Hi, everyone. And thank you very much for joining me while I discuss all of the laboratory tests for the oncology patient. So there are so many different ex like extra lab tests that we will use in oncology for our patients.
So I thought it'd be kind of, an interesting talk to put together just to help you guys get your head around it because it certainly took me a little while to get my head around all of these different tests that were available, and because we we all And if you're in general practise, it's not something that you would probably come across routinely. So, yeah, I just thought it would be an interesting talk for you, So I'm gonna kind of go through all of the the routine testing that you would probably see every day and the reason why we would actually do these and then some of the not so routine tests that are available as well. And the reason why there's so many different tests, and that it might seem that we're just sending so many samples off and doing this and doing that is so that we can fully diagnose that cancer.
We want to know exactly what type of cancer it is, what stage it's at and what the grade actually is. So we can then give a formulate a really good treatment plan for that patient and then give the owner some options about what they would what they actually want to go ahead with. And it gives the owners then a more accurate prognosis, for for their pet, so that they can then make their informed decision about what do they if they want to go ahead with treatment or what treatment, or if, in fact, no treatment at all.
So I'm gonna discuss the sampling, the preparation and what some of these results mean as well. So with all of these lab testing, so these tests are used alongside a thorough CLI clinical examination. So we need to remember and and diagnostic imaging as well.
So it's it's important that we look at everything and not just one part of, the process. And again, this just actually then helps us formulate the best treatment plan for our patient. So we've got these general tests which are the like the your haematology biochemistry, your cytology, your histopathology.
And then we start looking on on molecular level, which is, like the DNA and RN a proteins, antibodies and antigens. And then we've got some new novel, testing that that can be used for screening and early detection and for monitoring the cancer as well. So again, why do Why do we test?
We've OK, we've seen this dog. It's got, say, a mass. Why are we gonna start looking into everything else?
Because we want to identify any other concurrent diseases. So what treatment plan? We need to then consider concurrent diseases with that treatment plan and to identify any per plastic syndromes as well that we actually need to get on top of while we're still in the planning process of, making a treatment plan.
And it's to actually diagnose that cancer. We don't want to be going ahead with, chemotherapy just because we go, OK, this dog's got enlarged lymph nodes. It's got to be lymphoma.
Not necessarily. So we want to know that this dog definitely has lymphoma before we even start giving it some of some chemotherapy chemotherapy is not benign. Chemotherapy is, can be, Can is is Isn't isn't great.
The drugs are, can cause all sorts of side effects. If it's used incorrectly and for a tr for when it's used inappropriately as well. So we want to make sure that we're actually giving these these drugs to a patient that actually does need them.
So, yeah, we're diagnosing the cancer and actually there. We want to know that type. Is it a B or T cell lymphoma?
Because that will actually then affect what treatment protocol that we want to go ahead with. So we we do. We do lots of retesting as well to assess the response to treatment.
Just to make sure, actually, is the treatment that we're giving this patient actually working, Have we got a patient say that was hypercalcemic, and now it's come back down to its normal levels. That's perfect. Some chemotherapy medications are actually nephrotoxic or hepatotoxic.
So we want to make sure that we have, that we're checking their renal values, their liver values before we actually go ahead with that medication and then monitor ongoing as well, especially if we are using that medication. Long term. And as I said, just to make sure that that is that chemotherapy actually working.
And we need to assess that patient for suitability of some medications. You know, all of these, some of these breeds that do have the MD R one mutation, are suitable for some of the chemotherapy agents. So we either have to discontinue them completely or dose reduce, and it can completely affect the protocol that we would want to use for that patient.
So it's, it's good for us to know. If they do have that mutation, so just gonna go through like, the sample submission, over the next couple next two slides, just about the the the sampling, about the the actual pots that you're sending off and the form as well. And I know it might just seem kind of basic, but, just to make sure that you're including all of the information for that pathologist to have a look at, because that really helps them out.
So if we're sending off bloods, if we've got serum tubes, either the plain or the serum gel tubes, centrifuge them. If you've got the plain serum tubes separate that serum off from the the the the red cells at the bottom, and then label that properly as well. The smears, blood smears label the label that slide, as with the patient details and that it is actually blood, because if you're sending a whole pile of cytology slides off and then all of a sudden there's some more slides mixed into it, just give that pathologist a bit of a a bit of a head start on that so they at least know that it's blood.
And once you are, if you are doing these smears, either use the whole blood straight away from the syringe. Once you've popped the blood into the tubes or straight from the EDT a tube, and straight away, the longer it's sat in EDT a EDT A before the slides are actually prepared, can actually then affect the cells. So actually so immediately, it would be great for them.
And if that sample needs to be a frozen sample to be submitted, we need to process that immediately. We need to keep that sample chilled while it's been, as soon as it's taken. Centrifuge it if we need to, separate and freeze immediately.
We don't want that sitting on the counter for an hour before we go ahead and freeze. That let the pathologist know for urine. What was the method of collection?
Was it free? Catch? Was it a urinary catheter?
Was it a cysto centesis? And was there a sterile bowl for free catch as well? Because that will then give them if they're seeing, depending on, like, the types of bacteria they start seeing.
Is it just contamination? Is it an absolute actually? For Is that patient actually then got a UT?
I sorry. And if we have, if we're doing cytology and we've done a whole bunch of FNAs, we've got lots and lots of slides all laid out. Label them all patient name And where that mass, actually, where that mass was?
Just so that they have actually got they've got all of the information that they need so that they can actually give you all of the information back. And for biopsy or histopathology, make sure you keep the the formal in tubs away from cytology. Slides.
This can affect the, cytology slides. And make sure that you do label the pots with some with the sample location as well. And if and I'll go through a little bit more about marking biopsies, a little bit later on and for the form as well.
Make sure you put all of the details for that patient on the form. The name breed age gender. Has it been neutered?
A full clinical history. Has that patient been unwell? Has it been vomiting?
Absolutely everything to go with that. Pay to go with that paperwork, the duration of the the clinical signs. And if it's a cytology, how long has that mass been present?
Was it slow or rapid growing? Where abouts is that, whereabouts? Does that mass give them an exact location of where that is?
Sometimes even a little diagram to show where it is actually arising from that. Tell them how big the mass is again. The location.
And if they and if you see fit, send them some photographs as well. I'm sure they would love that. So, yeah, making sure that you've got and making sure you document on them forms as well Exactly where the samples have come from.
Especially if you're sending multiple samples. Especially for like, cytology. Was the blood sample fasted or non fasted?
Because, you know that can affect the results as well. And again, that method of urine collection so moving on to routine testing. So this is the stuff that you would probably be doing all the time every day in practise, as your basic PGA or just as a as a well check as well.
So we would be looking at some biochemistry, and we're probably quite We're looking at the mainly the renal values and the hepatic values just to make sure that see what's going on there. Make sure there's no evidence of, any disease starting to slide in there, and that we're then having to start considering that as a concurrent disease. And then we're looking at calcium as well, especially for some of our patients.
And I'll go more into that in a in a couple of slides. We're gonna look at haematology we're gonna look at that pack cell volume. We want to know what that P CV is, and the platelets to make sure that this patient gonna be able to, is it clotting?
OK, if we're gonna start doing samples and looking at that white, then white blood cell count as well, for the neutrophils, if this patient is already on a chemotherapy protocol, we wanna make sure that that patient isn't neutropenic. So has got a really low white blood cell count or neutrophil count before we go ahead with any more chemotherapy because they're then a a risk of, infection. Then lymphocytes as well are these sky high indicating that there could actually be some form of leukaemia?
Starting or ongoing. And we're gonna look at electrolytes as well, just to make sure that actually just this patient needs some supplementation, they come in, they're kind of off colour. They are, say they've been vomiting and they've got some diarrhoea.
They're now hypokalemic. So we actually need to start supplementing. So we're gonna look for cats.
We're gonna want to look at the FELVFIV status, so that has been associated with a less positive prognosis. If the patient has, FA LV. And does have lymphoma.
But it does actually then depend on that anatomical site and me. Feline leukaemia virus is most commonly associated with the mediastinal and renal lymphoma. So we all want to know, the status.
We will do some fresh blood smears again, just either straight from the syringe or straight from the EDT a tube as soon as it's pretty much as soon as it's gone into that pot. And we will do this just to assess the platelet count. Analysis just prior to sampling.
We're gonna look at a neutrophil count if our machines telling us that actually, we are neutropenic. So we will go ahead and do a manual neutrophil count, and then we're gonna look at cell morphology as well. So we're gonna look at these neutrophils.
Do we have degenerate neutrophils? Do we have bands which are like the immature, neutrophils, and looking at the red cells and everything else as well. So moving on to calcium.
So a hypercalcemia of malignancy is a Perrine plastic syndrome. That is, this can be secondary to a neoplastic process. When we see this, fairly commonly with dogs with the upper cr gland.
Anal sac, Adenocarcinoma. Lymphoma. Often the T cell lymphomas with dogs, multiple myeloma and sometimes with cats.
It's less frequently, but we can see this with lymphoma and squamous cell carcinoma, but it is mainly with the dogs. That we do see this, and this is caused by soluble mediators that are released by the tumour cells into the circulation. And then this acts on the bone and kidneys via the endocrine and paracrine pathways.
So this is one of the tests that we would monitor ongoing to in and because this can indicate disease status. So if we've got a dog that has come in, she's got, we've diagnosed lymphoma. She's also hypercalcemic, And we want to know that once we've started her on a chemo protocol that that lympho that that hyper calci calcium is actually then reducing down it should then come back into the normal levels as that start.
If that ever starts to rise again, this can be an indication that that disease is progressing. Or we've not actually then achieved a full remission. So we do this.
Yeah, we We have a little dog who comes to us every couple of, probably now I think she comes to us every two months. She was a Yeah, Like I said, lymphoma hypercalcemic. And we just monitor her.
We monitor her lymph nodes, and then we do her calcium as well. Because I said this can indicate disease. Relapse with anal sac adenocarcinoma.
If we are once we have, If we've got the initial calcium pre surgery or pretreat and they are hypercalcemic once that surgery has, been completed that calcium should go down immediately, into the normal levels. So if that calcium remains high post surgery, and we've seen it once where this was just kind of it just took a little while for it to start coming down. But usually this is because that there is some, evidence of malignancy left behind whether or not that is in lymph nodes that weren't, detectable or whether or not there's just been some tissue left behind as well.
So yeah, monitoring closely is essential. So if we are detecting the high, calcium, we also then need to make sure that we're checking them renal values as Well, Hypercalcemia can induce asthmatic diuresis and then inhibits the A DH and its action on the tubular cells of the collecting ducts leading into that patient, then becoming polyuria. So poly UIC, plus nausea and vomiting, can equal that dehydration, which will, kind of, as you'll know and then reduce that glom filtration rate, therefore then increasing the calcium reabsorption and worsening that hypercalcemia.
So this can then actually lead to some renal damage. And that is azotemia may not be reversible, so we need to be checking everything. A measurement of ionised calcium is more accurate than total calcium as well, But there are.
So if you're only able to do a total calcium, these calculations that you can use to convert it to ionised are not completely are not. Not as accurate as just running a a an ionised calcium. So we're looking at, plasma proteins now going on to electrophoresis.
So this is run on a serum sample, and this is used to separate the different protein fractions. So the albumin, the alpha globulins, the beta globulins, the gamma globulins so normal we should see a tall, narrow albumin spike to the left with three globulin fractions to the right. So with these patients that are hyper protonic, this is a relative or absolute increase in the protein concentration.
So this could just be hyper album anaemia could be hyper glob anaemia. Or it could be both, and we need to assess the albumin and the G globulins together. We there's no point in just doing one or the other.
You need to do them all. And then a gammopathy, is the abnormal proliferation of the B lymphocytes and then this results in an abnormal concentrations of the immunoglobulin production. So I said, the glam, the gammopathy, is an abnormal proliferation of the B cell lymphocytes, resulting in an abnormal concentrations of these immunoglobulin productions.
So if we have a monoclonal gammopathy, this is the increased production of one type of immunoglobulin by a single clone of cells. Abnormal protein produced is called a paraprotein or the M component, and this occurs in multiple myeloma, lympho, prolific and lymph and lymphoproliferative neo neoplasms. So this is a greatly increased serum protein concentrations that might actually then that may actually result in the hyperviscosity syndrome.
So if we have a biclonal gammopathy. This is usually a two narrow space 22 narrow based peaks, usually in they in the gamma region, they may indicate a neoplastic production of immunoglobulin. And if it's a poly polyclonal gammopathy, this is the increased production of C full, different immunoglobulins, and this can occur.
This can occur with a wide variety of infection, inflammation and immune mediated diseases. So just showing. Just put, I thought I'd just put these, charts in as well just to kind of show the difference.
So if you can compare back to what a normal one should be, we have the tall albumin spike to the left, and everything else should be kind of should be lower. Whereas if you look at this one for the monoclonal gammopathy with this dog with this dog that has a leukaemia and they have this massive spike in the gamma, in the gamma globulin region. And then this is a biclonal gummy with a mixed breed dog that has multiple myeloma.
So it's just interesting to kind of see the the difference between them so moving on to cytology. So I like to put this in. The sees.
See something? Do something. Why wait?
Aspirate. And this is from the, doctor Sue, she is an oncologist over in America, and she is really, really pushes for the if you've got if there If you see a mass if they if you tell, get your owners. If they're seeing masses and they are the size of a pea or larger and they've been present for a month or longer, do something about it.
The sooner we see see these masses, and the sooner we can actually diagnose and we can do something about them. It's always so, so sad when the dog comes to us and it say this mass has been left for months and they it's we can't then do anything about it. Whereas if we educate our owners say, Hey, if you see it, do something about it, and then hopefully if they obviously, if they don't want to, then go ahead and do anything absolutely fine.
But most people like if they're wanting to do something, it gives us a bit of a bit of bit of a better chance if we diagnose it earlier rather than later. So for taking some samples, we gonna need some, some kit for this. So some microscope slides and make sure again remember that you fully label which what?
Where these slides are coming from. What, The sample site actually is a pencil. For labelling.
Obviously, don't be writing on these slides in Sharpie, because once they start staining them, the Sharpie will come right off, and get into that stain and onto themselves. We use a different variety of needles. Depending on the size of the mass, the size of the patient.
How big the mass is. So I put 20 gauge there, but actually, we probably go from, like, the 20 threes, 20 ones 2320 threes and the twenties as well. You may need a spinal needle, especially if it's for a larger dog.
And we're going for, like, an internal organ and a five millimetre five mil syringe. For as for either aspiration or for pushing that sample out onto the slide. So just remember to consider before sampling, could this be a mast cell tumour and has that pa patient had a mass cell tumour before.
If this patient has had, mast cell tumours before, and it's got a new mass likelihood potentially could be another mast cell tumour. So we do want to then pretreat these patients with an antihistamine. Just to prevent any de degranulation and record as well.
So I like to We like to record absolutely everything. And this, this gives us a starting point for where, Where That masses at that point And then if we're going to surgery or if we're not going to surgery, it gives us an able we're able then to monitor response. So if this is a patient with lymphoma, we want to measure all of them lymph nodes because once we start treatment, we want to be able to measure them lymph nodes in the future at every single treatment and know that them lymph nodes have reduced in size, so cytology can be done in a couple of different ways.
So we have cytology, which is the FN A with aspiration. So you are. If you have got a needle and syringe attached, the needle goes into the mass.
And when we use that syringe to pull back on the plunger to aspirate some cells. And then you can use that syringe, then to squirt that out onto the microscope slide. And I saw that I did when I was doing a bit of a a Google search for some images, I saw this fine needle aspiration, device using an aspiration gun.
It's not something I've ever seen or ever used, but I thought it was interesting just to kind of include that on on there as well. Whether or not it just makes it a little bit easier for the way that you can hold it. And it holds a syringe as well.
We can do these FNAs without aspiration. So we will just use the needle. We will hold on to the needle hub.
And then we will just get hold of the mass and then use the needle in a couple of different directions, in and out and in the different directions as well. And then straight out, attach the syringe, with air into the end, and then squirt that out onto the slide so you can then make your smears. We can make some smears from, just by squeeze that directly onto the mass.
So anything that is alterative or any anything that has got any extra date come into it. If it's a mass that we've removed and we can cut that in half and do some impression smears if it's, alternative or like or discharging, we should give that a little bit of a clean before we start taking some samples, as all we're going to see is, well, everything, so yeah, so we can we can cut masses in half, to and to put them onto the slide as well. And we should then again let them dry.
So for fluid as well, we would want to put some. So if we've got a nice sample of a pleural effusion, we're gonna put that some of that into EDT a tubes. If them samples are potentially going to sit for a little while, we could drop a put a little bit of formalin.
Just one drop of formalin into one of the EDT A tubes. So you'd have one fresh one with a bit of formalin and make some fresh smears as this preserves the cell so they can be evaluated better. As I said, a smear is made from EDT.
A after arrival at the lab can have some artefacts in these cells that can occur in transit. So, we want to make sure that we've done some nice fresh smears and we could also do a sediment smear as well. Using that fluid where we would centrifuge and then that would actually just give us a larger concentration of cells in that sediment to smear as well.
So there's a couple of different smearing techniques. So if we have a solid tissue like we have FNA a lymph node, for the squash preparation of the slide over technique, it is you pop the the sample. You squirt the sample out from the syringe onto the sen onto a microscope slide, and then you would use the another microscope slide to on top of that and then pull them gently apart, and then you would create a slide across each other, and then you will come up.
You will make a nice smear. You can use the blood smear technique. So the same way that you would do a blood smear, a sample at one end of the slide second slide that comes back into it and then push up the slide to make your smear.
Or you can use make it using the star preparation or a needle spread technique, which is not That's not something we we seem to do an awful lot of, it's probably a little bit if they've got more of a a chunkier sample sample in the middle of the slide and then you actually then use the slide the the needle to spread this out. I think one of the the problems with this is that you actually then end up with a really thick sample, which can then be diff difficult to, examine properly under the microscope. So for fluid filled sample.
So for fluid samples, either from a mass or from a body cavity, we can again use the blood smear technique. Or we can use the line concentration smear technique where you will have start off the same way out that you would do for the blood smear. So a te a sample the dot of the sample at one end of the slide top slide, then comes back into it.
So you get that whole the sample going in along at the bottom at the at the top slide, and then you push up, as you would normally have done for the blood smear technique. But instead of going all the way, you stop part way and lift up. So then you end up with a con like a line of concentrated cells rate.
At the very end, with cytology, we can we can actually end up with a whole bunch of artefacts. And a lot of some of these can be due to poor sampling or smear preparation or staining techniques. Or even if they've just, I said, if they've not.
If the the sample's been sat in EDT a over the weekend, and then they're making it fresh at the lab on the on the Monday So if there's too much blood on the sample, especially if it's been a splenic sample, that can affect your The visualisation smears are too thick. If there's ultrasound gel contamination, if we've got dirty slides starch Granules from surgical gloves, if there's excessive stain, precipitate or even if they've got you've got dirty stain. We change our stain out, every week, just to make sure that it's not contaminated or there's nothing growing in there which could then give us a false, false bacteria on on our cytology slides.
There could be water artefact or crushing artefact. If so, if you've been a little bit too, overzealous with your smearing. So again, just going to go into the submission of the samples, Make sure we are fully labelling these samples with the name the date, the location of that sample.
We don't need to stain them before we send them. Or we we don't really need to fix them before they're going off to the lab, either with alcohol or any other preservatives. If if the smears been done and it's dried before packing, they will be fine with that.
So, yeah, allow that smear to air dry before you pop that into the boxes before you send it. And we've remember never to actually send unstained smears with histopathology samples. So we have to set.
You have to send them separately so formal INF can fix the sales on cytology smears, and it then inhibits the staining with romanowski stains. So it makes them actually uninterpretable. So you end up having to do resample and pack slides well, so that they don't break in transit.
So, yeah, the the the slide containers are usually available from your lab. So there's a couple of different stains, that we would that we could use in in practise. We tend to just use Excuse me, the three part, like D quick, the Roman D, quick stains or a variety of that where you have the 33 different stains.
But there's a couple of different ones that you can also use in house. But when when it's these are kind of the ones that you would be using in house. But once they start going off to an external lab laboratory, they will then advise if they want to start doing different stains, or whatever.
And it's whatever that pathologist is actually more comfortable using as well in, in, in in their lab. So the the more samples you send to them, the more the happier they will be. So for Histopathology, this we should send all the tissue removed to be, we either submit it all to the lab or we keep hold of it.
yeah, we we end up with a bit of a hoarding box. Sometimes. I'm sure you guys in practise do as well of masses that owners don't want to be submitted.
But, actually, let's just keep hold of it, you know, just in case it reoccurs. I know, actually, we we kind of want to know what that is. So if we're gonna have to move forward with a second surgery or surgery revision, just so we know.
But ideally, we want to take it. We want these samples going off so we can know exactly what's going off, because again, we're not gonna do any form of chemotherapy, or treatment, unless we know exactly what that that mass actually is. Because treatment options vary greatly depending on what that mass actually is.
If it's a really large sample, it may need to have some representative samples taken. The mass can also be if it's quite a large sample, it would need to then be cut in half or cut into pieces just so that they can, that it can be fixed properly. Bony samples may need to be decalcified at the lab prior to cutting, and actually preparing the histo slides.
Because this this can actually then take a little a little bit longer for you to actually start getting results. So some if it's a a bit of a tricky mass, I don't I'm sorry. This picture is not great.
This was a, a lymph node. So the surgeon actually marked, the mass by placing a suture at one end of that mass. Or they can actually use some different colours of Indian ink to kind of mark.
What which? Which orientation this was. So if they're getting, clean or dirty margins, we know exactly where this where Whereabouts.
The surgeon will need to go back in and again send the send the pathologist the full history, size, location, duration. Rapid growing, slow growing. Does it fluctuate in size?
Just just so they've got all of the information. So we will want to assess. So the pathologist will be assessing the margins to determine if they're clean or dirty.
And when I say clean, it means that we have got the whole mass. There's no cancer cells, in the margins or even close to their margins. If it's a dirty margin or an incomplete excision or positive margin, that means that there's actually cancer cells going right up to that margin.
All very, very close to it. Which means then we've got We've got the potential then for reoccurrence. Because of this, this tumour cells actually been left behind.
So this is either then when they need to follow up with a different surgery or electro chemotherapy or radiation. So these samples need to be fixed. The biopsy samples need to be fixed in a 10% neutral buffered saline, and that's one part sample to 10 parts formalin.
So any has said before any tissue that is greater than one centimetre thick needs to be in size to allow the formula to to penetrate that deep tissue. And remember, formula is carcinogenic. So if you are actually handling the sample, handling the pots, making up these pots, remember to actually wear gloves so moving on to culture and sensitivity, we can send a whole variety of different things.
For this, we could send some tissue, a tissue sample wrapped toid and sterile sterile saline soap swab. If it's a biopsy, charcoal swab. If we have a mass or fluid or if we have a culture bottle as blood culture bottle as well.
So if we're sending off, either blood or we have, say, joint fluid. Or we have, liquid form a mass or an effusion. And with the charcoal swab as well, we can either use use it straight into the liquid, or we can do sometimes.
What we will do is a centrifuge, some of that fluid to get that sediment at the bottom. Because then that's super concentrated with all of the cells, bacteria, everything in there. And then they're more likely to get, a culture AAA proper growth.
So we need to make sure that we are aseptically sampling, any tissues or fluids that we are wanting to centre for culture just to avoid contamination. Just so we're not getting a false positive result when, actually, it's because we've caused that. So you're in collection, for these patients, so cysto Centesis is the gold standard, but we have a lot of patients that are coming back to us every month, and we're not going to start cyto in them every single time.
So free catch is, is, is fine. For what? For some of the tests that we need.
If we're wanting a culture, it's culture sample. Ideally, cyto we could. We could do it by a urinary catheterization or free catch into a suitable, sterile container.
So we have these little pots, and I think we can get these from JAK. And they are the pets. So they come with the full kit, and it's it's been great for our owners, because the whole kit comes together and it's sterile.
The sample pot on the left on the right, which is the Jagermeister. This is what we had a urine sample brought in at one point. We only needed to dipstick it, thankfully so.
But I just put that in there because it made me and pretty much everyone that in our hospital chuckle for a little minute. So for the urine, we're looking at dipstick analysis, and we want to make sure that you're reading it at the correct time that you're not just covering it with urine wandering off and then coming back 10 minutes later, you need to read it specifically your 30 minutes 30 seconds. 40 seconds, 60 seconds To actually make sure you're getting a correct result.
We will do the dipstick test prior to cyclophosphamide. And we're just assessing that patient, for any evidence of a UT I before we go ahead with cyclophosphamide as that can irritate the bladder lining and cause a sterile hemorrhagic cystitis. We're wanting to assess for protein.
Is that patient proteinuric? Is it treatment related? Is it potentially developing an early chronic kidney disease?
And then are we detecting it there? So move it. So once we've got them results, we can then determine what else we actually want to do.
So microscopic sediment examination can be done on plain urine, fresh or warmed, if that sample has been previously refrigerated and that with this we're observing for red blood cells, white blood cells, bacteria, crystals or casts and it even in this picture, you can see all of that bacteria. On a patient that had a UT I So once we're looking at my going for the microscopic sediment examination on a stain sample, so in for the cytology. And we would do this if there's an abnormal or increased numbers of epithelial cells on the standard microscopic examination, so the urine should be concentrated.
So this is a slow centrifuge, and then we make a sediment smear and we stay in this, so this would then actually give us, let us visualise if there are bacteria or actually be able to diagnose if there was, any form of neoplasia as well. So you're in cultural sensitivity again. As I said, cysto centesis is, the gold standard for culture because we don't want any contamination.
As if there is a high risk of contamination, or false positive if it if it is free, catch even into a sterile bow so we can look at the protein creatinine ratio. And we do this as a baseline before any treatment as well. And when we're actually monitoring when these guys are on palaia or mat, this would give us a An idea is if that patient is we're gonna do this if that patient is showing elev, any elevated OC creatinine and we're just wanting to investigate if there's any potential kidney disease.
It's used to quantify the degree of protein in your ear. So you've done a dipstick. You've got protein on that dipstick.
We want to know the degree of protein in your ear. It's not affected by hydration. So it compares, so it quantifies the protein lost related to the amount of creatinine in the urination as well.
And this actually then But, well and it's not. I said it's not affected by the hydration or the urine specific gravity, so we can't be doing the protein creatinine ratio ratio. If there's evidence of any gross hematuria any inflammation present in the urine sediment that's including any white blood cells as well as this can actually falsely elevate the protein creatinine ratio.
And this is just run on plain urine as well. We don't need to do anything fancy with that. So there's some fairly newer, newer testing, available, and this is the cadet bra or bra plus sampling.
And this is actually used to detect transitional cell carcinoma or urethral cell carcinoma in dogs. It's a free catch urine sample. That's all we need.
And it can identify, 95% of TCC or UC cases it can actually help to diagnose them and then monitor for any reoccurrence as well. So it evaluates for the presence of cells, in that urine contain containing that bra mutation that is associated with, that TCC or UC. And it allows an early detection, due to the low limit detection of so it only needs about 10 mutating bearing mutation bearing cells in that sample.
But actually then to give you a positive. So we if if it does come back as a negative, But you are then I, I am convinced that this patient has a TCC. There is a second sample that you can run, which is the BR a plus.
And then that further evaluation actually detects more than two thirds of the positives. That were not actually identified for the regular test. So that R test, I think, actually does need quite a large volume of urine.
And so if we're gonna if you're sending it, make sure you send them a decent, a decent volume Check with which lab that you're using, just to find out exactly how much urine that they do need. So we go and go on to bone marrow, which is the aspirate and biopsy. So we'll use the biopsy, which is the histopathology, and this would then assess the bone marrow cellularity.
And as assess any disruption of the marrow architecture and just to see are the cell Is the marrow still there or is it all been infiltrated by fat? And it's not actually doing much. The cytology could assess the presence of blasts.
So, like these immature cells, and additional testing might actually be needed on these cytology and histo cells just to actually determine the cell lineage. So when we are sending off biopsies and cytology for bone marrow, we should send an EDT a whole blood sample, an EDT a and a fresh blood smear as well, because they will compare the the results of a haematology to what they're seeing on the sample, of the site of the bone marrow as well. So moving into some of the more specialised tests that we do in oncology, we have a flow cytometry.
So this test should be interpreted in conjunction with all the testing. So it shouldn't be used alone to make a diagnosis. So we would do this.
We have enlarged lymph nodes. We've now, then got a diagnosis of lymphoma. We want to know exactly what type of lymphoma this is.
Is it a B cell T cell? What is actually going on there? Because it gives us a better treatment, better prognosis and treatment option for that patient.
So flowcytometry involves the uses of the use of anti, sorry antibodies to detect specific antigens associated with that cell. So these antigens are either on the surface of the cell, They're in the cytoplasm, or are they in the nucleus of that cell as well? We can use this test to actually confirm immune mediated diseases.
Especially, new mediator thrombocytopenia. As well. So this test needs to be processed pretty quickly.
So we ideally want to get that sample taken and then straight in the post. Get to that lab the following day or couriered to it gets there the following day. We don't want that sitting, on the desk and missing that post.
And then, going off and then it taking three days to get there because it needs to be run on live cells. For them to actually get the result. If you're sending it off on a Friday, make sure that it's gonna speak to your lab.
About whether or not it it's OK for you to post it on a Friday for it to actually get to them on a Saturday. Because they might say, actually, no, keep hold of it, do something different with it before you actually submit it. Because what you're gonna do if you send it in the post Friday, it's gonna sit there till Monday, and even over bank holidays as well, just so that they actually get a decent sample.
So for for cytometry for lymph nodes. So we're looking for a lymphoma. So this is the FNAs of lymph nodes or tissue.
So we could use, like, liver spleen, any of the peripheral or internal lymph nodes. So we want to get a multiple, get multiple samples from multiple nodes. I said we need a suitable number of live cells in there, and we're gonna pop.
We're gonna get our samples, and we're going to submit them them themselves in a suitable transport medium. Previously, lab the labs have supplied us with a flow cytometry medium, which they've prepared beautifully for us. Now, we just make it ourselves.
And this was on the advice of one of the lab that we use. We make it up, using half a mil of EDT a plasma and half a mil of sterile saline mixed together and then in a fresh EDT a tube. And just make sure I like to make sure that I always use a one that's got a screw cap on there, because the the effort that goes into making this medium then getting this all of them samples, you don't want that cap to be popping off.
So I like to use the screw cap screw cap tubes. And we also then want to submit their lymph nodes, some lymph node smears or tissue smears for cytology as well. They will use them together.
So we This is one of my little one of our little tips what we do. We pop the tube into a roll of tape just to stop it falling over again. The effort that goes into making that medium and all of that samples, these tubes, they will fall over.
If you look at them sideways, they like to fall over. So yeah, just give yourself a a bit of a helping hand. So for flow cytometry for blood is when we're trying to differentiate the leukaemia.
And the the lineage from there. So this is EDT a whole blood. Nothing fancy there.
And then check with your lab about volume. I know previously, a long time ago, they wanted between five and 10 mils of EDT a blood. It's definitely a lot less now, but just do double check with your lab.
And again, it is rapid testing, so they want that sample as soon as possible. Don't be, holding on to it for a week before you decide to send that. So moving on to a PC R for antigen receptor rearrangements or par?
So this helps to determine the clonality of the cells to determine. Is it a monoclonal? And that because if it's a monoclonal set of cells, this can actually indicate neoplasia.
Or if it's polyclonal. This can indicate an inflammatory inflammatory or a reactive process. It can be used to immunophenotype so for, like the BC LT cell lymphomas.
But it is less reliable, so we tend to use flow for that. But but we can use this. So if if we're limited for sampling or we all the slap the slides are already at the lab, we can use that.
It examines DNA. So it's the live cells are not required. It can be run on stained or unstained cytology, slides, blood or any other fluids, or even a biopsy of whole of like, full tissue as well.
And again, this is to be used alongside other tests such as cytology and histopathology. Because we will we will. We we want all of the information so moving on to some of the different staining and stuff as well.
So we have, immuno history, chemistry or immune immunocytochemistry. So some tumour origins can be really difficult to interpret to interpret because they have they're either poorly differentiated. They have some reactive processes going on or if it's a mass that is actually demonstrating multiple features.
So, they will want to, some do some different testing, some different staining, so we can actually then get the full lineage of that cell and actually then make a full diagnosis. So this is a process again of detecting antigens specific to a certain cell type by using antibodies. So these antibio anti bodies bind to the antigens, causing an antibio, the antigen complex, which can then be visualised by the pathologist.
It's like it just blows my mind like all of these different tests and how they can actually determine everything. It's so interesting. And then these neoplastic cells are identified by positive or negative staining.
So the immuno histochemistry is run on tissue, so your his your histopathology cytochemistry is run on a monolayer of cells, so on your cytology slides, and then the pathologist would usually advise on the suggested stains for each sample. So if you're going to send them for your routine history or your C cytology once they've done that, they would usually then on your report, say we would advise staining with blah, blah blah four stains five stains, however many stains and then you can move forward with that just to get your full information. So we have K I 67 for canine mast cell tumours and I always think these are so pretty on slides.
With the purple Granules, they're just lovely, But you're just like, Oh, you dirty little things. So the K I 67 is Is immuno histo chemical sing staining? So it's one of them stains for the proliferation marker K I 67.
It's this is an unidentified antigen which to which the monoclonal antibody MIB one binds to. So K I 67 is a very large nuclear protein that is expressed excessively by cells during the cell cycle. So if that cell is expressing K I 67 it is actively replicating.
So the sample that we are gonna send is either is a formal and fixed paraffin formal and fixed tissue. So it's we we run this on biopsies, biopsies or the hist theathers. Well, so it's shown to be a prognostic factor for all grades of canine mast cell tumours.
So if there is a if we think it's a if there is an increased proliferation rate, or AAA, if it's actively replicating, it's like crazy. We will end up with a higher K I 67 which is then indicates a higher risk of local reoccurrence. And metastasis, which is actually then, indicates a worse prognosis.
So there is AC KC kit mutation, which again is for the mast cell tumours. And this is a molecular diagnostic parameter. So this this is a genetic analysis for the C Kit gene mutation, which activates the kit thoen kinase receptor.
So the CK mutations in Exxon 11 can indicate a more aggressive tumour. However, these tumours are actually pretty responsive, usually to the, tyrosine kinase inhibitors or the TKIS, or you'll more likely know them as mat or Pavia. So the CK mutations in Exxon eight so correlate correlate with a real pretty with a good response to a KIS.
However, this mutation isn't really associated with any prognostic information at so K nine mas cell tumours with a This kit dysregulation may behave in a more aggressive fashion. And there are three types of kit pattern as well, so feline mast cell tumours don't tend to reveal don't reveal a prognostic association with ca with the kit expression. So we tend to, I think, really use it more with for the dogs.
So we have a a bunch of new testing kind of starting to become more available. And this is used for the liquid. So we call it liquid biopsy.
And this is for identifying cancer markers using a fluid sample. So we can either do this in blood or urine. And we've already discussed one earlier on which was the bra test for urine, for detecting, transitional cell carcinoma or urethral cell carcinoma so it can be used in older dogs.
If we're just kind of doing a screen as well as the the at risk younger dogs So you know, your, Scottish terriers. The West is, Shelton sheepdogs. Your Bernese mountain dogs.
Gold. The golden retrievers, your flat coat retrievers. This can help if it's, if we If we're doing it for the urine, we can do this.
We can detect if there is any evidence of a urethral cell or transitional cell carcinoma a lot earlier than when actually signs start to develop. There's a couple of tests, and I found we We have some information on Kanan Diagnostics or new Q vet cancer test, which is available through nationwide. And they are blood samples.
But so check with the lab, on sample requirements for, like, volume of blood and urine that they actually want. And, this is something that's interesting to see how it's going to be used moving forward in the coming years to see if it's something that actually, people are starting to do as a routine thing. I myself, I went for my I've got older cats.
I've got cats and I would routinely do, blood tests annually just to kind of test just to make sure everything's kicking over. OK, is this something that we could actually start adding into our general health checks? If people are wanting to do this annually, blood and urine test to actually detect if there is any evidence of cancer, and then we could actually pick that up sooner rather than later.
So I just put a bunch of references down there, and I'm also gonna put all put all of these and, more in the notes as well. So in, have a little look through them and thank you very much for listening. It's quite a lot to probably get through and a lot of different tests.
And as I said, it's It's a lot of different tests to actually get your head around. It's took me a little while to kind of learn all of these different tests. But if you do have any questions, I am very happy to answer them.
I've put both my email and the the enquiries email for the hospital I am at, which is north. A veterinary specialist. I am happy to help.
And as would our oncologist, if you have any if you want any advice on testing or your cancer treatments, but also use your labs as well. If you're sending samples out to them, speak to them. Don't just wing it with some of your samples.
Ring them. Ask them exactly what they would like if it's not clear. Have a volume.
And what different if they want it freezing chilling room temperature. Anything like that use your pathologist so that you can actually get the most from your samples. So you can get the best results, and then you can actually then give the best, prognosis and treatment plan for your owners and for your patients.
So thank you very much. I hope you enjoyed this.
