The kidney Wk 7

I mentioned in the past that we or my lab has personally used tissue sections to understand exactly how platelets are involved in the separation of the blood and lymphatic vasculature. And as part of that work we certainly used histology to help us understand what was going on when we had some genetically modified mice.

So for today we are particularly focusing on the kidney and starting off just a little bit about the structure of the kidney. So some of this is likely to be familiar to you but we're going to try and link that structure to what we see in the histology.

So the necro is the structural unit of the kidney and that really is the crux of the filtration of the blood. And there's a huge amount of blood that actually flows through the kidney but even higher than the brain, the liver and the heart. So there's a huge amount of blood that actually passes through the kidney to help it do that filtering role.

And that filtration is really important for the removal of waste products and as part of that filtration process an awful lot of good things also pass through. There's lots of reabsorption that also happens within that structural unit of the nephron.

So this is a kind of an overview of the kidney. So the vast majority of people have two kidneys. You can function with one and you have a blood supply to the kidney that supports that massive amount of filtration that happens.

So we have your aorta that feeds blood into the kidney and then the blood is returned via the vena cava. So as I said we have two kidneys and they're usually vital for that filtration of your blood. So what does it actually look like if you were to take a cross-section through that kidney? So this is a kidney and we have a cortex and this cortex is surrounded by a fibrous cap and that fibrous cap allows the kidney to be protected with some respect and that protection just helps kind of protect those nephrons that are really important for the filtration of the blood. So within the cortex this is where you'll find all the little filtering units. So the main part where the filtration happens and that occurs in the cortex and then the nephrons and we'll talk a little bit about the structure later.

There are these loops that go down into the medulla and then as waste products are produced and collected they are collected in the pelvis area and then passed to the ureter and then they'll go through and be collected in the bladder.

So this is the kind of filtering organ. The filtering happens in the glomeruli which are located in the cortex of the kidney and then ultimately you get reabsorption of good things and then secretion of things that are waste products and that will form urine. And you'll see there's these kind of pyramidal structures within the kidney as well. Again it's how these filtering units come down and the urine kind of collects within these ducts and ultimately being delivered to the ureter.

So this is a schematic of a nephron. So I mentioned about the major filtering kind of function of the kidney and all that filtering initially happens in the glomerulus and that's this structure here and again we'll look at the actual histology of that structure a little bit later on. So we have the entry of blood into that filtering unit and then a lot of kind of filtration happens at this point so huge amounts of liquid are passed through and the structure of the cells around and in this filtering unit prevent large kind of cells from passing through so red blood cells, platelets, they all get kept behind in the capillaries and continue their journey in the bloodstream and they also prevent the kind of filtration of large molecules.

So large molecules such as albumin for example, antibodies, coagulation factors, they're all prevented from being filtered by the actual structure of the cells within that filtering unit.

So it's small things that are actually passed through at quite large filtration rates.

So all that filtration happens here and then in the Bauman's capsule then you have that filtrate that then passes into a series of tubes. So immediately after the the kind of filtering unit that glomerulus we have these proximal convoluted tubes and actually that's where the vast majority of water is taken back up into the bloodstream.

Molecules such as glucose and some salts are also taken up and then that filtrate passes through the loop of Henle and again further solutes are taken up back into the bloodstream. So you can see around the loop of Henle we still have this kind of vascularized kind of tissue that helps that diffusion and active transport of some of those solutes back into the bloodstream because the body wants to keep those things and again waste products continue on their journey.

After the loop of Henle we then have some distal convoluted tubes and again there's further uptake of solutes for example again overall maintaining those happy balances that happen in the body.

So the key message here really is with respect to histology is where these two kind of structures lie.

So again in the cortex that's where you'll find all the subunits those glomeruli so the glomerulus will be found in the cortex a cortex you'll also find those convoluted tubes so the proximal ones and the distal ones and then in the medulla you'll find the loop of Henle and also the kind of collection ducts that will collect the urine that will then pass eventually to the bladder. So what do these actually look like with respect to histology? So can we actually differentiate some of these tubular structures because the tubes are tube right but can actually we differentiate exactly what they look like using histology. So here I've got a histology image of some convoluted tubes and I've got here a mixture of proximal convoluted tubes and distal convoluted tubes and essentially they do have some subtle differences in how they appear in histology section.

So here we just have staining with an H and E stain so just as a reminder H and E staining is the kind of gold standard of a stain that happens clinically so every tissue that's taken one of the sections will be assessed and stained using an H and E stain so that's what this is an H and E stain of some convoluted tubes.

So we have proximal convoluted tubes and we have distal convoluted tubes so I'd just like you to kind of look at these and see if you can see any differences between these kind of tubular structures.

kind of tubular structures. So I'll just give you a minute just to kind of think about that and feel free to chat amongst yourselves. Can you actually see some kind of subtle differences between these convoluted tubes that will allow you to differentiate and identify a proximal convoluted tube versus a distal?

convoluted tube versus a distal? This might be in the slides so don't cheat. Right so there are two types of tubules here I've just highlighted these with the initials of the start so we've got two distal convoluted tubes and the rest are proximal convoluted tubes so what hopefully you could kind of start appreciating is that these distal convoluted tubes have a little bit of a clearer lumen and they appear a little bit more open than the proximal tubes.

tubes. The proximal tubules also are a little bit more synophilic so they've taken up more of the eosin dye compared to the distal convoluted tubes. So there are some subtle differences but being aware of them would allow you to then identify which is which in H&E histology say. So again here I've got a little bit more of a kind of zoomed out image of the kind of cortex of the kidney but here we also have that fibrous cap. So this is the fibrous cap that forms that protective barrier, protects your kind of nephrons from any potential damage and here we've got lots of tubules. So who would like to kind of tell me what the vast majority of these particular tubules are?

Proximal. Yeah absolutely brilliant. So the vast majority in this particular image are proximal convoluted tubes so they don't have that clear kind of lumen and they're a bit kind of messier. There's also one structure here that I wanted to point out and there's also lots of proximal tubes. So who would like to kind of suggest to me what those kind of cells or structures might be?

might be? So very pinky red aren't they? So within the kidney the kidneys involved in lots of filtration and that filtration is then anything that's good is taken up by a blood vessel right? So in between all your convoluted tubes you're going to have a blood supply. So actually these very kind of cherry pink kind of structures here.

So this is a blood vessel with lots of red blood cells. So these are actually red blood cells and you can also see in between the proximal convoluted tubes you can also see some red blood cells and in between those. So again the kidney is highly vascularized to support the function that it has.

So if you see kind of structures like this in an H&E stain and you might see these in the practical and these are red blood cells.

So let's focus a little bit more about the the actual filtering unit itself. So the glomerulus. So this is a kind of schematic diagram and it's a scaled back diagram to make it really clear about how this works. So within the glomerulus there's a huge amount of capillaries that are all kind of wrapped together. So this is a very simplified version. So we have entry of the blood supply through an afferent arterial.

It goes, passes through the glomerulus and then the blood exits by the afferent arterial. So this is the kind of the overall structure. So we've got the kind of capillaries and they are surrounded by an endothelial membrane and below the endothelial membrane there is a basement membrane. So that's here highlighted in green. And then on the other side of that basement membrane are these very specialized cells called podocytes and podocytes are and the kind of interaction between the endothelial cells it's this relationship that's really important for that filtration action.

And we'll talk a little bit about these specialized cells these podocytes and how they actually maintain their function and also kind of talk about how this may may become disturbed in some disorders.

So once the filtrate passes through the podocyte layer it's then collected in the Bowman's capsule here and then that filtrate will pass through the actual structure look like in a kind of normal kidney.

So hopefully some of you have kind of started to appreciate these structures when you're actually performing the practical session. So hopefully you actually have started to visualize these yourselves. So again we're in the renal cortex and we have some glomeruli.

cortex and we have some glomeruli. So how many glomeruli can you see in this image?

glomeruli can you see in this image? I think I there's two here this is one and this is the second.

So again we've got two glomeruli here and the Bowman's capsule is very difficult to identify but you can just about make it out here and it's a bit clearer here.

And again this is the filtering unit within the kidney and sometimes if the cells within that filtering unit are damaged then you start getting kind of a clinical outcomes.

So again based on your knowledge so I've just highlighted this little area here and again we've got some kind of cherry pink cells. Who would like to tell me what those cells are? Red blood cells absolutely yeah so here we've got another example and again you can see them in between those proximal convoluted tubes here as well. Okay so I mentioned about this very specialized cell the podocyte.

So the podocyte sits on one side of the basement membrane and on the other side we have the endothelial cells of the blood capillary. So the capillary here is indicated by C. Basement membrane is BM and then podocyte is PC. So what you can see so we've got these kind of penetrations within the endothelial cell and the same on the other side we've got these kind of structures for the podocyte.

So this is a transmission electron microscopy image. So again a section has been kind of sliced and then using different contrasting agents it's been imaged under an electron microscope. So these podocytes are really specialized and they form these kind of finger-like projections that kind of interlink between their neighboring cells and this is really kind of beautifully represented here with a scanning electron microscope image.

So here we've got several podocytes actually. So we've got a podocyte here and can you see these like finger-like projections that are kind of forming these kind of links between neighboring cells and it's that action that actually forms part of that filtration barrier and prevents very large things such as red blood cells, platelets, large proteins such as immunoglobulins, their antibodies, coagulation factors, they're all prevented from passing through by that very specialized kind of filtration barrier and I mean I personally think this is such a beautiful image and if you pseudo-color the different cells you can really see how they're interlinked with their neighboring cells. So these are like podocytes really specialized cells that form that key filtration barrier prevent the passage of large things passing through in conjunction with the endothelial cell. So those are your podocytes, incredibly important and we'll talk a little bit about, more about podocytes a little bit later on when we consider the case study where we can use histology.

So let's take a little bit more of a focus on kind of normal kidney histology.

So here we're in a completely different area and we're kind of further down into the medulla, kind of into the pelvis area of the kidney and again we've got another H and E stain. So I'd like to now kind of draw you back to how we started classifying different types of epithelial cells. So we've got a couple of different types here so I just want to kind of see how you're feeling about identifying different types of epithelial cells. So if you remember back I talked about those four kind of tissue types.

Actually let's see so who would like, so we've got epithelial, what are the other three? Can't hear anything clearly, sorry.

No no so endothelial cells are a type of epithelial cell. So that's the different classifications within epithelial.

So that's the different classifications within epithelial. What about the main four types of tissue?

four types of tissue? Muscle, yeah. Sorry. Connective, yeah. That's the type of muscle. No yeah okay those are the four main tissue types.

No yeah okay those are the four main tissue types. Okay so we're here focusing particularly on epithelial and how and then we can rightly classify those by their kind of morphology. So here we've got a duct, it's surrounded by some epithelial cells. So what kind of, how would you classify those? So we've got different types of multiple or single layers and then we've got the tell me what this might be. So what would be a single layer? What would be what would be the term based around there?

Simple, yeah absolutely. So it's simple and what's the shape here?

Do you think it's flat? So is it squamous?

So is it squamous? No. Is it like a big column? No.

So what does that leave? Okay I'll put you out of your misery. It's simple cuboidal.

So they've got this very kind of square shape and it's simple because it just has one single layer. And we have also some different types of epithelial cells.

So here we've got simple, so still one single layer, but these are very flat kind of tire-like cells.

So these are squamous and you'll find simple squamous surrounding kind of blood vessels. And here we've got two different types. We've got a venial and an arterial. So this, so that gives you just a bit of a brief overview of the kidney and the kind of major aspects of it and how it may look in a histology section.

So then that takes us on to the case study that's really linked and kind of brings together quite nicely all the different techniques that are available for histology.

So we're going to be specifically focusing on nephrotic syndrome. So this is kind of classified by a variety of different clinical outcomes.

So we have increased cholesterol in the bloodstream of patients who have nephrotic syndrome. And actually this is quite unknown really about how, why patients actually have this increased level of cholesterol. And it's not sure, like clear exactly what the mechanism is behind that. And obviously increased cholesterol can cause their own, can cause its own kind of challenges. These patients also have very low levels of the major protein that's found in your blood, which is albumin. So these patients have very low levels of fat because that protein, which is really abundant in your bloodstream, actually passes through the glomeruli and into the urine. And that leads to something called protein urea. So protein urea just means there's lots of protein in the urine. So normally you have relatively low amounts of protein and in patients who have elevated.

And it's elevated to the extent that the albumin levels are so low that these patients start getting swelling within their peripheral, a periphery. So they start getting swollen hands, legs, because that balance has been disrupted. So normal kind of osmosis doesn't happen and diffusion rates change because the protein from the blood is being secreted through the urine. These patients present with, with swelling and that's edema.

And the kind of main way of diagnosing nephrotic syndrome, obviously there are clinical visual kind of things you can see, but actually linking to the cause, the diagnosis is very dependent on histology. And if a patient is suspected of having nephrotic syndrome, they will have a kidney biopsy. So a very thin needle will pass through into the kidney and take a small amount of tissue. And then that tissue will be divided into three.

Some of it will be fixed with a kind of formaldehyde based solutions such as formalin.

Some of it will be frozen and some of it will be prepared using or fixed using glutaraldehyde. So if you remember back, this is the first step of preparing a tissue section. We have to fix it in as near native form as possible. So we have these three different ways of fixing the sample and that's really there to enable us with the next stage of processing and how we can then actually visualize the tissue.

So for anything fixed in formaldehyde, then these can be stained using H and E stains, specialized stains such as the past stain, for example, and then they can be imaged using light microscopy.

The frozen sections, this kind of preserves the tissue in a different way, which makes antibodies kind of preserves those and then immunophorescence can be used to then see if there are antibody deposits, for example, in the kidney.

And then glutaraldehyde is a fixative that can be used for electron microscopy.

So the reason why we've kind of introduced this kidney case study is it really does nicely use all the approaches that can be used for histology. So it's got tissue processing for light microscopy, tissue processing for immunophorescence, but also tissue processing for electron microscopy.

And this is actually performed clinically as part of the kind of understanding what the underlying causes of the nephrotic syndrome.

So there are a whole variety of different things that can cause nephrotic syndrome. We have primary and then we have secondary causes.

And actually today we're going to be focusing particularly on primary nephrotic so where the kind of dysregulation is happening at the level of the kidney. So secondary nephrotic syndrome, examples of that are things such as diabetes, for example, where very high glucose levels can actually damage the kidney subunits. But we're looking specifically at primary today and then how histology can be used to help identify the primary nephrotic syndrome.

So there are kind of four main types of nephrotic syndrome. And the most common in children is something called minimal change disease. And we'll talk a little bit about how those different types of histology can help actually diagnose minimal change disease.

There is also a focus on glomerular sclerosis.

And this is basically where the kidney glomeruli becomes scarred and there's a lot of increase in kind of fibrosis within the glomeruli.

Again, that leads to a decrease in their ability to filter out things that are unwanted, but also it damages them.

So blood proteins that normally kind of that no longer happens and that you get albumin passing through into the urine. And then we also have different types of kind of nephritis as well.

So again, this is inflammation from different causes, but inflammation of the actual glomeruli themselves. And I've kind of grouped those tubes together today, just to give you an idea. But really the outcome for today is how can we use histology to help diagnose each one of these kind of underlying causes of nephrotic syndrome.

So just to kind of give you a little bit of background about secondary nephrotic syndrome, and we're not going to talk about this today, there is a whole variety of different conditions that actually can cause nephrotic syndrome.

So swelling in peripheral tissue.

And again, we're going to be talking specifically about disorders that are primary, so specifically impacting the kidney itself.

So there are a whole variety of different stains that can be used to actually assess renal biopsy. So these are tissue sections that have been fixed in the formalin and then further processing down the line. So I mentioned that HNE is the gold standard. This always be done, but also the clinician may request specific stains as well, depending on what they suspect might be happening.

So for example, Congo red, particularly good for looking at amyloid deposits. So again, that would be a secondary cause, but there are other other stains as well, which we talked about quite a few weeks ago. But one that is particularly useful for staining renal biopsies is the PAS stain. So that's what these two glomeruli are stained with. So they've been stained with the PAS stain. The PAS stain is particularly good at picking up the basement membrane. So you can see that here around the edge.

And here we have a normal glomerulus and a minimal change disease glomeruli. So I'd just like you to kind of sit back and have a look to see if you can see any differences between the left and the right.

between the left and the right. So talk about yourselves, but I'd like you to just kind of think, can you actually see anything using the PAS stain? Are there any kind of changes that you can see using this kind of stain?

can see using this kind of stain? So using light microscopy. So I'll give you a couple of minutes to think about that.

So hopefully you can see no difference whatsoever. So actually under light microscopy for patients who are suspected to have minimal change disease as the underlying cause of vernofrotic syndrome, light microscopy doesn't help. You can see no overall differences between a normal biopsy and a biopsy from a patient for minimal change disease. So here, light microscopy gives you some information that there is no kind of scarring, for example, of the tissue, but actually with respect to minimal change disease and the underlying kind of histology doesn't help you thinking about it using light microscopy. However, that's where electron microscopy comes into play. So this is the only way that minimal change disease can be determined using histology.

So here we've got a normal biopsy and here again we've got the basement membrane, we've got those fenestrated endothelial cells, and here we've got the podocytes on the other side. And what you can hopefully quite clearly see is those podocyte kind of processes are completely gone with the sample from someone who's got minimal change. And what's really interesting is that minimal change disease probably affects about 70 to 90% of lymphotic syndrome patients with the kind of children. And actually it's not quite clear exactly what the underlying cause is if that minimal change, but it is treatable with steroids and it may be linked potentially to a reaction to an infection, for example.

So often in children, children will have an infection, then they will just remain kind of swelling a week or so later. So there may be some kind of links there between the presentation of this kind of lymphotic syndrome and potentially infections within children.

But the only way to actually specifically diagnose minimal change is using electron microscopy. And hopefully you really can see a clear difference between these two images.

So this is just kind of a schematic of what you might see from the electron microscopy. So here we've got a normal capillary. We have those really clear kind of finger-like projections that are happening between those podocytes. And with minimal change, these finger-like projections are just, they've gone. And that's called effacement. And they're no longer kind of clearly presented. But the only way you can actually visualize this is using electron microscopy. Like microscopy, you can't see those very subtle changes. It's only when you go down to the resolution of electron microscopy that you can actually see them.

So let's kind of move on to FSGS because it's very long term.

So this is where we can start seeing scarring of the glomeruli.

So again, this is an example of a past stain. So again, we can really see that kind of nice basement membrane. But you can also see there's lots of the stain that's picking up within kind of segments of the glomeruli.

So again, light microscopy in this case can actually be used to identify the kind of underlying cause of the kind of swelling within these patients. So again, we've got an example here where light microscopy can actually help with the diagnosis. And then finally, sometimes patients present with a kind of abnormal immune response.

And that can lead to kind of nephritis. And that's where we start getting kind of a build up of immune complexes within the kidney.

And it's that build up of immune complexes that actually then compromise the function of the glomeruli impacting that filtration that happens. And again, there are multiple kind of potential reasons for this. But again, it's not very clear in the literature exactly what those kind of underlying reasons are. They often are linked to an infection. But is it an infection linked to people's genetics, you know, that is still a big area of research. But for what you need to know at the moment is that we can actually use histology to actually help determine if the underlying cause is from a build up of immune complexes.

So these immune complexes have antibodies. And those antibodies can actually be visualized.

But this is also supported by light microscopy.

So again, there are changes that are caused on the kind of the level of that can be visualized by light microscopy.

And following this build up of immune complexes. And again, this is just a kind of example of how that might look like.

So here we've got a normal glomerulus stained with the past stain. So we can really nicely clearly see that basement membrane and the basement membrane here, underneath all those little capillaries that kind of squashed into the glomerulus.

And then here, we've got thickening of that basement membrane.

So again, there's a visual difference between the glomeruli based on light microscopy.

However, this doesn't tell us whether we have immune complexes.

And that's where we can then use immunofluorescence to help us determine if the underlying cause of the nephrotic syndrome is from build up of immune complexes. And that's why we need to preserve the tissue using kind of freezing fixation so that tissues are frozen. And they can then be processed to determine whether there are immune complexes that have been kind of built up within the glomeruli of these patients.

So hopefully, you can kind of really see that with respect to the case study of the kidney, and the presentation of nephrotic syndrome, that you need to have those three different types of processing and imaging to help clinicians then to diagnose exactly what the underlying cause is.

the underlying cause is. So does the patient have minimal change?

have minimal change? In which case, the light microscopy will look normal.

But when visualized using electron microscopy, then you'll start seeing the effacement of those podocytes.

the effacement of those podocytes. Is there kind of fibrosis happening?

kind of fibrosis happening? Again, the light microscopy will enable the clinician to see that.

microscopy will enable the clinician to see that. And then finally, is there a build up of immune complexes?

complexes? In which case, the frozen section can be used, and then it can be determined whether there are antibodies present in the glomerulus.

So hopefully, it kind of really gives you an idea of how those different arms of histology can actually be used to help with the actual diagnosis of a specific condition within the kidney.

So I'm going to give you maybe five minutes, or would you prefer 10? Five? Yeah, so I'll give you five minutes, and then you can just sit back and listen to what the future might hold. And so yeah, I'll give you five minutes. So shall we start back at five, two? Let me resume the recording. Okay, right.

Okay, so really, this is a great opportunity for you to just kind of sit back and kind of really listen about how the future is with respect to histology. So histology is quite an ancient kind of approach that's used, and it's still used incredibly frequently, both in a clinical setting, also in a research setting.

However, despite it being quite a kind of ancient art as such, there's actually innovation that's happening within that particular field. And I really wanted to just kind of share this with you, because it may hopefully give you some inspiration, not just linked to histology, but also other areas that you might listen to, or hear about as part of these lectures that we deliver as part of your programs.

So there are kind of areas that are particularly advancing with respect to histology. So these are actually approaches that are kind of used clinically, you know, yet.

However, they are being developed, and I just wanted to make you aware of these kind of areas of innovation. So you probably, or you will be aware of this a little bit later on, that some of the actual chemicals that are used in histology are actually quite nasty chemicals. They have to be used in a fume hood. And so there are actually researchers out there who are trying to investigate and improve safety linked to histology sectioning, and actually the preparations of those samples. So I'll talk a little bit about improved safety. There's also advancements in how the actual images are analyzed.

And again, that's not just in how they're analyzed, also how they're captured. And as you probably can appreciate, there's this massive drive to use artificial intelligence to help in the diagnosis and characterization of the tissues that affections that are actually visualized. So again, this is a kind of real area that's starting to really exponentially increase. And again, there's lots of possibility to help with supporting diagnosis. So increasing the speed of diagnosis, but also kind of preventing or missing clinical features that might be missed potentially with the human.

So at the moment, this is very much at the kind of level of looking at comparing the human scenario and how they analyze tissue sections, and how this is actually compared to the use of artificial intelligence and algorithms to help identify features and help with that kind of diagnosis process. As you probably, or you certainly will appreciate, because you'll do this yourself, staining is quite a laborious process.

There is a lot of optimization that's involved. There's an awful lot of processing involved, a lot of time it takes to actually stain samples. So actually, there are researchers out there who are trying to develop label-free approaches. And again, I'll talk a little bit about that later on in this section. And also, there is like a massive drive, and this is a big area, particularly within research, where you can actually get kind of molecular biology information and integrate that to a histology gives you kind of an overview of the kind of tissue, but actually, very little understanding can be gained and linked to molecular biology. But there are approaches that are being applied now that enable researchers to actually take a section, look at specific features within the tissue, and then apply molecular biology approaches. So I'll end on that bit.

So safety is a big thing in research. We all have to write risk assessments. For all the practicals you do, there's a risk assessment.

But with particularly tissue sectioning, one of the major risks is linked to something called xylene, which is a particularly toxic compound, must be used in a fume hood. So there are some significant health and safety concerns linked to xylene.

So there is research out there trying to use approaches that no longer require xylene specifically.

So there are some xylene replacements that have been developed. And actually, we've started using some of those xylene replacements as part of the practicals. So again, really increasing and improving our health and safety kind of considerations of the practicals that we give. And again, throughout, if you ever have a career in research, there will be part of that role that will be always driven behind making things safer.

Because everyone has a responsibility, not for their own safety, but also the safety of those around them. And as a group leader, also have responsibility for the safety of the people who work with me.

So there are drives to the practicals you do, it actually still requires to be used in a fume hood, but it's much safer than the standard xylene. There is research out there trying to replace xylene with isopropanil alcohol.

Again, it would still have to be used in a fume hood, but it's not quite as nasty as xylene. So there's certainly a lot of scope to develop kind of that processing step that involves in dehydrating and clearing the tissue samples. So that's a little bit about safety. There's obviously a lot of scope there, potentially for the future.

So the next area is really thinking about imaging. So when you look down a microscope, and you probably realize this when you're in your practical sessions, looking down the microscope, you can see one field of view. You move your tissue section and you can see a different field of view. And they could look so different. And I think some of you really appreciate some of those tissue sections that you can look in one area and see something, look in a different area, and it looks completely different.

So that's really where kind of automated image analysis can come into play.

So here I've got an example of a piece of software that can be linked to a microscope that allows the researcher to basically take multiple images and then the computer algorithm will stitch them together. So originally you'd only see a small area, but because the image analysis is taking multiple images, and these are called tiles, and those tiles can then be stitched together. So you actually have a full appreciation of the tissue that you have available.

So this is one area. And again, with that automated capture and that real kind of clear overview of a larger area of the tissue, that can also help with diagnosis. And there's a real drive as well to help with sharing these kind of big datasets between different countries. So some countries have more expertise in actually understanding the histology that they're seeing compared to others, but through kind of data sharing, then that could be addressed and expertise and can actually be shared across countries where they are.

So with these kind of large kind of datasets also comes that kind of understanding of image recognition systems.

So actually there was a really nice piece of work where they took an algorithm that was designed to identify volcanoes on Mars.

So volcanoes on Mars look very much like the nuclei of a cell. So there was a piece of work where they took the algorithm that's used to kind of look at volcanoes on Mars and they applied it to count the number of cells within a tissue sample. So again, that's a really nice example of kind of interdisciplinary work and actually the best innovation and the best ideas actually come from cross interdisciplinary directions.

So that's just one kind of example I wanted to share with you because I found that quite fascinating. So yeah, so we have kind of advances in kind of image recognition systems. Within kind of research, there's also a big drive to support researchers with access to tissues. So there are things such as biobanks and actually biobanks are something that I personally have also kind of benefited from as part of my own research. So I collaborate with some guys at Warwick who have access to a biobank and we can actually get access to patient samples through the biobank. There's obviously a lot of paperwork involved but it's a great way of kind of supporting research and ultimately helping people.

And also there are kind of moving towards opportunities for a bit of more point of care testing linked to histology but that really is in its infancy as you could probably appreciate the kind of complexities with taking a tissue section, preserving it and then actually analyse it.

So I mentioned that staining is a really kind of laborious aspect of tissue processing. It takes quite a lot of time, takes a lot of optimization to make sure that you have the right actual outcome that you're interested in. So there are researchers out there who are starting to develop kind of alternatives to staining such as the Asian e.

So and again if you are interested in this I will put this paper or you can look it up within Blackboard so it's really available.

So it's quite old now and I think this kind of highlights how challenging it is to start actually implementing some of these changes into kind of regular practice.

So here this is a liver section but actually they haven't taken sections, they've actually taken x-rays throughout the tissue section. So they haven't actually physically made slices, they've just used x-rays to kind of pseudo slice their tissue section.

So this is what it comes out with and again then following that they use algorithms to then try and pseudo stain their samples and this is what the outcome is from that particular piece of work.

So this approach didn't involve any physical sectioning of the liver.

It actually used x-rays to kind of pass through the sample and this has the potential to then give you a real 3D visualisation of that piece of liver. So there are also and more recently kind of researchers who are trying to develop ways by looking at just an unstained tissue can you then pseudo colour it so it then looks like an H&E stain.

So this approach would then avoid completely the need for an H&E stain.

So this is just from their kind of scientific abstract. So the tissue processing would be the same at the start so you'd still have to section your tissue into sections and put those onto slides and again this is something that you'll have the opportunity to do as part of a practical and then just like you would you guys are planning to do they'll do an H&E stain, actually develops an approach that can just take these unstained slides use some computational kind of power and then they can pseudo colour these images from these slides and then they can be then looked at by a person at the end. So this would avoid all the kind of optimisation that's needed for an H&E stain, the time that's also involved in that process and streamlines that kind of part of things and actually this is what they came up with. So this is the unstained tissue so when they did their sections and this is what they imaged in their piece of kit and then they did their virtual stains that's using those algorithms to then pseudo stain that piece of tissue and that's actually what it came back with and then they went back and then did a true H&E stain and that's what they call the ground truth here. So this is the H&E stained, this is what it looked like originally and this is the pseudo H&E stain.

So again with the examples they give in this particular paper it's pretty accurate I would say and however there's still lots of optimisation that needs to be done in different kind of disease settings for example can the algorithm actually support this transition from staining them physically to having the virtual stain and actually how does that kind of support the conditions and the but really this is a kind of big area particularly that researchers are quite excited about is that can you take your histology sections and then can you actually then get some kind of more in-depth molecular biology information from it.

So this is kind of some examples that kind of currently form quite a bit so we have our standard histology so we get our sections we stain them with our stains same with H&E and this is what it looks like but we might want to know how transcripts change within a tissue so that could be done in bulk so here we've taken a big chunk of tissue and then done some RNA sequencing in the whole tissue so while that has some kind of benefits but it gives you an idea of what's happening across the whole tissue rather than potentially specific areas that might be more informative. There are also kind of single cell RNA sequencing approaches that can be done and again that's helpful gives you information about individual cells but again it doesn't give you any information about the environment and then really the kind of an exciting kind of development is something called spatial transcriptomics that's where you take your tissue section so you have an idea of what's happening and kind of benchmarking what's happening in different areas and that allows you to then look at the transcripts and how things are changing in specific locations. So here the resolution isn't quite down to single cells but it gives you an idea of how different areas have changed with respect to their biology and it's really great because then it allows you to kind of give a suggestion of how the architecture of the tissue section relates to potentially changes in the biology.

So that's really a kind of big area that is kind of expanding and quite exciting for researchers and underlying that is an awful lot of kind of big data so being able to appreciate and handle big data is actually a really good skill and that's something you'll be exposed to as part of your degrees.

So that's the kind of the end for me.

So today we've talked about the kidney so having a bit of an appreciation of the structure of the kidney but specifically actually using the kidney case study of how we can have different types of sectioning and tissue processing to help researchers and clinicians understanding the cause of nephrotic syndrome because those patients will all look similar with the presentation but actually the underlying causes are very different and the only way that they can be diagnosed is actually through those three types of tissue processing and then the bit that again I've got to stress you're not assessed on this second part is just really about how something that's actually quite an ancient experimental procedure actually how innovation is helping support development in