Enhanced Automated Multiplex Capabilities on Leica Biosystems BOND Research System

Hello everyone and welcome to the Enhanced Automated Multiplex Capabilities on the Leica Biosystems BOND Research System. I'm Susie Meyer, stepping in for Jennifer with LabRoots, and I'll be your moderator for today's session. Now let's get started. I now present today's speakers, Damien Cockfield and Dean Talia. Damien is a Global Product Manager at Leica Biosystems, and Dean is a Senior Scientist at Leica Biosystems. For a complete biography on the two, please visit the full biography card for them in the presenter box on your right. Damien and Dean, you may now begin your presentation.

Thank you, Jennifer. There is an ever-increasing demand to derive more information from limited biopsy samples. Multiplex staining provides a means of visualizing many targets in a single tissue section with the ability to accurately resolve spatial relationships between targets. It's a vital tool in establishing expression profiles of immune checkpoint markers in the tumor microenvironment. The BOND RX and the BOND RX research systems from Leica Biosystems capabilities have been expanded to further support the need for automating chromogenic multiplexing in tissue. Over the next hour, my colleague Dean and I will go over the multiplex capabilities of the BOND RX research systems and how it supports chromogenic and fluorescent multiplexing, show you how to use the new features of the BOND RX 7.0 research software to support multiplexing through a demonstration on the instrument of the new software, and Dean will present a worked up examples of how to automate six-color chromogenic multiplexing on the BOND research systems.

As a brief introduction to the BOND Research System, the real value of automated standards in research comes from two key areas. Flexibility, the ability to design assays in your own way, and performance, which is around the ability of ease of use, rapid processing, and consistency of performance. If all designed correctly, it allows you to build assays that you can trust. While the BOND RX research system can support a myriad of applications, including IHC, ISH, and emerging tests, today we'll focus more on how it can support your research using multiplex technologies. So as a starter, we'll go over what is multiplexing, which I'm sure you're all aware of, but just so we're all on the same page throughout this discussion. Multiplexing allows multiple markers to be stained within single tissue sections. It uses combinations of different markers, chromogens or fluorophores to build a more complete image of the tissue structure and multiplexing methods can visualize multiple target antigens, DNA or RNA or a mixture all of within a single tissue sample and can be further sub categorized as either sequential staining, which we'll go through a bit more later or simultaneous or also known as parallel staining. Why multiplexing is important is because it provides new insights into what is occurring at the cellular level. It allows you to see cell to cell interactions, learn functional states, understand the direct relationships, as I was sort of explained before, between the DNA, RNA and protein. You can show all of those within a single cell or on the same slide. And it allows you to examine areas of interest within that tissue architecture, so spatial arrangements, interactions, co-like It's really the cellular phenotype that's becoming the area of greater interest these days that we're seeing in the research space. In the sort of the original workup of multiplexing, you know, one of the main reasons why people started to use it was just the ability to acquire data from single tissue samples or if you look at some of the popular multiplex applications in the past, especially in the clinical space, they're very much more related to understanding the spatial relationships that are going on. So where, for instance, where the tumor ends and the healthy tissue begins, or where you're seeing different cells staining side by side, for instance, your ratio, like in your Kappa and Lambda stain shown there. Whereas what we're seeing now with the recent advances in multiplexing is this focus on cellular phenotypes.

One good example is of a popular panel or marker combination that we see used in research at the moment is a panel such as PD-L1, CD8, CD68 and PAN-CK type marker. The CD68 by itself will identify macrophages, whereas if you have a co-localization of a CD68 and a PD-L1 within that cell, then you can identify that cell as immunosuppressed macrophages. So that's something that you wouldn't have been able to do previously by doing single stain, multiple single stain applications with all those different markers. So multiplexing is allowing researchers and future pathologists the ability to derive much more information from the cellular, from the tissue structures than what they've previously been able to do when just looking at single stains. In that example I just gave, you'll see a really beautiful stain of a skin melanoma with all four of these markers done using Leica RTUs. That will be shown by Dean's presentation later on.

Multiplex staining historically has been a long process. When done manually, it is generally performed over two or more days, depending on the number of markers and visualization combinations used. Automated chromogenic multiplexing applications have largely been limited to two or three-plex stains. And to conduct more than three plex has required a combination of manual steps, like pre-mixing chromogens, or conducting several processing runs. You stain your first two stains, then you take it off the instrument, re-put it back on the instrument, and stain two more stains as an example, on automated systems to get your desired plex outcome. As a result of these, these workflow inefficiencies often hindered optimal assay design and staining quality. When we think of multiplexing going forward, there is a need to automate the assay itself, but actually when you, and that's where the BOND RX comes in, if you can see on this slide, the part on the left-hand side with the stain. When you think about multiplexing as a whole, digital or the need to derive a far greater amount of information from that section is required. And that's where digital imaging and therefore analysis really becomes far more important. This is relevant for both chromogenic and fluorescent and if anything of the current times has shown with COVID is there's actually been a broader adoption now of scanning of slides and the ability to share those slides widely with other groups by using high-quality scanners and high-quality slide management systems to share those slides and to get that input from a large number of people. And obviously a scan slide looks great if you're going to put it into a research publication or something similar.

When thinking of multiplexing, it's not just the automation, it's not just the assay that we're considering, it's also how are you going to analyze that data and record that data. So, it's really looking at that total solution for an assay application. If we think of BOND RX to start off and how that supports a multiplexing application, it does this in a number of ways. First is around flexibility. Now, flexibility is key when you're creating new assays and new technologies. You want to be able to design an assay that works, or an automated assay that's... Sorry, let's try that again. You want to be able to take your assay and automate it such that it works within the parameters that you define. To do that, you need a product that allows that flexibility to change those key parameters. For instance, being able to choose your protocol steps, being able to use different times, different temperatures, and different arrangements of protocols and steps and assays that you need to run. On top of that flexibility, usually when you're automating, you're not just running the one test. You may be running a multiplex in one slide, you may be running a single stain in another, and you could be doing something completely different on the other side with FFP on one and CTCs on the other. So, you need that flexibility of broad access. From a usability point of view, you then get around to the speed point. You know, as a researcher, principal investigator, whoever you happen to be, your time is valuable. Doing the manual stain, those of us who have done it, it's fun when you first start doing it, but over time you actually just want to hurry up and get to the results so you can kind of move forward. The speed of the instrument becomes very key in terms of how you can then get that processing done. So where you're taking something that used to be done over multiple days, you can now run it in a workday, or potentially you load it up when you're going home for the night, let it run overnight and come in the next morning. And as part of that speed, you also get things like the ability to have plug-and-play reagents and so forth. That it all helps with that usability and speed of use of your system. That's further supported by an efficient system. So the ability to access your reagents at any time when you start to multiplex, you know, if you're having five or six different markers, for instance, you've got multiple detection systems or fluorophores that you're needing, you need to be able to access your instrument continuously so that you can switch between, you know, one test to the next. And that's what the BOND RX really provides, is the ability to continuously be interacting with the system to adjust, but also making it so that those interactions that you have are the ones that are value-added. There's a lot of things within the system that are designed there so that you only need to interact when it's necessary and it tells you when to do that. So, for instance, at EDSC bottles that are lit up you can tell when you need to top up your reagents without you know it'll give you a visual indicator versus you having to go and check on the instrument every half an hour.

And finally, when you get into consistency as you start to get into more and more complex applications like multiplexing every variable that you add it becomes a much harder test or activity to design an assay that's going to be consistent. So a system that is consistent in the way it dispenses reagents in terms of timing and temperatures, the ability to provide consistent flow by use of things like a bond covertile, as well as using, you know, the bond covertile adds a lot of different benefits because it helps to preserve tissue by not having a very vigorous dispense profile, as well as it can be used as an incubation chamber. For instance, if you're having long hybridizations, it gives you that ability to leave that slide and not worry about it drying out or anything like that. So, if you look at everything that's available now, all these little bits and pieces combined help to create an environment that that supports the movement into multiplexing. Multiplexing's kind of broken out into three key areas in what we're sort of seeing now. You've got your fluorescent multiplexing, and we'll go through some ones there, but they're ones that you can see with this sort of the Ultivue technologies, the Akoya technologies, the Crea technologies. You know, and they work both for IHC and all of these have several applications, all very similar applications, but different technology types that are available on the BOND system.

Today we'll talk through some of the chromogenic offerings as well as some of the new supporting chromogens that have been provided by Leica Biosystems now to help support the ability to multiplex up to a six-plex stain on the BOND RX system. And then the other type is sort of what we call multi-analyte High Plex, which is essentially the ability to do sequencing on the slide to gather that next level of genetic information and technologies like the NanoString ones are great for providing those. So, you can see here that a lot of these Leica Biosystems has several open innovation partners. All of these are there to provide new solutions to the multiplexing or to the problems that you're trying to solve when trying to create new data or new test types. So, all of these are similar multiplex applications, but the underlying technologies are different. Ultimately, it's up to you as a researcher to decide which technology is the one that you want to go with. Each of them has their valuable points depending on what the application is that you're trying to do. Our goal as a company is to provide you with all the different applications, as many as possible that are all the different quality applications that are available, get them to be automated so that you can choose the right test for the right application.

So, as I said we'll sort of focus a little bit more on the chromogenic side because this is where the real new advancements have come in for what we're offering here today. So chromogenic multiplexing predominantly as I said earlier in the clinical space you often see either the single plex or a dual plex, the two images on the left-hand side. The new functionality that we're providing now allows up to six-plex chromogenic staining on the same slide on the BOND RX system. So, what that does is that can be used for both just IHC, just ISH, or a combination of IHC and ISH. So, you can be doing a mixture of stains, essentially up to six chromogens. These, by doing chromogenic, they can be viewed under a bright field microscope or a standard scanner like the GT450 or the AT2. So, they can be much more accessible to the broader research environment because most people have a brightfield microscope and they may not necessarily have a fluorescent one. So potentially they're a little laboratory to have the wanting to move into multiplexing but they haven't been able to get that way because of the extra resources required to move into fluorescent. There's now that opportunity to do that chromogenically. Both chromogenic and fluorescent have their values in the research space. It just depends on what your application is. Sometimes fluorescent is the right application for what you're trying to do, sometimes chromogenic is a better solution.

So, when we go through what is new, the BOND RX 7.0 software is really built around this multiplex toolbox. So, it has this chromogenic, enhanced chromogenic and fluorescent multiplexing functionality. It allows six individual markers to be allocated to the slide. And as within that it allows six chromogen applications to be used, but this application can also be used fluorescently. So, you can have standard protocols in there for each of your different fluorophores and you can use those protocols and just switch out the markers versus some other applications where you may have had to design a single protocol for a single panel. Now you can have the same type of protocols per fluorophore and just use your and alternate the markers depending on what it is that you're trying to do. To further support that, you can now, you can also run the chromogenic and fluorescent multiplexing in parallel on both the BOND RX and the BOND RXm automated systems. And to further support the chromogenic side, we've released a Bond HRP Plex detection system and two new ready to use blue and green chromogens to augment the existing DAB and red chromogens that will allow up to four plex or four different chromogens from the Leica system and on top of that a standalone hemotoxin and fast red counter stain to support any type of staining combination that you want to do. I'll show you; I won't spend too much time on the actual slides here because I'll be giving you a demonstration of the software itself very soon. But essentially what this shows you is the initial screens of when you're adding a slide onto the BOND system to allow you to do that multiplex application. So, you can see here it allows the single staining. Sequential multiplex which we consider of doing for instance a stain followed by an enzyme like HRP and then a chromogen and then you might put your next marker on followed by your next enzyme next chromogen. So you're doing them sequentially which usually means within that sequential steps if you're doing something like a for mouse antibodies, for example, and for HRP enzymes, you would probably need to employ either a blocking step or a stripping reagent stripping step to remove those previous markers, but keep the chromogenic stains kept there. By doing a sequential step, what we'll see a little bit later on is these also have inbuilt retrieval protocols and different protocols enabled in them, such that if you want to, for instance, build in different stripping steps throughout your protocols, you can build those in. And they can be different types. So, you can use a blocking step in one application, a stripping step in another, or you can use a combination thereof.

The other type of staining is what we consider a parallel multiplex. A parallel multiplex is when you're dispensing all your markers at the one time. So for instance you may be using a multi-species marker set so maybe one of your antibodies is a mouse, one of your antibodies is a rabbit and so therefore they're not going to cross react and then you follow on with them with your secondaries that are specific to those primary markers so you can move through rapidly. Another example of parallel technology might be they're all mouse antibodies, but they've got individual hapten tags associated with them. So, they can be all dispensed onto the slide together because your subsequent enzymes are targeted to those anti-haptens. So usually what you find is a parallel multiplex is a little bit faster than a sequential. Doesn't necessarily mean one is better than the other. There are just different technology types that you can use. Another one that you may all be familiar with is the Ultivue technology is for fluorescent, is much closer to a parallel multiplex. The Akoya Opal technology is much closer to a sequential multiplex. And both have quite a lot of value depending on what type of application it is that you're trying to use.

We'll see this a bit more soon, but you can see here that what it does is it allows you to have multiple markers added to your one. So, in this instance, you can see a PD-L1 would be your first marker, followed by your first protocol, a preparation protocol, like a DUAX for instance, or deparaffinization, followed by your main heat retrieval. Your second protocol could be your next antibody, in this instance on the screen, it's a PD-1, followed by your next chromogenic stain, with the option of doing a light heat retrieval, for instance. And when Dean presents his part, you'll see some of the things that we've found from some of our exploratory testing around this and how heat retrieval can support same-species multiplexing, same-species marker multiplexing. So, when you get into chromogenic multiplexing, one of the important parts is you want to be able to use several different chromogens. So, Leica offers these plug-and-play chromogens with the DRB, the red, the blue, and the green. And there's some good examples of the individual stains there on the right-hand side, as well as the combination stains, so you can just see the different performance. On top of that, you can see the different counter stains. Some of you may think that a blue Chromogen works best with a red counterstain. It does get good contrast, but we’ve also found that because of the brightness of the blue Chromogen itself and the lightness of the hematoxylin counterstain, that when you're doing your multiplexing, sometimes a hematoxylin counterstain also works very well as well. Again, the purpose is the flexibility. So, you can choose the counterstain that suits your needs. The other important part is the instrument will mix the chromogens for you automatically, and these chromogens are mixed off the slide and so that when they're dispensed onto the slide, they're in their working state, ready to interact with the enzyme, so you get automatic chromium conversion. As we know, some chromogens, they need to go through multiple mixes, depending on the complexity of them. So, it's much better if you can get them into their ready state prior to going onto the slide versus having them mix themselves onto the slide in their sub-components.

The new chromogens themselves. I think everyone would be familiar with the standard DABs in red and the Leica red is a great chromogen and you can find that when you combine the Leica red chromogen with this new green chromogen is they co-localize quite well. So, when they co-localize you get a new color, it's sort of a purple color, so you can see where there's co-localization of staining. We've also seen that the red chromogen works well with some yellows on the market where if they co-localize you can also get a bit of a green stain as well. The blue chromogen both the two new chromogen 100 tests as I said they're ready to use the plug and play the instrument will mix them for you to ensure that they expense onto the slide in its stable state. The green chromogen is compatible with permanent mouthing media, so it means it can go through, and it's soluble in, or insoluble, sorry, in alkyl enzyme. So, you can move it through a, I guess, a standard clinical lab process, which is usually dehydrating the slides and cuff slipping. The blue chromogen, however, like most blue chromogens on the market, is incompatible with alcohol and xylene. So that's just something to take into consideration. If you're doing a multiplex with the blue chromogen included, then we would recommend using either an aqueous mounting, or air drying, or drying in an oven, and cover slipping that way. You can see all the images that are throughout this presentation, they've all been stained on the BOND RX with our chromogens, and they've all been scanned on either the GT450 or the AT2 for the chromogenic stains. And you can see that if cover slips appropriately, the chromogen stain performs well. So, it's understanding the differences in workflow and how they suit your needs and what it is that you're trying to do.

Now I did say that the BOND can deliver up to six mixed Chromogens onto the slide and Lyca are offering four new Chromogens that are plug and play. So, to allow you to get to that extra level, one of the fundamentals around the BON system is around that flexibility allowing you to choose what is right for the application that you're trying to do. So, on top of that we've also included this Open Chromogen Toolbox functionality and what that does is it allows third-party Chromogens to be run automated onto the BOND system. So, they'll be loaded into containers onto the bond system in their sub-components. The BOND can then take them and mix those chromogens. So, if you look at several third-party chromogens, probably the most predominant mixing ratio that you see is about a 50:1 mixing ratio. The example here is showing that there's a 50:1 mixing ratio that will mix and apply that reagent within 10 minutes of mixing. So, you know that you're going to get a very the chromogen won't have had time to degrade or anything by the time it's interacting with the with the compatible enzyme. And this is what allows you to move from that 4-plex offering with the like chromogens to move up to a 6-plex stain and there's some examples again here in this presentation as well as what Dean is presenting to show you how we've actually been able to get up to 6-plex staining onto the BOND RX all in one run.

So further supporting that flexibility on top of that is these products called what we call the Bond HRP Plex Detection Kit. And this is essentially a paired back detection kit with a post-primary. Peroxide blocks are a post-primary and a HRP polymer. So, it essentially doesn't have a chromogen or a counter stain that's within that kit. This is done on purpose because the blue and the green chromogens are both HRP or work with HRP. So, the idea being is that you can pick and choose the chromogen so that you have slots available. As you know, when you get into automation, there's only a certain number of slots available for reagents on the system. So, we wanted to make it as modular as possible so you can pick and choose what you're doing because as you start to add six markers, six different detection systems, you can run out of room very quickly. So, the best way to allow researchers to chop and choose and pick and choose the type of tests that they want to do and do as many tests as they want to do, creating a modular system allows that level of flexibility. The green and blue chromogens can also be used with the bond polymer refined detection. This includes the DAB chromogen, but you can design protocols such that use all the components within that defined kit, refined kit, sorry, but you just substitute, the protocol can substitute the DAB for the green chromogen or the blue mixed chromogen. So, there's full flexibility there depending on what you have.

And finally, you know, once you have your stain, your multiplexing stain, then it's really looking at those benefits of digitization of multiplexing with IHC and ISH. Obviously, permanent copies are valuable. It means you can go back to them, you can look at them at home. You know, one of the great things about remote working these days is, you know, I work very closely with Dean and I can hassle him all the time when he's in the office. He goes and scans something for him and I can go and look at it at home if I need to check something and vice versa. So, we're able to collaborate much more closely. We also have sister sites throughout the world that were included as part of this development program because our assays come from our UK site, and our instrumentation and software comes from our Melbourne site, and the ability to digitize multiplexing images really enabled us to share and progress these projects far faster than what we would have had to do in the past if we were shipping slides around the world. So, it's a really, really great way of collaborating. Plus, you can then go and dig into far deeper by being able to zoom in and look around and use image analysis and so forth to gather that extra information around your multiplexing application. And compare different slides to each other, different case types to each other, all on the same screen versus trying to switch between different slides on your microscope. So, there's so many new opportunities that are becoming available because of digital imaging of slides. So, as I said, everything that we've shown throughout here has either been scanned on the GT450, the AT2 from a chromogenic side, and the fluorescent ones we've also done through the Aperio Versa. Each of these systems have different values depending on what it is that you need on your laboratory yourselves. all of which are easy to use and deliver really great outputs which you've seen throughout this hopefully.

Okay, and so what I did focus mostly on the chromogenic side, obviously the Aperio Versa Scanner is a brilliant scanning system for capturing that fluorescent detail. Obviously, a great part about fluorescence scanning is you can do deconvolution, you can look at one marker at a time, you can do zed stacking. So, there's a lot of things that you can do that are really important when you start looking at fluorescence. But to summarize, I guess, everything we're trying to do with the total solution is, you know, the BOND RX kind of sits in the middle of that automation of moving into that next phase of multiplexing and discovery. We offer great applications on the fluorescent side in terms of lots of different technology types, all of which are supported by fluorescent scans like the Aperio Versa. We also are now offered the full complement of chromogenic multiplexing applications, which are further supported by the GT450 and the AT2. And then in the next-gen technology of things like the High plex, like the NanoString DSP technology, which is semi-automated on the BOND RX. And we're all constantly expanding our offerings in terms of new technologies and solutions that are provided, especially in the multiplexing space. It's such an exciting area to be in.

So that's my presentation. I'll now move over to doing the demo on our instruments, so please bear with me just whilst I switch. So hopefully everyone is familiar with the BOND RX system. If they are not, this is the BOND software that is used to set up tests and applications on our Bond systems. This software, the BOND RX 7.0 software, was released this year and it works for both the Bond RX and the Bond RXm instruments. You can see from the main screen here, I am showing a BOND RX instrument that is connected. And what we have here on the left is what we call three slide standing assemblies of 10 slides each. So, these are where you can load up your different test applications. So, for instance, you could be doing the chromogenic multiplex on the first, SSA, you could be doing a fluorescent test on the next, and some other type of test on the third. And so there are 30 different slide positions. On the right hand side, you can see that there are 36 reagent slots which you can kind of pick and choose what goes on them and they are fulfilled by four slots of nine reagent containers. And you can see I've sort of pre-loaded three products here and I'll just remove one and put one back in so you can see how they register. You can see there one of them has just been removed and then these are scanned by the system through just a handheld barcode scanner. And I'll show you how to do that in a moment.
You then load it on the instrument which will scan the system to recognize what it is.

And there we go. And it recognizes it as a bond polymer refinery detection system. So, the instrument knows exactly what it is, and within this you can check, for instance, the volume of the application, the lot number, expiration date, et cetera. You can see on the bottom there's also the bulk reagents. These are the reagents that you use regularly as part of the stain and stain, and you can see the visual indicators showing the different volumes. And if they were low, for instance, and they needed attention, there would be a, you know, a notification on here as well as what you can't see on the instrument next to me. The instrument bottles themselves are lit up and if they needed notification they would change from a white backlight to a red backlight or a blinking backlight depending on the level of urgency of what needed to be changed. So, you've got those visual cues in terms of what it is that you're trying to do.

We then move to the home screen, and this is where you'll set up your different slides. Now, as setting up the slides, usually what you want to do first is figure out what type of stain you want to do, and you'll add your markers or your markers, which could be a primary antibody, or it could be a DNA or RNA probe. You'll then need to add them to the system so the system knows what they are, and then design your protocols for the application you're trying to build. So, what I'll do is I'll walk you through some of those steps so you can see how the software is and how kind of easy it is to use. So as a starting point, for instance, I will show you, for instance, you have a Bond RTU. Well on that Bond RTU there is the barcode, you scan it as you can see there and the information becomes pre-loaded. So, you can see here all I did was scan a barcode and I popped the product names, the PDL1, the volume, the lot number, etc. And I can add that now and that is now registered on the Bond system and when I load that reagent onto the instrument it'll be recognized automatically.

This is if you want to use BOND's products, so BOND RTUs, and these work really well for multiplex applications because they're already kind of ready to go. They're in a good sort of working solution and they can be used for your stains. Alternatively, what you may want to do is create your own markup. for your different applications. Now you can add a primary antibody, you can add an ancillary, so for instance it could be a secondary antibody or it could be a secondary or a subsequent your own polymer that you're using, you can add DNA and RNA probes. What I'll do is I'll add an antibody here right now, I'll call it CD68, so on the side, CD68. I can choose in terms of if I want to have that marker set up as a sequential or a parallel. So, for instance, if it was a, you know, If I was applying a Pin 4 cocktail, for instance, I may have called it that because all the markers were getting employed would be dispensed at the same time. I can then set up protocols for each of these, so either for a single stain. For instance, if I wanted to stain this just as a standard IHC, and you know what I can't remember what CD68 retrieval is, but we'll just put it down as an ER2, ER1 sorry, Epitope Retrieval 1, which is like a citrate buffer for 20 minutes. I can then also choose stains whether I want it to be a preliminary stain, which is essentially not the last stain or the final stain, which is the last stain. And the reason why we call them preliminary and final is because if you're multiplexing, your protocols are going to look very similar between a preliminary and final, except your final protocol is going to use the counter stain. So that's where they commonly use the counter stain, so that's where the difference might be. And again, there are several protocols that are already loaded in that you could just use preloaded. So, for instance, a preliminary DAB or a red or a blue. I'll just choose a protocol. And I can also choose a final or I can choose not to use a final if I know I'm never going to use this as a final. 
That marker is now set up. And then I can take what we call an open container or a titration container. I can scan that, which you can see here, bond open container. I can then choose from the list of pre-loaded products.

CD68 marker, so the one I defined before. I can give it a lot, if I have a lot, and I can also give it an expiry date based on what it is I want to do. So, for instance, first of the first, 2025. And that will now will be registered so when I load it onto the instrument, let's do that now.

We'll get back to the screen so you can see. I shall remove that kit as before. Load it back on. And there you see it. Now what you can hear behind me is the instrument. So, when you put on new reagents, you can also choose to have those reagents to be level tested. So, we have a liquid level sensor on our system. So, when we dispense slides on the bonds, the way it works is we count dispensers, like most other automated instruments, but we also have a liquid level sensor on our system as well as a secondary level of protection. So, what that does is it'll go into the reagent containers and can confirm the volumes there. The advantage of that is, for instance, let's say you were working one day and you accidentally dropped your reagent, you spilled half of it on the floor, didn't realize, you put the instrument. If your instrument was just detecting by steps or number of previous dispensers, it might think that that reagent is still full. It'll try and dispense onto your slide, you'll get no reagent and you'll get a failure. Our system doesn't do that. It continuously checks for a liquid level sensor.
So, it always knows what reagent is available and it will adjust accordingly. So, if it goes from seven meals all of a sudden, it then goes and checks and goes, hang on a minute, I thought I had seven, I'm now only seeing two. It'll notify you of that and it'll adjust the reagent volume. And if it thinks there's not enough, it won't let you start the slide. Very important because obviously once you start a run, you want to make sure that slide is done, especially when we talk about conserving precious tissue. No one wants to afford it. You can't afford to lose any section, especially if you have very few of them. So, protecting that section is very important.

Okay, so I've set up my reagent. The next thing I may want to do is design a protocol. So, this is where you can come in here and find a bunch of pre-setup protocols. So, there are ones in here, for instance, for the DAB, there's a red protocol, blue protocol. And what I can do, if I copy the blue, for example, I can come in here and copy it. I can then create my own protocol. Damien's we'll call Better Blue protocol, no offense to the people who first created it. And within this I can design my own protocol that suits what it is I'm trying to do I can choose my own ancillary agents for example I can use a post primary that is from a detection system so you can see here that this protocol is linked directly to a Plex detection kit but I could also create my own research detection kit if I wanted to I'm not going to go through that today but the Another option is we have research kits, which is essentially the ability for you to build your own detection system.

So, you can use all your own reagents, your own enzymes, whatever it is you want to do, you can build your own detection system. Once you have that detection system built, you can then build your own protocols that use that detection system. So, for instance, I may have wanted to create a protocol that used you know a wash buffer for instance instead that was a part of that detection system. Within that I can then go and also add washers as I need to you know bomb wash or sort of DNIs water washers depending on what it is I want to do. I can change the temperature of each of my slides so I can change it from an ambient suspense. I can set that to 50 for instance. I can change my incubation time. So, zero minutes essentially just means it's going to sort of cycle through quite quickly or I can have a two-minute incubation, and I can change my different dispense types. So standard 150 microliter dispense to fill the decavitol chamber. We also have these other dispensers called open dispensers, which is decavitol moved off. Yeah, that's useful for instance if you've had a previous step that's had a reaction that's created bubbles, and you need to remove those bubbles. There's also an intermediate step. This is what we call almost, it's a closed chamber one, so good for long incubations. So, it moves the cover tile up so that you don't get any fluid flowing off the back of your slide. So good for incubations there. So, you can change a lot about your different tests to suit what it is that you're trying to do.

So, I can build a protocol that way and I can allocate this as a preliminary. I can also use it for a single or whatever else that I want to do, and I could change the detection system if I wanted to. So, once I've done that, I can save it. And I've now created my new reagents, my new protocols. That's a staining protocol. I can also do the same for pre-staining. So, as I said earlier, there is the ability to use a heat retrieval protocol in multiplexing to support reagent stripping steps. So, there are several preloaded protocols that are on here. You know, if you're doing a standard heat epitope retrieval, you're often using a citrate buffer like an ER1, or you're using an EDTA buffer like our bond epitope retrieval 2 solution here. And you know, if you're retrieving, you're usually doing it at 100 degrees to get the best retrieval possible. We often know from other testing is that once you get below sort of say, let's say 90 degrees, the retrieval that's occurring is minimal. So potentially what you want to do is continue to add different reagents or different retrieval steps into your protocol but not over retrieve your tissue. So, you can come here and you can choose different retrieval times.

So maybe if I'm doing cell lines for instance, I may not want to do 100 degree retrieval because of the different cell lines can often be a bit more susceptible to temperature and I may want to set that at 95 degrees. I can change also the time for how long I want that on there for, or I could go down to, you know, if I wanted a really light one, I could do it at 60 degrees, for instance, since we know, you know, a number of things may dissociate from the tissue that way. So, I'll do 95 in this instance, rename it ER195 degrees C, ER195, and then I can put a description in here about what it is I'm trying to do. Oops, I'm saying ER1, but I've chosen ER2. Let's just correct that just to... I'll change that to 15 minutes. Save that. And I now have a new product that I can use.

So, you can do that for your pre-staining protocols. So, you can change your dewax protocols, your heat retrieval, there's enzyme retrieval protocols available. There's also hybridization, denaturation protocols that are available for if you're doing ISH. So, all of these different protocols are available as part of what it is you're trying to do. So, you can see you've got at the bottom here, IHC and ISH staining options. You can filter by the different protocol types. There are also preloaded protocols in here for your brown, your green, and your blue chromes-ISH stains as well, so you can kind of pick and choose. And then lastly, and then after once you've got all your reagents set up, you obviously want to set up your slides. So, you can see here I've created a few already, but what I'll do is I'll show you how easy it is by creating a new one. So, I'm doing a new research study. I then just click on new study. So, a number of people using these computers, I can have a study per person. Okay, this one. I can assign my own researcher name if I wanted to. So, I didn't have to put it in my study ID. I can assign researchers to it so I can identify my studies that way. I can give it a study ID name. I can also have comments. So, if I have several studies, maybe this one is focusing on an exploration test. Can choose to have the preparation protocol pre-loaded so, for instance, the standard dewax and I can set that up there.

After that I then will want to add a slide and we can see here you can either choose from your sequential or multiplex. I'm going to go through a sequential and I can choose the number of stains that I want to do as part of that multiplex stain. So, for example I may want to build a slide that is a fourplex I can choose IHC or ISH. I'll just create a random example at the minute. FOXP3 with a DAB followed by an ISH stain for whatever reason with my probe followed by another IHC stain with a blue and finish off with an IHC stain which markets have I used. I'll use that one. with a with a green protocol and you know maybe I want to put a retrieval protocol into there and an enzyme protocol. So, I've got my first one with my chromogen-stained air being dewax. I'll choose a heat retrieval. My second one which is an ISH stain. I've got a two hour hybridization in there. My third one and with a blue and let's choose no retrieval just for the sake of it. And finally in my fourth one another IHC with a retrieval and enzyme and different markers. I can then add that slide, and I can add multiple slides if I want to and I can also add slide comments so second slide so I can go back and differentiate between them. You can then see from this screen, if you go away and you want to check what your different reagents are or what different sites you've set up, trying to fit in six different markers in here is going to get very busy very quickly. So, there's this drop-down box here, so you can see your different markers, your different protocols that are set up in shorthand. Once the slide's been created, you can also double-click on it and you can go back in here and edit that slide and those protocol information.

And so, here's a few examples where I've done before and you can see here with these ones here are ones where you can print. So, I'll do it, I'll print, I'll reprint this one. So, let's see, I've shown you how to add a reagent, create a different protocol, add the slide that I want to stain and now what we'll do is we will put a slide on the instrument and get it to start. So, what I'll do first, I showed you how to add a marker before. I'll also just show you how easy it is to add a Chromogen. So, I have a green chromogen here in front of me, and the green chromogen is a two-part chromogen. So, I register for the part A, register for the part B, and that's going to get loaded on. And whilst that is loading, what I'll also do is I will add that printed slide that I just printed there onto the instrument. just bear with me for a few seconds. And if this all worked correctly, what we should see is you can now see. I've got a brown kit here, I've got a flex kit here with the blue chromogen, a three-part blue chromogen, I have a red counter stain, I have the green chromogen that I just added. I have a hemotoxin counter stain, PD-L1, a CD8, and a AE1/AE3 CK marker. So as this just loads, you can see what the system does is it takes an image of each of the slides, which comes in handy for traceability, and on that slide is a barcode and it's able to register it.

So, you can see one of the slides I added is a multiplex slide, and you can double click, yes. Double click on that slide and you can see the information. So, the one I've loaded up here is a PDL1, a CD8, and a cytokeratin slide, and I've got all the reagents on for that thankfully which is good. And then it's all loaded, and I could hit play and the run could start now. What I could also do is for instance you know, it's going to be my lunchtime soon, or dinner time soon, or I'm going home for the day, depending on what it is I'm doing. I may want to get this to start overnight so that it will finish when I arrive at work the next day. So, I can also do this thing called a delayed start. And I can select the time or the day that I want to do. And for instance, I may, it's Friday night even, and I want to get it to start on Sunday the 31st, or any other day of the year. I can then choose the time. So, if I know it's a four-hour protocol, for instance, and I'll be in the office at 9 a.m., I may want to start it at 5 a.m. And then once I've done that, I can hit OK and the run will start. Which, oh, sorry, I've done it too far ahead. Let's just do that. Hit play and the run will then, the run is now scheduled to start later on. And now it'll schedule the run and it'll be ready to start. What I'll do is I'll abandon this run because obviously we don't want the instrument to be making a bunch of noise whilst we move on to the next presentation. So, this is now scheduled to do a processing run. So hopefully that gives you a good demonstration of how to use the system and how easy it is not only to set up a multiplexing run, but also the flexibility of designing your own multiplex application. So, what I'll do now is I'll just prepare to hand over to my colleague, Dean, and he will then present to you the next part. And thank you.

Thanks, Demi. I'll be presenting this poster which describes the method that was used to achieve chromogenic multiplexing up to six-plex on the BOND research platform as a fully automated protocol. So multiplexing, it's a versatile tool. It can tell us a lot about protein expression profiles while maintaining morphological context. And this is something that can't be said for similar techniques such as flow cytometry. So, the more biomarkers that can be stained simultaneously, the better, the more information you can derive from a single tissue section. And there are a number of approaches that can be taken to achieve this. commonly sequential multiplexing is performed for the flexibility in the panel design and choice of antibodies in comparison to the parallel alternative. The downside of the sequential method is the longer turnaround time and when performed manually, this is quite a lot of steps, so it's a technique that lends itself to automation. The BOND research platform until recently has permitted fully automated chromogenic multiplexing up to 2-plex. With additional stains possible, however, this required some manual intervention for the mixing of multi-part chromogens. And once mixed, these have a limited stability. So, this would limit overall the ability to use this application for higher plex staining. However, with the new functionality, as Damon has outlined, I'll be detailing the method of how up to six-plex chromogenic multiplexing was performed fully automated on the BOND RX instrument.

So, all of the stains were performed were on formalin-fixed paraffin-embedded sections of human tissue, and these were fixed to charge slides. The standard Leica-validated preparation and pre-treatment protocols for BOND were applied with all of the staining on the BOND RX instrument. So, the primary antibodies that we utilized were all Leica BOND primaries in the ready-to-use format. with all of the antibodies being raised in either mouse or rabbit. Most of them were in mouse. And the detection method utilized was an enzymatic detection with the BOND polymer refined detection kit and the BOND polymer refined red detection kit, which used the HRP and the alkaline phosphatase enzymes respectively. So, chromogens were chosen which would react with either of these two enzymes. So those familiar with BOND would know the DAB brown chromogen and the red chromogen. However, for this higher Plex staining, we required the use of additional chromogens, different colors, which were also utilized in the multi-part format. These open chromogen functionality supports the use of a number of mixing ratios so that we can accommodate chromogens from a number of manufacturers.

So, the staining method. As I mentioned, it was a sequential chromogenic staining method. The difficulty with having the primary antibodies mostly derived from the same species and utilizing multiple enzymes multiple times is that without any kind of intervention, this would cause a great deal of cross-reactivity with the detection antibodies picking up the same markers. So, there are a number of approaches that can be taken to circumvent this. The method that was applied here was a heat mediated antibody stripping, which acts to denature and presumably wash away bound antibodies so that additional stains can be performed while leaving the chromogen intact over the multiple staining rounds.

So let me show you a graphic representation of this procedure. So, the samples, as I mentioned, underwent the standard dewax and heat retrieval protocols. In this instance, these were all bond epitope retrieval 2 for 20 minutes. And following this, the first primary antibody is applied. It binds the tissue. Then subsequent steps with detection antibodies are applied and form a complex to include the enzyme. Then following this, the chromogen is applied, keeping in mind that by this point, the chromogen has already been mixed on board the instrument. The chromogen reacts with the enzyme, forming a precipitated product, which then binds to residues in the tissue adjacent to where the marker has bound. So that's the end of the first staining protocol. Once that's complete, then in the place of a heat retrieval protocol is a similar protocol, which is the heat mediated antibody stripping protocol. Then this is applied to remove those bound antibodies. and then a second staining protocol can be applied at this point. So, this sequence of events has been repeated up to six times with the final staining protocol also including the counter stain. So, keep in mind that previous staining protocols were modified in order to remove that counter stain. So, at this point that the slide is then air dried, and mounted, cover slips, and imaged.

So let me go over to the results. So all of these stains have been performed with the sequential method that I just described, with a range of tissues represented, both tumor and those without pathology, with in the chromogenic sequence that was applied, and also there were different counterstain options applied to achieve the optimal contrast with the chromogenic signal. The turnaround time achieved for the four-plex was around eight hours and 20 minutes and this is conceivably something that could be performed in a single shift provided everything was ready to go at the start of the day. While going through to the five and six-plex, the six-plex was achieved in 10 and a half hours on the BOND RX, and this is something that could be performed as an overnight stain. So, to achieve this multiplexing, there are a number of optimization considerations that you need to take into account when planning your assay. This includes the order of the chromogens being applied, with the more stable chromogens preferably being applied up front, and the less stable chromogens found in green to be more prone to this being introduced later in the sequence. Also, it's important to consider the chromogen choice and that it's a good contrast to other chromogens, particularly for biomarkers that are in close proximity to one another. And this is an important consideration, especially for co-localization applications. When optimizing the elution protocol or the stripping protocol, we utilized a range of temperatures and times. all of which here appeared to be equally effective in preventing any noticeable cross-reactivity between the stains. However, the shortest one utilized was a 12-minute incubation of epitope retrieval 2 at 80 degrees. So, this is unlikely to cause any significant epitope degradation or signal loss of your biomarkers over multiple rounds of stripping. and this may in fact reduce the overall optimization burden required for your optimization of the primary antibody sequencing. So, the methods presented here are an example and As Damien has outlined, they can be modified to facilitate the needs of the lab, and there's a great deal of flexibility in the ability to optimize specifically for a panel. So this extension of functionality will give users from a wider research base to access chromogenic multiplexing, and the advantage is it doesn't require specialized imaging that's utilized in fluorescent multiplexing, so these images can be viewed by anyone who has access to a bright field microscope. So, in summary, the data I presented here is an example of how to achieve 4-2-6-plex chromogenic multiplexing, which was fully automated on the BOND Research Platform. Thanks for listening and happy multiplexing. Thank you, Dean. And I think that's the end of the presentation from Dean and myself today, and we can now stop for questions.

All right, I'll start with questions. Dean, one for you to start. You mentioned ER2 a bit in your presentation. Is ER2 the only solution that can be used for stripping? Sorry, I think we might have a problem. Tammy Dean. I've lost emails. While Steve sorts himself out, good old technical, we had to move to get quiet, I can answer a different question while Steve sorts himself out there. One of the questions that came through was, if there are limitations with the BOND RXm and how does that compare to the RX from a purely technical point of view?

So basically, do the different technologies work on both RX and RXM? The answer is, essentially we have not seen any technologies not work on the RXM because of the way the systems work they share a very similar design structure in terms of you know the cover tile and the way they dispense heat slides remove reagents and so forth. So most of the technologies that we've assessed do work with the partnership or the partner technologies like the ones by Akoya, Ultivue, NanoString, et cetera, some of those partners have access to RXM, so test on both RXM and RX. In some instances, they will go to us to do some of that testing for them and there'll be evidence for it. And in some instances, that evidence may not exist yet, so whilst we think it'll work, we may not have anything to back that up. So, in those instances, I guess what I would ask is, if you're looking at a partnership technology, then work with the technology provider like your Akoya, your Ultivue and your NanoStrings, and they will be better able to guide you on that. If it's a Leica product, then usually we pretty much test across everything, so it's much easier for us to support those. Might have to fumble my way through here with any of these technical questions if Dean isn't able to get back in.
Let's have a look here.
See some more.
Hey, you hope you can hear me now.
Yep, we can hear you now. Excellent.
Great. Sorry about that. I'm not sure what happened there. So, there was a question about... Sure. Yeah, so we've utilized both ER1 and ER2 for stripping. We've demonstrated that both can work more or less equally effectively. We aren't limited in the software to choose just one of those solutions as a stripping option. So, it's actually possible to, if you wanted to, change between ER1 and ER2 as a stripping method between each staining protocol. Some epitopes, of course, are more well optimized for binding when using either ER1 or ER2, so it may be a possible method to improve staining for a particular antibody if it has already been optimized for ER1, for example, under single-plex standing condition. So, we haven't explored that option too much, but it might be a possible method.

Next one is for you, Dean. Have you got any experience in multiplex chromogenic IHC and fluorescence combined?

Right. We haven't performed that specific combination, but there's nothing preventing you from doing that in the software. You're able to combine both chromogenic mixing on board along with a premixed fluorophore and use those protocols in combination. Definitely possible, but might take a little bit more optimization to get it right but yeah there it's actually possible to perform additional fluorescence multiplex above six plex because of the pre-mixing off board prior to commencing the processing on the instrument.

And I guess just to add to that, the stuff that we can talk about and show where we've done it ourselves, but also, we hear back from customers yourselves who take other people's technologies and work them up. So we have, I guess we've had feedback from yourselves about a number of people who have done similar types of tests using either just sort of off-the-shelf Leica products or I can think of an instance where I think they've done it with an ACD product line with their fluorescent and chromogenic one as well. So, it's definitely we've seen possibilities of it. I just haven't seen an off-the-shelf product that's designed specifically for that much at the moment.

Got one here for you Damien. So, is there a need to update the version 7 software to use the new Chromogens and Plex Kit?

So, the Chromogens and Plex Kit will work as a single or double stain on the 6.0 software, so I guess the previous version of software up until this new one. And so, if you do want to do a single or double stain with the new chromogen, then you can do that on that software version. If you want to Plex higher than the two Plex, then that's where 7.0 would be required.

Next one, I can probably take this one Dean. Will there be any point be considered to make rat or goat antibodies? I'll answer that with, I guess, a little bit of a non-answer. We're always looking at other opportunities for what we're releasing and constantly expanding our menu or our partnerships or partners that do provide those products. What I would say though is there is, with the openness of the system, you can use anybody else's secondary on there or other markers on the RX system. There's also the research kit so you can create a, you know, take someone's manual kit for instance and automate and work it up yourself and automate that on the RX. So, the flexibility to run RAT and go to antibodies is there on the system now. We don't necessarily have any kind of RAT or go primaries that we offer specifically ourselves. Right next one you're doing can the chromogens be used to co-localize targets or is it better to apply those on different cell targets?

Yeah well I guess it depends entirely on what you're looking for what amount of contrast you're looking for I mean you will have an easier time staining for targets in separate cell compartments but given that it is possible to do co-localization that is also an option so it depends on which chromogens you use but we've utilized the Leica red and the Leica green chromogen in combination to produce a purple signal as a third product. So that's an example of where we've applied co-localization for targets that are in the same cellular compartment. And there are also other combinations that we've attempted. I believe a red and a yellow to produce a green. Although there's any number of combinations that you could try to see what works best to produce a good contrasting signal between the two biomarkers and the third co-localized product.

Okay. I have another question for Damien. Currently, I have BOND software, can I upgrade my system to get the new software?

Yep, so how that would occur would depend a little bit about the system that you currently have at your laboratory. You know, kind of like your business laptop, they need to be updated every number of years. We recommend around three years you should be updating your BOND controller so that it's got the It's got sort of the latest firmware and hardware and so forth that comes with that. So really it would be, if you are a BOND customer now, just speaking to your account manager or your local FSS or FSC support person, they would be able to have a look at your controller and see if it's updatable. Ones that are fairly recent should be updatable. Ones that are a bit older may require replacement.

For you again, Dean, what about the mounting medium when you're using different chromogens or fluorophores?

Yeah, so we've used a couple of different mounting mediums. I find that it's best to get your most troublesome Chromogen and try to optimize for that and the other chromogens tend to work. So, we've utilized the like a CV mounts and that's compatible with the blue and the green like a chromogen and we haven't had any problems with any of the other chromogens as well. So, for the multiplexing I find that that's worked well. Can't speak to what might be appropriate for fluorophores in combination with chromogenic. We haven't attempted to optimize that. But yeah, like the CV mount has been working quite well across the board so far.

Let's see. Time for another question. Well, this is quite a complicated one. Using 6.0 software and the new chromogen kit, could you make a standalone protocol encompassing all steps, so that's your pre-treatment and your stripping and staining, and run an ISH as well? This is what we're currently doing for opal staining protocols. Okay. Damien, is that something?

Yeah, I can do that one. So, the system, trying to forget how to work. So, in the 6.0 system, it can only, because you're doing chromogenic, the parallel protocol can mix two chromogens. And if you're doing sequential, then each sequential protocol can only mix one at a time. So, if you want to build in pre, if you want to build in multiple chromogen steps, then you're limited to two. That's where the 7.0 software comes in, in that you can parallel, you can mix six chromogens within a protocol, and within that parallel protocol between each chromogen, you could build in subsequent heat retrieval steps, like is done with the Akoya test, the opal test. Or you can do it sequentially. So, you can choose, the upside is I guess you could potentially design protocols that are unique to the fluorophore, for example. And then you can then choose the different retrievals for each subsequent sequential stain. So, you know, you may not always use the same stripping steps between each Chromogen or marker primary or fluorophore. Anything to add to that one, Dean?

No, I think, yep, summarize it there. There can be some limitations in terms of which combinations you run across different SSAs, though. It can be hard to predict sometimes where the line is in terms of what combinations work and which don't. But most combinations for the higher-level complexity protocols that we've attempted on two SSAs, both on RX and RXM, have worked well. You do have some difficulty sometimes on RXM when attempting three SSAs worth simultaneously though.

I probably should add one bit I missed on the question. It was directed around ISH. So again, the 6.0 software can do a double ISH. The 7.0 software could do a six ISH so it's the same for IHC or ISH so you can tax like that one.

Yeah, I think we've got time for one more, yep so the interesting presentation is there any option to run multiplexing on BOND 3 and BOND MAX and what software needs to be installed?

So currently on the clinical system, the BOND 3 and the BOND Max, they're limited to the two-plex functionality. We're only at the moment offering the chromogenic one for the research. Essentially, this is part of a journey into multiplexing. The idea is, you know, we will work on it and refine it and, you know, if there is a real need for it in the clinic, then we would look to transferring there with, you know, a product line. Obviously, just enabling the functionality into the clinical space, there's certain limitations in what we can and can't do because of things like IVDR and so forth. You know, they need to be bundled with a product that has an intended use and so forth. as we work through that we will look we will you know we'll look at when the time is right for that for that solution okay so we've tackled pretty much up most of the questions and we can probably wrap it up there I guess then if there's no more yes.

Yep, that's all we've got time for yeah so thanks for listening. Thank you. I appreciate you all taking the time. If you do have additional questions, please continue to type them through throughout the subsequent presentations or go into the main rooms and talk to their representatives there from Leica and from other places so you can get that additional feedback or discussions. and I hope it was you know you're able to learn something so and it was informative in some way and Dean and I were understandable enough with our antipathy in accents so appreciate you all taking the time.