Selecting the right detection for your IHC/ISH project (Fluorescence vs Chromogenic Staining)

Webinar Transcript

Hello and welcome to the Leica Biosystems presentation on why quality matters and my little segment on selecting the right detection method for your IHC and ISH assays. My name is Sheena Washington and I am a senior field application specialist in advanced staining for life science with Leica Biosystems. I've been with Leica for 5 1/2 years. Before that I worked in research Histology for approximately 17 years doing IHC/IF standard staining microscopy, fluorescent microscopy, confocal microscopy, all kinds of different things for both human and animal tissues and I've worked in academia and CRO's and now in a commercial industry. I want to clarify before we get into the meat of this presentation that the presentation does assume some basic knowledge of IHC and ISH concepts. Basically, antibodies, antigens, probes, RNA, DNA relationships, heat and enzyme antigen retrievals, antibody titrations dilutions, things like that. To gain the most from this information covered in this presentation, you will need some prior background and experience with IHC. We will jump into it here. Summary of this is how to select the most appropriate detection method for your work. We’ll talk about some initial considerations first. Chromogenic versus fluorescent labeling. We'll move into single versus Multiplex labeling, which everybody gets very excited about and available technologies. And finally, some best practices. Choosing the appropriate detection method for your targets will allow researchers to produce the most compelling visual images to support their research goals. We'll discuss some of the major factors that contribute to effective single and Multiplex standings with focus, of course on quality and then we'll talk about some best practices for assay development. Talking about initial considerations, these are going to be some key concepts you want to think about before you even before you purchase anything. Before you spend any money. What are the research questions that I need to answer first and foremost, that's what you need to be thinking about. The desired endpoint typically determines what kind of detection method will work best for you, so ask yourself what is it that you really need to demonstrate you know, there are lots of fun things out there, and maybe a fun idea to put 20 markers on the same piece of tissue. But do you really need that? And in the end, are you going to be able to analyze that effectively. Next good question is to know what's already in your lab that you have at your disposal. What kind of microscopes do you have access to? What kind of reagents do you already have in house. That leads directly into the next to the next question of how much time and how much money do I have? What is your budget for the project? Can you afford to try something new? What is the timeline for the project, and do we have time to develop something completely new? Those are big considerations, you know, with most people don't want to waste a lot of time and money. The next one is who will be doing the work. Do we have the manpower to develop a new assay? Do we have personnel with the skill and experience to develop that assay? So those are some big considerations too. That leads to how will you analyze the data? Do you have the right analysis methods for the type of detection method that you are selecting? If you have, obviously if you've only got light microscopes and house fluorescence detection assay is probably going to be pricey because you will also have to find a fluorescent microscope to use somewhere or buy a whole new one with all the different filters and different things that go along with that too. If you've only got light microscopy in house, you may want to go with a bright field chromogenic detection assay. Who will analyze the data? So that's always fun to know. Like, will it be student helpers doing the analysis of the data? Will it be a pathologist doing the analysis of the data. Knowing who these final slides are going to be read and analyzed will be key in making it useful as well. Another big consideration is what kind of support do you have access to. Do we have access to resources to support our potential knowledge gaps? Do we have access to resources to assist with any issues that may arise with developing the assay or analyzing the assay, the outcome of the slides? Those are some important things to think of before you even get started with purchasing the newest, latest and greatest detection methods out there on the market right now. So now we'll get into the differences between the 2 main detections we're going to talk about today. The chromogenic staining. So chromogenic detection assays. They first and foremost are the predominant detection technology in clinical settings and primarily it's, it's just like what we're looking at right here in this photo that the DAB dining open scene substrate with its familiar brown color. This is still widely used in research immunohistochemistry as well for assay development and proof of concept. Nothing else. The nice thing about this, like I said, it's widely accepted, so acceptance is the number one top benefit of this. People know how to read AB stains. It's why they accepted. They know what they're looking at. 2nd is the cost. It's a relatively inexpensive detection option. There are many different options out there, so availability is another. There's a range of different enzymatic detection. Commercial vendors commonly used the DAB test and demonstrate their IHC products and antibodies, so lots of options out there highly available. Microscopy. It is a highly accessible detection technology as it can be visualized by using standard bright field light microscopes which most labs already have available to them. The range of substrates and chromogen colors, and that's also a benefit, the DAB substrate works with horseradish peroxidase enzyme, which is a robust enzyme, now has several color substrates other than the standard AV. You can do yellows and Blues and greens and purples and Reds. There are also a couple of other enzyme options for detection. The one we see most often is the AP. The alkaline phosphatase enzyme. The AP and Detection also offers a range of different color substrates. Red Blues, greens, etcetera. Fluorescent detection is not as widely used and accepted as the chromogenic detection methods. It has been growing in popularity, and we do see it quite often in research. And there are just as many fluorescent detection reagents and options out there. But most of the time we see people choosing fluorescent detection out of necessity because the option for direct labeling is one major reason this can be fast and efficient for optimizing and visualizing certain markers. Direct detection often requires less development and skill to produce high quality results, so if you've got, if you've got less experienced people working in the lab, this may be a good option for you to take. Alternatively, you know indirectly, labeling is another method you can use both for chromogenic and fluorescent detection, but alternatively you know it gives you introduces more variables, but it also provides a bit more flexibility in the sense that you can use the same primaries or probes with different colors. You can mix and match a bit more, but direct labeling is a nice option there with the fluorescence. The most common reason we see groups choosing fluorescence in research right now is because they are looking for multiple markers that co-localize, and by co-localize I mean they present in the same cell or the same cellular compartment and when chromogens overlap it can be very difficult to visualize and analyze the staining results. This is not really an issue for fluorescent labeling so as long as you are using 4 fours that are far enough apart on the emission spectrum, you can easily visualize and distinguish between different targets on the same cells with fluorescent detection. Fluorescent detection technologies also offer the ability to detect more targets on the same sample. As I said, if you've got Co localizing things, you don't have to worry about overlapping. We very often will see, you know, seven or more targets being labeled on the same. Not all of them overlapping, mind you, it’s still quite a challenge to try to see 7 targets overlapped in the same space, but you can get 7 and beyond targets on the same tissue sample on the same slide. So that has become a very huge benefit of fluorescent detection. Recent advancements in imaging technologies have made visualization of both chromogenic and fluorescent much easier than it used to be, so it’s not always a huge concern, but it's something to keep in mind. Multiplexing consideration. If you are looking to detect more than one marker on the same piece of tissue. We've got a couple colorful photos there, demonstrations of multiplexing. Any kind of multiplexing assay requires a solid comprehension of the basics of basic IHC, basic ISH and automated platform. If you are using one. If you're using an automated Stainer needs to have a solid comprehension of its function as well, what it's capable of doing or not. With any Multiplex assay, there are many variables that can require intensive development work, so tissue based multiplexing assays can be as complicated as they look here, so you look at this and it's a little boggling to the eye. And it can be just as complicated to try to get a beautiful outcome like this. There are many variables to consider, so it's important to start with those fundamentals. Primary antibody compatibility is the primary concern, and we've got optimizing your antibody or probe titration, optimizing your pretreatment conditions, chromogenic and fluorophore efficiency changes in signal through subsequent processing. We will take those one at a time here. So primary antibody and primary probe compatibility. These things and primary antibody quality and when I say quality, I'm talking about things like affinity which means the specificity for a particular antigen. Avidity is another concept, so strength of the binding to the target and then prevalence within the tissue. So those are some things to keep in mind when we're talking about antibody quality. Lots of choices out there are commercially available as far as antibodies can be challenges when we've got antibodies that are generated in house, but quality is certainly a big concern. Species is another concern, so the host species for your primary. Mostly we think about cross reactivity, which can occur when antibodies from the same host species are being put on to the same tissue, and then the other thing we worry about is cellular location where we can have issues of stearic hindrance or just hindrance of binding due to the previous antibody that's been placed on. Or even just obstructive deposits left by substrates, especially if you're working with chromogens. So those are some concerns with your primaries. Depending on the technology you choose, you know this may or may not be a major factor and have a major impact on your assay development. Some technologies are made to be species-agnostic and ISH probes can be very specific to sequence. The success of other detection methods may depend heavily on compatibility of antibodies and targets. You know, clones or sequences, but some things will be species agnostic, so that's just a couple considerations with your primaries. Optimizing your antibodies and probe concentrations, so this is key. Certainly, if you were going to like I said earlier, try to throw you know 20 markers at a piece of tissue at the same time, you're going to need to know the optimized dilution concentration of those primaries and the probes as well. People often attempt, you know, right out-of-the-box to throw all of the desired markers onto a piece of tissue at once, which can provide some baseline information, but rarely produces the desired quality of outcome. Optimizing the individual antibodies with probes prior to combining them, we'll provide a good sound baseline for understanding the expected performance of each marker, and it will aid in the decision-making process for tweaks that may need to be made later in the assay processing building process. So once the markers are combined. Optimizing pretreatment conditions. Once you've got your optimal, you're working out your optimized titration for your antibodies or probes. You will also want to work out your optimized conditions for pretreatment, so each marker in a Multiplex assay may require a different pretreatment to produce the best quality staining results on its own. Whether this is higher than the heat epitope retrieval or enzymatic retrieval, it will be crucial to determine this for each marker. So to determine the retrieval conditions necessary to incorporate into the Multiplex assay. Chromatin and fluorophore efficiency. Reagent solution stability is the first thing I think of when I talk about efficiency, so it refers to the general performance of qualities of the chromogenic substrates, or the fluorescent dyes. Some color substrates or floors may be much more robust than others, so for chromogens, when we look at stability in terms of the region itself, so once solutions are mixed, we can see precipitates. It may be sensitive to time, to temperature or light. Whether the label deposit that you get from the final product can hold up to the high temperature, the changes in pH to solidity. And whether it is solvent resistant in the end. Once you've got the slide stained and you are going to mount them permanently or with aqueous mounting medium some different chromogens they may vary in mounting needs. We may have some that are always much stronger than others and we may see a loss of signal due to photobleaching over time. That's the primary consideration with the floors. Potential changes in signal through processing, so as we are going through multiplexing and different rounds of different solutions going on to the piece of tissue onto a slide, are we seeing changes with subsequent rounds? We only really need to consider this most of the time with sequential assays. If you are doing a parallel multiplexing assay, this is not going to be such a huge concern. Changes to staining over the course of subsequent processing with heat, with different pH solutions, different salinities, can really change the end staining. Is this common and sequential but a bit less with parallel, the effects makes determining the optimal order of application for your markers and their associated staining conditions, so epitope retrieval stripping methods make it crucial to the quality of the final staining result. Chromogenic multiplexing stains. Here's a little example. UM, this comes from my own portfolio and my own experience. I couldn't tell you what the exact markers are anymore. I can't remember and did not have it labeled on the photo. But I know that this is a CD marker. You've got probably a fox P3 or Tibet and a Ki 67 I believe at the time or BOND RX. This took me probably a few months in development and these markers this is on a human tonsil, so this was just my test run here. The image was also produced in a Leica microscope, so considerations here were the limitations of the automated platform at the time. Is it capable of doing Multiplex chromogenic right off? Does it have features that lend itself to that solution? Stability was a huge issue here, so you kind of see that that blue-green color. That chromogen was not stable overtime. Once it was mixed, it was stable and it would start to precipitate out of solution after about 15 minutes. We must keep that in mind. Cellular location so you can see here that the brown color is very ubiquitous all over the place in there. We wanted to put that one on 1st. The DAB was a more stable chromogen and held it better through subsequent rounds of staining. We put that DAB brown color on and it did take some balancing. We did have to back off on concentration to get it to be that nice light brown so that you could get the red color to kind of show through it. And same thing with the blue-green. We wanted to balance the colors. So that took some tweaking of the antibody concentrations and the epitope retrieval conditions. Determining cross reactivity. It's always nice to be able to do some testing with your chromogens and stuff, so you'll do them, work them up one at a time and then you'll work them up in combination. And there are some tests you can do to determine whether you're getting cross reactivity or getting real signal with multiple markers. And then visual contrast compatibility. You can kind of see by looking at this that the brown and the red are very close in the color family so can be tough to distinguish red from brown and especially if red and brown are on the same cell, if they're Co localized, can be very tough to see this, whereas the blue-green has a nice strong contrast against those colors. And to keep that in mind as well and then order of staining kind of discussed that already where we decided to put on the CD marker, the very ubiquitous and Brown first because it held up better through subsequent rounds of staining. Then the red went on and then we had the blue-green in the end. Moving on to our fluorescent Multiplex. I believe this is the same assay, the same markers on a human tonsil, but this was done with fluorescent detection reagents. I developed the IHC assay first and then I tried to move it into a Multiplex Immunofluorescent assay. And that really sped up the process. Having the whole thing worked out in Chromogens first, and there were some processes involved. Excuse me, that was quicker with the IIS. Chromogenic development was not a consideration. Some things went a little more smoothly. But in the entirety of the developments, it still took about a month to transition to this IF assay from the original IHC assay. The microscopy and imaging were a challenge at the time. All we had in the lab that could do before us and analysis and detection was a confocal microscope, so it was a Leica confocal. And then the biggest challenge for me at the time was that we in the lab did not have a good way of analyzing this data. We did not have software in the lab that we could use to separate out all and do cell counts for us or anything like that. After months and months and months of doing all this multiple Multiplex detection work, both the chromogenic and the fluorescent it sat on the shelves and couldn’t be read right away because we did not have the analysis in House. Available technologies. There are lots of things out there when we talk about enzymes, we talk about the prevalence of HRP, the horseradish peroxidase enzyme and then the alkaline phosphatase enzyme. We see those regularly. There's a beta gal, the beta galactosidase, that one is a little bit more obscure. Amplification. A lot of people want to see nice dark or nice bright staining of their markers, so they like to try to amplify the signal polymers oligos TSA haptens. Lots of stuff out there on the market. Break-apart technologies are another new thing that allows spatial relations and quantification. There are different technologies out for that as well. And then service labs can be a great option for those who do not have the resources to build up their own assays in House. There are lots of groups out there now who will do the staining as a service and as well as the analysis as a service. So as far as best practices go, early inventory of needs and resources is crucial. Always good to take stock of what you have at your disposal and then a good hard look at what you need. Review of reagent specifications. Make sure that you know what you need to work with your reagents, so know the stability of your reagents. Know what they are compatible with and not compatible with. And once you know that, go back and take stock of what you already have and what you don't have and what you may need. Specifically, a specific example of that would be chromogen substrate, so some of the substrates are not solvent stable or solvent proof. They will dissolve in different organic solvents. You can't run them down the way you normally would. Just a DAB stain or something like that. They may you may and get some products in the end with absolutely no standing on them because it dissolved into your mounting media. Automated platform compatibility. There are lots of automated platforms out there right now. They have different capabilities, different specialties. Pros and cons about all of them. A lot of people will decide they want to do something and then find out that their instrument cannot do that fully automated or cannot do it at all. It's good to know the actual limitations of your automated platform, if that's throughout, you're going to go or that you need to go. And then the final one absolutely, hands down being diligent development, so optimizing things. One thing at a time, good scientific method and adjustment of you know just one variable at a time as you're going along will produce the best quality outcome. Some good references here. Something that an author that really helped me when I was going after information on this and doing it myself in the laboratory, Chris van der Loos, so a couple different publications here of his and that will end my presentation, and I would like to thank my life science applications team and my mentors and thank you. We'll take some time for some questions.