Peripheral blood film evaluation: a critical component of a complete blood count (CBC)

Hi everyone, uh, thanks for joining today. Uh, thanks to Franchesco for having me speak. Uh, my name is Danny Heinrich, and I am a clinical pathologist at the University of Minnesota College of Veterinary Medicine. Um, I'm an assistant professor here and the lab director for the Clint path lab. And today I wanted to go over a peripheral blood film evaluation with you guys and just go over some case examples of why this is so important and an often overlooked part of our CBC, or our complete blood count.

And I'm not going to keep this camera on the whole time, just because I find it distracting. Um, and I don't want to look at myself give a speech. Um, just a little bit awkward, so I'm gonna hide myself. Um, but hopefully you guys will still be able to hear me just fine. All right, so let's get going, and I will try to keep this under an hour. That is the goal.

So I first wanted to start off by giving a thanks to my, um, two of my residency mentors, Jet Overman and Leslie Shy. Um, we have all contributed to this lecture, um, and used it in the past to some degree, and so I certainly wanted to acknowledge their contributions. And I did not, um, have all of these cool cases come across my desk myself.

So what are we going to cover today? We're going to talk about why we're performing a blood film evaluation. We're going to talk about the goals of in-clinic blood film evaluation. We're going to do some general recommendations, um, and approach to in-clinic blood film evaluation. And then what I think is the exciting part are the case examples. And I have six or seven cases that I hope to get through, um, with you guys, kind of showing the importance of blood film review. Um, what are we not going to cover? Well, well, first of all, I'm operating under the assumption, um, that most of you guys have, you know, some degree of training, whether you're veterinarians or nurses, technicians, students, assistants, um, have probably all had some exposure to hematology. So this is not going to be so introductory that, you know, I'm reviewing what's a neutrophil, what's a lymphocyte, um, you know, what's a CBC, things like that. I, I am expecting some operating, um, working knowledge, um, just, it'll make this a little bit easier. But, um, um, other things that I'm not going to cover, we're not going to do an exhaustive list of morphologic changes or infectious agents, um, that, you know, can be, um, you know, numerous lectures in in of itself. But you certainly can find a lot of that information in the plethora of veterinary, um, hematology atlases and textbooks that are out there. We're not going to go over analyzer methodology. So, you know, all of the hematology analyzers that are marketed, kind of work slightly differently, um, and again, kind of going through how each one of them works is beyond the scope of this talk and would take a long time.

The other thing we're not going to go over is scatter plot interpretation. So you may have noticed that a lot of your, um, CBC machines, um, spit out little tiny graphs that I know, at least when I was in general practice, I ignored them, um, and really didn't know what they meant at all. Um, but they actually are quite helpful. They can help, um, you look for errors with the analyzer. They can help you, um, identify some potential diagnoses even before you've began to look at the blood film. But again, um, that could be, you know, a few hour lectures in of itself. Um, so this is an example of the scatter plot that our analyzer spits out at the University that we utilize. But again, if you're, if you're interested in that, um, tell, um, Franchesco, and he can, um, hopefully, um, organize some more talks related to that, um, because it is an entire kind of science in of itself.

So why do we evaluate blood films? I personally think it's fun. Um, it's a little bit monotonous sometimes, but I do think it's fun. And this is, um, you know, a veterinary cytology Facebook page, right? And so when I was talking to Franchesco about organizing this, um, you know, I asked him, would it be okay to give this talk? Because this isn't technically cytology, but, you know, blood smear review is kind of cytology to some degree, right? We are, you know, methodically kind of looking at, um, cells under the microscope and classifying things and grouping things to get to a diagnosis. Um, and I kind of feel like sometimes, you know, hematology is like the redheaded stepchild, um, of cytology, but, um, it can be very diagnostically beneficial, um, and it really has an important part in veterinary practice. It's a critical component of a truly complete CBC. It can provide immediately clinically and diagnostically relevant information. Um, and something to remember is that hematology analyzers, even, you know, the really big expensive ones like the ones we have here, you know, that weigh, you know, hundreds of pounds, they, they don't provide all of the information that we need. They just can't, okay? Um, they tend, and they also tend to be the most erroneous in our sickest patients, which is really unfortunate because that's when we want the analyzer to work the best.

So a truly complete CBC includes both numerical and microscopic data describing the cellular elements of blood. Most numerical data is generated by a hematology analyzer, and then information regarding morphology, things like, you know, infectious agents, you know, plastic cells, shape change, that should truly come from blood film evaluation. This is actually, if you haven't seen these, this is like the size of some of our reference level analyzers, so they're quite big. So, you know, your, the little in-clinic analyzers, you know, are maybe, you know, this big for for size comparison. So even these massive, really expensive, heavy things can't do everything that we need them to do.

So generally, hematology analyzers are good for counting our cellular elements. So it can do things like our white cell count, our red cell count, um, our hemoglobin concentration, our hematocrit, platelets, questionable, right? Because we all know that platelet clumps are the bane of most veterinarians' existence. Um, so if you get a low platelet count from your analyzer, the first thing you need to do is check your, um, slide or your tube for platelet clumps, because if there are some clumps present, that consciously drops your platelet count. Data regarding morphology, um, including, you know, your white blood cell differential, the presence of things like bands or metarubricytes, nucleated red blood cells, is less accurate to unreliable. Um, so you can see here, this is, um, some analyzer data, and it's growing an asterisk next to the differential counts here and to, um, you know, the presence of bands, a morphologic change, and it's saying suspect, and then I don't have it shown here, but it probably says confirm via blood film review. So some analyzers can give us a little bit of morphologic information, but it can't confirm it, and we need to do it with a blood film.

All right, so again, these expensive analyzers or, you know, the more inexpensive point-of-care ones still are not perfect, and we need to be aware of that. So it's not going to look for things like red cell morphology. It can't definitively identify a left shift, you know, toxic change, organisms, you know, atypical cells. It can suggest the presence of certain things, but it cannot definitively identify them.

So if you're doing kind of in-clinic hematology, so if you're doing your own in-house CBCs, it is up to you to make this a standard part of evaluation. I am a fan of doing a blood film review for every patient, every time, which is, we have the luxury of being able to do that here at the University because we have a, you know, a small army of medical technologists. But, um, you know, it is really important out in general practice, and I understand, I was in general practice, you know, for a few years before I went back and did my residency, and so I know that we all get busy, um, but it is really important to look at this stuff. If you're sending a sample out to a reference lab, generally they're going to look at the blood film. Um, there are some exceptions where you can, you know, um, select just for automated data only for a cheaper panel, but be aware that that data is not being corroborated with a blood film review, um, because you're not, you know, paying for that service. Um, so, um, just be aware. And even if you are sending out blood, um, you know, take a minute, make a blood film before you send the blood out, because that may give you, you know, clinically relevant diagnostic information that's going to change how you treat this patient and, you know, potentially, you know, may save its life and save the owner a lot of headache and a lot of money.

So general recommendations and approach to blood film evaluation. You know, make blood film reviews a standard part of all clinic CBCs. So, you know, this includes well patients and ill patients, okay? And the biggest thing is to be consistent and methodical with your approach. So just like a physical exam, so if you do things the same way every time and in the same order, it's less likely that you're going to miss something on the film. And that also goes, this is a cytology group, right? So this also goes for cytologic evaluation. You know, I tend to to evaluate my my cytology slides kind of the same way every time, um, with some exceptions, but, and that's so I don't forget things. You know, I, I miss things if I don't look at the entire slide, um, and I focus in too fast on one area. And that same thing can happen with, um, hematology. So just keep that in mind.

I'm not going to go through into, um, kind of exhausted exhaustive detail about, you know, what do we do when we're actually approaching the blood film. Again, you know, I, I think I'm, I'm, I'm assuming a kind of, um, introductory level of of background that you guys have with this at least already. Um, so, you know, certainly when you're doing blood film evaluation, you know, you want to make sure you do your evaluation in the monolayer, right? Which is kind of generally about this area of the slide, um, where about 50% of the cells are about to touch but not overlapping. If you're onto your feathered edge, then you can get some disruption, and if you go into the body, everything just kind of clumps. Certainly, there are still diagnostically relevant things to find at the feathered edge and body, but morphologic identification occurs in the feathered edge of the slide.

We have different objectives out there, um, that, you know, most scopes come with a 10, maybe a 40, and maybe a 100 oil. Um, if you are super interested in pathology, it may be your benefit to get a 20x objective and a 50x oil, um, but again, they're not a must. Um, they are expensive. They're also really good for cytology as well, um, but, um, just be aware that they cost a little bit more money and are not standard.

So at the 10x objective, I'm usually looking for big things. So I'm looking for platelet clumps. I'm looking at my leukocyte distribution, so I want to make sure that all of my white cells aren't pulled out to the feathered edge, because if they are, um, then I can't do an accurate, um, differential and I can't accurately look at morphology. We're going to look at our red cell density. Um, you know, it's hard to identify mild anemias on a blood film, but we can certainly see more moderate to marked anemias. So you can corroborate analyzer data. You're going to want to look for large atypical cells. You're going to look for infectious agents like microfilaria that are quite big, and you're going to estimate your white cell count. Um, there are various estimate formulas out there. This one is using the average number of white cells per, let's say, 10 fields, 10 10x fields, and multiplying by 100 to 150. You can compare then that data to your analyzer count. If you decide to use a 20x objective, there's a slightly different estimate count as well.

And then if, um, you use the 100x objective or oil, you know, this is really where definitive evaluation occurs. So I see a lot of kind of beginner microscopists, um, you know, mostly here students, um, who want to do all of their cell identification and everything, you know, at, um, 10x or 40x right away, and they're really struggling to kind of say, is that a neutrophil? Is that an eosinophil? You know, is that a monocyte? Is that a lymphocyte? They're missing, you know, platelets. It's all just because that's too far away when you're just learning. So really spend time on oil, and then as you get comfortable, you can back out to, kind of, um, a lower magnification. Um, and then as you get comfortable, you'll be able to move more, move faster.

All right, so at 100x, I look for things like small platelet counts. I'm going to estimate my platelet count. Um, so it's usually your average number of platelets in 10 100x fields, and you multiply that by 15 to 20,000. If you're more of a glass half full person, multiply by 20,000. If you're more of a glass half empty, maybe multiply by 15,000, and that'll give you a platelet estimate. Um, you also can look, and you'd want to see maybe a minimum of 8 to 10 platelets per 100x field in order to be feel that it's adequate. Again, you can't just look at one field, though, right? You need to look at multiple fields across the slide. And at 100x, we're also going to evaluate morphology of all of our cell lines. So we're going to look for abnormal red cell morphology, nucleated reds, organisms, inclusions. 100 AES where you perform your 100 to 200 white blood cell differential. So, you know, we break our white blood cells out into neutrophils, you know, um, bands, monos, eos, things like that, and we're going to look for, um, other kind of morphologic changes like toxic change.

All right, so let's go through some case examples. I know they can be scary because we don't know all this stuff yet, but, you know, my goal is to just inspire you to become more interested in doing blood film evaluation.

So this first case is a 12-year-old male neutered Maine Coon cat. This is not the actual cat. When I was in vet school, I was so naive and thought that all of these photos were the actual animals, um, but I have since grown up. Um, so a 12-year-old male neutered Maine Coon cat presenting for further evaluation of polyuria and polydipsia, so increased drinking and urination, hypoglycemia, so elevated blood glucose, vomiting, and inappetence. The referring DVM, uh, diagnosed diabetes mellitus and initiated therapy for this diagnosis. Here is a, not a full, but a limited CBC from this patient. So you can see the differential is missing. Um, so our white cell count is within reference interval at about 13,000. Um, our three red cell parameters, so red cell, hemoglobin, and hematocrit are decreased. All right, so we have an anemia in this patient. Um, we're going to ignore right now, um, MCV and MCHC, so this corresponds to cell, red cell size and hemoglobin. Again, not the focus of this talk, um, but those were within reference interval. Our reticulocyte count, again, not the focus of this talk, but reticulocytes are, um, kind of immature red blood cells that are kind of precursor cells, and they are, um, used to define whether or not an anemia is regenerative or not, meaning is the bone marrow responding to the anemia? Okay? And so regenerative anemias are usually due to, um, red blood cell loss or red blood cell destruction, um, and so if we see regeneration, we think about those things. If the anemia is non-regenerative, then there's a bunch of other differentials that we think about, like chronic illness, um, neoplasia, infection in the bone marrow, immune-mediated destruction, things like that. But again, the specifics of that is not necessarily the focus here, so we're not going to spend a lot of time talking about that. Um, but this anemia does look mild, and it's non-regenerative. I would suspect that this anemia is an anemia of inflammation or an anemia of chronic disease related to the diabetes. Our platelet count is within reference interval, and we have a little bit of a decrease in our serum plasma protein, um, but I don't have a chemistry to know which fractions are low.

So these are some representative images from this patient's blood film. All right, um, on the right, this is a normal, um, best I could do to find a normal cat. We don't have a lot of normal cat blood here because everything seems to be coming in sick lately, um, but this is a fairly normal segmented neutrophil. Um, so remember, you know, a mature segmented neutrophil should have these nice, what I call, like, you know, thin nuclear sauc. It reminds me of like sausage links with the little connections between the links, but these are little nuclear bridges, um, going on here, and then we've got like a nice clumpy chromatin, um, to our nuclear chromatin pattern.

So here we have another segmented neutrophil. We've got our nice little thin nuclear bridges or our little nuclear connections, okay? And we still have a fairly clumped chromatin, but something that is going on here is there is a lot of basophilia to the cytoplasm. So this cytoplasm is more blue, um, and I know this isn't the best example because usually the cytoplasm would be more clear, um, but it's bluer compared to that cytoplasm. Okay, so we've got cytoplasmic basophilia. All right, and that is one indicator of toxic change. All right, and so what toxic change is, is toxic change is when the bone marrow is generally responding to, most often, an inflammatory stimulus. It kind of ramps up production, and, um, when it does that, it starts to release, um, cells with kind of some immature characteristics. Um, and toxic change are, um, some components of those immature characteristics, with cytoplasmic basophilia being one of the toxic change characteristics. Okay? And so why do we care about toxic change? Because it usually means that there's inflammation in our patient.

Here is another white blood cell. Okay, looks different than the last one. It looks different compared to this neutrophil. All right, so it definitely has that cytoplasmic basophilia going on, um, so toxicity. And it also has a little bit of foaminess to the cytoplasm, which is also an indicator of one of the other indicators of toxic change in this patient. But then you can also see that the nucleus is pretty kind of, um, U-shaped or band-shaped. So this is a band. Okay? So band cells are kind of a step before your segmented neutrophil in kind of production. All right? And when we start to, um, release bands into circulation, that is called a left shift, and that means that the bone marrow is trying to keep up with an inflammatory stimulus, and our normal mature neutrophils are being kind of consumed, and so, um, the bone marrow all can do is kind of release immature, pre-immature precursors. And these left shifts are usually associated with, um, inflammatory conditions as well. So there's some kind of inflammation going on in this patient.

All right, um, now there's a lot of, this not a lot, but, um, there's some disagreement amongst pathologists about what constitutes a band and what doesn't. You know, some people say these walls should be perfectly parallel of the nucleus, and if there's any constriction at all, like maybe there's a little bit right here, then it's not technically a band. Other people, you know, say that this constriction can't be, you know, less than two-thirds the area of any other part of the nucleus. Um, so it's really just being about, it's about being consistent with your, um, kind of morphologic identification. Um, something that's a little bit harder to see sometimes with bands is their chromatin tends to be a little bit less clumpy and mature, um, and they may be slightly larger cells than segmented neutrophils, um, but again, those are kind of slightly harder characteristics.

Here's another cell. Okay, we've got this kind of band-shaped or U-shaped nucleus. So we've got a band here. We've got cytoplasmic basophilia here and foam. And then we're also pointing out some of these kind of irregular, kind of, um, aggregates of kind of blue-gray material, and these are Dohle bodies. All right, Dohle bodies are another criteria of toxic change, um, in patients. It is important to be aware that in cats, small numbers of Dohle bodies can be okay, and actually, um, the longer blood sits in a tube, so even during transit to a reference lab, Dohle bodies can start to form on their own. But because we've got other indicators of toxic change here, um, I think these Dohle bodies are consistent with toxic change in this patient.

Okay? And then here's another, um, band neutrophil. So we had significant numbers of band neutrophils, so we had, you know, a pretty notable probably left shift when we did our differential. Um, and we also have pretty notable toxic change in this patient, all indicating inflammation.

All right, so here's what we got when we did our actual, um, manual differential. Um, we actually got more bands than segs. So some people would consider that a degenerate left shift. Um, some people don't like that term, um, but it's what's still in the literature in some areas, um, which can imply kind of significant inflammation and potentially a poorer prognosis for this patient. All right, we also had small numbers of metamyelocytes, which are a neutrophil precursor that's kind of the step before, um, our bands. Okay? So we had a pretty notable left shift in this patient. Whether or not you want to classify it as regenerative, um, or degenerate, um, that's up to you, um, but a notable left shift, um, that our analyzer couldn't have identified.

Lesser numbers of lymphocytes and monocytes, and then we're seeing significant toxic change in this patient, again, something that our analyzer wouldn't have identified. Another important thing to note, right, is our, um, white cell counter is within reference interval, right? So we don't even have, you know, an increase in white cells, but we're still seeing this notable morphologic change. So if you had just gone by your analyzer count and not looked at a blood film, you could have missed all of this clinically relevant information.

So what did we find on this patient? Um, there was severe pancreatitis on abdominal ultrasound with a severe diffuse peritoneal reaction, and there was mild, um, abdominal effusion. And they were actually concerned for, um, um, carcinoma or neoplasia in this patient's abdomen, although we didn't get a definitive diagnosis. We didn't get any cytology. Um, and so the takeaway from this first case is that there, there can be evidence of significant inflammation, meaning, you know, a left shift and toxic change on a blood film review, that A, the analyzer may not be able to identify, and B, it can be present when our leukocyte count is within reference interval. All right, so if we don't look at a blood film, we don't know that that's going on in that patient.

All right, case two. This is a 5-year-old female spayed domestic short hair cat who presented for evaluation of progressive lethargy over the past few weeks and had an increased respiratory rate over the last few days. The patient was diagnosed with FeLV and leukemia at one year of age, and on physical exam, um, a mildly increased heart rate was noted, respiratory rate was increased, the patient had pale gums and was slightly dehydrated. Our, um, limited hematology data that I'm going to show you first, we have a white cell count that was, was within reference interval, so 4,380. Okay? Our red cell lines, so red cell parameters, excuse me, so red cell count, hemoglobin, and hematocrit are all notably decreased. So we have a marked anemia in this patient. This patient actually has an increase in its MCV, so that's its red cell size, um, and this increase in size in the absence of any regenerative response, um, has been associated, um, with feline leukemia infection in cats, um, as a potential, um, dysplastic or abnormal change. So, um, that's kind of fitting with the history in this patient. And then we also have a pretty marked thrombocytopenia, so low platelets, and an elevation in total plasma protein. Now, obviously, the first thing we want to do is make sure that there's no platelet clumping. Cats love to clump their platelets, and I can tell you that we did, you know, obviously do a blood film review, and there was no platelet clumps, so this was a real finding for this patient. Okay, so we have a bicytopenia, so two cell lines being low, which is concerning.

Here's a representative, kind of, um, image of the blood film for this patient. Okay, so when we do blood film evaluation, um, I like the analogy, um, for those of you that have red, white, and blue in your, in your country flags, um, so, you know, America, red, white, and blue, um, think of your cells as red, red, white, and blue. So reds being red cells, white being white cells, and blue being, um, uh, platelets. Okay? So that way you won't forget to look at your three cell lines.

Okay, so the first thing I'm noticing with my red cells is there's a lot of white spacing in between them, and there, um, so that is kind of fitting with the marked anemia in this patient. Okay? There is some variability in red cell size, so, you know, this cell compared to that cell, um, this cell compared to that cell, so there is some variability, but I'm not seeing, um, what's called polychromasia, which is kind of, um, indicative of immature red cells, which I will, um, cover at a case later, so we're not going to focus on that right now. The other thing I'm seeing is a segmented neutrophil up here. There may be a faint Dohle body on the edge of this cell, but this isn't looking too toxic right now. The cats can have small numbers of Dohle bodies, and we're not going to make grand diagnoses from just a single cell to look at, okay? Okay, I don't see any platelets in this film area. Okay, so that corroborates the thrombocytopenia or fits with what we had already said.

Other things that I'm noticing are these cells, nucleated cells, right here. Okay? And you may be asking yourselves, what are these? Like, they kind of look lymphoid, but they kind of don't look lymphoid. Like, the color is a little bit weird. What's going on? So let's take a closer look. All right, so we've got two of these right here. This is an example of a typical small lymphocyte from a cat. All right, so it is about the size of a red cell. It has scant basophilic cytoplasm, so not a lot of amount, and then this slightly eccentric, kind of round to oval nucleus with fairly clumped chromatin. Then we have these cells which have more cytoplasm than our typical small lymphocyte. They've got a very dense nucleus that I can barely see, kind of the characteristics of, um, because it's so dense, and then we've got this variability in cytoplasm color that looks, um, varies from looking like the same as a red cell cytoplasm to kind of this more blue-purple in color. Okay? And so you may be asking yourself, well, what exactly are these? Or maybe you are, what are these? Or maybe you already know, which would be, um, sweet if you do, um, good for you, um, these are metarubricytes, okay, or nucleated red blood cells, also abbreviated NRBCs. These are, um, precursors, um, to red blood cells. Okay? And so, um, what's interesting, not only are we seeing nucleated red blood cells in this patient's blood, but also their abnormal nucleated red blood cells. So by the time a nucleated red blood cell matures, um, to having, um, cytoplasm that is full of hemoglobin, so the same color as a normal red cell, it should spit out its, um, nucleus, and so this nucleus is still present, and that's abnormal. So that's called dysplasia.

All right, we also have a little platelet right here. So we're able to find one. Here is another area of the film. So again, we've got another nucleated red blood cell here, and we've got, um, this is kind of bordering on like a rubricyte, kind of nucleated red cell as well, but again, the cytoplasm is starting to get more kind of fully hemoglobinized, and it has not lost its nucleus yet, so that's a little bit weird. All right, we're also seeing these crescents in the background. These are protein crescents. It's just because the patient had a high protein, and they fold on each other. I like to gross the students out and say that they look like toenail or fingernail clippings if that helps them remember, but, um, most people don't like that. And then here's another area, um, again, one of these nucleated red blood cells, and then lots of protein crescents in the background.

All right, so this patient has a pretty notable, um, metarubricytosis, or increase in nucleated red blood cells in circulation. So when we actually did our differential, look at that, our white cell count, um, was not 4,000, it was actually 1,000. This patient is actually leukopenic. And why is that happening? Well, the analyzer counts nucleated cells as white cells, and so what it's doing is, it's, it's classifying these nucleated red blood cells erroneously as white blood cells and including them in the white cell count, and potentially classifying them as lymphocytes or monocytes, depending on the analyzer.

All right, so what we need to do is we need to, um, correct our white cell count for the presence of nucleated reds. And in this patient, this patient had 340 nucleated reds per 100 white cells, which is a ton of nucleated reds. And what you're doing is, is while you're doing your 100 cell or 200 cell white cell differential, you also want to keep a tally of how many metarubricytes you're counting as you go. Those don't count as part of the 100 cell count, but they are important to know. And so there is this correction formula that you guys can, you know, use and look up. Don't memorize it, it's not worth it, um, and that corrects your white cell count, um, for the presence of nucleated reds, and then we do our differential off of that. So not only is this patient anemic and thrombocytopenic, but it's actually, um, notably leukopenic and profoundly neutropenic. So it actually is pancytopenic. All three cell lines are down, which is very concerning for underlying bone marrow pathology in this patient.

All right, and again, the analyzer may have said NRBC suspect, um, but it's not going to correct the white cell count, um, because it doesn't know for sure whether or not they're there, and it doesn't know how to classify them out from the other cells. All right, so something that's very important for you to be able to do. So this patient actually ended up having feline, what we think was feline leukemia-associated myeloblastic syndrome or some kind of bone marrow aplasia. Um, it received a transfusion, and the hematocrit rose to 17%, but it presented about, um, a week later with a hematocrit of 5%, so pretty anemic again. And so based on prognosis and quality of life, euthanasia was elected, and no necropsy was confirmed, was performed. Um, so this is just a great, um, example of correcting your white cell count for nucleated reds is really important.

So what are nucleated reds? Um, so they're abnormal in circulation. Again, they are red blood cell precursors that get released from the bone marrow. They can be present due to a variety of underlying causes. Regeneration, so they're associated with regenerative anemia sometimes, but do not indicate regeneration. Um, they can be seen with bone marrow pathology of various causes, lead poisoning, chemotherapy, splenectomy. Um, it's important to again remember that the white cell count from the analyzer is including all nucleated white, nucleated cells, and nucleated red cells are a nucleated cell. We generally correct for, um, the presence of these nucleated reds if there's more than five to 10 per 100 white cells. And then, um, the type of leukocyte that your analyzer makes these, whether it makes them, you know, lymphs or monos, is variable and depends on the analyzer.

All right, next case. Hopefully you guys are getting interested and exciting about and excited about this stuff. So this is a 13-year-old female spayed Labrador Retriever. Over the previous one to two weeks had become increasingly lethargic and reluctant to walk and had some difficulty rising and a decreased appetite. The patient is on non-steroidal anti-inflammatories and some joint supplements for orthopedic issues. On presentation, temperature was elevated, decreased range of motion was noted in her hind limbs, and she had mild to moderate effusion of both tarsal joints.

This is some, um, data from the analyzer. So our white cell count was within reference interval. Um, the patient had a very mild normocytic, normochromic, non-regenerative anemia. So again, probably anemia of inflammation in this patient, and we had a moderate thrombocytopenia. Okay? And I can tell you that there was no platelet clumping on this patient's blood film, so the thrombocytopenia is real. This is our differential. All right, so, um, all of our leukocytes are within reference interval except for our lymphocyte count, which is a little bit low. Um, this is indicative of a lymphopenia, which is usually associated with like a steroid or stress response, um, in this patient from being ill, but not super clinically relevant.

All right, these are some representative, um, films. Um, for the sake of time, I'm not going to focus on our red cell morphology because it's not important to this case. Um, we've got one platelet here, starting to get a little bit big, but it's not huge. And then here we have our segmented neutrophil, okay, and it's got some inclusions in the cytoplasm. All right, so these inclusions are, um, kind of, um, a more like lilac-purple color than the chromatin of the, um, nucleus of the neutrophil, and they are distinct from the nucleus, so they are not nuclear fragments. If we were able to focus up and down through them, they would actually kind of look like little packets of granules. Okay, so each one of these would have little, kind of, like, little stippled dots making them up. Here's another example of this cell having some here. And then, um, we're not going to go through the band, whether or not this is a band argument. Um, some people may not call this a band because that constriction's getting a little bit too severe, but some people would, but again, I'm not going to go down that, um, open up that can of worms. Um, but again, more of these kind of purple inclusions in our neutrophils, and then here is another one right here. They're just much smaller. Okay, okay, this neutrophil is a little bit toxic, so its cytoplasm is maybe a little bit foamy, but these are not Dohle bodies. Okay, they're kind of more purple, um, they have more sharp margins if we were able to focus in and out, and again, they kind of have this granular, kind of distribution.

So what are these? These are actually, um, um, bacterial morula. So this patient has little packets of bacterial morula inside of its neutrophil cytoplasm. Okay, we ran a 4DX SNAP test on this patient to look for antibodies, um, against, um, the bacterial morula that make these little, um, kind of inclusions, and they are more most commonly, um, um, Anaplasma species or an Ehrlichia species. Technically, this is written wrong because it's not just E. canis or Anaplasma phagocytophilum that reacts, but that's okay. Um, but this patient was negative for antibodies to, um, these organisms, even though, um, they were in circulation. And so that's just an important point that, um, it takes time to develop antibodies to these organisms, and so we can certainly have them in circulation and be 4DX negative or be serology negative of of various serologic tests. Okay? And so again, another reason to look at our blood film review.

So these morula were most likely Anaplasma phagocytophilum, given that's the most common, um, bacterial morula that we see in this part of the United States. Technically, Ehrlichia ewingii can appear similar, but we don't see that as often here in Minnesota. This is important to note that morula can precede blood seropositivity, and because we were able to find these on this patient, we were able to start appropriate therapy, um, and kind of get this patient feeling better. And if you had not done a blood film and had just been relying on your 4DX test, um, to decide whether or not to treat this patient, you would have missed the diagnosis.

Case five. This is a 7-year-old male neutered Cavalier King Charles Spaniel, um, who presented for evaluation of acute lethargy and collapse. On physical exam, the patient had iatrogenic ocular discomfort on abdominal palpation, a grade three of six systolic heart murmur, and decreased mentation.

Here is some selective, um, analyzer data. So we can see here that the patient has a moderate, um, increase in white cells, so moderate. Pretty markedly anemic, so our red cell, hemoglobin, and hematocrit are all markedly decreased. Our MCV is up a little bit, so it's a little bit macrocytic, but not markedly so, and the patient currently is not regenerative. Okay, so our reticulocyte count is normal, and our platelet count is within reference interval. So, um, again, notable findings include this marked anemia and this moderate.

This is a representative, um, kind of area. So let's focus on our, kind of, we'll ignore these arrows for a second, um, but you can see here that there's a lot of spacing between our red cells, so there is a confirmation of the anemia. Okay, we do see some polychromasia to our reticulocytes on the analyzer, so these are the indicators of regenerative cells, and so they are, um, red cells, but they're larger than normal red cells, and they have this more blue-purple, um, color to their cytoplasm, just because they haven't, um, kind of, um, lost, um, kind of their RNA yet, and so, um, or and like they're the kind of fragments of nuclei, and so, um, this is, um, you may think that this patient is regenerative because there's polychromasia here, but based on our analyzer, it actually is not, and sometimes that just happens.

Other findings, we've got, you know, a platelet here, and a platelet here, and a platelet here. Okay, so platelets are fine, even based on the analyzer count. And then if we look at our white cells, I see a segmented neutrophil here, but then I see these other neutrophils that don't have that nice segmentation. So if we look closer at our white cells, um, it looks like there's bands, just like before. All right, and then we also have toxic change, so we've got this cytoplasmic basophilia, kind of foaminess going on. All right, um, so we probably have a left shift in this patient, and we've got toxic change indicating inflammation. Again, here's another arrow pointing to polychromatophilic changes that I have some better photos of, so we're not going to focus on them just yet.

So if we go back and do our differential, all right, this patient, um, has a, um, leukocytosis and a neutrophilia, um, so kind of supporting inflammation, and we do have some toxic change present, again, supporting inflammation. I didn't show you any pictures of monocytes, but the patient also did have a monocytosis, part of the inflammatory response.

All right, other red cell morphologic changes that you may not have noticed. All right, so this right here is like, let's say this is a typical red blood cell. All right, so it's a little bit, technically has a little bit of artifact going on, but, um, the central pallor is a little bit too prominent because the cell's punched out, um, but the cell has central pallor, and this is about the normal size of the red cell. So we're seeing a couple of things. We're seeing a bunch of these red cells that are smaller and have lack of central pallor and are almost like a, I like to describe them as like a little bit more kind of orange in color, than like the typical red of, um, our normal red blood cell. A lot of these cells also have these kind of irregular, kind of, blobs coming off their margins. All right, and they're kind of a little bit lighter in color sometimes, sometimes about the same size as, same color, excuse me, as the rest of the cytoplasm, but hopefully you guys can appreciate kind of these knob-like projections coming off. Okay, here are some more examples of them, so these knob-like projections coming off of our red cells. All right, um, again, these red cells are a little small, they lack central pallor. Okay, we also have, um, these other cells that, um, are ghost cells. Okay, so they've lysed, and so all of their hemoglobin has leaked out of them, and they just have a residual cell membrane, and they also have these little, kind of, um, projections coming off of them.

And so, um, these are actually Heinz bodies. All right, so these are oxidized, um, hemoglobin that has been exposed to an oxidative agent, and they have oxidized and precipitated and kind of gotten stuck to the red cell membrane. All right, and so when we see these Heinz bodies, um, that means that there's oxidative damage in our patient. The presence of ghost cells, um, means that there's some degree of red cell hemolysis going on, and also these red cells being kind of smaller, denser, and lacking central pallor, these are actually spherocytes. Okay, and some of these spherocytes have Heinz bodies. So we're seeing a combination of spherocytes, we're seeing, um, Heinz bodies, and we're seeing ghost cells in this patient who has a severe anemia.

Here's some more examples, um, of the Heinz bodies. That one's really nice. Um, you can see it, kind of, this knob-like projection. Here's one here. Here's one right here. This is one. This is one. So lots of Heinz bodies. Here's another ghost cell that maybe it's a little, kind of, rough shape, rough shape, and then has, um, a little Heinz body. And then this is a platelet, and that is a platelet. Okay, so, um, then we, um, were like, whoa, this patient has Heinz bodies, and this patient has spherocytes, and that combination should immediately make alarm, alarm bells go off in your head, for, um, zinc toxicity. Zinc, you know, being in various metal objects, and so what zinc does is it damages the red cell membranes, and kind of causes protein abnormalities, which then induces, um, an immune-mediated response, which causes the ghost cells and the spherocytes. And at the same time, um, zinc is an oxidant, and so it causes oxidative damage, leading to Heinz body formation. All of these things, um, kind of drop your red cell count, either by destroying your red cells or making them more prone to removal.

We took x-rays of this patient, and there was a whole bunch of coins in this patient's stomach. Okay, right here, here's the lateral. All right, so the combination of Heinz bodies and spherocytes suggested a hemolytic anemia due to zinc toxicity, and it prompted x-rays of the abdomen in this patient, um, which got us our diagnosis. Multiple coins and medallions were removed from the stomach via endoscopy, and a few pennies that were dated 2008. Blood film review is often helpful in differentiating causes of hemolytic anemia, and so this is a great example where we were able to, um, give the clinicians something to go on and make a diagnosis. Um, and so just to be aware of, um, zinc toxicity as a potential differential, um, for your patients.

What are Heinz bodies again? So I said, you know, they're aggregates of precipitated hemoglobin from oxidative damage. There's lots of oxidants, so zinc, acetaminophen, onions, garlic, skunk musk, or skunk spray, naphthalene, which is in mothballs. Any of these things, if you get enough of them, they can cause oxidative damage and hemolytic anemia. It is important to note that because cats are weird, and they're just crazy weird animals, that normal cats, um, can have up to 5 to 10% Heinz bodies, and that can be typical for that species, and that's okay. They have, um, kind of a different hemoglobin conformation. Their hemoglobin is very reactive, and they tend to lack, kind of, um, a large amount of endogenous antioxidants compared to other species. Um, increased numbers of Heinz bodies in cats are not just associated with hemolytic toxins. You can also see them with some metabolic diseases like lymphoma, hyperthyroidism, and diabetes.

How do we confirm Heinz bodies? Because I, as you can imagine, these can be kind of hard to see sometimes. We can, and here's actually, this is a clear one sitting right here that you can barely see. Um, we can do a methylene blue stain. So this is an example. What I like to do, um, here's a patient. These clear, this patient actually got into, um, caramelized onions. It's a cat, and here's a whole bunch of these clear spots. These are the Heinz bodies. All right, but they're kind of hard to see. So what you can do is you can take a drop of blood and a drop of new methylene blue and let

them sit in a tube together for like five minutes and then make a blood film. All right. And then what's nice is the new methylene blue really highlights the Heinz bodies. So all these blue circles are Heinz bodies. It is tricky. This is actually a polychromatophilic erythrocyte. We used to use new methylene blue, um, manual preps to do reticulocyte counts. Um, so these are not, um, Heinz bodies, but all of these nice blue inclusions that are quite big in this case are Heinz bodies, and we were able to confirm the presence of Heinz bodies in that patient. So just a little pro tip. All right.

To more cases. I think, um, this is a five-month-old female, spayed Corgi mix. All right. Originally from Alabama, but was rescued to Minnesota. The patient has a history of lethargy, fever, vaginal discharge, abdominal pain, and there was some concern for a stump pyometra, um, because of the vaginal discharge and the fact the patient had been neutered at the rescue, like, I think, a month prior to, um, coming to Minnesota. Um, diagnostics at the RDBM revealed a, um, so a marked increase in white cells and a possible intra-abdominal tubular structure in the abdomen on their ultrasound. So that's why they were concerned about a pyometra. Um, the patient was put on antibiotics, um, and after 24 hours, not improving, was referred to the U for additional diagnostics. On physical exam, the patient was 5 to 7% dehydrated, had ocular discharge, and mild abdominal discomfort. So we got, um, a, uh, CBC on this patient. That patient has a marked increase in white blood cells, so 61,000 white cells, which is a lot. So anything over 50,000 is usually considered marked. All right. The patient had a kind of mild anemia. Um, this is not, um, a five-month-old puppy reference interval, but by five to six months of age, they should have about a normal red cell count. So, um, we are anemic here. All right. Non-regenerative, um, and our platelets were within reference interval. There wasn't enough, um, or sorry, we did not get a total plasma protein. So this ND stands for no data. Okay. So when we did our differential, our manual differential, um, the majority of our white cells were, um, segmented neutrophils, but there was a left shift of bands. We also had a mild lymphocytosis, monocytosis, and eosinophilia. So taking this all together, this is probably just a marked inflammatory leukogram in this patient. All right. We did see reactive lymphocytes and we saw toxic change with fits with the inflammatory response. So when I see marked neutrophilia and this is borderline, like again, 50,000 is marked, but this is coming up right on the edge there, think of the P's. Okay. This is what we teach the students. So, um, pyothorax, peritonitis, whether it's septic or non-septic, pancreatitis, um, parasitic, specifically Hepatozoon, pneumonia, pyelonephritis, pyometra, prostatitis, um, depending on this on the, um, sex of the patient, a paraneoplastic response, or primary neoplasia. Okay. Primary neoplasia is a, um, diagnosis of exclusion, so you have to kind of rule out all of this other stuff. And this is not an all-inclusive list, but if you think of the P's, um, it will help with your kind of diagnostic algorithm in these patients. And certainly, there was concern for pyometra in this patient. Other clinical chemistry findings, again, not the focus of this talk, but for those of you with some experience, this may clue you in onto what the diagnosis for this patient was. So the patient had a low albumin, blood protein, its globulins and inflammatory markers were increased. It had a mild elevation in its alkaline phosphatase, um, and a mild to moderate elevation in CK, indicating some degree of muscle injury. Okay. Again, not the focus of this talk, though.

So then I got a blood film, and this is actually at the feathered edge of this patient's film. All right. So, um, not the best place to do white cell morphology. These are, um, this is a neutrophil, this is a neutrophil, this is a neutrophil. Here's a monocyte, um, and then this one, like I can't 100% tell. There's some argument whether or not these are inside of monocytes or neutrophils. Um, so I'm not going to go there, but let's pay attention to what we're seeing here. So we're kind of seeing this kind of oval to tablet-shaped kind of, um, clear structure, okay? And that immediately got me worried. And so, um, but it was the only one that I found on this patient's blood film. So I asked for a buffy coat, um, and don't ever forget the importance of the buffy coat. So it's really easy to perform and if you're looking for small numbers of organisms or small numbers of neoplastic cells, the buffy coat can really help concentrate and look for it. So doing a buffy coat prep, this isn't the best example, but I was able to find like a whole two more. Um, this is one kind of overlying, um, the cytoplasm, or sorry, the nucleus of this cell right here. Okay. So not the best example. This one's a little better. So, um, you may be, you may or may not know what this patient's diagnosis is, um, but this patient had a marked neutrophilia and organisms consistent with Hepatozoon. So those were those clear tablet structures. There's two species of Hepatozoon: Hepatozoon canis and Hepatozoon americanum. Hepatozoon is a tick-borne, um, protozoan that usually is in tropical climates. So we do not see this in Minnesota. It gets way too cold here. Um, but this dog was from Alabama, and so that, um, you know, is close enough to a tropical climate, and they get this organism from tick ingestion. Um, the patient did not have a stump pyometra. They did an abdominal ultrasound, here everything looked normal, and that tubular structure, um, was not identified. And the medicine service was more worried that the patient probably just actually had a little bit of vaginitis. So what's cool in this case is that, um, we were able to give these people, uh, this owner a rapid diagnosis with a, um, a blood film and buffy coat review. Um, if you're not able to find the organism, but you're suspicious of it based on, um, where the patient is from, um, these patients have been described as having mild anemias, ocular discharge, elevations in globulins, elevations in ALP, potentially elevations in CK. All of that was fitting the diagnosis for this patient. Um, and so if you can't find the organism, you can send out for PCR, um, or you can, um, do a muscle biopsy depending on, um, the species. And, um, we tried to send out PCR, but, um, this came in over a weekend, so by the time it went out, the blood, um, was like almost five days old, and so the PCR did not work. Um, but we were able to, um, find this organism. I just couldn't speciate it between canis versus americanum. Um, it's probably more likely to be americanum given, um, the ocular discharge and the very limited numbers of organism in blood. Hepatozoon canis tends to have, um, significant blood parasitemia, but it is not, um, it certainly could still be that. And the puppy's still alive, but unfortunately, um, it's been about a year since we made the diagnosis, and they often don't live much longer than a year, and they have waxing and waning, um, kind of, um, clinical signs and kind of illness despite being on medication, unfortunately. So we'll see kind of what happens to this puppy.

All right. Last case. Um, another Cavalier King Charles. So a three-year-old male neutered Cavalier King Charles Spaniel, um, who had an appointment for pre-surgical blood work prior to a routine dental cleaning with our primary care service. The patient had a normal physical exam, no historical illness, and only medications are flea, tick, and heartworm preventative. This is our data from the analyzer. So normal white cell count, um, with a normal differential, it's just not shown here. Um, normal red cell count, and then a platelet count of 71,000, so a moderate thrombocytopenia. The first thing we want to do is look for platelet clumps, which we did not see. But as we were looking through the blood film, um, we were seeing these large purple structures that were, um, larger or the size of red blood cells with kind of this granularity, and they kind of have these little projections. So these are actually platelets. These are, um, large platelets or what are termed macroplatelets. They are quite big. All right. So they are, in order to be termed macroplatelets, they need to be about the size of or larger than red blood cells. Okay. Okay. Um, here is another macroplatelet, quite large. We've got some typical segmented neutrophils here in the background, okay? But macroplatelet again here as well. So we ended up seeing large numbers of macroplatelets and large platelets and again, this mild to moderate thrombocytopenia. So given signalment, this was most consistent with a diagnosis of macrothrombocytopenia of Cavalier King Charles Spaniels. Okay. So, so what is this? This is a, um, inherited condition. It's a mutation in, um, a gene encoding beta-1 tubulin, although there are other, there are other mutations, but this is the one that we can test for, um, in these patients. Your platelet count is often, um, in the range of 30 to 100,000. Um, there are some patients who can have a lower count, but it's not usual for them to get below that. Um, you will generally see large platelets that are frequently observed on blood film review. Now, certainly something that I didn't bring up is the analyzer will give you a mean platelet volume or your MPV, and your mean platelet volume will increase because these platelets are big. But the reason why you need to look at a blood film is because platelet clumps, platelet clumps can get counted by the analyzer as a single platelet, and if it does that, it's going to drive your MPV up and look like big platelets. So again, you can't just rely on your analyzer data, um, to kind of prioritize this diagnosis. You need to make a blood film. All right. There is another, um, analyte that some analyzers give you called the platelet crit. Um, and that is analogous to the hematocrit. So it's a measurement of your platelet mass. Um, and in these dogs, um, their platelet crit tends to be normal. Okay. There are some dogs that will have a low platelet crit, but a lot of times they have a normal platelet crit. Um, and that is because, um, these dogs, even though they have a low platelet count, they don't bleed. They don't have clinical bleeding, and that's because, um, the really what's the most important for the platelet part of coagulation is not the number, but your overall platelet mass. And these platelets are so big that these dogs have an overall normal platelet mass, and so they're not predisposed to bleeding from this condition. There's no treatment necessary. There's no clinical signs. Okay. And we can do confirmatory genetic testing. Auburn, um, Dr. Pete Christopherson, um, he's a, a platelet expert. He can do, um, confirmatory genetic testing. If it's the beta-1 tubulin mutation in these patients, um, it is not specific to Cavalier King Charles Spaniels, so other breeds can have it. This is just kind of the poster child breed that we think about. And I think this is just again, the last two cases, a point to the fact that we can get, you know, really important diagnostic information from the blood film that the analyzer can't tell us, especially for this last case. We get, um, every year we get a couple Cavalier King Charles that are referred for, um, low platelets and potentially immune-mediated thrombocytopenia. Okay. Something to remember, immune-mediated thrombocytopenia, they generally have pretty dramatically low platelet counts, like 10,000 or less. Um, but, um, certainly they can be in the 20 to 30 range. Um, but we get patients referred like for concern for, um, either immune-mediated thrombocytopenia or potentially like anaplasma infection, but they don't actually have this. They, they just have macrothrombocytopenia of Cavalier King Charles Spaniel. Okay. And so it's another, um, important morphologic change that we can identify by doing a blood film review.

All right. So in summary, um, blood film review should be completed for every patient, every time, in an ideal world. Analyzer data does not tell the entire story every time. Blood film review can help prioritize, um, definitive diagnosis, um, rapidly, or it can prioritize differentials and kind of lead additional testing. Okay. And so what we're looking for here are infectious agents, things like neoplastic cells, or morphologic changes that support underlying pathology. All right. So, um, hopefully, I mean, we'll, I think we'll stay around for any questions with this streaming, but, um, hopefully this has given you guys kind of just the taste for, um, kind of the power that blood film review holds. And, um, you know, I kind of when I'm bored and kind of having, you know, a day where I need to entertain myself, I'm like, just imagine myself as a detective, and I'm, you know, looking at the blood film and really trying to, you know, figure out what's going on. And there's nothing more exciting than, you know, calling a clinician and saying, hey, I have a diagnosis based on the blood film, and that's all we needed to do. Um, so there's a lot of power in this, um, diagnostic. And so with that, we'll take any questions. Thanks for your time today, and, um, I appreciate it. And, um, stay safe and stay healthy with all of the COVID stuff that's happening throughout the world. All right. It's