Thanks to everyone joining us for our members webinar this evening. Tonight we have Doctor Sarah Stewart, who will be presenting an optimising cytology in your practise. Tips and tricks to get high quality samples and maximise your diagnostic abilities.

Doctor Seward received her DBM degree from the University of Saskatchewan in 2008. She completed her internal medicine residency at the Animal Medical Medical centre in New York City. In 2012, which provided extensive training on urology and nephrology cases thanks to this specialised renal medicine service supported by a dialysis unit and interventional radiology department.

She spent 3 years in private specialty practise in San Fran San Francisco, followed by 6 years at the Royal Veterinary College as a lecturer in internal medicine and oncology. During her time. At the RBC she was the co-chair of the antimicrobial stewardship group and has a special interest in antimicrobial resistance and multi-drug resistant urinary tract infections.

Doctor Stewart now works as an international locum clinician in academic and private practise hospitals. She is a Vin consultant in urology and nephrology and editor in chief of the journal Topics in Companion Animal Medicine. She is passionate about urology and nephrology, antimicrobial stewardship, infection disease, medical oncology, feline medicine, and gastroenterology.

If you have any questions throughout the talk, please use the Q&A box. Thank you, Doctor Sarah and welcome. Well, thank you so much for the lovely introduction, Theo.

So excited to be speaking to all of you tonight about utilising cytology in your practise. So, as Theo mentioned, although my board certification is in internal medicine, for the last 6 years when I was at the Royal Vet College, I spent half of my time working as an oncologist, and largely in that place where we are struggling to, get a diagnosis in a lot of cases, so. Is it an inflammatory process, an infectious process, or a cancerous process?

And cytology was really one of the most important tools that I used every single day, in practise and still use, every day I'm in the hospital. So I hope by the end of tonight, you guys are all gonna feel much more comfortable, utilising this in practise and helping to get good answers for your patients as well as, more revenue for your hospital. So, without further ado, let's plunge in.

So to introduce cytology, this is essentially microscopic examination of tissue cells or blood cells. So we can collect samples via fine needle aspiration. You might also see this termed fine needle aspiration biopsy or fine needle biopsy.

All of these terms essentially mean that we are using a needle that is smaller than 20 gauge. So we're not gonna be pouring out tissue, we're just stucking out the cells themselves. Cytology has a lot of advantages.

It's very quick to do. It's very inexpensive, and it's quite easy to perform in most patients. We consider it quite minimally invasive, so for obtaining diagnoses, we always want to try and start with the test that is the, the least invasive and the least costly, so cytology is great for ticking those boxes.

In most patients, certainly for aspiration of cutaneous and subcutaneous masses, we don't typically need to perform sedation. But obviously if we're doing intracavitary sampling, we will need those patients to be very still for that. Cytology can help us generate some really helpful information on the types of cells that are present in masses or lesions if we're dealing with inflammation, infection, or cancer, as well as a lot of useful information about their morphology.

And in some cases like the, the lovely beautiful mast cell tumour we have in the image on this slide, and lymphoma, we can actually get information about tumour grading as well. So we have a long list here of all of the kinds of things that we can sample. So cytology, you sort of ways that we can use it in practise are almost limitless.

So where we most commonly encounter it is sampling of cutaneous, subcutaneous masses and internal masses that we can diagnose on imaging. We can get, samples from bony lesions, things like suspected osteosarcoma or osteomyelitis. We'll talk about that at the end of tonight.

All of our internal organs, all of our lymph nodes, both internal and external, we can sample airway fluids, trachea washes and bronchialveolar lavage. We, if any fluid is accumulating in the cavity, we can sample it. If we have cutaneous lesions that are oozing, having discharge, that can often be helpful for, cytology, especially diagnosis of things like leishmania or some cutaneous fungal infections.

We can do skin scrapes to look for parasites like Demodex. We can sample bile. We'll talk about that at the end.

Urine, of course, for of infection and things like a transitional cell carcinoma, prostatic washes, bone marrow sampling, even aqueous humour. So, for some, forms of ocular lymphoma, FIP, and fungal disease, actually sampling the fluid in that front chamber of the eye can help give us an answer in a relatively non-invasive way. And then finally our cerebrospinal fluid and synovial fluid.

So anything that we can stick a needle in, we can do cytology on. So one thing we I always like to to caution on every single lump and bump that an owner or a veterinarian finds should always be sampled, even if it feels just like a lipoma, I've lost track of the number of masses that we have diagnosed as mass cell tumours that come in and feel identical to. 95% of all the other lymph lipomas that we see out there.

I've also diagnosed 2 millimetre tiny little unexciting skin tags and warts as mast cell tumours. They can look and feel like anything. So if we find something, we always recommend to that owner, we are gonna stick a needle in it and find out, then we know.

I also caution on some owners will say, oh, why don't I just wait and see if the mass grows and then we can deal with it if it becomes a problem. Here we have a nice example of a 2 millimetre slightly red and erythematous mammary mass. We left that or these owners didn't want to do anything.

This was left for about 2 months, and then lo and behold, when we worked up the dog for having some respiratory distress, we had significant pulmonary metastasis already. So even a small cutaneous or subcutaneous mass can be the tip of the iceberg for a serious neoplastic process. So benefits of cytology, why should we consider doing it?

We have a lot of benefits for the pet. So, early detection of neoplasia, we have a much better chance of getting a cure for that patient or prolonged survival if we diagnose it early. We can confirm non neoplastic disease processes for peace of mind knowing that that is just a epidermal inclusion cyst and not something we have to worry about.

It's good for the pet. We don't have to put them through a big procedure, and it's a lot less invasive than jumping straight into a biopsy that might require general anaesthesia, and sort of surgical recovery. So always start with the least invasive, least costly thing.

It can quite be beneficial for owners as well. If we can sample a mass, I can give them peace of mind, we don't have to worry about it, or we know that we've discovered something early and now we have the best chance of being able to deal with it. We also have benefits for your veterinary practise, so it can help you ensure that we're doing the optimal workup for patients.

So, approaching oncologic conditions, planning surgery, knowing how far disease has already spread by sampling regional lymph nodes, all of these things can help make sure that we're going to make, recommend the best treatment plan for a patient, and sort of make Sure that if there are, say, you know, God forbid client complaints in future, we've done everything sort of by the book. So cytology is really good for helping to make sure we can thoroughly and cheaply do a full staging, and it all can also help generate additional income for your hospital doing good quality gold standard medicine, you know, we're doing tests that we should be doing that can help improve the, the bottom line overall. So there is a nice paper in fact that has specifically looked at this and how incorporation of cytology in our daily practise and appropriately charging for it, can really help to improve the hospital's, oh, you know, sort of overall bottom line.

So feel free to look that paper up if you'd like to, to hear more. And one of the reasons I draw attention to this is that we know cytology is significantly underutilised as a diagnostic modality in the UK. So we had a study in JSOP back in 2009, that found over half of samples were submitted to diagnostic lab without having any.

How cytology done beforehand. We're gonna talk a lot tonight about how important that is and making sure that samples we send to the lab have a decent chance of giving us an answer. 1 in 5 of the samples sent to the lab in this study were non-diagnostic.

There was nothing on the slide, that pathologists could say anything about. The authors of this study concluded that cytology is quite underused in the UK. They found that on average a practise is submitting about 4 samples a week, so that's less than 1 a day.

We had a range between 0 to 10 samples, but still, when I think about how many cytologies we run in a day at the RVC, it's probably, probably about 50 across internal medicine and oncology. So one of the other things they noted is that there was some frustration from veterinarians, in general practise about cytology sometimes not, not giving an answer, and that making them a bit less reluctant to want to reach for it in the future. So one of the other take homes.

Tonight we'll hopefully be getting a better idea of the things that cytology can tell us and the things where it may struggle a bit. Every test has positives and negatives, so just having that idea what cytology is really good at, and making sure that we can use it in those situations. So although this study was back in 2009, chatting with some of my colleagues at Zoetta who are really trying to work to, make cytology more easily accessible in general practise by, sort of doing things like having virtual slide scanning that we can, sort of send samples in quickly to get a look.

They found from their in-house surveys that things really haven't changed too much as far as how frequently people are running cytology and practise here in the UK. So, hopefully we'll be able to help get those numbers up. So, the reasons why some people might be frustrated with submitting cytology can be that we get a non-diagnostic sample back.

That's frustrating for the owners they've got to come back and, you know, repeat the test or do something more expensive, and it can be frustrating for us because we don't get an answer yet. So running through a few of the reasons why samples might be non-diagnostic, the most common. That we don't have enough cellular material on the slide.

So that might be because we're dealing with, types of tumours that don't exfoliate well when we sample them. So most of our sarcomas, like fibrosarcoma can fall into that category. We will talk about some tips later on for what we can do to increase the chance of getting good yields in those tough samples.

We might not have good sample collection technique, so we'll give you some tips to help with that tonight. And then we might have excessive hemodilution or blood contamination in the sample. If what we send in is 90% peripheral blood and we only have a few scattered cells, it can make it really hard for the pathologist to find the needle in a haystack.

We might have the opposite problem as well, but the cell layer is actually too thick. If we haven't spread the cells out into a nice even monolayer, if they're all clumped on top of each other, the pathologist can't actually distinguish the cellular detail with enough clarity. We can also damage the cells if we're not careful when we're preparing slides.

Neoplastic cells especially are quite fragile and it's very easy to break all of them. So I'll show you some examples of what that looks like, later on and some tips on how to avoid that. We may also run into samples that are necrotic, so I'm gonna show you some images of what that looks like later.

And then finally, if, if the samples we're collecting are not necessarily representative of the lesion, we may not be able to get a definitive diagnosis. So some masses will be very heterogeneous. We have pockets of fluid and then areas that are more solid.

It's not that common that we will get a definitive answer from sampling only the fluid component of lesions like that. We need to sample both the fluid and the solid tissue area to maximise the chances of getting an answer. So there are a few risks and complications of cytology to to raise.

Obviously it's the least invasive of all of the ways that we can sample masses, but still important to flag up to owners what could happen. Biggest issue potentially is bleeding, oftentimes that can just be minor if it's a cutaneous issue, let's say if that patient has low platelets or dysfunctional platelets, we could end up with quite significant bruising or hematoma, so just letting owners know that could happen. The bigger risk though is if we're sampling internal organs, so intracavitary lesions like liver, spleen, kidney, mesenteric lymph nodes.

If I am gonna perform any of those samples, I always want to have a platelet count on that patient, ideally from, you know, sort of 48 hour period, ideally right before you're gonna go and do that sample. An in-house smear is fine if you Where your nursing team is confident looking at platelet counts. I do get asked quite often, you know, do we need to do a full coagulation panel or PT and PTT for FNAs?

Most of the time, the answer is no. The exception for me is if I'm sampling the liver of a patient where I am concerned that they may have significant hepatic dysfunction that could be affecting. The production of their, clotting, clotting factors.

So if a patient is visibly eeric or jaundiced, they've got an elevated bilirubin on blood work, or I can see that some of my hepatic function parameters, so albumin globulin, urea cholesterol are low in my biochemistry, with a patient like that, I would want a coagulation panel before sticking a needle in the liver, but otherwise we're we're not too fussed. We could cause pain that's usually quite minimal, and certainly if a patient is quite uncomfortable, say we're sampling a mass like what we think is an inflamed mast cell tumour and they're quite sore, I'll probably sedate them, or give them some local pain control to help with that. Some of our topical ELA or numbing sprays, you know, can be, can be something that we can reach for there, and going for a slightly smaller gauge needle can also reduce discomfort.

If we're aspirating a lesion that could be a mast cell tumour, there's a risk that that could degranulate. So we'll usually advise pre-medicating those patients. So, I'll have some info on that in the next slide.

We always have a risk with cytology that we might not get an answer, so it's important owners know this could just be the first step in our diagnostic workup. We hope it will give us all the information we need to, but if it doesn't, we sort of have them already prepared from the beginning, there might be other steps coming. We may have risks that we haven't stay in the slides correctly to, to get an answer.

I'll give you some tips for how we can get around that. And then finally, one of the rare complications, but one that can be serious, is if we are sampling certain types of epithelial tumours, so malignant carcinomas, there's a chance that we could pull some of those cancerous cells out along the needle tract, and because these are often aggressive, processes, those. Cells can kind of be transplanted and seed new tumour along the needle tract.

So we this is most associated with transitional cell carcinomas in the bladder as well as renal carcinomas, but it has rarely been reported with a lot of other forms of carcinoma, so pulmonary and pancreatic lesions. Generally though, our feeling is. It's a very rare complication.

It's gonna be more benefit to the patient to get an answer and know what we're dealing with than not sample a patient because we're worried that there's a very small chance this could happen. So we always tell owners it's a possibility, but the most important thing is that we get an answer and hopefully they're they're good with that, but making sure we've documented that in the record so that we, we have ourselves covered should God forbid anything happen. So if we suspect an aggressive mast cell tumour, I'm gonna wanna take a few extra precautions before sticking a needle into that.

So things that might clue me in that I could be dealing with this, if I've got a mass that the owner's report has been erythematous, intermittently swelling and fluctuating in size. If we have a prior history of mast cell tumours in that pet, we will Have patients getting recurrent mast cell tumours, or if I've got a mass that's come up suddenly in a location that we know high grade mast cell tumours like to like to show up, so mucosal surfaces, inguinal region, we've got an image here of a a prepetial high grade mast cell tumour in a male dog. We could also see vulva mast cell tumours in a female dog.

So if I think one of those is, is present, I'm gonna make sure that I pre-treat with an injection of an antihistamine, something like Piriton to try and minimise chances of mast cell degranulation happening. But even with those precautions, sometimes with the high grade tumours, it will still happen. So I always warn the owners this mass might swell, bleed or look worse after the sampling than it did before.

But again, we have to get an answer to know what we're dealing with. So on the right here we have A lovely cytology image of a high grade mast cell tumour here, so we can see lots of criteria of malignancy, significant variation in nuclear size and cell size, so I need a cytosis and anisoaryosis. We've got a trinucleate cell right in the middle there, that's absolutely ginormous, and the granules in these mass cells, there's not quite as many as we would normally expect and they're a little bit unevenly distributed.

So whenever I see things like that. It can worry me that I'm dealing with more of a high grade process and conversely, sometimes the most aggressive mast cell tumours, we can't actually detect their granules on standard, sort of in-house stains, so, we always have to entertain that possibility that we could have a mast cell tumour where the granules just aren't showing up in-house. So moving on to all of the bits that we need to be able to perform cytology successfully in practise, of course we're gonna need needles to sample.

It's a little bit up to sort of personal preference, what size of needle you use generally will go somewhere between 2021 and 25 gauge. I am often reaching for for 23. The smaller the needle.

The less pain and bleeding we're likely to cause, but we're forcing the cells through a narrower end of the needle. So there's a higher chance that we can traumatise cells. It's also if you're doing ultrasound guided aspirates, the thinner the needle, the harder it's gonna potentially be to find the needle tract when you're scanning with your probe.

So larger board needles getting onto our sort of 2120 gauge. These can be useful if you are initially trying to sample a lesion with a smaller gauge needle and we're not getting much. So things like that, you know, sort of suspect soft tissue sarcoma that's not exfoliating well, you can try going up to a larger gauge.

These needles are easier to see with your ultrasound probe, and because the hole that we're forcing the cells through onto the slide is wider, will cause less trauma to the cells, but the risk is the needle might. A bit more traumatic, so it might be a bit more painful for the patient and we might risk more hemo dilution. So it's all weighing up the pros and cons.

There's a number of studies, we'll talk about a few of them throughout the evening, trying to determine is one needle size definitely better than the other, and really the results of all of those studies have been slightly different for, different sampling sites, different from study to study. So for me, the, the basic take home is, you know, you use the needle size that works best for you and your patient in that situation. For syringe size, we generally recommend, my, my favourite is a 5 cc syringe, we don't recommend smaller than 5 cc because the amount of sort of suction pressure we can get with the smaller syringe might not be enough to fully expel all of the cellular material in the needle hub, but if we start getting too big.

Again, we run risk of traumatising the cells a little bit, if we have that much pressure, expelling out onto a slide, and if you are doing an aspiration technique where you have the needle, sort of attached to the syringe, it gets a little bit harder to manoeuvre the bigger the syringe gets. We want to have a good supply of high quality slides that are frosted at one end so that we can easily label them. And good to have clippers and chlorhexidine, and alcohol sort of easily available, but for most of our dermal lesions and subcutaneous lesions, we don't necessarily have to clip as long as we can, sort of easily get our, our hands around the mass, immobilise it, sort of in our hands and get the needle in there.

It's not necessary to, to remove the hair. So here we have a couple of diagrams of techniques that we can use. So the general first approach we recommend is the needle only, what you call rapid jab or cutting technique, where we will immobilise the mass, take the needle with our hand and then sort of very rapidly jab in and out, while moving the needle in a few different planes of the tissue.

It has been proven in studies, in human medicine that this manoeuvring of the needle to try and get multiple tissue planes will result in better yield because we're able to get a few more cells stuck into the hub of the needle. If we do that and we don't get good diagnostic yield when I'm expelling the material on the slide, you can then move on to what we call a gentle aspiration or suction technique where we are, doing the same jabbing motion with the needle, but then applying gentle vacuum suction pressure with the syringe to try and sort of vacuum up a few more cells into the needle hub. The risk with this technique is that again we may potentially risk traumatising the cells a little bit more.

But in some cases with poorly exfoliated lesions, we have to do that to get a diagnostic sample. So I always personally start with the less invasive jabbing method if that gives me all a good quality side, I can stop there. If it doesn't, I move on to aspirating.

Once we have the material in the syringe, we wanna act fast because often there's gonna be some blood in there. It can clot quite quickly and if all of your lovely cells have clotted with the blood in the hub of the needle, we won't be able to expel anything. So we should move as quickly as we can once we have the sample removed from the patient.

When we're spreading again, he is as gentle as possible. So I'm gonna show you what some of the spreading techniques look like here on this slide. What we do for the vast majority of our samples is something called the horizontal squash spreading technique, or the pulling technique, so you can see an image of that in the upper right hand corner, but essentially we have a drop of our sample on our slide.

We will very gently put a spreader slide on top, and then very gently with applying minimal pressure, slide that's along the, specimen slide until we get a nice uniform sort of splotch which will represent a monolayer of cells. What we don't want to do is really sandwich these two slides together. Firmly, sometimes if you have a moderate amount of, sort of a liquid sample on the slide, you can almost feel them stick together like there's kind of a suction effect, that's not good for the cells because all of that extra pressure can traumatise and rupture them, so I want it to just sort of be as light of a whisper of pressure as possible to avoid traumatising cells.

If we're dealing with a very poorly cellular fluid, so something that I've taken from a body cavity, or a sample that really looks like it's got a lot of hemodilution, maybe I'm sampling the liver in an anaemic patient and I'm just getting a lot of watery fluid out. We can do something called a line prep technique. I also do this when I'm looking at urine sediment cytology sometimes for intracellular bacteria or cells, rather than doing our sort of standard drop of sediments, kind of without stain, you can do this technique and then just quick it as normal and it can be a bit, quite a bit easier to see intracellular bacteria and cellular morphology.

So to do a line prep, we essentially just put a little dot of fluid at the sort of frosted end of the slide similar to preparing for a blood smear, and then we will put the slide second slide at a sort of 45 degree angle, pull that back to, suck up the, the drop, very similar to making a blood smear, but instead of pushing. For a standard blood smear and getting that sort of lovely halo effect for the line prep, we stop at the very end of the slide, pause for a second, and then lift the spreader slide up and that will sort of concentrate all of the cells and the heavier elements along that line, and then you can use that to sort of examine your cytology closely under the microscope. So some dos and don'ts, when we are spreading slides, occasionally labs will get samples where there hasn't been any spreading done at all.

We've just squirted the stuff onto the slide, that's what we would call sort of a spray or a splat slide. The cells in those cases will all still be stacked on top of each other, so we can see an image of that in image A, you can see it's impossible to distinguish any cellular architecture here. If you don't spread your slide and get a monolayer, it's not going to be diagnostic.

We also don't want to do something called the vertical squash technique for good cytology samples that we can see in image B and that's essentially where we've just sandwiched two slides on top of each other, and then pulled them apart again without spreading. We can rupture cells, but we can also just not spread them out enough, so they all stacked together. What we want is what's in image C, a nice uniform monolayer where we haven't broken any of the cells.

And we want to try and do our best to put the sample on the centre of the slide. If we do find that most of it ends up concentrated on the very edges, say if we're spreading and a lot of it gets concentrated at the, the edge of the slide, it can be, almost impossible for the pathologist to get the microscope down at high power on those edges, just because of how the sort of mechanics of the microscope will work. So, the obviously sampling cutaneous and dermal and subcutaneous masses is gonna be pretty straightforward as long as we can isolate it, with our hands and get a needle in, we're fine.

But if we detect masses internally in the thorax or the abdomen, we're gonna need imaging guidance to help us sample those. So ultrasound guided FNA is something you can absolutely do on your own in general practise, if you. Confident with your ultrasound skills.

The most important thing when we're doing ultrasound guided FNA is that we want to always be able to visualise the needle when it is in an organ or in a mass. So we can see in the ultrasound image here below. I can very clearly see my image tracked.

I know where I am. There's no risk that I'm gonna accidentally be lacerating blood vessels, arteries, bile gallbladders, all of those sorts of things we don't want to poke. We also want to make sure we can, sort of predict where our needle throw is going to end up.

Ultrasound probes are quite pricey, so the last thing we want is to find that we have accidentally aspirated the end of the ultrasound probe. So the best thing is practising, with, sort of. External ultrasound setup.

So if you, sort of look online, they'll give suggestions for materials you can sort of put together, so basically you could have a a great sort of inside of plasticine that sort of thing and just put an ultrasound probe on there and try and guide your your needle in. Making sure we're confident doing it outside of a, a real live patient before we move on, that will help build those skills. And we have an example here on the, the right, of, of what good quality liver FNA that has been collected.

We don't have a lot of hemodilution. We've got a nice centrally collected sample that has been uniformly spread, so that's what we want to see if we're doing it well. There's certain masses that we can't easily access with ultrasound, so thoracic masses, and some spinal masses if I'm, say I have a suspicion of multiple myeloma involving a vertebral body.

Often I need a lot of precision being able to introduce a needle into there. So in a referral basis, we can do CT and fluoroscopy guided FNA. So if you say you did diagnose the challenging to access lesion in general practise, that is something you can absolutely refer in for to do.

So, if we are finding masses and lesions internally, the next question is where is the best place to try and get a sample from. So in the ultrasound image here on the right, we've got a large heterogeneous liver mass that is partially cavitary in some areas. So what I want to do to maximise the chances of diagnostic success.

Here is take a few samples from different areas of this mass, so I may try and sample somewhere around the sort of pocket of what looks like fluid or hypoechoic tissue, but often those areas are, you know, if it's an abscess, that will be the, the sort of the, the best possible sample we'll we'll get some purulent debris, . But for neoplastic processes, often we, we just get back a non-specific sort of modified transitate type fluid. So trying to get some samples from the surrounding solid tissue, if there's areas of marked heterogeneity in tissue, and other areas that look a bit more normal, I'll try and get samples from both of those for comparison.

And making sure I've labelled sort of which of those sites is which for for the pathologist, so I might say I have liver 1, liver 2, and liver 3, so that they know all of those slides are from slightly different areas. It's also really important when we're taking samples to take enough, so quite often we'll get submissions to the lab where we only have 1 or 2 slides sent in, and sometimes the lab will receive 6 or 7 slides and we only get a diagnostic sample on one of them. So most labs will have a sort of maximum slide number for their submissions where you can submit usually up to 5 slides without incurring any extra charges.

So for me, I want to maximise my chances of getting answers as much as possible. So if it's safe for the patient, we're not having any excessive bleeding or pain with the sampling, I'm gonna try and get as many good quality samples from that, from that patient as we can. There's nothing worse than sort of having to sedate a patient for ultrasound, the cost of that getting someone in to help, and then finding that we didn't get an answer and we have to come back and do it all over again.

So submit quite a few slides, and if you're finding that, you know, you've done 2 or 3 in-house, you're just not getting great return, keep going, try and collect as many good quality samples as we can. We also often see the slides that are sent to spread the material to the lab. So if you didn't get very many samples, it was a difficult to aspirate lesion then absolutely send everything you have, but if you've got 7 or 8 slides to choose from, you're gonna send the best 5.

Often the spreader slides are less diagnostic. We don't tend to have as many cells on that that side. So often I will save those for myself to look at in-house before I'm sending the sample out, and not necessarily send them to the lab until I think, unless I think that there's going to be something really helpful on there.

So, alluding a little bit to what we mentioned on the last slide, how do we determine if we, if we've gotten a good quality sample to send to the lab, do I need to keep poking? It's really essential to get in the habit of performing your own brief in-house cytology, before we're sending samples externally, because it helps to make sure that what we're actually gonna send out has something diagnostic on the slide. So we want to bring down that number of 1 in 5 samples going to the lab being completely non-diagnostic.

So this can really help to reduce client cost and frustration. So I have seen some cases where, you know, clients have had to come back 2 or 3 times for aspirates and they just say, I just, you know, can't can't keep coming. I'm just gonna stop and not continue working it up and that's always of course really, really sad.

We haven't been able to to get an answer for that client, so if we can do everything possible at the first visit to maximise the chance of getting an answer that will help. I also always recommend charging for your in-house cytology. It's a skill, so it's something else that can help to generate a bit of, useful extra income for the hospital.

So, the use of in-house cytology or what are our goals, we're not gonna be pathologists, we're not gonna usually make a definitive diagnosis ourselves in most cases. The most important thing is for us to determine is the sample we got diagnostic. Can I send it to the lab and have a decent chance of getting an answer back?

If so, then, you know, we sort of accomplished our, our main goal. It's also helpful for us initially determining does the lesion look more inflammatory or neoplastic. If it looks neoplastic, well I have that patient sedated anyway, I might say I'm gonna go ahead and aspirate what looks like the draining lymph node here to kind of complete staging that way the client won't have to come back a second time if my suspicion of this being a mast cell tumour is confirmed.

And it can also be helpful for us deciding, do I think it's actually not gonna be possible to get an answer with cytology here? Am I gonna have to get a true cut biopsy? Do I need to get a culture if I think there's something infectious and I'm just getting necrotic debris.

All of those things can be helpful. So for certain masses, if I have a high suspicion for a soft tissue sarcoma, or, you know, certain types of masses that don't exfoliate well, if my in-house cytology is just looking really crappy and I know I've done, you know, I've tried to aspirate from the syringe. I've used a larger gauge needle, nothing's working.

I may just tell that owner while I still have their pet under sedation. Let's just go ahead and get a true cut incisional biopsy while we're here, then we've maximise our chances that we'll get those answers. I'm just trying to advance this slide, there we go.

So moving on to in-house staining, so, the typical staining method we use is called diffquik, which is a modified Romanowski stain. What we recommend is the rapid dipping technique where we are, moving the slide up and down in the fluid repeatedly, rather than just dunking it in and leaving it there. The actual process of picking it up, having the the liquid drain off and putting it back in, helps to enhance staining and speeds the process up.

So generally I recommend about 10 seconds in the blue fixative, then 10 1 2nd long dips in stain one, the pink one, and then stain to the purple one. We do want to avoid understaining and overstating. I'll show you what that looks like shortly.

Once we've gone through the three stains, we just rinse gently rinse the slide off under with room temperature water. It's fine to use tap water for that or distilled water if your practise has it, it's fine. So, if we don't leave the slide in long enough, we won't be able to highlight any internal cellular detail.

So we can see that here on the left. If that happens where you just can't discreetly distinguish between nucleus and cytoplasm, just repeat the staining process, that's absolutely fine. If we've overstained though, unfortunately there's not anything we can do about that.

So here on the image on the right, it's become very difficult looking at these cells to distinguish cytoplasm and nucleus because everything just looks dark blue. So we kind of have to start over if we've overstayed. So drying slides, if we're looking at things in-house on a sedated patient that's in ultrasound, we wanna try and make this process as quick as possible.

So you can just let slides drip dry, put them vertically right after you finish rinsing them, and usually they'll dry in about 2-3 minutes, but often I'm a bit more impatient than that. I've got people waiting an ultrasound, so you can, Use a small handheld fan to dry the slides. It'll take about 60 seconds, or you can use a small blow dryer, just set on kind of a low heat speed, while heat fixation, where you're actually putting a direct flame under the slide that will damage cells, using a small blow dryer for in-house cytology is just fine.

You're not going to impact the cellular morphology too much. So, once we have, sort of used our, our staining equipment, we wanna make sure that, it's going to be good for, you know, the next person and for ourselves when we come back in a week. So, it's recommended that you replace all of your stains weekly.

If we don't replace them, we can start getting bacteria growing in them that can create confusing artefacts. So if you're looking to see if a patient has a septic abdomen and we see stain precipitate on there, it can be difficult to tell what's a bacteria and what's a stain precipitate. If you're doing a lot of dirty staining, so, ear smears, faecal cytology, it's best to have a dirty and a clean set of, of dips.

I do recommend wearing gloves when you're handling the stains and slides and oil, just because they, there's some, sort of concern with prolonged exposure to these compounds, so, just to minimise any risks to yourselves, your staff, just always get in the habit of using gloves. Stains will evaporate really quickly, so if you forget to put the lids back on, you're going to be going through a lot more supplies, so as soon as you finished with them, pop the lids back on. When you're maintaining your microscope, really important to not run your 4DX through oil.

If we get oil on that lens, it's gonna make it blurry, and if we keep doing that, essentially will become unusable. But it's also a good habits to, when you are using your oil lenses, wipe off. The excess oil right after you have finished doing your your slide inspection.

So, if you have some little sheets of lens paper, as soon as you've finished doing your evaluation, as you can see in the image here on the slide, just wipe those off, and then you'll be good to go and have good microscope health in the long term. So, when we are evaluating a slide in-house, initially it it's always tempting to go straight down to high power and have a look at the cellular architecture, but it's really helpful to look at low power first, so I always want to do a general overview of the slide. It's much easier to observe behaviour of certain types of neoplasms at lower power, so epithelial neoplasms that are trying to where the cells are forming sheets and clusters, mesenchymal cells.

Where we've got those lovely kind of spindle effects, often that can be easier to detect at low power, and at low power I will also go and look at the edges of the smear. So if I have heavier things like cells that have intracellular organisms in them, parasitic larva, so I've had a bronchoevela lavages where we've only found the larva on the very periphery of the slide, as well as some larger neoplastic cells, that can be a good place to look for high yield. And then once we've done that initial survey and I've identified some areas of interest, that's when I'm gonna zoom in closer either with my 40X where I'm not using oil or my 50 to 100X with oil, and we can see on the bottom of the slide here we've got some examples of what that sort of degree of zooming in looks like.

So, when we're preparing samples both in-house and to go to the lab, we want to do everything possible to minimise artefacts. So some of the things that can really trip up your pathologist, is going to be if we have excessive ultrasound gel or lubricant, sort of getting the contaminated into the sample. So if you're doing ultrasound guided FNAs, try to use minimal gel, wipe off the exces.

Before we're gonna introduce a needle through that, if we're collecting sort of catheterized urinary samples for sort of traumatic catheterization of urinary bladder masses, try to minimise the amount, you know, we want to make sure that the catheter is appropriately lubricated, but don't go too crazy because we have too much lube that's introduced on the tip of that catheter, it can contribute to this granular magenta. Debris we see in the upper image here, and these granules can look a bit like bacteria, they can look a bit like degranulated mass cells or granular lymphocytes, so it can definitely trip things up, and the sort of granular material itself can actually impact cellular morphology, so overall want want to minimise those. We can also see if you use powdered gloves, the starch granules can look a lot like a nucleate squamous cells.

We can see that in the bottom image here, so we generally recommend, working with non-powdered gloves if you are collecting for cytology samples, and the most common artefacts that we see is people pressing too hard when we are smearing slides out and rupturing everything. So here we've got a low power image of completely ruptured sample from a lymph node with lymphoma, so we. See there was a lot of cellular material on this slide, but it is all just a a schmoo all of the cells have been ruptured, and then here we've got a slightly closer up version of one of those, you know, we can see things that, you know, maybe are a bit suggestive of a lymphoblast, but everything here is so, so damaged that a pathologist isn't gonna be able to get you an answer.

So once we have done our in-house cytology, we're confident the samples we have look like they're going to be diagnostic. We're going to get those off to the lab. We, we sort of have the crude analogy garbage in equals garbage out.

So if we don't give the pathologist good samples, and a good description of what's going on, the Information they give us back may not be as helpful as it could be and in some cases isn't that helpful at all. So spending those extra minutes to really give them a full history on what's going on, especially if it's an unusual case, supply some additional physical exam findings, how big is the mass? How quickly did it come up?

Is it erythematous or swollen? Often with masses, photographs are really helpful, so, often when I'm doing oncology consultations, I want a photo of the mask that's coming along and the pathologists really appreciate that. So, if you're submitting digital samples for, things that could be a little bit unusual, snap a photo and send it along.

All of those things will help make sure that the information you get back is useful. The other thing I sort of recommend doing, and kind of getting your, older dog patients in the habit of is body mapping. So, many of these old Labradors will have, you know, 1015, 20, subcutaneous masses and dermal masses everywhere, and it can get confusing to keep track of what is what.

So the best way to do that is with body mapping. I'm just gonna flip ahead to this slide, so, . We're talking about body mapping, we have these lovely images you can download these for free from the IDEX website for all species, but essentially rather than describing a mass as, you know, we've got 4 masses, one of them is on the lateral flank, one is 3 centimetres up, that gets very confusing, but if we have a body map, you just draw on the map with a little X, which mass it is, what the measurements of that mass are, and it makes it very easy for you or your colleagues in.

Practise of seeing that patient for a recheck in 6 months, to go back and reevaluate that mass and see is it growing? Do we have a new mass, the 13th mass that has appeared after having 12 before. So what we do when we're body mapping is we'll measure every mass, ideally with callipers so that we have sort of accurate measurements, length, width and height.

We're going to record their location on the body map and then we're gonna FNA each one. So in many cases in her cytology alone might be adequate, so if you confirm that mass is a lipoma, I stick a needle in it and I just get back fat, and when I attempt to stay in that slide, all the fat immediately dissolves. I don't have to send that to the lab.

If I'm confident that it's an epidermal inclusion cyst or if it's. Patient that has multiple recurrent mast cell tumours, and I'm confident confirming that that is another mast cell tumour on cytology. You don't necessarily have to send all of those out, but it's, it's something if there's any suspicion that you've got something unusual or neoplastic, then I would, would put those on.

Body mapping can be a great thing to get your nursing team involved in. It is fairly time consuming and of course should be charged for appropriately, but it is something that, you know, it may take 20-30 minutes having a nurse working with a patient care assistant, getting all of that done, good for the patient and you can focus your time on seeing, seeing more new patients and when that's happening. So, for sending in the best possible samples, we want to make sure that we've got all of our slides labelled with pencil, pen will come off when we're automatically staining slides.

We're going to air dry the slides quickly, that's sort of the, the optimal way to prep samples. We're going to, if you've stained things in-house and you think they're diagnostic, you can send those stain slides along, but I'll always put them in a separate slide box so that if you have any immersion oil on them, it won't contaminate the other unstained slides. If we have a fluid sample, best to, get at least 2 tubes, one with EDTA for cytology, and then a sterile plane tube in case we need to culture anything, and those samples have to live in the fridge.

If I have taken something like an endotracheal wash or a prostatic wash, those cells will degrade quite quickly if they're just sitting around in fluid. So the best thing is make a few fresh smears from anything that is a fluid sample and send those along to the lab along with the tubes for optimal quality. Don't submit your slides, in, in bags that also have formalin pots.

The fumes will seriously damage the cells. We've got an image on the left of what that looks like, so don't have cytology slides anywhere near, formalin pots in the lab or in your transportation, sort of mailers. The other thing I always recommend is, you know, be, be friends with your clinical pathologist, so if you get back a result that doesn't really fit clinically or is really weird and was not something you were expecting, I pick up the phone, give them a call.

Most I've never met a pathologist who didn't love talking to, to vets in practise. And going through the case and it may be that getting more details from you about, you know, why you were surprised with the results, what didn't fit, it can make them think about some other differentials that maybe they didn't consider. So definitely give them a call if you've got any, any questions about results.

I also strongly recommend putting in appropriate charges. It's really common that vets don't charge for in-house cytology. It is a, you know, valued skill, so make sure that we bill for it.

I recommend, three tiers for, for charging. You can, if you're just doing a check to make sure that slide is going to be diagnostic, we don't have to, you know, the client's gonna be paying for the external samples, so just a minimal charge that covers a bit of your supplies and your time. If that sample doesn't have to be sent out, you're confirming a lipoma, or recurrent mast cell tumour that you know has got to, got to come off in a patient that's had many before.

If you clearly can see that sample is gonna be non-diagnostic, then maybe we do this slight charge up, and then usually body mapping will be our sort of highest tier because that will involve a lot of time and a lot of in-house cytology. So if we're doing in-house cytology, one of the other things that often our our interns working with will will ask is, you know, if we've looked and we're pretty confident, I think this looks like a lymphoma. Should we tell the clients that while we're pending results?

Most of the time I will, I try to avoid it even if I am very confident in, in my diagnosis cause ultimately I am not a clinical pathologist, and sometimes we get surprised there's atypical round. Tumours that might, might be something else. So most of the time I will just tell the owner I've looked at the slide, I've confirmed it's diagnostic.

We're gonna get this off to the lab and then we'll have our answer. But there are some situations where, you know, we have a life or death, sort of potential. We have to make a treatment decision based on what we're seeing under the microscope.

So the most common ones there would be diagnosing a septic abdomen or a very sick lymphoma patient that we think you're not gonna survive until Monday if I don't start some sort of emergency chemotherapy treatment now. So in those cases, I will explain to the owner, I think this is my, my best guess for what is going on, but I'm not a pathologist. The final diagnosis could change, but I recommend we start treatment based on our preliminary suspicion in the meantime and I document all that in the medical record just to cover ourselves and I think, you know, that's the approach that personally I always try to go with.

So, when we are looking at the slides, initially we want to first Determine, do we think we have an inflammatory or an infectious process going on. So we might see one of our three common types of inflammation, so predominantly neutrophilic, granulomatous, where we've got macrophages and neutrophils, or lymphocytic plasmacytic inflammation. If I start, if I, obviously, if I see organisms, then I know I've got an infectious process, but sometimes we don't see those, especially if maybe the patient received antibiotics in the day or two before, they came in to see you.

If I see high numbers of degenerate neutrophils and toxic change, then my, my index of suspicion for something infectious that I just can't see will go up. Maybe I say I'll try and collect a culture sample there at the time. So here we have a picture querying is this infectious or neoplastic, so obviously we have some big looking sort of scary lymphoblasts, but if we look closely there, you can see there's some encapsulated organisms in a few of these macrophages, so this is a highly reactive inflammatory sample where we've got, histoplasma fungal organisms present.

In there. So this is, well, we might have some cells that look scary. This is an infectious process.

We do see some highly reactive inflammation and infectious processes can result in cellular changes that can very closely mimic criteria of malignancy. So we always want to do that really thorough thorough hunt and thorough history to make sure we're not missing anything infectious. So deciding if we should submit a sample for culture.

Again, if I'm seeing lots of degenerative neutrophils or certainly if I obviously see bacteria, I'm gonna culture those. This is another area that in-house cytology can help you with. If I can see that I've got a high suspicion, looking at the slides myself, I can collect samples right away and I can start empiric antibiosis right away so we can start treatment for that patient as quickly as possible.

If we are on a hunt for infectious organisms, things like leishmania, other unusual protozoal diseases or fungal diseases, the best places to look for those are the organ, the organs in the body where the immune system will probably be trying to pull things out. So sampling the spleen, the peripheral lymph nodes for. Mania, and for fungal disease, we will also look in the lymph nodes and spleen, but also considering the liver, if we've got lesions there and even the lungs, if we've got a pulmonary nodule that's right on the periphery of the lungs and we can safely sample with ultrasound without having to go through normal lung parenchyma, then absolutely I will try to stick a needle in there.

If I'm suspicious for infection, I will write that in big letters all over my submission form. Every clinical pathologist I've ever worked with, they love finding infectious disease, so if they know you're on the hunt for it, they will do the hunting for you and we'll spend extra time looking at that slide. Since we're in the UK, we have a lot of mycobacteria here, so I do recommend, keeping that in mind, especially for purulent draining cutaneous lesions in cats.

So if we are doing in-house cytology and you see anything that looks like the image on the left where we have these negative. Space, lesions that sort of look like the shadow of a bacteria that puts us on red alert for possible mycobacterial organism. So we need to confirm that with an acid fast stain at the laboratory.

We can see one of those here on the right with the mycobacteria showing up bright pink. All my suspicion level will be raised for mycobacteria if I have a sample with pyogranulomatous inflammation, lots of macrophages there. This disease obviously is, is zoonotic.

We have risk of, transmitting that to humans, so we've got to be quite careful handling the samples and again let the lab know that that is what we might be dealing with on your forms, and again if I see something like this on cytology. I'm probably gonna want to think about collecting some extra samples to submit for PCR, which is our most helpful way of confirming specifically what type of mycobacteria is, confirming a diagnosis and the prognosis, certain types of mycobacteria will be much, much worse than than others as far as zoonotic and public health risk and whether we recommend trying to treat or recommend euthanasia given the the human health concern. So we're looking at septic abdomens, this is one of those life or death situations often if you have a patient coming in very sick, looking shocky, septic, you know, recent surgery or an older patient or we might have a perforating abdominal mass, you know, it's, it's down to us to identify that and often.

Make a call if we have to go for emergency surgery or not. So I'm gonna spend lots of time scouring the slide looking for intracellular bacteria. That's the, the slam dunk.

So here in the bottom we've got two images here of sort of small low numbers of intracellular bacteria. Extracellular bacteria will be highly suspicious but not as definitive cause there's a chance that we, you know, might have sampled into an intestinal loop potentially and gotten a bunch of, you know, extracellular bacteria that way, but if the clinical picture really fits, then certainly that alone may be enough to say yes, we've got to go to exploratory surgery. If we are suspicious for sepsis, but either you don't have an ultrasound in-house, or we're not getting very much fluid directing at sort of the one pocket you see, you can absolutely perform what we call a blind four quadrant tap.

So that's where we've got the patient in lateral recumbency, you can see in the image here on the left. We sort of divide the abdomen into 44 rough quadrants. I will slowly introduce a needle in.

If we put the needle in really fast, we might go straight through an organ or an intestinal loop that's on the other side, but if we introduce the needle slowly, there's a good chance that any organs that are there will, will move out of the way. So that can be quite useful for detecting, sort of small, smaller volume effusions or if you don't have an ultrasound probe available. The other thing we can do, often patients that are coming in shocking and septic, we're dehydrated and we hypovolemic.

So if I'm not seeing any fluid initially on my AAST or getting a negative blind four quadrant tap, I'll repeat that 4 to 6 hours after I've done my fluid resuscitation, and I might have to keep repeating it. If I c clinical suspicion remains, I keep looking to see if fluid is going to be accumulating. So for necrotic samples, if we have really large tumours, they can grow so fast they outstrip their blood supply and we get necrosis internally.

And if we're sampling those, it often results in a non-diagnostic sample. It's just kind of pink pink, purple debris and schmoo. So got a picture of what this looks like.

This was a renal mass that we could sort of see on ultrasound, but on sampling, we just have a few normal renal epithelial cells and this kind of indistinct pinky purple schmoo. Wow, we can't really clearly distinguish a nucleus from a cytoplasm, and we've got a bit of a picnotic nuclei there. So if I see things like that.

Those are situations where I might say I might have to consider an incisional biopsy versus going straight to surgery to remove what this is. The good thing with cytology is if we were dealing with a round cell neoplasm that we might not have to treat surgically. U FNA will be enough to get me that answer.

So if I, all I get on an FNA is necrotic debris and schmoo, it's less common that round cell tumours will do that and I can still feel confident moving to my next level of invasiveness or surgery. So if we're looking for neoplasia, we're looking to see, do we see criteria of malignancy without a lot of surrounding inflammation that might make the cells look angrier than they are, if they are neoplastic, am I looking to see, do I have a round cell population where all the cells are are circular but kind of independent from each other? Do I have a mesenchymal population where they look sort of spindoloid where the ends are tapered out?

Or do I have an epithelial cell population where either I'm forming sheets of cells, or clusters, and we can see here sort of the cluster formation variant or an adenocarcinoma of the pancreas and the right. So, it's important to remember neoplastic lymphocytes are really, really fragile. They're the most fragile type of cell, and it's very easy to burst all of them if we are sampling a neoplastic lymph node, liver, or spleen.

So this image is from a high grade lymphoma, but every single cell has pretty much has been ruptured. I can't clearly see nucleus and cytoplasm, so this is a non-diagnostic sample, but obviously suspicious for lymphoma, but not enough that we would be able to start chemotherapy. We have to resample, so.

Be gentle, if you got one of this, something that looked like this on in-house cytology, we would need to go ahead and, repeat that, before the patient wakes up or leaves the hospital. One thing we can also see with lymphoma is something called lymphoglandular bodies. These are the small, round dark blue cytoplasmic fragments from ruptured lymphocytes.

If we see those again in a sample, it raises our suspicion that we're dealing with lymphoma. So, it's pretty straightforward to diagnose lymphoma if we have a monomorphic population of large lymphoblasts, but if we have an emerging lymphoma, where we're earlier in the disease process, it can be really hard to differentiate between that and a very infectious inflammatory reactive lymph node. So we have to see at least 50% large lymphoblasts to confirm a diagnosis of lymphoma.

We classify a a large lymphoblast as being bigger than a neutrophil, so that's our internal ruler on the slide. And the situation is often the hardest to diagnose is if we're dealing with a low grade or indolent lymphoma, where we have a cancer of the small lymphocytes, so it can look virtually indistinguishable from a lymphocytic inflammatory process. So here's a lovely picture of a classic monomorphic population of large lymphoblasts.

We can see we've got an eosinophil here, but the same size as a neutrophil. All of these cells are bigger than that. We've got some lymphoglandular bodies scattered around here.

That's a very easy lymphoma diagnosis to make. This is not quite as easy. So we've got some really big scary looking cells, but there's less than 50% of them, and actually most of what's here is small to intermediate lymphocytes.

This was a highly reactive lymph node. If we are in one of these situations where the pathologist says, I just can't tell you if this is inflammation versus lymphoma, we can do some molecular tests to help us get to an answer. So, immuno phenotype is, the term for kind of the cell.

Signature of the cells. So we look at what unique receptors it's expressing, and those cell surface markers can tell us if we're dealing with a clonal population where all those cells are expressing the exact same markers, or a reactive population where they're all gonna be expressing different markers. So there's 3 ways that we can sort of determine immuno phenotype using our cytology samples, flow cytometry, immunocytic chemistry, and PAR.

So flow, we are labelling cells with some fluorescent antibody markers and then sorting them based on the fluorescence pattern, cell size, and nuclear complexity. So this is the most helpful method because it gives us a huge amount of information. We look at lots of cellular markers all at once, but the main limitation is we have to.

Performing on cells that are alive, so it's quite time sensitive, and we have to collect enough cells in the sample to have it be diagnostic. So it can be really useful, but you have to check with your lab about how they want you to submit samples, and it's not a test that we can submit on a Friday morning. By the time it gets to the lab on on Monday afternoon, the cells will be dead.

Immunocytic chemistry works on a similar principle, but we can only apply one marker at a time, and we've got to have unstained slides, and we need one slide for each marker that we're gonna look at. So limitation can largely be we, we need lots of unstained slides. Most of the time when we send multiple slides to a lab, they'll stain all of them automatically for the pathologist to look at, so we'd have to collect more, and it can be expensive if we're having to run lots and lots of separate stains.

So the other method we can use is something. Called PAR, which is PCR for antigen receptor rearrangement. This is essentially looking to see if the variable regions of the T cell receptor and B cell receptor that recognise different antigens in the environment.

If all of those little sequences are genetically clones of each other, it's all identical versus if all of the different markers that we are isolating with PCR are all different, then we have a reactive population. The nice thing about par is that we can do it on pretty much anything. We can do it on cytology samples by just scraping all of the stain material off.

We can do it on histopathology samples that are already in a tissue block, and there's no time time factor, unlike flow, which we have to do right away, we can run par weeks or even months after samples have been collected. The limitations though, it's not as reliable as flow cytometry is, and we can have some false negatives and positives. So what to run and when flow cytometry is our preference if we can, if you've got live cells and you can get samples before treatment started, but if we can't do that, then you can add par on to absolutely everything.

So, that's kind of our, our fail-safe backup if we can't run flow cytometry. So if we're dealing with mediastinal masses, it can be hard to differentiate between mediastinal lymphoma and cytology or lymphoma and thymoma and cytology alone. So what we can do in that case, in-house cytology is really helpful for these patients.

So I want to look at my slides. If it looks like there's not, there's not enough cells on the slides to tell the difference or it really looks like . Cytologists not gonna be able to get an answer.

Things look a little bit necrotic. I might say that I'm gonna collect samples while that patient is still under anaesthesia, for flow cytometry and maybe even do a true cut incisional biopsy of the mass for, for histopathology. So it can be quite frustrating and costly for owners of pets with mediasinal masses to have to re-anesthetize them multiple times to sample these masses.

So we really try and do one shot and you're done, get every sample we need. So, just finishing up talking about sampling some of our specific internal organs, so we can FNA the kidneys if we're looking for things like renal lymphoma, FIP in cats, renal masses, and confirmation of infection. It has been shown for renal FNA that the aspiration technique where we suck back a little bit, rather than just jabbing can get slightly.

More consistently good samples. My one, note of caution when we're sampling kidneys, only pick one sample, pick the most abnormal of the two. If we had bleeding occur when we FNA, the bleeding will often happen under the capsule of the kidney and that can cause swelling and in worst case could cause aneuric renal failure.

So only ever sample one kidney, so we have a backup one that will still be working OK. So for positioning needle, we don't ever wanna poke a needle into the medulla. It bleeds like stink and very rarely gives us a diagnosis.

So we have some images here on the right of the best places to place your needle. If you suspect renal lymphoma, it's very classic that we see these, hypocellular, infiltrates just around, just above the cortex of the kidney. So I will actually try to introduce my needle directly into this plane and get some of that subcapsular infiltrate to try and maximise the diagnosis.

So for hepatic FNAs, it's important thinking about what it can tell us versus what it can't. So it's very good at telling us if we have hepatic lipidosis or vacular changes. It's very good at diagnosing round cell neoplasias, and it can be helpful if I'm suspicious of things like immune-mediated hemolytic anaemia, if I can see erythrophagousytosis in the liver as well as the spleen, that can be useful.

But the liver is not as good as telling us about things like inflammation, copper storage diseases, anything that we need the cellular architecture and scaffolding of the cells to get an answer. So don't overestimate what liver cytology can tell you. It can't give us an answer for everything, but it can help us rule out things like around cell neoplasia before I may proceeding to doing an invasive liver biopsy on that patient.

So I will almost always start with cytology of the liver before I'm going to biopsy. So for liver FNA techniques, we want, we have confirmed that not doing the aspiration technique, the opposite of the kidney is gonna be the way to go, just jabbing the needle into the liver, will result in less hemodilution and less damage to the cells, in the liver. Especially important, we make sure we can see the tip of your needle with the probe at all times, because this is the organ that is most likely to, want to bleed when we're, when we're poking it.

There's a lot of blood vessels in there, and certainly for me, this is the organ that if anything is going to bleed when we're doing an internal FNA, this is the one that does. You can also sample the gallbladder, and while we think that this would be associated with high rates of complications, it is very well tolerated, so I think we have a lower complication rate than with sampling the liver, for example. The main risk is that we might, possibly have a leakage of bile into the abdomen.

What we do to minimise that is try and completely drain the gallbladder if we're sampling it. So we just hook up a 3. Stop cock and a long, sort of fluid set, but I have knock on wood, never had a complication with gallbladder sampling in, in 10 years of doing this.

So if I see an image, on the, like I do on the right here where I've got a diffusely thickened gallbladder wall, that's a patient that I would want to sample, often things like, infectious cholangitis, that might be, the easiest way to get a diagnosis easily. We want to submit samples for cytology and culture. It's always best to make fresh samples, fresh smears of bile, because it can be a little bit harder to smear, hours later after it's been in the fridge.

And then urinary tract, so you can get a confirmed diagnosis of things like transitional cell carcinoma on free catch samples. We just need to process them properly. I'll show you how to do that on our next slide.

We can do catheterized samples where we are visualising the tip of the catheter going into a bladder lesion to try and scrape a few samples off. Generally, we avoid doing FNA of bladder masses again due to that risk of tumour seeding, and then if we have any prostatic pathology, you can aspirate the prostate directly or you can do a prostatic wash. So here we have just an image of a diagnostic urine sediment cytology confirming a diagnosis of transitional cell carcinoma.

If you're submitting a urine sample, don't send it in just like a normal urinalysis. Spin it down in-house, and then make smears from the sediment that will give you a much better quality sample for the pathologist to look at. And the combination of doing.

Sort of urine sediment cytology, if it's suspicious or, you know, cytologically confirmatory for TCC, we can absolutely confirm the diagnosis by doing the BRAF PCR test. So it's a lovely useful test that will, if it's positive, 100% confirms that you have TCC, so it can be a really inexpensive, easy way to confirm this diagnosis. You can also confirm diagnosis of osteosarcoma with FNA of bone.

So if we have a lytic lesion that we see here on the left, if, if the patient you can palpate the lesion externally, you can just FNA the surface, you basically just put the needle in until you feel like contacting the periosteum drawback. The lesions are actually quite exfoliated. It's one of those exceptional.

Sarcomas that exfoliates well, but the best way to collect these is, is with ultrasound guidance. So if we put an ultrasound probe over these lesions, we will see irregularity in the surface of the bone as we can see here on the right. So, obviously we, before we do an amputation, it's ideal to try and confirm the diagnosis cytologically, so, we recommend doing this in all of our suspect osteosoric cases.

Here we have a lovely image of what that cytology looks like, osteosarcoma cells don't look like, as classic spindoloid mesenchymal cells. And then if we are taking bone marrow aspir samples, obviously we could do a whole lecture just on this, but essentially this can be really helpful if we are suspicious for a leukemic leukemias, multiple myelomas or peas. So, use useful technique, you can absolutely do it in general practise.

And that is it. So thank you guys so much for sticking with me and apologies we run a few minutes over and so much cytology to share. I will just stop sharing my screen here, but if anyone has any questions, you're very welcome to put them in the chat or turn your microphone on.

Happy to, happy to answer anything about cytology. Thank you, Sarah for a great webinar and your time this evening. If anyone have a question, please put it on in the Q&A box.

And I just want to remind everyone to don't forget about our quarterly CPD competition for unlimited members. So the first quarter of the year ends this month. So watch as many webinars as possible and you could be, you could be the winner of 1000 pounds.

Yes. So, yeah. OK, so it seems that we don't have any questions coming through, but Sarah, thank you so much again for your time this evening and thank you for a really great topic and presentation.

See you. And everyone in the next webinar. Bye.

Sounds great. Bye everyone.