8 FOCUS HAEMATOLOGY Veterinary Times

From Aardvark to Zorilla: understandingthe haematology of exotics

PROFESSOR John Cooper oncewrote, in an obituary for DrOliphant Jackson, how DrJackson exemplified that inorder to understand an animal -and to provide optimum care forit - one needed to know, and fullycomprehend, its natural historyand biology.Haematology is an important facet ofan animal's biology.With an increasingnumber of endangered species, andthe growing number of exotic animalskept as pets, it is vital that as muchknowledge as possible is obtained sothat through careful management andveterinary care the welfare and healthof these animals is assured.Haematology can provide muchinformation to aid the differentialdiagnosis in these animals.The dictionary definitions of exoticare: (1) introduced from, ororiginating in, a foreign country; and(2) attractively or remarkably strangeor unusual, bizarre.These definitionscover all the exotic species that couldbe seen by any veterinary surgeoneither in general practice or specialistpractice.It has been calculated that there areapproximately one million extantspecies with blood, of which thereare about 42,000 species of mammal,bird, reptile, amphibian and fish.It has been estimated (Hawkey, 1991)that reference ranges of the cellularcomponents of blood are currentlyavailable for about 600 species ofmammal, 350 species of bird and 80species of reptile and around two percent of amphibians and fish.Thereference ranges currently availablespan a considerable number of yearsand were derived in many cases withmethodologies that had not beenstandardised or validated.Many are to be found in obscurejournals and may prove difficult totrack down.The effects of handling, stress,immobilisation and anaesthesia are

mostly missing from the data andeven such obvious physiologicalvariations such as sex, age and theclinical status of the animals are oftennot accounted for. It may be possibleto predict ranges in an unstudiedspecies by referring to the ranges of aclosely related species.Where possible, all haematologyreports emanating from a laboratoryshould provide species-specificreference ranges.Veterinary surgeons should be awareof these shortcomings wheninterpreting data, especially fromlaboratories that have not developedtheir own ranges or do not usereliable sourcesand validatedmethodologies.Yet all is notdoom and gloombecause of anunderstanding of,and referral to,the basichaematologicalprinciples asdescribed byWintrobe in 1933,which show arelatively small variation inhaemoglobin (Hb), packed cell volume(PCV) and mean cell haemoglobinconcentration (MCHC) in mammalsand birds (Table 1).There is, however, a great deal ofvariation in red cell count (RBC),mean cell volume (MCV), mean cellhaemoglobin (MCH) and white cellcount (WBC). For example, goatshave many small red cells but have anMCHC similar to an animal with fewlarge cells, such as elephant.An understanding of the basic

principles of Hb, PCV and MCHC canprove very useful in differentialdiagnosis or health monitoring,especially in assessing anaemia,polycythaemia and hypochromia.These conditions can be identified inany adult mammal or bird withoutconsulting reference ranges or wherereference ranges are unavailable.These basic principles do not apply toreptiles, amphibians or fish.There is considerable variation in thetotal white cell count and in thenumbers of the various cell types thatmake up the white cell populationamong orders, classes, families andspecies.Again, referring to general

principles and noting the white cellmorphology on a stained film, anassessment of the clinical status aspresented by the haematology can bemade.

Physiological variations• Age. Certain age-relateddifferences are recognised in man, forexample Hb, RBC, PCV andneutrophil numbers are lower - andIymphocytes and mono-cytes higher -in children than in adults. Similar age -

MIScT, AIBMS, CBiol, MIBiolMIKE HARTof Greendale Veterinary Diagnostics, Knaphill, discusses theinterpretation of blood samples from exotic species

TABLE 1. Variation in haemoglobin, packed cell volume and mean cell haemoglobin concentration

Mammals Birds

Standard Standardmean deviation mean deviation

Haemoglobin 14.8g/dl ±2.1 15.2g/dl ±1.7

Packed cell volume 14.8g/dl ±2.1 15.2g/dl ±1.7

Mean cell haemoglobin 14.8g/dl ±2.1 15.2g/dl ±1.7concentration

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related differences have been found inother mammals and birds.• Sex. In man, and in somedomesticated species, adult maleshave higher Hb and PCV levels thanfemales.There is a tendency forhigher Hb and PCV levels in males ofmany exotic mammals and birds.• Stress. Excitement, fear, thepresence of humans and herd orgroup interaction can all result in therelease of stress hormones(catecholamines and cortico-steroids)which, in turn, affect both the red andwhite cells.It is the red cell numbers that aremost notably affected bycatecholamines.Catecholamines induce spontaneoussplenic contraction in animals withreactive spleens, for example canidae,felidae, camelidae, cervidae, bovidaeand equidae.These animals have large,reactive spleens capable ofsequestering up to 25 per cent oftheir total number of red cells.This contraction releases red cellsinto the circulation resulting in highRBC, Hb and PCV levels. Splenicrelaxation takes approximately 45minutes after the stress stimulus.Many exotic species need to beanaesthetised prior to examinationand blood sampling and the use ofadrenolytic immobilising drugssuppresses the action of cat-echolamines and the red cells areresequestered.This does not occur inprimates or birds.Care must be observed whenassessing the RBC, Hb and PCV levelsin animals that have beenanaesthetised with adrenolytic agents.It could be argued that theanaesthetised state is more"haematologically normal” than thestressed state.The effect of stress on white cells isless pronounced, is slower and usuallylasts much longer than that of the redcells.White cell changes are mainlyeffected by corticosteroids.Stress may produce neutro-philia,Iymphopaenia and eos-inopaenia andin dogs a mono-cytosis. Eosinopaeniais considered as diagnostic of stressin dolphin but the effects on whitecells of the majority of exotic species

is unknown, but it is possible thatmammals react the same as for manand domestic mammalian species.

ProblemsOf the 42,000 or more species with"conventional" blood, the averageveterinary surgeon or veterinarydiagnostic laboratory will see only anextremely small percentage, usually afew of the more common exoticspecies such as grey parrots, commoniguana, Greek tortoise and perhaps anoccasional snake.Even these common species needspecial care with haematology andsuitable reference ranges. It is notsufficient to have ranges for"tortoise" as even the usual speciessuch as Greek or Hermann's havediffering ranges.Veterinary surgeons should bemindful that not all laboratories havethe necessary skills, experience andappropriate instrumentation andmethodologies to produce meaningfuldata.

Materials and methodsAll the technical requirements thatapply to domestic species applyequally well to exotics, but with someadditional requirements.The site ofvenepuncture is sometimes animportant factor, for example whenbleeding tortoises from the tail vein

haemodilution with Iymph very oftenoccurs resulting in erroneous results.It is known that the blood of somespecies, for example penguin, clotsvery quickly and it is recommendedthat the venepuncture needle isflushed through with heparin solutionto reduce the risk.The choice of anticoagulant for the

majority of exotic species is EDTA,but for several species EDTA causesrapid and progressive red cell lysis,for example crowned crane, woodduck and tortoises: for these speciesheparin is the anticoagulant of choice.However, it should be borne in mindthat heparin may cause white cellaggregation making the total whitecell count inaccurate, if notimpossible.The author advises taking anothersample into EDTA solely for the totalwhite cell count.

Red cell countsAutomated, orsemi-automated, cellanalysers were developed for humanblood but several are suitable formany exotic mammals.However, there are some species thatthe majority of analysers are unableto produce accurate results for, suchas animals with MCV < 25fl, forexample goats, and those that havered cell counts above 9.0xl0 ^ 12/1 asmost cell analysers are not linearwith counts above 9.0, for thesefurther dilution is required.The most suitable instruments forcounting these species, and for non-mammalian species, are those thatemploy the impedence methoddeveloped by Coulter Electronics andthat have manually adjustableaperture currents and threshold

settings - or have “hunting”thresholds - built into the soft-ware.These instruments are now either nolonger manufactured and/or aredifficult to obtain in the UK.Many workers in avian haematologystill prefer to count red cells manuallyusing suitable microscopy (Samour,personal communication).

Figure 1. Bactrian camel oral red cells Figure 2. Genet showing sickling

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Haemoglobin estimationAny cell analyser capable ofmeasuring human haemoglobin will besuitable for exotic mammals.Theseinstruments use a modified Drabkin'ssolution which haemolyses the redcells and converts the releasedhaemoglobin to cyanmet-haemoglobin.This method is unsuitable for non-mammalian species as a turbidsolution results which interferes withthe light path in the instrument,producing grossly elevated results.The turbidity is caused by the releaseof the red cell nuclei which areunlysed.Several workers suggest a manualmethod using Drabkin's fluid withcentrifugation to spin down the nucleiand to read the clear supernatant in aspec-trophotometer or haemoglo-binometer.Avian red nuclei containhaemoglobin (Campbell, 1996): thiswould be lost to the measurement ifthe preparation is spun.The nuclearhaemo-globin represents one to twoper cent.The conversion ofhaemoglobin to oxyhaemo-globin wasthe method of choice prior to thedevelopment of theazidemethemoglobin method(Vanzetti, 1966).

White cell countsAt present there are no automatedcell analysers capable of reliablycounting non-mammalian white cellsand thrombo-cytes due to thepresence of the red cell nuclei. Someworkers are investigating thepossibility of using flow cytometry.White cell counts of these species

are best performed manually with acounting chamber and micro-scopewith a suitable diluent solution (Nattand Herrick, 1923) or fluid (Rees andEcker, 1952), or by phase contrastmicroscopy.Whatever method is used, difficultieswill be encountered as no method isperfect.The white cells of mostmammalian species can be countedusing automated or semi-automatedcell analysers.Thrombocyte numbersare estimated from a stained bloodsmear with reference to the totalwhite cell count.

Red cell morphology• Mammalian species. The red cellmorphology of most mammalianspecies is very similar, i.e. bi-concavediscs which usually vary only in size,degree of anisocytosis andpolychromasia.However, a few species show verydifferent characteristics: for instance,all the camelidae species have oval,anucleate flat cells (Figure 1), whileothers, such as the cervidae andsome felidae, show an in vitro sicklingtendency (Figure 2).Heinz bodies are often seen inrhinoceros and in some New Worldprimates.• Non-mammalianspecies.All animals inthese classes havenucleated, ovalred cells whichare larger thanmammalian cells.Their immaturered cells are

round, which become more oval asthey mature, and are morepolychromatic: up to five per centpolychromatic cells in birds isconsidered as a normal degree oferythropoiesis. Some species aremore, or less, ovoid than others.Many reptile species show intra-erythrocytic inclusions variouslyidentified as pirhemocyton, iridovirusinclusions or degenerate organelles.

White cell morphololgy• Mammalian species. The whitecells of the majority of mammals isvery similar, but there are somevariations. Elephant monocytes arelarge with bi- ortri-lobed nuclei(Figure 3).The neutrophils of camels oftenappear left shifted (the Pelger-Huetphenomenon) as a normal finding.Guinea pig Iymphocytes often show alarge basophilic inclusion known as aKurloff body (Figure 4).Seals and dolphins often have Dohlebodies in their neutro-phils.Theneutrophil granules vary greatly intheir amount and in their stainingcharacteristics, varying from agranularto heavily granulated, and from palepink to deep purple, and the nucleivary in the degree of lobulation.Thenumbers, size and shape of granulesand the intensity of staining is alsovariable in eosinophils and basophils.• Non-mammalian species.There is considerable variation in thegranulocytes of these classes ofanimals, especially in the heterophils.The granules of these cells aregenerally fusiform or rod-shaped andstain in a range of colours frommuddy brown to bright orange inbirds (Figure 5), but many reptiles

TABLE 2. Cell types seen in animals

Mammals Birds Reptiles

Erythrocytes Erythrocytes ErythrocytesNeutrophils Heterophils HeterophilsLymphocytes Lymphocytes LymphocytesMonocytes Monocytes Monocytes

AzurophilsEosinophils Eosinophils EosinophilsBasophils Basophils BasophilsPlatelets Thrombocytes Thrombocytes

Figure 3. Indian elephant showing tri-lobed monocyte.

Figure 4. Guinea pig showing Kurloffbody in lymphocyte

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have oval or round reddish granules(Figure 6).The eosinophils of birds normallyhave small, round, brightereosinophilic granules although a smallnumber of species have rod-shapedeosinophilic granules, but theeosinophil granules of parrot, macawand cockatoo spp are round, blue(Figure 5) and occasionally blue andpurple.The eosinophil granules of reptilesvary in shape but are usually roundishand paler staining. Iguana, and veryrarely snakes, also have blue stainingeosinophil granules. Reptiles areunique in that they have a cell calledan azurophil, although some workersreport them as atypical monocytes,reactive monocytes or azurophilicmonocytes (Figure 7). They are

rarely found in tortoises, but arecommon in most snakes.Fish granulocytes are often classifiedas Type 1, 11, 111, IV and thereappears to be considerablemorphological variation betweencartilaginous and bony fish, andpossibly between fresh water, seawater, warm water and cold waterfish.

Platelets and thrombocytes• Mammalian species. Althoughplatelet numbers vary betweenspecies their morphology is fairlysimilar but, of course, there areexceptions for example elephant haverelatively large numbers of very smallplatelets, and echidna often have twodistinct populations of platelets.Granularity may vary from species tospecies and the platelets of some,such as felidae, may become activatedand aggregate rapidly.Activated platelets, non-activatedplatelets and aggregates may all beseen together on stained smears ofany mammalian blood. Large increasesin numbers in a given species willoften indicate a disease process orother abnormality occurring.• Non-mammalian species.There are many varieties ofthrombocytes in these classes ofanimals but they have one feature incommon: they are all nucleated.Theyare easily mistaken for smalllymphocytes, especially if they areactivated when there is oftenincreased cytoplasmic basophilia, orwhen they have had prolongedexposure to anticoagulant causingthem to swell.Thrombocytes mayalso increase in number as a result ofa disease or abnormality occurring.

FibrinogenA few species of animal do not alwaysshow a leucocytosis in infection butwill often show a large increase infibrinogen, for example wallabies.Therange across the animal kingdom isapproximately 1.0-5.0 g/l.Fibrinogen is a very useful test in allspecies as a possible indicator ofinfection/inflammation.

ConclusionHaematology is already a useful toolin the diagnosis of disease indomesticated animals and is becominga very useful diagnostic tool for manyexotic species.However, much still needs to bedone, more reliable reference rangesof unstudied species need to beestablished, and existing ranges needto be increased.Improved and standardised methodsfor cell analysis need to be developedso that more reliable data aregenerated.With an increasing amount ofinformation available on the Internet,and with interested and importantorganisations such as the BritishVeterinary Zoological Society and theAssociation of ComparativeHaematology in the UK and severalAmerican and Europeanorganisations, the future of exoticanimal haematology as an importantdiagnostic aid is assured.

• Mike Hart can be contacted atGreendale VeterinaryDiagnostics, telephone 01483797707, fax 01483 797552, e-maillab@greendale.co.uk.

ReferencesHawkey, C. M. (1991) The value ofcomparative haematological studies.Comp. Haematol. Int. 1: 1-9.

Wintrobe, M. M. (1933) Variations inthe size and haemoglobin content inerythrocytes of various vertebrates.Folia Haematol. 51: 32-49.

Campbell,T.W. (1988) AvianHematology and Cytology. Iowa StateUniversity Press/Ames

Vanzetti, G . J . (1966) J. Lab. & Clin.Med. 67(1): 116.

Natt, M. P and Herrick, C. A. (1952) Anew blood diluent for counting theerythrocytes and leucocytes of thechicken. Poultry Sci. 31: 735-738.

Rees, M. and Ecker, E. E. (1923) Animproved method for counting bloodplatelets. Jour. Amer. Med. Assoc. 80:621-622.

Figure 6. Common iguana showingheterophil.

Figure 7. Reticulated python showingazurophils (x2).

Figure 5. Lesser sulpher crested cockatooshowing heterophil (H) and eosinophil (E).