HEMATOLOGY Sponse to Blood Loss, Which May Be a Reflection of Differences in Blood Volume Or Extravascular Fluid Depots

valuation of the avian hemogram involves counting the various blood cells per mi- CHAPTER croliter of blood as well as cytologic evalu- E ation of the cells. The techniques involved in the evaluation of the avian hemogram are easily performed by in-house veterinary laboratory person- nel. Because avian blood does not store well (eg, during transport), hematologic results obtained soon after collection are preferred over those performed 9 several hours later.6,18,34 Blood volume in birds depends on the species and varies from 5 ml/100g in the Ring-necked Pheasant to 16.3 to 20.3 ml/100g in the racing pigeon. In general, birds are better able to tolerate severe blood loss than mammals, which is due to their greater capacity for extravascular fluid mobilization. However, there is a marked variation among avian species in re- HEMATOLOGY sponse to blood loss, which may be a reflection of differences in blood volume or extravascular fluid depots. In healthy Mallard Ducks and racing pi- geons, a blood volume equivalent of up to three percent of the body weight can be collected. In Passeri- formes, pheasants and Psittaciformes, up to one percent of the body weight can be collected with few ill effects (0.9 ml from a 90 g cockatiel).

Blood can be collected from a variety of sites in avian Terry W. Campbell patients. 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 of blood needed. For best results, venous blood should be collected for hematologic studies. Blood collected from capillaries (eg, blood from clipped nails) often results in abnormal cell distributions and contains cellular artifacts such as macrophages and material not normally found in peripheral blood (Figure 9.1). Blood to be used for hematology should be collected into a collection tube containing EDTA (ethyle- nediaminetetraacetic acid) as the anticoagulant. Other anticoagulants, such as heparin, interfere with cell staining and create excessive cell clumping, resulting in erroneous cell counts and evaluations (Color 9.3)6,18,34 177 CHAPTER 9 HEMATOLOGY

Processing the Avian Hematologic Sample

Blood Collection Jugular venipuncture is a procedure that can be used for collecting blood from most avian species.6,18,34,38,71 It is the method of choice for small birds that do not have other blood vessels large enough for venipuncture. The right jugular vein is usually chosen over the left for blood collection because in many birds it is the larger of the two. To collect blood from the jugular vein, the bird is properly restrained with the head and neck extended (Figure 9.2). Extending the neck encourages the highly movable jugular vein to FIG 9.1 Blood for hematologic evaluation should be collected from fall into the jugular furrow. In many species, there is a free-flowing venous source. Blood collected from a toenail clip a featherless tract of skin (apterium) overlying the may yield abnormal cell distributions and cellular artifacts. jugular vein; therefore, lightly wetting the feathers with alcohol in this area will aid in the visualization Venipuncture of the medial metatarsal (caudal tibial) of the vein. Blood is collected into a syringe, and the vein, which lies on the medial side of the tibiotarsus size of needle is governed by the size of the vein. at the tibiotarsal-tarsometatarsal joint, is another Complications of jugular venipuncture include diffi- common method for blood collection in medium to culty in proper restraint of the bird or stabilization of large birds (Figure 9.4).6,18 The primary advantages the vein and hematoma formation. Improper atten- of this method over other methods of blood collection tion to technique and hemostasis can cause a large is that the surrounding leg muscles protect the me- hematoma to form during or following jugular dial metatarsal vein from hematoma formation and, venipuncture. However, jugular venipuncture be- in some species, the leg is more easily restrained comes a skill perfected with practice, and complica- than the wing. tions are infrequent in skilled hands. Blood can be collected from the occipital venous sinus Venipuncture of the ulnar or wing vein is a common of birds. This technique should be reserved for birds method for obtaining blood from medium to large used in research or for blood collection prior to eutha- birds. A needle is inserted into the vein, which is nasia,6,78 because of the potential for injuring the found crossing the ventral surface of the humero-ra- brainstem. When properly executed, however, this dioulnar joint (elbow) (Figure 9.3). Blood is either method can be safely used for collecting repeated aspirated into a syringe or allowed to drip from the blood samples from birds. The bird must be com- needle hub into a microcollection device. Collecting pletely restrained. The head is held firmly in a flexed blood in this manner reduces but does not eliminate position in a straight line with the cervical vertebrae. hematoma formation. A variety of these devices is The occipital venous sinus is just below the skin in available.a-c These collecting tubes contain EDTA for the space between the base of the skull and the first hematology studies, are plain (with or without a cervical vertebra. To collect blood from this sinus, an serum separator) or contain heparin (lithium hepa- evacuated tube with needle and holder is required. rin is the preferred form) for blood chemistry studies. The needle is passed through the skin at a 30 to 40° Hematoma formation, which can be severe, is com- angle to the cervical vertebrae on the dorsal midline mon when the ulnar vein is used for blood collection. just above the sinus. Following penetration through A needle with an extension tube, such as a butterfly the skin, the evacuated tube is advanced in the catheter,d aids in stabilization during sample collec- holder, allowing penetration of the tube stopper by tion to minimize tearing of the vein. the needle within the holder. The needle is advanced slightly downward to penetrate the venous sinus. 178 SECTION TWO PATIENT EVALUATION

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 is larger than the left jugular vein. The neck and head are held in extension, and the mid-cervical area is lifted slightly to improve the angle for 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.

When this occurs, blood will rapidly fill the evacu- chambers) and frequently results in staining arti- ated tube. Cardiac punctures should be used only for facts (Color 9.3).6,34 When preparing a blood film, the blood collection prior to euthanasia.1,45,77 standard two-slide wedge technique used in mammalian hematology usually works well with avian blood.6,12,67 It is advisable to use precleaned, bevel- Laboratory Techniques edged microscope slides to minimize cell damage After the blood is collected, a blood film is made. The during preparation of blood films. Peripheral blood film can be made either from blood containing no films can also be made using a two-coverglass tech- anticoagulant (especially if blood parasites are sus- nique. A drop of blood is placed in the center of one pected) or blood containing EDTA. EDTA will cause coverglass;16,17 a second coverglass is placed on top of hemolysis of erythrocytes in some birds including the first, and the two are pulled apart as the blood Corvidae, currasows, Crowned Cranes, hornbills and begins to spread between the two surfaces. A similar Brush Turkeys. Prolonged exposure to EDTA may technique using a microscope slide and a rectangular result in increased disruption of cells in the blood coverglass (24 mm x 50 mm) can be used to prepare film in some species (Color 9.3). Therefore, when a film on a microscope slide rather than on cover- using an anticoagulant, a blood film should be made glasses, making the sample easier to stain.6,18 Using immediately following blood collection. Heparin the two-coverglass or microscope slide-coverglass should be avoided whenever possible for hematologic methods should be considered if the standard two- studies. Heparinized blood contains artifacts such as slide wedge technique creates excessive smudging of clumping of cells (especially leukocytes in counting the cells. Most veterinarians and technicians accus- 179 CHAPTER 9 HEMATOLOGY

has been the standard in veterinary hematology, and all cell descriptions and illustrations used in this text are based upon that stain. These descriptions also apply to a great ex- tent to the other commonly used quick stains, which essentially are modifications of the classic Wright’s stain procedure.e,f The use of an auto- matic slide stainerg simplifies the staining procedure and provides a means for consistency and high qual- ity staining by removing variations that occur with hand-staining proce- dures.

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

The red blood cell (RBC) count is obtained either by automated or manual methods. The automated cell countersh provide a quick, reliable method for obtaining a RBC count in birds. The two manual methods that can be used are the erythrocyte Unopette systemi (standard method FIG 9.3 The cutaneous ulnar vein should be considered an inferior site to the right jugular in mammalian hematology) or Natt vein for blood collection. The vessel (top) is easy to access on the ventral surface of the wing, but hematoma formation is common. Note that the bevel of the needle is up and the and Herrick’s method. The latter brachial vein is being occluded with the thumb. A small gauge needle (bottom) is used to method requires the preparation of a minimize hematoma formation and is threaded into the vessel to decrease “wobble” and methyl violet 2B diluent.55 A 1:200 endothelial damage from the needle tip. dilution of the blood is made using this solution and a diluting pipette. tomed to performing the two-slide wedge technique After mixing, the diluted blood is discharged into a with mammalian blood have little difficulty in using Neubauer-ruled hemacytometer and the cells are al- the same technique to prepare proper avian blood lowed to settle to the surface for five minutes before films. enumeration. The red blood cells are counted using the four corner squares and one central square of the A variety of hematologic stains can be used to evalu- central large primary square of the hemacytometer. ate the air-dried blood film. Romanowsky stains, The number of red cells counted is multiplied by such as Wright’s, Giemsa, Wright-Giemsa, Wright- 10,000 to obtain the RBC count per microliter of Leishman or May-Grunwald and their combination, blood. Appropriate secondary squares are counted on are preferred6,18,34 (see Chapter 10). Wright’s stain each grid and the counts are averaged. 180 SECTION TWO PATIENT EVALUATION

interfere with the counting of white blood cells using electronic cell counters. The current methods of choice for obtaining a total leukocyte count in birds are the indirect method using the eosinophil Unopette brand 5877 system or the direct leukocyte count using Natt and Herrick’s method.6,13,18,55 Estima- tion of leukocyte numbers from a blood film should be reserved for those occasions when a quantitative count is unavailable or when there is suspicion of error in a value obtained from the other methods.

The indirect eosinophil Unopette brand method involves the filling of the 25 µl pipette with blood, mixing FIG 9.4 The medial metatarsal vein can be used to collect smaller quantities of blood from the blood with the phloxine B diluent medium- to large-sized birds. This vessel is supported by the soft tissues of the leg and in comparison to other blood collection sites, hematoma formation is rare (courtesy of Kathy in the vial provided in the system Quesenberry). and charging the hemacytometer chamber for cell counting. The blood- phloxine mixture should not be al- The mean corpuscular values can be calculated using lowed to stand in the Unopette vial for longer than the PCV, hemoglobin and RBC count values. The five minutes or erythrocytes may also be stained. The mean corpuscular volume (MCV) and mean corpus- charged hemacytometer should stand undisturbed cular hemoglobin concentration (MCHC) are useful for at least five minutes to allow the cells to settle to in the characterization of the erythrocytes, especially the surface of the counting grid. It is advisable to in the evaluation of anemia. keep the charged hemacytometer in a humid cham- PCV x 10 ber to prevent dehydration of the sample if the cham- MCV = ber is going to sit for longer than five minutes. The RBC count granulocytes that stain distinctly red (heterophils MCH (pg) = Hemoglobin x 10 and eosinophils) are counted in both sides of the RBC hemacytometer (representing 18 large squares). Therefore, a total absolute heterophil and eosinophil MCHC = Hemoglobin x 100 PCV count is obtained. A WBC count is obtained by determining a leukocyte differential from the peripheral A reticulocyte count can be useful in the evaluation blood film and using the formulas in Table 9.1. of the red cell regenerative response. The erythro- A total thrombocyte count can be obtained using the cytes are stained with a vital stain, such as new Natt and Herrick’s method; however, thrombocytes methylene blue stain, and the reticulocytes are iden- tend to clump, making an accurate count difficult to tified as red blood cells that contain distinct rings of achieve. A subjective opinion as to the number of aggregated reticulum encircling the cell nucleus thrombocytes present can be made from the peri- (Color 9.1).6,34 Other erythrocytes may contain vary- pheral blood film. An average of one to two thrombo- ing amounts of reticulum, but those with the distinct cytes are present in monolayer oil immersion (100 x) ring of aggregated reticulum surrounding the cell fields in blood films of normal birds. Numbers less nucleus appear to be cells that have recently entered than this suggest a thrombocytopenia and those the peripheral circulation, and thus reflect the cur- greater suggest a thrombocytosis. An estimate of the rent regenerative response. thrombocyte count can be made from the peripheral The white blood cell (WBC, leukocyte) count of birds blood film by obtaining the average number of throm- is obtained using manual techniques because the bocytes in five monolayer oil immersion fields. This presence of nucleated erythrocytes and thrombocytes 181 CHAPTER 9 HEMATOLOGY

represents the average number of thrombocytes per of birds is the rupturing of cells during preparation 1000 erythrocytes in most species of birds. of the film (Color 9.3). The majority of these cells appear to be erythrocytes. The free red cell nuclei A more accurate method would be to count the num- appear as amorphous, pink-to-purple material on the ber of thrombocytes per 1000 erythrocytes in the film. Other abnormal findings include variations in blood film. The number of thrombocytes per 1000 the location of the cell nucleus within the cell and erythrocytes is multiplied by the erythrocyte count nuclei having indentations, constrictions or protru- and divided by 1000 to obtain an estimated thrombo- sions (Color 9.3). Perinuclear rings are usually arti- cyte count per µl of blood. If the actual erythrocyte facts of staining (eg, a form of cellular crenation). count is not known, then 3,500,000 can be used to Cytoplasmic basophilic stippling is also indicative of represent the average number of erythrocytes per µl abnormal erythrocyte morphology. Hypochromasia is of blood in most species of birds having an average indicated by pale-staining cytoplasm, cytoplasmic PCV of 45%. If the PCV is below 40 or above 50, the vacuoles and round, pyknotic nuclei (Color 9.2). Ag- estimated thrombocyte count (est T) can be corrected glutination of erythrocytes in the blood film is a rare, using the following formula: abnormal finding.

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

TABLE 9.1 Formulas for Determining WBC counts Cell Identification The total heterophil and eosinophil count T(h+e) is obtained by Erythrocyte Morphology using the formula given for the eosinophil Unopette brand system: The normal mature avian erythrocyte is oval with a T(h+e)/mm3 = cells counted x 10 x 32 /18 centrally positioned oval nucleus. The cytoplasm is abundant and stains a uniform orange-pink, resembling the cytoplasm of mammalian erythrocytes The total leukocyte count (TWBC) is obtained using the leukocyte differential and the following formula: (Color 9.1). The nucleus of the mature erythrocyte is condensed and stains dark purple. The nuclear chro- TWBC/mm3 = (T(h+e) / % heterophils + eosinophils) x 100 matin is uniformly clumped. The red cell nuclei vary with age, becoming more condensed and darker staining as the cells age. The TWBC can be calculated using the formula: TWBC/mm3 number of cells counted x 10 x 32 x 100 Variations from the typical mature erythrocyte are = (% heterophils + eosinophils) x 18 occasionally seen in the peripheral blood of birds. Avian erythrocytes frequently demonstrate diffuse This formula can be simplified by using the formula: polychromasia. Polychromatic erythrocytes demon- 3 strate cytoplasmic basophilia and have nuclei that TWBC/mm = number of cells counted x 1.111 x 16 X 100 % heterophils + eosinophils are less condensed compared to mature erythrocytes (Color 9.1). Immature round erythrocytes (eg, rubri- or cytes) may also be found in the peripheral blood of 3 number of cells counted x 1778 birds. These developmental stages have been de- TWBC/mm = % heterophils + eosinophils scribed in this chapter with the discussion of the evaluation of hematopoietic tissue. Occasionally, round erythrocytes with oval nuclei may be found, The Natt and Herrick’s method is a direct method for obtaining a especially in anemic birds. This is suggestive of an TWBC and utilizes the same dilution and charged hemacytome- asynchronous maturation of the cell nucleus and the ter used to obtain a RBC count. The dark-staining leukocytes are counted in the nine large squares of the hemacytometer cham- cytoplasm, probably owing to accelerated erythropoi- ber. The TWBC is obtained using the following formula: esis. Anucleated, oval erythrocytes (erythroplastids) 3 are rare findings in peripheral blood films of birds TWBC count/mm = (total leukocytes in 9 squares) x 10 x 200 (Color 9.2). The shape of the red blood cell may 9 or simplified to: appear irregular, or smudging may occur as a result of artifacts created by the preparation of the film. The TWBC/mm3 = (total leukocytes in 9 squares + 10%) x 200 most common artifact found in peripheral blood films 182 SECTION TWO PATIENT EVALUATION

Hematology

Illustrations for Colors 9.1 to 9.10 are com- g) Anucleated erythrocytes are observed puter-generated reproductions of blood infrequently. These cells also are called cells originally printed in Lucas AJ, Jamroz erythroplastids. C: Atlas of Avian Hematology, USDA Monograph 25, Washington DC, 1961. Color 9.3 Common erythrocyte artifacts resulting Color 9.1 from improper collection or preparation of Normal erythropoiesis (Wright’s-Leishman blood smears. stain unless otherwise noted). a) Smudge cell resulting from traumatic a) Rubriblast with prominent nucleolus, disruption of a blood cell, usually an eryth- finely granular chromatin pattern and rocyte. dark-blue cytoplasm. b) Diffuse, non-refractile, cytoplasmic b) Prorubricyte with moderately granular vacuolation suggesting cytoplasmic edema chromatin pattern and dark-blue cyto- from loss of membrane integrity. These plasm. A nucleolus is not present. changes often result from cellular damage during blood smear preparation. c-g) Various stages of developing polychromatophilic erythrocytes. As maturation c) Refractile artifact caused by water or air progresses, the nuclear chromatin pattern trapped between the cell membrane and condenses, the cytoplasm becomes less ba- mounting medium or immersion oil. This sophilic and the nuclear and cell shapes artifact is commonly mistaken for a transform from round to elliptical. The hemoparasite. presence of these cells in the blood indicates d) Staining artifact seen most commonly in polychromasia or erythrocyte regeneration. smears from blood collected in heparin and h,i) Appearance of polychromatophilic subjected to Romanowsky staining erythrocytes as reticulocytes following new (Wright’s or Giemsa staining). methylene blue staining. Ribosomes are e) Intact erythrocyte nucleus following cel- stained and aggregate as particulate material around the nucleus. lular disruption. j,k) Mature erythrocytes contain abundant Color 9.4 hemoglobin, which imparts an orange color Normal thrombocytopoiesis. to the cytoplasm. As erythrocytes continue a) Thromboblast containing an indistinct to mature or age, the cell and nuclear nucleolus, finely granular chromatin pat- shapes become more elongate, and the chro- tern and basophilic cytoplasm. matin pattern is extremely condensed. b,c) Immature thrombocytes containing l) Early polychromatophilic erythrocyte in round nuclei, a moderately granular chro- mitosis. These cells are observed most com- matin pattern and moderately blue cyto- monly in bone marrow smears but are rare plasm. in peripheral blood. d) Late immature thrombocyte. As matu- Color 9.2 ration progresses, the cellular and nuclear Common erythrocyte abnormalities in the profiles become more elliptical. Cytoplas- stained blood smear. mic vacuolation and granules may appear. a-c) Poikilocytes are misshapen erythro- e-g) Mature thrombocytes are elliptical cytes. These cells may assume a variety of with a round-to-oval nucleus, condensed shapes including a unipolar-to-bipolar, chromatin pattern and light-blue vacuo- spindle appearance. Cytoplasmic constric- lated cytoplasm. One to three cytoplasmic tions also may be present. granules may be present, but granules may d) A macrocyte is an enlarged erythrocyte vary from absent to abundant. with voluminous cytoplasm and a con- h,i) Shrunken, degenerating thrombocytes densed, displaced nucleus. These cells may with pointed cytoplasmic projections or a be observed with certain forms of anemia. condensed nucleus. These cells are observed more frequently in old blood speci- e,f) Microcytes are small erythrocytes with mens. a minimum of cytoplasm. These cells are associated with lack of iron or iron deficiency anemia.

185 CHAPTER 9 HEMATOLOGY

Hematology

Color 9.5 j) An immunocyte is an antigenically Normal lymphopoiesis. stimulated lymphocyte. These large cells contain a round-to-scalloped nucleus, con- a,b) Lymphoblasts with a round to slightly densed chromatin pattern and dark-blue irregular nucleus, fine chromatin pattern, cytoplasm. Scattered immunocytes may be multiple nucleoli and basophilic cytoplasm. observed in the blood smear following anti- These cells may be found within the bone genic stimulation from immunization or marrow in health, but suggest neoplasia disease. when observed within the peripheral blood. Color 9.6 c) Small lymphocyte with a scant rim of Monocyte development. basophilic cytoplasm. These cells are often confused with thrombocytes by an inexpe- a-c) Monocytes with oval to slightly in- rienced microscopist. dented nuclei, a variable nuclear-to-cytoplasmic ratio and gray cytoplasm. These d) Typical small lymphocyte with an eccen- cells often are misidentified as lympho- tric nucleus and scant basophilic cyto- cytes. plasm. d,e) Mature monocytes with an indented to e) Small lymphocyte with cytoplasmic bilobed nucleus, abundant gray cytoplasm azurophilic granules. In mammals, these and occasional pseudopodia. cells are “killer” lymphocytes. f,g) Monocytes with cytoplasmic vacuola- f) Large lymphocytes are observed infre- tion or increased dust-like eosinophilic cy- quently in the blood. Usually, monocytes toplasmic granules. Cytoplasmic vacuola- are misidentified as large lymphocytes. tion usually occurs if the blood specimen is g,h) Degenerating lymphocytes with cyto- allowed to stand before making blood plasmic blebs or broad pseudopodia. These smears. The eosinophilic granules are cells are seen most frequently in old blood lysosomes. specimens. h,i) Two degenerating monocytes are pre- i) Plasma cells are the ultimate expression sent. The first monocyte has nuclear edema of a B-lymphocyte. These cells are round- and cytoplasmic vacuolation. The second to-rectangular with an eccentric nucleus, monocyte has broad pseudopodia or cyto- condensed chromatin pattern, abundant plasmic blebs. These cells are observed royal-blue cytoplasm and a pale golgi zone. most frequently within old blood speci- Plasma cells are observed frequently in cy- mens. tologic preparations, but are rare in blood smears. 186 SECTION TWO PATIENT EVALUATION

Leukocyte Morphology TABLE 9.2 Characteristics of Blood Cells Using Wright’s Stain The granulocytic leukocytes of birds are heterophils, eosinophils and basophils.6,18,34,36,44 The heterophil is Erythrocytes Dark purple nucleus; orange-pink cytoplasm a round cell with distinct eosinophilic cytoplasmic Heterophils Violet, lobed nucleus; colorless cytoplasm, orange-red, rod-shaped granules in most species granules (Color 9.9). These granules are oval to spin- Eosinophils Violet, lobed nucleus; blue cytoplasm; red- dle-shaped and often contain a distinct refractile orange, round granules in most species body in the center of the granule. The mature hetero- Basophils Purple, non-lobed nucleus; dark purple phil nucleus is lobed, usually containing fewer lobes cytoplasm granules than mammalian neutrophils (Color 9.8). The nu- Monocytes Purple nucleus; abundant, finely granular, blue- clear chromatin contains heavy chromatin clumping. grey cytoplasm The cytoplasm of normal mature heterophils is color- Lymphocytes Dark purple, non-lobed nucleus; pale blue, homo- less and nonvacuolated (Color 9.8). geneous cytoplasm Thrombocytes Dark purple nucleus; colorless cytoplasm; red Avian eosinophils are round granulocytes and contain granules distinct round-to-oval cytoplasmic granules that lack the central refractile body seen in heterophil gran- amount of cytoplasm compared to lymphocytes. The ules (Color 9.9). The cytoplasmic granules of eosino- cytoplasm generally stains darker than the cyto- phils typically stain brighter or differently from hetero- plasm of normal lymphocytes. The cytoplasm of phil granules in the same blood film. The intense monocytes has a finely granular, blue-gray appear- eosinophilic appearance of eosinophil granules is most ance and often contains vacuoles. Often two distinct likely related to the high concentration of arginine.19 cytoplasmic zones can be seen in monocytes: a light- The cytoplasm of avian eosinophils stains clear blue. staining area adjacent to the nucleus and a darker The eosinophil nucleus is lobed and generally stains staining area on the periphery. The cytoplasm of darker than the nuclei of heterophils (Color 9.9). monocytes may occasionally contain fine, dust-like There is variation in the morphologic appearance of eosinophilic granules. The nucleus of monocytes gen- the eosinophils of several avian species.34,41 erally contains less nuclear chromatin clumping as compared to mature lymphocytes. The shape of the The normal basophil is slightly smaller than the monocyte nucleus is variable, ranging from round to heterophil and has a colorless cytoplasm that con- bilobed. tains strongly basophilic granules (Color 9.10). These granules often dissolve or coalesce in alcohol-based On occasion, abnormal-appearing leukocytes are stains, such as the Romanowsky stains. Avian baso- found in the peripheral blood films of birds (Color phils have round-to-oval, non-lobed nuclei that are 9.5). Immature heterophils are abnormal findings in often hidden by cytoplasmic granules (Color 9.10). avian blood films, and their appearance has been described in the evaluation of avian hematopoietic The mononuclear leukocytes found in the peripheral tissue at the end of this chapter. The immature blood of birds are lymphocytes and monocytes (Color stages most commonly found are heterophil myelo- 9.5, 9.6). The mature avian lymphocytes are round cytes and metamyelocytes. In general, immature cells that frequently “mold” around adjacent cells in heterophils have increased cytoplasmic basophilia, the blood film. These cells have high nucleus to cyto- nonsegmented nuclei and immature cytoplasmic plasm (N:C) ratios. The nucleus is usually centrally granules compared to mature heterophils (Color 9.5). positioned and round with a scant amount of homo- Usually when immature heterophils are found on a geneous blue cytoplasm appearing as a small band blood film, mature heterophils can also be found. surrounding the nucleus (Color 9.5). Avian lymphocytes often vary in size, and the larger lymphocytes Mature heterophils appear to show toxic changes in that have pale-staining nuclei may be confused with a manner similar to the toxic changes identified in monocytes. The nuclear chromatin of mature lym- mammalian neutrophils.6,12,67 Signs of toxicity in- phocytes is densely clumped. Occasionally, the cyto- clude increased cytoplasmic basophilia, vacuolation, plasm of small mature lymphocytes may contain ir- abnormal granules, degranulation, and degeneration regular projections. of the nucleus (Color 9.18). The degree of toxicity is reported subjectively on a scale of +1 to +4, where the Monocytes are the largest leukocytes found in the lower rating reflects slight change and the higher peripheral blood films (Color 9.6). They vary in shape indicates severe change. A +1 toxic heterophil shows from round to ameboid. Monocytes have an abundant increased cytoplasmic basophilia (Color 9.8). An 187 CHAPTER 9 HEMATOLOGY

overall assessment of the staining of the blood film Cells that contain large granules that fill the cyto- must be made to assure the hematologist that the plasm are frequently found in blood films of birds. film is not overly stained blue, giving the impression Often these granules fail to stain or may stain that the heterophils have increased basophilia. A +2 blue.6,34,41 These cells are common in blood films of toxic heterophil has increased cytoplasmic baso- some species of birds (eg, cockatoos) and suggest philia, vacuolation and partial degranulation (Color either staining artifact or represent variation owing 9.11). A +3 toxicity shows a deeper cytoplasmic baso- to different cytochemical properties of these cells philia, vacuolation and abnormal granulation (Color compared to other avian species. The differential for 9.8). the type of cell involved includes eosinophils, basophils and rarely, Mott cell variant of plasma cells. Abnormal granulation is indicated by the presence of granules that vary in appearance from the typical Careful examination of the blood film most often rod-shaped eosinophilic granules (eg, large, pale, reveals normal staining basophils and no evidence of round eosinophilic granules and small, deeply baso- lymphoid reactivity (which may support the possibil- philic granules). A +4 toxic heterophil resembles a +3 ity of Mott cells being present), but there are no toxic heterophil except the cell nucleus has under- eosinophils present that stain normally. Based on gone karyorrhexis or karyolysis. The number of toxic these characteristics, the majority of these cells have heterophils present is an indication of severity and been identified as eosinophils. 6, 34, 41 suggestive of duration of an inflammatory response. A slight number (25% or less) of toxic heterophils Thrombocyte Morphology may be present in the early stages of disorders re- Birds have nucleated cells (thrombocytes) rather sponsible for their occurrence. As the disorder be- than cytoplasmic fragments as platelets that partici- comes increasingly severe, the number of toxic het- pate in blood coagulation. Thrombocytes are derived erophils will increase. A marked number (greater from a distinct line of cells found in hematopoietic than 25%) of toxic heterophils is common in birds tissue. Mature thrombocytes are small oval cells that showing this heterophil abnormality. It is common appear more rounded than the erythrocytes (Color for birds with toxic heterophil changes to have all of 9.4). The nucleus is pyknotic and the cytoplasm is their heterophils affected on the blood film. Clini- colorless in mature cells. The cytoplasm may contain cally, these birds will be severely compromised. one or more red granules and small vacuoles or clear spaces (Color 9.4). Thrombocytes, like mammalian Cytologic indications for reactivity in lymphocytes platelets, tend to clump in blood films. Thrombocytes include increased cell size, increased cytoplasmic ba- are differentiated from small, mature lymphocytes sophilia, the presence of azurophilic cytoplasmic by having a colorless, nonhomogeneous cytoplasm; granules and smooth nuclear chromatin (Color 9.12). small, round, red cytoplasmic granules; and a Blast-transformed lymphocytes have a deeply baso- smaller N:C ratio. Small mature lymphocytes have philic cytoplasm and smooth nuclear chromatin high N:C ratios with a scant amount of blue, homoge- (Color 9.5). Blast-transformed lymphocytes may also nous cytoplasm (Color 9.5). have nucleoli and distinct Golgi. Occasionally, plasma cells can be found in the peripheral blood of Abnormal thrombocyte cytology includes the pres- birds. These are relatively large lymphocytes with ence of reactive and immature thrombocytes. Reac- eccentric, mature-appearing nuclei; abundant, tive thrombocytes are usually found in aggregates, deeply basophilic cytoplasm; and prominent Golgi have a diffusely eosinophilic cytoplasm (suggesting adjacent to the nucleus. Lymphocytes containing release of chemicals from the granules) and irregular azurophilic granules (large purple cytoplasmic gran- cytoplasmic margins. Reactive thrombocytes tend to ules) are considered abnormal in birds. Lymphocytes be more spindle-shaped than nonreactive thrombo- having scalloped cytoplasmic margins are found oc- cytes (Color 9.4). casionally in avian blood films; however, large numbers of these cells are considered abnormal.5,62 Imma- Immature stages of thrombocytes are occasionally ture lymphocytes in the peripheral blood films are found in the blood film of birds (Color 9.4). The also considered to be abnormal (Color 9.5). mid-immature and late-immature thrombocytes are most often seen when immature cells are present. An occasional monocyte having a few cytoplasmic vacuoles is normal, but the presence of large numbers of highly vacuolated monocytes is abnormal. 188 SECTION TWO PATIENT EVALUATION

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

Blood-loss Anemia Interpretation (Appears regenerative except in the peracute stage) of the Avian Hemogram 1. Traumatic injury 2. Parasitism (ticks, Dermanyssus mites, coccidia) 3. Primary coagulopathy (rarely reported in birds) Interpretation of the Erythron 4. Toxicity resulting in a coagulopathy (aflatoxicosis and coumarin poisoning) The normal PCV of birds ranges between 35 and 55 5. Organic disease (ulcerated neoplasm, gastrointestinal ulcers, percent. A PCV less than 35 percent is indicative of organ rupture) anemia, and a PCV greater than 55 percent is sug- Hemolytic Anemia (Regenerative) gestive of dehydration or polycythemia. An increase 1. Red blood cell parasites (Plasmodium, Aegyptianella and, rarely, in red cell polychromasia is indicative of red blood Haemoproteus and Leucocytozoon) cell regeneration (Color 9.17). In normal birds, the 2. Bacterial septicemia (salmonellosis and spirochetosis) number of polychromatic erythrocytes (or reticulocytes) found in the peripheral blood film ranges be- 3. Toxicity (mustards and petroleum products) tween one and five percent of the erythrocytes. An 4. Immune-mediated (rarely reported in birds) anemic bird with a five percent or less degree of Depression Anemia (Nonregenerative) polychromasia (or reticulocytosis) is responding 1. Chronic diseases (tuberculosis, chlamydiosis, aspergillosis, neo- poorly to the anemia or there has not been enough plasia) time for the bird to demonstrate a significant re- 2. Hypothyroidism sponse. An anemic bird showing a ten percent or greater degree of polychromasia is exhibiting a sig- 3. Toxicity (lead poisoning and aflatoxicosis) nificant regenerative response. The presence of im- 4. Nutritional deficiencies (iron and folic acid deficiencies) mature erythrocytes (eg, rubricytes) in the periph- 5. Leukemia (lymphoid leukemia and erythroblastosis) eral blood along with an increase in polychromasia is indicative of a marked regenerative response. are expected to be the causes of this condition in birds Some common causes of anemia in birds are dis- as well. cussed in Table 9.3.

Hypochromasia can be associated with certain nutri- Interpretation of the Leukogram tional deficiencies in birds, especially iron deficiency. Hypochromasia has also been seen in lead toxico- There is wide variation in the normal leukograms sis.34,42 Lead toxicosis may also create a dichotomous among birds of the same species. Therefore, values of population of erythrocytes in the blood film of a diagnostic importance must differ greatly from nor- nonanemic bird. In such cases, small senescent, ma- mal reference intervals, which are generally much ture erythrocytes with pyknotic nuclei and young broader than those obtained from domestic mam- erythrocytes (eg, rubricytes) are present in the blood mals. Preparing normal reference values on healthy film without the appearance of normal, mature individual birds is the best method for evaluating erythrocytes. This condition resembles the inappro- blood parameters of a bird during illness. When these priate release of nucleated erythrocytes in the blood specific values have not been determined, the avian of nonanemic dogs suffering from lead poisoning. clinician must rely on reference intervals obtained Basophilic stippling in the cytoplasm of erythrocytes from several birds that are presumed to be healthy. is a rare finding with lead poisoning in birds.42 Baso- It is best to use values obtained in the laboratory that philic stippling may be associated with erythrocyte routinely performs the clinician’s avian profiles. Pub- regeneration and hypochromic anemia. lished values obtained from other laboratories can be used as a guide, but may differ from the avian clini- Polycythemia is rarely reported in birds.74 Increases cian’s routine laboratory. in the PCV (relative polycythemia) are usually associated with dehydration in birds; however, absolute In general, total leukocyte counts greater than µ polycythemia can also occur. The conditions often 10,000/ l are considered suggestive of leukocytosis in associated with absolute polycythemia in mammals tame, adult psittacine birds. The total leukocyte 189 CHAPTER 9 HEMATOLOGY

count in the blood of normal psittacine birds not pecially associated with bacterial toxins affecting the accustomed to handling may be high (greater than microenvironment of the hematopoietic tissue). The 10,000/µl) owing to a physiologic leukocytosis. The greater the degree of toxicity, the more severe the general causes of a leukocytosis include infection condition. The presence of a marked number of +4 (general or localized), trauma, toxicities, hemorrhage toxic heterophils indicates a poor prognosis for sur- into a body cavity, rapidly growing neoplasms and vival in birds (Color 9.18). leukemias. The leukocyte differential aids in the assessment of the leukocytosis. Because a leukocytosis The general causes of leukopenias in birds are deple- is often caused by inflammation, a heterophilia is tion of peripheral leukocytes and depression or de- usually present. generation of leukopoiesis. Leukopenias associated with heteropenias can be associated with certain Although avian heterophils lack the myeloperoxi- viral diseases (eg, Pacheco’s disease virus) and over- dase and alkaline phosphatase of mammalian neu- whelming bacterial infections.58,64 A leukopenia and trophils, studies of their ultrastructure, cytochemis- heteropenia associated with immature heterophils is try and function suggest they perform a similar suggestive of exhaustion of the mature heterophil function in the inflammatory response.14,35,46,52,60 The storage pool owing to excessive peripheral demand magnitude of the heterophilia usually indicates the for heterophils. A depression of the hematopoietic magnitude or severity of the initiating inflammatory tissue is indicated by a leukopenia, heteropenia and process. Although avian heterophils do not produce few, if any, immature heterophils. A degenerative hydrogen peroxide during phagocytosis, they do con- response is indicated by the presence of a leukopenia, tain lysosomal enzymes and have a bactericidal func- heteropenia, immature heterophils and toxic hetero- tion.23,50,59,60,75,76 A leukocytosis and heterophilia can phils. This degenerative response can be differenti- be associated with infectious agents (eg, bacteria, ated from depletion only by the presence of toxic fungi, chlamydia and parasites) and noninfectious heterophils or by following the decreasing leukocyte etiologies (eg, traumatic injury and toxicities). A count with serial leukograms. Leukopenias associ- slight to moderate leukocytosis, heterophilia and ated with lymphopenias have been reported in early lymphopenia can result from either an exogenous or response to corticosteroids in some species of endogenous excess of glucocorticosteroids (stress re- birds.5,15 A lymphopenia also may be expected with sponse).3,15,31,56,69,83 Species that normally have high certain viral diseases; however, viral causes have not numbers of circulating lymphocytes may develop a been well documented in birds. leukopenia and lymphopenia in the initial stress response, but up to 12 hours later show a leukocytosis A lymphocytosis may be expected with antigenic and heterophilia.15 Birds that normally have higher stimulation associated with certain infections. The numbers of circulating heterophils than lymphocytes presence of many reactive lymphocytes is also sug- often show a less dramatic change in the leukogram gestive of antigenic stimulation. An occasional reac- initially. A marked leukocytosis and heterophilia are tive lymphocyte may be found in the blood film of often associated with chlamydiosis, avian tuberculo- normal birds. A marked lymphocytosis with or with- sis and aspergillosis. out the presence of immature lymphocytes can occur with lymphocytic leukemia. A marked lymphocytosis, Immature heterophils occur rarely in the peripheral with the majority of cells appearing as small mature blood of most species of birds. When present, they lymphocytes with scalloped cytoplasmic margins, is generally represent an overwhelming peripheral de- suggestive of lymphoid neoplasia.5,6,62 mand for heterophils and a depletion of the mature storage pool in the hematopoietic tissues.73 The pres- A monocytosis can be found with certain diseases ence of immature heterophils in the peripheral blood that produce chemotactic agents for monocytes. usually indicates a severe inflammatory response, These conditions include avian chlamydiosis, myco- especially in association with a leukopenia (degen- tic and bacterial granulomas and massive tissue ne- erative left shift) (Color 9.18). A marked number of crosis.32 It should be emphasized that although these immature heterophils may be associated with a disorders can create a peripheral monocytosis, it may granulocytic leukemia, a rare condition in birds. not always occur. A monocytosis can also occur in birds on a zinc-deficient diet.82 The presence of toxic heterophils is also uncommon in the peripheral blood of birds. When present, they The function of the avian eosinophil is unclear.54 suggest the presence of a septicemia or toxemia (es- Although this avian granulocyte was given the name 190 SECTION TWO PATIENT EVALUATION

eosinophil, there is evidence that its function may The diagnosis of Haemoproteus is made by the de- differ from the mammalian eosinophil. Thus, condi- tecting characteristic intraerythrocytic gametocytes tions responsible for inducing avian eosinophilias in peripheral blood films. Only the gametocyte stage most likely differ from those causing mammalian of this organism appears in the peripheral blood, eosinophilias. Eosinophilias associated with gastro- whereas schizogony occurs in the tissues (eg, lung, intestinal nematode infections have occasionally spleen and liver).70 The mature gametocyte contains been reported; however, it has been difficult to induce yellow-to-brown, refractile pigment granules (Color this condition experimentally using parasite anti- 9.21). The typical mature gametocyte occupies gens.47 Studies suggest that avian eosinophils may greater than 50 percent of the red cell cytoplasm, participate in delayed (Type IV) hypersensitivity re- partially encircles the host cell nucleus forming the actions.51 Idiopathic eosinophilias occur sporadically classic “halter-shape” and causes little displacement in birds, and more research is needed to clarify the of the red cell nucleus. It is rare for more than one meaning of this condition. mature gametocyte to occur in a cell. Macrogameto- cytes stain blue with Romanowsky stains and have As with avian eosinophils, the exact function of baso- pigment granules dispersed throughout the cyto- phils in birds is unknown. Avian basophils are simi- plasm of the parasite. The smaller microgametocytes lar to mammalian basophils in their ability to pro- stain pale blue to pink with pigment granules ap- duce, store and release histamine.9 Basophils appear pearing in spherical aggregates. If blood containing to participate in the initial phase of the acute inflam- Haemoproteus organisms is allowed to stand at room matory response in birds, but this is not always temperature for a few hours prior to preparing a reflected as a basophilia in the leukogram.8,54 Be- blood film, gametes may be released from the cells cause basophils appear to play a role in early inflam- and found in the extracellular spaces of the blood mation and possibly hypersensitivity reactions in film. The macrogametes appear as spheres that re- birds, a peripheral blood basophilia may suggest the semble the macrogametocytes within the red cell presence of these conditions. cytoplasm. The microgametes appear as small spindle-shaped structures. When gametes are found, it Interpretation of Thrombocyte Changes should be considered as an artifact of blood film preparation because these structures normally leave Avian thrombocytes play a primary role in hemosta- the host red cell following ingestion by the interme- sis in a manner similar to mammalian platelets. diate insect host (hippoboscid flies). They may also have a phagocytic function and participate in removing foreign material from the Leucocytozoon is easily identified from blood films blood.19,30 A normal thrombocyte count ranging be- because it grossly distorts the host cell (usually im- tween 20,000 and 30,000/µl of blood or 10 to 15/ 1000 mature erythrocytes) that it parasitizes. Like Haem- erythrocytes can be used as a general reference for oproteus, only the gametocyte stage of Leucocytozoon most birds.6,19 Thrombocytopenias are usually indica- occurs in the peripheral blood of birds (Color 9.22).70 tive of excessive peripheral demand for thrombo- The large, round-to-elongated gametocytes cause the cytes, although a depression in thrombopoiesis host cell to enlarge and appear to have two nuclei: the should be considered. Thrombocytopenias are often host cell nucleus pushed to the margin of the cell and seen with severe septicemias, where a combination of the parasite nucleus, a pale-pink nucleus within the excessive peripheral demand for thrombocytes and parasite. The parasitized cell usually has tapered depression of thrombocyte production may occur. A ends with the remnants of the cell membrane trailing thrombocytosis may reflect a rebound response fol- away from the cell. The macrogametocyte stains lowing hemorrhage or recovery from other conditions dark blue with a condensed nucleus and occasional associated with excessive utilization of thrombo- cytoplasmic vacuoles. The microgametocyte stains cytes. Often a regenerative response can be detected light blue with a diffuse, pale-pink nucleus. Gameto- by the presence of immature thrombocytes in the cytes of Leucocytozoon lack the refractile pigment peripheral blood film. granules found in Haemoproteus. The intraerythrocytic gametocytes of Plasmodium Identification of Common Blood Parasites spp. are often confused with those of Haemoproteus For a complete review of avian parasites see Chapter spp. because they also contain refractile pigment 36. granules. However, Plasmodium gametocytes usually occupy less than 50 percent of the host cell 191 CHAPTER 9 HEMATOLOGY

cytoplasm, and those of some species alter the position of the red cell nucleus (Color 9.26). Two key features that aid in the detection of Plasmodium are Evaluation of the the presence of schizogony in the peripheral blood and gametocytes or schizonts in blood cells other Hematopoietic Tissue than erythrocytes.70 Schizonts appear as round-to- oval intracytoplasmic inclusions that contain dark- staining merozoites. The number of merozoites produced depends upon the species of Plasmodium. As Hematopoiesis occurs primarily in the bone marrow with Haemoproteus, Plasmodium macrogametocytes of post-hatch birds; however, hematopoietic activity stain darker than the microgametocytes. Both Plas- may also be found in various internal organs (eg, 4,19 modium and Haemoproteus infections may reveal liver and possibly spleen). A bone marrow sample small, ring-like forms (trophozoites) in the cytoplasm should be obtained for cytologic evaluation in avian of infected erythrocytes. In rare cases, only these patients with persistent nonregenerative anemia, forms may be seen, and it is impossible to identify the thrombocytopenia, panleukopenia and heteropenia. parasite involved. In such situations, resampling a Bone marrow evaluation is also indicated for sus- week or more later will often reveal the developed pected cases of leukemia or if unexplained abnormal forms having the characteristics described for either cells are found in the peripheral blood. An evaluation Plasmodium or Haemoproteus. Mosquitos (Culex and of the hemogram should accompany any bone mar- Aedes spp.) are the intermediate hosts for Plasmo- row evaluation to properly assess hematopoiesis. dium. Bone Marrow Collection Microfilaria are frequently found in the peripheral blood of a variety of birds. In general, the proximal tibiotarsus just below the femoral-tibiotarsal joint (knee) is the preferred site Atoxoplasma sp. is identified by its characteristic for bone marrow collection in most birds.6,79 After sporozoite within the cytoplasm of mononuclear leu- surgical preparation of the skin either on the cranial kocytes, especially lymphocytes (Color 9.27).40 The or medial aspect of the proximal tibiotarsus, a small sporozoites appear as pale-staining, round-to-oval stab incision through the skin is made using a scalpel intracytoplasmic inclusions that compress the host blade. A bone marrow aspiration biopsy needle is cell nucleus and create a characteristic crescent pushed through the thin cortex and into the marrow shape to the nucleus. This organism can be found in space using clockwise-counterclockwise rotational the per-ipheral blood films or imprints of tissues such movements. Once the needle has entered the marrow as the lung, liver and spleen. space, the stylet is removed from the needle and a syringe is attached to gently aspirate a small amount Aegyptianella can occur within the cytoplasm in one of marrow into the needle lumen. Excessive pressure of three forms: 1) anaplasma-like initial bodies ap- during aspiration should be avoided to prevent per- pearing as small (less than one micrometer in diame- ipheral blood contamination of the sample. Following ter), round, basophilic inclusions; 2) intermediate aspiration, the needle is removed from the bone and stages resembling Babesia and measuring between the syringe is detached from the needle. The syringe one and two micrometers in diameter; and 3) large, is filled with air and reattached to the needle hub. round-to-elliptical forms measuring between two and Using the air in the syringe, the marrow within the four micrometers in length.70 Aegyptianella spp. are needle lumen is forced onto a microscope slide. A considered to be pathogenic to many species of birds second slide is placed across the first on top of the (primarily Passeriformes) but may be difficult to de- marrow sample. As the two slides are pulled horizon- tect because the parasitemia stage of the disease is tally apart, two marrow films are made for cytologic often very short and easily missed. examination.

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

Bone marrow biopsy needles commonly used include pediatric Jamshidi bone marrow biopsy-aspiration 192 SECTION TWO PATIENT EVALUATION

Hematology

Color 9.7 cells are ignored during the leukocyte dif- Early myeloblast with a fine chromatin pat- ferential count. tern, nucleolar ring and dark-blue cyto- m) Smudged heterophil with dissolution of plasm. The myeloblast is the progenitor cell the outer granule matrix leaving intact for the heterophil (Color 9.8), eosinophil round central cores. Granule dissolution (Color 9.9) and basophil (Color 9.10). may cause an eosinophil-like appearance; Color 9.8 however, smudge cells are ignored during Heterophil development. the leukocyte differential count. a) Myeloblast with fine chromatin pattern, Color 9.9 nucleolus and light-blue cytoplasm. Eosinophil development. b) Promyelocyte with oval nucleus, pale- a) Late myeloblast with condensing chro- blue cytoplasm and round to slightly ir- matin pattern and light-blue cytoplasm. regular metachromatic cytoplasmic gran- b) Eosinophilic myelocyte with an oval nu- ules. cleus and scattered, variably sized, round, c) Heterophil myelocyte with an oval nu- secondary granules. cleus and a mixture of metachromatic granules and scattered round-to-rod-shaped c) Eosinophilic myelocyte with a slightly indented nucleus and round, red-orange eosinophilic granules. granules. d) Heterophilic metamyelocyte with a d) Eosinophilic band with U-shaped nu- slightly indented nucleus. cleus and round, red-orange granules. e) Heterophilic band with U-shaped nu- e) Segmented eosinophil with a lobulated cleus. nucleus and abundant secondary (specific) f,g) Mature, segmented heterophils with granules. numerous needle-shaped granules. The f) Disrupted or smudged eosinophil. These granules may obscure nuclear detail, making assessment of lobulation difficult. cells are ignored during the leukocyte differential count. h) Heterophil with mild toxic changes including cellular swelling, partial degranu- Color 9.10 Basophil development. lation and a basophilic cast to the cytoplasm. a,b) Basophilic myelocytes with round nuclei and round, variably sized, metachro- i,j) Toxic heterophils with progressive matic granules. granule dissolution leaving the round granule core intact. The cell is slightly swollen c,d) Basophils with round, intensely and has basophilic cytoplasm. This cell may stained, metachromatic granules. Basophil occasionally be confused with an eosino- granules have high affinity for Roma- phil, except for retention of a few needle- nowsky stain, often resulting in poor stain- shaped granules. Stain-induced heterophil ing of the cell nucleus. In addition, cyto- degranulation is not associated with cytoplasmic granules may obscure nuclear plasmic basophilia. detail. k) Heterophil with non-staining cytoplas- e) Basophil degranulation may occur with mic granules. This may be an artifact re- disease or as an artifact of blood smear sulting from exposure of the blood smear to staining. formalin vapor during transport or mailing. f) Disrupted basophil (smudge cell). These l) Disrupted heterophil showing typical cells are ignored during the leukocyte dif- needle-shaped granules. These smudge ferential count.

195 CHAPTER 9 HEMATOLOGY

Hematology

All photographs on this page were provided metocyte (0.7% of the erythrocytes con- Color 9.19 courtesy of Terry W. Campbell. tained these gametocytes) are demon- An adult African Grey Parrot was pre- strated by Wright’s stain. sented with a history of an intermittent Color 9.11 seizure disorder and was successfully An adult female Red-tailed Hawk was pre- Color 9.15 treated for hypocalcemia. The hemogram sented with clinical signs of lethargy, a A Blue and Gold Macaw was examined 93 revealed: PCV = 49%, RBC = 2,940,000/ marked reduction of the pectoral muscle hours following severe blood loss that re- mm3, WBC = 15,740/ mm3, heterophils = mass and diarrhea. A diagnosis of salmo- sulted from a traumatic injury to the foot. 3 3 6,453/ mm , lymphocytes = 8,814/ mm , nellosis was made based upon necropsy and The hemogram revealed: PCV = 21%, RBC 3 3 monocytes = 315/ mm and eosinophils = bacterial culture two days later. The hemo- = 1,660,000/ mm , Hb = 5.8 g/dl, WBC = 3 3 3 157/ mm . Eosinophils with large, blue cy- gram revealed: PCV = 28%, RBC = 19,457/ mm , heterophils = 12,255/ mm , toplasmic granules are demonstrated by 1,950,000/ mm3, WBC = 31,876/ mm3, het- lymphocytes = 6,615/ mm3, monocytes = 97/ Wright’s stain. erophils = 28,050/ mm3, lymphocytes = mm3 and basophils = 389/ mm3. Twenty- 3 1,594, monocytes = 638/ mm and eosino- two percent of the erythrocytes exhibited Color 9.20 phils = 1,594/ mm3. A toxic heterophil (2+ polychromasia, and an occasional imma- An adult Red-tailed Hawk was presented toxicity) and two eosinophils are shown. ture erythrocyte was noted. Polychromasia with multiple gunshot wounds and open Wright’s stain. and a round, immature erythrocyte (early fractures involving the right radius and polychromatic rubricyte) are demonstrated ulna. The hemogram revealed: PCV = 16%, Color 9.12 by Wright’s stain. Hb = 5.3 g/dl, RBC = 1,300,000/ mm3, WBC A Blue-fronted Amazon Parrot was pre- 3 3 = 15,740/ mm , heterophils = 6,453/ mm , sented with multifocal, depigmented, non- 3 Color 9.16 lymphocytes = 8,814/ mm and monocytes raised lesions involving the skin on the feet. The hemogram from a Harris Hawk that 3 = 472/ mm . The reticulocyte count was Serum chemistries and erythrocyte pa- died three days later from an acute pneu- 20% and there were many immature eryth- rameters were within normal limits. The monia revealed a moderate anemia, leuko- rocytes present. A mid-polychromatic ru- leukogram revealed the following: WBC = cytosis, heterophilia and left shift. Marked bricyte is demonstrated by Wright’s stain. 7,543/ mm3, heterophils = 1,886/ mm3, lym- anisocytosis and polychromasia are illus- 3 phocytes = -4,601/ mm , monocytes = 830/ trated as well as a binucleate erythrocyte Color 9.21. mm3 and eosinophils = 226/ mm3. Typical (which were frequently seen in the blood Haemoproteus sp. gametocytes in a blood reactivity of lymphocytes demonstrated by film). A heterophil, eosinophil, two lympho- film stained with Wright’s stain from a Wright’s stain. cytes, a thrombocyte and an immature Great Horned Owl. erythrocyte (basophilic rubricyte) can also Color 9.13 be seen by Wright’s stain. Color 9.22 A critically ill Green-cheeked Amazon Par- Haemoproteus and Leukocytozoon spp. ga- rot was presented for evaluation. The bird Color 9.17 metocytes in a blood film stained with was extremely weak and unable to perch. A juvenile Bar-headed Goose was pre- Wright’s stain from a Great Horned Owl. Severe uveitis was present in both eyes. The sented for weakness and weight loss. Six bird was housed in a room where varnish other geese in the group appeared normal. Color 9.23 Haemoproteus sp. microgametes in a blood was being applied to furniture. Important The hemogram revealed: PCV = 28%, Hb = hematologic findings included: PCV = 40%, 9.0 g/dl, RBC = 1,950,000/ mm3, 11% poly- film stained with Wright’s stain from a 3 Screech Owl. total protein = 5.0 gm/dl, WBC = 16,140/ chromasia, WBC = 39,556/ mm , hetero- mm3, heterophils = 13,880/ mm3 (10% of the phils = 31,645/ mm3, lymphocytes = 5,645/ 3 3 Color 9.24 heterophils were myelocytes and 20% were mm , monocytes = 1,187/ mm and baso- Plasmodium sp. gametocytes and schizonts 3 3 metamyelocytes), lymphocytes = 807/ mm , phils = 791/ mm . Blood lead levels were in the cytoplasm of erythrocytes from a 3 monocytes = 1,453/ mm and thrombocy- normal. The erythrocyte morphology re- Skua stained with Wright’s stain. topenia. A heterophilic myelocyte is demon- vealed polychromasia, hypochromasia and strated by Wright’s stain. stippled basophilia. Wright’s stain. Color 9.25 Plasmodium sp. gametocytes in the cyto- Color 9.14 Color 9.18 plasm of erythrocytes and extracellular A juvenile Red-tailed Hawk was presented A juvenile Red-tailed Hawk was presented space in a Wright’s-stained blood film from with marked reduction of the pectoral mus- with open fractures of the left radius and a Skua. cle mass. Radiographic evaluation revealed ulna and poxvirus lesions along the mar- a fracture of the left coracoid bone. The gins of the beak and on the feet. The bird Color 9.26 hematologic findings included: PCV = 37%, was extremely depressed and died within Plasmodium sp. gametocyte in the cyto- Hb = 11.5 g/dl, RBC = 2,250,000/ mm3, 4% 24 hours of presentation. The hemogram plasm of a thrombocyte in a Wright’s- polychromasia, total protein = 2.6 g/dl, showed: PCV = 35%, Hb = 11 g/dl, RBC = stained blood film from a Mississippi Kite. WBC = 32,932/ mm3, heterophils = 25,687/ 3,020,000/ mm3, WBC = 42,240/ mm3, 3 3 3 Color 9.27 mm , lymphocytes = 1,976/ mm , mono- monocytes = 3,802/ mm , eosinophils = Numerous intracytoplasmic Atoxoplasma cytes = 988/ mm3, eosinophils = 3,952/ mm3 1,267/ mm3 and basophils = 422/ mm3. The 3 sp. inclusions within lymphocytes from a and basophils = 392/ mm . A heterophil, majority of the heterophils appeared ex- lung imprint of a Siskin. eosinophil, basophil and Haemoproteus ga- tremely toxic (4+). Wright’s stain. 196 SECTION TWO PATIENT EVALUATION

needlesj and disposable Jamshidi Illinois-Ster- nucleus may appear oval with irregularly clumped nal/Iliac aspiration needles. Disposable spinal need- chromatin. The penultimate stage of erythrocyte de- les can be used to sample small birds because they velopment is the polychromatic erythrocyte, which contain a stylet to facilitate passage of the needle resembles the oval, mature erythrocyte except for the through the cortex without occlusion of the needle cytoplasmic basophilia and nuclear chromatin that lumen with bone. appear less condensed than the pyknotic nucleus of the mature cell (Color 9.1). Erythropoiesis Granulopoiesis The terminology describing the different stages of erythrocytic development varies in the litera- Avian granulopoiesis appears to follow developmen- ture.6,19,34,44 In general, there are six recognizable tal stages similar to those described for mammalian stages involved in red cell development. The earliest granulocytes.6,34,44 These stages are the myeloblast recognizable stage is the rubriblast (proerythroblast) (granuloblast), progranulocyte (promyelocyte), (Color 9.1). This cell has large, prominent nucleoli or myelocyte, metamyelocyte and mature granulocyte. nucleolar rings. The round nucleus is centrally positioned within the cell. The coarsely granular chroma- Myeloblasts are large, round cells with a narrow rim tin is atypical for most blast-type cells. The abundant of cytoplasm that appears less basophilic than that of cytoplasm stains deeply basophilic and contains fine, rubriblasts.44 In general, the nucleus is round with a clear spaces (mitochondrial spaces). Rubriblasts delicate reticular chromatin pattern and distinct nu- have high N:C ratios, typical of immature cells. cleoli. No cytoplasmic granules are present. The myeloblast stage is common to all the granulocytes The second stage in erythrocyte development is the (Color 9.7). prorubricyte (basophilic erythroblast). This cell resembles the rubriblast, but the nucleoli are either The next stage toward maturation is the progranulo- absent or indistinct, and the cytoplasm lacks the cyte. These are large cells with cytoplasmic granules mitochondrial spaces of the rubriblast (Color 9.1). and light blue cytoplasm. The granules are variable in appearance. An attempt has been made to differ- The next three stages are the rubricyte stages. These entiate progranulocytes into their respective granu- are round-to-slightly oval cells that are smaller than locytic cell lines based upon the appearance of the rubriblasts and prorubricytes. Rubricytes are di- cytoplasmic granules.44 Heterophil progranulocytes vided into three groups based upon their appearance contain orange spheres (primary granules) or rings in the cytologic sample. In order of increasing matu- and dark magenta granules or rings. The ring forms ration they are the basophilic rubricyte (early poly- are thought to be characteristic of the heterophil cell chromatic erythroblast), early polychromatic rubri- line. Eosinophil progranulocytes lack the dark ma- cyte (late polychromatic erythroblast) and late genta granules and rings and contain only brightly polychromatic rubricyte (orthochromic erythroblast). staining orange spheres (primary granules). Baso- The basophilic rubricyte has a high N:C ratio, homo- phil progranulocytes have magenta granules that geneous basophilic cytoplasm and round nucleus appear smaller than those of heterophil progranulo- with distinct chromatin clumping. cytes and have fewer ring forms. The nucleus of progranulocytes is typically eccentric in its cellular The early polychromatic rubricyte appears smaller position, has a delicate reticular chromatin pattern than the basophilic rubricyte and is the first stage of and often has indistinct margins. red cell development in which hemoglobinization of the cytoplasm can be detected with Wright’s stain. The myelocytes are smaller than the progranulocytes The hemoglobin gives the cytoplasm a gray, slightly and contain the specific granules (secondary gran- eosinophilic appearance. The nucleus appears ules) for each cell line. Heterophil myelocytes are smaller with increased density, and the cytoplasm is round cells with light blue cytoplasm containing pri- more abundant when compared to the previous stage mary granules, magenta granules and rings and the of development. The late polychromatic rubricyte is definitive rod-shaped heterophil granules. The de- a round-to-slightly oval cell with an eosinophilic finitive granules occupy less than 50 percent of the gray-to-weakly eosinophilic cytoplasm (Color 9.1). cytoplasmic volume. Eosinophil myelocytes contain This cell appears to have increased cytoplasmic vol- primary and secondary granules. The specific or sec- ume when compared to the previous stage, and the ondary granules occupy less than 50 percent of the 197 CHAPTER 9 HEMATOLOGY

cytoplasmic volume. The basophil myelocyte has ma- nucleus usually has marked chromatin clumping. genta granules and mature basophil granules (secon- The late-immature thrombocyte is an oval cell that dary granules) that occupy less than 50 percent of the has the appearance of the elongate, mature thrombo- cytoplasmic volume. The nucleus of myelocytes is cyte, except the cytoplasm is a pale blue and the round and has coarsely granular chromatin. nuclear chromatin is less condensed.

Metamyelocytes resemble myelocytes, except the cell nucleus is slightly indented and may have distinct Lymphopoiesis chromatin clumping. Heterophil metamyelocytes Lymphocyte development may be seen occasionally have definitive, rod-shaped granules that occupy when evaluating hematopoietic tissue (Color 9.5). greater than 50 percent of the cytoplasmic volume. Three distinctive stages can be identified for lympho- The primary granules and magenta spheres and cyte development: lymphoblasts, prolymphocytes rings may be present, but fewer in number than the and mature lymphocytes. Lymphoblasts are large, previous stage. The definitive granules of the eosino- round lymphocytes with high N:C ratios. The nucleus phil and basophil series also occupy greater than 50 has smooth chromatin, in comparison to the mature percent of the cytoplasmic volume in their respective cell, and contains distinct nucleoli. The cytoplasm of metamyelocyte stages. The basophil myelocyte nu- lymphoblasts stains deeply basophilic. cleus remains round. Prolymphocytes resemble lymphoblasts but are The granulocytic cell series will occasionally reveal a slightly smaller, lack nucleoli and have a less baso- band cell stage similar to that described in mammal- philic cytoplasm. In normal lymphoid tissue, lymph- ian granulocytes. However, the cell nucleus is often oblasts and prolymphocytes represent less than ten hidden by the cytoplasmic granules (especially in percent of the lymphoid cells. Thus, the majority of heterophils), making it difficult to differentiate the the cells should be mature lymphocytes with the band cell from mature cells. Mature avian basophil heavy nuclear chromatin clumping, high N:C ratio nuclei do not segment. and scant amount of blue, homogeneous cytoplasm.

Thrombocytopoiesis Other Bone Marrow Cells The developmental stages involved in thrombopoi- Other cells frequently encountered in bone marrow esis are the thromboblast, early-immature thrombo- samples include osteoclasts, osteoblasts, monocytes, cyte, mid-immature thrombocyte, late-immature plasma cells and mitotic figures. Osteoclasts are 6,44 thrombocyte and mature thrombocyte (Color 9.4). large, multinucleated cells that are ameboid in As the cell develops toward maturity, the cell size shape. The abundant cytoplasm is weakly basophilic decreases, the N:C ratio decreases, the nucleus be- and often contains vacuoles and small red granules comes increasingly pyknotic and cytoplasm becomes of various shapes. The nuclei are round-to-oval and less basophilic. usually contain distinct nucleoli. Osteoblasts are large cells that vary in shape. The oval-to-round Thromboblasts are large, round-to-ameboid-shaped nucleus is eccentrically positioned in the cell. The cells with a narrow rim of deeply basophilic cyto- abundant, foamy, basophilic cytoplasm contains a plasm surrounding the round nucleus. The nuclear prominent clear space (Golgi) that is located a dis- chromatin often appears punctate, making nucleoli tance from the nucleus. difficult to detect. The cytoplasm may contain small clear spaces.

The early-immature thrombocyte is smaller than the Products Mentioned in the Text thromboblast. It has a round-to-oval nucleus and a. Microtainer - Becton Dickinson, Rutherford, NJ smaller N:C ratio than the previous cell. The cyto- b. Samplette, Monoject, Sherwood Medical, St. Louis, MO c. Capiject, Terumo, Elkton, MD plasm is basophilic with small, clear spaces or vacu- d. Abbott Hospitals Inc, North Chicago, IL oles. The nuclear chromatin is irregularly clumped e. Diff Quik, American Scientific Products, McGraw Park, IL and nucleoli are absent. The mid-immature thrombo- f. Hemacolor, Miles Laboratories Inc, Elkhart, IN g. Hema-Tek, Ames Division of Miles Laboratories Inc, cyte appears slightly elongated or irregular with a Elkhart, IN pale blue, vacuolated cytoplasm. Specific red cyto- h. Coulter Counter, Coulter Electronics, Inc plasmic granules may be seen at this stage. The i. Unopette System, Becton-Dickinson, Rutherford, NJ j. Kormed Corp., Minneapolis, MN 198 SECTION TWO PATIENT EVALUATION

References and Suggested Reading

1.Andrews FX: Simplified heart punc- associated reactions in the avian 45.Mac Arthur FN: An improved method sion body hepatitis in a sulfur- ture in poultry diagnosis. J Am Vet host. J of Protozool 14:224-254, 1967. of obtaining blood from the chicken crested cockatoo. J Am Vet Med As- Med Assoc 16:38-39, 1950. 23.Ericsson JLE, Nair MK: Electron micro- heart. Poult Sci 23:542-544, 1944. soc 179:1273-1276, 1981. 2.Assoku R, Pehale W, Buxton A: An im- scopic demonstration of acid phos- 46.MacRae EK, Powell RE: Cytochemical 65.Rosskopf WJ, Woerpel RW: Erythre- munological basis for the anemia of phatase activity in the developing reaction for cationic proteins as a mic myelosis in conures: The “hemor- acute Salmonella gallinarium infec- and mature heterophils of the marker of primary granules during rhagic conure syndrome:” a prelimi- tion of chickens. Clin Exp Immunol chicken. Histochemie 37:97-105, 1973. development in chick heterophils. nary report. Proc Assoc Avian Vet, 7:865-874, 1970. 24.Ernst RA, Ringer RK: The effect of Histochemistry 60:295-308, 1979. 1984, pp 213-228. 3.Bhattacharyya TK, Sarkar AK: Avian DDT, Zectran, and Zytron on the 47.Maxwell MH: Attempted induction 66.Ritchie BW, et al: Avian leucocytic responses induced by packed cell volume, total erythrocyte of an avian eosinophilia using vari- polyomavirus: An overview. J Assoc stress and corticoid inhibitors. Ind J count, and mean corpuscular volume ous agents. Res Vet Sci 29:293-297, Avian Vet 5:147-153, 1991. Exp Biol 6:26-28, 1968. of Japanese quail. Poultry Sci 47:639- 1980. 67.Schalm OW, Jain NC, Carroll EJ: Vet- 4.Campbell F: Fine structure of the 643, 1968. 48.Maxwell MH: The production of a erinary Hematology 3rd ed. Philadel- bone marrow of the chicken and pi- 25.Fite RW: Diagnosis and control of “Heinz body” anaemia in the domes- phia, Lea and Febiger, 1975, p 21. geon. J Morph 123:405-40, 1967. Leucocytozoonosis in captive water- tic fowl after oral ingestion of di- 68.Siccardi FJ, Rutherford HO, Derieux 5.Campbell TW: Lymphoid leukosis in fowl. Proc Assoc Avian Vet, 1984, pp methyl disulphide: A haematological WT: Pathology and prevention of an Amazon parrot: A case report. 193-198. and ultrastructural study. Res Vet Leucocytozoon smithi infection of tur- Proc Assoc Avian Vet, 1984, pp 229- 26.Flammer K: Clinical aspects of Sci 30:233-238, 1981. keys. J Am Vet Med Assoc 158:1902- 234. atoxoplasmosis in canaries. Proc 1st 49.Maxwell MH: The effect of dietary 1902, 1971. 6.Campbell TW: Avian Hematology Intl Conf Zool & Avian Med, 1987, pp rapeseed meal on the hematology 69.Siegel HS: Blood cells and chemistry and Cytology. Ames, Iowa State Uni- 33-35. and thrombocyte ultrastructure of of young chickens during daily versity Press, 1988. 27.Fry DM, Addiego L: Hemolytic ane- the adult fowl. Avian Pathol 11:427- ACTH and cortisol administration. 7.Campbell TW, Dein FJ: Avian hema- mia complicates the clearing of oiled 440, 1982. Poult Sci 47:1811, 1968. tology: The basics. Vet Clin North seabirds. Wildlife Journal 10:3-6, 50.Maxwell MH: Histochemical identifi- 70.Soulsby EJL: Helminths, Arthropods, Am 14(2):223-248, 1984. 1987. cation of tissue eosinophils in the in- and Protozoa of Domesticated Ani- flammatory response of the fowl Gal- 8.Carlson HC, Hacking MA: Distribu- 28.Galvin CE: Approach to the anemic mals 7th ed. Philadelphia, Lea and tion of mast cells in chicken, turkey, patient. Calif Vet 2:12, 1978. lus domesticus. Res Vet Sci 37:7-11, Febiger, 1982. pheasant, and quail and their differ- 29.Gaskin JM: Psittacine viral diseases: 1984. 71.Stevens RWC, Ridgway GJ: A tech- entiation from basophils. Avian Dis A perspective. J Zoo Wildlife Med 51.Maxwell MH, Burns RB: Experimen- nique for bleeding chickens from the 16:574-577, 1972. 20:249-264, 1989. tal stimulation of eosinophil stimula- jugular vein. Poult Sci 1966, 45:204- 9.Chad N, Eyre P: Immunological re- 30.Grecchi R, Saliba AM, Mariano M: tion and of eosinophil production in 205. lease of histamine and SRS in domes- Morphological changes, surface recep- the domestic fowl. Res Vet Sci 41:114- 72.Sturkie PD: Blood: Physical charac- tic fowl. Can J Comp Med 42:519-24, tors and phagocytic potential of fowl 123, 1986. teristics, formed elements, hemoglo- 1978. mononuclear phagocytes and throm- 52.Maxwell MH, Trejo F: The ultrastruc- bin, and coagulation. In Sturkie PD 10.Christie G: Hematological and bio- bocytes in vivo and in vitro. J Path ture of white blood cells and thrombo- (ed): Avian Physiology. New York, chemical findings in an anemia in- 130:23-31, 1980. cytes of the domestic fowl. Br Vet J Springer-Verlag, 1976, pp 53-75. duced by daily bleeding of ten-week- 31.Gross WB, Siegel HS: Evaluation of 126:583-592, 1970. 73.Tangredi BP: Heterophilia and left old cockerels. Br Vet J 134:358-365, the heterophil/lymphocyte ratio as a 53.Miller RE, et al: Leucocytozoon si- shift with fatal diseases in four psit- 1978. measure of stress in chickens. Avian mondi infection in European and tacine birds. J Zoo An Med 12:13-16, American eiders. J Am Vet Med As- 11.Christie G: Hematological and bio- Dis 27:972-979, 1983. 1981. chemical findings in an experimen- 32.Hawkey CM, et al: Normal and clini- soc 183:1241-1244, 1983. 74.Taylor M: Polycythemia in the blue tally produced hemolytic anemia in cal haematology of captive cranes 54.Montali RJ: Comparative pathology and gold macaw: A case report of eight-week-old brown leghorn cocker- (Gruiformes). Avian Pathol 12:73-84, of inflammation in the higher verte- three cases. Proc 1st Intl Conf Zool & els. Br Vet J 135:279-285, 1979. 1983. brates (reptiles, birds, and mam- Avian Med, 1987, pp 95-104. 12.Coles EH: Veterinary Clinical Pathol- 33.Hawkey CM, et al: Haematological mals). J Comp Path 99:1-26, 1988. 75.Topp RC, Carlson HC: Studies on ogy 4th ed. Philadelphia, WB Saun- findings in healthy and sick African 55.Natt MP, Herrick CA: A new blood di- avian heterophils, II: Histochemis- ders Co, 1986, pp 53-54. grey parrots (Psittacus erithacus). luent for counting erythrocytes and try. Avian Dis 16:369-373, 1972. leucocytes of the chicken. Poult Sci 13.Costello RT: A Unopette for eosino- Vet Rec 111:580-582, 1983. 76.Topp RC, Carlson HC: Studies on phil counts. Am J Clin Path 54:249, 34.Hawkey CM, Dennett TB: Color Atlas 31:735-738, 1952. avian heterophils, III: Phagocytic 1970. of Comparative Veterinary Hematol- 56.Newcomer WS: Factors which influ- properties. Avian Dis 16:374-380, 14.Daimon T, Caxton-Martins A: Elec- ogy. London, Wolfe Medical Publica- ence acidophilia induced by stresses 1972. tron microscopic and enzyme cyto- tions, Ltd, 1989. in the chicken. Am J Physiol 194:251- 77.Utter JM, LeFebure EA, Greenlaw JJ: chemical studies on granules of ma- 35.Hodges RD: The Histology of the 254, 1958. A technique for sampling blood from ture chicken granular leucocytes. J Fowl. London, Academic Press, 1974. 57.Newell SM, McMillan MC, Moore FM: small passerines. Auk 88:169-171, Anat 123:553-562, 1977. 36.Hodges RD: Normal avian (poultry) Diagnosis and treatment of lympho- 1970. cytic leukemia and malignant lym- 15.Davidson TF, Flack IH: Changes in haematology. In Archer RK, Jeffcott 78.Vullaume A: A new technique for the peripheral blood leucocyte popula- LB (eds): Comparative Clinical Hae- phoma in a Pekin duck (Anas taking blood samples from ducks and tions following an injection of corticot- matology. London, Blackwell Scien- platyrhynchas domesticus). J Assoc geese. Avian Pathol 12:389-391, 1983. Avian Vet 5:83-86, 1991. ropin in the immature chicken. Res tific Publications. 1977, pp 483-517. 79.VanDerHeyden N: Bone marrow as- Vet Sci 30:79-82, 1981. 37.Jacobson ER, et al: Epornitic of 58.Olson C: Avian hematology. In Bi- piration technique in birds. Proc As- 16.Davidsohn I, Henry JB: Todd-Sanford papova-like virus-associated disease ester HE, Swarte LH (eds): Diseases soc Avian Vet, 1986, pp 53-60. Clinical Diagnosis by Laboratory in a psittacine nursery. J Am Vet of Poultry 5th ed. Ames, Iowa State University Press, 1965, pp 100-119. 80.Wainright PO, et al: Identification of Methods 15th ed. Philadelphia, WB Med Assoc 185:1337-1341, 1984. viruses from Amazon parrots with a Saunders, 1974. 38.Law GRJ: Blood samples from the 59.Osculati F: Fine structural localiza- hemorrhagic syndrome and a chronic 17.Dein FJ: Laboratory Manual of jugular vein of turkeys. Poult Sci tion of acid phosphatase and arylsul- respiratory disease. 1st Intl Conf Avian Hematology. Association of 39:1450-1452, 1960. fatase in the chick heterophil leuko- Zool & Avian Med, 1987, pp 15-19. cytes. Zeitschrift furr Zell Forschung Avian Veterinarians, East Northport, 39.Leighton FP, et al: Heinz body 81.White J: Protocol for the rehabilita- 1984. und Mikroskopische Anatomie hemolytic anemia from ingestion of 109:398-406, 1970. tion of oil-affected waterbirds. Proc 18.Dein FJ: Hematology. In Harrison crude oil: Primary toxic effect in ma- Assoc Avian Vet, 1990, pp 153-163. GJ, Harrison LR (eds): Clinical Avian rine birds. Science 220:871-873, 1983. 60.Penniall R, Spitznagel JK: Chicken neutrophils: Oxidative metabolism in 82.Wight PAL, et al: Monocytosis in ex- Medicine and Surgery. Philadelphia, 40.Levine ND: The genus Atoxoplasma perimental zinc deficiency of domes- WB Saunders Co, 1986, pp 174-191. phagocytic cells devoid of myeloper- (Protozoa, Apicomplexa). J Parasitol oxidase. Proc Natl Acad Sci 72:5012- tic birds. Avian Pathol 9:61-66, 1980. 19.Dieterlen-Lievre F: Birds. In Rawley 68:719-723, 1982. 5015, 1975. 83.Wolford JH, Ringer RK: Adrenal AF, Ratcliffe NA (eds): Vertebrate Morphology of the eosi- weight, adrenal ascorbic acid, adre- 41.Lind PJ, et al: Ploucha JM, Scott JB, Ringer RK: Vas- Blood Cells. Cambridge, Cambridge nophil in raptors. J Assoc Avian Vet 61. nal cholesterol, and differential leuco- University Press, 1988, pp 257-336. cular and hematologic effects of hem- 4:33-38, 1990. orrhage in the chicken. Am J Physiol cyte counts as physiological indica- 20.Djojosugito AM, Folkow B, Kovach 42.Lloyd M: Heavy metal ingestion: 240:H9-H17, 1981. tors of “stressor” agents in laying AGB: The mechanisms behind the Medical management and gastro- hens. Poult Dis 23:366-385, 1978. rapid blood volume restoration after 62.Purchas GH, Burmester BD: Leuk- scopic foreign body removal. J Assoc osis/sarcoma group, In Hofstad MS, 84.Yuassa N, et al: Isolation and some hemorrhage in birds. Acta Physiol Avian Vet 6:25-29, 1992. characteristics of an agent inducing Scand 74:114-122, 1968. et al (eds): Diseases of Poultry. Ames, 43.Lothrop C, et al: Miscellaneous dis- Iowa State University Press, 1978, pp anemia in chicks. Avian Dis 23:366- 21.Dorrestine GM, van de Hage MN, eases. In Harrison GJ, Harrison LR 418-468. 385, 1978. Zwart P: Diseases of passerines, es- (eds): Clinical Avian Medicine and 63.Rigdon RH, et al: Anemia produced pecially canaries and finches. Proc As- Surgery. Philadelphia, WB Saunders soc Avian Vet, 1985, pp 53-70. by chloramphenicol in the duck. AMA Co, 1986, pp 525-536. Arch Pathol 58:85-93, 1952. 22.Dresser SS: Schizogony and gameto- 44.Lucas AJ, Jamroz C: Atlas of Avian gony of Leucocytozoon simondi and 64.Rosskopf WJ, et al: Chronic endo- Hematology. USDA Monograph 25, crine disorder associated with inclu- Washington DC, 1961.