1 Jun 2009
Lesa Longley
Job Title
Owners of exotic pets frequently expect veterinary clinicians to take blood samples and interpret results (Table 1) when dealing with ill animals, and understand normal values in healthy individuals. This article will cover some haematology basics in exotic species, focusing on birds and reptiles.
Before any analysis can be performed, a sample must be obtained. Depending on the species and patient size, this may be the most difficult part of the procedure. Birds may require general anaesthesia (with isoflurane, for example) for phlebotomy (Table 2). Smalldiameter needles should be utilised in birds to avoid damaging fragile avian veins. Gentle pressure should be applied after venepuncture, to reduce the risk of haemorrhage.
Long needles are required to access the ventral coccygeal vein in larger lizards (such as the green iguana, Iguana iguana). In reptiles, sampling from several sites may produce a sample contaminated with lymph (most notably in the dorsal coccygeal vein and subcarapacial sinus in Chelonia), with the dilution affecting the results obtained.
A maximum of 10 per cent of the total blood volume may be sampled safely from a healthy individual. In birds, blood volume is approximately 10 per cent of bodyweight and, thus, a maximum of one per cent of bodyweight may be sampled (such as 3.5ml from a healthy 350g parrot). The blood volume is five to eight per cent of bodyweight in reptiles. Therefore, a maximum of 0.5 per cent bodyweight may be sampled (such as 0.5ml from a 100g snake). Smaller samples should be taken from ill animals that may be unable to cope with such large acute blood losses. It can be useful to inject a similar volume of fluid intravenously after sampling to help maintain circulatory volume.
EDTA is used as the anticoagulant for blood from most species. Partial haemolysis may occur with some species (consult other references for species lists), including some birds and Chelonia. If in doubt, use a heparin sample of whole blood for haematological assessment. In all cases, prepare a fresh blood smear for morphological analysis and differential white cell count. In very small patients, a basic health check can be performed on a microhaematocrit tube and blood smear (estimated and differential white cell counts).
While several UK laboratories offer excellent haematology services for exotics, there are times when it is useful to perform in-house tests (such as when dealing with an emergency case at the weekend, or with an acutely ill bird). In these cases, it is advisable to run at least a screening panel (possibly before sending a sample to a commercial laboratory).
• Preparation of blood films
A small drop of fresh blood directly from the syringe (with the needle removed) is applied towards one end of a clean microscope slide. A “spreader” slide is used to produce a bullet or flame-shaped film. Alternatively, two slides may be crossed and pulled apart to produce a blood smear of even thickness. The smear is air-dried by being shaken rapidly. Smears are stained in the same manner as other species — with Romanowsky stains, for example.
• Whole cell counts
Automated counters used for mammals cannot be used for avian and reptilian haematology, due to the similar cell sizes and the presence of nuclei in all blood cell types. Manual blood counts can be performed using various techniques, such as a Neubauer-ruled haematocytometer chamber.
A rough estimate of the white cell count (x109/L) can be obtained from a smear. A monolayer of cells is assessed (in bullet-shaped smears, this is near the feathered edge), counting the number of white blood cells (WBCs) in 10 fields under medium (x400) magnification. The average is calculated per field, and the result is multiplied by two.
To assess cell morphology and obtain a differential white cell count, the smear is examined under oil immersion (x1,000). Red blood cells (RBCs) and WBCs are assessed on the basis of size, shape and colour. Nuclei, cytoplasmic granules and cellular inclusions should also be described. A packed cell volume (PCV) value can be obtained after centrifuging blood in a microhaematocrit tube. Several factors affect PCV values, including RBC size and number, and plasma volume (dehydration). PCV is normally relatively high in birds, with anaemia present when it is less than 35 per cent, and haemoconcentration when it is more than 55 per cent. The normal reptile PCV is 20 to 40 per cent (Frye, 1991).
•Factors affecting haematology
Stress and disease processes will affect haematological values. Species, gender, age and environment (particularly in reptiles), diet and physiological variations are also seen.
In reptiles that hibernate (including many tortoise species), haematological variations during hibernation may include a fall in the total blood count, lower numbers of heterophils and lymphocytes, and higher numbers of eosinophils. There is usually little seasonal variation in basophil and monocyte numbers.
Many male reptiles have higher erythrocytic values compared to females – for example, PCV and haemoglobin concentrations in African hingeback turtles (Kinixys erosa). Conversely, female green iguanas have higher haemoglobin, PCV and mean corpuscular haemoglobin concentration counts than males.
Peripheral blood cells comprise erythrocytes (RBCs), leukocytes (WBCs) and haemostatic cells (these are called thrombocytes in birds and reptiles, rather than platelets as in mammals). Blood smears are important, as cell morphology (including size) varies greatly between taxonomic groups, species and disease.
• Red blood cells
These cells are elliptical and, unlike their mammalian counterparts, are nucleated. The nucleus is centrally situated, oval or round, and contains densely clumped chromatin. The nucleus may have an irregular outline in reptiles. Avian RBCs are larger than in mammals, and reptilian cells larger again – size also varies between species.
RBC lifespan varies between species. It is much shorter in birds (28 to 35 days in chickens – Sturkie, 1976) compared to mammals, and longer in reptiles (600 to 800 days – Sypek and Borysenko, 1988; Frye, 1991).
Basophilic inclusions are commonly seen within the cytoplasm of reptilian RBCs. These may be infectious agents (haemoparasites or viral inclusion bodies) or degenerate organelles.
Immature RBCs are often seen in the peripheral circulation of birds and reptiles. These cells have marked polychromasia and round nuclei. Reticulocytes have a ring of aggregated reticulum at the periphery of the nucleus. Large numbers may be associated with regenerative anaemia.
Regenerative responses may also be seen in reptiles waking from hibernation, and those with inflammatory disease or malnutrition. Changes include moderate to marked anisocytosis and poikilocytosis. Increased polychromasia and immature RBCs may be seen in young reptiles or those undergoing ecdysis.
• White blood cells
The aim of blood smear analysis in veterinary practice is often to identify changes in WBCs that reflect disease processes. However, it can be difficult for the first-opinion veterinary practitioner to become familiar with WBCs from birds and reptiles, as there are great inter-species variations in the appearance of normal cells. Relative and absolute numbers of WBCs also vary between species (the clinician is referred to other texts for normal values).
– Heterophils. In most species, these are the predominant WBCs. In normal mature heterophils, the cytoplasm is clear and contains eosinophilic granules. The appearance of these granules varies between species, and also with disease processes. In most reptiles, the heterophil nucleus is spherical or oval, but is lobed in birds and some lizards – all contain coarse, clumped chromatin.
Immature heterophils have increased cytoplasmic basophilia, non-segmented nuclei and granules occupying less than half the cytoplasmic volume, and they display pleomorphism. The presence of such immature heterophils in the peripheral blood (comparable to the “left shift” in mammals) suggests inflammatory disease. An overwhelming inflammatory response (often with infection) may result in these immature cells and heteropaenia.
Heterophils are functionally equivalent to mammalian neutrophils. They participate in inflammatory lesions and have phagocytic activity. Heterophils respond to inflammation by undergoing changes referred to as “toxic change” – the presence of these changes is typically associated with a worsening prognosis. Changes seen may include altered granulation (such as degranulation and deeply basophilic and/or coalescing granules), cytoplasmic basophilia, vacuolation in the cytoplasm and/or the presence of a band nucleus.
– Eosinophils. Granules within the cytoplasm of eosinophils are typically round and strongly eosinophilic. However, the granules may be oval or elongate, and in some species (such as iguanas and African grey parrots) are basophilic. The cytoplasm shows some basophilia. Nuclei are usually lobed in birds, and elongated to lobed in reptiles. There is great species variation in eosinophil numbers.
– Basophils. These small, spherical cells have a non-lobed nucleus. The granules are large, round and basophilic (sometimes obscuring the nucleus). Normal cell numbers and size vary greatly between species.
– Lymphocytes. These cells are round, as is the (occasionally indented) nucleus, which is surrounded by a rim of weakly basophilic cytoplasm. The cytoplasm does not normally contain any vacuoles or granules. On blood smears, lymphocytes are often seen moulded around or adhered to neighbouring RBCs. As with other WBCs, morphological changes are seen when lymphocytes become activated in disease (such as antigenic stimulation in infectious disease or lymphocytic leukaemia). These “reactive” changes may include clumping of nuclear chromatin and a deeply basophilic cytoplasm.
– Monocytes. These are large, round or amoeboid in shape, with a speckled or basophilic cytoplasm that contains vacuoles or fine eosinophilic granules. The nucleus is centrally placed and varies in shape. Compared to lymphocytes, monocyte nuclei have less chromatin clumping and there is more cytoplasm.
In a similar fashion to mammalian monocytes, in birds and reptiles these cells are phagocytic and process antigens.
Reptile monocytes with azurophilic staining (commonly called “azurophils”) are functionally the same as those without this staining.
Reactive monocytes have phagocytic activity and migrate into tissues to become macrophages. They have an important role in antigen processing.
• Thrombocytes
These cells are functionally similar to mammalian platelets. They are ellipsoid in shape, with a centrally located dark nucleus and nonstaining or weakly basophilic cytoplasm. Thrombocytes usually contain less cytoplasm than RBCs, and may clump on smears. Activated thrombocytes may show changes, such as irregular cytoplasmic margins and cytoplasmic pseudopodia. Nuclear and cytoplasmic changes may occur, and vacuoles and degranulation may be present.
Protozoan parasites are commonly seen on fresh blood smears from birds and reptiles. Many are asymptomatic infections. In cases where clinical signs are seen, they usually relate to anaemia, and include lethargy and anorexia.
• Birds
Haemoproteus are common in many wild birds. The mature gametocytes wrap around the RBC nucleus with a characteristic “halter shape”.
Plasmodium infection may result in clinical malaria in some species, including canaries, raptors and pigeons. Several life stages are seen in RBCs, WBCs and thrombocytes. Trophozoite stages result in a “signet ring” appearance in the host cell.
Gametocytes from Leukocytozoon parasites are often seen pushing the host cells’ nuclei to the edge of the cell in many wild birds. Some species, such as young waterfowl, are highly susceptible to disease. Microfilaria may be seen extracellularly in peripheral blood.
Atoxoplasma is uncommon but highly pathogenic. This coccidian parasite affects passerine birds. Sporozoites are seen mostly within lymphocytes as intracytoplasmic inclusions indenting the host cell nucleus.
• Reptiles
Haemogregarine gametocytes are seen as intracytoplasmic sausage-shaped structures in RBCs. Trypanosomes are large extracellular protozoa. Microfilaria are a common incidental finding, particularly in wild-caught chameleons.
Gametocytes of Plasmodium have refractile pigment granules. Schizogony may be seen, and trophozoites are signet ring structures in the cytoplasm of RBCs. Infection may result in severe haemolytic anaemia.
Right: a blood smear from a Fischer’s chameleon, showing a microfilarial parasite among red blood cells.
Most haematology profiles can be run on a blood sample stored in EDTA. In some species, EDTA causes haemolysis and heparin should be used as the anticoagulant.
TABLE 2. Phlebotomy sites in birds and reptiles
Lesa Longley
Job Title