So, hi, my name is Sophia Saleria. I am a GI pathologist here at Vanderbilt University Medical Center. I really truly do love GI, and especially within GI, my favorite is the esophagus. So I am going to be talking to you about EOE from my perspective, from the person that gets the tissue and gets to analyze and contribute, I will say, to the diagnosis. So the objectives are overall to outline the different causes of having eosinophils within the esophagus, it's not just EOE, and then to characterize the criteria that I use as a pathologist to contribute to this diagnosis, and then discuss how we put the entire picture together with the clinical team and what we use to ultimately reach this diagnosis. So before I get into that, I just would like to highlight what pathologists do, who pathologists are, and then that is going to be through the lens of someone that is a GI pathologist and observing the esophagus. So pathologists are medical doctors that have specialized in laboratory medicine. For the most part, we can be broadly classified as those that look at liquids, such as CSF or blood, and those that kind of then specialize, subspecialize into more anatomic or surgical pathology and look at tissues. And then further to that, you could subspecialize into whatever subspecialty you would like that kind of mirrors internal medicine. So cardiac, GI, breast, I have chosen GI liver and pancreas. But once tissue is acquired, and that tissue could be through a resection and surgery, or of course for EOE, it's more often than not a biopsy, it undergoes multiple steps before it reaches the slide. So first and foremost is the preservation of that tissue, so it must be placed in a fixative, and it undergoes multiple steps thereafter, which includes being embedded in a wax paraffin block and then cut in a very, very thin section to be placed on a slide. And I specifically am using this example here because these are GI esophageal biopsies here. This is at the point of acquisition once it's reached the lab, and you can see that they're very tiny, tiny pieces that have to be placed into a small tissue paper that is compatible with our processing to be placed further into a cassette. And they're so small that if they aren't placed in some sort of a holder, if you will, they can kind of pass through those little holes in the cassette. After that, the tissue is fixed, embedded in paraffin, and then put onto a slide. I like to show this because it shows how a tissue that's very, very small to begin with can become even smaller once it's dehydrated through formalin fixation and then placed on the glass slide. But paraffin tissue, once placed on the slide, is actually clear. It does not have any color. So the bedrock, if you will, of all pathology staining for interpretation on slides is the hematoxylin and the eosin stain. The hematoxylin tends to outline the nuclear features. So it's a bluish, purplish stain, and it highlights the nuclei and the nuclear features and the nucleoli. The eosin is a very pink stain that tends to highlight the cytoplasmic components. And together, these form our basis of interpretation of the cells, how the cells act together with other cells. Are they tightly joined? Are they single cells? What structures they're forming and how those structures can be aberrant based on any kind of pathology that's going on. We're going to talk about the star of the show, which is the eosinophil. So it is an inflammatory cell. It's characterized by these very two distinct bilobed nuclei and these glittery granules that you can see. Those granules contain different enzymes and proteins that react to different inflammatory effects. The two main arenas that we really see them come into play are allergic reactions and parasitic infections. And this is kind of what they look like in tissue. So the background here is actually esophageal squamous mucosa. And then you can see these very characteristic bilobed, very kind of raspberry pink cells with bilobed blue nuclei interspersed between the squamous cells. This is just for orientation. This is a whole mount, full thickness section of the esophagus. That kind of shows, and I will walk you through how we use this interpretation on a biopsy sample. Of course, we don't get a full mount for a biopsy sample, but we start out up here. This point here is the luminal surface. This is the point of impact, if you will, where food impaction happens. So you have the mucosa, which is this layer up here, this is squamous mucosa. This is the first point of contact for all luminal contents. Moving down, you would have the subepithelial tissue. And this is usually loose connective tissue with some small lymphatics. Sometimes you can have some glands in there. And then you have a thin muscular layer underneath that known as the muscularis mucosa. That's followed by the layer called the submucosa that really harbors the medium-sized to larger lymphatics, blood vessels, and also some glands. And then underneath that are the thick muscle layer, which are responsible for the motility of the esophagus. But like I said, for the purpose of this discussion and for the diagnosis of EOE, we're going to focus on just the mucosa because different iterations of the mucosa is what we receive as pathologists to make this diagnosis. So what does that look like? It looks like this little bit here. So again, just to highlight, this here is the luminal surface right here. This is where the luminal contents would pass by. This is the squamous mucosa. They're oftentimes described as squame. I'm not fluent in Greek, but tight fish scale is, I guess, the latter iteration of this word. And they have a pretty tight junction with each other. Moving forward, you can see that the bottom portion of the epithelium is quite dark. And this is the proliferative compartment. This is where the most immature, the baby cells, hang out. And as they move forward and up towards the top, they get more mature. Their nucleus decreases in size and their cytoplasm increases, so they tend to become a lot more pinker. And they are also eventually, with time, sloughed off into the lumen. The lamina propria, again, is mostly some lymphatics and some glands, but it's just kind of loose connective tissue. It's meant to just be kind of spongy and loose. But when we look at a biopsy, the initial evaluation usually occurs at low power. And so when I mean low power, I mean looking at it really from a bird's eye perspective instead of going and honing in very, very high power. And that's to give you an idea of what's going on through all these layers that I've described. Is there epithelium or is it just ulcerated? Is there inflammation? If there is, is it just hanging out in the epithelium or is it percolating down into the lamina propria? What kind of inflammation is it? Is it just deacinophils or is it a bunch of other inflammatory cells? How bad is the inflammation? And if possible, can we see the perpetrator? Are there any fungi? Are there any drugs? Are there any pill fragments embedded or near this inflammatory response? That's kind of how you initially evaluate a pathology specimen. So you start out at low power and you move higher. So now let's talk about eosinophils. When it comes to the presence of eosinophils in the squamous mucosa or in the mucosa of the esophagus, what's normal? How many are too many? Well, some studies have said having a few is okay in the lamina propria, but that you should have none in the epithelium. And this concept is really not that clearly defined. Moreover, in the colon, you tend to have seasonal variations regarding the presence of eosinophils, whether the summer months or the late fall, but that does not exist for the esophagus. This chart here kind of highlights all the reasons, not all, it's not a comprehensive list, but many reasons why you can have eosinophils in your esophagus. And you can see here, they range from very, very common reasons like reflux, to some more rare drugs, to sometimes being a part of a more systemic process like inflammatory bowel disease. Thankfully, most of them tend to have very distinct clinical features and histologic features, but sometimes there is some overlap. Moving now to the definition of eosinophilic esophagitis, it's defined as a chronic immune-mediated esophageal disease that's characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophil predominant inflammation. And the three main things I like to highlight about this definition is that it's a long-standing condition that has both distinct clinical and histologic features that should alert both the clinician and the pathologist to the awareness of this diagnosis. Many people have already talked about the incidence and prevalence of this. It does have an increasing incidence and prevalence, and in fact, the incidence is higher than that of eosinophilic gastroenteritis and colitis, more in which for males than females, it can be seen in the pediatric population, but also in adults, and more enriched for those that have Northern European or Caucasian ancestry. Risk factors include modes and modality of birth, prematurity, and then exposure to antibiotics. And like most allergic diseases, it tends to have an association with other allergic diseases. So if you think about the atopic march theory, it parallels that allergic diseases tend to occur in a time-based order, and if you have some kind of allergic condition in the skin, over time it can, for example, progress to the esophagus. Further to that, I hope that you will forgive me just taking a drink of water because we are in peak summer allergy season here in Nashville, and so my throat is a little bit dry. So moving on, there is definitely a genetic component. Again, this has already been highlighted, that EOE is more common in twins. Further, it's more common in dizygotic twins than siblings born at different time points. But in addition to genetics, we also do have an environmental allergen component. Certain food allergens permeate through a permeable mucosa where they're presented to antigen-presenting cells, and this leads to the activation of a whole cascade that I'm sure has already been discussed, mediated by Th2 interleukins that lead to the attracting of eosinophils. What's important to know about the chronicity of this disease is that over time, this reaction leads to the activation of TGF-beta, which then leads to the fibrosis, the fibrotic component of the disease that is actually responsible for the fibrostenotic complications that we see in EOE. There's a whole host of diagnostic criteria that, again, include both clinical and pathology features, all these taken together, given that there is no other cause for the presence of eosinophils in the esophagus due to the diagnosis of eosinophilic esophagitis. And again, I'd like to point out that this is not a diagnosis made in isolation just based on clinical features or only some pathology features. It really is a clinical pathologic diagnosis, and I hope that as I progress through this talk that will become clear as to how important the collaboration with our clinical team is for us as pathologists. So how do we make the diagnosis? If I get a biopsy, what are the features that I use? Well, before I progress, I just would like to highlight again, just to go briefly through the layers of the mucosa that we're seeing here, we have the squamous mucosa here, which is this top layer that, again, has a somewhat darker appearing basal layer, if you see here, and that is where the immature cells are. Usually for the most part, this is just one to two cell layers thick. As the cells mature, they become more pinker because their cytoplasm becomes more enriched and their nuclei tends to decrease in size. The lamina propria is, again, kind of loose connective tissue, so a light pink in color. And then you sometimes have these imaginations of lamina propria going up into the epithelium, and these are known as papillae. So it's okay to have a few papillae here and there, and they're usually not the entire thickness of the mucosa, they tend to be present in the lower one third. And this orientation is important when I will go to talk about how we make this diagnosis. So again, just looking at this normal esophageal biopsy that really has no significant histopathologic change. Let's move on to how we would make this diagnosis. So it's eosinophilic esophagitis, right, which means we're going to have eosinophils. These eosinophils can be present in numerous iterations. So they can be present as small clusters, medium-sized clusters, individual cells. You can see here towards the top portion of this photomicrograph, they tend to be forming a small little cluster here of maybe five to six cells here. And then they can also be present as cells that have degranulated. And the stars, the dark stars in this photomicrograph highlight those areas where we see just the granules themselves percolating through the cells, but not really any intact eosinophils. And it's important to note here that when we as pathologists are reviewing these slides and we're looking for the foci of peak, the highest density of eosinophils, we do not count degranulated eosinophils in any shape or form. You certainly can mention them in your report, but they do not, there's no way to count whether this cluster of granules here represents one or four degranulated eosinophils. So they are, degranulated eosinophils are a feature, but they're certainly not used in the counting of the eosinophils. Moving on here, this is again a mucosal biopsy. And one thing I'd like to highlight here is at the beginning, a few slides ago, I presented possibly the best oriented mucosal biopsy that you could get, where you had a nice quotient of squamous mucosa with lamina probria. More often than not, that's not what we get. What I'm showing you in these examples are again, somewhat better real life examples, but this is really what we get. Sometimes just a superficial fragment of superficial epithelium. Sometimes you might get some papillae, but oftentimes the lamina probria is not present. Again, as I had shown you through the processing of this tissue, sometimes it's not just the acquisition, but it's how it's oriented by the lab that is actually embedding them. These are very, very small pieces of tissue. It's hard to get them to, it's hard for the person even embedding them to know which side is mucosal and which side is the basal portion of it. But moving on here, one thing I'd like to highlight to the trainees when we're looking at esophageal biopsy all together is when you put down the slide, an EOE slide is just really blue. Why is it blue? It's blue for the most part, because the inflammatory response that is elicited by the presence of a eosinophil driven inflammation within the squamous mucosa, it tends to cause that basal layer that I had highlighted before where the immature cells hang out to just proliferate quite rapidly. And because of that, you have a thickening. It's called basal hyperplasia. And normally that's three to four cell layers thick. And here you can see that it's about eight to nine. Sometimes it's even more. And so an EOE biopsy is just very, very blue looking. And that is mostly because of the basal cell hyperplasia. Eosinophils can be present not only in the surface layer of the esophagus, but in EOE, they tend to percolate throughout all layers of the mucosa. And so that phenomenon is known as layering. That helps differentiate it from some of the other causes of eosinophilia in the esophagus, because they don't tend to have this trans epithelial layering. In addition to that, when this disease gets severe, especially when there's areas of impaction or if there's some kind of erosion or ulceration happening, it's not uncommon to have clusters of the superficial portions of the squamous mucosa just kind of slough off. And when they slough off, they tend to also have eosinophils aggregated within them. One thing I'd like to point out here just based on some of the questions that I heard answered, one of them I think referred to the clinician saying it's just too inflamed. And that stands to reason here that any cause of esophageal ulceration, whether it's caused by a drug, whether it's caused by EOE, whether it's caused by really bad reflux, whether it's caused by just a pill impaction, any kind of trauma that elicits a very dire response like ulceration, even though, for example, in EOE, there would be eosinophils, they're going to attract other inflammatory cells there too. And so I assume that is what's being said there, that at this point, because that's what happens when that sample reaches the pathologist too. And the question is, well, is this EOE? And it's hard for us to make that diagnosis because it's just too inflamed. And what that means is, yes, there's a predominance of eosinophils, but this tissue is so inflamed and so necrotic that there's a whole bunch of other players too here in terms of inflammatory cells. And usually at that point, you might need to biopsy a little bit away from the actual point of trauma or let that heal and see what the subsequent biopsies show. So I just wanted to kind of point that out. Another thing to point out here, and this is actually not a feature that is very specific to EOE, but it is commonly seen in EOE. And that is the presence, again, this inflammatory response, whether it's eosinophil driven or driven by any other inflammatory components can cause a presence of edema or fluid between these really tight squamous cells. So remember, they were like fish scales. They formed this really protective layer, but once there's inflammation, you tend to see them spread apart. So those cell bonds tend to spread apart and you can see almost through most of these, there's a whitish outline around all of them. This is known as spongiosis. Again, not specific for EOE, but a finding of inflammation within the esophagus. And just this insert here is to highlight how tight these bonds are usually, where you just see the outline of pink cell membranes touching each other and not this clear fluid in between. Well, moving on now to a feature that's quite classic for EOE, and then we don't see it almost anything else, pathology of the esophagus. And that is the presence of really robust hundreds of eosinophils forming almost these small little pustules, if you will, within the mucosa. And these are actually known as microapses. And this feature is pretty cool because whenever I see this, I always tell the resident or the fellow, pull up the endoscopy report because it really nicely correlates with what the clinician, the endoscopist is seeing. And it's these punctate tan white studding across the esophageal mucosa. But I want to highlight here, especially since when we go back to the definition of EOE, that this is a chronic longstanding disease. And the hallmark of that damage that occurs when a disease is longstanding in the esophageal mucosa is not always apparent in the superficial surface, epithelium, but more in the lamina propria and subepithelial areas. And unfortunately, that's not something that we always get in the tissue that we're observing. But if you go back to my, think back to my earlier slide that I showed, the lamina propria is kind of this loose, spongy tissue that doesn't really have much inflammation. And these two biopsies, what I wanted to highlight is, although they're not the best oriented, but what you will see here is that the lamina propria looks a bit thicker. It's a darker pink. There's inflammatory cells. And so there's real damage. This is a sign of chronicity that this damage has been ongoing. And then there's fibrosis. And so that's one of the hallmarks that really is helpful when it's present in the biopsy. Well, based on this, you would think, my gosh, if you have all these criteria, that's all you need. This diagnosis is a piece of cake, right? It's not. And let's get into why. First and foremost, I know that this was already touched on before I came on to speak, but it's just numbers. So the peak eosinophil value is 15 eosinophils per high power field or 60 per millimeter squared. And this is to be assessed by the pathologist at the focus of the highest density of intact eosinophils within the esophageal biopsy. And this is certainly very advantageous. It's a marker of the ongoing activity. It's objective. It's quantifiable. If I look at it and if my other colleague looks at it, there's a lot of agreement among us because it's a count. And so people love this. And I understand the merits of this, but it shouldn't be the only criteria and it shouldn't be entirely what you hang your hat on. And it does have limitations. So let's discuss some of those limitations. First of all, overall, a decrease in the count of eosinophils in the mucosa has correlated with the improvement of symptoms and the presence of better endoscopic outcomes. But really, if you look at the studies, there's a lot of variability among the data when it comes to this stuff. Secondly, is a limitation that is very close to home for me. And that is that if you look at some of the older studies, I will say that this limitation has largely been overcome now with modern manufacturers of microscopes. But at some of the earlier studies, the field of view was not really clearly defined or it was different among different makes of manufacturers. We have corrected this now because a 40X field is largely referred to as a high power field. Moreover, the definition also now includes millimeter squared in its diagnosis. And again, I want to point out here, when it comes to this count, we do not count degranulated eosinophils. Another limitation is what's response. So 15 is the criteria. 15 or greater eosinophils for high power field is a criteria for establishing the diagnosis of POE. But what does response mean? We don't really have a number for that. Is it absolutely zero eosinophils? Is it just a slight decrease? Is it a decrease of the features that are EOE related, such as the basal cell hyperplasia, the fibrosis? There's really not much data on this right now from a pathology perspective. And then of course, there is the biggest limitation, which is tissue variability. So these two photomicrographs that I'm showing you, and I think you can appreciate one of them has all the things that I've talked about, the spongiosis, the clusters of eosinophils. It's very blue looking. It's got basal cell hyperplasia. And then the one next to it here is a little bit more pink. There's a few scattered eosinophils here or there, but overall, I would say that the basal cells are still about two to three cell layers thick. Very different looking, right? Well, they're from the same patient, from the same sampling in the same area. And that's because this disease is very heterogeneous. And so increased sampling leads to increased sensitivity in diagnosis. And moving on, I know I discussed this already, but I have to point out that sometimes the samples that we're getting are just not a complete measure of the disease process. So again, you see here a biopsy, a superficial epithelial biopsy of EOE. Here is a unaffected sample of squamous mucosa just to contrast that. But the true activity sometimes, especially in longstanding patients, is going on in the area that is underneath the epithelium. So it's this thickened, enlarged, fibrotic lamina propria. And that's usually what you see in patients that have fibrostenotic clinical presentations. And I have to add in one other bane of our existence as pathologists when it comes to distinguishing is this EOE or is it something else? And that is the top of this list, which is reflux. So how do we tell the difference? Well, as you can see here through this chart, there are a lot of commonalities when it comes to some of the patient-centric features, such as the age and the gender of the patient. But then there's a lot of overlap otherwise. Moreover, some of the symptoms that have been reported in patients classically that have reflux can also be present in patients with EOE. And then some of these symptoms also don't always apply to children that tend to present very differently. Reflux is the most common cause of esophagitis in the United States. Well, I'm still gonna be a bit more academic about this but there are certainly some features that you can look at. And I think just by looking at this slide here, you can say, well, she's already talked about three or four of them and those are the ones that are present in EOE and that's the truth. There are a lot of, there's a lot of overlap and that's because the esophageal mucosa is not a very creative part of the body. It only has a few ways that it can react to different insults. And so there's a lot of overlap. I will highlight here that eosinophils along with other inflammatory cells also tend to be recruited when it's an injury based on reflux. Whereas EOE tends to be in the mild to moderate stages mostly just eosinophil driven. Well, let's talk about some of these features here. So this is a, again, a more robust biopsy of reflux esophagitis. And what I'd like to show here just based on what I've been talking about is that it is not a very blue biopsy. So it's tend to still maintain that hematoxylin-eosin balance. There's some, definitely the presence of some eosinophils up top here, you can see. But the other point I want to make out here that in mild to moderate reflux, the components of the actual proportion of these layers. So the epithelium, the loose spongy lamina propria and then the muscular mucosa here are normal proportions. There's no atypical fibrosis or thickening at least in this sample here. Moving on, again, a low power view of reflux esophagitis. One of the things to highlight here is that, like I mentioned before, the eosinophils at low power and at this power, they're just going to look to you like kind of magenta colored dots that you're seeing. They're definitely present there. I think at low power, you can appreciate the spongiosis that's occurring, but they're not as robust and the biopsy is not as blue. The basal hyperplasia is mildly expanded, but not really a prominent feature of the biopsy. As we move into higher power, one thing I have to point out here is that I had pointed out early that you can have the lamina propria kind of pooching into the squamous epithelium. Usually it's just towards the lower third of the squamous mucosa, but in times of chronic damage that occurs due to reflux, these papillae tend to hypertrophy. And so you can see here, there's this really elongation of the lamina propria papillae, and they are projecting almost through the full thickness of the biopsy. Another point here is that these cleared out cells, so similar to getting fluid in between the actual epithelial cells, you can also get accumulation of plasma proteins and fluid in between the actual squamous cells themselves. It tends to impart this very pale look to the cytoplasm, so they almost look like they're clear with a thick pink boundary. And this is known as balloon cells. It's a feature that is slightly more common in reflux than in other causes of esophageal injury. Here again, just trying to highlight the presence of the eosinophils. They tend to be scattered and not really clustering as much. But I think one point to highlight here very easily is that if I went to a higher field of view, there is easily greater than 15 or at least 15 eosinophils per high power field here. All in all, what I'd like to point out here is that, and I hope I've highlighted that in showing you these reflux biopsies of the esophagus, is that if you rely solely on the eosinophil count when you're observing the features of an esophageal biopsy, it will really lead to either over or under diagnosis. So what are some of the things that can help us? Well, one thing that I pointed out was the features, especially when it comes to reflux as GI pathologists, the presence of eosinophils is not required to make the diagnosis. Prominent eosinophils are only present in about 30% of patients with reflux. It's also important to note that the severity and extent of the histologic features don't really parallel what the clinician is seeing or what the patient is experiencing in terms of their symptoms. So that's why a GI pathologist don't rely entirely on the presence of eosinophils when we make this diagnosis. If all the other histologic criteria are fit, and this biopsy is a classic example because it's very pink, you're not really seeing those magenta dots of eosinophils present throughout the biopsy, but what you are seeing is these really elongated papillae, you're seeing some spongiosis. And so if all the other histologic criteria are in keeping with reflux, and if the clinical presentation also parallels the findings that we're seeing on a biopsy, then the lack of eosinophils should not deter one from making a diagnosis. I'd also like to add in here, for those of you that are just interested, that you have some glandular mucosa that is showing Barrett's mucosa here, so the presence of intestinal metaplasia, which also should help you think, this probably, what's going on here is chronic reflux-associated changes. This chart here is simply to show you that, and if you look at this, you'll say, well, a lot of the features are the same. And in fact, many of them are the same. The only weird, real distinction I would say is that in EOE, the features, the histologic or the microscopic features tend to be a lot more robust. So you're going to have a lot more inflammation. You're going to have more basal cell hyperplasia. You're going to have more diffuse involvement of the inflammatory cells throughout the mucosa. You're going to have microabscesses, right? The eosinophils are playing all kinds of inflammatory games within that squamous mucosa that is not always the case in mild to moderate reflux. And I use the caveat of mild to moderate because severe reflux can look like anything. But the two are not always a clear distinction. And also because reflux is so common, it's inevitable that these two will not always be mutually exclusive in the same patient. Well, now let's move out of the realm of what I use at my disposal at the scope and when I need to go to call my clinician or look at the electronic medical record to help me distinguish between the two. First and foremost is review of the history. It's really, really important to find out what is going on with the patient holistically. Do they have any other illnesses? Do they have any exposure to weird parasites? Are there any kind of any medication that could be eliciting this response? And most importantly, have they been treated for something before coming to our institution? Or is there something in the history that I need to check that they've been treated for EOE even? When it comes to a review of the histology, it is so, so important for you to get the prior pathology if it's present. And I think these two biopsies highlight exactly why that is. The changes that are seen in the earlier part of the year tend to be mild EOE. The eosinophil count isn't even all that robust, but as we progress forward, you can see that all those features that I had highlighted that can lead to, that are the physical manifestation in the biopsy of those fibrostenotic luminal narrowing changes, which is the presence of this really thick lamina propria, which some could argue, I just didn't have lamina propria in my January biopsy. But again, this gives me a progression of what is going on. So to compare two different time points, not only through history, but also through the prior biopsies is very important to see how this disease is progressing. Location, figuring out where the biopsy was again acquired from. For the most part conventionally, the distal portion of the esophagus tends to be enriched for more reflux associated changes. And as we move more proximally, you can see still an increase in eosinophils for an EOE patient. And then again, nicely highlighted here are the endoscopy findings that I always love to correlate. And also if I see this on the requisition that there's a felonization or a furrowing or rings of the esophagus, it is always really fun to kind of have that differential in my mind before I even look at the slide. When present, seeing the, or comparing with the endoscopic ultrasound is always really great. It tends to show an expansion of all the layers of the, especially the layers of the esophagus rather than just the superficial portion. And so when I can correlate with that, it's always helpful. But I think it's important for me to end my talk really in highlighting that distinguishing between EOE, reflux, or EOE or any other cause of eosinophils in the esophagus, I use reflux because it's the most common differential, is really sometimes impossible based on just one single time point and one single biopsy. And in the absence of clinical features and endoscopic features to correlate, sometimes the only thing that can help in making that diagnosis is just the tincture of time and seeing how these patients progress on their treatment and other subsequent samples to see if the entity or the disease or the etiology actually declares itself. And so I'll end with some diagnostic tips and pearls. I think I've highlighted here that the peak eosinophil value of 15 is a diagnostic feature, but it shouldn't be the only thing defining a pathologist diagnosis for making that disease, that you need a combination of all the features that are present and also those that are absent to really drive what helps you make this diagnosis, that it's really, really critical to have past and present history, especially when it refers to treatment modalities that have been instituted in that patient to help make this diagnosis, and that the disease can be very, very patchy. And you can see here that the sensitivity of the biopsy and hence the diagnosis increases by the increased numbers of biopsies across the esophagus. And again, to correlate with prior samples and prior histology is always useful. So I am happy to take any questions. Thank you for your time. Great. Thank you, Dr. Salaria. Okay, if you have questions, please raise your hand. I'll recognize you, and I think if you speak into the microphone, Dr. Salaria can hear your question. Okay, Dr. Patel? Hello. My question is, basically, mostly I hear this from the field. Can you hear me properly? Sorry. It's a little low, but I can hear. So you mentioned earlier in the presentation about the stages of how the biopsies are received and then they're reached to you. It's about the fixating jar. I hear this in a community a lot more, that if the gastroenterologist sends too many samples in one fixating jar, the community pathologist thinks the quality gets compromised. And so what is your take on if there is a number that can be sent, the biopsy samples in one jar, or it should be divided, that impacts on the quality of leading to the proper diagnosis since it's a patchy disease? Yeah, I mean, as a pathologist, I don't set the standard of what goes into a jar, but I feel like, and maybe some of the endoscopists can give their view here, the sampling has to be really from across both the proximal and the distal. And so usually for the most part, I think the tissue that I showed you that was in that small little sample, that is from one jar. So that one jar would be mid or proximal. And I think it was not more than four or five pieces. That's what goes in, that's what it's transported in. And that's kind of our criteria. We don't like to have too many of them. And especially if the preservative fluid in the jars is being overwhelmed by the number of samples, I think that's a big issue. I don't know if, and this is kind of counterintuitive, if the outside clinician is putting all their samples from all across the mid, the distal into one jar, because then it's impossible for the pathologist to figure out where they're coming from. Okay. Debra? You mentioned looking in high power field. I have two questions. One is related to EOE, one is related to your practice. How do you determine what random area to choose for high power field assessment of eosinophil counts when you're looking from low power field? And the other question is related to other inflammatory cells that you mentioned. It's not often just pure eosinophils. How often do you see neutrophilic or lymphocytic inflammation in the presence of eosinophilic inflammation? And what are the determinants or what are the common features, maybe clinical features associated with this mixed inflammation type? Is it the disease duration or other comorbid atopic diseases or comorbid IBD? What are the factors that might be explaining to you that yes, it makes sense that I see more neutrophils here or more lymphocytic inflammation here along with the eosinophils? Right. So to answer your first question first, when I said that we start looking at low power, that was just kind of like the routine of when you put down a slide. So you will always ultimately go down to high power, especially in a patient with EOE. And so the selection of the field is never random. It is actually your bird's eye view gives you an idea of, and again, it's because these cells are so pink and glittery that they will let you know where to focus on. But the low power field is just a perusing field. Going down to actually make any of these counts is never a random selection. It is based on your low power field, narrowing down to where the peak eosinophil value is. And sometimes if things are kind of close, I will count one field. And if it's 60 and the other is around 70, then I have to do that to go to the high power field that is the most representative. In terms of your second portion of your question of the presence of other inflammatory cells, that's why I use the term mild to moderate. For the most part, EOE is an eosinophil predominant disease. Now that doesn't mean that if there's surface erosion, so if the severity of the disease, or if the sample is taken right next to where there was some food impaction that kind of eroded the surface layer of the squamous epithelium, that's going to recruit some eosinophils. If that's there, if I'm looking at the clinical history and I'm like, okay, they sampled this close to the impaction, or this patient had luminal narrowing for a long time, so there was probably sloughing going on for a very long time. And that led to the erosion. If I cannot explain away the other factors, and if it's still within the realm of mild to moderate disease, then I know that based on the histology and based on the clinical pathology that this is EOE. But when you bring up other cases, like for example, IBD, IBD patients can definitely have some smattering of eosinophils throughout the esophagus. But if I'm not seeing all the other histologic features that I pointed out to you, having a few eosinophils, and usually there are, they are almost always less than 15, does not make me jump towards EOE. Moreover, some with IBD can also have reflux. So if that biopsy is more distal and the proximal biopsy isn't showing anything, then I have to take the presence of these other inflammatory cells with the baseline disease that this patient might or might not have, and the severity of the disease that might lead to the recruitment of other inflammatory cells, even if the patient has an underlying EOE. Because you can certainly have neutrophils in a patient that has an ulceration because of EOE, because ulcers bring neutrophils with them. Does that answer your question? Yes, thank you very much. Okay, one other question in the back here. Okay, hi. My name is Jeanette. I'm an MSL in the West. I'm curious, I hear from my community-based folks that there seems to be some variability in how pathologists will look at a slide. Sometimes they'll just say, oh yeah, you have EOE, and they won't count the number of eosinophils. Is there a standardized approach among pathologists, like guidelines or something for EOE? And then another question that I have is, why don't you count degranulated EOs? Right. I'm going to answer your second part of your question first. We don't count degranulated because there's no way to account for how many eosinophils those granules are coming from, right? So there's, I can mention them, that there's 30 eosinophils per high-powered field, plus there's a robust presence of degranulated eosinophils. But I can't count something that I can't account for the number of eosinophils those granules are actually coming from. So that's why we don't count for eosinophils. As far as the standardization of practice, I practice in an academic setting, but I, we have our huge referral center. And so we do see a lot of slides from the outside community. I have rarely seen a pathologist diagnose just a top line of eosinophilic esophagitis. The count is actually a standard of care. So when you are going to make a top line diagnosis of eosinophilic esophagitis, I would argue that it kind of has to come with what you are basing that with. And for the most part, people do rely on the presence of eosinophils per high-powered field. I know someone had mentioned here that, you know, now people are not going to go, and even myself, I'm not going to count like 220 eosinophils, right? Usually it's over a hundred. We will say with over a hundred per high-powered field. But I would argue that it is standard of care to have the number there, especially if that, if you're, even if you're in the community practice and you're like, wow, this is a lot. If you're going to top line your diagnosis as having, you know, this patient having eosinophilic esophagitis pattern, then you have to have the number per high-power field accompanying that. If you are not going to top line that, but the eosinophils are robust, usually it's, it should be there in a comment, but I would, I would argue that I think it needs to be there. It is, it is standard of care. Dr. Solari, is there a standardized quality reporting system nationally for pathologists to, to have those criteria in any, whether it's EO counts or any other findings that go with EOE? Is there a standardized report structure that is recommended as best practice among pathologists? You know, there's no standardized report structure, but I would say, you know, and this goes back to the question prior to yours, that having that eosinophil count is standard. If you're going to enter that, I mean, we also sometimes, you know, because it's, again, it's, it's kind of a, a diagnosis that is between us and our colleagues. And if I have, if this is a, of someone that's new to our practice here and they're being spoke for the first time and I, and the history is not quite in there in terms of our, our EPIC system, then I will say, you know, you have to have a EPIC system. Then I will say, you know, there's 30 eosinophils here, there's 45 in the, in the mid. In a comment, I'll put these findings have been associated with eosinophilic esophagitis, but I won't top line it because I am at a deficit in that I don't know the history. And maybe this patient just has really bad reflux and my clinical colleague needs to, needs to kind of interpret that based on the history that they know. But in terms of standardized reporting I you're, you're correct. There is no standardized reporting of EOE other than the mentioning of the eosinophils per high power field. Some practices we hear will put basal cell hyperplasia, sometimes degranulated. We definitely mentioned the micro abscesses when they're present. But I don't think that that is common practice across different, different practice models. Question here in the front. Hopefully you can hear me. I was just curious when you do receive samples, if, if you're receiving it from a provider who has not specifically ordered, indicated that, that they're looking for a diagnosis of EOE or asked for anything eosinophilic or ES related, is that something that you're naturally inclined to look for and, and comment on? In your report naturally or is that going to limit how you report what you find in the pathology? So if, if it's not coming on the requisition to look for this, you know, honestly, if the findings are not of EOE, I don't, on routine biopsies for dysphagia, for difficulty swallowing, for whatever, I don't comment on the presence of eosinophils because the features aren't there. As a pathologist, what drives me to comment on them is the presence or absence of these features that I've outlined here. So if I'm seeing clustering, if I'm seeing degranulation, if I'm seeing abscesses, there's no way that I, I mean, I have to mention them, right? And so even if I am at a, at a, you know, at a point where I either don't have or have limited information clinically from the rec of what the, what the endoscopist wants me to look at, that's going in my diagnosis. There's, there's, that's what the pathology is. That's what I have to bring up. But if I have no information and there's limited, you know, information on the requisition and I'm not seeing anything, I'm not counting eosinophils or, I mean, I would just, I would just mention the features that I see. Okay. And the other thing I was just curious about is it, it sounds like when you do see eosinophils present that you're inclined to want to kind of do either, either, or, you know, it's, it's reflux or, or it's EOE. No, no, I mean, I, to coexist, they can definitely co-exist. And so, you know, this is my personal practice and it's, it's kind of followed me from, from where I trained my top line. If it's not slam dunk or there's no history of EOE, but if it's, I mean, if I'm in this nebulous space, if I don't have enough history, it looks like it could be EOE, but it's not really progressing proximately. And I don't have a history, maybe they were treated partially or whatever is eosinophilic esophagitis pattern. And in parentheses, I will put the count of eosinophils per high power field. And I do that for each and every sample taken from that patient. And then I really do in that way, I'm kind of presenting the data. And when I use pattern, because my colleagues here are very used to the basal cell hyperplasia, the spongiosis, it just adds a lot of words to my top line that I don't, in my, in my particular practice here, I don't need to do my GI colleagues know what I'm talking about. And I provided them with the pqs and info value for each of the points for each of the biopsies that were provided to me. And they kind of to date know what to do with it or how to handle it. Okay. Awesome. And then your points about history, like atopic, other atopic disease and stuff would play in as well. I'm sure. Sorry, I cannot hear you. I think, I think she was just making a final comment that clinical correlation and other clinical features certainly factor. Yes. Awesome. Thank you so much. Question here. Hi, you mentioned that eosinophil count in the colon can vary seasonally. Did I catch that correctly? Yes. Yes. That there's different, different reasons for it. But usually from in the colon, you can see, I think there's, and again, this is not like a robust, like eosinophil colitis, but there's what my point was that there's more studies on the variability, slight, even if it is of the prominence of eosinophils in the lamina propria of the colon, usually around January or August time, it might have to do with allergies or other things that are seasonally affecting the patient population, but there's no such thing in the esophagus or the data are evolving or not really present there for us to know what is normal. Is it normal to have one eosinophil in the lamina propria or is it normal to have a cluster? That data is not really out there. We kind of, when we see an eosinophil, we kind of just jump to, you know, oh, this has to be reflux or this has to be EOE or something parasitic or drug related. My whole point was to correlate with that. Why it is, is probably due to a whole host of things based on the studies that I've read. So sometimes it's a seasonal variation again, like based on the environment. Thank you. Any other questions? One thing that really is very evident both in Dr. Gonsalves' discussion and Dr. Solari's discussion is that the relationship between the endoscopist or the clinician and the pathologist is really quite critical. That exchange of information to have clinical correlation with what they're seeing under the microscope is always paramount. That's true for a lot of diseases that we see. I did want to praise Dr. Solari, that great clinical correlation of the endoscopic findings of the exudate correlating with the eosinophilic microabscesses. We really should be educating our endoscopists to target those exudates to help our pathologists see those microabscesses a little bit more frequently. It's really a great clinical correlation. I'm also a GI fellowship director. So whenever the residents or we have a GI fellow that rotates through our service, I like to pull up the endoscopy report. Sometimes even before I put the slide down, if there is something that is so distinct in the images that they just scream EOE, I'll point to the electronic medical record and I'll say, before I put the slide down, what should your differential be? I think that's helpful both ways. We have GI fellows, clinical fellows that rotate on our service. They spend a week in pathology. By the end of it, they know EOE looks really blue. I think it's this hand-holding that we have to make this diagnosis. I don't have to be a gastroenterologist and certainly these people that are clinicians don't have to be pathologists. But when you are subspecialized in this way or when you're going to go out into the community, just to know what the basics are, I think are always really helpful.

eosinophilic esophagitis

pathologist's perspective

diagnosis criteria

tissue preparation

hematoxylin and eosin stains

inflammatory cells

coexisting diseases

clinical correlation

accurate diagnosis