Hello everybody, welcome to this presentation on the investigation of the anaemic patient. This is aimed at how you can rapidly get a diagnosis in the anaemic patients, how you can approach the treatment of that patient, and we'll touch a little bit on the appropriate blood products that we can think about. My name's James McMurra.

I work as an advanced practitioner in small animal internal medicine and veterinary cardiology out of vetsinary referrals in Manchester. So on to the introduction. Anaemia can be defined as a condition in which there is a deficiency of red blood cells or of haemoglobin itself within the red blood cells, and ultimately this will result in clinical signs that the owner and or the clinician will detect, primarily those of weakness and lethargy, in combination with mucous membrane pallor.

Now the mucous membrane pallor can vary in its severity and is sometimes readily detectable by even a client, if the patient is quite profoundly anaemic, but it can be really quite subtle in other patients as well. And we have to bear in mind that some forms of anaemia or reduced red blood cell mass can be physiologically normal for that individual. And one example includes splenic relaxation in which there is a transient anaemia because of red blood cell sequestration within the spleen.

These red blood cells are then released back into the bloodstream and therefore this form of anaemia is physiological er and transient. We also should bear in mind that young animals have a physiologic anaemia as well, so their red blood cell counts are often below normal reference ranges for adults. So it's important to bear that in mind when you're running bloods in-house or submitting them externally, that the age is recorded and an appropriate reference range is used.

When we think about looking at anaemia, we're going to talk about the diagnosis of anaemia primarily based on combining our physical examination with estimation of the red cell mass. And the most common ways that we do that in clinics are either with the packed cell volume or the hematocrit. Now, the packed cell volume is something that we use quite a lot in the emergency situation.

It allows a very quick test with a very small amount of blood to tell you the red blood cell mass in that individual and is really quite reliable and certainly more so in some patients than the hematocrit, and particularly patients who have homolysis. The hematocrit is, a measurement that we get from an analyzer, and is somewhat interchangeable with the PCV but can vary a little bit, when compared to the PCV in the individual. One thing that we should consider is that when we run a hematocrit on an in-house analyzer, if that hematocrit is normal, it's probably pretty reliable and that we don't need to worry too much about that patient being anaemic.

However, when the hematocrit is abnormal, it's certainly worth doing a PCV firstly, because the PCV could be more accurate and the hematocrit could be erroneous. But also when we're monitoring patients who have anaemia, the PCV is a nice, cheap and easy way to do that, so it's good to have a PCV at baseline. So certainly I recommend hematocrits in all patients as they'll be part of the hemogram profile that we run on our analyzers, but I'd like to sort of highlight the importance of often running a PCV alongside that.

We also need to bear in mind that we want to interpret the PCV and hematocrit in light of the hydration status. The reason for this is that a patient who is dehydrated will have an increase in their PCV or hematocrit because of a reduction in plasma volume that those red cells are circulating in. So we might end up with a situation where we have a patient that presents who actually does have a reduced red cell mass.

However, they're also dehydrated, in which case they're. Hematocrit or PCV might actually be at the low end of the normal reference range, and once we hydrate that patient normally, their red cell indices, PCV and hematocrit might actually drop below the reference range and they might be mildly anaemic. So to be able to interpret them in light of hydration status, we want to combine our PCV and hematocrit with a physical examination.

Looking for things like skin tenting and tachy mucous membranes that tell us about dehydration as well as how sunken their eyes are, but also looking at their circulating volume status by assessing heart rate, pulse quality, capillary refill time, and importantly, their total solids, also known as total proteins. So whenever I run a PCV. I'll get my, capillary tube and I'll snap it, and, I will measure the total solids of the plasma, on a refractometer.

And that then allows me to sort of look at the red cell indices in light of the total solids, and that can help point me towards whether or not the patient is volume depleted because of course the less plasma a patient has, the higher their total solids will be. So a take home message certainly is whenever you're running a PCV, always run a total solids at the same time. We need to be aware of what we consider normal reference ranges, and they do vary between cats and dogs, and that's one important thing to be aware of.

Cats generally have a lower normal hematocrit or PCV than dogs. So dogs, we expect their range to be between about 37 and 55%, whereas cats between 27 and 45%. And when we're looking at an anaemia, we like to think about how severe the anaemia is for a few reasons.

Firstly, it can tell us about the necessity for transfusion in that individual. Of course, the more severe the anaemia, the more likely they are to be transfusion dependent, which is a term we'll get on to a little bit later on. Therefore, they're more likely to need blood products.

However, milder anemias may be anemias that we can simply monitor and investigate but not require blood products, immediately as part of their treatment plan. The other reason why we can grade the severity of an anaemia is it can give us a bit of information about what the underlying problem is. And as we'll get to later on, some diseases will typically cause mild anemiasas, whilst others more commonly severe anemias.

And whilst we can't always use these bandings to differentiate the causes of anaemia, it can help push us in the right direction. So a mild anaemia in a dog would be classified as a hematocrit between 30 and 37%, a moderate anaemia between 20 and 30%, and a severe anaemia below 20. In cats, a mild anaemia might be in the order of sort of 20 to 27, not, not 25, a moderate anaemia, 15 to 2025 and a severe anaemia below 15.

When we perform a hemogram, which we'll talk about quite a bit throughout this presentation, there are some of the red cell indices that we can look at as well to help guide us with the type of anaemia that our patient has got. And by knowing the type of anaemia they have, again, it will allow us to sort of create a list of differentials and therefore investigate them appropriately. And to help guide us, we'll combine smear analysis, which we're going to talk about in a few slides with some of the red cell in disease from the hemogram.

And two of the important ones to be aware of are the mean corpuscular volume, which is the actual average size of the red blood cells. So bigger cells increase the MCV, smaller cells decrease it. And but we also can look at the mean corpuscular haemoglobin concentration.

So this is the average amount of haemoglobin within each red blood cell. And again, this can vary between individuals with different causes of anaemia. So when we're looking at smears and looking at the MCV and MCHC, we start to think about some of these terms that are listed here, and these are ways that we can describe the sort of morphology, the appearance, if you will, of the red blood cells to help determine what type of anaemia they've got.

So it's important to understand these definitions. So firstly, we can refer to the size of the red blood cell. So a normocytic red blood cell is one that has a normal size.

It will therefore have a normal MCV, but it may not have a normal mean corpuscular haemoglobin concentration if the amount of haemoglobin within those normal sized red blood cells is decreased. We then have macrocytic anaemia, so these are red cells that are increased in size. And this is not a particularly common form of anaemia, that we see in small animals, but it can be seen for instance in cats with FELV infection.

Microcytic anaemia for patients who have small red blood cells and have a reduced, mean corpuscular volume. And that really is it for talking about size. They're either normal, increased in size, or decreased in size.

We can then think about the colour or the hue of the red blood cells, particularly when we're thinking about how they appear on a smear, and most normal red blood cells appear normochromic, so they have a normal amount of haemoglobin within them. And have a normal red colour or hue. And these patients generally will have a normal mean corpuscular haemoglobin concentration.

We then have patients with hypochromic anaemia, and these individuals have reduced red blood cell size, so a decreased . Sorry, a decreased amount of haemoglobin within their red blood cells. So they have, a reduced red colour or hue to them, and these individuals will have, a decreased mean corpuscular haemoglobin concentration.

So when we've defined a patient as being anaemic based on their physical examination and a reduced PCV or hematocrit, we then want to try and group our patients into the different types of anaemia and to help us carry out the right set of tests to get to a diagnosis as quickly as possible in those individuals. And this is the way that I like to think about my anaemic patients as falling into one of these three groups. These are either patients with regenerative.

Non-regenerative or primarily iron deficient anemias. So firstly, with regenerative anemias, these are patients in which they have a reduced circulating red cell mass, and their bone marrow is effectively producing new red blood cells at an increased rate, so a hypercellular marrow. Producing juvenile red cells to replace the ones that aren't there in the bloodstream.

And these patients largely have one of two problems. Either they have blood loss, and that blood loss can be through the gastrointestinal tract. That can be through the urinary tract, it can be third spacing into the pleural or peritoneal space, or it could be external blood loss because of trauma.

Now these patients generally are easy to diagnose because the owner may present the pet because they've noticed bleeding in the stool, for instance, or. From, the gums, or when you carry out some basic investigations, you might note a hemo abdomen, for instance, a third spacing of blood. And some patients, however, can have slightly more slow and steady bleeds and be a bit trickier to diagnose, but we'll get to those a little bit later on.

Homolysis is the second cause of regenerative anaemia. And these patients essentially have destruction of red blood cells that are either destroyed in situ within the blood vessels, which we refer to as intravascular hemolysis, or they're removed from the bloodstream and destroyed in other organs, most commonly the spleen. So this is great.

Using this approach allows us to sort of determine, quite quickly, whether a patient might have blood loss or hemolysis simply based on analysis of a smear, er, and a hemogram, if they have an evidence of regeneration. We then have the non-regenerative patients. So we'll talk about how we diagnose that in just a couple of slides, but these are the patients in which there is a lack of or at least an inappropriate production of new red blood cells from the bone marrow.

And the first sort of group of patients that we need to think about within the non-regenerative anemias are those that essentially might be pre-regenerative. So these might be patients that have a reduced red cell mass, but it's happened quite acutely. For instance, they've just experienced a splenic rupture because of a splenic mass, and they've just bled into their peritoneal cavity.

These patients will take a few days to develop a regenerative response because it takes that period of time for new red cells to be produced at an increased rate from the bone marrow. So these individuals at day one will have what appears to be a non-regenerative anaemia, but if we repeat their testing a few days later, typically 3 to 5, they will then have developed a regenerative anaemia. So these patients, are ones that need monitoring and need serial, PCVs and, assessments of their smear over a period of several days.

And if, however, the patient has a truly non-regenerative anaemia and they don't develop any signs of regeneration, then they likely have one of the following 4 sort of groups of diseases. So the first one is anaemia of renal disease. So these patients have kidney disease, and as a result of their kidney disease they have reduced erythropoietin production from the kidneys and that lack of erythropoietin.

And reduces stimulation of the bone marrow in new red blood cell production. So these patients are generally pretty easy to diagnose because they present, primarily because of their chronic kidney disease, and we often find that they have an anaemia that goes along with it, and it's often non-regenerative, and in the investigation of their kidney disease we often sort of rule out other causes of non-regenerative anemias, and simply chalk it down to their kidney problems. Typically anemias of renal disease are quite mild and don't require specific therapy.

However, in some individuals, the anaemia can be at least moderately severe and may warrant some other therapies. And we then have patients that have got anaemia of chronic disease, otherwise known as anaemia of inflammatory disease, and this is any individual that's got a disease that's severe enough and that has been there long enough to essentially suppress bone marrow production of new red blood cells. Examples of these patients are those that have, for instance, cancers.

Or patients that have long standing inflammatory diseases, like inflammatory bowel disease, and typically these individuals have quite mild non-regenerative anemias as well, and their anaemia often responds to treatment of the underlying disease and doesn't require specific therapy itself. We then have anaemia of endocrine disease, and we're really thinking about dogs when we're thinking about anaemia of endocrine disease, and primarily, we're thinking about hypothyroidism, and hypoadrenal corticism. So underactive thyroids can cause patients to have sort of a variety of symptoms, but when we do our blood work on these individuals, there's two things they quite often have that's a hypercholesterolemia on the biochemistry and a mild non-regenerative anaemia on their CBC.

And these patients again don't require. Specific sort of investigations other than thyroid testing, and their anemias often respond to treatment of the underlying thyroid disease with thyroid supplementation. Canine addisons can cause, a mild non-regenerative anaemia, and that might be detectable at presentation and again as we treat the endocrinopathy, that will normally, improve and normalise.

But a lot of Addisonians that we see, can present in Addisonian crises. In which they're quite volume depleted because of mineralla corticoid deficiency and their electrolyte derangements. And because of their hypovolemic state, these patients quite often actually present with normal PCBs or hematocrits.

And as we volume resuscitate them with intravenous fluids, that can then cause their PCB or hematocrit to drop below reference range. So again highlighting the importance of interpreting PCB and hematocrit along with hydration status and total solids. We then have bone marrow diseases, and this is quite a large group of different disorders that through a variety of different mechanisms can essentially reduce red blood cell production from the marrow.

Now, it might be because the patient has a lack of any red blood cell precursors in their bone marrow, because they, medullary cavity has been replaced by other disease processes like myelofibrosis or, it may be due to a myelothysis because the bone marrow is full of, cancer cells, for instance, which we might see in a stage 5 lymphoma. But there are other reasons as well why the marrow might be diseased and not be able to produce new red blood cells, and these include things like myelodysplasias in which the production of new cells from the stem cells in the bone marrow is dysplastic and ineffective, and those cells are therefore not. Produced appropriately or not released into the bloodstream, or we may have an immune mediated attack on the bone marrow which can stop red blood cells maturing within the bone marrow beyond a certain level.

And this is a group of patients that we sometimes refer to as those that have the precursor targeted immune mediated anemias, otherwise known as PEMA. A disorder that is very much, considered, similar to IMHA in which the immune system attacks the red cells. However, the red cells being attacked are actually within the bone marrow before they're released into the bloodstream.

So, bone marrow patients, can have really quite severe anemias in, in a lot of cases. And they may well have other cytopenias as well. So by cytopeniaass, meaning two cell lines are reduced, or pancytopeniaass in which typically neutrophils, platelets and red cell counts, are all reduced.

And I think whenever we see a patient who's got a bio a pancytopenia, it should always point us towards the bone marrow as the cause, of that problem. We then finally have the patients who have iron deficient anemias. So these are patients that have a lack of iron stores within their body and are therefore not able to produce appropriate amounts of haemoglobin to fill the red blood cells.

And the most common reason for iron deficiency anaemia is chronic blood loss, and most commonly, the blood loss that we see in these patients is low grade gastrointestinal bleeding. And most commonly, it is due to ulceration of the GI tract associated with small intestinal tumours. So these patients bleed at a really slow and steady level.

And during that time, they initially will have regenerative anemias because the body's losing blood, the bone marrow produces new red cells. However, that high rate of red cell production over a protracted period of time causes the body to use up its iron stores, and it's therefore not able to produce haemoglobin appropriately, and the red cells that are being produced will not have enough haemoglobin within them. Other causes of iron deficiency anaemia as well include chronic, lead toxicity.

Copper and iron deficient diets and hemoglobinopathies. So hemoglobinopathies are much better described in humans than animals, but they're generally patients who've got a genetic problem in which they're unable to produce enough haemoglobin or the haemoglobin that they do produce isn't functioning normally. We then have to bear in mind our other causes of anaemia as well, so these sort of need ruling out in the investigation of our patient, and these include oxidant injury.

So this can occur because of commonly toxicities, including things like onion, garlic, toxicity in cats, and oxidant injury is generally something that's relatively easy for a pathologist to diagnose on a smear analysis. We then have fragmentation injury as well. So these are disorders in which the red blood cells are damaged within the bloodstream, often associated with vasculitis or disorders like hemangiosarcomas in which the red blood cells pass through the bloodstream through the tumour, and they're damaged by the irregular vascular endothelium.

We then have histiocytic disorders and particularly one to bear in mind is the hemahagocytic histiocytic sarcoma of the spleen, and a cancer of the spleen that causes it to remove, and destroy red blood cells from the bloodstream. We then have inherited red blood cell defects, so, pyruva chinase deficiency in cats and phosphocto chinase deficiency in dogs. There are certain breeds that are overrepresented, so absinian cats, West Highland white terrier dogs, and essentially I'd always recommend an anaemic patient just thinking about their breed and double checking if they could have, any increased risk of having an inherited red cell defect because these things can be tested for relatively easily, with genetic testing which is, available.

Various labs in the UK. We then have patients with oestrogen excess, so they're hyper secreting insulin and insulin, sorry, hyper secreting oestrogen, and oestrogen essentially suppresses the marrow and can not only lead to quite profound anaemia because of aplasia of the marrow in patients, but also other cell line deficiencies as well, causing neutropenia and thrombocytopenia as well. And we then have various drugs, and one of which is worth mentioning is erythropoietin.

So erythropoietin is available from the human medical profession and can be used to treat anaemia of renal disease in cats and dogs. It obviously will stimulate the red blood cell production from the bone marrow, but in some individuals administering human erythropoietin. To that animal can actually cause them to produce alloe antibodies against their own red blood cells and actually lead to a worsening of their anaemia because of red cell destruction.

We then have hypophosphatemia as well. So hypophosphatemia can cause a form of hemolytic anaemia when it's particularly acute. And severe.

And the most common situation that we tend to see this is in the patients with diabetic ketoacidosis, in which their phosphate levels can drop really quite low, really quite quickly and lead to a hemolytic state in that individual. And these patients are simply treated with blood transfusions as needed. Their ketoacidosis is treated appropriately.

And their hypophosphatemia is supplemented with phosphate-containing products and that should reduce the ongoing destruction of their red cells. So just a little bit on transfusion medicine. So we're talking about anaemia and the best way to treat anaemia is to treat the cause of the anaemia, whether it's blood loss, homolysis, bone marrow disease, iron deficiency, etc.

Etc. But some patients are too poorly to undergo investigations at presentation because of how profound their anaemia is. Or their treatment plan, can take so long to work that they simply, aren't able to cope without a red blood cell transfusion, that period of time that it will take for their treatment plan to work.

So these individuals, might benefit from, red cell transfusion. Now when we're thinking about who specifically needs red cells, we can think about two things. Firstly, the absolute PCV or hematocrit, so how severe it is, knowing that generally it's patients with moderate to severe anemias that require blood transfusions.

And we can sort of have some sort of cutoffs in mind as a percentage for when we should transfuse, but I think really these numbers are somewhat arbitrary because various factors, most importantly, including the creicity of their anaemia, can really determine how well they cope with that PCB or hematocrit. And it's not uncommon for instance for a dog with a splenic rupture who has a very acute anaemia that might not actually be that severe to be profoundly weak and collapsed and in need of a blood transfusion, whereas we sometimes see, for instance, cats presenting with chronic non-regenerative anemias that are really quite bright, alert, and responsive and may even have normal heart and respiratory rates with PCBs below 10%. So the number is important, but it must be interpreted in light of the patient's clinical signs, particularly heart rates, respiratory rates, and their demeanour level of lethargy.

And by combining these two, we can really determine the answer to the question, is this patient transfusion dependent or not? It shouldn't just be based on the PCV alone. So if we do want to transfer a patient because they are transfusion dependent, we want to type them in advance.

Now in dogs, largely speaking, we think about whether patients are, DA 1.1, positive or negative. And in cats, there are several different blood groups to consider.

Now dogs. Don't have naturally occurring aloe antibodies, meaning the first transfusion in a dog that's never had a transfusion before and that has never had a litter of puppies, it's an important question to ask, and is unlikely to have a significant transfusion reaction to their first product. But we still want to do blood type them nonetheless, because it is always sensible to give the right blood group to the right patients.

But also because with stored blood, certainly in the UK, there is a relative abundance of DA 1.1 positive compared to negative blood. And if we have a patient.

That is DEA 1.1 positive, it would certainly be beneficial to blood bank stores in the UK to give them positive blood rather than de facto give all of our dogs DEA 1.1 negative blood, which is a trap that people sometimes fall into because we know that negative blood is the least antigenic blood and if you didn't know a patient's blood group, it is in theory the safest type of blood to give them.

But essentially we should really always. Type of dogs and definitely cats before any blood transfusion. Cats in particular, because they do have, naturally occurring antibodies against other blood groups, and giving the wrong blood group to that patient could be, really serious.

Once we're happy that we've typed the patient and we we know we can get hold of the appropriate blood group, and we then consider whether or not we need to cross match them. Now, largely speaking, in dogs, we often don't need to cross match. As I've said, we don't have these naturally occurring alloe antibodies, which is great.

And when we transfuse a dog, they can then start to develop these antibodies, but it often takes a good few days, typically 3 to 5. So during that 1st 3 to 5 days of treatment of that patient, it is safe to administer several blood transfusions without the need to cross match them. However, in cats, we typically always recommend cross matching because of their increased risk of transfusion reactions.

So cross matching kits are readily available now. And they, these are tests that are, that are quite easily to perform. And essentially the idea is that we're taking the donor's red blood cells and we're suspending them within the recipient's plasma to see if there will be a direct antibody sort of reaction to the epitomes on the surface of the red blood cells.

And of course if there is a cross-match reaction, then we don't want to transfuse that patient with that product. So we can then think about which product is best for our individuals and largely speaking, we look at one of two products, and those are packed red blood cells in which a proportion of the plasma is removed from donated blood and this is normally stored blood and these transfusions are quite red cell rich for their relative volume. We then have whole blood transfusions in which blood is taken from a patient and donated straight to another, and the right blood transfusion for the individual patient can be determined by thinking about their type of anaemia, their severity of anaemia, their volume status, and their need for plasma proteins as well.

Essentially, the majority of patients, it is often appropriate to administer them, packed red blood cells. If they then need plasma proteins that can be administered via plasma transfusions. Whether they be, fresh or frozen, or, there's also cryoprecipitate available as well for patients with coagulopathies.

And if that individual does need volume, then that can be given to them with the use of intravenous fluid therapy. So essentially, think about the individual patient, think about what their individual needs are, and if you're at all unsure, discuss, with the pet blood bank, the appropriate nature of, their products relative to that individual patient. We then need to calculate the volume of blood that's needed for the individual.

Going into this is kind of beyond the scope of this lecture, and there are calculations that are very readily accessible, very easy to perform, and ultimately, we look at calculations, for the individual based on what their current red cell, count is, what we'd like to get their red cell count to. And the concentration of red blood cells in the products that we're giving them, so this is about twice as much in pet cells, as it is in whole blood. We then want to administer the transfusion safely with appropriate monitoring.

So again, this is beyond the scope of today, but there's lots of information available out there, particularly if you go on the Pet Blood Bank's website in the UK. There's lots of information and also monitoring forms that can be downloaded and filled out, and it has all the information about the patient receiving the transfusion, all the information regarding the product that's been donated to them. And how to monitor that patient appropriately, how to administer that volume of blood, at what rate, over what period of time, etc.

Etc. And it's important to bear in mind at this point as well that, whilst transfusion reactions can occur, severe transfusion reactions are really quite uncommon. But nonetheless, appropriate monitoring is really, important in those individuals.

So now we've talked about transfusions and the different types of anaemia. We're then gonna go specifically into our testing of these patients. And as with any classic medicine sort of presentation, we're gonna think about history, physical examination, what blood tests we're gonna want to run, what forms of imaging we might want to do, and whether or not there's gonna be any sampling.

And then the further testing as well, which might include endoscopy if they've got GI bleeding or bone marrow sampling, if they've got evidence of bone marrow disease. So history and physical examinations and blood work, we're gonna kind of cover, under the different categories of anaemia, but I first wanted to just talk a little bit about making blood smears and how to interpret them in practise. So I think this is one of the key skills that we can all improve upon.

And getting comfortable with making smears and looking at them really help you very rapidly differentiate the types of anaemia in practise and really get to the bottom of that problem as quickly as possible. So we recommend whenever you take a blood sample to run a PCV or a hemogram, you immediately make a fresh smear. I would typically make two fresh smears, one for myself to stain and look at in-house, and one to be left unstained to be sent with the EDTA blood to the external lab.

This is really important because there are changes to red blood cells. That occur during storage and transport, and that can affect the ability of the pathologist to interpret the smear if it's then made at a later date. So please get into the habit in all of our patients of making these blood smears at the time of running PCBs and hemograms.

So to make the blood smear, we'll take a very small amount of blood, typically, using a capillary tube, dipped into the blood, in, an EDTA tube. So we do want to use blood with anticoagulant in it. And then we'll dab the capillary tube on one end of the glass slide and we have a little blab of blood.

And we'll take another glass slide and we'll hold it at an angle of about 30 to 45 degrees to the glass slide that's got the blood on it. We'll draw it backwards until that slide meets the blab of blood, and then we'll slowly spread the slide along, creating this nice sort of smear of blood. And your blood smears should look like the example in the bottom left.

So this is one that's been made at our clinic and stained. And that's a really nice looking blood smear that's gonna allow us to look at the red cells and determine their type of anaemia really quite quickly. And if you don't have enough practise making blood smears, you might end up having blood smears that look like one of the examples on the right, and there's various different problems with these smears, and that vary for things like slide F, where we have grease and dirt on the slide, slide D, where we've not used enough blood, slide B, where we've been sort of hesitant, with our spreading slide.

But essentially if you end up making a blood smear that looks like one of these. Before wasting your time diff quicking it or packaging it up to the external lab, just make another blood smear. Keep practising until you get a good technique, because it's really one of the, Most important skills in being able to investigate anaemia, making a smear and analysing it.

So we're going to talk about how we look at these red blood cells using a microscope to determine that patient's type of anaemia. We're not really going to concentrate on white cells and platelets, that sort of thing. We're just looking at the red cells because we're talking about anaemia today.

So this is a patient who's got a non-regenerative anaemia. And the terms that we would use to describe its appearance are normochromic and normocytic. So they essentially mean normal colour.

And normal size. So these red cells look, well, they are normal adult red blood cells and are very uniform in their size and their colour. Looking at this smear, each red cell, when compared to the others around it, all look very, very similar.

And so if we see this on a smear, we can describe this as normochromic, normocytic, and determine that that patient at that point either has a non-regenerative anaemia or as we said earlier, they could have pre-regenerative anaemia. And if you think we've got a pre-regenerative anaemia, we're going to repeat that smear in 3 to 5 days. And when we run their blood through the analyzer.

We'll get a normal mean corpuscular haemoglobin concentration and, a normal mean corpuscular volume in these individuals, OK? So, pretty straightforward. We then move on to regenerative anemias.

So just take one second to look at this smear and immediately appreciate the difference between that and the smear before. What are the things that stand out to us? Well, firstly, there's evidence of polychromasia.

So there is a variation in the colour or hue of the red blood cells. Some of them, particularly those arrowed, look much more blue or basophilic than the other red blood cells, don't they? And we also quite clearly see a difference in the size of the red blood cells as well, so we refer to this as anisocytosis.

So again, these arrowed red blood cells are much larger than the non-arrow red blood cells, and there's a significant difference there. It really isn't that subtle, so we can tell that these patients have got anisocytosis. If you're at all unsure about variation in size, one of the things that I do is look at the smear, look at some of the largest red cells, and then compare them to some of the smallest red blood cells.

And often by doing that, you can more easily appreciate a significant difference in the size. So these patients are those that have regenerative anemias, so due to hemolytic disease or blood loss. And these larger blue basic illic red cells are essentially juvenile red blood cells being released from the bone marrow.

Another thing we can do as well is look for a reticular cytosis. So this is something that's typically done at the external lab where the pathologist will stain the smear with new methylene blue and count the number of reticular sites which are juvenile red blood cells within the smear. And when we of course have an increased amount of reticular sites, we have a regenerative anaemia.

So when we look at the smears, sorry, when we look at the hemograms for these patients, they often have an increased mean corpuscular volume cos these more juvenile red blood cells are larger than the mature red cells. Now one thing you have to bear in mind when looking at regeneration is there are different levels of regeneration, mild, moderate and severe. And therefore we must interpret that level of regeneration in light of their PCB and hematocrit.

So for instance, if we have a patient who has mild polychromasia and anisocytosis. But they have a PCV, let's say it's a dog of 10, they've got a very severe anaemia and yet only mild evidence of regeneration. That would be considered non-regenerative.

OK? So look at the level of anisocytosis and polychromasia relative to the patient's PCV and hematocrit. So you can see from a very simple task that's very cheap to perform, looking at a blood smear can really very easily differentiate regenerative anemias from non-regenerative anemias, and as we discussed earlier, it's a completely different list of causes of regenerative anemias.

And it will totally change how we approach the investigation of the patient once we've had a look at their smear. So it's often one of the first things that we want to do. And again, let me highlight whenever you have a patient who has got a reduced red blood cell count on a hemogram, the first thing that you should do is look at that smear, OK, for the red cell morphology, as well, of course, as send that smear off to an external lab for a pathologist's appraisal too.

We then have iron deficiency anaemia, and these patients are described as having hypochromic, meaning reduced colour or hue in the red cells, and microcytic anaemia, so small red cells. So when you look at these, they look like they have a much larger area of central pallor than a normal red blood cell. They look like they're lacking in haemoglobin.

And these are patients that will benefit from an iron panel. So again, that's a blood test that can be sent to the external lab, and the pathologist will look at various indices and help determine if their iron panel is also consistent with a iron deficiency anaemia. These patients typically have a reduced MCHC and also because of a lack of haemoglobin in their cells, and also a reduced MCV because of these smaller microcytic red cells.

So that's it really for smear analysis, and you can see from just these three examples how easy it can be to just take the time, look at a smear and really differentiate these three groups of anaemia in primary care practise. So once we know our type of anaemia, we're then gonna investigate further. So if we've got a regenerative anaemia, we know that it's either blood loss or IMHA and there is certainly quite a bit of crossover in the investigation of these patients.

But certainly if they have evidence of blood loss anaemia quite early on, these are things that we're going to sort of think about. Well, in their history, we may have had blood loss, described or a reason why they might be at risk of having blood loss, such as having been in an RTA. Their physical examination, of course, they will be pale, in their mucous membranes, and bear in mind that patients that are anaemic but have a normal volume status often have an increased or rapid capillary refill time.

Rather than a prolonged capillary refill time, which in combination with pallor can indicate reduced peripheral perfusion, often because various causes of shock. Blood loss anaemia, as as many anemias will often cause bounding pulses and increased heart rates, etc. Etc.

But on our physical exam, we definitely want to check. Areas like the mouth, do a rectal examination, and check the prep use, check the skin, and for any evidence of blood loss in those areas. We're then going to perform pretty standard blood work, looking at biochemistry then haematology.

And bear in mind when we have a patient that's got blood loss anaemia, we might have reductions in total protein because of loss, total protein through the blood, and that might also be combined with a thrombocytopenia, because of course when we bleed, we lose platelets and proteins and red cells as well. And we may have an increased urea if we're bleeding into the gastrointestinal tract, and we may have other things as well that can point us towards a cause of blood loss anaemia, such as they might be azotemic and we might start to think about uremic gastritis and bleeding in the stomach. We're gonna do urinalysis, of course, to look for evidence of bleeding in the urinary tract.

And then we're going to image these patients. So we're going to look for gastrointestinal lesions, lesions within the urinary tracts that could be bleeding. We're going to look for evidence of bleeding into pleural space, the peritoneal space.

Maybe even bleeding into the lungs. And then some of these patients will, warrant endoscopy, and this is most commonly when we think the patient has got blood loss anaemia from the GI tract and particularly if it's quite severe bleeding. We might want to get into there and say, OK, does this patient have diffuse erosions which are quite sort of superficial bleeds of the GI tract that we could be seen with lots of different disorders including things like inflammatory bowel disease, or do they have?

More discreet, sort of focal deeper ulceration that they're bleeding from that might warrant, some sort of surgical intervention, potentially. So just to bear in mind some of our differentials when we're looking at blood loss anaemia, one of the things we always have to consider are coagulopathies. So we're going to ask in the history, is there any evidence of rodenticide exposure?

And is the patient wormed, because of course in the UK and just stronggulospozorum can lead to coanulopathies. But we're also probably gonna consider testing, of our coagulation, whether that's looking at, the absolute platelet count. Whether it's checking clotting times to assess secondary coagulation, whether it's viscoelastic testing to look for hyperfibrillinolysis, which is not readily available in primary care practise, but certainly something that can be considered as well as other things like bu mucosal bleeding times, etc.

Etc. I mentioned erosion and ulceration of the GI tract. Bear in mind there are various different causes of that.

They include things like non-steroidal toxicities, neoplasms of the GI tract, particularly carcinomas, sorry, adenocarcinomas and liomyomas and liomyosarcomas. We might think about canine Addison's disease as an uncommon but important cause of GI ulceration. It may be due to chronic inflammatory disease like IBD.

It might be due to portal hypertension in a patient who's got end stage liver disease, and we might want to look into their liver in a bit more detail. So just bear in mind that GI ulceration and erosion is itself not a diagnosis, but almost a symptom or a consequence of many different diseases. Intracavitary bleeding we've sort of talked about, and that could be caused obviously by things like coagulopathies, trauma, ruptured neoplasms, etc.

Etc. And then we have this weird, disorder that's been more recently described in animals but has been described previously in humans called angiodysplasia. There's an image of this on the right here, a disorder where we have these abnormal blood vessels within the GI tract that are impossible to see on ultrasonography and advanced imaging.

But can cause quite profound bleeding in the GI tractive animals and can sometimes only be detectable on standard flexible endoscopy or even capsule endoscopy. So the anaemic patient that's got blood loss, these guys are generally not the most challenging to investigate in the world, but nonetheless, you do need a pretty sort of standardised approach. Then on to patients with hemolytic anaemia.

So these guys are often picked up honestly quite early in their investigations. And that's for a variety of reasons. Their, history will be sort of quite remarkable, although we do need to check in a patient with hemolysis, any recent drugs that they may have started, .

Whether or not they've been vaccinated recently, although the level of evidence surrounding vaccination and IMHA is really quite lacking, and we need to check whether or not they have a travel history to any endemic parts of the world where certain infectious diseases could cause a hemolytic anaemia, etc. Etc. Their physical examination will often be pretty similar to many other anaemic patients, but they might be jaundiced at presentation.

And that's. Not exclusive to IMHA as a cause of anaemia, you know, we certainly could have a dog with end stage liver failure that has an anaemia of chronic disease and jaundice because of hepatic dysfunction, but certainly a significant portion of IMHA patients will have hyperbilirubinemia on their blood work or be jaundiced, and that's simply because of the massive increase in haemoglobin released from red cells that's then converted. Into, bilirubin by the liver, and unfortunately, the liver, is unable to, excrete that bilirubin quick enough in, into the GI tract, and, and levels in the bloodstream go up, as a result of it.

Otherwise, the blood work of these patients can be really quite unremarkable or certainly quite non-specific, other than their smear analysis. So when we perform a smear in an IMHA patient, we typically expect a regenerative anaemia, certainly in dogs, sometimes less so in cats, but they are typically quite profoundly regenerative. But there are other things as well that we'll see on a smear, and one of those is spherocytosis.

So spherocytes are small red blood cells that lack the area of central pallor, and this is because they've had their red cell membrane damaged by the immune system, and as a result they've lost that sort of biconcave disc shape and become spherical. So these are really important to look for in a smear and if you're not sure if a patient's got spherocytes, we're definitely gonna send the blood to a pathologist and see if they think there are spherocytes, cos that is certainly specific to hemolytic anaemia. And we're then going to potentially consider looking at whether or not there's any auto agglutination.

So we're going to perform an in saline agglutination test, and this is where we put the patient's blood into saline and we combine those sorry on a glass smear. And we will look at it with the naked eye for macroscopic evidence of agglutination, but also we'll look at it under the microscope for evidence of microscopic glutination. So that's important to do.

Don't forget when doing in saline agglutination to look under the microscope as well, because there are many patients that can have microscopic agglutination that don't have macroscopic. Bear in mind as well. The amount of saline that we use in this test can help increase the specificity of our test as well by using more saline relative to EDTA blood, and we will get a more specific test results.

We can also look at the smear as well for any evidence of ghost cells. So these are, seen in the second image at the bottom here, these very pale, red blood cells, and when we see ghost cells, they're quite uncommon, but certainly can be seen in patients with intravascular IMHA. If we're suspecting IMHA, we'll consider running a Coons test as well.

So this is a test looking for a direct anti erythrocyte antibody. And a significant proportion of IMHA patients, about 2/3 are Coombs positive. So for further information on diagnosing IMHA I definitely recommend you check out the ICVIM consensus statement on the diagnosis of IMHA in dogs and cats, which is open access through the Journal of Veterinary Internal Medicine, and they essentially propose a combination of, Two out of three of the following to make a diagnosis of IMHA Sperocytosis, positive Coombs in saline agglutination.

So if you have 2 or 3 of those, that supports a diagnosis of IMHA. If you don't, it's not impossible to have IMHA but you certainly have to investigate other causes, as well. So when we're happy that our smear analysis and Coombs testing and in saline and glutination looks like the patient's got hemolysis, we're then gonna carry out your analysis to see if there's any evidence of hemoglobinuria.

Or more commonly bilirubin urea, so we see those with intra and extravascular homolysis and respectively, we're going to look for any evidence of active sediment and see if there's urinary tract infection that might warrant therapy, and we're going to image our patients as well, and we're going to look on our imaging for any evidence of neoplastic causes of IMHA. Now again in this consensus statement. They looked at the evidence surrounding neoplasia as a cause of IMHA, and it really was quite lacking.

It's really quite uncommon for neoplasia to cause IMHA, but it's certainly sensible to screen for it in individuals that can sort of afford and are stable enough to undergo those investigations. And do bear in mind as well, of course, our infectious causes in the UK. Of IMHA, there's a very low level of evidence of infectious diseases, certainly dogs causing, IMHA, and other than Birbezia cannis in the UK.

And it's often quite prudent in a patient's got IMHA to at least consider running umiresia PCR on an EDTA sample when investigating IMHA. And in cats they're really quite different in a lot of ways. They're not always as regenerative.

Sphherocytosis is not as evident in these individuals, and of course infectious causes like mycoplasma hemophilus are much more common as causes of anaemia in cats than a primary autoimmune disease. I just lifted this flow chart from the ACBIM consensus statement. So again, if you've not read this paper before, it's definitely worth checking out loads of useful information there on how we can help diagnose our patients with hemolytic anaemia.

And then on to non-regenerative anemias. So this is quite a wide group of patients, as I've said earlier. Those who have anaemia or renal disease are very easy to diagnose based on aotemia on their biochemistry.

Again, those that have endocrine disease typically quite easy to detect, the Addisonians, the patients with hypothyroidism. And then we're essentially screening the rest of our patients for either evidence of, well, does this look like it's an anaemia of chronic disease? Or is it a bone marrow problem?

So much like with our other causes of anaemia, we're going to perform a complete physical examination. Blood work and often imaging with your analysis to say, OK, is there evidence of a significant comorbidity here that could be causing this non-regenerative anaemia, such as cancer, as I say, inflammatory bowel disease, and etc. Etc.

And bearing in mind, as I said earlier, that most patients with anaemia of chronic disease is quite mild and often their other cell lines are normal. If we've done all of those investigations and not found any specific changes on any of those further tests to tell us that there's an underlying chronic disease that's causing their anaemia, or the patient has abie or a pancytopenia, that will actually point us towards the bone marrow, as our final step in the investigation. And typically bone marrow sampling is something that certainly I and others have found like a challenging technique to learn.

I do find it easier in dogs and cats, and the bigger the dog, the easier it is. But typically the things to know are that bone marrow sampling, once you're comfortable with the technique, is something that's certainly able to be performed in primary care practise. It's typically performed under general anaesthesia.

And there's several sites that you can look at, the proximal humerus, proximal femur, the wing of the ileum, for full bone marrow sampling, or if we're just doing bone marrow aspirates, we could look at a rib aspirate. Now just to quickly explain the difference between aspirates and core biopsies, essentially an aspirate is a sample, of, fluid that's aspirated. From the medullary cavity and submitted to the external lab on freshly made smears, but also as an EDTA sample if you've got enough.

And this is generally the most useful test when we're looking at bone marrow disorders. It allows us to assess the, to some degree the cellularity of the marrow, how well the cell precursors are maturing in the marrow, whether there's evidence of. Dysplasia, whether there's evidence of a lack of, maturation of the red blood cells, but really importantly, whether or not there's evidence of cancer as well, OK?

Once we've taken an aspirate, we then take a core biopsy, which is essentially, once the biopsy needle is introduced into the bone and we've drawn out some fluid, we advance that biopsy needle further into the medullary cavity and it calls out. A section of the medullary cavity that sits inside our needle and then we can simply push that out at the end and we submit then a core biopsy for histopathology, and this is really quite useful for looking at overall cellularity of the marrow, whether there's evidence of aplasia, where we get large amounts of fat in the marrow replacing the normal precursors, and for evidence of myelofibrosis as well. Now, do you consider contacting a a medic or you know, a pathologist when you're thinking about doing a bone marrow, just to double check the very sort of specific sample requirements for submitting to the lab, as well as what tests you might want to run, because cats, for instance, we might commonly consider retroviral PCRs on the bone marrow when we're investigating non-regenerative anaemia in those patients as well.

But often having taken, an aspirate or or a core biopsy or a combination of the two, having ruled out all the other causes of our anaemia of chronic disease, we should be able to comfortably diagnose all the various bone marrow disorders and then offer treatments and gauge owners's expectations appropriately. Iron deficiency anaemia, as we said earlier, these are patients that most commonly have chronic GI blood loss, but they could be bleeding anywhere. So we're gonna do a physical and take history, to look, you know, quite closely, for any evidence of bleeding.

The, blood work will include an iron panel, as I mentioned earlier. And these patients are gonna have the microcytic hypochromic, types of anaemia. We're going to look at urinalysis to make sure there's no bleeding into the urinary tract, and imaging is really important in these individuals, to look at the urinary tract, in particular in the GI tract, for any evidence of lesions that could be bleeding.

And as I said earlier, the most common cause of iron deficiency anaemia that we that we see in animals is ulcerated, small intestinal neoplasia. And some of these individuals will require endoscopy, . If we're still unsure of their cause of iron deficiency anaemia after their imaging, so flexible endoscopy or capsule endoscopy, as I mentioned earlier, and then, patients in which, a bleeding, particularly GI lesion is identified, are often then candidates for surgery to have that source of the bleeding removed and have that lesion submitted for histopath to see what the underlying cause is and what the treatment options are, from there.

So as you can see, there's 3 quite distinct diagnostic pathways that we go down depending on whether the patient's got regenerative, non-regenerative, or iron deficiency anaemia. That's why it's so important to be doing these blood smears as early as possible as we can in the investigation. So finally, just a little bit on nursing the anaemic patient, really very sensible, straightforward stuff.

And consider those patients that are profoundly anaemic administering oxygen supplementation to help with their hypoxemia. Definitely limit exercise in the clinic and then when the animal goes home as well to make sure that they're not at risk of collapse or syncope because of their severity of anaemia. Monitor the red blood cell in disease during treatment, as I said, right back at the beginning of the lecture.

That's often best done with a PCV in combination with total solids. And we may consider the use of gastroprotectants in patients who have evidence of GI bleeding, so looking at combining sacralfate with antacids. We need to consider as well how we're gonna nurse patients who have bi or pancytopenias as well.

So it's really important to make the whole team aware that if you've got a thrombocytopenic patient in the hospital, you know, we're. Not going to perform blood samples from the jugular vein. We're gonna use small needles and go through peripheral veins and keep pressure on for long periods of time afterwards.

We're going to ask the people nursing that patient to monitor for any evidence of spontaneous bleeding. And any patient who is neutropenic, certainly if they're profoundly neutropenic and or have a high temperature, they may require prophylactic anti biosis. And, or reverse barrier nursing so that we don't put them at risk of picking up an infection whilst in the hospital.

And as I said earlier as well, we'll consider the use of transfusion medicine as well in these patients, whether that's to get them well enough to undergo anaesthesia for their investigations or as part of their treatment plan to get them home and happy as an outpatient whilst their medications do the job. So that's the end of the talk. I hope there are a few sort of top tips for you guys to take home that you can put into practise in the primary care setting to really get the very most out of the investigation of the anaemic patient.

I really very much enjoy investigating these patients and enjoy treating them as well because there are a lot of diagnoses in these patients apart from generally the bone marrow diseases in which they can have a good outcome. So thanks once again for listening and everyone, take care. Bye bye.