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valuation of the avian hemogram involvescounting the various blood cells per mi-croliter of blood as well as cytologic evalu-ation of the cells. The techniques involved

in the evaluation of the avian hemogram are easilyperformed by in-house veterinary laboratory person-nel. Because avian blood does not store well (eg,during transport), hematologic results obtained soonafter collection are preferred over those performedseveral hours later.6,18,34

Blood volume in birds depends on the species andvaries from 5 ml/100g in the Ring-necked Pheasantto 16.3 to 20.3 ml/100g in the racing pigeon. In gen-eral, birds are better able to tolerate severe blood lossthan mammals, which is due to their greater capacityfor extravascular fluid mobilization. However, thereis a marked variation among avian species in re-sponse to blood loss, which may be a reflection ofdifferences in blood volume or extravascular fluiddepots. In healthy Mallard Ducks and racing pi-geons, a blood volume equivalent of up to three per-cent of the body weight can be collected. In Passeri-formes, pheasants and Psittaciformes, up to onepercent of the body weight can be collected with fewill effects (0.9 ml from a 90 g cockatiel).

Blood can be collected from a variety of sites in avianpatients. The choice of a blood collection site is influ-enced by the species of bird, preference of the collec-tor, physical condition of the patient and volume ofblood needed. For best results, venous blood shouldbe collected for hematologic studies. Blood collectedfrom capillaries (eg, blood from clipped nails) oftenresults in abnormal cell distributions and containscellular artifacts such as macrophages and materialnot normally found in peripheral blood (Figure 9.1).Blood to be used for hematology should be collectedinto a collection tube containing EDTA (ethyle-nediaminetetraacetic acid) as the anticoagulant.Other anticoagulants, such as heparin, interfere withcell staining and create excessive cell clumping, re-sulting in erroneous cell counts and evaluations(Color 9.3)6,18,34

EC H A P T E R

9HEMATOLOGY

Terry W. Campbell

Processing the AvianHematologic Sample

Blood Collection

Jugular venipuncture is a procedure that can be usedfor collecting blood from most avian species.6,18,34,38,71

It is the method of choice for small birds that do nothave other blood vessels large enough for venipunc-ture. The right jugular vein is usually chosen overthe left for blood collection because in many birds itis the larger of the two. To collect blood from thejugular vein, the bird is properly restrained with thehead and neck extended (Figure 9.2). Extending theneck encourages the highly movable jugular vein tofall into the jugular furrow. In many species, there isa featherless tract of skin (apterium) overlying thejugular vein; therefore, lightly wetting the featherswith alcohol in this area will aid in the visualizationof the vein. Blood is collected into a syringe, and thesize of needle is governed by the size of the vein.Complications of jugular venipuncture include diffi-culty in proper restraint of the bird or stabilization ofthe vein and hematoma formation. Improper atten-tion to technique and hemostasis can cause a largehematoma to form during or following jugularvenipuncture. However, jugular venipuncture be-comes a skill perfected with practice, and complica-tions are infrequent in skilled hands.

Venipuncture of the ulnar or wing vein is a commonmethod for obtaining blood from medium to largebirds. A needle is inserted into the vein, which isfound crossing the ventral surface of the humero-ra-dioulnar joint (elbow) (Figure 9.3). Blood is eitheraspirated into a syringe or allowed to drip from theneedle hub into a microcollection device. Collectingblood in this manner reduces but does not eliminatehematoma formation. A variety of these devices isavailable.a-c These collecting tubes contain EDTA forhematology studies, are plain (with or without aserum separator) or contain heparin (lithium hepa-rin is the preferred form) for blood chemistry studies.Hematoma formation, which can be severe, is com-mon when the ulnar vein is used for blood collection.A needle with an extension tube, such as a butterflycatheter,d aids in stabilization during sample collec-tion to minimize tearing of the vein.

Venipuncture of the medial metatarsal (caudal tibial)vein, which lies on the medial side of the tibiotarsusat the tibiotarsal-tarsometatarsal joint, is anothercommon method for blood collection in medium tolarge birds (Figure 9.4).6,18 The primary advantagesof this method over other methods of blood collectionis that the surrounding leg muscles protect the me-dial metatarsal vein from hematoma formation and,in some species, the leg is more easily restrainedthan the wing.

Blood can be collected from the occipital venous sinusof birds. This technique should be reserved for birdsused in research or for blood collection prior to eutha-nasia,6,78 because of the potential for injuring thebrainstem. When properly executed, however, thismethod can be safely used for collecting repeatedblood samples from birds. The bird must be com-pletely restrained. The head is held firmly in a flexedposition in a straight line with the cervical vertebrae.The occipital venous sinus is just below the skin inthe space between the base of the skull and the firstcervical vertebra. To collect blood from this sinus, anevacuated tube with needle and holder is required.The needle is passed through the skin at a 30 to 40°angle to the cervical vertebrae on the dorsal midlinejust above the sinus. Following penetration throughthe skin, the evacuated tube is advanced in theholder, allowing penetration of the tube stopper bythe needle within the holder. The needle is advancedslightly downward to penetrate the venous sinus.

FIG 9.1 Blood for hematologic evaluation should be collected froma free-flowing venous source. Blood collected from a toenail clipmay yield abnormal cell distributions and cellular artifacts.

CHAPTER 9 HEMATOLOGY

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When this occurs, blood will rapidly fill the evacu-ated tube. Cardiac punctures should be used only forblood collection prior to euthanasia.1,45,77

Laboratory Techniques

After the blood is collected, a blood film is made. Thefilm can be made either from blood containing noanticoagulant (especially if blood parasites are sus-pected) or blood containing EDTA. EDTA will causehemolysis of erythrocytes in some birds includingCorvidae, currasows, Crowned Cranes, hornbills andBrush Turkeys. Prolonged exposure to EDTA mayresult in increased disruption of cells in the bloodfilm in some species (Color 9.3). Therefore, whenusing an anticoagulant, a blood film should be madeimmediately following blood collection. Heparinshould be avoided whenever possible for hematologicstudies. Heparinized blood contains artifacts such asclumping of cells (especially leukocytes in counting

chambers) and frequently results in staining arti-facts (Color 9.3).6,34 When preparing a blood film, thestandard two-slide wedge technique used in mam-malian hematology usually works well with avianblood.6,12,67 It is advisable to use precleaned, bevel-edged microscope slides to minimize cell damageduring preparation of blood films. Peripheral bloodfilms can also be made using a two-coverglass tech-nique. A drop of blood is placed in the center of onecoverglass;16,17 a second coverglass is placed on top ofthe first, and the two are pulled apart as the bloodbegins to spread between the two surfaces. A similartechnique using a microscope slide and a rectangularcoverglass (24 mm x 50 mm) can be used to preparea film on a microscope slide rather than on cover-glasses, making the sample easier to stain.6,18 Usingthe two-coverglass or microscope slide-coverglassmethods should be considered if the standard two-slide wedge technique creates excessive smudging ofthe cells. Most veterinarians and technicians accus-

FIG 9.2 The right jugular vein (left) is the preferred site for blood collection in most species of birds. The vessel is easy to visualize and islarger than the left jugular vein. The neck and head are held in extension, and the mid-cervical area is lifted slightly to improve the anglefor venipuncture. The vessel is occluded at the thoracic inlet (right) to facilitate distention and blood collection. Note the featherless tract(apterium) overlying the right lateral neck and jugular furrow. The vessel can be entered from either a cranial or caudal direction.

SECTION TWO PATIENT EVALUATION

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tomed to performing the two-slide wedge techniquewith mammalian blood have little difficulty in usingthe same technique to prepare proper avian bloodfilms.

A variety of hematologic stains can be used to evalu-ate the air-dried blood film. Romanowsky stains,such as Wright’s, Giemsa, Wright-Giemsa, Wright-Leishman or May-Grunwald and their combination,are preferred6,18,34 (see Chapter 10). Wright’s stain

has been the standard in veterinaryhematology, and all cell descriptionsand illustrations used in this text arebased upon that stain. These de-scriptions also apply to a great ex-tent to the other commonly usedquick stains, which essentially aremodifications of the classic Wright’sstain procedure.e,f The use of an auto-matic slide stainerg simplifies thestaining procedure and provides ameans for consistency and high qual-ity staining by removing variationsthat occur with hand-staining proce-dures.

After making a blood film, the re-mainder of the blood sample is usedto obtain a packed cell volume (PCV),hemoglobin concentration and cellcount. The PCV is obtained by centri-fuging a microhematocrit tube full ofblood at 12,000 G for five minutes.The hemoglobin concentration ismeasured spectrophotometrically byusing the manual or automated cy-anmethemoglobin method after cen-trifugation removal of free red cellnuclei and membrane debris.

The red blood cell (RBC) count is ob-tained either by automated or man-ual methods. The automated cellcountersh provide a quick, reliablemethod for obtaining a RBC count inbirds. The two manual methods thatcan be used are the erythrocyteUnopette systemi (standard methodin mammalian hematology) or Nattand Herrick’s method. The lattermethod requires the preparation of amethyl violet 2B diluent.55 A 1:200dilution of the blood is made usingthis solution and a diluting pipette.

After mixing, the diluted blood is discharged into aNeubauer-ruled hemacytometer and the cells are al-lowed to settle to the surface for five minutes beforeenumeration. The red blood cells are counted usingthe four corner squares and one central square of thecentral large primary square of the hemacytometer.The number of red cells counted is multiplied by10,000 to obtain the RBC count per microliter ofblood. Appropriate secondary squares are counted oneach grid and the counts are averaged.

FIG 9.3 The cutaneous ulnar vein should be considered an inferior site to the right jugularvein for blood collection. The vessel (top) is easy to access on the ventral surface of thewing, but hematoma formation is common. Note that the bevel of the needle is up and thebrachial vein is being occluded with the thumb. A small gauge needle (bottom) is used tominimize hematoma formation and is threaded into the vessel to decrease “wobble” andendothelial damage from the needle tip.


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The mean corpuscular values can be calculated usingthe PCV, hemoglobin and RBC count values. Themean corpuscular volume (MCV) and mean corpus-cular hemoglobin concentration (MCHC) are usefulin the characterization of the erythrocytes, especiallyin the evaluation of anemia.

MCV = PCV x 10

RBC count

MCH (pg) = Hemoglobin x 10 RBC

MCHC = Hemoglobin x 100 PCV

A reticulocyte count can be useful in the evaluationof the red cell regenerative response. The erythro-cytes are stained with a vital stain, such as newmethylene blue stain, and the reticulocytes are iden-tified as red blood cells that contain distinct rings ofaggregated reticulum encircling the cell nucleus(Color 9.1).6,34 Other erythrocytes may contain vary-ing amounts of reticulum, but those with the distinctring of aggregated reticulum surrounding the cellnucleus appear to be cells that have recently enteredthe peripheral circulation, and thus reflect the cur-rent regenerative response.

The white blood cell (WBC, leukocyte) count of birdsis obtained using manual techniques because thepresence of nucleated erythrocytes and thrombocytes

interfere with the counting of whiteblood cells using electronic cellcounters. The current methods ofchoice for obtaining a total leukocytecount in birds are the indirectmetho d using the eos inophi lUnopette brand 5877 system or thedirect leukocyte count using Nattand Herrick’s method.6,13,18,55 Estima-tion of leukocyte numbers from ablood film should be reserved forthose occasions when a quantitativecount is unavailable or when there issuspicion of error in a value obtainedfrom the other methods.

The indirect eosinophil Unopettebrand method involves the filling ofthe 25 µl pipette with blood, mixingthe blood with the phloxine B diluentin the vial provided in the systemand charging the hemacytometerchamber for cell counting. The blood-phloxine mixture should not be al-

lowed to stand in the Unopette vial for longer thanfive minutes or erythrocytes may also be stained. Thecharged hemacytometer should stand undisturbedfor at least five minutes to allow the cells to settle tothe surface of the counting grid. It is advisable tokeep the charged hemacytometer in a humid cham-ber to prevent dehydration of the sample if the cham-ber is going to sit for longer than five minutes. Thegranulocytes that stain distinctly red (heterophilsand eosinophils) are counted in both sides of thehemacytometer (representing 18 large squares).Therefore, a total absolute heterophil and eosinophilcount is obtained. A WBC count is obtained by deter-mining a leukocyte differential from the peripheralblood film and using the formulas in Table 9.1.

A total thrombocyte count can be obtained using theNatt and Herrick’s method; however, thrombocytestend to clump, making an accurate count difficult toachieve. A subjective opinion as to the number ofthrombocytes present can be made from the peri-pheral blood film. An average of one to two thrombo-cytes are present in monolayer oil immersion (100 x)fields in blood films of normal birds. Numbers lessthan this suggest a thrombocytopenia and thosegreater suggest a thrombocytosis. An estimate of thethrombocyte count can be made from the peripheralblood film by obtaining the average number of throm-bocytes in five monolayer oil immersion fields. This

FIG 9.4 The medial metatarsal vein can be used to collect smaller quantities of blood frommedium- to large-sized birds. This vessel is supported by the soft tissues of the leg and incomparison to other blood collection sites, hematoma formation is rare (courtesy of KathyQuesenberry).


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represents the average number of thrombocytes per1000 erythrocytes in most species of birds.

A more accurate method would be to count the num-ber of thrombocytes per 1000 erythrocytes in theblood film. The number of thrombocytes per 1000erythrocytes is multiplied by the erythrocyte countand divided by 1000 to obtain an estimated thrombo-cyte count per µl of blood. If the actual erythrocytecount is not known, then 3,500,000 can be used torepresent the average number of erythrocytes per µlof blood in most species of birds having an averagePCV of 45%. If the PCV is below 40 or above 50, theestimated thrombocyte count (est T) can be correctedusing the following formula:

Corrected est T = est T X observed PCV normal PCV (averages 45%)

Cell Identification

Erythrocyte MorphologyThe normal mature avian erythrocyte is oval with acentrally positioned oval nucleus. The cytoplasm isabundant and stains a uniform orange-pink, resem-bling the cytoplasm of mammalian erythrocytes(Color 9.1). The nucleus of the mature erythrocyte iscondensed and stains dark purple. The nuclear chro-matin is uniformly clumped. The red cell nuclei varywith age, becoming more condensed and darkerstaining as the cells age.

Variations from the typical mature erythrocyte areoccasionally seen in the peripheral blood of birds.Avian erythrocytes frequently demonstrate diffusepolychromasia. Polychromatic erythrocytes demon-strate cytoplasmic basophilia and have nuclei thatare less condensed compared to mature erythrocytes(Color 9.1). Immature round erythrocytes (eg, rubri-cytes) may also be found in the peripheral blood ofbirds. These developmental stages have been de-scribed in this chapter with the discussion of theevaluation of hematopoietic tissue. Occasionally,round erythrocytes with oval nuclei may be found,especially in anemic birds. This is suggestive of anasynchronous maturation of the cell nucleus and thecytoplasm, probably owing to accelerated erythropoi-esis. Anucleated, oval erythrocytes (erythroplastids)are rare findings in peripheral blood films of birds(Color 9.2). The shape of the red blood cell mayappear irregular, or smudging may occur as a resultof artifacts created by the preparation of the film. Themost common artifact found in peripheral blood films

of birds is the rupturing of cells during preparationof the film (Color 9.3). The majority of these cellsappear to be erythrocytes. The free red cell nucleiappear as amorphous, pink-to-purple material on thefilm. Other abnormal findings include variations inthe location of the cell nucleus within the cell andnuclei having indentations, constrictions or protru-sions (Color 9.3). Perinuclear rings are usually arti-facts of staining (eg, a form of cellular crenation).Cytoplasmic basophilic stippling is also indicative ofabnormal erythrocyte morphology. Hypochromasia isindicated by pale-staining cytoplasm, cytoplasmicvacuoles and round, pyknotic nuclei (Color 9.2). Ag-glutination of erythrocytes in the blood film is a rare,abnormal finding.

TABLE 9.1 Formulas for Determining WBC counts

The total heterophil and eosinophil count T(h+e) is obtained byusing the formula given for the eosinophil Unopette brand system:

T(h+e)/mm3 = cells counted x 10 x 32 /18

The total leukocyte count (TWBC) is obtained using the leukocytedifferential and the following formula:

TWBC/mm3 = (T(h+e) / % heterophils + eosinophils) x 100

The TWBC can be calculated using the formula:

TWBC/mm3 = number of cells counted x 10 x 32 x 100 (% heterophils + eosinophils) x 18

This formula can be simplified by using the formula:

TWBC/mm3 = number of cells counted x 1.111 x 16 X 100

% heterophils + eosinophils

or

TWBC/mm3 = number of cells counted x 1778

% heterophils + eosinophils

The Natt and Herrick’s method is a direct method for obtaining aTWBC and utilizes the same dilution and charged hemacytome-ter used to obtain a RBC count. The dark-staining leukocytes arecounted in the nine large squares of the hemacytometer cham-ber. The TWBC is obtained using the following formula:

TWBC count/mm3 = (total leukocytes in 9 squares) x 10 x 200

9or simplified to:

TWBC/mm3 = (total leukocytes in 9 squares + 10%) x 200


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Hematology

Illustrations for Colors 9.1 to 9.10 are com-puter-generated reproductions of bloodcells originally printed in Lucas AJ, JamrozC: Atlas of Avian Hematology, USDAMonograph 25, Washington DC, 1961.

Color 9.1 Normal erythropoiesis (Wright’s-Leishmanstain unless otherwise noted).

a) Rubriblast with prominent nucleolus,finely granular chromatin pattern anddark-blue cytoplasm.

b) Prorubricyte with moderately granularchromatin pattern and dark-blue cyto-plasm. A nucleolus is not present.

c-g) Various stages of developing polychro-matophilic erythrocytes. As maturationprogresses, the nuclear chromatin patterncondenses, the cytoplasm becomes less ba-sophilic and the nuclear and cell shapestransform from round to elliptical. Thepresence of these cells in the blood indicatespolychromasia or erythrocyte regeneration.

h,i) Appearance of polychromatophilicerythrocytes as reticulocytes following newmethylene blue staining. Ribosomes arestained and aggregate as particulate mate-rial around the nucleus.

j,k) Mature erythrocytes contain abundanthemoglobin, which imparts an orange colorto the cytoplasm. As erythrocytes continueto mature or age, the cell and nuclearshapes become more elongate, and the chro-matin pattern is extremely condensed.

l) Early polychromatophilic erythrocyte inmitosis. These cells are observed most com-monly in bone marrow smears but are rarein peripheral blood.

Color 9.2 Common erythrocyte abnormalities in thestained blood smear.

a-c) Poikilocytes are misshapen erythro-cytes. These cells may assume a variety ofshapes including a unipolar-to-bipolar,spindle appearance. Cytoplasmic constric-tions also may be present.

d) A macrocyte is an enlarged erythrocytewith voluminous cytoplasm and a con-densed, displaced nucleus. These cells maybe observed with certain forms of anemia.

e,f) Microcytes are small erythrocytes witha minimum of cytoplasm. These cells areassociated with lack of iron or iron defi-ciency anemia.

g) Anucleated erythrocytes are observedinfrequently. These cells also are callederythroplastids.

Color 9.3 Common erythrocyte artifacts resultingfrom improper collection or preparation ofblood smears.

a) Smudge cell resulting from traumaticdisruption of a blood cell, usually an eryth-rocyte.

b) Diffuse, non-refractile, cytoplasmicvacuolation suggesting cytoplasmic edemafrom loss of membrane integrity. Thesechanges often result from cellular damageduring blood smear preparation.

c) Refractile artifact caused by water or airtrapped between the cell membrane andmounting medium or immersion oil. Thisartifact is commonly mistaken for ahemoparasite.

d) Staining artifact seen most commonly insmears from blood collected in heparin andsubjected to Romanowsky staining(Wright’s or Giemsa staining).

e) Intact erythrocyte nucleus following cel-lular disruption.

Color 9.4 Normal thrombocytopoiesis.

a) Thromboblast containing an indistinctnucleolus, finely granular chromatin pat-tern and basophilic cytoplasm.

b,c) Immature thrombocytes containinground nuclei, a moderately granular chro-matin pattern and moderately blue cyto-plasm.

d) Late immature thrombocyte. As matu-ration progresses, the cellular and nuclearprofiles become more elliptical. Cytoplas-mic vacuolation and granules may appear.

e-g) Mature thrombocytes are ellipticalwith a round-to-oval nucleus, condensedchromatin pattern and light-blue vacuo-lated cytoplasm. One to three cytoplasmicgranules may be present, but granules mayvary from absent to abundant.

h,i) Shrunken, degenerating thrombocyteswith pointed cytoplasmic projections or acondensed nucleus. These cells are ob-served more frequently in old blood speci-mens.


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Hematology

Color 9.5 Normal lymphopoiesis.

a,b) Lymphoblasts with a round to slightlyirregular nucleus, fine chromatin pattern,multiple nucleoli and basophilic cytoplasm.These cells may be found within the bonemarrow in health, but suggest neoplasiawhen observed within the peripheral blood.

c) Small lymphocyte with a scant rim ofbasophilic cytoplasm. These cells are oftenconfused with thrombocytes by an inexpe-rienced microscopist.

d) Typical small lymphocyte with an eccen-tric nucleus and scant basophilic cyto-plasm.

e) Small lymphocyte with cytoplasmicazurophilic granules. In mammals, thesecells are “killer” lymphocytes.

f) Large lymphocytes are observed infre-quently in the blood. Usually, monocytesare misidentified as large lymphocytes.

g,h) Degenerating lymphocytes with cyto-plasmic blebs or broad pseudopodia. Thesecells are seen most frequently in old bloodspecimens.

i) Plasma cells are the ultimate expressionof a B-lymphocyte. These cells are round-to-rectangular with an eccentric nucleus,condensed chromatin pattern, abundantroyal-blue cytoplasm and a pale golgi zone.Plasma cells are observed frequently in cy-tologic preparations, but are rare in bloodsmears.

j) An immunocyte is an antigenicallystimulated lymphocyte. These large cellscontain a round-to-scalloped nucleus, con-densed chromatin pattern and dark-bluecytoplasm. Scattered immunocytes may beobserved in the blood smear following anti-genic stimulation from immunization ordisease.

Color 9.6 Monocyte development.

a-c) Monocytes with oval to slightly in-dented nuclei, a variable nuclear-to-cyto-plasmic ratio and gray cytoplasm. Thesecells often are misidentified as lympho-cytes.

d,e) Mature monocytes with an indented tobilobed nucleus, abundant gray cytoplasmand occasional pseudopodia.

f,g) Monocytes with cytoplasmic vacuola-tion or increased dust-like eosinophilic cy-toplasmic granules. Cytoplasmic vacuola-tion usually occurs if the blood specimen isallowed to stand before making bloodsmears. The eosinophilic granules arelysosomes.

h,i) Two degenerating monocytes are pre-sent. The first monocyte has nuclear edemaand cytoplasmic vacuolation. The secondmonocyte has broad pseudopodia or cyto-plasmic blebs. These cells are observedmost frequently within old blood speci-mens.


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Leukocyte MorphologyThe granulocytic leukocytes of birds are heterophils,eosinophils and basophils.6,18,34,36,44 The heterophil isa round cell with distinct eosinophilic cytoplasmicgranules (Color 9.9). These granules are oval to spin-dle-shaped and often contain a distinct refractilebody in the center of the granule. The mature hetero-phil nucleus is lobed, usually containing fewer lobesthan mammalian neutrophils (Color 9.8). The nu-clear chromatin contains heavy chromatin clumping.The cytoplasm of normal mature heterophils is color-less and nonvacuolated (Color 9.8).

Avian eosinophils are round granulocytes and containdistinct round-to-oval cytoplasmic granules that lackthe central refractile body seen in heterophil gran-ules (Color 9.9). The cytoplasmic granules of eosino-phils typically stain brighter or differently from hetero-phil granules in the same blood film. The intenseeosinophilic appearance of eosinophil granules is mostlikely related to the high concentration of arginine.19

The cytoplasm of avian eosinophils stains clear blue.The eosinophil nucleus is lobed and generally stainsdarker than the nuclei of heterophils (Color 9.9).There is variation in the morphologic appearance ofthe eosinophils of several avian species.34,41

The normal basophil is slightly smaller than theheterophil and has a colorless cytoplasm that con-tains strongly basophilic granules (Color 9.10). Thesegranules often dissolve or coalesce in alcohol-basedstains, such as the Romanowsky stains. Avian baso-phils have round-to-oval, non-lobed nuclei that areoften hidden by cytoplasmic granules (Color 9.10).

The mononuclear leukocytes found in the peripheralblood of birds are lymphocytes and monocytes (Color9.5, 9.6). The mature avian lymphocytes are roundcells that frequently “mold” around adjacent cells inthe blood film. These cells have high nucleus to cyto-plasm (N:C) ratios. The nucleus is usually centrallypositioned and round with a scant amount of homo-geneous blue cytoplasm appearing as a small bandsurrounding the nucleus (Color 9.5). Avian lympho-cytes often vary in size, and the larger lymphocytesthat have pale-staining nuclei may be confused withmonocytes. The nuclear chromatin of mature lym-phocytes is densely clumped. Occasionally, the cyto-plasm of small mature lymphocytes may contain ir-regular projections.

Monocytes are the largest leukocytes found in theperipheral blood films (Color 9.6). They vary in shapefrom round to ameboid. Monocytes have an abundant

amount of cytoplasm compared to lymphocytes. Thecytoplasm generally stains darker than the cyto-plasm of normal lymphocytes. The cytoplasm ofmonocytes has a finely granular, blue-gray appear-ance and often contains vacuoles. Often two distinctcytoplasmic zones can be seen in monocytes: a light-staining area adjacent to the nucleus and a darkerstaining area on the periphery. The cytoplasm ofmonocytes may occasionally contain fine, dust-likeeosinophilic granules. The nucleus of monocytes gen-erally contains less nuclear chromatin clumping ascompared to mature lymphocytes. The shape of themonocyte nucleus is variable, ranging from round tobilobed.

On occasion, abnormal-appearing leukocytes arefound in the peripheral blood films of birds (Color9.5). Immature heterophils are abnormal findings inavian blood films, and their appearance has beendescribed in the evaluation of avian hematopoietictissue at the end of this chapter. The immaturestages most commonly found are heterophil myelo-cytes and metamyelocytes. In general, immatureheterophils have increased cytoplasmic basophilia,nonsegmented nuclei and immature cytoplasmicgranules compared to mature heterophils (Color 9.5).Usually when immature heterophils are found on ablood film, mature heterophils can also be found.

Mature heterophils appear to show toxic changes ina manner similar to the toxic changes identified inmammalian neutrophils.6,12,67 Signs of toxicity in-clude increased cytoplasmic basophilia, vacuolation,abnormal granules, degranulation, and degenerationof the nucleus (Color 9.18). The degree of toxicity isreported subjectively on a scale of +1 to +4, where thelower rating reflects slight change and the higherindicates severe change. A +1 toxic heterophil showsincreased cytoplasmic basophilia (Color 9.8). An

TABLE 9.2 Characteristics of Blood Cells Using Wright’s Stain

Erythrocytes Dark purple nucleus; orange-pink cytoplasm

Heterophils Violet, lobed nucleus; colorless cytoplasm,orange-red, rod-shaped granules in most species

Eosinophils Violet, lobed nucleus; blue cytoplasm; red-orange, round granules in most species

Basophils Purple, non-lobed nucleus; dark purplecytoplasm granules

Monocytes Purple nucleus; abundant, finely granular, blue-grey cytoplasm

Lymphocytes Dark purple, non-lobed nucleus; pale blue, homo-geneous cytoplasm

Thrombocytes Dark purple nucleus; colorless cytoplasm; redgranules


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overall assessment of the staining of the blood filmmust be made to assure the hematologist that thefilm is not overly stained blue, giving the impressionthat the heterophils have increased basophilia. A +2toxic heterophil has increased cytoplasmic baso-philia, vacuolation and partial degranulation (Color9.11). A +3 toxicity shows a deeper cytoplasmic baso-philia, vacuolation and abnormal granulation (Color9.8).

Abnormal granulation is indicated by the presence ofgranules that vary in appearance from the typicalrod-shaped eosinophilic granules (eg, large, pale,round eosinophilic granules and small, deeply baso-philic granules). A +4 toxic heterophil resembles a +3toxic heterophil except the cell nucleus has under-gone karyorrhexis or karyolysis. The number of toxicheterophils present is an indication of severity andsuggestive of duration of an inflammatory response.A slight number (25% or less) of toxic heterophilsmay be present in the early stages of disorders re-sponsible for their occurrence. As the disorder be-comes increasingly severe, the number of toxic het-erophils will increase. A marked number (greaterthan 25%) of toxic heterophils is common in birdsshowing this heterophil abnormality. It is commonfor birds with toxic heterophil changes to have all oftheir heterophils affected on the blood film. Clini-cally, these birds will be severely compromised.

Cytologic indications for reactivity in lymphocytesinclude increased cell size, increased cytoplasmic ba-sophilia, the presence of azurophilic cytoplasmicgranules and smooth nuclear chromatin (Color 9.12).Blast-transformed lymphocytes have a deeply baso-philic cytoplasm and smooth nuclear chromatin(Color 9.5). Blast-transformed lymphocytes may alsohave nucleoli and distinct Golgi. Occasionally,plasma cells can be found in the peripheral blood ofbirds. These are relatively large lymphocytes witheccentric, mature-appearing nuclei; abundant,deeply basophilic cytoplasm; and prominent Golgiadjacent to the nucleus. Lymphocytes containingazurophilic granules (large purple cytoplasmic gran-ules) are considered abnormal in birds. Lymphocyteshaving scalloped cytoplasmic margins are found oc-casionally in avian blood films; however, large num-bers of these cells are considered abnormal.5,62 Imma-ture lymphocytes in the peripheral blood films arealso considered to be abnormal (Color 9.5).

An occasional monocyte having a few cytoplasmicvacuoles is normal, but the presence of large numbersof highly vacuolated monocytes is abnormal.

Cells that contain large granules that fill the cyto-plasm are frequently found in blood films of birds.Often these granules fail to stain or may stainblue.6,34,41 These cells are common in blood films ofsome species of birds (eg, cockatoos) and suggesteither staining artifact or represent variation owingto different cytochemical properties of these cellscompared to other avian species. The differential forthe type of cell involved includes eosinophils, baso-phils and rarely, Mott cell variant of plasma cells.

Careful examination of the blood film most oftenreveals normal staining basophils and no evidence oflymphoid reactivity (which may support the possibil-ity of Mott cells being present), but there are noeosinophils present that stain normally. Based onthese characteristics, the majority of these cells havebeen identified as eosinophils. 6, 34, 41

Thrombocyte MorphologyBirds have nucleated cells (thrombocytes) ratherthan cytoplasmic fragments as platelets that partici-pate in blood coagulation. Thrombocytes are derivedfrom a distinct line of cells found in hematopoietictissue. Mature thrombocytes are small oval cells thatappear more rounded than the erythrocytes (Color9.4). The nucleus is pyknotic and the cytoplasm iscolorless in mature cells. The cytoplasm may containone or more red granules and small vacuoles or clearspaces (Color 9.4). Thrombocytes, like mammalianplatelets, tend to clump in blood films. Thrombocytesare differentiated from small, mature lymphocytesby having a colorless, nonhomogeneous cytoplasm;small, round, red cytoplasmic granules; and asmaller N:C ratio. Small mature lymphocytes havehigh N:C ratios with a scant amount of blue, homoge-nous cytoplasm (Color 9.5).

Abnormal thrombocyte cytology includes the pres-ence of reactive and immature thrombocytes. Reac-tive thrombocytes are usually found in aggregates,have a diffusely eosinophilic cytoplasm (suggestingrelease of chemicals from the granules) and irregularcytoplasmic margins. Reactive thrombocytes tend tobe more spindle-shaped than nonreactive thrombo-cytes (Color 9.4).

Immature stages of thrombocytes are occasionallyfound in the blood film of birds (Color 9.4). Themid-immature and late-immature thrombocytes aremost often seen when immature cells are present.


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Interpretationof the Avian Hemogram

Interpretation of the Erythron

The normal PCV of birds ranges between 35 and 55percent. A PCV less than 35 percent is indicative ofanemia, and a PCV greater than 55 percent is sug-gestive of dehydration or polycythemia. An increasein red cell polychromasia is indicative of red bloodcell regeneration (Color 9.17). In normal birds, thenumber of polychromatic erythrocytes (or reticulo-cytes) found in the peripheral blood film ranges be-tween one and five percent of the erythrocytes. Ananemic bird with a five percent or less degree ofpolychromasia (or reticulocytosis) is respondingpoorly to the anemia or there has not been enoughtime for the bird to demonstrate a significant re-sponse. An anemic bird showing a ten percent orgreater degree of polychromasia is exhibiting a sig-nificant regenerative response. The presence of im-mature erythrocytes (eg, rubricytes) in the periph-eral blood along with an increase in polychromasia isindicative of a marked regenerative response.

Some common causes of anemia in birds are dis-cussed in Table 9.3.

Hypochromasia can be associated with certain nutri-tional deficiencies in birds, especially iron deficiency.Hypochromasia has also been seen in lead toxico-sis.34,42 Lead toxicosis may also create a dichotomouspopulation of erythrocytes in the blood film of anonanemic bird. In such cases, small senescent, ma-ture erythrocytes with pyknotic nuclei and youngerythrocytes (eg, rubricytes) are present in the bloodfilm without the appearance of normal, matureerythrocytes. This condition resembles the inappro-priate release of nucleated erythrocytes in the bloodof nonanemic dogs suffering from lead poisoning.Basophilic stippling in the cytoplasm of erythrocytesis a rare finding with lead poisoning in birds.42 Baso-philic stippling may be associated with erythrocyteregeneration and hypochromic anemia.

Polycythemia is rarely reported in birds.74 Increasesin the PCV (relative polycythemia) are usually asso-ciated with dehydration in birds; however, absolutepolycythemia can also occur. The conditions oftenassociated with absolute polycythemia in mammals

are expected to be the causes of this condition in birdsas well.

Interpretation of the Leukogram

There is wide variation in the normal leukogramsamong birds of the same species. Therefore, values ofdiagnostic importance must differ greatly from nor-mal reference intervals, which are generally muchbroader than those obtained from domestic mam-mals. Preparing normal reference values on healthyindividual birds is the best method for evaluatingblood parameters of a bird during illness. When thesespecific values have not been determined, the avianclinician must rely on reference intervals obtainedfrom several birds that are presumed to be healthy.It is best to use values obtained in the laboratory thatroutinely performs the clinician’s avian profiles. Pub-lished values obtained from other laboratories can beused as a guide, but may differ from the avian clini-cian’s routine laboratory.

In general, total leukocyte counts greater than10,000/µl are considered suggestive of leukocytosis intame, adult psittacine birds. The total leukocyte

TABLE 9.3 Causes of Anemia in Birds2,10,11,20,24,32,33,39,48,49,61,63,84

Blood-loss Anemia(Appears regenerative except in the peracute stage)

1. Traumatic injury

2. Parasitism (ticks, Dermanyssus mites, coccidia)

3. Primary coagulopathy (rarely reported in birds)

4. Toxicity resulting in a coagulopathy (aflatoxicosis and coumarinpoisoning)

5. Organic disease (ulcerated neoplasm, gastrointestinal ulcers,organ rupture)

Hemolytic Anemia (Regenerative)

1. Red blood cell parasites (Plasmodium, Aegyptianella and, rarely,Haemoproteus and Leucocytozoon)

2. Bacterial septicemia (salmonellosis and spirochetosis)

3. Toxicity (mustards and petroleum products)

4. Immune-mediated (rarely reported in birds)

Depression Anemia (Nonregenerative)

1. Chronic diseases (tuberculosis, chlamydiosis, aspergillosis, neo-plasia)

2. Hypothyroidism

3. Toxicity (lead poisoning and aflatoxicosis)

4. Nutritional deficiencies (iron and folic acid deficiencies)

5. Leukemia (lymphoid leukemia and erythroblastosis)


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count in the blood of normal psittacine birds notaccustomed to handling may be high (greater than10,000/µl) owing to a physiologic leukocytosis. Thegeneral causes of a leukocytosis include infection(general or localized), trauma, toxicities, hemorrhageinto a body cavity, rapidly growing neoplasms andleukemias. The leukocyte differential aids in the as-sessment of the leukocytosis. Because a leukocytosisis often caused by inflammation, a heterophilia isusually present.

Although avian heterophils lack the myeloperoxi-dase and alkaline phosphatase of mammalian neu-trophils, studies of their ultrastructure, cytochemis-try and function suggest they perform a similarfunction in the inflammatory response.14,35,46,52,60 Themagnitude of the heterophilia usually indicates themagnitude or severity of the initiating inflammatoryprocess. Although avian heterophils do not producehydrogen peroxide during phagocytosis, they do con-tain lysosomal enzymes and have a bactericidal func-tion.23,50,59,60,75,76 A leukocytosis and heterophilia canbe associated with infectious agents (eg, bacteria,fungi, chlamydia and parasites) and noninfectiousetiologies (eg, traumatic injury and toxicities). Aslight to moderate leukocytosis, heterophilia andlymphopenia can result from either an exogenous orendogenous excess of glucocorticosteroids (stress re-sponse).3,15,31,56,69,83 Species that normally have highnumbers of circulating lymphocytes may develop aleukopenia and lymphopenia in the initial stress re-sponse, but up to 12 hours later show a leukocytosisand heterophilia.15 Birds that normally have highernumbers of circulating heterophils than lymphocytesoften show a less dramatic change in the leukograminitially. A marked leukocytosis and heterophilia areoften associated with chlamydiosis, avian tuberculo-sis and aspergillosis.

Immature heterophils occur rarely in the peripheralblood of most species of birds. When present, theygenerally represent an overwhelming peripheral de-mand for heterophils and a depletion of the maturestorage pool in the hematopoietic tissues.73 The pres-ence of immature heterophils in the peripheral bloodusually indicates a severe inflammatory response,especially in association with a leukopenia (degen-erative left shift) (Color 9.18). A marked number ofimmature heterophils may be associated with agranulocytic leukemia, a rare condition in birds.

The presence of toxic heterophils is also uncommonin the peripheral blood of birds. When present, theysuggest the presence of a septicemia or toxemia (es-

pecially associated with bacterial toxins affecting themicroenvironment of the hematopoietic tissue). Thegreater the degree of toxicity, the more severe thecondition. The presence of a marked number of +4toxic heterophils indicates a poor prognosis for sur-vival in birds (Color 9.18).

The general causes of leukopenias in birds are deple-tion of peripheral leukocytes and depression or de-generation of leukopoiesis. Leukopenias associatedwith heteropenias can be associated with certainviral diseases (eg, Pacheco’s disease virus) and over-whelming bacterial infections.58,64 A leukopenia andheteropenia associated with immature heterophils issuggestive of exhaustion of the mature heterophilstorage pool owing to excessive peripheral demandfor heterophils. A depression of the hematopoietictissue is indicated by a leukopenia, heteropenia andfew, if any, immature heterophils. A degenerativeresponse is indicated by the presence of a leukopenia,heteropenia, immature heterophils and toxic hetero-phils. This degenerative response can be differenti-ated from depletion only by the presence of toxicheterophils or by following the decreasing leukocytecount with serial leukograms. Leukopenias associ-ated with lymphopenias have been reported in earlyresponse to corticosteroids in some species ofbirds.5,15 A lymphopenia also may be expected withcertain viral diseases; however, viral causes have notbeen well documented in birds.

A lymphocytosis may be expected with antigenicstimulation associated with certain infections. Thepresence of many reactive lymphocytes is also sug-gestive of antigenic stimulation. An occasional reac-tive lymphocyte may be found in the blood film ofnormal birds. A marked lymphocytosis with or with-out the presence of immature lymphocytes can occurwith lymphocytic leukemia. A marked lymphocytosis,with the majority of cells appearing as small maturelymphocytes with scalloped cytoplasmic margins, issuggestive of lymphoid neoplasia.5,6,62

A monocytosis can be found with certain diseasesthat produce chemotactic agents for monocytes.These conditions include avian chlamydiosis, myco-tic and bacterial granulomas and massive tissue ne-crosis.32 It should be emphasized that although thesedisorders can create a peripheral monocytosis, it maynot always occur. A monocytosis can also occur inbirds on a zinc-deficient diet.82

The function of the avian eosinophil is unclear.54

Although this avian granulocyte was given the name


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eosinophil, there is evidence that its function maydiffer from the mammalian eosinophil. Thus, condi-tions responsible for inducing avian eosinophiliasmost likely differ from those causing mammalianeosinophilias. Eosinophilias associated with gastro-intestinal nematode infections have occasionallybeen reported; however, it has been difficult to inducethis condition experimentally using parasite anti-gens.47 Studies suggest that avian eosinophils mayparticipate in delayed (Type IV) hypersensitivity re-actions.51 Idiopathic eosinophilias occur sporadicallyin birds, and more research is needed to clarify themeaning of this condition.

As with avian eosinophils, the exact function of baso-phils in birds is unknown. Avian basophils are simi-lar to mammalian basophils in their ability to pro-duce, store and release histamine.9 Basophils appearto participate in the initial phase of the acute inflam-matory response in birds, but this is not alwaysreflected as a basophilia in the leukogram.8,54 Be-cause basophils appear to play a role in early inflam-mation and possibly hypersensitivity reactions inbirds, a peripheral blood basophilia may suggest thepresence of these conditions.

Interpretation of Thrombocyte Changes

Avian thrombocytes play a primary role in hemosta-sis in a manner similar to mammalian platelets.They may also have a phagocytic function and par-ticipate in removing foreign material from theblood.19,30 A normal thrombocyte count ranging be-tween 20,000 and 30,000/µl of blood or 10 to 15/ 1000erythrocytes can be used as a general reference formost birds.6,19 Thrombocytopenias are usually indica-tive of excessive peripheral demand for thrombo-cytes, although a depression in thrombopoiesisshould be considered. Thrombocytopenias are oftenseen with severe septicemias, where a combination ofexcessive peripheral demand for thrombocytes anddepression of thrombocyte production may occur. Athrombocytosis may reflect a rebound response fol-lowing hemorrhage or recovery from other conditionsassociated with excessive utilization of thrombo-cytes. Often a regenerative response can be detectedby the presence of immature thrombocytes in theperipheral blood film.

Identification of Common Blood Parasites

For a complete review of avian parasites see Chapter36.

The diagnosis of Haemoproteus is made by the de-tecting characteristic intraerythrocytic gametocytesin peripheral blood films. Only the gametocyte stageof this organism appears in the peripheral blood,whereas schizogony occurs in the tissues (eg, lung,spleen and liver).70 The mature gametocyte containsyellow-to-brown, refractile pigment granules (Color9.21). The typical mature gametocyte occupiesgreater than 50 percent of the red cell cytoplasm,partially encircles the host cell nucleus forming theclassic “halter-shape” and causes little displacementof the red cell nucleus. It is rare for more than onemature gametocyte to occur in a cell. Macrogameto-cytes stain blue with Romanowsky stains and havepigment granules dispersed throughout the cyto-plasm of the parasite. The smaller microgametocytesstain pale blue to pink with pigment granules ap-pearing in spherical aggregates. If blood containingHaemoproteus organisms is allowed to stand at roomtemperature for a few hours prior to preparing ablood film, gametes may be released from the cellsand found in the extracellular spaces of the bloodfilm. The macrogametes appear as spheres that re-semble the macrogametocytes within the red cellcytoplasm. The microgametes appear as small spin-dle-shaped structures. When gametes are found, itshould be considered as an artifact of blood filmpreparation because these structures normally leavethe host red cell following ingestion by the interme-diate insect host (hippoboscid flies).

Leucocytozoon is easily identified from blood filmsbecause it grossly distorts the host cell (usually im-mature erythrocytes) that it parasitizes. Like Haem-oproteus, only the gametocyte stage of Leucocytozoonoccurs in the peripheral blood of birds (Color 9.22).70

The large, round-to-elongated gametocytes cause thehost cell to enlarge and appear to have two nuclei: thehost cell nucleus pushed to the margin of the cell andthe parasite nucleus, a pale-pink nucleus within theparasite. The parasitized cell usually has taperedends with the remnants of the cell membrane trailingaway from the cell. The macrogametocyte stainsdark blue with a condensed nucleus and occasionalcytoplasmic vacuoles. The microgametocyte stainslight blue with a diffuse, pale-pink nucleus. Gameto-cytes of Leucocytozoon lack the refractile pigmentgranules found in Haemoproteus.

The intraerythrocytic gametocytes of Plasmodiumspp. are often confused with those of Haemoproteusspp. because they also contain refractile pigmentgranules. However, Plasmodium gametocytes usu-ally occupy less than 50 percent of the host cell


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cytoplasm, and those of some species alter the posi-tion of the red cell nucleus (Color 9.26). Two keyfeatures that aid in the detection of Plasmodium arethe presence of schizogony in the peripheral bloodand gametocytes or schizonts in blood cells otherthan erythrocytes.70 Schizonts appear as round-to-oval intracytoplasmic inclusions that contain dark-staining merozoites. The number of merozoites pro-duced depends upon the species of Plasmodium. Aswith Haemoproteus, Plasmodium macrogametocytesstain darker than the microgametocytes. Both Plas-modium and Haemoproteus infections may revealsmall, ring-like forms (trophozoites) in the cytoplasmof infected erythrocytes. In rare cases, only theseforms may be seen, and it is impossible to identify theparasite involved. In such situations, resampling aweek or more later will often reveal the developedforms having the characteristics described for eitherPlasmodium or Haemoproteus. Mosquitos (Culex andAedes spp.) are the intermediate hosts for Plasmo-dium.

Microfilaria are frequently found in the peripheralblood of a variety of birds.

Atoxoplasma sp. is identified by its characteristicsporozoite within the cytoplasm of mononuclear leu-kocytes, especially lymphocytes (Color 9.27).40 Thesporozoites appear as pale-staining, round-to-ovalintracytoplasmic inclusions that compress the hostcell nucleus and create a characteristic crescentshape to the nucleus. This organism can be found inthe per-ipheral blood films or imprints of tissues suchas the lung, liver and spleen.

Aegyptianella can occur within the cytoplasm in oneof three forms: 1) anaplasma-like initial bodies ap-pearing as small (less than one micrometer in diame-ter), round, basophilic inclusions; 2) intermediatestages resembling Babesia and measuring betweenone and two micrometers in diameter; and 3) large,round-to-elliptical forms measuring between two andfour micrometers in length.70 Aegyptianella spp. areconsidered to be pathogenic to many species of birds(primarily Passeriformes) but may be difficult to de-tect because the parasitemia stage of the disease isoften very short and easily missed.

Evaluation of theHematopoietic Tissue

Hematopoiesis occurs primarily in the bone marrowof post-hatch birds; however, hematopoietic activitymay also be found in various internal organs (eg,liver and possibly spleen).4,19 A bone marrow sampleshould be obtained for cytologic evaluation in avianpatients with persistent nonregenerative anemia,thrombocytopenia, panleukopenia and heteropenia.Bone marrow evaluation is also indicated for sus-pected cases of leukemia or if unexplained abnormalcells are found in the peripheral blood. An evaluationof the hemogram should accompany any bone mar-row evaluation to properly assess hematopoiesis.

Bone Marrow Collection

In general, the proximal tibiotarsus just below thefemoral-tibiotarsal joint (knee) is the preferred sitefor bone marrow collection in most birds.6,79 Aftersurgical preparation of the skin either on the cranialor medial aspect of the proximal tibiotarsus, a smallstab incision through the skin is made using a scalpelblade. A bone marrow aspiration biopsy needle ispushed through the thin cortex and into the marrowspace using clockwise-counterclockwise rotationalmovements. Once the needle has entered the marrowspace, the stylet is removed from the needle and asyringe is attached to gently aspirate a small amountof marrow into the needle lumen. Excessive pressureduring aspiration should be avoided to prevent per-ipheral blood contamination of the sample. Followingaspiration, the needle is removed from the bone andthe syringe is detached from the needle. The syringeis filled with air and reattached to the needle hub.Using the air in the syringe, the marrow within theneedle lumen is forced onto a microscope slide. Asecond slide is placed across the first on top of themarrow sample. As the two slides are pulled horizon-tally apart, two marrow films are made for cytologicexamination.

Marrow can also be obtained from the sternum (keel)of some birds with the biopsy needle inserted into thewidest part of the sternal ridge.

Bone marrow biopsy needles commonly used includepediatric Jamshidi bone marrow biopsy-aspiration


191

Hematology

Color 9.7 Early myeloblast with a fine chromatin pat-tern, nucleolar ring and dark-blue cyto-plasm. The myeloblast is the progenitor cellfor the heterophil (Color 9.8), eosinophil(Color 9.9) and basophil (Color 9.10).

Color 9.8 Heterophil development.

a) Myeloblast with fine chromatin pattern,nucleolus and light-blue cytoplasm.

b) Promyelocyte with oval nucleus, pale-blue cytoplasm and round to slightly ir-regular metachromatic cytoplasmic gran-ules.

c) Heterophil myelocyte with an oval nu-cleus and a mixture of metachromatic gran-ules and scattered round-to-rod-shapedeosinophilic granules.

d) Heterophilic metamyelocyte with aslightly indented nucleus.

e) Heterophilic band with U-shaped nu-cleus.

f,g) Mature, segmented heterophils withnumerous needle-shaped granules. Thegranules may obscure nuclear detail, mak-ing assessment of lobulation difficult.

h) Heterophil with mild toxic changes in-cluding cellular swelling, partial degranu-lation and a basophilic cast to the cyto-plasm.

i,j) Toxic heterophils with progressivegranule dissolution leaving the round gran-ule core intact. The cell is slightly swollenand has basophilic cytoplasm. This cell mayoccasionally be confused with an eosino-phil, except for retention of a few needle-shaped granules. Stain-induced heterophildegranulation is not associated with cyto-plasmic basophilia.

k) Heterophil with non-staining cytoplas-mic granules. This may be an artifact re-sulting from exposure of the blood smear toformalin vapor during transport or mailing.

l) Disrupted heterophil showing typicalneedle-shaped granules. These smudge

cells are ignored during the leukocyte dif-ferential count.

m) Smudged heterophil with dissolution ofthe outer granule matrix leaving intactround central cores. Granule dissolutionmay cause an eosinophil-like appearance;however, smudge cells are ignored duringthe leukocyte differential count.

Color 9.9 Eosinophil development.

a) Late myeloblast with condensing chro-matin pattern and light-blue cytoplasm.

b) Eosinophilic myelocyte with an oval nu-cleus and scattered, variably sized, round,secondary granules.

c) Eosinophilic myelocyte with a slightlyindented nucleus and round, red-orangegranules.

d) Eosinophilic band with U-shaped nu-cleus and round, red-orange granules.

e) Segmented eosinophil with a lobulatednucleus and abundant secondary (specific)granules.

f) Disrupted or smudged eosinophil. Thesecells are ignored during the leukocyte dif-ferential count.

Color 9.10 Basophil development.

a,b) Basophilic myelocytes with round nu-clei and round, variably sized, metachro-matic granules.

c,d) Basophils with round, intenselystained, metachromatic granules. Basophilgranules have high affinity for Roma-nowsky stain, often resulting in poor stain-ing of the cell nucleus. In addition, cyto-plasmic granules may obscure nucleardetail.

e) Basophil degranulation may occur withdisease or as an artifact of blood smearstaining.

f) Disrupted basophil (smudge cell). Thesecells are ignored during the leukocyte dif-ferential count.


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Hematology

All photographs on this page were providedcourtesy of Terry W. Campbell.

Color 9.11 An adult female Red-tailed Hawk was pre-sented with clinical signs of lethargy, amarked reduction of the pectoral musclemass and diarrhea. A diagnosis of salmo-nellosis was made based upon necropsy andbacterial culture two days later. The hemo-gram revealed: PCV = 28%, RBC =1,950,000/ mm3, WBC = 31,876/ mm3, het-erophils = 28,050/ mm3, lymphocytes =1,594, monocytes = 638/ mm3 and eosino-phils = 1,594/ mm3. A toxic heterophil (2+toxicity) and two eosinophils are shown.Wright’s stain.

Color 9.12 A Blue-fronted Amazon Parrot was pre-sented with multifocal, depigmented, non-raised lesions involving the skin on the feet.Serum chemistries and erythrocyte pa-rameters were within normal limits. Theleukogram revealed the following: WBC =7,543/ mm3, heterophils = 1,886/ mm3, lym-phocytes = -4,601/ mm3, monocytes = 830/mm3 and eosinophils = 226/ mm3. Typicalreactivity of lymphocytes demonstrated byWright’s stain.

Color 9.13 A critically ill Green-cheeked Amazon Par-rot was presented for evaluation. The birdwas extremely weak and unable to perch.Severe uveitis was present in both eyes. Thebird was housed in a room where varnishwas being applied to furniture. Importanthematologic findings included: PCV = 40%,total protein = 5.0 gm/dl, WBC = 16,140/mm3, heterophils = 13,880/ mm3 (10% of theheterophils were myelocytes and 20% weremetamyelocytes), lymphocytes = 807/ mm3,monocytes = 1,453/ mm3 and thrombocy-topenia. A heterophilic myelocyte is demon-strated by Wright’s stain.

Color 9.14 A juvenile Red-tailed Hawk was presentedwith marked reduction of the pectoral mus-cle mass. Radiographic evaluation revealeda fracture of the left coracoid bone. Thehematologic findings included: PCV = 37%,Hb = 11.5 g/dl, RBC = 2,250,000/ mm3, 4%polychromasia, total protein = 2.6 g/dl,WBC = 32,932/ mm3, heterophils = 25,687/mm3, lymphocytes = 1,976/ mm3, mono-cytes = 988/ mm3, eosinophils = 3,952/ mm3

and basophils = 392/ mm3. A heterophil,eosinophil, basophil and Haemoproteus ga-

metocyte (0.7% of the erythrocytes con-tained these gametocytes) are demon-strated by Wright’s stain.

Color 9.15 A Blue and Gold Macaw was examined 93hours following severe blood loss that re-sulted from a traumatic injury to the foot.The hemogram revealed: PCV = 21%, RBC= 1,660,000/ mm3, Hb = 5.8 g/dl, WBC =19,457/ mm3, heterophils = 12,255/ mm3,lymphocytes = 6,615/ mm3, monocytes = 97/mm3 and basophils = 389/ mm3. Twenty-two percent of the erythrocytes exhibitedpolychromasia, and an occasional imma-ture erythrocyte was noted. Polychromasiaand a round, immature erythrocyte (earlypolychromatic rubricyte) are demonstratedby Wright’s stain.

Color 9.16 The hemogram from a Harris Hawk thatdied three days later from an acute pneu-monia revealed a moderate anemia, leuko-cytosis, heterophilia and left shift. Markedanisocytosis and polychromasia are illus-trated as well as a binucleate erythrocyte(which were frequently seen in the bloodfilm). A heterophil, eosinophil, two lympho-cytes, a thrombocyte and an immatureerythrocyte (basophilic rubricyte) can alsobe seen by Wright’s stain.

Color 9.17 A juvenile Bar-headed Goose was pre-sented for weakness and weight loss. Sixother geese in the group appeared normal.The hemogram revealed: PCV = 28%, Hb =9.0 g/dl, RBC = 1,950,000/ mm3, 11% poly-chromasia, WBC = 39,556/ mm3, hetero-phils = 31,645/ mm3, lymphocytes = 5,645/mm3, monocytes = 1,187/ mm3 and baso-phils = 791/ mm3. Blood lead levels werenormal. The erythrocyte morphology re-vealed polychromasia, hypochromasia andstippled basophilia. Wright’s stain.

Color 9.18 A juvenile Red-tailed Hawk was presentedwith open fractures of the left radius andulna and poxvirus lesions along the mar-gins of the beak and on the feet. The birdwas extremely depressed and died within24 hours of presentation. The hemogramshowed: PCV = 35%, Hb = 11 g/dl, RBC =3,020,000/ mm3, WBC = 42,240/ mm3,monocytes = 3,802/ mm3, eosinophils =1,267/ mm3 and basophils = 422/ mm3. Themajority of the heterophils appeared ex-tremely toxic (4+). Wright’s stain.

Color 9.19 An adult African Grey Parrot was pre-sented with a history of an intermittentseizure disorder and was successfullytreated for hypocalcemia. The hemogramrevealed: PCV = 49%, RBC = 2,940,000/mm3, WBC = 15,740/ mm3, heterophils =6,453/ mm3, lymphocytes = 8,814/ mm3,monocytes = 315/ mm3 and eosinophils =157/ mm3. Eosinophils with large, blue cy-toplasmic granules are demonstrated byWright’s stain.

Color 9.20 An adult Red-tailed Hawk was presentedwith multiple gunshot wounds and openfractures involving the right radius andulna. The hemogram revealed: PCV = 16%,Hb = 5.3 g/dl, RBC = 1,300,000/ mm3, WBC= 15,740/ mm3, heterophils = 6,453/ mm3,lymphocytes = 8,814/ mm3 and monocytes= 472/ mm3. The reticulocyte count was20% and there were many immature eryth-rocytes present. A mid-polychromatic ru-bricyte is demonstrated by Wright’s stain.

Color 9.21. Haemoproteus sp. gametocytes in a bloodfilm stained with Wright’s stain from aGreat Horned Owl.

Color 9.22 Haemoproteus and Leukocytozoon spp. ga-metocytes in a blood film stained withWright’s stain from a Great Horned Owl.

Color 9.23 Haemoproteus sp. microgametes in a bloodfilm stained with Wright’s stain from aScreech Owl.

Color 9.24 Plasmodium sp. gametocytes and schizontsin the cytoplasm of erythrocytes from aSkua stained with Wright’s stain.

Color 9.25 Plasmodium sp. gametocytes in the cyto-plasm of erythrocytes and extracellularspace in a Wright’s-stained blood film froma Skua.

Color 9.26 Plasmodium sp. gametocyte in the cyto-plasm of a thrombocyte in a Wright’s-stained blood film from a Mississippi Kite.

Color 9.27 Numerous intracytoplasmic Atoxoplasmasp. inclusions within lymphocytes from alung imprint of a Siskin.


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needlesj and disposable Jamshidi Illinois-Ster-nal/Iliac aspiration needles. Disposable spinal need-les can be used to sample small birds because theycontain a stylet to facilitate passage of the needlethrough the cortex without occlusion of the needlelumen with bone.

Erythropoiesis

The terminology describing the different stages oferythrocytic development varies in the litera-ture.6,19,34,44 In general, there are six recognizablestages involved in red cell development. The earliestrecognizable stage is the rubriblast (proerythroblast)(Color 9.1). This cell has large, prominent nucleoli ornucleolar rings. The round nucleus is centrally posi-tioned within the cell. The coarsely granular chroma-tin is atypical for most blast-type cells. The abundantcytoplasm stains deeply basophilic and contains fine,clear spaces (mitochondrial spaces). Rubriblastshave high N:C ratios, typical of immature cells.

The second stage in erythrocyte development is theprorubricyte (basophilic erythroblast). This cell re-sembles the rubriblast, but the nucleoli are eitherabsent or indistinct, and the cytoplasm lacks themitochondrial spaces of the rubriblast (Color 9.1).

The next three stages are the rubricyte stages. Theseare round-to-slightly oval cells that are smaller thanrubriblasts and prorubricytes. Rubricytes are di-vided into three groups based upon their appearancein the cytologic sample. In order of increasing matu-ration they are the basophilic rubricyte (early poly-chromatic erythroblast), early polychromatic rubri-cyte (late polychromatic erythroblast) and latepolychromatic rubricyte (orthochromic erythroblast).The basophilic rubricyte has a high N:C ratio, homo-geneous basophilic cytoplasm and round nucleuswith distinct chromatin clumping.

The early polychromatic rubricyte appears smallerthan the basophilic rubricyte and is the first stage ofred cell development in which hemoglobinization ofthe cytoplasm can be detected with Wright’s stain.The hemoglobin gives the cytoplasm a gray, slightlyeosinophilic appearance. The nucleus appearssmaller with increased density, and the cytoplasm ismore abundant when compared to the previous stageof development. The late polychromatic rubricyte isa round-to-slightly oval cell with an eosinophilicgray-to-weakly eosinophilic cytoplasm (Color 9.1).This cell appears to have increased cytoplasmic vol-ume when compared to the previous stage, and the

nucleus may appear oval with irregularly clumpedchromatin. The penultimate stage of erythrocyte de-velopment is the polychromatic erythrocyte, whichresembles the oval, mature erythrocyte except for thecytoplasmic basophilia and nuclear chromatin thatappear less condensed than the pyknotic nucleus ofthe mature cell (Color 9.1).

Granulopoiesis

Avian granulopoiesis appears to follow developmen-tal stages similar to those described for mammaliangranulocytes.6,34,44 These stages are the myeloblast(granuloblast), progranulocyte (promyelocyte),myelocyte, metamyelocyte and mature granulocyte.

Myeloblasts are large, round cells with a narrow rimof cytoplasm that appears less basophilic than that ofrubriblasts.44 In general, the nucleus is round with adelicate reticular chromatin pattern and distinct nu-cleoli. No cytoplasmic granules are present. Themyeloblast stage is common to all the granulocytes(Color 9.7).

The next stage toward maturation is the progranulo-cyte. These are large cells with cytoplasmic granulesand light blue cytoplasm. The granules are variablein appearance. An attempt has been made to differ-entiate progranulocytes into their respective granu-locytic cell lines based upon the appearance of thecytoplasmic granules.44 Heterophil progranulocytescontain orange spheres (primary granules) or ringsand dark magenta granules or rings. The ring formsare thought to be characteristic of the heterophil cellline. Eosinophil progranulocytes lack the dark ma-genta granules and rings and contain only brightlystaining orange spheres (primary granules). Baso-phil progranulocytes have magenta granules thatappear smaller than those of heterophil progranulo-cytes and have fewer ring forms. The nucleus ofprogranulocytes is typically eccentric in its cellularposition, has a delicate reticular chromatin patternand often has indistinct margins.

The myelocytes are smaller than the progranulocytesand contain the specific granules (secondary gran-ules) for each cell line. Heterophil myelocytes areround cells with light blue cytoplasm containing pri-mary granules, magenta granules and rings and thedefinitive rod-shaped heterophil granules. The de-finitive granules occupy less than 50 percent of thecytoplasmic volume. Eosinophil myelocytes containprimary and secondary granules. The specific or sec-ondary granules occupy less than 50 percent of the


196

cytoplasmic volume. The basophil myelocyte has ma-genta granules and mature basophil granules (secon-dary granules) that occupy less than 50 percent of thecytoplasmic volume. The nucleus of myelocytes isround and has coarsely granular chromatin.

Metamyelocytes resemble myelocytes, except the cellnucleus is slightly indented and may have distinctchromatin clumping. Heterophil metamyelocyteshave definitive, rod-shaped granules that occupygreater than 50 percent of the cytoplasmic volume.The primary granules and magenta spheres andrings may be present, but fewer in number than theprevious stage. The definitive granules of the eosino-phil and basophil series also occupy greater than 50percent of the cytoplasmic volume in their respectivemetamyelocyte stages. The basophil myelocyte nu-cleus remains round.

The granulocytic cell series will occasionally reveal aband cell stage similar to that described in mammal-ian granulocytes. However, the cell nucleus is oftenhidden by the cytoplasmic granules (especially inheterophils), making it difficult to differentiate theband cell from mature cells. Mature avian basophilnuclei do not segment.

Thrombocytopoiesis

The developmental stages involved in thrombopoi-esis are the thromboblast, early-immature thrombo-cyte, mid-immature thrombocyte, late-immaturethrombocyte and mature thrombocyte (Color 9.4).6,44

As the cell develops toward maturity, the cell sizedecreases, the N:C ratio decreases, the nucleus be-comes increasingly pyknotic and cytoplasm becomesless basophilic.

Thromboblasts are large, round-to-ameboid-shapedcells with a narrow rim of deeply basophilic cyto-plasm surrounding the round nucleus. The nuclearchromatin often appears punctate, making nucleolidifficult to detect. The cytoplasm may contain smallclear spaces.

The early-immature thrombocyte is smaller than thethromboblast. It has a round-to-oval nucleus andsmaller N:C ratio than the previous cell. The cyto-plasm is basophilic with small, clear spaces or vacu-oles. The nuclear chromatin is irregularly clumpedand nucleoli are absent. The mid-immature thrombo-cyte appears slightly elongated or irregular with apale blue, vacuolated cytoplasm. Specific red cyto-plasmic granules may be seen at this stage. The

nucleus usually has marked chromatin clumping.The late-immature thrombocyte is an oval cell thathas the appearance of the elongate, mature thrombo-cyte, except the cytoplasm is a pale blue and thenuclear chromatin is less condensed.

Lymphopoiesis

Lymphocyte development may be seen occasionallywhen evaluating hematopoietic tissue (Color 9.5).Three distinctive stages can be identified for lympho-cyte development: lymphoblasts, prolymphocytesand mature lymphocytes. Lymphoblasts are large,round lymphocytes with high N:C ratios. The nucleushas smooth chromatin, in comparison to the maturecell, and contains distinct nucleoli. The cytoplasm oflymphoblasts stains deeply basophilic.

Prolymphocytes resemble lymphoblasts but areslightly smaller, lack nucleoli and have a less baso-philic cytoplasm. In normal lymphoid tissue, lymph-oblasts and prolymphocytes represent less than tenpercent of the lymphoid cells. Thus, the majority ofthe cells should be mature lymphocytes with theheavy nuclear chromatin clumping, high N:C ratioand scant amount of blue, homogeneous cytoplasm.

Other Bone Marrow Cells

Other cells frequently encountered in bone marrowsamples include osteoclasts, osteoblasts, monocytes,plasma cells and mitotic figures. Osteoclasts arelarge, multinucleated cells that are ameboid inshape. The abundant cytoplasm is weakly basophilicand often contains vacuoles and small red granulesof various shapes. The nuclei are round-to-oval andusually contain distinct nucleoli. Osteoblasts arelarge cells that vary in shape. The oval-to-roundnucleus is eccentrically positioned in the cell. Theabundant, foamy, basophilic cytoplasm contains aprominent clear space (Golgi) that is located a dis-tance from the nucleus.

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Elkhart, INh. Coulter Counter, Coulter Electronics, Inci. Unopette System, Becton-Dickinson, Rutherford, NJj. Kormed Corp., Minneapolis, MN


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