Anaemia

Anaemia means a decrease in red blood cells (RBC), haemoglobin (Hb) and/or haematocrit (Hct). Anaemia is not a disease, but a frequent laboratory finding to which a good diagnostic algorithm must be applied in clinical practice.

The state of anaemia translates mainly into a reduction of the capacity of an organism to transport oxygen. The clinical signs, albeit non-specific, manifesting this state include tachycardia, lethargy, intolerance to physical exercise and pale mucosae. The mucosae can sometimes become jaundiced in the case of haemolysis. The severity of the clinical signs is closely related to the degree of anaemia, its cause and speed of onset.

Anaemia can be classified according to:

• the degree of the anaemia
• morphological criteria
• pathogenic mechanisms    
• bone marrow response

DEGREE OF ANAEMIA

Tab.1. Classification of anaemia according to the degree of severity.

MORPHOLOGICAL CRITERIA

Anaemia can also be classified morphologically on the basis of two red blood cell indices, mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), as follows:

• normocytic normochromic (MCV and MCHC within the norm)
• normocytic hypochromic (MCV within the norm, MCHC decreased)
• microcytic normochromic (MCV decreased, MCHC within the norm)
• microcytic hypochromic (MCV and MCHC both decreased)
• macrocytic normochromic (MCV increased, MCHC within the norm)
• macrocytic hypochromic (MCV increased, MCHC decreased).

The state of hyperchromia (increased MCHC) is not included since, physiologically, red blood cells are already saturated in haemoglobin and cannot contain a higher concentration of this protein. However, since MCHC is an index calculated as Hb x 100/Hct (where Hb is the value of haemoglobin and Hct is an expression of red blood cell volume calculated as MCVxRBC/10) artefactual increases in haemoglobin (e.g., due to lipaemia) or reductions in haematocrit (e.g., due to haemolysis) can cause false increases in MCHC.

Some causes of anaemia, divided according to morphological criteria, are reported below.

• Normocytic normochromic: anaemia of chronic inflammation and dietary deficiencies. Chronic inflammation is the most common cause of anaemia. Various inflammatory cytokines are involved: in particular, it is suspected that interleukin 1 (IL-1) reduces the half-life of red blood cells; the same cytokine, together with tumour necrosis factor (TNF) and transforming  growth factor beta (TGF-β), can cause inappropriate use of Fe_²⁺ as well as make erythroid precursors refractory to erythropoietin. Anaemia due to dietary deficiencies is related to reduced availability in the diet of vitamin B12  and folates, fundamental constituents of DNA.
• Normocytic hypochromic: a rare condition which should first suggest the need to re-evaluate the reference ranges or consider a laboratory error.
• Macrocytic normochromic: chronic hypoxia or regenerative events, deficiency of vitamin B12 and folates, erythroid dysplasia, race-related, hypernatraemia and stored samples (in vitro). During chronic hypoxia (e.g. brachycephalic breeds, chronic heart and lung diseases) or regenerative events an increase in red blood cell volume can occur as a way of compensating for the oxygen deficit. Deficiency of B12 and folates can cause macrocytosis because the resulting defect in DNA synthesis leads to a lack of mitotic division. The macrocytic normochromic anaemia of malabsorption typical of humans is found only in the giant Schnauzer, since in other breeds malabsorption results more frequently in normocytic normochromic anaemia. In erythroid dysplasia, the macrocytosis is a manifestation of defective DNA synthesis. In cats infected by feline leukaemia virus, the virus-induced ineffective erythropoiesis is frequently expressed as a macrocytic anaemia. A breed-related macrocytosis is seen in poodles. Hypernatraemia can cause instrumental macrocytosis. This is because a patient with hypernatraemia has hyperosmolar blood. Samples of blood aspirated by a blood counter are diluted in isotonic solution in order to enable laminar flow: if the sample is hyperosmotic, the hypertonic red blood cells absorb water to create an osmotic equilibrium. The consequence is an increase in the laboratory-measured MCV (this alteration is not seen on the blood smear) sometimes combined with a decrease in the MCHC (MCHC = Hb x 100/MCV). In stored samples the aging red blood cells are more susceptible to the osmotic pressure exercised by the isotonic fluid during the aspiration phase of the cell counter. Once again, there is an increase in the laboratory-measured MCV and a possible reduction of the MCHC.
• Macrocytic hypochromic: advanced stage haemorrhagic or haemolytic anaemia, hypernatraemia and stored samples (in vitro). In the case of haemolytic or haemorrhagic anaemia, 3-4 days after the bleed, polychromatophilic and macrocytic red cells are seen in the peripheral blood as evidence of the ongoing regeneration. These cells have a higher MCV thus causing macrocytosis with consequent hypochromia. As regards hypernatraemia and stored samples, see the section above.
• Microcytic normochromic: due to chronic inflammation (++ liver failure), associated with breed, iron-depleted anaemia and hyponatraemia. In anaemia due to chronic inflammation it is common to find low levels of iron in the blood when inflammation is present: this finding, known as pseudohyposideraemia, occurs because of an IL-1-mediated effect of sequestration of iron in macrophages in the bone marrow, liver and spleen. Some breeds (Akita Inu, Shiba) have a constitutional microcytosis. This condition is the result of the persistence of sodium/potassium pumps in the red cell membrane: because of the osmotic effect, the water follows the sodium out of the cell, reducing this latter’s volume. In iron-deficiency anaemia, during states of iron deficiency there is a reduction in the concentration of haemoglobin within the red blood cells, which is manifested by greater pallor of the cells and a reduction in their volume. In fact, the mechanism that interrupts mitosis of erythroid precursors is saturation of the cytoplasm by haemoglobin: in the case of delayed synthesis of this protein, mitosis is protracted over time in order to enable cytoplasmic saturation, thus inducing a reduction in the MCV. In the case of persistent haemoglobin deficiency, saturation does not occur and hypochromia develops. Studies carried out in the dog showed that a reduced content of haemoglobin in reticulocytes (CH_retic) and a decrease in the mean reticulocyte volume (MCV_retic) were associated with haematological and biochemical alterations indicative of iron-deficiency. Hyponatraemia can cause instrumental microcytosis. This is because patients with hyponatraemia have a state of hypo-osmolarity. When a sample of hypo-osmotic blood is aspirated into the blood counter, it is diluted in an isotonic solution to enable laminar flow: the hypotonic red blood cells release water in order to achieve an osmotic equilibrium. There is a consequent reduction in the MCV and increase in the MCHC (these changes are not evident on a blood smear).
• Microcytic hypochromic: iron-deficiency anaemia, chronic inflammation, vitamin B6 deficiency. For iron-deficiency anaemia and anaemia due to chronic inflammation, see the paragraph above. Vitamin B6 deficiency causes inappropriate use of haemoglobin, with a consequent development of iron-deficiency anaemia. It is distinguished from real iron-deficiency anaemia in that the concentration of iron is within the norm.

PATHOGENIC MECHANISMS

According to the underlying pathogenic mechanism, there are three types of anaemia:

• Haemorrhagic anaemia
• Haemolytic anaemia
• ‘Impaired production’ anaemia

Haemorrhagic anaemia: this can be distinguished into acute and chronic and into internal and external. The initial phase of acute haemorrhagic anaemia is characterized by a sudden fall in blood pressure, which is followed by activation of the sympathetic nervous system and the renin-angiotensin-aldosterone system with the aim of restoring intravascular blood volume and blood pressure through peripheral vasoconstriction and recovery of water from the kidneys. The result is haemodilution and a reduction in the haematocrit in the first 24 hours. At the same time, peripheral hypoxia activates the renal production of erythropoietin to promote erythropoiesis and inhibits apoptosis in the bone marrow. About 4 days after the haemorrhage, the morphological changes typical of regeneration occur in red blood cells in the peripheral circulation: macrocytosis, anisocytosis, polychromasia and nucleated red blood cells (Fig. 1).

In the case of chronic haemorrhagic anaemia there is an iron-deficiency anaemia, because of the chronic loss of iron and consequent defect of haemoglobin synthesis. The reduction in haemoglobin concentration within red blood cells translates into greater pallor of the erythrocytes and a reduction in their volume (see microcytic hypochromic anaemia) (Fig. 2). The most common sites of chronic bleeding are the gastrointestinal tract (ulcers, neoplasms, haematophagous parasites) and urinary tract (stones, bladder cancers).

Haemolytic anaemia: this occurs when there is increased destruction of red blood cells with a consequent reduction in erythrocyte half-life. Haemolysis may be acute or chronic, may occur in the presence of normal or impaired bone marrow function, and may be intravascular or extravascular. Intravascular haemolysis is more severe because haemoglobin is released into the blood circulation; given its iron content, free haemoglobin has a strong oxidative potential, which can cause cell necrosis (e.g., acute renal tubular necrosis).

The haemolytic anaemias can be divided according to the cause of the haemolysis as follows:

• immune-mediated
• due to haematic parasites
• due to metabolic defects
• due to mechanical damage

Immune-mediated haemolytic anaemia is characterized by the production of antibodies that can bind to red blood cell membranes causing a reduction in haematocrit by direct lysis of the cells or through the removal of the complexed red cells by the reticulo-endothelial system.

Blood parasites such as Babesia spp. (Fig. 3), Mycoplasma haemofelis, Mycoplasma haemominutum (Fig. 4), and Cytauxzoon spp. (Fig. 5) can cause haemolytic anaemia by direct and/or immune-mediated lysis of red blood cells.

The metabolic defects can be divided into congenital and acquired. The congenital forms include deficiencies of pyruvate kinase and pyruvate phosphofructokinase [see congenital red blood cell disorders]. The acquired forms include oxidative damage to the red cell membrane or to haemoglobin. The former are more common in the dog and are associated with the production of eccentrocytes (Fig. 6), while the latter are seen more commonly in the cat and can cause the formation of Heinz bodies (Fig. 7). Feline haemoglobin has eight sulphydryl groups (whereas other species have two groups) and is more sensitive to oxidative damage. The most common causes of oxidative damage include ingestion of garlic and onions, poisoning by rodenticides, use of paracetamol, continuous infusion of propofol, diabetic ketoacidosis and lymphomas.

Mechanical damage is manifested as fragmented red blood cells and can be caused by disseminated intravascular coagulation, vasculitis, microcirculatory alterations, neoangiogenesis (e.g. haemangiosarcoma) and defects of heart valves.

The haematological alterations vary depending on the mechanism involved. In cases of an appropriate bone marrow response, the usual alterations found in regenerative anaemia will be seen, such as anisocytosis, polychromasia, macrocytosis, and nucleated red blood cells (Fig. 1). In the case of immune-mediated mechanisms there can also be spherocytes (Fig. 8) and/or crescent-shaped cells (Fig. 9). Schistocytes (Fig. 10) and keratocytes (Fig.  11) are more commonly associated with mechanical damage. Evidence of parasites within or near to red blood cells enables a direct diagnosis.

In order to manage haemolytic anaemias correctly, it is essential to distinguish immune-mediated forms from other forms; although morphological characteristics are of use, supplementary diagnostics techniques, such as the direct Coombs’ test or flow cytometry, must be used to determine the presence of antibodies to the red cells.

‘Impaired production’ anaemia: there are various different pathogenic mechanisms of impaired production anaemia. The most common is the anaemia of chronic inflammation, in which there is a reduced production of red blood cells due to diminished availability of iron, a shortened half-life of red cells and ineffective erythropoiesis because erythroid precursors are refractory to erythropoietin; these effects are caused by inflammatory cytokines. Impaired production anaemia classically presents as a mild-to-moderate normocytic normochromic anaemia, although rarely microcytes can occur. Nutritional deficiencies (e.g., vitamin B12 or folate deficiency) and metabolic disorders (e.g. hypoadrenocorticism [14]and hypothyroidism [15]) can also cause normocytic, normochromic, non-regenerative anaemias.

Anaemia due to chronic renal failure, besides having the same pathogenic mechanisms as anaemia of chronic inflammation, is also caused by inadequate production of erythropoietin by the failing kidneys. It is generally more severe than anaemia of chronic inflammation. Anaemia due to hypoplastic or aplastic bone marrow can, unlike the above conditions, be associated with peripheral cytopenia of a single cell lineage or multiple cell lineages. The primary cause of the bone marrow failure may be chemotherapeutic agents, abnormally high levels of oestrogen, or infectious agents. Primary bone marrow neoplastic disorders (leukaemias[16]) or metastatic bone marrow disease (e.g., leukaemic lymphomas, carcinomas) can cause severe medullary infiltration with disruption of the bone marrow architecture, to the point of complete replacement of the haematopoietic tissue by the neoplastic proliferation. This category of anaemia also includes the aplastic anaemias, pure red cell aplasia and the myelodysplasias; in these last conditions there is altered proliferation and differentiation of stem cells leading to hypercellular bone marrow with blast cells accounting for less than 20% of the total stem cells and single or multiple peripheral cytopenias.

BONE MARROW RESPONSE

In the case of substantial anaemia it is important to determine whether the bone marrow responds appropriately to this state. Depending on whether it does or does not, anaemias can be divided into regenerative and non-regenerative types, respectively. This distinction can be made on the basis of direct and indirect criteria. The former include laboratory indices, such as variability in red cell size (anisocytosis – measured by the red cell distribution width, RDW) (Tab. 2) and haemoglobin (anisochromia – measured by the haemoglobin distribution width, HDW) (Tab. 3) and morphological criteria that demonstrate peripheral regeneration (macrocytosis, polychromasia, anisocytosis, nucleated red blood cells) (Fig. 1).

Tab. 3 [18]) puts the number of reticulocytes into relation to the degree of anaemia, using the following formula:

CRP = RP x Hct of the patient/normal Hct of the species (dog, 45%; cat, 37%).

The reticuloctye index (RI), which can only be calculated in the dog, takes into consideration the half-life of the reticulocytes as a function of the degree of anaemia. It is calculated by dividing the corrected reticulocyte percentage by the reiculocyte maturation time (RMT) (Tab. 6, Tab. 7). In fact, if the reticulocyte maturation time in the bone marrow is about 24 hours, in the case of anaemia, the duration is reduced, thus increasing the number of reticulocytes in the peripheral blood. The use of the reticulocyte index avoids overestimating the regeneration.

Tab. 6. Reticulocyte maturation time (RMT) expressed in days as a function of the haematocrit (Hct).

Tab. 7. Classification of anaemia according to the reticulocyte index (RI).

In the clinic, the combined use of different criteria in a diagnostic algorithm (Fig. 12) can be helpful for determining the most probable pathogenic mechanism of the anaemia and, therefore, the underlying disorder.

Fig. 12. Diagnostic algorithm for a correct approach to anaemia (Fe_²⁺: iron; Tot. P: total proteins, NRBC: nucleated red blood cells, CHr: reticulocyte haemoglobin content, ACI: anaemia of chronic inflammation).

Suggested reading

1. Feldman B.F., Zinkl J.G., Jain N.C. – Schalm’s Veterinary haematology, 5° edition, ed Lippincott Wlliams & Wilkins, 2000.
2. Fry MM, Kirk CA.Reticulocyte indices in a canine model of nutritional iron deficiency. Vet Clin Pathol. Jun;35(2):172-81, 2006
3. Harvey J.W. Atlas of veterinary hematology – Blood and bone marrow of domestic animals –  Saunders 2001
4. Stockham S.L., Scott M.A. – Fundamentals of Veterinary Clinical Pathology – 1° edition, Blackwell 2002,
5. Willard M.D., Tveden H. – Small Animal Clinical Diagnosis by laboratory Methods – 4° edition, Saunders 2004.