Webinar 4 - Granulocyte cases

Welcome everyone. My name is Dr. Ali Mahdi. I'm a hematologist working in South Wales in the United Kingdom, and on behalf of the Hematology Interest Group and Blood Academy, I welcome you to the fourth webinar in our six-part series on blood cell morphology. Our focus today is going to be on granulocyte cases with plenty of neutrophils and interesting cases to go through. We've got three cases, um, to go through, and we really want to make this as interactive as possible, get you to engage into the cases, uh, stimulate discussion and debate as well. I'm delighted to be joined by Sam Doughey today. Um, we we've got some other things to go through before we start. Just to remind you that this webinar series has been kindly supported by the company Uber Mannheim, and the whole webinar series, including this webinar, is accredited by the Royal College of Pathologists in the UK for CME. Um, you can still register for your CME certificate. Um, I've already posted a link on the comment section, so if you haven't done so so far, then feel free to register, and hopefully we will be able to email you the certificate in the next few days. I'm sure you want to watch this webinar again, or if you've missed it, then you can do so via the Blood Academy YouTube page. We've also got the other recordings from our three previous webinars that should hopefully be available in the next day or two as well. Now, these webinars will give you an overview of blood cell morphology. If you do want to know more detail, then we do have an exclusive online course on the Blood Academy website. It can consist of video tutorials, online e-modules, and quite extensive assessments as well. And again, this course has been accredited by the Royal College of Pathologists, and you'll be issued with a CPD or CME certificate after completion of the course as well. Uh, we do have a special 20% discount, um, which is linked to the Hematology Interest Group. Uh, if you're interested in checking, uh, the course out, there, I'll, uh, post a link for the the course on the comment section, and if you want to save 20%, then just use the discount code HIG20 at the checkout.

So, moving on to today's, uh, webinar, I'm delighted to be joined by, uh, Sam Doughey. She is based in the, uh, in the United States. Uh, she is a senior medical lab scientist in hematology. She's currently working in Norton, uh, Brosboro Hospital in Louisville, Kentucky. Hopefully, I pronounced that correctly. She completed her undergraduate studies in Bellamy University. She is a certified medical laboratory scientist and specialist in hematology, and she has the interesting responsibility of overseeing the operation and training, as well as quality assurance in hematology, coagulation, uh, as well. And she's also one of the hard-working admins of the Hematology Interest Group. So, thank you, Sam, for joining us today. We have three interesting cases. Um, before we hand over to you, Sam, just to let everyone know, we, uh, we would, uh, appreciate, uh, discussion and comments, as I've already mentioned, in the comments section on Facebook. And before each case, I'll be reminding you, what do you think the, uh, the diagnosis is. There we go. Welcome, Sam. Thank you for joining us today. Thanks. Um, I'll just allow you to show your screen. Perfect. Excellent. So, hopefully, you should be able to share your screen. Um, so our first case today is a 31-year-old male who's presenting with abdominal pain. He has a slight anemia, hemoglobin of 129, and, uh, significantly increased white cell count, 67.1 on the automated analyzer, with a mild thrombocytosis of 538. Um, so if you could just go over the the main sort of morphological features of this case, that would be great.

Yeah, um, so leukocytosis. Um, we have an increased WBC. Uh, so the first thing I always ask myself when I see a WBC of like 67.1 is, what type of cell is causing this leukocytosis? Are they granulocytes, lymphocytes, blasts? Are we dealing with cells in various stages of maturation, or is it more of a monomorphic picture with primarily like lymphocytes or blasts? And to answer these questions, we have to go over the CBC data and review the peripheral blood film in order to identify the cells that are causing this. And I always do that first by looking at low power, and we can see pretty quickly, and the most striking feature of this blood film is obviously the marked leukocytosis. The white blood cells make up a good fraction of the total cells in this film, and normally the WBCs make up approximately 1% of the total cells, but in here, we can see that the WBCs are more seven or eight times that. We can also notice on low power that the chief cells are in the granulocytic series, specifically, um, the neutrophilic, uh, myeloid cells, and that they are at various stages of maturation. Uh, so we're not dealing with, uh, predominantly blasts here, but mostly granulocytes and in various stages. So, after I've looked at low power and kind of gotten an idea of the overall slide quality and identify the cells that are causing the leukocytosis, I usually, uh, go in a little closer to get more details about the RBC and the platelets. See, and over here, we can assess the RBCs and the platelets. I didn't want to go or spend too much time on these because we already spoke or covered these topics in the other webinars, but I did want to mention them that it is part of the peripheral blood film, and that we do assess all three lines whenever we're reviewing a CBC and a peripheral blood smear. And for the RBCs, we always want to assess them for abnormal variations in size, shape, and color. Microscopically, these cells are a nice salmon pink color. Um, they are normally around seven or eight microns and there are micrometers in size and diameter, and normally we can use a lymphocyte to compare the nucleus of a lymph to the RBCs and get a general idea of the size of our cells. Um, let's find a nice lymph for comparison. Um, this is a lymphocyte here. It is a small, round nucleus with, uh, it's darkly clumped and coarse chromatin, and we can compare the nucleus of this lymph to our RBCs and get a general idea about the size. Um, the other morphology, kind of morphology that I see here is, uh, teardrop cells here and here. And as Nicole mentioned in her webinar discussing RBC morphology, that you want to, you can kind of differentiate whether or not it's true teardrop cells from maybe, um, an interference or just an artifact by the fact that the teardrops face different directions and that they're not all pointed in the same way. The other thing I see, I saw some polychromasia in just general poikilocytosis. For platelet identification, we can usually get some platelets here and here. These are just normal platelets. They are nucleated cells that come from the megakaryocyte, and they represent the cytoplasmic remnants of these cells. They do not have a nucleus, so they would not can be considered true cells, but we do refer to them as cells because they're derived from the megakaryocytes. They're small, generally two to four micrometers in diameter, and they're oval, round shaped, and blue in color, and usually you can fit two to three in a red blood cell. So, once we have assessed, uh, both the RBC and the platelet morphology, we can continue, uh, on assessing our granulocytes.

I'm just going to mention, Sam, that we've already got some interesting comments coming in on the, uh, comment section, so, uh, we can discuss those when you've gone through the, uh, the slide. Okay.

Um, so, so far, just looking at the slide in general, um, I was feeling that this was kind of suggestive of chronic myeloid leukemia, just because of the various stages of maturation and what we saw in the RBC morphology with the teardrop cells. I also had noticed, just when we were scanning, that we had a lot of eosinophils and basophils, but we can continue on with reviewing our WBCs and see if we, we still feel that way at the end. Um, due to all of the WBCs in the white count, I usually try to count, if I was doing this at the bench, three to four hundred just to increase accuracy and precision when there's so many cells, but here we have some nice segmented neutrophils. Um, they're about three to four lobes in general. Um, the chromatin filaments may be obscured, um, sometimes, but here it's really nice, and it's obvious to tell that this is a normal segmented neutrophil with its numerous pink, tan, uh, granules and its dark clump, uh, chromatin. They're a medium-sized cell, about 10 to 15 micrometers in size, with an N:C ratio that is about 1 to 3, which is the lowest of the myeloid series. Here's another segmented neutrophil down here, and here we also have a basophil here. And I've noticed sometimes, and I think even in this case, I had seen some basophils that had washed out granules, so just keep that in mind with the staining because you want to make sure you identify those cells correctly and you don't accidentally call them segmented neutrophils. Um, see, we also have some bands here. Um, I know are a question of debate. Uh, some places call them, some places don't. Um, I think the Clinical Laboratory Standard Institute even recommended not, um, differentiating between bands and neutrophils, but actually calling them together due to the fact that they are not reliably differentiated. At our facility, we still call, we still differentiate them, but here's two great bands here, and the band had kind of it's overlapped here. Uh, they can be S-shaped or C-shaped. They're the same size as the segmented, uh, neutrophils, and they're still considered mature. We also have some myelocytes, and there are a lot of myelocytes here that also kind of makes me think that maybe we're dealing with CML, due to all the myelocytes and that myeloid bulge that you can kind of see or sometimes get in individuals with CML. This myeloid cell here has an oval, uh, nucleus, some chromatin clumping. One of the size is starting to flatten out, but it is not indented, and it has some of those secondary granules, and some more bands and segs. Here's a nice eosinophil. Eosinophils are usually bilobed, and they have those nice red, large granules that are uniform and shaped. They have a dense, uh, purple, uh, dark chromatin, and, um, we do not differentiate the different stages of these cells for maturation because they're so few. So, if I see any, I usually just call them eos. Same with the basos. Here's some more segmented neutrophils here and a lymphocyte. More segs. Another basophil here and a monocyte. Um, and the monocytes are the largest of the, uh, the white blood cells, and sometimes I'll even see, uh, they have rougher borders that sky blue and sandy, uh, cytoplasm. There aren't too many blasts, which I didn't expect to see too many in this case to begin with. Usually, it's less than 2% or less than 5% in CML, and that's what I [Music] am leaning towards at this point for this case. Still have some more segmented neutrophils, and these cells don't have very many toxic features. So, um, I don't see very many Doly bodies, toxic granules, or toxic vacuoles present in the segs, which would kind of help me differentiate between maybe a CML or a leukemoid reaction. Here's another monocyte here, some more segs, another mono, a myelocyte, a meta, um, with the differentiate the meta myelocytes from the band, you usually go with the, uh, how far indented the nucleus is. So, when it's more than halfway, it would be considered a band. When it's less than halfway, it would still be considered a, a myelocyte. So, this cell here, it's, um, it's not quite halfway, and some more segs and bands and another. Oh, we haven't seen very many lymphocytes. Here's a nice lymphocyte there, and another eo with its bright red granules, a myelocyte there with its nice round nucleus. It has a darker cytoplasm, more blue, basophilic than its, uh, segmented, more mature, uh, cells around it, and a nice big horseshoe-shaped nucleus for our monocyte. Some. We've got one comment which is quite interesting, and I'm sure we'll go through this later, but, um, someone has commented about the, um, the significance of the monocytes. Could this be something else like, uh, chronic myelomonocytic leukemia or CMML? Um, and what's more leaning you towards CML in this case?

Just, it has the classical features. There's a lot more segs and not quite as many monos, um, as I would usually see in the CMML. And sometimes in CMML, I've seen some like other, um, more dysplastic features on occasion, and these are all pretty, uh, normal. But really, to diagnose this, it would be the cytogenetic testing, Bcr-Abl. So, that would be, I guess, the true way to differentiate it, since it's not based on morphology.

Definitely. I, I, I completely agree. I think the other thing is the classical features which you've already described, and, um, the lack of dysplasia, um, is very typical, really. Is there anything you wanted to add?

Well, I, I was going to add that from, if from a clinical perspective, we all know that, uh, chronic myeloid leukemia, which this is a classical, um, case of chronic phase chronic myeloid leukemia, there are three main phases: chronic, accelerated, and blast phase. Um, and the, the, the film like this, especially if you're starting off looking at morphology, is really good to appreciate the different stages of neutrophil maturation. You, you pretty much get all of the, the cells apart from the, um, um, promyelocytes. Um, so it's really good just to review the different stages. And if you want to look at promyelocyte morphology, then look at cases of, um, APML. Um, from what we can also see from the blood count is that this is unlikely, uh, to be an acute leukemia because the platelet count is, is normal, if anything, it's slightly higher. And we're always guided by the clinical features as well. So, abdominal pain may suggest splenomegaly, which is very typical in, in chronic myeloid leukemia. And I think the, from a lab perspective, and I think it can be very challenging, but a very good differential count is really important, because we, we need to know what the blast count is, because as we'll go through later, that will define what stage of disease that you have. Anything more than 20 is acute leukemia. Whether there's any abnormal lymphocytes there as well, because you can get a lymphoid, uh, accelerated lymphoid disease or blast crisis on the background of CML. And a basophil, I, I'm very, uh, very interested and really want to know what the basophil count, because it tells us what whether there could be a possibility of accelerated disease. And there are some prognostic scores, um, for CML which rely on the basophil count. Um, there's one in particular which I, I tend to use, is something called the Utz score. So, from a sort of clinical perspective, those are the main things. But I think you, the, um, the features that you've described, and, um, the case, uh, has been excellent, really. I'm just going to have a look at the, um, whether when to call it. There's, so there's a comment here, Sam, about, um, when to call this a leukemoid reaction or CML. Maybe we can answer that question after looking at the next case.

Yeah, that's okay. It's funny that you mentioned the, uh, basophil count, because, uh, it actually happened at the facility I was working at, and a case that was CML where the techs were so overwhelmed with all the segs and the immature cells that they, and I think the count was 400, so even at 100, they, the fields were just filled, and they would skip all the basophils, so they weren't reporting the basophils at all, even though they're around three or four percent. And the pathologists and the residents were really confused as to why this patient that they thought was, while they were waiting for cytogenetics, was a cut and dry CML, didn't have any basophils. And it really threw them off, but it was mostly because the techs were performing a 100-cell count diff instead of a three or four hundred cell count diff, and they didn't know the significance and why it was important to count all the cells and not just the ones that they thought were important, which were the more immature cells.

Yeah, I think that's a really good point. I think the, in in these cases, the 300-cell count diff is relatively straightforward because there's so many of them, so many cells, so it shouldn't take you too long. And I think a big basophil, yes, an eosinophil count and a blast count is really important. So, that that's excellent, really. Um, should we, um, actually, what one final point is that, as you've already mentioned, Sam, that this, this kind of case should prompt us to start looking for the classical genetic abnormality related to CML, which is a translocation between nine and chromosomes 9 and 22, which leads to a fusion product between two genes called BCR-ABL1. Um, and we, we tend to do that quickly, uh, via, um, FISH, so fluorescence in situ hybridization. There are some labs that will also do it via PCR as well. So, it's really important to get those tests done quickly because treatments for this condition are really, really good, and the prognosis, provided the patients respond, is very, very good. So, it's good to to get those confirmatory tests done, uh, straight away, get the clinical team involved, uh, immediately, and, um, start treatment, which is, um, for the benefit of the patient, really. So, perfect. Should we, I think that's a good point to move on to case two.

Um, so case two is a 61-year-old lady. The, the clinical information that we've been given is chemotherapy for breast cancer. She has a slightly low hemoglobin, it's 109, but as with all these cases, there's a leukocytosis, 41.5, and the platelet count is normal at 281. Um, so it's quite interesting moving from that case to this case and see the subtleties between them. I'll hand over to you, Sam.

Okay, so, um, microscopic review is essential whenever we have a leukocytosis or an abnormality on our analyzer, and our flag on our analyzer flags leukocytosis at twenty thousand, and we just have to keep in mind to review all three cell lines, um, when we're reviewing the smear. I had seen that question on Facebook quite a few times where they had asked if it flagged just for leukocytosis or left shift, did they still have to review the RBCs and the platelets, or if it just flagged the RBCs, do they still need to review the WBCs? And the answer to that is yes, anytime you look at a slide, you need to review all three cell lines. And I always like to start all my cases on low power to see what is actually causing this leukocytosis. And this doesn't have as many WBCs as our last, which would correlate with our WBC count. It still has quite a few, and we can see that the predominant cell type here are the neutrophils, and that they are in various stages of maturation, but we can also see, I see a lot of monocytes here, um, a lot of immature cells, so maybe we have a left shift, so to speak. I don't see a ton of, uh, blasts, maybe just a rare blast, um, but we do have cells in all different stages of maturation. So, once I get an idea of the slide quality and I know which cell is causing or which cell line is causing the leukocytosis, I will usually, um, kind of look at the RBC and the platelets after that. And by slide quality, I mean the color, the distribution of the cells. You want to always check the feathered edges and the lateral edges to make sure that the cells are not all pushed off to the sides or the to the end of the smear, which can be termed as a snow plow effect when that happens. And you want to make sure that there's, uh, not more than four times, uh, the WBCs on the sides and the end of the smear as there are on the monolayer, which is hard to do on the virtual cases, but it's just something to keep in mind. For the RBCs of this case, we again want to try to find a lymph for reference for the general size of the RBCs, and the central power should be about one-third of the cells are at the center. Uh, here's a nice lymph here we can use for reference and look at its nucleus for the RBC size. And this, uh, lymphocyte, it's round or oval and has a slightly indented, uh, the chromatin is clumped and dense. There is no nucleoli present, and it has scant cytoplasm. It's very basophilic. The RBCs, there are some polychromasia, but nothing really stands out, maybe some anisocytosis. Also, the platelets are generally normal in size here. Here's some more platelets. Um, they're usually two to four micrometers in diameter and again with that purple color. Here's some more hair. Some larger platelets here, but we wouldn't call them giant unless they were larger than the RBCs. So, uh, the WBCs, um, most of these neutrophils have a lot of toxic features. You can see there's very dark, um, granules here. There's also Doly bodies present. We have quite a left shift present with a lot of myelocytes and immature cells, a lot of bands, more myelocytes, metamyelocytes, another band, and just all these toxic features. These are very dark granules. You can see a nice big Doly body right there in the cell. Some more segmented neutrophils here with dark, coarse granules, a mono here. One thing that helped for this case to help me differentiate maybe between the CML and the leukemoid reaction is that lack of basophils. There aren't nearly as many basophils or eosinophils present in this case like we saw in the last, and we are also seeing a lot of toxic features in this case, which we were not seeing in the last case, with these, uh, toxic granules and Doly bodies. And usually, if I see, um, toxic granules or Doly bodies, I look for the other one. They're not always present together, but more often than not, you'll see both of those features. You do want to be careful though with toxic vacuolation because that can sometimes be an artifact. So, again, more segs. Um, these are medium-sized cells, like I said, they're the most mature cell in the myeloid series. Their N:C ratio is very low, and these have a darker cytoplasm than what we would normally see. So, I have a myelocyte here. It's starting to indent a little bit. You can even see they call it the dawn of neutrophilia where it has that, uh, kind of clearing by the nucleus. Bands here.

Sorry to interject. The, the question is coming up about whether this could be CMML. I, I, I think you've already answered this, um, question, uh, in the previous case, but I think it's worth just going over. Was to could this be CMML? What are the features that suggest that it isn't or could be?

The toxic features, um, there's a lot of toxic features. And usually, uh, even in her case, the fact that she was on chemotherapy for breast cancer, sometimes when I see that, I'll even look at the previous counts to see what the white count was before, what it is now, and if I see a big jump, sometimes I'll look to see if they had put those patients on any sort of medication while I'm doing the diff. So, I do all of this kind of like while I'm doing my differential. If I'm kind of on the fence about something and kind of digging into the the history, um, and just the toxic features, there are a lot of really dark granules in the neutrophils, a really dark, uh, big large Doly bodies, along with a lot of bands, not a lot of basophils, not a lot of eosinophils. The monocytes, those threw me off a little bit, but again, that's why I would, uh, look in the chart and see maybe if the patient was recovering from chemotherapy, because monocytes are usually some of the first cells we see first coming back, or if, uh, someone's given growth factor, we see a lot of monocytes.

Excellent. Thank you. Uh, so, I, I, I mean, I, I think I've been busy scribbling away the, the main points that you've made. I think the one you've just made about looking at previous counts is really important. Um, the clinical information is really important because we're given the the information that this patient is, uh, uh, receiving chemotherapy for malignancy, so it could be due to their underlying malignancy, it could be due to, as you've already mentioned, growth factors, things such as G-CSF. And if you've got that information with these kind of with the morphological features, then you're pretty much home and dry, I think. And, uh, it, you very much avoid all the unnecessary investigations and panic that that follows afterwards, uh, if you, if you have that information. And the other points that you made were the, the toxic features, which were really significant. Um, the monocytosis is a bit unusual, but it can, can happen, especially in patients on G-CSF. And the, the key thing that you've mentioned, um, to distinguish this was with chronic myeloid leukemia, on top of the clinical information, the previous counts, etcetera, is the absence of basophilia and eosinophilia. I think that those are really good distinguishing features, but between a sort of what we call a leukemoid reaction and, uh, and CML, as well. So, that that's really, that's really good. Um, I don't know if you've got any further comments on cases like this or any sort of previous experience of dealing with cases like this in the lab.

Uh, it's, it's the toxic features, uh, and looking at that history. If you see a huge jump, and I know we have a television at our facility, so sometimes if I see something like this, it's kind of at first, like when you look at it, it's, it's a little scary, um, because the cells do look so angry, um, and toxic, um, and there, there are a lot of immature cells with those very dark granules, um, so you do get concerned. And usually, I'll even look at the cell vision cases, uh, in prior, and even look at the cells to give myself a better idea of what was going on, what is going on, and just get a more well-rounded idea. And the medication is, uh, and when I see a picture with the monocyte, monocytes with those toxic features, I always look to see if they were given growth factors, specifically because you'll see this type of picture time and time again in patients who are given growth factor, especially if you already know they're on chemotherapy.

I think, um, one, one thing that I've just thought of is that having a good relationship with our, with our clinicians as well is really important because sometimes we don't get any clinical information at all, and without that clinical information and correlating that with the morphological features, it's very, very, very difficult. And it's, um, it's really important to educate, um, you know, all the healthcare professionals who are requesting these tests, um, because that information is really important so we can give an accurate, uh, interpretation and an issue a valid report. I think I don't know whether you've got any experiences in trying to get, you know, healthcare professionals to give us more information or because in cases like this, it's really important.

I think, yeah, um, I, I, I think it is, uh, we have a pretty good charting system, so most of the information is in there, but I, I could imagine it would be, it could be hard. I know sometimes with getting a prior bone marrow results, like from 10 years ago, when they want to correlate them, they've had issues finding those or being able to get a hold of those slides to make the best, you know, report.

Perfect. Excellent. Should we move over to case three now? Um, I don't think we've got any any further comments.

So, case three is a 69-year-old male who's presenting with fatigue. His hemoglobin is a little bit low, it's 121. Uh, again, we've got leukocytosis. The white cell count by the automated analyzer is 30.2, and the platelet count is normal at 273. Um, I'll hand over to you, Sam, if that's okay.

Okay. We go through the slide. So, again, we want to know what's causing our patient's leukocytosis. Um, so we can look at low power and again, we see a lot of neutrophils here, but they are not in various stages of maturation. Uh, they're almost all exclusively, like mature neutrophils, either in the segmented, uh, stage or band, um, but there are not a lot of immature cells here. Um, all of these neutrophils look like pretty much, uh, segmented or band in the band form, maybe some myelocytes. Um, and we can see that our analyzer count does correlate with our slide. We do have an increase in leukocytes, as we can see, and our slide quality, uh, is acceptable. So, we'd always want to make sure to review that on, uh, 10x also. As far as RBC morphology, uh, is concerned, I didn't see much when reviewing this case besides a little bit of a basophilic stippling. I was very fine, um, in the cytoplasm of some of the RBCs. Usually, if I'm on the fence on a on television or a virtual slide, I would pull the slide and check for that under the scope because it can be hard to tell on the virtual films if that is present or not, but I did see some, uh, polychromasia, basophilic stippling for the RBCs. Platelets, again, are pretty. Here's some platelets here, normal platelets, normal size, maybe a slightly larger one, but I would still consider it. It's just a regular platelet. It's not larger than the RBC quite yet. A polychromatic philic cell there, another larger platelet here and here. Um, so next for the WBCs, um, again, these are segmented neutrophils. Uh, we have a nice eo here, a lymphocyte, some more segmented neutrophils. Um, again, they're medium-sized cells with very thin filaments, and they're filled with these secondary granules. You don't see as many, you don't see those toxic features we saw in the prior slides in these cells. A typical lymphocyte here, where it's kind of wrapping around the RBCs in a darker cytoplasm where it touches the RBCs. I also saw some of these, um, almost pyknotic or necrotic RBCs where they're starting to undergo apoptosis. I saw several of those. For the neutrophils here, uh, they just start to kind of, um, round up, they're spherical, they're very, they almost have a shiny kind of, um, look to them. Here's another band here. It goes in more than half the distance of the nucleus, and another lymph here. Here's a larger platelet up here that's about the same size as the RBCs. This cell right here is probably a myelocyte. It's got a nice round nucleus. It's not indented.

Do your facilities still call the band or differentiate, or are most calling, um, lumping them both together? I know that was a big debate and they were trying to phase out the calling bands in the laboratory, but I wasn't sure, uh, if labs were actually phasing that out or if most places were still differentiating between the two.

Here's some nice classical bands here, and that's C-shaped with no segmented, no segments, no filaments. We, we don't, um, repo, we, we report them together. The two cells. I'm not sure what other people do. Be interesting to see people's comments in the, uh, the comment section.

Yeah, I was interested to hear that, because at all, I've always had to differentiate, and I have noticed, again, when I look at prior results, my results, um, that there's a lot of variation in what people call a band. So, I would see as a clinician that that would be confusing if one day it was 25 bands and the next day it was eight, and the day after that it was 25 again. We even got calls about that in the lab just because it's very hard to kind of differentiate those, and it's tech to tech, and even the same tech could call a cell band once and then, uh, seg the next time. So, I was, uh, kind of wondering what the other, what other people were calling or doing, uh, in their practices.

I think we've had a couple of comments, and, um, I think the the consensus on those is that they're reporting them all together. Um, you mentioned a guideline that suggested that we should be counting them together. Which guideline was that?

Yeah, that was the Clinical Laboratory, uh, the Clinical, uh, Laboratory Institute had recommended not differentiating the two because it was not easy to correlate and there was a lot of discrepancies. Even like, if your facility has criteria of what they call a band, some is, uh, some criteria is stricter than others. So, even facility to facility, it can be different, and then you have tech to tech differences. Um, so they were suggesting not calling it anymore. I know in our API surveys, we stopped differentiating them for our lab surveys, our unknown specimens that are sent, they have us lump them together because they also saw that discrepancy, so they stopped separating those two out.

I think it would be really nice if we didn't call it, but I think our, our physicians want those, uh, to be called. Um, as a physician, do you, do you feel like it's a useful parameter to have to know if someone has a left shift?

Well, I think it's useful in, in some, some disorders. Um, I think because we, the comments here and what you've already mentioned, that this case is a sort of a mature, uh, neutrophil leukocytosis, neutrophilia. And if we're thinking, is this a leukemia reaction? Is it CMML? CML in particular, then is there a more of a left shift? Um, but generally, if it's left shifted, you're probably going to see more myelocytes, and that's a better distinction of calling something left shift and, you know, numerous band forms. I think, and I think that's where the these sort of consensus comments from, from big groups have come from, really. So, um, it's, um, it's an area of debate, but I think from a clinical perspective, if you're going to see left shift, you're probably going to see more cells which are immature than the band form stage. I think.

I don't know if you agree, Sam.

Oh, I agree. I hate even differentiating between the two, like at my facility, because it is in our policy that we do so to correlate. I have to do that, um, but it, it really, it bothers me. I hate trying to differentiate between the two because it's just not very reproducible, and it's sometimes it's even hard to tell.

So, what would you, I mean, just coming back to this case, what, what would you do? You've got the, the blood count, so obviously it's abnormal, and we've correlated it with a clinical history that's just fatigue, and we assume that, you know, there's no other clinical features to suggest a leukemia reaction, things like infection, malignancy, growth factors, etcetera. Doesn't look like CML. What kind of things maybe should we be thinking about and how we take things further for this case?

This would be, there isn't very, there's not dysplastic features. Um, I, as a clinician, I guess they would, uh, they could do further studies. There are additional cytogenetic tests available. And I saw in our laboratory, um, when they suspect any of the myeloproliferative neoplasms at all, they can order a panel that includes the new colony or colony stimulating factor 3. It's in that panel, along with Bcr-Abl, JAK3, and some of the CALR genetic tests, and they can send one sample to get all of those tested. And this one, I would see if they had an increase in neutrophils, uh, for maybe one time. It would be, you know, if there was no reason to suspect infection, and you had done, uh, maybe, um, like blood cultures, um, and some other, like septic tests like PCTs, lactic acids, and there was no indicator of infection, maybe then look towards, uh, what was causing this leukocytosis.

Yeah, I, I, I agree. Yes. Um, I, I, we, um, I've got an interesting table that I can share with you, and we can just sort of, um, we can compare all the different, um, diagnoses, uh, that is possible from morphological features like this, as well as the ones that we've seen previously. If I, let me share my screen.

Okay, so, so hopefully, can you see this table, Sam?

I can. Yeah. Perfect. Okay. So, this is a very, very busy table. I'll break it down and try and go through the main points from it. So, of all the cases that we've seen, there's a predominant neutrophilia. There's a leukocytosis, which, uh, we also see some of the granulocytes, the basophils, and the eosinophils. I think this is a useful table, um, to differentiate all the different diagnoses that this, these features could be due to. Of the two most common things that we would see, and common things being common, we should always think of of common things before thinking about really rare things, the leukemoid reaction and chronic myeloid leukemia. So, Sam has beautifully already gone through the the features of leukemoid reaction. So, these are left-shifted neutrophils. We get, we get reactive features, toxic granulation, vacuolation. We may even see some Doly bodies as well. And this will be supported by the clinical history, infection, malignancy, growth factors, etcetera. And usually, we shouldn't see any cytogenetic abnormality associated with that. These are acquired causes. And then less common, but more common than the other conditions, is chronic myeloid leukemia. And as Sam has gone through, the classical features, left-shifted to neutrophilia, you get that myelocyte bulge or peak, so there's an abnormal increase in myelocytes from the, um, all, if you count all the immature, uh, neutrophil precursors, you'll see that eosinophilia, basophilia as well. And the classical thing that we'd see on a genetic level is the presence of the Philadelphia chromosome. It's important to know the other features of accelerated disease as well, which are all down there. Um, and then we've got really weird, wonderful things which are extremely rare, and we don't really know what's the the best treatment for these because often because of the rarity. So, the, the last case that we've got, I think it's useful to consider because it doesn't look like a leukemoid reaction, it doesn't look like CML. Think about, could this be atypical CML, which is a type of myeloproliferative neoplasm, where the white cell count is above 13, you will get neutrophil precursors, uh, constituting 10% or more of the white cell count. The total white cells. Dysplasia is, is a really useful feature because it's very common in atypical CML, and you don't tend to get that basophilia, you don't get monocytosis as well, and obviously, you won't get any a significant increase number of blasts. So, just remember that the magic number for blast percentage is 20 for all these conditions, because as soon as you hit 20 in the blood or bone marrow, then you're looking at acute myeloid leukemia. There are no specific genetic changes that we see in atypical CML, uh, but as you mentioned, Sam, that we'll often see, uh, rearrangements, uh, we'd need to exclude rearrangements of the JAK2 gene, any other associated mutations related to myeloproliferative neoplasms like CALR and MPL. And there are really rare things which are associated with a gene called platelet-derived growth factor receptor A and B. Um, so, a lot of the time, morphological features and, uh, excluding all the other really rare and wonderful myeloproliferative disorders, then chronic neutrophilic leukemia, which this case turned out to be. So, we'll get, uh, an increase in white cell counts. It's more than 25. These, these are, this is a mature neutrophilia. So, you'll get 80, more than 80% which are segmented or band neutrophils, and that's why it's quite useful to lump these together, uh, these two stages of maturation. Um, and, um, we shouldn't see a huge number of immature forms. They should constitute less than 10%. Dysplasia shouldn't be there, so that's a good distinguishing feature between atypical CML and chronic neutrophilic leukemia. We shouldn't see very marrow blasts, very rare in this condition, and monocytes, um, should be less than 1 times 10 to the 9 per liter. Um, and as all, as you mentioned, Sam, that the key genetic factor, um, driving chronic neutrophilic leukemia is the CSF3R mutation, and there are various different types of mutation. It's important because this mutation has been proven to be sensitive to various treatments, um, so JAK inhibitors such as Ruxolitinib and tyrosine kinase inhibitors as well, drugs that we tend to use for chronic myeloid leukemia as well. Again, you want to exclude all the other, um, rare and wonderful genetic abnormalities related to rare myeloproliferative neoplasms. And then CMML is more common than we, than atypical CML and chronic neutrophilic leukemia, but we, I think we've already gone through the main features of CMML and distinguishing the the cases that we've seen from this condition. But really, what you're looking at is a persistent monocytosis. It has to be more than 1 times 10 to the 9 liter, and the monocytes should constitute more than 10% of all white cells. Uh, it has to be, uh, persistent in terms of monocytosis for more than three months. Less than 20% blast. Remember, blasts in CMML, you count monoblasts and and promonocytes as well. So, it's really important to get that distinguishing feature, and dysplasia is a, as a typical feature that we'll see in CMML, because CMML is a, is a bit of an odd disease. We've got dysplastic, uh, features of the condition as well as a myeloproliferative features as well. So, we get high white cell counts, and we get that dysplasia as well. And again, we wouldn't see those, uh, genetic changes that are associated with, uh, rare myeloproliferative neoplasms. And then there's an even rarer condition that we see in the pediatric population called, uh, juvenile myelomonocytic leukemia, which is typically associated with other sorts of clinical features which I've listed here. Um, so, I think it's, uh, this table, I'll make available after the webinar, but I think it's a useful overview of, uh, of, um, the different types of conditions that these cases should prompt you to think about and, uh, differentiate as well. So, um, I don't know if you've got anything further to add, um, Sam, um, any sort of, uh, pills of wisdom from, from a lab perspective and a lab manager and trainer as, as to how to approach these cases as well?

I just always review the peripheral blood slides in a systematic process and try to evaluate all the morphological features, including like overall cell size, the nuclear characteristics, the cytoplasmic appearance, because it is important that we appropriately identify and classify these cells to help the clinician determine diagnosis and further evaluation.

Excellent. Thank you. So, um, I think we're dead on one hour now, so we've, we've done really well. Thank you so much, Sam. The really great overview of three really interesting cases. I'm, let me just bring my slide again. Here we go. There we go. So, thank you, Sam, for again for an excellent overview of granulocyte cases. Uh, thank you for everyone who's joined into the webinar today, and thank again our sponsors, Uber Mannheim. Um, you can still, um, uh, register for your CME certificate, uh, provided you've registered, we'll email that to you in the next couple of days. I will put a, a survey for this webinar in the comments section, and that will be emailed out with a certificate. Um, if you want to watch, if you want to watch the webinar again, then it will make it available, hopefully, in the next day or so on the YouTube channel, and we'll copy a link onto the Hematology Interest Group. And, uh, hopefully, we'll see you again in two weeks' time where our fifth of, um, part of the webinar series, we're going to be joined by Joe Side from Australia, slightly earlier than planned, at nine o'clock in UK time, where we're going to be discussing another three interesting cases, but with a focus on platelet disorders. So, we look forward to seeing you there. Look, look out for the announcements on the Hematology Interest Group, and thank you again, uh, Sam, uh, everyone for watching.