Thank you very much for the kind introduction. Can everybody hear me and see my image on my screen? Is it working?
Yeah, that looks good to me. Excellent. So thank you very much.
It's a great pleasure, for me to talk about these topics and thanks for the kind invite, to present. I always like, I, I love to talk about haematology, but actually this topic has now to me become a bit more important because, Practically in the UK there has been a massive push to to have haematology analyzers and probably 80+% of the practises now have one. And what, on the other hand, what I'm seeing a little bit though is that how to really use those machines is, is much less known.
So probably anybody knows how to, to work with an X-ray machine, but when it comes to the haematology analyzer, I do see quite a varied experience and when I run my online course on haematology I told you here at the college and we have discussion forums. This is a very interesting topic that some people just blindly believe every number that comes out of this machine. Others have been kind of either burned or they see all the asterisks and then they just kind of stop trusting the machine at all.
And, and I don't think neither of those is a good approach. So unfortunately, I think what's important is That you kind of have to start to understand a little bit what this machine really does, what is it we can't trust, and what is it we shouldn't trust. So, so I'd like to kind of give you a couple of the of these topics that if you're aware of it, you will start to understand a bit better when the machine is throwing, for example, wrong results at you or or you have a better understanding of what you can trust.
But also we're gonna have to come to a conclusion that actually to use one of these haematology analyzers is practically not possible without at least a 2-3 minute quick blood smear exam. So I'm, my goal is to give you some really practical tips of how to do this quickly and efficiently and, and just kind of highlight them later on with the platelets, red cell, white cells, and some of the things that what you could look for even in this very short period of time. So now in terms of samples, one of the biggest disadvantages of sending a blood out to a big reference laboratory like ours, the commercial ones is that there will be changes in that sample and one is very typical that the red cells will continuously swell with time.
So if these red cells become larger, the machine measures the average size of the red cells that you probably all remember it's called the MC. And this is going to falsely increase. Now, if you think of a hematocrit 2, how tall your column of red cells going to be, i.e., how high is your PCV or hematocrit, is going to be intuitively a question of how many red cells you have packed in there, but also how big they are.
So if they're all swollen up, this hematocrit or PCV will actually falsely increase. The machine actually calculate. It's this from basically multiplying the red cell numbers with the average size, the MCV, or with the XL volume with the PCV to obviously we do this manually.
But either way, this will be, it can be a significant like a 3 day holiday, you could literally wipe out an anaemia like a PC or 38 and a dollar 37 by Tuesday morning when we look at the book could be 4142. So this is something that can be significant. Now, also organisms can fall off of red cells in 8 samples.
So for example, mycoplasma is famous for this because that's on the surface of the red cells. So a fresh smear, you can see it, by the time the sample comes to the laboratory, they all fallen off, it's gonna be in the background, mixed up the same precipit and very hard to recognise that. Another very big issue is that we struggle a lot is that the leukocyte morphology, especially neutrophils, which you pay a lot of attention to, they don't like to sit in that fluid and after a few, even a few hours, they start to have like a fake foamy cytoplasm, fake doy bodies that start to look like toxicity, which is a very, very, very big issue.
The nucleus starts kind of swelling up and starts looking like a band or even like a metamylocyte, but actually if you look at the fresh smear, these neutrophil look perfectly happy and really nice and, and had no signs of toxicity. So if you ever work with an outside laboratory, I cannot beg you enough to always make a fresh blood smear and try to send it along with your EDTA sample. This will really be a benefit for both sides.
We will be able to give you better information. Of course, you're gonna get better information. And you don't have to fix it or or do anything other than just dry it with the air and preferably with no heat, no hair dryer.
This will really nicely preserve the white cells and the red blood cell morphology. And the only other little caveat is that you probably want to store your the blood tube in the fridge, but unfortunately glass doesn't work well with cold and then coming into warm rooms. So those of you glasses know what happens, water will condense on it when you take it out of the fridge.
And then that will lie your red cells. But other than that, you don't really have to worry too much about any hair dried blood smear, just send, please, please, please do send it along with your EDTA sample. Now, in terms of the actual analysis, whether you do it in-house or whether you do it.
Of course we have the some kind of automated analyzer. It could be a small one, it could be a big one, but as I already mentioned, I cannot emphasise, you know, how critically important is to actually do look in the microscope and, and look at the blood smear, so we have an idea of whether the machine is doing the right thing or was it confused by something. Now, in terms of talking about confusion, probably the most important confusion is with platelets and, and, and, and as I mentioned, why this topic to me become more and more important because sadly here at the, for example, the vet College, we do see now patients being referred to for very simple machine errors, such as like the platelet counting.
And, and it is just a bit heartbreaking to see people, you know, driving all the way up here, spending a day here and then we just send it through the blood and then tell them that there was nothing. Wrong with the dog. So, and, and this now become actually not infrequent that just very simple machine errors end up being kind of over exaggerated and, and, and patients being treated for them or or referred for them.
So, by far the killers of most haematology analyzer is going to be the platelets and actually realistically, most of these analyzers are glorified particle counters, so they have absolutely no idea what a platelet or what a red. So what they do is they see particles. So in an ideal situation, what you see is a lot of small particles and then you see big particles.
So if you actually kind of plot this in a graph where the x-axis is the volume of the particle or the size of the particle, the Y-axis is the number, ideally what you want to see is a small blip here. These are the small particles and then you can't see anything in between and then the big particles come, which is gonna be the red blood cells. This is an enlargement of that.
Small area. Now this is relatively common in a dog, but for example, if you look at a cat, what the machine will see is we'll see a very small particle and a bit larger one and a larger one and a bit bigger and bigger one, and then all of a sudden now we're in the exact same area of the large particles. So when you plot this, what you see is just a continuum of sizes of particles and even on the enlargement, there is absolutely no valley in between those two.
So, so the machine will have no choice, but it has Cut it somewhere and then it will call anything left of that a platelet and anything right of it a red blood cell. So you can see that in cats, for example, this can definitely cause issues. Now, on the other hand, if you look at the blood smear and you can tell that that's a platelet and that's a red blood cell, then you can actually estimate the number of platelets from the smear more reliably than what the machine was doing with these animals like horses, sheep or sheep and as well, where the red cells are very small and cats.
Now, so it's so many you can can always important to just confirm what the machine was doing. And if you actually start counting platelets as red blood cells, which is the more common, instead of red cells being counted as platelets, then you start to drive the MCV to the size value again down so that will kind of give you a false calculations as well. Now, Probably the number one problem, and, and we may see a bit bit more here at the college because a lot of students are trying to take or or learning how to take the blood samples, but, but here at the college, at least 98 out of 100 samples we got has platelet clumping.
And of course, the platelet's job is to, to plug up the hole. So when we activate them, all they know is they want to adhere to each other so that they can actually plug up the hole that to prevent blood loss. So, so they have no idea whether they're in the circulation or they're in a tube.
If they've been activated, they're gonna start to to adhere to each other and form these what platelet plugs, the primary hemostasis in a blood vessel. Now, when a machine wants to actually measure the platelet concentration, intuitively you probably will all agree with me that what's very important that those particles have to be uniformly distributed in that tube of fluid. So when the machine comes in and takes the 100, 150 microliter or even less sample, what you want is that that's very representative, so the distribution in that should be the same as in the tooth.
And look what happens if you have big clumps. So the machine comes in, takes a sample, and there's going to be a lot less platelets in that sample because these platelets are all in these big clumps and arguably, if a big clump is within that little sample window, guess what? It's going to be a big particle, so the machine is gonna count it as a red blood cell.
So anytime you have clumping, the machine will always, always falsely Decrease device. It will underestimate the platelets. Now why is that little trivia important?
So we can use the number, but we can only use it as a minimum estimate. So whatever the machine, if we detect platelet clamping, I'll talk about that in a minute, from that point on, whatever the machine measures, it's going to be a minimum estimate. So we don't know how much more platelet we have, but we can use that number as a minimum estimate.
Now in terms of how do we know if the platelets are activated, again, it's vital to have a quick look on the, the blood smear and the fat of that is a nice area where we can relatively quickly scan for, for the anything that's large will ship out there so platelet comes easy. You can find there. And then this large man you can see that this is the very, very end of the smear.
There's a large platelet club here where you can actually see that this little purple blob is made up of individual platelets. It's important to spot that these are the individual platelets in there. So, now one thing that you can actually do is you can do a manual estimate.
And this is a, it's a kind of a bit of a cool topic that if you look, think of a blood smear, I think about it just for a second, how in the earth is it possible that I'm gonna look down a microscope and I look at an image, just, you know, like a painting on the wall. And I'm gonna start estimating the platelet concentration. Now, any of you who know very old, you know what the word concentration means.
It means basically the number of particles per volume of fluid. So how can I look at a painting and talk about particles per volume of fluid. So the concept is here that if you actually look at these bloods here and magically turn it to its side, this is What it's going to look like.
This is where we put the blood drop. And as we smeared it out, it becomes thinner and thinner and thinner and thinner. So when I actually use the microscope and I use the 100 X, the highest magnification, and I looked down the microscope, actually, what I'm looking at is a small column of fluid.
So, so, and I'm going to be actually counting particles within that small column of fluid. Nobody knows what really the volume of this little column of fluid is, but people worked out that one platelet in this field is about 15 to 20 times 109 per litre. Now, the one very important aspect of this though that look what happens if you go to the wrong area of the smear.
So if my smear is twice as thick, and, you have a relatively uniform distribution of platelets, then my column of fluid will be twice as tall, so odds are you're gonna have twice as many platelets in there. So if you kind of do your estimate in the wrong area of the smear, in a thicker area of the smear, you're gonna get an overestimation of your platelets, arguably, if you come too close to the fat edge, then you will underestimate it. So it's very, very important that we do this always in the proper monolayer and The monolayer from smear to smear to smear should have relatively uniform thickness.
So this is why this method can actually work even though we kind of looks like that we're looking at a 2D image. So I'm going to talk about it in a minute of how to. Become an expert of quickly finding a monolayer, but this is very important that you always have to strive to do this in the monolayer, which with a normal PCV that could be literally 100 X or field.
So it's not necessarily as wide as many people think. And, and now, of course, if, if especially if you have very low platelet count, and you know, I look at one field and I had one platelet, and then I look at the next one I had 4, and then look at one has 0 and then the next one has 5 course which should I use? All best is to kind of every out and with low numbers, we would always average out at least 10 fields.
If you have normalish numbers. And you count it 2 or 3 fields, that takes literally 10 seconds, and that's enough to, to get a quick idea of what the platelets are doing. But with low counts where you worry about cytopenia, we would do at least 10 fields every amount, and then we multiply with 20.
That's a very easy number to remember, so just, multiply with 20 and that's it. So, here's kind of a question just to, I want you to think with me for a second. Should I do a manual estimate if I have something?
Is there a point to do this? And of course, I cannot really see you guys answering this now, but, but, but, but the, the question is that, is it gonna be reliable? So, Now, if I go into that tube instead of the metal needle of the machine, and I'm going in with my little capillary tube and I take a drop out from that tube where I showed you that there was no uniform distribution of the particles, then in my one drop, it won't be either.
So when I put that on the blood smear and I smear it out, I'm not gonna have an an adequate distribution of the particles. So this It's not the fault of the fault of the machine, it's the fault of the sample. So, so when I make that smear, I won't be able to do an actually an accurate estimate.
Of course, estimation is, is, is an estimation, but it's not going to be accurate. Guess what? It's going to be another minimum estimate.
So I will underestimate the platelet, but Let's say if my machine gave me a number of, let's say 10, I looked on the blood smear, I still pumping, so, OK, I know that I have more than 10, but, you know, 20 is more than 10, versus I want a blood smear and I look at it and I think, OK, my minimum estimate is now 100. Now from a laboratory analytical perspective, both of these numbers are complete rubbish, but clinically, knowing That you have more than 100 or more than 150 is definitely much more valuable than knowing that, OK, I have more than 10, but I don't know how much more. So actually, even though I'm ending up with a completely erroneous value, again, we very commonly will do a manual count on the platelet or on the smear, hoping that we can give a higher minimum estimate.
I hope that concept makes sense. Yeah. So, now, in terms of other problems like hemolysis, it's relatively intuitive that if you like the red cells, then and then I'm trying to count those particles, the machine, of course, is not gonna be able to count it, so I'm gonna have a a false decrease of my red cell concentration.
Excuse me, and then of course, when we calculate the hematorate of how many red cells we had and how big they are, that's not going to be counted and also in the PCV tube, those light cells are not going to take up space. So all of this is going to be falsely decreased. Now when we start using these calculated values such as how much haemoglobin we have per red blood cells or how what's the concentration of haemoglobin in an average red blood cells, of course, the red cells are missing from there.
So we will have the haemoglobin but not the cells, so we're gonna be overestimating and we're gonna get false increases of these values. Now, haemoglobin is a tricky topic, and just a little bit of the trivia most machines measure haemoglobin by basically lying all the red blood cells. So first they can them and then they lice them all, liberate the haemoglobin, and we measure it optically in the spectrophotometer.
So if you have in vitro hemolysis, in other words, your sample got a bit frozen or, or it was shaken too much while it was taken to the lab and utilised half of the red blood cells, you know, that's gonna be a problem because none of these values that I have high up on the slide. Will be accurate, but that haemoglobin will be in the tube, so my machine will just slice the other half of the red blood cells, and I have all the hemoglobins. So actually, I'm gonna get a reliable value, and that's going to be the only reliable value basically in a hemolyzed sample.
On the other hand, if you have in vivo hemolysis, that's a bit trickier because basically in your tube, you're gonna have haemoglobin from the cells that were lied in the circulation, and we're gonna have hemoglobins that brought in with the red blood cells. So Ilyze those red cells, liberate the haemoglobin, but there's gonna be actually more. Glow in then what would be expected for the number of red cells.
So it's gonna be an accurate measurement in terms of the actual haemoglobin concentration, but clinically it's not gonna be accurate because it's gonna be kind of overestimating the plate or the, the, the hemato page for us. Now agglutination is another very common problem with IMHA with the immune hemolytic media hemolytic anaemia, which is one of the probably more common hemolytic or or or diseases of the hemolymphatic system where you think you want to have a haematology analyzer. Now, the analyzers basically, as I mentioned earlier, can particles, which means dilute the blood and a single particle goes to an aperture.
And it counts them and then the machine will know how big those particles are. That's where the MCV comes in. But guess what happens?
So if, if 6 or 10 red cells are stuck together and they go through the aperture as one, the machine will count it as a very large red blood cell, and instead of counting 10, it will only count 1. So you can immediately intuitively work it out that the red blood cell concentration is gonna fall to decrease. My MCV, the average size of the cell.
It's going to falsely increase. So funnily, when we take a hematocrit, I'm gonna multiply a completely erroneous MCV with a completely erroneous RBC concentration, but because these two errors are in the opposite direction, many times they kind of somewhat cancel each other out. So the hematocrit sometimes will actually be not far off from the true value, but it's a basically complete wrong number or or it's a multiplication of two completely wrong numbers.
So we generally recommend using PCV. If you have really marked glutination, you may not be able to pack them down as tightly as you should, but overall, pretty much in any blood that's agglutinating, PCV is gonna be your best bet, and most of the other value is gonna be all falsely affected. Now this is one just a little side note that we always love to do absolute ridiculous side concentration instead of the corrected or the percentages, but this is one time when you kind of have to go more with the corrected one because the RBC concentration that we use is going to be falsely decreased.
I'll come back to this in a second. And in terms of the errors like as I mentioned, we measure haemoglobin optically, so if there's any kind of optical. Obstruction, then that will give us a false increase of the haemoglobin.
So the most common would be leukaemia. But if you have a lot of nuclear red blood cells, a lot of Heinz bodies, those can still float after the lysis of the red cells happen. So they will block the light, so the machine will think that it's haemoglobin that's giving that opacity, so we're gonna get a falsely increased value and of course then the numbers we calculate from haemoglobin such as MC MCHC will be erroneous as well.
So what I would really recommend is that it's always useful to do a a on PCV even if you have a fancy haematology analyzer, because you can compare the hematically to the PCV so that can already rule out some of the issues. And also you're gonna get a the you're gonna get to see the colour of the plasma that can be very informative as well. When the patient has too low sodium or too high sodium, the red cells get accustomed to that, and you put them in a machine with an isotonic fluid, those cells that actually start swell up or or shrink down.
So then you're gonna get again false false values, so the PCV in that context should be more reliable. Or if you have actually a very short sample, it's a tiny cat, you only get a few drops, the salt, the EDT salt will actually suck the fluid out of those red cells. So the PCV is gonna be actually.
Falsely decrease. Funny enough, if you put that sample into the machine, those red cells will regain their volume when there's lots of sheet fluid in the machine. And actually that's the only time when I will actually trust the hematocrit more than than the PCV.
So that's something that you can do that every time you run haematology, ask your techs or, or nurses to, to, to run a PCV because that will help you. And, and one little trick is that I mentioned that, you know, sometimes it's only the haemoglobin value that should be reliable. The haemoglobin hemato ratio should be basically 1 to 3.
So if you have a haemoglobin value of 15, then you can pretty much bet that the PC or hematoca is going to be 45. So that's kind of a good way to, we, we veinarians, we love thinking in hematocrits versus all the human doctors, they always talk about haemoglobin, but so if you want to convert the haemoglobin to hematocrit, just multiply it by 3. Now, lastly, in terms of the machine errors, the white blood cell concentration is probably one of the most robust aspects of the machine that I like if I had to work with the haematology analyzers that that in practise, probably the low white cell count would be where I would really want to have an analyzer because that's the one that's difficult to reliably estimate from the smear.
And, and overall, the total white cell concentration is can only be food binucleate red blood cells, which again, you're never gonna know unless you have a quick peek on the blood smear. Conventionally we count them as per 100 white cells, so we count them separately while I do the 100 white cell differential. So, and then we can actually correct the white cell concentration because basically, so for example, if my white cell concentration was 14 and I had about 40 nuclear red.
Per 100, that means out of 140 cells, only 100 is a white cell. So 100/140 is the ratio that we need to multiply the 14 with, and then we're going to get a correction of the value. So if it's below, if your nuclear reds is less than 10 or 5 per 100, then I wouldn't worry about this, but sometimes you see that actually there's more nuclear red blood cells than white blood cells.
So in those cases, you could get a significant error from the machine. So, so with all that said, these are some of the more common problems that the machines deal with, and actually most of these that I listed were not the machine's fault. So these were all the samples fault.
In terms of the machine fault, it's, it's a whole other topic that I certainly don't have time to go into, but the quality control. All of it is a big issue, and that's when they sell the machine, they don't really sell you the expense and the know how and how to do it. But, but that's something that keeps the machine running, adequately versus these problems that are listed these problems in the sample.
Now, if you come to the conclusion, OK, have a, a quick smear, look at the the blood smear quickly to, to just check what the machine was doing, then you can very quickly realise that a good quality blood smear is essential and these are just kind of my . Favourite collection and my most favourite is this artistic one, but none of these are useful. Like these top two needs like an X-ray machine, not a microscope to look through them.
Here they made lots of little pitstops for them, basically you don't have a nice uniform monolayer and this made it actually starts to shape up quite well, almost the bullet shape, but then see how the blood shoots in front of it. This is what happens if . Somebody's too tentative and for a millisecond they lift the glass or tilt it a bit and then blood gets in front of it.
And instead of pulling it with capillary reaction, you're basically smashing through those red cells. So again, that disrupts the smear. So a nice quality blood smear should look something like this.
It doesn't need to be long, or some of the automated machines don't even stain all the way, so it's ideal just to have it in the middle. And what's very important is that you want to have a nice distance from the side. Notice how actually these glasses even have these cut corners.
So, so, so basically you have to prevent the blood spreading to these inner edges, of the smear, . Sorry, apologies. So, when you come into the blood draw, it's actually important to just go through it as a squeegee.
Like sometimes people get really tentative and just stop at the very front of it. Then many times you end up just using a little bit of it, not the whole drop, which adds another layer of variability. So, but if you kind of go through it, then.
And let it, leave it spreading along this edge, but you have to start the smearing before it goes all the way out to the edges because that's when the white cells prove their momentum, they go out there and then white cells will all end up on the foetid edge and not in the monolayer. So again, then that's, unfortunately makes the blood smear less usable. And also notice how nice and uniform this smear is, and then we have the nice fe there that we're gonna examine.
So here is a video or, or I'll show you quickly two videos of how to do it. It's very useful to smear away from us. Sometimes people like to put their fingers in here, which is OK, but it's very important not to stop the smearing.
The smearing action has to be smooth and kind of off the glass to make sure that you have a nice smooth end of the smear. Notice how it's important to keep about a 45. 5 degree angle for the spreading.
It's very common that people just kind of, lower their wrist and then even if they start at 45 degrees, they will end up smearing at a very low angle. That creates a long, thin smear that again is not great. So, so we come into the drop, let it spread.
Now, how high the PCV will have an effect. If it's a very high PCV, it will be a very, very slow spread. If it's very anaemic, it's going to have a very quick, spread.
So you have to make sure you start the smearing before that. So, notice how small, how a small amount of blood I'm using. This is critical, like that's probably the most common problem that people just use way too much blood and, and that, is, is, is an issue.
So a microhematic cap tube, like the long one is the best to allocate just a very small amount and then just one nice. With motion, with the hand. Now, another method that actually is my favourite, I'm right-handed, so I hold the spreader slide in my right hand, and then the bottom slide is in my left hand.
I put the drop, down here. I can actually practise a little bit with the glass, so I put this, again, very small amount of blood. And then I can kind of see how the glass spreads on the or or slides, then I'll go into the job and then I'll smear it towards myself again, .
So again, just a small amount of blood, keeping that 45 degree angle. I have my thumb on the frosted bed and then my pointing finger at the edge of the smear, and then I just smear it and I make sure I hit my finger, to, to have that smoothness of the smear. So, if we have a nice uniform blood smear, then in the next little section, what I want to share with you is is kind of a very systematic quick approach to how to examine a blood smear very efficiently, very quickly.
So just like when you were taught to look at a radiograph and you kind of always do it in the same order, I think that's very useful for the blood smear as well, so it makes you less likely to forget about anything and then just helps you to get through it very quickly, systematically. So when I put a blood smear on the microscope, historically, most of us would put the fat dash toward my left hand. So when I looked down the scope, it's at 3 o'clock is the thin bit of the smear and 9 o'clock, towards 9 o'clock is the thick bit.
So, while I'm going to be heading up to this top corner, at very low magnification, then we already start looking at the distribution. Is it nice and uniform or is there a lot of agglutination, is a lot of clumping, and then on the feather edge we're going to look for the platelet clumps. So this is important, as I mentioned, the particle distribution has to be nice and uniform for anything to be reliable when we estimate things or when we analyse it.
Then we're gonna go through the feathered edge and this is basically about a 10 or 22nd, 32nd maximum. With a bit of practise, you should be able to do this at a 10x magnification and just one slow, continuous motion across the feathered edge. This is where we're gonna find the platelet clos, but anything that's abnormal low.
Large that will shift out here. So it's a really nice way to concentrate potentially abnormal cells. Cells do get damaged, so we don't use this area for morphology, but we use this for picking up potentially abnormal things.
Mast cells is something that we can commonly find potentially if they're present in the side of that. And then we need to find a monolayer which is where we're gonna have the best cell morphology. This is where we do a differential by the zigzagging kind of pattern to avoid recounting the same cells.
And this is where, this is probably the most important aspect of being able to be good at finding monolayer quickly. Most people, if I look at when they're looking at a blood smear, they're not in the right area, and then you just make your life more difficult. So you're going to be efficient, spend your time in the right monolayer.
And also, I always do it in the same order. So I always look at platelets, then red cells, and white cells. If you don't, if you have a normal microscope with a 40 dry lens, I would probably look at the white cells first with that oil, and then I would go put oil on the slide, and I would look at the platelets and the red blood cells.
It doesn't matter what order you do, but if you always do it in the same order, you're less likely to forget about one or the other. It's actually quite easy to just jump on some nice. Red or white cell abnormalities and that actually completely miss the fact that there is not a single platelet on the smear.
So that's why I always look at that first. Now, in terms of how to orient on the smear, and how to be an expert of finding the monolayer, notice how, if I go back for a second, this was the very end of the smear. So you see this fettering, we call it the feathered edge, but as I start moving a bit more inwards, you see how the pattern of the red cells starts to change.
And first they start to kind of coalesce and form these little islands of red cells and then those islands start to kind of coalesce into each other and as you go in and in, eventually instead of the little red cell island, but you're gonna end up with little white bubbles between the red cells and then as you keep going in, there comes a point where Some of the cells start touching each other, but they're very nicely and uniformly distributed throughout, your viewing field. And as you go further and further in, then you're going to start to have the red cells piling. If you remember that cross section I showed them the blood smear, the more we go left or to the thicker part, the more, depth it's gonna have thickness it will have, so the red cells start piling.
On each other. Now, if I stop you for a second and you looked at this picture from the monolayer where we have really nice morphology, 10-20% of the cells you touch each other, but only couples. You don't want to have 34, 5s touching each other.
If you look at the red cells here versus on these two pictures, the question is, can you spot two key differences? There's two very important difference in the appearance of the red blood cell. So, I pause for a second, but if you haven't spot it, notice how none of the cells here have central pallor versus in the monolayer, we have really nice central pallor.
This is very true for dogs, but sometimes in cats we do see it, even though we teach that cats less likely to have central pallor. And another thing is that if you look at these cells, they're very angular, they kind of mould into each other. Versus when you come to the monolayer, they stop that moulding and they more like overlap each other.
So, so basically, and then of course in the thick part they they kind of there's too many piling on each other. So if you follow these few bits of tips, you could actually look down any microscope and without even touching it, you could tell where you are. Are on a bloods.
Now the bloodst needs to be high quality for this to work well, but, but, but just based on the appearance, if you see the central pallor and there's no angulation, then you're in the monolayers. There's not too much piling. If they're piling, then it's too thick.
If you start to see the angulation and the disappearance of the central pallor, then you're getting too thin, you're too close to the centred. Unfortunately, there is no line on this. There's a, there's a grey zone between these two where the central pallor starts to come in and let's say half the cells have central pallor, the other half don't.
That's a dangerous area because any small cell or a cell that's relatively small and happens not to have central pallor, then people call them spherocytes. So you need to make sure that you properly come into the monolayer and this. This is where the cells that we use the fried egg versus the boiled egg analogy, the white cells will look really nice and flattened out, so you see the detail of them, versus if you go to the thick areas, the cells bow up and so you see much less detail and it's much more difficult for the untrained eye than to see the even recognise the cells, but especially to see the details, for example, of the cytoplasm.
So I strongly recommend to pay attention the next time you have a nice smear, just kind of go to the federal agent and start coming in and you will see this change of appearance of the red cells and then become an expert of where the monolayer. So the quicker you go there, the more efficient you're gonna be. Notice how the, the distribution or the, the spacing between the red cells actually is going to be directly proportional to your PCV.
So for us and for the For exam, you have to practise that I can look at this and say that looks like 25 to 30 or is it 40 to 45. You don't need to know that, but paying attention to the spacing is nice because that immediately trigger, oh, there's quite a bit of spacing. Oh, that's anaemia.
So then I'm going to start looking for regeneration and so on. So that's my little sermon on orientation, but then of course, this is when it comes to real life. So if you have terrible quality blood smears, There's not going to be any kind of monolayer or that you can examine.
Here's a smear that was one of those very flat angle where they just basically the whole smear is the fact that. Not that pattern that I showed you that should be at the very end of the smear, it just really goes all the way through to the back of the smear. So then you cannot examine and remember for those platelet estimations, we need to have that monolayer for it to be reliable.
Now, just one very quick word that the staining is, is a very common issue. People, not using the correctly or you have all these, for example, water artefacts, or if you didn't dry the blood smear enough or you have the alcohol jar was not closed or replaced enough, then it attracts water. So then of course it's next impossible to to look at your your cells if you're not doing the staining well.
And in terms of the microscope, another very common problem is not having the microscope set up correctly. For cytology or haematology, we need to have the condenser high up. And even on the condenser, there's usually a little lever or a dio that does the same thing as lowering the condenser.
So people, when they use the scope for urine, they usually need a lot of contrast to show the crystals and count the cells. But that High contrast will destroy the image. Now, I couldn't really take a good enough picture because the camera somehow just kept, compensating for it.
But when you have this high contrast setting, you have this really vibrant image where you just don't see the detail and you need a flat image to reliably examine the cells for cytology or blood test. So the condenser needs to go all the way to the top and just a little bit below that. So, now, if we have a good quality smear, we have a good quality staining, and we have a, well sort of microscope.
Those three pillars are extremely critical. I could have a whole hour just talking about those 3. But if you're in practise and you don't have massive experience, those 3 is something you want to make sure you get the, right, because That way you can make yourself in a better position to actually see the things you need to see.
If any of those fails, the quality of the smear, the staining, or the microscope set up, your job is going to be immediately more difficult and with less experience, you can just waste a lot of time and not get the right answer. So it's important to make sure you start at right. Now, as I said, the feed that is a very quick exam, and, and, and we can spot things that with a little bit of practise, worth practising by with 10Xs you should be able to spot even 3 or 4 or 5 platelets stuck together with a little bit of practise.
Things that can mimic them a little bit like stain precipitate. You can see in this picture. Or squam.
So this is, for example, from just going through the, the skin to with the needle to take the blood. These big squams end up in the foetid edge, but they're very angular and thin and blue turquoise colour instead of the actual platelet lumb that I showed you where you can see the individual platelets within. Mast cells is something that it's not uncommon that we pick them up on the feathered edge and in cats that's almost always neoplasia, so it's important that the spleen is full of mast cells.
In cats and dogs is the opposite. It's, it's hardly ever neoplasia, but, but again, the feathered edge is a good place to spot them and organisms sometimes because they get laid large, they will shift out to the feathered edge or parasitized cells. So for example, there is a more like monocyte.
Or, or even the red cells with organisms will end up being a bit more on the feed just because they're heavier. So, so that's again, that's a very quick examination of the feathered edge. It takes about 1020 seconds maximum, and we look for the platelet counts and then we're going to start moving in to the monolayer.
So then, as I said, I'd like to look at platelets first and then red cells, white cells, but again, whatever order you do, up to you. Platelets, we actually don't have to worry too much about things as long as we validated the concentration that's very important. And of course we look for low numbers or high numbers, and there are relatively few morphologic abnormalities.
We do look for giant ones, if they're as big as a red cell, then we call them large platelets, that meant to be an increased production. This is a super giant one. And then, also these elongated forms, we call them proplatelets.
That's again, meant to be, an, an increased production, but that doesn't mean it's tromocytopenic or it could be at a normal count if the marrow is being, triggered to produce more platelets. And that's kind of it. So with platelets, pretty much the numbers and looking.
For the platelet com. So again, if I go into, I looked at the feathered edge, go into the monolayer, and the platelets to me is going to be I count 2 or 3 fields. I had 2022, 25, so that's a multiply the average about 20 times 20, so that's a 400 platelet count, that's it, and I'm moving on.
So, and then I'm gonna look at red cells. Of course, most of the times we, we worry about anaemia, but less commonly we could look at too many of the good things if you have erythrocytosis, and looking for signs of regeneration, I think is a very useful tip with a bit of practise. This takes like 3 seconds, 5 seconds to tell if an anaemia is regenerative or nondegenerative.
I'll show you pictures in a second. And then I want to share a few of the more specific morphologies that actually can trigger a specific kind of diagnosis for you. So in terms of regeneration, the most important aspect is the polychromatophys.
You see these larger cells, or it doesn't have to be larger, but the cells that have a purplish colour, they still have the cell machinery. Produce haemoglobin, even the smallest tinge counts. These circulate for about 24 hours as such and then become mature cells.
It's a really good indicator of how many young cells are coming into the circulation. If they have a tiny bit of colour, that means they've probably been there for 23 hours. If they're really dark like this one, then it's probably just came into the circulation.
Notice how, for example, probably this would be the normal red cells, so there's a lot of size variation, what we call anisocytosis. And we have these large, young red cells are still large, and that's the macrocytosis. You can have nuclear red blood cells that haven't extruded the nucleus yet, or we may have nuclear remnant how jolly bodies, increased number of these again tells us that the regeneration, and a very severe regeneration, you may have the what's called the basophilic stiffening.
So, so picking these up actually takes only a little bit of practise and then it's, it's a question of just seconds to do it. Now in terms of reticular size, unfortunately, the machine values can be really mixed up with different things. So, I don't necessarily recommend using them.
The classic method is, is using a new methylene blue stain. Actually, in practise, what you can do, instead of doing the proper thing where we mix it, what you can do is make an actual blood smear, dry it, and then put a drop of either new melene blue if you have, or Or actually just a drop of the blue stain from your death qua, put a cover slip on it, and it will really beautifully stain up everything and you can count the actual, reticular site in there very easily. We typically count, at least 500 but 1000 cells or technicians, but you don't probably have to do that just to get a quick idea.
You probably all learn about in school how you have to correct it. If you have, if you don't, if you're lacking all the mature cells, then the percentage will appear. It is much higher than the normal.
Nowadays we almost always use the absolute ridiculous high concentration where we basically take the percent, multiply with the red cell concentration, and if it's more than 60 or 80,000, then we think the anaemia is regenerative. Now, clinicians always like numbers and that's something you can actually track over the different days, but, but looking at a blood smear, is a, is a picture is a 1000 words. So to actually tell qualitatively that this regeneration, it's very easy on the smear, but you need the the reticular side if you want to have a a quantitative and a number.
On the other hand, if you look in the blood smear where there's no regeneration, where you're gonna have very uniform red cells, none of them have the purple colour, notice how there's a lot of spacing. This is the cats are probably a PCU about 1015, and there's not much going on. There's not much variation in the sizes.
All these cells. Now, in terms of the more specific morphologies, sphere sites and ghost cells are very useful to be able to recognise them, and, because these are hallmarks of, of IMHA. What's very important that if for example in this field.
Let's say that red cell is a normal red cell or maybe this one. Notice how the, excuse me, the spherocytes are appear much, much smaller and they need to be really dark and dense because you're looking through a lot of haemoglobin, it's not a disc like the normal red blood cells. And then those cells, they can be annoying because you can completely miss them and you see one and then all of a sudden you start to see all of them, but that's basically these cells lost the haemoglobin, holes are poked into it and then just literally the membrane left, so they kind of it's a really good name for, they look like those cells.
But, but again, both of these can tell you that there is an immediate destruction of the red cells. IMHA also very commonly goes hand in hand with the glutination, which is the red cells kind of form these grape-like clusters which you have to, differentiate from ruleloin where you have a stack of coins and that's an artefact. And you can do a rat mount where you put a, a very, very small amount of blood on the glass and then you flood it with saline.
It's OK to do a big dilution and, and we Actually then look under the microscope and this is what it's going to look like. So I just put this fat man is sitting in a microscope, I blow on it just with my mouth and then you can see these agglutinating cells just rolling all together and, and, and, and that way we can confirm that we have agglutination, which is pretty much almost always means immunit hemolytic anaemia versus rulo is more like a stack of coins. Now, another concept that we, we see sometimes that can be pathogonomic is the iron deficiency anaemia where you have the microcytosis.
And if I wanted to fool you, and I would ask you, is this a microcy or not, probably not all of you would think that that actually is a microcyte, but it is. And the concept here is that with iron deficiency, what we have is leptocytes, very, very thin cells. So if you look at this picture of the, thinking in three dimensions, not only two, a leptocyte, a thin cell can be the same diameter as a normal cell, but instead of having the full thickness, it's going to be very, very thin, which is why it looks so pale in the microscope.
So actually, even though the diameter is normal, but the volume of this cell is very, very small. So that is a microsy, which is the opposite for sphero side. The spheroy looks very, very small in the scope because it has a small diameter.
If you looked at it from the side, it is a sphere, so the actual volume of it is, is pretty much almost normal. And then the small kind of cells with normal thickness is more what we see with Akitas or Shibas, the Japanese dogs with born as such, not the iron deficiency type picture. Shear injury, if you have any kind of surface where the endothelium is not smooth, for example, hemangiosarcoma or vasculitis or bad heart valve, then you're gonna start to The damage to the cell.
This is an amazing picture where you have disseminated intravascular coagulation where fibrin strands are within the bloodstream. You can see how it literally cuts the red cell through, and the red cell memory is a lipid bilayer. It's like a soap bubble bursts or it may reform smaller bits, and then it will be in the circulation as schisto side like the fragment.
One very typical or nice looking feature that we can see, especially with heosarcomas are the acantoy, which will have these narrow base kind of blunted projections unevenly spaced around the red cell. Notice how the norm or the the typical crenated. Cells have either broad base or even if it's a narrow base, but kind of spike projections they get around the whole cell, versus the cant side will have these little tennis racket like a golf club type, it's more slender projections with a narrow base, and the more blunt end of that.
The oxidative damage, if you oxidise the haemoglobin, we form hinds bodies. These take a bit of practise or experience. It's very easy to completely miss them.
I've seen these being diagnosed like, for example, like onion toxicity or zinc, like when animals, swallow little coins. And I've seen people call this like IMA changes because they didn't look. The smear and carefully enough, but you can see the the hind's body is basically oxidised haemoglobin, that's almost the same colour but one not lighter than the red cells, and if they start sticking out of the cells, it's easier to to recognise them.
You can see them even freely in the background. So again, that's a very useful hallmark and the machines are not going to tell you that. If you oxidise the membrane of the red side instead of the haemoglobin, then you end up with this fusion of the two sides of the membrane and pushing the rest of the, the haemoglobin into a sphere.
And these are what we call eccentricytes. The problem with this. That this will tear away and then form what we call a picnicide, which is basically a spherical object.
So a lot of people misidentified it as a sphero side, but they're usually not as perfectly round and you may see a little tag of what's left from this membrane. And then lastly, you can pick up some organisms as well, in, in, in the blood, here in the UK we don't necessarily see a lot unless the dog went to France, but, it's, we have now better technique like PCR, but again, if you can pick them up during a quick examination. So, so that's kind of what I wanted to talk about, the importance of a good quality smear, staining and a microscope set up, then looking quickly the feedex, then coming into the monolayer, look at platelets, look at red cells, and then I just want to have a few minutes to talk about leukocytes.
So the total white blood cell concentration, as I said, it's one of the most reliable things from the machine unless you have nuclear red blood cells, but then we need to come to the neutrophil lymphocyte dose concentrations. A lot of the machine differentials is is absolutely a hit or miss. It could be spot on, but it could be completely lost and literally reverse.
So again, you cannot really trust. Them without having a quick look. And while in the laboratory, we will do a typically 100 cell differential, 100 consecutive cells.
You may not have time for this, but at least just have a quick look and look at, is it about mostly neutrophils and a few lymphs and monocytes, or do I have mostly lymphocytes? So that again takes like 20 seconds to look in there in terms of the numbers. But the morphology is something I think is a very useful skill to practise.
So being able to recognise lap shift and toxicity, these are clinically very important. The animals are very sick when they have these, and none of the machines, no matter how sophisticated they are, they're not gonna be able to tell you that. So the neutrophils and a high quality blood smears when it's fresh, they will have a nice clumpy chromatin segmented nucleus and a nice uniform cytoplasm with the neutral staining granules.
You need to have a nice fresh tooth quick. Please do replace it every 2 or 3 weeks. Don't use it for a month, to be able to see that kind of detail of the neutrophils.
Now, the left shift basically means When we have the earlier forms out, the bands, for example, where you have these parallel walls, and, and also a less mature chromatin pattern, but toxicity, so when you have this frosty foamy cytoplasm or diffused base of the blueness, and you can have dolly bodies in them. Toxicity means the cells are produced very fast, so that bone marrow is always on its edge and it's barely keeping up. If you have a normal neutrophil concentration, but you have toxicity or left shift, the normal will flag up as green on your machine.
But if you see toxicity in left shift, that animal is in a major trouble because it's not even able to mount a neutrophilia. So that could become neutropenic any minute. So recognising these features, again, a nice frosty cytoplasm, the do the body is, I think, useful, very useful clinically.
Just for the last minute, monocytes is, is what students hate the most. They can really mimic neutrophils. When neutrophils become toxic, I showed you they can become bluer, they can become larger, and the nucleus is less clumpy chromatin, so then they start to look a bit like monocyte.
But monocytes tend to have a more blue-gray cytoplasm. They may have the vacuoles and the chromatin is, is always lighter. So that takes a bit of experience, but If you recognise toxicity in left shape, but you cannot tell one or two cells if it's monocyte or toxic ban, that's fine.
Very last minute lymphocytes, we can see nice reactive lymphocytes. You want to see how they look, look at a puppy. You can even see mitosis in their dark blue cells, don't get scared from them, unless you have a, a high number of them.
So when you have a leukemic blood fixture, a lot of immature blastic living cells, it's, I think it's very easy to recognise. That there's something really, really wrong to be able to tell the minute details that is an acute lymphoid leukaemia or acute myeloid or myelomonocytic leukaemia, you need a lot of expertise and even we will tell you need a full cytometry, but to recognise this, just in the blood smear is actually relatively easy. There's a lot of very abnormal looking cells.
So, so again, like I don't need, I don't think you need to spend too much time on it, but, but I would definitely kind of have a quick look at the white cells, the distribution, the numbers, and then I would look at like 2050, 100 neutrophils, especially if you do a differential, and then look for these features of toxicity and left shift because again to me that's something that you can pick up some very severe condition and you as a veterinarian, you have to pay special attention to those animals that have left. For toxicity, especially if they don't have any neutrophilia. And just last slide background, we can pick up some like protein crescents if you have very high protein, or if you have a very blue or, or, or, or, or background that that could give away like a very high globulin like when you have the multiple myeloma or if you have likemia can have a lot of little lipidvacules in the background.
Generally this is not as critical as the other findings were. So, so my goal was to kind of give you some really practical tips, and, and, and kind of highlight a little bit of how the machines are not foolproof and you kind of need to start to work with them. But then you can start trusting them, I think, better.
There are some excellent references here that you can use. The, the bottom one is a really nice colour atlas. So there's lots of pictures.
And if you have that next to the microso. So you can look up the different type of red cells, different kind of neutrophil changes. The, the other one is a really great textbook where you get a lot of very useful information to look up.
So, so I would strongly recommend if you can afford the time and you have a haematology analyzer, then I do beg you to please try to have a 23 minutes of this very systematic, very concise. Focused blood smear exam that I think will really benefit you, both clinically, and, and your patients as well. So I hope you kind of like this is a very different topic than the, the surgery that we heard before, but, equally, I think it, it, it hopefully is, is helping you in the practise.
So, thank you very much for your attention and we have some minutes left for questions. That's great. Thank you so much, Balash.
If anybody's got any questions, if you want to pop those into the Q&A box for us, and then we've got a few minutes left to answer those, I've got a quick question for you actually just want to come through. I find that some Of the slides that I use are much better. I get much better smears with them than others.
Do you have a particular preference when it comes to what, what sort of slide you should choose? Definitely. And the sad bit is that the more expensive, the better.
So I would, the one thing I would definitely avoid at all costs is those really cheap glass that doesn't even have the frosted egg. The, the better ones will say that they kind of pre-clean and they have grounded edges. So that means they, they.
Not just the glass that they cut it, but they, they finally grind it. So those slides were much nice, nicer, slide on each other. So, I would strongly recommend them.
And then with the frosted, you can write the numbers on them. It, it definitely helps to make the smear, better. And, and also it's from, I mean, I've seen funny things where people like break out those corners of the slides and then use one slide as the.
Spread a slide. You really shouldn't do that because blood dries on it and that will make the smears very streaky. you can use a slide as a spreader slide and then you can put it down and use it as your next slide if you want to.
But, but don't reuse slides to spread them. And as I said, a better quality lens like these pre-cleaned, grounded edge slides and frosted will definitely give you better quality smears. Fantastic.
That's good to know. I'll make sure I check that I've got the right type in the future. Brilliant.
Well, we've got lots of thank yous, but I've not got any more questions coming through from anybody. So I think we'll move on to the next one. So, thank you very much for that, Balash.
It was really interesting. Again, you somehow managed to make this, this topic fun.
