Welcome to the virtual congress and this session on adrenal gland testing. And when I was asked to do this, I thought, oh this, this, this will be quite a short talk. But actually, as you start to realise how many things we areas we have to cover, I realised that there's quite a lot to cover, not just the diagnosis of Cushing's and Addison's, but also thy chromocytomas, hyper aldosterism.

And not just the diagnosis, but also the monitoring. So we, we've got quite a little bit to, to get through, but I think before we start, I think it's important, to talk a little bit about, how good the tests are and, relative to the prevalence in a population. So when we talk about, a quality of a test, we talk about several things.

We talk about the sensitivity, we talk about the specificity, but these are innate functions of the test and don't really reflect what the usage of the test is. What really Matters in clinical practise is the positive predictive value, which is to say the, the likelihood that when you get a positive result, it's right. And the negative predictive value is the likelihood that when we have a negative result, it really is, a negative.

These can be put into, mathematical formulations like this. But, but the really important point about this diagram is, is not how you calculate these things, but the fact that for any test, you need to have some sort of gold standard truth to, by which to compare that, that test. And therein lies, lies the rub is, is that it sometimes can be difficult to get to the truth.

and so in clinical practise, we have to, to rely on these tests, knowing that the truth, may, may, may not be so easy to, to, to find. Positive predictive values increase as negative predictive values decrease. And, and I think that's a fairly obvious statement, but the relationship is not linear, and, you can get quite big increases in positive predictive values, for a particular given proportion in the population.

So on this, graph here, the proportion on the, The, the X axis here is the proportion of animals in a given population. And, and so, as a result of that, you can see that as the proportion of diseased animals in the population increases, so the, these test characteristics change. To put it more in a, in a, in a more clinical cir circumstance, to make it easier, I think it's helpful to look at a case like this.

This is, this is Gaston. He, he has clearly got some problems here in, in life. He's a Pyrenees mountain dog, and yes, he's, he's lacking a certain amount of coat here.

As well as that, he's PUP. He's polyphagic, he's lethargic, and when we do, haematology, biochemistry, we find changes that we would expect with, high liver enzymes, maybe slightly high glucose, high cholesterol. You're in specific gravity, following on from Tim's talk there, would be, about 1.02.

Er, there might be a bit of protein in there. There might even be some glucose in there if he's becoming diabetic on top of everything else. And on radiography or or abdominal ultraography, we see big livers, lots of fats, and indeed there may be adrenal pathology.

Put all that together and I think most of us would be saying, well, we're pretty sure what this animal has. and contrast that with this dog here, which has just got polyure polydipsia. No changes on haematology, no changes on biochemistry, no changes on, urinalysis, no radio radiographic features, particularly suggesting Cushing's.

And at this point, we have a much lower level of, of confidence and of, suspicion that this dog has, Cushing's disease. So, When, when people ask me which test should I use for, for Cushing's disease, I, I think you have to start from the assumption that we've tried to do some basic work up first, that we, we've tried to philtre these cases, and then we're going to ask the question, how do we prove that a dog has Cushing's disease? So in Gaston's case, we clearly want to prove this dog has Cushing's disease because really nothing else fits, and we want to do, and my, my A suggestion would be that the most appropriate test in Gaston's case would be to do an ACTH stimulation test.

Whereas in the second case, we want to prove that a dog probably doesn't have Cushing's, we're not confident that perhaps very small number of dogs that present like that will turn out actually to have Cushing's disease. And then in those circumstances, we're probably trying to rule out the diagnosis. And then I think a low dose dexamethaone suppression test starts to come in.

So then I stepped a stall out now. Let, let's, let's see why, that, that is the case. If we look at the ACTH stimulation test, then we see a test that, that really is a test for, adrenal reserve.

And dogs with Cushing's disease, have, a significant amount of adrenal reserve. And so when we stimulate them, we, we can produce levels that, that are higher than, than normal. But by no means all dogs, with, Cushing's disease do, achieve high levels of cortisol with an ACTH stimulation test and the test is not really used that much, for example, in humans.

If you look at the actual figures, you can see that there are significant overlaps of, of Cushingo dogs with normal dogs, and there is around here a number of dogs here that would be described as having a sorry, a number of dogs here that, that have a, a low level of cortisol after ACTH stimulation test, despite having, Cushing's disease. And even a few dogs, that, that, have a, that have a high level of cortisol, despite not having Cushing's disease. These are often dogs that are extremely metabolically stressed, and in this study, it was dogs with things like diabetic ketoacidosis and, and tumours and pyometras and things like this, which were coming through here.

And this is where I think it's important that, that routine buds and, and routine urinalysis is done before we start. Are using ACTH stimulation tests to try to decide whether the dog does or does not have Cushing's because these dogs would never be tested in normal clinical practise with an ACTH stimulation test, because you would have found out that it had diabetic ketoacidosis and therefore, you, you would treat that before trying to, to diagnose Cushing's disease. Of course, the other problem with ACTH, as we know, is that its availability internationally is very variable.

In the UK we used to have synaxin, which was extremely cheap and readily available, but that then went off the market. DeA brought out a tetracos Ata, which is ACTH, in, in a, in a. In a bottle and tetracosactide is, is currently in, in somewhat short supply.

So we're having to go back to using synaxin, which is, which is expensive. In the US, Ctrain has been available for a long time, but it's always very expensive. And in mainland Europe, synactin remains cheap.

So the availability of ACTH is a big factor in determining whether you are going to be using ACTH in, in your diagnosis. The, the, alternative, the low-dose dexamethasone suppression test, is, is more of a test of the pituitary's control, of, of the adrenal gland. It is a, it is a test of the hypothalamic pituitary axis.

And by administering dexamethasone to a dog, it acts on the pituitary, and the pituitary production of ACTH goes down, and as a result, cortisol production is reduced. And that's what should happen and that's what does happen in, in normal dogs. However, in dogs that break out of suppression, this is because presumably the pituitary is overproducing ACTH or that the adrenal gland is no longer responding normally to, to ACTH.

And that can be caused by Cushing's, but that can also happen with, stress, and with other diseases. So the low dose dexamethasone suppression test is great at telling you that, the, the, the dog is normal, but it's not so good at telling you what the cause of it's, it's illness is. The other, one advantage of low-dose de methadone suppression test is that if the cortisol level goes down, to, to less than 40 and then comes, above that threshold, then it's likely that this is a pituitary dependent disease.

This is an interesting study here looking at the effects of stress on low dose dexamethasone suppression tests. And it was published in 2004. They only had 6 normal dogs, but I think the results are nonetheless quite important.

And what this, this group of researchers did was that they performed the low dose dexamethasone suppression test 5 times on each of their test dogs. And, so they had 30 tests from 6 dogs, and During the process of doing 4 of these tests, they did a, an abdominal ultrasound and took samples at random, at about 20 minutes later to see what effect that abdominal ultrasound had. Now, repeatedly bringing these dogs into the hospital environment, you thought would have some effect, but in fact, that was not significant.

But short term stress turned out to have a significant effect, so much so that all 6 dogs on one test or another were actually diagnosed as having Cushing's disease, if you do, and this is the problem with the dexamethasone suppression test, is that if you use it in, in normal animals as a diagnostic test for the condition, you're going to get a high rate of, of, of false positives. So the ACTH stimulation test is a specific test, but it's not very sensitive. It misses cases.

The dexamethasone suppression test, on the other hand, is a sensitive test but lacks specificity. So it tends to overdiagnose, but it rarely misses the cases. There are differences in the price of these tests, and depending on your supply of ACTH, and the cost of doing cortisols in your, practise, the, the price differential between those two tests can be quite significant.

But what I'd like to do now is to look at the effect of prevalence on the performance of these tests. So if we take the situation of looking at the positive predictive value, and we look at the situation here with Gaston, where we're 80% sure that this, this, this dog has Cushing's disease, then the positive predictive value of an ACTH stimulation test is really very, very high indeed, we can be pretty confident that if it comes back positive, that we've got the right disease. On the other hand, if you look at the negative predictive value, it's actually pretty poor.

And what that means is that if you really think the dog has Cushing's and you get a negative on the ACTH stim, it's the test that's wrong, not you. At the much lower end of the spectrum, so this relates to the dog that has very few changes suggested, then an ACTH stimulation test has a quite reasonable negative predictive value. It can still rule out the condition quite well, but it's positive predictive value is pretty low, pretty good too.

So, at either end of the spectrum, we seem to have quite a good test here. In contrast, if we look at the low dose dexamethasone suppression test, then it's positive predictive value, when they really very high anyway, isn't any better than the ACTH stimulation test. But at the lower end of the range, it's positive predict value is really rather poor.

And that's, and that's a dangerous figure. So if you're not confident the dog has Cushing's, doing a low dose dexamethasone suppression test might well give you some false positives. Whereas in contrast, if you think that the dog does not have Cushing's and you really want to rule it out, a 98% negative predictive value, it's very, very reasonable.

And it's these sorts The figures that that lead us to these suggestions that the ACTH stimulation test is good for positively diagnosing when you really think it's, it's there, as long as long as you're prepared to accept is negative predictive value is poor. But the dexamethasone suppression test is better for ruling out the condition. Have these dogs ever been compared?

Have these, dogs have these tests ever been compared? Well, well, yes, back in 1995, then Kaplan and others, took 21 normal dogs, 20 dogs with pituitary dependent disease, and 59 dogs with, non-adrenal, disease. Of those, dogs with non-adrenal disease, only 14% came back as false positives on the ACTH stimulation test.

And that was, not significant. 56% came back positive on the low dose Xs, and we haven't talked about it yet, but 76% came back positive with a high urine cortisol creatinine ratio. Which is quite worrying figures, I think, until you consider that that really means is that the prevalence in this population was only 20%, which is really quite a low population, quite a small number.

If I, if I started my We and I did 100 tests for dogs with Cushing's and it actually turned out that only 20 of them did have the, did have the disease. I, I think I'd be, be rather over testing. And that's the problem with over testing, is that it starts to produce some bizarre figures.

As well as worrying about the actual test choice, I think we also have to worry about the tests that we choose. So the, the cortisol measurement, is, is important and the method's important. There are, variability between assays.

There are variabilities even within the same assay. There are variabilities between laboratories, and there are variabilities in the ability of these tests to quantify. So in other words, to tell the difference between 10 and 20, and also to tell the difference, and, and between 0 and 10, which is the limit of detection.

This has been a study, a study that's been ongoing now for several years through the European Society of Veterinary Endocrinology, and it's basically where the same sample is sent to a group of laboratories around Europe, all using different methods for measuring cortisol. And you can see here that there is quite a widespread of reported values. From this same sample.

Now, some of these laboratories will have different reference ranges, so it doesn't mean that there's one test that's good and one test that's not good. But there are certainly tests in there that seem to be giving some fairly unusual results. The, method 6 here in green shows you that, depending on where the laboratory is, seems to have more influence on the, result than anything else.

No that was not included in here are in-house analyzers, and the performance of these can really depend on your local quality control, and, the, the actual analyzers themselves. And for the most part, these analyzers have significant issues with the quantification. So the ability to discriminate between, concentrations that are very close together, and, and also some significant in as a variabilities.

So as a result of this, when we're making a diagnosis using a very narrow cutoff point, then it is not really appropriate to be using one of these machines unless it's an emergency. for, for, for the record, the, the method 8 here, which is the Semenzimulite is the one that's most widely used, in, in the UK in the main reference laboratories. But there are, of course, other good, good methods out there as well.

So what happens if we have a dog that has clinical signs of Cushing's but on an ACTH stimulation test and on low dose dexamethasone suppression test is negative. This is sometimes termed atypical Cushing's disease, and this brings us to two other tests that are available for Cushing's disease, naming the urine corticoid creatinine ratio and 17 hydroxyprogesterone. First of all, to talk about urine corticoid creatinine ratio, this, this is measurement of cortisol metabolites in urine, which is an approximation of the 24 hour total urine cortisol.

It's important when we're doing this to obtain 3 samples of urine at home, for reasons that will become clear, and it's in these should be obtained in the morning. This test has been regarded as being, sensitive, and, but not specific. And, and we hear a lot of people saying that you can use urine, corticoid, creatinine ratios to rule out, Cushing's disease.

Recently, there's been, some publications coming out looking at this. And again, the, the thing that, that worries, worries, me about this test is that there is significant lab to lab or method to method variation. These are, 5 assays, for urine corticoid, creatinine ratio, in, normal dogs and dogs with Cushing's.

And you can see here that on the right, the gas chromatography mass spectrometry method, which might be regarded as being the gold standard as, as, as it were, gives you the, the, the positive control. And you can see. That there are significant differences between these assays with the normal dogs on the left of each pair, having an assay A, quite high levels of apparently urine, cortico, creatinine levels, whereas the B and D are lower, C and E are somewhat in between.

So as a specific reference ranges are absolutely vital if you're going to, to, to use this, and to use your own encoded creatinine ratio to rule out Cushing's is, is, is difficult, as you see from this graph, there are many dogs with Cushing's that have a urine corticoid creatin ratio that's actually within the reference range. And the reason why that there is this difference is that these laboratories use different methods. They use different antibodies to measure, and some are measuring urine cortisol, and some are measuring urine corticoids.

Some are using chemical extraction techniques, some are not. And that produces a degree of variation. The essay used at Utrecht University, which is the essay that, most is published about, but is actually relatively, it's relatively, more difficult to send to, to, to, to Utrek to, to get this measured if you, if you live in the UK, so as a result of, of that, we're tending to send it to, to laboratories who haven't published, so much stuff on their method.

So I think we have to say that urine corticoid creatinine ratios do come with a certain health warning. They, they, they, they can miss cases, and they can, at times give you false positive diagnoses. The alternative to doing this would be to measure 17 hydroxyprogesterone.

This is a cortisol precursor that's stimulated by ACTH and this really came to the fore in 2002 when a group of us published a group of dogs that were normal. Some had confirmed Cushing's, and some had Cushing's confirmed, but only by their response to treatment. And, and that's obviously has, has some issues with the formal whether they really did have Cushing's.

And what they found was that the load, that the 17 hydroxyprogesterone worked quite well at identifying some of these low dose, dexamethasone negative, ACTHM negative dogs that still had, Cushing's disease. The, I showed you this paper, paper before. These are the 17 hydroxyprogesterone results from that same paper.

And, and again, you can see that there's a very clear overlap between other diseases and the Cushing's. So it's, it's not a very good test for telling you whether why an animal's ill, but if you're not sure whether it's got Cushing's or it's actually just normal, then, then there is, quite a clear, clear. Cut off.

But I would again remind you that, that these other diseases included diseases such as diabetes mellitus and hepatic disease and GI disease and diabetes incipidus, which one might expect to have been diagnosed, and certainly the urinary tract disease one might well have been diagnosed without doing a test in a normal clinical practise. And so this, this study is somewhat weighted, towards, an unrealistic situation. If you put these into, receiver operating curves, which match the sensitivity against the specificity at virtually all concentrations, 17 hydroxyprogesterone is not as good as cortisol in an ACTH stimulation test in this study.

And therefore, one would never suggest that the 17 hydroxyprogesterone was measured in preference to the cortisol, but there are cases where it is, it is useful, where the ACTH stimulation test has not given us a positive. So, we, we interpret this, as being, 7 hydroxyprogesterone levels over about 13 here, something like that, are, are clearly abnormal. Less than 13, they can be normal, and below 8, they nearly always aren't.

Normal. But once you have a dog that has a 79 droxyprogesterone, greater than 8, then you really don't know whether it's got typical Cushing's, atypical Cushing's or other diseases, and you'll need to use other methods to rule those, those out to arrive at a diagnosis. I think one of the problems is that we, we are wedded to the idea of this idea of a cutoff.

And, and in 2015, Linda Frank shown this rather nicely that in dogs that had a atypical hypergena corticism shown with the dash middle line here, these dogs had over the course of the day, an abnormally high serum cortisol compared to control dogs. They weren't as as high as the typical pituitary dependent dogs, but they were increased. And, and so really we should be looking at the clinical signs of the dog to interpret the biochemistry and the cortisol results.

If there's Lots of clinical signs. Linda Frank concluded that you can reduce the threshold. You can reduce that cutoff.

The more clinical signs you have, the more you can reduce the threshold. The threshold goes up if you have fewer and fewer clinical signs. So if we can put that into a summary then, and then we'll talk a little bit about monitoring treatment and feline Cushing's.

When we want to make a diagnosis of Cushing's disease, the history and the physical exam, the urinalysis, the routine biochemistry, and the routine haematology are absolutely vital to gain evidence of, of, yes, this dog has Cushing's or no, it does not. By putting all that evidence together, we can then go and look at, ACTH stimulation tests and low dose xyone suppression. If we have a, a lot of yeses, then we can probably go and, say that going straight to the ACTH stimulation test is the right thing.

Whereas if there's a lot of no's and there are a lot of reasons why you don't think it's Cushing's, then maybe going first to the low dose dexamethasone suppression test to confirm that it does not have Cushing's might be a more, sensible way, to, to go forward. Obviously, the order of ACTHM and low dose tacks will again be, determined by local factors such as the availability of these, things, of ACTH, and of local costs. But, all other things being equal, then, an ACTH.

Ation test being used in the yeses and the low dose where there's lots of nos, makes sense. I should draw your attention then to the, the, we're not going to talk about it today, but once you confirm the diagnosis of Cushing's, we should be taking some measures to address whether it's adrenal or pituitary dependent. So, one last couple of comments, on, on which test should I use in Cushing's.

I, I think it's important that we, we stop trying to talk about, trying to prove, that dogs have or have not got, got, got Cushing's. It's not a legal, legal proof, it's not even pure science. And really what we want to be doing is looking at, at the client communications, and understanding that, any diagnosis.

It is only very based on a certain amount of evidence, and that we will always be taking a little bit of a chance when we treat a dog for Cushing's or decide not to treat it for Cushing's, that it does not have or does have Cushing's. And, and I think that a client, client communication to explain that, difficulty may well get us out of this problem of always talking about proving something. The second point I would make is I think that it's important when we talk about these tests that we think about how you prepared to do another test, because that really does, influence your choice of tests.

If, if you want the one test that does everything, you're probably not going to find it. You need to be able to prepare to say, I'm right, the test is wrong, I'm discarding that result, and I'm going to do another test. If you rely on laboratory testing to make a diagnosis totally, then you are lacking the experience with the test.

If you don't do any laboratory testings, then that's probably not very safe, safe. It's important to use both the test and your clinical judgement in these cases. So just a brief word then before moving on to talk about cats about control of hypergena courseism and what tests we should use.

Let's be clear that as far as I'm concerned, controlling Cushing's is taking a dog that looks like this and turning it into a dog that looks like this. And anything that we do in the way of monitoring, use of, trilasta in these animals is guided towards achieving that end, without at the same time over suppressing the animal and risking Addison's disease. But for, for, for my preference, my selection would, would be, not to do an ACTH stimulation test anymore in dogs with Cushing's, that are on treatment, but rather to do a pre-veteral cortisol, taking, a sample about, Time of trying to stay in the administration, but, making sure that the owner hasn't given the drug when we do this, and sending the sample to a, an external laboratory that is using a siemensimulate, to measure the cortisol.

It's important this is done in unstressed dogs. If they are, traumatised or ill or, have a, a bad attitude, then, then the, the stress levels may well affect the, the, the results. This is available, how to interpret this is available from, from Decker, as a flow chart, but it starts with a good history and a good clinical examination.

We then go on to say that if the animal is ill, we should stop the veterol anyway because, sick dogs, no matter why they're sick, need cortisol. So we will stop the veterol and obviously, because we will be worried here about, significant. And over suppression, it, it could be sensible to do an ACTH stimulation test.

And if the values are high, then we know it's not Addison's, and so we can investigate why the animal is ill. But of course, if the cortisols are low and we've got clinical signs of hypercortisolism, then we can, assume that the drug has done it, and we need to withhold that drug, for some time, and reintroduce at a lower dose. If the dog doesn't have clinical signs of Cushing's, what we're really worried about here is to identify those dogs that have a low level of preveteral cortisol because they're the ones that are in danger of becoming ill.

And so we should lower the dose in those cases and recheck in a month's time. If the dog has no clinical signs of Cushing's, but has a high preveterral cortisol, that would suggest to me that we're probably not controlling it, that, that well. We should reevaluate the case, discuss with the owner, and consider, switching to a twice daily dosing if we're not already doing that.

If there are clinical signs of Cushing's, then really we're just using this test to decide how much we increase the frequency or how much we increase the dose of veterol too. And if, you get a low result with one of those, then, of course, that's, somewhat unexpected, and you probably should, contact Dera for some help. What about the diagnosis of feline Cushing's?

Well, we don't see very many cases of feline Cushing's. It's a relatively uncommon condition. Within that population, diabetes is very common.

About 80, 85% of cats with Cus Cushing's have diabetes. about 30% have, hyper fragility of the skin with spontaneous tearing. And those cases are considered to be more advanced and the prognosis is worse.

The diagnosis, based on clinical history and examination, is, is really important. So looking for that skin fragility, looking for it in your diabetic cat population. And being aware that endocrine testing for the CushingoA is, is more difficult.

This is the sort of skin changes that we can see with, bruising at sites of venupuncture, with thin skin, loss of elasticity and skin, and so forth. And if you come to look at the, endocrine tests that are available for, cats with, hyperadrenal corticism. Then the, the ACTH stimulation test, it actually does not too bad.

And because it is a quicker test than dexamethasone suppression, I, I would commend this test, but don't be too surprised to find that the post ACTH cortisol is never as dramatically high as it is in the dog. In cats, we typically see values only between sort of 400 and 600, something like that. A normal cat will rarely stimulate to more than 300.

it, it, it, the, the, the, the whole response is just a little bit less than in, dogs. So providing we change our, criteria, then the ACTH stimulation test is pretty successful. It's become, popular to do, what might be considered a high-dose dexamethasone suppression test in cats, with using a 0.1 milligramme, per kilogramme.

But the problem with that is, that test is that it does miss some cats. And so, if you miss, if you do a dexametone suppression test and the result is that the cat is normal, but you still think this cat has diabetes, it's got skin hyper fragility and so forth. It is reasonable then to go to do either an ACTHT or to do a genuine low dose dexamethasone suppression test, because it is a very sensitive test in the cat.

This test, a test is, is also something that can be used to rule out Cushing's in the cap. Endogenous ACTH is increased in quite a large number of cats, which is, helpful we're making this diagnosis. And evidence of adrenal pathology in cats is, really quite prevalent, where it is in the dog, that figure would instead of being 92%, would probably be down at about 80%.

in cats, they do, do get morphological changes in their adrenal glands, far more readily. So those are, those are the options. None of them have been proven to be consistently, the best, and they all have their, merits.

So what about Addison's? Well, again, it's important to philtre cases of Addison's, and in particular, I sometimes hear people say that that the dog can't have Addison's because it has a tachycardia. In fact, bradycardia is really quite unusual in these cases.

And so using heart rate to rule out Addison's is a bad idea. The signs of Addison's are very non-specific, and so we should be thinking about it quite commonly. What does mark out Anderson's is that there is a very high rate of electrolyte abnormalities and azotemia.

And if you want to rule out Addison's, then you could do a lot worse than simply say, I'm going to look at the urea, the creatinine, the sodium, and the potassium. And if they are all normal, the chance of this dog having Addison's is extremely small. The sodium potassium ratio is another alternative, but unfortunately this, again, there is significant overlaps with other diseases and only at the very lowest levels of sodium potassium ratio does it start to become a diagnostic test to, to use.

So I personally don't, don't use it. I look at the sodium and I look at the potassium separate from the ratio. I should point out that only of those 37 endocrine cases, only 27 actually had Addison's.

The other 10 had diabetes, which gave us a hit rate of about 17%, which is pretty poor for a sodium potassium ratio. It's become popular to do basal cortisols to help rule out dogs with Addisons. But I actually think that if you, if you've ruled out Addison's on the basis of the sodium, the potassium, the urea, and the creatinine.

And you still think the dog's got Cushing's Addison's disease, then really a basal cortisol is, is not going to be terribly successful, and you probably are going to have to do an ACTH stimulation test. But, for, for those of you that, find, find that ACTH is extremely expensive, it is reasonable to do basal cortisols, and, as, as a rule out, but it doesn't rule it in, and that's the problem. There are significant numbers of dogs.

With non-addrenal illnesses that have undetectable cortisols and, and so as a rule out, this, this basal cortisol can work to show that there doesn't not have Addison's, but it doesn't show the dog does have Addison's. So we need to do an ACTH stimulation test in all those cases where we think that the dog might have Addison's if, if, if we can. And I, and I think to avoid doing ACTH stimulation tests is, is a waste of time and, and money unless the ACTH is extremely expensive.

Normal dogs will stimulate to greater than 16 animals per litre. Dogs that are ill may stimulate to, to, as we said, about 50, 600 quite, quite happily. So, so we need to be aware of that.

Dogs that have Addison's disease will typically stimulate to less than 40. Should note that concurrent use of steroids will also produce this pattern. So it's really important that you check the history very carefully to make sure that this animal has not had steroids.

Those steroids would include things like Delmodone for the treatment of prostatic disease, progestogens for the treatment of, for er suppression, and as well as, as more classically steroids like dexamethasone and prednisolone. So we've talked about the diagnosis of Cushing's disease and Addison's disease. Let's just finish off with it, with a couple of comments on the diagnosis of pyochromocytoma and hyperaldosterone.

Fichryocytomas are, are adrenaline secreting tumours of the adrenal medulla. The clinical signs are often very mild and, and variable, and it can be difficult to, to pick, to pick up. Sadly, one of the commonest clinical signs in published studies is sudden death.

And these animals, need to, try to be recognised if, if we can. These days, these cases can look like, many other diseases. They can look like hyperdosterism, they can look like Addison's.

But the one that they really do look like is Cushing's disease. Clinically, they both suffer from weakness, both get PUPD, both have panting, both can have an adrenal. Mass.

Both can be hypertensive. Both can be stressed. And the fact that these animals are so stressed can drive the low dose dexamethasone suppression test to becoming positive.

And indeed, in some cases, even the ACTH stimulation test can become positive in these pyochromocytomas. So to raise the index of suspicion of pyochromocytomas, we would want to look at a consistent and chronic level, chronic history of consistent clinical signs. And we would want to identify an adrenal mass, and that will require some ultrasound.

And many of these animals are hypertensive. This may take, repeated measurements of the blood pressure to confirm that they are hypertensive. But, that is an important finding.

It should also say that these animals will not have any skin changes. So if an animal has significant alopletia, then it's more likely to have Cushing's disease. And, that as hyperthyroidism, although it's quite rare in the dog, can look very much like this, it's important to palpate the neck for a thyroid mass, as well and probably measure the T4.

To diagnose this, we wish to do a urinary normetanephrine. this is a fiddly test to do, but it is possible to do it in practise. One would take 10 mL of urine and add some concentrated hydrochloric acid, and that's probably the thing that you will have to, buy in, to do.

acidify it until the pH is less than 2. And for that, you'll need some pH, paper, and, then keep the tubes like protected, and either freeze them or, keep them in. Fridge and do this quickly.

And then you have to send either in dry ice or as a frozen pack to Zurich University. It is a fiddle, but we have been doing quite a few of these at Glasgow, Glasgow now, and I have to say so far they've been pretty successful. The method relies on the fact that as adrenaline is produced, it is metabolised to these metanephrines and nor metanephrines, and researchers out of Zurich have shown that dogs with pyochromocytomas shown here on the right, have significantly higher levels of normeinephrines compared to dogs with Cushing's disease or healthy dogs.

Hyperaldosterism, the last disease to cover, is more seen in cats. It's usually caused by an adrenal tumour and it is, is something that has become more and more recognised. The aldosterone secretion by these tumours causes hypokalemia, in these animals causing extensive, weakness.

It's still relatively rare, but, the diagnosis is becoming more common as more and more people are measuring electrolytes, we're recognising these low levels of, potassium, and, and it's becoming easier and more, frequent to measure aldosterone. So hyperaldosteroneism is, is a bit like a pyochromocytoma. It's one of these diseases that you often write in differential lists, but you can rule out fairly quickly and is quite rare.

We see a weakness, these cats will have a change in voice. They will develop cervical ventral flexion, they may become quite distressed, they may become quite lethargic and inappetent. Remembering that these low.

Potassiums are also seen with, renal disease, and in that rare, rare Burmese cat with hypokalemic polymyopathy, a, a genetic disease of Burmese cats, which fortunately is on, on, on the decrease now. To diagnose, the hyperaldosterinism, we want to, demonstrate a hypo, kalemia, but this may take several samples. Sometimes the initial, the initial presentation is of a kind with normal potassium levels.

we may want to measure the CK levels because of the myopathy that's induced by the, low potassium, and we will, measure CK levels to demonstrate this myopathy. We'll need to demonstrate an adrenal mass, sometimes by radiography, but more often by ultrasonography. And we'll need to demonstrate a hyper aldosteroneism.

And at the same time, we need to probably exclude hyper, adrenal corticism or hyper hypercortisolism, as a cause of, of the, the problem. And so an ACTH stimulation test, would be, reasonable or a dexamethasone suppression test. Having made this diagnosis, it would be nice to measure renin levels to see if, if they are increased.

But, but sadly, assays for renin are fairly hard to come by and fairly expensive. But if you can do that, that, that is better because it gives us a better idea of the source of the, aldosterone and the reason why, the, the cat has a high aldosterone level. So if I can bring this to, to, to a summary now, we've talked about hyperadrenal corticism, about the use of the ACTH stem versus the low dose dexamethasone suppression test, and when we would want, use one, when we would use the other, and the importance of, of thinking about the percentage.

Prevalence in the population, and we can alter this percentage prevalence in the tested population by filtering the cases better before we do the test. The more we philtre them, the more valuable the ACTH then becomes, the less we philtre them, the more the low dose dexamethasone suppression test becomes the most appropriate case test to use. We've talked about feline Cushing's disease, about the relative merits of ACTH stimulation tests, and the two dexamethasone suppression tests.

We've talked about hypoadrenal corticism and ACTH stimulation tests, about pyochromocytoma and that test for the urinary normetanephrines and hyperaldosterinism or CO syndrome, the, the measurement of aldosterone. And throughout all of this, we've talked about it's important to think about the question, what are you trying to show? Are you trying to prove that the or show that the animal has a good chance of having Cushing's or has not a good chance so that it's less so, and you need to think about that question before you do the test.

Try to increase the prevalence in your population, of your, in your tested population, by filtering better using clinical signs, clinical pathological changes, and so forth. And that will get you the sort of performance in tests that you see in the literature. If you do ACTH stimulation tests on every dog that comes through, your, your clinic, then you're going to be disappointed.

And, and I think we can all accept that. But, but if you miss out the filtering, then the performance will be different. And always in this, be prepared to ignore a result that does not fit.

If your clinical acumen tells you that this dog has or cat has a disease, then you get a result that doesn't fit. Be prepared to ignore it, not necessarily to the exclusion of everything else, but be prepared to ignore it and use other tests. OK, I hope you enjoyed that quick run through adrenal gland testing and it gave you some food for thought.

And, so I'd be happy to take any questions now. OK, thank you very much. I it's absolutely brilliant talk.

And I think you've done pretty well to fit so much information into just one hour. Yeah, it was quite a run run through. I appreciate that many people will be a little bit shell shocked after that.

But it was quite a lot to get through in one hour. No, it was really good. Thank you very much.

We do have one question so far from Lucy. She says, any recommendations. In testing for hypoadrenal corticism with the current unavailability of tetracosecttide, and what should we do in a likely Adisonian case, which has not had an ACTH stim test while awaiting the tetracosectide being available again.

OK. So let's do, let's just backtrack what happens if you have not got tetracozactide. Well, well, to my understanding, tetracozactide is available and on imported, you can import tetracozactide.

It's quite expensive to do that in the UK, but for an A Soian dog, it's worthwhile. They'll only have the test done once. I mean, I think the problem with, with ACTH and tetrachosactide is, it's very expensive to use this as a monitoring tool in Cushing eye dogs, but as a diagnostic test, I mean, compared to an ultrasound exam or something like that.

ACTH, even, even a sort of 50 or 60 or 70 pounds a vial, which is really expensive, but it's still a worthwhile test to do. So I, I would encourage you to try hard to get hold of some tetracosactide or ACTH because it is out there. I know Dero have not got their product out yet.

I, I understand it is coming back soon, but do try and get hold of some synack them, OK? That aside, assuming that you can't get synactin. I, I think it is reasonable to use basal cortisol in this circumstance, to, to, to, as, as an ex to help rule out.

And the second thing that you can do is when you measure the basal cortisol, measure ACTH itself. Now, that involves taking an EDTA blood sample, spinning it down. Taking the supernatant, putting the supernatant into a serum tube, and putting the serum tube then into your freezer and doing that, process in about 15 minutes, which is very easy to do.

You then phone the laboratory and get them to send you a pack to measure ACTH in your sample. And that pack, you then freeze and you then send the frozen pack with the serum tube inside it back to, to the laboratory, and they will measure the ACTH for you. In a dog that's been, if they've got a low cortisol, and it's, as it were, naturally low because that dog just had a low cortisol that time, you'll find the ACTH is low as well.

Whereas in an Addisonian dog, the dog's pituitary is saying, come on, adrenals, wake up, wake up, and it's producing a whole load of ACTH, massive levels of ACTH. You get more ACTH out of an Addisonian dog than you do out of a Cushingoid dog with a pituitary tumour. There's absolutely humongous amounts of ACTH in these dogs.

And so that's a very good way of adding good corroborating evidence that this dog has a primary adrenal gland failure, i.e., it has Addison's disease.

Incidentally, it's also quite useful if you think the dogs had steroids, because of course, the steroids have suppressed the cortisol. They've done so through the medium of ACTH. So you'd expect the ACTH to be low as well.

So learn to measure ACTH is, is my, my, my single biggest answer to that. Once you've got the cortisol and the ACTH done, then it is perfectly reasonable to start steroid treatment. And if the dog is in a crisis, then it's reasonable to give hydrocortisone.

and, and once the dog's through its crisis, it's reasonable, to continue with prednisolone, continue with salt supplementation, and, and, to wait for your results to come back. If you are worried about the dog, and you think that, you know, I'm really, really sure it's got Addison's disease, then I would be quite comfortable injecting DOCP into one of these dogs or using fludrocortisone, even though I'm not confirmed the diagnosis. As long as I know that the tests are on their way, that the result, we've done the, taken the right sample.

Has and they're being processed and we're just waiting for the results to come back. I would have no worries about using fludrocortisone to keep the dog going, or indeed, zycotal if I couldn't get hold of flutocortisone. One injection of Zycotal into an otherwise healthy dog is not going to hurt it that much.

And, and indeed, if it does have Addison's, then you may save its life. I, I would remind you, you should not use Zycortal in a dogging crisis. The correct treatment is IV fluids, hydrocortisone or dexamethasone.

We don't inject, Zycortal until a dog is well hydrated and, and going home. Excellent, thank you very much. And we have one more question from Sarah asking, she's said that you can freeze synactin, is this true?

Yes, yes, it is. Guarantees, of course, that never is, but this is a, a simple peptide. It responds very well for freezing.

Many people have done studies looking at various formulations, not enact them, but various formulations of tetrachos tide, and all of them have shown that you're able to freeze it. So, so we, we've been freezing, freezing it at Glasgow now, for some time. I think we probably set a limit of about 3 months, on, on the frozen ones, but no more than that.

OK, and another one from Sarah. Would you recommend fludrocortisone over Zycotone? No, I, I, I, recommend, zycotal, first, if you've confirmed the diagnosis.

the, the problem with Iortal, as you know, is it's a 28 day injection. Now, if you haven't confirmed the diagnosis, then obviously, if you've injected it turns out not to have Addison's, you've, you've, injected something that you, you're not going to be able to suck out. And in that circumstance, Fludrocortisone may give you some mineralla corticoid support for a short term whilst you're waiting for that, diagnosis to, to, to come in, and, and to get the results.

And so it'd be reasonable to use fludrocortisone for the those 2 or 3 days if you have some in stock that you can use. If you don't, the, the OCP but for the general management of Addison's disease, confirmed Addison's disease, Zycotal is, in my view, a better and, more importantly, in the UK is licenced and so it should be your first, port of call. Excellent, thank you very much.

And other than a couple of comments saying what a great talk this was, that seems to be the end of our questions. So I'd just like to thank all of our listeners for logging in. I hope you have time to listen to some more webinars on virtual Congress, and thank you very much to you, Ian, for such a great webinar, and I hope you can enjoy the rest of your day.