BLOOD

The blood consists of specialized cells derived from the bone marrow that are suspended in a liquid called plasma. In an adult animal, the blood volume is about 8 to 10% of the body weight or approximately 40 mL of blood for each lb. of body weight. Thus, the total blood volume in a 1000-lb. horse is estimated at 40 L, whereas that of a 10-lb. dog or cat is only about 400 mL. Blood volume of some laboratory animals such as mice may be as low as 6% of body weight. The plasma component represents 55% of the blood volume, with the formed or cellular elements (red blood cells, leukocytes, and platelets) making up the remaining 45%.

The blood has several important functions. First, the hemoglobin contained within red blood cells carries oxygen to the tissues and collects carbon dioxide to facilitate its removal. Blood also conveys nutrients such as amino acids, sugars, and minerals to the tissues, and it is a conduit for byproducts and toxic substances that may be removed by the liver and kidney. Hormones, enzymes, and vitamins make their way to tissue targets by means of the blood. As a result of the phagocytic activity of leukocytes, the killing potential of their granules, and the humoral and cellmediated immune responses mounted by lymphocytes, the blood provides a defense system for the animal. Finally, the platelets are tiny cellular elements that play a major role in hemostasis, preventing the entire blood volume from being lost during hemorrhage. When blood is freshly drawn into a test tube without anticoagulants, platelets and coagulation factors in the fluid portion are responsible for the formation of a clot.

Plasma

The addition of an anticoagulant such as ethylenediaminetetraacetic acid (EDTA) to a test tube of freshly drawn blood will prevent the formation of a clot. The plasma, or fluid portion of the blood, can be separated from its cellular components by centrifugation, resulting in plasma at the top of the centrifuged blood, the buffy coat in the middle, and the red cell mass at the bottom of the tube (Fig. 4-1). The plasma is colorless to lightly yellow depending on the animal species, and is a slightly alkaline fluid consisting of approximately 92% water and 8% solids or dry matter. About 90% of dry matter is organic substances such as glucose, lipids (cholesterol, triglycerides, phospholipids, lecithin, and fats), proteins (albumin, globulins, fibrinogen, and others), glycoproteins, hormones, amino acids, and vitamins. The inorganic or mineral portion of the dry matter of plasma is dissolved in ionic forms that can dissociate into positive and negative ions.

FIGURE 4-1 Blood before and after sedimentation. The volume of packed erythrocytes is almost 45% of the total blood volume. The leukocytes and platelets form a buffy coat, accounting for approximately 1% of the blood volume. The remainder of the blood is the supernatant plasma.

The cellular elements of blood form distinct layers following centrifugation. The lowest layer is red in color and represents about 45% of the blood volume. This layer consists of erythrocytes and represents the packed cell volume (PCV) or hematocrit. The PCV is a measure (%) of the volume of erythrocytes relative to the total volume of whole blood in a sample. The buffy coat is an off-white layer identified on top of the packed red cells. In the normal patient, the buffy coat accounts for about 1% of the total blood volume and consists of platelets and leukocytes. The platelet layer is whiter and is located at the top of the buffy coat. This layer can be distinguished from the leukocyte layer, which is below and slightly pink due to an admixture with erythrocytes of low specific gravity, including reticulocytes. Although the numbers of leukocytes and platelets vary in domestic animals, 8000 to 12,000 leukocytes/µL of blood and 200,000 to 400,000 platelets/µL of blood are usual.

Erythrocytes

In general, the size of an erythrocyte, or red blood cell, in domestic animal species ranges from 3 to 7 µm. The red blood cells of the dog are largest at about 7 µm in size, whereas those of sheep and goats are only 4.5 µm and 3.2 µm, respectively. The shape of the erythrocyte and presence of a nucleus varies among animal species. Although mammalian red cells are reported to have a biconcave disk shape that results in a slight central pallor visible with light microscopy, this feature is only easily seen in the red blood cells of the dog (Fig. 4-2). The erythrocytes of family camellidae, including camels, llamas, and alpacas, have a characteristic oval shape. Red blood cells of amphibians, reptiles, and birds are also oval and, unlike mammalian cells, retain a nucleus in maturity. Fragments of the nucleus, or Howell-Jolly bodies, are normally seen in a few circulating red cells of the horse and cat. An increased number of circulating red cells with nuclei or Howell-Jolly bodies in other animal species may suggest that splenic function is abnormal or that the animal may have been splenectomized.

Red blood cells are rich in hemoglobin, a protein capable of binding oxygen and transporting it to the tissues. The lack of a nucleus allows more room for hemoglobin, and the biconcave shape of the red blood cell helps increase its oxygen-carrying capacity. The latter is likely related to increased surface area.

FIGURE 4-2 Scanning electron micrograph of erythrocytes from a clinically normal dog. The erythrocytes are biconcave disks. (From Jain NC. Schalm’s Veterinary Hematology. 4th Ed. Philadelphia: Lea & Febiger, 1983.)

Erythrocytes are the most numerous of the formed elements in the blood. About 5 to 10 million red blood cells are typically present in every µL of blood in a healthy adult animal. In contrast, the number of red blood cells may be as high as 12 to 15 million/µL of blood in sheep and goats.

A few young polychromatophilic red blood cells (reticulocytes) are normally found in peripheral blood smears of dogs. In contrast, these cells are normally absent from the blood of horses and cows. The reticulocyte is slightly more bluish than the mature red blood cell in a Wright-Giemsa-stained blood smear, due to the presence of residual ribonucleic acid (RNA). The ribosomes, polyribosomes, and mitochondria retained in these cells are aggregated into a reticular mesh when stained with vital stains such as new methylene blue. The enumeration of reticulocyte numbers in the peripheral blood is used as an indication of bone marrow erythropoietic activity.

The lifespan of the red blood cells in circulation is variable in veterinary species. In mice, the lifespan of red blood cells is about 30 days, whereas the red blood cells of the cat and dog are reported to survive 68 days and 110 days, respectively. A longer lifespan has been reported in adult goats, cows, and horses, approaching 125 days, 145 days, and 160 days, respectively. The loss of these cells is continually balanced by the release of reticulocytes and mature red blood cells from the bone marrow.

A slight variation in the size of red blood cells, or anisocytosis, is a common finding in a peripheral blood smear. Although the presence of poikilocytosis, or variation in shape of red blood cells, is a normal finding in goats and sheep, it is uncommon in other animal species. Crenated red blood cells, which have numerous short, evenly spaced, blunt or sharp projections, are also a common finding. These changes are usually artifact, but may occur in animals with lymphoma and in patients that are uremic. Rouleau formation, or aggregates of red blood cells resembling “stacked coins,” is a common finding in the blood smear of the horse. In the dog and cat, the presence of rouleau may suggest that certain total protein concentrations, such as immune globulins or fibrinogen, are increased. Agglutination, or irregular clumping of red blood cells rather than “coinlike” stacking, is abnormal and must be distinguished from rouleau formation.

Leukocytes

The blood leukocytes are divided into two categories: granulocytes and agranulocytes. The term granulocyte relates to the presence of specific granules in the cytoplasm of these cells, which can be used to differentiate the neutrophil, eosinophil, and basophil. The differing affinity of the granules for neutral, acidic, and basic stains gives them their characteristic color. Agranulocytes lack distinctive granules.

Neutrophil

Neutrophils are produced in the bone marrow and released into the blood once they mature. The mature neutrophil is approximately 12 to 15 µm in diameter and is distinguished by a segmented nucleus, often comprised of three to four lobes containing clumped or heterochromatic chromatin (Plate 4-1). Granules of the neutrophil contain many hydrolytic enzymes and antibacterial substances needed to inactivate and digest phagocytosed microorganisms. The cell cytoplasm is rather transparent because the granules are small and neutral-staining in most mammalian neutrophils. In contrast, the cytoplasmic granules of the neutrophil in rabbits, guinea pigs, birds, amphibians, and reptiles are large and red, and the cell is called a heterophil (Plate 4-10).

Electron microscopic and cytochemical studies have revealed that neutrophils contain an active Golgi complex but few mitochondria. The cytoplasmic granules of the neutrophil vary in size and peroxidase activity. Primary granules (azurophilic granules) are larger and peroxidase-positive, whereas secondary granules (specific granules) are smaller and lack peroxidase activity (Fig. 4-3). The primary granules are membrane-bounded and contain enzymes such as acid hydrolases, neutral proteases, and elastase. Microbicidal elements, including myeloperoxidase, lysozyme, defensins, and bactericidal permeability–inducing protein, are also contained in primary granules. Interspecies variation in the cytochemical reactivity and content of the primary granules has been reported. The secondary (specific) granules of the neutrophil contain enzymes such as alkaline phosphatase, collagenase, and C5a splitting enzymes. Neutrophils also possess oxygen-dependent and oxygen-independent systems for destroying internalized microorganisms within their secondary granules. These antimicrobial elements include cationic proteins and enzymes such as hydrolases, proteases, lactoferrin, lysozyme, and defensins as well as enzymes that generate toxic metabolites of oxygen.

In most species, neutrophils are the most numerous of the circulating white cells, accounting for 40 to 80% of the total white cell numbers in most animal species. They function as the body’s first line of defense against microbial infections.

FIGURE 4-3 Electron micrograph of a mature neutrophil from canine bone marrow stained for peroxidase. The neutrophil contains large, spherical, peroxidase-positive primary granules (pg) and small, pleomorphic, peroxidase-negative secondary granules (sg). Some mitochondria (m) are present. The nuclear lobes show chromatin condensation. (From Jain NC. Schalm’s Veterinary Hematology. 4th Ed. Philadelphia: Lea & Febiger, 1983.)

After spending a brief time of approximately 8 hours circulating in the blood, neutrophils enter the surrounding tissues and body cavities to carry out their specific functions. Thus, the entire circulating pool of neutrophils is turned over about three times daily. Physiologic stimuli, such as stress (corticosteroids), fear (epinephrine), infection, infarction, or trauma, can increase either the production or the release of neutrophils from the bone marrow.

The circulating neutrophilic cells in the normal, healthy animal include mature, segmented neutrophils with a few band neutrophils. The band neutrophil is approximately the same size or slightly smaller (12 to 15 µm) than the metamyelocyte. The nuclear chromatin pattern of the band is slightly less condensed and the nucleus is nonsegmented with a smooth contour and parallel-appearing sides. The mature neutrophil (segmented neutrophil) has features similar to the band neutrophil, but the nucleus has two to five distinct lobes connected by fine nuclear filaments. The cytoplasm of both band and segmented neutrophils is clear and contains pale- or neutral-staining granules. The numbers of band neutrophils and less mature cells of this series may increase in response to a disease process. If the numbers of bands are increased, but still less than the numbers of mature neutrophils, this type of blood count is called a “left-shift.” Other morphologic indications of an inflammatory blood picture may include the presence of blue cytoplasmic inclusions known as Dohle bodies (aggregates of ribosomes and endoplasmic reticulum), cytoplasmic basophilia, and cytoplasmic vacuoles.

Eosinophil

Eosinophils are approximately the same size as the neutrophil, but are easily distinguished by the presence of bright reddish granules in their cytoplasm. The granules of the eosinophil have an affinity for eosin, a red acidophilic dye found in Wright stain. This staining characteristic is attributed to a high content of arginine-rich, highly basic proteins that attract the acidic dye. Another feature that is helpful in identifying the eosinophil is the presence of a nucleus that rarely has more than two lobes, whereas the neutrophil nucleus usually has three or four lobes.

The size, shape, number, and staining characteristics of the granules in eosinophils vary among the different animal species. In the dog, the granules rarely fill the cytoplasm of the cell and may vary from small, homogenous pinkish-orange to a vacuolated granule. The granules in the eosinophil of the cat are rod-shaped and numerous and stain reddish. Large, bright reddish granules are found in the horse, whereas smaller, less intensely stained granules that almost completely fill the cytoplasm are seen in the sheep, goat, cow, and pig (Plates 4-2 and 4-3).

Ultrastructural studies of the eosinophil have shown that the Golgi complex elaborates many primary granules (azurophilic granules). These granules are larger than primary granules in the neutrophil. The secondary granules (specific granules) of the canine eosinophil vary from dense homogenous granules surrounded by a narrow rim of lighter matrix to a clear vesicle with only a cap of dense material. A few mitochondria and remnants of rough endoplasmic reticulum are also found in the mature eosinophil (Fig. 4-4). Feline and guinea pig eosinophils have char acteristic crystalloid specific granules, whereas other species, including the cow and horse, have only homogenous granules.

FIGURE 4-4 Electron micrograph of a canine eosinophil. The cell contains pleomorphic granules. Dense and light homogeneous granules and clear vesicles are present. A few mitochondria (m) and remnants of rough endoplasmic reticulum (rER) are scattered throughout the cell. (From Jain NC. Schalm’s Veterinary Hematology. 4th Ed. Philadelphia: Lea & Febiger, 1983.)

Eosinophils usually account for only 0 to 8% of the total leukocyte count, giving absolute numbers of 0 to 500 eosinophils/µL of blood. The intravascular lifespan of the eosinophil is extremely short, estimated at less than 1 hour in the dog. The eosinophil plays an important role in acute inflammatory, allergic, and anaphylactic reactions, and in controlling infestations by helminthic parasites. In the process of regulating allergic and inflammatory responses, this cell phagocytizes immune complexes and inhibits the release and replenishment of histamine and other vasoactive amines. The phagocytic and bactericidal capabilities of eosinophils are limited when compared to those of neutrophils. It has been demonstrated that eosinophils may also induce damage in tissues and play a role in fibrosis when excessive numbers of eosinophils are present. Substances such as antigen–antibody complexes, fibrin, fibrinogen, and factors released from stimulated T lymphocytes, basophils, or mast cells may act as chemotactic factors for eosinophils. Histamine, released by both mast cells and basophils in response to tissue injury or allergic reactions, is the major chemotactic factor for eosinophils.

Basophil

The basophil measures 10 to 15 µm and has a segmented nucleus. Characteristic deep purple granules often fill the cytoplasm and obscure the nucleus. The purplish staining of the granules is ascribed to their content of sulfated glycosaminoglycans. Granules of the feline basophil are rod-shaped and usually stain dull purplish-gray due to lack of sulfated glycosaminoglycans, whereas granules of the canine basophil stain reddish violet (Plates 4-4 and 4-5). Basophil granules in the dog do not fill the cytoplasm of the cell, in contrast to the granules in basophils from cows, horses, and cats. In some species, the granules are water-soluble and may be partially dissolved or lost during the process of staining the blood smear.

The basophil is the least numerous granulocyte in the peripheral blood, rarely accounting for more than 0 to 1.5% of the total leukocyte count or 0 to 200 basophils/µL. Evidence supports the role of the basophil in allergic conditions, including urticaria, allergic rhinitis, allergic conjunctivitis, asthma, allergic gastroenteritis, and anaphylaxis caused by drug reactions or insect stings. Another function of the basophil is to promote lipolysis. Heparin released from the granules of the basophil promotes the release of lipoprotein lipase from endothelial cells of the blood vessel wall. This process causes clearing of chylomicra from the blood and facilitates the metabolism of triglycerides. Basophils also play a major role in mediating inflammatory responses. The anticoagulant actions of heparin and the procoagulant effects of protease-generated kallikreins that are secreted by basophils may antagonize and promote hemostasis, respectively.

Monocyte

Monocytes are the largest leukocytes in the blood and are closely related to the neutrophil, sharing the same precursor cell (CSFGM). They are 12 to 18 µm in diameter and have a pleomorphic nucleus, which may appear elongated, folded, indented, horseshoe-shaped, and even lobed. The nuclear chromatin of the monocyte is lacy or reticular with some areas of condensation, and nucleoli are inconspicuous. The cytoplasm is abundant and grayish blue in color, often containing a few discrete vacuoles and/or fine azurophilic granules (Plates 4-9 and 4-11).

Monocytes account for 3 to 8% of the total leukocyte count. Thus, the absolute number of monocytes is approximately 200 to 1000 cells/µL of blood. The circulating half-life of monocytes in the blood is variable among the species. They transiently circulate in the peripheral blood, exiting the vasculature either randomly or in response to an inflammatory stimulus.

Upon leaving the blood, monocytes differentiate into longlived macrophages under the influence of specific tissue factors. The young macrophage is similar in morphology to that of the circulating monocytes, but with time, they become activated, increasing in size, phagocytic activity, and lysosomal enzyme content. Electron micrographic studies often show many short microvillus-like projections along the surface of the macrophage and the cytoplasm is frequently vacuolated (Fig. 4-5). Although tissue macrophages are capable of cell division, most of the macrophages at an inflammatory site have been recruited from the blood. The exact lifespan of macrophages in tissues is unknown. It appears that resident macrophages are long-lived; however, macrophages that accumulate in response to inflammatory stimuli are shorter lived.

Circulating monocytes and tissue macrophages comprise the mononuclear phagocyte system (MPS). These cells are widely distributed in tissues and serosal cavities throughout the body and include specific cells such as stellate macrophages (Kupffer cells) in the liver, alveolar and interstitial macrophages in the lung, synovial cells in the joints, and microglia in the brain. Phagocytosis and digestion of cellular debris, microorganisms, and particulate matter are major functions of the macrophage. The MPS also plays an important role in the presentation of antigens to lymphocytes, which initiates an immune response, and in the regulation of granulopoiesis and erythropoiesis through the action of cytokines secreted by the macrophages.

FIGURE 4-5 Electron micrograph of a canine monocyte. The cell contains many lysosomal granules (gr), several small to large vesicles (v), abundant ribosomes (arrow), and prominent rough endoplasmic reticulum (er), especially along the cell periphery. This cell also has many microvilluslike projections along the cellular outline. The nucleus appears bilobed and shows areas of chromatin condensation. (From Jain NC. Schalm’s Veterinary Hematology. 4th Ed. Philadelphia: Lea & Febiger, 1983.)

Lymphocyte

Lymphocytes are variable in size. The smaller cells are 6 to 9 µm in diameter, which is only slightly larger than a red blood cell, while larger lymphocytes measure up to 15 µm in diameter (Plates 4-1, 4-6, 4-7, and 4-8). Small lymphocytes are the most numerous and may be found in the blood, lymphatic circulation, and lymphatic tissue. In the blood of dogs and cats, smaller lymphocytes are most common, whereas both small and large lymphocytes are present in the blood of cows, sheep, and goats. These large lymphocytes are mature and lack nucleoli.

The cell profile of the lymphocyte is normally round and smooth with light microscopy, but short microvilli may be observed on their surface with electron microscopy (Fig. 4-6). Lymphocytes have a round to slightly indented nucleus with clumped heterochromatin. Although lymphocytes are classified as agranular, some of them may have a few azurophilic granules in their cytoplasm. The nuclear-to-cytoplasmic ratio of small lymphocytes is high, with only a modest amount of pale blue cytoplasm.

The number of lymphocytes in the peripheral circulation varies among the species. These cells account for 20 to 40% of the total leukocyte count in dogs, cats, and horses, but may be 50 to 60% of the leukocyte differential in cows, mice, and pigs. Lymphocytes are key components of the adaptive immune response. They play an important role in cell-mediated (T lymphocytes) and antibody-mediated (B lymphocytes) immunity.

FIGURE 4-6 Electron micrograph of a canine lymphocyte. The cell has a moderate amount of cytoplasm surrounding a large nucleus with nucleolus. The cytoplasm is rich in free ribosomes, has some profiles of rough endoplasmic reticulum and scattered mitochondria, and contains dense granules (azurophilic granules). (From Jain NC. Schalm’s Veterinary Hematology. 4th Ed. Philadelphia: Lea & Febiger, 1983.)

Platelets/Thrombocytes

Platelets vary in size from 5 to 7 µm in length and 1.3 to 4.7 µm in width among the animal species. A slight variation in platelet size is present in most species, but is greatly accentuated in the cat. Larger platelets, or proplatelets, up to 20 µm in length can be observed in the blood of the horse. In stained blood smears, platelets are discoid, oval, or elongated fragments of cytoplasm that lack a nucleus and have fine, reddish-purple granules. The term platelet and thrombocyte are often used interchangeably; however, when describing the nucleated platelet in fish, reptiles, and birds, the term thrombocyte is preferred (Plate 4-12).

When observed by electron microscopy, resting platelets have a smooth surface with random indentations that represent the membranous invaginations of the open canalicular system (OCS). This system opens to the platelet surface and is used for externalization of platelet secretory products and internalization of substances from plasma into the platelet. Bovine platelets are devoid of an OCS and secrete their contents directly to the exterior. Normal platelets are covered with amorphous material that forms a thin external glycocalyx. This glycoprotein-rich layer is responsible for the platelet’s adhesive properties.

Beneath the surface membrane of the platelet, some microfilaments and a bundle of microtubules are observed. These cytoskeletal elements maintain normal platelet shape and make up the contractile system. Thus, they are responsible for the change in shape of platelets following activation and the subsequent secretion of platelet granules. Another series of channels, the dense tubular system (DTS), is found deep to the superficial cytoskeleton. The DTS provides a site for sequestration of calcium and localization of enzymes for prostaglandin synthesis. The internal structure of the platelet is composed of many α granules, electrondense granules, and glycogen particles, with only a few mitochondria and lysosomes. The α granules are membrane-bounded and contain platelet factor 4, coagulation factors I (fibrinogen) and V (proaccelerin), platelet-derived growth factor, and many other components. Electron-dense granules are a storage pool for adenine nucleotides, histamine, serotonin, catecholamines, and calcium (Fig. 4-7).

The number of platelets in circulation ranges from about 200,000 to 400,000/µL of blood. In general, the lifespan of circulating platelets in domestic animal species is about 8 to 12 days. A key role of the platelet is maintenance of primary (platelet plug formation) and secondary (coagulation) hemostasis.

BONE MARROW

FIGURE 4-7 Schematic drawing of the fine structure of a platelet.

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