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Biopsy Technique & Sample Preparation: Tips for Accurate Pathological Diagnosis
Faith Taylor • Updated Jan 29, 2025 • 32 hits
Accurate pathological diagnosis relies on obtaining high-quality tissue samples, a process often complicated by the variability of lesion types and biopsy techniques. Low-quality biopsy samples can delay diagnosis, reduce accuracy, and ultimately impact patient outcomes.
In this article, pathologist Dr. Andrew Sholl shares strategies to improve the diagnostic accuracy of biopsies, focusing on what to do before the tissue sample reaches pathology. With input from interventional radiologist Dr. Christopher Beck, the diagnostic duo cover biopsy device selection, collection techniques, and sample preparation. This article features transcripts from the BackTable Podcast. We’ve provided the highlight reel here, and you can listen to the full podcast below.
The BackTable Brief
•The choice of biopsy device should be guided by lesion type, location, and operator preference, with fine-needle aspiration (FNA) being preferred for fibrotic tumors like those in the pancreas.
•Smaller gauge needles, such as 22 or 23, are often used in procedures like endoscopic ultrasound-guided biopsies due to anatomical constraints and provide more accurate diagnostic material..
•In thyroid biopsies, using a 25-gauge needle is preferred to minimize blood contamination and fragmentation, ensuring better diagnostic clarity and adherence to Bethesda criteria.
•Time spent within the nodule should be minimized (no more than 10 seconds) during FNA procedures, as prolonged contact can lead to compromised samples, especially if relying on blood return in the needle hub.
•Optimal biopsy sample collection involves obtaining multiple cores (ideally 2 to 4 cm in length), with adjacent normal tissue included to serve as a reference for staining and tumor marker identification
•Proper tissue preservation methods, such as formalin fixation or RPMI medium, should align with the intended diagnostic techniques to maintain sample integrity.
Table of Contents
(1) Choosing the Right Biopsy Device for Accurate Sample Collection
(2) Thyroid Biopsy Needle Selection & FNA Technique
(3) Optimizing Biopsy Sample Collection & Preparation for Pathological Analysis
Choosing the Right Biopsy Device for Accurate Sample Collection
The choice of biopsy device is often guided by lesion location and operator preference. For specific lesions, such as fibrotic pancreatic tumors, smaller biopsy cores or fine-needle aspiration (FNA) may be more effective. Techniques like endoscopic ultrasound-guided biopsy are often limited to smaller gauges, such as 22 or 23, due to anatomical constraints. In these cases, FNA may provide better diagnostic material, particularly when dealing with fibrotic tissue or chronic pancreatitis. The biopsy device used can directly impact tissue sample quality and the accuracy of subsequent pathological analysis. Therefore, open communication between pathologists and clinicians can better align procedural decisions with diagnostic requirements, ensuring the most effective approach for each case.
[Dr. Chris Beck]
I want to talk to you a little bit about biopsy devices. I'm just curious from a pathologist perspective, can you ever tell-- surely you can tell the difference between whether we're using an FNA or a core, but can you tell the difference between when we give you an 18 gauge versus a 20 gauge core?
[Dr. Andrew Sholl]
Yes, I can tell the difference, yes.
[Dr. Chris Beck]
In theory, if you had a side-cutting needle, for some reason, if we thought-- because in IR, there's-- it's not a big debate, because a lot of times your biopsy device is dictated by where you're biopsying, operator preference, critical structures nearby but sometimes in some situations, you'd prefer to use a side-cutting needle or a notched needle. From your perspective, that looks like, if it's an 18-gauge is pretty close because in theory, the full core would actually get a fuller core.
[Dr. Andrew Sholl]
It's the same size. It's really more about what your preference is and what you feel you can get in there and get a good specimen right.
[Dr. Chris Beck]
Whatever device gives you the most comfort to get a good sample, that's which device.
[Dr. Andrew Sholl]
Yes. As long as it's there. We always laugh at you guys because you're like, "Well, my needle was in the lesion." I was like, well, the lesion's not in the needle.
[Dr. Chris Beck]
We're like fighter pilots. We always think we hit, it's always like, hey, but look, it's in there. It's got to be.
[Dr. Andrew Sholl]
That's how I always joke. I was like, I understand that the needle is in the lesion, but the lesion is not in the needle and there's nothing I can do.
[Dr. Chris Beck]
That's a really good line. How about the difference between what GI and interventional pulm is giving? Are their samples any different? Because I think they'll use a 20 gauge sometimes, but I think they do different advanced techniques, like a cryo freeze then to pull it out or something.
[Dr. Andrew Sholl]
Given that they are accessing lesions that are deep via an endoscope and an ultrasound probe, the cores have to be small. To be honest, what I found, and I'm probably biased because I am trained as a cytopathologist, I would prefer they just FNA them. I really would. Particularly the pancreas.
[Dr. Chris Beck]
That probably draws an important point here to drill down on. That's what you would definitely prefer, but it's good to open up a lot of communication with your pathologist and say, what's going to help you get the best answer.
[Dr. Andrew Sholl]
That's absolutely true because-- and some of these lesions, particularly the fibrotic ones in the pancreas to get cells out of there, it's just better to use a 22, 23 gauge endoscopy needle and FNA it. Because a lot of the times too, you got chronic pancreatitis in the background and you just got fibrosis on the cores and it's not very helpful.
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Thyroid Biopsy Needle Selection & FNA Technique
The effectiveness of thyroid fine needle aspiration (FNA) heavily depends on proper technique and appropriate needle selection. Dr. Andrew Sholl recommends using smaller gauge needles, such as 25 or 27 gauge, for optimal sample quality and adherence to established cytological classification systems like the Bethesda criteria. Larger needles, such as 20 or 18 gauge, are discouraged due to their tendency to produce samples that are overly bloody or fragmented, compromising diagnostic clarity.
Proper FNA technique involves minimizing time within the nodule—typically no longer than 10 seconds—and avoiding reliance on visible blood return in the needle hub as an indicator of a successful sample. Dr. Sholl recommends prioritizing the collection of cellular material without excess blood contamination and discourages substituting core needle biopsies for thyroid FNAs, as cores often lack the cytological and architectural features required for accurate lesion categorization and diagnosis.
[Dr. Chris Beck]
At our institution, interventional radiology does a large majority of the biopsies, like of course, exclusional biopsies. For the percutaneous biopsies, it's mostly interventional radiology, the breast radiologists.
Then we have a smattering like that come from Neil Lynan, his interventional pull. Where else do some of the small tissue biopsies coming from at Turo?
[Dr. Andrew Sholl]
You guys do most of the thyroid FNA. Sometimes we see those come from the endocrinologist across the street. Neil's doing EBUS, Joshi, and a few of those. The GI guys will do an endoscopy and do FNAs and cores of pancreatic head masses and that sort of stuff.
[Dr. Chris Beck]
I'll just say that at our practice, and it can vary from provider to provider, we do 18-gauge cores of just about everything with the exception of lung, where it's either going to be a 20 or an 18-gauge. That can vary depending on operator experience, comfort, and what we're biopsying. If we think architecture is needed, if we think it's adenocarcinoma in situ, then we try and push for bigger biopsies.
All our thyroids-- I want to say all of our thyroids that we do coming from IR, FNAs, but I think some of my partners are core, maybe a combination of cores and FNAs. I just mean to say that in general, we're coring most of what we do, and FNAs are reserved for thyroids. Can you speak anything to what you're--
[Dr. Andrew Sholl]
What size needles are you typically using for your thyroids?
[Dr. Chris Beck]
25 gauge as recommended by you.
[Dr. Andrew Sholl]
Yes, excellent.
[Dr. Chris Beck]
You want to comment on that at all? Because my partners, I don't know if they've really bought into--
[Dr. Andrew Sholl]
Yes, I think some of them are just using that core needle and then just scraping it on a slide and calling it an FNA.
[Dr. Chris Beck]
Yes, exactly.
[Dr. Andrew Sholl]
I don't know what size they're using.
[Dr. Chris Beck]
20 gauge.
[Dr. Andrew Sholl]
20, okay. Yes, that can make it bloody. To be honest, the cores of the thyroids are not really helpful because things fall apart. You're not getting the architecture and within a subspecialty side of pathology, you have these parameters that you use to put it into these Bethesda categories. When you have chunks of core on a slide, that just totally mucks up the Bethesda category and so it's blood and tissue fragments. Really you want that nice fine 25 gauge smear on those slides to apply the Bethesda.
[Dr. Chris Beck]
Let me ask you this because this actually comes up and I don't know if it's all that common knowledge, but if you have like a 20 gauge or let's say even an 18 gauge core of a thyroid nodule, is there any way to get-- say we take it and then I know that some people will smear it around a slide and they call it an FNA, but is there any way to extract like smaller cells from a bigger core or you have what you have?
[Dr. Andrew Sholl]
Yes, you have what you have, but smearing it on a slide, it does it. The thing with the papillary thyroid carcinoma is that you're using nuclear features and architectural features. Those are best coming out on a slide from an FNA and not so much from a core that's just full of blood.
[Dr. Chris Beck]
Any advantage at all to taking a core of a thyroid biopsy?
[Dr. Andrew Sholl]
I find none because the way that you're categorizing these is utilizing years and years of categorization via Bethesda on a cytology smear. The cores, yes, they might have some papillary architecture. Yes, they might have that, but you're going to get that from the FNA anyway. When you're looking at follicular lesions, adenoma versus carcinoma, it's the capsule that you're really worried about, but you're not getting a capsule on a core.
Again, that doesn't help you further categorize any of these lesions. The PTC that you were going to see on the cytology is not going to be enhanced by the core in any way. I actually discourage cores as much as possible.
[Dr. Chris Beck]
Hold on, how about this? Why 25 gauge? Why not 23, 21, or 27? 25 is just that [crosstalk].
[Dr. Andrew Sholl]
Yes, 25, 27, 23 is fine. Sometimes those nodules are a little bit more fibrous and to get through that fibrosis, you need a 23 to push through it, and that's fine. I don't discourage that, but, 20, 18, that's too big.
[Dr. Chris Beck]
I'll tell you, I think a lot of interventional radiologists, so like the way we train, it's like you're left with the general sense that bigger is better. Then also you may go through a training program for interventional radiology, and you may not actually do a lot of thyroid FNAs just because that complexity is low. You may spend your time training, doing these other different procedures, and then you have to do a lot of thyroid FNAs in actual private practice. Actually, would you actually--
[Dr. Andrew Sholl]
Is it just ingrained in your head that a core, just-- I core this.
[Dr. Chris Beck]
Yes, core equals better is really-- and so that's what I wanted to get at today is that, hey man, that's not always the case.
[Dr. Andrew Sholl]
No, it's not.
[Dr. Chris Beck]
Yes, but I will say, just so everyone knows, it's not like I was-- I knew this inherently, my practice changed whenever you came and gave us a pathology crash course about like, "Hey, just do the smaller gauge needles. You're going to get better core or you're going to get better FNAs and better--"
[Dr. Andrew Sholl]
Yes, you get better stuff. Oh, they look fantastic at Turo now. They really do.
[Dr. Chris Beck]
Oh yes?
[Dr. Andrew Sholl]
Yes. They talk about getting blood in the hub too and that drives me insane because the first piece of the specimen that's coming up is going to come through that needle. It's going to get up into the hub and then everything in front of it is just blood. The blood in the hub is actually the sample. Then when you put it down on the slide, you're just pushing the blood that was in the tip of the needle onto the slide.
[Dr. Chris Beck]
Yes, so you don't need any of the blood, you just want--
[Dr. Andrew Sholl]
No, you don't want the blood, you want the sample. The sample-- so I always advocate that don't stay in a lesion longer than 10 seconds. I don't care if blood in the hub or not because the sample is going to be in that needle.
[Dr. Chris Beck]
When you're saying blood in the hub, just for people who may not have a perfect visual on this, you have a 25 gauge needle. I usually hook it up to the back of a syringe, like a 3 or 5 CC syringe, but just stay in the nodule long enough. You don't have to see blood return into the hub of the needle. You just stay in it for five, 10 seconds and then whatever else, put that onto the slide.
[Dr. Andrew Sholl]
Yes, exactly. That's the best way to do it.
Optimizing Biopsy Sample Collection & Preparation for Pathological Analysis
Proper collection and preparation of core biopsy samples directly impact their diagnostic utility. For optimal analysis, Dr. Andrew Sholl recommends obtaining three to four 2-centimeter cores, with two cores typically used for diagnosis and the others reserved for additional tests such as next-generation sequencing. When collecting the sample it is optimal to include adjacent normal tissue as it provides a valuable reference point during immunohistochemical staining. This approach enables pathologists to verify proper staining and accurately identify tumor markers. Then separating well-preserved cores from those with necrotic or compromised tissue streamlines the evaluation process and enhances the quality of pathological analysis.
To further ensure diagnostic effectiveness after sample collection, tissue preservation methods should align with the intended diagnostic procedures. Formalin fixation, a widely used method for maintaining tissue architecture, cross-links proteins, which prevents its use in flow cytometry. Flow cytometry requires live, individual cells; therefore, fresh samples should be placed immediately in RPMI—a cell culture medium that preserves cellular viability. Adhering to these practices ensures that tissue samples are maintained in a condition suitable for accurate pathological evaluation.
[Dr. Chris Beck]
Okay, that's a good tip. See, we're already getting into it. All right, so let's get a little bit out of thyroid FNAs and get in a little bit to core biopsies. Once a biopsy is taken, whether it's interventional pull, GI, or more likely IR, the pros, can you talk a little bit about what happens when we put it in to-- or actually talk about the different ways you can send out a sample because you don't just have to put in formal, you have options. When does it change? It may not be formal, which is the default, I would say.
[Dr. Andrew Sholl]
Yes, formal is typically the default. Usually, you need some clinical history in order to triage things. If you're going into a lymph node in a patient who's got lymphadenopathy and you're thinking lymphoma, you got to go not put some in formalin so you can get some flow cytometry out of that sample.
[Dr. Chris Beck]
Why can't you get flow cytometry out of a formalin sample? I know that you can't, I just never really knew why.
[Dr. Andrew Sholl]
Formalin fixation cross-links proteins. Once those proteins are cross-linked, they stick together. Flow is a procedure in which each individual cell has to flow in a laminar fashion through an aperture. You attach different signals to those cells. Then a laser reads, okay, this is signal X and this is signal Y. This is a B cell, this is a T cell, this is a macrophage. If you have cross-linked those proteins, they're not going to flow through that aperture.
That's why you need a fresh sample where you can have individual cells that are still alive and healthy in the flow cytometer.
[Dr. Chris Beck]
Whatever you're putting it, getting a sample, and you think you have a lymphoma, you have a high pre-test probability, what's the preferred-- because I've done different things with them, RPMI, just having fresh sample on a wet Telpha pad. What's the preferred--
[Dr. Andrew Sholl]
Immediately into RPMI is preferred.
[Dr. Chris Beck]
Immediately into RPMI?
[Dr. Andrew Sholl]
Yes. You're going to want to fix some too. You're going to want formalin fix and RPMI but yes, immediately in RPMI, which is basically a cell culture medium that just keeps the cells alive.
[Dr. Chris Beck]
Got it. Why do we put everything in formalin? Why is that the default? What's so great about formalin? Why aren't we just putting everything in saline?
[Dr. Andrew Sholl]
Tissue after being exposed to air and saline--
[Dr. Chris Beck]
I feel like you're thinking about like a way that you can actually dumb this down for interventional radiologists that are listening. For people who don't see the audio, Andrew looked up to the sky and was like, how can I explain this to this guy?
[Dr. Andrew Sholl]
How can I explain this? Right. Basically, tissue dies when it's left away from its blood supply. In order to preserve architecture and the integrity of that tissue, you need to cross-link the proteins, cross-link the DNA, the RNA in that specimen. Formalin is a substance that basically cross-links proteins and DNA, RNA in the cells, in the individual cells. That preserves the architecture.
In that process, basically a water molecule is produced. You're kicking out water and cross-linking proteins. That's what's going to preserve that specimen for a long period of time so it can be processed and put under a microscope. Lung, for example, so lung has alveolar spaces, it's got the interstitium, it's got the blood vessels in there. If you let that sit on a Telpha pad, it's all going to disintegrate. The alveolar spaces are going to collapse. The tissue is going to degrade.
Then the architecture that, we as a pathologist require to see, okay, this is an alveolar space, this is a tumor cell, this is a pneumocyte, this is a blood vessel, that just melts into itself. The formalin is what allows that architecture and that integrity to be there.
[Dr. Chris Beck]
In terms of samples, and I know it's hard to paint everything with a broad brush, but I just want to see where this takes us. The way I think about it is bigger samples are better. That means not only by gauge but on length. I want to get your thoughts on that outside of thyroids.
[Dr. Andrew Sholl]
Yes, I agree with that.
[Dr. Chris Beck]
Okay, bigger is better. All right.
[Dr. Andrew Sholl]
Yes, bigger is absolutely better when you're talking about cores of tissue. Your trade-off is, am I going to give this person a pneumo? Am I going to, give this person a hemothorax? Yes, bigger is always better.
[Dr. Chris Beck]
All things being equal, bigger is better.
[Dr. Andrew Sholl]
Yes, if you can get a bigger sample safely, then you absolutely have to do that.
[Dr. Chris Beck]
More samples, always better?
[Dr. Andrew Sholl]
Yes, absolutely.
[Dr. Chris Beck]
How about if you're getting though-- sometimes I'll take 10 or 12 cores. The reason I'm doing this is sometimes I'm in a big lung lesion, and I feel like the needle, I'll be a little bit too central and so I'm getting what appears to me, on--
[Dr. Chris Beck]
Necrotic stuff.
[Dr. Andrew Sholl]
Yes, necrotic or I just feel like I'm not getting a good sample, but I'm putting it all into the same formula. I end up with 12 passes, but maybe only three or four of those are really good tan white tissue. Sometimes I think would it be better if you separate it out the "good passes" for the "bad passes" in two different containers.
[Dr. Andrew Sholl]
Yes.
[Dr. Chris Beck]
All right. Talk about that a little bit in that.
[Dr. Andrew Sholl]
If you're getting 12 course and say, seven of them are bad.
[Dr. Chris Beck]
Yes. Little shitty mushy fragment.
[Dr. Andrew Sholl]
In that sense, if you put that into one single formula and container, the processing of that sample has to go through a fixation process. We basically embed it with paraffin and then it has to be put in a flat plane into a mold where then we cut sections on it. Those tiny little cores, they're not 2D, they're 3D. They flow up and down a little bit and getting 12 pieces on the same plane is much harder than getting four good ones.
If you feel that your first couple of passes are bad, put those in one container formula and then the good ones that are separate. If you separate them out, then you can get the good stuff in a single plane much easier than trying to combine good and bad cores into a single cassette.
[Dr. Chris Beck]
How about if you have a big lesion and so all things being equal, you can get into any component of it safely? Is it at all helpful to have-- let's say we're in liver, a little bit of what looks like by imaging, normal liver on the periphery. If you're getting into a lesion and I don't think it's necrotic, so I can either bullseye and what you get is all core, all tumor, or I can get a little bit to the side and give you 90% core, a little bit on the periphery or, maybe 20% healthy liver and then 80% tumor. Does any of that make a difference?
[Dr. Andrew Sholl]
Yes, I think it would. I didn't know that you really could be that precise with it.
[Dr. Chris Beck]
Yes, we're like snipers. Not always and sometimes you can't be, but then sometimes you have total control of the situation and that is totally possible.
[Dr. Andrew Sholl]
The way that our brain works is that, the first thing that I look at when I get a slide is what's the specimen? Is it lung? Is it liver? My brain can turn on, what is the background supposed to look like. When my brain can turn on like what the background is supposed to look like, then it's much easier for me to pick out stuff that's not appropriate or not supposed to be there. Same thing with you when you're looking at a lung, you know from top to bottom what it looks like and okay, there's a lesion there. It comes up and it disappears.
That's not a vessel. It's not part of the normal background lung process. You can see it. The same thought process works for the pathologist as well in that if I know you're supposed to be in liver, okay, that is not a bile duct. That is not a hepatocyte. It helps me. In that sense, if you are as good as you claim you can be, then having a little bit of background liver in there would be helpful because then when I want to categorize that type of tumor, so is it HCC? Is it a metastatic colon? Is it something?
We have stains that we can put on it and I know that a hepatocyte doesn't stain with this, whereas a tumor would. If I have that background benign liver there that picks up the stain in the expected manner, then A, I know it worked. B, I know that the tumor is in fact real staining for this colon cancer marker. Yes, having normal stuff back there is super duper helpful. We call it an internal control. TTF is an immunostain that we use, for example, that's a marker of lung.
If you have a metastatic lung and it goes to the liver, the TTF will stay in the nuclei of the lung tumor-positive. What's interesting about hepatocytes is that the cytoplasm of the hepatocytes takes up that TTF1. I can see in the background that that TTF1 is staining the hepatocytes in a granular cytoplasmic way appropriately and the tumor in the nucleus appropriately, so I know that my stain worked, it covered the tumor, and it's a real positive stain.
[Dr. Chris Beck]
Got it. Right. Rather than a false positive. It's like an internal control there.
[Dr. Andrew Sholl]
Yes. The internal control is super helpful if you guys were that good.
[Dr. Chris Beck]
Like I said, depends on the day, depends on the doc. All right. We said bigger is better. How about-- and I know it's hard to say, but if you're getting good high-quality samples, how many samples would you say-- what's the bare minimum? What's like, all right, you got it. That's fantastic. Then what's like, all right, man, if you're taking 20 great cores, I just don't need that many. You know what I mean?
[Dr. Andrew Sholl]
Yes.
[Dr. Chris Beck]
I bet if I had to guess, it's probably on a spectrum. Too few is too bad. Too many it's--
[Dr. Andrew Sholl]
How long are your cores? They're 1 centimeter long, basically.
[Dr. Chris Beck]
Let's say, let's say 2-centimeter cores, 2-centimeter 18 gauge cores.
[Dr. Andrew Sholl]
2-centimeter cores, yes, you need 3.
[Dr. Chris Beck]
Three?
[Dr. Andrew Sholl]
You need three to four to do what you need to do, like in a lung tumor.
[Dr. Chris Beck]
That includes next-gen sequencing?
[Dr. Andrew Sholl]
Yes, exactly. Two of those I'll need for, the diagnosis. Then the two can go for PL1, next-gen sequencing, all the adenocarcinoma, BRAF, whatever you need. Let's say four.
Podcast Contributors
Dr. Andrew Sholl
Dr. Andrew Sholl is a pathoogist at Delta Pathology Group in New Orleans, Louisiana.
Dr. Christopher Beck
Dr. Chris Beck is a practicing interventional radiologist with Regional Radiology Group in New Orleans.
Cite This Podcast
BackTable, LLC (Producer). (2024, March 5). Ep. 422 – Pathology 101: Solid Advice for Percutaneous Biopsies [Audio podcast]. Retrieved from https://www.backtable.com
Disclaimer: The Materials available on BackTable.com are for informational and educational purposes only and are not a substitute for the professional judgment of a healthcare professional in diagnosing and treating patients. The opinions expressed by participants of the BackTable Podcast belong solely to the participants, and do not necessarily reflect the views of BackTable.
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