avian haematology and biochemistry 1. haematology

510 In Practice November/December 2014 | Volume 36 | 510-518

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The class Aves contains more than 9000 species of birds classified in 199 families. While few species-specific haematological characteristics have been described, there are clear intra-class variations and differences from vertebrates. This article, the first of a two-part series on avian haematology and biochemistry, concentrates on haematology. It includes guidance on how to collect blood samples, how to assess the sample and information on the haematological disorders most commonly found in birds.

Birds typically hide clinical signs of disease until pathogenesis is advanced. Haematology and biochemistry are diagnostically informative and are minimally invasive techniques. Correct interpretation is affected by physiological factors (sex, age, reproductive status, season, and postprandial and diurnal alterations), pathological factors and handling factors.

Avian blood cells include erythrocytes, leukocytes, and haemostatic cells. With minimal exceptions, red blood cells are nucleated and oval in shape, due to a cytoskeleton imparting reversibility following traumatic deformation. Nucleated thrombocytes and heterophils with acidophilic granules are found rather than the platelets and neutrophils found in mammals.

The presence of nuclei in most avian blood cells prevents the use of electronic counters, and favours flow cytometry counters (laser counters), which are still not able to replicate manual cell counting techniques and differential count determined by light microscopy. Also, the actual lack of a standard avian antibody panel does not allow immunophenotyping across all species (Beaufrère and others 2013).

HaematopoiesisBone marrow haematological characteristics are reduced to a few studies in chickens, Japanese quail (Coturnix japonica), ducks and black-headed gulls (Larus ridibundus) (Clark and others 2009).

Primitive haematopoiesis occurs in the blood islands of the yolk sac of avian embryos (starting the second day of incubation in chickens), which remain haematopoietic until bone marrow, spleen, bursa of Fabricius, and thymus assume this function. Haemoglobin could be detected 24 hours after fertilisation (two to four somites stage) in chickens (Claver and Quaglia 2009).

Definitive haematopoiesis begins in the dorsal mesenchyma (aortic and para-aortic foci) from days five to eight of incubation. New stem cells from these areas

Mikel Sabater graduated from UCH-CEU Valencia, spain, in 2005, gained his CertZooMed in 2013, and completed a three-year European residency in avian medicine at Great Western Exotics in swindon in 2014. He currently works at Loro Parque, Tenerife.

Avian haematology and biochemistry1. Haematology

Mikel Sabater, Neil Forbes

doi:10.1136/inp.g5870

colonise the spleen, which is haematopoietic from days nine to 18, and finally the bone marrow from day 12 of incubation into adult life (Claver and Quaglia 2009).

Erythropoiesis occurs within the lumen of the vascular sinusoids in the bone marrow. Avian erythropoietin, a glycoprotein synthesised by the kidneys, showing no cross-reactivity with mammals, is necessary for the multiplication and differentiation of precursor stem cells into eryth-rocytes. Five stages of erythrocyte maturation could be observed: rubriblasts (proerythroblasts), basophilic rubricytes (early polychromatic erythroblasts), late polychromatic rubricytes (orthochromic erythroblasts), polychromatic erythrocytes and mature erythrocytes (Campbell and Ellis 2007).

Granulopoiesis occurs in a similar way as in mammals, but the location of precursor cell origin varies. Avian hetero-phils derive from stem cells present in the extravascular spaces of the bone marrow, whereas mammalian neutrophils develop from precursor cells within the vascular spaces. Extramedullary haematopoiesis is also observed in the bursa of Fabricius (unique source of B lymphocytes in adults), thymus (avian T lymphocytes), and spleen. The developmental stages include myeloblasts, pro-granulocytes, myelocytes, meta-myelocytes, band cells and mature granulocytes (Fudge 2000).

Thrombopoiesis appears to develop from mononuclear cells in the bone marrow and not from the mammalian cytoplasmic fragments of large, multinucleated megakaryocytes. developmental stages include early, mid, or late immature and mature thrombocytes (Fudge 2000).

Blood collection Safety aspectsAvian handlers must be mindful of ‘danger points or behaviours’ of different individuals (kicks from Struthioniformes, bites from Psittaciformes, eye-targeted beak attacks from cranes, herons, darters, and bustards, talon injuries from raptors, as well as any contagious or zoonotic risks (circovirus, herpesvirus, bornavirus, avian

Neil Forbes leads the team at Great Western Exotics. He graduated from the royal Veterinary College in 1983, gained his rCVs specialist status in 1992, his FrCVs by examination in Exotic Bird Medicine in 1996, and his European diploma in 1997. He was President of the European College of Zoological Medicine from 2006 to 2009 and President of the European Board of Veterinary specialisation from 2010 to 2011.

Sabater P1 - final.indd 510 06/11/2014 12:35

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511In Practice November/December 2014 | Volume 36 | 510-518

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Fig 1: Preparing a blood smear

flu, chlamydiosis, salmonellosis, etc) in order to avoid injuries or minimise risks of infection.

Restraining versus anaesthesiasedation or anaesthesia may be used to minimise the stress effects of handling and the risk of patient movement during blood sampling.

The percentage of heterophils, and the levels of creatin-kinase and glucose, more than doubled whereas the percentage of lymphocytes and uric acid decreased after handling pigeons for three hours (Lumeij 2008).

A study of the haematological effects of isofluorane anaesthesia on American kestrels (Falco sparverius) demonstrated a mild decrease in packed cell volume (PCV), basophil concentration, and plasma proteins in anaesthetised kestrels. However, haematocrits of red-tailed hawks (Buteo jamaicensis) that were administered ketamine hydrochloride intramuscularly, assessed 10, 20 and 40 minutes post-administration, showed no significant differences from the control group (Clark and others 2009).

Starvation and postprandial differencesHyperglycaemia has been reported in pigeons when starved for three days, as well as postprandial hyperuricaemia and increased bile acids. in penguins and raptors, 24 hours of fasting is recommended to avoid postprandial elevation of urea and uric acid (Lumeij 2008).

Volume of bloodThere is intraspecies variation in blood volume (67±3 ml/kg for common pheasants, 62±5 ml/kg for red-tailed hawks, 106±3 ml/kg for galahs, and 111±3 ml/kg for red-head and canvasback ducks).

The avian spleen is not a reservoir of erythrocytes. However, studies have shown rapid recovery from experimentally induced blood loss probably due to a better capacity to mobilise fluids from extravascular compartments than mammals (Lumeij 2008).

The safe collectable blood volume varies with respect to body weight (blood volume) and the health status of the patient. However, in general terms, one can safely remove a volume equivalent to 1 per cent of the bird’s body weight, ie, 10 ml/kg body weight in a healthy bird (Clark and others 2009).

Blood sampling sitesin practice, the right jugular vein, basilic vein and medial metatarsal vein are used as blood sampling sites. Foot web, chorio-allantoic vessel, skin puncture, comb, venous occipital sinus, toenail clipping (for dNA analysis only) and cardiac puncture (reserved for approved research settings or for pre-postmortem examination sampling) may be used in research, but, in general, are not recommended as routine sites by the authors.

Sampling methodremoval of feathers should be limited, but it may improve access to the vein. Also, cutaneous application of alcohol aids visibility, although excessive volumes may provoke sample haemolysis, and increase patient skin heat loss.

in most species, occlusion of the veins is not necessary and may contribute to haematoma formation.

The sterile needle attached to the syringe with the bevel dorsal is aligned parallel with the vein at 15° to horizontal,

and advanced until the vein is pierced. Once the sample is collected (with minimal negative pressure), the needle is removed and haematoma formation controlled by topical pressure application.

The needle is removed from the syringe and the blood is expelled into an appropriate anticoagulant container together with smear preparation from remaining drops.

Anticoagulantsn EdTA: 1 to 2 mg/ml of blood is ideal for manual

haematology cell counts in mammals and most species of birds, and for fibrinogen determination. However, it can provoke osmotic changes in the cells, especially in erythrocytes, provoking inaccurate cell volume size when determined by automatic cell counters. Ostriches, corvids, crowned cranes, jays, brush turkeys, and hornbills’ erythrocytes tend to lyse in EdTA (Lumeij 2008) so heparin is preferred. in the UK, fibrinogen and lead assays are normally performed using EdTA.

n Lithium heparin: 25 iu/ml of blood may provoke staining artefacts. Plasma is ideal for blood lead assays (not centrifugated), and gene-based diagnostics (viral, Chlamydia species, and gender-dNA analysis), as well as being useful for haematology, biochemistry, zinc, PCr, and serology.

n Citrate: ideal for cell counts by laser flow cytometry and fibrinogen determination.

immediate centrifugation after obtaining the sample will separate plasma from cells avoiding artefacts related with the intracellular movement of potassium from plasma (Lumeij 2008). The absence of anticoagulants will provide serum, which is useful for biochemical determinations.

Blood smearsBlood smears should be prepared directly from the syringe immediately after blood collection without anticoagulants. Coverslide-to-coverslide (preferred), coverslide-to-slide or slide-to-slide (Fig 1) techniques could be used.

StainsHaematology stains for avian blood smears include the romanowsky stains (Wright’s, Giemsa, diff-Quik, Hemacolor, etc).

Reference valuesresults should be compared with values obtained from similar clinically healthy specimens. reference values for many avian species can be found in literature. However, several factors (eg, the analytical method) can considerably influence the reference values, making them often not comparable, and resulting in a low diagnostic value.






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ErythrocytesAvian erythrocytes are larger than mammalian ones. They are oval to elliptical, with orange cytoplasm and an oval purple nucleus that is situated centrally (Fig 2). Their half-life is short in comparison with mammalian cells (28 to 35 days in chickens, 35 to 45 in pigeons, 42 in ducks). The major function of erythrocytes is oxygen transport. However, despite not being immune cells, they are able to participate in some immune responses that contribute to host defence (Claver and Quaglia 2009).

Although mature erythrocytes are the predominant blood cells in healthy birds, different stages of erythroid development might be encountered in blood smears (Fig 3):

Polychromatic erythrocytes are the penultimate stage of the development of erythrocytes. Their cytoplasms are bluish in colour due to increased amounts of ribosomal rNA. Their nuclei present less dense chromatin than in mature erythrocytes. They typically comprise 1 to 5 per cent of the erythrocytes in healthy birds (Clark and others 2009). increased numbers may be observed in neonates or patients with regenerative anaemias when haematopoietic rates are increased.

Rubricytes are the most commonly encountered immature stage of erythroid forms. They are typically smaller and more rounded than mature erythrocytes. Their nucleus is typically round and composed of coarsely clumped chromatin, which is less dense than that of mature erythrocytes. Their nuclear to cytoplasmic ratio is greater than in mature erythrocytes, with a small to moderate rim of markedly basophilic cytoplasm (Clark and others 2009).

Reticulocytes are slightly larger than mature erythrocytes. When stained with new methylene blue, they exhibit granular basophilic aggregations of rNA forming an incomplete ring around the nucleus. They typically comprise 2 to 10 per cent of the normal circulating red blood cells (rBCs) and correlate with the number of polychromatophilic erythrocytes (Clark and others 2009).

Other erythroid forms observed in healthy birds are (Clark and others 2009):n Erythroid cells undergoing division in the peripheral

blood, observed as mitotic figures, may occasionally be encountered in healthy birds;

n Erythroplastids or anucleated erythrocytes typically

represent less than 1 per cent of all erythrocytes. They are of unknown significance and could just be an artefact;

n Poikilocytes are erythrocytes that exhibit a non-ovoid shape (round, fusiform, drop-shaped, etc) and typically comprise a very small proportion of the total erythrocyte population in healthy birds. The presence of haemoparasites such as Haemoproteus species, and Plasmodium species may alter the shape of the cell, enlarge its size, or displace its nucleus.

Assessment of erythrocytesErythrocytic indicesThe packed cell volume (PCV) is gained from the measurement of a blood-filled microhaematocrit tube after centrifugation at 11,000 G for five minutes.

The mean cell volume (MCV) indicates the average volume of individual erythrocytes. MCV (fl) = (PCV/rBC) x 10. increased values (macrocytosis) may arise through artefact (erythrocyte clumping or agglutination, storage-related changes, hyperosmolality [hypernatraemia]), or associated with regenerative anaemia. decreased values (microcytosis) may be artefactual (excess of EdTA, hyponatraemia), physiological (young animals), or related to liver disease (Fudge 2000).

The haematocrit is calculated from measured mean corpuscular volume and rBC count when derived by automatic cell counters and is more accurate than the PCV (Fudge 2000).

The RBC count can be determined manually using the erythrocyte Unopette system or the Natt and Herrick’s method and a Neubauer-ruled modified haemacytometer. The blood is diluted, stained then placed in a haemacyto-meter to settle for 5 to 10 minutes. The red blood cells are then counted according to the standard enumeration procedure. The total count is multiplied by a factor of 10,000 to obtain the number of erythrocytes 1013/ml.

Haemoglobin (Hgb) can be estimated using automatic or manual methods. With automatic methods, after

Fig 2: Mature erythrocytes in a chicken

Fig 3: A mature erythrocytes (arrow), an anucleated erythrocyte (or erythroplastid) (arrowhead), and polychromatophilic erythrocytes (stars) in an African grey parrot






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lysing of the erythrocyte, the free nuclei tend to interfere with the photometric reading. With manual methods, colorimetric results tend to be low, unless estimations of cyanmethaemoglobin or oxyhaemoglobin are performed.

Haemoglobin can be estimated directly as oxyhaemoglobin using a commercial haemoglobinometer. small and portable haemoglobinometers that use a spectrophotometric double-wavelength azide-methaemoglobin method to correct for sample turbidity resulting from lipid particles, cell stromata, and large proteins that tend to interfere with the cyanomethemoglobin method are commercially available and provide reliable results in avian species. A proportional relationship between Hgb concentration and the PCV has been demonstrated and could be estimated by using the formula Hgb (g/dl) = 0.30 x PCV in members of the orders Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Psittaci-formes, sphenisciformes, and strigiformes, and the formula Hgb = 0.217 x PCV + 6.69 in members of the order Phoenicopteriformes (Velguth and others 2010).

The mean cell haemoglobin (MCH) represents the absolute amount of Hgb in the average erythrocyte. MCH (pg) = (Hgb x 10)/rBC

The mean cell haemoglobin concentration (MCHC) indicates the capacity of the bone marrow to produce erythrocytes of normal size, metabolic capacity, and haemoglobin content. MCHC(g/dl) = (Hgb/PCV) x 100.

The red cell distribution width (rdW) is the electronic equivalent of anisocytosis in smear examination. rdW = (standard deviation/mean cell volume) x 100.

Abnormalities indicative of disease processesMorphological or distribution pattern abnormalities in avian erythrocytes that may indicate a disease process are:n Anisocytosis, the variability in cell size, occurs normally

in peripheral avian blood. However, it increases with response to anaemia (younger, larger and rounder rBCs) and decreases with lack of response to anaemia.

n Nuclear abnormalities are usually attributable to dyserythropoiesis but occasionally occur because of markedly accelerated erythrocyte production. Nuclear abnormalities can include karyorrhexis, pyknosis, Howell-Jolly bodies, nuclear shape changes, and binucleation (rarely reported in birds and higher numbers are considered abnormal) (Mitchell and Johns 2008).

n Agglutination: clumping of erythrocytes usually mediated by antibodies ‘bridging’ cells indicates immune-mediated haemolytic anaemia (Mitchell and Johns 2008).

n roleaux: linear arrangement of erythrocytes mediated by plasma viscosity indicates increased protein concentration, such as acute phase proteins. These are not commonly encountered (Mitchell and Johns 2008).

n Hypochromatic erythrocytes have an area of decreased colouration due to decreased haemoglobin content and indicate decreased haemoglobin production as seen in iron deficiency cases (Mitchell and Johns 2008).

n Ghost erythrocytes are pale due to the disrupted cell membrane permitting loss of haemoglobin and may be indicative of haemolysis, contact with EdTA in some species or artefact in aged samples (Mitchell and Johns 2008).

n Basophilic stippling: small, dark blue staining ‘granules’ within erythrocytes, usually due to the presence of residual aggregations of rNA, but occasionally due to the presence of iron aggregations, indicate increased erythropoiesis, as seen in regenerative anaemias (Mitchell and Johns 2008).

n Heinz bodies are rounded, eosinophilic (romanowsky stain) or blue (new methylene blue stain) projections from the surface of erythrocytes that represent denatured haemoglobin indicative of oxidative injury (dimethyldisulfide in fowls, oil spill ingestion, etc) (Fudge 2000, Clark and others 2009).

n Erythrocytic ballooning: cells with bulges in their normal elliptic shape, often accompanied by areas of hypochromasia. This is a frequent finding in lead toxicosis and in conure bleeding syndrome (Fudge 2000).

Disorders of avian erythrocytesPolycythaemia and erythrocytosis Polycythaemia, the increase in the circulating mass of erythrocytes and haemoglobin, and erythrocytosis, the increase in the circulation concentration of erythrocytes, could be:n relative: due to haemoconcentration of the blood

(dehydration). Assessing the colour of the mucus membranes, and measuring uric acid, calcium, total protein and glucose levels, enables exclusion of dehydration. restoring hydration will reverse polycythaemia (Fudge 2000).

n Absolute: due to an increased production of erythrocytes that results in an absolute increase in the number of erythrocytes. Primary polycythaemia refers to autonomous production of erythrocytes, whereas secondary polycythaemia is used when increased erythropoiesis occurs in response to hypoxia and the consequent increased output of erythropoietin (cardiovascular, pulmonary or renal disease) (Fudge 2000).

Those species of birds that have evolved to undertake high-altitude flight posses haemoglobin with a relatively

Fig 4: Severe anaemia in a palm cockatoo (Probosciger aterrimus). The skin under the eye should be red






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increased affinity for oxygen, but they do not exhibit an increased erythrocyte concentration in response to hypoxia when compared with other species showing increased erythrocytosis in response to the hypoxia of high altitude. some diving and dabbling species present with a greater blood volume than non-aquatic birds, but have similar erythroid characteristics (Clark and others 2009).

Treatment of polycythaemia involves treatment of the underlying cause and periodic phlebotomy (Mitchell and Johns 2008).

Anaemia Anaemia is the result of a decrease of circulating mass of erythrocytes. in general, in birds, anaemia is defined as a PCV lower than 35 per cent. A PCV between 25 and 35 per cent indicates mild to moderate anaemia, whereas a PCV of lower than 20 per cent indicates severe anaemia (Mitchell

and Johns 2008) (Fig 4). in general, smaller avian species tend to present smaller erythrocytes, and hence higher total rBC counts. reported reference ranges for avian rBCs, PCV and Hgb vary significantly among reports and among species sampled (Fudge 2000) (eg, many psittacines have a normal PCV of higher than 45 per cent) (Mitchell and Johns 2008). Whereas important seasonal variability has been reported among certain species of wildlife related to moulting, reproduction, migration and food supplies, it does not appear to occur in pet birds (Fudge 2000).

Anaemia should be investigated as soon as it is detected due to the short half-life of avian erythrocytes meaning that non-regenerative anaemia patients deteriorate rapidly. Also, birds respond rapidly to acute blood loss (Fudge 2000).

Haemoglobin is more reliable than PCV or rBC as an assessment of the degree of anaemia when the blood sample quality has deteriorated due to aging. in general, birds with values below 12 g/dl should be considered anaemic (Fudge 2000).

Anaemia classification is explained in Box 1.

LeukocytesAssessment of leukocytesTotal leukocyte countTotal leukocyte count can be estimated by counting the number of leukocytes per high power field (x 40) in 10 different fields, averaging them, and multiplying by 2000 to get the number of leukocytes per ml of blood.

However, preferred methods of total leukocyte count include the direct Natt and Herrick’s or rees and Ecker’s methods. These methods require the availability of a Neubauer haemocytometer (Fig 5). Using the Natt and Herrick’s method allows for enumeration of both leukocytes and erythrocytes simultaneously. The resulting number is adjusted, based on cell counts in the differential, to produce a final total leukocyte count. Leukocytes appear as dark blue cells in a haemacytometer using the rees and Ecker’s method (Fig 6).

Differential leukocyte countAvian leukocytes include lymphocytes, monocytes and

Box 1: Classification of avian anaemia

Avian anaemias can be classified with respect to:n The morphological changes observed under the microscope (anisocytosis and

polychromasia), or by measurement and calculation of erythrocytic indices (more precise) (Fudge 2000) .

– Hypochromic microcytic anaemia: decreased mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), increased polychromasia, and normal to increased anisocytosis: A result of nutritional iron deficiency (experimental), chronic haemorrhage, blood-sucking parasites (Fudge 2000).

– Hypochromic macrocytic anaemia: decreased MCHC, increased MCV, and polychromasia, and normal to increased anisocytosis: Response to acute haemorrhage, toxins (including lead early stages [erythrocytic ballooning is also present]), haemolytic anaemia, conure bleeding syndrome and, in some cases, when the medical cause of a non-regenerative anaemia is alleviated (Fudge 2000).

– Normochromic normocytic anaemia: the most common of avian anaemias. In general, it is non-regenerative. It is characterised by presenting normal MCHC, normal MCV, slight to absent polychromasia, slight to absent anisocytosis. This is the anaemia of chronic and inflammatory disease: aspergillosis, tuberculosis, chlamydiosis, chronic bacterial infection, organ inflammation, yolk peritonitis, haematopoietic neoplasm, mesenchymal neoplasm, viral disease (circovirus), starvation, drugs (cyclophosphamide, corticosteroids) (Fudge 2000).

n The degree of response observed in: – Regenerative anaemia: monitoring of packed cell volume (PCV) quantifies

regeneration. An increased red blood cell distribution width (RDW) percentage means there is increased variability in MCV measurements and regeneration. An increase in anisocytosis microscopically corresponds to an increase in RDW percentage. Regenerative anaemia causes include:

n Improvement of non-regenerative anaemia due to successful treatment of chronic or inflammatory disease (Fudge 2000).

n Accelerated bone marrow response to: (a) Blood cell loss (haemorrhagic anaemia) due to: trauma, parasites

(Dermanysus species, coccidiosis, etc), coagulopathies (usually acquired and associated with toxicities (aflatoxicosis, cumarin poisoning, etc) or severe liver diseases such as papovirus), ulcerated neoplasms, and haemorrhagic lesions of internal organs such gastric ulceration or rupture of the liver or spleen.

(b) Blood destruction (haemolytic anaemia): haemolysed plasma is observed in correctly collected and processed blood samples. It could be:

– Immune-mediated: only reported in one Eclectus parrot (Johnston and others 2007); or

– Associated with systemic or haematogenous bacterial infection (septicaemia) – salmonellosis or spirochaetosis; infectious disease – fowl typhoid (Fudge 2000); haemoparasites – Plasmodium species, Aegyptianella species, etc; toxicities – phenylhydramine, copper (Fudge 2000), petroleum derivatives (Mitchell and Johns 2008), aflatoxicosis, plant toxins as such those found in mustards, acute lead or zinc toxicosis, etc; and transfusion reaction.

– Non-regenerative anaemia: decreased RDW percentage. The differential diagnoses include: anaemia of chronic disease (especially chlamydiosis, mycobacteriosis, aspergillosis, West Nile virus, or neoplasia), toxicity (eg, lead toxicosis, aflatoxicosis), iron deficiency, hypothyroidism, and leukaemia (Mitchell and Johns 2008).

Fig 5: Instruments for measuring leukocytes. (1) Cell counter. (2) Rees and Ecker’s solution. (3) Improved Neubauer haemocytometer. (4) White blood cell pipette






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granulocytes. Granulocytes posses distinct cytoplasmic granules and include heterophils, eosinophils and basophils. Furthermore, acidophils include eosinophils and heterophils due to the fact that their predominant cytoplasmic granules exhibit affinity for acidic stains (such as eosin). Lymphocytes and monocytes may be collectively referred to as mononuclear cells.

Heterophils are typically the most commonly encountered granulocyte. They are typically irregularly round cells with a bi/tri-lobed basophilic nucleus and prominent acidophilic cytoplasmic granules, characterised in shape as fusiform in longitudinal section and round in cross section and ‘brick-red’ to brown in colour (brighter red-orange in eosinophils) (Fig 7). Characteristically, their nucleus is less basophilic than the eosinophil nucleus. in some heterophils, the granules may exhibit a central granular body (a round to ovoid, pale or refractile structure located in the mid-section of the granule). Mature, segmented heterophils are normally counted on most blood smears (Clark and others 2009).

Eosinophils are the second type of acidophilic granulocytes that may be encountered in avian blood. They are irregular round cells with a bi-lobed nucleus composed of dark basophilic chromatin and a pale to moderately basophilic cytoplasm containing bright eosinophilic cytoplasmic granules (dull brown, aqua, grey or pale blue colour in some species) (Mitchell and Johns 2008) (Fig 8).

Basophils are typically round cells presenting a single lobe nucleus and darkly basophilic cytoplasmic granules at such a high density that the individual granules cannot

be discerned and the nucleus is partly obscured by them. diff-Quik staining can provoke artefactual round vacuoles in a pale cytoplasm with occasional basophilic granules (Clark and others 2009).

Lymphocytes are the most commonly encountered mononuclear cell and, in some species, the most commonly encountered leukocyte (Fig 9). According to Clark and others (2009), lymphocytes could be classified depending on their morphology as follows:n Typical small lymphocytes are the smallest of the

leukocytes and have a round nucleus composed of dense, coarsely clumped chromatin and a small, often incomplete ‘rim’ of cytoplasm that is moderately to deeply basophilic.

n Medium-sized lymphocytes are larger than small lymphocytes, often similar in size to granulocytes, and have an irregularly round nucleus composed of moderately dense, irregularly clumped chromatin with a moderate amount of agranulated, moderately basophilic cytoplasm.

n Large lymphocytes are typically larger than granulocytes and may be of similar size to monocytes. Large lymphocytes typically have a round–ovoid nucleus composed of moderately dense, irregularly clumped chromatin with a moderate amount of agranulated, moderately basophilic cytoplasm.

Lymphocytes may exhibit several punctate, azurophilic to basophilic granules in their cytoplasm.

Monocytes are large, pleomorphic leukocytes (Fig 10). The nucleus might be ovoid, indented in shaped or irregularly shaped and composed of fine to reticular chromatin. The cytoplasm is a moderate to large amount of grey to basophilic colour. Granules are not typically evident in the cytoplasm; however, when present, they appear smaller and eosinophilic. One to several small vacuoles could sometimes be evident in the cytoplasm (Clark and others 2009).

Leukocytes with atypical morphologyTypically, morphological atypia is more common in heterophils and lymphocytes, less common in monocytes and rare in eosinophils and basophils.

According to Clark and others (2009), inflammation-related changes in the morphology of avian leukocytes may occur as a result of: n The release of leukocytes from sites of haematopoiesis

before they are mature;n The direct actions of toxins on the cells in the peripheral

blood; or

Fig 6: Leukocytes and thrombocytes appear as dark blue cells in a haemocytometer using Rees and Ecker’s method

(a) (b) (c)

Fig 7: Heterophils seen in (a) a hornbill, (b) a blackbird, and (c) a quaker parrot






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n By increased synthetic activity by the cell.

Less mature heterophils (band heterophils, metamyelocytes and myelocytes) may be observed in response to significant inflammatory demand. n Band heterophils: lack of nuclear segmentation or

constriction greater than half of the nucleus. Cells at the penultimate stage of granulocyte development leave the bone marrow prematurely when needed. An increase in the relative number of immature heterophils describes a left shift (Fudge 2000, Clark and others 2009).

n Metamyelocytes have a reniform-shaped nucleus, whereas myelocytes have an ovoid nucleus; both are indicative of inflammation (Clark and others 2009).

Left shifts of avian heterophils result in younger peripheral forms. Heterophilic left shifts are most commonly seen with severe bacterial infections, mycobacteriosis, aspergillosis, chlamydiosis, and severe tissue necrosis from traumatic or neoplastic lesions.

Other heterophil changes indicative of inflammation include increased basophilia of the cytoplasm, decreased density of granules, larger and rounded granules, or an abnormal colour (amphophilic or basophilic) (Fudge 2000, Clark and others 2009).

Morphological characteristics of avian leukocytes that may indicate a disease process include:n Leukocyte degranulation, which is rare and normally

artefactual.n reactive lymphocytes presenting darker blue cytoplasm,

nuclear changes, and prominent nucleoli. These may be a consequence of antigenic stimulation (and immunoglobulin production) from viral infections (eg, polyomavirus and herpesvirus) and possibly chlamydiosis (Fudge 2000, Clark and others 2009).

n Toxic heterophils, characterising by presenting basophilic cytoplasmic granules, nuclear hypersegmentation (more than three segments) etc. These result from toxaemic processes in birds, mainly associated with bacterial infections (mycobacteriosis, chlamydiosis, fungal and some viral infections) (Fudge 2000).

According to the severity of the changes, toxic heterophils could be classified as:n Mild: mildly decreased numbers of granules, slightly

rounded granules and increased basophilia of their cytoplasm.

n Moderate: a greater decrease in the density of granules and rounded granules.

n severe: round granules, basophilic granules, large granules, ‘foamy’/vacuolated cytoplasm and karyolysis. The number of abnormal heterophils may be graded as ‘few’ (5 to 10 per cent), ‘moderate’ (11 to 30 per cent) and ‘marked’ (greater than 30 per cent) (Clark and others 2009).

The presence of haemoparasites (Leukocytozoon species, Plasmodium species and Hepatozoon species) may alter the morphology of the host cell by distorting its shape, enlarging its size, or displacing its nucleus (Clark and others 2009).

Fig 8: A heterophil (left) and an eosinophil (right) in a blackbird

Fig 9: Mature lymphocytes in a quaker parrot

Fig 10: Monocytes seen in (a) a hornbill, (b) a blackbird, and (c) a blue and gold macaw

(a) (b) (c)






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Fig 11: Two thrombocytes (star) and a heterophil (arrowhead) in a Harris hawk

Changes in the leukocyte concentration observed in avian disease include (Clark and others 2009):

Heterophilia increased concentration of heterophils is commonly indicative of inflammation or stress:n inflammation: air sacculitis, arthritis, septic,

aspergillosis, chlamydiosis (acute/active), dermatitis, Gram-negative and/or positive inflammatory lesions, hypersensitivity, neoplasia, necrosis of tissue, nephritis, pneumonia, septicaemia, tuberculosis, zinc or organophosphorates toxicity, yolk peritonitis and sometimes viral diseases.

n stress: causes of physiological stress include anaesthesia, cold stress, conspecific conflict, crowding, exogenous corticosteroids, fear, heat stress, haemorrhage (internal), hypoxia, nervousness, pain, restraint, road transport, starvation, trauma and wetness. some studies suggest that the heterophil:lymphocyte ratio may be a more reliable measure of physiological stress than even corticosterone assays. Young psittacine birds frequently show a heterophilia, which may be physiologically normal.

Heteropenia decreased concentration of heterophils is uncommon in birds and indicative of decreased granulopoiesis or marked inflammation resulting in depletion of bone marrow granulocyte reserves. it is normally associated with a leukopenia (eg, circovirus fatal infections, Gram-negative septicaemia, etc).

Lymphocytosis increased concentration of lymphocytes is indicative of immune stimulation. Non-viral immune-mediated causes of lymphocytosis are not well documented in pet birds.

Lymphopenia A decreased concentration of lymphocytes can occur in conjunction with stress heterophilia and is a common indicator of stress also found in some viral infections.

Monocytosis increased concentration of monocytes is typically related to chronic inflammation including granulomatous or histiocytic-related forms (aspergillosis, tuberculosis, chlamydiosis, other bacterial granulomas, chronic bacterial dermatitis, fungal granulomas, salmonellosis, etc). it is less frequently observed in stress response in birds than in mammals.

Monocytopenia is not clinically recognised in birds.

Eosinophilia An increased concentration of eosinophils is uncommon. it may be an indication of generalised inflammation, parasitic infection (Galliformes), and perhaps allergic reactions.

Eosinopenia decreased concentration of eosinophils has been shown in response to corticosteroid therapy and physiological stress. suspected allergies and hypersensitivity reactions based on histopathological diagnosis showed no effect on the eosinophil count.

Basophilia Clinical observations in pet birds revealed the presence of basophilia in some chlamydiosis cases (eg, amazons, budgerigars, cockatiels), and as a transient change 48 hours after tissue trauma (self-mutilation, postsurgical). inconsistent basophilia has been seen in air sac mites and other respiratory infections.

Leukaemia Leukaemia is uncommon in birds. However, it should be suspected with significantly elevated leukocyte

counts and when a large number of mast-like cells are seen in the peripheral circulation:n Granulocytic leukaemia: large numbers of

progranulocytes and myeloblasts.n Lymphocytic leukaemia: increased lymphoblasts in the

peripheral blood.

High leukocyte counts due to severe inflammation processes and accompanied by profound morphological changes could be suggestive of leukaemia. Haematopoietic neoplasms may reside primarily in the bone marrow or be sequestered in extravascular locations.

Viral aetiologies have been demonstrated in some poultry haematopoietic neoplasms.

ThrombocytesThrombocytes are the nucleated haemostatic cells of birds. individual thrombocytes are typically smaller than both leukocytes and erythrocytes. They have a very dense, darkly staining, ovoid nucleus with a small to moderate amount of pale grey or pale basophilic cytoplasm (Fig 11). sometimes, fine cytoplasmic projections (pseudopodia) may be evident. several small, punctate, eosinophilic or azurophilic granules may be observed in the cytoplasm of some thrombocytes. in many cases, the aggregation of thrombocytes into variably sized clumps throughout the blood film making it difficult to discern individual cells (Clark and others 2009). Like leukocytes, they appear as dark blue cells in a haemocytometer using the rees and Ecker’s method (Fig 6).

Estimate thrombocyte count (thrombocytes/µl) = Mean of thrombocytes in five immersion oil fields x 3,500,000/1000. if the PCV is outside the normal range (35 to 55 per cent), the estimated thrombocyte count should be corrected. Corrected thrombocyte count (thrombocytes/μl) = estimated thrombocyte count x observed PCV/ normal PCV (45 per cent).

ReferencesBEAUFrèrE, H., AMMErsBACH, M. & TULLY, T. N., Jr (2013) Complete cell blood count in psittaciformes using high-throughput image cytometry: a pilot study. Journal of Avian Medicine and Surgery 27, 211-217CLArK, P., BOArdMAN, W. & rAidAL, s. (2009) Atlas of Clinical






Birds

Quiz: Avian haematologyCan you identify the cells in the following pictures? in picture 7, what might be happening in this cell?

1. 2. 3.

4. 5. 6.

7.

Answers: 1. red blood cells at different stages of maturation in an African grey parrot.2. One monocyte (arrow) and one immature erythrocyte (star) in an African grey parrot.3. Heterophils (stars), two thrombocytes (arrowhead) and one monocyte (arrow) in a cockatoo.4. Heterophil (star) and lymphocytes (arrowheads) in a chicken.5. Four thrombocytes (One activated [arrowhead], three non-activated [arrow]) and one lymphocyte (star) in a chicken.6. Two mature erythrocytes and one anucleated erythroycte in an African grey parrot.7. An erythrocyte in amniotic cell division in an African grey parrot. These erythrocytes are a sign of an accelerated erythropoiesis, which in combination with the hypochromic microcytic erythrocytes in this figure, make it conceivable that this parrot was suffering from peripheral tissue oxygenation.

Avian Haematology. Wiley-BlackwellCLAVEr, J. A. & QUAGLiA, A. i. E. (2009) Comparative morphology, development, and function of blood cells in non mammalian vertebrates. Journal of Exotic Pet Medicine 18, 87-97CAMPBELL, T. W. & ELLis, C. K. (2007) Hematology of birds. in Avian and Exotics Animal Hematology and Cytology. 3rd edn. Eds T. W. Campbell, C. K. Ellis. Blackwell Publishing. pp 3-50FUdGE, A. M. (2000) Laboratory Medicine: Avian and Exotic Pets. saundersJOHNsTON, M. s., sON, T. T. & rOsENTHAL, K. L. (2007) immune-mediated hemolytic anemia in an Eclectus parrot. Journal of the

American Veterinary Medical Association 230, 1028-1031LUMEiJ, J. T. (2008) Avian clinical biochemistry. in Clinical Biochemistry of domestic Animals. 6th edn. Eds J. J. Kaneko, J. W. Harvey, M. L. Bruss. Academic Press. pp 839–872MiTCHELL, E .B. & JOHNs, J. (2008) Avian hematology and related disorders. in Veterinary Clinics of North America Exotics Animal Practice. Ed T. L. Hadley. saunders. pp 501-522 VELGUTH, K. E., PAYTON, M. E., & HOOVEr, J. P. (2010) relationship of hemoglobin concentration to packed cell volume in avian blood samples. Journal of Avian Medicine and Surgery 24, 115-121





HaematologyAvian haematology and biochemistry 1.

Mikel Sabater and Neil Forbes

doi: 10.1136/inp.g58702014 36: 510-518 In Practice

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