#pathologysaturdays

2) How do you do a necropsy?

Everybody has a slightly different technique, but in the end they should reach the same result. This is a quick run down of how I approach it (this is where we’ll get into some discussion of actually dissecting bodies, so skip this if that makes you queasy).

1) External exam: The first step is to look at everything you can see without opening the animal. Same as a physical exam on a live animal, we assess the fur/feathers/scales, skin, eyes, ears, inside the mouth, claws, and so on. This is important because it can start you down the right track as to what you’re going to find inside. For example, a cat that has been hit by a car usually has shredded claws, because they try to grab onto the road at the point of impact. Or if you find blisters in the mouth and between the toes of a pig you might want to quickly get on the phone with the state vet because you suspect foot and mouth disease! We also take note of identifying features of the animal (colour, brands, microchip number, ear tags etc) so that the findings can be matched to the right animal (which can be contentious in insurance or legal cases).

2) Opening the animal: So now I’m ready to start cutting. How you position the animal to start depends a bit on the species, but for your average mammal you’ll lay them on one side. I then get the legs, skin, muscles, and ribs out of the way so that the abdomen and thorax are open for inspection.

3) Stop and look for a minute: Before we take anything out it’s important to take a moment to look at how things are sitting. Are all the organs in the right place? What colour are they? Is there any fluid there shouldn’t be?

4) Examine the organs: Now that we’ve taken note of how things were, we can start taking things out and getting a more detailed look at them. Every organ gets its moment in the spotlight. Tubular organs get opened so we can look at the inside, and parenchymous (solid) organs get sliced into so we can see anything going on in the middle of them. And while we’re doing this, we take a piece of everything. Some things we’ll keep fresh for microbiological or toxicologial testing - lung, liver, spleen, and kidney usually, occasionally brain if it’s indicated - and everything else goes in formalin to preserve it for looking at under the microscope later.

5) Tidying up: After we’ve looked at everything, it’s time to clean up. This depends on what the owner wanted. Animals are often sent off for cremation after this process, but if the owner wants to animal back we can do what is called a “cosmetic necropsy”. When we do this we just get into the animal through one incision along its belly, which we stitch back up when we’re done. Overall, we try to be as respectful as possible, and if you do have concerns about the animal you are submitting for examination, you should talk to your pathologist! We’re happy to answer questions and will try to accommodate your needs during the process.

6) Writing a report: Every necropsy performed should be followed by a report lisitng everything we saw while we did it. This is a legal document which includes everything that was abnormal, everything that was normal, and anything needed to identify the animal. 

3) So wait... why are we doing all of this?

For a lot of people, it can be hard to understand why we are still looking into what was wrong with an animal after it has died. And I get that, we’re a bit past the point of helping that animal once it reaches the necropsy floor, so who cares?

Well for one, in a significant number of cases the animal dies without us knowing why, and that makes it hard to find closure. Maybe your pet died suddenly, or it was being treated for something but died anyway. With a necropsy we can either give you a solid answer as to what caused your animal to pass away (maybe there was a tumor you didn’t know about, or a clot) or we can tell you that there’s no evidence of anything that you could have helped or prevented. That knowledge and reassurance can help owners process the death of their animal, and I’ve seen it make a big difference.

Secondly, if there are other animals involved or at risk, we can find out how to help them! This reason is most often applied to production animals where we can figure out herd health problems, but it can apply to pets from a household with other animals as well. If your dog dies from getting into something toxic, it’s good to know that happened so you can remove the poison and possibly save your other dogs from the same fate. Or if there’s an infectious disease moving through your herd of cattle, now you know how to treat or what to vaccinate against. It may be too late for the animal on my table, but that animal might still be able to help the others around it.

Thirdly, that information could help save animals you don’t even know. The information we find at necropsy can educate the vet who was treating it, students present for the examination, and the veterinary community at large. Maybe your animal presented unusually for a known disease, so your vet didn’t recognise what was happening. That’s sad, but vets are human too and can’t know everything. But now that they know what killed your pet, they’ll know to check for that in the next patient with the same presentation. Veterinary students see and participate in necropsy at school so that they can know what lesions look like before going out into practice. The more they can see, the more knowledge they are equipped with before your sick pet is in front of them. And maybe your animal had something wrong with it that nobody knew happened. If we can see that at necropsy we can publish information to teach vets all over the world that this can happen and we need to find out how to fix it.

The difference between the two specialties comes down to the types of things we look at and the methodology we use to come to a diagnosis. This affects not just what we do, but where we work and how quickly we can give you an answer.

3) What does an anatomic pathologist do?

Anatomic pathology covers two main diagnostic methodologies I’ve already written introductory posts on - necropsy and histology. Those posts are linked about but the quick summary is this:

Necropsy - dissection of a cadaver and examination of the tissues with the naked eye to look for lesions (changes associated with disease or injury) and collect samples for bacteriology, toxicology, and of course, histology.

Histology - microscopic examination of slides made from thinly sliced tissue specimens collected at necropsy or surgery (biopsies). With this we can look at the types of cells in the tissue and how those cells fit together (the architecture).

Structure is an important feature assessed in anatomic pathology, so the techniques we use maintain that. As opposed to...

4) What does a clinical pathologist do?

Clinical pathology includes interpretation of blood tests (biochemistry and the complete blood count), urine tests, and cytology. Now I’m not an expert on any of this but I’ll quickly explain cytology because it’s where the two specialties tend to get confused.

Cytology - microscopic examination of slides made with cells where the tissue architecture is not maintained or assessable. These cells are collected through aspiration (sticking a needle in something), scraping, or pressing someting against the slide. You get a random selection of whatever cells can be picked up by the method that you choose. You can also look at fluids (which may or may not contain cells) this way. 

5) What else is different?

Residency - from my understanding, many anatomic pathology residencies will take you straight out of vet school (how I got into this). Clinical pathology residencies tend to prefer that you have experience in clinical practice, and often like you to have completed a rotating clinical internship as well. In some places a pathology residency will cover a bit of both specialty though, so things can get complicated!

Where are things done? - Parts of each specialty can be done by a general practice vet in clinic, but obviously there are added benefits to consulting a specialist. A lot of GP vets will do some of their own clinical pathology - in-house blood analysers are becoming more common, where you can get point of care results for quick analysis, and most clinics have a microscope so they can take a quick look at cytology to make the decision to ask a specialist or not. GP vets can also do their own necropsies, which is a good way to reduce costs. However GP clinics are not set up to do their own histology - that requires a lot of processing that isn’t plausible within a GP clinic, and vets aren’t routinely trained in much histopathological interpretation.

I have heard of specialist clinics with in-house clinical pathologists for convenient consultation by other specialists. This allows them to work closely with internal medicine over blood and urine results, and cytology of fluid pulled from abomens and chests; and both surgeons and medics can benefit from other cytological examination. Anatomic pathologists, largely because of the need for histology processing equipment, tend to be more affiliated with diagnostic labs, although there are many of these located close to or associated with teaching hospitals.

So that’s the basics of the difference for you guys! Any clinical pathologists out there, feel free to chime in and correct me because I obviously don’t have a lot of experience with the specifics of becoming and being a clinical pathologist. 

Histology is defined as being “the study of the microscopic structure of tissues”. What that amounts to practically is this: we receive a tissue, whether that’s a biopsy specimen or a piece of tissue we’ve collected at necropsy. That tissue is fixed in formalin, then dehydrated in alcohol, rinsed, and set in a block of paraffin. Then that block is sliced very, very thin, aiming for just one cell layer. That is stained with whatever we need to use to see what we’re interested in (there are a lot of stains, the most common is hematoxylin and eosin, usually known as H&E). That provides us with a slide we can put on the microscope to see something a bit like this:

This is a piece of horse tongue stained with H&E I examined recently. We look at the shape, size, colour, number, and other characteristics of the cells in a section to decide if they’re normal or if there’s disease going on. 

2) That’s all cool, but why do we do it?

I’m glad you asked! There are a few different things we need histology to identify. This isn’t an exhaustive list, but it should cover most of the big ones.

a) Histology provides more information about a lesion we can see grossly. Say an animal shows up to the local vet clinic with horrible ulcerated skin. We know it’s probably inflamed, but why? And what cells are involved? Taking a piece of that skin for a pathologist to look at under the microscope can tell you a lot more about the underlying etiology (inciting cause) and disease process that is resulting in your dog suddenly being kinda gross. And this information can be very important for guiding treatment and gauging prognosis.

b) Histology is best for telling you how much you need to worry. This ties in with a above but I felt it needed its own section. A mass on an animal could be a lot of things. It could be inflammatory (granuloma, abscess etc) or it could be neoplastic. And within neoplasia, it could be benign or malignant (scary). Cytology can tell us a lot, but to truly gauge how scary most tumor types are, we need to look at their structure, infiltration, mitotic rate, and more. And on that...

c) We need histology to see if you got the whole thing out. This is mostly a concern with neoplastic masses, but can be relevant in the inflammation family too. Only histology can truly assess the size and quality of surgical margins. A mass may look discrete grossly, and be a nice clear round thing, but you don’t know what tendrils it has snaking out into apparently normal tissue. When we perform histology on a mass, we purposely take sections that allow us to assess the margins a surgeon has left and tell them how likely it is any scary neoplastic cells have been left behind.

d) Histology can find disease where you couldn’t see it grossly. We see this more with our necropsy cases because usually something normal looking isn’t biopsied, but it’s an important thing to remember! We get a fair number of necropsy submissions where the owner is adamant they don’t want histology done. Which I can understand from some angles, it can be expensive! But we can’t tell you anywhere near as much if we don’t do it. I’m currently working through a case that we thought the answer to would be in the brain, instead we stumbled across a mesenteric vasculitis (inflammation of the blood vessels within the stuff that holds the intestines in place) which has changed our whole perception of the case and changed the differential diagnoses (possible diseases) entirely. And below is a case I saw with biliary hyperplasia, a change that can be indicative of liver disease but you can only see when you do histo:

3) So when should we be ordering histology?

This can be a tricky question, because as I said above it can be expensive and I know that not everyone can afford this type of testing, especially when it may or may not provide any answers (casual disclaimer there, while we can find a lot of things, sometimes we also... don’t).

My opinion is that any mass you are taking off an animal is worth histo. With maybe the exception of a lipoma, which can be pretty happily diagnosed by gross characteristics and cytology. My recommendation is: if you’re worried enough to take it off, you’re worried enough to know what it is. Where I work, submitting a surgical biopsy for histology is $60 USD. It’s not cheap, but it’s not crazy expensive either. We may ask for more testing (like IHC) which would add to the price, but we can give you a lot for just that $60 starting price. 

Most necropsies should also get histology done. ESPECIALLY if nothing is grossly evident, or the gross findings are non-specific. Occasionally the cause of death is pretty obvious, but usually it’s not. And when we give you a gross morphological diagnosis, we completely reserve the right to change that when we see the histo! Redness may suggest inflammation, but it could also just be congestion. A normal looking kidney might be acutely inflamed and full of calcium oxalate crystals. We just can’t see enough without looking at the cellular make up of the thing, so the money you spend getting the necropsy done is going to be worth a lot more if you let us do the histo as well. Or it might be cheaper for a clinical vet to do a necropsy and send us a jar of tissue. That’s still likely to give you a better result than paying a pathologist to do a necropsy and not letting us use our more powerful, specific tools. 

That’s it for today folks! If you’re still reading, thank you! I’m writing these really hoping to help both clients and veterinarians get a better look into what we pathologists do, and why we’re suggesting all this testing. If you have any questions, definitely send them my way, you can do that through a reply to this post, a direct message, or an ask. I do like receiving them as asks so I can share with everyone, because as all our teachers say “if you have a question odds are somebody else had the same one”. 

I have a few topics I’m considering for next week, let me know if you have a preference or I’ll just pick what I’m feeling like. My current options are:

1) Anatomic pathology vs clinical pathology 2) The pros and cons of cytology vs histology 3) How to get the most out of your biopsy submission 4) To ink or not to ink? That is the question!

2) Fix your sample well! A poorly fixed sample will not only be delayed at the lab while we wait for it to fix, it’s also not going to give results. Why is it delayed? Unfixed biopsies are hard to trim well. We have to cut anything you give us into thin slices to even start the process of making a slide, and if it’s not firmly fixed we can’t do that well. Why is it going to give bad results? The longer we have to wait for something to fix, the more the cells are going to autolyse (break down). There is far less information in autolysis than in a sample where the cells look exactly how they did when they were still attached to the animal.

How do you fix a sample well? You should have a formalin:tissue ratio of 10:1 (if using 10% buffered formalin). That means ten times as much formalin as tissue. It’s annoying, I know, but it’s worth it. Also, if the sample is really fatty, consider using more formalin. Fat will partially dissolve into the formalin and make it less effective. For samples that are really bloody (spleens!) try to minimise how much blood makes it into the jar, because this fills up the formalin and then the formalin is wasted fixing all those loose blood cells instead of the tissue you actually want us to look at.

3) Use a sensible container: A good container should be hard to break (for posting, plastic is usually better than glass) and water tight (screw on lids are best). Most importantly, it needs to have a wide opening! If you have to squish your sample through the top when it’s fresh, we are not going to be able to get it out fixed. Samples fix to be very firm, so it won’t squish any more, and they often fix in weird positions - so if it’s something long and thin, it might fit through fine when you can hold it out straight but it won’t when it’s fixed curled up in a ball. Ideally pick a container where the opening is the same diameter as the container. That way you know that if the container is the right size, so is the opening. Good containers look like this:

Bad containers look like this:

4) Make margins or areas you are concerned about clear: If you are particularly worried about one part of a sample, that needs to be easy for us to figure out, and it needs to take into account that things look different once they’re fixed. For example: if you are submitting a uterus you just took out, and you think one ovary looks a bit off but it’s not 4 times the size of the other, find a way to show on the sample which one you’re worried about. Telling us left or right isn’t helpful when it gets to us and isn’t in the animal any more. It’s safer to assume that we won’t know what’s what by the time it gets to us. So you should utilise things like sutures, ink, or separate jars (cut the funky ovary off and submit it in its own pottle!) to make it obvious. And write down what that means on your submission form!

4a) Using sutures well for identification: Write down the colour and look of the suture you’ve used (eg: blue, smooth nylon or purple woven). This is especially important if you’re trying to mark out several things. You can also use different lengths of ends (but make it obvious, cut them very short and very long). More knots is hard to interpret, but more pieces (if they’re nice and close together) can work. We can take the sutures out once we’ve got all the infomation we need so it’s a good way to tell us things without risking negatively affecting what information you’ll get back.

4b) Using ink for identification: Ink can be great if you know what you’re doing. We ink things all the time to identify margins, so you need to make sure that what you’re doing won’t impede how we can then interpret it. If you really want to use ink, I would suggest calling your diagnostic lab and asking for advice. One thing you can do is ink a non-critical surface. What does that mean? If you’re submitting a skin mass, put ink on the haired skin side only. We don’t need any diagnostic marking on that surface because the top side isn’t a surgical margin. So if you want to put some ink on the hairy side, showing us which margin is which, that’s fine! Avoid putting ink on any aspect of the sample you have cut through, because those are the ones where ink can give us serious diagnostic information, so if it’s not done right you are going to lose out. In general I would say that the best markers you can use are ones that we can take off. Ink is permanent so again, if you’re not sure, don’t do it.