Erythropoiesis
In the foetus erythropoiesis (production of red blood cells) occurs in the liver and spleen. In the adult
the bone marrow is the main production site however the liver and spleen maintain erythropoeitic
capacity (extramedullary haematopoiesis). Erythropoiesis has three essential components; stem
cells, cytokines and appropriate microenvironment. The microenvironment includes an adequate
supply of nutrients such as iron, amino acids, vitamin B12 and folate also an adequate supply of
oxygen. Erythropoiesis occurs in the bone marrow under the influence of cytokines including
interleukin (IL)-3 and erythropoietin. These cytokines act on the surface receptors of erythroid stem
cells: BFU-E (burst-forming unit – erythroid) and CFU-E (colony-forming unit – erythroid).
Erythropoietin has 3 actions: to increase the number of committed erythroid stem cells, to enhance
the survival of erythroid cells and to promote the release of maturing red cells.
The effect of erythropoietin is enhanced by hormones such as thyroxine, corticosteroids and
androgens. The effect of erythropoietin is reduced by down-regulation of the expression of the
surface receptors on erythroid stem cells. Suppressive factors include inflammatory cytokines IL-1
and tumour necrosis factor (TNF)-α, these are released during inflammation from macrophages.
Oestrogen also suppresses erythropoiesis.
Normal red blood cells are biconcave discs shape; this gives a large surface area to allow
deformation without rupture when red blood cells are squeezed through capillaries. Red blood cells
transport oxygen to tissues and remove carbon dioxide. The large quantity of carbonic anhydrase
within red blood cells allows the water of the blood to transport large quantities of CO2 in the form
of bicarbonate (HCO3-) from the tissues to the lungs. Haemoglobin is a good acid-base buffer. Red
cell life span is approximately 110 days in dogs and 70 days in cats. The major route of red cell
removal is via phagocytic macrophages with recycling of the components.
History
Anaemic patients should be triaged effectively and efficiently and action taken if they are critically
unstable. Otherwise, a detailed history should be taken. History should include the onset and
duration of clinical signs; an acute bleed is likely to result in more significant clinical signs than a
chronic, progressive anaemia. Owners should be questioned regarding any evidence of external
haemorrhage, particularly melena and haematuria which can easily be overlooked. Potential
exposure to toxins such as lead, vitamin D and onions should be discussed. Recent vaccination or
medication could increase the risk of immune-mediated diseases. Previous foreign travel or tick may
raise the suspicion of exotic or tick-borne diseases respectively.
Clinical Signs
There are many other causes of the signs seen in anaemic patients therefore a thorough history and
clinical examination is paramount to prevent a misdiagnosis. Common clinical complaints in an
anaemic patient include lethargy, exercise intolerance, and collapse. The classic pale mucous
membrane could also be due to reduced peripheral perfusion, a prolonged capillary refill time (CRT)
is suggestive of poor perfusion. CRT is important to interpret alongside hydration status since
dehydration can cause hypovolemia, hypotension and vasoconstriction. Mucous membranes may
appear icteric in haemolytic disease due to the breakdown of haemoglobin. Heart rate and
peripheral pulse quality are important parameters to monitor for response to treatment. A ‘new’
heart murmur may be noted, this could be a physiological murmur (not associated with structural
defects in the heart). Skin, eyes and mucous membranes should be examined for evidence of
bleeding such as petechiation, ecchymoses and haematomas, particularly after venipuncture.
Bleeding can also be appreciated on rectal examination or could be associated with an ascitic fluid.
Diagnostics
1) Packed cell volume (PCV) is one of the quickest and easiest diagnostic tests to confirm the
presence of anaemia.
PCV: dog = 37-55%, cat = 28-44%.
Fill half to ¾ of a microhaematocrit tube with blood from an EDTA (Ethyinene Diamine Tetra Acitic
acid) tube. Centrifuge at 10,000 rpm for 5 minutes to separate blood into packed red cells, buffy coat
and plasma. Assess the colour of the plasma; icteric plasma is often seen in haemolytic anaemia
(bilirubinemia) and red plasma represents intravascular haemolysis (haemoglobinemia).Sight hounds
have a higher PCV (45-65%). PCV can be increased due to dehydration leading to
haemoconcentration. Using a refractometer, the plasma can be used to assess total protein (TP).
TP: dog = 55-75g/L, cat = 55-78g/L
Total protein can remain normal during a bleed however usually drops during intravascular volume
expansion during recovery. TP can also be lowered due to conditions such as hepatic disease causing
hypoalbuminemia.
2) Blood smear evaluation
To make a blood smear take a drop of blood using a microhaematocrit tube from a well-mixed EDTA
tube and place on a clean slide. Bring a spreader slide at approximately 30˚ into contact with the
blood, allow the blood to spread along the edge of this slide. Finally move the spreader slide quickly
and smoothly away from the drop of blood until the feathered edge is complete. Air dry rapidly and
stain (Diff-Quik). Examination of a blood smear allows systematic evaluation of red cells, white cells
and platelets. Assess the red cell morphology in the monolayer, look for evidence of regeneration.
Neutrophils are used to assess red cell size, always check the feathered edge for platelet clumps and
white cells.
Red cells: Microcytosis (small red cell size) can be seen in iron deficiency anaemia (chronic blood
loss, portosystemic shunts and liver disease). Macrocytosis (large red cell size) is often associated
with a regenerative anaemia due to the larger size of polychromatophils but can also be seen in
myelodysplasia and feline leukaemia virus (FeLV). The size of red cells on the smear should be
interpreted with the mean cell volume (MCV) value from the laboratory haematology.
Polychromatophils are more immature red cells that are larger and bluer, they are seen in
regenerative anaemia. New Methylene Blue (NMB) stain can be used to show the reticulin, the cells
are then called reticulocytes. A high prevalence of polychromatophils creates anisocytosis (variable
red cell size) which is a feature of regenerative anaemias. Nucleated red cells and Howell-Jolly bodies
represent early release of immature red cells which is seen in a regenerative response.
Hypochromasia (low haemoglobin concentration) is associated with an increased central pallor of
red cells, this should be interpreted with the mean cell haemoglobin concentration (MCHC) from the
laboratory haematology. Spherocytes are formed with immunoglobulin marked red cells are partially
phagocytosed by macrophages. They appear as slightly smaller, darker spherical cells that lack
central pallor. Spherocytes are difficult to detect in feline blood. Heinz bodies and eccentrocytes
occur due to oxidised haemoglobin being pushed to the cell margin. These can be seen in
paracetamol toxicity in cats and onion ingestion. Acanthocytes and schistocytes are seen when red
cells have ‘shear injury’ from being forced through small vessels for example in tumours. Babesia
species can be seen on blood smears as paired pair-shaped (B. canis) or single angular bodies (B.
gibsoni) within red cells.
Platelets: A platelet count should be performed. The number of platelets per high powered field
multiplied by 15,000 gives an estimated platelet count (per µL). Spontaneous bleeding can occur
with <50,000/µL. Ensure to check the feathered edge for platelet clumps.
White cells: Assessment of white cell morphology to look for evidence of neutrophil toxicity and
bands which are observed increased demand states.
3) External haematology including reticulocyte count
Haemoglobin is directly related to the oxygen carrying capacity of the blood. It can be falsely
increased in lipemic samples if it is measured using a colorimetric method. Haematocrit (HCT) gives
the same information as the PCV but it is a machine calculated value therefore depends on red cell
count and red cell volume readings having been accurate. PCV is a more accurate measurement.
Mean cell volume (MCV) shows if the anaemia is normocytic, microcytic or macrocytic. Red cells
swell when stored in EDTA therefore can falsely increase the MCV. Mean cell haemoglobin
concentration (MCHC) shows whether the anaemia is normochromic or hypochromic.
Hypochromasia is associated with iron deficiency and in regenerative anaemia where red cells are
not fully haemoglobinised.
Reticulocyte count can be performed using NMB stain. Add 1 drop of NMB to 1 drop of EDTA blood
and leave for 20-30 minutes. Smear as normal and dry. NMB shows the reticulin in cells
(reticulocytes). Cats have aggregate reticulocytes (with large amounts of reticulin) which mature to
punctate (with 2-6 spots of reticulin). Feline reticulocyte counts show record aggregate for a picture
of what is happening now.
4) Slide agglutination
Strong cell to cell bridges are formed by adherent antibodies giving three dimensional aggregations
of red cells. To perform a slide agglutination test put one drop of EDTA blood and one drop of 0.9%
saline on a glass slide and gently rock. A positive result cab be seen grossly as flecking on the smear.
To differentiate from rouleaux (normal in cats) apply a cover slide and look microscopically.
Agglutination is a disorganised aggregation whereas rouleaux is stacks of red cells. A positive result
gives evidence of a strong immune-mediated haemolysis. A negative result does not rule out a
haemolysis as a cause of the anaemia
5) Serum biochemistry
Serum biochemistry is performed to look for the underlying cause of the anaemia. Raised liver
enzymes and bile acids can suggest hepatic disease. Hypercholesterolaemia is seen in
endocrinopathies such as hypothyroidism. Hyperbilirubinaemia is seen in haemolytic anaemia.
Azotaemia may be present due to dehydration but could also reflect a renal disease. Electrolytes and
basal cortisol should be measured to investigate hypoadrenocorticism.
6) Clotting function
Platelet number should have been assessed already on a blood smear. Platelet function can be
tested with a buccal mucosa bleeding time (BMBT). Activated clotting time (ACT) or
thromboelastography (TEG) assesses both the intrinsic and extrinsic clotting pathways. Activated
partial thromboplastin time (APTT) and prothrombin time (PT) assess the intrinsic and extrinsic
coagulation pathways respectively.
7) Imaging
Thoracic and abdominal CT (or thoracic radiographs and abdominal ultrasound if CT is not available)
is helpful to look for any underlying internal abnormalities not detected on physical examination.
8) Infectious disease
Canine: Ehrlichia and Anaplasma (often combined with thrombocytopenia), Leishmania and Babesia
Feline: Mycoplasma and FeLV.
9) Bone marrow biopsy
Indicated if the anaemia is none-regenerative.
The cause of anaemia can be divided into 3 categories: haemorrhage, haemolysis or
ineffective/decreased haematopoiesis. Both haemorrhage and haemolysis are usually strongly
regenerative anaemias.
Haemorrhage
Haemorrhage can be internal or external. External haemorrhage is likely to be discovered from the
clinical history and clinical examination. Examples include melena, urinary tract bleeding, epistaxis,
post trauma/surgery and marked ectoparasitism. A regenerative response requires 3-5 days after
acute haemorrhage to be apparent, before this the response appears poorly regenerative or ‘preregenerative’. Chronic haemorrhage may lead to thrombocytosis. Protein levels will be normal in the
acute stage but decrease as fluid re-equilibrates. It is important to check the iron status in suspected
chronic bleeding. Internal haemorrhage can be associated with bleeding tumours, trauma, bleeding
disorders and surgery. As red cell breakdown products are available for recycling, iron deficiency
does not result.
Haemolysis
This can be intravascular or extravascular. Examples include immune-mediated haemolytic anaemia
(IMHA), Babesia, Mycoplasma, phosphofructokinase deficiency (Springers), pyruvate kinase
deficiency (Basenjis, Abysinnians) hypophosphataemia and toxins (lead, copper, zinc, onions and
paracetamol). IMHA can be primary or secondary.
Non-regenerative anaemia
The most common cause of a non-regenerative anaemia is anaemia of chronic/inflammatory
disease. The anaemia is usually mild and becomes non-regenerative due to iron sequestration.
Diagnosis and reversal of the underlying cause is the aim of treatment. Endocrinopathies such as
hypothyroidism and hypoadrenocorticism cause a non-regenerative anaemia as thyroid hormone
and cortisol both have a facultative effect on red cell production. The kidneys produce
erythropoietin and uraemic toxins reduced red cell lifespan therefore renal disease can be
associated with a non-regenerative anaemia. If bone marrow disease such as neoplasia and
myelodysplastic disorders are suspected a bone marrow aspiration and core biopsy are indicated.