Hello, it's Anthony Chadwick from the webinar Bet welcoming you to another of our lunchtime webinars. Really super pleased today to be able to introduce Stacey Newton to you, but we're gonna be talking about blood proteins and their use in equine biochemistry profiles, . Obviously really pleased that this is a free webinar courtesy of nationwide laboratories and They're obviously doing some super work, but I was particularly excited to to talk to Alina before the webinar began and obviously saw on the video as well some of the fantastic work they're doing to be more sustainable, with their wildflower garden and also I know they're very careful around .
They their use of . The renewable products, you know, the, the, the tubes and everything, and, and how to become more circular in that as well. Consumables, that was the word I was after.
I lost the word for a second. So Stacey, graduated from the University of Bristol and she actually did a residency in equine internal medicine and earned a PhD in equine neurology at the University of Liverpool, which is my old alma mater. And her PhD focused on head shaking in horses and was obtained in 2002.
I remember, Derek Notten bell giving some very good lectures on head shaking, so . Stacey was in very good hands there. She then locumed for a a variety of veterinary practises before joining Nationwide Labs.
She's a diplomat of the Royal College of Pathologists, a fellow of the Royal College of Pathologists, and she's written various papers. And has talked extensively at Beaver conference and other venues at providing excellent CPD. She's currently working at Nationwide Laboratories, and she's a head veterinary clinical pathologist.
So, Stacey, super to have you on the webinar, as I say, brings back happy memories of being at Lee Hurst watching . Doctor Nottton belt explaining about head shaking and so on, so, really looking forward to today's topic though on blood proteins. And their use in equine biochemistry profiles.
Thanks, Stacey. Thanks very much, Antony, and thank you very much, everybody for joining at, busy lunchtime. I'm going to, let's see, hopefully you can see my screen here.
Today I'm going to talk about, blood proteins and their use in equine biochemistry profiles. Now, it will specifically mainly be on equine, but I will mention occasionally other species. So, I currently work at Nationwide Laboratories, as Anthony said, I'm senior or head clinical pathologist, and I've been there for a number of years.
And there's been a wide variety of, different sort of species that we look at there. And horses do, form a smaller but significant part of it. So we do see those on a regular basis.
So samples submitted from them. So Just as a brief introduction. I'm going to talk about what are proteins.
So just, you know, just remind you what they are. And then, you know, talk about and, explain a bit more about the main ones we use in the biochemistry profiles and, and what their use are. And I'll particularly focus on acute phase proteins and then talk a bit more about globulins, which are part of the, the proteins that we measure.
And The use of doing serum protein electrophoresis and the method there, and what, what is the use of dividing these into different fractions? And then towards the end, I'll be looking at main reasons why we might get different values of albumin and globulin, just as a, you know, a sort of ballpark thing, you know, what does that mean? What could it indicate what sort of pathology?
So we take a blood sample. And We submit it generally as part of a biochemistry profile. Now, you can see that we've got proteins, but we also have a whole load of other, parameters and markers that we will be looking at that, you know, looking, at pathology that might be involving the liver, skeletal muscle, obviously the renal enzymes, and also various, electrolytes.
And this will give information on sometimes function and also if you've got any damage, etc. Pathology involving a particular organ, and the proteins form quite an important part here and can indicate various changes along with what we're seeing on the changes in the other parameters of what might be going on in your individual. So what are proteins?
Now there are a large group of molecules and there's over 100 present that are involved in doing a huge or wide variety of biological functions, and they're in they're essential for the normal function of an individual. Now, collectively, they're involved in important thing like nutrition. They also maintain colloidal osmotic pressure.
They're involved in immune and inflammatory responses. Also, clotting, which I'm not going to talk about here really. It'll get mentioned, but that requires a separate talk.
And also, it's involved in acid-base balance and other things. So the proteins are known as various different things. So enzymes, we have hormones, obviously, the antibodies, we have various carriers of other, substances, the coagulation factors, which again involved in clotting, and the acute phase proteins, which I'm going to go on to talk about in more detail.
So when we look at a biochemistry profile, in most domestic animals, we're looking at our, this includes the horse, obviously, the globulins, and then we have acute phase proteins, which are not in our profiles, but in most cases are added to give more information. And then we have the clotting factors. So the liver is the main producer of most proteins.
So it's a very important organ here. Now there are other tissues which are listed underneath there that can produce proteins under certain conditions, and they may include various organs like intestine, lung, etc. And just to remind you that serum.
In contrast to plasma, does not contain fibrinogen. Or some of the clotting factors. So the difference between serum and plasma, just, I mean, it's probably basic, but I just thought you remind you of that.
So when we do our biochemistry profiles, we start off with the total protein. And this is a combination of albumin and globulins. We can also look at total solids, which can be done in the practise.
And this is done by refractometer in comparison to what we do in the lab here, which are chemistry methods for the album and the total protein. So the refractometer, you can read that, putting some of the, serum on there, and have a look and get an estimation, which is similar to total protein, but will more, in more cases and not have be slightly higher. And this is because, well, actually, if we have fibrinogen there, you're probably looking at, plasma.
So, but other substances, such as glucose, urea, some of the electrolytes, etc. Are all gonna add to the total solids that we're looking at, on the refractometer. Now, if you have significant lipemia, this can actually cause false elevations in the readings of total solids.
But actually with our assays in the lab, if you have significant lipemia, this can also have an effect on the values of albuminin globulins, so the total protein. And also bear in mind if you've got significant homolysis that can cause difficulty in actually reading the refractometer. The acute phase proteins are classified into positive and negative.
Now, positive ones, tend to increase. Well, that's what the meaning is. And the major one in the horse is serum amyloid A or SAA.
The moderate one is fibrinogen, we've also got haptoglobin, and then we've got a couple of minor ones, which are actually more important than other species. So the alpha one alpha glycoprotein and the C reactive protein. Now albumin is a negative acute acute phase protein, so it decreases when we have an inflammatory type response.
So acute phase proteins or APPs, what's the definition of these? They're actually proteins, whose plasma concentration will increase or decrease by at least 25% after an inflammatory stimulus or an acute phase response or APR. And as I mentioned, positive ones go up, and they will increase quite rapidly, usually within a couple of days.
And they form part of the APR which is a complex system, systemic early defence system. And this is part of the innate immune response, which is activated by a number of different factors, which include inflammation, but also you've got trauma, infection, stress, neoplasia, etc. Can activate this and then get, production of APPs.
So, as I said, positive and negative reflect the relative increase or decrease. And then as I mentioned as well, that the positive AVPs are also further categorised into major, moderate, and minor. So in the horse, just to show you here, the major one is serum amyloid A.
And moderate ones include fibrinogen and haptoglobin, and then the minor ones, alpha 1 a glycoprotein and C reactive protein, are not really used in the horse, and I'll come to that in a minute. But there we have our major negative acute phase protein there on the end as albumin, so that will decrease. So why are we classifying them as this?
So the major acute phase protein, serum amyloid A, is present in low concentrations or not measurable in the serum of healthy individuals. And then when it's stimulated by an APR or various stimulus, it can increase quite rapidly and also very markedly, so up to 1000 times. And it peaks about 24 to 48 hours after the insult, but then rapidly decreases once that's gone.
The moderate APPs, which in the horse currently that we use is fibrinogen. And this is present in the blood of healthy animals, but not to a high level. And when we get an APR or a stimulus, we get an increase that's much lower in amounts, so 5 to 10 times, and it peaks at a slower time.
So we're looking at 48 to 72 hours. And then once that insult's gone, it decreases at a slower rate back down to normal values. Now the minor APPs are not terribly useful in the fact that they only increase by about 50 to 100% above the resting levels, and they're very much slower, slower increasing as well as decreasing, so they're not really relevant as to what's going on in the current time.
So the increase, the rate of increase and decrease and whether something's a major or moderate APP will actually vary between different species. So the APPs form part of, mainly part of the globulins. Most are in the alpha fraction.
Now, I'll come to where we're getting these fractions from slightly later, but I just wanted to include this now. And there are some in the beta globulin fraction. As I said, they're mainly synthesised by the liver and usually within 24 hours, after acute tissue injury, but also you're going to get other factors that can stimulate the production.
So stress, estradiol, corticosteroids, and you can also have, as I've mentioned before, sorry, the extraopathic production by the tissues in some cases. So the synthesis is stimulated by interleukins, so IL1B, 16, and also tumour necrosis factor, which are released mainly from macrophages. And then we get the APPs being produced, which then have a role in immune response, they're anti-inflammatory, and they're also anti-infective.
So this is why they're useful because once we get information or some trauma or some injury for some reason or whatever the cause of it, they increase quite rapidly and they increase faster than any changes you might see in the hemograms, so the white cell count. And high values can correlate with the severity of the original insult, but it doesn't necessarily reflect what's going to happen, in the, the outcome. So you can get very high levels, but if the original insult resolves, this can go back to normal and you get complete resolution.
So looking at APPs is also important in monitoring whether we've got a response to treatment and also whether we're getting a resolution of the original insults, so tissue inflammation or trauma. And if you have sustained high values of these APPs, this may indicate a poorer prognosis. So obviously, you've got ongoing pathology for some reason.
Now, it can also be, they can also be increased with various tumours, mainly ones that are associated with significant inflammation. So we're looking at something like lymphoma. And just bear in mind that APP values in some of the references have suggested that they don't always correlate with response to chemotherapy, if that's being used.
So they may be, may, may not be the best thing to use for monitoring in that sort of situation. Now, you can measure APPs at other sites. So we're looking at body cavity fluids, synovial fluid.
CSF So if we just move to albumin for a minute just to go through this. So Almon is a very important protein. It forms the majority of plasma proteins, so up to 50%.
It's water soluble produced in the liver. And as I've said, it's our major, well, I should say it's our most important negative APP. So its production is regulated by lots of different factors, which include colloid osmotic pressure, which is quite important, and then various hormones, which include the typical hormones, you know, used for other things.
You're looking at insulin, we're looking at cortisol, thyroxine, etc. And it has a long half-life. So we're looking at nearly 20 days.
One of its major important, functions is to transport different plasma constituents, and it's very important, with respect to getting things around the body. It also has various other functions now that's got more than I've listed on here, but for example, it dampens platelet aggregation and augments antithrombin II, which obviously is involved in the coagulation, cascades. Now in the lab, we measure it with various dye binding assays, so we use bromorestal green, and there are other methods that can be used to measure it.
Our normal reference interval that we use at the lab for a non-thoroughbred adult is between 27, 36 grammes per litre. Now, this will vary between different labs and references, but it's roughly about that for a normal adult. When we get an APR or inflammation, the almond can decrease, but it usually remains above 20 grammes per litre.
So it's usually a mild decrease. Now some people might say, oh, if it's gone from 27 to 20, that's not mild. Well, it may not be.
It depends exactly what's going on, but it doesn't go down to the levels where we're starting to worry about, you know, where, you know, what's happening, where is it must be loss, you know, must be losing proteins. So things like intestinal disease, malnutrition, chronic blood loss. Various chronic inflammatory disorders, will usually result in decreases in album, but it can be, you know, it's, it remains usually above 20.
When we start to get severe losses, so I'm talking about less than 20, and you're actually probably require in most cases less than 15 before you start to get things like effusions occurring or peripheral edoema, etc. Then usually we're looking at gut, pathology. So protein losing enteropathy.
So the globulins here, we have those, they're measured from, we measure total protein and albumin in the lab by, chemistry methods, and we actually take the albumin from the total protein to get our globulin total. So it's not actually measured, it's inferred, basically, or calculated, shall we say. It includes the APPs as well as the immunoglobulins, and we can further look at what is making up these different globulins by doing serum protein electrophoresis, which I'll go on to just explain briefly, shortly.
So the globulin fractions we can get by separating them out in this, with this method are the alpha 1 and 2 fractions, beta 1 and beta 2 and gamma. Now, alpha and beta are mainly synthesised by the liver, and they include RAPPs as I've mentioned. And there's also IGM and IGA.
The gamma fraction is mainly immunoglobulins, and these are produced by B lymphocytes or plasma cells. So if the globulins are increased, various reasons, obviously, inflammation, this can be acute or chronic or and chronic. It can be infection, we can have an immune response.
And also bear in mind, if we've got a ppatic disease or, you know, patetic failure insufficiency, you can actually get increases, and this is usually due to the livers involved in the degradation of globulins. So once the function becomes significantly impaired, it globins can actually be increased. But obviously, if you've got inflammation as well occurring, you know, there may be some sort of, difficulty in differentiating and that's where we come to serum protein electroresis.
So we'll come to that in a minute. But decreases can also occur. And most importantly in the horse, we're looking at the neonates.
It's a failure of passive transfer. So, you know, within 24 hours, they've not received enough lostrum, they may have low globulins. There's also various inherited disorders and, protein losing and thropathy, as I mentioned before.
If this is particularly severe, you can get low globulins. So if you move on to talk about serum protein electrophoresis. This is most commonly done by an Agarose gel method.
And what we technically do here is the serum is put in the gel. And the various fractions and proteins present get separated out. The gel is then stained and dried, and then we put it into a densometer which translates the different fractions or the protein fractions into this nice little graph, which we have here on the right.
So an electroferriogram. So what we can see there is we've got Amin on the left, this giant peak that's in white, and then the other shaded area are all the different globulin fractions. Now they're not labelled alpha and alpha 2, but 1234, and 5, sorry, are the different fractions of the alpha, beta and gamma.
So, when we have the, serum in the gel, we actually pass a current through it, running from, negative to positive. And then the proteins separate out based on their size and their charge. So we get this spreading out through the gel.
And at the same time we, we measure our total protein on the analyzer, so we've got an estimate of that, and we also measure albumin and you'll probably find in most cases the albumin measured by the lab, so the chemistry method will be different. And one of the reasons is that some of the dye binding methods can bind some of the globulin fractions and therefore you get an artificially high result. So the albumin might actually be higher compared to what you're seeing on your trace.
We do the serum protein electrophoresis. Now, more recently, in the last couple of years, we have the cap capillary zone method for doing so in protein electrophoresis. And this is a more automated method of doing this, and it involves using an electrolyte solution in a capillary, and you do the same thing, you pass a current for a certain length of time.
And you actually end up with a similar trace as you can see on the right, with this method. Now, the measurements are typically very similar. There are some slight differences.
Almin can be a bit higher than the gel method. And some of the globulin fractions can be different, and you can actually separate out more, what looks like more apparent fractions. One of the things that you might also see is two peaks in the albumin fraction, which I couldn't find a trace for this, but that can occur and it can occur in normal animals as well as abnormal individuals.
It's also quite a common finding in the exotics of the avian species, but you can see here on this trace, we've done an albumin value from the analyzer in the lab, and that's 34. 35.2 grammes per litre.
And what we've got on the trace is 29.5. So you can see there is a difference, but it's still at this moment is remaining within the reference interval.
The resolution with capillary zone, protein electrophoresis is slightly, greater on the negative side, so towards the albumin side, and then it's slightly less as you move towards the gamma fraction or the positive side. And bear in mind that if you've got significant hemolysis, you can actually get haemoglobin haptoglobin complexes forming. And this can result in a peak in the B22 region, which I don't have a pointer, unless you can see my arrow, which would end up being somewhere here and could end up being an artefact.
So, That last second to last fraction with the narrow lines there. So just remember, you can get things affecting your trace, that are actually, artefact. So just to show you a normal trace.
And as I mentioned, Amin is there on the left. It's the forms usually the largest part of this, unless of course you've got massively increased globulins. And then you have the alpha one fraction, which I've show show you there that serum amyloid A is present within there, as well as our alpha 1 alpha glycoprotein.
Haptoglobin is generally present in the alpha 2 fraction. And then the beta and gamma fractions can contain CRP which, as mentioned previously, is not particularly useful in the horse, but just to mention where you might find it. And then you've got gamma fraction there with our immunoglobulins, so IGG.
So what happens after an APR? I mean, what happens to the APPs, what happens to the serum protein electrophoresis trace. Now, it depends on the degree of information and what's going on, but the total protein may remain normal or it may show mild increase.
Your album may remain normal also, or a mild decrease. And then you get variable increases in the alpha 1 and alpha 2 fractions, particularly if this is an acute inflammatory process. Now, the APP is found in high concentrations, which interestingly, haptoglobin, which we don't use commonly in the horse, but is a moderate, acute phase protein, will actually significantly increase the alpha 2 fraction of the alpha globulins.
And the serum amyloid A, which though it's our major protein, is found in much smaller amounts normally or not at all in a normal animal. So when it increases, though it can increase quite massively compared to what it is at the resting level, it actually in most cases does not change the fraction seen in the trace. So you're not going to probably get an obvious increase there that you can say, oh yes, that's probably due to that.
So, With the APPs, We're going to look at them separately. And obviously, that sounds sensible. It's much more sensitive test to actually look at them separately in some cases, rather than trying to rely on the trace, even though we know which fractions you might find them in.
So we measure the, the various, APPs separately in the positive ones, and we look at the changes, and, and seeing, you know, different conditions, you might get various increases. So we'd be looking mostly at alpha beta region if you were looking at a trace, and then we can measure these separately. So if we look at serum amyloid A, As I've mentioned, this is our major acute phase protein.
It's actually quite highly conserved amongst our domestic species and so useful in other species. So you can use it in the dog, the cat, etc. It has various functions including binding optimised and optimised gram-negative bacteria.
It's also antiviral. It's involved in leukocyte, chemotaxis, and obviously it's also a precursor of amyloid A which can be involved in various conditions where we get deposition of that. There are various assay methods that are used.
We currently use latex agglutination method, but they're, you know, they're different between different species as well. Ours can be used for not only the horse, but also, the cat and dog, etc. It has a short half-life.
And it increases pretty rapidly after an inflammatory stimulus of 6 to 24 hours, and then decreases again within 12 hours of resolution of the insult. The normal reference interval we tend to use is between 0 and 20 mg per litre, and it can go up to like 1000 or more Migs per litre. And as mentioned before, it's more sensitive indicator of inflammatory, process compared to white cell counts and fibrinogen.
It's non-specific, so I can't say, oh, this is increased to this amount and therefore means I've got this disease. It should be used in context with other changes in your biochemistry profile and obviously clinical presentation. And we can measure it in other substances, so, synovial fluid, body cavity fluids, saliva, we can also use that.
So this is quite an important protein that we use in the horse. If we look at fibrinogen, Now, I've put this trace on here just to show you what would happen if we did a trace on plasma with, with fibrinogen. So if you did a trace on serum, you're not going to see fibrinogen.
It appears when we do a trace, as you can see on the right. As a peak in the beta 2 region and it often looks monoclonal, so quite a narrow sharp peak in the beta 2 region. So if you did a trace on the plasma, that's what you would get.
And it's basically, it's, doesn't mean it's got some pathology there. It means it's got fibrinogen. So you need to turn around and do, look at the serum sample, and then you can compare.
So fibrinogen is a moderate acute phase protein. It's produced in the liver, and there's various assay methods that are used. We currently use the Claus method, which is a modified thrombin clot time method.
And the reference interval we use for fibering currently, and it will vary between labs, is about 0.5 to 4 grammes per litre. And after an APR we get a 1 to 10 times increase in the value, and that's within 24 to 72 hours with a peak that is longer than SA, you know, serum amyloidase, around about 3 to 7 days.
And again, fibrinogen actually the increase may also precede changes in the hemogram. It is the largest proportion of our plasma proteins. So it is quite important.
And therefore, it, you know, when we've got a significant inflammatory response, you can, it's measurable. So it is quite useful in looking at in, you know, it's included in the profile. So it increases with inflammation, also neoplastic processes, which may be involved with inflammation.
Now, I put dehydration in there cause there's various references that talk about the interference of dehydration with measuring fibrinogen. Now, I've not really ever used this. I tend to look at the hemogram and the red cell parameters, but they suggest that you should do a ratio between plasma protein and fibrinogen and determine whether you've got any interference.
So is it a true increase in fibrinogen or you've just got dehydration? But as I say, I tend to look at the hemogram. You can also get low fibrinogen, and this is most commonly seen with DIC, so when you've got thromboembolic disease and also liver failure, but as I've come on to, it needs to usually be quite severe, failure in function before you start getting significant decreases.
Now, I wanted to talk about haptoglobin, which no one uses currently in this country, which they probably do, but today, unless somebody can tell me in the chat or whatever, the, there is no lab as far as I know, that currently measures this in the UK. Now, Germany, has Labilin doing this assay, and, it's a moderate acus face protein, so you'd think it'd be quite useful. It is actually quite laborious to, to do the assays.
And there's various different ones that have been developed, but I think partly because we've got serum amyloid A and it's easy to measure fibrinogen, and because the assay is so difficult, no one seems to have really started to use haptoglobin. And I think it probably involve would, would, you know, be interesting to see if we can have some further investigation into the use of this protein. Because one of the interesting factors here is that it actually binds free haemoglobin and myoglobin.
And macrophages phagocytose this to prevent iron loss. So if you get free haemoglobin occurring, so you've got a hemolytic episode, then this will stimulate the production of haptoglobin. And the, when we have these occurring, so intravascularmolysis and also I mentioned myoglobins, so you've got exertional rhabdomyolysis or other myopathies, significant muscle trauma.
Then this will actually, bind haemoglobin, sorry, haptoglobin, and also the free haemoglobin myoglobin will stimulate its production. So what happens to the actual level, level of haptoglobin? We don't, in some cases, it's difficult to determine whether it's actually going to increase or decrease.
So it depends on what's going on. If you have no evidence of, of hemolysis or rhabdomyolysis, then it can be a very useful protein. But also, if you've got low levels for some reason, then it could suggest to you, hey, look, look at the hemogram, etc.
Have we got this also occurring? Why is the haptoglobin low? But obviously, we don't measure it on a regular basis, so we wouldn't know.
So obviously we need to look at hemogram. But it increases within 12 to 24 hours post-inflammatory stimulus, peaking similar to fibrinogen, so 72 to 120 hours. And we can see it increased in both acute or chronic inflammatory processes.
Just to mention briefly the minor acute phase proteins, we have alpha one acid glycoprotein. So this is not very useful, as I mentioned, it's slow to increase, slow to go back down to normal, and so therefore not typically representative of what's going on. Now, there are various references out there that have looked at it in, you know, whether it's useful in looking, post castration or gut surgery.
And how long it takes to return back to normal. And you can see there that in, that particular one that was taking up to a month. So in most cases, it's not terribly useful in the horse.
Now, it's actually a moderate APP in the cat, particularly when you're looking at FIP or other inflammatory processes. It's obviously not specific for FIP but, it's one of the useful proteins in the cat. C-reactive protein is a major one in the dog, and some references actually suggest that Duncan and Price suggests that it's also moderate in the horse, but most of the others say not.
And it's got various assays, and we currently measure it in the lab, particularly, just for the dog. Our assay only does it for the, measures in the dog, so only validated for that. But in the horse, it again, it's quite slow to increase and decrease, and there's been various references looking at increases with arthritis, various other inflammatory conditions, including, RAO, various asthma type, conditions.
So to briefly mention other proteins that could be of use and are probably useful, but just not measured very often, we have transferrin, which are actually measured when we're looking at parts of the iron, profile. And this is a negative acute phase protein like albumin, and it will increase with acute liver disease. We also have the low density lipoprotein, so LDLs, VLDLs, which, will increase with nephrotic syndrome.
Completing components, involved in various inflammatory and clotting increase with, acute liver disease, and troponin, which we measure in the lab, is a marker of myocardial damage and so therefore quite useful. And then I just mentioned er the plasma with copper transport, and then obviously the clotting proteins, which are a whole massive thing to talk about, but are not included in this current talk. So the final part of the talk here is to look at what does albumin and globulin mean when they're at different, you know, increased or decreased.
And then I will just go on to towards the end, looking a bit more at the traces of the globulins with the serum protein electrophoresis. So if we have low albumin and globulin, what does that mean? In most cases, it means loss, and this could be blood loss.
This could be lost through various organs. So we're looking at prot enteropathy, you've got significant gut ulceration or parasitic disease, neoplastic processes, etc. Involving the gut, you can get lost through there.
And there are all, sorry, other organs such as the skin, which is a massive organ, you get significant damage to this. You can obviously get exudation of proteins through this. And also, various effusive disease, so loss into the body cavities for various reasons, you know, peritonitis, pyothorax, pleurritis, you're going to get protein loss in there.
Also, remember, vasculitis, which again, can result in their decrease. Remember also, if you've got a patient, particularly an inpatient that might be on fluid therapy, this could cause a dilution effect and you'll get a lot, well, apparent loss, but it's not. It's just a decrease in albumin and globulins.
And then you have other edoema dis disorders, including secondary to congestive heart failure or nephrotic syndrome. If you can see that. And then excess ADH secretion.
So anything that's involving, increase in ADH, can also cause a decrease. So if we have low albumin and normal or increased globulins, what does this mean? This tends to mean, in most cases, a loss in albumin alone.
And for example, you can get lost through the kidneys or if you have a glomerulonephritis, and you should have other biochemical biochemical changes occurring, which include, protein urea, hematuria, and also you can get elevated cholesterol. This can also occur secondary to decreased albumin production, but in this case it's quite minor. It's usually secondary to an APR.
And unless you've got severe liver failure, so 80% loss of functional mass, or more, then you're unlikely to see a change in the albumin. And this is basically because the almond's got such a half, a long half-life. Now, you will also see other changes in the biochemistry.
We do expect to lower your rear. Low cholesterol, you might also see evidence of, cholestasis, so increased bilirubin. And then also, if you've got prolonged starvation or malabsorption, then you can also see a loss in album and usually there's GI tract signs associated with the malabsorption.
Now, if we have normal to increased albumin or and low globulins, then it normally indicates, sorry, decreased immunoglobulins. Now, the most important one, as I mentioned previously, neonatal falls with the failure of passive transfer, so lack of colostrum or inadequate lostrum, you can get, low globulins. Now, this could be masked by inflammation and dehydration, etc.
So we actually can measure the IgD separately. And look to see if this is adequate. So it should be greater than 8 grammes per litre.
So in, in that case, that's more useful than trying to look at our total globulins. We also have various immunodeficiency disorders, and they can be both inherited or acquired. And we have severe combined immunodeficiencies that you see with Arab falls, also fell pony syndrome.
And then we have various selective deficiencies that might involve different immunoglobulins. If we have increased almin, in most cases, it's because we've got dehydration. And just remember this, effect of the BCG dye method, pseudohyperalbumia, where we're getting, some binding, sorry, of parts of the globulin fraction and therefore, we're getting an artificially high albumin.
If you've got increased almon and globulins, again, usually due to dehydration, you should also have increase in red cell parameters, which you can use to confirm this. However, if you've got an animal that's got coexisting anaemia, remember that can look normal and as soon as it's hydrated, it'll actually fall and then you can see that we have an anaemia present in that individual. But usually increases are due to dehydration.
So if we look at the globulins, we'll start with the alpha fraction. So the alpha fractions have alpha 1 and alpha 2. And the acute phase proteins are present in these.
So we've got serum amyloid A present in, alpha one, and these will increase with acute inflammation. However, as I've said, Serum amyloid A by itself. There are other, proteins there.
So alpha and acid glycoprotein will also increase. But serum amyloid A in most cases, as I say, even though I've put it in there, is not really going to cause a huge change there. The alpha 2 protein contains haptoglobin, so fraction, sorry, can, can increase there, if you have increased haptoglobin.
And also I mentioned there, the alpha 2 lipoprotein, which is involved, you see an increase with nephrotic syndrome, and that's usually due to an increased production because you're getting loose loss of albumin it's trying to control the osmotic effect and also you get, defects in the actual, excretion of the protein. So you just basically get increase of these proteins circulating and cholesterol is another one that will go up, secondary to nephrotic syndrome. But you can see in these fractions here, the alpha 2 fraction, there's a peak there, that second peak that you can see after the albumin.
And then in the second trace here, you've got peaks in the alpha 2 and quite a big peak in that beta one. Which if we move on to the beta fraction, so we've got beta 1 and beta 2 are the main ones. I mentioned CRP which again, we don't really use in the horse, but beta one increases can be seen with, strongal activity or strongal infections.
Again, we can have, various inflammatory processes including increasing beta1, beta2, particularly beta 2, and it can be polyclonal, where you get these broad peaks there. And if you get a monoclonal peak, which you can see this on the second diagram, you've got this large tall, blue shaded peak, which is quite narrow. This is not a monoclonal peak and usually due to neoplasia.
So we're looking at lymphoma or multiple myeloma, also sometimes chronic lymphocytic leukaemia. We can also get increases in beta2 with liver disease, and also, in some of the references, they talk about, the, a monoclonal peak, which should be similar to this one that you can see on the second diagram, due to intestinal, pathology by strong Ilu species of vulgaris and Vesta. This is usually due to an increase in IGDT which is a specific subclass in the horse, that can increase when we have infections by these parasites.
And it also, as I mentioned, can be seen in, in the alpha 2 region. So just to mention beta gamma bridging, this is a loss in, definition between beta and gamma peaks, as you can see in this graph. You've lost the division here almost.
I mean, it's almost just one peak, but you've got a slight dip. Classically, this is due to liver disease, but it can also be seen with various infectious processes. And the reason for the association with liver disease is due to decreased removal of IGA.
And antigenic material and therefore you get a regulation of IGM IGA and IgG and so you get increases there that, that can occur. so just to mention one of the reasons why you might see that in a, a trace. Now moving on to the gamma globulins, these mainly have the immunoglobulins.
I've mentioned CRP, which obviously, is not as important, but it follows a similar thing with the beta fraction. If you get a polyclonal increase, which actually this is a normal trace, but if you had an increase that was polyclonal, you'd see a broad curve like that. You see at the end there.
And this is usually due to, infectious disease, chronic inflammation, and, and you'll just get a, a broad increased peak in that area. If you get a similar narrow peak that we saw back in the beta fraction, it means a similar thing, usually means neoplasia. And this is usually due to, as I said, B cell lymphoma or multiple myeloma, and also chronic lymphocytic leukaemia.
Also bear in mind the oligoclonal peaks, which are actually also known as restricted polyclonal, and these are actually polyclonal and the fact that there's lots of little pro proteins that are very similar size and charge, and they migrate to a very similar area and they mimic a monoclonal peak. So this is usually due to infectious disease and is not neoplastic. So that's one of the things you've got to bear in mind when you're looking at these things.
It may not always be neoplasia and you'd have to, correlate it with other, clinical findings and changes in your biochemistry. So just to summarise, as I mentioned, proteins are very important, and there's lots of them, and they have a huge variety of functions that are very important. Now, we've only really touched on a few of the important proteins we use in the horse, and there's plenty of others out there, I think that probably would benefit from further investigation.
As to use in helping us diagnose various processes, but we can see that looking at these proteins, we can determine whether we've got an acute or inflammatory process. So serummolo and fibrinogen, most often seen with active acute inflammation. Remember our album, can remain normal or decrease with inflammatory processes.
And then we have our beta and gamma globulins, which are more commonly increased with chronic inflammation. And we can also see by looking at our serum protein electrophoresis, we can investigate other different pathology that might be present, particularly neoplasia, but also liver disease. And one of the things that people say or ask is we've got, a measurement of globulin, it's not elevated.
Is it useful at all to do so in protein electrophoresis? And actually, I think you probably should start doing that in any case because if we get changes in different fractions, it can suggest something else is going on, particularly if you've got early stage neoplasia and you might start to see these, you know, narrow peaks occurring in the particularly the beta or the gamma fraction. And, and various of the changes that, you know, may suggest we've got an abnormality.
Now, obviously, you could have, if there's only, it's very very mild, each individuals can have a slightly different trace and is it significant? You just have to use it to monitor and see if there's any changes and also correlate it with what you've got in your biochemistry profile. And you can see here with just looking at albumin and globulins, as basic values, they can be important in telling you what's, you know, possibly going on.
And can indicate certain disease processes depending on the, the different values. And monitoring of the proteins is quite important. You know, we can see whether our treatments working, has the original insult or whatever is going on that we've identified, hopefully gone away, or is it still, you know, present.
So, this is just a brief thing. There's a whole lot of other proteins we could talk about and, thank you very much. For listening to this talk, I've put a list of references here, that are quite useful, with respect to looking in particularly proteins, but a whole load of other, factors on, clinical pathology.
And hope you find those of use. And If you have any questions, please shout and thank you very much for giving me your attention, at this sort of quite busy time. Thank you very much.
Thank you so much, that was excellent. We have got a few questions, so I will feed those over to you now. We have a question, which is not about horses, but I don't know if you don't, if you don't mind answering it, the first one up was, what is the importance of haptoglobulin haptoglobin, sorry, in mastitis diagnosis in cattle.
Is that just something that we, we monitor and look at at all? I don't have enough information on that currently, but I would think haptoglobin would increase in cattle. As far as I know, we have used it.
As I say, most labs don't measure it, but I think it would be probably useful. I'd have to go back and look at references, Antony. I don't, I don't know offhand.
But I know that they have measured it in, in cattle. So anything that's involving inflammatory process, and particularly something like mastitis, which can, unless it's full blown, and obviously, you know, you can see it obviously clinically, then yes, it could be of use for sure, yes. Yeah, it would seem to make sense that within mastitis, it might go up, doesn't it?
Yeah. Mahesh is also asking what conditions might cause hyperglobulinemia in equines. Hyperglobulinemia, hyper.
So what particular conditions? So, well, I would mainly, I mean, you'd be looking at, something more chronic inflammatory process, anything that's resulting in immune stimulation. If you've not done a trace, it's going to be difficult to say which fractions are causing this increase.
But, if you had, also, also neoplastic processes, particularly you want to try and exclude. So I would particularly suggest, I mean, particularly if you had increased globulin that you do a trace, a serum protein electrophoresis and look to see whether you've got, any particular peak, you know, like, in the gamma or the, the beta fractions that may suggest you've got a neoplastic process. But, by themselves, you know, it's usually something, it's either inflammatory, immune stimulation, usually.
Yeah. It's not, it's not specific for any particular disease. I can't say that it's got this level of globulin, it's got this.
So yeah, it's those are the main processes that increase them. Fantastic, . To sort of two questions coming together here, how would you interpret a hypoglobulinemia with normal albumin in an adult animal presented with weight loss?
And then as a secondary, is there any value in looking at the albumin globulin ratio? I'm not sure the album globulin ratio helps a huge amount. I like to look at things individually.
I think in the horse, if you're finding slightly low globulins, and we're looking at, you know, probably a young. Particularly any, any sort of young horse we're looking at anything maybe up to a year or so, you may have slightly low globulins. And this is also something that you see in the dog, you know, younger dogs and, younger, you know, that, that sort of, those species.
If it's particularly severe, you've got normal al and you've got, you know, and it's particularly severe, then you might have one of these immunodeficiency. Type problems and obviously you should look at the breed, you know, exclude it's not an Arab, etc. But you could have an immunodeficiency of some sort.
Now if you're getting significant protein loss somewhere, you would expect the album also to be low. The globulins are not going to decrease without the albumin, so you can probably exclude that. Though I suppose if it's early stages, you couldn't, but our low globulins by itself usually, indicate that you've got some sort of, decreased production for some reason.
So we're looking for one of those. Thank you. I do like your horse on your slides, by the way, Stacey Porcelain horse, I presume it is.
Yes, it looks lovely. Anna is asking a question, when monitoring for subclinical overuse, stress injury, what cut off levels of SAA would you use in poor performance cases? Right.
So this is something actually, they were talking about this in Beaver last September, but, I don't have any current, a lot of information on, on that. And the, the levels are that, you know, vary, because the assays are slightly different because there's a lot of assays that they use on, you know, on site, and they're different from the ones we use in the lab. And their cutoffs of what might be normal or abnormal.
I mean, I can't remember the guy's name, sorry, that we're speaking. He was actually referring to quite a lot of these cases, particularly the endurance ones, and he was using cutoff levels that were higher than we class as normal. So I think anything less than 100, he was suggesting.
You know, the horse is probably fine, which I thought was quite huge. But he said the values he was getting in some cases that were obviously showing, you know, evidence of poor performance were much higher than that. They were getting, you know, 200, 300 up into the, you know, you know, top 100s.
So it does vary between the essays and, you know, what you, what you sort of look at, but I, reference range, isn't it, whatever. Yeah. No, that's really interesting, and then Tim's saying not a question but a comment about haptoglobin, haptoglobin.
He thinks he's offered by SRUC up in Scotland, at least it's in their price list, he said. So thanks for that, Tim. Right, yeah, thanks for that.
But I think this is what I'm, this is why I might be wrong, but I remember looking for this and when we talked to them, they didn't actually do it in-house. I might be wrong. I think they send it away.
Yeah, yeah. But I might, they might have changed it since then because it was a couple of months since I've looked into this, but I did do some investigation and I'm pretty sure they send it out. No, that's useful.
So I mean, with any, with that, it's obviously gonna be a, a longer time to report back on which sometimes reduces it, the reason to do it in the first place, doesn't it, I suppose. Yeah, exactly. I mean, if they can find a better way of economic way of measuring it and sort of do it where they can persuade the quiring community that it's really useful, then, we, you know, we can start, start it up and using it.
Yeah. Sharon's saying thank you, really interesting. Obviously just like to thank you, Stacey, on behalf of, everybody listening, and Nationwide Laboratories for, for doing the webinar today.
The only problem with webinars is you don't hear those tumultuous applause that you might hear at Be giving a talk, so apologies for that. Perhaps just giving people a couple of minutes just to think before we finish, perhaps, You would let us know where you're listening in from. It's always interesting to hear where people are listening in from.
Jaws said that he's listening in from Lisbon. Hopefully it's good weather over there in in Portugal, Jo, but if anybody else wants to pop in where they're listening to, it's, it's always nice to hear, cos, you know, webinar vet's mission is to make veterinary education more accessible and more affordable to vets across the world. Obviously nationwide is a, is a UK company, but I think the beauty of having set this up 15 years ago is that it allows, The education that we're producing in the UK to go, you know, all over the world.
And it's great to see here, we've got Malaysia, Azalea from Malaysia, we've got Anna from Germany, Mark from Dallas, Texas. Great to see you, Mark, and Sina from Hoboken. In New Jersey and Lola in North Carolina.
Daniela in Malta had a very nice week at Easter in Malta a couple of years ago, so, bit windy Daniela, but it was, it was lovely. And then Anne's from the Wirral. So you're not far, I'm in Waterloo and Crosby at the moment, Anne, so we can probably wave at each other.
Sharon's saying boring County Durham. I think County Durham's lovely, so don't be, don't be saying that, Sharon, it's, it's all good. So thanks everyone for listening.
If there are any other questions, speak now or forever hold your peace. And I just love you, well, sorry, I'd love you. I've just heard Sharon saying I love it really, so that confused me from County Durham.
It is a beautiful part of the world. If those of you listening from abroad, County Durham, they've got great accents as well. So.
Thank you all for listening, it's great to see you listening in from all over the world. Thank you again to Stacey for a a really great presentation, and of course nationwide for making this possible. So if there are no more questions, Danielle is saying thank you very much.
We've really enjoyed that webinar, Stacey, and hope to see you. Soon on a webinar with Nationwide or indeed one of our other webinars as well, and I hope you have a great rest of the day, evening, morning, wherever you are. Take care and have a great day, bye bye.
