Features of regenerative and nonregenerative anaemia

Regenerative Anaemia

Haemorrhagic Anaemia

This may be acute, transient severe haemorrhage or chronic, persistent haemorrhage. There may be evidence of blood loss, such as external haemorrhage from wounds, lesions or body orifices (nares, anus); may be associated signs of trauma (RTA) or possibly defective haemostasis. Internal haemorrhage may be intracavity or into the GI or urinary tract and evident in excreted material (urine, faeces, vomit). It can appear as fresh or altered blood. Small amounts of blood may not be visible to the naked eye but may be visualized microscopically or detected chemically.

Immediately following acute haemorrhage the PCV and other RBC indices remain relatively stable, whole blood has been lost and haemodilution, due to fluid shifts from the extravascular to the intravascular compartment, takes time to develop. Splenic contraction may also helpmaintain the PCV. After 2-3 hours, haemodilution becomes evident, leading to reductions in red cell indices and plasma protein concentrations. The full magnitude of blood loss may not be evident until 24hrs after the onset of haemorrhage. Red cell indices may be less affected following haemorrhage into body cavities because two thirds of the erythrocytes may return to the circulation; plasma proteins and iron from the remaining RBCs are retained and can be utilised for erythropoiesis. Anaemia is initially normocytic normochromic but after 2-4 days there is a significant erythroid response, which is most marked by day 7, and features polychromasia, reticulocytes, Howell-Jolly bodies, nucleated RBCs, increased MCV. If reticulocytosis and thrombocytosis, with reduced plasma proteins persist after 2-3 weeks, continuing haemorrhage should be suspected. PCV should have returned to low normal by 2 weeks following a single episode of haemorrhage.

By the time chronic haemorrhage causes clinical signs of anaemia, regeneration should have begun, The regenerative response is more marked with acute haemorrhage. With persistent chronic haemorrhage, particularly with blood loss from the body, the RBCs may be microcytic and hypochromic due to iron deficiency. There is initially an increased reticulocyte count which then falls as iron deficiency develops.

Even after haemorrhage the regenerative response may be poor or absent if the bone marrow is damaged or activity is suppressed by, for example, neoplasia or infection.

Leucocyte changes. Acute blood loss is usually accompanied by a leucocytosis with neutrophilia and a left shift (in- creased band cells). This is not associated with infection but may reflect inflammation secondary to hypoxic damage. Immediately post haemorrhage there may be a mild to moderate transient thrombocytopaenia due to increased platelet consumption; this is followed by thrombocytosis with large immature platelets, reflecting the bone marrow response to increased demand.

Chronic blood loss is often associated with thrombocytosis (platelet count 500-1000x109/l).

Biochemical changes. Plasma protein and albumin levels will be low after severe acute haemorrhage and may be low with chronic haemorrhage.

Increased blood urea nitrogen with normal serum creatinine can be seen with gastrointestinal haemorrhage.

Haemolytic Anaemia

Usually presents as a markedly regenerative anaemia without hypoproteinaemia or other evidence of blood loss. Haemolysis may be intravascular or extravascular.

• Extravascular haemolysis relates to the pathological phagocytosis of erythrocytes by macrophages in spleen, liver and bone marrow and is the most common form of

haemolytic anaemia.

• Intravascular haemolysis relates to destruction of erythrocytes within the circulation and often results in more acute and severe haemolysis then extravascular haemolysis. Haemoglobinuria is often a feature. The most common causes are complement fixing immune-mediated haemolytic anaemia and Heinz body anaemia.

• When both forms occur together, the haemolysis is classified by the predominant type.

• Haemolytic anaemias often result in a degree of hyperbilirubinaemia which, if sufficiently severe, will be evident as icterus. RBC destruction leads to an increased level of unconjugated bilirubin which exceeds the rate of hepatic excretion. Acute severe anaemia may also cause hypoxic or toxic hepatic injury, resulting in decreased bilirubin metabolism and cholestasis. Although hyperbilirubinaemia results primarily from increased unconjugated bilirubin, conjugated bilirubin also increases and leads to bilirubinuria. Evaluation of unconjugated versus conjugated bilirubin is often not helpful when trying to differentiate between pre-hepatic and hepatic jaundice.

Immune mediated haemolytic anaemia (IMHA).

Primary IMHA results from the formation of antibodies which are specifically directed against the hosts’ erythrocytes. Secondary IMHA occurs when antibodies directed against non-erythrocyte antigens also attach to RBCs. Antibody coated erythrocytes have a decreased lifespan, they are prematurely removed from the circulation by splenic and hepatic macrophages.

Antibodies which fix complement can cause intravascular haemolysis.

Primary IMHA is idiopathic. Secondary IMHA, the most common form seen in cats, may be secondary to certain drugs (potentiated sulphonamides, anti-thyroid drugs), lymphoid neoplasia, infection (bacterial, FeLV, haemotrophic Mycoplasma spp).

Haematological findings include a moderate to severe anaemia (PCV<16%), marked reticulocytosis, moderate to marked polychromasia, normal or slightly increased plasma proteins, marked leucocytosis, often with a left shift, significant spherocytosis, autoagglutination and thrombocytopaenia. A positive Coombs test supports a diagnosis but the test is subjects to false negative and false positive results. Autoagglutination which is not dispersed by saline is virtually pathognomonic for IMHA, usually caused by surface bound IgM antibodies or compliment fixation.

Coexisting thrombocytopaenia may also be immune-mediated (Evan’s syndrome) or result from disseminated intravascular coagulation.

Some cases of IMHA are nonregenerative due to the destruction of red cell precursors in the bone marrow.

The diagnosis of IMHA in cats is more problematic than in dogs. Spherocytosis and leucocytosis generally are not evident. Haemotrophic feline Mycoplasma spp., particularly Mycoplasma haemofelis, induce IMHA. Blood film examination is poorly sensitive; PCR assay is indicated in all cases of suspected feline IMHA. FIA related anaemia is strongly regenerative unless there is coexisting FeLV infection.

Neonatal Isoerythrolysis (NI)

In cats there are 3 blood groups type A, type B and type AB. Type A is dominant. Most non pedigree cats are type A, however, in pedigree cats there is a higher incidence of type B, particularly in the British Shorthair. All type B adult cats naturally have high titres of anti-A antibodies (alloantibodies). If a type A male mates with type B female, the type A kittens receive maternal anti-A antibodies in the colostrum. Kittens which appear healthy at birth fade due to haemolysis with icterus, haemoglobinuria and severe anaemia. Some may die in shock. Some cases are less severe with mild anaemia, these kittens may recover.

NI is also seen in newborn foals caused by blood group incompatibility between the foal and dam, mediated, as in the cat, by maternal antibodies directed against foal erythrocytes, absorbed from the colostrum. The disease most often affects foals of multiparous dams since sensitisation usually occurs late in gestation, or during parturition of an incompatible foal. A primaparous mare can produce a foal with NI if she has received a prior, sensitising blood transfusion or developed placental abnormalities early in gestation, which allowed leakage of foetal RBCs into her circulation.

Heinz body Anaemia

Oxidative toxins damage the sulphydryl groups of the globin chains of haemoglobin resulting in the formation of Heinz bodies (HBs). These are seen as unstained projections on the cell membrane with Romanowsky stain, but stain with new methylene blue.

Oxidative injury may also result in eccentrocyte formation +/- methaemoglobinaemia (see below). Many substances can cause oxidative damage. Affected cells may be phagocytosed in the spleen or, if severely damaged, may undergo haemolysis within the circulation - there may be both extravascular and intravascular haemolysis. This type of anaemia is regenerative. In dogs HBs are not normally present. Canine Heinz bodies are often small irregular and multiple. The most common cause of Heinz body anaemia is ingestion of onion or garlic.

In cats HBs are naturally present since feline haemoglobin is more susceptible to oxidative damage and the spleen is less efficient at removing affected cells. Healthy cats can have up to 5% of HB’s within their erythrocytes, therefore, to determine their significance the number of Heinz bodies, PCV and reticulocyte count should all be considered. Oxidative toxins reported to cause HB formation in cats include acetaminophen, propylene glycol and salmon based diets.

They also occur in disease conditions including diabetes mellitus, hyperthyroidism, renal failure and lymphoma, possibly due to altered metabolism generating oxidative metabolic intermediates.

Methaemoglobinaemia

Methaemoglobin is a brownish compound formed by the oxidation of iron in haemoglobin from the ferrous to the ferric state. When present in quantity it leads to a muddy cyanotic discolouration of mucous membranes. Usually it accounts for less than 1.1% of haemoglobin. It is increased due to oxidative damage caused by toxins which may also cause Heinz body and eccentrocyte formation. Methaemoglobinaemia results from either increased production due to oxidative injury or decreased reduction of methaemoglobin to Hb. Congenital methaemoglobinaemia has been reported due to deficiency of the RBC enzyme NADH-methaemoglobin reductase.

Pyruvate Kinase deficiency

This is an autosomal recessive genetic disease in dogs causing severe persistant extravascular haemolysis. There is usually moderate to severe anaemia (PCV18-25%) with marked reticulocytosis, possibly due to rapid RBC turnover and splenomegaly. PK-deficient RBCs have a shortened life span with inefficient energy production. Affected dogs are young and usually die by the age of 3 years with, in many cases, myelofibrosis and osteosclerosis. PK deficiency is transmitted as an autosomal recessive trait in many breeds of dogs with the highest prevalence in Basenji’s and Beagle’s. PK deficiency has been reported in the DSH, Abyssinian and Somali breeds of cat.

Phosphofructokinase deficiency

Has been reported in English Springer spaniels and Cocker spaniels. Commonly there is recurring haemoglobinuria, splenomegaly and/or icterus. Intravascular haemolysis is precipitated by respiratory alkalosis associated with stress induced hyperventilation. A haemolytic crisis may develop following exercise.

Haemotrophic mycoplasmas

(Haemoplasmas) are small bacteria that attach to the external erythrocyte membrane. There are multiple species which vary in pathogenicity. In the cat Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ are seen most frequently; a further species, ‘Candidatus Mycoplasma haematoparvum-like’ has been reported in the literature. Mycoplasma haemofelis is the most pathogenic, producing anaemia and clinical signs of disease. ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ may cause some changes in red cell parameters but anaemia is only seen in cats with concurrent disease (Retroviral infection), immunocompromised cats or those receiving corticosteroid therapy.

In dogs Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’ have been identified and may cause haemolytic anaemia in splenectomised or immunocompromised dogs. The method of choice for diagnosis is PCR using appropriate controls on EDTA blood samples.

Previously diagnosis was by examination of air-dried blood smears (not EDTA smears) to search for the organisms. Even in the acute phase, parasitaemia is not always present so it may be necessary to take blood and make smears on 5-7 consecutive days. The organisms are very small and must be distinguished from stain precipitate in Romanowsky stained smears, Howell-Jolly bodies and background debris. They may be seen as very small organisms on the periphery of the erythrocyte or form chains and rings. May-Grunwald or acridine orange stains may increase the sensitivity of blood film examination.

Leptospirosis

Haemolytic anaemia is one of the features of Leptospirosis.

Babesia canis

Until recently this was not a problem in the UK. Since the introduction of the Pet Passport scheme cases of this tick transmitted disease are being seen more frequently. Infection is associated with both intravascular haemolysis and extravascular haemolysis (due to infected cells being sequestered and lysed in the spleen). Diagnosis is by PCR or by demonstrating the intracellular teardrop shaped organisms, particularly in smears of capillary blood (ear margin).

Microangiopathic haemolytic anaemia

This is usually a mild, often subclinical anaemia unless there is concurrent haemorrhage from, for example, a tumour. RBCs are damaged as they pass through abnormal vessels or regions of turbulent blood flow, giving rise to schistocytes. These fragmented red cells are phagocytosed by the mononuclear phagocytic system, causing anaemia. Numerous schistocytes in a smear are always significant.

Non-Regenerative Anaemia

An inadequate degree of reticulocytosis is the essential diagnostic feature. Some reticulocytes may be present but fewer than required for a given degree of anaemia, after sufficient time has elapsed for an erythroid response to have developed. These anaemias are mainly normochromic normocytic.

Primary Anaemia

Bone marrow diseases

A method for bone marrow aspiration can be found in the cytology section. Where bone marrow hypoplasia is suspected a core biopsy in formalin should also betaken.

Bicytopaenia or pancytopaenia

Reflects poor marrow production of two or three cell lines. This suggests marrow disease. Because of the long life span of RBCs, anaemia is usually detected

later than neutropaenia and thrombocytopaenia.

Aplastic pancytopaenia and myelofibrosis

These conditions require bone marrow histopathology to confirm the diagnosis. A core biopsy is essential to differentiate between a poor bone marrow aspirate sample and poor cellularity. In aplastic anaemia the haematopoietic bone marrow is replaced by fat, in myelofibrosis by fibrous tissue. Causes include drug toxicity, viruses and immune-mediated processes. Pure red cell aplasia causes severe nonregenerative anaemia with in the marrow, normal myeloid and megakaryocytic cells but no erythroid precursors. It is thought to be immune-mediated and may respond to steroid therapy.

Dyserythropoiesis

There are a variety of causes including B12 or folate deficiency, iron deficiency, drug toxicities. These are nonregenerative anaemias with normal to increased erythroid precursor cells in the bone marrow. There are blast cells, binucleate RBC precursors, macrocytes megaloblasts and asynchronous maturation of the cytoplasm and nucleus.

Proliferative disorders

Can also cause nonregenerative anaemia as neoplastic cells over-run the bone marrow (myelophthisic disease).

Drug-induced haematological dyscrasias

Many drugs have been found to cause blood dyscrasias in both dogs and cats. Drugs causing irreversible aplastic anaemia include phenylbutazone, meclofenamic acid and oestrogens; oestrogen toxicity may also be reversible. Griseofulvin has been associated with reversible aplastic anaemia. The prognosis for these conditions is generally poor.

Oestrogen toxicity whether due to drug administration or a Sertoli cell tumour can cause thrombocytosis 5-7 days after administration/exposure followed by thrombocytopaenia and neutrophilia for 2-3 weeks followed by neutropaenia. Pancytopaenia develops 3-4 weeks after administration/exposure.

Infections

FeLV. Responsible for most cases of nonregenerative anaemia in cats. There may be macrocytosis in the absence of reticulocytosis. FIV. May be associated with nonregenerative anaemia possibly with concurrent neutropaenia.

Secondary anaemia

Anaemia of inflammation

This is the most common cause of nonregenerative anaemia and may occur with inflammatory or neoplastic disease. It is usually mild to moderate. Macrophages involved in an inflammatory response release cytokines including interleukin-1, interleukin-6 and tumour necrosis factor. These not only initiate fever but cause iron sequestration within macrophages thereby reducing serum iron and restricting iron availability for erythropoiesis.

Anaemia of chronic renal disease

Ineffective erythropoiesis, shortened RBC life span and blood loss may contribute to this anaemia which may become severe. The mechanism appears to be more complex than a relative deficiency of erythropoietin (EPO), but this type of anaemia may respond to EPO therapy.

Anaemia of chronic hepatic disease

Coagulation defects due to reduced synthesis of clotting factors in severe hepatic disease may result in haemorrhage. Reduced hepatic function may also lead to deficient levels of nutrients required for haematopoiesis. In dogs with portosystemic shunts, microcytosis is common due to a functional iron deficiency. These dogs have increased hepatic iron but low serum iron, normal total iron binding capacity and a low percentage of transferrin saturation.

Hypothyroidism and hyperadrenocorticism

These endocrinopathies may result in mild, clinically insignificant anaemia.

Iron deficiency anaemia

Typically hypochromic microcytic, initially regenerative but may become nonregenerative. This has already been discussed.