Transcription
Welcome everyone. Um, welcome to this, uh, blood cell morphology webinar series, the first of its kind, uh, a collaboration between the Hematology Interest Group on Facebook and Blood Academy. My name is, uh, Dr. Ali Mahdi. I'm a consultant hematologist in South Wales in the UK. I'm really delighted to introduce the webinar series and get things started with with our speaker today, Dan Pelling. We have been very fortunate to get a group of experts in blood cell morphology around the world, and they will hopefully guide you through the basics and more advanced aspects of this essential skill for a whole range of different people working in the hematology field, from biomedical scientists to pathologists and hematologists as well. And really, to allow us all to learn from each other and sharpen our skills in in this important skill.
Uh, before I introduce our speaker today, um, I just want to go through some basic things about the webinar series. And and just to remind you all that this, uh, series has been kindly supported by Urban Mannheim, uh, and, uh, we are very grateful for their support, uh, in delivering the the webinar to to all of you, uh, on a free basis. So, how will the webinar series work? As you can see, and hopefully you've all logged into Facebook, we'll be running this live on Facebook through the Hematology Interest Group page. There will be case-based discussions. As you've probably already seen, there are links which are which will direct you to the Blood Academy website, and on that, you will find some digital slides where we will be discussing, um, the the cases around the digital slides as well. Like I've already mentioned, we've, uh, we've managed to get some expert speakers from all around the world. Uh, we'll be running these webinars every two weeks, and we'll have a total of six, um, six webinars. Uh, they will range from just from an introduction to blood microscopy today, all the way up to things such as specific lymphoid disorders and acute leukemias. This has, this whole webinar series has been accredited by the Royal College of Pathologists in the UK. It's really important that you register for your certificate before the start of the of each individual webinar, and provided that you've registered, you will be emailed that certificate over to you after the webinar finishes. So, if you haven't registered for your, uh, CME certificates, then make sure that you do. You've still got time to do it now, and you can find the form on the website where the of the individual cases are found as well.
If you've missed the webinar, um, if you're, if you're watching us now, then you just hopefully you're going to be watching the whole thing. But, uh, if you, if you have missed it, if you've missed part of it, or you just want to see the webinar again, the webinars will be available on the Blood Academy YouTube page. We'll also make this available on the Hematology Interest Group as well. So, um, hopefully, if you found the webinar useful, which I'm sure you will, then you can go back and revise all the the key learning points that we've covered, uh, at a at a later time, at your own convenience.
This is just a small plug. I'm the co-founder of Blood Academy. We're an e-learning website dedicated to biomedical scientists, um, and to hematologists and pathologists. We run online courses and various other online material as well, is available on our website. Um, the webinar series will give you an, uh, an overview of a whole range of different things. It would be very ambitious for us to cover everything in blood cell morphology, but if you do want a more detailed approach and work through video tutorials, online modules, and assessments, and get accredited CPD or CME points, then you can register for the Essential Blood Cell Morphology course on the Blood Academy website. And we're running a 20% discount for people who are participating on this webinar series by just inputting the HIG20 code to get your discount.
So, without further ado, um, I just, I'm delighted to have Dan Pelling. He is a senior biomedical scientist in hematology. He's the lead for educational development at Imperial College in London in the UK, and he's also a very active, uh, administrator on the HIG group as well. So, I'm absolutely delighted that we can get Dan, um, to speak to us today. We've got two digital slides to to to review. Um, we'll give things away in that, the the first case is a normal film, um, and the second case is a degraded sample, an aged sample. But really, it's, it's about, uh, talking about the the things that go around, especially for people, um, who are at the start of learning blood cell morphology. So, I will, um, I'll just allow you to share your screen now, Dan, if I stop sharing. There we go. Hopefully, you should be able to share your screen now, Dan.
Okay. Okay. So, I believe that I'm sharing my screen now. I hope that everyone can see it. It is a low-power view of a film. But before I go any further, I'd like to thank Dr. Mahdi there for the introduction. As he said, my name is Dan Pelling. I, um, lead for the blood sciences services at St. Mary's Hospital, which is part of Northwest London Pathology, which is co-hosted by Imperial College Healthcare NHS Trust in London. I have been working in hematology for about 30 years now, maybe a little bit over, and there are just a few things that I've learned along the way that will help me, I hope, deliver some of those learning points to you, uh, our wide audience that I hope that we have from across the globe. So, welcome to everyone. This is a great opportunity to share the resources between Blood Academy and the Hematology Interest Group, to share the knowledge base that we have and the teaching skills, and I'm certainly pretty excited to be delivering this first in this webinar series. Hopefully, technology won't let us down. There will be quite a bit of jumping around between screens in order for me to emphasize some of the points that I'm talking about, um, and I hope that you'll be able to follow me on this journey, which is all about the approach to the blood film.
We, we often see on many Facebook groups, many other websites, in learning documents from all manner of sources, interesting cases being put forward, cases for acute leukemias, lymphoid disorders, red cell disorders as well. And it is very easy to get caught up in the abnormalities of these slides. In fact, that's what we see mostly in our day-to-day work. But for those people, um, and certainly our participants on here who are just starting out on their morphology journey, being able to understand where some of these decisions come from, that such and such a cell is abnormal, can be a little bit tricky because at the start of that journey, people aren't always aware of what the range of normal actually is. And understanding that range of normal is key to being able to distinguish, well, then that feature that I'm looking at probably is abnormal. So, we're going to focus on that today. And we're not going to jump straight into looking at cells. We, we will be looking at different cells when we magnify the images later. But one thing is very important to remember, and that is normal can only be identified from a blood film, a two-dimensional blood film, if you understand to start with that you've got a good quality smear and good quality staining.
So, I'd like to spend just a couple of minutes at the beginning going through how you can identify good quality smear and good quality staining. And if we take just this first case now, I'm going to expand this image so it fills my entire screen. I hope it fills your entire screen too. We can see a blood film. We can certainly see the tail part of a blood film. Now, up in the top right-hand corner, you may be able to see a navigation pane that shows me in the little red box where I'm looking at on the blood film. I'm looking at the tail end, and we can see that a lot of cells seem to be forming lines and stripes, and there are a significant number of gaps between the lines and stripes of the cells. And as we move back down the film, we can see the cells get more numerous. Well, what that tells us is that we have a nice feathered edge to our blood smear. The feathered edge allows us to identify, we're going to be putting in a lot of terminology here, the monolayer. And the monolayer of a blood film is the region in a blood film that we want to use to assess cell morphology. This will become more apparent as I magnify the images. So, I'm now zooming in on the image. Once we've identified the monolayer part of the blood film, that is the part of the blood film where the red cells are just touching, maybe slightly overlapping in some areas, or maybe just some small gaps between them. That's the monolayer. Then we can begin to look at the staining quality and determine whether or not we've got a good quality stain, and that will allow us to be confident in identifying any abnormal features that we might come across.
The blood film has this feathered edge, and is often very useful to look at the blood film. And if you can identify the feathered edge by eye, you might notice occasionally that it has a slight prismatic effect to it, almost as if a small rainbow effect can be seen in the monolayer. That's a sign of a well-made smear. Additionally, you always want to be able to look at the edges of a blood smear. So, the blood smear itself should be slightly narrower than the slide on which it's made. And that's because larger cells can often preferentially distribute to the edges of the film or to the tail of the film. And of course, you don't want the smear to be too long because you want to be able to look right towards the feathered edge. Equally, you don't want the smear to be too short because if the blood smear is too short, then it will go from two thicker parts of the film to to thinner parts of the film too quickly, and you won't have sufficient space to look at enough cells.
So, if I scroll up, you can see that gradually the space between the cells is getting larger and larger, and noticeably, you can see that the area of central power within the individual red cells seems to be getting less frequent, almost as if the red cells are being squashed. Well, when you're looking at this part of the film, then you're generally looking in a part of the film that is too thin because you can't look at the red cell morphology without this sort of splayed out distortion. If I were to zoom out and go to the bottom part of the film that we're looking at and zoom in again, then we can see that there is quite a significant amount of overlap between the red cells here, and it's becoming a little bit tricky to look at individual cell morphology. Well, that tells us that we're in two thicker parts of the blood film. So, it's finding that Goldilocks zone, that zone in the middle where the cells are in this monolayer and just touching or ever so slightly overlapping. And here is a pretty much an ideal space. And what we notice straight away is that the areas of central power can be seen clearly in nearly all of the red cells.
So, looking at this part of the film, I begin to think, right, can I say that this is a good quality stain? What do I want for a good quality stain? Well, I want my red cells to have that traditional maybe salmon pink, slight orange color. Occasionally, as we've got here, there can be a slight beige tint to the red cells. That's absolutely fine. And when we see that small range of colors, we know that the stain is good quality, certainly for the red cells. And that's because of the pH. pH of the majority of peripheral blood films staining is about 6.8, give or take about 0.02 maximum either side of that. If you find your red cells are too vibrant orange, almost red, then it usually indicates that your stain is too acidic. If your red cells have got too much of a blue-gray, almost slightly green tinge to them, then that usually indicates that your stain is too alkaline, usually around about the pH 7.1, 7.2 and above. So, you're looking for those ideal cell and pink, maybe slight beige tinge to the red cells to know that you've got the right pH.
You can then look at some individual components of some of the cells to make sure that the cytoplasm of some of the small mononuclear cells, such as the lymphocytes, are staining a nice blue color, and that the chromatin within the nucleus is a nice purple color, purple, slightly mauve color. It does change depending upon what type of cell you're looking at, but it was usually within that mauve, purple range. You may then want to try and find some of the granulocytes and have a look at their staining. One particularly sensitive indicator of stain quality are the neutrophil granules, and you want to be able to see the neutrophil granules. They, they stain in a time-dependent way and take up both components of the general Romanowsky stains, the eosin components and the basic components. So, if your neutrophils are showing a nice degree of granulation, then that is something else that tells you you've got a good quality stain. And your platelets in your normal patient are showing granularity as well. And we can see our platelets here, dotted around in between the red cells, quite nicely. Nice size, nice degree of staining. Equally, we can see here an eosinophil, and we can see that its granules, or sometimes they're referred to as globular granules in the cytoplasm, in a slightly more vibrant orange color. That's because they take up the eosin component of the stain. And again, seeing that is a good indicator of stain quality overall as well.
Looking between the cells at some of the the bare blank white patches, I can see there's no background staining, no blue tinge, and I can see that there's no stain deposit as well. So, that tells me I've got a good quality stain. And I can't see any shiny bright refractile bodies or curves within the red cells either, which tells me that my stain is good quality and that there's not too much methanol or too much water in the methanol. We find it quite useful sometimes to have a tick list of things to look for daily in slides, so almost as if you QC your slides and your stain every day, and you look for all these components, their presence or their absence, and via that combination, determine whether or not you've got a good quality stain, because stain can vary between batches and also within batch as well.
So, once I've determined that I can find the monolayer on my slide and that the stain is good quality, I can begin to work through the slide in a standard fashion. And really, this is what this first webinar is about: the basic approach to any blood film. Now, it really doesn't matter how experienced you are, whether you're just starting out on your morphology journey, or whether you've got 30 or more years experience behind you, having a standard approach to reviewing a blood film and assessing the morphology is key to making sure that you don't by accident overlook a critical feature. Sometimes we can get some really exciting, very beautiful, very florid films passed on to us to review, and it's very easy to get caught up in in the interesting features or the abnormal features on the blood film, and then forget to look at some of the less interesting stuff, which actually might be equally as revealing to the end user. And at the end of the day, after all, we're looking at blood films because we're making comments that add value to the rest of the results coming out of the laboratory. So, having this standard approach makes sure that you look at all lineages and make the relevant comments.
And when we break it down, we really can see that there are four main components to consider in this approach, and that would be platelets, white cells, red cells, and then a bit of a catch-all, a sort of other, another bucket. Now, I can pass on the experience that I've had over the years and things that I've learned and been taught, but of course, you will have local policies and procedures that have been developed between the laboratories and the clinicians to make sure that some sort of standard approach and blood film reporting format is followed. So, I would like to caveat everything that's being said with, you must always follow your local procedures. But maybe today's webinar can give people a little bit of extra food for thought to incorporate into procedures, or maybe to review procedures, or it may be that it provides information to help people validate and give themselves their own assurance that their procedures really are working as well as expected.
So, when I approach the film, I make sure that I've got the full set of full blood count results in front of me, not just the full blood counts that pertain to the blood film, but also the patient's history as well, and as much of the patient's clinical details, because after all, you need to know precisely why you're looking at a film. The vast majority of times, it will be because there's been an unexpected or unexplained result coming from one of the analyzers in the laboratory. It may be an unexpected or unexplained low platelet count, or maybe a flag off an analyzer, such as a blast flag or a variant lymph flag or a platelet clump flag. So, you need all that information around you to make sure that you're looking at the film with the right sort of mindset and the right sort of approach. And once I have that information, including the patient's demographics, then I decide what's going to be the easiest lineage to report on and then put to one side so I can move on to the next lineage. And for this particular case, what we have is a 34-year-old patient with relatively normal results. Doesn't need to be any further explanation than that.
And scanning, just to start with, at low power, because it's always important to scan at low power to get a feel for the slide. I can't see anything that stands out as particularly abnormal. I can't see any large clumps of white cells or large clumps of platelets. I can't see any particular shape or size of red cell that stands out. And that low-power scan is really important because sometimes, paradoxically, it's easier to see any abnormalities at a lower scan than it is at a higher scan. So, so nothing stands out there. I've got a few white cells dotted around. We can see them here. That looks quite normal. And if I zoom in in my monolayer region, then I can see I've got quite a large number of platelets dotted around between the red cells, and generally they look about the right size and the right color. They look like they're well granulated. And there's one thing. So, there's me sitting here saying, well, they look about the right size and they look about the right color. And there may be people out there listening now saying, well, how do you know? Well, there are some rough rules of thumb that can be followed when you're looking at a film.
One thing to remember is that a normal red cell is approximately six to eight microns in diameter, and the area of central power for a normal healthy red cell is about a third to a half the overall diameter of a red cell. And that's generally what we can see here. That looks about right. But people may still be asking, well, okay, how can you tell that's about six to eight microns across? Well, there is another rough rule of thumb, and this is something really that can only be learned and practiced by looking at many, many normal sort of healthy volunteer cells, and that is a normal red cell is roughly the same size as the nucleus of a normal small lymphocyte. It's really important to take note of that sentence because sometimes people say, okay, so a normal red cell is roughly the same size as a normal small lymphocyte. No, that's wrong. A normal red cell is roughly the same size as the nucleus of a normal small lymphocyte. Having said that, sometimes it can be a little bit tricky to find the normal small lymphocytes, but hopefully, if you have a normal patient, there will be some around that you'll be able to to find and look at and make a comparison against. That's a slightly large lymphocyte there. So, again, it's all down to practice, practice, practice, and eventually you'll be able to get your eye in and be able to say, well, the MCV, mean cell volume, on this patient looks looks normal, looks within the normal reference range, or the cells do look a little bit on the small side, or the cells, the red cells do, look a little bit on the large side. And I mentioned about the platelets, but they look about normal in size as well.
But what does that mean? Well, platelets are usually about a quarter, maybe about a fifth, a quarter to this compared with the diameter of a normal red cell. Even in a completely normal healthy person, you will find the occasional slightly larger platelets. But platelets can be classified on their size as again being normocytic, although they're not really cells, but they are normal size platelets. That are larger than that are usually known as large platelets. And there we have a good example of a large platelet. They don't have a nucleus. They don't have a nuclear membrane. They are not well defined, and that is how we can identify platelets. They're fragments of megakaryocyte cytoplasm. This one we can see compared with the size of the red cells around is is much larger than these normal sized platelets here, and we call that a large platelet. But what happens when a platelet gets to the same size or even bigger than a red cell? Well, that's when their name changes, and they're called giant platelets. And there is a distinct difference between those two terms, large platelets and giant platelets, and that's the criteria.
As we move around this slide as well, some of you may have noticed that not all the red cells are the same shape here. Hopefully, what's in the middle of my screen, I hope it's in the middle of the screen for you guys as well, then what we see is, uh, the majority of red cells are nice and round. They're disco sites. In fact, if you think about a red cell, it's it's donut-shaped, except the hole doesn't go all the way through in the middle. But here we have a slightly elongated red cell. It's sort of elliptical in nature. Well, okay, I don't see very many of those. I see a couple of other red cells that are slightly misshapen as well. But really, in this patient, the vast majority of red cells are normal, round with their area of central pallor. They're normocytic and normochromic. Normocytic being normal cell size, and normochromic meaning normal in color. Nothing particularly stands out as an abnormal shape, i.e., a a certain type of poikilocyte. That's another bit of terminology. Poikilocyte means abnormal shape. And nothing particularly stands out as particularly pale, so with a large area of central pallor, that would make the cells hypochromic. And as I said before, there will always be a small degree of variation even in normal healthy patients. As the film reviewer, you have to make a decision. It's a subjective decision, semi-subjective decision, on whether or not a particular feature or change in shape or change in color is sufficiently prominent for you to make a comment on.
So, if I were approaching this film, I would have thought, okay, platelets. Platelets are unremarkable. There are no notable features. Yes, we saw one large platelet. Um, one large platelet. A comment doesn't make the majority of platelets look completely fine. So, I can comment on those, say they're unremarkable, or there are no notable features, and then move on to the red cells. The red cells look unremarkable to me. Slight variation in shape, but nothing that stands out that to me would be of any clinical significance. And remember, as I said before, we are making film comments that add value to all the other results coming out from the laboratory, the full blood count or the CBC, complete blood counts, and maybe even the chemistry results as well.
So, then I might just want to move on to my white cells. So, my white cells here, we have a neutrophil. It's not particularly well granulated, I must say. But the neutrophil has, um, neutrophil is also known as a polymorphonuclear cell, and that's because its nucleus has lobes. And we can see the cytoplasm, and we can see the chromatin of the nucleus, that purple color in the middle there. Let's come out and find some more. We have another neutrophil here, roughly in the middle of the screen. So, the neutrophil is the cell that has, that falls into the granulocyte category. The granulocytes are the neutrophils, the eosinophils, and the basophils, and they are in that category because their cytoplasm has granules. Their nuclei have lobes, generally in normal healthy people, and two to five lobes is generally considered normal. The majority of neutrophils in a normal healthy person will generally have maybe three or four lobes connected by fairly thin strands of chromatin. If we look closely at the chromatin, we can see that it looks a little bit blotchy. Some parts of it look darker, some parts of it look lighter. Well, that's called having a heterogeneous appearance. And a heterogeneous or blotchy or condensed appearance is a sign of chromatin maturity, nuclear maturity, and that's what we want to see in our normal healthy person, nice number of healthy mature cells circulating around.
But what we always need to bear in mind as well is that we're looking at a blood film that's been spread in two dimensions. These cells are are in three dimensions. So, we often have to look at the cells, especially the cells that have got lobes to their nuclei, and putting our mind's eye, a sort of three-dimensional image, because sometimes we might have to imagine that a lobe could be turned towards us in three dimensions and not always clearly distinct. Here we have one of the other granulocytes, the eosinophil. Now, what it can often be seen down a microscope slightly better than it can be seen on a digital image, like any digital image, is that the granules in the eosinophil aren't always point granules like they are in the neutrophil. They often appear as actually small little globules that shine. Sometimes they're likened to small orange glass beads in the cytoplasm of the eosinophil, and they should be this nice refractile orange color. So, if you focus up and down, they have a shiny appearance to them.
Out of our five white cells, we've seen that three can be cast as granulocytes. Well, the other main two, the lymphocyte and the monocyte, are in an overall way classed as agranulocytes. They're the mononuclear cells, the agranulocytes. That classification is because generally they don't present with granules in the cytoplasm, such as this cell here. However, having said that, depending upon the quality of your stain, you may sometimes see a very faint hint of granulation in the monocytes, and you may occasionally see small discrete granules in the lymphocyte as well, in just the occasional cell dotted around here and there, that is completely normal. There are certain criteria, um, that will be talked about in some of the later webinar series that allow you to say whether the degree of granulation is normal or abnormal. But let's take this cell here now. I've magnified it as much as I can on my screen. We can see that this is quite a large cell overall compared with the red cells surrounding it. It seems to have a sort of slight dusky blue, it's slight, maybe gray-blue appearance to the cytoplasm, and the chromatin in the nucleus is slightly irregular, still got some blotchy bits in it, and we can see a faint hint of some small, pale vacuoles in the cytoplasm. Well, this is a monocyte, and it's pretty much a typical example of a monocyte. Monocytes tend to have slightly convoluted nuclei, and I would imagine in three dimensions, this would be slightly convoluted, um, in a sort of north-south orientation towards us, almost as if it's coming out the slide slightly. The monocytes are often described as having a gray-blue cytoplasm or a cytoplasm that looks like a ground glass appearance, and the occasional vacuole is completely normal for a monocyte as well, with its overall large size as well.
Now, this is quite a useful screen. I'm not going to magnify it fully because I think we can see the features that are slightly better at a slightly lower magnification. We have on the right-hand side of the screen a monocyte, overall large cell, a slightly gray-blue cytoplasm, a sort of convoluted, slightly irregular appearing nucleus, sometimes they're described as reniform, so having the shape of the kidney or horseshoe-shaped, and just the very odd occasional small vacuole. And on the left-hand side of the screen, we can see a lymphocyte, a slightly larger lymphocyte with its slightly more blue cytoplasm and its nice purple, condensed, mature chromatin in its nucleus. But if we look at those two cells and we flip backwards and forwards between them, we can see that the amount of space taken up within the cell by the nucleus is different. In our monocyte on the right-hand side, the nucleus takes up probably a half, maybe just more than the entire size of the cell. In our lymphocyte on the left-hand side, it probably takes up about two-thirds, maybe even three-quarters of the overall size of the cell. Now, the amount of space that the nucleus takes up within the cell is called the nucleocytoplasmic ratio. And we find in general that as cells mature, as they go from their immature stages through all their intermediate stages to their most mature stages, that the nucleocytoplasmic ratio decreases. And it can be a little bit difficult again when people are just starting out on their morphology career to understand and align what that means. Well, to put it in its basic terms, is as cells mature, the nucleus and the nuclei take up less space within the cell. There's more cytoplasm around. So, when we're looking at cells, we're looking at the degree of granulation if there are granules present.
I'm going to stop here. The color of the nucleus, how blotchy and condensed, and therefore mature, the chromatin is within the nucleus, the color of the cytoplasm, the shape of the nucleus, the overall size of the cell, and this new concept, this nuclear cytoplasmic ratio, to determine what type of cell we're looking at and where on its maturation pathway it actually sits, from immature right up to mature.
Now, just fortunately, I've just stopped on this screen here just to emphasize one point. Here we were talking about platelets before and how we can distinguish different terminology, large platelets from giant platelets. Remember I said when a platelet, here we have a platelet with its slightly raggedy edge but no clear sort of nuclear boundary because they don't have nuclei. When they get to the size of a red cell or greater, they are called giant platelets. That's what we have right in front of us there. Nice example of a giant platelet. So, so there we really, we really have a really good example of a normal film and how it could be approached, looking at the platelets first, and then maybe looking at the red cells, then looking at the white cells, and making a small relevant comment on each. I tend to leave the most complex lineage till last because that leaves me sufficient time to really sit and think about what I'm reporting on. But films don't always look this nice. Okay, this is a well-stained, well-spread smear. We're going to compare it with something that you shouldn't really be reporting on now.
So, I'm going to go to a slightly different film now. And while you do that, I just, um, I should have made this comment earlier. Um, if anyone's got any, uh, additional comments or questions, then, uh, please feel free to add it all in the the chat, and we can go through them at the end. Hopefully. Yeah, that'll be great. I mean, this, this is really, you know, very informal, um, I think really, especially for this first one, um, and it'll be nice to hear other people's thoughts and opinions and have input. Okay, so we're looking at another film here. This does not look the same as the film we were looking at before. What is the main thing that stands out? Look at the edges of the red cells now. Believe it or not, I am in what for this particular smear is the monolayer. So, it would be the normal part of the film that I'd be looking at. But the cells look all crinkly and crenated. There we go. Another term that's being thrown into the mix, almost like they've shrunk away, and, and they just don't look very nice and smooth. In fact, the majority of the cells look like that as well. Look at some of these platelets. It looks like some of the granules are a little bit bigger, and they're not entirely filling all the platelets. Don't look very nice at all. Look at some of these other, these are meant to be white cells. Well, they don't look like any of the white cells we've just been looking at. Um, they look almost a bit squashed out, as if they were fragile and got damaged while the film was being made. Hmm. Okay, let's have a look at some other features here. What's this now in the middle here? That looks like one of the other white cells we were looking at on the nice film. It looks like it could have been a neutrophil, but look at the boundaries, the edges of it. It's almost like it's burst open, and some of the granules aren't fitting the cytoplasm, and the cytoplasm has become more ragged at the edge.
Well, this is an example of a blood film made from an aged sample. So, we know that for the vast majority of labs, our CBCs, our FBCs, are stored generally at two to eight degrees. Now, I know some labs will have different policies, that's absolutely fine. But blood changes within the tube. Red cells can swell, white cells can mature and degrade, nucleated red cells can lose their nuclei, reticulocytes, the slightly less mature red cells, can mature within the tube as well. And that process is certainly ongoing, and it continues at quite a normal pace when full blood counts or those EDTA samples are stored at room temperature. Those degrading processes are slightly slowing down when samples are stored at between two to eight degrees, but they will still happen in time. And that's why it's really important to make sure that blood films aren't made from aged samples. And again, laboratories will have their own local rules and regulations. But for the vast majority, really, blood films begin to show these signs of degradation as little as about two to four hours after the sample has been drawn. And after about 24 hours, the changes, these red cell crenation changes and white cell degradation changes, become quite prominent. So much so that it would be unwise to make a morphological evaluation, because if there were any true signs of dysplasia, they would be masked by cell degradation. What we often find is that white cells are the most sensitive to these changes, and we see them, especially in lymphocytes and neutrophils, most quickly. There are various papers out there that will give different opinions, but experience has shown that neutrophils and lymphocytes do show nuclear changes quite quickly in stored samples.
When we're assessing from a blood film, if or not the change we're seeing is genuine, so down to some sort of pathology or due to storage, we have to consider the degree of change, and are we seeing it on every film and every slide that we look at? For example, and this is something that crops up probably most frequently, there are certain conditions that can lead to what's called red cell crenation, crenation being the development of these little spines and bumps around the edge of the red cell. Renal disease is one big pathology that we come across quite a lot that can lead to red cell crenation. Certain enzyme defects within the red cell can also lead to red cell crenation, but those are usually present in specific subpopulations of the red cells, and where they're not present, all the other red cells generally look quite nice and normal. But in this example, we can see this bumpy, lumpy crenation in almost every red cell, and that tells us that no, this isn't due to a pathology. And in conjunction with the degradation seen within the white cells, this is due to an aged sample, a film being made from an aged sample. If I were to see this, then I would not report the film. I would probably make some comment, "Film made unable to report due to aged sample. Please repeat on fresh sample," or some other appropriate comment, or certainly something that follows the local agreed policy and procedure. So, being able to identify the good staining or the inappropriate aged film is key to not falling into that pitfall to start with.
Now, we've only got a few minutes left, and this is the first of the webinars looking at the approach to the normal full blood film. But I just want people to maybe think, and we haven't got a polling slide here because this isn't being done by PowerPoint, but maybe you just want people to take 30 seconds or so, maybe if you're watching this in a group or even if you're by yourself, just to think, what do you think, in light of all the things that we've said over the past 50 minutes or so, is the biggest pitfall with erroneously reporting a blood film? Okay, so we'll just have a little bit of quiet and think about that for about 20 seconds or so. We can encourage people to put their their comments in the chat as well. That'd be really good if we can get that. Okay. Yeah, absolutely. I will only be able to see that chat once I go down from full screen to to normal screen, so I'll be sitting here waiting. It'd be really interesting to see what people think the biggest error made for reporting blood films. So, um, Ali, can you, can you actually see the chat? I can see the chat. Yeah. So, we, we've got some comments coming in. We do have a slight delay. There's a delay of 20 seconds from what we're broadcasting over to Facebook, but we're getting some, uh, some interesting comments coming now. So, uh, okay, so have people made some comments what they think are the the biggest, the biggest pitfalls? We haven't had any comments regarding that question, but it's still early days, um, considering the delay, but, um, we're getting some now. So, poor staining and film preparation, um, we're getting degradation of white cells and platelet clumps. That comes the wrong diagnosis, which, which is interesting and very important as well. So, okay, very interesting. So, so a range of things already discussed. And the wrong diagnosis, um, again, yes, the wrong diagnosis can be made from the film if, if people aren't, um, familiar with the range of normal and therefore what's abnormal. But the biggest error made with film reporting is not checking the name on the blood film against the name of the CBC or FBC results that you're looking at on your computer screen. So, we've got some very clever people who've managed to to state that as well. So, reporting on the wrong patient, transcription error, really important. Yeah. I, I, in my career, I've had the privilege to work with some particularly well-known St. Mary's hematologists, and I have even known them to make that mistake as well, and it's very easy to do. So, we can have as advanced knowledge as you like and been practicing, but that is before you even check your smear quality and your stain quality, check your name on your slide against the name of the patient who you're reporting out, especially if you work in a pediatric hospital where you might have twins. And I think one other thing to add to Dan, I've been caught out by this, is that in some places, names are, they can be repeated, and patients can share the same names as well. So, looking at that identifier, the, the specific lab identifier on that specimen is really important as well. So, I've been caught up in in some issues with this, but yeah, it's, it's such an important point to make.
Okay. All right. I'm going to come out of full screen and just go back to our normal patients just for a couple of minutes because we're going to round up. So, we have some time for questions, so, so I think the key learning points really are to have your approach, your standard approach to reporting any film. Now, I've given what my approach is and the approach that I've taught many people over the years, but anyone out there, as they get more experience, you will develop your own standard approach. The key thing is that you have a standard approach and that you stick to it, that you don't get distracted by the the wonderful range of of cells and precursors in a new, new chronic myeloid leukemia that comes through, but you forget to look at the red cells and miss the malaria that might be there. So, have your standard approach. Make it a habit that you always use it when you, uh, sit down with a blood film. Um, and, and don't be afraid to actually send a film back and, and get it restained if you don't think the colors are appropriate and that it's not well stained, because you don't want to miss Auer rods or Döhle bodies or Pappenheimer bodies or some of those other inclusions that I'm sure are going to be talked about in some of the the upcoming webinars in this partnership. So, it has been a whirlwind tour. I'd like to end there and thank you very much. And I think we can have some time for some questions if there are any, which I hope I'll be able to answer. I'm going to stop sharing my screen now. I think that's the process we mentioned. That's right. Yeah.
That's great, Dan. That's a really, really good start to the webinar series, um, covering the basics is so important before we go into the details of specific pathologies and, and the things around it as well. So, I, I've been busy scribbling away the main points that you've made. So, I'll just summarize in terms of, um, what, what you've said. The first thing, really, is making sure that your slide corresponds to the, the correct patient, really important, identifying that. Um, the second thing is the sample quality, making sure that the stain is adequate and that you've got a good enough sample for you to to make a, a proper, uh, interpretation and provide a, a, a report from the laboratory. Having a holistic approach, so checking that, making sure that we get the clinical details as well, so that's really important to correlate the morphological features with the clinical details of the patient, as well as the blood count and any other investigations that we may have to hand. Reporting in a checklist fashion and identifying consistently abnormal features in the blood film, so that we don't miss the really abnormal things, but making sure that we have a list of things that we need to go through as well. And I think that the point that you made was that the report that is sent out from the, from the lab should provide value, and I think that's really, really important that we have to provide that value, uh, for the, for whoever's looking after the patient, um, and, and adding to that patient care, really. So, I think that's an absolute fantastic start to the, uh, the webinar series.
We've got some questions come in. We've got one, one question. I'll go right to the top. Um, a comment about, can you base your interpretation about the number of platelets and neutrophils just on morphology, how good it is, saying whether a patient is thrombocytopenic or neutropenic, and the other way around as well?
Okay. Um, uh, um, from a personal, um, perspective, um, I can, but that's because of 30 years practice looking at these and seeing hundreds and hundreds of thousands of films go through across the range. But also, within my group of hospitals, we have a number of training slides that have platelets on certain patients set at certain levels, a patient with a platelet count of 20 or 50, somewhere between 50 and 100, somewhere between 100 and 150, and we look at those slides time and time again. So, we, we almost imprint it on our memories what the frequency of platelets on a on a film looks like when they're at those certain levels. There are some rough estimations that can be performed on patients that have a normal red count or normal hematocrit. If you're looking under times 100 oil, you, you can perform estimated platelet counts. It's, it's not that straightforward. There are a number of, um, well-known textbooks out there that help you through that sort of three or four.
step process, but what is important is to know that if you have an unexpected or unexplained low platelet count, you have to look at a film urgently because you have to determine is it genuine thrombocytopenia, and if it is, is it then associated with other features such as fragmentation, and are you looking at a potential sort of a thrombotic microangiopathy situation, or is it because you've got platelet clamps?
Now, if you have platelet clumps, you cannot perform a platelet estimate from the blood film. It doesn't matter who tells you you can, you can't, because you cannot take into account differences of platelet comp size. All you can do is remove the automated platelet count and maybe put a comment out there that says, "Platelets, platelets appear adequate or normal or still reduced or increased." What you can do, if platelets are large and they haven't been counted by the analyzer, then you might be able to actually perform a manual platelet estimate from the film, and it's fairly similar with white count as well. You get your eye in over a period of time.
Excellent. Um, we've got a lot of people, um, who may be at the start of their journey looking at blood films or blood smears, however you call it. Um, on the other side, for those people in positions of management, looking after a laboratory, uh, for a hematology lab, what kind of things would you do you have in place, or just generic things, just to ensure that your staff are competent at looking at blood films?
So, so we have, um, a number of, well, I say we, anyone really could have a number of processes in place. That always starts with an initial training program, and that in itself will always start with having new staff looking at normal blood films and seeing how they would report them, because it is very easy to over-report and sort of over-confuse when you're starting out in films because you want to comment on every little thing that you've seen to let your, your supervisor or trainer know that you're able to spot these things, which, which is good. But over-reporting can be as misleading as, as under-reporting a blood film. So, you looking at a lot of normal films to start with to get that bench air benchmark is, is a good way to start. And then to introduce some of the more regular things that are seen on a day-to-day basis, the, the iron deficiencies, the thalassemias, the B12 and folate deficiencies, the glandular fever patients or, um, infectious mono patients, and maybe some of the, the slightly more frequent pathologies such as the chronic lymphocytic leukemias, introducing some of those. But other features as well, uh, putting out slides where a patient is humanizing and seeing if, if staff in training know the importance of commenting on polychromasia, the, the difference in cell color as an indicator of sort of bone marrow response.
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So having that training program there is, is really important. But I am, as a, I mean, I run a number of services. It's not like it was in the old days. We don't always have the resources and time to train people anymore. So a lot of that training has got to be driven and directed by the individual. But there are lots of EQA programs out there or proficiency testing programs that give out known slides or known images that you can use, you can anonymize, and you can use to, to test and train staff with as well. So there's a lot of resources out there, um, to set people up, um, with a good sort of base, a known base against which they can map their knowledge.
Definitely. I think, um, HIG certainly adds to that, that training in that, in a very good way as well. So I think we've been going for just over an hour. I'm gonna close things now. I'm sure we'll be, uh, continuing the conversation on the HIG page. Um, I'm just gonna just finish off with the, the last slide that I have to share, and I just want to first of all thank you, Dan, for an insightful and an excellent overview of blood microscopy. I think this has given us a really good and important foundation for the discussion of all the cases that we'll see in the remainder of the webinar series.
Um, just some things, uh, to, to finish off with, to remind you all that if you've registered for your CME, your CME or CPD certificate, that will be emailed to you hopefully in the next five to seven days. Uh, there will be a post-webinar survey that will be available, uh, within that email with a certificate, as well as being available on the HIG Facebook page as well. And remember, if you want to see this webinar again, it will be available on the Facebook page as well as the Blood Academy YouTube page. And I'm sure a lot of people who have missed this will find this very useful. And hopefully, we'll see you again in two weeks' time where we'll have a discussion about specific red cell cases, uh, again in the same place. And we really look forward to seeing you. Please again, if you want to register for your CPD, CME certificate, um, look out for the, the link on the Facebook page. And, uh, again, thank you to Dan, thank you to everyone for being involved and watching, uh, in the, this webinar today. And, uh, hopefully, we'll see you again in a couple of weeks' time. Thank you very much.
Excellent. Thank you.