit about the BOND RX platform and how that works with open innovation to allow researchers like Alex to bring things onto their system that are brand new. Let's get started and I'll show you a little bit about the system and how it works with our open innovation partners like Ultivue. 

The Freedom to Discover

One of the principal things about the platform that we bring to our customers is it gives them the opportunity to explore new ideas, accelerate test programs, and then, if you're talking to people like Ultivue or some of our other open innovation partners, they look at it to commercialize discoveries. That's something that the open software that we're going to talk about in just a little bit allows them to do with the system. 

Explore Your Ideas

Now let's start with, explore your ideas, and when you're talking about a piece of automation, what do you mean when you, say, “explore your ideas?” 

Tests Automated on BOND RX

One of the things that you can do is if you talk about a system that you can bring your third-party assay onto, and if you think about it, you see lots of little things in the check boxes. You see in many fluorescents, you see circulating tumor cells. You see TSA, which is tyramine, we're going to talk about that in a little bit. You see TUNEL. You see multiplexing. All of those different things. All of those things can be done on the same platform. 

Do you see that unique little box up in the corner that says “your test here?” Well, when we go to talk to an open innovation partner, this is somebody that's got something new and novel that not many labs are running right now in the research world. We go and actively look for those people and what we want them to do is to push the instrument to their limits and try to get their assay automated on the system and their assay becomes the next “your test here.” In this case it happened to be Ultivue that has the “your test here” button. And that's what we've done with them and you're going to see some other partners that we've done that through the years with and there's a whole pipeline of those partners that play on the system.

Things that we put on ourselves are things like the Novocastra antibodies. We've put Kreatech probes on there and we have our own detection chemistries that we run on the system that are the exact same between the clinical instrument and the research instrument. What we're trying to do is find new ways to bring things onto the clinical system through exercising what we can do on the research platform. 

Technology Introduction Timeline

There's a long history of doing things like that. If you look back to when the very first BOND RX was released in 2011, over the last seven years or so, we formed many partnerships and we've gone through many iterations of software, each time making the system a little bit more flexible. 

Open Possibilities

That opens all kinds of possibilities if you happen to be a researcher. It allows you to do things like break apart probes. It allows you to do all kinds of fluorescent multiplexing. It allows you to do IHC. People have tried all kinds of things on the system. 

Endless Customization Options

You get an endless amount of options that you can play with. That's one of the things that makes my job easy. I can walk into a laboratory and talk to a pharmaceutical company or a biotech company that's got this great idea, and ask them, “have you thought about automating it?” We can kind of play around to see if there's some way that we can make that happen. 

Pre-Staining Preparation Customization

You can do all sorts of freestanding customization. You can play with heat. You can play with, deparaffinization. All of those are things that Ultivue had to look at as they were putting together their new assay. Each company goes through things a little differently, and each one works to get a protocol that they can put on the system and make work. Some of them start with assays that are manual. Some of them start from the ground up and build an assay. 

Pre-Staining Antigen Retrieval Customization

Sometimes it's antigen retrieval that makes all the difference. Sometimes it's being able to change incubation times and temperatures. Sometimes it's the way that you wash your slides and each one of those things contributes to how well an assay will work on an instrument. 

Staining Customization

Sometimes it's bringing able to put a third-party marker onto the system, or sometimes it's not being able to run a detection chemistry that you want to run that makes a difference. Now one of the things we lean on a lot with our open innovation partners is they don't start out running a detection kit that we create. If you look at someone like Ultivue, what they do is they take open containers and they actually pour their reagents in it and they put them on the system. Things don't come in a container that we make. That's what each of our open innovation partners does. As a researcher, Alex has the same ability to do something that one of our open innovation partners does.

If he decided he wanted to run a detection color tomorrow but we as a company don't make, he could go anywhere and buy that detection color and he could give that a whirl if he wanted to. That's openness in a system that researchers like to play with. I come out of a research lab and being a researcher, I like to tinker. The ability to tinker on the system is something that makes most researchers happy. You want to be able to put what you want on the system when you want. A lot of times it's about being able to do that more than a lot of things to get your assay to work. 

There were a lot of things that when we started working with Ultivue had to be customized. Some of their reagents didn't like water. You must customize to that. One of the things at the very end of their procedure was that it couldn't sit in water. There's customization that went into their procedure and that's what happens with our open innovation partners. They helped us create our new software that came out and that's one of the things that's so wonderful about working with researchers is they come through and they help us become better. 

Accelerate Your Test Program

The “Accelerate Your Testing Program” is something that we value a lot when it comes to our customers and our researchers and our partners. 

Speed

A lot of times, speed is not something that's as important in research as it is in the clinic, but it does help us get better. A lot of times with the antibodies in the clinical laboratory, you can get those done in about 2 ½ hours. One of the things that the instrument does well is each of the drawers run independently. If we're running something like Ultivue, we can run that on one tray and run something completely different on another tray. That lets the instrument function in like two or three separate instruments. 

Efficiency

It also makes things very efficient. Now the new instrument that just came out has a way of relieving some of the strain on the laboratory by adding some visual management, which is something we didn't have on its predecessor. It has both bottles that flash colors. If you have an instrument that has a bottle fluid that's low, first it will change colors in white, and then if it really gets irritated at you, it will turn the bottle red and then it expects to get some attention for that red bottle. 

The instrument is small now. There is a bench top version of it, but the top-of-the-line instrument is a floor model. Given the size of some of the other instruments on the market, it tends to be a little bit smaller. The footprint is not extremely huge. 

One of the newest things that came on the instrument was a change in the software GUI. Now again a GUI which is a front screen that you look at and it was redone to give a little bit more visual management. You can see your bulk bottles on the front screen. You can see all the reagents that you have loaded over on the right hand side and you can see each individual drawer and what you have on it. That helps you keep track of things on the instrument a little bit better. You also get better auditing controls, which is really nice and it makes it easier to set your runs up.

These were the bulk bottles that we were talking about. This is the visual management. If you've got a bottle that is decommissioned, it looks gray. If you've got one that's in a natural state, it looks just a little bit backlit. If you've got one that needs attention, it'll have a white light behind it, and if you have one that's really in trouble and it gets very aggravated with you, it turns red. 
The bottles can have fluid added to them on the fly. You don't have to stock your instrument to do anything to the bulk bottles, you can just remove them and add fluid to them. Or unscrew the lid and add the fluid to them. That can help keep the instrument running without you having to stop and adjust anything. 

Consistency

One of our hallmarks of the instruments that has worked so well for the researchers over the years, and one of the things that our open innovation partners have found to be a key to success has been the covertiles. These let us do some unique things. One of the partners I work with works with circulating tumor cells. The thing that they have found that helps them the most is the way the covertiles work, because it preserves those very rare cells in place on the slides. You get excellent retention of the cells. It holds everything in place and keeps things from moving around. It also provides a place for the antibodies to incubate. It keeps the cells from drying out, so it's kind of a hallmark of the technology. 

They went back and redid the way that part of their cell spreader works to suit the covertile that was on the instrument rather than asking us to make a change to the instrumentation. It's an interesting way that these things sometimes work together.

Access to New Tests From a Range of Partners 

One of the things that's interesting about the instrument is the way it functions and the way that we've been able to form partnerships based off the technology that we bring to the table so that we can go out and seek other companies that have good technology to bring to researchers. Ultivue is the one that we're talking about today, because they have such wonderful technology, but we have a history of forming partnerships with great companies. One of the companies that we have a partnership is PerkinElmer, who does multiplexing. We have a partnership with a little company called RareCyte, which is a circulating tumor cell company and they are fully automated on the system as well. The company that we're also very excited about today is Ultivue. 

Ultivue has their UltiMapper reagents and they can do their five plex on the system as well in a fully automated fashion. This is one that we were very excited to be able to bring to our researchers and they've been a wonderful company to work with as well. We also have a partnership with a little company called Clearbridge Diagnostics. Clear Bridge is a company from Singapore and they are a circulating tumor cell company. 

Our longest lived partnership is with Advanced Cell Diagnostics out of California and this was our first company that went all the way through our open innovation program and they started out on the BOND RX just like all of the other companies that you just saw. Now they have partnerships based on the research side and on the clinical side with us, so they're on more than one instrument platform with us. That's how these companies go from the open innovation on the research and the openness of the system all the way through to the ability to commercialize on the clinical side. It's a wonderful path for companies and a great collaboration for us as a company, which is why we're so excited to have Ultivue as part of that family of companies. 

The Ideal Balance

It's a wonderful way for us to be able to take the freedom and flexibility that the BOND RX offers and the consistent results that we're able to offer to our partners in conjunction with their wonderful reagents to be able to bring something very special to the researchers today. 

It creates a unique pathway; a pathway from open innovation, being able to offer something unique to the researchers, and hopefully a path forward to bring something very special to the clinic at some point in time. That lets us provide a commercial path for some of our wonderful partners and hopefully provide something unique to our customers and our researchers and hopefully to their clinical patients in the future. Now, Alex, I'm going to hand it over to you and let you tell them a little bit more about our special partner Ultivue.

Thanks, Traci. Let me first just speak to my disclaimers. Leica Biosystems did not sponsor the studies that I'm about to talk to you about. And the opinions expressed in these presentations are my opinions as a scientist, having experience with the equipment and the reagents that are being presented and are not considered an official endorsement by Boehringer Ingelheim of any of these products or services described. 

I'm excited to be here today to talk to you about one of my favorite topics, which is multiplex fluorescence immunohistochemistry, and also to relate to our recent experience using this new Ultivue InSituPlex multiplex fluorescence immunohistochemistry platform on our Leica BOND RX auto stainers. 

Multiplex immunohistochemistry is a very powerful tool. It allows us to visualize the context of target protein expression within human disease tissues. This context is provided at multiple levels from a histopathological context and being able to understand and visualize the expression of an individual target with, say, a singleplex assay with respect to features of disease. As well as adding additional layers of context on top of that by adding additional target stains. 

I'll just point your attention here. This is a piece of Crohn's disease tissue and the histopathological feature that we're looking at is an abscessing crypt. These epithelial cells in the center of the image are starting to lose their integrity. There are immune cells starting to invade into the lumen of the crypt, unlike the other epithelial cells that you see in other parts of the image. When we layer on top of this our biomarker expression you can see in red that our biomarker of interest is highly expressed in those epithelial cells, just the ones associated with that lesional crypt. They're also associated with the lamina propria cells underneath that crypt.

With multiplexing, we can start to understand a little bit more about the context of this expression. For instance, we can look at what type of cells are expressing our target of interest. If we look here with CD68 and 163 in the same channel, look shown in green, we can see that most of these target expressing cells are phagocytes. They express these markers. We can see that there's a high degree of coexpression beneath this lesional piece of tissue. If you look over to the left or to the right of the image, and you look at the phagocytes there, they don't co-express this marker of interest. It's very specific to this feature of disease. 

Now if we want to delve even further into this, we can start to look at maybe some of the molecular biology that's going on in these cells. Here we've layered on top of that Phospho stat 1. Now we can start to understand the state of these cells. Perhaps they're responding to a local burst of interferon gamma and this helps us build a complete picture of the molecular biology of our target with respect to specific features associated with human disease.

Similar to what we see with flow cytometry, the greater that we complex, the more flexibility we have to ask different biological questions of our tissues. It really is an immense power and this grows exponentially with every marker that we have. However, the devil is really in the technical details of how we optimize and develop and run these IHC assays.

To realize the dream of a robust and reproducible accurate multiplex fluorescence IHC assay, it does require a lot of assay optimization. This image here took us three months to develop just as an example. 

Multiplex fluorescence IHC using sequential antibody application and detection

To understand why we need this optimization and some of the technical challenges associated with this, we really need to understand a little bit more about the methods and the reagents that we use. While there are a number of different methods and variations that can be used for multiplex fluorescence IHC, one of the more powerful methods that's out there and the one that my lab relies on routinely is in opal style sequential visualization of tyramide fluors. To better understand the asset optimization challenges and limitations, we need to understand the chemistry associated with this method.

Here I've just shown you a picture. You can see epitope one for our first target antibody and epitope two for our second. Say we're trying to look at Co expression of a biomarker of interest with CD20 as a marker of B cells. And so. We add our first primary antibody to CD20. We add a secondary reagent conjugated to HRP to detect that. And then we utilized tyramide chemistry to covalently link tyramide fluors through an HRP assisted reaction with tyrosine residues in the same subcellular compartment that our primary antibody has bound. 

This creates a covalent bond linking the tyrosine residue to the tyramide fluor. I've represented that here with the green circle as our, say, maybe an FTC tyramide converting to this triangle. Then this covalently binds to the tyramide residues in that subcellular compartment. Now, using an opal cell method, we'll use heat-induced epitope retrieval to strip away the first set of antibodies, leaving behind only the covalently bound tyramide fluors. Now we can start our second or look for co-expression of our target of interest. Here we apply the primary antibody, our second secondary reagent, and our second tyramide fluor. Once that's covalently bound, we can counterstain and visualize co-expression in the same subcellular compartment here of our red target and our green target.

My lab primarily uses this on using a homebrew developed method, very similar to the OPAL Multiplex IHC workflow, using our Leica BOND RX autostainer. It starts out very much like a standard immunohistochemistry assay. We run through deparaffinization, antigen retrieval, and then we start with a first primary antibody, our first secondary antibody, our first and then we use heat-induced epitope retrieval for approximately 10 minutes to remove that first set of antibodies, leaving behind only that tyramide fluor, and then we repeat this cycle one or two more times, depending on the level of multiplexing that we're looking for. We counterstain with Hertz, and we melt for fluorescent microscopy. Now, this is a powerful system. We use it routinely. It does have some limitations. Now, we don't normally run into many of these limitations as, say, sequential heat retrieval effects on tissue and epitopes. As you heat retrieve a tissue, you can start to destroy the histology or the morphology of the tissue, and you may be impacting these epitopes negatively. But with a two or three target co-expression assay, we're not going above 40 or 50 minutes of heat and just epitope retrieval, so it's not really a big problem. There's also a limit to the number of fluors that can be resolved. You know, once these tyramide fluors are covalently bound to the tissue, I'm not sure that it's possible to easily get them off. Once you put your PTC down, it's taking up that spectral area, and you're going to be able to resolve this against the other fluors that you're adding. Certainly, you can do this with things like multispectral imaging, but if you're like me and you have a standard fluorescence microscopy whole-slide imaging setup, you're really limited to DAPI, FITC, Cy3 and Cy5 channels. This is not really a big issue.

What does impact everybody, and what I'm going to focus on, is assay optimization. And this comes down to that chemistry that I talked to you about. When you’re multiplexing, you're layering one Tyramide assay on top of another. And because the Tyramide fluors covalently modify the tissue, you can have some adverse interactions. In the next few slides, I'll walk you through some of these challenges. The first, and this has to do a lot with the type of work that we do in the lab, where we're really looking at understanding the expression, the cell type specific expression of our targets of interest. We use cell type specific protein biomarkers. For anyone familiar with doing IHC on these, such as CD3 for T cells, CD20 for B cells, or pancytokeratin for epithelial cells, you know that they have a robust immune reactivity. Because they are a biomarker of that cell type, they're highly expressed on that cell type. And, because they're so well studied, there's been an evolution of reagents, and we now have very high affinity antibody reagents available to us.

We get these beautifully robust immunohistochemistry assays. Now this can provide some challenges when you're building a multiplex assay, and I'll just describe that here. Imagine we're looking again at CD20, we put our primary antibody down, we put our secondary reagent, and now we add our tyramide fluor. And so, this tyramide fluor is now going to covalently bind to tyrosine residues in its local subcellular compartment. And because we have such robust demeanor reactivity, we've now taken up the majority of these free tyrosine residues. And so, after we strip away with heat, our first set of antibodies, we come in with our second set, our target of interest here in red, and our second secondary in Tyramide, because we have a limited number of tyrosine residues available, we may get this false impression that we have very low immune reactivity or low expression of our target of interest within this CD20 positive subset of cells. In addition, there's another way in which tyramide chemistry can interfere with subsequent steps, and this has to do with the modification of epitopes. Even if we have a nicely balanced CD20 assay here, which is not taking up all the free tyrosine residues, if our second or third epitope contains a tyrosine residue within that epitope, it can now become covalently modified. And so, after heat stripping away our first reagent, our first antibody set, and we add our second antibody, now it may not be able to recognize this tyramide-modified epitope, or it may recognize it with a lower affinity. And again, this may give us a false impression that we have weak expression or no expression of our target of interest in this, say, CD20 compartment. Now, how often do you think you have tyrosine residues in an IHC epitope? It's more often than you think.

There's a nice study by Somperin and colleagues a few years back, and they were interested in better understanding what makes a good IHC epitope and the nature of IHC epitopes. And so, what they did is they did epitope mapping study on the most used antibodies for breast cancer diagnostics and treatment, including estrogen receptor clones, progesterone receptor clones, the D07P53 clone and the MIB1Ti67 clone. When we take all those amino acids from all those epitopes and you put them together and you look at the frequency of each of those amino acids in these successful IHC antibodies, you see that's not evenly distributed across all the amino acids. And there's particularly an enrichment. And so, what you can see is that tyrosine residues are, in fact, the third most highly enriched or frequent constituent of an IHD, a good IHD epitope. This is something that my lab is currently working on trying to better understand it and further optimize this so that we can better understand how to create epitopes specific for the generation of IHD antibodies.

The bottom line is it doesn't matter which of those challenges you're facing, you end up with a similar outcome. And you can see that outcome in this example. And I'll just quickly preface this. For the rest of the images in the presentation, you'll see that I've changed all the black pixels in the background and inverted them to white. And I find this a lot easier to see low-level immunoreactivity and to better be able to see more sensitively co-expression. So here, when we look at red and green co-expression, instead of looking for a shade of yellow, it comes out as black, and so it's very easy to see. What we're looking at in the left panel is an assay, and we're just looking at our target of interest here, but we started out with an anti-CD20 antibody, We did the heat stripping, and now we're looking at the anti-target one. And we're just visualizing target one in red. And in this piece of ulcerative colitis tissue, if you haven't looked at the controls, you'd think that our target of interest was most highly expressed in the area of mucosal damage and perhaps in some of the vessel structures deeper within the tissue. Whereas, in fact, when we run our control -- and here, this was running an isotype control instead of anti-CD20, followed by our target 1 -- you can see that the highest degree of immune activity is, in fact, in the germinal center B cells in this gut-associated lymphoid tissue, and it's really striking the difference between these two images.

Now, certainly, there are several different ways that we could have troubleshooted this -- or troubleshot this. Sorry. What we ended up doing and what worked well was simply diluting out our anti-CD20 antibody, which was added first. So, we went from using a one-to-one concentration in a ready-to-use antibody, followed by target one on the left, to diluting it down to a 1:15 dilution of the anti-CD20 on the right. And you can see the big impact that this has had on our visualization of our target of interest in red. This did not have an impact on our CD20. As you can see in green, the CD20 immune activity still looks nice, even as it's diluted. And we can see that nice black co-expression that I was talking to you about on the right panel, showing that our target is highly present in CD20 positive cells.

While this tyramide-based sequential visualization is a really powerful assay system, it does have some drawbacks. Certainly, using these tyramide fluors to develop co-expression assays, particularly when you're looking at co-expression in the same Sub-cellular compartment requires a substantial investment of time in assay optimization and troubleshooting for any given combination of antibodies, and so you're optimizing things like your primary antibody, the order of the primary antibody application, which fluor am I using for which target, the concentration of your primary antibody, your tyramide fluor, and... I think most unfortunately is sometimes you have to re-optimize one of your really well-developed three-target assays when you want to replace one of those targets with a new target, and you realize that putting that new antibody in that position just doesn't work with the rest of your assay, and you're getting some of this interference, and so you have to redesign and re-optimize the whole thing. There are also certainly some limitations as to how many targets can be visualized, particularly, but you're not particularly susceptible to these if you're using, again, as I mentioned, the standard fluorescence chemistry whole-slide imaging setup, as we do, where we're really limited to, you know, a DAPI channel, a FTC, Sci-3, Sci-5. There are some ways of getting around, you know, the limitations around targets -- number of targets. These require more time and specialized equipment, such as multispectral imaging, microscopy, or something like multi-slide ion beam imaging, not something that we will be using in my lab right now.

What can we do to streamline this process? What can we do to increase our ability to multiplex using standard equipment? This is where the Ultivue InSituPlex assay comes in. So, this assay system, which has been optimized for use on the BOND RX autostainers, is an alternative fluorescence multiplexing platform. And the big difference here is that this system replaces the use of a secondary antibody reagent, as well as the tyramide fluors, and it replaces this by barcoding the individual antibodies. So here on the left, you can see the different colored antibodies representing antibodies that are specific to individual targets. And each of those target-specific antibodies has a unique DNA barcode. And this barcode is what we're going to use to visualize eventually our signal as the antibodies bound within the tissue. You put this on the BOND; it uses a very normal dewax and retrieval system. Then you can add all your antibodies all at once, because they are unique in that they have these distinct barcodes on each of them. So, a single staining step, which simplifies, you know, putting your reagent wand together. Next, we have amplification.

Here's an enzymatic amplification of the DNA barcode. And again, this is a single step that we're doing as opposed to sequential rounds of stripping away your primaries and secondaries. And then finally, the detection step is also a single step. Now you're looking at a mixture of barcode-specific probes that are each labeled with a unique fluor and in this case we're looking at four fluors that can detect our four targets of interest all in one step in the coverslip and we can image this in a fluorescence microscope. As it stands this is a nice assay system. It's a powerful system that allows us to look at four targets simultaneously using standard microscopy setup and on an autostainer like the BOND RX. There's additional power in this assay. And this comes as the company is developing more barcodes. You can imagine you have more barcodes available. Now you're able to put on eight unique antibodies all at the same time in the first step. You're able to amplify all eight at the same time. And then when it gets to detection with the probes, you're adding four at a time because that's the limit of what you can distinguish with your fluorescence microscopy setup. You image the first four, you remove the coverslip. You remove the original probes. You add a second set of probes to the second set of antibodies that are already in place and already amplified. And now you can visualize those next four targets using the set of probes on your standard fluorescence microscopy setup. Take those images, merge them based on the common nuclear counterstain, and now you have eight targets, nine color, or nine-channel immunohistochemistry. There's a lot of potential with this assay that I'm really excited about. I think one of the other things that this technology does by removing that secondary antibody step and the tyramide step is it avoids the challenges that we associate with tyramide chemistry, which enables effective co-expression of targets within the same subcellular compartment.

If we're looking at something in the membrane compartment, we can now layer on two or three targets without having to worry about that interference that I showed you with our CD20 assay. And so, all you need here is no special equipment. You can use your BOND RX auto stainer. You can use your standard fluorescent slide scanners. All you need are the Ultivue-specific reagents, so your barcoded DNA -- or, sorry, your barcoded antibodies and the reagents associated with the amplification and detection. Two InSituPlex kits have recently been released with predefined antibody combinations. They've been validated and optimized for use on the BOND RX  autostainers, and they are the UltiMapper Immuno-Oncology PD-L1 kit, which looks at PD-L1, CD68, CD8, pan-cytokeratin and SOX10 in the same channel, so that this assay is useful both for carcinomas as well as melanomas. There's also the PD-1 kit, which looks at PD-1, CD45RO, CD3, and pan-cytokeratin SOX10.

Over the next few slides, I'm going to walk you through our experience being involved in the alpha and beta testing of these two kits, as well as the development of some of the newer kits that LCDU is planning to release. I'll start out with a summary of our feelings on how it was to use this assay system. And this is not all from me. This is also from discussions with Diane Mears in my lab, who performed all this beautiful immunohistochemistry using the LTV platform. And so, from her perspective, the LTD method certainly streamlined multiplex IHC workflow on the BOND RX. That first assay that I showed you with the tyramide, that was an overnight assay that she would run. She'd come in in the morning, and then she'd manually intervene and do the final target in the morning. Now we're down to about a five-hour assay that looks at four targets instead of three. So, dramatically reduced the amount of time involved. It also streamlined how you build your assay on the bond, and that it was very simple to add all your detection antibodies in one reagent, your amplification, and your visualization. It made setting up the bond bonds a lot easier as well. What I was really excited about was the fact that the LTVU methods allow for the use of a Cy7 or a near-infrared channel, which is not typical or easily detectable using HRP and something like Tyramide Cy7. So, I have to say, as soon as I saw Ultivue present for the first time, I came right back to the lab and I thought, we can do this. And I purchased some Tyramide Cy7, and we tested it out in a standard HRP assay using a pan cytokeratin antibody, and we couldn't see anything. So, something about how Ultivue is developed is the use of that channel really allows us to take full advantage of that near-infrared range. The validated OT-View assay kits were very easy to use, very well optimized. And I say this, that Diane looked at the instructions, and was able to very easily go through. And the optimization, I think we can speak to based on the imaging. We imaged several different tissues, including inflammatory bowel disease tissue, inflamed skin, inflamed tonsil, diseased lung tissue, as well as cancer tissue. And it didn't matter which tissue that we were using or where its source was from, we were able to use the same exposure times and get very comparable results. So, in that respect, I think it was very well optimized, and we were able to see three target co-expression in the same subcellular compartment, which really impressed me.

So that brings me to the next point, which is that yes, co-expression of multiple targets in the same compartment does not appear to suffer from some of the challenges we've already with Tyramide-based multiplexing. The caveat here being that my lab did not develop this assay, and so I don't know what was involved in the optimization of the assay at Ultivue. But having spoken to a few people in the lab at Ultivue who had also had a lot of experience with Tyramide chemistry, it was a lot simpler to set up this assay. But that's certainly a question that you can bring up with Ultivue in the future. Also, you're not just getting a kit. The Ultivue support staff were responsive and highly skilled in both the arts of municipal chemistry and whole slide imaging. And most importantly, the data that we generated is beautiful and interesting. I'll walk you through in the next set of slides some of the examples of the results that we were able to obtain during alpha and beta testing.

So first off, I'm going to start, and really the focus here is going to be on the co-expression of targets within the same compartment. Because again, this is one of the things that we do on a routine basis in the lab. And this is something that we really have challenges with and spend a lot of time optimizing with ceremide. So, we're starting out, we're looking in some gut-associated lymphoid tissue here in a Crohn's disease patient. So, this is a tertiary lymphoid structure. There's no epithelium in the area, so we're not looking at pan-cytokeratin in pink. But what you can see right now with PD-1 immunoactivity in red, that there are some high-expressing cells with more robust immunoactivity, and there are some low-expressing cells, and sort of a range in between. When we layer on top of that CD45RO, you can see this is co-expressed in the same membrane compartment in these same cells. There's a high degree of correlation between PD-1 expression and CD45RO expression in terms of the same cells. But what's most impressive is when we look at the CD3. So, this CD3 signal is exceptionally robust. And yet, despite that, we can see that these CD45RO PD1 positive -- CD3 positive T cells maintain this dynamic range of expression. We can see very high expressors and very low expressors. We haven't bleached out that signal like we saw with that CD20 assay that I showed you earlier. You get this beautiful co-expression in the same subcellular compartment of three different markers with varying degrees of immunoreactivity. And so, we can see this at a higher magnification in another piece of Crohn's disease tissue underneath some epithelial barrier damage. Here we can see, again, you have some cells that have high immunoreactivity for PD-1 in red, as well as some low immunoreactivity. And you can see these beautiful panchic membrane spots of the assay. When we layer on top of that CD45RO, you can see again that there's a high degree of correlation between a cell expressing PD1 and CD45RO, suggesting that they're memory cells. And again, we layer on top of that the CD3, and you can see that beautiful co-expression, all in the same compartment within the same cell type.

Shifting gears. We now move to the PD-L1. And so now we're looking at some mucosal tissue from Crohn's disease. And so, we have epithelium, and you can see the pen cytokeratin nicely identifies this in pink. This is in the Cy7 channel for those who are interested. And so really nice immuno-activity, really clear signal here. And so, what I'm showing you here outlined in yellow are areas of a loss of epithelial barrier integrity. So, the mucosal barriers breaking down in the lumen of the gut and the host tissue are now allowed to interact more directly. And so, there's an inflammatory response associated with this. And you can see the epithelial cells have a very disorganized structure as well. On the right-hand side, we're just a few sections below where the actual epithelium are, but I have outlined in yellow where there is a loss of epithelial barrier integrity. And so now when we layer on top of this our CD8 signal in dark blue, you can see these cells don't particularly accumulate around the areas of barrier injury. And this is contrasted by the CD68 positive phagocytes. And here you can see that they're highly concentrated in these areas of epithelial barrier loss, and they're clustered right around the epithelial cells on the left. And what was most interesting to me is that when we look at TDL1 immunoreactivity in green on top of that, it's highly correlated with the cells that are clustered, the CD68 positive phagocytes that are clustered around the epithelial barrier injury. And as soon as you start to move away from the barrier injury, the CD68 positive cells are now PD-L1 negative. What's also nice to see is that you have weak immunoreactivity to PD-L1 in the actual epithelial cells themselves. If you look at the top of that crypt structure, there's a very faint immunoreactivity for PD-L1 in the epithelial cells, in these inflamed and activated epithelial cells. And this is something that we've seen with other markers that these activated epithelial cells start adopting some antigen presenting cell phenotype. They start to express a little bit of CD68 and MHC class 2, and now we're able to show that they're also able to express very sensitively some degree of PD-L1. And so, this is just another example of a lower magnification of an epithelial barrier injury and so you can see the pink epithelium moving across the screen and then where I start that yellow line that's where we've lost the epithelial barrier almost completely and if you follow that yellow line that's the interface between the host tissue and the lumen of the gut and moving out into the lumen of the gut are a number of immune cells trying to create a barrier and heal this wound. And so now if we look at CD8 positive cells, you can see they're sort of clustered in and around that area of injury as compared to how they are -- they're at a higher concentration there than they are in the more regular lamina appropriate at the bottom left of the screen. But that's nothing compared to what you see with the CD68 positive phagocytes. They're highly concentrated in that wound bed at that interface between the host and the lumen of the gut. And just as we saw in the previous image, these cells are all PD-L1 positive. And this is robust immune interactivity for both CD68 and PDL1, and we're still able to see the nice black co-expression. There's no interference between those two channels, even though they're both exceptionally robust here. And despite this robust immune interactivity, if you look at the image, you can still see those little blue CD8 positive cells interspersed quite cleanly in that huge concentration of CD68 and PD-L1. So really beautiful for co-expression in the same subcellular compartment.

That's not to say that all your PD-L1 positive cells in inflamed tissue are CD68 positive. If we take a look just a fraction of a millimeter away from that area of tissue injury, and we look at some gut-associated lymphoid tissue, this is a tertiary lymphoid structure, so we see an accumulation of CD8 positive cells in here. We also see some CD68-positive phagocytes. And I think, interestingly, you see PD-L1 immune reactivity. But unlike what we saw at that area of mucosal barrier damage, what we're seeing here is that while there are a few CD68-positive PD-L1-positive cells where there's co-expression, the bulk of the PD-L1-positive cells are CD68-negative. I want you to use your imagination. Instead of using this four-target assay, we were using eight targets, so we could better define what those PD-L1 positive cells are. And so here we're just going to move down slightly in the tissue. I don't, unfortunately, have serial sections of this tissue, but if we look at a gut-associated lymphoid tissue just a little bit down, using the new ATP antigen-presenting cell, TIP that's being developed by LTVU. You can see this beautiful CD20 meter activity in this gut associated lymphoid tissue identifying B cells in pink. We have CD68 and CD163 now in the same channel as a pan phagocyte marker. 
You can see they're all clustered in this area as well. We also have CD11C in dark blue. And you can see there's some beautiful dark purple co-expression between some of the CD11C cells and the CD68 cells in the gut-associated lymphoid tissue. There are also some CD11C-only positive cells in this area. We add on top of that our green MHC class II. While there is some co-expression between MHC class II and the CD11C and CD68-163 positive phagocytes, By and large, the bulk of that MHC class II immunity reactivity is in a CD68 negative population, very similar to what we saw with the PD-L1 positive population in gut-associated lymphoid tissue. If we were running these assays on top of each, now we would merge this together, and we'd be able to better understand, is this MHC class II CD68 negative cell population the same as our PD-L1 positive population?

This is just a low magnification view, looking at the tissues that we were using for the alpha and beta testing of these kits. So, we were using matched non-lesional and regional Crohn's disease ileum, as well as Crohn's disease colon. At this low magnification, you can nicely see the pan cytokeratin in pink. Where you see lots of pan cytokeratin, you see this green PD-L1 immune interactivity. You can see it in both the Crohn's colon as well as in the Crohn's ileum. And you can also see, at this low magnification, PD-L1 immune reactivity in gut-associated lymphoid tissue. It's most prominent when these galls are very close to that epithelial barrier injury on the bottom right in the Crohn's disease colon, and it starts to get weaker as you move further away, as you see in the lesional Crohn's disease ileum and the non-lesional Crohn's disease colon. So, what we see here is that PD-L1 is expressed in CD68 positive cells proximal to epithelial barrier disruption, but that PD-L1 is also expressed in CD68 negative cells within gut-associated lymphoid tissue.

Just to summarize, I think you can tell I'm very excited about this InSituPlex technology. It was a very easy-to-use kit to run on the BOND RX, which allows us to increase our throughput. using automation and increase the reproducibility of the assays. It reduces the complexity of running multiplex IHC's assays, particularly in our lab where, you know, the methods that we've developed took quite a while to put together. It gives us that additional target in the near-infrared range, which is a powerful addition. You know, adding that fourth channel, the fourth target really gives you additional insight into your tissue biology. I'm also very excited because it addresses some of the challenges that we associate with PureMine co-expression, particularly when you're looking at co-expression in the same subcellular compartment. While we didn't develop these assays, and so I don't have a good hands-on experience showing that it's streamlining the development of co-expression assays in conversations with all of you, that does appear to be the case. This enables more flexibility to obtain flow cytometry-like cell expression analysis in tissue, just like you saw with those PD-1 positive memory T cells be able to look at three targets in that numbering compartments to find a unique cell type. We're working to develop a custom 4-Plex kit with Ultivue to look at some of our targets of interest using InSituPlex and define workflow ahead of the development of additional barcodes in this greater than four target IHC that we're very much looking forward to. 
I'd like to acknowledge Diane Mears in the lab who did all the immunohistochemistry that you saw from that first tyramide assay all the way through to this last image. And to thank the people at Ultivue the Director of Business Development, Louis Levy, for building this relationship with us and allowing us to work with them around the alpha-1 beta testing on the BOND RX, as well as some of the scientists we've worked with from Ultivue, Amanda Bars, and Kitir Patel. And with that, I think we can open the floor to questions, except perhaps something about these PD-L1 positive CD68 negative cells, which I'm still not sure what they are.

Thank you, Traci and Alex, for your informative presentation. We will now start the live Q&A portion of the webinar. If you have a question you'd like to ask, please do so now. Just click on the Ask a Question box located on the far left of your screen. It will answer as many of your questions as we have time for. Let's get started. Our first question is, why wouldn't you use four conjugated primary antibodies or four conjugated secondary antibodies to avoid some of the challenges associated with tyramide for multiplexing?

I think I can field that question. I think this really comes down to being able to visualize targets very sensitively. A lot of the work that we do using looking at inflammatory bowel disease and some of the targets associated there, they are very low weak expression assays. If we couldn’t use the amplification that we get with tyramide or with the Ultivue we wouldn't be able to detect these using a multiplex assay in formal and fixed paraffin embedded tissue. This may be possible, maybe, using frozen tissue where you can do confocal microscopy and maybe sensitively detect, but using a higher throughput formal and fixed paraffin embedded assay, it's just not feasible.

Now our next question. How do I know if the Ultivue kits will work on my fluorescence microscopy setup?

I'll go ahead and take that one. When you work with Ultivue, they have a set of guidelines that they can provide to anyone who would like to stain with the in-situ plex, and that provides the filters and all the information needed to set up a microscope. For actual reading of the stains, they can provide you with the names of the microscope vendors that they worked with, as well as all the filters set at.

If I could just add to that, Traci, I think it's also important to understand whether the light source that you have and the camera that you have are efficient in that near-infrared range. to ensure that you're able to detect in that Cy7 signal. And just to add to that as well, we've tested this on several different microscopy platforms in the lab. We're using a Leica Versa instrument, and it works nicely there. We've tested this on an Aperio ScanScope FL from Leica. This works nicely, but we're just not able to use the Cy7 channel. And in working with Ultivue, they've scanned slides for us on the Zeiss ActioScan as well as the of the Polaris multispectral imaging microscopy setup. So, really, we've seen that it works nicely on several different setups.

Now, how much time does it take to optimize a multiplex IHC assay using the Ultivue technology?

I can field this one, Traci. Well, right now, as it stands, the commercial kits are preset. And so right now it takes no time at all. You buy the kit and you have this beautifully optimized assay. And as I mentioned, we found in our hands, looking at several different tissues, that it was well optimized assay and that we had similar immunoreactivity on many different tissue types from different sources. At this point, I think that's what you're limited with. If you want to start optimizing or building assays that are using your own targets of interest, that’s something that I think you can do right now, working in a partnership with Ultivue and their services lab. But as it stands, yeah, the assays are out-of-the-box.

We are getting so many good questions, and today it looks like we have time for a couple more. Let's move on to our next one. Are the commercially available kits only for use in human tissue? And, if yes, are there plans to develop kits for mouse tissue analysis?

I'll go ahead and take this one, Alex. The commercial kits today are only available for use on human tissue. In talking with the development team from Ultivue, there are plans to work on kits for mouse tissue. One of thing to keep in mind if you talk to the development team at Ultivue, they do provide services where they will develop custom antibodies and custom kits for anyone who is interested. If you have special needs, one of the things that you can do is reach out to Ultivue directly and they'll work with you to develop a kit that suits your need.

Would this technology allow us to identify dim versus bright expression of T-cell activation?

That's a great question and I think you can if you look back into one of the previous slides looking at those CD3 positive PD-1 positive, CD-45RO positive cells, I tried to emphasize that you can see some stronger expressing cells and weaker expressing cells, both for PD-1 as well as CD-45RO. I think with some image analysis, which we haven't gotten around to doing yet, you should be able to identify different populations of T cells based on the TIF as is. Very similar to what you can do with flow cytometry.

We have time for one more question. How does this fluorescence multiplex system compare to the Ventana 5 target chromogenic assay?

That's an excellent question. One of the things to keep in mind is that the Ventana five-plex chromogenic assay is tyramide-based. There is a very big difference between the way that the InSituPlex and the five-plex chromogenic work. The five-plex chromogenic originally came out for their research platform. It does have its basis in the tyramide fluors. That is the biggest difference between the two kits. If you think back to the original slides in Alex's presentation that show some of the pluses and minuses of working with the tyramide fluors, that's where you're going to find the largest difference in the two technologies.
Thank you again, Traci and Alex. Do you have any final comments for our audience?

Thank you so much for attending, and please feel free to keep questions coming and let the elaborate team route those to us if you have any additional questions that we didn't have time to answer today, and it was a pleasure to present for you.

It was a real pleasure to be able to present to you, and yes, I'd be happy to answer any additional questions. I'd like to thank the audience for joining us today and for their interesting questions. Questions we did not have time for today and those submitted during the on-demand period will be addressed by the speakers via the contact information that you provided at the time of registration. We would like to thank LabRoots and our sponsor, Leica Biosystems, for underwriting today's educational webcast. This webcast can be viewed on demand through May of 2019. LabRoots will alert you via e-mail when it's available for replay. We encourage you to share that e-mail with your colleagues who may have missed today's live event.