Hirschsprung Disease: Pathology Aspect

We are going to have many questions throughout the show, so just answer the first one. Considering a premature baby born in your hospital, at what gestational age is a suction biopsy a good, reliable tool for your pathologist to make the diagnosis of Hirschsprung's disease so everybody can vote, and then Dr. Collins will comment on the answer. We're gonna wait a second because the, the, the numbers are changing here. So most people are voting. I struggled with this one. What is your struggle? I couldn't figure out the answer. You know, I, I'm trying to imagine a 28 weeker is probably usually. 500 g, I mean, probably not even probably 0.5 kg. I didn't look at the answer yet. I was probably gonna guess 32 weeks. But I randomly guessed. I have no idea. So I think in the poll we have mixed answers, and most people voted from 1 to 4 and nobody voted at 5. Is that correct? But actually when we were in Berlin, many people said that their pathologists don't make the diagnosis before 6 weeks after birth, so I was surprised that nobody voted on this one, but I'll let Dr. Collins answer. Thank you, Andrea, and I wish to thank very much uh my colleagues Doctor Raj Kapoor and Miguel Reyes Mujica. Uh, our thoughts were collectively so close together that we were able to synthesize our responses into one, mostly one, slide, and so. As part of our training as pediatric pathologists, um, we perform autopsies and during the course of those autopsies, we examine sections from the bowel and so we know that there are ganglion cells present in the rectal submucosa at 28 weeks gestation normally. They do not necessarily look like mature ganglion cells, but an experienced pathologist, especially an experienced pediatric pathologist, will be able to recognize immature, um, ganglion cells. Suction rectal biopsies to rule out Hirschprung's disease on 28 week gestation newborns are extremely rare in our um collective experience, but, uh, at least in theory, an, an experienced pathologist would be able to recognize ganglion cells in the submucosa. So I, I'm still not quite clear on this. Only 6 weeks after birth in a premature baby. That's what we're saying. Explain the 6 weeks thing. No, the 6 weeks was just, it came as, as a question in when we were in Berlin. Some people saying that in normal babies, their pathologists would only interpret a suction rectal biopsy after 6 weeks after birth, so. Which is irrelevant at that point. I mean you're, that's why we said there is so much discrepancy on how people practice medicine all over the world, and in this case we are dependent on the pathologist's experience to let us know if it's Hiprung or not. So the next question, and again just type any question that you have, if you want to ask more about that, just type that's the time to do it. Until what age is a suction rectal biopsy a good reliable tool for the diagnosis of Hirschsprung's disease? Everybody can vote. Option 16 months of age. Option 21 year. Option 32 years. Option 45 years. Option 6, at any age. Everybody can vote. And I think we have answers everywhere, but number 4. I think nobody said 5 years, but all the others, we have answers. So, Doctor Collins, We believe that the suction rectal biopsy is diagnostic, can be diagnostic for a patient of any age. However, the failure rate for the suction rectal biopsy increases after one year of age. Potential reasons for the failure rate are, um, that beyond. Um, infancy, there is increased separation of the ganglia, um, as a result of the growth of the baby. There's increased toughness of the stroma, making it more difficult to obtain a good suction rectal biopsy. The anal canal becomes longer and thicker, and I'm sure there are other possible reasons to explain. Um, the failure rate, um, but it is not an unreasonable, um, first option, and if it doesn't work, um, then a deeper or full thickness rectal biopsy is, uh, is, is called for and maybe, uh, maybe the, the first option to consider in children over the first, uh, year of life. So given that, what age would you? Start, when would you say I'm not even gonna try a suction rectal biopsy? Jason, what age would you say they're too, they're too old. I'm not even gonna try a suction rectum, or is it size, not age? Uh, for me, I think it's, I, I was trained about 6 months of age. Once I start, get to, once the patients get to that age, I typically just take them to the operating room. Um, it's certainly worth a try if they're in your office in the clinic. It's, it's a procedure that if you could avoid taking them for an anesthetic, it's obviously beneficial, right. But I, I, I've found once we get to 6 months of age or so, I've been taking them to the operating room. Usually I do suction biopsies when it's the newborn period, 1 month, 2 months, and then after that I also take them to the operating room. OK, I don't remember. I don't remember doing any suction biopsy on a baby older than 6 months old, and most. Perhaps the reason is the fact that after that age, the clinical picture of the few cases that we see is so obvious that you don't need something like that, you know, and anyway, we don't, I personally don't believe that the suction rectal biopsy is the main element for the diagnosis, and it's one extra piece of information. That is very useful, particularly in newborn babies, but it's not the only, the in order to do the operation you need to, you must move forward with a full thickness biopsy, and I don't remember doing a suction biopsy in an older child, you know, the clinical picture is so obvious that it's kind of irrelevant. So we have a newborn patient with symptoms of Hisprung and a suction rectal biopsy with absent ganglion cells. What should we as surgeons do? Option one, proceed with the operation. There is enough evidence that we have Hirsprung's disease. Option two, proceed with a full thickness rectal biopsy at the time of the operation to confirm the diagnosis prior to start the resection. Everybody can vote. So, are, are you deliberately leaving out anything about hypertrophic nerves or anything else in this? No, not on purpose. I think we'll be having questions a lot about those, but if you want to comment, Dr. Collins, a little bit. Uh, certainly having the presence of, of, uh, hypertrophic nerves is helpful in the setting of no ganglion cells in an otherwise adequate, um, suction rectal biopsy, and I certainly invite my colleagues, um, Doctor Kapoor and, and Reyes Mujica to, uh, comment, um, if, if they wish, um. There are a number of features that, that must be taken into consideration when arriving at the hopefully correct diagnosis and as Doctor Pena has already pointed out, there are clinical um scenarios as well that must be um considered in, in deciding whether to proceed or not. This is an area in which there must be communication between the surgeon and the pathologist. One of the many areas in which um we need to know the clinical setting and they need to know what we're thinking and what we have found and I and I and I wanna clarify this now and by the way. We're close enough now that you can say shut up Ponsky. So if I'm talking too much, stop me. But, but I, you know, you guys are the experts and I'm not. So I wanna ask, when you say proceed would go to a full thickness, and let me make sure I'm reading this right, at the time of operation, um, so you're saying. Go to the operating room, do a full thickness biopsy and wait for a frozen section before doing anything else at the beginning of operation. That's one of the options. OK, not go to the OR, do a full thickness, and, OK, same time. So it's interesting is that we're almost split exactly down the middle here. That's very interesting, and I think it has to do also with how your pathologists work. If you have pathologists available in your. Hospital at any time for a frozen session. Remember, if you are practicing in a place that you don't have pathologists available to do frozen sessions, sometimes you already did the many biopsies and you already know the level of your egg bionic bowel to do the put through. So I think it also depends on your circumstances to decide which one is the best option. Let me, let me say something the There are different scenarios that pediatric surgeons all over the world will be facing. Number one. We start the study of the patient by the clinical signs, right? And second, we order a contrast enema, and sometimes the clinical signs and the contrast enema are perfectly obvious of Hiton disease. In those cases, the suction rectal biopsy is just a confirmation for me because I'm sure that the baby has Hirschber disease. Now. The other scenario is a patient that has clinical signs that are kind of in between. We are not sure. And then a contrast enema that sometimes is not clear, you know. In that case, the suction biopsy is more important, but even more important would be a full thickness biopsy, and I may not book the case because I'm in doubt, you know. And then there, another scenarios when the pediatric surgeon is working in a place where there are, there are pathologists, but they are not experts in Hibone disease. And in those cases, the strategy that the pediatric surgeon must follow must be completely different. I remember working in a place where we used to do a laparotomy, take multiple biopsies, and then send the full thickness biopsy and send the specimens to different pathologists, reliable expert pathologists, before making a decision of an operation. And then I work in another place in which there was only one pathologist that was really knowledgeable about Hishbone disease, and we never booked a case for Hishbon when that pathologist was on vacation. So, so because we had previous bad experiences. So, so it's not, it's not black and white. every pediatric surgeon must act according to his own circumstances. There are cases so clear from the contrast enema and the clinical picture that. That the, the, the suction biopsy is just a confirmation and it comes from a good pathologist. That's all what I need as far as I'm concerned. And there are other things which if the pathologist is against my impression in contrast enema, the clinical symptoms, then I will go for a full thickness. I see. OK, so you just answered my question. I never really. Understood or I've, have seen a contrast enema change my management. I know that's like, what are you talking? I mean, I feel like you're gonna do the biopsy. You're gonna do leveling in the operating room. The contrast enema is fun to do. You get a little idea, but it's never changed my management. But this sort of gives a little. Spectrum on where it might actually. I think we're gonna talk more about that later in the radiology session, but actually, depending on if you have a contrast enema that it's very suspicious for a rectosigmoid transition zone, that's the most common one. So for sure I'm going to start transanally now if I cannot wait to talk about this. OK, good. Now if I have a contrast. that looks like a total colonic ganglionosis, then I think it might change or not your approach, but we're going to discuss that later. One last question, not a full, and then I want to actually ask you this, not a full thickness rectal biopsy, but what about I do these laparoscopically. What about a just at the peritoneal reflection, my first biopsy is not a leveling, uh, but to get one right at the peritoneal reflection, so serum muscular, uh, and then that I do. Before I do anything, instead of, instead of from below, I do it from above at the perineal flexion. What are your thoughts on that? For what purpose? For confirming my, my, that's the question that pathologists ask me. Why are you doing this? Just to be positively sure if there was any question about my suction rectal biopsy. And you say you're looking at. Your eyes are looking at me like you're crazy, which is exactly what my pathologists look at me like. So maybe I should take a hint. So because the pathology could be distal to the peritoneal reflection, you could be misled. You could have a false positive reading that everything is OK, and actually there is Hirschsprung disease, but it's distal. Um, and, and a short segment Hirschprung disease as well as, um, you could be in transition zone, and the only, uh, uh, the better way to evaluate for transition zone is not a serum muscular biopsy. It's at least a full thickness, um, biopsy, and, uh, in response to the original question, I think I've said everything that's on, um, this slide concerning the adjunctive studies and so on and the communication between the surgeon. And the um pathologist, I think this will come up again um later, um, it, it is not a good idea to base an entire surgical procedure on the frozen section of a biopsy obtained intraoperatively, right, um, and so, um, this scenario, a full thickness rectal biopsy, the time to confirm. Prior to the start of the resection, if the idea is to start the resection at that surgical procedure, that's not generally a good idea, and a lot of us will not, will not, uh, sanction that approach if the idea is to get a good full thickness rectal biopsy. Not ask for a frozen section. Allow the pathologist to do permanent sections, adjunctive studies if those are necessary, and then plan the surgical, um, resection to begin at a future date. That's a much, much better approach, um, to, to the entire, uh, situation. Doctor Kapoor, Doctor Rayes, do you wanna comment a little bit of how surgeons makes you very mad? I can't. Now, can you hear me now? Can you hear me? M be a problem with her earpiece. Yeah, we can hear you, um. So this is Raj Kapoor. Yeah, um, very interesting discussion, I think by and large everyone seems to be pretty much in line in their thinking. I, the, the comments I guess I would, uh, bring up are the following one, the way I approach suction rectal biopsies or full thickness biopsies is that. When I'm making that diagnosis, I'm committing that child to losing at least some rectum, because if I firmly state that this patient has Hirschprung disease and saying consistent with Hirschprung disease is the same thing in my mind, it's sort of a, a, a use of language that really is irrelevant, you're saying the kid has Hirschprung disease if you. Say there are no ganglion cells confidently in a suction rectal biopsy and the other ancillary studies that you do at your institution fit with that. You're committing that child in my mind to losing at least a part of their rectum because if the surgeon goes in as was described and does a biopsy as far distal as they can in the peritoneum. Uh, uh, yeah, at the level of peritoneal reflection and finds ganglion cells, I would still contend if I've rendered that diagnosis on the suction rectal biopsy that that patient has short segment disease and then the surgeon at our institution anyway, uh, will usually do a one stage procedure and it's gonna be pulling through a short length of, of rectum, and I'm not gonna know. That patient has or confirm my diagnosis until that section of rectum has been resected and I I'm looking at the uh the resection specimen and confirming the absence of ganglion cells so from a, from a, from a practical standpoint, um, I wanna be very confident when I'm diagnosing Hirschberg disease on that biopsy and recognizing that if I blow it, I'm probably gonna get sued and so I think that. If there's any equivocation or iffiness to the biopsy, that's where this rapport with the surgeon becomes so critical that. There's a clear understanding that there's something suboptimal in either the clinical story or more often in the material that I have available to look at and or the methods that I have at the institution I'm at to look at that material and in those situations it may be necessary to rebiopsy or think of other. Uh, strategies. I personally, I have not seen at our institution instances or maybe that I could count on one hand where someone has intraoperatively biopsied the distal rectum to confirm the diagnosis. Of course, in patients with longer segment disease that extends proximal to the peritoneal reflection, you'll get intraoperative confirmation when that serum muscular biopsy is done at the peritoneal reflection, but. There are many patients where a gangliosis is a shorter segment than that, and the initial biopsy of the peritoneal reflection has ganglion cells, so at least that's, that is my feeling about how uh. I guess serious the analysis of suction rectal biopsies or full thickness rectal biopsies is and how careful and confident one has to be and then it all comes back to experience. A lot of it comes back to experience and the panel's already made that point that, you know, an experienced pathologist can do things like, I think, recognize immature gangon cells probably even at 28 weeks, although I can't claim I've ever been asked to do that. in a suction biopsy, and I think knows how to apply and interpret ancillary studies when necessary to Arrive at a confident diagnosis and most importantly knows when. This the material. Uh, is suboptimal and and has the confidence to talk to the surgeon and say we need to get more biopsies or approach this case a little differently. Thank you very much, Doctor Pur. We're gonna now proceed with the next question. If you find in a specimen absent gunian cells, and I think that's exactly the question that Doctor Ponsky just asked, and no hypertrophic nerves, do you consider it his room or not? Everybody can vote yes or no. And I'll let the pathologists comment on that one. And I think we have our polls mixed again. People saying yes and people saying no. Dr. Collins, and while we're waiting for these, I just want to, before we move on, make sure we're all in agreement here on the panel. You have a frozen, I want to make sure I'm understanding this. You have a suction rectal biopsy. Let's say it's perfect. It's no ganglion cells, hypertrophic nerves, everything you can imagine. That patient's getting an operation. There's nothing intraoperative that you're going to do that's going to potentially say that this patient, we were wrong. This patient's getting a pull through or a stoma, right? Is that, am I, am I understanding that correctly? If the clinic is his prone, yes, contrast enema, and suction biopsy, prone, they're getting a pull through. They're getting a pull through. Wow, OK, cool. OK, you, you think different. I am, I'm gonna talk. Look, the reason I love these shows because I always change my management because I don't know what I'm doing. I by what you said that you do. It's what I do. So what happens when it comes back normal? You just at that point I would stop. It hasn't happened yet, but at that point I would stop the operation. I would go talk to the family. I would say. And then you would all say, look at, I'm sure you're all saying that's crazy, but I, it's like I feel like I want just to be positive. Yeah, that's definitely her sprungs because I don't know, but you're right, there's probably no. I would stop the operation and then I would do a full thickness rectal biopsy, not frozen section. Actually, I would do it at that time. I would do it. I'm going to say to the mom, we did a peritoneal biopsy, serum muscular. It's not the best biopsy, but it showed ganglion cells. I'm not, I'm not totally sure. I did a full thickness. I'm going to do a full thickness rectal biopsy. We're going to wait a few, wait a few days, get the biopsies back. If it comes back, Hirschman's, we'll come back in a couple of days and do the. So hopefully at the end of the show, you will be convinced to start transanally, and if this will never happen. Right, because you're gonna start from where the disease starts. That's on the bottom. I've been burned more. We'll talk about, OK, we'll get to that. Good. So Doctor Collins, you can comment on that. Well, our responses maybe. Um, large nerves are not present in the submucosa of all cases of Hirschberg disease. So it is possible to have a suction rectal biopsy that shows, uh, a lack of ganglion cells and does not demonstrate, um, large nerves in the submucosa, total colonic being a, a classic example. Um, so, uh, but we need to take into consideration then the strength of the clinical findings as, as we've just said, whether we believe the biopsy is adequate, we need to know the age of the patient. Um, nerve hypertrophy may be less apparent in the very young as well as in older, um, children. The gender of the patient is important, the results of contrast enema, family history. And then the ancillary stains that we may perform and we'll speak more about um those in a moment. But again, this is a scenario in which communication between the surgeon and the pathologist is, is very, very um helpful and a rebiopsy may be indicated. Um, the compulsion to um do something immediately maybe, uh, should be actually, um, Uh, secondary to having a more firm diagnosis before going into the operating room. So the other question, and that's just for the pathologist to answer, and I'll ask Doctor Reyes also to speak up and Doctor Kapoor, uh, what is the added value for the diagnosis of Hiprung on carretinin, hematoxylin, and osin, ACE nerve measurement, any other? What can you see in a permanent section that you cannot see in a frozen section? Jim, can we open up the phones so we can hear the panelists? I actually want to answer that. Sure, again, this is consensus that we, I think, all agreed on, at least the 3 pathologists who are involved here, so this isn't just my view. I think these ancillary studies or ancillary findings are extremely helpful in two contexts, one of which, hold on a second, I lost my screen here, sorry, one of which is, there we go, is. When you have uh some conflicting or ambiguous results or suboptimal adequacy of a specimen, a good example is that last uh slide that we just showed where you have a patient who has no ganglion cells, maybe what looks like an adequate amount of submucosa in the suction biopsies, but doesn't have hypertrophic nerves. In that instance, if you have a very. convincing uh abnormality in cal retinin immunoreactivity fitting with the pattern you see in Hirschprung disease or acetylcholinesterase staining fitting with the pattern you see in Hirschsprung disease that can be enough to tip me over to to discount the fact that I don't see the hypertrophic nerves and call, uh, call and make the diagnosis of Hirschprung disease. I think that nerve measurements, uh, I personally do not. Do nerve measurements in getting out an ocular micrometer or another digital calipers or something to do that on a routine basis. I think that an experienced pathologist gets a real feel for sort of a gestalt of what are too many, too big nerves. And although there's this 40 micron rule that we that Monforti Munoz and others made popular that basically says, particularly in a young infant under 6 months of age. You shouldn't see in the distal rectum nerves or a nerve that is greater than 40 microns in diameter. That is generally true at that young age, but there are rare exceptions to that, and certainly in older age kids that rule does not hold. So I don't get too hung up on finding one nerve that is greater than 40 microns. It's this pattern of. Sort of a shift in the caliber of all the nerves so that you're seeing more intermediate moderately enlarged and and markedly enlarged nerves in a greater concentration of the submucosa than normal. All of this comes down to experience more than anything else. Doctor Reyes, do you wanna comment on anything? Well, I think that Margaret and and Rash has summarized our collective thinking. I would just like to emphasize the need for ample communication and uh to, to change our mindset from an active physician looking at the patient and then a a consultant down in the basement which is remotely looking at a small piece of tissue. If we bring the pathologist more into the equation of making the decision and teach your pathologist to uh take into consideration not only the clinical picture, but also the uh radiological studies and uh bring your own experience combined with ours, that will reduce the number of difficulties and possible errors significantly. And I cannot overemphasize the need for complete communication. The receptions that we receive are big operations, and I have the feeling that many of our relatively inexperienced junior colleagues go into an operation sometimes because there is some evidence, and they They want to operate and resolve the issue along the way with additional things that are not within a protocol like, well, let's let's order a frost section now. Let's see what happens with this. An operation like this is too big a deal to be made on a flimsy basis, and I think that if we dialogue with our surgical colleagues, we pathologists will learn a lot from what you need to know, and you will learn a lot about what we can offer in everything. Margaret, we had a question from the audience about how many pathologists should look at the slides. Well, one experienced pediatric pathologist is generally enough, um, actually, if, uh, it comes up, I would say equally in frozen sections as well as in suction rectal biopsies that, um, we may have questions about, um, a frozen section from what might become a margin of resection, um, if it's absolutely normal or not. Um, in which we consult with each other, um, and, and we do that freely, and also on the suction rectal biopsies, we freely pass them around among ourselves, um, to, um, to get opinions from other of our, uh, colleagues on staff, so. Um, I think in, in most, um, in all of the hospitals I've worked in, that's been the scenario, and, and I'd say it's probably true for at least most of the rectal biopsies that they are seen by more than one, pathologist before a, uh, a diagnosis is rendered. And then in the um frozen section room, um, we just one point I'd like to make, we have a, a multi-headed microscope and which is very common with an arrow, and some years ago, we got a stage micrometer and we measured the length of the arrow, and now we know the length of the arrow and we've got it posted next to the microscope. The length of the arrow at different magnifications and so we don't take out calipers or make a big deal out of measuring um the nerves. We've got the arrow and we use that arrow to take our um nerve measurements, especially during um frozen sections so it's something we can do um quickly and easily um during our evaluation. So, we would like to know what do you consider characteristics of transition zone. Uh, so again, I invite my, um, colleagues. We, we were, uh, in remarkably close consensus on all of these points. Probably the least controversial is partial circumferential a ganglionosis, which of course implies that you've got the whole circumference of the bowel to look at. So, transition zone contains ganglion cells, but they're not in their normal distribution completely. Uh, in the circumference of the, um, bowel, um, there is hypoganglionosis by definition, um, and there are hypertrophic nerves that can be evaluated or and brought out by a glute one stain, um, more in the submucosa than in the, uh, myenteric plexus with or without associated, um, ganglion cells. A bit more controversial is a feature known as submucosal hyperganglionosis with many ganglion cells in one ganglion, at least 10, I believe is the usual um definition, in which case, um, the submucosa can look like the IND type B. We'll get into that, um, in a future question. Um, there can be ectopic ganglion cells actually in what we think are normal biopsies and normally innervated, um, bowels, so that's a bit more controversial. ganglion cells in the lamina propria, um, in particular. And then, um, additional features that can be helpful to, um, to recognize transition zone include a cal retinin stain performed proximal to an a ganglionic segment. If the calretinin stain is positive, meaning that it shows nerve twigs in the um lamina propria, that's a sign that there are ganglion cells, even if they are not present in that um particular section. So the other question is that in Europe, neuronalintestinal dysplasia seems to be an established entity and why not in the United States? And Doctor Collins loves this question because we always ask her. So there are a number of reasons. Um, one is that the methodology that has been used in Europe and it's largely in two centers in Europe, hasn't been adopted in the United States. 15 micron thick sections are Required, which is at least 3 times the thickness of the normal sections, the sections normally cut, um, in the United States. Those are very thick sections. Um, the histochemical stains that are used are not commonly used in the United States, um, either. There have been inconsistent diagnostic criteria. The definition for the various forms of IMD have changed several times over the last several decades. Perhaps the, the most important is inadequate control data, inadequate control, um, specimens. One cannot look at biopsies from constipated children and conclude that Any of the features there are responsible for the constipation if one does not have matched controls, age matched, and so on from children who are not constipated, in which case the, the same features um may be present and, and, and that lack of, of control data has resulted in um a disorder of uncertain clinical significance. The recommendation is that the diagnosis should not be made in infants. Um, the disorder is outgrown by the age of 4. It's not a disorder that requires surgical therapy. It is, um, self-correcting. Um, so the clinical significance of it is, is unclear. And in addition, multiple papers, multiple authors have challenged if in fact, the diagnostic criteria represent one end of a normal spectrum, and that's where having good control data. Uh, would be very helpful, um, and if the histopathological phenotype is actually a consequence for an adaptation of a downwards, uh, downstream rather, um, dysmotility rather than the cause of the dysmotility, um, in those, um, cases. Cases. So, um, there are a number of reasons why in the United States, um, we, we speak much less about, um, IND and tend to think it's transition zone. Um, many of the features could be transition zone and Hirschprung disease. Um, if my colleagues would like to expand or I another thing I also wanna make a comment. You know, everybody does. When I, when I heard about the neuro and intestinal dysplasia for the first time, I became extremely interested in that because I thought that perhaps it would be represent the answer for many, many problems that we see in the management of his disease patients with persistent symptoms after a perfect or almost perfect operation. Then I made a special trip to Switzerland to meet Doctor Mayor Ruge personally and have long conversations with him. And then, then I invited him to the, to the United States. But then I went further and reviewed the entire literature and could not find a single paper, a single study with a topographic um study of neurointestinal dysplasia. In other words, And the, the reason why we depend so much on pathology in Human disease is because we try to determine the extension of the defect. We need, we, the pathologist will tell us that, that the abnormal portion goes from here to here and that's where we have to resect. That's the basic principle of our operations and there is not a single topographic study. Of so-called neuronal intestinal dysplasia, and yet people are doing resections, treating with laxatives, treating with enemas, and but they don't, they don't describe that they resected the, the histologically abnormal portion of the colon. There is no such study. And then, as Doctor Collin said, some people believe that this condition disappears with time and there's all kinds of clinical confusion and that's why. The concept is still irrelevant. I'm sure that there are many secrets in the in histological characterization of the many motility disorders that have to be studied in the future. I'm very interested, but at, uh, at, at present time, the concept is not clinically relevant from my point of view. Uh I think that uh the Problem with intestinal neuronal dysplasia arose out of the need to explain the subset of patients with symptoms as Alberto has mentioned, and it was a good effort. It was a good theoretical frame put together by Professor Maya Ruga, who I also. Conversations with and then it was championed by not a pathologist, but a surgeon doing. Pathology. And that is probably not appropriate, just as inappropriate as if I was going to operate your patients. So I think that we should, uh, put intestinal neurondysplasia in its right place as a probably transitional period of uh developing in a, in a subset of patients, but not a condition that requires surgery as Alberto has emphasized. Thank you very much. Do we have a moment? Do you have a moment more for one quick comment? Sure. This is Raj Kapoor. So, uh, my dear friend Miguel will not like me for this one, but he, he, uh, and I think Luis wrote a beautiful editorial called IND Rest in Peace. and I, and it was accompanied an article that appeared in pediatric Developmental pathology that was written by Maya Ruge and Elizabeth Bruder where I was able to put questions to them regarding IND and they were very forthcoming, I thought, and useful responses that they provided. And at the conclusion of that piece and and others that have been referred to here, I was very much and have been very much a skeptic of IND. I have to say, having done some of what Dr. Pena asked for in terms of topographical mapping of patients with short segment Hirschsprung disease, I am not as convinced as I once was that IND has been put to death yet. And um I would say that I would still remain open minded as to whether or not there is an age-related change in the density of giant ganglia in the submucosa and whether or not that is shifted in the patients with Hirschsprung disease so that. In many of those patients proximal to the ganglionic segment, one sees a greater proportion of giant ganglia than expected for age. The unfortunate part is it takes a lot of patients, a lot, both patients with a T, uh, to study, as well as patience on the part of somebody counting ganglion cells in order to get the data one needs to. To figure this out and then it takes a huge effort to try to correlate it with outcome and especially with all the other variables that are involved in potentially affecting outcome and if I can make one quick plug for the Hirschprung Disease Research Collaborative that's a multi-institutional. Endeavor to try to get this kind of data that will require lots of patients and good perspective follow up. I would do that and encourage people to visit their website, the Hirshprung Disease Research Collaborative, and get involved to get their institution involved. Thank you very much, Doctor Kapur, and the other question is how do you establish the diagnosis of hypoganglionosis? The uh best way is to only consider the myenteric um ganglion cell density, uh, which means that, um, one has to be dealing with a resected bowel, not just a suction, um, biopsy. And we currently only confidently diagnose severe hypoganglionosis. We don't do counts, um, but we base it on long stretches of the myoteric, uh, plexus containing small ganglion, meaning one or two ganglion cells per Ganglia with minimal um neuropy. Um, and for research purposes, only um less severe forms of hypoganglionosis um can be resolved with a dedicated count of ganglion cells using a particular, um, antibody as a marker. But there's naturally normal variation that is huge and there's a need to count um large areas to get an accurate assessment which limits the clinical value of, of making a diagnosis of less severe um hypoganglionosis. Um, usually this um concept enters the differential diagnosis for patients who have had multiple surgical procedures and, and, uh, poor outcomes. So I think that ends our pathology session. I would like to thank the