Good evening everybody and welcome to tonight's webinar. My name is Bruce Stevenson and I have the honour and privilege of chairing tonight's webinar. A little bit of housekeeping for those of you that haven't been with us before.
If you do have a question for us, just move your cursor over the screen. For those of you, watching on Zoom, your control bar will pop up at the bottom. There is a Q&A box.
Just click on that, type in the questions. They will come through to me and, we will get to as many of those as we can at the end of the evening. For those of you who are watching on the website, Dawn will pop her email address in the chat box in a second.
If you've got questions, you can email those to her and she will feed those through to me as well. And we will cover all of those, or as many of those as we can at the end of the evening. Also, please remember that we can't go back on slides, but these sessions are all recorded and it's, will be up on the website in the next 24 or 36 hours.
And you can go back and rewind and watch as much as you want to. So, the first thank you tonight is obviously to all of you for attending. We really appreciate your time, and a huge big thank you to Antech for sponsoring tonight's session.
And tonight's session, I'm very pleased to say, is going to be presented by Holly Brown. Holly received a veterinary degree from the University of Georgia. She worked in private practise in Colorado and North Carolina before returning to UGA in Georgia for a residency and PhD in clinical pathology.
Doctor Brown remained a faculty member at UGA where she served as a diagnostic clinical pathologist, an instructor, and collaborative researcher. She then joined a large general referral and emergency practise in State College, Pennsylvania, as an on-staff clinical pathologist. There, she sees a wide variety of diagnostic submissions and enjoys playing an important role at the interface of diagnostic testing and patient care.
Holly has been, always been very passionate about and actively involved in the delivery of continuing education around maximising laboratory testing. She has recently transitioned into her new role as chief veterinary educator for Antech Diagnostics, and she will share her latest, passion toy, shall we call it, of a podcast, with you at the end. Holly, welcome back to the webinar, vet and it's over to you.
Thank you so much and thank you all for joining me tonight. As you can hear from my accent, obviously I'm a US veterinarian and so please take that into consideration as you hear about some of the diagnostic results. I think I converted everything appropriately to the SI units, but if there's any treatment or testing recommendations that differ by locality, just understand.
So when we talk about performing laboratory testing, right, why do we perform it? To get objective information about our patients, right? Understanding that clinical signs can be very non-specific, right?
If a patient's not presenting with porcupine quills coming out of their nose or holding up a broken leg, right, a lot of the signs are otherwise fairly non-specific when we talk about lethargy, inappetence, right? There could be a variety of underlying conditions. As well, a lot of behavioural changes we see in our pets may be just that, they may be behavioural because it's fireworks.
We recently had 4th of July here in the US or they're doing construction next door, or you have a new pet in the household, or you've lost a pet in the household, right? So sometimes it can be hard to say whether that behavioural change you're seeing is because of those behavioural conditions or whether there's truly something underlying, that's medical. As far as obtaining objective data, you know, we think that our physical exam is part of our objective data as we go through our, our ruleouts, right?
And we're doing our physical exam on our patients, but even things like heart rate, something that's numerical, right? Something like respiratory rate that's numerical. If we take a patient like this, right, in your practise, And this cat has an elevated heart rate, they have an elevated respiratory rate, and they're reactive to palpating their abdomen.
It may be that they have a GI foreign body and they're really ill, or it may be they have a really stressed cat in your practise, right? So when we obtain objective data, Doctor Dennis Deacola is Really a world renowned clinic pathologist, and he always says animals can't hide their objective data, right? And we think that's especially true in our feline patients, how much they try to hide their illness, right?
But when we can get true objective data during lab work is when we can get more accurate diagnosis, thus make more appropriate treatments, right? And ultimately all of our goals to have our patients live longer, healthier lives. So we talked about the why we perform laboratory testing, but when do we perform laboratory testing, right?
I mean, I'll argue all the time. Right? Certainly in in cases of sickness, right?
When our patients are unwell or they have a myriad of these clinical signs we talked about. But also in health, right? And not just for these older patients, right?
Where we wanna look at their lab data and hopefully find some changes before we see the clinical signs, right before they've sort of crested into the elevated through to we see overt clinical signs and they might be, you know, keeping it under the clinical radar, but also in our younger patients, right? Because if we think about looking at laboratory data, right? And here's an example of a CBC we could be looking at a chemistry.
Example, but notice that reference intervals, you know, are really wide, inherently, they're wide. These reference intervals of health are determined from, you know, 30, 50 different animals, maybe, maybe more. Let's say we're talking about dogs.
It will be from a variety of of breeds, a variety of sizes, whether they're male or female, they're intact or they're spayed or neutered. They can be anywhere from like 1 year of age to 7 or 8 years of age. Right, a whole variety of animals.
Even if they are clinically healthy, that gives you a very wide range of what's considered normal, right? With that reference interval. But for an individual patient, right, that's a, that's a much more narrow reference interval of health.
So we can get some baseline testing when they are young. We know what their target is during health. So again, we can much more sensitively detect those changes, right, as they fall, as they start to rise or fall even before they fall outside of the reference interval.
So what I've done today is I put together 3 clinical cases, ones where maybe what they showed at presentation wasn't ultimately what we found when we did our diagnostic workup, right, to sort of underscore the importance of looking at laboratory diagnostics to get that objective information to best help our patients. So the first case I have is Allie. So Allie is a 3 year old female intact beagle.
Allie belongs to a gentleman who lives in rural Pennsylvania. I believe he's a hunter because he has 3 other hound dogs who live on his property. They do free range, a lot of time during the day, although they do, he does bring them in in the evening.
He does treat them as pets otherwise. He has a lot of property, but it is not fenced in, so while they range during the day, it's possible they get off of his property. Generally, they all seem healthy.
They're all relatively young. And on this given weekend leading up to this presentation at the beginning of the work week, he said he noticed that Ali had he saw dripping blood from the quote back end. And so he really wasn't comfortable talking more about the anatomy of the back end or where that dripping was coming from, but he said he noticed it once when he was outside over the weekend, and then once when she, he was inside, she just come in from outside and he saw what he thought was a drip of blood back there.
So as we think just on history alone, right? We think what might be going on with Allie. Well, she's intact, right?
So maybe that, maybe she's in heat and we talked to the owner about this, and he's actually well aware of her heat cycle. She is not in heat on physical exam, she had no swollen vulva, or obvious discharge. He the vulva.
She has no overt rectal anal disease. The anal glands palpate normally on using a gloved finger on a digital exam, there was no blood detected. So the clinician on the case said, I've seen this before.
I wonder, given that Allie's this beagle with a nose that might have her lack some dietary discretion with her having free range on a lot of property, he wondered actually if she had gotten into. An an anticoagulant rodenticide that may be sometimes we see them present with the hemothorax, right, and respiratory distress, he's seen them present with the hemoabdomen. He's also seen them present with hematuria from that.
So that was actually his first thought process that maybe there's drips of blood from the back end might be urine. So he collected a urine sample on Allie to start her workup and indeed, this is certainly what was considered the dripping blood from the back end. So I think the doctor at the time was spelling a little bit smug like he called it just based on this history and his physical exam.
But as we look at this urine sample, that's the first sample we have, right, to bring into the lab. We run her urinalysis, right? Of course, it is red and opaque.
It's highly concentrated. There's protein in there, right, as we could probably see from the pigmentation, but notice that there are no red blood cells. That's really interesting, right?
This, this overtly what looks like hematuria, right? But as we look at an image from the urine sediment, there, there really aren't any significant numbers of red blood cells, right? So we have been calling this hematuria, right, when he recognised it and we collected the sample, but it turns out this is not blood in the urine, right?
And this is Allie's urine that spun down, right, in the Eppendorf tube. And if she had hematuria and we spun it down, right, the red cells would have pelleted at the bottom and cleared the urine. So now we realise that we were, we were overspeaking, right?
And that we should not have called this hematuria but probably pigmentaria, right? So she had pigmented urine, but it wasn't indeed blood in there. So if the pigmenturia doesn't clear, right, then it's either because of the protein of haemoglobin or myoglobin.
Both of those can come from the blood, concentrate through the kidneys into the urine to make this dark red urine. So then we have sort of a nice pivot test that we can do to distinguish between the two, whether it's myoglobin area or haemoglobin area, and that is to spin down a micro hematocrit tube. So we spin on the micro hematocrit tube, we see in this image that the plasma is pink, right?
And we know that hemolysis causes pink plasma, right, especially if it's iatrogenic, right, associated with our phlebotomy, right? And when we have hemolysis, we recognise this pink pigmented plasma. Right, so this is free haemoglobin that comes from a liced red blood cells.
If it was myoglobin that was pigmenting her urine that came from the blood, it actually clears from the blood much faster and so you don't get this pink pigmentation. So now, in sort of the CSI of what's going on with Allie, it turns out she does not have hematuria, but she had pigmenturia, right? That is causing, is caused by hemoglobinemia, right?
Excess haemoglobin in her blood. So now we think about where that comes from. As I mentioned, it really comes from hemolysis.
That's the only place that haemoglobin comes from is within the red blood cells. So we're keeping that up to date, you know, during the workup of her case and When we first see, we first go back in the room to show him that it was actually urine that was pigmented and so then we said we're gonna check that out and it could have been a urinary tract infection, right? It could have been a bleeding bladder tumour right in there.
It could have been the coagulopathy, right? But it turns out it Wasn't blood, so now we're giving you an update and saying, oh, it's myoglobin. This is coming from within her red blood cells.
Let's figure out what's causing this presumed hemolysis. So the next step that we're gonna do is run a CBC, right? In particular here, we're focused on the erythrogram and we used Nex's labmate CBC here for the data.
And we'll notice, of course, I'm looking at the red cell mass that it is indeed significantly decreased, so she is anaemic. And when we think of our causes of anaemia, we already think we know what's going on as far as hemolysis, right? But if we were to back up and be a little more objective, right, or more thorough, I always think of things sort of chronologically.
When the cell count is low, are we not making the cells, right? Are we licing them in circulation or we losing them from the body? Right?
And so the initial thought was we actually thought she had hematuria, but that she's not losing blood. We now believe it's hemolysis and so it would be appropriate for that anaemia to be regenerative if it's not a bone marrow problem. Now, on labmate CBC and the majority of our in-clinic haematology analyzers, they don't have an automated reticulocyte count, right, reported.
So we can use the red cell indices to help us get some more information about if this appears regenerative. The first that we use is the MCV or the mean cell volume, right? So, younger red blood cells are bigger, so if the mean of all of the red cells has increased, the most common reason would be the contribution of more younger red blood cells or a regenerative response.
The MCH is the main corpuscular haemoglobin and it's a, it's a weight-based thing that's based on the volume of the red cells, so it really doesn't provide much more information beyond the size of the cell. Bigger cells have more haemoglobin, right? And smaller cells have less.
But the MCHC, the mean corpuscular haemoglobin concentration, right, it takes into account, regardless of the cell size, is the haemoglobin concentration appropriate. And in this case, while it falls within the reference interval, it's at the lower end. And so having these results of a contribution within this mean of larger cells with less haemoglobin concentration, that pattern fits well with having an increased number of reticulocytes or young red blood cells.
As well, the RDW that's reported off of labmate CBC, that's the red cell distribution with, and it's the degree of anisocytosis or cell size variability of your red cells. When that falls outside the reference interval, it's either because you have a lot of bigger cells or a lot of smaller cells or both, and again, the most common reason being because of a reticular cytosis. So we can use those red cell indices to help us assess for a generation.
A gold standard, of course, we don't have her ticks reported is to look at a blood film, right? So in this routinely stained blood film, so using a diff quick or other three-part stain, we see these larger bluer cells, those are the young red blood cells. Polychromatophils is what we call them with this routine stain, the stain that the analyzers use, they'll call them reticulocytes, same blood cells.
So we have this regenerative anaemia, right, which fits with a suspected hemolysis that's releasing haemoglobin free into the blood. It's causing the pink pigmentation of the plasma. It concentrates through the kidneys into the urine as a dark red urine that dad thought was dripping blood.
So as they look at her blood to try and figure out what's causing the hemolysis, right? So from this low power view, it probably stands out that indeed this is regenerative, right? We see all these polychromatophils in here.
These bluer cells, and there are some smaller cells mixed in here, right? As we go to a higher power view, I don't know if your thoughts are like mine initially, but my first thoughts were, oh, are these spherocytes, right? So we have smaller red blood cells that lack central pallor, as you look more closely at each of these spherocytes or spherocytoid cells, notice that they have little noses on them.
It's a little protuberances. So I don't know who remembers what these are, right? But these smaller spheres that excels with protuberances, those protuberances are Hinds bodies.
OK, so remember this. So when Hinds bodies, it it's caused by an oxidative injury to the red cells, it causes cross-linking in the red cell membrane, right? So that oxidant, there's these sulphur sulfhydro groups on the inside of the red cell membrane.
So think of a sulphur group with a hydrogen ion on it, and then when the oxidant enters the red cell, that oxidant grabs the two hydrogens from neighbouring sulfhydro groups. It pulls them away. And the sulphur groups form a disulfide bond and they cross link across the membrane.
What happens is you've taken a red cell that has this biconcave disc, right, with this excess membrane for the contents, right? Relative to the contents. And now we cross this cross-linking, right?
And now it's turned that floppy disc into this sort of taut balloon of fluid, right? Because we've got this cross link with that little nose perturbance on it. And now what happens, you know, the red cells are made with this excess membrane and this biconcave discs, so they can move so freely through circulation, all the bends and twists and alterations that happen as they move through the vasculature and particularly the microvasculature, right?
But now that we have these top bags of fluid, right, they're more likely to rupture, right? And we see here, there's, this is a ghost cell, and it's just the red cell remnant after it ruptured. So that contribus to the hemolysis.
OK, so the body's been exposed to an oxidant cause cross-linking of the red cell membrane that made these cells more fragile as they move through circulation that results in hemolysis. Her regenerative anaemia, right, as well as this release of free haemoglobin concentrates through the urine, haemoglobin area, and it looked like dripping blood from the back end. So now, we think of, you know, what, OK, let me back up.
When we're looking on here, I can understand that it might be hard for the average person or even the skilled person at the microscope to pick up on those little noses. And when Allie's case came across my desk, you know, I got so excited, we don't see a lot of, we see a lot of hemolytic anemias, not a lot of Heinzbo's hemolysis. So I got pretty excited and I was showing my technician and she said, I can barely see those, and I said, oh, we have something that we can do.
So new methylene blue stain. It is a very inexpensive stain. It comes in a small bottle.
It's very shelf stable. I think we replace ours like every 6 months. It's a different protocol than using our typical diff quick or Romanowski type stain because it's called a supra vital stain, meaning we put it on top of the living cells, right?
Versus fixing the cells and then putting a stain on top of it. So we're gonna mix equal drops of our EDTA blood with the new methylene blue stain. Gonna mix them in the Ebendorf tube where we let them sit at room temperature for about 10 to 15 minutes.
Two things are going to happen. One, it's gonna cause aggregation and staining of any RNA that's in there. OK, it's gonna cause a re of the RNA as well, when it stains the protein, the haemoglobin, the haemoglobin in the nose of the hinds body, it denatures that that protein denatures cause it doesn't have the rest of the physiology of the cell to stay healthy and it's going to stain that differentially from how it stains the haemoglobin that's healthy in the cell.
So after about 1015 minutes, you'll make a blood film with a drop of that blood that is stained, and then make a film, look at it under the microscope. It'll look very, very pale when you look at it grossly. But as you look under the microscope, and you do see some significant changes, right?
First, maybe you see this dense aggregate material in here, and that's the aggregated RNA in our young red blood cells, right? Those are reticulocytes, right, that we can visualise. That's another tip you can use if you're in clinic haematology analyzer, doesn't report reticulocytes, you can look for polychromatophils, but there's a tendency in our clinics that we Over stain when we use diff quick, we often get too much of the blue stain on there and then it's really hard to see polychromatophiles, which are supposed to be bluer than the rest of our red cells.
So new methy blue stain is a nice mainstay to use to really see those young cells, but it also shows all the blue noses, right? Those Heinz bodies on our red cells. So a nice another trick we can use to identify Hein's body anemias.
So again, what causes Hein's body, as I said it was oxidative damage. So how does the body get exposed to this oxidant, right? As I think of our rule out this, so we've been keeping dad up to date, you know, oh, I got so excited I went and say, oh, it turns out she has a Heinz body anaemia.
This is really exciting and He was a little less enthused, you know, but I, I was delighted and I said, well, let us, let me talk to a clinician, and we'll get some rollouts for you. So the clinician said, what are my rohus again? I reminded him, you know, exposure to onions and garlic are the other braska species of plants.
They are strong oxidants. Acetaminophen or Tylenol, it's especially a problem in cats for two reasons. One, there's a dose dependent response, of course, right, a 10 pound cat is affected more by acetaminophen at 325 milligrammes or whatever versus, you know, an 80 pound dog, right?
But also because they have more of those sulfhydro groups, so they are more susceptible to Hein's body formation, that cross-linking. Oops sorry, zinc, right? So in the US we think of pennies that are minted after 1982, have a high zinc content, but there's zinc, of course, in some zippers and other cogs.
We had a dog present who had ingested, some cog thing from a bike, that presented with a Heinz body anaemia. We have zinc in a lot of creams, skin creams or like those baby butt pastes. They have zinc in them so dogs who either lick our skin or get into diapers or eat the tube, can get exposed.
We have repeated propofol administration in cats. I had a, got to see this back at the University of Georgia. We had a fractious cat who had a deep loving wound, and they had to sedate the cat each day to fix the wet to dry bandages, right, to replace those.
And so they gave propofol day after day. I can't remember how many days we got in before we started to see this regenerative anaemia from Hein's body formation. There are some inherited enzyme deficiencies that can result in that, but I have yet to see one in practise, so I think it's unlikely you'll see that.
Something I have seen more of in practise has been some endogenous production of oxidants. And we see this, I see this most commonly in sick cats. Sick cats with unregulated diabetes mellitus.
Right, so especially those that come in in DKA. Cats with overt lymphoma. So not a GI lymphoma, maybe a small cell lymphoma, but a typical large cell lymphoma where they are sick with it, as well as cats with unregulated hyperthyroidism.
In all of those cases, there are sometimes they will develop a progressive anaemia while they're hospitalised with us before we get their treatment under control. And it's an unfortunate consequence of the underlying disease process because I I can't fix it. I see the Hinds bodies there.
I see the appropriate response of the marrow. We have to fix this condition for that to right itself. So I go over this with the clinician.
He goes back in the room, talked to the owner, and he said, so we know she's been exposed to an accident. Our goal now is to make sure we stop this, right? So any chance she got into any onions and garlic?
Do you cook with them? Does she get in the garbage? Maybe the compost at your house or our neighbours, and he did not think so, not, he did not cook with them.
He did not have a compost pile. He does not use Tylenol or have it in the house. We asked about zinc and, and things get a little weird here.
He brings up zinc and mentions that in the US and coins minted after 1982, have a high zinc content. And when he brought up pennies, I'm sorry, pennies specifically after 1982, the owner says, oh, my cat ate pennies last year. I like, what?
I just, that just seemed really weird to me, but at least it got him on board. I think he was maybe critical about this whole process. So we asked to take a lateral radiograph.
And indeed, as well as their cat from last year, Allie had 4 coins in her stomach. So we started with endoscopy to try to remove the four coins. I don't know if you can appreciate, but these 3 here were actually trying to wedge down into the pyloris, and they were really challenging for the doctor to grab.
So he took her to surgery for a basic gastronomy and removed. These four coins, and this is the other interesting twist to the story in that, well, I see the nickel, the US nickel has nothing to do with zinc. It doesn't have a high zinc content.
It's more like your euros, your coins in the UK, but here on the pennies, so the, the high zinc content is those pennies minted after 1982 and it's on the inside. So they actually have to sit in the GI tract for a bit of time till the, the stomach acids erode the surface of them, and then it exposes the zinc and it causes a lot of gastritis, and that bleeding in the gut allows that easy translocation of the oxidant into the blood. But these were not the pennies that caused it.
As a matter of fact, I rinsed them off and looked and they were all minted before 1982, which I thought Also really unusual. Like, what are the chances that the ones she got into was so old, but also, what are the chances she has a Heinz body anaemia, known penny ingestion, right? And that they're not related.
So in further talking to the owner, he did say she had noticed twice over the weekend that she had vomited. She could have vomited more than that. She's unregulated a lot when she's outside on her own.
So my guess is she either vomited up or maybe even pooped out the implicated pennies that actually were minted after 1982 that the stomach acids had broken down the outer coating had exposed the zinc, exposure to the oxidant, caused that cross-linking in the red cell membrane. That Heins body formation, the rupture of the red cells as they move through circulation, release of all that free haemoglobin, hemoglobinemia, right? And then the hemoglobinuria that we saw.
So to remember when we see red urine, right? Remember that we should call it pigmentary until we know otherwise, right? We spin that down.
If it doesn't, if it clears and you see a red cell pellet, you are right to call it hematuria. If it doesn't clear, it's either myoglobinuria or hemoglobinuria, and they come from the blood. So now we knew she had hemoglobinemia.
Right was we spun down her microhematica tube and saw the pink pink vented plasma. We know that comes from red cells. We do a CBC and we did have evidence for hemolysis, a regenerative anaemia.
As we look for the cause of that, we did see Hein's body formation, right? And we found out it was because of the same toxicosis. All right.
Next case. Bella, Bella is a seven year old female state Labradoodle, and she has a history of seizures. She's been on phenobarb and recently they increased her dose cause she had a couple of breakthrough seizures.
She hadn't had seizures actually in years, but she had a couple of breakthrough seizures. I believe she must have called into the practise. She talked to the doctor on her case.
They did say you can increase the dose and they made an appointment for a follow-up visit. She comes in for this particular visit for an exam and because they had run out or they're running out of their phenobarb now that they'd increase the dose, so they were here for prescription refill. Now, the doctor noted on physical exam that Bella had gained weight since her last appointment.
He noticed some thinning of her hair over the dorsum, and the owner reported she was polyuric and polydipsic, right? So she was urinating more and drinking more, that they attributed to as they remembered when they loaded her on her enobar dose, they thought it was, they attributed to the increased dose of phenobarb because they remember her doing the same thing years ago when they started medicating her. Now the clinician in the case said, I mean, maybe, maybe it's because of your phenobarb dose, but maybe something's changing in that.
Like why the breakthrough seizures, right? And what if the PUPD, the weight gain, the hair thinning? What if it's because of something else, right?
I think we should do diagnostics rather than just continue to prescribe her phenobarb at this increased dose. So the doctor recommended doing blood work and a urinalysis. So they drew their blood and they bring her back to ultrasound where they do the ultrasound guided cystocentesis.
So here's the table that they use, the trough that they used to use ultrasound guided scisocentesis. This is where I was sitting at the time at my microscope and my computer. I'm sitting here looking at cases and I overhear the technicians talking to the doctor about the case.
And they were saying how she was PUPD. The owner was noting he said, yeah, I'd feel better if we check the urine in her blood and see what's going on. And he's struggling for a bit because he says I can't find her bladder.
So he's looking at this is one of our skilled clinicians and so he's quite seasoned with ultrasound. I'm trying to say he can't find her bladder and, and I was just sort of listening to this over my shoulder and I'm looking at Bella's case trying to wonder maybe what's going on. And I found images from last year where we had captured images from her urinary bladder.
It's not like she had a pelvic bladder or something weird like that. So we've seen it before. And yeah, I hear him say, oh well, she Must have urinated before she came in.
You can bring her back to the room. They'd already had a blood sample. And I said, I mean, she is polyure and poly deficit, not having a urine sample to even get a urine specific gravity to me seems like it'd be such a mess.
So I, I bought it in a little bit, of course, as the clinical pathologist, and I said, could we try to even get a few drops of free catch urine just to even get a urines specific gravity because she's P PPD and what if, you know, her Renal values are elevated and we don't, we can't pair it with a specific gravity, a paired time sample. So he said that's fine. Technicians go out to take her, see if she'll even go to squat a little bit to mark or anything to grab a few drops of urine, and they returned to the lab with this, and they said, and that wasn't all Doctor Brown, she peed so much more.
So we call the doctor back into the lab and we said, this is Bella's urine, and he said, what? He said, go grab me a lateral radiograph, like, what's going on? And this is Bella's lateral radiograph, and this is the first time I had seen this.
This might be something that you're familiar with. Obviously, this is her bladder here, but this is mp hy se ma t ous cystitis. So she's got a bacterial infection.
It's gas-producing bacteria in the lumen or in the wall, right? And so they're producing gas and it's showing up as this opacity in here. So on ultrasound, it matched the soft tissues around it and we didn't get that typical black fluid-filled bladder.
So really glad that we grabbed that urine sample. Obviously very pathologic. So as we process that urine, her free catch sample, it's red and cloudy, there's protein there because of course, all of the blood in there, and she does have a lot of white blood cells as well, so a pyuria and rods were seen.
Now, we Sometimes can feel confident when we're looking at a urine sediment about seeing bacteria, but my preference of any bacteria suspected is that we do a line prep, right, and we stain it cytologically like we're used to looking at our cytology specimens. So we take a drop of the sedimented urine, right, and we act like we're making a blood smear, but instead of pushing it all the way out and making like a fed. Edge, we wanna push all those formed elements and then lift straight up and it'll deposit all the formed elements of the urine in one line, right, that we can stain routinely like with the quick stain that we're used to looking for bacteria, right?
And then as we look at that microscopic umbella, of course, we see all of those rod-shaped bacteria to confirm that the red cells, the white cells, etc. So she definitely has a bacterial infection. As it turns out, this was not her first UTI and not her first UTI of late.
So just this is maybe in early spring of the year and just in December of that year, when I shown the other image from her ultrasound, she had been in for urinary tract infection at that time as well. So they submitted urine culture and sensitivity. It grew E.
Coli, not surprisingly, and fortunately, it was sensitive to all of our commonly used antimicrobials for the urinary tract. But because this wasn't her first rodeo, I think we looked a little more critically at her chemistry panel. So we have this chemistry data off of Ante's labmate CMXT.
We noticed here we look at the liver enzymes specifically. The LT is minimally elevated, right? But alkaline phosphatase is significantly elevated.
And we had seen that in her previous flutter, but again, it had been attributed to her being on phenobarb, right? But now we're looking at a little bit differently, right? We have a dog who has recurrent infections, right?
Who's poly polydipsy, who has hair thinning over the dorsum and some weight gain, it's sounding a little bit like an endocrinopathy. Right? So in performing a low dose depression test where we're going to, you know, get a baseline cortisol.
We get an injection of a low dose dexamethasone. We do a 4-hour cortisol check and then an 8-hour cortisol check, and if we graph out her results, her results started on the higher end. She did, so I'm sorry, so normal is here in red, this is Bella here in blue.
She suppresses to more than 50. Percent of what her baseline is at the 4 hour mark, but at the 8 hour mark she comes out of suppression, right? And so that pattern of suppression, less than 50% of her baseline at 4 hours, but coming out of suppression at the end is classic for pituitary dependent hyperadrenal corticism or Cushing's.
So good thing we did this workup, right, or she's probably gonna continue to have both the clinical signs as well as the recurrent infections. And as well as we look back at her chemistry profile, we see her elevated cholesterol, right? The most common reason in an ageing dog for an elevated cholesterol is gonna be hypothyroidism, right?
So, on the labmate IA we're able to measure a total T4 and it came back low. Right, but we have to remember, right, if it came back normal, we could rule it out, but coming back low, that could be hypothyroidism, but it also could be you thyroid sick. She has other concurrent conditions.
Right, so then after the reference side, we requested a free T4 by equilibrium dialysis and that also came back decreased. So that combination of a low total T4 and a low free T4 by ED with those clinical signs that we are seeing that could be attributed to hypothyroidism supported the diagnosis. So for Bella, while they mentioned PUPD they were gonna write that off as being her on her medication and the increased dosing and so they were just wanted to refill and take her home, right?
But the clinician delved a little deeper to look up under that PUPD what could be going on. Found out she did have a urinary tract infection. It's amazing that they never saw any of that.
Bloody urine in the house, she obviously had no accents and they weren't able to look at it outside. Because it was recurrent, we looked a little more critically at our chemistry profile, right? That elevated alkyn phosphatase and mildly elevate ALT that had been attributed to the phenobarb.
We're now wondering, is it because she's cushionoid, right? And the cholesterol level being high, we wonder is it because she's hypothyroid. So indeed, we do the low dose dexamethasone which supported the Cushing's diagnosis and the total T4 and free T4 which support her hypothyroidism.
And the last case that I will do for you guys, you get my time, is Mac. So Mac is a 12 year old male neutered mixed breed dog, and whenever cases take an interesting twist, I always look back to think, what did they think was going on, right? Like on Allie's case, right?
And so for Mac, I looked back and said, OK, he was referred from the referring veterinarian for an irregular spleen, which I'm going to guess is based on ultrasound imaging based on what we saw, but He was actually quite a thin dog and it's possible they may, it's this irregular, they may have palpated it. And they said possible lymphoma, and he had no peripheral lymphadenopathy, so I don't know if the possible lymphoma was based on the irregular spleen looking like it had an infiltrated disease. That is my thought process based on what we ended up finding out.
So on a history of increasing anorexia and lethargy, by the time Matt got to us, he is so uncomfortable in his abdomen. He remains in prayer position, like elbows down, his bottom in the air, right stretching his very painful abdomen. Really throughout his physical exam, we left the room, we came back, he's still in that position, right?
And when they were palpating him abdominally, he would just grunt. It was really, really sad. So presenting to us as an acute abdomen, perhaps appeared differently when he first was at the referring vet, and I think I have a story for that.
So in looking at Max haematology data from labmate CBC, right, there are most significant changes. They look like they're up here in the leukogram, right? So as we focus on the leukogram specifically.
Now, things that I want us to pay particular attention to. I love the CBC as our first test of overall health for a patient, right? So I like it in wellness and I like it in sickness because, you know, the CBC is not one test, right, of course.
So the gives you 20 to 25 different variables about the blood, right? It gives us cell counts and it also tells us about the nuances of the red cells or any significant changes patterns in the leukocytes, right, or any thrombocytopenia for us tick-borne disease is big in our area. Right, so there's so much data that we can glean from the CBC itself, especially if we pair it with cytograms that are available.
Labmate CBC has some wonderful scattergram for the WBCs that you can start to recognise patterns on to help you see an automated look at the cell morphology as well as histograms to look at red cell changes or platelet changes, right? So we add the, it's haematology, it's really not just the CBC data, right? But augmenting it with those cytograms and then with the blood film, right?
And between those three, I, I don't think that you could find a sick animal that didn't have some significant change that we could find in there, right? So I love it as a screening test and, and affordable, and if you do it at point of care really rapid, right, so we can get, especially now Mac got referred to as a sick patient, right? So that affords us the opportunity of an owner that's expecting to do a diagnostic workup.
Right, but potentially, he had had lab work done, you know, a day before or two days before or whenever he came from the referring vet, right? But with the CBC this changes in hours, right? So we absolutely are going to want to repeat it to see what's going on right now.
And there are some significant changes and nuances to Max that are worth discussing. One pattern that's so important is recognising a lymphoenia and eosinopenia. So that combination of low lymphocytes and low eosinophils is a glucocorticoid response or stress response, right?
So not a stress response like an excitement, epinephrine-induced response like a cat who's just been travelling, who's on. Or a challenging phlebotomy, right, those sort of excitement responses, they cause a lymphocytosis, right? Because you get the smooth muscle and the spleen contracts and so all those lymphocytes from the spleen get pushed out into circulation, right, as you do your phlebotomy.
So in this case, we're talking about a stress response like the body's going through something big deal, right, significant, and the cortisol levels then have risen in an attempt to survive, you know, this battle, whatever they're dealing with clinically. And so, in this case, recognising the lympopenia mopenia tells us we have a sick dog, right? And what's interesting, pair with that a monocytosis, right?
So a monocytosis, especially a solitary monocytosis, but also not just a marginal one, right? This is significantly increased is that should always be concerning. I'll tell you for a bunch of reasons.
One is, of course, if read accurately, monocytes, you know, they become macrophages in the tissues and they're there, there's tissue destruction remodelling, right? So that's really concerning for some tissue destruction, right, that we need to find on imaging that always prompts me to do imaging when I see a monocytosis. Monocytes are our youngest, most immature cell that that's released in health.
If you had an acute leukaemia or other young immature cells, they're often counted as monocytes by our automated technology. So that's another thing that would help you catch is if you had maybe some immature cells, leukemic cells in circulation, as well, . Yeah, I said those are reactive lymphocytes might be counted as monocytes, any larger, more immature cells, so they should always raise concern, tissue destruction, immature cells, what's going on, and it always prompts me to tell the ultrasonographer to start looking, right?
Finding that site of tissue destruction if it's not outwardly apparent. And then as you learn about Mac's cases, we walk this, Mac has profound inflammatory disease, but note that his total white cell count and his total neutral count are within the reference interval. And this is not because of poor performance allowing CBC.
These are accurate, right? But it's an understanding of what we're sampling is a point in time, right? These blood cells, somewhere between what the tissue demands are and the bone marrow's attempt to meet those demands.
So we take an example, like if you draw my blood today and you look at my total leukocyte count, my total neutrophil count, right? And then some, then I'd have marked trauma, right? Let's say I But traumatically, I don't know, amputate my arm.
OK, so major inflammation, right? Then you check my blood soon after and guess what, even with a lot of blood loss, those numbers will stay the same, right? Because it's the concentrations.
So you lose blood, they stay the same. But then the Whole tissue area starts crying out with inflammation, right? So the cytokines calling the cells there to help fight all this destruction in the area.
So, for a moment when you check my blood hours later, those cell counts are likely low because they're all heading to that tissue focus of inflammation. Right, but as we start to catch up now, the bone marrow is going to release, we have the circulating pool of neutrophils, right? There's an equal number of mature neutrophils waiting the bone marrow, right, the storage pool of mature neutrophils.
They get released pretty readily. So now they're gonna replace those ones that you lost to the tissue. Right.
So then we check my numbers again and they might be right within the reference interval back to where they were, right? But those mature ones that came from the marrow, they're all still going to the site of inflammation cause that has certainly hasn't slowed down, right? But now the bone marrow is gonna start releasing young ones, right, to try and meet the needs cause the inflammatory cytokines are still so high.
So the total white cell count and the total neutrophil count can stay in the reference interval even when there's intense inflammatory disease, as we will see here from Mac. So we want to look at a blood film for further information and the one thing we'll note is that he does have bands, right? So bands are truly the hallmark of inflammation when they're caught in the blood.
Right, because it means you've exceeded the mature neutrophil circulating pool, right? They've already been used and gone to the tissues. The, the storage pool of mature neutrophils, they've already been utilised in the blood and I've gone to the tissues.
Now we're still calling for more cells such like young ones, bands, sometimes metamylocytes are being released from the marrow and they're in circulation. So catching those in circulation lets you know you have intense or overwhelming inflammation. Notice also there's some changes to the red cells, some anthocytes or poiulocytes, right, those misshapen red blood cells.
To me, they say these red cells are moving through some. Highly vascularized tissue that has some significant endothelial changes, right? Something within that vasculature is causing a disruption of those red cells, alterations of their membrane, and so again, the more I see of those, the more I tell the ultrasonographer, find me that tissue, right?
Because it's gonna be GI tract, liver, spleen, kidneys less commonly, one of our highly vascularized tissues, right, has some significant abnormality. In this other area of the film we see more of those acanthocytes throughout, right, as well as this young band, right? If we look closely at his cytoplasm, there's some toxic change there.
So toxic changes include a cytoplasmic greying or basophilia, doli bodies, this angular glue inclusion in here, and then this foamy evaculation, right? So those are signs of toxic change which means we have a systemic inflammatory process. And so this is not just inflammation that we would Get with surgery with some trauma, right?
But this is gonna be inflammation like a bacterial infection, right? Fulin and autoimmune disease, things that cause high fevers, right? And so I always worry about a gram-negative bacterial infection first and foremost, because often they're GI in origin and often they're surgical, right?
So keep an eye out for toxic change. And then I came across one of these and I was like, what is that cell? Right, so, big young cell with big pink purple granules in there, big round nucleus, and it's not a basophil, right?
Cause the baso has a ribbon-like nucleus. So my first thought, maybe I was swayed a little bit because they thought he might have lymphoma and he has irregular spleen, and I thought, well, maybe this is like a large granular lymphoma. Maybe it's LGL lymphoma.
That was my first thought. And then as I looked on a little more out at the feathered edge, I saw three more of these and I thought, oh no, you're getting a lot more like mast cells. So mass cells in circulation, what does that mean?
Well, there was a retrospective study done that was published in JVIM back in, 0, 2007, 2009. And it looked at mastocythemia in dogs and cats, so mast cells in circulation in dogs and cats, and the dogs, they found they compared dogs with mast cells in circulation that had mast cell neoplasia and those that didn't have mast cell neoplasia, and the majority of mast cells in circulation came from dogs without mast cell neoplasia. And as a matter of fact, those had higher numbers.
There was just nonspecifically associated with other inflammatory diseases, versus those with mast cell neoplasia only had the lower numbers in circulation. And in cats, all the mast cells, all the cats with mast cells in circulation were cats with mast cell neoplasia. And my thought might be on that is that those cats had like that visceral form or that splenic form and so maybe more likely to come out in circulation.
So for Max's case, right, he has a mastocythemia. He has known inflammatory disease, but I had came across a bunch of them. So I actually didn't know what side of the table he was going to fall.
Mac's abdominal ultrasound showed this indeed irregular spleen, right? So it's billowing in here, something infiltrated with this scalped margin throughout. And also we found a pocket of ascites, right?
That's marked as ascites and what an opportunity when we see some free fluid in the abdomen to tap that fluid to get some pretty non-invasive but informative details about what's going on inside the patient's abdomen. So you can run your fluids, assuming they, you put them in EDTA so they're not clotting through really any of your haematology analyzers to get a nucleated cell count, right? So we ran a fluid through the lab mate CBC.
Again, it's not validated for that, but what we're using, we don't want this whole CBC report. What we're really using it for is to get a total leukocyte count, a total white cell count or inflammatory or nucleated cell count, which case markedly increased here at 21,000, right? So a fluid in health would be if you could tap it like up to 2000 cells per microliter nucleated cells is up at 21,000.
Right? And the majority are identified as neutrophils. We need to confirm that, but that's not like a marked inflammatory focus in the abdomen and not too much blood.
We'll also get a red cell count off that as well. So notice if you do run, if you do a labmate CBC and you run a non-heme fluid through, just note it'll always give you a flag of a low sample volume unless it's a really bloody fluid because it uses to identify the sample volume being appropriate, it actually measures the haemoglobin in there. So there's not a lot of red blood cells, it'll save a low sample volume even though that's not the case.
So as you make a direct smear, we do appreciate that it is a majority of neutrophils with some large mononuclear cells in here, and we make a sediment smear as well and we look in closely and we do indeed see these rod-shaped bacteria. Intracellular rod-shaped bacteria, most common place of origin is gonna be the GI tract. So now he, we know he has some compromise of his GI tract, and something going on with the spleen and a mastocythemia.
We don't know how they all tie together. Mom allows us to do an exploratory surgery. We take out his spleen and look at that.
I've never seen a spleen that diffusely nodular throughout. We grabbed one of those nodules. We made a scraping from the inside of that, put it on a slide, stained it routinely, and those are just wall to wall mast cells, right?
Obviously, the origin of those we saw in circulation. So Max's acting more like a cat, right, with a splenic mass on neoplasia. And then we did see he had a perforation in his pylorus that caused the septic abdomen.
We trimmed that tissue around there and we sent out for histopathology and it looked like it was just a perforation, probably from an ulcer from all the histamine from his mess on the plasia. So Mac presented with an acute abdomen. We saw a toxic change in a mastocythemia on the blood.
We had an effusion in the abdomen. We ran that through the analyzer to find out that he had significant inflammatory focus. We looked at it cytologically.
It was septic. He had a GI perforation noted as a cause for septic abdomen as well as splenic mass cell the plasia. So if you've enjoyed the stories, I hope that you will check out our podcast.
So together with my right hand person from the hospital, Jessica Wilson has, she's an amazing technician who also now does stuff for Antech with me around point of care diagnostics. We put together tales from the lab, so we tell these stories and other similar ones. It would be awesome if you guys wanted to listen and learn more along with us.
Ali, thank you so much. If you will just leave that slide up because I'm sure the audience are as enthralled as I have been listening to you and they need a little bit of time to come back to Earth and grab their phones and scan that QR code and that. So, that is fantastic.
Thank you so much. As always, your, your cases are brilliant and your insights into reading the results is just amazing. So thank you so much.
Thank you. I do, I've been a vet for a very long time. I'm not gonna say for how long because it'll embarrass me.
but one of the things for me with Clin Path, why it is so fascinating yet frustrating, is because 1 + 1 very seldom equals 2 until people like you get hold of it. And I'm hoping that these podcasts are going to start shedding a bit more light like you have tonight. I do think we are also fortunate that we are using a lot more AI in the field, right?
And so we are taking things from where we have to teach it in our classroom, where these advanced nuances off of a blood film or changes that now we can train AI a bit to pick up on some of that with us much more sensitively, right? Because if we use AI. Look across blood clones or blood samples, you can look at tens of thousands of cells, right, where historically we've been looking at, you know, 100 on a nucleated cell count.
And a start into that is to try and use any of your cytograms if you have them at point of care because they do look at a lot more cells and we can start to recognise the patterns that will help. Yeah. And I think that's where a fantastic support like a company like Antech comes into this.
And a huge big thank you to them for sponsoring tonight. But when, when you get great companies like Antech starting to delve into this, and helping and supporting fantastic clinicians like yourself, It's, it starts to really build and develop the understanding of the field. And I think the AI is just a, a cherry on the top that, that gives us that bigger sample that you're talking about.
That's right. Yeah, that's fantastic. So folks, I would encourage you all to scan that QR code.
Make a note of the, the web, address for the podcast homepage, and go and listen, to Holly and her partner to, hear more and more of these fascinating stories where, of course, 1 + 1 seldom equals 2. Also, Dawn, thank you very much, has just dropped the link to the survey Monkey in. Some of you that have been on with me before will have heard me say that the webinar vet is our channel.
It's your channel, it's my channel. And the way we know, and I say we, or the way the office knows and Dawn and, and her fabulous team with Anthony and them knows what you want is to complete a survey, monkey. And fill in information on, yes, please, yes, please.
Let's get Doctor Holly Brown back again. And other topics that you want. Because really if you give that feedback to the office, they can then get more and more fantastic speakers like Holly on board to be listening, For us to be listening to and, and gaining information from.
So, yeah. Shameless plug for the survey monkey. But it really is a valuable tool, as Holly was saying with with the, the Clint Pasta.
It's a valuable tool for the webinar vet. Let's move on, Holly, to some questions, if you would. .
We have one that's come through that says, how do you distinguish how jolly bodies from other red blood cell inclusions such as Mycoplasma foetus? Yeah, great. So when I think of what, so in addition to sending it to a clinical pathologist, right?
I do think that new methy blue stain is a nice trick, right? It's really inexpensive to have in practise and it's quite specific for looking for Hinds bodies, reticular sites, young red blood cells, but as far as inclusions for really identifying those Hinds bodies because they do stain differentially from the rest of the cell. Now, what can be a real challenge with mycoplasma is those are much smaller, right?
And I think sometimes one thing that can be really confusing is if you have any water artefact on your slide, when you look at it with some oil, you're gonna see some refraction around the outside of the red blood cell that can look blue at different densities. And so being careful. That we have no water in our stains.
That's often where it comes from, making sure the sides are dry before we stain them, so that if there's really blue cocy around that edge and they'll be much smaller and much denser, right, than anything like the Heinz body on new methylone blue stain, that will help you pick those up and distinguish them from the water artefact. Make sure they aren't refractile, right, as you scan up and down. And then it turns out those mycoplasmas, they're hard for everybody, especially if there's any stain precipitate.
And fortunately, there's some good PCRs. So if you have a case of regenerative anaemia in any cat, it's not a bad idea to send off to the reference lab for PCR for mycoplasma because it can be hard to see. Yeah, they're a nightmare.
I, I did most of my, well, all of my training and most of my formative years in South Africa. And I had to say I got a bit caught out on that last one. I went, oh, I wonder if we go down the Elia line here.
Because they do, you know, your mind starts to, to wander along those lines. But fortunately, PCR is there to help us in a lot of those strange cases. Absolutely.
And also wonderful people in the, the reference labs. I think we all, as clinicians in practise, we forget about the fantastic resource that is the clinician in the, in the reference lab, like yourself, that can, you know, just, back, back up what we're thinking and, and have a discussion and, and, you know, you can get lost in, in a lot of time on the telephone sometimes, but it's, it's fascinating stuff. Yeah, and you really do get to move the needle some, right, when you have those conversations, so.
Yeah, well, it's, it's not only benefiting the patient. You as a person and a clinician, you grow because you're learning, you know, again, 1 + 1 is not always 2. Yeah.
Right. And of course I, I learned from the conversations as well, right? So I always want to know what you all are seeing in the field as well.
Yeah, yeah. Another great question that's come through. Would testing straight for free T4 rather than total T4, then free T4 help confirm hypothyroidism?
So starting first with free or just total? I missed that. Yeah, so we just don't, to my knowledge, there is not a free T4 available for in-clinic testing.
So we are talking, we're talking about screening our patients and we are doing our lab work in clinic first. Total T4 is your first screening option, right? And it's nice because we can do it for both dogs and cats, right?
We're looking for hyperthyroidism or hypothyroid. But free T4 at the reference lab is, is certainly the next reflex test, right, to further investigate. So often we'll screen with a total and then go to free T4 and sometimes even TSH if needed, because sometimes you have to tie that all together because as Brusa said, 1 and 1 doesn't always equal 2, right?
Sometimes there are some exceptions to those. Yeah. I must be honest, I, I've, always been a big fan of dumping TSH in there because sometimes you get one of those results and you just go, hmm, that's not even a whole number anymore.
And then, and then when you start putting TSH in, all of a sudden it starts to get a little bit more. Clarity coming through. I agree and I think especially, you know, I'm such a fan of doing in clinic diagnostics, but when you have to send the reference up, if the owner's already waiting for that expertise or advanced testing and you're gonna be calling them back with it, you'd like to be able to tell the whole story, right?
So having both of those would be nice. Yeah, fantastic, fantastic. Folks, just a reminder that we, we are running a little bit low on time here.
But we have recorded this webinar, and Dawn and the fantastic crew back in the office will have it up on the website. I used to say within 24 hours and put a bit of pressure on them, but let's be nice. In the next 24 to 36 hours, it will be up.
So you will be able to go back and listen to Holly again. And, and re go through those cases, rethink them through while you've got them and you can pause them and everything else, which obviously we can't do, live in these sessions. Holly, I think sadly, we have run out of time.
I could talk to you all night about this. But a huge big thank you to you for your time tonight. We, really do appreciate it and I always love it when I see your name pop on the list, because I know it's going to be a fascinating webinar.
So thank you to you. Thank you so much, first. I appreciate it.
A huge big thank you to our sponsors tonight, Antech. They really are a very, progressive company when it comes to this sort of thing. And, they do, really support us.
So, we should be supporting the companies that do support us. So thanks to Antech for all their support for tonight. And remember that Tales from the lab podcast.
I know that, my first one is going to be tonight. And, yeah, if I don't get much sleep tonight, Holly, it's your fault. OK?
Hopefully a lot of laughs and some knowledge too. Yeah, fantastic. To all of you, as always, thank you so much for your time.
I hope you enjoyed it as much as I did. And last but definitely not least to my wonderful controller, Dawn in the background for making everything happen seamlessly. From myself, Bruce Stevenson, it's good night.
