Webinar 3 - Lymphoid Cases

Uh, welcome everyone. Um, my name is, uh, Dr. Ali Mahdi. I'm a hematologist working in Newport in the United Kingdom. Uh, I'm also the co-founder for, uh, the website Blood Academy. Uh, and along with the Hematology Interest Group on Facebook, I'm delighted to welcome you to the third of our six-part webinar series, uh, where we'll be discussing lymphoid malignancies. And I'm, uh, really delighted to be joined by Dr. Audi Setiadi, uh, who's based in Canada. Um, we have three very interesting cases, and there's a lot to learn, uh, during this session as well. So please stay focused, uh, on the cases, and hopefully, we'll bring up some interesting discussion points as well.

Just to remind you that this webinar is supported by the company Urban Mannheim, uh, and that, uh, the webinar series, uh, is, uh, approved by the Royal College of Pathologists for CPD or CME points. You can still register if you haven't for your CPD points using the website page where the cases can be found. Uh, in addition, if you want to see this webinar again, then this, uh, webinar is being recorded, uh, and will be available on the Blood Academy's YouTube page, and we will be posting a link to that video, uh, over the next couple of days on the Hematology Interest Group as well.

These webinars are hopefully going to give you an overview of blood cell morphology. We can't cover everything, although we'd like to. We'll try and cover as much as we can, but if you're interested in learning more and supporting your education, then we, Blood Academy, does have an online on-demand course where constituting video tutorials, modules, assessments, and all the content. And the whole course is certified by the Royal College of Pathologists in the UK. Along with the Hematology Interest Group, we have a 20% discount using the discount code HIG20 for anyone who wants to learn a little bit more.

So, um, back to today's session. Our focus is on lymphoid cases. Uh, we're going to go through some really interesting cases. We've got three, as I've already mentioned. I am really happy and delighted to to be joined by Dr. Audi Setiadi. Um, she is, uh, she completed her undergraduate medical studies in McMaster University in Ontario, Canada, in 2012. Then she went on to complete her specialist postgraduate hematopathology training at the University of British Columbia, and she currently works as a hematopathologist at British Columbia Children's Hospital in Vancouver, or in Canada. Her interests are lymphoid malignancies, flow cytometry, and obviously medical education, and hence, really happy to have her involvement here today. She's lectured widely in all sorts of places and different backgrounds as well, including, uh, participating in lectures for the Canadian Association of Pathologists as well. So, thank you for joining us today, uh, Audi.

Thank you for the introduction, Ali. You're very welcome. Uh, right, so you should be able to show your screen now, Audi. The hardest thing with these things is the technical side of it, trying to get things all in place. Have you got it all? Perfect. Excellent.

Um, so we, uh, just as an introduction to the case, um, the first case is an 81-year-old female. The blood film was done as part of the annual hypertensive screen, probably reflex from the, uh, increase in the white cell count. The hemoglobin is normal, the platelet count is normal, and as overall dimension, the white cell count is slightly higher than what it should be. But before you start, Audi, I'm just going to say that if anyone, um, if anyone wants to give a guess as to what could be going on whilst, uh, Dr. Audi presents the case, you can do that in the comments. And if you do have any questions or further comments, then please use the comment section too to make those. I'll hand over to you.

All right, let's make the screen bigger. There we go. So let's go through the blood slide. Um, so in accordance to the indices, uh, as you see here, there is an increase in the lymphocytes. And as we scroll through the slides, we're also noticing these, uh, forms of, uh, like it looks like cells that are being squashed, right? Um, and they, they look like they have been broken or squashed during this slide preparation. So, um, does anybody want to take a guess what these, uh, forms are called in the chat section, in the comments section?

So we've already got some comments here in that these are small mature lymphocytes and smudge cells. Very good. So these are called smudge cells that she correctly identified. And these are actually the fragile lymphocytes that are smashed during the slide preparation. They, they're broken, they're not like this in the body, but when you're making the slides, uh, these fragile lymphocytes are more readily squashed. So they're called smudge cells, and we'll talk a little bit more about that. And then as we go through the slides, um, you also see these lymphocytes. And in normal individuals, usually the lymphocytes should be variable in size. There are some small and mature ones, but there are also some bigger ones with the, uh, more abundant cytoplasm, right? So we call that pleomorphic, uh, normally. But in this patient here, you, you see that although there's some variability in size, a lot of the lymphocytes are mostly small with, uh, scant cytoplasm, and they also have this, uh, sort of like chromatin, clumpy chromatin look, uh, in their nuclei. So it's almost like a soccer ball. Might have to use your imagination a bit, but for example, like this lymphocyte here. Um, so the pattern of the chromatin clumping looks like a soccer ball with a darker, uh, clumpy, uh, chromatin in the nuclei. So, um, we call this more monomorphic lymphocytes, right? So they're instead of pleomorphic like in normal individuals, they're somewhat monomorphic in this patient, and they have this soccer ball look, and they have smudge cells. So does anybody want to take a guess on what the diagnosis is in this patient? I think people have already tried, um, given the diagnosis. I think the, uh, there you go, vast majority of people are thinking that this could be CLL or chronic lymphocytic leukemia. Very good. All right, you guys are on a roll here.

So, um, okay. So otherwise, the, the platelets look good. The red cells look, uh, unremarkable. Uh, there is no evidence of hemolysis. So in CLL, sometimes I will talk about a bit more about this disorder. Uh, I've prepared some PowerPoint slides, uh, in a moment, but, uh, sometimes it can be associated with hemolytic anemia, more specifically autoimmune hemolytic anemia. So in that case, in addition to this lymphocytosis with this morphology, you will also see, uh, increased spherocytes in the red blood cells and increased polychromasia, right? But in this case, uh, the red blood cells actually look nice, uh, and they, they're unremarkable in morphology. You don't see increased spherocytes, you don't see increased polychromasia. Um, and another thing that we look for in a CLL, so there are three things that I always look for as a hematopathologist in CLL. So number one, we talk about the red cells: is there any any background, uh, autoimmune hemolytic anemia? Number two, is there any large cell component, uh, that might suggest that the CLL has transformed into a more aggressive type of leukemia, like diffuse large B-cell lymphoma, sorry? So, uh, we'll talk about the morphology a bit more, but like as you see, there are some variability, uh, in the lymphocytes, but this, uh, slightly larger lymphocytes that you see here, they are, uh, still very mature looking. They, uh, they have a bit more abundant cytoplasm, but they don't look, uh, large and with immature chromatin that you might see in diffuse large B-cell lymphoma, for example. So, so this looks like it's still within the morphological spectrum of chronic lymphocytic leukemia. Um, and and in diffuse large B-cell lymphoma, uh, you would see like very, uh, irregular, uh, nuclei, right? Um, and and usually like they can be vacuolated as well, and immature chromatin, and you don't see necessarily that in this larger lymphocyte. So this looks like it's still within the morphological spectrum of CLL. And the third thing I'm looking at is whether there's any increased prolymphocytes. So in CLL, it's common to have a small subset of the lymphocytes, uh, having a prolymphocytic morphology. So prolymphocytes are cells that are more medium-sized, uh, slightly abundant cytoplasm, and they have usually prominent nucleoli. And I'll have some slides to give you, uh, some examples of, uh, prolymphocytes. And it's okay to have a small, small subset of the lymphocytes having the prolymphocytic morphology in CLL, but you cannot have more than 55%, right? So that's a cutoff we use. And if you have more than 55%, then we'll be worried about transformation or prolymphocytic leukemia, for example. So, so we don't see that here. Uh, this looks like a straightforward case of chronic lymphocytic leukemia in this 81-year-old. So we'll switch to, uh, my presentation slide about CLL. There you go. You can see my slide. Okay. Yeah, it's all there. Are they? Okay. Perfect.

All right. So what is CLL? Uh, according to the WHO definition, it's a lymphoid malignancy. It's a mature lymphoid malignancy. Uh, com, although it's called leukemia, it's not, uh, leukemia in a sense of acute lymphoblastic leukemia, right? Where, uh, it's composed of immature blasts. Uh, in CLL, these are malignancy of mature lymphoid cells that's composed of, uh, B cells that are positive for CD5 and CD23. So these B cells aberrantly express, uh, the CD5 marker, which is usually a T-cell marker, as well as CD23. And it's actually one of the most common leukemias in, in Western countries in adults with an incidence of five to more than 20 in 100,000. And interestingly, it's more rare in Asian countries, which may indicate some genetic basis of this disease. But what exactly causes this, uh, is still widely unknown. Uh, and a median age of diagnosis for CLL, it's more commonly diagnosed in older adults with a median age of 70 years, and there is a slight predilection to male than female. And normally, this, this patient presents without any symptoms, and it's mostly, uh, discovered through incidental findings on CBC done for other reasons, such as in this case. Um, so usually the patient, uh, gets a blood work for, uh, some other, uh, reason or some other symptoms, but then incidentally, they're found to have increased lymphocytes. Um, but in, in some, uh, subsets of the patient, they can also present with, uh, your usual B symptoms or what we call B symptoms, or, uh, in, in patients with lymphomas, they have weight loss, fevers, night sweats, or fatigue. That, that can also happen, and you can have some lymphadenopathy as well. In, in CLL, Um, and the, the malignant cells can involve blood, bone marrow, spleen, as well as lymph nodes. So there's a different spectrum of the disease, and it's widely classified depending on their monoclonal B-cell count in peripheral blood. So in CLL, you have more than five monoclonal B-cell counts. So, note that this is not total lymphocytosis, we're talking about. It's actually the, the count of the malignant B cells, and we derive that number from using flow cytometry to diagnose a disease and count how many percent of the B cells are malignant by flow cytometry, and then multiply that by the absolute number of the lymphocytes to get this number. And in CLL, we have more than five. Uh, and and there are some precursor conditions called monoclonal B-cell lymphocytosis, uh, that, uh, presents with a lower, uh, monoclonal B-cell count of less than five. And then there's another one called small lymphocytic lymphoma or SLL, and in WHO, it's classified together with CLL. So it's used often called CLL/SLL. And the only difference is that in CLL, you have more than five, and in SLL, you have less than five malignant B cells circulating in peripheral blood. And then in, in both CLL and SLL, you can have tissue infiltration as well as lymphadenopathy or splenomegaly, whereas in monoclonal B-cell lymphocytosis or MBL, you, by definition, you do not have any of this organ involvement. And in more recent updates of WHO, MBL is actually divided into a low count versus high count. Uh, so low count MBL is less than 0.5, uh, and high count MBL is more than 0.5, so between 0.5 to 5. Um, and that has to do, uh, it's thought to that the low count MBL has even lower risk of progression into CLL than high count MBL. So this patient can actually be followed less frequently. Patients with high count MBL. All right.

So in CLL, we have lymphocytosis, as we talked about. It can be very high. I can be more than 100, and I've seen CLL, uh, with white counts or lymphocyte counts more than 200. Um, and you can have cytopenia because of these malignant lymphocytes infiltrating the bone marrow and suppressing other cell production, like your red cells and your platelets. Will be low, and we talked about that. You can also have a complication in the form of autoimmune hemolytic anemia, and that's usually DAT positive. So if you see the morphological signs of hemolysis, you can do your hemolytic workup, including the DAT. Peripheral blood smear, of course, essential for recognizing this condition. And then I highlight and read what we really will be very useful for, uh, diagnosis of CLL. So flow cytometry is very useful to demonstrate the, uh, aberrant expression of 5 and 23 in the, uh, B cells. And sometimes you can see, uh, increase in LDH and beta-2 microglobulins, uh, as a more like a marker of higher cellular turnover. And often in CLL, you can have low immunoglobulins. So like the immunoglobulins are actually suppressed, and some of these patients require monthly IVIG infusions to keep their immunoglobulins up. Bone marrow biopsy is usually not required for diagnosis because we can make the diagnosis with peripheral blood smear and flow cytometry of the blood. But it may be done before beginning treatment as a baseline and to see how, how heavily involved the bone marrow is. And sometimes they do either tissue biopsy if there's any tissue involvement or in large lymph nodes. And genetic studies in CLL is important for prognostication, as we'll talk about a bit more later. So we talked about this mostly while we go through the slides. Um, so in CLL, classically, you have small mature B cells with soccer ball chromatin, uh, and smudge cells, and it may be at mixed with these prolymphocytes. So in black arrow showing some cell, uh, examples of prolymphocytes with a more moderate amount of cytoplasm and prominent nucleoli. So we talked about smudge cells. I would like to like talk a bit more about that. Um, it doesn't always mean CLL. So like we talk about smudge cells classically in textbooks, uh, as associated with CLL, as in this case, but it can also be seen in other reactive conditions such as viral conditions or chronic inflammatory diseases. So what I really want you to take home from this lecture is that like although it's a classic finding in CLL, whenever you see smudge cells, don't always jump into a conclusion that this is a CLL. Keep in mind that you can also see that in reactive conditions. It has to be to diagnose CLL, you have to like take everything into account that we already talked about. It's also called basket cells, uh, if you see here in, in some cases, it resembles the, the weave of the baskets. And it's also called Gumprecht shadows, uh, after the German physicians who discovered this in the context of CLL.

So how, as you know, as a tech, how do we deal with smudge cells, right? And often it, it can present a problem with your manual diff and it can make it discrepant with an automated diff. The thing is, your hematology analyzer will accurately count for gel cells, right? Like so it doesn't look like this in, in the body. So your hematology analyzer will only look for cells. But when you actually make the slice manually, then this is where the cells are smashed, uh, and squashed, and it makes much sense. So if you don't count these smudge cells manually in your slide diff, then you, you will have this discrepancy between your manual and automated diff. And how to address that is we have to make albumin slides, right? So albumin preparation will help preserve the morphology of these lymphocytes, so they actually will look, uh, intact, uh, and and so you, you actually do, you can actually do your manual diff in your albumin preparation, uh, and and you can also report your diff, uh, using the automated diff instead of your manual diff. But if, in, in some centers or in some places where you may not, uh, have automated diff available, uh, it is for you to be aware that smudge cells should likely be counted as lymphocytes. Of course, you should make albumin slides or put it in a separate category. And in places where you use Tel-Aviv, for example, the Tel-Aviv usually puts the smudge cells in a separate category. So that's something you have to be aware of as well, that these are actually lymphocytes that are put in a separate category, but they have to be differed as lymphocytes. Okay.

So we talked about how CLL can transform into a more aggressive form, and there's a term for that, it's called Richter's transformation. It's most often diffuse large B-cell lymphoma. So is it actually an example of that where, uh, there are these large cells with, uh, markedly convoluted, uh, nuclear contour, dark blue cytoplasm, some with vacuolation? So it's different from the morphological spectrum that we see in this case. If you see cells like this, you have to be really worried that the CLL has transformed into a more aggressive form, and you have to notify your clinician. And, uh, we talked about it can also transform into B-cell prolymphocytic leukemia, and there's, uh, this example, uh, of, uh, prolymphocytic leukemia where mostly the cells are medium in size with a moderate amount of cytoplasm and prominent nucleoli, accounting for more than 55%. Okay.

And let's talk a little bit about flow cytometry in CLL. But before that, I really like this approach to cell lymphoid malignancies in general. I teach my residents this. I just, uh, conceptualize your thinking. And of course, in real life, it's more complicated than this, and there are always exceptions, but this is a good place to start to think about this way. So if you have CD19 clonal B cells, the first thing you have to ask, like, does it express, does it co-express CD5? If yes, the next thing you ask is, does it co-express CD23? And if it expresses both 5 and 23, there goes your CLL, right? That's a classic phenotype for CLL. And if it does not express 23, so CD5 positive B-cell malignancy that does not express CD23, there's mantle cell lymphoma. And if it doesn't, uh, express CD5, the next thing you, you ask is whether it expresses CD10. And if it expresses CD10, the differential for that includes Burkitt lymphoma, leukemia, the LBCL, and this is more immature form of B-cell lymphoid malignancies, your BALL, uh, B-cell lymphoblastic leukemia lymphoma, which also expresses CD10. Uh, and if your CD10 is not expressed, then you think about everything else. And in that sort of like, uh, garbage can category, you think about hairy cell leukemia, lymphoplasmacytic lymphoma. I didn't put it here, but also think about marginal zone lymphoma. And, uh, hairy cell leukemia is actually associated with like, uh, other, uh, uh, flow cytometry markers like CD103, uh, and CD25 and 11c, okay? So in CLL, typically CD19 positive, and the CD20, uh, is typically dim in CLL. So it's dimmer than your, uh, usual mature B cells. Uh, and so let's look at this flow cytometry plots here. So we're looking at the blue population here that's expressing bright CD45 and low side scatter because they're lymphocytes. They're supposed to be, uh, low side scatter because like they, uh, they're very simple, they, they're not like neutrophils where they're very complex. Remember that side scatter reflects your cell complexity. And, uh, this blue population of cells, they express CD20, then your mature B cells that are, uh, highlighted in the, in the black circle. Okay. And it's co-expressing CD5, and also note the, uh, light chain expression in CLL. It's actually dimmer than your, uh, mature B cells. So in, in black circles are your mature, uh, B cells like polyclonal, some expressing kappa, some expressing lambda, but there's this kappa restricted or kappa clonal B-cell population that's a bit dimmer, and that's your CLL cells there. And it does not express, uh, FMC7. Um, and in contrast to mantle cell lymphoma, which is the biggest differential diagnosis for CLL, it, it's, uh, CD5 positive but CD23 negative and FMC7 positive. Okay. So it's a bit of a challenge for you. Uh, there is a, uh, cell population that's co-expressing CD19 and CD5. There are actually two, uh, populations, blue and red, and both are co-expressing CD5 and CD19, and both are co-expressing CD20, uh, and some are more variable than the other. Um, and both are also expressing CD23. Okay. Uh, but look at your kappa and lambda plot. One cell is kappa restricted, and the other cells were lambda restricted. So what is your diagnosis here? Does anybody want to take a shot in the comments? And Ali, you might want to help me read the comment section because I can answer.

Yeah, there's a slight delay, uh, with our broadcaster with Facebook. So we'll get a couple of, uh, couple of seconds for. So we've got B-CLL as one comment. I think that one interesting point from the plot is that the expression of kappa and lambda is quite weak as well, where that's right, more aggressive lymphoid malignancies are usually a little bit higher up, aren't they? May help them a little bit. That's a very good observation. [Laughter] Yeah. So there were some possibilities here, right? So we talked about it could be a transformation to a more aggressive, uh, B-cell lymphoma, but usually then, um, your, uh, your light chain expression might be, uh, brighter and your CD20 might be brighter. Um, [Music] and and there, there's also a possibility that there might be, uh, another type of, uh, concomitant lymphoma, like another, uh, lymphoma into the CLL, but the thing is, like it actually expresses exactly the same CLL phenotype, right? So 19, 5, 23, and then kappa and lambda. So this is actually a case of a biclonal CLL, which is very interesting. It does happen, and I've seen it. So whenever you, you see a cell population that co-expresses 19 and 5, it's never normal. Um, so even though the kappa and lambda look polyclonal, always check on your kappa and lambda population and see if they're co-expressing, uh, aberrant markers, right? In this case, CD5. So, uh, so you should be careful, uh, not to call this an, um, polyclonal or normal lymphocytes because they look polyclonal in the kappa and lambda plot, but they're actually, uh, expressing aberrant markers, and this is actually a case of biclonal CLL, rare but can happen. Something I can't, I want to bring up clear attention. Okay.

So, um, briefly about genetic studies. Can be detected in about half of CLL cases, and it informs prognostication and treatment choices for patients. And there are some higher risk cytogenetics like CD38 expression, Zap70, unmutated IGH immunoglobulin heavy chain status, and deletion 17p. That these patients will be treated more aggressively, and there are some newer agents, therapeutic agents that they start on these patients in comparison to if they have lower cytogenetics. Okay.

So let's take a quick take-home point. Um, so any lymphocytosis, especially five, uh, more than five is usually the cutoff, but I'm sure in different centers, you guys use different cutoffs, should be flagged and smear should be reviewed. Smudge cells don't always mean CLL. Always make albumin slides to assist with your manual diff, and it's important to differentiate CLL from other causes of lymphocytosis, including other B-cell disorders or infectious causes. And if you have persistent lymphocytosis of unknown cause, uh, they should trigger flow cytometry assessment, uh, to, uh, look for lymphoproliferative disorders like CLL. Okay.

Excellent. Thank you. That's a great start to the webinar. Um, yeah, you've answered all the questions that I wanted to ask you, and, uh, I think you've really highlighted the point that flow cytometry is essential in the diagnosis of, uh, lymphoid malignancy. So that, that's excellent. Let's move on to case two, which is a 59-year-old male, uh, who is, uh, presenting with back pain, and he's anemic. He's got a normal white cell count and platelet count, at least by the, uh, automated differential count. Again, if people want to put their comments of what they're seeing on on the slide, please do so in the comments section.

All right. So as you see here, this patient has anemia, quite marked anemia, and, uh, let's look at the blood smear. So I'm going to zoom on this. All right, now we are in focus. So, um, we're actually pretty close to the edge of the smear, but as we move, uh, deeper into the smear, we're actually noticing, uh, one of the red cells that are, uh, giving this sort of, uh, coin stacks. Sorry, I'm just looking for a good field here. There you go. There's some more white cells. All right, this is better now. So, uh, which highlights that to assess the red cells, we always have to go to a good, uh, field of the film because otherwise, they all will look like they're on top of each other. But in this area of the film, uh, you see that although a lot of the red cells are apart from each other, but there are a group of red cells that look like they're lining up, right? They're making this sort of stack or like coin stack. So anybody wants to put in the comment section what this form is called? It's coin stack of the red cells. We've got some comments saying that this could be agglutination. Some people are saying that this is, uh, rouleau formation as well. That's right. And we'll talk about the difference between the two. But, uh, the majority of what we see here, uh, is actually rouleau formation because they're actually making more of a coin stack instead of like grape-like, uh, clump of the red cells like you see in true agglutination. They're lining up pretty nicely. So these are actually rouleau formation, uh, and which highlights the importance of like really picking a good field of the blood film when you're assessing, uh, your slide because like if you go too thick, uh, in a blood smear, you can start seeing the red cells being, you know, on top of each other and they can all look like they're agglutinating. But here, actually, it's mostly rouleau rather than agglutination. And as we cruise along here, uh, so we, we see some of the lymphocytes that look like they're a bit, uh, plasmacytoid in morphology. So their nuclei is polarized to one side, and they have a little bit of a halo or like clearing around the nucleus, uh, so-called like plasmacytoid lymphocytes. But I haven't really seen any real, uh, plasma cells yet in this slide. So there's another form of plasmacytoid lymphocytes. So, um, whenever you see rouleau formation, you have to think about two things. And the most important thing you have to think about is whether, uh, this patient has abnormal paraprotein or M-protein that causes, uh, rouleau formation. What, so what happens in rouleau formation is that you have your normal, your red cells normally have a negative charge or what we call zeta potential, where they repel each other because of that negative charge. But if you, in the presence of abnormal proteins or immunoglobulins or fibrinogen, for example, they bring the red cells closer together, and so then they actually stick to each other, and that's what causes rouleau formation. Uh, so that can happen in abnormal, uh, paraprotein anemia like in myeloma, but it can also happen in, uh, just reactive causes like in, uh, infection or inflammatory causes where you have increased, uh, fibrinogen, you have increased, [Music] polyclonal proteins that can also cause this picture. But you, you have to evaluate the patient for abnormal paraprotein if you see rouleau formations. Okay.

So this is actually a case of plasma cell myeloma, and I'm going to talk about that in my current PowerPoint slide. So, uh, what is plasma cell myeloma? So, uh, your plasma cells are made in the bone marrow, where, uh, they produce antibodies or immunoglobulins to help protect us against various types of infections. And normally, they produce different, uh, immunoglobulins or antibodies with different specificity to antigens. But in multiple myeloma or in plasma cell myeloma, these antibodies are non-functional. They, they result from proliferation of a single clone of plasma cells, and they all produce the same type of antibodies, and they're non-functional. And that's what they're what's called M-proteins or paraproteins, and they can be detectable in the serum, uh, or urine of the patient, and they can as well, they can accumulate in different organs and lead to organ damage, like in your kidneys, for example. And they can occupy your bone marrow and leading to anemia and thrombocytopenia and neutropenia in the presence of these abnormal plasma cells. And it, it happens. It's, it's fairly common, uh, diagnosis in adults, especially at 10 to 15% of hematopoietic neoplasms. And, uh, in 90% of cases, it occurs in, uh, again, more than 50 years old, and median age of diagnosis of 70. And, uh, so we'll talk about the clinical features of myeloma. So in, in myeloma, the tumor cells affect the bone marrow environment in such a way that it has higher bone resorption activity than the bone building activity. So it looks like your, your bone is being eaten from inside. So actually, they lead to osteoporosis, lytic lesions, uh, in the bone, and high turnover of the bone leads to high calcium in the blood, right? And, uh, the abnormal, uh, immunoglobulins can also accumulate in your kidney tubules, leading to, uh, renal failure. And as we talked about, it can also lead to anemia. And there's some mnemonic for that, uh, called CRAB, that's like a classic mnemonic in myeloma that's, uh, that we teach in medical school. And, uh, and it can also lead to, uh, infections due to depressed, uh, normal immunoglobulin production and bleeding, for example, because you have low platelets, and neurological symptoms due to, uh, pathological fractures, uh, in your spinal cord, uh, so you can have some neurological symptoms for that. And this is a picture of a lytic lesion in the skull, where you look like there's some hole being punched in the skull, like punch lesion, we call it in the radiological terms.

So, uh, how do you establish clonality? How do you tell if you have a proliferation of plasma cells in a bone marrow that produces this abnormal protein? So this is a diagram of immunoglobulins that's composed of heavy chain and light chain, right? Held together by disulfide bonds. So your light chain can be kappa or lambda, uh, and and normally, like we talk about, like usually lymphocytes produce both kappa and lambda, and plasma cells as well, they produce both kappa and lambda, uh, in about two to one proportion. But when you start producing only one or the other, that's what we what we call clonal, uh, population of lymphocytes or plasma cells in this case. So we can do protein electrophoresis, which which is done by agarose gel or capillary zone electrophoresis. And in a normal individual, because they produce all kinds of, uh, proteins and all types of different immunoglobulins, there's a smear, right? So this first peak here is albumin, and then in the gamma globulin region, there's like a, it's more like a smear or like more like a, uh, like a hill pattern. Whereas in a patient with myeloma, if you do serum protein electrophoresis, you see like a spike or like a, or like a sharp peak in the gamma globulin region, and that's your, uh, M protein, right? So this actually quantifies, uh, or not quite actually identifies, uh, uh, this whole antibody structure that we talk about. Both the heavy and light chain will be, uh, represented here. But then when you overlay it with, uh, different monospecific antibody towards heavy or light chain, and then do a protein precipitation, is what we call serum immunofixation, then you can actually identify, uh, the exact type of this abnormal antibody. So in this case, it's IgG kappa, right? It can be different types as well. So that's how we demonstrate that this is abnormal, uh, paraprotein. And there is also another assay that only detects the light chain, right? Instead of that whole antibody molecules, it only, uh, identifies the light chain portion. And plasma cells normally produce excess light chain compared to heavy chain, and, uh, and there's antibody-based systems that can detect the light chain concentration, uh, and the normal ratio kappa:lambda usually about 0.25 to 1.65. But in myeloma, you can have more than 100 to 1, for example, ratio. And in, in a proportion of myeloma, it's only light chain, they don't actually have a heavy chain, and it will be used to monitor progression or treatment as well for the patient. And you can also do immunohistochemistry using kappa and lambda antibodies in, in the tissue or the bone marrow. And of course, you can also do flow cytometry where you identify your plasma cells, which is CD38 bright, and demonstrate kappa or lambda light chain restriction by flow cytometry.

So, uh, for the interest of time, I'm just going to quickly show you some of these. This is how we used to diagnose myeloma in the old classification. So there's also precursor lesions called monoclonal gammopathy of undetermined significance or MGUS, there's smoldering myeloma, and the only difference is that in MGUS, you have less than 10% of clonal plasma cells and less than 3 grams per deciliter of M-protein. And in a smoldering myeloma, it's more than 10%, but in plasma cell myeloma, you have this association, associated CRAB symptoms, whereas in the other two precursor conditions, you don't have any CRAB symptoms. But in the more recent WHO, in addition to those, you can now diagnose myeloma in patients even without the CRAB symptoms as long as you have more than 60% of clonal plasma cells in a bone marrow, or if you have serum free light chain ratio of more than 100, or you can also do MRI and identify more than one focal bone lesion in the MRI. So if any of these myeloma-defining events are present, you can diagnose plasma cell myeloma regardless of the CRAB symptoms. And this is interesting. So in myeloma, you have like, uh, peripheral blood smear, what happens is like you have excessive immunoglobulin produced by the plasma cells that are acidic, and it takes up the basophilic stain in the blood smear, so it appears really blue compared to your normal blood film. So whenever you see this blue stain, uh, you have to suspect that there is like increased, uh, protein, uh, production. And we talked about rouleau formation, and, uh, you sometimes can see plasma cells circulating in the blood as well. So like whenever you see rouleau, make sure you look for any increased plasma cells in the blood, which can indicate plasma cell leukemia if it's more than 20% in the peripheral blood.

So we talked about the, uh, rouleau versus agglutination, uh, and we talked about the mechanism of the rouleau formation and agglutination. Uh, so as opposed to coin stack, you have this grape-like clusters, and so you can see, uh, rouleau not only in myeloma, but you can also see this in infection or autoimmune conditions and even chronic liver disease as well. And this can present a problem in, uh, blood banking, if any of you are in blood bank, in pre-transfusion testing, it can cause false positive blood grouping and crossmatching as well in red cells, as well as increased ESR. Uh, so do you guys know what technique might be useful to help differentiate rouleau versus agglutination? Just whilst we wait for some of the, uh, information to come through. I think, uh, it's, uh, really, we've got an extreme case of myeloma here in the peripheral blood, but it's trying to make sure that the patient is safe as well by, if you see these kind of features with such significant rouleau, looking at the renal function of the calcium, because the, the threat to the patient of course, compression and renal failure is quite high. So, so we've got some, some answers to, uh, your question, Audi. So saline replacement. That's right. Yeah, I think I accidentally clicked on it to give a clue. That's very good. So, uh, saline replacement is a useful technique because it can, uh, dissociate rouleau formation by dilution of the red cells, whereas true, uh, red cell agglutination will persist despite saline dilution.

All right. So just to show you, uh, some morphological findings in the bone marrow. So normal bone marrow, usually you have different cell types, and in multiple myeloma, you have proliferation of plasma cells, uh, that look very abnormal with expanded, uh, basophilic cytoplasm, and you can see this halo that signifies the plasma cells. But not all plasma cells look like this in real life. You have to be aware of variation of morphology in plasma cells, and they can look quite scary, actually. So, and I've seen different types of plasma cells, and there are plasma cells that even resemble, uh, small lymphocytes. They look, they look very, uh, small, and they, uh, they have, uh, scant cytoplasm, and the halo may not look so obvious, right? And they can have like more immature chromatin pattern that may resemble blasts, and they can look quite, uh, huge with, uh, multi-lobulation, and they can also have this pinkish cytoplasmic fraying that resembles like a flame. So we call it flame cell, that's classically described in IgA myeloma. And then you can have like multiple different inclusions as well, like these plasma cells have cytoplasmic vacuoles, granules, or crystals that sometimes resemble Auer rods even, and have been reported in literature. And there are terms to describe these cytoplasmic immunoglobulin inclusions called Russell bodies, and if it's inside the nucleus, it's called Dutcher bodies. But like you have to be, and they're actually made of, uh, immunoglobulin inclusions in the plasma cells. Uh, and plasma cells that contain multiple vacuoles, there's also a term for that, it's called Mott cells, Mott what cells. So for the interest of time, I'm going to skip that, but just to be aware that like there is, there are some plasma cells that are that resemble lymphocytes. So you have to be careful and, uh, make sure to establish the, uh, identity of the plasma cells using flow cytometry and immunohistochemistry. And M-protein can also be found in other conditions, not just plasma cell myeloma, but also in some lymphoma like LPL and also CLL as well. And the clinicians might order albumin and serum beta-2 microglobulin, and that's usually, uh, useful for, uh, prognostication of myeloma as per this international staging system. And the cytogenetics in myeloma is also useful for, uh, prognostication because some cytogenetics are indicative of higher risk than others. So I'm not sure if some of the tests here are in blood bank, but I just want to, uh, briefly, uh, bring it to your attention that there are some newer, uh, therapeutic agents for myeloma like anti-CD38 called Daratumumab or Elotuzumab that may potentially interfere with your lab testing, especially in antibody identification for transfusion. What happens if your red cells actually express CD38 as well? So the anti-CD38 drug will bind to the red cells, and if you add the anti-human globulin for antibody identification, it will agglutinate the red cells, resulting in pan-reactivity of your antibody panel. It can also, uh, uh, interfere with your, uh, SPEP as well as flow cytometry for detection of plasma cells by CD38 gating. So there are some techniques that people use to resolve this, for example, using DTT, but it can also denature your Kell antigens. So you have to basically, the number one solution for this is like you have to phenotype your red cells before you start this treatment. It's the, uh, the bottom line.

All right. So rouleau formation should always be reviewed and trigger investigation, as Ali highlighted. Uh, and lab testing in myeloma is important for diagnosis, prognostication, monitoring for treatments. Be aware of morphological variation in plasma cells, and any newer therapeutic agents for myeloma that can interfere with lab testing. All right. So the last part will be much quicker than the other two.

No, that that's great, Audi. Um, I, I just highlight my experience in patients with plasma cell leukemia. There's one question about how you just, how you distinguish myeloma from plasma cell leukemia. And I've just made some comments in the description, but I don't know if you've got any thoughts about the morphology of plasma cell leukemia, the how they sort of stray away from the usual appearance of that sort of fried egg appearance of normal plasma cells that we'd expect.

Yes, so morphology, like the difference between plasma cell leukemia, uh, and just straightforward myeloma, really by definition is that you have more than 20% or more than 2 gigaliters of plasma cells in the peripheral blood. Uh, so morphologically, I don't think they look different from each other. Uh, it's more, uh, the percentage of the plasma cells in peripheral blood. So the plasma cell leukemia can have very, uh, variable morphology as well, just as myeloma does. So it's really more a percentage of the cells in peripheral blood.

Excellent. So let's move on to case three. Um, so this is a 68-year-old female who's presenting with breathlessness, and she's anemic, but she has a very, very high white cell count and very scary white cell count if you see it, with a mild thrombocytopenia as well. Uh, so if you could just go over the slide, Audi, that'd be great.

Yes, so, uh, so this patient has, as you see, they're markedly increased lymphocytes, and this is above 100, uh, if you differ, and they look pretty monomorphic, right? And they also have, uh, if you see here, some of the cytoplasm have this sort of bleb or cytoplasmic protrusion, uh, in, in a lot of these cells. So, so this cytoplasmic protrusion, uh, is often seen in this condition along with very high, uh, lymphocyte counts, and they have, they're like scant cytoplasm, and they don't have such, uh, prominent nucleoli, uh, like you see in your B-ALL prolymphocytes. But the hallmark is really they have this cytoplasmic protrusion. All right. So anybody want to take a guess what this is? It's a very rare condition, but very interesting. Does anybody, uh, put in the comments of what this might be?

The comments are coming in, uh, slowly. Uh, yeah, we don't have a specific diagnosis as such, but, uh, we've got hairy cell leukemia. We're wondering, are these cells mature or not? They are mature, yes, because they, they look mature because they have condensed chromatin, right? Just like in us mature lymphocytes, as opposed to finer chromatin like you see in blasts. But that's a good question because they have very high N:C ratio, that might make you wonder whether your cells are blasts. So we've got some, uh, some diagnoses come in now. So the, uh, the main three, I think are hairy cell leukemia, MEG, could these be megakaryoblasts, or could this be T-prolymphocytic leukemia?

Very good. That's a very good, uh, differential diagnosis, actually. So this case is actually a case of T-prolymphocytic leukemia. And those people who guessed megakaryoblastic, uh, leukemia, they might notice the blebbing in the cytoplasm, which is also a hallmark of this condition. That's why whenever you see cytoplasmic blebbing, you think about megakaryoblastic leukemia as well as this more rare condition called T-prolymphocytic leukemia. Uh, I think the two big differentials for cytoplasmic blebs, hairy cell is not a, not a bad guess. They can have some morphological variation, but normally the classic hairy cell, they have more abundant cytoplasm and more spiky like, uh, but but the hairy cell leukemia, they can also have different type of hairy cells, so to speak. And and I've seen that some hairy cells also have more like blunted projections, almost like blebs. So that's actually not a bad guess at all. So those three, uh, are very good differential diagnoses. So T-lymphocytic leukemia is a very aggressive leukemia, uh, that happens in about 2% in mature lymphocytic leukemias in adults, and characterized by very high lymphocyte count, usually more than 100. And the patient often has this diffuse skin rash infiltration, and you see like this edema or fluid collection around the, uh, orbits. And you see the prolymphocytes, just like we talked about the prolymphocytes, similar morphology, but they can also have some variation like a smaller cell that you can have like cerebriform where it's more, it's more convoluted, the nuclei. But the common feature is the cytoplasmic protrusions or blebs. And in terms of the immunophenotype, these cells are, uh, uh, usually expressing CD3, CD25, and usually have bright CD7. In contrast to some other cell malignancies where they're often losing CD7, in T-PLL, it's actually pretty bright. And in a significant proportion of the cases, it's actually positive for both CD4 and CD8. And classically, cytogenetics characterized by translocation 14:14, inversions 14, and genetic mutation in TCL1 and in some other genes as well. They're always exceptions, like in this case where CD3 is a bit dimmer. And it is a very aggressive disease, unfortunately, with no curative therapy. And this patient only survived for usually about one to two years. Sometimes they get transplanted if they're eligible or well enough for transplant. And there's a newer drug, uh, anti-CD52 or Alemtuzumab, that may result in in better response for this, uh, patients with this unfortunate disease. And that's all I have. If you have any questions at all, feel free to shoot me an email. I realize with all the technical difficulties and all, there's not much time for questions, but I'm opening it for questions right now in case anybody has burning questions.

Thank you. I did that. That was absolutely fantastic. Really enjoyed it. Um, I, I think it sort of highlights the, the range of different conditions, uh, that patients with lymphoid malignancies can present. And as you've highlighted in all the three cases, really the importance of, uh, flow cytometry and immunohistochemistry, because I always tell the trainees that you can be very good at morphology, but the diagnostic tests are the other things as well, but you need to know what panels to use and antibodies and things. So, thank you so much for that. That was, uh, very, very interesting. And hopefully, if you don't mind, Audi, if it's possible to share the slides to the, uh, to the rest of the people as well, we can make them available as well as the video.

Yes, definitely. I will do that. I will send it to you, and you can, uh, distribute to the HIG group. Perfect. Excellent.

So I'm just going to reclaim the host so I can just, uh, okay, share my screen. Make you a host again. There we go. So, um, again, thank you, Audi, for an excellent webinar review of three excellent cases. Uh, thank you to everyone who joined us today, uh, on the webinar, uh, on Facebook. Uh, thank us also like to thank our sponsors, Urban Mannheim, for providing the support to to facilitate these free webinars. Just a reminder to everyone that the CME certificate will be emailed to you in the next couple of days. I will be posting a link to a survey after this webinar as well, and also be included in the email with the CME certificate. Again, if you want to watch the webinar again, then you can do using the Blood Academy YouTube page, and hopefully, we'll see you again in two weeks' time. Uh, we're going to be joined by Sam Derwe. Our next webinar is going to be focusing on lots of neutrophil cases, granular sites, in two weeks' time. So we look forward to seeing you in, in two weeks' time at the same place and roughly the same time as well. Uh, we will be joining, uh, hopefully Audi will be joining us again in September, where we'll be talking about, uh, acute leukemia cases. So, uh, we'll be delighted to have you back in a couple of weeks.

Awesome. Thank you very much. I look forward to seeing you all again.

Excellent. Thank you very much, everyone. And thank you, Audi. Thank you. All the best. Bye.