Today, I'm going to talk about hypercalcemia in dogs and cats. So just as a brief introduction, I'm going to focus mainly on the ETopathogenesis and then talk about diagnosis. And then clinic, well, clinic in that order, as we've got in an introduction, but focused mainly on the ET pathogenes and diagnosis.

And then go through a few clinical signs, etc. And then talk about treatment right at the end. Now, the treatment, I don't really have any current experience of just what we know from textbooks, references, other clinicians.

So that will just be Sort of a summary of that. So to start off with, with respect to calcium. Just going to run you through exactly what calcium is composed of.

So the total calcium that you measure in blood, so the total serum or occasionally plasma calcium is made of 3 different parts. The main part, which is about 50 to 55% is ionised calcium, and that's the active part, and this is the part that we need to look at. The other two parts, which one is the protein bounding, bound one, which is mainly albumin.

And the complex calcium, which is complex to other sort of ions like phosphates, lactates, carbonates. Form up less than half and are not important with respect to the calcium levels in the body. So in the dog and the cat, the ranges for total calcium and ionised calcium are fairly similar, and they're quite tight.

The cats have a slightly lower bottom normal range, and the dogs are narrow range for the ionised calcium. So in the lab, total calcium is usually measured by spectrophotometric methods, usually stum, but you can do it on plasma. And just to remind you, you need to avoid using the anticoagulant tubes, so EDTA, citrate, etc.

Because they will chelate the calcium and therefore, you, you end up with a low result. And it's often a low calcium, it's often an indication that you've got EDT contamination, EDTA sorry, contamination of the sample. So we need to avoid that, so serum, preferably.

And then ionised calcium, you can actually, there are machines obviously in-house that can do this and the blood gas machines are one of the ones that does, that does do this and, and they measured by an iron-specific electrode. The sample collection ionised calcium is different. We don't want gel tubes because this can contain calcium.

And also, it needs to be collected anaerobically and preferably separated rather than left as whole blood. We do have correction equations with respect to changes in pH, which will alter the the ionised calcium. So it's separated serum excluding air, and it doesn't need to be frozen because in some cases, if you do freeze it, it will actually artificially lower the ionised calcium.

You can keep it at room temperature to chilled for quite a long time without it deteriorating. So going on to pathogenesis, the, I'm just going to go through calcium regulation and just, you know, go through the actual hormones that are involved, and then how we end up with this state of hypercalcemia. So the main hormones involved with calcium regulation are the parathyroid hormone, which is the main one.

And we had the dog, in particular, has two sets of, or two pairs in the neck, which are quite tiny, and they sit in the thyroid gland. And they are responsible for the minute to minute control of the calcium. They've the bone and the kidneys to increase calcium.

The next important one is calcitriol or vitamin D3. And this is the active form of it is, is, at the kidney. So, in its its effect is on the day to day control, so it's more longer term control of the calcium.

And it is activating the kidneys and in dogs in particular, and cats, the liver can store vitamin D, and this is in the non-active form, and then it goes to the kidneys to be activated. Calcitrol or vitamin D3 affects 3 different sites. It affects the intestine as well as the bone and the kidneys to increase the levels of ionised calcium.

The calcitonin I've mentioned at the bottom there is not particularly important in our animals, so we don't tend to, to look at it or refer to it. And the diagram on the side is just a, a sort of like illustration looking at PTH and showing the three main sites there that we've got. We've got kidney, bone, and intestine.

So just a diagram, the parathyroidro is labelled there as a tiny gland on each side, and it sits in the thyroid gland. And it's obviously one of the things that could be at risk of being removed if you're doing surgery for hyperthyroidism in a cat and therefore a risk of post-op, low calcium. So primarily, hypercalcium, hypercalcemia results due to either an increased bone mobilisation of calcium.

Or decreased urinary calcium loss. Now, I've put the increased intestinal calcium absorption as in a smaller font there because it, it does have variable contribution, and it is mainly the other two that result in the, the pathogenesis, of the hypercalcemia. But obviously, if you get vitamin D toxicity, then that sort of, that source is obviously a much greater contributor.

So hypercalcemia, by definition. Though total calcium is usually increased, total calcium is an insensitive method as indicated before, due to the fact that the ionised calcium is the fat, is the part that we're looking at, and that's the active part. So by definition, if you get a high dose of calcium, you should really confirm it with ionised calcium.

So hypercalcemia, high ionised calcium, and with the state of hypercalcemia, What, what should happen is you get decreased PTH or parathyroid secretion from the parathyroid gland and you get decreased calcitriol or vitamin D production or activation, I should say from the kidneys, and therefore it should result in an increased urinary calcium excretion. And also decreased mobilisation of calcium from the bone. So, the diagram here, I don't know if you can, yeah, you can see the top bit.

So this is just to show the, the negative feedback. In the, in the normal animal that has a functioning parathyroid gland, the, The parathyroid, is negatively, there's a negative feedback effect of it by calcium and phosphorus. So if you increase calcium and phosphorus, this has a negative feedback on the parathyroid gland and therefore decreased production of parathyroid hormone.

The calcium and phosphorus also has a negative feedback on the kidneys and the activation to the active form of vitamin D3. And there, it just shows vitamin D3 have an effect on the, has an effect on the bone and the intestines, as well as the, kidney. So just to go through a bit about vitamin D.

I was, I was looking in this and I, looking at this and I thought, oh, vitamin D, yeah, vitamin D3, that's just, you know, vitamin D. But vitamin D is a group of compounds and there's not just vitamin D3. The two most important ones, well, vitamin D3 is the most important and then the next important is vitamin D2.

And these two are important with respect to possible pathogenesis of hypercalcemia in our animals. Now, unlike humans, dogs and cats have a limited or no ability to synthesise vitamin D in the skin, and they're sort of quite similar to us in our country where we get sun. Not the last time, and therefore require vitamin D from another source, usually orally.

So the two main sources of vitamin D, D2 and D3, so holy calciphol, which is vitamin D3. This is found mainly in animal products. And then we've got ergocalci calcipherol, vitamin D2, which is usually a plant origin.

Now, cats and dogs have different diets and therefore, dogs, which are omnivores, can deal with both of these vitamin D's. So if they consume both of these, they can metabolise and activate both of them. The cats, unfortunately, are carnivores and therefore, they can only utilise vitamin D3.

So if for some reason you were supplementing a cat, For whatever reason, though it's usually, it's probably unusual with vitamin D2, it probably would have minimal effect on the actual calcium levels. The Both those two, vitamin D2 and D3, sorry, they are converted in the liver to this non-active form of D3, which we can actually measure, which is called calcidiol, another name for vitamin, another different name to. So I think we just call it active and inactive D3.

It's just a diagram to show that there's a, there's the D3 choliccalciferols actually inactive, and then it requires a hydroxylation process, which occurs in the kidney to form the active form of it, which is 125 dihydroxy vitamin D3. And the, the, in the humans, where it comes from the skin, it is actually, again, it forms the inactive form prior to being activated. So just to, there was just a brief table there so you could compare them, but D2 tends to be plant origin and D3 is the main one that we use for supplementations, particularly in humans as well.

So just to go through, vitamin D is metabolised in the liver to the 25 hydroxy vitamin D, which is calcidol, and then this goes to the kidneys and becomes hydroxyized to, which is the form of vitamin D. This then results in the three processes there to a varying degree. The hydroxy enzyme as I sort of alluded to previously, is also controlled by PTH to a degree and also the levels of calcium and phosphorus as to its activation.

So you'll get an increased level of hydroxylation and vitamin D with increased PTH, and you'll get a decrease if we have high phosphorus for some reason or other, particularly, say, in kidney disease. So, coming to hypercalcemia, the, there are two groups of causes which are named either primary hyperparathyroidism. Or secondary hyperparathyroidism, or they name them also PTH dependent, which is the primary hyperparathyroidism, or parathyroid or PTH independent hypercalcemia or hyperparathyroidism.

So there's all long, long names. So basically, we have a primary and we have a secondary, causes of the hypercalcemia. The primary hyperparathyroidism is an abnormality of the parathyroid gland.

It's usually unilateral and most commonly it's an adenoma involving one of the thyroid, sorry, parathyroid glands. Occasionally, you'll get hyperplasia and less commonly, you'll get a carcinoma. It causes an overproduction of parathyroid hormone, which is not negatively, fed back by the calcium levels that it increases.

So, though the calcium levels are increased because the PTH has increased, They don't have a negative feedback effect on the parathyroid gland because it's abnormal. Now, in unilateral cases, the normal gland will obviously have a normal function and negative feedback and therefore it will often become very tiny, and the big, you know, the abnormal gland remain large or bigger. So, in these cases, we have a high parathyroid hormone level, and we have a high ionised calcium.

In secondary hyperparathyroidism or parathyroid-independent hypercalcemia cases, there are lots of different processes that, that can result in this. And then one of them is to mimic the PTH and form these parathyroid-related proteins. And there are also interleukins, and things like tumour necrosis factor, various cytokines which are produced that can affect vitamin D and therefore affect calcium.

So, in some of the cases, obviously, we have excess vitamin D causing hypercalcemia and that's a direct effect of the vitamin D. The, in these cases, the ionised calcium is high and the PTH because it's been negatively affected by the high calcium is low or very low. Now, in occasional cases, we'll get PTH, which is in not low, as in not low, low, but in actually in our reference interval that we have.

Now, when we look at these cases, it needs to be in the lower third of the reference range for the PTH for us to say that this is not a primary hyperparathyroid case, which sounds a bit sort of very close cut, but that's, that's how it works. So there are transient causes of hypercalcemia, which may result in a high total calcium, but the ionised calcium is not affected, and this may include Things like dehydration, you get a sick animal that comes in dehydrated or it's got hyperproteonemia, etc. The increase in albumin and protein levels and that can cause increased binding to that and therefore resulting in an increased total calcium.

And then I put on the the hypogenalum, which actually Does seem to be one of the more common causes of, of increased calcium. And some of these, about 50% have a high total calcium, but only about a quarter will actually have a high ionised calcium if measured. And then there's some nonpathologic causes, particularly remember, young animals may have higher calcium.

And then there's the lab error. You need to try and rule that out. I do know for a fact that if we do get significant hemolysis in some of the samples in the lab, our actual assay is affected rather than increasing calcium, it tends to lower the total calcium to a slight degree.

So we need to rule out any spurious or labris. So, just going, now going on to the actual causes in a bit more detail. So primary hyperparathyroidism is our, our primary cases and the PTH dependent.

And then we've got these independent cases, which are not related to a high PTH and have other mechanisms. And neoplasia is probably the most common. And then we've got various other causes include Addison's renal disease, usually chronic kidney disease and idiopathic, quite common in the cats.

So there's a picture of an anal sac adenocarcinoma, which can result in hypercalcemia. So, in order of most common neoplasia by far is the most common of the PTH independent types, hypercalcemia. And then in the dog, we've also got Addison's and then we have primary, which is less common, and renal failure, which, as I say, in most cases in renal failure and in, in acute, sorry, in chronic renal failure, it was actually the total calcium that's high and not the ionised calcium, but there are some cases where the ionised calcium does increase.

So less common causes just listed a few here. But there's also things like, local osteolysis from infections, and mediated disease. There's also, overdosing if you've been using calcium, containing phosphate binders, say, for in treatment of chronic kidney disease.

And also, occasionally, there's reports of grape or raising toxicity, which in most of these cases, actually, by the time they're found, they've actually consumed quite a lot of this and the result is quite severe disease or even fatal. So it, it's not common, but it does occur. So just a bit more detail on neoplasia.

This, as I've said before, it's an independent PTH independent hypercalcemia. There's a high ionised calcium, and there's usually low or low normal PTH. Now, PTHRP we can measure that, and that has increased in some of the cases, but not all.

It's often, high with, or in in most cases, I said, would say anal sac, adenocarcinomas, but also lymphoma. And then there are other sort of hematologic type malignancies like multiple myeloma that can result in high calcium and metastatic tumours to the, to the bone. In cases of malignancy, it's usually the hemoral causes that are resulting in this high calcium.

And one of them is to mimic the PTHRP and some of the tumours actually secrete a similar protein, which can be measured, which is we call PTHRP. And this has the same effect as PTH on the, the receptors to increasing calcium in the, the kidneys and bone. There are also cytokines, which I mentioned before, which can have, similar effects, particularly in activating or making vitamin D and therefore affecting calcium.

So in lymphoma, probably about The 3rd, or round about that will have hypercalcemia, and they're usually associated with a T cell type lymphoma. Anal sac adenocarcinoma, as I mentioned, they often show hypercalcemia. So, as I say, look at this, only 50%, in the references that I've found actually demonstrated.

So there are cases that you can have that and they will not have the, co-existing hypercalcemia. The other neoplasmins I've mentioned before, and they do include things like carcinomas and melanomas, they can be, they can result in high calcium. So the local osteolytic effect, as I say, it tends to be more the metastatic tumours that cause this.

It's very rarely that you'll see primary bone tumours resulting in a hypercalcemia. So, you know, if you have an osteosarcoma, it's unlikely to, if you go and measure calcium in those cases, for that to be high. So it's mainly, as I say, metastatic, causing the local effects with releasing calcium from the bone.

And then we've got Addison's, which I've mentioned. It does tend to be quite mild and obviously, it's reversed when you have adequate treatment and of the, of the Addison's itself. The chronic kidney disease in most cases have a normal or low ionised calcium.

And it's the total calcium that's elevated. And then I'll come on to it, but I've mentioned at the bottom, and I'm sorry, on some of these slides, there's quite a lot of detail. So don't, don't worry about, you know, trying to copy them.

I, you can have a copy of the slides if you would wish. But there, in chronic kidney disease, you can get a form of tertiary hyperparathyroidism which becomes more complicated, and the exact cause is not completely, understood. So in chronic kidney disease, just a bit more detail, you can actually get hypercalcemia secondary to the treatment of it, as I alluded to before, with respect to, if you're using calcitriol for some reason, or you've got these, phosphate binders that contain calcium, and this can result in increased calcium.

However, if the treatment is monitored and everything, this should really be, not happen that, that commonly. And usually you can, you can use them at quite low doses with reasonable effect. The tertiary hyperparathyroidism is usually the result of these chronic kidney cases going through a stage where the low or low, sorry, the normal or low calcium, ionised calcium causes an increase in PTH.

And then, Combined with the uremic toxins, you get altering and decreasing the number of the actual receptors in the PTH for the calcium and And also with it affects the vitamin D activation with when you have renal, you know, tubular disease, and we end up with this low calcium affecting a high, causing a high PTH, which then for some reason, sets the calcium, it increases the calcium and then sets the calcium level in which is normally set by the hypothalamus, hypothalamus at a higher level and therefore it thinks that this higher level of calcium is normal and therefore, these cats, these cats and dogs become. Hypercalcemic, but they also have high PTH. So just to remind, in most cases of chronic renal failure, the reason why they have high calcium, it's the total calcium that's high.

And for some reason they, in these cases, they will have an increased complex part, which is the one that's complex to the phosphate citrate or lactate. So if you measure ionised calcium, if you see on this diagram, they're still the same. So primary hyperparathyroidism.

It is uncommon. And as I say, most of the time there, it's an adenoma affecting a single parathyroid gland. The ionised calcium is high and the PTH is usually high or high normal.

And in most of these cases, you, you can't palpate a mass, and ultrasound in some cases can be helpful, but in some, it doesn't actually, identify mass either. In some cases, you can actually have both glands affected. And then in those cases, they may be easier to, to detect, but that just depends on the, the actual, case individually.

The keyshound appear to be genetically, predisposed to this disease, and there are reports of these cases, these animals developing primary hyperparathyroidism, and then receiving treatment that's worked, and then it's reoccurred several years down the line with another. Case of primary hyperparathyroidism. So vitamin D, this is mainly an overdose of some compound or some, some plant or anything that has a source of vitamin, vitamin D that, that's excessive intake.

And it can be due to, as I say, overdose with, calcitriol, which is, one of the treatments when we use with hypoparathyroidism. So we're now talking about the reverse, so a low calcium. And also, if you ingest certain plants, which cats tend to like things like that.

And also the psoriasis creams, the anti psoriasis creams often contain vitamin D, and cats and dogs seem to love them in some cases and therefore can result in toxicity. The warfarin antagonists or vitamin K antagonist type poisons, if you look at these, most of these in the recent years have been had a lot of vitamin D added to them, and they use this as part of the way of causing the lethal effect to the, you know, the animals they're trying to kill. So you need to be careful with that because we, we can also get significant vitamin D toxicity and hypercalcemia from that, as well as trying to, cause a coagulation problem.

So just a bit more about the vitamin D, but the vitamin D toxicosis can actually be very severe and it is related to the amount that is consumed by the actual individual. And the more that they consume and the more rapid the effects and the increase in calcium, the clinical signs are therefore more severe. And you often see high phosphorus in these cases, and they can, if they're not detected early enough, can result in tissue mineralization and therefore, the prognosis becomes poorer.

So these cases are the, the PTH independent, they have a low PTH and they they usually have, in, in most cases, it depends on the source, a high 25 hydroxy vitamin D or the, the calcidol. They, in some of these cases, particularly the plant and sometimes the psoriasis creams, the 25 OHD or the calcidol will be normal and therefore you need to measure the calcitriol, which is the 125 dihydroxy vitamin D, if that's normal. Sorry, if the other one's normal, the 25 hydroxy, vitamin D.

Just brief mention granulomatous disease. This is always one of the differentials that's thrown in, and it does occur. The, the mechanism appears to be to do with cytokines and macrophages and increasing the synthesis of calcitriol or vitamin D3.

And any granulomas disease could be classed as Animals with injection site reactions or a widespreadendiculitis or something like that. The blastomycosis I mentioned, I mean, these are obviously going to be more common in, in other countries rather than the UK. It's not really a common, presentation that we get systemic fungal disease.

Obviously, in these cases, the underlying disease process may actually present as, that's the, the actual presenting clinical signs rather than the, the high calcium, which may be found incidentally. So in cats, just, I just mentioned them briefly because in cats, the courses are all similar in cats. And the same things apply.

Again, neoplasia, probably the most common, and then chronic renal failure again can be related to, obviously, as I keep saying, it's mainly total calcium in these cases unless they have that coexisting tertiary hyperparathyroidism. And then in cats, What's, what seems to be most common, and, and they do report it as the most common in the states, but it's probably getting quite common in this country, is idiopathic. Now, this is really a diagnosis of rule out where you, you've excluded all the other causes, and we can't find the reason why this cat has high calcium and therefore, it Just has to be managed.

Now. Luckily, most of these cases actually show no clinical signs, or the ionised calcium itself is only minimally elevated. So they tend to do quite well if it can be controlled.

I don't know why it's caused. They, they say there's some sort of increased sensitivity to vitamin D in some of these cases, or there's some dysfunction in the, the calcium receptors. It's not, it's not really known, exactly what causes it.

So just a brief thing on signalment, just remember. Addison's in the dog does tend to affect younger animals. Now, obviously, it can be at any age that's affected, but it, the mean, sorry, the median age is about 4 years.

And there's various breed predilections, predilection, sorry, and genetic basis. So just need to remember when, when this might be a possible underlying cause, if you've got a hypercalcemia that's picked up. Primary hyperparathyroidism.

Though, and it is less common, is an older dog, disease. And as I said, there is a genetic basis in the keyo and other, breed predilections, as well. And some of them are listed there.

And as I've mentioned before, idiopathic in the cat, wide range, no sort of pre-liction to any particular breed or any age, and apart from possibly long hairs, maybe overrepresentative. And again, primary hyperparathyroidism is quite rare in the cats. I think some of these might get overlapped with older cats that have chronic renal disease and they have the tertiary hyperparathyroidism.

So, it again, like the dog, primary hyperparathyism, hyper, sorry, hyperparathyroidism, is not that common. So going on to clinical signs, I've listed some clinical signs here. We talk about dogs first, but in a lot of these cases, these animals have come in for another reason.

And when you've done the screening on the blood, you've picked up a high total calcium or it's only marginally. You know, increased and therefore, they're presenting signs aren't always the signs that you see here like a PUPD case. So coming thinking back to it, quite a lot of presentation of older or not necessarily the older animals, but animals coming into the vet is, yes, it's PUPD.

So remember, it's always a, a differential for that presenting sign. It can also be very vague, clinical signs, they're off colour, or food, etc. And if they've got a particular cause, not just obviously primary hyperthyroidism, but other causes, it may be that disease process that's more obvious than, you know, the, the actual results of the high calcium.

And remember that obviously Addison's normally has, can have a very variable presentation and it can be difficult to diagnose. But primary hyperparathyroidism may only present with, say, a dog that's got repeated or a cat, sorry, with repeated UTI infections, or you've got urus, so calcium oxalate, urulus. So without any other clinical signs.

In the cat, they often show very little, and as they do with most diseases. They're often anorexic is the most common clinical sign, and then you can get the, the various vague signs that cats show, which is not eating, vomiting, etc. And which can be, you know, the clinical signs of lots of different diseases.

So it's quite vague and not particularly specific. The, as I said, the idiopathic hypercalcemia, which is becoming quite common. May have no clinical signs, and it's, it's only identified incidentally.

Again, they can get stones secondary to, to high calcium. And in the, in the cat, vitamin Dtoxicy similar to the dog, quite vague signs, depends on the, how much they've ingested again with rat poisoning. And again, primary hyperparathyroidism, clinical signs can be mild or non-existent.

And in these cases. So clinical signs depend on how high the ionised calcium is and how fast it increases. So I've given you levels there at the bottom.

You usually don't see any clinical signs until the ionised calcium is greater than about 1.75 and 1.8, and I can say that most of the cases and a lot of the animals or, you know, patients that we have that are measuring calcium.

They, a lot of them are not that level when we, when we first diagnosed them. I mean, some, some different, I'll go on to later, but some specific cases, particularly neoplasia, can often result in quite high levels. And those are more like the levels and also vitamin D toxicity, like the levels that you see where they should be critically ill, which is greater than 2, 2.2 for the ionised calcium, which is very high.

So the diagnosis of hypercalcemia is made when you can make it on total calcium, but that's unreliable because great then if you took those total calcium, a lot of those, if you don't went on to measure ionised calcium, that the ionised calcium would be normal. So we need to look at the ionised calcium. And in dogs, once you're above the reference range, same in cats, once you're above the reference range, that is hypercalcemia.

That's how tight this reference range is. So anything above those levels is considered a hypercalcemia. And if you get those on a repeated basis, then you should, you know, take them seriously.

So diagnosis of hypercalcemia, preferably ionised calcium. Obviously, if you have a total calcium that's really high, like 4 milli per litre, and it's on a, you know, persistent basis, then your ionised calcium will probably be high as well. And then once you've measured your ionised calcium and you've confirmed that it's high, you need to go on a measure of parathyroid hormone.

And preferably, this needs to be done with a repeated ionised calcium measured on the same sample. If you go back to what I was talking about, the control path, you know, The actual physiological control, the PTH is involved in minute to minute control. So if we take a sample on one day and then take an ionised calcium a week later, they aren't necessarily going to correlate.

So we need to do it on the same sample. The, the PTHRP obviously, you can measure that with suspected cases of malignancy, and they do tend, quite a lot of cases of anal sac adenocarcinoma. And some of the lymphomas will have an elevated PTHRP.

But you must remember that if your PTHRP comes back low, it doesn't exclude malignancy, particularly if your, PTH is also low. So remember that it's not, you know, a, a normal PTHRP doesn't exclude, a neoplastic cause. And we can also obviously measure the two different types of vitamin.

D. So we've got hydroxy vitamin D, and we've got 125 hydroxy with vitamin D, which is, calcitriol. We normally do the first one.

You can measure the first one, the, the 25 hydroxy vitamin D first, and if it's normal, then go on and look at calcitriol, you know, depending on the, the actual suspicion of what's causing the vitamin D, toxicity. So to remind you, ionised calcium, serum sample, not gel, separated. No, EDTA or anticoagulant type pla plasma samples, they're, they're not, they will result in completely, you know, spurious results.

The sample needs to be collected, sorry, anaerobically and you exclude air and then you separate it and then put it into a separate tube again, excluding air. Anything like chemolysis or the exposure of cells and so, you know, phosphorus from cell, etc. They will affect the, the ionised calcium.

So, and it often what it does is it actually lowers the, the actual ionised calcium. And if you're trying to confirm a high calcium, and you've got a hemoly sample or some, or a sample that's been exposed to actual cells, and you, you get a result that's sort of like in the reference range near the top, but not above it, then you don't know. So you need a separated sample, and send that.

The PTH and the PTHRP have a completely different sample collection. There are special kits that we can send out to you on request from the specialist laboratory where it's measured. And these are plasma samples collected into EDTA which are, and the, the tubes are, are cooled prior to sampling.

Then the sample is separated. And then once it's separated, it should be frozen immediately and kept frozen until the sample is sent by a courier on ice packs to the lab for analysis. If you do not collect the sample like this, or the sample defrosts and goes up to room temperature and instead.

Like that for any particular length of time, then what it results in is a degradation of the PTH and the PTHRP. The A protein is supposed to help decrease the, degradation speed of this. And the PTHRP is particularly sensitive to this and you'll end up with low levels.

So the sample must arrive at the lab frozen. So a chart that we actually have in our book that we send out with the, the, well the specialist came, lab, sorry, they have this in the, in part of the, the manual there. And it's just showing a comparison of ionised calcium.

So, the labels gone over there, but the PTH is at the left hand side and the ionised calcium obviously is labelled on the bottom, just showing normal and, you know, the, the two different causes. Now, Primary hyperparathyroidism obviously is up in this right-hand corner because you've got high PTH and high calc ionised calcium. And the other two here, they've separated them, so they've got secondary hyperparrothyism and PTH independent hypercalcemia.

Those really are in, in similar groups. It just depends on the, the cause there, because the secondary. In most of these, in these cases, if you, if you go back to what I'm saying, they present as a high, they can have a high total calcium and a normal ionised calcium or they have high ionised calcium.

So they've split these, split these up. But, as I say, there's two different, groups, primary and secondary. The vitamin D measurement, which the specialist lab deals with, actually, there, there aren't any current places as far as I know.

And if anybody knows, please correct me. But we send them over to the states and it's serum sample and it should, if you're going to submit it to the lab, it should be sent, you should freeze it prior and and just send it chilled, and we normally send it over on ice packs. It doesn't have to be, kept at minus whatever, but it just needs to be kept chilled, and that's, that's a serum sample.

So this was just the approach to diagnosis of hypercalcemia to use. I mean, it's quite useful if you're, just for a quick look, if you have a case. So you start with the total calcium and then you confirm that with ionised calcium and then you go on to measure the other, you know, PTH, etc.

Depending on what you find. And, and that's quite useful as an, an overview to help. So treatment I'm just going to go through just a few, slides with this, but it's, it's really, I have no experience on this, and this is all what's gathered from the references and what's normally, used.

You need to consider, first of all, when you have a hypercalcemic case and you initially diagnosed that, does it actually need treatment now or can we go on and diagnose it and find out what's causing it? It does depend on the level of calcium that's present. And also the rate of development.

So, if the levels aren't that high, you're not going to need any of this immediate treatment, like trying to, correct the levels to avoid, adverse effects on, you know, your, your neuro neurological system, cardiac, renal dysfunction, etc. And those are, as I say, they're usually when you've got high levels of calcium. So we're looking at total calcium greater than 4, ionised calcium greater than 2.2.

OK. So if we have these cases that, that That, sorry, if we, we have a case of, hypercalcemia. When we, when we go to treatment, initially, we need to try and diagnose what's causing it, because obviously, the treatment of the and removal of the cause will result in resolution.

So it does depend on the cause and depending on the levels of high calcium, whether we need to do some initial supportive therapy, like fluid therapy or diuretics, etc. To try and decrease the levels of calcium. The steroids that I've mentioned there are more a long-term treatment in hypercalcemia, and they really shouldn't be used in initial, treated, you know, treatment of cases.

One, because if you think about it, cases of Addison, you start treating with steroids, you're gonna have, if you haven't actually diagnosed it at that, that stage, it's on this, you know, it's a, it's an acute crisis case that's presented. The fluid therapy itself will help resolve the clinical signs and therefore you can go and do your test prior to actually starting any steroids and therefore interfering with the test for Addisons. So the, the steroids should be left until later, I think, in most of these cases.

And also, if you have neoplastic causes, particularly lymphoma. They can interfere with trying to diagnose that, particularly if you're going to do, cytology, say if they've got enlarged lymph nodes as well, that will interfere if you've used steroids prior to that to make a diagnosis, and it will also interfere with the histology. And also chemotherapy at a later date, though some people do advocate using, steroids prior to doing chemotherapy, it can actually decrease the efficacy of certain chemotherapy, Protocols.

OK. So we try and use that as a, as a later on in the disease, particularly if we've not actually, either reached the diagnosis or it's a, it's a cause of hypercalcemia that cannot be resolved easily. Calcitonin can also be used in the treatment.

It's usually used in combination with other therapies. It's quite expensive and it's, in some dogs in particular, they can have severe side effects secondary to it. The bisphosphonates, are quite common.

They are usually, they're used if the steroids aren't working or you're going to get like, you know, the animal or particular patient has side effects secondary to the steroids. So they can, they can use, be used quite, readily and there's lots of different types. They, and that, as I say, often when you don't know the actual underlying cause or you, you can't resolve it.

And also specific treatment for Addison's obviously will, should resolve your hypercalcemia. And then I just mentioned aotemic chronic kidney disease. I mean, there's other texts for that, but this, as I mentioned prior, sorry, previously, the low dose calcitriol therapy can be helpful in these cases to, to improve quality of life, but you have to be careful and monitor the actual doses to, to avoid resulting in, high, levels of calcium and therefore getting the secondary hypercalcemia.

And then a specific treatment for vitamin vitamin dose D obviously stop them ingesting it is the main one. And then it depending on how level, how high the levels are, you may need aggressive therapy to try and reduce the ionised calcium initially. And then you can use calcium phosphate, non, sorry, calcium containing phosphate binders to try and, you know, just to reduce the, the level of calcium.

In the case of primary hyperparathyroidism, the main treatment of choice seems to be surgery. It does obviously depend on the, the, the, the clinicians or the, the institution, etc. As to what they might use.

But surgery tends to be the, the main treatment of choice and seems to be fairly, well, I should say easy, but it's, it is reasonably, straightforward. If you, if you're fairly experienced. There is an issue that you may have, a hypercalcemia that results secondary or immediately after surgery, but bear, bear in mind this is not that common.

You have to remember the other thyroid gland, if this is a unilateral case, will be very tiny and probably won't be active. So in some cases, you may have to supplement, for, or, you know, to try and avoid the low calcium. So pre-treatment with calcitriol.

Vitamin D can be helpful in some of these cases. There are other ways of, of removing the, enlarged gland and you've got ethanol ablation, which sounds a bit old school, but, and also ultrasound guided radio frequency heat ablation has been used. So, prognosis, the Depends really on the underlying cause.

So cases of primary hyperparathyroidism have usually quite a good prognosis, particularly if you can remove the gland, except in the keysho, you've got to bear it or keysound, you've got to bear in mind that you've got to warn the owner that some of these cases that actually can reoccur. So they, they may develop it several years down the line. And Addison's obviously has a, has a good prognosis, if it's well-controlled and hypervitamosis D if you, you know, you get rid of the actual cause and you can, you know, determine what's actually resulted in the overdose.

Cats with idiopathic hypercalcemia, it's quite tends to be quite a low level of hypercalcemia, and they often do well with just, treatment, medical treatment. And primary hy parathyroidism again, quite rare, but it's quite, the prognosis is quite good in cats. I've just put this little table to the, to the side.

Obviously, The neoplastic causes and various, if they have chronic renal disease and they've got this tertiary hyperparathyroidism, the prognosis can be fair to guarded depending on to what's going on and, you know, where the tumour is and whether it's responding to chemotherapy, etc. Etc. So, You know, it just depends on the cause, but quite a lot of the causes, if you can identify them, have a reasonable prognosis.

So just to summarise, the clinical signs are often mild, and I'd say quite a lot of the cases present as incidental and the fact that you've picked it up because they've been presented for another reason or other clinical signs. And the ionised calcium in these cases is important to try and confirm that we have a hypercalcemia. Once you've confirmed that the, the ionised calcium is high, we then need to measure a PTH plus a minus a PTHRP and the PTH should be measured with a repeat ionised calcium done on the same sample.

Sample collection, remember the serum only, no air, ionised calcium, and remember the, the PTH has a special collection, protocol and kit, which needs to be followed to avoid falsely low levels of either of these two PTH, either the PTH or the PTHRP. And then treatment ideally is to remove the underlying cause and identify it. Now, I know we've had a lot of cases and I've talked to other vets and our clients when they've been trying to determine what's causing the high calcium.

It's not always as easy said, you know, we've ruled out primary hyperparathyroidism and then we were trying going for a fishing trip to try and find out what's causing the, the, the calcium. And I would say that neoplasia tends to be the most common, but in some cases, you don't find anything. And then I think you just really need to, to monitor and try and control the, the levels.

A lot of these animals can be older animals and therefore, you must remember they may have other underlying disease processes which could be affecting the, the calcium and particularly older cats, which have multiple things like chronic renal disease. So in, in all, the prognosis is fairly good if we can. Determine the, the underlying causes and try to, to manage it.

So, That's the end. So if you've got any questions, hopefully not lots, then, then feel free. Tonight's talk is, as you can see, basic cytology.

Some of you will know me better, possibly as dermatop pathologist, but I've been doing cytology, as Bruce said, for the last 35 years, and we have a, a good backup cytology service within, within the group. This is very basic, and it goes from the word from the first word go. Setting up a cytology laboratory and then looking at your samples.

I apologise for people that are very experienced at this already, but if you can pick up two or three pieces of information from almost any, talk, now, that's probably a success. So I'm hopefully you'll pick up a lot more than that. Right, what you're in for.

I'm going to spend some time taking the sample and preparing the slide, and probably half of this, this, presentation goes up to number 3 on this list. The reason for this is that we get a lot of, samples in from practises, and I would say, off the top of my head, about, something up to 25% of samples are really unsuitable for interpretation. So if you don't get a good preparation, there's no point in looking at the slide or learning how to interpret it.

So that's very important. Then we then go on to examining and interpreting the slide. I've been probably a bit ambitious here, interpretations for 80% of practised cytology, but.

I think there's probably about 10 or 12 situations in practise where you would need cytology, where you use cytology frequently. We're not talking about the esoteric tumours that you can get every now and again once in a blue moon. This is for most, most of the presentations or most of the cytology samples that you take will be covered about 80% of those today, all being well.

I just put this in just to say that there's two routes to examining, particularly lumps and bumps, cytology and histopath, and there's pros and cons for each of these. Cytology is relatively cheap and quick. But you don't get something for nothing, and the price you pay for that is that they're often non-diagnostic.

Histopathology almost always will give you either a diagnosis or the process that's occurring in the tissue, which will then, inform your, care of the, of the patient. I've always been of the view that practitioners ideally would like a diagnosis, but also what they really want is the next step in their clinical process for that particular animal. And that's basically what we try and do with cytology and histopathology.

I'll just put this in quickly, just to say that, cytology often is a, a rule out for some of your differentials. Now, that presupposes you've got some differentials. We do see quite a lot of samples coming in that may have been better examined some other way, initially, but it's very important to get a list of differentials before you do any laboratory testing.

OK, the types of samples that we can see, skin scrape I'm not dealing with because that's a dermatology. . That's, that's for the dermatologists to deal with.

Impression smears. I'm not gonna deal with impression smears today. The reason being that I personally, and some of these are my personal views, I personally think they're a waste of time.

Impression smears are usually taken from an ulcerated surface, and on that surface, there's almost always some serrocellular exudate, and that's almost all you get on an impression smear. You very rarely get the cells that are underneath it that are causing the problem. The only exception to this is if you take off a a mass for histological examination before it's fixed, if you actually cut.

A surface, blot it on some blotting paper, get rid of some the the blood. And then just dab the cut surface onto some slides. That gives you some cells from that mass.

Now, if you're doing your own cytology, and you look at that, it'll take you, what, 10 minutes, maybe to 1015 minutes to fix stain and look at the look at the slide. You can give the owners some sort of idea what you think is going on with this mass when they pick the animal up. You don't have to wait for the site, the histology to report to come back.

It also gives you good practise at looking at cytology and then comparing it with the report that you subsequently get from the histology. So that's for me is the only real use for an impression smear. Now an FNA this is usually for lumps and bumps, mostly on the skin, but also in the internal organs.

We'll be dealing probably mostly with that today. Bone marrow I'm not going to mention very much about, and I'm not gonna do any of the fluids, I'm afraid. The basic principles that we will go through in interpreting cytology will apply to all of these.

But I'll. Restrict it today pretty well to the FNAs. Simply because of time.

These are the standard sites that we see, we get sent to us, skin mass is the most common lymph nodes, liver, kidney, thyroid, and then we have the, the fluids. Right. For those that you are setting up cytology.

This is a list of what you'll really need. Obviously you'll need a microscope. Our microscopes cost somewhere between 5000 and 7000 pounds, and you're obviously not going to buy one of those for practise use.

You do get what you pay for with respect to microscopes. Anything under about 1000 British pounds probably is not very good. You may be lucky, but it may be a false economy to go cheap because you won't get good results, you'll get fed up with doing it and then you won't do it anymore.

So it's a false economy. The Chinese have made some very good microscopes somewhere between 1000 pounds of 1500 pounds, which is well affordable for most practises, and I would advise you to shop around, try some out, but go in that sort of bracket if you can't go any, any higher than that. You'll get an oilymersion for, for, for, for about 1500 pounds.

And some of the quality of the lenses are very good now. Hairdryer and fan, that's just to dry the slide which fixes it. Frosted slides, please, and a pencil, you should label everything.

With frosted slides, you can just write on the end with a pencil. You don't have to scratch, with a diamond marker. You should always label.

Pencil doesn't come off in any solvent, as far as I know, and it doesn't fade with time, with sunlight. So it's very useful for collecting a library of slides. The stain will do within a minute.

Needles. 21 gauge and 25 gauge. The clue's in the name, it's an FNA fine needle aspirate.

Please don't be tempted to use a grey or a purple needle of 15 or 17. Because what you end up with is a piece of tissue, not separated cells. The tissue is too small to process for histology, and it's too large for cytology, so don't go any bigger than 21 gauge.

A syringe and textbooks. I just put this in because I had this fairly recently last week, these are just large pieces of something. Can't make very much detail out of these.

But when you come up into higher power, you can see that. There's some nuclei. These cells are long and you'll just see here there's some cross striations.

This is muscle. And the practitioner who took this, I think, must have thought to themselves, I'll use a bit of a needle today, I'll get more cells. Well, this was completely waste of time.

Textbooks, The top two on here are excellent. They cover everything. They are very well written, they are very friendly, there's some good illustrations in them, and either of those will be very good on your practise shelf.

The 3rd 1 on the list, the clinical atlas, is an atlas, so there's very few words in it. It's all pictures, and I simply put it in because the pictures are fantastic. I don't know how they've done it.

I have a devil of a job getting really good photomicrographs of cytology, but these are just brilliant. So if you get into this in a big way, it's really nice to look at these, at this atlas. I've also included the Merck veterinary manual.

Two reasons. One is that. Most practises have this summer on their shelf.

There's a very, very basic cytology section. Which basically takes in roughly what I'm gonna say tonight because I wrote that section and this is all based on that, essentially. So if you've got that on your shelf and you want to start cytology, you could have a look at that.

Right. Basics of preparation. Please be gentle.

You're dealing with something that's very fragile. Cell membrane, as you will remember from your histology, I'm sure, is somewhere between 7.5 and 10 nanometers thick.

That is incredibly thin. It's very delicate and cells burst very easily, and it's one of the biggest, problems with cytology, biggest artefacts. Aim for a monolayer.

If you can't get a monolayer. Then don't press too hard to force a monolayer. Because if you do that, you're not being gentle and you'll burst the cells.

So it's a bit of a compromise. Don't cover slip. It stops the cells from from fixing.

We get cover slip slides quite often actually sent to us. Air dryer, no heat. Always label a slide.

At that stage, you must decide whether to examine it in-house or sent to a laboratory. And in fact, certainly if you're setting up, I would advise you to do both. You can then look at your own slides and compare them with what, with the report that comes back from the one sent to the laboratory.

You then stain And examine I've just put a little note on here, because even if you get into cytology, you'll still send some things away. And certainly fluids, I always, I personally think are better dealt with by a professional laboratory, mainly because they have machines, we have a machine called a cytospin. And the Cell preservation and preparation on cytospin samples is far, far superior to a standard er smear.

Cyto spins essentially, spin round slides that are upright, and the fluid is forced onto the front of the slide. Fluid part is absorbed by absorbent paper, and you're left with, just the cells stuck on the slide in a small area. So it's a good way of concentrating cells.

However, if you send fluid like that away, and I'm talking mainly about BALs, urine, prosthetic washes, Cells deteriorate quickly. There are some preservatives. EDTA is one, which I don't think is very useful.

Boric acid is commonly used for urine. I don't think that's very useful either. Formalin, I think, is brilliant.

So my personal, and I have to emphasise, personal preference for fluids when they come to the laboratory, is that they've had a few drops of formalin. This is not an accurate dilution of formalin. You just add a few drops, you usually take in fluid, I don't know, 5 to 20 mLs maybe.

Just add a few drops, that's all you need. It fixes the cells straight away. But you must tell your lab you've done that because they can't use the standard stains that you usually use for cytology.

They don't work. Right, I'm sorry about this, this is a bit corny, but 3 important tips are practise, practise, and yep, practise. So it's a technique that looks easy.

But to get really good consistent results, you do need to practise. So it's not something that you think, oh, I'll just do one and away we go. To get good consistent results, you must practise.

Right, take an FNA my simple diagram. Put the needle into the lesion. You Direct re keep redirecting it in different areas after you've put a made a small suction into the syringe, that's probably too much suction, I have to say.

When you think you've got a sample, you let the suction go, don't keep the suction on. And then you can withdraw the needle, you can then express it onto a slide. If you get a lot of blood, it may be, it is commonly said that it's because this suction is too heavy, you've got too much suction on it.

And if you do get a lot of blood, a lot of practitioners, even in, without the, without a lot of blood, as a standard technique, only use a needle. They don't put the syringe on it. So you can put the needle in, redirect it, redirect it.

And then take the needle out. Attach your syringe and express the material. I put this in, just to show.

The, obviously this has been expressed onto the slide. If you get reports back from a laboratory or you yourself see a lot of nuclei that don't have any cytoplasm, that is naked nuclei. It's possibly because you're expressing material through out from the syringe, through the needle again.

Because if you have a reasonable amount of material in the syringe, then if you're pushing that through the needle, you're forcing it into a very small hole. And that crowds the cells and it can strip the cytoplasm. So you might be better off if you have a reasonable size sample to take, to suck the material from the needle into the syringe.

And then take the needle off and put that on the slide. Simple diagrams on how to smear. There's your sample, layer glass slide on the top.

The weight of the slide may spread this out sufficiently er for your purposes. If it doesn't, you can apply gentle pressure but don't apply too much. You'll burst the cells.

Then you slide the slide. Along the the sample slide. And you end up with a smear like that.

In fact, you don't only end up with D, you end up with two smears, because you also have cells on this slider, smearing slide. You get 2 for the price of 1. I just put this in to say, this is a monolayer.

It's an inflammatory process with lots of different inflammatory cells. This is not a monolayer. This deeply staining area here.

You can't see any cells in there, hardly. And that cannot be interpreted. But even if you get this, around the edges of these, they do tend to separate out.

And you can see here there's a lot of neutrophils. There's the odd lymphocyte mononuclear cells, and this is an inflammatory process, there's neosinophil there. This is an inflammatory process.

So even though you haven't got a nice monolayer, you've got, some interpretation here. Now, stain. Standard stain, you see these in practises.

I wouldn't mind betting that 90% of you got diff quick in your practise. I have to say from the outset, I personally don't like diff quick. It's possible that I get a a skewed representation of diff quick cos every, almost every diff quick stain sample I see coming into the lab is not suitable for examination, it's too pale, it's not well differentiated.

And diffquik, by the way, doesn't stain mast cell granules very well, in my view. And mast cell tumours are one of the most common ones that you will do cytology on. It's a simple Quick technique, but There are a number of other rapid stains, not only diff quick, and these I think are cheaper.

I think they're better. This is one that we use, which is TCS Biosciences Limited, but, you know, there are a lot of, there are a number of these. I would look them up on the internet if you can.

And if you're going to do this seriously, you should write to them or contact them and get them to send you some samples. They often will send you some free samples, so that you can, try them out. So that you can, see which ones suit your practise best.

I think one of the problems in practise with staining, and it may be the case for diffquik, is that they're not changed often enough. They're left to go to run out. The more you use them, obviously, they use up the stain.

Also, they absorb water from the air, and that degenerates them. So Get your stain. That suits you best.

Right. Interpretation. This is a simple algorithm for interpreting.

Cytology preparations. I said earlier that I think practitioners want the next step, that's the minimum they really want from any of these diagnostic tests. And it's possible that you may only need to go to this first layer on the algorithm, Neoplasia or inflammation.

That may be all you need to know to know your next step in the clinical management of the case. If you need more, you can have a look at the inflammation to see if it's acute chronic granulomatous. There are different cells often in these different stages, although I have to say acute and chronic can be very difficult to differentiate.

We then have the neoplastic side. If you feel that you're dealing with the neoplastic process, you can then look for epithelium ws and chimal round cell, which may then determine how you treat this, this case, or whether you need to, go further with histopathology, for example. And I do think most samples that you do cytology on, particularly neoplastic ones, should have histology done subsequently, but I'm also a histopathologist, so I would say that.

And then you can look at features that decide whether it's benign or malignant. But as I've already said, you may not need to go that far. You may just need to go to the top part of this algorithm.

And then to these if you want a bit more detail, and then to this area, if you want even more detail. But I think if you're getting to this side, you're gonna end up doing a histology anyway. Right.

Basic interpretation, we're gonna deal with some basics of interpretation of cells of cytologies. No cells is a big problem. Quite a number of quite a small, a significant minority of cases that we see sent to our laboratory, but from practises have no cells.

And you'll see a number of cytologies yourself if you're looking at them, no cells and think, oh, no cells, that's a waste of time. Well. It's possible that your technique's not good enough.

If, however, you've done this a lot and you think your technique is pretty good. Then the absence of cells may give you an indication as to the type of mass. That you may be dealing with.

Cells in the body have different strengths of adhesion depending on their function. The round cells that you see listed there, they're all part of the inflammatory process. And therefore, they move around the body.

They communicate with, with each other with cytokines and chemicals, rather than, usually rather than direct, attachment. So they don't attach to very much. So, therefore, they tend to exfoliate really well with the cytology preparation.

So if you get a lot of slides. They're often round cells. Epithelial cells do adhere to each other, not super strong, most of them.

So again, you get a moderate number of cells, and you usually get a reasonable cell harvest with epithelial cells. Mesenchymal cells, however, they're the cells that hold your body together. So they need to stick together and they don't like being drawn out into a needle.

They are very Strongly adhered, so no cells may well mean that you're dealing with a mass or proliferation composed of mesenchymal cells. Blood. Blood is another problem.

Quite a lot of cytology preparations have a lot of blood and some only have blood. You can get something out of blood. In that I'll come on to that in a minute.

Keratin is another common result. Round cells, epithelial cells, and median chimal cells, which I've talked about already, which we'll deal with in a minute. Right.

Basic interpretation, no cells I've mentioned. Keratin. And blood Keratin is a very common contaminant on cytology preparations.

It can also come from lesions, particularly the keratin filled cysts that you get in skin, commonly in skin. This is. Very common Scattered single squams fairly uniformly distributed across the.

Across the Oops, across the slide. This is contamination. This is come this has come off of the skin of the animal, off of your hands, off of the nurse's hands.

They may be in the air, they are, after all, a major component of house dust. And they're everywhere, so this is a very common and with that number spread fairly uniformly, small squams typical of contamination, you wouldn't interpret that. Oops Daisy.

If you get keratin flakes like this, which are a bit larger, but they're most, most importantly, they're in a dense clump. This is not what you get from the skin. This is a real change, and this is typical of the keratin-filled cysts that you get in skin.

These are benign or non-neoplastic. Some of the benign adnexal tumours will produce keratin. I don't think they produce quite enough to actually give you this pattern.

So I think this is pretty well diagnostic for a, for a, non-neoplastic cyst, which, as you know, ris usually from different parts of the follicle, mainly. Blood. It's another contamination.

This blood smear looks like a blood smear. This was a cytology preparation. You've got white cells scattered within the preparation here are the neutrophils, and you have to make a decision at this stage whether you're dealing with blood contamination.

Or whether you're dealing with a contaminated in a sample of inflammation. You'll hear pathologists or see on reports that they say there are cells here in hemic proportion, so that's what you have to do. Does this look like a an an increased inflammation cells, inflammatory cells, or does this look like circulating blood?

The other thing you get is these, these are clusters of thrombocytes. If you see these, it's normally indicative of circulating blood. You've hit a vessel, it's artefact.

I have to say, I think pretty well every 95% at least of samples that have blood are simply artefact rather than from a blood-filled space like a hemangioma or something like that. The other thing about blood, if you think, oh well, I've got a blood I've got a smear of blood here, that's a waste of time. Just have a look at the edges.

Because surface tension effects on the surface of the slide tend to attract the heavy items to the edge of the smear and also to the feathered tail at the end of the smear. And that's where you might see some cells that are not quite right for blood. And this was actually a lymph node aspirate, and there are a few cells here that are quite big.

These are large lymphoid cells. And this Would give you an indication that you might be dealing with a lymphoma. Even though you've got mostly blood.

The other thing is that if you do have blood and only blood. Then it means you haven't sampled the cells and you've basically back to the same sort of interpretation that you'd have for no cells. OK.

Basic interpretation. The first part of the algorithm is it inflammation. I've put this on because you're all.

Probably know what inflammatory cells are, because you look at blood smears, and you get neutrophils. Most of the, obviously, the white cells in the blood are inflammatory cells. You get neutrophils, as we have here.

There's some lymphocytes, yeah, probably here, here. But you, I've put this in because of these big cells, and I think this confuses some people because these actually are macrophages. That probably is, that probably is.

They just, those two just haven't become activated enough. These contain lysozymes, of course, lysosomes full of enzymes that degrade, engulfed material. And these are seen in damaged tissue, almost irrespective of the damage.

They're not specific for a particular cause. So that's Here's an H&E stain cytology preparation, again, these are macrophages. And again, these have engulfed debris.

So just be aware that they're there and again. Obviously, when you, See an inflammatory process, you then may want to know, look for a cause. And of course, the two most common causes of inflammation in the skin, at least, are infection and foreign body reaction.

Mainly, the latter mainly being phunculosis, that was the most common type of foreign body. The hair and the keratin, as you know, acts as foreign material in the skin when it's released from, follicles. This is inflammation associated with bacteria.

You can see in the cytoplasm of these rather degenerate granulocytes, these structures, these are bacteria. You will also see them in. Samples that have been sent or been delayed, I should say, before they're looked at.

Sometimes you'll see them in the background of the smear. If they're in the cell. They are almost certainly pathogenic, neutrophils do not passively engulf.

Bacteria that are contaminants, or they simply, you've simply left it too long before you look at the slide and they've proliferated in the in the fluid or milieu. So look in the cytoplasm of the of the cells. Here's another one.

These are rods obviously in what are very degenerate neutrophils. And here we have a lot of cockeye. In this, these are Staphylococci.

So you look for a cause. I very rarely see the hair or the keratin with the er cytology preparation. The material may be too big, I don't know to be adequately sampled by FNA certainly the hair would probably be too big.

The other thing that We see occasionally are these. These are obviously different to the other inflammatory cells, most of which are neutrophils here. These are spindle cells.

As the name suggests, there's a spindle cell. These are fibroblasts. Strictly speaking, if you just had this small group of cells, you would not be able to distinguish differentiate inflammation or reactive spindle cells from those of a low grade tumour.

They have the same very similar morphology. We then look therefore at the company they keep, as it were. And the company these spindle cells are keeping is inflammation, and inflammation is the most potent stimulus for fibroplasia.

So you'd be pretty certain to say this is all just reactive. In my experience, it's very rare, and I use the word rare, it's very very uncommon. For tumours to have this amount of inflammation in the centre of them.

On the ulcerated surface, that's fair enough, but actually in deep into the tissue, this is quite rare to have this sort of inflammation in my experience. So if you see this inflammation with with spindle cells, it's almost certainly, reactive. Right, round cells.

These are the basic types of round cell that we see. How do we know we're dealing with a round cell? This is low power A lot of blood here, but there's a lot of cells in this preparation.

And they're quite separate. You can see they're lying very separate, and I said earlier, they do not adhere to each other very well. You might get a little bit here and there, but most of these cells are lying separately from each other.

And even under low power, you're looking at that and saying, this is a round cell tumour, and then you make the differential from These 5 different types. This is a higher power view. Eccentric nucleus quite often, quite a lot of cytoplasm, a bit variable, some variation in size.

This, these are histiocytic cells. This is a histiocytoma. And so is this.

They do often have this eccentric nucleus in the cytoplasm. The other cells that mainly have this are actually osteoblasts, such as you get with osteosarcoma, but. It's an illustration that you obviously have to take the clinical picture into account because you wouldn't expect an osteosarcoma to occur on the lip or the ear, whereas you would expect a osteocytoma to be there.

So you have to look at the whole, not just the cytology, and you'll know the case if you're doing these yourself, you need to look at the whole picture. And here's some more and much higher power and binucleate cell here too. These are round cells again, you can see they're mostly separated within the preparation.

There's actually two types of round cells in there's inflammatory cells here, earsinophils. You wouldn't really probably call that a round cell, but there's earsinophils here. And if you see that, particularly with round cells, you would have to look to see if these are mast cells, because they do, as you know, tracks and fills in dogs, not in cats.

These cells are densely staining. That's densely staining. You can just about see a nucleus here, probably here, possibly here.

But they're densely staining, and there are granules packed into these cytoplasm that are obscuring the nucleus. And this is a common pattern. This is absolutely diagnostic for a mast cell tumour.

There's no equivocation here. If you see more than probably actually 3 or 4 mast cells in a high power field, you're almost certainly dealing with. You can almost certainly say diagnosis mast cell tumour.

They do occur in inflammation but not in large numbers. Here we are again. You can see in the background here these little dots, these are granules that have been released from burst cells, these are not looking very healthy, I think they've burst, and that one there, that one there, they're releasing their granules.

The granules vary a bit in their colour. These are purple. That's the metachromasia that that people talk about.

And it's slightly different colour to the blue that actually is the the stain colour. They change the granules change the colour slightly. And when they're very densely packed like this, they're very dark.

Here's another one. This Shows some of the features of malignancy, and I'll put this in because we have here a huge cell, one here. One here These are smaller There's a binucleate cell.

If you look at this binucleate cell, it's a bit dusty here. Slightly, slightly granular, slightly purply, same here. This is a mast cell tumour.

Now there are, there is a paper in the literature about grading mast cell tumours on cytology. I have to say I wasn't entirely convinced of the. Conclusions, but.

It depends on counting about 200 cells and comparing the diameter of the nuclei and looking at the variation. I think if you get anything that looks like this, with this variation, one of the features of malignancy is trem is this is variation of cell size and of nucleus to cytoplasm ratio. And binucleate cells can also be included in that, to a lesser extent.

They're features of malignancy and this would be almost certainly classified just on that basis, on that field of a a high grade tumour. Here we are again, very faintly granular these, very faint. In fact, not many of the cells have granules.

So you sometimes have to look very closely for them, but mostly it's they're, they're in your face. Here we have another one, and again in this case we have another cell type. This is a spindle cell, obviously, and this elongated.

And some mast cell tumours will produce quite a marked fibroblastic response. So don't be, don't mistake these for neoplastic process. These are just part of the reaction to all of these mast cells.

There was a paper, about probably about 25 years ago in the Human pathology journal looking at my cells in spinal cell tumours, because you do get them. They were looking at them to see if they were a prognostic indicator, which they actually weren't. But you'd never get this sort of number.

You'd be lucky to find one. I put this in because again you've got spindle cells here. You've got some ma cells.

There's 2 here. You'd be slightly suspicious, even with just two ma cells that you're dealing with a ma cell tumour and this is its fibrous reaction. But you wouldn't probably make a definitive diagnosis on this smear because the cells, there's just not enough of them.

And again here, that's a mast cell size that and that and that, that. And that there's no other inflammation here. And although we only have what, say 6 cells here.

That's pretty diagnostic from our cell tumour, particularly as there's no other cell type present. This is also a round cell tumour. I'm afraid this the .

The photograph seems to show granules, but it, these aren't gran these aren't sort of metachromatic granules. This is a plasma cytoma. I think you'd be hard put to make a good diagnosis for a plasma cytoma and cytology if you're not very used to looking at them.

You could easily think these were histocytic cells, I think. You got a reasonable amount of cytoplasm and eccentric nucleus, but the nuclei aren't quite the same, and I think you'd have difficulty making that diagnosis on this. These are lymphocytes, also round cells separated out, big.

There's a red cell here. And if you compare lymphocyte nuclear size with red cells, if they're more than about 1.5 times the diameter of a red cell, you're probably dealing with a neoplastic process.

This is neoplasia, this is lymphoma, this is from a lymph node. That's a that's a mitotic figure. So is that, don't see them very often in cytology, even though histology may say it's a high mitotic index.

They don't crop up too often in cytology. Multiple nucleoli. The size, nucleolar characteristics, mitotic activity, all very characteristic of neoplasia.

Lymphomas, as you know are malignant. One of the features of malignancy that I mentioned earlier is variation in size, pleomorphism of the cells. This is an exception.

Lymphoma Usually doesn't produce tremendous pleomorphism. They're fairly uniform in size. And nucleus to cytoplasm ratio.

So that's one of the exceptions to the rule. I just put this in, because this was also, this was a submandibular mass that was FNA. And these are the cells that came out.

That big. There's a nucleus, all of this is cytoplasm, nucleus, all lots of cytoplasm here. If you see this in a submandibular mass.

This is salivary glen. We see these regularly. So you either have missed the lymph node that may be enlarged and you've just hit the salivary gland that's in the vicinity.

Or you're dealing with an enlarged hypertrophic salivary gland, which is a condition that's generally described as syadonnosis or silosis. Cause of that's unknown, by the way. So don't think these are big lymphocytes.

These are actually fairly uniform because the nuclei are very uniform in size. This is salivary tissue. Oh, and this is just a TVT .

I think they're difficult to diagnose on cytology. You probably wouldn't be able to do that very easily. They do have, tend to have quite a lot of cyto vacuoles in the cytoplasm.

Nuclei are sort of very granular in their appearance with indistinct nucleoli usually, on cytology. These only occur in some parts of the world, but we're seeing a few of these now, particularly from dogs from Eastern Europe, especially Romania. They seem to have a problem there.

I've seen some cases in this country, but they may have been from dogs that have come from abroad. Epithelial cells. I'll speed up a little bit.

Clusters. They are a bit more adherent, not super adherent, like spindle cells, but you get clusters which are usually often fairly well demarcated. With a few cells spreading out.

So that's a, if it's adherent like that, forming clusters, you'd think of epithelium. These are Basoid cells. Fairly uniform nuclei.

This looks benign. This also looks benign and with basal cells sometimes you get structures like asinine and sometimes kind of sading. And again here, however, this one.

And this one are from perianal masses, and this is the other exception, one of the other exceptions for malignancy. Apocrine anal glands. When they produce carcinoma, when they develop carcinoma, have cells that are fairly uniform.

They don't show t usually show tremendous pleomorphism. So if you see a basalloid looking tumour in the perianal region, then beware that this may well be a a perianal apocrine gland adenoma, adenocarcinoma. I put this in because this is the other perianal tumour.

Again, a cluster. They're adherent, big cells, lots of cytoplasm, but uniform nuclei, so the size isn't probably gonna be associated with malignancy, and this actually is a hepatoid adenoma, an anal adenoma. There's another one, there's some big cells in it, some smaller cells which are the reserve cells.

But that's a very typical anal adenoma. I put this in because simply cos this is liver, and they look hepatoid, hence the name. Mesenchymal cells are the two major ones obviously are spindle spindle cells and fat cells.

Spinddle cells also cluster. So if you get a decent cell harvest, if you do manage to suck them out, as it were, then they often are in clusters in my experience, but the cluster. Those aren't nice, sharply demarcated, they sort of, almost as if they're sort of trying to get out, they're flowing away and this irregular outline is very characteristic, I think of spindle sole.

Proliferations and you also get this magenta matrix which is also seen predominantly in mesenchymal spindle cell proliferations. And you can see around the periphery, that's a nice tail on that one, that's a spindle cell. It's a nice spindle cell there, there, these are, this is a spindle cell tumour.

You wouldn't know whether this is benign or malignant. Some benign spindle cell tumours such as the old fashioned hemangioparicytomas. Sometimes they binucleate.

You can see some, in some cases quite variable, morphological characteristics. But as a general rule, sarcomas do behave in the usual way, differentiating benign from malignant with the malignant characteristics. Here we have another one, this is a spindle cell tumour as well.

Nice spindle cell there. A lot of these are rounded. They look polygonal and.

When you take spindle cells out of the tissue context, they can become polygonal. But you have a look around and you will eventually find one or two that are like that. The spindle cells.

They also have very rather indistinct boundaries, not very sharp, particularly on this this micrograph. This is not a very sharp boundary. There's no sharp boundaries in there.

It's another feature of spindle cells. Most epithelial proliferations give you fairly sharp boundaries between the cytoplasm between each cell. And that's another one.

Fat cells common. Big balloons of fat. This is a classic picture, classic classic example.

That's what you usually get. That would be ideal. And sometimes you might only get one or two of these in the whole preparation.

They are mesenchymal cells after all. They do stick together. They don't like coming out as a cytology.

They don't exfoliate well. So you might only get a few of these scattered around. And then you have to decide whether those are.

A part of the mass, whether that's representative or whether this is simply mature body fat, because from a cytology point of view, you cannot distinguish those. This is exactly the same as you'd get with fat contamination, so it depends how confident you are that you've got the cells from the mass. That's what they usually look like, folded over on top of each other, but when you go down, you can see there's a lot of vaculation in here.

And you'll pick out a few classic fat cells. So that's a very typical picture. Another class, another little cluster of fat cells, but also this, this is the extracellular fat.

As is this, sometimes that's all you see. And then again, you can't make a diagnosis on that, but if that's all you've got and you're confident that you're in centre that you've taken this from the centre of a soft mass, then you're fairly home and dry. So we've done this.

We've looked at inflammation, we've looked at these different types of neoplasia. I'm not gonna go into this at this stage. You may be able to make a differentiation, if you've got a cytology of inflammation, you may be able to say if it's acute to chronic or granulomatous on the clinical picture, on your clinical picture.

But it would depend a little on the, Obviously on the cell mixture that you see in the cytology. So I'm not going to go here, just a very quick one on problems. And I'll whiz through this because we're running out of time.

I apologise. Right, too thick. We've dealt with this already, you can sometimes get something out of that.

This is completely destroyed. These are, this is nuclear smearing. These strands of nuclear material have been dragged across the slide, and you're left also with cell nuclei that haven't burst, that have no cytoplasm.

These are what I describe as naked nuclei. That it's a waste of time trying to exam, trying to identify these. You can't do that because as soon as they lose their cytoplasm, they start to swell, osmotic factors come into play and they eventually burst.

Here we are again, very common. This is lymph node, this is quite common as well, you've got lymphoid cells scattered around, but you've got all also these. Without cytoplasm, they're becoming different shape, they're starting to explode.

That's their naked nuclei. That's not uncommon with almost any lymph node cytology. I put this in because it's pretty.

This is haemoglobin. This is just crystallised out on the slide. I had this last week.

Ultrasound gel, very common. This is pink stuff, lots of purple material, lots of little dots of purple all collected together, that's ultrasound gel. And you can see some cells underneath being completely obscured by this.

Ultrasound gel, ultrasound gel, beware. Screams we've dealt with. Just an indistinct green stain final, finally.

These are smears that are normally stained. This is obviously very pale, it's like a pale bluey greeny colour. That's the type of pattern that you see when cytology have been exposed to formalin.

So be careful if you have formula on the bench near your cytology because these don't stain well. You don't get good detail in the cells. You have to keep them separate.

And if you're packing stuff off for a lab, make sure that they're nicely packed up so you don't get form infu contamination of the smears. So far we've looked at most of this, believe it or not. And that probably accounts for pretty well 80%, I would think of the things that you'll lumps and bumps you'll see do cytology on.