Thank you very much for the kind introduction and it's a great pleasure for me to join the webinar speakers and also to, to share some of my top tips with all the audience. . I, I, of course, hopefully all the people who joined today love cytology, and I hope you won't get disappointed that this talk is not all about the different colours and the shape of the cells, but what I would really like to do is to give you a solid foundation that if you want to start looking at cytology, how to get the best results.
So what I'd like to do is give You lots of very practical tips. And of course, if there's interest, I would be more than happy to go in a future seminar into more specific syology. So just before I start going into the details, I would appreciate just a tiny bit of feedback to get a feel for the audience.
So the first polling question would be, how often do you take FNA's in your practise? Right guys, so those members, you know what to do on your screen is are the questions, sorry is are. Simply click on the answer which best suits you and we'll give you about a minute or so to answer those.
And as soon as all those answers are in, we will then reveal the answers. So simply just click on the answer that best suits the way you feel about answering this question. Right, come on guys.
10 seconds more. There's a couple of you sitting on the fence that haven't. Thank you very much.
Let's get those votes in. Few more, come on. Not everybody's voted, but we've got the majority in.
So let's end that poll quickly and let's share those results for you. Can you see I can see the accent. So, so the majority of people do it commonly, not necessarily every day, but certainly a few of us, I guess hopefully, probably by the end of this talk, maybe people will shift a little bit, more upwards, but, .
We'll see and hopefully you will enjoy these practical tips that will help you in your clinical work. And then the second polling question would be that if you take an FNA, how often would you actually look at it yourself versus just sending out to a laboratory, which is obviously perfectly fine as well. Right, there you go guys.
Pole is launched. Ah, that's better. Yeah, nice and quick coming in with the answers on this one, Bolash.
Thanks guys. Couple of stragglers there. Just give us a click on the answers.
Please remember all these questions are anonymous. So don't feel embarrassed. Just answer truthfully, .
And nobody knows who's answered what. Right, so 5 more seconds and then we're gonna end that one. Thank you, thank you.
There's a few more coming in. Right, Polash, let's end that quickly and share those results with you. OK.
So it looks very similar, although definitely looks like a a bit less people looking at them. So, but anyway, so I think that's, that's a great and thanks very much for that feedback. That's good for me to know.
So basically, this is the, the plan for tonight. What I want to talk about is give you my top tips for smear quality. I think a lot of us have been taught simply just wrong how to do this whole thing.
I'm hoping that most of you already is in the different category who has either been retrained or, or young enough to be correctly trained, but I do see a lot of problems. So in my everyday life, this is probably what hinders cytology the most people not taking the samples correctly. If you want to look at your slides as well, then, I've done lots of, continuing education and one of my main thing is what I find all the time is people not, using the stain adequately, so the colours are all wrong, which then I think it just makes it much, much more difficult for people to, to get the kind of answers.
And similarly with setting up The microscope. So as much as I love to talk about the cells and the shapes of them and the colours of them, I do feel very strongly that people will have a much easier time to gain benefit from cytology if they have a good quality sample, they stain it properly and they have their microscope set up correctly. Now, if we can get to that foundation, which is really important to build the house on, then I also like to share you a couple of microscopic skills that are worth kind of considering or thinking about and practising because practising those will make you more efficient, and I know that probably most of your time in practise struggle with time the most.
That's probably the most expensive aspect of your work. So, so being more efficient in a microscope, I think is, is a key for being able to do the microscopy as opposed to just trying to spend too much time and not getting the answers. And I'd like to finish a little bit about just with the submission with the lab to the laboratory, how to communicate with the labs and a couple of top tips on, on common mistakes or how to, to avoid those.
So pretty you all know that cytology is a very quick, inexpensive, minimally invasive tool, and it's a great screening tool, and I want to emphasise the word screening tool because I, I've been hammered, this is when I was, I did my residency training that you have to think of cytology as a screening tool. Knowing your expectation from cytology will make you a lot. More appreciative of the results of cytology.
Some people want always a final diagnosis, which is not what cytology for. Some people just don't believe in it at all because they never get good samples. So what they get back results is meaningless.
But you need to understand that you try to screen a lesion with cytology. If you think about it like that, cytology will almost always be. That it will take you further along the line of your diagnostic.
Yes, it can give you final diagnosis. Other times it may just bring you more differentials in or it may help you to rule out a few differentials, but we'll leave it still open. So it's not uncommon that we're gonna have to go to more confirmatory tests, such as, for example, histopathologic examination.
In terms of doing cytology in practise, it really relies on the sample quality, as I said, that's the skill of the collector. This is really dependent on the individual. So even within our hospitals, I hate to admit like surgeons give us very poor samples, oncologists or medics who do this all the time, they give us beautiful samples.
So it is really just to follow those few. Tapes, how to get good samples. If you do those, your slides is gonna be great.
If you don't know those, then, then generally you will get back results of non diagnostic or too bloody, that kind of results. Overall, then the smearing qualities, I'm gonna talk quite a bit about it it's not just not enough to collect the samples, but you have to make the preparation correct. And obviously some tissues will not exfoliate, will not give up the cells as well as some other tissues, but I would overall say that this is much less of an importance in, in terms of limitation.
Now then of course, who looks at the slide is also going to be an important aspect. If somebody does it all the time, obviously they're gonna be able to tell more than somebody who just starting it, but obviously it's an experienced question, so the more you put effort into it, the better you will be. Now.
Overall, one of the biggest issues or lack or, or, or limitation of cytology that we will not see the tissue architecture. So we just take cells out of a lesion and then we extrapolate back to that lesion. So this is kind of very important in tumour cytology, for example.
And there's a few diagnostic challenges in cytology that, I usually go into more detail when we talk about the nitty gritties of the actual cytology. So probably a lot of you used the word a fine needle aspiration, and so my first question would be, or my next polling question would be that how much vacuum should we really use when you take an FNA, let's say, using that terminology. Right, the pole is open, guys, you know what to do.
Go mad. 5 more seconds for the stragglers to come through. Come on guys, give us an answer.
As I said to you before, it's all anonymous. Thank you. Thank you.
Yeah, thank you. Right, let's end the poll quickly and share those results. There you go.
Excellent. So, so we kind of have a majority of using about 2 CCs, but there's about 21% who doesn't like to, use any vacuum, which is kind of where I'm heading. So, this is great feedback for me.
Many thanks for that. So, as I mentioned that a lot of us, including myself, we were taught to use a syringe and to aspirate and then instead of just, you may laugh at me, but whenever we work back at the vet school in Budapest, we would even nick these special devices from human hospitals because they used it as a single-use item and of course, we would just sterilise them and use them over and over. But I don't know if any of you are old enough to remember this where this is meant that you can put 4 fingers in.
There are at least 2 and literally suck as hard as you can on the lesion. And this is how most of us have been taught. But by now we realise that this is absolutely not helping the sample and it will just introduce blood to it and damages the cell.
So, surprisingly or quite actually counterintuitively, actually just using a needle will provide a much better sample, probably 90+% of the time. So this little movie, I can promise you that no animals were hurt during the filming of this movie, but, I meant to show you how, so I'm right-handed. So with my left hand, I would stabilise the lesion or let's say it's a lymph node, and with my right hand, I would go into it and saw in the video how I would redirect the needle without coming out, which makes Help us to kind of make the sample more representative.
Now, when I came out of the lesion, that's when I would attach the syringe and relatively gently squirt it out, and then comes the spreader slide. So we're gonna spread the slides and I'll come back a lot more about the actual spreading and, and the smear making aspect of it. So, in other words, it's probably even the terminology like a fine needle biopsy would be better than, than keep saying the aspiration, aspiration because that really kind of makes people want to aspirate.
But overall, what is also kind of counterintuitive is that what you really need is just what the amount of material you need is what's gonna be actually within the needle. That's more than enough material. If you actually use a.
And you, when you suck in the lesion, you start to see any kind of material within the hub of your syringe, you probably ruined the sample. So counterintuitively, what you need is just what will be in that needle, that's more than enough. And relatively small gauges tend to be enough.
If you didn't get a good sample, you may go, a bit thicker with the needle, but that would, typically what we recommend. As you saw it on the video, like going into the lesion, multiple times to different direction is a great scale, a great tip because it's not uncommon that like on this picture, you can see a beautiful necrotic picture which you can diagnose really well in cytology necrosis, but it's not uncommon that you have a big lesion, you will have a necrotic sample. So if you just go into the middle of it, take the sample, that's where you're gonna get this necrotic cell material.
But by redirecting the needles. Several times, you can actually make your sample much more representative. And interestingly, I have seen now several times when where clinicians, when they follow up with a true-cut biopsy with one of those triggering needles, they don't have the advantage that they can go to different or they tend not to take lots of those.
So, so with cytology, we do have a bit of disadvantage that we can actually make a more representation of a particular lesion by using this simple tip. And then we're gonna put the syringe on, as I mentioned. Now, if you tried 2 or 3 times, even with a bit, thicker needles and you still haven't got anything, that would be the only time when I would say that you could potentially try to do an actual aspiration, but most times they're not, when you have to go there, the sample is probably not gonna be great anyway, but if you tried it, overall the About whoever said 23 CC, that's probably what I would use.
Similarly, I would redirect the needle a couple of times, always goes at place, pull back the plunger, then move it about, pull it back again, and it's very important at the end, let the plunger back as you come out of the lesion, so, you're not coming out under suction because then of course your sample will end up, inside the syringe. So now with that said, I hope, all of you especially who was in the evening I had a nice dinner because I want to talk a little bit about the gastronomic background of smear making and you may wonder that what is this guy talking about, when I have a picture of a fried egg versus a boiled egg, but this is a very important concept that in cytology, when cells are spread out in a thin area, they will look like a fried egg versus Any thick area on the slides, the cells will kind of look more like a boiled egg when you cut it in half. So if you compare these two pictures, look and imagine these are cells, look at how large the cell or the egg on the left looks like compared to the one on the right.
Also, I think this picture worked out really well. If you can look at the detail of the cytoplasm of the cell versus how much little. You're going to see if you have the boil.
So on the bottom, you can actually see a simple i in the full granular side, which is very easy to recognise, obviously, but see the difference if that cell is in a thick area of the smear where it's bowled up versus if it's a thin area where it's nicely flattened out. So you can obviously see that this is an important aspect of making smears that we get this flattening of the cells so you can see the details of that. Now, if you look at, for example, these are two pictures from a lymph node cytology, and then in the lymph node world, it's all about size.
So we describe lymphocytes if they're small, medium, or large, purely based on their size. Now, if on the picture on the right, it shows you a very thick area, the Cells are not flattened out, they will look smaller. So a medium lymphocyte will look like a small lymphocyte, and that's the difference between making a diagnosis of a lymphoma versus a normal lymph node.
So having this flattening is an important aspect of making the smear. Now, if you look at this picture and think just for a second that do you think this is a good picture, is a, or a good cytology or a bad cytology, I'll let you think of it for just about a second. But those of you who think that this is not great, I would agree with those because actually, even though we do have quite a few, cells, not a lot of blood cells, red blood cells, but actually, if you look at how many cells do you have cytoplasm, this is one, in this area.
So I can circle this, and most of these other structures are just basically free nuclei. So, these all lost their cytoplasm. There is a neutrophil here which is intact, but we get good quality sample, but actually we kind of damage the cell.
So this was my first attempt of frying the egg. Now, the next slide is not uncommon where we see this kind of structure and I refer to this as the scrambled egg, where basically there was just too much pressure and all these, all these purple strands you can see, these are actual nuclei of cells, so this is nuclear material and obviously the cells have been all damaged, so there's no chance to really get much of a diagnosis out of them. So, so when we make actually smears, the key goal is that this is not the other, so let me step back.
So most of us who've been trained to suck as hard as we can on the lesion, we were also told then to try to spray the cells as hard as we can. So that's how we were taught that suck really hard and then try to spray as hard as you can. And basically what you will end up is a small droplets, which will be too thick, so the cell's not gonna flatten out.
And instead, what we really want to do is when you attach that syringe, we just want enough force to get the sample out of the, core of the needle. As I said, that's the material that we have. And the, the more gently you can actually squirt it out into a small blob, the better your sample is going to be.
And instead of trying to spray those cells, we're gonna actually smear them and this is how we're gonna to attain that nice flattened image of the cells instead of the spraying. So in terms of the goals of making a smear is now, it's not possible to, with the blood, obviously with a, with a drop of blood, we can make a blood smhere where we have a perfect monolayer where we can see the cells exactly how they should be. Now it's not possible to create a cytology that's a, a monolayer, but what we want is to actually have some thin areas with good cells spread.
We can have thick areas and we're just gonna ignore them, but we want to make sure we get some nice areas. Where we get these cells flattening. We also do want to minimise the cell damage, so we don't want to, damage too many of the cells because again, that's gonna hinder the reading.
And during the sampling, of course, we want to kind of minimise the blood content because blood will be your enemy in cytology. So this is a characteristic picture when somebody did the old technique when spraying, and what you end up seeing is that each droplet is actually too thick. So in each droplet, the cells will look like the boiled eggs.
And there's not a nice area where the cells flatten out. So this is kind of heartbreaking for the cytologist because you can see that they got a good sample, but they just actually ruined it by making the preparation. So this, so in this kind of cases, what I would try to do is find the, the, the, the kind of the thinnest droplet and then hoping that the cells had a better spread in that area.
But, but basically all these areas and all these thick droplets will be completely unexaminable. So when we make the smear, now there's different techniques to it, and as you see on this picture, I show you the cross techniques. Some people like to do them parallel where you pull them apart and you end up actually material in both classes.
I personally find that the smears look much better when you do the cross type smearing where actually only. Only one smear will become, your cytologic preparation and the spreader slides, the one I hold in my right hand, which is, vertical on this picture, will become actually empty. Now, just, as you can see, I use a small drop of blood to make this picture, but if your sample actually looks like that, it's probably not going to be useful, but it's very difficult to make nice.
Pictures with just a clear fluid. So, that's why I use blood, but you do not want to have your sample looking like that, but it certainly helps to make the pictures better. So, so basically what we want to do is, as you saw already on a little bit on the video that I will kind of touch down with the spreader slide, and then the material starts spreading between the two glasses.
And before it spreads. All the way out, it's good to start the smearing action. If you wait too long sometimes and you will feel that the two glasses just like sticking like blue to to each other.
So starting the smearing before it spreads out and fills up the, the entire square formed by the two glasses is is a good idea. And then we just start the smearing action. Now, when you do this smearing.
There will be to some degree a minimal amount of fine adjustment that some sample needs a little bit more pressure, some needs a bit of less pressure, but what you want, and actually that's where looking at your own slides can really help you develop your skills or technique, because what you want to do here is to have enough pressure that you spread the cells out so we will have those fried eggs, but not to have obviously too much pressure, so we're damaging the cells or too many of the cells. So again, just a quick video. Now here I use more like a like a fluid type sample and then just as simple as that.
So it's a very easy quick technique. Now, the next video, what I want to share with you is an absolutely brilliant tip that I learned from the oncologist back at K State. Where you end up with a really good sample.
Again, I use a drop of blood just because it visually looks better, but again, you do not want your cytology to look like that. But if you kind of blur, like squirted out your material, and it's a fairly big blob, it's, it's, it's, it's probably a mistake to actually just make one smear of it because in fact, if you have too much material. What you may end up with the the the spreader slide will pretty much like float or like aquaplane on top of that fluid or especially when there's a bit of fat in it like lipoma and not actually really firmly smearing the cells out.
So, so if you got a bit too much material, this is a really cool trick where you take your spreader slide and you actually pick up a small amount of it. And then you go to a clean slide, you press it down and you make your smear. And if you did this well, then your actual spreader slide becomes clean.
So you can just keep going back, holding it in your dominant hand, and then go to clean slides again and again, and I can keep going back and then pick up a bit more material and go to another slide and smear it out and at the end when I only have a small amount left, then I would go back and then just smear it out. Now, if you think for a second, why is it that I will end up with 234 very uniform slides that are actually it's a big advantage. So those of you who thought that actually the, the trick of this thing is that when you pick up the material, you will pick up relatively similar amount of material.
So this is another reason why it's a great technique that you will end up with 23 or 4 very uniform slides. So you can actually take one of those for yourself. And stay in it, but you still have 2 or 3 other good quality slides with similar material that you can then send out, for example, to a laboratory instead of having just kind of one too thick smears.
So I, I strongly recommend this technique that it works really well when you get a bit too much material, to make multiple good slide. Now, the next segment, what I want to talk a little bit about, if you have any questions, please feel free to pass them on and, and I will try to get to them at the end. Obviously it's a live, an audience, we can do that as we talk, but here at the end, I will be more than happy to answer them.
The next segment, what I would like to talk a little bit about is, especially for those of you who sent out the slides. I want to give you a couple of tips of how to look at your slide if it's any good or not, because, obviously if you look in a microscope, it's easier, but there are some techniques that you can actually have a fairly decent idea about the slide, without even staining it. So in other words, which glass do you want to send away to the laboratory.
So if you look at these two pictures, to these smears, which do you think would be the better one and which would be the, the not so good one? So obviously, those of you who thought that the left one looks much better, I would agree with you. The one on the right is just way too much blood.
So if your sample looks like this, this is what it's gonna look like after staining. So if you look at the density of this, you actually need an X-ray machine, not a microscope to, to be able to look through this. So in fact, even if you look at this smear on the left, Basically, the vast majority of it is going to be examinable.
So all this area is gonna be probably way too thick, but what I will go to with my microscope is probably that little area there or like somewhere like along that area. So, and this small area that I'm just circling, you could actually look at that for like an hour, there's so much cells in there. So, but this is the area where I will probably have the nice spread out.
Elsewhere they will look like fried eggs, versus in this area, they will look like boiled eggs. So is this a good slide? Yes, even though a big part of it is probably unusable, but we do have some really nice areas, so this would be definitely my preferred slide to send away to a laboratory.
Now, another problem is when you introduce blood to your sample, if you think with me for a second, if you think of one drop of blood, half of that is going to be red blood cells. The other half of it is going to be plasma, but there's also going to be white blood cells, and most of you work in small animal practise, dogs and cats, the most numerous white blood cells are neutrophils, especially if the animal, for example, has a neutrophilia, you can actually introduce quite a few neutrophils into the sample, which is why I said earlier that blood is our enemy in cytology. If you A lot of blood in your sample bringing the neutrophils, it can be very difficult to judge that whether the amount of neutrophils there is proportionate to the blood or is a little bit increased.
Of course, if it's lots of them, then it's easier, but, but it can make the diagnosis more difficult when you introduce these inflammatory cells, what we consider them as inflammatory cells in the tissues. Now, here's a couple of other pictures that I hope you can see what I'm trying to show because it's very difficult to photograph it, but if you hold the glass into the light and and kind of look at the reflection of the ceiling light, what you can see actually on this picture that we have this really nice oval outline of the smear which you, you get with the cross sections. But notice how in the middle.
We have this kind of more whitish granular material. This is where your nucleate cell is gonna be and the outside is going to be a bit thinner and a little bit more the red blood cells. So if you look at the same slide stain, you can see how nice and there are all these nucleate cells that appears kind of dark purple here versus the outside area is going to be probably less useful, but that's a perfect, that's a beautiful slide for examination.
Now, here's another example again, how to evaluate your slides before you look at it. Now, The one on the right seems to have quite a bit of fat, and I know that's a fairly common worry of people when they aspirate lipomas are very lipid like masses, and they worry about that this is going to go away with the alcohol. Yes, the alcohol in a fixative will dissolve the, the fat, so that will go away, but the cells, if you smear them out, should still stick to the glass.
But as I mentioned a bit earlier, if you have too much fat and you just float. The spreader slide on top of it instead of actually smearing those cells to the glass, then you may end up without the cells. But for example, this picture shows you that this was actually they tried to aspirate a lymph node where they got a lot of the perinoal fat.
And after staining, you can see that, yeah, there's still a lot of nucleate cells that's left on that glass, even though there was lots of fat, which is now gone because the alcohol dissolved it. So, again, if you have any questions on how to, to, to, to evaluate these smears, please feel free to post a question, but, but I, I thought these were kind of the top tips in terms of the aspirate and, and, and, and how to, to kind of evaluate your smears. The next couple of minutes, I would like to spend on imprints.
Now, this is something that's arguable that like we're kind of lucky here at the Royal College that, you know, a lot of our patients are, are insured, so there is financial means that we can actually do imprints of an excise mass and you know, still, you know, pay for cytology and for histopathology. This may not work in your practise that you cannot justify both. Expenses or if you can do it yourself, it can be really valuable that you can get a quick answer about something a mass that you exercise before you can plan your therapy or, or, or, or potentially more excision around it.
So, so you can judge yourself whether this is something that you would do in your own practise or not, but we even do this like, in dogs when they're on the operating tables and a bit like in the big human medicine where you know. We can actually do intraoperative cytology and we tell the clinician while the animal is still under anaesthesia, if it's a really ugly tumour or other things like that. So in terms of getting good quality imprints, there's only a couple of top tips, but again, if you miss those, your sample is gonna be very poor versus following those will make your samples beautiful.
The key thing with if you have an excised mass, obviously as you handle the tissue, there will be tissue juices, blood on it. And it can be a bit damage from your handling it. So actually making a fresh cut surface is an advantage.
Obviously you don't want to chop it too far into bits because the histopathologist will not be happy, but making one fresh cut in the middle of it, for example, actually is advantageous because formalin will then penetrate better. Now, when you cut into that tissue, it's very likely that tissue juices or blood will kind of ooze to the. Surface.
So this is the most important aspect that you have to bloody dry. So I would very strongly emphasise this just as normal paper towel or quim wipes, you bloody dry as dry as you can, and then you will actually touch it with the glass. So, I would usually, it depends if it's a small piece of tissue, then you can hold it with the tweezer and then from the top you would touch down with the glass.
I'll show you with you in a minute. The good thing is doing it from the top down is that you can actually see through the glass and you can see how the tissue is, is, is imprinting onto the glass. If it's a small tissue, you can actually do 345 imprints on one slide.
If it's a big mass, then one glass will be basically 11 mass will be one glass or one glass will only have one sample on it. So here's the the same video and actually the Mandarin really works well in this particular instance because as you cut into it, of course you have the tissue juices. So I would take a paper towel and just blood it dry and you want to have this very dry surface, so then you don't actually need a lot of material.
If you let the fluid stay on there, then guess what, when you touch it with the glass, that's what you're going to imprint the blood and the tissue juices. So, I have a, in a second, like a bit more close up, so you, you want to see that very nice dry surface, and then actually a fairly small amount of pressure is needed to imprint the cells through the glass. You don't want to press down too hard because then again you're gonna start smashing the cells and this is a, it's a nice way to get a cytology sample from a tissue that you already have excised from the animal.
So if you look at these pictures, I'll give you a second to think about what do you think went wrong. Obviously this is the classic example. This was one of our top surgeons and this was a very interesting bronchial cyst that went malignant, so we even published this case.
But these were the kind of slides I had to deal with, where you can see that it's just all blood. So pretty much all of those slides you can completely forget about. There's only one slide here is the one where you could try to find some thinner areas, but it was definitely starting to look like looking for needles in a haystack because there's so much blood on them.
So all was forgotten in this case, as I said, just to bloody dry, take the blood off with the paper towel, and then you have the tissue that you can really make nice imprints of it. So again, if you have any questions on, on imprinting, feel free to pass them on. Where, as I'm moving on to the next little section is fluids because obviously, in practise you will take fluid samples and that's something that you can look at yourself or send it to a laboratory.
It's very important to try to use an EDTA tube, even if it's not a bloody fluid, but if it's inflammatory, it can still clot. Of course, if you're trying to do bacteriology, you would use a ster up pod. But the one thing that's very important with fluids is to try to make a fresh preparations.
When cells are in a fluid, and you send it to a laboratory, especially over a weekend, those cells are still alive. So for example, macrophages can eat red blood cells and neutrophils can eat bacteria, but also they will deteriorate. So there can be in vitro changes that can hinder diagnosis.
So if you can make a fresh preparation at the time. And then you send it along with your sample that gives you a huge advantage because then of course we will make another smear in the laboratory and comparing those two smears, I can immediately then tell if what happened in the tube in vitro during the shipping and what was already on there, in the animals. So, so I will want to share a couple of top tips again, how to make some good preparations from fluids that will be advantageous again for your diagnostic work.
We do struggle quite a bit with clots, and I wonder if this is people don't mix up the sample quite quick enough, when you put it in the EDTA, but those of you who kind of do a little bit of haematology, you know what happens, you hear a lot of platelet clumps, that will trap the, the platelets. So in this case, these clots will trap the nuclear cells. So this is what it looks like in a microscope that, those little clots that we see.
Get out on the glass, they have a lot of nucleate cells in them. So if you look a bit closer, you can see that in the other areas there's much less nucleate cells. So when you measure it with a machine, for example, the total nucleate cell count, which is one of the key ways to classify if it's a transited or an exitate or modified transitate, you will get a falsely decreased number.
So you want to try to avoid. Clotting because it traps the cell. Now in terms of making direct smears, that can be an advantageous, as I said, the fresh smear is is a huge advantage.
I put a picture in here as a blood smear, because it would be the exact same technique. Of course, if I put a picture of a clear fluid smear that you wouldn't see anything. So, but, but the technique should be the exact same thing.
So you put a small drop of the fluid. Use a 45 degree angle, come back to it and smear it out. So here's the video where, I have the fluid already.
I would, I'm a right-handed person, so I'm going to be holding the spirit slide in my right hand. And my left, pointing finger, which you may not see quite yet, that, that, that's hold the slide close to me at the frosted edge, and I will smear away from me. Some people put their hand.
At the end of the slide in this area which then kind of hinders them of smearing against it. So it's better to hold it down here so you're gonna smear away from yourself. So here is the technique.
I just, you want to control the amount of material that's probably even too much, just a very small amount, and then you come back to it and smear it out as a normal smear, it's very similar to to a blood smear. And then you just air dry it. You don't have to fix this preparation, just air dry it and then put it in a box to to, to preserve it.
Now, sometimes if, if fluids have very low solarity, it's important to make some kind of concentrated preparations and what we call a line preparation is a very cheap but brilliant way to achieve that where it basically starts similar as a normal blood smear type technique where you come into the. And then you start the smearing, but instead of kind of going all the way, you stop in the middle of the glass and you lift up your spreader glass. So you end up with an actual line where the larger, typically the more abnormal or interesting cells will line up.
So this is what it, this looks like in the the actual slide. This is the cover slip. This area is a cover slip, but this is my line preparation.
So you can see that it's quite dark blue, so that's where the nuclear cells are. This is what it looks like in the microscope, where, you have the, the cells, and here you can see that they, they, they actually spread out quite well. So this is the technique again, how to do it, the video like you control the amount of material, and you come back to it with the spreader slide, but instead of going all the way you stop in the middle and then you lift it up a little bit and then again you can kind of see here that this is my line and it's, it's I I like to kind of Lift it out a little bit so the fluid can flow back.
Now, if you use too much fluid, then the line can be a bit too thick. So then it, it may end up with a bit of the boiled eggs instead of the fried eggs. But if you control the amount of fluid you put down and you make that smear, then, the actual line will be relatively thin, so it will look nice, under the microscope.
Now, if you have some more particular matter in the fluid, then again, you can use the same technique that I showed you before. Again, I'm just using a small drop of blood to illustrate it, but you squash down if it's a small particle or something in that sample, and then you start the smearing as similar as I showed you before. There's just a video of it that's my fluid or if there's any particles in it, and then I come down with a cross type smear and then smear it out.
So that's pretty much I wanted to talk about how to get a good representative, good quality cytology sample, how to make smears that will be nice and where we're going to have that kind of spreading out those cells that you can actually evaluate them and judge them. And, and, and, and of course a little bit of those tips about how to make good imprints, but with the same ideas of getting good intact cells with good spread. So the next session, again, if you have any questions regarding any of the sampling, please, make sure you, you type it in and I'll, we'll try to get to that at the end.
So the middle section of the talk now, what I want to talk about it is staining, and you might find that, I hope you don't find this to be, it sounds too boring that who cares, but actually this is a lot of people make this wrong and, and I think it's, it's, it's, it will really help you if you use the staining properly. Of course in laboratories we have automated stainers, but most of you, I presume will use some kind of big quick type stain. So I wonder how many of you have seen this kind of preparation where most times when I do this to the live audience, people don't necessarily know what the hell is going on here, but you see all these inclusions in the red blood cells.
Basically what happens here is that the slide was not dried properly yet before staining, or the first jar, the alcohol jar, is too old and has attracted water. So this is an artefact that pretty much if you look at, for example. That's where it makes it impossible to examine it.
This is another slide where, for example, the staining is completely off. So even myself who spends all day in the microscope, I would have a hard time reliably tell if this cell is a plasma cell or a nuclear red blood cell. So this slide doesn't have adequate staining, doesn't have enough blue stain in it, and it has all the water damage in the red blood cells.
So again, like not having the right stain can make your cytological diagnosis much more difficult. So most times when I get people ask me about Divuk, one of the most common questions is how many times should I dip in and then actually realistically, the best to start is always what the manufacturer for that particular stain recommends. Note that Divuk is a bit like Kleenex, that's the original company who came up with it, but now there's lots of other companies like Emoolor or Quick De or.
It doesn't matter which one you are using, they're pretty much all the same. Now, the first jar is the actual alcohol the fixative. So, if you're in a hurry, then you need to kind of make sure that you dip like 67, whatever the packaging tells you times, but if you're not in a super hurry, then actually just putting the slides in.
There and letting them soak in there is the best. The longer you fix them, the better they will be. So what I would usually do is put the slides in there and then start opening the other jars, the lids from them, and then take some paper towels and things, so, so you get the good fixation.
Now the red jar is basically the the key trivia is that you cannot overstain with the rat. So if again, if you're in a hurry, you can understain it, so you have to make sure you dip enough times, but you cannot overstain with it, so you're safe from that side. And it's only the blue jar where you can both under stain or over stain.
So definitely you need to start with whatever the manufacturer recommends, kind of look at your slides, and you may actually need to go back and dip a couple more times. Now, you don't have to go through the whole line again, I wouldn't put it back in the alcohol in the red stain. You can't just, after you washed it.
And and dried it and you feel that it's under stained with the blue, then you can just go back and dip one or two more times. I didn't feel the picture of the 4th jar, which is basically water in an ideal world, it's distilled water. You can actually use tap water, to be honest, just relatively gentle flows so physically not removing the cells.
But either way, that's that's going to be defined. Another very important tip is that probably a lot of you have seen this, that when the, excuse me, in the morning you open up the blue jar, there's going to be like a mirrorry film on the top of it. Now, the worst thing you can do is to push that in with a slide because it will break up into small fragments and that will end up all on your future slide.
So what you have to do is take a small picture. Towel or bigger paper towel and literally blot it off. So you will lose some stain, but that's less of an evil than actually to have the film within the stain breaking up.
So you want to kind of blot it away. Also, one tip is important to keep the lids on, especially the first jar, the alcohol jar, because it evaporates quite quickly. So if you keep the lids on them, you can kind of control that a little bit better.
Now, Here is my polling question. How long can you use the death quake before changing it? So this is a very important aspect again of using the stain.
Right guys, by now you know how this works. So just click on the answer that best suits you, and then we will reveal all afterwards. Remember it is anonymous, so don't worry about incorrect answers or somebody knowing what you answered.
Right, Balash, good sign, everybody is still wide awake at this stage, which is great. So we're all done. That's fabulous.
Right, let's reveal those quickly. Excellent, so that, that's a great thing. Like most people, the, the most common answer was 2 weeks, which is the correct one.
And this is something that, again, this is why I feel so strongly about talking about it because a lot of people don't necessarily notice and then the staining, I mean, I have to say that probably 9 out of 10 slides that we got from outside or even sometimes our clinicians, the red blood cells look dark green and people go through these changes that they don't notice it because. Just got used to it. But if your red bloods are not orange but dark green, then guess what?
All the other colours will be wrong as well. So, so it's very important to upkeep your dick, and then it is a great stain actually. So, I would usually recommend to, to about 2 weeks.
Now, if you use cytology every day and you put loads of slides in it, then you can probably go shorter. If you rarely use it, then you may go a little bit longer, but overall it's, it's, that's how the stain is designed, and there's another issue that sometimes people ask me when you go from one jar to another to another, the stain is designed that you can actually have the dripping red stain on the slide and go into the blue jar, but of course it will start to deteriorate the stain a little bit. So I tend to kind of blot it a little bit, but wouldn't wait too long because then you lose the convenience of the whole stain.
People sometimes ask me if instead of doing the dipping, should they just Put a drop on the la, like have it in a horizontal and, and, and that way then you don't have to kind of waste so much stain. Now, yeah, that's true. You won't waste the steam, but you just completely lost the convenience of the whole thing.
And if you're willing to go to that mass, then you can actually find some, simple, recipes for white stain, which will be nicer colours. So the Deuk has this huge advantage that it's so quick, so easy to do. It's the convenience basically that what you get with it.
Now, another very common mistake is that when you have the, the alcohol jar, as it, because it keeps evaporating, and as I said, it attracts water, so that will cause all those water damage on the cells. It's not uncommon that people, even if, OK, like it's 2 weeks now, it's, it's a good idea to put the date on the top of the, the jar, so you kind of see when it's getting close to, to ready to change. But sometimes people feel that, OK, I just tipped topped off the topped up the, the alcohol like yesterday, so then they actually end up not changing it.
But that's a mistake because that water is still in there. So even though if you just topped it up, unfortunately you do have to chuck it and put new alcohol or new fixative in there. Another common mistake is that people will just pour it out and pour in the new stain.
Especially this is very true for the blue jar. If you left all the crud on the side of the glass, as soon as you put the new stain in, you immediately ruin it. So as But tedious as it sounds, you need, or at least not you, but hopefully your technician, you need a toothbrush, so, and you if you use glass jars, you have to keep like clean them sparkling clean, so you don't want to leave that material on there.
Now I, I found a really cool, I heard a cool tip from a Norwegian practitioner once who uses the slide boxes that we, the laboratories are more than happy to send you, get a Styrofoam to, to put. These in there so you're not knock them over. And this way you can actually use fairly low volume.
And if you only use a couple of slides a day, then you can get away with a low volume set up, and it won't deteriorate any quicker than that, let's say the two week window. And when you come to the end of it, you can just chuck the whole thing and, and we'll send you more slide boxes anyway. So you don't have to mess with the cleaning and And another reason why this low volume can be advantageous, especially for those people who don't look at a lot of cytology, because we do strongly recommend to have two sets of stain if you do like ear canals or skins or fecals, so you do not want to contaminate your blood smear or cytology stain, the stain you use for your blood smear cytology is with any dirty stuff.
Because that those malacasia and bacteria that come off from an ear smear will end up on your blood smears and cytology. So, so if you have two sets of stains and you use kind of relatively bigger jars and you kind of tip it out every 2 weeks, you start going through the bottle pretty quickly. So you need to kind of judge how much you use and, and, and you can kind of adjust a little bit of the volume of the stain.
You end up using, but if you follow these few tips and hope it was not too boring but but helpful for you, then actually this is a decent stain. It has very good penetration, so as long as you are patient enough to stain it, it will stain through some thicker areas. Nuclear detail, nucleolized stain actually quite well.
The one disadvantage unfortunately is that mass. Granules will probably 1 out of 1020 mass of tumours, they will not stain. So, I usually go more into the details of this when we talk about more specific cytology, but, but you have to factor that in that it doesn't always stain the mass granules which then can cause, cause a bit of a challenge in terms of your cytology.
If you use it for blood smears, you're probably not going to see many basophil granules, but realistically, you can go through a whole life of practise without blood smears on blood smears recognising basophils. I hate to admit that, but it's, I could count on one finger how many times I needed to recognise basophils when it was clinically relevant. So, so the mast cell granules would be probably the biggest disadvantage of DFA.
OK, so that was kind of my sermon, as I said again, I feel strongly about this because a lot of slides we get just don't look the right colours and it to me it just makes it so much more difficult to see the right things, the colours are off. So I, I strongly recommend to kind of use your stain the best way so make your life the easiest. So, again, if you have any questions on staining, please, post them and, and we'll I'll try to answer them as best as I can.
While I'm moving on to the next section, which is the microscope. Now, again, I hope you're not gonna get bored on this, but, but that's another thing I find, when we run our online CPD course like 6 weeks on haematology and cytology, and I ask people to take pictures. Of their blood smears or cytologies, it's 7 or 9 times out of 10.
I can tell from the pictures that the microscope is not set up for cytology. They probably always set on urine sediments and that makes it very, very difficult to see blood smears or look at blood smears or cytologist. So, so I wanna share a few tips with you about the microscope, how to, to, to set it up and what are the key question.
Now here's a, a, a quick polling question, which do you think is the most important part of a microscope? Where would you wanna spend your money on? Which part do you want to spend your money on?
Right guys, it's open, away you go and well lush, I think you've got them flummoxed on this one. Remember, again, it is anonymous, it's not a trick question. Just click on the one that you personally feel is the best answer.
I'm gonna give you another 5 seconds because Balash seems to have floored most people on this one. There we go. That's better.
Thank you very much. Right, let's reveal this one quickly. OK, so that's excellent.
So most people would spend the money on the objectives of the lenses, which is the most important. So any of you who like photography will know that that's where the image is basically created. So all the rest is what the people at the, the shops will try to tell you that it has LED light source it has this, it has USB ports, but they will sell you very cheap lenses that will make the image quality poor.
So what you need to know is that there are 4 levels of quality. Aroma or A, sometimes you see an A or ACH on the actual lens on the side of it you can see the writing. That's a very, that's what I would try to avoid at all costs.
That lens will only have the middle circle in focus and the outer half of the circle is out of focus. So if you were to try to look at the whole field or trying to count platelets in that one field, literally like half of your, the other half of your image is out of focus, so it will give you a headache very quickly. If you can afford to go to a plan acromat, so it will either say a P or PACH on the actual objective, that's a great upgrade because in that lens everything is in focus.
So I would really, really strongly recommend to use at least a plan acronym. For, for diagnostic use in, in practise it just makes you less headache, you know, gives you less headache, it makes it much easier to work with. Fluoride has a nicer colour and a much better resolution, but this becomes expensive now on this ladder, every time you go up, you double the prices.
It's, but, but realistically when you buy a microscope, the cost will come from the lenses. So it's a false economy to buy one with cheap lenses and then think that later you will upgrade the lenses because you will end up paying the same amount of money. So, and then manufacturers become never quite tricky that they will tell you semi plan and these kind of things that you want to look at that image and you want to see is the whole field in focus or not.
In terms of how many objectives you can put in a microscope, it will be a bit of a question of the nose pieces you can maybe, typically you can have 4 lenses in there if it's a bit bigger, then you may have 5 or 6 in big microscopes. It helps you to design how many and what kind of lenses you want to use. Now, I mentioned here the 50X oil, which is an amazing lens.
It's unfortunately relatively expensive, so it's kind of a decision to make to you want to invest in it or not. It will make your microscopy much, much more efficient, much more enjoyable, and because it's an expensive lens, the image quality is brilliant, so you will love it. You will not want to use any of the other lenses.
One thing that I want to share is the 40X lens because I see this 99 out of 100 times when I give talks or meet people, they miss this small piece of trivia that the 40X lens needs an extra layer of glass, such as a cover slip. So when you use it for your urine sediments, you have that cover slip on there. But if you put a microscopes like a haematology slide or a slide on there, and you don't have a cover slip on it.
It will be foggy no matter what, because it's designed to have another layer of glass. You don't have to glue down the cover slip the way we do it in, in, in our teaching sets, you know, that, but, but you can just drop on it. So next to your microscope, you want to have a small pot of, or like a small box of cover sleeves, and you just drop it on there and then you can use the 40 X lens and it will be nice and, and, and sharp, but without that glass, it will never be sharp.
Another big problem with the 40X lens is that it cannot see through oil. So if you put already oil on the slide, like you can see here there's a blob of oil, then the 40X you cannot use, it's not going to be able to create an image, but also what you can see that the clearance of the 40X is very, very small. So if you have oil on the glass, you have to make sure that you're not rotating the 40X lens through the glass because it will drag into the oil and this is kind of a.
Bags glass in there, the oil will get on there and it's very difficult to clean it. You literally have to unscrew the lens from the microscope, use a Q-tip with alcohol or a cleaning solution, and it's quite a, a, an elaborate, way to, to actually clean it properly. So if you go back to your practise and your 40X lens is foggy, the odds are that it has all dried on it, so you need to clean it off.
But also you need that cover slip to, to have a sharp. Image of it. So this is something that I found a lot of people missing this bit of information, but it's essential to have this.
Now if you can afford, you can convince the practise manager to invest in a 50X oil. It's brilliant because then you can go back and forth between 100 and 50 and you don't have to worry about the oil. The 20 or 10 or 4 Xs, they have a lot of clearance so they and they can see through the oil, so it's only the 40Xs that has this particular problem.
Now, one slide about eyepieces, it's very important that the diameter of the eyepiece, almost all of them will be 10x magnification, so that doesn't change, but the other number written on there is the actual diameter. Now an 180 millimetre is old microscope, it's like a peep show. It's a very, very small.
It's very difficult to work on it, so I would try to avoid it at all costs. 20 is nice, 22 is great, 25 is like an IMAX movie. Like it's an amazing picture, but it's not really realistic for practise.
So definitely try to shoot for at least a 2020 is enough. I mean, that's, that's good enough, to use in practise. And the last thing about the microscope is the condenser.
So this is what I mentioned earlier on that every Time I see pictures from people sending me, the condenser is typically set to the bottom, which is what you want to use to make the light go around objects. So for urine sediment that's perfect, it highlights the crystals, the cells, but for cytology, what you want to have is the condenser all the way at the top. So, and just a little bit below that.
So that's the, you're going to get a flat image. Versus if you lower the condenser, you get a very contrast image. So that kills the cytology or haematology because you don't see the internal details of the cells.
So you need to have that condenser very much all the way to the top, just a little bit below. There's a more elaborate way how to set it up, but, but for now's purposes just have it close to the top. Also, if you see on this picture.
There is a dial on the actual condenser or some smaller microscope. It has a little, oh sorry, little lever, where that's the same thing actually then lowering the condenser. So, so maybe even if your condenser is up, but if this little lever is.
Closed, then you will have the high contrast image that makes it very difficult to examine the cells. So you want to make sure that your condenser is up and you have to have this almost all the way open to get that proper flat image that you need for cytology. Excellent.
So that's kind of what I wanted to talk about the, the microscope. Now I, I'm kind of getting to the end of the time, so I hope you guys can still bear with me for a little bit. The last bit I want to talk a little bit about the essential kind of microscopic skills of how to make you more efficient, in, in the microscope.
So if you just think with me for a second and look at this picture, can everybody see that I have a large multinucleated giant cell in the middle. I have a binucleate cell here on the top right looks like a bug eye. I have some small dark like lymphocytes, like small cells, and I have a neutrophil up here.
Now if you get a good view of this, and I also have some macrophages with purple kind of granular. Material in them. Now this is 100 XL.
This is as close as it gets in the microscope. Now, if I go down to 40 X or 50 oil, look at, can you guys still appreciate that I have, I have my multinucle giant cell. Can everybody still see my bug eye here?
And then we have those small dark like lymphocytes and we still have those macrophages. So it's very interesting that if your brain, and in fact we still have the neutrophil, you can. Still appreciate that segmented nucleus.
So if you can like if you, if your brain processes an image, it's amazing when you look at it in a lower power, you can actually see the same amount of detail, but look at how much more I can see. So I can see here there's another big trinucleate cell. Here's another big binucleate cell.
So, and I can see a lot of those dark cells. So it is, you see a lot more and that, that makes you much. More efficient.
So anything you can actually do, one magnification less than what you would be comfortable with will exponentially increase your, efficiency in the microscope. So I'm going to push it a bit more forward, more, beyond. Can, still everybody see, oops, sorry, can everybody still see the, the big multinically giant cell?
And here's my bug eye. You can probably still. There's some dark blue blobs in there.
Probably the neutrophil is not really visible in a good quality microscope, you can still see it, but, but again, just see how much more you will see when you go a bit lower power and it's not we have to make the diagnosis at low power, but the more you can screen the slide, the better you can hone down to the right area quickly and, and the more information you will get in a short amount of time. Now, another important aspect is judging size. A lot of times people ask me questions, but how do you tell them plasma cells from a macrophage because they have the eccentric nucleus.
Yeah, but one is a small cell and one is a big cell. So, knowing a little bit of your sizes can be quite helpful and especially if you use textbooks to, to, to look at the pictures. So it's very difficult to put a needle in a dog or a cat and Get a cytology without having some red blood cells, which is a great way to measure sizes.
So we would usually use the red blood cells and you can say that let's say this nucleus is about, for example, 2 or 1.5 red blood cells across. That's more than enough.
Or you can actually use a neutrophil if it's a nice flattened area. This neutrophil that squashed, that's not a good size comparison. It's not fair to compare a build up neutrophil to a build up lymphocyte, but the neutrophil can be actually advantageous that, even for the last trained eye, you can probably recognise that this neutrophil is better flattened out versus with the, for example, lymphocyte, you need a bit of training to tell that this is a flattened out cell and this is not a flattened out cell.
So, but you can use a flatten out neutrophil again as as a size comparison that's helpful when you're judging these cells. Now, the last bit what I want to talk about is how to spend, how to find the area on the slide where you want to spend your time on, and this is again, it's really links into your efficiency. Now, if you look at this picture, and think for a second what's wrong here, you could probably appreciate that.
It's very, very thick. So I'm on very low power now. So I'm scanning the slides at 4 x or 10X, and I see an area like this.
So I would just immediately move on because actually the staining is not great. It's instead of purple it's blue, so it is a bit under stained because it's so thick. So then I moved to this area and it's a little bit better spread.
There's a bit more sun coming through, but again, and there's a bit darker staining, but it's still way too thick, so I would just keep moving and moving, and this is not the area where I want to spend my time on. Now if you come to this area, this is really nice and very thin, but those of you who remember said earlier, what's the problem here? Actually, most of these cells are just free nuclei.
So here's a small clump of cells that are intact. You can see the blue cytoplasm around them. Here are some that you can see them here as well.
So these are the only cells you can really judge in cytology we pretty much have to ignore live cells. Now, does anybody know what kind of cell this one is? If you said neutrophil that's brilliant, and look at that.
This is like really far away. So yeah, as soon as you start thinking along these lines of lower power, lower magnification, you will find that you can actually recognise a lot more than what you think you could at the moment. So this is a good area because it's, it's thinner, but it's just too many light cells.
So we want to go to an area where we have a lot of more intact cells so we can examine that. So here's an area that for example on this particular slide was much better. All these light purple blobs are again smashed cells, but all of these you can probably appreciate on the picture that there's a purple nucleus, there's a blue cytoplasm around it.
So this is an area where we do have a lot of intact cells, still have some light cells, a few red blood cells, but this is a lot better. Now this is a bit closer magnification. So this is what this would look like on a low magnification.
Now this is the scale to work on or develop. That at low magnification, you can appreciate that these light purple blobs are my light cells and in, when you look through a microscope, you will appreciate that these cells do have the cytoplasm around them. So if you can recognise, for example, this area as the best on this particular slide compared to the other pictures I showed you, this would be my window.
So the quicker you can scan a slide, let's say if you have a 4x lens, you can scan an entire glass in within like 20 seconds, then you want to find a nice area. This is stained really well, so we have nice purple colours for the nuclei and it's spread enough and we have a lot of intact cells. So the quicker you find the window, the more efficient you will be.
So this is again it's a very important skill to practise and it will make you much more efficient. So in summary, you really don't want to waste your time because it's so expensive and try to recognise more things at a bit lower power and use high power to confirm all of these suspicions. So don't lurk around on high power.
That's the best way to waste lots of time in the microscope. So that's just my quick tip. Now, I want, just, one mention of, looking for bugs, probably most of you feel comfortable doing it on a 100 X lens, the maximum magnification, but actually 40 X lens is much better.
So, you will see a lot more neutrophils and your eyes can learn this very quickly to find it, it's kind of like looking at a lot of orange a lot of yellow tennis balls and the one orange in between your eyes will go immediately to the one that's different. So if you have a lot of neutrophils that are normal and they have one with the bacteria in it, your eyes can, can be trained very easily to then start picking out the, the different pattern from the picture. So on a 100 X, you will have very few cells typically in an area where the cells flatten out versus in a 40 X you will get a lot more.
This is the same picture what I'm trying to show here is that concept of the boiled egg versus the fried egg, the fluid, especially like thick lets and. As a pertinacious background, the cells tend not to flatten out. So when you have a neutrophil, these are all neutrophils, when they build up, you can appreciate how much, how little detail you can see.
So on these slides, you have to try to find an area where you see these neutrophils flatten out, so you see the cytoplasm, so you have a better chance to pick up the bacteria in them. It would be next to impossible to find bacteria in an area where the, the, the slides are so or the cells are so balled up. Now, degenerative neutrophils is something that's very useful to be able to recognise.
If you look at this picture, probably if you're untrained, you have very very few of you would think that any of these are neutrophils. This is what a severely degenerative neutrophils look like. These are all bacteria in them.
It's basically the nuke the cell just slices itself as it releases those oxidative bursts to kill the bacteria. So if you've seen this a couple of times, it becomes much easier to recognise, but if you see degenerate neutrophils, you always want to keep looking around for bacteria. So here's the comparison.
We have degenerate neutrophils with bacteria in them versus these are really nice happy looking neutrophils. These an apoptotic one that's the old end of the life, the, the, the life of a neutrophil that was not on the battlefield. One little trick is that stain precipitation unfortunately is something you have to deal with.
There's no stain that doesn't cause it, so you have to be careful that like in this picture, it kind of looks like that this neutrophil is full of bacteria, but if you go to the, if you change the focal plane, you can actually see that there's a whole lot of this stained precipitate that's actually in a different focal plane. Stain precipitates will almost always be the same colour as the nuclei, like a purple colour, it can be. Dark or light, but bacteria, if you look on the picture on the right, it's a different colour.
So this here you can see it's like a dark blue, sometimes almost dark green. You need to get your eyes trained to the colour of a bacteria on your setup, your stain your microscope, and it should be a different colour than staying precipitate if you pay close attention to it. So just to finish up, I apologise that we went over, but I hope we still have time to, for the questions.
When you Send stuff to the laboratory, formalin fumes will destroy cytology slides or blood smears. Any formalin jar that was opened close to the slide will create this green hue where the cells just cannot be examined anymore. So you have to make sure that formalin is kept away from cytologies don't have any formalin open when you do cytology or blood smears.
And if you send them to the lab, you send a biopsy to the lab with, with cytology, make sure you double triple bag them so that there's No contamin even a closed jar will give enough fumes that will destroy the cytology slide. And then one last thing that people keep putting slides into the fridge, and those of you with glasses will know what happens when you take, when a cold glass goes into warm air. Water will condense on it.
Air drying the slide perfectly preserves it, but if you put water on there, that will lie the sauce. So you don't need to put slides into the fridge, so I would try to avoid that. Now, this is a couple of the pictures that are great for cytology, and this is something that's very useful if you want to do cytology to have one of these references next to your microscope.
And with that I'm more I'm very open to any questions I would be happy to answer any of your questions. Thank you very much for your attention. And I hope you found this practical enough that or that you can take home and and use in your work as soon as tomorrow.
Thanks very much. I don't think there's anybody that's attended that hasn't learned at least one thing and probably a half dozen. As Anthony always says, if we were in an auditorium, you would be hearing thunderous applause now because that really was an amazing talk.
So thank you for that. There are a few questions that have come through. I think your presentation was brilliant so you answered most of the questions as we.
But there are a couple, and there's a real hot potato for you to start with here. And it says if an FNA sample was sent to the lab and comes back as non-diagnostic because the sample was badly taken, do you refund the client the cost of the lab fee? I think obviously gonna depend from laboratory to laboratory and it's a commercial competitive environment, so you may find some labs to be lenient on it.
I mean, we are a university lab and if you actually think about it, when you send in a non-diagnostic slide, let's say if there's nothing on the glass because there's not even a single cells on it. I would be probably a bit more, lenient, but if you send me a really bloody slide that I had to spend like half an hour looking through and try to find if there's any cells on it, I would be very, less likely to kind of give a discount on because we still have to process those slides if you just think from our perspective, you know, or technicians have to book it in, they have to stain it, we have to kind of give it to the pathologist. I will spend my time on it.
I have to write that report to you. You have to spend it. So we still work quite a bit, not as much necessary as if it had lots of things to look at it, and I need to write a detailed report.
But again, some of the non-diagnostic report slides, we, especially here at the college, you know, we, we try to help as much as we can our clinicians and, and we sometimes spend a lot of time trying. To give something at least. So of course, if I was in a very busy commercial lab and I see that the 10X or 4x the slide is kind of poor, then I probably don't have the time to spend on it.
But so you will find potential different ways of how commercially competing companies deal with that or sometimes maybe they let you just resubmit. I, I wouldn't be surprised if some companies do that. I think that's a, I think that's a good point and I think there's a lot of practises and practise managers and practise principals out there that would have a fit if you started refunding fees to clients and that.
But I think besides a good moral standpoint, offering a client, a redo and submitting it at no extra charge rather than refunding the original one can also be a really great compromise. That's just my opinion. But I think just one more point though that I think it's always very important that as a veterinarian who's facing a client, I think it's always important to kind of make their expectation realistic and already tell them that, you know, cytology is great, inexpensive, very little invasive tool.
But sometimes we did not get the cells that we want. So, you know, when that result come back and it's a genuine, so it's, it's not because somebody doesn't know how to do it, but if you know how to do it, you follow these tips and you still didn't get a good sample, sometimes the lesion is like that. I had this sometimes that, for example, the whole leg of the dog feels like a huge tumour and it may just really like, such an edoema that feels like a hard tumour, but of course you put a needle in there, you don't get anything out of it.
So, so I, I think you kind of need to have the clients prepared for that potentially you don't get the answer that, and, and I think if they accept that, then you can go ahead with the using cytology. I think I would prepare the clients for that. Absolutely, that's a great point creating the expectation because otherwise it sounds afterwards like you're making excuses.
Exactly, yeah. Yeah. Excellent.
Jill wants to know, and this one came in when you were talking about blotting tissues and blotting the stains and everything else. She says, what do you blot with? I mean, we in the lab, we have a lot of chem wipes, you know, these little white paper, but, but, to blot the, you can actually use the hand, you know, if you have like where you wash your hands and you have those papers that come out of, to dry your hands, that's more than good enough for blotting tissues, or, to blot the stain off from the, the slides.
I mean, if you're An operating theatre when you're doing this, then you could use gauze, for example, but anything, it, it depends if you, if you're in a sterile environment or not, but, it's, as long as it's not dirty, like it's a clean paper, it's, it's fine. It's just something needs to be absorbed in paper. So like the one you use to dry your hands or in the laboratory, we have these skin wipes that are kind of absorbed in paper.
Excellent. Balash, I can't tell you how many comments we're getting coming through, saying thank you, lovely webinar, hugely practical. I've learned so much.
Awesome talk. And then, the one that, that keeps repeating, which I personally would like to, to bring to the fore and Matt, you should be listening in the background. Feedback into the the company tomorrow please is when are we gonna have the next webinar with the tips on cell recognition?
Because I think tonight was so awesome. We have all learned so much. The next step obviously is once we get the samples, the enjoyment of actually starting to read them ourselves instead of just sending them off.
Absolutely, I mean, I would be more than happy if, if the if, if, if, if there's interest in it, then I, I definitely, I, I do these talks quite a bit. So, so I have kind of similarly set up talks, but, but I hope people understood that to me after, you know, when I was young and I started to teach this and I was, I would go into the great depth of the detail of the cells and with the years of teaching this, I really realised that until you have these things right, there's really no point trying to talk about. So this is why I wanted to kind of start more with this very basic foundation that That, that then helps to start looking in the right area, which is stained well, which is a good preparation.
So then I'm, I'm absolutely more than happy to then kind of discuss the whole cytology, you know, what an inflammatory lesion, what a neoplastic, how do we go about examining those or for example, lymph nodes is a specific topic that again, I could be an entire seminar just to go through the. Lymph nodes and we could do even cases with poll questions. So I have a lot of those kind of materials that I've done in the past.
So if, if there's interest, I would be more than happy. If people find it useful, I would be more than happy to, to do that. There is definitely strong interest by the comments that are coming through.
So Matt, get on to it. Let's tie Balash up for at least one more but preferably more than that webinar. Balash, unfortunately we have run out of time.
We have run over tonight, guys. I do apologise. Nothing to apologise for a good sign for me is that not one attendee has left.
So even though we've run over, everybody has hung on every word you've said and I know I personally enjoyed it and I have learned a lot. So Balash on behalf of all of our members and all the attendees tonight, thank you very much for your time and excellent teaching. You really are a great speaker.
Thank you very much and thanks for everybody's attention and at this late of the night, especially in the UK so I much appreciate that. No, that's great. To the members tonight, thank you for attending and we will feed back to the office and we will certainly tie Balash up for some more webinars going forward.
From my side, I just left to say good night and have a good evening.