Amyloidosis Workup: What Pathologists Need to Know

Dr. Kay Washington:
Welcome to the latest edition of the College of American Pathologists' CAPcast. I'm Dr. Kay Washington, chair of the Center Guideline Committee for the CAP. Today we're talking with the chairs of the expert panel that developed the new evidence-based guideline on workup of amyloidosis, Dr. Dylan Miller and Dr. Billie Fyfe. Before we get into the questions, let's learn more about our guests. Dr. Miller, let's start with you.

Dr. Dylan Miller:
Hi there. I'm Dylan Miller. I'm a general surgical pathologist, but I also do renal and cardiac pathology, and I work at the Intermountain Health System Central Laboratory in Salt Lake City, Utah.

Dr. Kay Washington:
Thanks, Dr. Miller. Dr. Fyfe?

Dr. Billie Fyfe-Kirschner:
Hi, I am Billie Fyfe and I'm a professor of pathology in the Rutgers Health System, which now encompasses New Jersey Med School and Robert Wood Johnson Medical School and very happy to be here today and to have participated in this process.

Dr. Kay Washington:
Thank you. Dr. Fyfe. Let's jump right in. One of the challenges for pathologists, this guideline addresses is what to do with those fat pad biopsies for amyloid when we get them in the lab, and I'm a GI pathologist, but these get sent to me, what do I do? Do you have any recommendations or guidance for us?

Dr. Billie Fyfe-Kirschner:
Yes. I think one of the more frequent questions that I get asked is how do I do a fat pad biopsy? And that's even before it gets to the lab. So one of the first things pathologists need to know is how their laboratory prefers to handle these specimens. Do you want to do them as cytology specimens? If so, do you have an appropriately controlled amyloid stain set up to do? Do you have a plan for what you're going to do if you don't have enough tissue for fibril typing, which is imperative? Are you going to do cytology and cell blocks? Are you going to take these specimens and do them as formal and fixed paraffin embedded specimens? And so once that's decided, you can guide your clinicians on the best way to submit the specimens and there's benefits to cytology alone. It's quick, it's easy. You're looking at the entire specimen, but you might not have tissue left for fibril typing, which again is imperative and doing a surgical biopsy type of fat pair biopsy leaves you with additional tissue in most cases for doing fibril typing. But even those can be a little bit dicey in terms of the amount of tissue. So we all have to be very careful and be very careful with fat pad sections on your appropriate controlled tissue staining and your tissue thickness, which can significantly affect especially Congo red evaluation.

Dr. Dylan Miller:
Yeah, those are excellent points, Billie. And you mentioned Congo Red, which is sort of the mainstay or the workhorse of amyloid. Other institutions have successfully used other amyloid specific dyes or stains like ThT, ThioS, or Sulfated Alcian blue and some other things. The recommendation does since it is evidence-based and since most of the evidence is with Congo red does recommend Congo red, but does allow labs the opportunity to sort of validate whatever other stains they want to use as long as it shows an equivalent performance to Congo red and Congo red's a really interesting stain. Its history goes back more than a century and it was used in fabric dying processes kind of around the industrial revolution. It has a unique property of interpolating itself just based on the molecule shape between the beta pleated sheet regions that the amyloid deposits have. And because of that sort of molecular configuration of having these parallel arrays, it can change photons as they pass through the tissue.

And so one of the things we all learned in medical school with Congo red is that you polarize it and when you polarize it, it shows apple green birefringence in the discussion part of the guideline, we actually expand on that a little. Apple green is one of the colors that birefringence, but there are some other shades kind of in the yellow and orange part of the spectrum that you often see when you do polarization in terms of the birefringence. And so we use the term in the guideline characteristic birefringence instead of apple green birefringence. Another thing that the dye does is actually fluoresces. And so our recommendation number three in the paper is to use fluorescence either with a Texas red or TRITC filter, especially on the difficult cases of amyloid. So you should see fluorescence with a TRITC and Texas red on your cases and that can be helpful. Like I said, especially in the really subtle cases.

I think the next thing to talk about in terms of fat pad biopsies and what to do if you do have tissue remaining, you're only halfway there when you diagnose amyloid. Really the importance in terms of the workup of amyloid is subtyping and determining the fibril type that composes the amyloid deposit. Everything clinically sort of depends on what that fibro type is. And so our recommendation number four as well as we have a good practice statement there that talk about every time you need to do the subtyping for systemic amyloidosis and the method that's recommended is mass spectrometry just because it's open-ended and it has the highest sensitivity and specificity, you don't have to know what type of amyloid you're looking for. It will find the fis that are making up the, I think one last important thing, and Billie you can help me here, is what about when the fat pad biopsy is negative?

Are there comments you should put in the report or what recommendations should be made there? And I think that it's important to know fat pad is not a hundred percent sensitive so that if they still have a suspicion that there might be cardiac or renal or some other organ involved by amyloid, that they may want to do a targeted organ biopsy. Also, that if they've had another recent pathology specimen and there's an archive block available, it may be worthwhile getting that block to do more tissue, more sampling that they don't have to go back and re-biopsy the patient. They could go to an archive block and do amyloid studies on that. Do you have anything to add there, Billie?

Dr. Billie Fyfe-Kirschner:
Yeah, just about the concept of localized amyloidosis, especially when we're looking at fat pad biopsies because we know that medications such as insulin being injected into abdominal fat can lead to localized amyloid deposits. And so it's also important and another reason to do fibro typing, but it's important for pathologists to also be aware of the concept of localized amyloid deposits.

Dr. Kay Washington:
Thank you for that review. I think your point about the localized deposits is a really important one. Billie and I have forgotten about that, so thank you. You guys are cardiac and renal pathologists, so you see a lot of amyloid, but what are some take home points and tips for the rest of us?

Dr. Dylan Miller:
I'll start. I think one thing is just to realize, I think this is one of the hardest things we do in pathology. This is not a no-brainer. This is definitely a brainer. It's a challenging area. It's challenging to read Congo red and the other amyloid stains. So having some healthy respect for it's important. My approach is I actually always just start with the H&E in my view. You should see something on the H&E that looks suspicious for amyloid, some kind of deposit material that doesn't belong there. And on the H&E, you can see the texture and sort of pink color characteristics that suggest that it might be amyloid. So I think that don't go straight to the Congo red, but really look at the tissue and see if you think there is something suspicious for amyloid. Now it gets really tricky.

I wish that we had a method to detect the first amyloid fibril being laid down in the tissue, and it's those really early cases and the fat pads definitely fall in this category that are the most difficult. So another tip I have would be to look at the on slide controlled tissue, and typically that's a tissue with a lot of amyloid, so it's going to have very obvious and large deposits, and that might be different from your tissue, but you can at least compare the texture, the sort of color of the control amyloid to what you're seeing in your tissue, and then you should do polarization and if you can do fluorescence on the control to sort of see how it bio fringes and see how it fluoresces and match that up against what you suspect might be amyloid in your patient tissue. So really use that on slide control that will help you.

I think the last advice I would have is to, I share these cases with my other colleagues that do renal pathology and that see a lot of amyloid. This is hard and I struggle sometimes and I really find that input from colleagues helpful. Just one last point, since everything clinically really depends on the amyloid fibro typing and finding the amyloid in the first place, I really think of this almost like a predictive marker in cancer, that predictive markers change the therapy and sort of direct treatment for those patients. And in my mind, this is very similar to that.

Dr. Billie Fyfe-Kirschner:
Yeah, I'll add to that, Dr. Miller, that amyloidosis should be considered a critical diagnosis, especially the two most common types, AL and ATTR because 25% of patients with AL may die of disease within six months of diagnosis and 25% of patients with ATTR wild type may die within 24 months of diagnosis. And often these patients have had a delayed diagnosis and other specimens which have recently clinically been associated with an increased risk for patients developing cardiac amyloidosis like carpal tunnel specimens and lumbar stenosis specimens may have been taken in these patients and previously missed. So another really important point in our discussion for pathologists to take home is to have a high index of suspicion in clinical situations which have been known to be associated with further development of systemic amyloidosis in patients.

Dr. Kay Washington:
Thank you. Those are all really good points. My final question is how would I go about doing a fat pad biopsy in the unlikely event that I was ever asked to do one?

Dr. Billie Fyfe-Kirschner:
I don't do them actually, but for me, I'd probably do a large core needle aspiration and probably do a slight squash prep and then save some inform for processing, but that's just me.

Dr. Dylan Miller:
I also don't do them but agree that that's the most common method. The large or needle biopsies, there can be kind of incisional biopsies or you start with a punch biopsy like a derm specimen and then use a scalpel to go deeper.

Dr. Billie Fyfe-Kirschner:
And just a pitch for autopsies. Residents who want to learn and try and start looking at what fab pad cytology might look like can attempt these processes on fully, appropriately consented autopsies.

Dr. Dylan Miller:
So we definitely acknowledge the expense and the turnaround time delays that are involved with mass spectrometry fibril typing. I think that it still is the best test, and so it is the recommended method. I think if you do an extensive immunohistochemistry panel for amyloid, it's certainly possible to successfully subtype that way. I think when you get up to doing eight to 10 immunostains, then the cost is fairly equivalent to mass spectrometry and there are definitely sensitivity and specificity issues with immunochemistry and it only tests for the specific markers in the panel that you have. And so the possibility of missing a rare type of amyloid definitely exists.

Dr. Billie Fyfe-Kirschner:
I would add that another drawback is for each of those amyloid types, you have to have appropriate control tissue, which is going to be hard for some if you're testing for some of the rarer types. And also immuno-EM is also another technique that people may consider for typing, but that's similarly expensive and possibly even more limited in availability than mass spec is at this point in time.

Dr. Kay Washington:
I want to thank you, Dr. Miller and Dr. Fyfe, for joining the podcast to talk about this important evidence-based guideline coming from the College of American Pathologists. And I want to thank you all for listening to this CAPcast. To learn more about the updated guideline, visit cap.org. Thank you.