Hello and welcome for a new webinar together. Today we are going to discuss regenerative anaemia in dogs and predominantly we are going to focus on what to do when we are facing such an anomaly in our patients, and, investigate these anomalies together. So, first, in terms of clinical presentation, these patients are anaemic.

So the clinical presentation is quite classic for an anaemia with a lack of stamina from this patient, weakness, pale mucous membranes, as well as nothing really specific from the anaemia itself. The other might find that their behaviour is a bit changed, some of them might have collapse as well if the anaemia is very severe. We have a classic clinical presentation for an anaemia with tachycardia, tachypenia, when you do your physical examination, and these pale mucous membranes that are quite commonly seen.

A heart murmur might be present because if the red blood cell volume is reduced drastically, that can change the viscosity of the blood and lead to a hemic murmur. And we have hyperdynamic pulses, because these patients are huge volemic and well hydrated, so there is no alteration of their perfusion. Just as a quick reminder, the pulse quality can be used to kind of Differentiate or suspect a difference between anaemia and hypovolemia.

If you have a hypovolemic patient and there is a decreased overall blood pressure, the pulses are going to be weak versus in an anaemic patient where the dog is hevolemic, the patient, the pulses should be strong and even hyperdynamic again due to the difference in viscosity in the blood. So there is no strong clinical sign that they're gonna scream that the anaemia is there or even that the anaemia is non-regenerative, but all of that should make you suspect that your patient is anaemic, it's not pathognomonic of course and eventually you might have signs of bleeding associated with the presentation, bleeding in the urine, bleeding in the faeces, that might be reported by the owners or determined at the time of the physical examination. So initially we want to confirm the anaemia.

In humans, the gold standard is to use haemoglobin, but in veterinary medicine we use mostly the PCV or the hematocrit. The PCV is obtained by a centrifugal method, using a capillary tube just as indicated on the slide, whereas the hematocrit. Is a parameter that is given to you by your haematology analyzer, and therefore you obtain much more parameters along that, including your red blood cell count, your white blood cell count, and also in the indexes around the size of the red blood cells, and the regeneration as well.

You want to classify your anaemia when you are diagnosing a patient with the anaemia. And it's very important to take into account a lot of things. So clinical information such as like the breed or the age will affect the interpretation of the degree of anaemia of your patient.

For example, greyhounds are known to have different reference values for haematology, and they have a higher hematocrit compared to most breeds, with normals being between 50% and 65%. Therefore, maybe for them, an anaemia of 35% is going to be much more significant than for another dog. Similarly, puppies have a lower hematocrits than adult dogs, they also.

When they are very young, might have different indexes around the size of their red blood cells. So it's important to realise that a puppy could have a hematocrit of 26 to 30%, with no reticular sites, and yet that might be normal for him. The hydration status of your patient should be also considered because obviously the hematocrit represents the percentage of red blood cells present in the sample that was submitted.

Therefore, if your patient is dehydrated, you can suspect that this hematocrit is going to decrease as you re-expand the circulatory volume of your patient. We use other haematological indices to classify the anaemia further. What we want to know is the degree of the anaemia, mild, moderate or severe, for that we use as we just said, the hematocrit or the PCV.

You can use the red blood cell count, you can use the haemoglobin. We also look at the content in haemoglobin of these, cells, and for that we look at the MCH predominantly and that we will qualify as hypo or no chromesia depending if the MCH is normal or if it is decreased and if the content in haemoglobin is lower in the cells that are circulating in your patient. You also look at the size of the red blood cells, looking at the MCV, the mean cell volume, and that will lead us to decide if the anaemia is microcytic, normocytic, or macrocytic, so do we have smaller or bigger red blood cells compared to normal?

And then you want to look at the regeneration. The regeneration is assessed by the presence of reticulocyte. If your analyzer gives you a reticulocyte count.

You can also look at a blood smear and the presence of polychromasia. So, red blood cells with varying staining and the recognition of a reticulocyte as a polychromatophy on the blood smear is suggestive of regeneration. Anisocytosis, so a wide variation in size of red blood cells, it's also suggestive of regeneration.

Finally, the RDW is an index that can give you a hint that there is variation in size. However, it doesn't tell you if the variation is towards bigger or smaller cells. The RDW just represents the diversity of size that is present in the sample.

And so an elevated RDW just says that the size of the cells is very variable, but it doesn't tell you if it's like because they are more smaller cells or more bigger cells. We try to classify the IMHA kind of the anaemia story trying to use all of these parameters together, and that is one of the possible classification. Today we are going to focus on non-reative anaemia, and therefore we are going to look more at the first two columns of this classification.

So what is the definition of a non-regenerative anaemia or of a regenerative anaemia? Well, the definition is based on the presence or not of reticulocytes, and you can see on the table on the right hand side that there is different values of reticulocytosis that have been established to determine if the regeneration is present, weak, moderate, or strong. And that there is a difference between dogs and cats.

Firstly, I would say that personally I prefer to use absolute reticular side count because I find them easier and much more easy to compare from one time to the other. In dogs, we only look at reticular cys and there are different thresholds that are used to determine the quality of the regeneration of the bone marrow. In cats, we know that there are two types of reticulocytes, the aggregates and the punctate.

And that will depend on the quantity of nucleic acids that are left in the reticulocytes and depending on the analyzer that you have, they are going to amalgamate them together or they can separate these counts mostly if you send that to an external lab, they might be able to do that for you. And it's possible that sometimes a cat is non-regenerative, but when you do these special stains and you look at either the aggregates or the punctate, you might have a better answer for them. So back to our diagram, we said that today we wanted to focus on a anemias that were fairly or non-regenerative at all.

And when I say fairly, it's just because initially some of these anaemia might be regenerative, but over time there might be an exhaustion of the resources that are necessary to produce the red blood cells and the ultimate stage of the disease will be a non-regenerative anaemia. Obviously you know where I'm going with that, and the first one that we are going to discuss is iron deficiency anaemia. So the aetiology of iron deficiency anaemia is a lack of iron that therefore impairs the production of new red blood cells.

Low iron intake is an important cause of iron deficiency anaemia, mostly in humans, and more precisely even in women, and dietary iron requirements are known for dogs and cats, and they are estimated to be around 180 milligrammes per kilogramme of dry matter. And it is possible that in puppies and kittens, that intake is not sufficient, because they grow very rapidly and they might continue to drink milk, which is, doesn't contain any iron at all. So it is important to make sure that if you have a very young animal and it's time to transition to a proper balanced diet, it is done by the owners to ensure that their intake of iron is enough.

And inadequate dietary iron intake, rarely occurs in adult dogs or cats that are fed with a commercial pet food, because all of these diets are well supplemented in iron and their composition is fixed, and it can sometimes occur with home cooked diet or vegetarian diets, moreover, if it's done from the owner's proper, Kind of initiative and not supervised by a veterinary professional or veterinary nutritionist and these diets need to have an appropriate iron supplementation to be sure that the intakes are sufficient. Chronic blood losses are the other main cause of iron deficiency and. It seems logical that during an acute blood loss, the body stores iron, so there should be enough storage to replace what has been lost.

However, when you have, chronic, haemorrhage and chronic blood loss. Therefore, the iron that is contained within these red blood cells is lost to the body and it's not necessarily replaced enough by the intake to promote further production of more red blood cells. Iron deficiency anaemia though, as it makes sense now, will develop over weeks to months because you need a lot of these chronic recurrent blood loss, to have a significant decrease in your iron storage, to the point that your intake is not enough to compensate for them.

Massive causes of chronic external blood losses are ectoparasites, mostly in young animals, hematuria, epistaxis, hemorrhagic skin disease with like the skin oozing blood kind of chronically and without a lot of control, a chronic coagapulopathy, thrombocytopenia, thrombocytopathias, and gastrointestinal haemorrhage. Gastrointestinal haemorrhage can be associated with a mass, with inflammation, or with an ulcer, and that can be eventually promoted by ulcerogenic drugs such as NSAIDs or steroids if there is already an underlying pathology associated with this patient. Some inflammatory disease can mimic iron deficiency anaemia, and that is called anaemia of chronic disease, and we will dive a little bit further into that a little bit later.

It is also important to remember that chronic phlebotomies can lead to depletion of red blood cells and that for small size patients or for patients that are bled very, very regularly, it can be significant. So there are some studies that have shown that blood donors that were overused could become iron deficient. It's also been shown that in smaller patients, phlebotomies over 1% of the patient's body weight every week could create iron deficiency anaemia as well.

Now if we dive a little bit deeper into the pathogenesis of iron deficiency anaemia, there are 3 stages that are basically a progression from a storage iron deficiency, so the body starts to have used all of the iron that has been stored. The iron deficient erythropoiesis, where the erythropoiesis starts to be affected by the lack of iron and the difficulty to form. New functional heme, to have a proper haemoglobin, molecule, to, to uptake into these new red blood cells.

And finally, the iron deficiency anaemia, which is the ultimate stage where the individual is so affected that, the, the bone marrow is not able to produce enough red blood cells and the red blood cells that are produced are abnormal. The anaemia is very much exac exacerbated because at the later stage of iron deficiency anaemia, these erythrocytes are abnormal. They have a shortened survival due to their fragility, therefore they are either destroyed or sequestrated earlier than normal red blood cells.

They also are different in shape. They are smaller, so it's a microcytic anaemia, and they contain less haemoglobin, so they are paler. So it's a hypochromic anaemia.

So we have a low MCH, a low mean concentration of haemoglobin, and you have also low MCHC, which is the low mean concentration, mean cell haemoglobin concentration. So the concentration in each individual red blood cell is much, much decreased. You can see on the picture that they have very specific appearance and you can suspect it when you have such a dramatic blood smear, but that is a very, very advanced stage of iron deficiency anaemia.

So, from the three stages, it starts probably as a normalcytic normochromic anaemia and then these anaemia progresses to more hypochromic and eventually microcytic. It is also important to realise that the concentration in haemoglobin is one of the trigger to release mature red blood cell into the circulation. So the bone marrow is gonna try to reach the highest concentration in red blood cells before releasing them into the circulation, and that explained the decreased size of these red blood cells because to increase the concentration, if you reduce the size, you obviously increase that concentration of the molecule within the cell itself.

In terms of pathogenesis, so as I said earlier, iron deficiency state are difficult to suspect, because they will be eventually regenerative initially and then eventually become normocytic, normochromic, but there will be some reticulocytes present in the initial stage, but you can see that there is a discrepancy between the amount of reticulocytes and the actual, severity of the anaemia. And as I have mentioned in the previous slide, these red blood cells are not completely normal, and there is a degree of hemolysis, sequestration that will worsen the anaemia and make it, kind of, progressing even faster than you would expect with just the lack of production within the bone marrow. Functional iron deficiency is what we suspect is the mechanism associated with anaemia of inflammatory diseases.

And I've mentioned that a little bit earlier and they kind of mimic iron deficiency anaemia and they can mimic it to a very extreme degree. These are disease states in which a functional iron deficiency occurs and then iron becomes unavailable despite being stored in the body. It's unavailable for the synthesis of heme and the production of new haemoglobin molecule.

Among these anemias of inflammatory disease, you can have normal, Iron storage, but you will have decreased serum iron levels, and you have progressive insight of a normocetic normochromic, and then a microcetic and then eventually a hypochromic anaemia if that condition continues to procure and is not corrected. Microcytosis is also reported in other diseases where iron metabolism might be impaired, and that is protosystemic shunt. We know that they have low serum iron concentration, microcytosis, otherwise they are fine and their hepatic function is otherwise normal.

And we don't really understand the mechanism under this pathology, but we suspect that it's the same as anaemia of inflammatory disease, where there are some cytokine impairment of iron, mobilisation and iron release for it to be used and to form a heme and a normal haemoglobin molecule. Finally, in chronic kidney disease, we know that these patients can be abnormally anaemic, and that's one of the main mechanism is the decrease in renal erythropoietin synthesis. However, CKD is also considered as a chronic inflammatory disease and part of the anaemia could be enhanced and worsened by anaemia of inflammatory diseases and impaired iron and impaired copper, metabolism.

We also know that these patients are probably a bit more prone to have chronic GI bleedings with very minimal bleeding, but very, very chronic, and therefore there could be also an iron deficiency anaemia associated with it that would eventually worsen even further the picture of these anaemic cats. . Recommendations in these situations are to treat the underlying cause, and if we are facing something like CKD that we can't address, to eventually replenish the iron store or maximise the iron store and use erythropoietin to try to stimulate as much as possible the bone marrow in forming new red blood cells.

So if we go back to our physic our diagnostic investigations, the initial steps were taking a history, taking a physical examination, trying to identify any trigger or any underlying cause for the anaemia that you have identified. And if you suspect that the anaemia is probably an iron deficiency anaemia based on your MCV MCHC, your blood smear, you really want to insist in a few things, and you want to make sure that the patient has not been exposed to any drugs, either accidentally or because they were prescribed. You want to make sure that your patient has not been exposed.

Any toxin that could be toxic to the bone marrow, you want to know what diet they are on to make sure that it's complete and balanced. And you also want to question the owners about sources of blood loss. Have they noticed any blood in the faeces, any epistaxis, any hematuria?

When you do your rectal examination, is there melina on the glove, just to know, you know, is there any evidence, of blood loss that could explain a non-residentive anaemia. Your initial tests are the haematology with a blood smear. You're gonna have a reticulocyte count, most likely a biochemistry to rule out any form of metabolic disease to explain the non-regenerative anaemia, urinalysis for the same reasons, and a faecal sample because we know that faecal parasites can be associated with chronic blood loss and non-regenerative anaemia.

So all of that is really a, a very basic, first step. I would probably also include eventually coagulation testing, because you want to know if your patient has a coagulopathy to explain why he might have, chronic blood losses. Then If you have not identified an obvious cause that you could address, you want to advance a little bit further, and for that you are gonna probably do an ACTH stimulation test because Addison's disease has been associated with non-regenerative anaemia.

If you have any other. Concomitant clinical signs or clinical pathological changes for hypothyroidism. This is something you could consider in the right patient.

I would not recommend to do a T4, if you don't have more suspicion for hypothyroidism than just the non-regenerative anaemia. Then you want to probably perform an iron panel because you want to know if your patient is iron deficient and if the metabolism of iron is impaired in your patient and you want to do an abdominal ultrasound to look for any abnormality that could explain the chronic bleeding, or the source or the underlying cause for these bleedings. So how do we interpret an iron panel?

The iron panel will give you a lot of different information and it's important to know how to, what to make with it. So the first information that you're going to get in your iron panel is your serum iron concentration, and typically that concentration is very low in animals with iron deficiency anaemia. It can be mildly low, low normal, if you are at the beginning of the disease, but usually it's fairly severely affected.

In the contrary, if you are looking at anaemia or inflammatory disease, usually that zero iron concentration is only kind of low normal. Serum iron can be transiently elevated if you have red blood cell lysis, so that could give you like a false positive kind of. It might be changed if the patient has received a blood transfusion before you initiate your investigation, so that's very important to know.

Or if obviously the iron supplementation has started before you've sampled the patient to send for the iron panel, and that will make the the interpretation of the iron panel very, very difficult, so you might as well postpone that eventually, for a little bit later if the patient has been transfused or if you have initiated iron supplementation. Exogenous administration of corticosteroids have also been reported to increase serum iron levels, and the mechanism so far is remained unknown. TIBC is usually the second parameter that you obtain from your iron panel, and that's the total iron binding capacity, and it measures the capacity of the plasma to carry iron, and transport iron from storage places to organs where the haemoglobin is going to be produced.

And that kind of represents the maximum concentration of iron that can be bound to plasma transferrin, which is the plasma molecule, the protein, that will transport iron in the plasma. The clinical use of TIBC is fairly limited, because it doesn't tell you much about serum, or tissue iron level, like the actual availability of iron, and the TIBC doesn't change very much in disease state in dogs, so the interpretation we have of it is usually fairly, fairly limited. Other indexes that are associated with the TIBC are iron saturation, which reflects the amount of iron bound to the transferin and it's usually low, below 20% in case of iron deficiency anaemia.

Or you can have the UIBC which is the unsaturated iron binding capacity, which basically measures the transferring space where they could bind more iron but are currently unbound. And they are usually elevated in iron deficiency anaemia because obviously there is less iron to transport. Ferritin is another very important marker of an iron panel.

Ferritin can be measured in the serum and we correlate very well with the body iron stores. It is important to note that the availability of ferritin assays is very limited and it's not a widely available test. But also that ferritin assays are very species specific.

Therefore it's important for you to confirm with the laboratory that you're using that the assay that they have is meant and validated for dogs or cats. Ferritin is decreased in dogs with iron deficiency anaemia and will be increased if you have an elevated total body store of iron. So that's exactly the markers that we would like to have to confirm the presence of an iron deficiency anaemia.

Ferritin is an acute phase protein though, and therefore elevation of ferritin can occur if you have a very inflammatory process going on. So it can also help to differentiate between iron deficiency anaemia and anaemia of chronic disease, given that ferritin has been shown to be elevated with pancreatitis, neoplasia, liver disease, or hemolytic anemias. So low serum ferritin concentration are very helpful in differentiating iron deficiency anaemia, but the problem remains to find a laboratory that is easily accessible for you to measure ferritin.

Finally, body iron stores can be detected differently. They can be assessed in the bone marrow, the liver, the spleen, either by cytology or estopathology, but obviously all of these are much more invasive than. A blood draw and a blood sample that we could send to a laboratory.

So unless you have another good reason to perform a bone marrow sample, which we will discuss in a few minutes, usually these tests are not done just to check for iron storage. Briefly discussing the treatment of iron deficiency anaemia, as you can understand, it depends a little bit on the underlying cause. So I will focus on replenishing the iron storage, and I think there are two main strategies here.

One is if your patient needs a transfusion, you're going to administer iron through that transfusion. And so if the patient feels better after the transfusion and you have a good control of the underlying cause, that might be enough to replenish his iron, . Is iron status.

The other strategy is to give iron supplementation either parenteral or oral, and that will depend on what is available where you are working, obviously, and on the compliance of the patient. The oral absorption of iron is a bit intermittent, but there are good results with long term treatment. So now if we go back to the differential for the non-regenerative anemias, we have covered the iron deficiency anemias, we have covered the anemias of chronic inflammatory disease, and these two diseases are mostly associated with the decreased erythrocyte production.

Something impairs the bone marrow ability to form and to produce new red blood cells. Other pathologies of the bone marrow are more directly involving the bone marrow tissue itself, and they will involve like hypoplasia or aplasia, which can be secondary to infections and toxins or can be primary and associated probably more with an immune mediated disease. We also have more specific, bone marrow neoplasia such as mylodysplastic syndrome or leukaemia.

So if we go a bit more in depth with bone marrow primary pathologies, we have to remember that. There Easier way to be detected are cytopenia, which means that there is a defect in the production of these cells, or there is a consumption or destruction of these cells or a loss. Once you have eliminated the consumption, destruction or the losses from your differential diagnosis, you are left with a defect in production.

And that should prompt you to investigate a bone marrow disease. You can have the opposite where the bone marrow is so active that you have an increase, an abnormal increase in circulating cells. Could be the red blood cells, could be the white blood cells, could be the platelets.

And sometimes with some bone marrow disease, you also have an increase in globulins, and an elevation in the total proteins of your patients. So these are, . Clinical signs, clinical pathological signs that should hint and prompt you to investigate bone marrow disease.

We're going to go through a case to try to make this a little bit more pleasant and see what is the approach that we've had with that patient. So Bowie was a 4 month old male entire mixed breed dog who presented for a 5 day history of vomiting and diarrhoea and a very marked progression of a profound lethargy. The dog wasn't well 5 days ago and now the dog is like almost recumbent and very, very lethargic.

The dog has become completely anorexic after these 5 days of vomiting and diarrhoea. On physical examination, the dog was very, very pale. It was tachychotic, tachypnick, he had bounding pulses, so we suspected that this dog had an anaemia.

We performed a hematocrits, we performed a complete blood count, sorry, and the dog had a hematocrit of 9%, so very profoundly decreased even if we are with a puppy, and that might be even worse if we consider that it might be a little bit dehydrated with the vomiting and the diarrhoea. The reticulocyte count was low at 22, so within reference intervals, so very discrepant given the degree of anaemia that we had, and the platelet count was 90 degrees at 100. We performed a slide recognition test and the slide agglugnition test was very, very positive with a strong agglutination of the red blood cells in the peripheral blood.

So what would you do next? Well, next we decided to investigate with a biochemistry and a urinalysis to look for metabolic conditions that could lead to agglutination and hemolytic anaemia, which was what we were suspecting. However, the bilirubin was normal, which started to shift a little bit our concerns that we have no reticular site, no evidence of hemolysis as the bilirubin was normal.

There was no bilirubinuria either, so it started to become a little bit suspicious that things might not be happening only in the peripheral blood. We sent for a Coombs test, which is in like a An in vitro test for antibodies coating the red blood cells, and that test was negative. We sent for a 4DX to look for tick bone diseases associated with hemolytic anemias, which was normal, well negative, and then we did abdominal and thoracic imaging, which were both normal.

Bowie was hospitalised, received a blood transfusion, and we can see that he's kind of maintaining his PCV after the blood transfusion, but after 3 days, we still have no reticulocytes, to be seen anywhere. The bone marrow has started to produce some platelets, but has not started to produce any erythrocytes. Due to the suspicion of an immune-mediated hemolytic anaemia of some degree, we had started the dog on steroids at that point, and despite the daily transfusions, he was not able to maintain his PCV very high and obviously.

The bone marrow response was very, very limited, and so at day 4, we had still no regeneration and still giving more transfusions. And at day 7 we had even less reticulocytes than before. So what would you do next?

Well, obviously, Bo being a puppy, we measured a B12 to kind of rule out congenital abnormalities that could lead to very profound anaemia. But most importantly, we performed a bone marrow cytology and the biopsy and we also cross matched him to, initiate further transfusions. And yes, I said cytology and biopsies because I think it's very important to perform both when you suspect a bone marrow pathology, unless you are very, very confident that what you are looking for is a lymphoma or a leukaemia, which was not the case here, you're gonna obtain very different but very complementary information from both your cytology and your biopsy.

The cytology is ideal. To look at the precise morphology of the cell and identify which lineage they are coming from. So here you can see that we have myeloid precursors which have very clear cytoplasms, and we have erythrocytic precursors which have this very dark cytoplasm on the right hand side.

You can also see that there is a mitotic figure and you can also appreciate that there is a wide variation in the size of these cells. So if you are a trained pathologist, you are able to recognise the different . Stage of maturation and differentiation of both the mylo myloid and erythroid lineage.

The biopsy is gonna give you a much better idea of the architecture, OK? Because you do not preserve the architecture when you smear your bone marrow sample for, the cytology of the bone marrow. Whereas here, you can see on the left hand side that the bone marrow is very patchy.

And that there is actually a lot of empty space that was fat. So the bone marrow was not that rich in cells compared to the sample on the right hand side, which is a hypercellular bone marrow with, cells that are touching one another from one wall to the other. There is only cell, cell, cell, cell, cells everywhere.

And that is very important to know as well because depending on the age, the bone marrow should be more or less rich in fat. And depending on the pathology, the hypercellularity might be a criteria to diagnose certain disease of the bone marrow. You can also appreciate fibrosis with a biopsy and you can see here that you have this kind of pink mixoid material between the cells that is kind of separating the cells.

And these are fibroblasts and evidence of severe myofibrosis in the sample. So if we go back to Bowie, we performed a bone marrow, cytology and biopsies, and we diagnosed him with a precursor targeted immune-mediated anaemia. So we have a non-regenerative anaemia with evidence of both peripheral immune-mediated disease with that positive slide declination test and evidence of a bone marrow disease with a maturation arrest and the inability of the bone marrow to produce mature erythrocytes that will be released in circulation.

So what do we know about treatment and prognosis of that disease? So precursor targeted immune-mediated anaemia, or PIMA, is something that is relatively newly described in the veterinary literature. There is a recent retrospective study with a good, a good cohort of dogs, that showed that the dog most of the time will need a transfusion and half of them have needed at least 2 transfusions.

Most of the treatments that are used are immunosuppressive medication, which makes sense because we suspect an immune-medited destruction of the red blood cell precursors. So prednisolone, prednisone, azathioprine, cyclosporin have been reported. And there is not enough data to have one that we would favour over the others.

However, based on our experience with, just the regular peripheral immune-mediated hemolytic anaemia, prednisone and prednisolone seems to be the first line and for me I think should be the first line in this patient. The other one should be added on top of it as a second line to help, kind of being more aggressive with the immunosuppression. The breeds represented in that disease were different than the one that we currently know for IMHA and Boxer larchers, Dashund and Whippet were the most common breeds with that bone marrow disease.

Clinical signs were very similar to the one for IMHA except maybe for PUPD. Now when we look at the results, it's very interesting to see that there was persistent agglutination of erythrocytes after dilution in saline in about half of the dog, so that's kind of what we had with Bowie, and they also had some dogs that had a positive Coombs test, and And that was representing about 8% of the patient. The blood smears had spherocytes, also suggestive of peripheral destruction, in about 90% of the dog and goat cells in about 5% of the dog.

So even though they have a bone marrow pathology, there was evidence of peripheral destruction as well. We can see that they presented with a low PCV, compared to normal, and they had no regenerative cell whatsoever compared to normal. Platelets were not very exciting, white blood cells were, a little bit low but nothing too, too bad.

So really something focused around their red blood cell line and an abnormal erythroid production. Finally, the bilirubin was within normal limits for this patient. So despite the evidence of peripheral destruction, there was no evidence of hyperbilirubinemia for the majority or for 50% at least of these patients.

In terms of prognosis, of the dogs that had a complete follow-up available at 3 and 12 months, . 61% of them were still alive at 3 months and 43% of them were still alive at 1 year, which is a bit. Less than what we would expect for a peripheral IMHA, but it's still a decent survival time and it shows that it's worth trying to treat this patient and if you have a response, you could have a very, very good response.

The median duration before initiation of regeneration was about a month, and that is something that we see very often with bone marrow pathology. It's going to take a long time for the bone marrow to start regenerating, therefore, people have to be patient and be committed to treat the animals for at least a few months and eventually have to give a few transfusions along the way to support them until the medications kick in and the bone marrow starts to regenerate. Well Bowie, unfortunately the outcome was not as good and he was euthanized based on you know, welfare ground because he received so many transfusions and also because of monetary issues.

What was interesting with Bowie was that duality between bone marrow and peripheral disease which is reported in a precursor targeted immune-mediated anemias. However, we had, also, some, discrepancies with a negative coms but a positive saline negligenation test, so we were trying to understand and we know that there are a lot of pathologies that we don't. Know very well in veterinary medicine, but that could lead to different type of presentations.

So for example, we know that anaemia in a patient with malignancy can be associated with other mechanisms of destructions and the cell lysis might not be exactly the same as it is for IMHA in patients with leukaemia or myelodysplastic syndrome in terms of speed of destruction or mechanism of destruction. So despite prednisone, cyclosporin, IVIG, and 8 red blood cell transfusion, Bowie was unfortunately euthanized. Now if you want to discuss some other bone marrow pathologies, we can mention pure red cell aplasia.

And obviously I brought this one just after the precursor targeted immune mediated anaemia because this is just the ultimate form of it where the target is the very, very, very early precursor in the bone marrow, which means that there is not even . Maturation arrest, there is no maturation at all because there are virtually no red blood cell precursors that will be seen on your sample, either cytology or biopsy. It has been reported in both dogs and cats, although it seems to be, it's a very uncommon disease, but it seems to be more common in cats.

In dogs, it's suspected to be autoimmune in all of the cases. In cats, it has been associated with the use of recombinant human erythropoietin. So we used to use human recombinant erythropoietin before using darbopoetin to treat anaemia of renal disease in cats and dogs, and that explained why we went with, an analogue of erythropoietin called darbopoetin that seems to create less immune reaction and therefore no targeting of the red blood cell precursors because that would be kind of .

Counter effective to what we are trying to do by administering the double poietin or the erythropoietin. In cats that's also been reported to be associated with feline leukaemia virus infections. So the presentation is a severe, non-regenerative normocytic normochromic anaemia.

In cats, they are often less than 2 years old. In dogs, there is not enough case reports to have like any sense of an age distribution. All of the tests are negative, principally, the Coombs test is always negative in these dogs, and some patients seem to show a bone marrow lymphocytosis.

On the picture on the right hand side, this is a patient that I've seen a few months ago called Peaches, and she was diagnosed with pure cell plasia and as you can see on the bone marrow cytology that is presented to you, you only have cells with a very clear cytoplasmic cytoplasm. A very clear background and that is myeloid precursors progressing up to segmented neutrophils. You can see a few platelets, you can see a few mature lymphocytes, but there is no erythroid precursors whatsoever in that cytology, and so she was diagnosed with pure cellarplasia.

The mainstay of the treatment is immunosuppressive medication with prednisolone and usually we add up right away a second immunosuppressive agent such as chlorambuil or or cyclosporin. The response, as I said, is slow, and you can imagine that if you have no precursors at all, it's going to take a very long time for the body to produce some and for them to be able to produce enough red blood cells for the patient to be self maintaining their red blood cell pool. So it's important to prepare the owners for long term treatment and once we have a normal haematology and that may happen in some patients, then we can start to back off the medication progressively very, very slowly.

The few case reports, case theories on pur cellarplasia seems to show that there is a better prognosis if there is some evidence of regeneration after 1 month of treatment. For Peaches, she showed a very good response and she had a very nice regeneration at 1 month and her hematocrit became very normal around 6 or 8 weeks after initiation of the medications. Another type of bone marrow disease that you might have heard of is myelodysplastic syndrome.

It is also associated with non-regenerative anaemia, however, it can affect all of the other lines of cells that are produced by the bone marrow. It's an acquired clonal disorder. So if it's a clonal disorder, technically it's a neoplasia.

And in humans, it's a very well known pre-leukemic stage. So if you're diagnosed in human with a myelodysplastic syndrome because you have a chronic anaemia, you are going to be kind of monitored and treated as a patient that might develop leukaemia, in the near future. We are not yet there in veterinary medicine.

It's still something that is sometimes to be challenging to diagnose or to have a. To be confident on the diagnosis that we obtain, it's always associated with peripheral cytopenia. Most of them are anaemic, and the bone marrow in reaction is hyperplastic.

So there is a discrepancy between a bone marrow that is hypercellular and a peripheral cytopenia and a reduced number of circulating cells. You can see on the picture there from a cat with mylodysplastic syndrome that you have a big blast on the right and you have abnormal. Kind of early red blood cell precursor on the left hand side.

So it's associated with an ineffective hematopoiesis and a difficulty to release functional cells into the circulation. It's different from this mylopoiesis, which is a transient inability of the bone marrow to produce normal cells, usually associated with drug exposure, eventually hyperplasia in response to a profound cytopenia due to like peripheral destruction or a congenital disease with abnormal abnormal lineage, but as a congenital disorder and not an acquired clonal disorder. A bone marrow sample is often necessary for the diagnostic of my dysplastic syndrome, and these bone marrow samples have an elevated blast count, although not high enough to be called leukaemia.

So usually the total blast count is between 5 and 15% for this patient. And again, that ineffective hematopoiesis illustration there. Acute myeloid leukaemia, so in humans it would be the continuum of the same disease as your myelodysplastic syndrome.

It's the definition says that it's an aggressive hematopoietic neoplasm, with the clonal expansion of myeloid cell, and it's very important to realise that. Myeloid cells means all cells that are not lymphoid and therefore all cells that are produced in the bone marrow are considered as myeloid cells. So in alphabetical order, we will have erythroid leukaemia, granulocytic leukaemia, so red blood cell leukaemia, granulocytic leukaemia kind of any circulating white blood cell.

Neutrophil, eosinophil, basophil, mega karyocytic leukaemia, and mast cell leukaemia have been described in veterinary medicine. The diagnostic in humans is based on a bone marrow sample and genetic determination because they have a very specific genetic pattern between each type of acute myeloid leukaemia. There are about 9 different subtypes of acute myeloid leukaemia in human medicine.

In veterinary medicine, we use a modified classification based on bone marrow cytology and biopsy and the presence of a blast count of over 20%, . The treatment of choice is unknown. There are a few protocols that are described.

I think the one that has, the biggest favour at the moment is a combination of cytos R and doxorubicin. It's a disease with a very poor prognosis at the minute, although some patients seem to do better than others. So we might be able in the future to characterise it better and maybe tailor treatment a little bit better depending on the subtype of AML that we are treating.

Finally, the last disease process that I want to touch base about is myofibrosis, which can be primary or secondary. Primary myofibrosis can be idiopathic, where we can't find an underlying cause, or autoimmune. It's been very well described in humans and it's suspected in veterinary medicine.

It can be secondary to an infection or an inflammatory process at the level of the bone marrow and basically there is a mylotisis, so an invasion of the bone marrow space with fibrous tissue. So these long strands of pink, pale pink tissue, . Among the cells that you can see on the pictures are all fibrin fibrinous strands.

It will impair the expansion of the hematopoietic tissue and therefore the ability of the cell to replicate, differentiate and being released in circulation. As I said, there is a nice description of autoimmune myelofibrosis in humans, which encourage us to treat myelofibrosis as an autoimmune disorder with immunosuppressive medication. Usually we use a classic combination of prednisolone and cyclosporin.

However, some more advanced treatment have been described like anti-thymocyte serum, and some of these patients have shown complete reversion and resolution of the myelobros. So it's quite important to continue to investigate this disease and collect cases that we treat to see which outcome we can actually achieve for this patient, given that a few years ago we would have given them almost a death sentence with such a disease. So in conclusion, non-regenerative anaemia can come from a bone marrow impairment, either from a functional impairment like iron deficiency anaemia or a primary intrinsic bone marrow disorder.

There are a few tests that have to be performed in a non-invasive way, meaning blood tests to try to rule out in or out. Some of these pathologies such as iron deficiency anaemia or anaemia of chronic disease, and if you do not get the results with these investigations, or if you have other cytopenias present or abnormal cells in circulation and you suspect a primary bone marrow disorder, then you will need to perform a bone marrow cytology and a biopsy to continue to investigate these cases. Thank you very much.