Cellular pathology wk 5

They are made up of histology and cytology. So cytology, as it says there, is the science of cells. And histology is the science of tissue. Now first of, tissue is made up of cells. So why are the two of us? The main reason being, cytology, the science of cells, is just that the cells are scraped off a body, FNA'd out of the fluid, and then just dumped on a slide. Anna will kill me with the telling bear. Dumped on a slide, stained, and the individual cell is looked at to see if it's malignant, abnormal, or what may be going on. When we look at tissue, we're looking at those cells, but we're actually looking how they relate to their neighbour, to the connective tissue around them, and the whole architecture that is forming the organ to see what is going on, even if the cells are actually malignant. So I'll ask you this question first. What do you know about pathology? Anyway, no, no one raised us morning. So if we ask the public, this is what they know. Apart from the fact we're so important, that's about 95% of all clinical decisions are made on a pathology report. So doctors cannot treat you without our work, but still, they know nothing. If you're lucky, it's living with the dead, and it's all a bit CSI and silent witness, you get to go off to do investigations and become a bit of an investigator, and it's all great fun, and you can crack all the cases. As not practised by real doctors, and pathologists don't see patients. So within cellular pathology, yes, that statement is true. Our pathologists don't actually see the patients, but in blood sciences and microbiology, all the medical side, all interact with patients in clinics and things. So actually to talk about histology, and I've just put there underneath, there is sometimes some confusion about the terms, but histopathology is just the term given to the diagnosis side of histology. So, as I said before, it's all about tissue. Not these tissues, they're pretty boring to look at, even if they have got some nice bacteria in them, but these tissues. So the cells, I was talking about that they look at in cytology in isolation, all form together to form the tissues, which all form together to form our organs. So that's where we fit in, in the chain that we're looking at these in isolation to the organ, to see what might be going on because a patient is suffering from some sort of disease. So you can basically separate us into four sections. The first one is when we receive tissue, we have to fix it. It needs to be reserved so that all the morphology of the tissue and the architecture is exactly as it was in the body. So we can see exactly what was going on. It needs to be dissected and sample selection. I'll tell you a bit more about that in a minute. We then need to process and bed section cut the tissues. So these are techniques that we have to do to embed them into wax to give it a solid support so we can actually take nice thin sections off of the tissue. We then have the staining and the different types of microscopy we use. And then a section of special stains and immunohistochemistry which will come in the presentation afterwards. First thing first, in 2004, which actually was quite a long time ago now, but it still feels fresh, the Human Tissue Act came into force. So prior to this, we could do what we liked with tissue, basically, and that doesn't sound as bad as it sounds, but we could use tissue for all sorts of tests, control tissue, find something interesting, you could mount it and just have like a nice presentation, basically, of what things were like. With the Human Tissue Act coming in, that all stopped and the availability of tissue to show people things has really, really reduced over the years. But the Human Tissue Act covers the removal, the storage, the use and the disposal of anything to do with the human body. And it's all around informed consent. So patients have to know exactly what is going to happen to that piece of tissue, be it that we're going to test it to see what was wrong with it and then store it for 30 years, which is the legal requirement. Or if we're going to send it off for research because something interesting has appeared in it, we want to know a bit more about it. And then this becomes a potential problem in the case of post-mortems because no pathologist wants to go back to a family of a deceased member and ask for more permission to do something else to their tissue. So it has quite limited the research side of sending tissue off and finding out things, but we've got around it. So this is basically what we do as scientists in the histology. What is that? Yeah, it's a kidney. So that's what we get sent quite regularly in the lab, just like that. And this is what we produce. So this is a three micron thick H and E stain section of a kidney. You can see the glomeruli here. And you can see a few tubules here. And then the background is all the connected tissue, which is holding all the important components together. So that's as we see tissue down the microscope. You can also see here as well, we have some ball-like structures, which are completely pink. They are actually dead glomeruli. Blood vessels have been completely dissolved, completely disappeared. And this is scar tissue within the structure of the glomeruli. So this is identifying disease within this kidney. This patient has got sclerosed glomeruli, so they've got some sort of renal condition causing the glomeruli to die. So as I said before, the first part is fixation. So to do our fixation, we use a solution called 10% neutral buffer formulin, which is actually a 4% solution of formaldehyde. Formaldehyde is a horrible chemical to have to use, but it's the only one that does the job properly. So we still have to use it. That means we have big health and safety risk using this chemical because it causes, it affects your mucous membranes, so your sneeze, your ice water, your nose runs. And it's worse, it can cause scarring in your lungs if you inhale too much. So we have to use this with great care. And in labs nowadays, we actually have, and I'll show you some in a minute, we actually have big benches, which have downdraft systems on them to pull all the chemicals away from us, so we don't have to wear masks and things because that makes actually working in that environment quite difficult. We don't have the risk anymore as well. But the health and safety side of using these chemicals has really changed over the years and we have to be careful. We've been taught that we have to be careful. So it's essential to fix that tissue. As soon as tissue is removed from the body, it starts decaying. The cellular processes within the cells carry on for a little while, and then they start breaking down and the cells start being digested, autolysis. We can't do any sort of diagnosis on tissue that has just dissolved completely because there's nothing to see. So it's really important that as soon as that blood supply is cut, that piece of tissue gets into a fixative. So all of those processes can be stopped and the tissue is fixed. It's a bit like a pickle. However, although we don't want to do this to the tissue because we want it in its life-like state, it does mean we chemically alter the tissue by fixing it because the fixative has to create new bonds between all the proteins to hold everything in place, which is not ideal. And further on in the process, we then have to do various things that we have to start breaking those bonds to get other things done. But it's the only way it's ever been done and anyone has ever found that it can be done. However, this process, let's say, it chemically changes the tissue. It also causes certain elements within the tissue to move out of the cells, such as enzymes, glycogen, and it dissolves fats. So it's really important that we know before it goes into the fixative exactly what we are testing that piece of tissue for. So this is our cut-up room. It looks very pandemonium in there and very busy. So we have, these are the A-force benches. You can just about see the grids here. And these have motors which pull all the fumes down away from us and we don't have to wear masks. We have six benches within the lab and we have various cut-ups going on ranging from tissue transfer, which the band five BMSs can do, up to the pathologists who are in doing big tissue dissections of known or query malignant tissue where they need to do a much better description of what the specimen looks like than we can do as biomedical cyclists. So it's quite a busy room. I actually don't know what our work is in there at the minute, but it has grown quite considerably. So various people sit at these benches and whether they're dissecting, we have to dictate everything we are seeing, measure everything, what it looks like, color, texture, everything to be typed into the report. And then as the pathologists are going along, they like take samples of the areas that they think are going to be interesting. So they take samples of the tumor and then they will take samples of the surrounding tissue on the edge just to make sure any tumor cells have completely been removed. So here's the tissue, here's the popcorn then. It's been taken out. This is a BMS size dissection. They would describe this piece of tissue, measure it, see what the color is, and then slice it up so it can be embedded correctly at the next stage. When samples come to us, we check all their information matches on former pot. And when we're happy, we give them a unique number, which is this number is then printed on cassettes. And then there's a well here, which is where the tissue is put into for processing. So we only put through little bits. We don't put through whole breasts and legs and things. We can only cope with little samples. So in that room, as I just said, a representative sample is undertaken. And this is an example of what the pathologist would be doing. So this is a prostate. And little bits are usually taken, described and taken and placed into these normal sized cassettes here. The prostate is one that is slightly different because malignancies within the prostate tend to be multifocal in the vapor all over the place. So the practice is to actually just slice the organ hole and process it whole in these big mega cassettes, as we call them. So this is a whole section of the prostate. And this hole here is where the urethra passes through. So that equates to these two here, where the wax blocks has actually been made. And each of these, this is the representative HNE for these two pieces of tissue. And you can see where the pathologist has marked the areas of malignancy. And you can see, you can't really see that in the prostate. You can a little bit, the color difference is slightly different there. And there is a slight difference there. But essentially you can't tell the difference that's looking at that piece of tissue. So that's why they prefer to have it whole because there could be loads of little bits throughout it, which could quite easily be lost if they drop it down into smaller pieces. So it's back to front. So once cast up is done, the little cassettes of tissue are all collected together and counted and scanned and everything that we have to do to track them through the lab. And they then have to go through the processing, which is done on machines like this overnight. So to be able to cut those thin sections, you remember I said that the three microns stick, which is 3,000th of a centimeter and about the size of a cell. To be able to do that, we have to remove the water from the tissue and be able to inject something firmer into them to make sure they hold together properly. And we do that by processing. So we dehydrate the tissue through alcohol into a clearing agent because wax and alcohol don't mix. The clearing agent, we use the xylene, which is the nasty chemical, but we use that in between and then they sit in hot wax for a few hours just to start penetrating through the tissue. And that's done on machines like this, whereas where all the reagents are here, the tissue goes in here in this chamber and then overnight, using heat, agitation, and vacuum, the chemicals are taken into the chamber and drawn through the tissue to remove the water and get to wax by the morning, hopefully. So for normal pieces of tissue, for little biopsy bits, this takes about 15 hours. If we're doing big pieces of tissue, like the Megas I showed you just now, or fatty tissue, it takes about 20 to 25 hours. So this is just a photo I took the other day of our processing room. We've actually got another machine down here as well. I just couldn't fit it in. So to that one machine, we've got seven processors. Usually six are going every day. I did mean to look up how much they take, actually. I think they take about 200 blocks each. So we are processing between 600 and 800 blocks a day. So blocks are what the cassettes that I'm used to were in the first bit. So we're processing about 600 to 800 a day of those. Hopefully that works overnight and the machines don't have fit. So these are all new ones. They're very good. The next thing, the following, the next step, the following morning is to embed the tissue. So it comes off the processors. It's still a flimsy bit of tissue. It's not quite as flimsy as it was, but it's still quite flimsy. And to be able to hold that still so we can pass a knife past it precisely, we have to embed it in wax to give it a solid support around it. So they're removed from the processor. You can see the cassettes here with their lids on still. That opens up and we look at the tissue and we have to decide how to orientate it within these little molds, big molds. So molten wax is poured into these. Now most tissue isn't a major problem, but tissue like skin, it's really, really important that we orientate it correctly. And that is that when we cut the block, we will be cutting this bottom surface. So when we take a section from this, we want to make sure that the skin, you can see the epithelium, the dermis and any other underlying tissue clearly in all pieces. If we put it onto the surface, we will lose the lesion. If we put it on this surface, which you can see is actually inked to help us this one, we won't see the tumor and potentially it could be a negative diagnosis for something that is there because we haven't shown it properly. So it takes an extra level of training to embed skins and tissue which needs to be put on edge, as we call it. But it's really, really important because it can mean the difference between a diagnosis and not a diagnosis treatment and no treatment. So we take great care, trying to embed those correctly. Sometimes they are inked for us, which always helps because we can see the color and we know we do a way round to put it. But those will all be taken with the blue edge and you see there will be placed like that in the mold. They will then be pushed down to solidify slightly and then topped up to make the wax blocks left to cool and make the wax blocks that we actually produce. So just to reiterate that with skins, if we have like a punch biopsy, we have to make sure we put it on one of these edges. So as we cut it, we will cut into the lesion and on each slide that we put a section, you will see a different picture going through. The punch biopsies are sort of about that big and they look like tubes. So the danger always is that we will put it on one of the ends. So if you think of the tube, you need to see the lumen and then a punch biopsy put like that means we may take the epsilium off before it gets anywhere near a pathologist. So that's why we have to be really careful and know what we're looking at. Sometimes punch biopsies are really big. So they're bisected down the middle. They're not as hard to get wrong. And here we have a nice excision of skin where it is sliced up and then it's quite easy to see the epithelium, the tumor and any underlying tissue which is how we want to produce it on the slide. Section cutting. So three micron thick sections are cut. They're floated out onto warm water and then picked up onto glass slides. This is a typical cutting station over in the lab where we have the microtomes. Microtomes are just like glorified bacon slices. You stick the tissue in, you set it to what you want the thickness you want it to cut and you turn the wheel and it cuts the section for you. However, it is not a one, two, three centimeters it's at three microns. So this is a normal sort of set up our hot water, our water bath with warm water which is set just under the melting temperature of wax. So the sections can relax and spread out. So we don't get any creases or any artifacts in our pieces of tissue. And then picked up on a glass slide. Now this here, this is new for September last year. We have just had a tracking system put into the lab where we can follow a block through the whole process within the lab. But it means that each station we now have these big screens where we have to scan the barcodes that are printed onto the blocks. So the system can actually register where the block is at that time. I've not used it yet, I can't decide on it but I know it's caused a few issues. So you can see here a closeup of the knife area a very sharp blade is clamped in here. Here we have the wax block with the tissue and the flywheel here is just turned and a ribbon of wax and tissue just comes out in front of you, which you gently pull and then float onto the water bath. And then when you pick it up on the glass slide you can see the wax support around the outside and we've got two pieces of tissue there. So we've pretty much achieved what we're aiming to do. We have a three micron thick section on the slide. If only it was that easy. So the next step we have to do is to look down the microscope at that section. It's very gray and there's no contrast and you can't really make any sort of diagnosis on it. So we have to add color to the section to be able to see the features. So the stain every lab in the country uses in the world probably is the hematoxylin eosin stain which is the lovely pink and blue stain you see in most biology books. So we use a substance called hematoxylin to stain the nuclei a blue color and then eosin to stain the rest of the tissue pinky color. And it can actually differentiate tissue fibers, connected tissues and elastin and all different parts of the connective tissue system. So we can actually start looking into the architecture of the tissue and what may have happened. So hematoxylin itself is not actually a dye. It's not very good dye. To be a dye, the substance needs two features which I'll mention in a minute. Hematoxylin is slightly different in that it doesn't have an affinity for tissue or it has a poor affinity for tissue. So it has to use a mordant to help the dye flow into the tissue which is what I'm representing there with the binding to allow the tissue to stain. So the HNE is our bread and butter. Everything, every 600 blocks that is processed every day has to have at least one HNE done on it, if not more. So this is just an overview of the technique of what's going on. Here, I've got a picture of what it would look like unstained and you can see, you can't make out any sort of structure whatsoever there. You can see the shape, but you can't see any cellular detail as opposed to this one which has been stained. And you could clearly see the epithelial layer here and the ducts here, which now you know they're there, you can just about see. So that's a different staining makes. So to do staining, hematoxynilacin or water-based dyes. So to do that, we have to remove the wax. To remove the wax, we have to put it into xylene to dissolve the wax away. We then have to add water to the tissues so they are receptive to the water-based dyes. So we have to undo everything that we did with processing. So we do that through xylene and graded alcohols down to water. We have to do it, when it says slowly, it's not done slowly as in time, but it has to be done through each graded alcohol so it doesn't shock the cells. They don't like going from absolute to water. We then stain it in the hematoxylin, which actually stains everything, a redy-bluey color. And then because we only want it to stain the nuclei, which are the type, which are the structure, sorry, that has the highest affinity for retaining the hematoxylin, we have to put it in 0.5% acid alcohol just to take the background color out so that we can only see the nuclei. And that is a process known as differentiation. After that, we have to make the hematoxylin a little bit more stable. It's not very stable when it's red. But if you place your sections into a strong alkali solution, it turns the hematoxylin blue and it becomes more stable. And that is a technique just known as bluing when you're doing the staining. You then have to give it a really thorough wash because we don't want to transfer any acid or alkali into the following solutions because it will contaminate them and it will mess up the staining. So after that, it goes into the eosin, which stains everything else pink. And then we have to, because we have to keep the slides for 30 years, we have to be able to cover-slip them in a mountain that is going to go hard and protect that section for that amount of time. Most of those mountains available on the market are xylene-based. So we have to go backwards again or forwards again, depending on which way you're looking at. Remove all the water and take the slides into xylene so they can be cover-slipped in permanent mountains. And then that mountain then hardens off in ovens and hopefully you can keep your slide for many years. So we have two of these monstrosities. These are H&E machines. And I'm told by other students who come to us placement that like that just in all of these because they have to do it by hand and we have these huge machines. These do everything from removing the wax from the section, rehydrating the tissue, they do the hematoxin, they do the eosin, all the differentiation, all the blurring. They dehydrate the other end, put it into xylene, cover-slip and cure all of that and come out finished. So we put them in as wax sections. We take them out as proper H&E stained sections, stained slides, which is really good. We don't have to do any of that by hand. These take 20 slides at a time. You can see we've got three loading points here and there's three stainers all behind here. So they can take 60 slides each time we load. As soon as a rack has cleared a station in the staining, you can then load another. So we near enough have constant loading all day long. So after about, I think it's 10 minutes, we can then load another three racks of slides. That's another 60 slides. And that goes on all day long, just continuous feed through the machine. As I said, we have two of these. It uses minuscule amounts of the stains, which is efficient, as long as there's no air bubbles in the pipes. But all the stains are in bottles in here and it literally just pipettes them up out of the bottle and pipettes them onto the slide. So you've got about time and then washes them off. And here, because of those mega slides I showed you at the beginning, they don't fit on our staining machine. So we have to have a second staining machine to be able to stain those big slides, which is what this one is. This is more typical of an old staining machine. These are quite new. And you can see there the solutions. You've got the xylene through the alcohols, the hematoxylin, r acid alkylized solution, your eosin, and then your alcohols to xylene coming back up again. You can see the racks here waiting to be cover slipped. So we have to cover all the mega slides by hand. So that's the main part of histology in a nutshell. This is a slight section of a normal kidney where you can see the pelvis. Janan, the name has just gone from my head. The important bits, the main bit around the outside. And this is an example of a cut surface of a tumour within the kidney. So you can see here, it's slightly, there's this little wall around here. This is normal, if not slightly stressed kidney, but normal kidney tissue. And this is all tumour. So as tumours grow, they become necrotic. They die in the middle because they don't waste time keeping that bit alive when they want to spread out further. So they just cut the blood in spite of that bit. Concentrate more on the bits on the outside, which is why we've got brown here, and then white, which is a normal image of a tumour. And this is probably just blood. It's probably just a little congested or inflamed and just got some blood around it. And then that equates to when we section to it. This is a normal H and E with a normal glomerulus. Tubules, each side looking nice and healthy. And this is an example of a renal cell carcinoma where you can see the cells are quite bizarre. The nuclei are even bizarre. There's no sort of structure. There's no glom, there's no tubule, there's no structure there at all. So that means that kidney cannot function at all. And it just looks really bizarre. To us, it looks really lovely, but it just looks really bizarre. And that would be example of, as I say, a renal cell. This is actually a clear cell one because the cytoplasm is gone as well. So that technique of histology takes at least 24 hours, if not longer. We're probably talking about five days with all the work that we have as well. There are occasions when you may need a more rapid result than that. If a surgeon is excising a tumour from a patient who's still under anaesthetic on the table, it may come very close to one of its reception margins. So if you think of a breast, if they're trying to excite a tumour of the breast and it's really, really close to the muscle above the rib, they want to know how far they can take that before having to go into the muscle and the ribs and doing quite a major surgery that they probably had not prepared for. They can remove a little piece of tissue and they send it down to the lab as a frozen section. We do the same process, but instead of taking it all through the chemicals, we just rapidly freeze it to give it firmness and to make it solid. And then cut it on a microtome, which is in a freezer, which is what this is, and produce a frozen section. So we produce a three, four micron thick section of frozen tissue. We then do a rapid H and E stain and give it to the pathologists. They can tell, yes, there's tumour cells close to the margin, no, there's no tumour cells, and relate back to the surgeon as to how they need to proceed. We can also do it if, when surgeons are operating, if they come across something they weren't expecting, they can send it down to us and we can just do this test and within about 20 minutes, we can say, yes, it's malignant, no, it's not malignant. What we can't do is say, yes, you've got a lymphoma there. No, you've got an infected lymph node or something because the morphology is appalling when we do this. So we can't make any sort of diagnosis, I say we, the pathologists, can't make any sort of diagnosis on frozen tissue. All they can do is say, yes, the malignant cells there, well, no, it's not. So, on to the types of microscopy we use. We use bright field, the basic normal type of microscopy, polarising microscopy, which is where you have two filters. Polarisers are filters that have lots of lines all the way through them. Any photographers in the room will be familiar with polarisers. But if you put in a filter below the specimen and a filter above the specimen and turn them at right angles to each other, it blocks out all the light. So there's certain substances in nature that are birefringent and can actually turn the light so we can then see colour through the M-top analyser when it should be dark. So I have some images to show you all there. It's usually crystals, but there are other things in the tissue as well. We also use fluorescence microscopy, which is using ultraviolet light to excite electrons. And electron microscopy. So although I've included it because it is an important part of cellular pathology, not every lab has an electron microscope. It's very, very specialised. And as far as I'm aware, in the south of England, I think Southampton is the only one that's got one working. So just a quick diagram of a microscope, typical one that we would use in the labs today. This portion, the condenser, is the most important part of any microscope. And it's how microscopy has developed over the years because it takes the light beam and it focuses it into a really fine point. So anything that you look at down the microscope, this is where the specimen would be, is illuminated by the most amount of light in the smallest field as possible. So this one's just to show the light beam and how it works going through the condenser and then up to the eye. This is a very complex version of a polarising microscope. We don't have any, we think, quite as elaborate as that. But this shows the two filters. So when they're turned at right angles to each other, no light passes through. And if you happen to have a birefringent specimen in between them, it does turn the light. And then we start getting an image. Through sort of lit in half light. And this is how the fluorescent microscope works. And then straight away, health and safety, you'll see the light comes in from the side, not from underneath. This is so that the ultraviolet light, if anything goes wrong with the prisms in the mirror, the ultraviolet light never goes near the human eye because it's dangerous. And so the light comes in and it's bounced down and then reflected back up. So here's some images. Everyone recognise what this is an image? What type of microscopy? Come on, we're awake now. No. So this is an electron microscope image where you can see quite detailed, quite detailed detail. Within the cell, we can see the nuclei, the nucleolus, and mitochondria all within. Electron microscopy is used for renal diseases. I will show you the reason later, but it's quite an important part of the diagnosis of renal diseases. This is a polarising image of crystals. You can see against the dark background, you can see the shape of the crystals all over. This is just a dark ground, dark face photograph that I just happened to like. This is a normal microscopic image that everybody should really be familiar with. It's a piece of bough which is demonstrating mast cells here. It contains heparin. This is a fluorescent image of a skin condition called pemphigus where immunogoblins are laid down in between the cells, the skin cells, causing them to separate. And that's what you can see here, this chicken wire appearance, but all the cells have separated apart. Then these two go together, and this is how we use polarising microscopy in the lab. It's usually to identify a substance called amyloid, which is a protein. It's a natural protein in our body, but in certain conditions, it gets deposited where it shouldn't be, or it changes its structure slightly so it can't be removed as it should be, so it builds up within the organ. This is a kidney, I think it is, and you can see the amyloid here, nice bright red all around the blood vessels, which is where it tends to be deposited. The thing about amyloid is that it is birefringent, and when we stain it red with the Congo Red stain, we actually then put it in polarised light. We actually get this apple green birefringent as we turn the polarisers. So this apple green colour you can see here, and then you can see the rest is a little bit orangey-ready. So what actually happens as we turn those polarisers, that substance will come in green, and the green will go to red, so we can actually get a full picture of how it is turning the light as we turn the polarisers. That is a gold standard result for amyloid, and that's what has to be done, and it's the only way they'll say, yes, this is amyloid, and start the treatment for the patients. Not a lot you can do, though, because once it starts building up, it tends to carry on. It's usually associated with other diseases, sort of autoimmune diseases, but there are studies going on, and it has actually been linked with Alzheimer's as well now as well, so there's quite a lot of research into amyloid at the minute. And just because I need a drink, some pretty pictures of some of the things we can do. So we're going to go on to the special stains side. We're using special stains. We actually want to identify certain aspects of the architecture of the cell, what the cell is producing. We want to know it's doing it properly, and that's what we're doing when we do our special stains. We're using dyes and chemicals to react with the proteins, the carbohydrates, all the basic stuff within the tissue and with the cells to show us that they're there, and are they there in the right amount of where they should be? This is an image of a liver, which is demonstrating these black lines. These are very, very fine reticuline fibers, which are a type of collagen, and they form the scaffolding to hold the structure altogether within the liver and the lymph node. If that is broken, the structure is gone, and even though the liver has managed to regenerate, it leaves the echo behind that we can see that structure has gone, so there has been some sort of process that's gone on within this tissue. This is a blood vessel, a nice big blood vessel, where the black is staining the elastin, and the red is staining the collagen. This is in the lung. This is a stomach biopsy, a gastric biopsy, and it's stained to see if there's any heliobacteria pylori present, which is a very simple bacteria, but it has actually been linked to stomach cancer. Because it can actually live in the stomach, in all that acid, and actually metabolize the acid and produce ammonia, which neutralizes the acids and starts the gastric process. Okay, sorry, gastric cancer process. So this is identifying the bacteria, because if they're present, it's about a six, nine months treatment of three different antibiotics, and it's gone, and the tumor will go as well. This is to identify that the bacteria are here, and thus the patient will benefit from that particular treatment, and they're just these little black flecks, just along the epithelial surface of the stomach lining. And this is an immunohistochemistry picture of cytokeratins, which are present in epithelial tissue, and you'll see a few more of these afterwards when we do the immunohistochemistry. Oh, anyone got any questions? Was that all all right? So, special stains, a little bit of chemistry coming now. So we use dyes and chemicals. Dyes are essentially benzene rings that have two properties to them, the property of color that they can absorb in a bit light, so you can see it, and the ability to bind to tissue. Most of the dyes that we use were developed for use in the textile industry, so they're all synthetically produced. The only natural dye we use today is hematoxylin, and we said that's not much of a dye, but we class it as a dye. This actually comes from the logwood tree, which grows in the Caribbean, Mexico area. So, as I said, it has those two properties, a chromophore and an oxochrome. Chromophore. Is the group of atoms that give it the ability to absorb light, and they're usually meso, nitro, or carbonyl groups. And the oxochrome is the bit that gives it the ability to bind to tissue, which are usually acidic or basic, or amino groups. Amino groups being the ones in the proteins, which is why we can stain proteins. So, just to give you an example of how you can get to that fairly easily, if you take benzene, we all know the structure of benzene, and you add a nitro group, which is a chromophore, you get a substance called nitro benzene. Now, benzene is a colourless liquid. It emits light, absorbs and emits light in the ultraviolet region. So, to the human eye, it is completely colourless. However, by adding this one nitro group, we have made it pale yellow. If we add number two, we can make it quite dark yellow, but it's still not a dye. Because it still hasn't got the property to bind to tissue. And that's just the formula for the trinitro. Make us a good bomb, but it's not a dye. So, if we then add a hydroxyl group, which is the oxochrome, to the trinitro benzene, we get trinitrophenol, which is this lovely stuff here in the bottle. It should be kept saturated. You don't want to keep the dry crystals around because it is explosive. However, this lovely yellow stuff is actually picric acid that we use quite routinely. Another health and safety risk in the lab. But it's an important component of the van Giesen stain, and it stains very blood cells yellow. So, we do use it. And like I say, you have to look after it properly. So, as in all good chemistry, staining of tissue occurs by the normal bonding processes of everything else. It can be ionic, covalent, hydrophobic, van der Waals forces. It can be via mordens, which is how hematoxylin works. Or it can be by the molecular size and permeability. So, literally, as tissue is fixed and processed, some things shrink, some things grow, some things explode. And just depending on that, the various molecule sizes of the dyes can fit into those holes and don't come back out again. So, it's just some examples of how we use the different types. This is one that it works by molecular size and permeability. And by using the molecular size of three dyes from large to small, we can stain muscle, connective tissue, fibrin, red blood cells. These are van Giesen stain red blood cells. And connective tissue there. So, just by using different molecular sized dyes, we can get quite a colorful picture and identify quite a lot of the structure. The elastic van Giesen, which I showed you earlier, is another one where we use the molecular size to demonstrate the elastin and then the collagen and the red blood cells are separated out by the van Giesen solution. We can use a technique which will stain carbohydrates to demonstrate glycogen. So, as I said, it demonstrates carbohydrates. Carbohydrates are everywhere in the world. So, it stains carbohydrates pink. But to be able to actually conclusively say this is glycogen, we use an enzyme technique using amylase, which is present in your saliva, and it actually digests the glycogen out of the cells. We then do the same staining technique and we get a negative result. So, on that tissue, we can say that is glycogen. We can also use the same technique to demonstrate the carbohydrates in fungi or cell walls. This is one here where the hyphae are all stained. And also, we can also use a silver impregnation technique to stain the carbohydrates in the fungal wall as well. This one takes a little bit more time and a little bit more expertise, which is why we don't use it as routinely. PAS is quite simple to do. However, this one is slightly better because it will actually identify viable and non-viable fungi. So, you can see fungi that has died within the tissue. Whereas this one, when you stain viable fungi. And then just to show you a bit of how staining can fit in with diagnosis. This is just a normal image of a liver biopsy. You get little thin cores of tissue. You can't make it out really well, but you can see cores of cells all lining up. And there we have the triads of connective tissue and the three vessels within the liver. We can use a stain which uses the Prussian blue chemical reaction to identify iron within the liver. It should naturally be there because iron is processed in the liver and it's stored there. And that's what we can see here. So, the Prussian blue reaction has actually produced this bluish color where iron is located within the tissue. And then it can show the abnormal size of iron being deposited within the tissue. Iron is toxic to the body and it has to be really tightly controlled. And if you're starting to get it depositing like this, then there is quite a serious condition going on because we don't excrete iron either. So, this is a case of somebody who has hemochromatosis, which is a condition of iron storage. You store too much in your organs and your skin and you get quite a bronze kind of skin, which is probably quite the nice side of it. But it's not a very nice condition at all. And this is what the liver would look like stained with the pearls. And then, as I alluded to earlier, the staining of renal diseases. In renal disease, and this isn't tumors cancer, this is like renal diseases. What usually happens is proteins, immunoglobulins are deposited along this really thin membrane, stops the blood vessels being able to filter things across. To be able to see these, because this really, really is microscopically minuscule trying to look at it, we do a silver stain to highlight this membrane as black. Hopefully, it will look just like somebody who's gone around with a really fine black pen. Normally, as it starts to thicken, that means things are being deposited there. You, if you're lucky, you can see the deposits as just like little piles on the membrane, which is why we use the silver stain. That is why these diseases then go for electron microscopy because they can actually see the whole structure of those little piles of unwanted, it's ever been dumped on there. I can actually make the diagnosis better, for want of a better word. So we would do the initial gets there, and then it would then get sent from electron microscopy to actually see what's going on. So special stains are all done by hand. That's a slight lie. We do have a machine now, but principally they're all done by hand. We make up all the chemicals ourselves, we make up with all the various dyes and solutions. Again, that's a little lie. We don't anymore. Because everything has to be so standardized within a lab, because labs here have to perform the same as labs in Scotland or anywhere else. We've been pushed to move towards ready to use. So we have to buy in everything now. We can't make it up like we used to. But when this was taken, we were making them all up. And literally, just on a sink, you lay the slide out, and somebody would have to do all those different staining techniques by hand. We have about 25 that we can do at any given time. It's not always bad. Some days it is, some days it's not. We have to be quite organized to get them all done, because some take three hours, some take 10 minutes. The other thing that we're not so much involved in, but what cellular pathology is involved in on the pathologist side, because obviously they look at the tissue, are post-mortems. So there's three types in this country. You have a hospital post-mortem, where somebody has gone into hospital and maybe died in an operation or something. The hospital need to hold an inquiry. And as the living relatives, you have to give consent to that. If you don't want to give consent, it will go to the coroner who you cannot argue with. The coroner has the ultimate and final say on any post-mortem. And they are ones that are unwitnessed or are unexpected. So in this country, if you've not actually seen your doctor for a fortnight and you die at home, that is an unexpected death and you have to have a post-mortem. And finally, the forensic post-mortems, which have now been taken away and are done in major centers all around the country. They used to be done in individual mortaries, but not anymore. It's all very tightly controlled nowadays. Why do we still do post-mortems? Why are they interesting? Well, it's actually quite important to actually know the exact cause of death. Old age and heart failure are not allowed to be causes of death. There had to be something else that caused all of that. The deceased could be carrying or have evidence of some other disease, which has not been detected in their life. And that in turn may have effect on other members of the family if it happens to be a familiar or an inherited disease. They're used for audit reviews, obviously monitoring the death rate in certain conditions, particularly COVID. And they are helpful in research and teaching. So although because of the Human Tissue Act, we don't have the teaching benefit as much from the post-mortem tissue as we did in older days. They are still helpful in research and if you happen to get to go along and work alongside the people doing post-mortems, obviously you're gonna learn about the human anatomy like nothing else. So that's it for the first bit. Anyone got any questions? That's what we do as biomedical scientists. No. Do you want to have a break? Or do you want to come on? So 10 minutes. Don't have a 10 minute break and then come back. Okay. So I'm just gonna bend your brains for a little bit more. Next one I'm going to talk about is immunohistochemistry, which is a staining method we use, but it has become crucial within the department. We use it for most everything now. And as I'm gonna show you how it developed and how we can use it now, it's quite a boring technique to actually try to explain. And it's very much about counting for our blobs as they come up. You can see how we've made it much more specific nowadays. So at the end, I've just got two case reviews to show you how we would actually use it in the lab. So what we're gonna cover is I'll revise the structure of immunoglobulins and antigens, how the antibodies are all produced, and then explain the method that we actually use within the lab and the different ways that it was implemented and how it developed. Automation, this is one technique which is automated in the lab still. Not much is in histology, but this is one that is. Thank goodness, because thousands go through a day. But this particular technique, because we can't see what we're trying to identify, we can't be sure whether it is there or not, we have to use controls, positive and negative controls. And I'm just going to talk about their usage in this technique. And then at the end, I've just got some case reviews so immunohistochemistry is the localization of antigens or proteins in tissue sections. So all cells, just as in all bacteria and things have antigens on them, all cells have antigens, these protein markers, which stick up on the cell surface and can be recognized by antibodies. What is then done in the process that I'm about to explain is those antibodies, which are added onto the sections are actually labeled either by fluorescent dye or an enzyme complex. So we can actually identify that tiny cellular protein. Yeah, that's just repeats with us. So this technique was first developed in 1941. It became a new technique to try in the late 80s within the labs. And by the late 90s, it more or less has become routine in labs throughout the country. Like I said, we use enzyme labels that can all be visualized within a light microscopy. And electron microscopy use the heavy metals, obviously because they're dense in the electron beam and can be viewed that way. So first off, what is an antigen? It's a protein, it's a molecule capable of inducing an immune response. So it's a structure that the cell uses, be it bacteria, be it a human cell. And we can exploit the principle of the antibody antigen reaction to be able to actually identify that small antigen sitting on the cell surface. As cells develop, they all have particular antigens dependent on what that cell has to be, what that cell is programmed to be. So that's how we can really exploit it because if a cell is situated out of place, we can actually identify what it should be as opposed to what it's pretending to be. So these chains are called, protein chains are called epitopes rather than antigens. And what was actually discovered was the antigen, as you can see here, has loads of different epitopes. And what can actually be done now is antibodies can actually be raised against each individual epitope. So an antibody, is an immunoglobulin, typically thought of as a Y-shaped, so it can sit on a former little hat on any antigen. It has an antigen binding site at each fork. And that's just how it's made up, consists of constant regions and different immunoglobulins. And the antigen binding site contains this paratome, which is like a little hole. The easiest way to think about it is that it's like a lock and key. You put the key in, you turn it to lock. When the antibody is finished doing what it can do, you turn the lock, you take it out and move on to the next one. So they're not particular to certain epitopes or anything, they're multidisciplinary, form of a better word. And nothing is permanent within this structure. So just briefly, immunoglobulins are produced by plasma cells, which are fully differentiated lymphocytes at the end of their life, lymphocytes, sorry, B cell lymphocytes. And there's five different types. You have IgA, IgD, IgE, IgG, and IgM. They all have different functions within the immune system, such as IgE, which is the easiest one to remember, is the one that reacts to allergen, causes hay fever, asthma, and other diseases like that. So how do we get the antibodies? So there's two types. One is polyclonal, it's the easiest way to get an antibody. It's how every mammal's immune system works. If you come across an antigen, in this case, they immunize a rabbit against something, whatever way you want to identify at the end. And after a couple of days, you just harvest the plasma and harvest the antibodies which have been produced. It's termed as polyclonal because the antibody has been made by many types of plasma cells, which all vary slightly amongst one another. So it's polyclonal and it will attack a wide attack, attach to a wide range of epitopes on the antigen. So this means that they're very sensitive. They're highly sensitive. It can pick up an antigen just by having four or five different epitopes attaching on it, but it's not very specific because it's picking up five or six epitopes. We then have monoclonal antibodies. So these are made by only one clone of plasma cells. So all the antibodies will be exactly the same. They will only be matching to that one epitope that was present on the antigen that the mice were immunized with. It's usually raised in mice. And what happens afterwards is the cells are mixed with myeloma cells because we don't want to keep doing this because it means you have to keep the mice permanently clonal, which is not good for them. So it's then mixed with myeloma cells, which give it a immortal life. And we can then just keep using those cells on a turn to produce the immunoglobulins rather than going back to the animal at the time. So they're taken from the spleen in mice. And then they're mixed with the myeloma cells to give them a immortal life and an unlimited supply of an antibody. So I said these are clonal. These are made by only one particular type of plasma cell. So it's all going to be the same. And it will only bind to one specific epitope on that antigen, which makes them highly specific because it's really getting down to the nitty-gritty. But it's not very sensitive because for one reason or another, that epitope is not there. It will not pick up that antigen. So that's just an explanation to the way we use specificity and sensitivity. They're quite difficult to remember and I never get them around the right way. But in one case, an example of how we can use those is the PSA antigen, which stands for prostate-specific antigen for men with prostate cancer. It's only produced by the prostate. So it's pretty conclusive that if it's there, those cells come from the prostate. We can use, it comes as polyclonal and monoclonal. We can use the monoclonal antibody if the tumor cells are still located within the prostate to actually find which epitope is particularly active. And thus certain treatments are better for certain parts of the antigen, the epitopes which are reacting, which is why we have to sort of test down at this level. So we use a monoclonal for that one, different types of monoclonal ones. However, if the tumor has spread into the blood system or is lodged somewhere else in some other organ, all we need to know is that it's a prostate cell. We don't need to know which epitope is reacting because the disease has already moved up here. So we would then use a polyclonal antibody because it would pick up multiple epitopes and it will tell us that it is a prostate cell. Okay, so a brief history of the method. So in the simplest form, all we're doing is we have an antigen here, we have two antigens here. We want an antibody that will react with these antigens and we want it labeled so we can see it. That is known as the direct method because it's just one label, one antibody on the antigen. And that is what is used in immunopurescence. And hopefully you remember this picture before, that's how this is generated. It's very simple, very easy. However, it is not very sensitive because we've only have one signal here. You don't get any sort of, I have to put it, really strong signals as to how much is actually going on. The thing is with cells, when they overexpress these proteins, they can get loads of overexpression of it and you don't get any sort of feel for how overexpressed the cell or protein may be. And then as knowledge of proteins and all these cellular interactions developed, they found that the technique was not specific enough. It was not good enough to just be able to see this one signal. So they amplified the technique and looked at ways of trying to be able to make it more specific. And there, they aim to get as many markers on an antibody as possible. Labels, I should actually say, rather than markers. So this was the next stage that came in. We have our antigen here. We have our primary antibody here, which has to be, which was usually rabbit anti-human. So it would react. And they then developed the secondary antibodies, labeled, so we now have two blobs, which were usually cow or bovine anti-rabbit. So they would all bind together. And this is where it started getting quite complicated because every time we wanted to attach another antibody, it had to be the next species higher. So like I said, I've got a mouse there for monoclonal. The primary antibody, this one is mouse anti-human. And then the secondary ones, I've got goat there, goat, anti-mouse, and so on. We would then have to have bovine anti-goat if you want to come up and layer this up. But each time you did, you would add another two labeled antibodies onto it. So we've got one to two to four to eight to 16, depending on how big you made this layer until you ran out of animals. And then this is the basic method of the indirect, sorry, the best technique, no, no. The basic technique of the indirect method, which is what is used in all labs now. However, as I just said, because we're now using different animals, antibodies from different animals, this now then, this then produced a problem, is that you can have reactions of all the serum, of all those antibodies present in the serum, I can't speak anymore, in the serum of all the different species that you're now using. So what you then had to do was add another technique in to block all these signals and make sure we didn't have any non-specific binding going on. And then that's how it was done for a couple of years. But ever wanting to produce better signals and more specific signals, scientists kept working on the label side on that primary antibody. And they came up with these two enzymes, which are quite easily accessible, quite easy to get hold of. And there were actually two enzymes that could be attached to an antibody. And in the presence of certain chemicals, they actually produced a color compound as well. First one was the horseradish peroxidase, which is a plant, the horseradish plant. And it's just the peroxidase from the root of the plant. The second was alkaline phosphatase, not so easily to get hold of. It actually comes from the intestines of calves, but it still was in the presence of certain chemicals, it changes a chromogen to color. So it produced a signal that was visible. So it's the horseradish, which is the one that we still use today because it's easiest to get hold of in a plant. In the presence of hydrogen peroxide, it turns this chemical brown. And then alkaline phosphatase from the calf intestines can actually catalyze the reaction of fuxin substrates and fuxin is red. So it actually gives us a red signal. And this led to the two techniques, which in older textbooks, you'll probably see a lot, which is the PAP, peroxidase, anti-peroxidase, and the APAP, which is the alkaline phosphatase, and these stayed with us for quite a few years, they were best for that. And this is the sort of structure it was producing. So we have our antigen, our primary antibody, our secondary antibodies. And then on top, we have our third layer of antibodies, which was the labeled complex, peroxidized labeled complex is here, and these are the antibodies used to attach it. So we've gone from one to two, two, four, eight, 16 to six blobs. So we've got a stronger signal coming from this one little antibody doing its job attaching to the antigens here. And it's good enough, but it still wasn't good enough really. As cancer studies progressed, they want to be able to see more and more of these antigens. These antigens are actually present on the nuclear membrane as well. They want to be able to get into them as more and more is known about the processes of cancer and how it develops and what's going on within the cell. So this was good, but not good enough. So then they tried the ABC, which was using Avertin-Biotin, which is a complex, it's a big complex as well. Avertin found in egg whites, and Biotin is the soluble B vitamin. They bind together and Avertin can actually hold four biotins when it binds. So this then, the principle then was that we've got four attachments here, so it's much bigger. Again, it had its good and bad points. So this is an Avertin-Biotin peroxidase method. We have our antigen, our primary antibody, our secondary antibodies, and these are the Avertin-Biotin complexes. So straight away, you don't get four attachments because you have to use one attachment to the antibodies. So we only have three labels on here. However, you've got four sets of the complex there with three labels on them. We're hitting a much better signal now. Three, six, nine, 12, brown blobs there. So the signal has got stronger again. So this was, it was a good method. The only problem with this method is the sheer size of this complex, or four complexes actually, it caused a lot of stereotactic issues in that if you had smaller, less reactive antigens underneath, it actually pushed things out of place, which meant, although you had a stronger signal for your one antibody, you had very poor attachment to other antigens within the tissue. So it wasn't quite as specific as you want it to be. It was like giving a false image. So this is what we use today. This is known as the two-step polymer. And we have the antigen, the primary antibody, a secondary antibody, which is bound to a piece of string for want of better word labeled with the enzyme complex and DAB and multiple secondary antibodies. The benefit of this technique is because it's in the piece of string, it can bend, it can turn, it can twist, and all these secondary antibodies can twist round into the tissue and attach to those other weaker antigens without getting pushed out of place. So we get a massive signal for that one primary antibody. So there was one final discovery that had to happen in all of this because we fixed the tissue. If you remember, I said before, it causes chemical changes. It forms a cross link between the proteins within the tissue. This means it hides antigenic sites, protein links, stop them all and the antibodies cannot get into all these antigenic sites on the cell. So scientists developed ways that this could be done. The first technique they developed was an enzyme-based one. So using enzymes just to digest those bonds which had been created, those bridges that have been created amongst the proteins, just to digest them away and open up the antigenic sites. It had its benefits, it had its disadvantages, one being if you left it too long, you digested the whole section off the slide. But it worked for a certain amount of time. But again, as we start trying to find those smaller antigens, those less visible antigens, those antigens on the nuclear membrane as well, it wasn't good enough, it couldn't digest deep enough into those bonds to open up the antigenic sites. And what they actually developed, which goes against everything, everyone was taught in histology up to that point. This would have been the early 90s. They heated them in pressure cookers. He did not ever heat a section, it's too gentle. It's too susceptible to it. But no, they chucked them into pressure cookers of different buffers, usually citrate buffer or EDTA, and they pressure cooked them and microwaved them. And it worked, cells weren't affected. Everything was all good. The best thing was the protein links have gone from the fixation and all these antigenic sites suddenly became available for staining. So this is a method we use today. So the tissue preparation is exactly the same as before. The slides are then treated to antigen retrieval. We do still use enzyme antigen retrieval occasionally, but most of the time now we're using heat because it just opens up so many more antigenic sites. We then do the protein block to make sure we don't have any cross reaction with all the different theorems which are going on. And we then have an endogenous enzyme block. Yes, using horseradish peroxidase is good. It comes from a plant, there shouldn't be too much. There are certain cells within the body that do produce peroxidase. So we have to block them from reacting to make sure we don't get any cross contamination that way as well. The sections are then incubated in the primary antibody, incubated in the second complex. The stain is then produced by adding hydrogen peroxide. They're counterstained, the hematoxylin, so you get a good image to look at. It's not too hard on the eyes. And then dehydrated clear amount, which is the taking the alcohol output and it's resigning and then putting it into the glue, which hardens so we can keep them. So that's the basic method. Other than that one and that one, we have machines to do all of that for us nowadays, which is really, really good. Because as I say, there's thousands that go through a day now for various loads of different types of antibodies where antigens have been discovered on cells or very particular to the cancer pathways as the cells become disorganized and confused. They do things that they shouldn't do really. And those are the sort of antigens that we're looking at. So they'll start producing proteins that they shouldn't. So let's head at the beginning. Because we're actually looking for something that you cannot see at the light microscopy level, you can probably just about, but they would probably still need staining at the electron microscope level. We have to know that when we stain it, we've stained the right bit. We put the right antibody on and it has picked up the right antigen for what we're looking for. So we have to use control sections alongside doing the immuno and we have to have positive and negative control. Positive is easy. It's a positive case. It's a known positive piece of tissue that you put on with the slide. If it works, you know you've done your job. Negative is not so easy. You want a piece of tissue, which is not going to react with the serums that you put on and with the peroxidase that you've put on. So you need quite a generic bit of tissue, which is going to stain negative. So you can be sure that you've done all your blocking properly and there's no cross reaction between the antibodies. And to manage all this as well, as part of UCAS accreditation, that labs are expected to aim for nowadays to prove that they are maintaining their standards and all the standards are exactly the same amongst all the labs. We belong to UK NECRAS schemes, which are an external quality control. So as well as controlling the technique inside, the lab internally, we send slides off to an organization who actually employ people to look at the slides and to critique whether they're working correctly or not as well. I said that's part all labs have to participate in to get their accreditation to show that they are maintaining all their standards. So I thought it was boring. So I just do a few case reviews with you just to show you how we would use it. Before I start, there's a few terms that I'll use that you may not be aware of. So I'll just run through them. The first one is poorly moderate well differentiated carcinomas. So grading tumors, carcinomas come from epithelial tissue. They're a tumor from epithelial tissue, which can either be squamous or glandular. When we grade tumors, they're graded according to how normal they are behaving. So epithelial tissues go through a period of differentiation when they start as stem cells, those little babies, and then they mature, begin their function, whatever their function may be, mature before they die off. That maturing is known as differentiation. And the thing is with tumors is depending on how fast they are growing, they either carry on developing as normal and do as they should do, which means they're not benign, but they're quite slow growing. Or if they're very fast growing, then they don't ever get to that maturation stage. They're just babies all turning over and over and over. So the terms are, if it's still differentiating to an adult and still producing and behaving as it should do, that is well differentiated. At the other end, if they're just turning over as stem cells, then that is poorly differentiated. And that's the worst category because the cells are just reproducing all the time. Okay. I'm sure there's another one, but I've forgotten it. So we'll see if I come across this. So the first one, this was a case we had in the lab. It was a mass in the liver of a 59 year old man who had no other history. And just for you to compare it to, this is a picture of a normal liver, and this was his liver. So you can just about see, it's pretty normal-ish this side, but this doesn't look anything like this at all. So this is obviously tumor in this side. So we're then looking at it to say, is it a liver tumor? What is it? First off, you would sort of look at it and sort of say, well, these are trying to form into balls. Sort of. Which then immediately to a pathologist will say, it's a tumor of glandular tissue and adenocarcinoma. But it's not typical of a liver one. So we're looking at that. This has probably come from somewhere else into this gentleman's liver. And we need to be able to identify where those cells have come from. So we need to look at what their signal is, what their antigens are saying on their cell surface as to where they've come from. So the initial diagnosis was as a poorly differentiated adenocarcinoma. Poorly because let's say they're trying to form into balls, but that should look like a duct. So it should have a lumen in the middle and it should just be one cell ring around. That has no lumen and that is just so crowded cells. They're quite small and they're quite condensed. So they're poorly differentiated cells. They're not becoming anything near as odd as a human adult. Okay, sorry. And then immunohistochemistry was done to specify where this tumor had come from. So this was the panel that was used, the antibodies to the various bits we're looking at. The ones that are all positive are antibodies for cytokeratins, which are particular to epithelial tissue. So we know it's an epithelial tumor. The CDX2 is a marker of gastrointestinal differentiation. And the fact that this was positive means that it has come from the gut. The rest are negative of just testing for lung, prostate and liver tumors, which did come up negative. And yeah, just some images of what those markers would have looked like. This is a cytokeratin one. So from that, the pathologist deduced that it was an epithelial tumor. It was an adenocartinoma, so one of glandular tissue and likely to be from the gastrointestinal tract. So he reported it as consistent with metastatics. That means it has spread from the original of pancreatic or upper GI origin. So that's how we use immunohistochemistry to point out where those particular cells are coming from. If they've spread somewhere else. And then just a quick line. This is a mass in the auxiliary lymph node of a 66 year old woman who has a pretty extensive history of cancer both sides. She recently had a recurrence in the right-hand side and now has a large lymph nodes on the left-hand side, which were biopsied. Come on, where you got? So this is what the cores would have looked like when they came into us. You can see the slight white areas. And this is what it would look like down the microscopes. This is a normal breast and a normal duct. And this is the abnormal one where the cells are completely out of place. We then used ER and PR antibodies on all breast tumors because certain tumors are fed by estrogen. And you can be given the drug tamoxifen to block the estrogen and thus make the tumor reduce. It can't be given to every patient because it actually makes you quite ill as well. So it's only given to patients who are ER positive. And then there is another drug now available called Herceptin. It's been found out that HER2, which is a growth receptor, is overexpressed in certain types of breast cancer. You can see it here being demonstrated. Certain women who have the certain genetic predisposition to overexpress it, which is what is being demonstrated here, pink bits, benefit from this drug, which just shuts it down completely and stops the tumor growing or becoming any more aggressive. So we test the tissue for all of these markers to see if patients can actually benefit from certain drugs which are now available. Just quite a change in histology because we're not just doing the diagnosis. We're now actually looking at prognostic work, looking how tumors are gonna react, how patients are gonna react to drugs and things and monitoring the disease through. Because as tumors spread, they turn on things and turn off things and they change. And we're now looking at all of that down the line. So these drugs, they do improve survival. For this poor lady, it was ER positive. She was given tamoxifen, but she already has such an extensive history of breast cancer. They couldn't really do a lot for her. And yeah, than