 I have the pleasure of introducing Dr. Yasmeen Bhatt. She's an anatomic pathologist with subspecialty expertise in pulmonary and cardiac pathology. Her clinical focus includes interstitial lung disease, lung cancer, transplant pathology, cardiovascular pathology. She's interested in education and she is the current chair of the DLMP Education Committee and recently published a textbook, Atlas of Pulmonary Pathology, first edition of pattern-based approach. Thank you so much. So I think I know the answer to this question, but I feel like I need to ask it. Are there any other pathologists in this room? Nope, just me. Okay. Well, thank you so much for the invitation and I'm going to talk to you today about the role of biopsy. It's been mentioned in almost every lecture. So let's talk about the actual biopsies. Unlike many people here, I have no disclosures. Okay, so we'll talk about how to recognize amyloid in tissues and then the histologic differential of amyloidosis for us from the pathology side as well as some of the challenges that we face. And I'm also going to touch on light-chain deposit disease, which I think has been briefly mentioned once or twice today, and then briefly talk about hereditary TTR amyloidosis. So we've already talked about this, so I don't need to belabor this point, but amyloid is a misfolded protein that deposits in tissues and that is not a good thing. There are lots of different types. You just heard a lot about AL light-chain and that's one of the most common that you see across all organs and here are a couple of other types listed here, but there are many more amyloid types that exist. So you might think, okay, you sent the biopsy, it's easy, right? It's kind of a black box, like you send the tissue somewhere and then you get a report back afterwards. It's actually not always the case. So sometimes it's an unexpected finding. So I think one of the themes that I have seen arising out of the lectures today is that there's lots of different clinical symptoms that may eventually lead to a diagnosis of amyloidosis, but you're not necessarily thinking about it right away. And someone may have a GI biopsy for their diarrhea, but they're not thinking about amyloid, right? So for the pathologist on the other end, especially if when you, the clinician, are seeing the patient, you're not thinking about amyloid, we might not be thinking about it either. And sometimes on the actual tissue, the finding of amyloid can be extremely focal and can just blend into the background, especially if you're thinking about something from another side. I'll use GI biopsy again as an example. You're thinking, oh, this patient has diarrhea. I'm worried about inflammatory bowel disease. I'm looking for architectural distortion. You might miss the little slips of amyloid that are in the submucosa. So amyloid can be seen, of course, in any organ or any tissue. So it can be unexpected. And there are mimics. There are things that look like amyloid. One of those is light chain deposit disease, which I'll talk at the end. And there are other things too that look like amyloid that actually aren't. And then interpretation of the special stains like congo red can actually be technically challenging, especially if you don't have experience or you're someone who only orders it once every six months, once a year, once every five years. All right, so that initial tissue diagnosis. There are a couple of stains that I wanted to mention. So the first, of course, is congo red. So when we have something that's congo red, we want to see congophilia. Okay, so we want to see this kind of pinkish red color. All right, in this particular case, this is a myocardium wrapped around the myocytes. And then when we put it under cross polarized light, we want to see apple green by refringence, okay? So here's a nice example of that. You can see that nice apple green by refringence. This is actually quite beautiful under the microscope. It's extremely challenging to take photos of, so they always look really, really dark and not very pretty. But it's actually very nice under the microscope. Another stain I wanted to talk about was sulfatedalsium blue. So this is a stain you might not hear about as much. And this will stain amyloid in this very nice teal green, almost like a toothpaste, like that old toothpaste color. And then trichrome stains will stain amyloid in a grayish blue color. This is more of a blue, but it also can sometimes have more of a grayish tint. So those are the basic special stains that we use for amyloid recognition in tissue. So let's talk about the heart. So cardiac involvement, of course, as many people have said today, is very important for the prognosis of how these patients perform. Many different types can be in the heart, AL and ATTR, of course, amongst the most common. So let's look at an example. So here's an endomyocardial biopsy. And actually, can I, does this come off? Doesn't, okay. So this is an endomyocardial biopsy. All right, so, whoops, go back. So this would be the endomyocardium, all right. So if this was still in the patient, this is where the blood would be in the chamber. And then underneath here are all the myocytes. So I've given you an inset here on the right, a little bit higher power, sorry. Laser in your eye. And these are what myocytes should look like, okay. You shouldn't have really anything in between them. But if you look at this here, you start to see, well, okay, this is a cell, this is a nucleus. And then there's all this amorphous material around it, all right. And it has this almost like a little cracked structure. It's very light, light pink color. This is one of our standard stains, is a hematoxin and eosin. Everything that comes through surgical pathology is put with this stain. And you start to see that, okay, I wonder why this patient, I don't wonder why this patient might be having trouble on cardiac symptoms, because instead of a solid sheet of myocytes, we're seeing all this material replacing those myocytes. So if we go to, and here's another piece, this is actually, I wanted to show you the second piece because this is from the same biopsy. And it's a little less obvious, I think, compared to that last biopsy, where you see more myocytes and maybe a hint of something going on. But I could see someone who wasn't used to looking at these types of biopsies. And if this was what most of the specimen looked like, looked at it really quickly, they might miss this material that's edging in around the edges there. Okay, so I'm gonna start off with a sulfated alchium blue stain, all right? And this is a very nice stain for screening. And it can pick up very small amounts of amyloid very easily. So again, so our myocytes are this orange color here. And then you're seeing the sulfated alchium blue is all of this green, which is almost completely overtaking this piece of tissue. All right, and there's that other piece you can see now. It's really obvious there's green everywhere where there shouldn't be green. So the amyloid becomes very obvious on this stain. I'll say I really like this stain. We use this at Mayo for essentially all of our cases, but not all labs have this stain or a proficient in using it for this purpose. All right, so here's the Congo red, okay? And again, it looks kind of similar to the H&E because there's so much amyloid in this particular example, but it's a little more pink, a little more pinkish red. So let's polarize that tissue there. And then now we can see all of our green there, that kind of characteristic apple green birefringence. All right, so this is a nice case because there is quite a lot of amyloid, but it's not often the case. Sometimes you see just a little bit of amyloid or your biopsy isn't as generous as those that our wonderful cardiologists send our way. And so you may have trouble seeing the little bits. And one thing I also wanted to point out here is you see the apple green birefringence. And even in this case where there's a ton of amyloid, not every piece of amyloid is actually staining. And so that's something that I think people miss sometimes. It's not every single bit, it's a polarization. It's a physical polarization of how the light is refracting through that amyloid. So you don't always see the green absolutely everywhere. And when you have small specimens, this can be incredibly challenging. Okay, so we have amyloid, now what? Typing is of course very important for patient management, has been talked about in multiple talks today. There's lots of methods. There's IHC, immunofluorescence, electron microscopy. I will say probably the two most common ones I see people use are the mass spec, which is what we do at Mayo. We send it up to Rochester and then IHC. IHC has a lot of issues associated with it. It's not always sensitive, it's not always specific. There can be a lot of background, there can be false positives, there can be false negatives. Honestly, I would advocate against using IHC in identification of amyloid typing. So if that is what your pathologist is doing in your institution or where the lab where you're sending your samples, try recommending or asking, could we actually send this for mass spec typing? I think it's a far superior system and I have seen multiple cases where the IHC led somebody down the wrong road and then when we actually typed it, you get the correct diagnosis. Very sensitive, very specific. I'm always incredibly impressed with the amount of tiny amounts of amyloid that they can pull out with their micro dissection techniques in order to run mass spec. So I'd highly recommend that you use mass spec for typing rather than IHC or some of these additional methods. All right, so I would consider this to be the gold standard. All right, so for that particular case, the patient had transcyrheating type. This patient ended up getting a lung trans, lungs on my mind, a heart transplant. And this is a cross section, a short access cross section of what we're seeing from this patient's x-planted heart. And at first glance, when you look at this, you might think, well, probably for this group, you're not gonna think this is normal. But if I'm not talking to pathology residents and other trainees, I say, oh, it looks kind of normal. But it's actually not. So you have a diffuse thickening of the myocardium here. And you might think, well, is it hypertrophic because they have coronary artery disease? But then when you look at the coronaries, they're really clear and open. There's nothing going on there. Of course, you can get amyloid deposition in vessels. So let's look at a little bit higher. Take a piece here, all right? So this is that piece and you can see, even on low power, this is where the blood would be. You can see some blood that's left there. You have this modeled look to it. Certainly not what normal myocardium should look like on HNE. And all of those lighter pink areas is our amyloid deposition. You can see just kind of wrapping around the myocytes. So really, when you get this kind of visceral look at what amyloid looks like in tissues, it doesn't surprise you that these patients have clinical symptoms. OK, so let's move to another organ. So 65-year-old man, in history of hypertension, GERD, obesity, obstructive sleep apnea, he was endorsing progressive shortness of breath. And he was found on imaging to have multiple bilateral pulmonary nodules. So they eventually decided to do a biopsy on this case. And this is the biopsy. This is a needle core biopsy of one of those nodules. All right? So when you look at this, you're like, OK, well, I don't think that's lung. Is it lung? It just looks like a solid mass of pink. There's a few giant cells. There's some inflammation of the little blue dots or lymphocytes and a little bit of more normal lung here on the side. But even that has some evidence of hemorrhage going on right there. All right, so it's a light pink mass. Not too much cellularity. It's not cancer. Always a concern with patients is a carcinoma. So what are we going to do next? So when this case comes across to my desk, I'm going to want to identify what that material is. So when we see this material, which, of course, as you all can probably guess, is going to end up being amyloid, there are other things, though, that we have to consider that are much more common. So necrosis can be very similar in appearance. Fibrosis, so collagen fibrosis, some kind of scarring. And then, of course, amyloid. And then, very rarely, you might think about light chain deposit disease. So these are some examples of what some of these deferentials might look like. So on the upper left, you have necrosis. This isn't a lung biopsy. Necrosis has a little bit more of a granular falling apart appearance in contrast to amyloid, which has more of a cracked, almost like little sheet-like look. Fibrosis in the upper right. So again, I apologize, there's a lot of lung in here. So whoops, go back. All right, so here's fibrosis. So kind of similar to amyloid in that it's pink. But you can see that collagen substructure in fibrosis. Here's an example of amyloid in the lung. And then this is what light chain deposit disease looks like. It's very similar to amyloid, but it's a little bit more eosinophilic. OK, so we'll do our next step. We'll do some stains, a Congo red, sulfated calcium blue. We'll do what I call our special stains to look for bugs. So AFB, a zeal nilson stain, we'll look for acid fast bacilli, and then GMS to look for fungus. So here's that sulfated calcium blue, showing that very characteristic teal color. And then there's our Congo red. This is a nice example of what we call congophilia. So it's not enough to see something that looks like it might be polarizing. And that's a point I want to bring out here as one of the technical challenges is collagen, for example, will give you a little bit of polarized look. It'll look kind of refractile. And you might convince yourself that, is there a little touch of green there? Maybe that's what it is. But it's important that you're actually looking at congophilic material. And then that is what polarizes to show you the apple green by refringing. So it's not just one thing. You need to see both in order to feel confident in diagnosing amyloid on using this Congo red stain. So there's our polarization. Lots and lots of apple green by refringence there. All right, so this was a patient that we diagnosed with nodular amyloid. It's a more localized form, turned out to be AL type. Here's another example. This is actually a very recent case that I had. Again, patient had a nodule in the lung and the needle core biopsy was almost completely replaced by this pale amorphous material. And one thing I hope that is coming across in the photos, now all of these photos, most of these photos are a combination of in-house cases and consultation cases. So you can see there's a variability in what the stains look like in the colors. So it's not just a reflection of how they're projecting, that's the reality. So every lab is gonna look a little bit different. And so where you train, just thinking about pathologists in general practice, where you train what the few cases of amyloid you may have seen, it may look a little bit different than when you actually go into practice or if you move jobs. So amyloid looks a little bit different depending on what lab it's being looked at in. So here's a trichrome. So I mentioned trichrome earlier and you may say, why are you doing such a general fibrosis stain? And I'll get to that in a minute. And this trichrome on that same case is gonna show you this nice blue color. And I liked this picture because it also nicely highlighted that kind of cracked look, almost like a fish scale look that you can sometimes see in amyloid. There's our sulfated alchim blue. Again, that very nice teal toothpaste color. And then our Congo red. All right, so it's not as congophilic. The other stains fit, it's not as congophilic. And you polarize it and you're like, is there a little bit of green right there? Sometimes you honestly just need to turn the lights off in your office and turn the light way up on your microscope and play around with the fine focus to actually see that apple green by refringing. So this is actually more common than not in many cases. So even in a case like this where there is so much amyloid, it's like nodular deposits of amyloid, doesn't actually refract very well. And that's not uncommon. And that's what leads to some of these false negatives or equivocal stains that you might get. And you're like, well, everything fits, you're calling it equivocal. If you don't have the experience to call something, you might miss something. All right, so this patient ended up having indolent B cell lymphoma, which is often the case with the nodular amyloid deposition in the lung. Okay, so in the lung in particular, it can be quite subtle. It's important to always keep it in mind. You can have systemic or nodular forms in the lung. I won't go over this table, but essentially when you have diffuse septal amyloidosis, you're gonna think about an underlying systemic amyloidosis versus nodular. You might think more about an indolent B cell lymphoma or something associated with that. So when I talk to our trainees, I say, okay, if you find nodular amyloid deposition, the next thing you need to do is look for the lymphoma, right? So that's the second thing we do is a whole stained panel to see if there are a lymphoma around. Is it Kappa predominant, lambda predominant, what's going on? Okay, so next patient, 73-year-old man, increasing shortness of breath, and here is our biopsy, very short. Not gonna go too much into the details of the clinical side of things, but this is what his biopsy looked like. So this is lung, all right? So these empty spaces here are where air would be, and then these are those at the delicate alveolar is after where air exchange happens, all right? So this is where the magic happens, okay? I know I'm talking to a group of cardiologists, but this is where the magic happens. All right, but there's a little bit, there's something going on here. This is actually not quite as thin and delicate as you expect. This is probably closer to normal. You can see there's single red blood cells in here, nice little capillary, but there's some material that's interposing itself in between, right? That shouldn't be there. So not surprise, surprise, but here's our stains. This is sulfated calcium blue. It nicely highlights, it's actually a ton of material in there staining green, and then you can see the same thing on your congo red, and then that nicely shows you this apple green biorefringence. But again, in contrast to how much green you see here, and I just wanted to point that out again, you see there's a lot of green, right? On our calcium blue there, but then when you look at the amount of actual apple green biorefringence, just a little bit right there, maybe a little there, maybe a little there. So it can be challenging sometimes to make these diagnoses. So this patient actually did also have a history of ATTR-type cardiac amyloidosis that was previously diagnosed, and now it was in his lungs. Okay, so I wanna talk about amyloid as an incidental finding, because we mentioned sometimes there, you're not thinking about this from the clinical side, and they get a biopsy. So this is a patient who was coming in for a mitrovalve repair. It's some myxomatous mitrovalve disease. I got it as a appendage and as mitrovalve, all right? So here's his left atrial appendage. At first glance, does it look too crazy? You know, we always look to see if there's any fibrosis, any hypertrophy of the myocytes, anything along those lines. Let's look a little closer, all right? So again, a little bit of vascularization, and I wondered, is there something in there that looks a little bit off, and so decided to do a congo red on this particular one? And sure enough, there's these very wispy, wispy congophilic material in between, okay? And then if you polarize it, this one actually was a beautiful polarization. We had ton of apple-green birefringents there. I was like, okay. Then I looked at the valve, and I was like, okay, here's the valve. All right, all this deep purple stuff is calcification. There's some fibrosis here. There were some myxomatous changes and some of the other pieces, but I was like, you know, looking at some of the material, it just, I just, I worried about this case. And so I threw a congo red on this one as well. Let's look at this spot here. So this is the congo red, and it's a little hard to tell with all the calcification going on, but there actually is some congophilia, right here, right here, right here. And then when you look at the polarization, sure enough, apple-green birefringents right there. So I sent for typing on this particular case for actually both, and the left atrial appendage came back as atrial nitritic factor, ANF type amyloid, and then the mitral valve came back as an interminate type of amyloidosis. So, you know, it's probably out of the scope to talk too much about ANF type amyloid, but certainly can have some clinical implications and is important to know, but it's a different type of amyloid that was in that mitral valve. And likely what was in the mitral valve was probably just associated with a localized degenerative process. But we do see this type of amyloid. Dr. Rosen's all gonna test. I send her, end up sending patients her way by finding all this degenerative amyloid. So it's kind of interesting you can see that. But this highlights how you can have different types of amyloid in the same patient, depending on where you take the tissue in. So for this particular patient, it may or may not have had significant impact, but in other patients, for example, where you're worried about like an ATTR type and maybe you find something that's degenerative, looking at the actual organ that's impacted can be really important. So actually getting that cardiac biopsy. So you can have multiple types of amyloid. All right, so let's take a bit of a different path here. So let's look at a lung nodule in a 78 year old man. Okay, so here's his nodule, high power. So very pink kind of amorphous material. There's a couple of giant cells here and here. Okay, and you're like, okay, this looks kind of like amyloid. This was one of my consult cases actually. And so the outside pathologist said, okay, it looks kind of like amyloid. I'm gonna do a conga red. So they did a conga red, maybe conga philic, but nothing polarized, nothing there, okay? So they were like, the conga red is negative. I don't know what this material is. And so they sent it out, which is totally reasonable. So what do we do next? So this is where those other stains actually come in very helpful. So when I get cases like this, I will add a sulfated alchium blue and a trichrome. And so this is what the sulfated alchium blue look like. Now, this is not that pretty toothpaste green, right? So this is kind of like a salmon pinkish color. Very different than what we saw in amyloid. And then here's a trichrome. We're not seeing blueish gray. We're seeing this bright fire engine red color. All right, so what does this tell us? So this tells us that the patient has light chain deposition disease. So this is not amyloid, it's not amyloidogenic. It's a monoclonal Ig deposition disease. You can rarely see it in the lung and heart, but it certainly has been reported. Renal involvement tends to be more common. The nodular form is associated with chogrens, low grade lymphoma, plasma cell neoplasms. So light chain deposition disease I feel like is often missed. At least once every couple of months we get a case that comes through a consult where they're like, we don't know what this is. We're just lost because the conga red is negative. And it's one of the reasons why I really like seeing some of these additional special stains. I think I have a chart here, yes. So this is amyloid versus light chain. And what the very, very simple sulfated alchium blue trichrome, the sulfated alchium blue, not all labs have this stain available for use, but every pathology lab is gonna have trichrome. This is just a stain for fibrosis. It's used in a multitude of different scenarios. So everyone's gonna have a trichrome. So if you have a conga red and a trichrome, you can pretty reliably say if something's going to be light chain deposition disease or not. So the trichrome will give you that very distinctive crimson red in light chain in contrast to a blue-gray color that you see in amyloid. And if you do have the sulfated alchium blue, you get a salmon pink in light chain versus that bright teal. So very different colors. No chance of sort of misdiagnosing them because they look very, very different. And then of course conga red in light chain could be vocally positive or kind of weak looking or it could just be completely negative. As a side, you can have amyloid and light chain deposition disease in the same biopsy and in the same patient. That does happen and you get a very nice mix of the two colors. If you were to do electron microscopy, not as many people do EM anymore for this, but you would see a difference in an EM. You get the nine to 10 nanometers randomly oriented fibrils for amyloid, of course. And then light chain looks more of like this kind of dense powdery deposits. Okay, so I didn't wanna talk briefly about hereditary amyloid transirite and polyneuropathy since I know that this was a topic that was being pushed a bit here. I feel like it has been talked about in better detail than what I have on here. Certainly a progressive disease, very debilitating early diagnosis is key. What I will mention from the pathology portion is that biopsy is obviously very important in identifying these patients, but the biopsy is not always of the nerve, right? And even if the patient does have a nerve biopsy that's negative, there may still be amyloid there. And so there's a lot of value in considering other biopsy sites, particularly if they do have systemic amyloidosis that's being deposited in other organs that may be affected and might be easier to get a biopsy from. So certainly you can see that there. Oftentimes though, I think, fortunately the burden on thinking about this is on you all first to figure it out. If we can find it, that's great. We can confirm it and then send it for typing. All right, so again, same thing. This was actually, this paper was talking about ATTR amyloidosis as hereditary, but I thought in their table they nicely summarized essentially everything that I wanted to talk about with the issues associated with biopsy is that you can have false negatives depending on sampling, right? So amyloid is not everywhere. So even in that picture of the explanted heart that I showed you, not every piece of the heart was involved by amyloid, even though it was quite a severe case. In fact, I took a little piece of normal appearing myocytes from that picture to use as my normal myocytes for the picture from the biopsy from that patient. So sampling is everything, right? So if we were only getting one tiny piece, you may have missed the amyloid. So consider, and then the other thing I did wanna mention too, again, coming kind of harping on that point is if you don't diagnosis a lot or the laboratory that you're sending it to doesn't see this a lot, consider asking, could you send it out or did you do any additional special stains or maybe repeat the stain because I have seen more than one case come through our service where amyloid was either missed or it was called negative and it was clearly positive, things like that. So definitely there is a level of technical skill involved both in the laboratory side of running and creating these stains and then also in the interpretation side from the pathologist of interpreting those congo red stains. All right, so I think that's my last slide. Hopefully it caught us up in a little bit of time. Thank you.