Webinar 2 - Red Cell Cases

[Music] Welcome everyone. Uh, welcome to the second of our six-part webinar series on blood cell morphology. Uh, brought to you by the Hematology Interest Group and the Apology Blood Academy website. As well, glad to uh welcome you all to this webinar. Building on the previous webinar led by Dan Pelling, where we discussed the basic concepts and important factors involved in looking at blood film. I'm delighted to be joined by Nicole Starkey. We have three really interesting red cell cases to go through. But before we do that, I just want to bring up a few issues regarding the webinar series, which I'll start now.

So, as you all know, this webinar series has been kindly supported by Urban Mannheim. The webinar series is accredited by the Royal College of Pathologists in the UK. Uh, it's very important that you register for your CME certificate. You can do that on the Blood Academy website and by completing the online form. Uh, you will have until the end of this webinar to register for your CME certificate, which is specific to this webinar. There has been a question already being made on the comment section, uh, that states, uh, whether we have to register for every webinar. You do need to, so we can make sure that we send you the correct certificate to you. Uh, this certificate will be emailed to you in the next few days, provided that you have registered. If you want to see this webinar again, then we will make this available on the Blood Academy YouTube page, and that link will also be posted onto the Hematology Interest Group Facebook page.

Um, this webinar series, uh, is providing you with an introduction about blood cell morphology. We've got some really excellent speakers from all around the world. But if you do want to learn more about blood cell morphology, the more of the details of certain diseases, then Blood Academy does have an online on-demand course, made up of video tutorials, assessments, all of which are accredited by the Royal College of Pathologists. There is a 20% discount at the moment, linked with the Hematology Interest Group, which you can get by entering the discount code HIG20.

So, I am really excited to introduce Nicole Starkey. Nicole is one of the hard-working admins on the Hematology Interest Group. She is the lead biomedical scientist in hematology at the Royal Free Hospital in London, a very big teaching hospital in the capital of the UK. She is originally from Australia and moved to the UK very recently, I think last year. She's almost, she's managed to acquire almost a decade of experience as a specialist biomedical scientist, and as we'll see, she has a keen passion and interest in both blood film morphology and education as well. So, I'm just going to stop sharing my screen and just allow Nicole. Hi, Nicole. You should be able to share your screen now.

Hi there. Yes. Stop it going. Excellent. Um, so before, before you start, um, just a couple of points. Um, with regards to the cases, you can follow the cases live, uh, using, uh, the web link that I posted in the comment section. Uh, you can also add in any other questions as well. And before we start, I will be introducing just the case, um, in terms of the clinical history as well. So, we've, the first case is a 24-year-old female. The history, the clinical history that we have is very brief, which is often the case, uh, with the, the information that we're given in the lab. It's, uh, fatigue, and probably, uh, not unsurprising with the, uh, the hemoglobin that she has of only 68 grams per liter. So, I'll, I'll leave it up to you now, Nicole.

Sure. Thank you very much. Um, okay, can everyone see my screen? Should be shared now. Yeah, we can see it. Yeah. Excellent. All right. So, we've been told that this is a young female patient, uh, and they have a hemoglobin of 68. Um, so the first thing that I would look for in reviewing this film is, does what we're seeing on the film match a hemoglobin of 68? Uh, the answer, I think, unequivocally is yes. Um, so in looking at the film here, the first thing that I notice is the lack of red cells. So, this is a nice, actually quite thick area of the film that we're looking at that we're reviewing, and you can see a lot of gaps there. There's a lot of background. There's not a whole lot of red cells in this field. The patient's definitely anemic. That is a low hemoglobin concentration. And looking at red cells themselves, usually when we are looking at a patient with anemia or a patient with, you know, any kind of film that we're reviewing, uh, we want to look at the red cells in terms of a few different things. So, firstly, we want to look at red cell size. We want to look at red cell shape. And we also want to look at what I call some big picture things. And big picture things are things like rouleaux, where you have a coin stacking appearance to the cells, agglutination, where you have clumps of cells sticking together, and things like proteinaceous background, where you have a blue kind of, uh, appearance to the background of your film. All of those things can be present in numerous different types of diseases, but I find they're easier to look for on low power. So, zooming out a little bit, I'm looking for any of those types of things I just mentioned. I'm not seeing any agglutination where cells are clumping together in large groups, and it doesn't look like there's increased rouleaux. I'm actually in a quite thick part of the film, uh, which doesn't look that way because of the anemia of the patient, um, but overall, I don't think there's increased rouleaux. I don't think there's autoagglutination. So, I'm happy to go down and have a closer examination at the cells themselves.

What I want to do here is actually look around for a small lymphocyte. So, in our last webinar with Dan Pelling, he went over one of the ways that we review size of red cells, and that is actually by using the size of a small lymphocyte to compare. And by a small lymphocyte, I mean a nice normal small one like this one here, uh, where it has a very deep condensed nucleus and not a whole lot of cytoplasm. This is a nice mature small lymphocyte. And what I'm looking for essentially is the red cells should be about two-thirds the size of the lymphocyte as a whole, or roughly the same size as the nucleus of the lymphocyte. If they're bigger than that, obviously we're looking at macrocytosis or an increase in cell size. And if they're smaller, we're looking at microcytosis or reduced cell size, which in a 3D space translates to volume, or, you know, the overall size of the cell. So, looking around here at this small lymphocyte and the red cells surrounding it, you can see this one here in particular is very, very small, much, much smaller than that nucleus is. There's a little bit of variation. These cells over here are probably a little bit bigger. These ones probably a little bit bigger, but overall, they are smaller than that nucleus of the cell. They're all microcytic, um, but there is a little bit of what we call anisocytosis, which is variation in cell size. I would still classify all of them as microcytes, though, which is important.

The overall pattern of this film is one that I would call macrocytic. The other thing that we're looking for is hypochromia. So, often microcytosis and hypochromia go hand in hand. And so hypochromia, represented on a blood film, um, you have these cells where instead of being a big fat donut with an area of central pallor that's quite small, about a third of the size of the cell, we see cells where the area of central pallor is actually much, much bigger than that and can be most of the cell. And what you're left with really is this very, very light-staining, poorly hemoglobinized cell, where the central pallor is much bigger than it should be. And this represents essentially low hemoglobin concentration in that cell. It's also small. So, this is what we would call a hypochromic microcyte. And looking around this field, that's what the majority of those cells are. So, the overall pattern is hypochromic microcytic. And what I want to look then for is, are there any normal cells in here? Does it look like maybe this patient's had some treatment, or maybe had possibly a transfusion, something that would call a dimorphic picture? A dimorphic is where you have two specific types of cells in the field. You might have hypochromic microcytes and you might have normochromic normocytic cells. So, I'm going to look around and see, can I see a second population of cells? And I think I'm going to say no. The majority of these cells are all quite small. They all have that bigger area of central pallor than we would expect, that we would want. There's no cells in here that I'd really consider to be super normal. They're all still small. This one has probably better hemoglobinization than the rest, but it's still a small cell. So, I'm overall happy with the pattern, hypochromic microcytic pattern. And immediately when I see that, I think of a couple of different conditions. So, in a young female patient, most likely to be something like an iron deficiency. But there are lots of other types and causes of a hypochromic microcytic picture, one of which is a thalassemia or a hemoglobinopathy. There are some inherited disorders that you might have, um, and some acquired ones as well. So, we won't jump the gun and call this anything yet, but we will look around and see if there's any other evidence or any other things that we can see on this film to point out what might be the cause.

Looking around further, I'm looking for poikilocytes now. Poikilocytes is a catch-all term that means different shapes. And when we're talking about different shapes of red cells, some of them are commonly found in some disorders, and some of them are really, really non-specific. One that you can find very commonly in an iron deficiency is these type of cells here, where they are an elongated cell. These can have multiple different names depending on the lab you work in, the country that you work in. I've called them many different things myself over the course of my career. Um, a common one is a pencil cell. Here's another one here. Um, probably another one there. You can also hear them called elongated cells or elliptocytes, but the main premise is they are an elongated cell that essentially looks like it's been stretched on the axis, but they still have that area of central pallor that is more than what you would consider normal for the cells. So, they are still hypochromic, but they are that elongated appearance. And you see them commonly in iron deficiency, but they're not specific to iron deficiency. Don't get yourself into a situation where you think you saw pencil cells and therefore it must be iron deficiency. It's not always textbook, and you can still have variation. It might still be something else.

So, let's look around further and see, is there anything else that we can see on this field? So, together with the hypochromic microcytes, we've seen some pencil cells or some elongated cells. There are also a few of these type of poikilocytes. Now, this, this type of cell is called a teardrop cell, and you can see why it looks like a teardrop. It's actually got one end of it that looks like it's being drawn to a point. Um, and these are again, not a specific type of cell. You don't see them just in one type of disorder, but in my experience, you often see these in disorders that is affecting a patient's bone marrow. Now, that can be a number of different causes, um, but they do think that these type of cells might be formed when they're actually squeezing out of the bone marrow spaces. That could be due to increased destruction in the bone marrow or over, you know, turnover, increased production of cells, and they're trying to get them out early, they're pushing them out early, and they're actually getting a little bit squashed as they come out. Now, with teardrops, it's important to remember that they can occasionally happen as a smearing artifact. And when they're a smearing artifact, there's an easy way that you can tell when you're reviewing a film and it has teardrop cells. Look to see, are the tails all pointing in the same direction? If you have a clump of cells and they look like teardrops, but they're all pointing in one direction, it's probably a smear artifact, and it's the way that it was made. If you have teardrops where they're pointing in all different directions and they're not all clumped together in one area of the film, you probably have genuine teardrops.

Looking around further, I can see a few different types of cells, but they're becoming a little rarer. So, we have possibly what looks like maybe a target cell, um, which is a cell that has, again, an area of central pallor, but it looks like it has a bit of a bullseye in the center. Um, those ones are formed by, kind of, an abnormal lipid membrane metabolism process, which can happen when you have disordered erythropoiesis. But there's only a few of them. They're rare, and I don't know if they're significant enough in number that I would even comment on them in my film report. What I have so far is, I have a hypochromic microcytic picture with occasional elongated cells. I don't know even if I have enough teardrop cells to be significant enough to comment on. If I did, I'd probably say rare teardrop cells. And the last thing, really, that I'm looking for is any evidence of parasites and any evidence of polychromasia. So, polychromasia is an indicator that there is red cell turnover, that the body is compensating for, um, and in a patient who has a hemolytic disorder, you will have increased turnover and blood loss. You should have increased turnover where the body should compensate, and you should get increased polychromasia. That's an expected response. In patients who have a deficiency, they may not have that adequate response because they don't have the building blocks for the cells themselves. So, often in a severe iron deficiency, you will not see polychromasia, and in this film, I'm not seeing polychromasia, which is pointing more towards a deficiency than another type of process. And I think for the red cells, that's the majority of the changes that we can see there.

Looking briefly at the white cells, the neutrophil counts, the white cell count as a whole looks normal. It's in the normal range, and the neutrophils, to me, they look pretty happy and mature. I wouldn't say that there's anything too abnormal. There are a few neutrophils which have five lobes. Do keep an eye out for neutrophils which are hypersegmented, because you can have patients with combined deficiencies where they have B12, folate, and iron deficiency together. So, do check that your neutrophils are not hypersegmented, which is classified as six or more lobes, or lots of neutrophils with five lobes. So, looking around, I think there's a good mix. There's a few with five, there's lots with three, lots with four. I'm not convinced that they're hypersegmented. And looking around at the platelets, the platelet count for my laboratory range is high normal, but not what we would consider to be out of range. Um, and so the platelets themselves, they look mostly normal. They're of a normal size, and I'm, I'm overall pretty happy that there's, there's no issue in the platelets.

So, essentially, what we have here is a hypochromic microcytic pattern. We have occasional pencil cells and maybe rare teardrop cells. Putting all of that together, my particular diagnosis for this patient would be a severe iron deficiency. Let us know in the comments if you think differently or if you have things that you'd want to exclude. And do you have any questions so far about this case? I think we're looking at doing a case and then we can discuss and ask questions, and then when we're happy, we'll move on to the next case. So, anyone have any questions so far?

I don't think we've got any questions as yet. Um, I'm sure they will be coming in. I've got one question, Nicole. Patients with this anemia, where do you look to see whether there is true rouleaux or not, and how do you exclude it? Because we've got this field here, there are a whole load of cells which are stacked on top of each other. Uh, I, I always find it very difficult.

Mm-hmm. It is. And as you have a, a virtual slide here, it's a little bit less easy to show. Essentially, what I would do is, on the physical slide, I would look where I normally would cast my eyes for a monolayer, which is about two-thirds of the way down the actual physical smear, and I would check there to see what does the actual field look like. Um, and as we move further out, I can't show this. This is not the very tail of the film, but this is kind of where the monolayer would be, um, and I can see that they don't really look like they're they're stacked. They're quite distinct from each other. And also, if they had them available, I would look for an ESR. A lot of labs are still doing them, so you could check to see if there was increased rouleaux that way. Um, and also, I would check to see, not morphologically, but, you know, does the patient have an infection? Are there, you know, is there any reason why they would be rouleaux? I suppose, um, on the actual film itself, I find it would be easier to do with a physical smear rather than a virtual one. Virtual ones in that way can be a little bit limiting. Um, but I will say for this patient, who is a young woman, I will ask a question actually to the comments. Why do you think, or what would be the most common cause of, let's say, an iron deficiency in a patient of this age group and, you know, a patient group, I suppose? What, what do you think the main cause of her deficiency would be and why would it be so severe?

We've got a slight delay when we're transmitting over Zoom, so hopefully the, the comments of that question should be coming through. Just whilst we do wait for that, um, what kind of other clinical features, laboratory features would be important in a case like this?

Sure. So, the first thing that I would look for, if this was a patient of mine, firstly, is do we have any history on the patient? Um, if we saw the patient, you know, a few years ago, and their MCV, MCHC, MCH were normal, and now they've come into this range, um, it would say that this is more likely to be an iron deficiency than something inherited. Obviously, if it's something inherited, it will persist, and their previous one should have also been abnormal. I would also look to see, do they have any iron studies available? Um, you know, often if a patient has a symptomatic anemia, the doctor will order iron studies, hopefully, and they will have those available. So, you would be looking at a reduced ferritin level, um, you know, reduced transferrin saturation and indices overall. Um, also, if you had available any results of like a thalassemia screen, you could exclude the presence of a thalassemia or hemoglobinopathy, some other type of disorder that might cause this picture. Um, and the clinical details would be critically important. Um, so, you know, is the patient actually symptomatic with anemia? Do they have a history of blood loss? And, you know, those kind of clinical details could be important as well.

So, I think most people, uh, I think rightfully so, in a woman of this, this age, have, uh, explained the iron deficiency by menorrhagia or heavy, um, vaginal blood loss, menstrual blood loss. And we've got some other comments here in terms of acute blood loss. Do we tend to see this kind of picture in an acute blood loss due to trauma or anything? No. So, I think comparing this, uh, situation, which in my mind is probably a severe iron deficiency caused by chronic blood loss, as people pointed out in the comments, in an acute situation, you would not expect to see microcytosis because it is the iron deficiency that is causing the microcytosis. You would more expect to actually have macrocytic indices, and the reason for that is you would expect in a person who is young and healthy that their bone marrow would be able to compensate for a rapid drop in hemoglobin by pushing out polychromatic cells and nucleated reds. Now, those will push up your MCV, so you're more likely to have a macrocytic indices, um, and lots of polychromasia. You know, can be marked polychromasia in a patient who has had a major hemorrhage or a major blood loss incident. Um, this one, there's really very little polychromasia to indicate that there's, you know, um, an acute blood loss happening. It's much more likely in this case to be a chronic onset that's suddenly got to the point where the patient has decompensated to the anemia. I mean, also add that clinically, the patient, if you drop your hemoglobin from normal to to this level, you'll be very ill, whilst the, the human body is quite good at compensating over a long period of time and be able to just about function with a hemoglobin of this low. Uh, yeah, I find it, it's very nervous. Yeah. So, I have seen in children, especially children are very resilient. I have seen hemoglobins in the 20 grams per liter, and obviously they compensate very well to a point. But, you know, a long-term slow gradual process is much easier for the human body to compensate than a rapid drop. I don't think any of us would be standing, um, after a rapid drop down to 20.

So, for the interest of time, um, I think we need to move on to case two. Sure. We've got some other questions. If we do have time at the end, we'll try and cover them, um, but we've got a lot to get through with the other two cases. Um, so, case two is a 21-year-old male who's presenting with chest pain, and as a common theme, he is anemic with a hemoglobin of 71 grams per liter. Um, so, I'll, I'll hand over back to you, Nicole.

Sure. No worries. Uh, so we have a young male who is anemic with chest pain. Now, looking at this film, the first thing I notice is it's a bit of a mess. There's a lot going on in this field, and the first thing that my eye is drawn to is a lot of cells that are much darker staining, and some people will term these hyperchromatic, um, in that they appear to have a higher hemoglobin concentration than what is normal. Um, and if I zoom in and have a look, perhaps over here, uh, hopefully, let me know in the chat if you know what these cells are, um, but these, a lot of these are classic sickle cells, and they are a very, uh, sort of elongated and pointy, very sharp-looking cell, um, which is the, you know, pattern one excel of sickle type disorders. So, patients who have sickle cell disease, um, and combined inheritance of sickle cell with some other thalassemias and hemoglobinopathies or hemoglobin variants, they can have the appearance of these cells, which occur during low oxygen tension. Now, these cells are very problematic for the patient. They can irreversibly polymerize into this shape, and they get stuck in the microvasculature of patients' blood vessels. And looking at the clinical notes that were provided with this case, this is a classical case of acute chest syndrome, um, where these actually get into the microvesicles in the lungs and in the respiratory system, and they actually cause little blockages essentially, which are very, very painful, and is one of the main kind of morbidity events for patients with sickle cell disease. So, in saying that, let's look around and kind of have a look at the film and I'm going to look for evidence of sickle cell disease, because I'm almost convinced, and we can look around and see what else we can find.

So, we've seen some sickles, which are these kind of sharp, very, very sharp, um, sickle-shaped cells. We also see a lot of these what they call boat-shaped cells, which are, they're not completely pointy like this one was, they're a bit rounded on the end, but you can see there's no area of central pallor in there. They're a completely solid-looking cell, um, and there's a lot of them around. So, when I was classifying these, I would say that there were marked numbers or numerous, um, because, you know, in looking at one high power field, there are many, many of them. Um, looking further, also, it may be difficult to see on a digital scan, um, but there is marked polychromasia on this field. So, all of the cells that have that very bluish tint to them, they are all polychromatic cells, which are newer, younger cells that the bone marrow has pushed out, um, in response to low oxygen concentration. So, the body is attempting to, to, uh, compensate for low oxygen, low hemoglobin, and it's pushing out a lot of younger cells. Together with that, we can see there are some poikilocytes. So, there are target cells in here, which is to be expected from a sickle cell patient again. So, these are those cells where they have a kind of bullseye type appearance to them, and they, they are present in numerous hemoglobinopathies and thalassemias and many other conditions. They're not specific for this, but they are a common finding together with a few other things, um, that I'm specifically looking for, and I'll let you know when I find one, um, as we go around.

Here's what I'm looking for. So, this cell here has an inclusion body in it, um, which is this little dark blue staining inclusion here. This is called a Howell-Jolly body, and they are nuclear remnants. So, these are actually found in patients who have had a splenectomy or are asplenic for whatever reason. So, patients had an operation, they've had it removed, or through some other reason, their spleen has stopped working. Now, in sickle cell patients, by this age, this patient's 21, he will no longer have a functional spleen because all of these sickle-shaped cells, over time, they get stuck in the microvessels of the spleen, and they actually cause infarcts, and they cause the spleen to die. So, it's a common finding in sickle cell patients that you'll see evidence of hyposplenism, and that manifests itself in target cells and in Howell-Jolly bodies and in a few other little things that we'll go around and look for. Here's another Howell-Jolly body here, that's actually in a target cell. So, you can see we've combined two things here. We have an inclusion, and we have a target cell. And looking further around, there may be one or two little things. There are some kind of very strange irregularly contracted cells, um, that are around here, and here's another one here.

Looking at the actual size of the cells, it's difficult to characterize because there's a lot going on here, and it's quite a mess in general terms, um, but it's not a hypochromic microcytic picture. It is a hemoglobinopathy, and most of them cause hypochromic microcytic pictures, but this one does not. It's actually a hemolytic disorder, and it causes a picture that has, you know, marked poikilocytosis in terms of lots of different sizes and shapes, um, but the actual overall picture is not microcytic, and actually some of these are macrocytic. If you compare, this is probably the nucleus of a small lymphocyte, that's quite a large cell there, and these ones are quite big. Um, looking around, there's probably some normal-ish sizes, and there's a few small ones as well. So, what we would term anisocytosis, variation in cell size, um, without an MCV, it's difficult to say what this would actually be put at, but I think with all of the polychromasia, all of these blue, very large cells, it's probably presenting as macrocytic, um, in terms of the MCV. Um, and this is, that's what I was looking for. So, this is probably a little spherocyte. Um, so it is smaller, it is darker staining, more dense, um, because it has actually lost some of its membrane and collapsed on itself to become a round spherocyte, as opposed to being a biconcave disc. Um, and they can be seen in, um, patients with hyposplenism as well, only occasionally. We've only seen a few, but it all kind of fits together in that picture.

As for the rest of the field, um, the white cell count was in the normal range, um, which is not unusual. There is a little bit of toxic granulation or hypergranulation, um, in a patient with acute chest syndrome where there's obviously inflammation going on, that's not unexpected. Um, and in the platelets together, um, looking at hyposplenism again, we do have some quite large platelets that are floating around. This one's probably a large platelet, um, you know, larger than a red cell, I consider to be a giant platelet. Um, this one here, another large one, this one here, probably a megakaryocyte fragment, but a very, very large object there on the field. And so, all that put together to me says a sickle cell disease in a patient with probably acute chest syndrome. And if you work somewhere in the world where sickle cell disease is quite prevalent and endemic, you will see these a lot. I know in my hospital, we see many of these patients, um, because they are part of our local community. But if you live somewhere perhaps where I used to live in Australia, you might not see these as often, but they are actually quite prevalent in some parts of the world. So, um, I think that's what we have so far on this film. Do we have any questions, I suppose, from Ali or from the comments so far, or any, any other comments?

So, we haven't got any questions as such, but I'm sure they'll be coming, coming in. Um, I think most people, uh, sort of agree that this is sickle cell disease. They've also commented on active hemolysis, which I think is an important thing. They're features of hyposplenism. Um, I, I, when I was starting off looking at films, these kind of films did scare me, and one of the things that did scare me was distinguishing between a sickle cell and a schistocyte.

Mm-hmm. I just wonder whether you had any comments to, to sort of alleviate those fears and try and distinguish the two.

Sure. I mean, it can be difficult because patients do sometimes have both in one field, um, but I find with sickle cells, what you're looking for is, it is usually elongated, so it is not in the normal sort of shape, or, I suppose, constrained by that sort of shape that a normal cell would be. So, when you're looking at a fragment or a schistocyte, it still just has to be smaller than the red cell would be within the same sort of confines and shape. Um, they can be numerous different, um, appearance in terms of you can have some helmet type, you can have some that are just like a triangle, you can have one where it looks like it's been sheared in half, but they should all sort of be smaller than and can form within that sort of shape. With a sickle cell, you're looking at something which has elongated as the, the fibers have actually polymerized, um, and they usually have sharp edges. Um, though, as we said before, the boat cells don't necessarily have those completely sharp edges at the end. Um, they're also what we would call hyperchromatic, so they should appear darker than the surrounding cells. Um, so, if you look at the ones that we have here, comparing to the ones next to it, they should appear, they should stain darker. Um, whereas your schistocytes, your fragmented cells, necessarily shouldn't, because they're made, I guess, by mechanical stress, um, and they should appear the same staining quality as a normal cell, but, you know, they might be a different shape, I suppose, is how I would differentiate them.

So, we've got some other questions. Um, so, one of the questions, as you've already alluded to, uh, sickle cell disease is an umbrella term for a whole range of different, uh, genetic combinations. The most common one that we'll see is hemoglobin SS, um, so homozygosity for the sickle cell gene, but you can get, um, heterozygous states where you have the sickle cell gene on the beta globin gene, as well as co-inheritance of beta thalassemia and hemoglobin C. How do you distinguish those two? Well, those three conditions, sickle cell homozygous, homozygous sickle cell, SC disease, and, um, the beta thalassemia sickle cell, it's another sort of difficulty that I find about using additional tests. What kind of morphological features may, may help you?

So, firstly, I would look at, is there nucleated reds, macrocytosis? So, when I'm looking at a thalassemia picture, even if it's a HbS, uh, beta cell, um, it typically carries with it a hyperchromic microcytic picture and nucleated reds. Now, if you've got a patient who's been transfused, that can also complicate things, um, and so that can add an extra layer of difficulty to it. Um, sickle cell on its own should not be hypochromic microcytic. So, in the absence of iron deficiency, you should see this type of classical picture. Um, a beta thalassemia with a sickle, you can still have sickles on the film. In my experience, you have less, but that's not a hard and fast rule. You should, in a beta thalassemia, hyperchromic microcytic nucleated reds. In an HbSC, you expect to see more target cells. Hemoglobin C often carries with it, you know, an increased proportion of target cells compared to the other, um, cells and hemoglobinopathies. So, I expect to see more, um, target cells, but also you can see hemoglobin C crystals, um, which are sometimes hard to find, but they are, you know, a very, very solid mass that kind of looks like an oblong-shaped rod, um, which you can see in, here we go, and C disease. You may see them in hemoglobin SC. You can also see hemoglobin SC poikilocytes, um, which are very strange, little poikilocytes that are sort of, they can be like a triangular shape, um, and very, very dark, dense staining, like a sickle cell with the pointed edges, but more like a very condensed, almost triangular type of, uh, cell, and they are classical for HbSC. Um, so, if you saw those, I'd be leaning more towards that than a pure sickle or HbSS.

That's great. Thank you. Um, just to add that for me to distinguish them with confidence, use your additional tests like HPLC and gel electrophoresis. They're a lot more accurate. Um, so, the last question that I think is useful, uh, because I think this term is, is often shrouded in mystery, how do you distinguish between a spherocyte and an irregularly contracted cell?

Sure. So, to my mind, a spherocyte has to be perfectly small and round and smaller than the size of the rest of your red cells. If it's not, it's probably not a spherocyte. Um, so, spherocytes, the way that they're formed, they actually lose some of their membrane, and they shrink in on themselves, and they actually become a perfect sphere. They become spherical. So, remember when we look at films, we're looking at a 3D object in a 2D space. We're looking normally at a biconcave disc. A spherocyte, instead, is a round ball. So, when you view it from the top, it appears perfectly flat, which is why spherocytes look perfectly flat. And an irregularly contracted cell, then, is not perfectly round. They're usually quite an odd, unusual shape, um, and they may be formed by a similar mechanism, um, but they are a distinct sort of entity compared to a spherocyte. So, if I can find a nice small spherocyte, so this one here, I'd probably call an irregularly contracted cell, but it's not round. It's not actually a sphere in 3D space. It's just a very strange, abnormal little cell that's a bit contracted and not the right shape. If we look at this cell here, and that's as far as I can zoom in, unfortunately, but this one here, it is a perfectly round little cell that is darker staining than the ones around it, and that's what I'd consider to be a classic spherocyte.

Excellent. Thank you. We could keep talking about this film. There's so many other things that we could discuss, but again, for the interest of time, we'll move on to case three. Um, and so, again, this is a, a young patient, a 22-year-old female. Uh, this has been done as part of a booking blood test, uh, for a pregnancy. Uh, the blood count isn't too bad, actually. She's slightly anemic. The hemoglobin is 109, but you may expect that in someone who's, who's pregnant. Um, so, it's, I think it'd be useful to go over this film and sort of compare and contrast, especially with the, uh, the first slide as well.

So, sure. So, um, I will say upon looking at this film, apologies for the quality of this film, and it is something that we've run into in the laboratory. Um, sometimes you don't get the best smear, and sometimes if the film's too old, you might not be able to remake it, and you have to make do with what you've got. Now, the morphology on this, it is accessible, um, but it is somewhat a thick area of the film that we have to work with. So, apologies for that. When we start, it's also, um, quite pale, so hopefully you can see this if you're watching it on mobile or on the Facebook app. Um, it is a little bit washed out, so apologies for that. But what we're looking at here is the, uh, patient is a young female, booking bloods, as we said. So, this is being used as a screening test. The patient is not symptomatic. They don't have anything that would indicate that they have some kind of disorder. Um, what we want to look at here, as I said, it's difficult to assess due to the thickness of the film, so we won't be looking at rouleaux for now because I can't show you the, the feathered edge of the film, essentially, but we can look at the red cell morphology. So, I'm going to look and see, can I find a nice little lymphocyte? And I want to find a nice small one. There we go.

So, again, looking at the size of the red cells first, if we're comparing to the size of a nice small lymphocyte, they are probably a little bit microcytic, and not like the first one where they were markedly microcytic. Um, but these do look a little bit smaller than usual to my eye. They are smaller than that nucleus, certainly. And the area of central pallor of them, apologies, it is a pale film, but there is more central pallor. It's more than one-third of the cell, and therefore they are also hypochromic. Um, so, we have a hypochromic microcytic picture, but as Ellie said, in the beginning, hemoglobin of 109 is normal for pregnancy. So, this patient's not anemic. So, it's unlikely, uh, that this would be an iron deficiency, um, and more likely that it may be something else. And we'll have a look around and see if we can find other poikilocytes that might sort of help us to guide where our diagnosis is going to go. The other thing that I want to look at is, do the cells look mostly uniform, or is there anisocytosis? So, anisocytosis being a difference in cell size. And if we zoom right in here, there's actually quite significant anisocytosis. We have some cells which are just mildly microcytic, and we have some that are tiny, tiny, um, that are still microcytic, but they are significantly different in size. So, we do actually have some anisocytosis. We have a poikilocyte here, which is again, an elongated or a pencil cell. Um, we have one here that's kind of like an almost teardrop, but, you know, could go either way. Another one here, a pencil cell here. Um, what I'm looking for is, do we have any maybe target cells? And we do have one just here with that little bull's eye in the center. Uh, we've got another one just over here, another one just here, but there's not a huge number of them. I would probably say occasional, um, if I was reviewing this film myself.

So, putting it together, what we have is a mildly hypochromic microcytic with occasional elongated cells. For the target cells, I don't know if there's even significant numbers enough to comment on them. Do you remember that when you're commenting on films, if you look at a film for long enough, you will find one of every single poikilocyte. Whether or not it's in significant numbers enough for you to actually report is a different story altogether. So, do keep in mind when you're reporting, you don't want to over-report, um, and call out every single poikilocyte that you see. But, of course, we don't want to under-report. So, all of the significant poikilocytes you see, you should report. So, I think this one, hypochromic microcytic with occasional elongated or pencil cells. Um, looking around, we have, uh, I think we had a normal white cell count, um, and the white cells look, look pretty average to me. There's nothing there that screams anything unusual to me. And the platelet count was again normal, and most of these are normal size as well. There's an occasional little large one again. In pregnancy, that's not unusual, um, to have just an odd occasional large one. I wouldn't say that there are insignificant numbers, though, just occasional. Um, and so, for this case, this one compared to the first case that we had, the first thing that strikes me is, while this patient is microcytic and hypochromic, microcytic, they are not anemic, and that's an important thing to remember. Um, they do have something that is making their red cells smaller than they should be, but it hasn't caused them to actually become anemic. The other thing is, they don't have any symptoms of anemia. This is a booking blood, um, and it's, it's being used as a screening test for the patient. Um, so, compared to the first one that was a severe iron deficiency, I think this one is most likely to be a thalassemia trait or a hemoglobinopathy. My reasoning for that is, we have a hypochromic microcytic picture, and we have a patient who is not anemic and not symptomatic, and so the microcytosis is probably only mild, um, and it's not affecting them to the point where, you know, they're anemic or being diagnosed with anything. So, um, my question would be, why do they test for this in a screening blood at 12 weeks, and what's the significance of this in terms of, you know, what disorders could this woman's child be affected with if this is an inherited condition?

Where I'm from, the city I'm from, we have a large population that has, uh, thalassemia, particularly beta thalassemia. And so, screening programs like this in the UK and Australia are super, super common. So, I think I can't diagnose the type based on the film. It could be alpha thalassemia trait, it could be a beta thalassemia trait, or it could be a hemoglobinopathy trait, and that's kind of where my diagnosis would stop, and HPLC would take over. So, whilst we're waiting for people to, um, to give their comments for that question about the significance of these, um, the blood cell indices and that the possibility of a thalassemia trait, um, in pregnancy, um, what, uh, what, just out of interest really, what kind of things would you do in your lab to try and confirm this, this diagnosis?

Sure. So, what we would do essentially, and most of these, uh, patients already have this ordered, is we could do an HPLC to determine if the patient has a thalassemia trait or a hemoglobinopathy. Compared to the, the previous one, this patient doesn't appear to have any sickle cells, um, but you will not have sickle cells if you have sickle cell trait. So, there are ways that you can test for or screen for, um, sickle cell varieties in doing a sickle solubility test if you were leaning that way. Um, and you could do a full hemoglobin electrophoresis panel. You could also do iron studies, because it might be that the patient is iron deficient, but they haven't developed iron deficiency anemia yet. So, often your, your indices, you will become microcytic before you become anemic, because it does take, you know, the iron will run out as being used by your bone marrow as a building block, and eventually you'll get to the point where you'll become anemic. This patient is not, um, but you will need to also rule out a concurrent iron deficiency or a developing iron deficiency.

Excellent. So, I think we've got some comments. Rightfully so, people have mentioned, uh, the importance of testing the partner. And although the patient may not have any significant, uh, clinical issues, then they may, and they may that their child may inherit a clinically significant hemoglobinopathy if their partner has a coexisting hemoglobinopathy as well. So, absolutely. Yeah. So, I think that that's really good in terms of the comments. Um, just seeing there's one comment about basophilic stippling or punctate basophilia. Would you expect to see something like this here, and is it useful in distinguishing between coarse and fine basophilic stippling? And is it?

Sure. Sorry, go on. Sorry. So, um, I didn't see any in this film, uh, and I wouldn't really expect to see any in a, uh, thalassemia trait film of this level. So, um, if it's, uh, a hemoglobinopathy or a thalassemia trait, um, you don't often see basophilic stippling. You can definitely see basophilic stippling in hemoglobinopathies, in beta thalassemia, especially, um, and you can see it where there is compound heterozygosity where they've inherited multiple mutations. In my experience, most of the basophilic stippling is quite fine. I know that in the laboratory, we don't often distinguish between coarse and delicate basophilic stippling. Um, the reason, the only time I would really mention it is if you have a heavy metal toxicity, because that is genuinely very coarse. Um, but, uh, you can get basophilic stippling in any condition where you have hemolysis. So, certainly in the, uh, thalassemias, not trait, but thalassemia major and intermedia, and even in sickle cell, you can definitely see basophilic stippling. Yeah, but I wouldn't expect it in a case like this. Yeah.

Excellent. So, I think, um, we've got through most of the questions, um, and it's been a really excellent session that you provided us with today, Nicole. I'm just going to reclaim the host, sure, function from you, and if I could just share my screen. There we go. So, uh, I want to thank everyone for, um, their participation in today's webinar. Um, thank you, Nicole, for for delivering such a great session, and thank our sponsors, Urban Mannheim. Just to remind you again that your CMA certificate will be emailed if you've registered within the next few days. There will be a survey which will post on the Hematology Interest Group, um, to get vital feedback to make these kind of events, um, even better, although it's very difficult to do it from today's session. And the webinar will be uploaded onto the Blood Academy YouTube page, hopefully in the next day or so. And, uh, hopefully, we'll be able to see you again in a week's time, where we'll be joined by Dr. Uh, Audi Setiadi from Canada, where we'll be looking at lymphoid malignancies, again, three cases, which will be available on the Hematology Interest Group. So, thanks again to, to you, Nicole. Thanks to everyone who's watched and participated in today's webinar, and hopefully, we'll see you again in a week's time. Thank you very much.