Good evening, everybody, and welcome to this first of February webinar. My name is Bruce Stevenson, and I have the honour and privilege of chairing tonight's webinar. To start off with a huge big thank you to our sponsors at Antech.

It is really so, so nice to have you guys with us, and we really appreciate the sponsorship. It's through their generous sponsorship that we are able to bring you this webinar for free tonight. Little bit of housekeeping before I introduce our wonderful speaker.

If you have any questions for us, please just move your mouse over the screen. You'll see a little control bar pops up. It's normally at the bottom.

It's normally a little black one. And you just click on the Q&A, type your questions in there, and we will hold those over to the end. If we don't manage to get through all of them, please don't fret.

We often run out of time in fantastic webinars like we have tonight. Those questions will be sent through to the Antech team and Holly, to have a look at an answer, and you will get answers back via email within a couple of days. So don't stress if your question doesn't get answered, we will get round to it.

I promise you. So as I said, tonight is proudly sponsored by Antech, and they bring us Doctor Holly Brown, who received her veterinary degree from the University of Georgia. She worked in private practise in Colorado and North Carolina before returning to UGA for a residency and her PhD in clinical pathology.

Doctor Brown remained a faculty member at UGA where she served as a diagnostic clinical pathologist, an instructor, and a collaborative researcher. She then joined a large general referral and emergency practise in the State College, Pennsylvania, as an on-staff clinical pathologist where she sees a wide variety of diagnostic submissions and enjoys playing an important role at the interface of diagnostic testing and patient care. Doctor Brown has always been passionate about and actively involved in the delivering of continuing education around maximising laboratory testing.

And she recently transitioned into a new role as the chief veterinary educator for Antech Diagnostics. Holly, welcome to the webinar, vet and it's over to you. Thank you so much for the introduction, Bruce, and thank you all for joining me in your evening tonight.

So, As Bruce mentioned, I am so passionate about delivering continuing education around laboratory diagnostics, and I feel like my, my passion is really around haematology. So, in particular, so many nuances through the CBC that we get to look at next to our patients. And get to use it to trend the data to help us diagnostically.

And so what I thought I'd put together for you tonight, is a series of slides where we go through sort of advanced level of going through the CBC where we'll look through the erythrogram, the thrombogram. On the leukogram and we're gonna pair that with some of the graphical data that's available to us at point of care as well. And we're going to do it through the lens of a case of immune-mediated hemolytic anaemia, so one of our most classic haematology critically ill patients.

So we are so fortunate to practise in a time where we have these advanced diagnostics, available at, almost at our fingertips, right? When it comes to haematology, we have these wonderful large reference lab haematology analyzers that can have really high throughput, deliver a lot of advanced information. And a lot of quality around that information, but we are also so fortunate to have point of care analyzers to deliver some of that same quality, patient side, right?

So really learning how to maximise that information so that we're able to use it to help our patients now is so essential in our critically ill patients. Before we start the CBC, what I thought I would do is talk a little bit about the technology. So I think if we understand some of the technology behind the analyzers, it'll help us understand the data, where it's derived from and again that graphical representation that's gonna help us further augment our interpretation.

So, the classic technology, in our haematology analyzers for cell counting, as we're looking through the, the red cells and white cells and platelets, is impedance technology or laminar flow impedance. And then we have some more advanced technology around flow cytometry, and that allows Us to look at other facets of the cells. So other than just cell counting and who they are, give us some more idea of if there's abnormal morphology changes, if there are more immature populations, and really also help us tease apart, the different five-part leukocyte differential.

So to understand what's happening with that laminar flow impedance. So this technology is really excellent at cell counting, right? And so here's a little graphical depiction of it where we're gonna take the blood cells and we're sending them through this aperture or opening across which there's a charge differential.

And so this Cells using advanced fluidics are going to move through here individually. And when they cross through the aperture here, they impede that charge differential, right? The impedance technology, and that causes an electrical pulse.

And so these analyzers get a pulse for each cell that moves through there individually, and so it can rapidly count tens of thousands of cells really, really quickly. The data that's generated is not just the individual counting of the cells, but the size of the cell, is proportional to the pulse that is impeded and generated. So we can get a graphical depiction of what it's looking at.

And so we look at a non-nucleated cell run. So we're only allowing the erythrocytes and the platelets through the aperture. We get this histogram data generated from this technology.

And so here we have on X-axis, this tells us about the size of the cell, right? Because that was proportional to, the pulse that was generated. And then on the Y axis, it's the number of cells that certain size.

Right? So as these individual cells are coming through here, again, tens of thousands that it gets to count, it ties up, you know, the number of cells in these smaller size here, right? The large majority are here in this size, and then there's a few smaller ones and a few larger ones.

Then there's These events over here that are a lot larger, and there's numerous ones here in size and a few larger and a few smaller. And when you get a nice demarcation between the two, it may becomes obvious that the smaller vents or cells that are going through are going to be platelets, and then the larger ones are going to be our red blood cells. So this technology, again, really good cell counting, and when we get histogram data that we can trace like this, especially when there's a nice demarcation, right, between the two different cell populations, we can use that as a quality control mechanism to say we really believe in an accurate platelet count because the analyzer was able to count them and differentiate them from the red blood cells, two different peaks, right?

So they're counted separately. And the technology is wonderful, especially like in a healthy dog, where the red cells are always gonna be bigger than the platelets. They're almost always bigger, right?

But it's gonna have some challenges when there's pathologic changes to your red cells, maybe with fragmentation or a microcytosis, or cats who have smaller red cells. And in those same cases, if we have some larger platelets or platelet clumps, because it's going to see these events coming through this area, and you won't get this nice peak and trough. And so when You have impedance technology, and some analyzers will show you this histogram data and can use it as a quality control to say if these populations are well separated, we can trust the data that's reported those red cell count and those platelet counts.

If we see that and we'll show you some examples, tracing does not come back down but curves right into the red cell population. We know that there's going to be there some larger platelets or platelet clumps that are going to alter an accurate count. Now, we just use impedance technology alone, it's really inadequate to separate out the five-part leukocyte differential because again, it's using size here and a lot of our leukocytes will overlap in size.

So flow cytometry uses again these advanced fluidics and it's going to introduce the cells individually here to the flow cell but in front of a laser light. And the laser light's gonna shine on the cells, and then we're going to capture the refracted light. So there's gonna be forward or maybe low angle scatter.

You can get a high angle scatter and some analyzers, there's a side scatter, and the, the characteristics of the cells, so their size, but also the complexity of the cytoplasm, some other characteristics about the nucleus are gonna cause a diff a difference in that refraction or reflection of light. And so we capture there are these odes that are going to capture the light at these different time points and get objective information about the cells going from the laser. And as a result, we can plot the data here on this graphic or scatergram that we call it.

So if you saw the other histogram that comes from impedance counting, where it's just using size to identify who the cells are and enumerate them, if you have a scattergram like this comes from flow cytometry. So here, this is that low angle that is capturing this information, this objective information from the cells, and this will be here on the Y axis and it'll get a certain coordinate, right? And that's largely proportional to the cell size.

And here, the high angle scatter that's capturing off of a diode up here, that's gonna tell us about the complexity, really about the insides of the cell, and that's really gonna help us tease apart, our different leukocytes. So what happens is each cell that goes in front of the laser, it gets its coordinates, placed here on the map. So it might have A y axis value of using this arbitrary, but like a 5 and then only a 1 on the high angle and it gets a dot.

And another dot for the next cell, another dot, and it speculates this whole thing looking at again tens of thousands of cells, which each dot representing one of the cells that came in front of the laser. And then It knows, like on a healthy dog, for instance, it knows these different clouds are from the different cell populations, and these low angle ones that are smaller in size, these are going to be the lymphocytes, right? Not a lot of cell complexity, not a lot going on in the cytoplasm.

So it's going to colour this cloud of cells pink and numerate them as lymphocytes. And this example, this cloud in here, it knows that in health in a healthy dog, for example here, that monocytes fall in this area, right? So it's going to colour them green for you, label them as monocytes, and count as such.

Neutrophils up here, eosinophils up here, and actually a lot of these analyzers end up calculating basophils. So they're really hard for the analyzer to recognise. So in a lot of analyzers they're calculated.

And what's neat about learning these scadograms for a different species of what normal looks like, it really helps us to identify really objectively when there's abnormalities in our cells. So again, this is a healthy dog example. But if you have morphologic changes in your cells, one of the most prominent and important ones we think of is a left shift in our neutrophils, right?

If there's more immaturity to our neutrophils, it's going to affect their coordinates on the graph. It will appear different in the schematic, and then we have different interpretive, Abilities based on seeing those differences. So, also, if you notice that different clouds are not well separated, because there's events that are falling in areas that they shouldn't be because there is an abnormality in the cell population.

So again, it's a wonderful control, quality control to be able to look at these different populations here, be able to say that if the clouds are individually separated, then we feel really good about the data generated from that. We can trust that the lymphocytes are truly lymphocytes, monocytes are counted correctly, etc. If there's bending of any of the clouds, and I'll show you examples, or if you have any abnormal populations, often you're, you're, you will be qualified, sometimes with asterisks.

Depending on the analyzer, to say something's off with the location of those cells, meaning something's wrong with your cells. And what an opportunity at point of care, even before you get to the microscope, to know that something's off of your cells. It's not always the cell numbers that let us know that there's a problem, right?

It's about cell morphology that can help us as well. Toxic change or immaturity of lymphocytes, things like that, which would be abnormalities, and we can see those changes in the scattergram. So the added value in looking at these different cytograms or scadograms is that they give us objective information about the cells, right?

So even before we're gonna use our subjective lens at the microscope, it's gonna give us feedback about the cells that tells us if they're normal or abnormal. We can use that to validate the CBC results, right? So we don't need to just trust the whole numbers that are reported.

We can look at these scattergrams, look at the histograms, and say, yes, they were, you know, observed normally in their appropriate position. We're able to count them accurately. We're looking on the histogram, are the peaks well separated?

The scatergram are the cell clusters well defined? It does allow us to recognise morphologic changes that, you know, there's so much subjectivity on the microscope, but it objectively starts to recognise those changes in the cells and report them back to us. It lets us know then which are the, cases in particular that we need to make blood films on, right?

Because we already know there's an abnormality in that cell population, and we get to look for it and some directed look for it too, because we have an idea based on the scattergram, maybe what cell type that is or what concern there is. And then it facilitate. It's consultation.

So, you know, I look at these scattergrams every day in my job, and it's such, an opportunity to hone those skills to start to say, Oh, when that looks like that, I know this is going on. I know where reactive lymphocytes lie. I know where bands are, I know where toxic changes.

I know what a chronic lymphocytic leukaemia looks like, when there's activation of the monocytes, things like that. We really get really skilled at. Depending on your analyzer, you can really learn those ins and outs and As a clinic pathologist, it's a pleasure for me to get to consult on those.

So as I said, I'm gonna take a case of a critically ill patient, right, a hematologic emergency if you will, a case of IMHA that we had at our hospital, and then walk through the hemogram, walk through the CBC and talk about the nuances of both recognising normal and abnormal, and then what we do diagnostically, specifically, for IMHA. So Bella is a 5 year 5 year old female spade lab. She has a history of being lethargic and jaundiced for the owner.

She went to the referring vet. Her Excel volume, and a spun micro hematocrit tube was 18%, and she was referred for a suspected IMAJ. So we wanna, before we look at the data, make sure we understand what we're looking for if we think she does have IMHA, right?

So we talked about the pathophysiology here. The idea being that these red discs are supposed to be the patient's red blood cells. Something has been changed in the patient, right?

Something's been altered, whether it was an infectious age. Maybe it was a fever, maybe it was a drug administration. Something has changed the antigen presentation on the outside of the red cells such that a proteins recognised as abnormal now by the body, right?

So it might be adhered on there from a drug or might be revealed through a neoplastic cell or through fever or something like that. So we see a foreign antigen or antigens on the red cell surface and so the body's gonna make antibodies, right, to the foreign antigen there because red cells are so numerous and if we think about areas of the immunoglobulin molecule, the antibody that bind to the anti And we have sort of two parts to that, right? The, the white part, those arms there.

And so if each one can connect and make an antigen antibody complex, you can get a glutination, right? The neighbouring red blood cells. So that's what, what we think is happening in the face of IMHA.

With these oxidised red blood cells, meaning they have that antigen antibody complex on them, as they go through circulation, brown thing is supposed to be the spleen. They move past the spleen, and the spleen, the macrophages in the spleen, they recognise the other end of that antibody and, and it's that FC receptor, sort of the, the elbow end of things. And it's going to grab that, right?

Because the macrophages are antigen presenting cells. So it's going to grab that and then deliver that to our lymphocytes. To make more antibodies, or directed at whatever that antigen is.

Sometimes it grabs the entire red blood cell. We get that hemolysis from that phagocytosis, and sometimes it just nips off that antigen antibody complex. So it might just do a nip off of the membrane and then that red cell seals back up again as a sphere site, right?

So we call that extravascular hemolysis because it's occurring in the spleen, not directly in the vasculature itself. Now, intravascular hemolysis, the other arm of the immune system. The complement part of the immune system gets activated, right?

And that complement finds these opsimized red blood cells that have the antigen antibody complex, and it, what it does to destroy the cell is it pokes a hole in it. So it makes that MAC complex, it pokes a hole in that. It releases the free haemoglobin in the cell.

As a result, we get this ghost cell remnant, and that's intravascular hemolysis because it's happening within the vasculature. So as a result of these antigen antibody complexes, we may see agglutination, right, that we can see in the tube or in the microscope. We can see spherocyte formation because of this extravascular hemolysis where it nip off part of the red cell membrane, or we can see ghost cell formation.

And so when we look at our blood film, these are the things that we can look at to understand that this whole process is what's going on underneath it. As far as recognising tube agglutination, so again, and this is the crossing and red cells that we're looking for is we look at DT tube, so we shouldn't have any aggregation of the cells cause it has an anticoagulant in it. We'll rock it back and forth gently and then we'll watch the blood run down the inside.

The tube and I prefer to look at sort of the back side of the label where it's white, to look against that, to look for graininess of the agglutinated red blood cells, right? So that graininess in there tells me that we do have at least increased red cell association, so we would call that positive tube a glutination. And for Bella's case, this is what her blood tube looks like.

So this is her EDTA blood tube. Not only do we have the graininess of the red cell associations or glutination, it looks like she's also as well. My mom had said that she was jaundiced at home, so we would confirm.

Now, we can take that a little bit further because we see that grain is to know that the red cell associations are there, right? But it doesn't necessarily mean that it's from immune-mediated linkage. So we can have increased red cell association with high globulins.

We can have red cell associations that increase when we change the electrolytes, so it changes that zeta. Potential on the red cell, and they no longer repulse each other. And that can cause the red cells to team up together, right?

And look like that graininess inside the tube. So we will challenge that with some saline on the slide. So it's a saline agglutination test or a slide agglutination test, and we'll take just a drop of that patient's blood, that EDTA blood.

And we can add one or more drops of saline to try to separate out those red cells. We're basically trying to wash the red cells to see if it was a charge differential or because of the globulins. Maybe we can get them to separate out.

If it was true immunoglobulin, right, that was linking them together and crosslinking them like that, then we're not going to be able to separate them. So we add the saline. You can add up to 4 drops.

Usually we start to flood the slide a little bit, but the idea being we really want to try to wash them. We'll rotate it around. And in Bella's case, we can certainly see she still has agglutination.

We can take it a step further and look at it microscopically. And we'll see that while we were able to wash some of the red cells away from each other, we still have these these ape-like clustering, right? The red cells throughout.

And as we added more drops of saline, moved apart from each other, we still had these aggregates of red blood cells. So it would be a positive saline or slide agglutination test. Now, when we talk about the CBC and in Bella's example, I'll be using Nex labmate CBC haematology analyzer.

You know, we talk about the CBC as if it's one test, but as we know, the CBC can have up to 24 different variables that are inmates that are reported. And it's really nice. I don't expect you to look through the whole data now, but I appreciate how it's laid out.

I like that we start with the leukogram and we go through the five-part differential. We include percentages in here, but that's not really what we're going to look for reference intervals for, right? We use absolute numbers for those.

It goes through the erythrogram and then the thrombone at the bottom. It's keeping the leukogram separate from the red cells and the platelets because of the technology that's used and the graphics that come with that. So I like that not only does it report the data and the reference interval, I think sometimes more importantly is where my patient's data lies relative to the reference interval.

So sometimes things are reported as normal because it's within the reference interval, but it's a very high end, right, which may well be abnormal for that patient. And if something's high, is it a little bit high or is it really high? Because that tends to affect my interpretation.

So I, it's infrequent. I'm gonna look actually at the number and see where it is in the reference reference interval here. So I look at this again, graphical depiction of the data, see where those trends lie, right?

Because where they lie relative to each other matters too. You know.in is low and globulins are at the high end of the reference interval, but they're still within the reference interval.

I wouldn't call that normal. Right? Some things they usually trend together unless, you know, albumin is suppressed maybe or globulins are increased production, right?

Or they maybe creatinine falls a little bit out of the reference interval, but BUN is high normal. The fact that they're training together is significant to me, and I wouldn't call. Just one high and one normal, right?

So seeing where the patient's data lies relative to the reference interval, which is in the bold black here is also helpful. And then again, these essential graphics that we'll talk about individually to make sure that we understand that additional information that they have. So we'll start with her erythrogram, and we'll focus it on there.

So remember, she was referred to us for her anaemia, and I think at the referring vet maybe her hematocrit was 18%. When she gets to us and we run it through our haematology analyzer, hematocrit was reported at 16%. So depending on if you look at hematocrit or haemoglobin more often, regardless, you know, we look at those three together, the red cell count, hematocrit or haemoglobin, they're all quite low, right, because of her significant anaemia.

Then we go through the rest of the erythrogram. We look at these red cell indices. So MCV is the mean cell volume, right?

So that's saying of all of the red blood cells, right? Where is the mean? And when that falls high, when that's increased, one of the most common reasons for it to be increased is that she has a lot of young red blood cells.

So young red blood cells, young cells in general, they come out larger from the bone marrow and they get smaller with age. So we have larger red blood cells. The most common reason, especially in the face of anaemia, would be if there was a significant regenerative response.

So while we don't have reticulocytes reported on this case, we're able to use some clues in here that point towards her having a regenerative response, and the MCV being high is one of them. Now, this just isn't a little high, right? But if we look at the, the graphics of it, it's, it's way outside the reference interval.

And if you get up to 95 femiliters, I don't think that could be a mean for all of our red blood cells. So my concern there is that there's actually the glutination of the red cells. So if one or if more than one red cell came through that impedance counter, it causes one pulse, right?

So it gets counted as one cell, but it might have, you know, the overall size of 4 cells, right? And that's contributing to that mean and pulling it way far to the right. So when you get up over anything close to 905 litres and above, it's most commonly because of agglutination.

Now, the MCH that does not provide any additional information cause it always trends with the mean cell volume. It's the mean cell haemoglobin, but it's the haemoglobin content would depend on what the size of that that cell is, so that's just gonna trend with that. No additional information.

But the MCHC, that's the mean cell haemoglobin concentration. So it's saying regardless of the red cell size, is it made with the appropriate amount of haemoglobin. So if we think we have a regenerative response, creating a high MCV cause they're larger cells, the MCHC, the haemoglobin concentration, is actually usually on the low end.

So we have low haemoglobin concentration when not a lot. Iron is around, right? And our young red blood cells, they don't have their full haemoglobin content.

So a lot of young red blood cells, a lot of reticulocytes will contribute to a high MCV and a lower MCHC. But as you see, in this case, it's at the high end, right? And so that is unexpected, and it makes me think of different interference.

So when we measure haemoglobin in these analyzers, it licces all the red cells and then it measures the hemoglob. But if there are some background substances that can contribute. So in particular, lipemia is a big one for increasing the MCHC.

Having a glutination itself will increase the MCHC, and, any homolysis will add to that. So it's, I think, a little spuriously elevated in this case. I bet because of the amount of regeneration, it would more likely to be at the low end of the reference interval.

And then something maybe new to you all is looking at RDW, and that's the red cell distribution with, and I will talk about that, on the next slide. I wanted to again think about the graphical depiction of this data. We're using the impedance technology to count the red cells and the platelets through here.

This is how it's shown for to us on Bella's case, right? And we see this first, Its first peak here, it's barely a peak, right mound will be our platelets. As it comes down, the next peak is going to be our red blood cells.

It's certainly not robust, right? Normally, it comes up quite high like it does in this normal, depiction here. And we are really markedly anaemic, right?

At, what, 16%. Also, Notice that in health, you know, we get a nice Gaussian shaped curve where you could drop a line down the middle and you'd have equal numbers that are bigger and equal numbers that are smaller. So it would be symmetrical on both sides of the curve.

But that's not the case on Bellass, right? So we drop a line down from the peak. This curve comes down pretty readily, but this extends out quite far to the right, right?

And so my arguments and what I, what I assume is happening there. That I think probably in this area it'd be common to see an increased number of larger red blood cells, its size here across the x-axis, and that'll probably be your reticular cys or young red blood cells. The fact that it tracks all the way out here, close to 200 centilitres where there's no such thing as a red cell in that size, this would be all your agglutinated cells.

So we can just see in this depiction here and maybe if we just look. That just the hemogram, and just the erythrogram from her, we could guess maybe that she has IMHA. We see this significant, this marked anaemia.

We see evidence for regeneration with the high MCV in this area, but also to extends so far out to the right. MCV is up at 90 something centillis, you know, then we start to say, that's so high. I bet that has a glutination.

So we really get a wealth of information on there. I just need to move this picture side. OK.

So let's talk about that RDW. So the RDW is the red cell distribution with and when that is, it's basically tells you about an index of the degree of anisocytosis, or red cell size variability. So an increased RDW we can see on the histogram as a widening of that peak.

So it's the distribution in the size, so from the small on the x-axis to the right hand side, large on the x-axis, that distribution has been widened either because there's larger red blood cells in there and or smaller red blood cells in there contributing to that. So in this particular patient where the red cells have just a mild amount of variability, it gives us a nice peak like this where it's pretty well on both sides and it's not too wide. RDW falls there right within the reference interval.

So the distribution with of the red cell size, the degree of anisocytosis falls within the reference interval when it looks like this. In the case where we have a lot of red cell variability in size, right? We have these smaller ones next to these larger ones, we have a really different peak, right?

No longer do you sort of have that more narrow peak here. It is widened from the presence of smaller cells, these almost spheroidic looking cells, and then these larger cells as well, right, those polychromatopils. And so that will increase your RDW.

It's really an important thing to start to tune into because it actually takes a lot of variability to move that outside the reference interval. A low RDW is not significant, but a high RDW, you should look at the graph, look at your MCV and determine if you think that's because you have larger cells in there, which are often because of the presence of reticulocytes or young red blood cells, or if they're going to the the lower end, right, the left hand side of the curve, because that could be red cell fragmentation, that could be iron deficiency. That could be poorer systemic shunt formation.

So pairing that RDW to say it's a wider peak than expected, and then often I'll use that mean cell volume to tell me whether it's dragging them low cause lower ones are there or larger ones are there, and then we look at a blood film to take that a little bit further. And indeed for Bella, if we look at her blood film, we certainly see a lot of red cell variability in size. It's quite remarkable.

The larger ones are really large and the small do get quite small. So perhaps not surprising that we have this very wide peak through here and RDW that's far outside and increased over the reference interval. So, let's talk about other blood film changes we expect with IMHA and what we're gonna be looking for.

So first, you're anaemic, right? So we expect that we're looking at what would be the monolayer of the blood film that there are gaps between the red blood cells, right? There are spaces in there because the patient has more plasma relative to the number of red cells.

And then we need to assess whether it's regenerative, right? So, it's a hemolytic anaemia, so the causes for anaemia could be a production problem. We're not making them.

We're hemolyzing them in circulation or, or losing them from the body, right? So those are sort of the three buckets. And if the first thing to rule out is, are, are we making them, right?

If this is IMHA, it's not a production problem, right? We're hemolyzing them. So we want to assess for that young red cell population.

We had clues in the automated data based on an MCV being increased. We saw the increased RDW and larger cells apparent on the right-hand side of that histogram. But also when we look on the blood film, we look for polychromatophils, right?

So these larger bluer cells are our young red blood cells. And that tells us that we do have an appropriate bone marrow response, right? It is regenerative.

It's not a production problem that got her down to an 18% hematocrit. We should be thinking about blood loss or lysis. She had no overt evidence for bleeding.

She, on imaging, you know, on ultrasound, no hemoabdomen blood loss there, and indeed she was ictrix. So that really goes to support her having hemolysis over blood loss. There's another stain that we can use that helps us even more, look at the young red blood cells, and that is a new methylene blue stain.

What we do is take equal drops of the blood and the new methyne blue stain, we let them incubate together for about 10 to 15 minutes and we make a slide. And it stains the red cells really differently. This is a super vital stain, meaning it's put on the living cells, so we don't fix it first like we do for diffrec, and it's a really pale stain.

It won't look nearly as blue as this, but it looks quite different under the microscope to see the blue. Has is super vital stain, it's going to go inside the red cells, and it's gonna cause aggregation of the ribosomes or any residual RNA. And there's only residual RNA in our young red blood cells.

Those that were recently making proteins, right, before they lost the nucleus. And so while we're used to looking for polychromatophils on the Romanowski or quick stained slide to look at regeneration, the new methane blue, they show up as these aggregates, right? And they really stand out.

And so we call them reticulocytes when it's new methylene blue stain, this is polychromatophils here, and a lot of our automated technology that that counts the young red blood cells except reticulocytes specifically, those are the same cells, right? It just depends what stain you're using. Also in the blood film, we're looking for evidence of spherocytes, right?

We're looking for evidence of macrophage, you know, pulling off that antigen antibody complex off of the surface of the red cell, and the rest, it pulls some of the membrane off and the rest of the red cell seals up. What that looks like microscopically, you know, is that instead of having these biconcave discs that have central pallor, they appear smaller in diameter and they often stain a little more pink like we can appreciate here and often they can even be perfectly round and spheroid. So that's evidence for that immune hemolysis that helps to support it.

A word of caution is make sure when you're looking that you don't look too close to the feathered edge. Cause as you're making your blood film and you're rolling the blood cells along the slide, when you get out to the feathered edge, you really start to flatten them. And so then almost all the cells start to lack central pallor because you flatten them from those, discs those biconcave discs.

They're no longer biconcave. And so a lot more Cells will look like spherocytes. And, you know, there's that confirmation bias that we have inherently that if we think they have IMHA because of their marked anaemia, and then we make a blood film to look, we're gonna, we think we're gonna see them, right?

And I see all the time, if we look too close to the feathered edge, you want to move back into the body of the film where the cells can show their central pallor, right? And only in these areas where Many of the cells do show their central pallor. Could we assess for spherocytes?

So this is actually the same case, right? Not Bella's case, but one where they're lurking too close to the feathered edge and it looks like they are spherocytes. Please back up into what would be the monolayer.

Make sure the cells can show the central pallor if they have it. Only then could you assess if there are also spherocytes. The cells are from the complement part of the immune system and where they poke a hole in the cell, right, they cause osmotic lysis and the haemoglobin leaks out.

All you do is get a red cell membrane like a cell. So in this particular case, again, not blood, we have these spherocytes in here. We have the polychromatophils, we have the young red blood cells, and you see all these little ghost cells in here, right?

They're these ghost cell membranes that we see, and those are the ones that that complement poked a hole in them. So that's our intravascular hemolysis, and I love zooming on this picture cause you can actually see the haemoglobin just like leaking out of the cell. So these intravascular hemolysis is when our IMHA cases show this as well.

Those are the ones that become bilirubinemic, even the earliest, right, because of all that free haemoglobin. So, on Bella's blood film, indeed, you know, we confirmed that she does have these spherocytes, she's anaemic, she's highly regenerative, that checks all of our boxes of things that we're expecting to see in our traditional IMHA cases. With the leukogram, a little more challenging, right?

So there's a lot more things to look at, right, than just, just the red cells that we've been paying attention to. So, in Bella's case, when we look at just the automated data, she does have a leukocytosis, right? So leukocytosis that 4, almost 43,000 characterised by a neutrophilia and a monocytosis, right?

And that's a classic inflammatory leukogram, right? So when we get up over, you know, 40,000 leukocytes up here, for, well, for you guys, so I say 40,000 for microliter, they, we almost get into that leukemoid category, right? That's a lot of inflammation.

And immune disease is high on the list as a possible underlying cause. So again, inflammatory leukogram, that's what I expect with IMHA. Also look within here, I asked the monos here to remind me to tell you that we commonly see in our fulmin and IMHA cases, the monocytosis trends really well with how active the immune attack is.

So when they have fulminant disease, the monos are high, and before we get control of that immune response, they might continue to climb. But when our immunosuppressive drugs kick in and we start to get a toehold on this, and we're seeing less inflammation, we're seeing less, the clinical signs are not as febrile, maybe they're feeling better, less spherocytes, right? Maybe our hematocrit is starting to come back up.

That trends nicer with watching those monocytes come down, right? And I think that's because You know, monocytes become the macrophages and the tissues and the macrophages are what mediate, you know, this homolysis. And so I think when there's less demand for that, the monocyte count comes down in the blood.

So it's a nice value to trend for these guys. It's also a, in this case, a low-end lymphocyte count, right? And eosinopenia.

They're most absent. And that trend of decreased lymphocytes or lower end lymphocytes and decreased eosinophils, that's a stress response. So that's a great test for just overall health of a patient.

And when you see a stress response, something Really wrong, right? That's in response to increased glucocorticoids produced from the body. So again, classic IMHA case in the leukogram, we see an inflammatory leukogram, and watch that monocytosis as we're looking at how we respond to therapy and immunosuppression, and then a stress response with low EOS and lymphocytes.

When we look at the scattergram, we get even more information. OK, so if we remember the technology, right, it's flow cytometry, and then we're capturing objective information off of the low low angle and the high angle scatter that tells us cell size roughly here on the Y axis and cell complexity on the X-axis. We have the different clouds of cells, but remember when they're not well separated, means a lot more, right?

Then there's a pathologic change to your cells. If these clouds were nice and well separated, you could trust the counts and that this could be a healthy animal. This pattern looks really different.

I think I'll zoom in here. Yeah, to show a healthy dog here on the right and Bella's case on the left. So going maybe left to right, the first thing I'll see is that there's more pink dots here where they're denser, it's white in the middle and there's fewer pink dots, and that's because we're trending towards that lymphopenia.

Now, the monocytes in health are sort of this quiet oval cloud in here. It's really widen in here and it's a thing against that neutrophil cloud. So we get this sort of slab-sided junction here.

I don't know whether it's the left shifting of the neutrophils or it's the activation or left shifting of the monocytes, but this is classic for inflammation. These two would abut each other. We see that there are more neutrophils.

There's more white here in the middle because the intensity of all of those dots in there, the intensity of them, of that cloud, that's your neutrophilia. Up here, those are actually where we see bands. So we actually get a visual depiction.

They are blue, they will be counted as lymphocytes, but this lighter colour blue is where we see bands. And so that's really awesome for detecting a left shift. We compare it in health.

Bands up there, of course, are a couple of little dots up there. Also less orange dots than they're in health, and that's again that eosinopenia, lympopenia, and eoinopenia from that stress response. Inflammation we see is more neutrophils, changes in the monocytes, slab siding junction of the two of them because of morphologic changes in those cells, and then the bands we can visualise here.

Here's another picture where we see bands in this case they're falling more on top of the cloud. They can be in either location. And actually it seems that in the limited experience we have those ones where the turquoise dots fall right on top of the cloud are sometimes less left shifter or toxic, the more left shifter toxic than the ones that lift off the top, usually during recovery.

So I'll show, so one reason I have this objective data on the bands is that when we look under our microscope and we look at 100 cells in our differential 100 leukocytes, we're really insensitive at detecting and certainly in trending inflammation, right? Because what you call a band and what I call a band will probably differ, right, on a cell by cell basis. We have a definition, this horseshoe-shaped nucleus with parallel sides, and that works great when you have a segmented neutrophil right next to a band neutrophil classic shape, no problem.

Then we get a case like this we call that a band. I mean, it doesn't segment, but squeezes in some. There's alternate definitions that say, well, slight nuclear indentations are acceptable, or maybe a neutrophil where the thinnest portion of the nucleus is greater than a third of the, as if you're gonna take a cell, right?

And measure what's the thinnest portion and compared to the thickest and get a ratio in there. And that just doesn't happen, right? So it's really challenging as we're looking at a variety of the neutrophils to actually have confidence in the number of bands that are there because it's so subjective, you know, under the microscope of the human eye, right?

And that's without being a clinic pathologist. So, this objective information off of our analyzers really helps us both quantitate that left shift and then trend it as well. So in this case where I told you sometimes having turquoise dots right on top of neutral co can even be the more intense inflammation.

And here's a case, it was a pneumonia case and we tracked it and we followed it through, I think this is maybe 2 days later, far fewer dots on here, right? And that's cause there's less bands. It was an awesome way to trend our patients in response to therapy.

And then we'll talk about the thrombogram. Bella, so we're looking at her platelets here, she has a reported thrombocytopenia, and that's classic IMHA. They almost all have some degree of thrombocytopenia.

Not that they have Evans syndrome, not that they have both ITP with their IMHA because that would mean your platelets are like less than 15,000 in my mind. But it's really common just to have some degree of thrombocytopenia, and that might be because they're in that engorged spleen or there's non-specific clearance of them. There's a variety of, of suspected causes, but she does have You know, a, a mild a cytopenia.

She doesn't increased mean platelet volume. So whenever a cell count is low, and I'm trying to figure out is it a production problem or something happening in circulation, are we losing them, I like to see if the mean volume of that cell type is increased because If it's increased, it's probably cause it's a younger cell, and that's the increased mean platelet volume is actually probably part of that regenerative response of the platelets. So it's not like we're not making them, right?

Just something's happening either again, they're sequestered in the spleen, or maybe they're getting cleared by the immune system as well. We have a platelet crit is calculated based on the platelet number and the mean platelet volume. And so, this also is really helpful in cases where you have some platelet clumping because your platelet count might be low, but your mean platelet volume would trend high because of those clumps.

And so when we do the math of that, we get the CRI, the concentration, and that might put us back into the reference reference interval to know that our platelet mass is adequate. And then there's also a platelet distribution with mirrors a distribution with of our red cells that RDW we talked about, and being low is just like in red cells is of no consequence. If it's high, just because there's a lot more larger ones or maybe platelet clumps.

We look at, oh sorry, as we look at her histogram, you know, in health with normal numbers of platelets in normal sizing, it would come up and come back down and then we go through a huge red cell peak on the right, right? But we're very anaemic. Well, The count isn't very low.

We do have an increased mean platelet volume and so these larger ones that are more recently made are pulling that curve off to the right, so it's no longer the sort of peak through here, dragging out to the right, and maybe getting close to some of those small red blood cells. So it's a little bit of a challenge in this case, to read the histogram, but at least you can look at it to validate the data that yes, it does look like there, there's a lower peak and there's probably some thrombocytopenia in there. Other cases just to see what platelet clumps look like, on this analyzer, so you can get a reported thrombocytopenia and we look over at histogram and we see that rather than coming up and coming back down, it's coming up and then it's staying at that level because we have these larger clumps all through here that bleed all the way over until red cell peak starts, OK?

And then in this particular analyzer off of labmate CBC we do have a flag for like clumps and cats. So that's really nice that we'll get an automated flag. So when you have a reported thrombocytopenia, the first thing you can check and see if there's a flag to say that clumps are, are present.

So with a number that's not increased and the clumps present, you can assume that that mass is adequate. But it's actually not the histogram that gives us this flag. It actually comes from The scattergram, if there are large platelet counts, they get interpreted as leukocytes, and they're actually down here in the scattergram.

And so when the analyzer, you know, it, it uses its advanced software and not only first we count all the cells that and we put them here based on their coordinates on the graph, right? But then it goes in afterwards to say who's who and it knows down in this area, the only, there's no other cells in this area. It has to be platelet aggregates, so it's gonna colour them a different colour and again give you that flag.

So that's a really nice, attribute. We have reported thrombocytopennias. Those are some tools that we're gonna be able to use, but let me just tell you a couple for looking at the blood film itself.

Look on the feathered edge, right, for those platelet clumps. Report thrombocytopenia first, you need to verify it. So using the scadogram, using the flag, using the cytogram or the histogram to help us is one way.

Looking manually for platelet clumps on the feathered edge. There's a calculation we can use where we take the average number of platelets per 100 X field, multiply it by 15,000 for a dog, 20,000 for a cat to give you a total number per microliter, knowing that about 10 to 20 platelets per 100X field would be normal. On Bella's blood film, again, we've looked at this image before to confirm her anaemia, her spherocytosis, her polychromasia regenerative response.

We have an mm early monocyte, I think here. We have bands in here as well of her inflammatory response and then we go to 100 X, we could enumerate her platelets and I end up counting 8 through here and then so we can multiply that by 15,000 in a dog and get a set sort of the lower end or just low of the reference interval, which is what was reported. Coombs testing real quick, and I know I need to wrap up so I can take some questions.

But Coomb's testing, we always think we were taught, at least we were in the US in school, that's sort of our definitive diagnosis for IMHA, but it doesn't always work out that way. So the idea with Koh's testing is that we actually mix the patient's red blood cells with antibodies to that antibody. If the red cells are coated with antibody, antibodies will cross link.

An antigen antibody complex on the red cells. So we mix the patient's blood with the Coon serum and then we run it through these gel columns. It caused a glutination, it can't make it through the gel column.

They're too thick. It doesn't cause a glutination, they peel it down at the bottom. And we look from the top and this would be a negative coons and that would be a positive cone.

It turns out a positive Coombs test or direct anti-globulin test doesn't equal IMHA. There are reasons for false false positive reactions. There are reasons for false negative reactions.

And so in reality, I don't use it as my end all be all for making the diagnosis. I'd rather use all those other things we talked about. Is false cytometry at like a reference lab, and research lab setting, but we've had to make a decision at point of care whether we're gonna immunosuppress them before we ever get those results back.

In 2019, ACBM published a consensus statement on the diagnosis of IMHA. So it's something that you can look up. And those consensus statements, they usually take about 6 internists together, and they look at the variety of things that need to be positive, to give us a diagnosis of IMHA, supportive of, a suspicious for, or or not IMHA.

And it's really just a culmination of those different tests we talked about, right? Tub agglutination, saline agglutination. Look for hemolysis, look for evidence of sphere cytosis, all inflammatory grass all are gonna play into our diagnosis.

I usually just, it doesn't have to be all of them, right? I'm just trying to paint a picture to see if that makes sense for that diagnosis. Real quick, positive for tubal glutination, positive for her saline agglutination test as well.

On the CBC regenerative anaemia, inflammatory leukogram, stress response, thrombocytopenia, those are all classic, and on the blood film a sphere of cytosis, and her chemistry to boot, remember she was icic, she also has increased bilirubin as evidence for that hemolysis. And with that, I would love to answer some questions. Holly, thank you so much.

That was absolutely fascinating. Those dot plots always blow my mind. They really do.

But you make it sound so simple. I just know that tomorrow or the next day in the clinic when that dot plot arrives, I'm gonna go, what the hell did Holly say? But there is such a wealth of information, right?

They really give us a look at so many of those cells, and it's far more accurate than even a clinical pathologist at the microscope because of the sheer numbers and the objectivity of that day. I, I just need a copy of you on my shoulder, whispering in my ear when I've got it. Before we go to the Q&A, I'm just going to turn things around a little bit.

I'm going to hand over to Shelly first, and then we'll come back to the question. So, Shelly, if you would like to come in and, chat a little bit, to us about Antech, and once again, huge big thank you to Antech for your generous sponsorship of tonight. Yeah, thank you, Bruce.

Absolutely. No, I just think, obviously I wanted to thank Holly for her fantastic, lecture on haematology, as always. We're really excited to sort of introduce Antech Diagnostics to the UK market.

For those of you that haven't been involved or seen sort of much on Antech Diagnostics, Antech Diagnostics has been in the US since 1987. And have been basically leading the way in lab and, imaging diagnostics. In 2017, they joined the Mars Pet Care Worldwide family, and they've actually, basically are now the largest animal health company.

So, very exciting to be sort of part of Antech Diagnostics and to have launched into the UK market, so. For those of you that were at Spivs and have been sort of following our progress, we launched into the UK market in January, and we're bringing our diagnostic solutions to the UK market. Starting in the sort of point of care, but we'll be more to follow.

So, really excited. You know, our sort of our vision is really to be able to bring a flexible, solution to our customers that's customer focused, and that we're sort of leading on education. So, really pleased to be part of this.

Thank you. Thank you so much, Shelly, and it's great to have Antech and, and more of the Mars family in the market here. We know how fabulous they are.

And, I haven't clinically worked with you guys, but I have no doubt that the support, is going to be fantastic like it is with the rest of the Mars family. So welcome. Just to, before I start popping questions to you, Holly, I just want to say to people, we can't go back to slides and, and re-show things and that.

But what I can tell you is that we are recording these sessions, and, Dawn assures me that Our wonderful technical crew back at the webinar vet will have the recording up on the website by tomorrow. Maybe give them a bit of a break and say tomorrow afternoon. But, there is a full recording of this.

So if you missed something, log on to the webinar vet website, go and have a look at it. And the good news is there you can stop and rewind and fast forward and rewind, and you can listen to all those explanations that you are asking Holly to go back to. So, Holly, a couple of great questions that we haven't yet answered.

How jolly bodies, what is their significance, if any? Yeah, so I didn't have any examples to show you, but a ho jolly body will be a deep blue purple inclusion, a small one round, nearer to the outer part of the membrane, about 13 in maybe from the diameter of the red cell membrane, and it's a nuclear remnant. So a how jolly body is a little nuclear remnant, and we see a little bit more of it.

It's almost an air. And we see it more when there's rapid proliferation and regenerative response. So we do see them in our IMHA cases sometimes or other cases of hemolysis because we have so much red cell turnover and they're being so rapidly produced that sometimes the nucleus doesn't leave the cell as it should and so there's a little nuclear remnant.

So to not confuse it with a parasite, right, it's a dense blue dot. It doesn't have any clearing in the centre. It stays near sort of the outer third of the membrane, and no pathologic significance beyond that.

Fantastic. I'm going to paraphrase a little bit because there's a lot of questions coming through related to the trending of the monocytosis and the monitoring of the AMI put my false teeth back in. IMHA specifically in response to therapy.

Yeah, so we, it, as I was saying that, you know, all of our patients with these inflammatory leukograms, and they present with fulmin and IMHA. They always have a monocytosis, right? That is, that is classic.

And again, I think it's because those monocytes become the macrophages and the tissues that are mediating this whole hemolysis. And then when we get, we put them on immunosuppressives depending on how sick they are, you know, they may be hospitalised, getting injectables for a couple of days before they're transferred to, oral immunosuppressive drugs, they might need some other. For GI support.

They may have bleeding. They need IV fluids. Whatever we're doing medically in the hospital, in addition to immunosuppression, we're watching their CBC daily, right?

To keep an eye on that anaemia, right? And where their hematocrit is. And even before we see a significant rise, I think, in that hematocrit, we see the monocytes start to quiet down.

So again, watching them every day, that serial data. When the monocytes start to come down, then I start to feel like we are controlling that immune response. I feel like we're getting a better toehold on the disease.

Fantastic. Again, I'm going to paraphrase a lot of questions. By the way, Holly, loads and loads of absolute, awe with what you've done and thanking you for the fabulous presentation from all over the place.

We've got comments coming in from Canada and Germany and everywhere else, so that's absolutely fantastic. Thank you for that. There's a lot of questions coming through about, finding literature or study material on this topic.

Natalie wanted to know also about seeing this stuff and getting information specifically in rats. But where would you recommend that people can go to get a good source of this besides phoning you? Yeah, that's so, so especially in alternate species.

And I do think those consensus guidelines from ACVIM, they are available online. So they were published through the Journal of Veterinary Internal Medicine, JVIM, but they are accessible and free access online, these consensus statements. They go through a whole A whole lot about the pathophysiology and the diagnosis of IMHA, and they have a separate consensus statement on treatment of it as well.

So that would probably be a great reference for a starting point. And then alternate species, Shaw's haematology is a classic haematology text, and it does have a lot of the alternate species, so that might be another source. Excellent.

Thank you for that. Just a word, folks, to some of the questions coming through. this webinar tonight is not really about the clinical treatment of IMHA and, and the best drugs and everything else.

So, I apologise up front, but I am going to ignore those questions. We can't go into those. But something that is coming through, as a common question.

And I've noted somewhere along the line, I've missed the question. We do have a student vet nurse on, who was the first one who asked about it, saying, could you just explain the significance of bans and what you mean by bands? Oh, that's awesome.

OK. So, you know, when we talk about recognising inflammation in our patients, right? Well Most common conditions we diagnose, in our sick patients, right, is inflammatory disease.

Looking at whole cell counts, like the whole neutrophil count, right? Or the whole leukocyte count can be really misleading, and you can miss inflammation because you're capturing those cells in a, a snapshot in time as they move from the bone marrow where they've been produced through the bloodstream to the tissue that's calling them or lost through the gut. Right?

So, I, Give an example of, you know, inflammation where I could have a, a crushing trauma of my arm, let's say. And when that happens, you know, acutely, all the inflammatory cytokines are calling on the neutrals there. And if you check my blood when that happens right away, the neutrophil count first will be normal.

It'll actually go down, right? So we have an intense inflammatory disease, right? But the neutrophil count's actually low, not our typical neutrophilia we think of with inflammation.

And then there'll be a steady state where maybe my bone marrow is able to keep up because we have the circulating pool of neutrophils, mature segmented neutrophils. We have a storage pool of equal numbers ready to come out, mature neutrophils to take their place. So then there might be a moment, well, even though a lot of neutrophils are going towards this marked inflammatory focus, we're replenishing them from the marrow.

So they might fall within the reference interval. And it's Not because there is an intense inflammatory disease, right? But then when those ones from circulation, the mature neutrophils have now left to egress to the tissue, right, to get to that source of inflammation.

Now, if they're still calling for more, you're gonna reach back in the bone marrow and start spitting out the young red blood, the young neutrophils, and those are the bands, named because of that wider nucleus, because they're not fully matured down to that segment of. Nucleus. So those band neutrophils are more immature neutrophils that had to come out because we could not meet the demand with what we had in circulation or the mature pool.

And so regardless of what the total number is, because as I've described, it can go up and down, right? You go down and up and be in the reference interval. If we can detect bands in circulation, we know we have intense or overwhelming inflammation.

So bands are such a critical part of diagnosing. And then trending inflammatory disease, because knowing that it's there is the first thing, right? But then when we do our imaging or whatever our follow-up diagnostics are to figure out where the tissue focuses of inflammation and we start treating it, we need to watch those bands go down, right?

And we can't use just looking at a blood film because you saw that subjectivity of what the different bands look like. On a given day, we're gonna get different numbers and it's gonna look different the next day and it's just so, it's really hard to qualify over 100 cell count. But these automated cell counters are counting 10s of thousands of cells.

And if you're lucky enough to have an analyzer that will show you bands on the scattergram, you can watch that number improve or go away, or worsen, depending on the condition. So it's a really sensitive way to trend intense inflammatory disease. That's absolutely fantastic.

And you've, I'm sure, enlightened our student vet nurse significantly. She can go and show off in the classroom when they start talking about these things. Fantastic question that's coming through a number of times as well when you're talking about monitoring responses and everything else.

The gist of the questions is how often would you rerun the bloods to monitor? Yeah. Well, so it really depends on the condition, because things change within hours, right?

And it sort of depends on what, what we're watching and how closely we need to watch it. But at our hospital, for our hospitalised patients, we Just, we run it each morning. So, no matter when they come in the day before, right, we'll get a baseline of what they were on admission.

And then we're gonna, because of our workflow in our hospital, it's just, it's easier for hospitalised patients to draw blood in the morning. But we're gonna be checking them at least every 24 hours, right? To watch their conditions, because they change that quickly.

And it helps us very sensitively, both indicate if they're improving so we can get them out of. The hospital earlier when we realised we're on the right therapy because the inflammation's going down, something like that, the monocytes are coming down. Maybe you now go to orals and you can go home earlier, but also to recognise any complications, right?

Especially in a post-op patient or surgical patient, you know, being able to watch them, and make sure we're not seeing any complications, we're gonna check them each day until they go home. So 24 hours, sometimes less depending on when they were admitted the day before. Fantastic.

There are so many comments coming through here, thanking you and saying what a fantastic presentation it was. I know Anthony always likes to say, if we were in an auditorium, you would have heard thunderous applause. So let me just pass that on to you.

There is lots of love pouring in for you here, Holly. We are rapidly running out of time here. One last question that theatre is asks is, how do intracellular parasites modify laser measurements of the size of the blood cells and therefore modify the picture on the dots?

That is Awesome question and we don't know yet. So, my labmate CBC, we haven't done enough. We don't have enough experience with it to see.

We are hoping to be able to see some changes in there cause it's, it's quite sensitive, but I don't know yet. So unfortunately, I can't answer that. There you go, Beata.

So any of those Babezia cases and that, we will wait for Holly to update us on the next webinar that we're hopefully going to have with her. Folks, we have run out of time tonight. It is my, pleasure to thank Antech for their support of tonight.

And it is my absolute pleasure to thank Holly for an amazing presentation. And, yeah, Dawn, I know you're listening. Anthony, I know you're listening.

Please, please, please, can we have Holly back again? This was fantastic. Thank you, Holly, for your time tonight.

Really appreciate it. And to everybody that attended tonight, thank you for your time. I hope you enjoyed it nearly as much as I did and we look forward to seeing you on the next webinar.

Thank you. To dawn my controller in the background. Thank you for making things run seamlessly as always.

And from myself, Bruce Stevenson, it's good night. Good night.