Wk 6 cervical + andrology

kay, so, what is a screening program? So, a lot of the time people sort of go to hospital because they're feeling unwell, they're looking for a diagnosis, but in a screening program you're actually looking at a healthy population to try and identify disease before it becomes apparent.

So, it's easier, we all know how easy it is to treat cancer if it's caught early enough.

So, that's what the screening program does, it looks at the healthy individuals, but not all conditions can be used in a screening program.

There are certain conditions that things that diseases have to meet, so it has to be easily identifiable, so you can detect it easily, and if you do detect it, then actually you need to be able to treat it.

So, probably there has to be an effective treatment for that, and the screening program can't be too invasive, otherwise people won't want to participate.

The NHS runs a lot of screening programs, so these are just some examples of them here. So, last year 3.2 million women were screened for cervical abnormalities, people are screened for bowel cancer, men are screened for abnormal aortic aneurysm, a lot of babies have sort of anti-natal screening and postnatal screening. Those with diabetes have routine eye screening to make sure that if they have any degenerative changes in the eye, they're picked up early enough.

Obviously, pregnant women are screened for a whole host of diseases as well, and then there's the breast screening program as well, people have radiology on breast to see if there's any abnormal lumps and lumps in testing for fetal abnormalities. So, there's an awful lot of sort of screening that goes on, and there's a little link here at the bottom if you want to find out any more about any of the other sort of screening programs.

The one I'm talking to you about today is a cervical screening program, because that's something I used to be involved in when I sort of first started out in my career.

It's now kind of moved across into a molecular test, so there's a far more sort of way back in the dawn of time, a chap called George Papanicolo in 1941 first sort of said that he could, if you examine cervical cells, you could help the detection of cancerous changes in the cervix.

This was sort of, everyone agreed that this was a case, so, and the sort of national screening program started in 1964.

However, it wasn't very well organised, women just came along as of when they wanted. There wasn't a proper sort of call and follow-up, so if something was detected, you weren't necessarily sure that it could then be sort of managed properly. So, the NHS developed the cervical screening program in 1988, which has a centralised computer system, and every woman was kind of like logged on this system, and they were invited to participate in the program and have a smear test, which at the time was spread on the slide, and the cells were looked at by sort of humans, every slide was looked at by a human, and anything that looked abnormal, they used to be sent to the treatment. This kind of carried on like that until the year 2000, which introduced liquid-based cytology, which used machines to kind of make a really thin film of cells, like a monolayer of cells on the slide, which made it a lot easier to look at the HPV triage, because the human papilloma virus is what causes the changes in the cervical cells that continue to sort of precancerous and cancerous changes. So, the idea was, if as a human we detected something that was abnormal, then they would do an HPV triage on that to see whether they had a high-risk virus or not, and then they were also using that system to sort of, when the person has gone in for treatment, they were testing to see if all the virus had been removed, all those tests, it's like a test of cure. Then following on from that, in 2020, it switched to the human papilloma virus detection, was a primary screening tool, and then if they were positive, then actually then people would then look at the slides, so it kind of turned it on its head.

Okay, so I'm sure you guys have all been part of this, so you only had your HPV vaccine when you were at sort of the vaccine, so there are HPV viruses which are high-risk and HPV viruses that are low-risk. It's the high-risk of cancer ones that you get vaccinated against.

The low-risk causes sort of like genital warts and things, but doesn't actually lead to the progression to cancer. So, the vaccine guards against most of the need sort of like common high-risk viruses.

So, about your 25th birthday, you'll get a call inviting you to attend your first survival screening.

So, you have a smear done, and that will obviously be tested and analyzed, and then every three years, the system will call you back for a repeat test until you're 50, and then they call you back every five years until you're 65 when the screening stops.

So, in 2020, the World Health Organization set a target to try and eliminate cervical cancer by 2030, and their aim was to vaccinate 90% of all girls, screen 70% of all women, and treat 90% of those with cervical disease before it progressed to cancer.

They can't stop the sort of precursors to cancer, but they can stop the actual cancers from arising.

And this is just a little image of, so if 100 women went for screening, obviously 13 of them are likely to be sort of HPV positive, and then if you're HPV positive, your body can sometimes eliminate the virus on its own, so they won't treat everybody who's HPV positive on their first test. You'll be sent away and invited back in 12 months time. If you're still positive, then those people are referred to what happens at colcoscopy later.

So, what's the effect of all this? So, this is a type of, sometimes of more up-to-date information, but it's, so this is the effect of the different changes that I've talked about, about how we used to be doing the people looking at all the slides, and then looking at the monolayer of slides, and then adding in the HPV testing.

So, this is from 2016 and the prediction to 2032, what they think the introduction of HPV as primary testing. So, obviously if it had carried on the yellow line up here, which is it was just cervical screening only, so the rate per 100,000 women was likely to have stayed at about 16 women to developing spinal cancer.

So, when they added this, so primary HPV screening from 2008 here.

So, they did pilots before this, and that shows how the sort of the rates of cervical cancer are predicted to kind of decline using the primary HPV testing. So, all is going great here in the UK and a lot of the sort of modern world, but if we're looking at sort of worldwide and the rest of the world, cervical cancer still ranks forth in all of the cancers worldwide, and it's the highest cause of death, cancer deaths in women in 38 countries.

And in 2022, 348,000 women died from cervical cancer worldwide, and this is likely to sort of continue to grow, and most of these occur in low and middle income countries as they have sort of poor access to screening, and so the early detection isn't there, and you can sort of see the number of cancer, cervical cancer cases expected, and there's really sort of, you know, greater than 500,000 in areas like China, India, and certain countries in Africa.

And then, yeah, in a lifetime, you just have a look at the different demographics there. I know there's been a lot of work being done recently about trying to expand cervical screening to these countries, sort of like self-testing and having the tests sort of with AI and having the information sort of sent across the sort of regional centres, because I think in a lot of these places, the pathologists can't get to look at the results, but now with the sort of advent of modern technology, the slides can be basically sort of digitised, and the information sent via sort of the internet and people remotely can look at them, and then refer back to the women. So that's kind of a big, a big step forward, and that's where a lot of the work has been happening at the moment. So back to us over here.

So the cervical screening programme obviously uses the HPV testing, and the cervical smear sampling is done in a little pot called a thin prep tube and a brush. The brush is put into the cervix and swept around and around. There's a little area just here called the transformation zone where a lot of the, because one cell type is transferring from an ectoplasmic to endoplasmic sort of cell type.

So there's a lot of change happening already in that cell type and that makes them quite vulnerable to sort of becoming precancerous. So we tried to make sure that little area is captured on the smear. It's put into a little pot. All the cells are removed from the brush, and this is what a thin prep slide will look like.

So what are we looking at? We talked a bit a lot about the HPV virus and how it sort of causes carcinogenesis.

So this is kind of like the viral genome. You've got all the sort of require, you know, the usual kind of genes that are required, that a virus requires to kind of cause cancer.

And the HPV virus is a double-stranded DNA virus.

So it has two genes, E6 and E7, and what it will do, it will cut into your genome and insert itself into the genome and cause the expression of oncogenes in E6 and E7 is what causes the cancer to occur. So the way that it works is E6 targets P53, which is one of your genes, which binds to DNA and prevents mutations in the DNA. So actually it's one of your housekeeping genes, it stops your cells from kind of becoming cancerous. And if something is detected that's not right, it will bind to that and actually stop that cell from dividing.

But obviously E6 will bind to that and prevent it from doing its job. And E7 does something similar.

It targets the retinoblastoma gene, and the retinoblastoma gene will normally inhibit cell cycle progression until the cell is ready to divide. So in a lot of cancers you get kind of like genomes that have like more than one copy or partial copies. And if the cell notices that something's not quite right, again, it will stop its cell from dividing.

But again, when that is kind of like being targeted, and then that stops working as it should. So the assay that's done in this area, and I think it's kind of nationwide, we use something from a company called Hologic, and it's called Atima assay.

So what this does is rather than a DNA-based test, it will actually, if these genes are being transcribed, then actually they're going to be, if you have transcription, then obviously they're going to make mRNA copies of themselves.

And the idea of the Hologic test is that actually just because the gene's there, although that doesn't mean that it's going to be doing anything, but if it's actually actively transcribed, being transcribed, then it's having an effect, it's doing what it shouldn't be doing.

So that's why in this one it's targeting the messenger RNA, and it only identifies high-risk HPV infections that are present and active. And the studies are showing that the level of messenger RNA present identifies the activity of a high-risk infection. And obviously, the more infection you've got, the more mRNA, the more risk, the risk of it developing. So the test actually has a cutoff point, so it'll get to a certain level. If it breaches that level, then it says, yes, it's positive, because the person's at risk.

And it identifies the presence of 14 high-risk HPV types. So one of the things is there are 14, it's detecting 14 high-risk HPV types, and one of the risks of this that we're currently sort of seeing when we sort of get, we treat the people with cervical cancer, is there a new, we all know what happens to COVID, and the flu virus.

Every time there's new mutations, there's new things that come to the fore, and I think what's happening now is there's another sort of virus subtype called P73, which used to be really low level, didn't really cause anybody any problems, but we're now sort of finding that that's now creeping in.

So the assay will probably need to kind of evolve to include that to make sure it's capturing all the variations in the virus that happens.

So back to here. So here we are. So HPV DNA levels, this is the actual presence of the virus, but as it becomes integrated and active, it moves into the genome, and you're going to get mRNA levels increasing.

The little picture above it is the actual sort of skin of the cervix. So in normal cervical epithelium, you've got your basal layer at the bottom, which are like basal cells, which are the ones that divide. So like normal epithelial sort of things shed off the top, and the cells divide from the bottom, and then push their way to the top, like your skin sheds at the top, and this does the same thing. They start off at the bottom, and they divide and divide and divide. They mature as they get to the top. They become thinner and flatter until they shed off the top.

So if they become infected with HPV, again, you can sort of see this here. So this is quite a low level infection. It might cause minor changes, which may or may not be detectable by the assay, and as much as the mRNA levels here are quite low. As more cells become infected, you get something called low-grade or syn1 lesion, and they're starting to sort of be a little bit more frequent.

Then you can get, again, further progression.

You get syn2, so they become more abnormal.

There's more cells affected, and moving on to syn3 or high-grade, it's filling up the whole width of the epithelium.

In all these cases, all these are precancerous, and this is kind of what we're hoping to catch, because when it's at this stage or below, and all you need to do is basically take off that layer, and you're cured.

Because what it hasn't done is it hasn't, so underneath the basal layer of cells is something called the basement membrane, which separates the epithelium from the subdermal tissues.

If the cells haven't broken through that layer, then it's called an in-situ cancer.

So it's still confined, and it hasn't spread. Once it's broken through that basement membrane, it becomes a cervical carcinoma, which means, because once it's broken through, it has access to the rest of your body systems. It can reach ducts, it can reach blood vessels, it can sort of spread through the stroma. So the whole point is to try and stop it before it gets here.

Okay, so there are various other tests available, which you can look up if you wanted to.

So there's hybrid capture, there's the COBAS test, there's also the histologic one, there's a whole load of them out there, if you want to look a little bit more about the others. But I'm just talking about the one that gets done in this sort of area. And here, all the HPV testing is currently done by Berkshire Surrey Pathology Services, which is based in Chertsey along the road. And they use one of these little machines, which is a PCR machine, so polymer chain reaction machine. It's called the Panther. Okay, this little slide, just a little bit about all the different subtypes that are being tested and how often they're present in cancers they're detected. So HPV 16 is one of the main culprits, so you can get cancers in two parts of the cervix.

so you can get cancers in two parts of the cervix. There's the squamous cell carcinoma, which is on the ectocervix, and then the adenocarcinomas, which are in the endocervix, are more glandular in origin. I'll just have some pictures of those later. So how does the test work?

So how does the test work? So obviously testing for the messenger RNA, it's a qualitative test based on the direct expression of E6 and E7 from all the different subtypes. It doesn't discriminate amongst the 14 types.

If you want to know which subtype you've got to, we've got, you're going to have to go and do some further analysis and testing. It just tells you that one of those high-risk subtypes is there.

And it's got an internal control, so we make sure that the test is working as it should, and a negative control to make sure that there isn't anything in there that's giving you a false positive. So you test the control in against false positives and false negatives, and it can carry up 250 tests in approximately five hours. So there are a lot of tests that come in every day. There's probably several thousand that sort of come in to be done every day.

Some pictures. Okay, so this is what you'd see in a normal sort of endospichal, normal sphichal smear.

So you've got these little tiny little cells here, are white blood cells.

These are called squamous cells, and so these are the ones that near the surface, and they're sort of quite flat.

They change color depending on their maturity, so when they're sort of a bit more immature, they're sort of a blue. You can get some smaller ones, so they go from small to slightly bigger like this as an intermediate cell, and then they're at the very, very top. These are sort of superficial cells, and by the time they've got to the very, very top of the epithelium, they're kind of dead. So you can see the nucleus inside, so this nucleus is still sort of vibrant and active. It's got some chromatin, you can see the chromatin structure in there, but by the time you've got to the superficial cell, the chromatin is kind of all dead and degenerate.

These little things here are called the endospichal cells, and they produce sort of mucin and other secretions.

So you can see they're like a long thin tube with a little nucleus at the very, very top, and this is low-grade HPV infected cells. So here you can sort of see the nuclei become larger, more irregular.

The chromatin pattern becomes more coarse.

It's not like a fine lattice. Again, you can see some clumping, and these things here are called choilocytes.

You don't always see them, so sometimes a lot of the time you're looking for the nuclear changes, but what this cleared area here is where the virus has gone in and replicated, and this clear area is actually full of kind of viral particles ready to be when the cell lyses, they obviously sort of spread. The next slide is showing something with high-grade dyskaryosis.

So you can see these cells, it's infecting the cells that are quite superficial, and the changes are kind of, you can see those changes there. These ones are affecting cells that are much sort of lower down, and you can see they're much smaller, they're much rounder, they look more immature. The actual nucleus is much, much larger compared to the entire volume of the cell, and you can see on this one, the actual chromatin pattern has really sort of changed, and it's not looking very nice at all. You get degenerate cells, keratinized cells, a lot more inflammation, so obviously this person would have gone straight to kophoscopy. And this one is, if you remember the little rod-like cells, the endoservical cells, so they also can be affected by sort of HPV and the changes, and this is kind of, so rather than a nice, this is kind of a more normal section, they're actually very well behaved. They form little honeycomb patterns, so you can see rows and columns of cells, and they're also nicely orientated.

That's like a normal group, and then suddenly when they become like this, it's called sieging, so by called glandular intraepithelial neoplasia. So you can see they've lost their kind of like organizational structure, and they're all crowded in on each other, and the actual sort of shape of the actual cells themselves changed as well.

And again, like the others, the chromatin, the volume of the nucleus will increase, and the chromatin will have such changes as well.

So it's not just, if you're looking at the slides, it's not just kind of like the abnormal cells that you can see. You can also see infections, so the first one is Candida. You can see it's a yeast infection, you can see the hyphae, and sometimes you can even see some of the spores, you can see the hyphae sort of branching through, and it will actually kind of go through some of the cells as well.

A little bug called actinomyces, which is yeah, sort of a cross between the fungus and the bacteria.

It's like a little furry ball with little hyphae coming outside.

with little hyphae coming outside. Herpes, so that's a sort of fairly characteristic appearance. The nuclear material seems to sort of almost disappear, it's called like a ground glass appearance, and they form these things which they call tombstones for some inexplicable reason, where the cells actually kind of like mold, the nuclei all sort of mold into each other. One of the other things that we see is trichomonas, which is a nasty little thing, which a bug which goes around and nibbles away and eats away at these sort of cells themselves. Okay, so once you've had something abnormal, you will be sent off to coposcopy, and what they will do is they will put you down and they will actually have a look at your cervix, and then sort of one of the things they can sort of see, you need to be normal. This is kind of like a bit of the appearance of like low-grade dyskaryosis and high-grade and cervical cancer, so what they can do is if they're not quite sure, they can take a small biopsy, and the biopsy will obviously go to histology and then have the histology done on it, and they'll have a look at it there. If it's confirmed as a high-grade, then obviously you're going to go in and have treatment and have a sort of a large loop position of the transformation zone, which is basically they get a hot metal wire and scoop out the area that looks affected, and then they will check to make sure that it's all gone, or they can actually cut out that piece of the cervix to make sure that everything has been excised. If it comes back and it's query invasive or invasive, then obviously sort of more drastic measures are sort of taken depending on how far they think it's extended. You can start off with hysterectomy, and then there might be all the usual sort of cancer sort of treatments on that one as well. So that's the end of this one. I was going to say time for break, but I don't think you get one yet. There's no lecture yet. I can give you two minutes. Oh, do you have any questions?

Oh, do you have any questions? We're going to have any questions. Okay. So the next talk is going to be on diagnostic cytology. So we looked at the study of cells to kind of look at pre-cancerous changes.

So diagnostic cytology is looking at cells to sort of make a diagnosis.

So I know you've had the lecture about histology, so this is a little comparison as to why they sometimes use cytology and why they sometimes use histology.

So cytology can be quite quick. I know sort of in Reading, they run one-stop clinics, so rest, lumps and things, so you can come in. Someone will take a sample, you'll wait in the waiting room, and you'll come back and get a result within an hour. So yeah, to get the same day diagnosis of benign lesions, it's not modestly cheap because people just come in and it's quite a short inpatient session.

It's less invasive, so the patient can sort of recover faster.

Against it requires expertise because in histology there's a lot more architecture to look at. In cytology it's very much sort of single cells and you need to make sure that you're targeting the right lesion when you're taking a sample as well. It might not be as sensitive or specific and ancillary tests, so if you want further testing done that might not always be possible on the cytology sample. Obviously for histology it's the gold standard of our diagnosis and it makes all the additional testing a lot easier. You've got more material to work with.

You get things like immunohistochemistry and sort of receptor status of malignant lesions. You can do special stains and molecular panels, which you can't very easily do on cytology.

Against it, its same day results are difficult, but getting stuff through a histology lab will take a minimum of three or four days. It's a lot more expensive and it's usually far more invasive for the patient, so people will sort of weigh up what's the best test to do. So, as I said, cytology is basically the study of sort of three cells in a solution. You can obtain them by scraping a tissue surface. You can get cells from a urine or a sputum. You can get cells by brushing or washing or aspirating cells during such procedures, or you can aspirate cells from lumps and bumps with a blood taking needle. There's a lot of different sites that you can get cytology from, so you've got bladder, pancreas, barduct, chlorofluid, a whole list of them there. Some of these are sort of guided by radiology, so you have a sort of radiology assistive so you can actually sort of make sure that you're aspirating cells from the right region. So, when we get them, fluids.

So, things like urines, cerebral spinal fluids, serous fusion, and then from the lungs, abdomen, ulcers.

So, once we've got the sample, we're interested in the cells. It comes as a suspension, like a blood film or a urine. So, what we need to do is obviously place that microbiology, probably interested in their plasma, you know, human blood sciences are interested in the sort of chemistry of the blood.

We're interested in the cellular components. So, the first thing that's done is they're centrifuged. So, little samples here.

So, they're centrifuged, and what happens is obviously it's like a density gradient.

So, obviously, red blood cells have small ions, they're small and dense, so they will sort of spin down and sit at the bottom of the tube. The plasma is the lightest element, so it will be at the top here, and in between, you sometimes get a little white area, which is where the less dense cells sit, and these are the cells that we're interested in. That's called the buffy coat.

Obviously, if it's a very malignant sample, that can be quite large, but normally they all have them, because everyone will have some white blood cells in their sort of sample.

Something like a urine obviously don't get much blood, if it's more normal, and you'll just get a tiny little pellet of cells at the bottom, which we work with.

Once you've got the sample spun down, we're using a sort of a fine pipette. You will kind of go in and try and aspirate cells just from that little section there, and then put on a glass slide, and then spread.

So, you're trying to get a thin monolayer, so you can sort of see all the cells.

Sometimes you don't get many cells. The buffy coat is very, very small, or if you're spinning down the urine, you can get a tiny, tiny little pellet of cells. So, they are sort of further concentrated into a cytoscentrifuge, so the little cells are topped in there with some other plasma, and basically all the cells are spun down, placed on a little circle on the slide.

the slide. So, do you remember when Claire talked to you about hematoxynineosin staining?

you about hematoxynineosin staining? So, all histology has a hematoxynineosin stain.

H and E stains are first.

In cytology, we use different stains.

So, the two main stains are the papa nikolowski.

A lot of the fluids will have one of each done because they're giving the pathologist different information as to what they're looking at. So, the papa nikolowski is for wet fixed specimens.

So, things that have come in, and in the same way that histology is always fixed in and this sort of papa nikolowski is good for the nuclear detail. It's the same stain that's used in the cervical screening program, and it gives you an idea of the nuclear detail and the maturity of the cell. So, immature cells will kind of be like a dark sort of greeny blue. They'll go to a paleo greeny blue, and then they'll go to pink if they're keratinized. So, you get a little bit more of an idea of the maturity of the cell if there's keratin in there. So, if you've got something like a sperm cell carcinoma, a lot of the time that will keratinize, so the cells will have keratin in them. So, it's also sort of quite good for that.

So, that's what you can, and it's very nice to sort of see the nuclear outline. You can see the nuclear loss and various information like that.

The cells are quite small. So, one of the things that when you fix a cell, it's a sort of cell is a little round ball. If it's fixed, it will remain fixed in this little round ball, it won't sort of, it always stays small.

So, all the information is kind of quite sort of compact. If you've got an air dry specimen, so a lot of things like the thyroid, they'll spread the slides and the slides are just left to dry in the air. So, they're fixed by air drying, but what happens then is you've got a little round cell and it's sticking to the slide, so it becomes flat. So, you get a much bigger sort of cell and a lot of the nuclear detail and the cytopasmic detail. It's a lot easier to see. So, if you look at sort of here, have the difference in the cell size and type and any sort of evacuoles or clearings in the cytoplasm, kind of much easier to see.

So, often they have both, so you can get different bits of information from each cell.

I know Claire also probably talked about immunohistochemistry. Immunohistochemistry is a little bit more difficult on cytology, but it can be done. So, this is just an example of carcinoembryanogenic antigen in a plural fluid. So, this patient sort of had lung cancer and the person is accumulating fluid in the chest cavity, so they obviously drain that fluid from the chest cavity. So, again, we spin it down, pull out the abnormal cells and it's sort of gone through a panel of antibody staining.

So, normally, all of your cells lying in the cavity, in the body cavity, is called mesothelial cells. So, they're sort of small sort of round sort of cells and they have, they express their own sort of antigens, which is normal for them.

So, a normal mesothelial cell would not be positive carcinosinogenogenic antigen.

But some of these cells here are sort of being positive and they're sort of sailing up round.

So, this makes me actually, this looks like an adenocarcinoma because that antigen is always expressed in adenocarcinomas and they would use other antibodies at TTF1 to see if they could find out what the primary was.

They kind of suspect it might be lung, you know, it may be bowel, it may be anything. So, they use a panel of antibodies to try and narrow it down where their primary is because obviously once you know what the primary is, you're going to have a better idea how best to treat the cancer.

So, this is just a little example here.

So, they used two other antibodies, CK7 and CK20, to see whether the tumor is a primary lung or a metastatic lesion of the digestive tract.

So, this is what you'd expect. So, if it was lung, it would be positive for CK7 and negative for CK20. And if it was from sort of the GI tract, it would be negative for CK7 and positive for CK20. So, you've got an idea where these malignant cells have sort of come from.

So, that's general fluid. We also can have them from the cerebral spinal sort of space.

And it's a clear watery fluid that surrounds the brain and the spinal cord.

Not many cells get into, you wouldn't expect many cells in the CSF.

I mean, sometimes you might get the odd inflammatory cell, but actually you shouldn't normally have any cells in there at all.

But we have sometimes sort of sent them down and there's very few cells and again, we use a cytoscentrifuge to sort of see if there's any cells there. And this is an example of a CSF, which contains malignant cells from a cancer of the breast. So, it has spread from the breast into the CSF.

And obviously, there's a risk of sort of brain kind of metastases from that urine.

Again, this is quite a quick and simple sort of way of checking for bladder cancer. Sometimes if there's been blood detected in the urine, obviously the first sort of step would be to send a sample to the lab. They will also send that sample to microbiology to make sure that it's not bacterial inflammation that's causing the bleeding. Then we would get a sample as well to make sure we could see any cancer cells in there.

there. There used to be a big, when the dye industry workers urine to make sure that there wasn't sort of a risk of bladder cancer because the dyes and their intermediates such as benzidine can have carcinogenic effects on the bladder. And obviously, smoking also increases the risk of bladder cancer. What would we see?

What would we see? As you can see, all cells look pretty much the same. So, these are the endothelial cells, urothelial cells rather, and some sort of squamous cells are in there, and a few white blood cells as well.

So, this is some histology of the normal sort of benign urothelium that you get in a bladder. Because bladders stretch, so when they're empty, all the cells all scratch together. And then as the bladder fills up, the cells open up.

So, any sort of abnormal cells can be sort of shed into the urine. And sometimes it's kind of easier because it's the only way you're actually going to be able to get cells easily from all the different areas. Because if it's closed, you can have like cancer cells tucked away inside, so it's more difficult to access them. Yeah, so as I say, it's particularly sort of flat tumors which you can miss using the cystoscopy.

Because you can put a little wire up there to have a look and see, and to take biopsies and stuff, but you might always sort of see them.

So, most of the cancers you get in a urine are transitional cell cancers. And this is kind of what they look like.

See, again, they all follow the same sort of pattern. You get a much more irregular nucleus, the chromatin will be sort of disorganized. And they fill up an awful lot more of the cell. And sometimes because you've got so much more DNA, because the copy numbers sort of wrong that they become a lot darker, there's a lot of DNA present there as well. So, that's the transitional cells. You can also get a squamous cell bladder cancer, which is rarer, but usually invasive. And here, like I was mentioning, this is a pap stain. So, you can sort of see these are the sort of urethralia cells, again, not looking too abnormal, but in amongst them, you've got these kind of pink cells. So, these are actually keratinized squamous cell carcinoma cells. And they shouldn't be anything sort of keratinized or like that in the bladder. So, they're actually quite sort of obvious to see. Squamous cell carcinoma is quite rare in the UK, but it's common in countries where the heartseia exists, like the parasitic worm that lives in freshwater in some parts of the world. And that, if it gets into your bladder, what kind of actually sort of initiates sort of squamous cell carcinoma. So, it's far more common in Africa and Asia. You also get adenocarcinomas.

And it's kind of very rare. There's only a few glandular cells in the bladder.

There's only one little small area. And it's about, you know, only one or two percent of bladder cancers tend to be this type. You also get sarcomas and small cell cancers, which are even rarer. But any part of it can sort of become cancerous.

So, that's in the bladder muscle. So, a sarcoma is a muscle cell tumor. And small cell carcinoma of the bladder is a poorly differentiated neuroendocrine neuroplasia.

So, that's kind of hormone-producing cells. To more sort of invasive sort of ways of getting cytology samples. So, obviously, there's endoscopes or bronchoscopes.

So, a bronchoscope is a little device like this. So, it'll go through your mouth and down into your bronchus to take samples. An endoscope will obviously the same sort of thing, but it'll go down the esophagus and the GI tract from either end, depending on where they think the lesion is. So, what they can do is they can sort of like take little biopsies. So, they'll take little chunks and pop them into it to take them out and pop them into a tube. Or they can take scrapings at the surface as well as a little brush. Alternatively, if you're looking at something in the lung that's kind of like so far into the bronchi and stuff, what they can do is they'll push saline through the little tube and then suck the saline out and you end up with cells suspended in the saline. So, things are sort of more distal. You can also get some cells and samples from that as well. So, again, this is what I've sort of said.

So, it's called a bronchi alveolar lavage. So, again, we'll often share our samples with microbiology because it can be cancer or you can also get sort of infections in the lungs. If there's bacterial infections, you can get fungal infections.

I know sometimes they'll come up and do a CT scan and you'll get, I think, they'll see like a lump mass in your lung, but that doesn't necessarily have to be cancer. Sometimes it can be sort of aspergillosis or catatosis. So, you actually get concentrations of fungal tissue growing in your lung. It's not very nice, but it's not cancer. And it can also, so we can use it for deep-seated tumors and any sort of other opportunities and or they've got persistent cough or wheeze.

It's kind of one of those things that might do to actually sort of see if they can identify what's going on. So, donor microscope.

So, donor microscope. What would we see?

we see? So, these are some normal bronchial cells and you've got little ciliated cells. So, these look very much like they did in the cervix. They're sort of like columnar cells with a little nucleus at the bottom. The difference with these is they've got little cilia at the top.

So, they all sort of line your bronchus and then the little cilia beats upwards to make sure that all the gunk and publishing your lungs get kind of pushed out so you can cough it up.

You've also got this more little bronchial cells here. There's some bacteria and some red blood cells.

And again, that's kind of normal what you'd expect. And you also get sort of macrophages and white blood cells neutrophils here.

Again, they go around and sort of make sure everything's cleaned up, get rid of the bacteria. So, this is kind of what you'd see in a normal and obviously it's a mucus and various things like that. But that's normally what you'd see.

Like before, there's various sort of cancers that can arise in the lung. So, in the bladder, squamous carcinoma was probably not so common. In the lung, squamous carcinoma is very common.

They tend to be large tumors. And in the middle, as tumors get very large, sometimes the central area doesn't get enough oxygen or blood supply. So, the central can actually sort of become necrotic and die.

And squamous carcinomas.

And if you're looking at the cells here, again, very similar, you've got keratinized cells and this one's got three nuclei. Make sure you have one. So, in the lung, there shouldn't actually be any sort of squamous cells. There should all be those little bronchial cells that we saw, the little columns with the nuclei and the cilium.

But because of damage, let's say smoking being one, that's one of the reasons that lung cancer is so close to smoking, is that your little cells, those little sort of bronchial cells will, under stress, transform into, it's called metoplasia, when you transform from one cell to actual another. So, they'll actually transform into squamous cells, which are sort of more resistant and sort of give more protection.

And in that transformation, they're all a greater risk than becoming cancerous. So, adenocarcinoma is the most common type of lung cancer. And this is where there's actual little bronchial cells become malignant because, obviously, the number of sort of DNA mutations and in smoking, obviously, all the sort of chemicals that are in there will cause kind of sort of malignant causes.

But one of the things that they can't, so when we talk about screening program, lung cancer is one of those ones it's very difficult to have a screening program for because by the time you've got any symptoms, it's usually sort of quite well advanced by the time you've got to diagnosis.

And the last one is small cell, not that cell carcinoma. So, that's kind of sort of quite rare.

When they have their own little specialized appearance, they kind of mold into each other and have very, very little cytoplasm.

The adenocarcinoma, you can see that they're glandular because they're producing a whole lot of mucin, but they're all kind of other than being in a nice bordered line. So, they're all just bunching together in big clusters of balls. Okay, moving on to the next part of the body.

So, we get a lot of thyroid samples and obviously they're taken by fine needle aspiration, which is literally what it says. They put a needle in and they suck out cells to see what it might be.

So, obviously, the thyroid is in your neck.

If it gets causes of swelling, you've got a goiter, so your neck sort of big swelling in the neck.

It sort of produces and stalks hormones and that affects sort of metabolism, sort of heart rate, pressure or the temperature and how efficiently you use energy.

It produces hormones like T4 and T3. So, one in 10 people will develop a thyroid nodule by the time they're 50. Not all of them nasty. Some of them are just there.

So, less than 10% on ligament. So, what would we expect to see? So, a normal sort of thyroid would have follicular cells, again, which is like monolayers.

On cytology would appear like a monolayer sheet of nice neatly organized cells all respecting their own space.

The thyroid also has colloid, which would sort of stain with blue or green.

You can see that in there, and again, that's a normal experience you sort of see there.

Too much of this colloid will also kind of cause maybe cause a goiter, but that would be a sort of benign goiter. Also, it looks quite pretty on histology, and you can sort of see how the two cytology pictures relate to the histology, because you've got the colloid in the middle, and then you've got the cells lining the outside. So, you can sort of see how the structure would then sort of work. It's a very, very vascular organ.

So, a lot of, it leaves a lot when you kind of get your samples available, often quite sort of bloody.

So, goiter, when you get the swelling in the sort of like the iodine deficiency, and thyroid sort of goes hyperplasia, so it just kind of keeps growing and growing to try and sort of produce more sort of to compensate for the lack of iodine. There's also Hashimoto's thyroiditis, which is an autoimmune condition, where your body obviously is reacting against the thyroid tissue itself, and thyroid is sort of progressive being destroyed, and then you become sort of, have low levels of thyroid hormone and have to take supplements.

A lot of people with this tend to get quite tired and cold, usually what takes them to the GP first.

You can get thyroid carcinomas.

Again, you've got overlapping cells there, with all the usual sort of characteristics that we saw in the other ones, and a medullary carcinoma.

and a medullary carcinoma. Again, each body type has its own different types of cancers. Can you still with me?

Can you still with me? Okay, nearly it. So, breast cytology. So, obviously there's the breast is sort of made up of the ducts, the milk ducts, and the lobules, and a lot of fatty tissue. And at the moment, it's the leading type of cancer in women, which is 25% of all the cases of cancer. It also affects men as well. So, it does have a peak incidence of between 55 and 69, which is the one which you get to 50. You end up being part of the screening program, which can obviously try to detect sort of the cancers earlier in our treatment. So, the breast is composed of fat and stroma that supports the glandular tissue, and a branch and ductal system that leads to 60 main ducts, which open up with a nickel.

So, signs of breast cancer, you can get like a lump in the breast, a change in the breast shape, some dimpling of the skin, patchy or red scaling, more inverted sort of nipples.

So, about 5% of the cases are a result of inherited genetic predisposition to cancer, which includes a BRCA gene, mutant mutation, which I'm sure is where the name goes.

Angelina Jolie, that's why she had double mastectomy, because she was fat, but she carried double. She was kind of like this for this gene, so she kind of went for elective kind of mastectomy to try and stop the cancer. Okay, so cancer as always can develop anywhere. Those that develop in the ducts are known as ductal carcinomas. One of those in the lobules here are lobular carcinomas.

carcinomas. So, in cytology, what would we see?

what would we see? So, this is kind of what we'd sort of see in a normal breast. So, you either only got fat than a few benign ductal cells, and benign normal breast tissue is really aspirated, less lesion is completely mischievous by the needle. This is kind of like a normal breast. It's kind of quite cohesive.

These are some fat cells, so they're virtually completely empty, which is tiny little nucleus at the end, and they've got a single background of myopoeia cells, which are single cells popping on their own. Sometimes, in younger women, you can get like lumps, which are suspicious, but in a lot of cases, these are things called fibroidinomas.

They're just sort of benign ductal cells with some epithelial cells.

Sometimes, they form really cohesive groups, and I think that one looks a bit like a poo.

Okay, so ductal carcinomas make up 80% of all the breast cancers. And in the same way as we talked about in the screening program, you can have invasive or you can be in situ. So, if it's just sitting in the duct and it hasn't moved to the rest of the breast, it's called ductal carcinoma in situ.

And again, in the same way, just that one little section can sort of be removed and you're probably going to be okay.

And again, same sort of pattern. You've got your cells, the nuclei become more irregular.

In this case, they're more discontinuative. So, the treatment of breast cancer depends on its hormone receptor status. And the most common one is HER2, which controls cell growth, division, and repair. So, high levels in some cancers and tumour grows faster if it's HER2 positive.

But conversely, it will also respond better to drugs that target that protein. So, a lot of breast cancers that we get will also then go on to have histology and be sent away for sort of their HER2 status to be known.

The lobular carcinomas make up the rest of the breast cancers. They respond well to treatment, but can be difficult to detect as it rarely forms a lump.

And as you see, they're sort of like quite widely spread along the ducts and the lobular sort of there. So, it can be easily missed due to low serially limited atypia and cells present in the cords or clusters. And again, that's usually only detected if you've actually had sort of an omen told you get 10 minutes.

omen told you get 10 minutes. Okay, any questions? Okay, you ready?

Okay, you ready? Right. So, this is the last lecture of the morning. And this is on something that's completely different.

And this is on something that's completely different. It's on Andrology. Does anybody know what Andrology means?

know what Andrology means? No? So, Andrology is a study of male fertility.

So, over in where I work in Buckinghamshire, we've got quite a large Andrology Centre.

So, obviously, we have histology, cytology, and Andrology.

So, it's completely different to anything else that we do. So, a lot of hospitals will run fertility clinics, and they're usually based in either gynae or urology.

And Andrology provides information on male fertility as part of this process. One of the main differences is we actually provide an outpatient service. So, it's probably one of the few areas in sort of cell path where we actually have contact with patients.

And this requires a whole different sort of skill set. So, obviously, there's patient bookings. We have to deal with sort of GP referrals. We have to talk to patients and do patient interviews, as well as so.

Okay. So, obviously, it's sort of a growing field because a lot of couples are now sort of having sort of difficulties in conceiving.

So, I think it's about sort of 12% at the moment. So, I think it's something that's kind of there's a big need for, and it's all growing as well. So, sperm production.

It's hormone driven by the hypothalamus and anterior pituitary gland. So, there's a lot of hormones here that you find sort of men and women share. So, there's follicle stimulating hormone and luteinizing hormone. Obviously, they do slightly different things.

So, the hypothalamus secretes the gonadotropin-releasing hormone, and it acts on the pituitary gland, and stimulating it to release the follicle-simulating hormone, and luteinizing hormone.

They're released into the bloodstream, and they act only on the testes, and encourage spermatogenesis within the sort of testes. Okay.

Just a little bit of a little diagram there.

So, how do the little sperm develop? So, obviously, it grows from the outside inside.

So, on the outside, within the testicle, there's 700 feet of tubing, all very neatly packaged.

And these contain, these little tubes, they're called terminiferous tubules, in which the sperm are made.

Sperm are made from precursor cells, sort of here, called germ cells, which divide by mitosis, and they can reduce up to 120 million sperm per day.

The busy little bees. But, obviously, these have a sort of, the normal sort of genome. So, there's two copies, two N of the DNA.

So, and obviously, to produce a germ cell, you kind of can't have that because it needs to combine with the DNA from the egg to have two N copies of those chromosomes.

So, actually, it needs to split in half.

So, you go from having two copies to only one copy. But, before it sort of does that, to increase kind of genetic variation, it will actually sort of replicate, but it will kind of twist and make sort of, you do the replication crossing over a very sort of genetic material. So, actually, its sperm will have a completely different sort of set of genetic material. So, you don't get clones of half clones of yourself. Each one is increasing the sort of variety.

This will then split by meiosis. So, each copy will only have one copy of the sort of genome in it.

And then, these will sort of then continue to divide into sort of chromatids, and then they will sort of grow into bromatozoa. And so, diploids of bones will then, you say, divide into the haploids of 64 days.

64 days. So, it's not quite. So, even though there are high numbers of sperm produced, there are a lot of quality control checkpoints throughout the process, which ensure that the biological and genetic integrity of the sort of ejaculated sperm is as it should be. So, what is classed as sub-fertility?

So, what is classed as sub-fertility? So, infertility is defined as a couple being unable to conceive for 12 months or more, and approximately one in seven couples. That's three and a half million people in the UK may have difficulty conceiving per year. The causes of infertility can include sort of, we just don't know, disorders of ovulation, which is about 25%, tubal damage in the ovaries and sort of tubular.

That's about 20% male factor infertility, which makes up 30%, or uterine or peritoneal disorders, which make up another 10%. So, obviously, one of the first things that's considered is let's have a look at the male fertility, because that's probably one of the easiest sort of quick tests to do. So, in our lab, we get referrals from the GP, but patients who've been having sex with that contraception for a year or more and have not conceived. We get referrals from gynae and sometimes we also get referrals for post-radiation and chemotherapy treatment, sort of check the fertility in cancer patients. In some places, they also run sperm banks, so someone's going in to have cancer treatment, which they know could affect their sort of their fertility later on.

They're actually sort of stored in sperm banks for later, if needed. Okay, so after all this hard work of 64 days, what does the end product look like? So, it is actually, even though there's sort of millions and millions of them produced every day, it's a remarkably complex metabolic locomotive and genetic machine. So, it's approximately 60 microns in length and it can be divided into three sections. So, you've got the head, the mid-piece, and the tail.

So, the sperm head obviously contains the sort of DNA, the genetic material, and around it, there's a fluid called the acryso, which contains enzymes required to penetrate the egg for fertilization.

So, we'll kind of dissolve a little bit of the eggs so the DNA material can get inside. The neck obviously connects the head to the tail, but it also is where all the mitochondria sort of exist, that give the little sperm energy to sort of move, and the tail is made up of an axial filament, which is powered by, obviously, the little bit here in the mid-piece.

So, it's got one of the sort of standard microtubule sort of formation, which is like the sort of 9 plus 2, so you've got central core with some sort of centrioles around the outside.

Okay, in total there's about 200-300 proteins to enable all this to sort of function.

So, it's kind of quite a complex little beast and the tail will obviously sort of help it to propel it forward. You've got the energy that gives the mitochondria, because it needs to actually break into the egg, and they travel a long distance, considering how small they are.

So, any defects within this can be sort of associated with sort of infertility.

So, when we've got our sample, one of the things we look at is what they look like under the microscope.

A morphology describes the size and shape of the sperm, and you can sort of see they have defects. They can have defects in the head, the mid-piece and the tail.

So, obviously, they can kind of be different in shapes. They can have no acrosome, or they can have a very small head, different changes, baculated.

It's a very small acrosome area.

The mid-piece can be absent or inserted in the wrong place, and obviously, there's a tail defects as well. So, looking at them, actually, a lot of them tend to be not quite right.

So, the World Health Organization, which produces our manual, which kind of dictates what we consider abnormal and abnormal and abnormal, considers that if four percent or more of sperm look normal, that should be sufficient for fertility. So, that's our cutoff point.

So, one of the differences between this and the people in the lab actually do the diagnosis and write the reports. So, the heads obviously can have misshapen heads, they can have large or small heads, or sometimes even an extra head.

Tails can be bent, coiled, or stumpy, or not attached to the correct location.

One of the things they've sort of discovered is increased testicular temperature, toxic chemicals, infection, or some genetic traits can increase the percentage of abnormal sperm. So, looking down our microscopes, what do we see? So, this first picture is what a normal sperm would sort of look like. They've got an intact mid-piece and a nice uncoiled single tail, and the headpiece looks right.

These would normally have the correct number of chromosomes, and when you're looking at them swimming, they will swim in a progressive manner, which means they will move forward.

Sometimes you get them with macrocephaly, so they have a giant head, and sometimes these will carry extra chromosomes, and I think in the research that they've done, this can be a genetic condition, so there's a new mutation in the uroicinase C gene. In this one, you can get abnormal head shape, so these are sort of tapered head sperm, so they're often sort of cigar-shaped long and thin. They sometimes have abnormal chromatin or packaging of the DNA, and again, sort of high temperatures will tend to make this kind of more prevalent.

prevalent. This is an example of some of the abnormal sort of sperm tails, so they're kind of coiled here, coiled up into tiny bundles, so obviously they can't swim, and they won't sort of be progressive, so they've identified that this is sometimes caused to incorrect seminal fluid conditions, sometimes if there's sort of bacteria or infections present, and obviously, if someone is a heavy smoker, this is something that we see a lot more frequently in those patients. So what do we do in the lab?

So what do we do in the lab? So patients often need to be referred to us because we're not allowed to give results out to two patients because that needs to be done by their clinician so they can talk about the clinical impact of other steps that might be needed.

So sperm are quite delicate especially toxins in plastic, so what we need to do is any of the pops and consumables that we sort of prepare have to be sort of tested for toxicity to make sure that they don't actually kill the sperm before we get to them.

So each patient gets a set of instructions and we need to have a sample within 50 minutes of production because otherwise we're not getting an accurate sort of result of what's happened again because of the toxicity of various consumables and stuff that we do.

Patients book appointments online and then we sort of meet the patient, talk to them, explain the process and take some sort of more relevant details to make sure that we've got all the information we need. So first thing we sort of do is describe what it looks like, so semen itself will liquefy in 30 minutes and has a distinct odour. The abnormal appearance could include if the colour is sort of red or brown indicating sort of like presence of blood, if it fails to liquefy, and it's kind of very viscous, so obviously that would impact how well a sperm can swim and has a strong odour and it's described as either normal or viscous. One of the other measurements is the pH, so because we're actually diagnosing everything that we do has upper and lower reference limits, what's normal and what isn't and the normal will be within that sort of range. So we would expect it to have a pH of about 7.2, a lower pH can indicate a blockage of the seminal oesicles and a high pH can indicate an infection, and again that's report put into the report as well.

The volume is measured by weight, so a lot of the pots that we send out as well as being toxicity tested also weighed, but also weighed when we get them back and then we do the calculation. You're clever people so you know how that works.

Okay so I've looked at the morphology, we've looked at sort of the pH, we've looked at the appearance, we've looked at the viscosity. One of the other tests that we do is to determine how fast they can how well they're moving and the number of progressive sperm that is the ones swimming in a positive direction is the most significant predictor of fertilisation and pregnancy. We would look at a minimum of 200 sperm on two slides and they're counted and then divided into e.

either sperm that are progressive, i. the ones that are moving forward, non-progressive which is sperm that are moving but they're not really getting anywhere, they're kind of either just twitching or moving on a small circle, and immotile sperm which are not moving. One of the big problems in Andrology is the uncertainty because obviously you're looking at a system that isn't static, which is why we always do a minimum of 200 on two slides and a lot of the analysis of Andrology is trying to reduce the uncertainty. So the little table here is sort of saying actually well actually what's the acceptable difference between your two results and if the results are not acceptable, if the difference is beyond the acceptable difference, then it's repeated. When we report it, so if progressive motility is under 32 percent or the total progressive and non-progressive motility is under 40 percent of this counts as low motility and that would be referred to a fertility clinic, it may be that if the sample has arrived late or there have been other factors like the patient sort of they're asked to abstain sex for sort of three days prior to sort of producing their samples, if any of those categories have not been reached then they may really ask them to receive repeats about the sample just to make sure that they're getting an accurate representation.

And again if they repeat again shows the same thing then they would be referred to the clinic and they would kind of look at that as well.

So the last thing that we do is try to work out what the concentration is. So how many sperm per mil are there and as there's a lot. So we use a dilution of one in 20 on the pipette and we have something called a counting chamber here which has grid lines on it. So again it's duplicated to sort of reduce the sort of error rate. So a quick look we're sort of determining which dilution should be used obviously if you're looking at down there and there's too many sperm then actually you can have to dilute it even further and actually to calculate your concentration depending on obviously your dilution factor. If there's very few sperm then you will probably use a slightly sort of lower dilution to sort of try and get enough sperm.

Again you're trying to count about 200 of them. So this little device is called a hemocytometer and only holds sperm accounted and the ones that look sort of normal sometimes you get tiny pinheads they're not very ignored and you're counting the ones that inside the grid. And this is like a little dilution factor here so greater than 200 you'd probably dilute it 150 and obviously once you've got your numbers of sperm you would then take into account the dilution factor and get your concentration in sperm per mil. Again so both sides of counting chamber are counted and using the graph to determine if the difference between the two numbers is acceptable. So in the same way that we did before we tried to work out is it that acceptable difference are the other two readings is it perhaps the sample hasn't been kind of agitated enough you've got like clumps in one and not clumps in the other so you could be getting a different reading which isn't kind of quite right for the patient.

And in this case a low sperm count is classified as a total sperm of 15 million per mil.

Sometimes you get AZ sperm examples where no sperm is seen at all in which case that sample is centrifuged and a drop of the pellet at the bottom is added to a slide which is assessed for in the presence of any sperm at all.

So the concentration is sort low the patient it may be repeated if there's no sperm seen or the patient can be referred directly to the clinic.

And then what happens next? So obviously anyone on the fertility pathway they'd be both sort of the man and woman would be there together.

So once it's been identified what the problem is it's obviously sort of assisted reproductive technologies that they can sort of participate in.

There's the interuterine insemination which induces ovulation would collect and prepare the semen and increases the number of sperm so that site of fertilization and the success rate of this is that sort of 10 to 12 percent per round.

You can get IVF which again induces super ovulation of a woman and the eggs are harvested.

The sperm are prepared and cleaned and several thousand sperm are introduced into a petri dish with the eggs and these one of these eggs are sort of fertilized they can then be planted in the uterus and excess rate of success rate this is about 25 to 50 percent.

The last one is the intracytoplasmic sperm injection which again induces super ovulation and halves eggs but then it actually injects a single sperm directly into the egg and again the success rate is about 25 to 50 percent and if the sperm is sort of fairly motile then obviously this is the method of sort of choice and then the last one is obviously