Transcription
This webinar series has been kindly supported by the company Uber Mannheim, and the webinar series is also accredited by the Royal College of Pathologists in the UK for CME points. This webinar is for one CME point. Uh, it is important that you register for your CME certificate before, and which you can also register at this time now as well, before the end of this webinar. I will post the link to the registration form in the comment section on the Facebook group. If you still haven't registered for your certificate, uh, we'd like to think that you want to see this again. This webinar, um, all the webinars of this series will be available on the Blood Academy YouTube page, um, so it'd be an opportunity to revisit the cases and the learning points that I discussed as well.
This webinar series is an introduction to blood microscopy. We'll try and go through as much as we can, but if you do want to learn more in a more detailed and work at your own pace, then the Blood Academy website has a, a essential blood cell morphology course, which consists of video tutorials, online modules, and assessments. And again, this is also accredited by the Royal College of Pathologists. We are running a, a discount with the Hematology Interest Group where you can save 20 percent. So if you're interested, go and check that out on the Blood Academy website and use the code HIG20 for your 20 discount.
Uh, okay, so today I'm going to be joined by Joe Sidey. Um, he is based in the eastern part of Australia. Um, just a little bit of background before, before we start about Joe. He gained his, uh, bachelor's degree in Medical Laboratory Sciences, uh, from Wollongong, hopefully I've pronounced that correctly, and Charles Sturt University in East Australia. He's got over 10 years' experience as a hematology scientist, especially in the field of blood cell morphology and microscopy. He is now working as a, as a senior laboratory supervisor at Coffs Harbour Hospital, just north of Sydney, and he's responsible for training and professional development in the team that he leads. He's also one of the hard-working admins of the Hematology Interest Group. So welcome, Joe. Thank you, Ali.
Excellent. Um, so, but before we crack on with the cases, I'm just going to put it out there to everyone that, um, we want to try and make this webinar as interactive as possible using the comment section. Um, and we, uh, we would really appreciate to get your opinion on what the diagnosis is from the cases that we've discussed. So I'll just allow you to share your screen now, Joe.
There we go. That working? Perfect. Excellent. Uh, so should we, um, we start off with, uh, with case one? Uh, we've got a 42-year-old female who's presenting with slurred speech. Um, she's anemic. She's got hemoglobin of 90. We're not given the, the differential count for the white cell count, but it's 4.1. The total leukocyte count, and the platelet count is 41. Uh, so I'll, I'll leave it over to you, Joe, just to go through some more. We can discuss some of the, uh, the interesting, uh, issues around it.
Okay, no worries. Um, so we've got a 42-year-old female, and she has slurred speech. She has a mild anemia of the Hb of 90. The white cell count is 4.1. Uh, the normal range for a woman this age is, well, four or 4.5 is the lower side of normal range, so it's mildly reduced. But we need to keep in mind that it may actually be a normal white cell count for, for the individual. And she has a moderate thrombocytopenia of 41.
Okay, so let's have a look at the slide. Uh, before we look at the slide, actually, if this is a real patient, you would definitely want to be checking the sample for a clot, uh, in case there was a difficult collect or the tube wasn't inverted, uh, sufficiently post collection for the EDTA to stop being to act as an anticoagulant. Um, so when a film is reviewed, uh, you first want to start on low power, and because the patient has the lower platelet count, you definitely want to be looking in the tail section of the film. And unfortunately, the tail section's been cropped out a little bit in the making of this virtual slide, but we'll do what we can. So looking at low power, and with a low platelet count, sometimes you can get platelet clumping that can form, which could either be due to a traumatic collection or poor collection technique. Maybe the sample is collected in a needle and syringe, and the sample's left in the syringe for a prolonged time before transferred to the anticoagulated tube, and you might get some platelet activation, not enough to make a clot form, but the platelets still might aggregate together. So we're just having a look at low power and look around our sort of tail to see if we can see platelet clumps, and we can't see any. So what platelet clumps would look like would be, uh, light blue aggregations of platelets, and maybe like, from this, like small bundles of grapes, kind of. So we can't see any platelet clumps, so we're going to believe this platelet count is accurate. And also, when you're looking at low power, it's good to assess the whole slide to see the quality of the smear. You don't want to see, you probably don't want to see large bridges, no, you can't, large bridges of thicker areas of film where the red cells and white cells clump come together more, and then thinner areas, because when you do your differential, you may not get an accurate differential of your white blood cells. But now we've assessed that the white cell, it's a, we've assessed that it's a well-made smear and that there's no platelet clumping. So we'll go into a higher power for probably something about 40 times or 20 times that used to in your laboratory. And the very first thing that would jump out at you is the quite irregular red cells, as you can see here. There's this red cell that's, we call this hyperchromatic, where you, you no longer see an area of central pallor, which is a lighter area of the red cell, and it's also lost its circular shape and it's become this sort of, um, two pointed ends at each side. And here's another cell with pointed ends, and it's got a slight area of central pallor, but it's starting to lose its central pallor. And we'll also see there's lots of these crenated cells or echinocytes, as they should be called. So the most prominent features for the red cells that we can see are definitely these cells that have pointed ends and with a lacking central pallor, hypochromic, and these are called fragmented red cells or preferably schistocytes. And there's lots of these cells with crenation. And also on the blood film, there's this cell here that has this dark blue, dark blue, dark purple, looks more like a miniature nucleus. That's called a Howell-Jolly body. Other red cell features that are presented on the film, present film, uh, here is a cell that has no central pallor next to a normal-looking red cell. And so this would potentially be a spherocyte. I'm sorry, I'm a little bit nervous, so I jumped a little bit ahead of myself. When you're looking at low power, you should also, before you jump, dive down and look at the more high-powered field, you want to find the ideal area thickness or a monolayer area of the blood film. Dan Pelling talked about this in the webinar number one. Um, so the ideal area of thickness is an area on the film where the red cells are evenly distributed, perfectly distributed, and you start to get some overlapping of the red cells, and it's the best area to examine for red cell features, white cell features, and also platelet morphology. So we'll dive back down into the high power field, and we can see lots of these cells that have that are hypochromic with pointed edges, and they also can be quite speculated and crenated as well. These schistocytes, which is not uncommon to see when they're formed. And there's also some polychromasia. It's not overly prominent, but there is a higher than normal amount of polychromasia. So every, every high power field, we're seeing a polychromatic cell or two. And reviewing polychromasia, polychromatic cells, let me just start again. Polychromatic cells are these cells that have this, red cells that have a purple appearance, and they've got basophilic and eosinophilic staining characteristics due to the higher RNA content. Blood cell, and they're immature red cells. And in the red cell maturation sequence in the marrow, you have erythroblasts, they mature down into nucleated red cells, and then the nucleus undergoes like an apoptotic process, and it gets extruded from the cell, and you still have red cells that have, right, lots of ribosomes and RNA in them. And on a peripheral blood film, they're immature, but they would stain this polychromatic appearance. So they would have eosinophilic pink, red, and basophilic blue and purple staining characteristics, unlike mature red cells, which have this, uh, eosinophilic staining characteristic. And the degree of polychromasia on a film doesn't always directly correlate to the number of these immature red cells, which are called reticulocytes. So a reticulocyte count, whether it's manual or automated from an analyzer, is the best way to report the actual reticulocyte concentration and percentage for a full blood count. So at my laboratory, we don't get too carried away with grading the reticulocyte level. We just report if we see enough that we believe it's significant, we just report that there would be significant polychromasia present on the blood film.
So to summarize the red cell features that we've been seeing on this blood film, uh, there's significant numbers of schistocytes, and schistocytes are these cells with pointed ends, and they're hyperchromatic, and they can also be a little bit crenated. So don't let that throw you off that, like this cell, it's got two pointed ends on the polar ends of the cell, but it's also quite a couple of bumps along it that are crenation. And say, say this one, there's still a schistocyte. Lots of crenated cells. Crenated cells can be the product of abnormal renal function, like acute kidney injury. They could be due to improper sample storage, like heating the sample, or the sample being left at room temperature too long. It can be an artifact. Now, that would be an artifactual crenation. Another artifactual change is if it was a difficult collection and there wasn't much blood in the EDTA sample, the EDTA, uh, may cause crenation of the red cells. Generally, in those situations, you get a uniform appearance across the whole blood film of crenated cells. Schistocytes, that is the other most significant thing. They're, uh, the herald of a microangiopathic process or mechanical hemolysis occurring in vivo, inside the person's body. That could be due to damaged heart valves, and the high forces applied to the red cells when they're passing over a damaged valve, causing the red cells to tear. Or a prosthetic valve that has fibrin or is damaged or has fibrin strands to form on it, and then the high shearing force, the high flow across it from the heart pumping will start tearing the cells, and the cells get a tear in them. And because they're a bi-lipid membrane on there, they'd love to reform after they've been damaged, but they've lost their structural integrity, and they form this, uh, elongated cell with two pointed ends. And there's a, the other thing I talked about was a microangiopathic hemolytic process. So microangiopathic is a dysfunction of the small vascular system. Hemolytic, just rupturing or destroying of red cells. And anemia is a low hemoglobin or red cell count, or just process, just it's a dysfunction of the microvascular system that's causing mechanical hemolysis, essentially. And I won't go too much into why we're seeing these on the film to the end, because we, I don't want to give too much away. But one thing to remember when you're looking at a blood film is we're getting quite excited that we're seeing all these very significant red cell morphologies, and this might be an actual textbook case or something very significant that every good morphologist should know. However, we shouldn't get too carried away and forget to look at the white cells and the platelets, because every single blood film review, a good morphologist should be reviewing red cells, what are the specific morphologies we're seeing? Is there any? If there's not, should we be reporting these minor morphologies that we might say that might not have any clinical usefulness to the clinician referring for the full blood count? What's going on with the white cells? Is there any significant morphology we can see? Is there any cytosis, high cell counts, any cytopenias, low cell counts for the white cells, and what are they? Is a manual differential required? Can we trust the analyzer's results? And the platelets, is the platelet count accurate? Is there something going on like platelet clumping that we looked at straight away because the platelet count was low? Can the platelet count be trusted? Is the analyzer's platelet count accurate? If, if the method used for the platelet count to determine the platelet count is an impedance method, an analyzer, large and giant platelets might be inaccurately counted as red cells, or even very small red cell fragments or microspherocytes. Could they, they may be counted as platelets? And in a case like this, where we've determined yes, there's no platelet clumping, so we want to trust the analyzer's result, is there enough very small fragmented red cells like this? Is this potentially causing a falsely high platelet count for this case? And that's where maybe if you have an analyzer that can do an optical or fluorescent platelet count, it might be able to give a more accurate platelet count, and you might not have to go down the rabbit hole following an indirect platelet count or a platelet estimate. You can trust the superior methodology of the analyzer doing an alternative platelet count method, that's if you're lucky enough to have a laboratory with optical platelet count or support.
So like I was saying before, I'm digressing. Uh, I was saying before, it's best not to try not to get too swept away with the very significant red cell morphology you're seeing in a case like this, and remember you always need to assess your white cells and platelets. So we'll move into white cells. So the white cell count was 4.1, which would be mildly reduced for a woman or 42-year-old, 42 years old, at least for my laboratory would be, um, maybe not for yours. But the most prominent cell, most numerous white cell we're seeing when we're looking across this blood film is mature neutrophils. And I'm not going to go ahead and do a manual differential because that'd be a little bit awkward and difficult, but it looks to me like there's about 80 percent, like four out of five maybe, or maybe a little bit more of the white cells are mature neutrophils. So it's pretty safe to say that this patient has a normal neutrophil count. Where a neutropenia would be something below 1.5, and neutrophilia would be above 77.5 or something. And the white cell count is 4.1, so it's probably around about the 3.3.5 mark. So it's a normal neutrophil count. Now, the neutrophils that we're seeing now, we have, we have to examine them morphologically. So we can do that by the lobulation. Have they got the, I mean, a mature neutrophil normally has two to five lobes. Is most of the neutrophils have two to five lobes? Then that would be considered normal lobulation for the neutrophil. And going off this film, it looks like that is the case. And then there would be granulation for the neutrophils. Is there toxic or hypergranulation or increased granulation, which might be a reactive process to infection or inflammation, or maybe a drug therapy that they've been given? Or is there hypogranulation or agranulation? That's when a neutrophil has reduced numbers of primary, tertiary, secondary granules, or an absence of granules. And that would be a dysplastic feature, which suggests that something has gone wrong in the granulocytic maturation sequence in the marrow, which would point towards a dysplasia, a mild or myelodysplastic syndrome potentially, or a mild myeloproliferative neoplasm like, uh, MDS or CMML. But this, the neutrophils in this patient, on this patient's blood film, have normal granulation. It's not overly increased or darkly staining. You can definitely see this granulation there. It's not hypergranular. So I'm happy to say that these neutrophils look happy and healthy and normal for an individual. Now, um, there's not too many lymphocytes that we've seen, but the ones that I have seen have been well differentiated. There's been a couple of lymphocytes of a bit more abundant cytoplasm, and there's been a couple of small, small mature lymphocytes. Um, I haven't seen many much reactive processes. And episodes, sorry about, uh, I've got some optimal internet at my home, so we might get some buffering issues from time to time. So I was just going to mention that we've got some, uh, comments coming in about the diagnosis. The, um, but we've got a range of things in that this is, uh, could be TTP, HUS, DIC. And, um, we've got some, uh, comments also saying that this could be, um, as you've mentioned, microangiopathic hemolytic anemia. And, um, one interesting comment, question really, is "noticed some red cell agglutination." Is that significant, or could that just be due to where the, well, could we just be not looking at the right spot on the film? But, yeah, that's a keen eye. I completely overlooked that. Kudos to the person that pointed that out. So I was a little bit nervous when I first started, and I jumped straight into it and didn't talk, didn't talk much about looking at the ideal area thickness or the monolayer of the blood film. So, uh, on low power, so to low power, you want to be able to see some of the white cells and red cells. So we want to go down and look at where the red cells start to overlap, but they're evenly distributed, as or as fast as we can get, especially a patient that has a hemolytic process going on or anemia. They can be sort of passively like little gaps every now and then in a blood film. It's hard to really find that area that is a good ideal area of thickness. But I'm happy about, I'm about here where there's some, I stopped pointing my finger and I start using the cursor, so you can see where I'm looking at. Sorry, guys. Um, there's some red cell standard overlap, but the majority of red cells are sitting by themselves. And as you can see, they're not really evenly distributed, but there's sort of areas here that, um, there's a gap. But and we can't, like, I think where I was looking at before was too thick in the blood film where you start seeing this pseudo-rulo formation, which is like rulo formation is when, think of it as like cells sticking together end to end, looks like a pile of coins that has been tipped over on a desk, or poker chips that have just been knocked over in a sort of form a stack, or like Oreo biscuits laid down on, um, on a serving platter. So we're not seeing that. But if you go too far in the area of thickness, you might start seeing agglutination, which is different to rulo. Red cells sticking together like rulo, but it's more haphazard and like a bundle of grapes, or, um, frog spawn, or fish eggs. Uh, so I don't believe there's agglutination in this film. So I always find a challenge, um, Joe, that when you're, so anemic that the film, depending on where you're going to see what the, what the monolayer is, where that sweet spot is. But I think it just keep going up to the, the thinner part, and you'll see the agglutination disappear really. So, yeah, I think there, yeah.
Um, so, I mean, in this, looks like a really scary film, and, yeah, it's, it's something that needs to be actioned straight away. Um, in, in terms of the sort of, um, would you put a percentage? Is there a significant number of schistocytes? Oh, yes. Um, so my laboratory, uh, our standard operating procedures for reporting blood films, uh, we don't grade red cell morphologies. We only comment on when their significant morphologies have been identified. But I am, comment, I am familiar with, like, the grade, the grading system that hopefully most laboratories are using, but is it ICHs or ICSh? Yeah. Um, so schistocytes sort of fall in their own sort of category where you can only see, if you see a small number of schistocytes with a low platelet count, it's considered very significant. Like this case, this, as a couple of the viewers have pointed out that, they believe that it's a microangiopathic hemolytic anemia, and some people pointing towards DIC, disseminated intravascular coagulopathy, or TTP, thrombotic thrombocytopenic purpura, or HUS, hemolytic uremic syndrome. Or TTP, HUS, and DIC are scary things, and they're all three genuine hematological emergencies where you really want to action these results quickly and accurately to the referring clinician, and hopefully give them some good advice. Um, so, I mean, if I had a moderately low platelet count with two to three schistocytes per high power field, I'd be very concerned. And this, this, this film, I'm seeing like dozens, dozens and dozens per field. What were you, what do you want that? Sorry for cutting.
So, no, I was just going to say about the, the Howell-Jolly bodies as well. I mean, it's a bit atypical for, for a MAHA process. Is there any way we can explain that? Oh, yeah. So, I, for, like, this looks like an acute process. So it's something that's suddenly onset, a patient. And so I wouldn't normally expect to see Howell-Jolly bodies, um, in a film like this. You could see Howell-Jolly bodies in the film with a patient with, uh, acute or a hemolytic process where the spleen function is to filter damaged and aged red cells in the body, and it's sort of getting overwhelmed. So you get a small number of Howell-Jolly bodies performed naturally from production erythropoiesis in the marrow. They come out of blood supply, but in a healthy individual, they get the Howell-Jolly bodies get extruded by macrophages and spleen quite quickly. But the spleen is getting overworked and tired due to all this hemolysis and damaged red cells that are about. I would expect some to start slipping through the cracks, and you'd get, might be able to see a couple on the blood film. Or this patient might have a complicated medical history, and they might be like splenectomized previously, and it's just a coincidental finding on this blood film. Maybe, I don't know. Do you have anything to add, Alan?
No, I don't. I mean, I, I think, um, just answering some more, more comments on the, uh, the comment section, the, the significance of the percentage. I think one percent generally is significant of all red cells. Um, and in cases that I've seen, at least, this is very, very obvious. But in some cases, it can be quite subtle. There was, there was a move previously to monitor the percentage of, of, uh, schistocytes with treatment, but now that that's been superseded by other techniques really in patients with TTP, really. So, okay. I don't know if, if I could just give my comments on the sort of the clinical side of it, because, yeah, definitely the, you see a film like this, really, it needs to be actioned straight away, and the team is looking after the patient needs to be contacted because the differential diagnosis is so wide, and really have to correlate that with the, the clinical picture. And there could be things like, as you've already mentioned, things like DIC, um, TTP, HUS, even things like pre-eclampsia can cause this. So the clinical history is really important to sort of differentiate what we do next, really, and what it could be. And then you're looking at your, your basic tests to, to, uh, confirm hemolysis, your reticulocyte count, LDH, etc. And then, um, if you're strongly suspecting that this could be TTP, and a good differentiator between that and other conditions is, is doing an ADAMTS13 level, really. So, yeah, I know in some places it's not that easy to get, um, to get an activity level. So really trying to get, get things rolling, because if, especially in TTP, TTP is a very, um, treatable condition, and the outcomes are very good, really, if provided the patients are treated quickly. So time is of the essence. Uh, I think, as you mentioned, this is a medical emergency, and get things moving quickly, really.
So I think, so I, I think that's an excellent start, Joe. Should we move on to case two?
Yep, if you're happy.
Um, so case two is a 70-year-old female who's having a routine blood count as part of the, um, pre-op for knee replacement. Uh, hemoglobin looks okay, 115. The white cell count is within the normal range at 8.5, and the platelet count is low. We've got a common theme here. Um, yeah, it's reported by the analyzer at least at 31. So I'll hand it over to you, Joe.
Yep, no worries. Okay, so the platelet count was 31. So like I talked about the first, uh, case, uh, should we be trusting this result? There's a couple of checks we can do. Number one, check the sample for a clot. Is the sample clotted? Look for an alternative EDTA sample that would may have been collected and see that's clotted and run it. Or if it's clotted and you don't have another sample to use, organize a recollection. Or maybe the platelet count may be inaccurate because there might be platelet clumping or platelet satellitism. That's where there's a, the platelets stick to the outside of white cells, mostly that you see, and they're stuck to neutrophils in the sample, just on the blood film. And when they go through the analyzer, they don't get picked up during the platelet count. And platelet clumping, one big clump of 10 to 15 platelets is counted as one platelet, or might even become a white cell or something in the white cell count. Um, and you get a falsely low platelet count. So if the sample's not clotted, you make a peripheral blood film, dry it, stain it, examine it. And when you examine it first, then low power, and you would look at the feathered edge, and you'd be looking for platelet clumps. And on low power, and I mentioned before, they look like irregular shaped light blue, purple splotches or bundles of grapes. And this is a textbook case of platelet clumping. And we'll zoom in on high power just to have a look at it, and wait for my slow internet to buffer and load it so we can see there's lots of little platelets all aggregated together in this big clump. And when the analyzer has done the hydrodynamic flow for like an impedance platelet count, these would go through as these big clumps, and probably not only get counted as platelets, probably it can be counted as white cells or nothing because they're even too big. And you would get this falsely reduced count. So the platelet count of 31 is definitely not accurate.
So a good morphologist is a hematological scientist for a low platelet count, that's a new presentation, should always be reviewing a full blood count, a peripheral blood film, just because if you're inaccurately report a low platelet count, a clinician may make a clinical decision on that and start treatment. If they believe the patient might have something like ITP, they might start on corticosteroids or something, and you don't really want that occurring. Or maybe the platelet count was five or something, and, uh, they might have, they might send them to an emergency department, an ED, and order a platelet infusion transfusion. And we don't, and the transfusions, regardless of the red cells, platelets, or plasma, always have an inherent risk associated with them. It's not a perfectly safe practice, and we want to avoid this best we can. We want to provide quality work, high quality results, quality. Yes.
Okay, so this is a textbook case of platelet clumping, and platelet clumping can be due to two things predominantly. So it could be due to, [Music] an in vitro phenomenon where the platelets aggregate together due to an autoantibody. It, I'm sure everyone who has done a bit of study has in medical laboratory science and hematology has heard about EDTA-dependent platelet clumping, and that is where there's an autoantibody that inside the individual, inside the body, doesn't cause the platelets to clump, but when it's exposed to EDTA anticoagulant, the, like the antigen on the platelets that causes the antibody to cross-link and make all these aggregates to form, gets changed slightly with the presence of EDTA and it allows this autoantibody to actually cross-bind the platelets together and of course forms clumps inside an EDTA tube. There's also clumping that can occur, um, platelet clumping that can can occur in vitro that is not due to, it's not EDTA dependent, and I'll talk, we'll talk about that later. But you can also get platelet clumping due to, uh, improper phlebotomy technique or a difficult collection, like someone's got a hard vein to access and someone's really struggled and tried their best, so and they've gotten some blood, but it's been traumatic, or it's been delayed transferring to the EDTA sample, and some platelet activation has occurred, and partial coagulation has occurred. It might not be enough to form a physical detectable clot in the sample, or check for sticky finger streams, like the way you detect, look for a clot in an EDTA is with an applicator stick, one or two or three of them, stick them to the sample, so go fishing about, pull them out, have a look, and you can might pull out a big nasty clot or a couple of little clots, or maybe the blood's just extra sticky, and you know some sticky filaments that, uh, stringy strands that are sticking to the wooden sticks, and they're called fibrin-like fibrin streams, and that's a clot is started to develop. And, um, I can, I would highly recommend you how you action and troubleshoot these. Follow your standard operating procedures or use procedure manuals for your laboratory, because how to troubleshoot platelet clumping varies wildly from laboratory to laboratory, and we get this on the Hematology Interest Group when people discuss this, and some people are very, uh, relaxed about it, and they would give a qualitative analysis, like report of, "I think platelet clumping," or there might be a hospital lab, and you have quick access to a patient, and you would organize a recollect pronto, like you can. And if you think it might be, and collect it in conjunction with a citrate sample, maybe, or alternative blood tube. But, um, I'm sort of diving into the discussion of the case, and Ali asked me not to. No, okay.
So, let's just get back into reviewing your blood film. So, um, as I mentioned in case one, uh, it's best, you got to remember not to get swept up and just thinking about the platelets in this case, because there's so much prompting, we need to assess. Like, we've got a blood film here, and we're probably going to be reporting results. So, um, we need to review this film because there's, there can sometimes be some insidious morphology that can be lurking in a case, and we don't want to get too relaxed just because we think, "Oh, yeah, my review and blood film because platelet count's okay," which is this clumping. It could be something going on. Like, you never know, like there might be malaria hiding in some of these red cells, or they come, it might be a couple of blood cells, and someone's developing a myeloproliferative neoplasm and leukemia, or they might be abnormal lymphocytes, and they might, you might catch something, you might find Sezary syndrome or something. Like, you never know. So it's, try, like, be a diligent morphologist and always review your three cell lines when you're looking at a blood film. It will add a little bit of time, but one day in your career, you will pick something up, and you'll be pretty proud of yourself and pretty chuffed. So, um, okay, so we're looking at this on low power. We've detected this platelet clumps. So now we're going to assess the quality of the smear. This looks like, disregarding the platelet clumps, quite a well-made smear. Um, the white cells look evenly distributed, uh, the red cells look evenly distributed. There's no big columns of thicker bands of wet red cells and white cells. So now let's zoom in a little bit more to try to find this monolayer or ideal area of thickness we're talking about before. So we want to start seeing that red cells being evenly distributed and also some regular standard overlap. And I think I've got it now. So the red cells are mostly distributed and separated, but we can see that there's some red cell overlap. So this is the perfect area to perform a differential, where it's the white cells to be evenly distributed, and the red cells will be able to see the morphology clearly. If you go too thin of an area of blood film, you will start the red cells start to lose their central pallor, and you might start, you might be overcalling spherocytes or fault in inappropriately reporting schistocytes. If you go too thick in the film, you might start seeing rulo formation when it's not necessarily there, it's not significant, or you might start seeing what you think might be agglutination, and you start, you wait, then you go troubleshoot it and follow your procedure manuals, and you start warming samples, cold agglutinins, and you waste the 20, 30 minutes of your time. So it's always good to look at the ideal area thickness.
So let's start off with the red cells. The red cells here are so normal in appearance. They're circular in shape, they've got a central pallor. Uh, there's occasional crenated cells and irregular poikilocytes, and there's a little fragment here. But as Ali was talking about before, like, you gotta look at the whole picture, like every field of view here, we're looking at, there's a couple hundred red cells, and we've seen one crenated cell in a couple of fields, we've seen one irregular fragment and one irregular poikilocyte that I couldn't name because, so it's less than one percent for these things. So they're not really significant, and the red cells appear normal in size and shape. So to find, so I said the normal size, to do that, I would find a small lymphocyte because a MCV wasn't wasn't provided in the full blood count results. And here's a small lymphocyte. It has a small amount of cytoplasm, which is ideal, and it's got a dense and compact nucleus. So a normal red cell, I believe, is about seven to nine micrometers in diameter. I may be wrong there because, yep, I haven't really looked that up recently, and something I don't use in day-to-day running and reporting of blood films, running a laboratory reporting blood films. But a red cell should be the same size as the nucleus, or maybe a little bit smaller. And here's the nucleus that's about from here to here, and this red cell is about the same size, just a little bit smaller. But I'm confident to say that these red cells are normal in size, normal in shape, and normal in hemoglobin concentration because they've got a small area of central pallor. So we would call these normal, normal acidic, normal chromic red cells.
So, uh, now we'll go looking at the white cells. So the white cell count for this patient was, I believe, 8.5, which is upper to offer the normal range. So it's a normal white cell count. Zoom out a little bit just so we can quickly assess the numbers of different types of white cells. The predominant, most common white cell in this patient is neutrophils. These multi-lobular, uh, white cells with granulation. Uh, there's one, two, so it looks like that, three, four, the white cells are neutrophils. And a normal neutrophil count is anywhere from about 1.5 to 7.5 ish, like the ranges vary from laboratory to laboratory. But I'm happy to say that this neutrophil count is normal. So they don't have a, neutrophilia or a neutropenia. And also for neutrophils, you would look at the lobulation, the chromatin pattern, and the granulation to determine whether there might be a reactive process going on there, where you would get an increased granulation in neutrophils or vacuolation, or if there might be a deficiency state or a medication given to the patient, or a dysplastic feature where you may get a hypolobulation, so reduced number of lobes of neutrophil. And you can also get a hyperlobulation where you get band forms in a reactive process. A band form is where there's one lobe and it's stretched across the cell, and it often forms a C shape or an S shape or even a straight line. So, so these cells, there's actually, there do appear to be a few bands. But given the other neutrophil features I'm seeing, where there is a little bit of granulation, it's not increased, the granulation isn't overly stained dark, so I would say the granulation is normal for these neutrophils. There's definitely, there's, I can see granulation in most of the, in majority of neutrophils. So I wouldn't say it's hypogranular, which would be maybe heralded a dysplastic process going on in the marrow. But it doesn't seem to be any obvious reactive changes in neutrophils, and there's not an overly high number of band forms. So I would be comfortable saying not going into reporting those if you was part of your procedures. Sorry, it's going to take a little bit of buffer.
So, and the lymphocytes, uh, the majority of lymphocytes are small mature lymphocytes. Um, I haven't seen any reactive lymphocytes, and the monocytes look normal. So I'm happy to say that the lymphocytes, uh, the white cells are, there's no significant morphologies that I can see with the white cells. And the platelets, we saw the platelet clumping. But however, you should also have a look at the platelets to see if there's significant numbers of large platelets, and the granulation of the platelets, you can, which might point towards a required condition like mild dysplasia or some dysplastic process or a myeloproliferative process, but you're getting large platelets forming, or maybe a congenital inherited condition where you can have large platelets and granulation. You can have hypergranular platelets or irregular granulation in platelets where it all gets pulled, your large platelet or gets pulled to the center, pulled in the center of the platelet, not evenly distributed. So, yeah, it's quite tricky. But the platelets appear relatively normal as well. Like I saw a couple of large ones, but it wasn't enough. Like, uh, a good rule of thumb is large. I should also just explain what a large and a giant platelet is, because a large platelet is a platelet that is, I think of it as greater than a third of the size of a red cell, up to the size of a red cell, a normal red cell. And a giant platelet would be larger than a normal red cell, size of a white cell or larger. And you only need to see a couple of giant platelets for it to be a significant finding that you may want to report on or refer to, refer to a hematologist or pathologist for a second opinion and let them know that you've seen it. And large platelets, it's recommended, uh, by up to 20 percent of the platelets, one in five, if one in five of the platelets are large platelets, then it's something significant and should be commented on or at least referred to pathologists for a review. Um, so, yeah, so now I guess, Ali, did you want to start discussing troubleshooting or how we would, uh, think about reporting the platelet count for a patient that has platelet clumping in this fashion?
So, I mean, in what we, this is a really obvious case. I mean, this is quite an extreme one, similar to the previous case. Um, a lot of cases of platelet clumping probably won't look as extreme as this, and you may see the odd clump here and there. Yeah. But generally, it's like you said to Joe, that for the clinical team, you want to make sure that they get a, you want to find out why they've done it, you know, what, what could there be a clinical reason? It's not mixed properly, or there was a traumatic venipuncture. And really, getting another sample in a citrate, in a citrate or heparin bottle, using an alternative anticoagulant. Um, I mean, sometimes, I mean, I have been in, uh, some scenarios where you can get, um, platelet clumping even in alternative anticoagulants, and they're really challenging. There's a question about what, what happens if you get clumping in a citrate or heparin. Um, there's no clear answer as to what to do, because even things like platelet counts by flow cytometry, they'll still be clumped because you're still using the same thing. Um, and it's really just doing the, that there is some evidence that there could be a difference between optical and, uh, impedance analyzers, and whether they're, they're better. But it's a real challenge. I think you're looking at morphology to see whether the platelets are actually there or not, and you have to put your hands up and say, well, we can't give an accurate platelet count. So, yeah.
Um, from a clinical perspective, I, I would, the only thing that I would add to your, you know, your really good point that you don't want patients to be treated unnecessarily for things like ITP, but equally, you don't want patients not to receive some treatment. So I've seen some cases where, um, physicians have not given things like, um, heparin as thromboprophylaxis when they're in hospital because they think the platelet count is too low. So there could be harm by giving treatments and not giving as well from this. So it's, um, a good relationship with the, um, the guys who are looking after the patients to get another sample, really. So, yeah.
Should we move on to case three?
Yes. Were there any questions for this case? I don't think there was any other questions. We've got some really good, um, comments about what, what we would do afterwards in terms of using alternative anticoagulants. Um, and, um, we've got one comment saying that, um, sometimes cold samples that haven't been warmed up properly can show clumping as well, but most people got that this was, um, uh, EDTA dependent agglutination. So that's really good. Um, perfect. Should we move on to case three?
Okay. Uh, so case three is a, um, 13-year-old, uh, girl, um, who's the clinical details that we've been provided are bruising. She has slightly anemic, which could be relatively normal in this age. Hemoglobin 109. Uh, white cell count is within the normal range at 6.1, and the platelet count is, not surprisingly, low. We're going to have a theme going on here. So I'll hand over to you.
Yeah, okay, no worries. So just like with the other ones, uh, you want to be, just first thing you want to determine is, is the platelet count accurate or not? Is the sample clotted? Is it platelet clumping? Is it platelet satellitism? So, yeah, I'm sure you're all getting the gist of it by now. So, um, when you review your full blood, peripheral blood film, stain film, uh, you want to look on low power, and you would like start looking at the tail, the feathered tail section of the film, looking for platelets. So, of course, you haven't really got much here, but it's a little bit here where this is the side of the tail, um, that will have a bit of a look over here. So there's no obvious, no, so we can't really seem to platelet clumping here. So, um, and also looking on low power, you would, you should be able to notice platelet satellitism if it was forming. That's when there is a similar autoantibody that, uh, only causes the platelets to adhere to the white cell walls of white cells in the presence of EDTA or other anticoagulants. And on low power, you would see these, uh, rosette sort of rings of platelets around the white cells, and we're not seeing that here. So we can safely say we can relatively trust this analyzer count of 81 for the platelets. So that's a moderate thrombocytopenia. So then you would want to look for the ideal area thickness or monolayer on the blood film. Start from the feathered tail end of the blood, blood film, and work your way thicker to the body of the blood film, that where you see the red cells begin to overlap but are still uniformly distributed. And you'll zoom in a little bit or go to a higher power objective and start having a scan about to see what you're working with. So when a patient has a low platelet count, before you even start looking at them, you start thinking about what are some potential causes. A common cause of like platelets would be a transient viral infection, and you may cause a decrease in thrombopoiesis or just usage of the platelets and aggregation of platelets in the body, and a decreased platelet count. And you may expect to see reactive lymphocytes. So reactive lymphocytes might be something before, maybe in an infection or inflammatory process, you might get some sequestration of platelets and reduced platelet count. So let's start, have a look at the white cells first. And so this white cell count was 6.1, which is normal. And the most common white cell we're seeing is mature neutrophils. So we can say that, uh, and a normal.
The neutral count is about one point five to seven or seven point five. So, in the white scale, six point one. So they definitely can't, they cannot have a neutrophilia. And there's enough difficulty here that definitely don't have a neutropenia. But, um, there's neutrophils. So it looks like this slight increased granulation, but maybe not enough for me to say it's or are hypergranulated. Uh, they've got normal lobulation, but there's this area here which is light blue colored staining, basophilic staining area of the neutrophil. And these, uh, when when you say them, they're called Döhle bodies or Döhle bodies. I pronounce them Döhle bodies, uh, which is a rough endoplasmic reticulum in the cell, and it stains a basophilic blue color. And it's due to a very, a normally a reactive process. Neutrophils, it wants to make a make a lot of granules, fight an infection, get involved in from an inflammatory process, and you get this light blue staining splotchy area in the neutrophil. So, yeah, so the granulation does look a little bit increased, but, uh, this can change from different, uh, stains to different stains. So it may be normal in this case. Um, so we'll move on to assessing the red cells. And if I see any, um, white cells that I believe have significant morphology when I'm scanning about the film, I'll stop and I'll point out. But the red cells, um, there's, uh, a small central central pallor area, central pallor. So they look normal chromic. Um, and we don't have MCV, but, uh, it might take me a bit of time to find a small lymphocyte. So they, we're just going to presume for now that they're normal civics. So they're normal in size. Uh, the patient was mildly mimic or could be normal for their for the individual. But, uh, we're just gonna. And there's no obvious point dif, uh, abnormal poikilocytes. There's the occasional cell here that looks a little might be a spherocyte, but there's not very many of them. So if I wasn't seeing a couple, you know, go to better area thickness up in the film, I'd want to see one or two per field for me to be start getting quite like concerned thinking that maybe there might be an autoimmune hemolytic process going on, which spherocytes may entail like might point you to walls. And one way to troubleshoot that would be to do a Coombs test, a DCT, and if that came out positive, then maybe there might be an autoimmune hemolytic process going on. But I don't think that's the case. So there's no, um, no significant numbers of abnormal poikilocytes such that poikilocytes is a abnormal cell for a red cell. And they're normal acidic and normal chromic. So let's move on to the platelets. So I mentioned in the case before that, um, large platelets are about a quarter to a third size of the red cell to the size of a red cell. And you need to see about 20 or one in five of the platelets need to be a large platelet for to be deemed significant. Well, that's in my professional opinion. But, um, so these are definitely large platelets. And just go, these are, yeah, they're getting onto large platelets. So one, two, three, four, five out of, yeah, like over 50% of the platelets that I'm seeing are large platelets. So that's quite interesting, really. So, and we're also seeing what was it? The clinical notes was a bleed, uh, bruising. So the doctor's, the clinician is querying that's a patient that may have a bleeding tendency, unexplained bruising. So they order full block out, probably with a barrage of other tests like coagulation studies and LFTs for objective liver function to see maybe we can find a cause for this bruising. So large platelets and neutrophils with these Döhle-like bodies. But, um, Döhle bodies are generally, uh, irregular in shape, but normally round or oval or a amoeboid sort of appearance like a splodge. But if you have a look at these neutrophils, oh, this one's a bit of a splodge, but they're more, uh, elongated rod-shaped is basophilic light blue staining, which is interesting. Sorry for my poor internet, takes a while to buffer sometimes to load the image. I'm not really convincing myself here. I'm looking at these ones, I'm like, it's a bit of a splodge, but yeah, so, yeah. So it's not, there's the interesting morphological features I've seen in this film are the large platelets. And we should talk about the granulation. So they're well granulated. They're great. We're not getting a pooling of granulation in the center of the cell with a granular ring of cytoplasm in the platelets. So normal granulation. So now we're going to be asking ourselves, what, oh, here's a nice giant platelet. What could be going on here? Could this patient have ITP and idiopathic thrombocytopenia thrombocytopenia? Or could there be something else going on? They're a 13-year-old female, so adolescent. They probably haven't had many blood tests in their life. This might be a first finding. So I don't give away the answer here. Ally, did you, was has there been any comments about what maybe might be going on here? There have been. So, I mean, we, um, initially when we started off, I think the, uh, the comments were very much, could this be ITP? Could this be infection? And I think as we zoomed in and looked at the the details of the the platelets, this the large size, good granularity that they they display, uh, but especially with the the neutrophil and the the Döhle bodies, um, we're getting more of, uh, that possible May Hegglin. Yeah, Döhle-like bodies, Döhle-like bodies. Um, and I think we, um, it's a really sort of interesting case, this one, because I think it's the the point that you made is well, have a look at a previous count. Is it, has it been low before? And what else is going on? Um, well, maybe this is a verb like we've seen for this individual as well. Yeah. Um, and, you know, it can be difficult, but just looking at the, then if you just go back to the neutrophil again, um, what, what make it, because I mean, one of the possibilities that we see Döhle-like bodies and things like sepsis, inflammatory disorders, and things, is anything that may suggest a sort of reactive process as opposed to sort of congenital? Well, um, yeah, like Lynn, I first, when I first looked at film, like they do look like they have increased granulation. And so it's quite a difficult slide because it could be, I, it definitely could be a reactive process here going on here, because the neutrophils look like they have increased granulation, thus here and here. There's, uh, band forms. So that's when the marrow is starting to pump out more neutrophils, and they're not as matured, but they're still a fully functioning mature neutrophil. It might be an inflammation, infection process. But, um, just my academic interest, I would definitely be referring this one to a hematologist at my laboratory, just going, query May Hegglin anomaly. Um, and patients with May Hegglin anomaly, um, do have bleeding tendencies. Uh, it can be, I'm not trying to speak for you because you're the expert, you're the hematologist, but, um, they not necessarily, uh, severe bleeding tendencies, but they can bruise easily and have, uh, prolonged, uh, clotting times, like stop bleeding. And some patients may require their like, uh, drugs to increase their platelet production to try to prevent these bleeding tendencies, or maybe in a crisis, like when they have, uh, need a platelet transfusion to stop bleeding. So, yeah, I know it's a tricky film. It's tricky, but interesting. When I think it's, it's, um, so this patient did have May Hegglin anomaly. And okay, like you said, that generally these patients don't have a huge amount of problems with bleeding, and if they do, it's very mild. But I think, um, the modern modern way of describing these conditions is related to a mutation in MYH9. I'm not sure whether you've come across these kind of issues. Yep. Yeah. If you have any comments. Oh, so yeah, it's a mutation in MYH9, which is a non-muscle myosin, uh, heavy chain, uh, uh, pro-like structure. And it, like, the, uh, defect in MYH9 gene causes, uh, these, uh, non-muscle myosin, uh, tubular structures to form in the granulocytes, and they stain a lot like, uh, Döhle bodies, but often in like a rod or elongated shape. And it also causes, uh, the platelet dysplastic megakaryopoiesis. So that's the formation of platelets, and you get a reduced platelet count and larger platelets and giant cells in some cases, but they have a normal function. So they do, that's where I would come into. They patients with May Hegglin anomaly often have mild bleeding tendencies, and often doesn't disrupt their day-to-day life or cause, um, but because they're large platelets and you reduce count, uh, I guess when you're trying to, you do get a damage to a blood vessel and you start a bleeding occurring, it will be a less efficient platelet plug that would form. And so I guess that's where the mild bleeding tendencies come into play. Definitely. Anything that, no, I think, um, originally they thought that these were distinct subgroups, but, um, so things like May Hegglin, there's conditions like Sebastian syndrome, really rare things. There's another one called Epstein, yeah, syndrome, but they very much run on a spectrum of, uh, different clinical associated features. So you can get things like deafness, yeah, neurological, uh, abnormalities as well, and variable sorts of bleeding tendencies. But, um, the, the family history is usually very useful, and as well as, uh, just looking at genetics. Sorry, Joe, is it autosomal dominant? So if the child has it, it's most likely, it's almost guaranteed a parent has it. Yeah, definitely. So, you know, okay, getting the full picture is, is, is important in these cases. We've got one comment saying that this could be, is this could this be gray, gray platelet syndrome? Okay. I mean, um, I've never seen a case of that. I'm not sure if you. No, I haven't, but I've read about it. Like, when we get a quality assurance surveys and you're looking into this, uh, macro congenital macrothrombocytopenias, like, and stuff, and yet you start reading, uh, Barbara Bain's textbooks and other wonderful textbooks about morphology, and you come across these rare congenital conditions. So gray platelet syndrome, uh, you would expect to see, uh, hypergranular or agranular platelets. That's where the name gray platelet could basically, it's the platelet would look like these, but you wouldn't get any of these basophilic darkly purple staining granules in the platelets. It would just look like, uh, sort of just like imagine, uh, some monocyte cytoplasm that just free by itself at this size or this size. And I'm like, I'm not sure the gray platelet syndrome even has, uh, macrothrombocytes, like large platelets, but don't quote me on that because it's been quite some time since I've read into it. But yeah, you, gray platelet syndrome, you would be the most, most prominent feature for the platelets would be is a granular platelets, and you'd be going in the line thinking, is this a dysplastic process or is this maybe a gray very pleasant? Could be a congenital inherited condition. Yeah, yeah. I think, um, in again, like I said, I've never seen a case of this in real life, but, um, at least from the textbooks, you can get medium to large size platelets, but as you mentioned, it's a predominant feature is the sort of lack of granularity and sort of empty appearance platelets. Excellent. So I think we've covered all the all the comments, um, that have been made, um, and any questions as well. Um, I'm just going to share my screen again. Um, do I stop? Yeah, if you stop sharing and, okay, if I share my screen, there we go. So, uh, Joe, thank you so much for for your time today and your insight into three interesting cases. Oh, no worries. Thank you. A little bit of a nervous break. No, not at all. You did it. Did really well. We really enjoyed it. Um, so plenty of discussion stimulated, and, uh, it's been absolutely fantastic. So thank you, Joe. Uh, thank you to everyone who participated in today's webinar. I'd also like to thank our sponsors Urban Mannheim. Just to remind you again that a CME certificate will be emailed to you provided that you've registered. I'm going to put a link into our post-webinar survey, and we're really grateful if you could complete that so we can use that as the basis for providing further educational content. Um, you should hopefully be able to watch the webinar again on the Blood Academy YouTube page, and we'll get that video uploaded in the next day or so. And hopefully, we'll see you in two weeks' time where we're going to be, uh, joined again by Dr. Audi Satardy from Canada. We're going to be discussing, uh, acute leukemia cases. We've got three interesting cases, and hopefully, we can, uh, stimulate more interesting discussion and comments as well from, uh, the guys on the, uh, hematology interest group. So, uh, thanks again, Joe. Thanks, uh, everyone else. And, uh, thank you. It's good in two weeks' time. Okay, excellent. All the best then.