Bone Marrow, Blood Cells, and the Lymphatic System*

• Hematopoietic tissue is highly proliferative. Billions of cells per kilogram of body weight are produced each day.

• Pluripotent hematopoietic stem cells are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells. The process of hematopoietic differentiation is shown in Fig. 13-3.

• Hematopoietic cells undergo sequential divisions as they develop, so there are progressively higher numbers of cells as they mature. Cells also continue to mature after they have stopped dividing. Conceptually, it is helpful to consider cells in the bone marrow as belonging to mitotic and postmitotic compartments. Examples of developing hematopoietic cells are shown in Fig. 13-4.

• Mature cells released into the blood circulation have different normal lifespans, varying from hours (neutrophils), to days (platelets), to months (erythrocytes), and to years (some lymphocytes).

• The hematopoietic system is under exquisite local and systemic control and responds rapidly and predictably to various stimuli.

• Production and turnover of blood cells are balanced so that numbers are maintained within normal ranges (steady-state kinetics) in healthy individuals.

• Normally the bone marrow releases only mature cell types (and very low numbers of cells that are almost fully mature) into the circulation. In response to certain physiologic or pathologic stimuli, however, the bone marrow releases immature cells that are further back in the supply “pipeline.”

Regulation of Hematopoiesis

Control of hematopoiesis is complex, with many redundancies, feedback mechanisms, and pathways that overlap with other physiologic and pathologic processes. Many cytokines influence cells of different lineages and stages of differentiation. The aim of this section is not to explain these regulatory pathways in detail but rather to provide a broad overview and thus a basic framework for understanding mechanisms of disease involving the hematopoietic system.

Erythropoiesis

Erythropoiesis—from erythros (Gr., red)—refers to the production of red blood cells (RBCs), or erythrocytes, whose main function is gas (oxygen [O₂] and carbon dioxide [CO₂]) exchange. The dominant regulator of erythropoiesis is a glycoprotein aptly named erythropoietin (Epo). Epo acts on early erythroid progenitor cells in concert with other cytokines, including interleukins (IL-3, IL-4, and IL-9), granulocyte-macrophage colony-stimulating factor (GM-CSF), and insulin-like growth factor. Epo is synthesized primarily in the kidney, and exerts its effects by promoting proliferation and inhibiting apoptosis of developing erythroid cells that express Epo receptors. The stimulus for increased Epo production is hypoxia. Because erythrocytes are packed with hemoglobin, of which iron is an essential component, erythropoiesis requires adequate availability of iron. Physiologic systems exist to conserve and recycle iron, as discussed further later.

The earliest erythroid precursor identifiable by routine light microscopy is the rubriblast, which undergoes maturational division to produce 8 to 32 progeny cells. Late-stage erythroid precursors known as metarubricytes extrude their nuclei and become reticulocytes, which are the cells at the differentiation stage immediately preceding the mature erythrocyte. Reticulocytes start maturing in the bone marrow and finish maturing in the peripheral blood circulation and the spleen. (Horses are an exception in that they do not release reticulocytes into circulation, even in situations of increased demand.) The normal transit time from rubriblast to mature erythrocyte is approximately 1 week. Unlike mature erythrocytes, which lack organelles, reticulocytes still contain ribosomes and mitochondria, mainly to support completion of hemoglobin synthesis. These remaining organelles impart a bluish-purple cast (polychromasia) to reticulocytes on routine blood smear examination and when stained with dye, such as new methylene blue, precipitate to form irregular aggregates of dark-staining material (Fig. 13-5). Cats also have a more mature form of reticulocyte in circulation, the punctate reticulocyte, which has a much finer staining pattern when stained with new methylene blue and does not appear bluish-purple (polychromatophilic) on routine blood smear examination (see the section Anemia).

Mature erythrocytes circulate for a long time compared with other blood cells. The erythrocyte mean lifespan varies between species: approximately 150 days in horses and cattle, 100 days in dogs, and 70 days in cats. Erythrocytes therefore must be highly resilient cells capable of enduring continual mechanical and biochemical stresses. In most mammals, erythrocytes have a biconcave disk shape, sometimes called the discocyte. Species in which the discocyte is not the norm include goats (flat, irregularly shaped RBCs), camelids (oval RBCs), and some cervids (sickle-shaped RBCs). In dogs, some breeds (Akitas and Shibas) have smaller erythrocytes than others; erythrocytes in these breeds also have a high concentration of potassium, unlike erythrocytes in other dogs.

One of the key properties of erythrocytes is deformability; they change shape as they move through the microvasculature. This deformability is a function of interactions between the cell membrane, the cytoskeleton, and intracellular contents. Mature mammalian erythrocytes lack nuclei and organelles and are thus incapable of transcription, translation, and oxidative metabolism. However, they do require energy for various functions, including maintenance of shape and deformability, active transport, and prevention of oxidative damage, and they generate this energy entirely through glycolysis (also known as the Embden-Meyerhof pathway). Erythrocyte antioxidant biochemical pathways are discussed in more detail in the section on Defense Mechanisms.

The concentration of circulating erythrocytes typically decreases postnatally and remains below normal adult levels during the period of rapid body growth. The age at which erythrocyte numbers begin to increase and the age at which adult levels are reached vary among species. In dogs, adult values are usually reached between 4 and 6 months of age; in horses, this occurs at approximately 1 year of age. In most species, erythrocytes are larger at birth, and their mean volume decreases as fetal erythrocytes are replaced.

Granulopoiesis and Monocytopoiesis (Myelopoiesis)

Granulocytes (neutrophils, eosinophils, and basophils) and monocytes have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages); parasiticidal activity and participation in allergic reactions (eosinophils and basophils); antigen processing and presentation; and cytokine production (macrophages). Inflammatory mediators, such as ILs and tumor necrosis factor α (TNF-α), stimulate fibroblasts, macrophages, and endothelial cells to produce cytokines, such as granulocyte colony-stimulating factor (G-CSF) and GM-CSF, that increase granulopoiesis and monocytopoiesis.

Granulocytic and monocytic cells are sometimes referred to collectively as myeloid cells. (note: This terminology can be confusing because “myeloid” also has other meanings. In the most general sense, it refers to the bone marrow. In the context of hematology, “myeloid” is also sometimes used to mean a hematopoietic cell of nonlymphoid origin, a distinction reiterated in the classification of leukemias as discussed later in this chapter. In neuroanatomy, the prefixes “myelo-” or “myel-” can refer to the spinal cord or myelin.) The earliest granulocytic or monocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division to produce 16 to 32 progeny cells. The normal transit time from myeloblast to mature neutrophil is approximately 5 days. In addition, a reserve of fully mature neutrophils is maintained in the bone marrow. The size of this so-called storage pool is species-dependent. (Species differences and clinical significance of the storage pool are discussed in more detail in later sections.)

The concentration of granulocytes measured in the blood depends on the rate of production and release from the bone marrow, the proportions of cells circulating freely within the vasculature versus those transiently attached to the endothelial surface (marginated), and the rate of migration from the vasculature into tissues. Neutrophils normally are the predominant leukocyte type in blood of most domestic species. Neutrophils are only in circulation for a short time (less than 12 hours).

Thrombopoiesis

Thrombopoiesis—from thrombos (Gr., clot)—refers to the production of platelets, which have a central role in primary hemostasis and also participate in coagulation and inflammatory pathways. Thrombopoietin (Tpo), synthesized primarily in the liver, is the dominant regulator of thrombopoiesis. Unlike Epo, which is upregulated in response to hypoxia, Tpo is produced at a relatively constant rate (constitutively). The mechanism regulating production is therefore different. Platelets and their precursors express the Tpo receptor, which binds Tpo in the plasma. When the platelet mass is decreased, less Tpo is bound to platelet receptors, and the increased concentration of Tpo free in the plasma stimulates thrombopoiesis.

Thrombopoiesis differs fundamentally from other forms of hematopoiesis. Platelets (sometimes called thrombocytes) are anucleate cells formed not by maturational division but by shedding of membrane-bound cytoplasmic fragments from precursor cells called megakaryocytes. (note: Birds and reptiles produce thrombocytes in the manner of other blood cells and their thrombocytes and erythrocytes are nucleated.) As the name suggests, megakaryocytes are very large cells, much larger than any other hematopoietic cells (Fig. 13-6). Megakaryocytes arise from a progenitor cell and undergo endomitosis to become polyploid (usually 8N-32N). As they mature, megakaryocytes increase in size and their cytoplasm becomes more abundant. They change in staining pattern (Wright’s stain of aspirates) from intensely basophilic to eosinophilic, and their nuclei change from round to highly lobulated. Megakaryocytes extend cytoplasmic processes into the lumina of the bone marrow venous sinusoids, where they shed platelets into circulation. The lifespan of a platelet in circulation is approximately 6 days in the dog.

Platelets normally circulate in a quiescent state, as flattened disks. Activation of platelets is triggered by soluble agonists, such as thrombin, or insoluble agonists, such as collagen. Platelets express surface receptors for fibrinogen, von Willebrand factor (vWF), collagen, and other ligands. Other key features of platelets include a network of membrane invaginations known as the open canalicular system (not present in cattle), which facilitates the expansion of surface area and release of granule contents in response to activation; specialized endoplasmic reticulum known as the dense tubular system that acts as a reservoir for calcium; and cytoskeletal components involved in mediating shape change, release of granule contents, receptor interactions, and clot retraction. When activated, platelets change to more spherical forms with pseudopodia and participate in a number of related processes at sites of vascular damage:

Phospholipids (especially phosphatidylserine) expressed on the surface of activated platelets also help to localize coagulation (formation of an insoluble fibrin clot) to sites of vascular damage.

Lymphopoiesis

Lymphopoiesis refers to the production of new lymphocytes. T lymphocytes and B lymphocytes are the key effectors of cell-mediated and humoral immunity, respectively. Lymphopoiesis is discussed in more detail in Chapter 5, but a brief overview is provided here. T lymphocytes originate in the bone marrow and migrate to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. B lymphocyte development occurs in two phases, first in an antigen-independent phase in the bone marrow and ileal Peyer’s patches (the site of B lymphocyte development in ruminants), then in an antigen-dependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosa-associated lymphoid tissue [MALT]). Trafficking of lymphocytes occurs under the direction of chemokine (chemoattractant cytokine) signals. Once they have migrated to the peripheral lymphoid tissue, lymphocytes may undergo clonal expansion in response to antigenic stimulation.

Unlike other hematopoietic cells, which circulate only in the blood vessels, lymphocytes travel in both blood and lymphatic vessels, continually recirculating between the two systems. In most species, the majority of lymphocytes in blood circulation are T lymphocytes. According to conventional wisdom, cattle normally have higher numbers of lymphocytes than neutrophils in circulation. However, recent work suggests that is no longer the case, at least in mid-lactation Holstein cows, most likely because of changes in genetics and husbandry.

Hemostasis (Coagulation And Platelet Function)

Hemostasis—from haima (Gr., blood) and stasis (Gr., “a standing still”)—refers to the cessation of bleeding. The main components responsible for hemostasis are platelets, blood vessels, and the coagulation system. Bleeding disorders may result if any of these systems are perturbed. Although coagulation factors are mainly synthesized in the liver, they are included in this chapter because of their inextricable link with the hematopoietic system.

Physiologic hemostasis involves coordinated processes: formation of a platelet plug at the site of vascular damage (primary hemostasis), development of a cross-linked network of insoluble fibrin that provides further clot stability (secondary hemostasis) and retraction and enzymatic degradation of the clot (tertiary hemostasis). The main components of these processes are summarized in Chapter 2 and Figs. 2-12 through 2-17. Signalment, history, and clinical signs are important in evaluating any patient with a suspected bleeding tendency, but laboratory testing is required to definitively identify specific defects in hemostasis. Routine assays for assessing hemostasis are summarized in Web Appendix 13-1.

Bone Marrow

Bone marrow responses to injury include abnormal patterns of proliferation of normal hematopoietic cell types, proliferations of abnormal hematopoietic cell types (neoplastic or nonneoplastic), replacement of normal hematopoietic tissue by abnormal cells or fibrous tissue, necrosis, and inflammation. Each of these is discussed further; the relationship between altered hematopoiesis and concentrations of blood cells in circulation is discussed in more detail in the next section on Blood Cells.

The classic nomenclature for describing different types of abnormal cellular proliferations applies to the hematopoietic system, with some exceptions and modifications. Hyperplasia, an increase in cell number, may be an obviously appropriate response to a stimulus (e.g., erythroid hyperplasia in response to hypoxemia, or granulocytic hyperplasia in response to inflammatory stimuli). In other situations, hyperplasia is a secondary phenomenon for which the specific stimulus may not be clear—for example, a number of conditions (inflammation, neoplasia, iron deficiency, asplenia, and others) are associated with reactive thrombocytosis, in which an increased concentration of circulating platelets reflects megakaryocytic hyperplasia. Sometimes hyperplasia is idiopathic. For example, splenic nodular hyperplasia, which often includes a hematopoietic component, is frequently an incidental finding.

Hypertrophy, an increase in cell size, is not a conventional description for hematopoietic cells or for bone marrow as an organ. Abnormally large hematopoietic cells are considered evidence of dysplasia, or altered cell formation (alterations to shape or organization of cells are also evidence of dysplasia). Dysplasia of hematopoietic cells, indicating dyshematopoiesis, may be primary (idiopathic) or secondary to a number of conditions, including infection, nutritional imbalance, and toxicosis. Unfortunately, the terminology is sometimes confusing. For example, myelodysplastic syndromes are clonal hematopoietic stem cell disorders that are in fact neoplastic (see the section on Hematopoietic Neoplasia). Splenic hypertrophy is usually referred to as splenomegaly and may reflect processes other than hyperplasia or cellular hypertrophy (e.g., congestion). Similarly, lymph node enlargement is typically referred to as lymphadenopathy or lymphadenomegaly and may reflect lymphoid hyperplasia or other processes (e.g., neoplasia, inflammation, or edema).

Replacement of hematopoietic tissue in the bone marrow by abnormal tissue, usually fibrous tissue or malignant cells, is known as myelophthisis. The term metaplasia, describing the replacement of cells normally comprising a tissue with another well-differentiated cell type, is rarely used to describe hematopoietic tissues in veterinary medicine, although in humans, the terms myeloid metaplasia and extramedullary hematopoiesis are sometimes used interchangeably. Similarly, the term atrophy, which means a diminution in the size of a cell, tissue, organ, or part, is seldom used to describe bone marrow. Instead, a decrease in bone marrow hematopoietic tissue is typically referred to as hypoplasia, as discussed later in the section on cytopenias. The absence of bone marrow hematopoietic tissue of a particular lineage is typically referred to as aplasia or, if affecting all lineages, by the misleading term aplastic anemia (more aptly termed aplastic pancytopenia). An exception is “serous atrophy of fat,” a condition associated with cachexia or starvation in which fat is catabolized and bone marrow reticular cells produce a mucoid ground substance (Web Fig. 13-1). However, neither hematopoietic cells nor fat is necessarily absent from the bone marrow in this condition, and other names, such as gelatinous transformation, have been suggested to describe it. Neoplasia of bone marrow or lymphoid organs may be primary or secondary (metastatic). Neoplasia may arise from any hematopoietic lineage. Specific types of hematopoietic neoplasia (leukemias, lymphomas, and others) are covered later in this chapter.

Bone marrow fibrosis (myelofibrosis) and necrosis are associated with many common underlying conditions, often occur in tandem, and are described most extensively in dogs. Conditions known or suspected to cause myelofibrosis and necrosis include sepsis, malignancy (especially hematopoietic malignancies), drug toxicity, and immune-mediated disease (especially nonregenerative immune-mediated hemolytic anemia [IMHA]).

Bone marrow inflammation can take different forms. Granulomatous inflammation in response to fungal infection (e.g., histoplasmosis) or mycobacteriosis may be evident histologically or as macrophage hyperplasia in cytologic preparations of aspirates. Phagocytosed organisms within macrophages are detectable with either type of sample. Dogs and cats with nonregenerative IMHA often have bone marrow inflammation, in addition to fibrosis and necrosis. The inflammation is evident as fibrin deposition, edema, and multifocal neutrophilic infiltrates; immune-mediated cytopenias are also associated with bone marrow lymphocytic and/or plasma cell hyperplasia.

Blood Cells

Responses of circulating blood cells to injury include decreased survival (destruction, consumption, or loss), altered distribution, and altered structure or function (or both). These responses are not mutually exclusive—for example, altered erythrocyte structure may lead to decreased survival. Often, but not always, these responses result in decreased concentrations of blood cells in circulation.

Methods for Gross and Microscopic Examination

Additional Tests

Hemostasis

Structural or functional abnormalities of blood vessels, platelets, or coagulation factors may result in a tendency toward hypocoagulability (bleeding), hypercoagulability (inappropriate thrombosis), or both. In veterinary medicine, there has been a great deal of work on specific mechanisms of hypocoagulability, whereas mechanisms of hypercoagulability are less fully characterized. Disorders of primary hemostasis typically result in “small bleeds” (e.g., petechiation, mild ecchymosis, bleeding from mucous membranes, bleeding immediately after venipuncture), whereas disorders of secondary hemostasis typically result in “big bleeds” (e.g., hemorrhage into body cavities/joints, marked ecchymosis, large hematomas, delayed bleeding after venipuncture). This chapter concentrates on primary disorders of hemostasis and also covers DIC, which is the secondary condition. However, it is important to note that coagulation disorders can also result from other underlying disease processes. For example, advanced liver disease can lead to abnormal hemostasis through decreased or defective synthesis of coagulation factors, or impaired clearance of fibrinolytic products that inhibit coagulation reactions and platelet function. Vascular disorders may also result in a bleeding tendency because of abnormalities of endothelial function or collagen-platelet interactions. Specific diseases involving abnormal structure or function of hematopoietic or hemostatic elements are discussed later in this chapter.

The CBC provides basic information about platelets, including numeric values for platelet concentration and mean platelet volume (MPV), subjective assessment of platelet morphology (size, shape, and granularity), and a rough estimation of platelet numbers based on examination of a blood smear. Some laboratories measure reticulated platelets (platelets recently released from the bone marrow), although this test is mostly used in the research setting at present. Increased MPV and increased numbers of reticulated platelets tend to indicate increased thrombopoiesis. Bone marrow examination is indicated with any unexplained cytopenia, including thrombocytopenia, to evaluate production. Tests to evaluate the components of the hemostatic process are described and listed in Web Appendix 13-1.

• Chemical agents

• Infectious agents

• Idiopathic (horses, cattle, dogs, cats)

Erythropoietic Porphyrias

Porphyrias are inherited defects of enzymes involved in the synthesis of porphyrins, precursors of hemoglobin, and other heme proteins. Porphyrias result in accumulation of toxic porphyrin compounds. Congenital erythropoietic porphyria, transmitted as an autosomal recessive trait, occurs in Holstein and shorthorn cattle and is characterized by red-brown discoloration of teeth, bones, and urine caused by accumulation of porphyrins (see Fig. 1-75). Because of the circulation of the photodynamic porphyrins in blood, these animals have lesions of photosensitization of the nonpigmented skin and hemolytic anemia. All affected tissues, including erythrocytes, exhibit fluorescence with ultraviolet light. The premature destruction of developing and mature erythrocytes is caused by the accumulation within these cells of excess porphyrins. Bovine erythropoietic protoporphyria is an inherited disorder of heme synthetase, a terminal enzyme of the heme synthetic pathway, resulting in the accumulation of protoporphyrins in tissues and erythrocytes. It is inherited as an autosomal recessive trait and is confined to Limousin or Limousin-cross cattle. Photosensitivity is the only clinical manifestation of the disease; there is no anemia and no discoloration of teeth and bone. A congenital porphyria described in Siamese and domestic short-haired cats resembles congenital erythropoietic porphyria in cattle. These cats have brown teeth, photosensitization, and hemolytic anemia.

Pyruvate Kinase Deficiency

Pyruvate kinase (PK) deficiency is an inherited autosomal recessive condition reported in many dog breeds and fewer cat breeds (Abyssinian, Somali, and domestic shorthair). The glycolytic isoenzyme that is deficient in erythrocytes of affected animals normally catalyzes the last ATP-generating reaction in glycolysis. Thus there is decreased production of ATP in PK-deficient individuals, which results in loss of normal membrane function and decreased erythrocyte lifespan. The disease is characterized by moderate to severe extravascular hemolytic anemia that is strongly regenerative. In dogs, PK deficiency typically also involves progressive iron overload, myelofibrosis, osteosclerosis, and cirrhosis. Dogs with PK deficiency usually die from complications of the disease between 1 and 5 years of age. In cats, PK deficiency is associated with milder hematologic abnormalities and is not associated with osteosclerosis; the prognosis is more favorable. Dogs with PK deficiency do not necessarily have the same genetic defect, so mutation-specific DNA-based assays are required. In contrast, a single DNA-based test is available to detect the common mutation affecting Abyssinian, Somali, and domestic shorthair cats.

Cytochrome b₅ Reductase Deficiency

Deficiency of cytochrome b₅ reductase (Cb₅R, also known as methemoglobin reductase), the enzyme that catalyzes the reduction of methemoglobin (Fe³⁺) to hemoglobin (Fe²⁺), has been recognized in many dog breeds and in domestic shorthair cats. It is probably an autosomal recessive trait. Affected animals may have cyanotic mucous membranes or exercise intolerance but are not anemic, usually lack clinical signs of disease, and have normal life expectancies.

Glucose-6-Phosphate Dehydrogenase Deficiency

Deficiency of the glycolytic enzyme glucose-6-phosphate dehydrogenase (G6PD), a common X-linked disease in people, has been reported in an American saddle bred colt with eccentrocytosis and persistent hemolytic anemia and in a partially deficient male dog without anemia or clinical signs.

Leukocyte Adhesion Deficiency

LAD is an inherited fatal autosomal recessive condition characterized by deficiency of the common β₂ chain (also known as CD18) of leukocyte integrins. Without normal expression of this adhesion molecule, leukocytes have severely impaired ability to migrate from the blood into tissues. As a result, animals with LAD are highly susceptible to infections and usually die at a young age. Infected foci are nonsuppurative because of the absence of infiltrating leukocytes. LAD is also characterized by very high concentrations of neutrophils in the blood. LAD has been recognized in Holstein cattle (known as bovine LAD [BLAD]) and Irish setter dogs (see Chapter 3).

Pelger-Huët Anomaly

Pelger-Huët anomaly (PHA) is a condition characterized by lack of normal segmentation of the nuclei of mature granulocytes. This condition results in granulocyte morphology similar to that of band neutrophils in an inflammatory left shift; however, in the absence of any other disease process, animals with PHA should not have clinical signs or other laboratory findings indicating inflammation. PHA cells can be distinguished from immature forms of otherwise normal band forms on the basis of their mature chromatin pattern (Fig. 13-22). PHA has been described in dogs, cats, horses, and rabbits, as well as in humans. Prevalence is higher in certain dog breeds. In Australian shepherds, the mode of inheritance is autosomal dominant with incomplete penetrance. Most cases of PHA are the heterozygous form and probably of no clinical significance. In these cases, the shape of most granulocyte nuclei resembles those of band and metamyelocyte forms. A rare homozygous form of PHA has also been reported in rabbits and cats. It is associated with accompanying skeletal abnormalities and stillbirths or early mortality in these species (there are also rare reports of homozygous PHA in humans, without associated skeletal abnormalities or shortened lifespan). In homozygous PHA cells, granulocyte nuclei are round or oval. An acquired, reversible condition mimicking PHA, known as pseudo–PHA, is occasionally noted in animals with infectious disease, neoplasia, or in connection with drug administration.

Chédiak-Higashi Syndrome

Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive condition characterized by recurrent pyogenic infections, bleeding tendencies, ocular and cutaneous hypopigmentation, and prominent cytoplasmic inclusions in blood cells. CHS has been described in several breeds of cattle and in Persian cats. The mutated gene encodes a protein called beige or LYST (lysosome trafficking regulator), the specific functions of which are still being investigated. Individuals with CHS have severely impaired cellular innate immunity because of neutropenia, impaired chemotaxis, and impaired killing by granulocytes and cytotoxic lymphocytes.

One of the classic features of CHS is a bleeding tendency caused by platelet dysfunction. Platelets in individuals with CHS lack the dense granules that normally contain key bioactive molecules involved in hemostasis, including platelet agonists, such as ADP and serotonin, and platelet aggregation in vitro in response to collagen, in particular, is severely impaired.

Glanzmann Thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited platelet function defect caused by defective expression of the integrin α_IIbβ₃ (also known as GPIIb-IIIa). The α_IIbβ₃ molecule has multiple functions but is best known as a fibrinogen receptor that is essential for normal platelet aggregation. Bleeding tendencies vary widely between affected individuals, and bleeding tends to occur mainly on mucosal surfaces. The condition is characterized by an in vitro lack of response to all platelet agonists and severely impaired clot retraction. GT has been recognized in Great Pyrenees and Otterhound dogs and in a quarter horse, a thoroughbred-cross, and an Oldenburg filly. To date, all reported cases in animals have involved mutations to the IIb gene. Molecular testing is available to detect affected or carrier Great Pyrenees and Otterhound dogs and to detect the reported mutations in horses.

CalDAG-GEFI Thrombopathia

Conditions characterized by inability to relay signaling information in response to most platelet agonists have been described in basset hound, Eskimo spitz, and Landseer-ECT dogs and in Simmental cattle. The molecular basis involves mutations to the gene encoding the guanine nucleotide exchange factor, CalDAG-GEFI. Platelet receptor expression in affected animals is apparently normal. All reported mutations have been associated with a bleeding tendency.

Oxidative Agents

Rather than presenting an exhaustive list, this section describes some of the more commonly recognized oxidants that cause hemolytic anemia or impaired hemoglobin function in common domestic species. Oxidative insult may result in extravascular hemolysis because of phagocytosis of damaged erythrocytes by splenic macrophages, or in intravascular hemolysis if the damage is severe enough. In horses, red maple (Acer rubrum) toxicity is a well-characterized, potentially fatal cause of acute intravascular hemolytic anemia and methemoglobinemia. Ingestion of sufficient amounts of wilted or dried leaves or bark causes Heinz body formation, eccentrocytosis, severe intravascular hemolysis, and methemoglobinemia. Common postmortem findings include icterus, splenic hemosiderosis, splenomegaly, brown discoloration and swollen liver, and swollen kidneys that can be dark red to blue-black. Histologically, the kidneys have characteristic red-brown tubular casts (hemoglobinuric nephrosis).

Phenothiazine can cause Heinz body formation in horses. In ruminants, Brassica and rye grass are associated with Heinz body formation, and nitrite toxicity causes methemoglobinemia.

Copper toxicity is a well-known cause of acute intravascular hemolytic anemia in sheep and may also occur in goats and calves. The condition occurs in animals that have accumulated large amounts of copper in the liver. The copper is released under conditions of stress (e.g., shipping, starvation) and is believed to cause hemolysis as a result of direct interaction with membrane proteins, lipid peroxidation, formation of reactive oxygen species, and enzyme inhibition. Affected animals are often markedly icteric, and hemoglobinuric nephrosis is a classic postmortem lesion.

Onions and garlic are most commonly recognized as a cause of Heinz body hemolytic anemia in dogs and cats but can cause oxidative damage to erythrocytes in other domestic animals, including horses and cattle. Other causes of Heinz bodies in dogs include acetaminophen, benzocaine, naphthalene, phenylhydrazine, vitamins K₁ and K₃, and zinc toxicity. Other causes of Heinz bodies in cats include acetaminophen, benzocaine, methionine, naphthalene, propofol, and propylene glycol.

Snake Envenomation

Hemolytic anemia that results from snake envenomation has been reported in horses and dogs. Some venoms contain phospholipases that cause intravascular hemolysis; spherocytosis, consistent extravascular hemolysis, has also been reported.

Babesiosis

Babesia spp. are intraerythrocytic protozoal organisms (piroplasmas) spread by arthropods (ticks, biting flies), transplacentally, and by blood transfusions. Evidence is accumulating that infection also is transmitted by dog fighting. Babesia spp. cause hemolytic anemia in horses, cattle, dogs, cats, and various nondomesticated animals. Babesia organisms are typically classified as large or small form, based on routine light microscopic morphology. Examples of large and small form organisms are shown in Fig. 13-23. Babesia equi and B. caballi infect horses and other equids in tropical and subtropical areas worldwide (B. equi, which also infects lymphocytes, is not considered a true Babesia but is phylogenetically more closely related to Theileria and Cytauxzoon). B. bovis and B. bigemina (small and large form organisms, respectively) are pathogenic Babesia spp. in cattle. These organisms have a worldwide distribution but have been eradicated in North America. Other, less pathogenic Babesia spp. may also infect cattle. In dogs, large form and small form Babesia spp. infections are associated with hemolytic anemia. The predominant large form is B. canis, which has three subtypes (canis, rossi, and vogeli). B. canis vogeli, considered the least pathogenic strain, is the most common one in the United States (US). In some areas, particularly the southeastern US, the seroprevalence of B. canis is high, and many infected dogs are asymptomatic chronic carriers. There are also reports of dogs with other types of large form babesiosis, including one caused by an organism with highest molecular homology to B. bovis. Small form Babesia spp. in dogs include B. gibsoni, B. conradae, and a form known as the Spanish isolate (formerly Theileria annae). A number of Babesia spp. (B. cati, B. felis, B. herpailuri, B. pantherae, and others) have been reported in domestic and wild cats worldwide. Little is known about the epidemiology of feline babesiosis; the severity of disease ranges from asymptomatic to severe.

Babesiosis may cause both intravascular and extravascular hemolysis and is also associated with a wide range of other clinical signs. The wide variety of clinical signs is a result of variations in pathogenicity of the organisms and susceptibility of the host. Infection with highly virulent strains may cause severe multisystemic disease. In these cases, massive immunostimulation and cytokine release cause circulatory disturbances, which may result in shock, induction of the systemic inflammatory response, and multiple organ dysfunction syndromes. Mechanisms of hemolysis may include direct damage to the erythrocyte by protozoal proteases, immune-mediated destruction, and oxidative damage. In animals with acute disease, signs often include fever, lethargy, pallor, hemoglobinuria, splenomegaly, and icterus. Animals are often thrombocytopenic, presumably a result of immune-mediated destruction of platelets. They also may have lymphadenopathy. Less common signs include edema, ascites, signs of central nervous system dysfunction, renal failure, rhabdomyolysis, stomatitis, and gastroenteritis.

Babesia organisms can usually be detected on a routine blood smear in animals with acute disease. Infected erythrocytes may be more prevalent in capillary blood, so blood smears made from samples taken from the pinna of the ear or the nail bed may increase the likelihood of detecting organisms microscopically. Buffy coat smears also have an enriched population of infected RBCs. PCR testing is the most sensitive assay for detecting infection in animals with very low levels of parasitemia. At necropsy, animals dying of the acute disease have splenomegaly, jaundice, hemoglobinuria, swollen hemoglobin-stained kidneys, and subepicardial and subendocardial hemorrhages. The cut surface of the enlarged, congested spleen oozes blood. The gallbladder is usually distended with thick bile. A striking feature of B. bovis infections is congestion of gray matter throughout the brain, which is readily visible compared with the white matter. Parasitized erythrocytes are best visualized on impression smears of the kidney, brain, and skeletal muscle. Microscopic findings in the liver and kidney are typical of a hemolytic crisis and include anemia-induced degeneration, necrosis of periacinar hepatocytes and cholestasis, and hemoglobinuric nephrosis with degeneration of tubular epithelium. Erythroid hyperplasia is present in the bone marrow. In animals that survive the acute disease, there is hemosiderin accumulation in the liver, kidney, spleen, and bone marrow. In chronic cases, there is hyperplasia of macrophages in the red pulp of the spleen.

Theileriosis

Theileria spp. are tick-borne protozoal organisms that infect many domestic and wild artiodactyls worldwide. Infection is characterized by intralymphocytic schizonts and pleomorphic intraerythrocytic piroplasmas (merozoites and trophozoites). The latter stages closely resemble Cytauxzoon and small form Babesia spp. Recognized Theileria pathogens in cattle include Theileria parva (the cause of East Coast fever in Africa), T. annulata (the cause of tropical theileriosis in the Mediterranean regions, Middle East, and Asia), and T. buffeli, which has recently been documented as a cause of hemolytic anemia in the US. Possible mechanisms of anemia in theileriosis include invasion of erythroid precursors by merozoite stages and associated erythroid hypoplasia (as occurs with T. parva infection), immune-mediated hemolysis, mechanical fragmentation because of vasculitis or microthrombi, enzymatic destruction by proteases, and oxidative damage. Clinical signs in a severely anemic cow infected with T. buffeli included recumbency, fever, pallor, tachycardia, and lymphadenopathy. Necropsy findings included splenic hemosiderosis, edema of lymph nodes and the subcutis, thoracic and peritoneal effusions, and pneumonia.

Trypanosomiasis

Trypanosomes are flagellated protozoa that normally survive and are nonpathogenic in wildlife reservoir hosts. They are transmitted by arthropod vectors. Some Trypanosoma spp. are recognized as causing hemolytic anemia in animals in tropical and subtropical regions outside of North America. Trypanosoma brucei and T. evansi affect horses. T. congolense and T. vivax affect cattle. Trypanosoma spp. that cause anemia in other species include T. simiae in pigs and T. brucei in camels and horses. Trypanosomes also cause nonhemolytic disease (e.g., T. cruzi, the agent of Chagas’ disease, or American trypanosomiasis) in many hosts, and nonpathogenic variants (e.g., T. theileri in cattle worldwide) are also recognized. Trypanosomal organisms do not infect erythrocytes but rather exist as free trypomastigotes (flagellated forms with a characteristic undulating membrane) in the blood (Fig. 13-24, A) or as amastigotes in tissue. The mechanism of anemia is believed to be immune-mediated. Cattle with acute trypanosomiasis have significant anemia, which initially is regenerative, but less so with time. The extent of parasitemia is readily apparent with T. vivax and T. theileri infections because the organisms are present in large numbers in the blood. This is in contrast to T. congolense, which localizes within the vasculature of the brain and skeletal muscle. Cattle with T. congolense infections develop chronic debilitating disease; they have scruffy hair coats, appear “potbellied,” and have fever, intermittent diarrhea, and exercise intolerance. Mortality is greater with T. vivax infections, usually because of intercurrent acute infectious diseases such as salmonellosis. Necropsy findings in cattle with trypanosomiasis include cachexia, generalized edema with increased fluid in body cavities, and generalized lymph node enlargement. Bronchopneumonia, a flabby heart, and serous atrophy of pericardial fat may be present. Liver and kidneys are symmetrically enlarged. Lymph nodes are enlarged up to four times normal size because of lymphoid hyperplasia, and most of the fatty bone marrow is replaced by red hematopoietic tissue. The spleen is enlarged because of lymphoid hyperplasia and is firm when incised.

Anaplasmosis and Ehrlichiosis

Anaplasma spp. are rickettsial organisms that may be transmitted by arthropod vectors (ticks, biting flies) or by blood-contaminated needles. Anaplasmosis is a cause of hemolytic anemia in cattle in tropical and subtropical areas of many parts of the world. Anaplasma marginale, considered the more pathogenic species, has a worldwide distribution. A. centrale is found in South America, Africa, and the Middle East. The genus names reflect the typical locations of the organisms when detected on examination of a blood smear, either on the periphery or more centrally within infected erythrocytes. A related species, A. ovis, affects sheep and goats in tropical and subtropical areas worldwide. Wild animals, such as deer, elk, and bison, may be latently infected and serve as reservoir hosts for A. marginale. Anaplasma organisms infect erythrocytes intracellularly.

Anaplasmosis causes anemia mainly by immune-mediated extravascular hemolysis. The severity of disease in infected animals varies with age. Infected calves under 1 year of age rarely develop clinical disease, whereas cattle 3 years of age or older are more likely to develop severe, potentially fatal, illness. The reason for this discrepancy is not clear. In clinically affected animals, common signs include lethargy or recumbency, pallor, and icterus. Animals dying of acute anaplasmosis have blood of low viscosity, pale to icteric tissue, an enlarged turgid spleen, and an icteric liver with a distended gallbladder. In animals with acute disease, it is usually easy to detect A. marginale organisms on routine blood smear evaluation (Fig. 13-24, B). However, in recovering animals, the organisms may be difficult to find. Surviving cattle become chronic carriers (and thus reservoirs for infection of other animals) and develop cyclic parasitemia, which is typically not detectable on blood smears. Splenectomy of carrier animals results in marked parasitemia and acute hemolysis. PCR testing is the most sensitive means of identifying animals with low levels of parasitemia.

Ehrlichiae are small, pleomorphic, Gram-negative, obligate intracellular bacteria that are transmitted by tick vectors. Some Ehrlichia spp. have a tropism for granulocytes, and morulae are sometimes found within the cytoplasm of neutrophils of affected animals (Fig. 13-25). Molecular analysis has shown that organisms previously considered to be distinct entities—E. equi, the agent of human granulocytic ehrlichiosis, and E. phagocytophilum—are genetically indistinguishable. All are now designated as Anaplasma phagocytophilum. Granulocytic ehrlichiosis occurs naturally in horses (A. phagocytophilum), dogs (A. phagocytophilum and E. ewingii), and cats (A. phagocytophilum). A. phagocytophilum survives within neutrophils by dysregulating key bactericidal functions, including the NADPH oxidase system, and delaying neutrophil apoptosis. Common clinical manifestations of infection include fever and signs referable to processes affecting cell or tissue types other than neutrophils (e.g., thrombocytopenia, anemia, immune-mediated polyarthritis). Neutropenia may occur in animals infected with A. phagocytophilum, but the mechanism is not clear. Judging from serologic evidence of exposure to A. phagocytophilum, most naturally infected dogs probably stay healthy.

Some Ehrlichieae have a tropism for mononuclear cells, but clinical manifestations are usually referable to other cell types or body systems. Morulae may be found in mononuclear cells of infected animals on routine examination of blood smears, and examination of buffy coat smears increases the probability of detecting the organism. In horses, E. risticii—the agent of Potomac horse fever (also known as equine monocytic ehrlichiosis)—infects monocytes and enterocytes and is primarily a diarrheal disease (see Chapter 7). In dogs, E. canis, the agent of canine monocytic ehrlichiosis, infects mononuclear cells. Infection with E. chaffeensis, the agent of human monocytic ehrlichiosis, has also been reported in dogs. Canine monocytic ehrlichiosis has acute and chronic forms. Acutely ill animals typically have a fever, lymph node enlargement, and splenomegaly. Thrombocytopenia and nonregenerative anemia are common findings. Untreated dogs recovering from acute disease develop a subclinical phase and may have persistent mild thrombocytopenia. A subset of these dogs develop chronic disease that may be debilitating and in some cases life threatening. Some studies indicate that German shepherd dogs with ehrlichiosis are predisposed to have particularly severe clinical disease. Severe cases are characterized by weight loss, lymph node enlargement, pyrexia, thrombocytopenia, and nonregenerative anemia. Thrombocytopenic animals may have severe bleeding tendencies. Some dogs with chronic disease develop pancytopenia. Necropsy findings vary with the stage of the disease. In the acute disease, there are widespread petechiae and ecchymoses, with splenomegaly and lymphadenomegaly. Chronically infected dogs are emaciated. The bone marrow is hyperplastic and red in the acute disease, but becomes hypoplastic and pale in animals with pancytopenia. Histologic findings include generalized perivascular plasma cell infiltration, which is most pronounced in animals with chronic disease. Multifocal, nonsuppurative meningoencephalitis, interstitial pneumonia, and glomerulonephritis are present in most dogs with the disease. Ehrlichia organisms are difficult to detect histologically; examination of Wright-Giemsa–stained impression smears of lung, liver, lymph nodes, and spleen is a more effective method for detecting the morulae in macrophages. Ehrlichiosis is often diagnosed on the basis of serologic testing, but PCR testing is more sensitive.

Anaplasma platys (previously called Ehrlichia platys) is a rickettsial organism that infects canine platelets, causing recurrent marked thrombocytopenia (the disease is also known as infectious canine cyclic thrombocytopenia). The disease is tick-borne and has been recognized worldwide. Evidence of megakaryocytic hyperplasia and organism-associated antigen within macrophages indicate that thrombocytopenia likely results from increased platelet destruction. Infection is generally considered to be asymptomatic, and morulae within platelets may be detected on blood smears incidentally, but there are rare reports describing more severe clinical signs in infected animals.

Clostridial Diseases

Certain Clostridium spp. may cause potentially fatal hemolytic anemias in animals. The mechanism of hemolysis involves a bacterial toxin (phospholipase C or lecithinase), which enzymatically degrades cell membranes, causing acute intravascular hemolysis. C. haemolyticum and C. novyi type D cause the disease in cattle known as bacillary hemoglobinuria. (The phrase “red water” has also been used for this disease and for hemolytic anemias in cattle caused by Babesia spp.) Similar naturally occurring disease has been reported in sheep and elk. In cattle, the disease is associated with liver fluke (Fasciola hepatica) migration in susceptible animals. Ingested clostridial spores may live in Kupffer cells of the liver for a long time without causing disease. However, when migrating flukes cause hepatic necrosis, the resulting anaerobic environment stimulates the clostridial organisms to proliferate and elaborate their hemolytic toxins, causing additional hepatic necrosis. Bacillary hemoglobinuria has also been associated with liver biopsy in calves. C. perfringens type A causes intravascular hemolytic anemia in lambs and calves—a condition known as yellow lamb disease, yellows, or enterotoxemic jaundice because of the characteristic jaundice. The organism is a normal inhabitant of the GI tract in these animals, but may proliferate abnormally in response to some diets. C. perfringens has also been associated with intravascular hemolytic anemia in horses with clostridial abscesses and in a ewe with clostridial mastitis.

Leptospirosis

Leptospirosis is recognized as a cause of hemolytic anemia in calves, lambs, pigs, and black rhinoceros. Specific leptospiral organisms associated with hemolytic disease include Leptospira interrogans serovars pomona and ictohaemorrhagiae. Proposed mechanisms of disease include immune-mediated (IgM cold agglutinin) extravascular hemolysis and enzymatic (phospholipase produced by the organism) intravascular hemolysis.

Leptospira organisms are ubiquitous in the environment. Infection occurs percutaneously and via mucosal surfaces and is followed by leptospiremia; organisms then localize preferentially in certain tissues (e.g., kidney, liver, and pregnant uterus). Leptospirosis can also cause many disease manifestations besides hemolysis (e.g., renal failure, liver failure, abortion, and other conditions) that are not discussed here.

In addition to anemia, common findings in animals with leptospirosis-induced hemolysis include hemoglobinuria and icterus. On necropsy, renal tubular necrosis, which occurs in part because of hemoglobinuria (hemoglobinuric nephrosis), may also be present.

Hemotropic Mycoplasmosis

The term hemotropic mycoplasmas, or hemoplasmas, encompasses a group of bacteria, formerly known as Haemobartonella or Eperythrozoon spp., that commonly infect RBCs of many domestic, laboratory, and wild animals. Hemotropic mycoplasmas affecting common domestic species are listed in Table 13-2. The mode of transmission is poorly understood but arthropods are believed to play a role; transmission in utero and through biting or fighting is also suspected. Effects of infection vary from subclinical to fatal anemia, depending on the specific organism, dose, and host susceptibility. Anemia occurs mainly because of extravascular hemolysis. Although the pathogenetic mechanisms are not completely understood, an immune-mediated component is highly probable. Hemotropic mycoplasmas (and nonhemotropic Mycoplasma spp.) induce cold agglutinins in infected individuals, although it is not clear whether these particular antibodies are important in the development of hemolytic anemia. Like other mycoplasmas, hemotropic mycoplasmas are small (0.3 to 3 µm in diameter), are Gram-negative, and lack a cell wall. They are epicellular parasites, residing in indentations and invaginations of the RBC surface. When detected on routine blood smear evaluation, the organisms are variably shaped (cocci, small rods, or ring forms) and sometimes arranged in short, branching chains (especially M. haemocanis). The organisms may also be noted extracellularly, in the background of the blood smear, especially if the smear is made after prolonged storage of the blood in an anticoagulant tube. A photomicrograph of hemotropic mycoplasma organisms in a peripheral blood smear are shown in Fig. 13-26.

Most hemotropic mycoplasma subspecies are more likely to cause acute illness in individuals that are immunocompromised or have concurrent disease. M. haemofelis infection is an exception, causing acute hemolytic anemia in immunocompetent cats. The disease in sheep and pigs has a seasonal incidence corresponding to the peak occurrence of biting insects. However, it can also occur at other times of the year as a recrudescence in a carrier animal secondary to another disease. In both sheep and pigs, unexpected death of one or two animals is often followed by anemia in other animals within the herd. M. wenyonii in cattle is less pathogenic than M. ovis and M. suis in sheep and pigs, respectively. Infection with M. wenyonii appears to be widespread, but it rarely causes disease. Clinical signs in animals with acute disease include lethargy, fever, and pallor. Affected animals usually have mild to moderate hyperbilirubinemia and may be icteric. Animals probably remain chronically infected after recovery, even if treated with appropriate antibiotics. Chronically infected cattle, sheep, and pigs may have decreased production. Chronically infected dogs and cats are typically asymptomatic. In dogs, M. haemocanis infection is usually subclinical in immunocompetent animals but causes acute hemolytic anemia when infected animals receive a splenectomy. Two forms of hemoplasmas are known to infect cats. As mentioned earlier, M. haemofelis, the large form variant, causes acute hemolytic anemia in immunocompetent animals. Cats infected with the small form variant, which has the proposed name M. haemominutum, are typically asymptomatic or have only mild disease. Organisms are often but not always detected on routine blood smear examination in acutely ill animals. PCR testing is the most sensitive means of detecting infection in animals with low levels of parasitemia. In animals dying of hemotropic mycoplasma infection, the findings are typical of extravascular hemolysis, with pallor, icterus, and splenomegaly, and distended gallbladder (Fig. 13-27). Microscopic lesions in the spleen include congestion, erythrophagia, macrophage hyperplasia, extramedullary hematopoiesis, and increased numbers of plasma cells. Bone marrow has varying degrees of erythroid hyperplasia, depending on the duration of hemolysis.

Immune-Mediated Hemolytic Anemia

IMHA is a condition characterized by increased destruction of erythrocytes because of binding of immunoglobulin to cell surface antigens. It is a common, life-threatening condition in dogs and also has been described in horses, cattle, and cats. Although the clinical picture of IMHA is variable, it typically has an acute onset and causes severe anemia. Some studies have shown that certain dog breeds (cocker spaniels and others) are predisposed to develop IMHA, suggesting the possibility of a genetic component, and the disease is more common in young to middle-aged female dogs. In most cases the reactive antibody is IgG, and hemolysis is extravascular (i.e., RBCs with bound antibody are phagocytosed by macrophages, mainly in the spleen). IgM and/or complement proteins may also contribute to IMHA. Complement usually acts as an opsonin (C₃b) that promotes phagocytosis. However, formation of the complement membrane attack complex and resulting intravascular hemolysis is also a recognized mechanism and more likely to occur with IgM autoantibodies. Most immunoglobulins implicated in IMHA are reactive at body temperature (warm hemagglutinins). A smaller portion, usually IgM, are more reactive at lower temperatures and may lead to a condition known as cold hemagglutinin disease—ischemic necrosis at anatomic extremities (e.g., tips of the ears), where cooling of the circulation causes autoagglutination of erythrocytes and occlusion of the microvasculature. Typically, IMHA targets mature erythrocytes and is accompanied by a marked regenerative response. However, as discussed earlier in the chapter, immune-mediated destruction of immature erythroid cells in the bone marrow may also occur, resulting in nonregenerative anemia.

In veterinary medicine, IMHA is usually idiopathic (also called primary IMHA or autoimmune hemolytic anemia), and the specific trigger for the autoimmune reaction is not clear. Factors implicated in secondary IMHA include infection, drug administration, vaccination, neoplasia, and bee sting envenomation. Diagnosis of secondary IMHA is most often based on circumstantial evidence and exclusion of other known causes of hemolytic anemia, rather than on controlled experiments proving a direct causal relationship. Infectious agents affecting blood cells are discussed in more detail later. Drugs or chemicals associated with or suspected of causing IMHA in animals include antibiotics (cephalosporins, penicillin, and sulfonamides), levamisole, propylthiouracil, and the insecticide pirimicarb. Most drug-induced IMHA is believed to occur because of drug or drug metabolite interacting with the erythrocyte plasma membrane. Other proposed mechanisms include binding of drug-antibody immune complexes to the erythrocyte membrane, or induction of a true autoantibody directed against an erythrocyte antigen. A case of suspected vaccine-associated IMHA has been reported in a cow. Certain vaccines used in cows have been incriminated in the development of a specific form of IMHA in newborn calves: neonatal isoerythrolysis, which is discussed later. Retrospective studies investigating the relationship between vaccination history and development of IMHA in dogs have yielded conflicting results and are inconclusive.

Other common clinical findings in patients with IMHA include hyperbilirubinemia and splenomegaly (see Fig. 13-9), pyrexia, and inflammatory neutrophilia. These abnormalities vary in magnitude depending on the severity and duration of disease. Dogs with IMHA are also predisposed to develop hemostatic abnormalities (prolonged coagulation times, decreased plasma antithrombin [AT] concentration, increased plasma concentration of FDPs/D-dimer, thrombocytopenia, and DIC). The severity of postmortem lesions in dogs with IMHA, including ischemic necrosis within vital organs (liver, kidney, heart, and lung) and the spleen as a result of thromboembolic disease or hypoxia, has been shown to correlate with the severity of leukocytosis. Intravascular hemolysis (IVH) plays a relatively insignificant role in most cases of IMHA, but evidence of IVH (hemoglobinemia, hemoglobinuria) is noted occasionally, presumably in those cases in which IgM and complement are major mediators of hemolysis.

Neonatal Isoerythrolysis

A form of IMHA whose specific pathogenesis is well understood is neonatal isoerythrolysis (NI), a condition in which a newborn has colostrum-derived maternal antibodies, which react against its own erythrocytes. NI is common in horses (Fig. 13-28) and has been reported in cattle, cats, and some other domestic and wildlife species. In horses, this situation occurs as a result of immunosensitization of the dam from exposure to an incompatible blood type inherited from the stallion (e.g., transplacental exposure to fetal blood during pregnancy or mixing of maternal and fetal blood during parturition). A previously mismatched blood transfusion produces the same results. Some equine blood groups are more antigenic than others; in particular, types Aa and Qa are very immunogenic in mares. Severely affected foals become lethargic and weak as early as 8 to 10 hours after birth or at any time during the succeeding 4 to 5 days. They have pale, icteric mucous membranes and may have hemoglobinuria. Serum bilirubin concentrations are usually increased, and foals that die during a hemolytic crisis are notably icteric and have splenomegaly. In cattle, NI has been associated with vaccination with whole blood products or products containing erythrocyte membrane fragments. In cats, the recognized form of NI does not depend on prior maternal immunosensitization but on naturally occurring anti-A antibodies in queens with Type B blood. NI has been produced experimentally in dogs, but there are no reports of naturally occurring disease. NI can be prevented by typing the maternal and paternal blood and not allowing neonates from incompatible matings access to the mother’s colostrum, or by not allowing animals with strongly incompatible blood types to breed.

Pure Red Cell Aplasia

Pure red cell aplasia (PRCA) is a rare bone marrow disorder characterized by absence of erythropoiesis and severe nonregenerative anemia. Primary and secondary forms of PRCA have been described in dogs and cats. Primary PRCA is apparently caused by immune-mediated destruction of early erythroid progenitor cells, a presumption supported by the response of some patients to immunosuppressive therapy and by the detection of antibodies inhibiting erythroid colony formation in vitro in some dogs. Infection with FeLV subgroup C is associated with secondary erythroid aplasia in cats, probably because of infection of early stage erythroid precursors. Parvoviral infection has been suggested as a possible cause of secondary PRCA in dogs. Administration of recombinant human Epo (rhEpo) has been identified as a cause of secondary PRCA in dogs, cats, and horses, presumably caused by induction of antibodies against rhEpo that cross-react with endogenous Epo. Experimentation with the use of species-specific recombinant Epo has produced mixed results. Dogs treated with recombinant canine Epo have not developed PRCA. However, in experiments reported thus far involving cats treated with recombinant feline Epo, at least some animals have developed PRCA.

Immune-Mediated Neutropenia

Immune-mediated neutropenia is a rare condition that has been reported in horses, dogs, and cats. The range of etiologies is presumably similar to that of the better characterized immune-mediated cytopenias (anemia and thrombocytopenia). The diagnosis may be supported by flow cytometric detection of immunoglobulin bound to neutrophils but is most often made on the basis of exclusion of other causes of neutropenia and response to immunosuppressive therapy.

Immune-Mediated Thrombocytopenia

Immune-mediated thrombocytopenia (IMT) is a condition characterized by immune-mediated destruction of platelets. There are numerous similarities between IMT and IMHA. IMT is a fairly common condition in dogs (it has also been described in horses and cats). It is more common in middle-aged animals, females, and perhaps some breeds of dogs. The disease is usually idiopathic and typically results in severe thrombocytopenia (often <10,000 platelets/µL), although less severe forms of the disease also occur. Affected animals have varying degrees of clinical bleeding tendencies. IMT also sometimes occurs together with IMHA, a condition known as Evans syndrome.

Underlying conditions associated with secondary IMT include infection (e.g., equine infectious anemia virus [EIAV] and ehrlichiosis), drug administration, neoplasia, and other immune-mediated diseases. Many types of infectious agents (viral, bacterial, fungal, and protozoal) have been associated with IMT, some of which may also cause thrombocytopenia by other mechanisms. Ehrlichiosis, for example, has been shown to elicit production of antibodies that are cross-reactive with platelet antigens in humans. Drugs suspected of causing IMT in animals include cephalosporins and sulfonamides.

Neonatal Alloimmune Thrombocytopenia

A form of immune-mediated thrombocytopenia, known as neonatal alloimmune thrombocytopenia, is recognized in neonatal pigs and foals. The pathogenesis of this disease is virtually identical to that of neonatal isoerythrolysis as a cause of anemia: a neonate inheriting paternal platelet antigens absorbs maternal antibodies against these antigens through the colostrum. In principle, a similar situation may occur after platelet-incompatible transfusion of blood or blood products containing platelets.

Hemophagocytic Syndrome

Hemophagocytic syndrome is a term used to describe proliferation of nonneoplastic (i.e., polyclonal), well-differentiated but highly erythrophagic macrophages. The condition is rare but has been recognized in dogs and cats. Unlike histiocytic sarcoma, which is neoplastic and in some forms also is characterized by highly erythrophagic macrophages, hemophagocytic syndrome is a secondary condition, occurring as a sequela of neoplasia, infection, or other underlying diseases. The macrophage proliferation and hyperactivation is a response to increased production of stimulatory cytokines occurring as part of the primary disease process. Macrophages are found in high numbers in the bone marrow and commonly in other tissues, including lymph nodes, spleen, and liver. Affected animals usually have cytopenias of two or more cell lines. Bone marrow findings reported in animals with hemophagocytic syndrome vary widely, ranging from hypoplasia to hyperplasia of cell lines with peripheral cytopenias, and may also include detection of macrophages containing phagocytosed hematopoietic precursor cells in addition to mature erythrocytes.

Lymphoma (Lymphosarcoma)

The term lymphoma (also known as lymphosarcoma or malignant lymphoma) encompasses a diverse group of malignancies arising in lymphoid tissue(s) outside the bone marrow. There are many different forms, with notable variability in the following:

As mentioned previously, there is an ongoing effort to develop an updated system for classifying hematopoietic neoplasia animals. This effort includes a specific project focusing on classification of lymphoma in dogs. The principle underlying this effort is that each category should constitute a distinct disease entity based on cell morphology, immunophenotype, tissue architecture, anatomic distribution, patient signalment, and full clinical history. A critical component of this effort is to test interobserver and intraobserver variability of interpretation.

Lymphoma is often suspected on the basis of organomegaly (e.g., generalized lymph node enlargement) or abnormal ultrasound findings (e.g., thickened intestines with abnormal echogenicity). Initial diagnosis typically is made based on aspiration cytology, biopsy and/or histopathology, or both, of the affected organ(s). In both cytologic and histologic preparations, lymphoma is characterized by a monomorphic population of morphologically atypical lymphocytes. Histologically, lymphoma is also characterized by disruption of normal tissue architecture. In general, lymphomas of small, well-differentiated cells with low mitotic rates are low-grade (indolent, slowly progressive) diseases, whereas lymphomas of large, poorly differentiated cells with high mitotic rates are high-grade (aggressive, rapidly progressive) diseases (Fig. 13-31). The type of tumor and extent of disease may be characterized further by immunophenotyping and clinical staging, respectively. Immunophenotyping is done to determine whether a tumor is of B lymphocyte or T lymphocyte origin (this distinction cannot be made reliably based on routine staining) (see Fig. 13-31). It is not necessarily the case that B lymphocyte lymphomas have a more favorable prognosis than T lymphocyte lymphomas because lymphomas of both cell types occur in low-grade and high-grade forms. Clinical staging includes examination of aspirates of bone marrow and other enlarged organs (e.g., spleen, liver, and other lymph nodes).

In cases of advanced disease, there may be marked bone marrow and blood involvement and distinguishing lymphoma from lymphoid leukemia can occasionally be difficult. The distinction can often be made on the basis of immunophenotyping, especially for CD34, which typically is expressed by neoplastic cells in acute leukemias and not in lymphomas. Other laboratory findings with lymphoma are variable. Mild-to-moderate nonregenerative anemia is probably the most common hematologic abnormality. Lymphopenia is noted more frequently than lymphocytosis, presumably because of abnormal trafficking of normal, nonneoplastic lymphocytes. Hypercalcemia is often associated with lymphoma caused by production of parathyroid hormone-related peptide (PTHrP) by the neoplastic cells. Such paraneoplastic hypercalcemia (humoral hypercalcemia of malignancy) may lead to pathologic calcification and related complications (e.g., renal failure).

Necropsy findings in cases of multicentric lymphoma include enlarged lymph nodes that bulge on cut section and are gray-white to light tan or reddish (see Fig. 13-80). In advanced cases, the normal architectural distinction between the cortex and medulla is obliterated. Infiltration of spleen and liver can result in diffuse enlargement or nodules. In alimentary lymphoma, affected portions of the GI tract are thickened and may be nodular. The lesions may be ulcerated. Enlarged mesenteric nodes may coalesce to form large masses. The gross appearance of thymic lymphoma is a large, gray, soft mass in the cranial mediastinum (see Fig. 13-49). Renal lymphoma often affects both kidneys and is evident as bilateral diffuse renomegaly. Central nervous system and bone marrow involvement of lymphoma can be difficult to detect on necropsy because of the similar gross appearance of neoplastic and normal tissue. In any of the various forms of lymphoma, large masses may have areas of central necrosis.

Lymphoma is the most common hematopoietic malignancy in animals and has been reported in all the domestic species.

Lymphoma is uncommon in horses, representing 5% or less of all neoplasms. Multicentric lymphoma is the most common form, with lesions occurring more frequently in the abdominal and thoracic cavities than in peripheral lymph nodes. Cutaneous and other forms may also occur. Lymph nodes in different anatomic regions (e.g., superficial, mediastinal, and alimentary) are often similarly affected, and infiltration of liver and spleen is common. Reports of the frequency of concurrent blood involvement are highly variable. The alimentary form of equine lymphoma is a wasting syndrome, presumably because of involvement of the small intestine, resulting in malabsorption. Cutaneous lymphoma in horses, as in other species, is a chronic disease without blood or internal organ involvement. A recent report of 37 cases of equine lymphoma found T lymphocyte lymphoma to be more common than B lymphocyte lymphoma. Weight loss and ventral edema were the most common clinical signs. Laboratory abnormalities present in at least one-third of cases included hyperfibrinogenemia, hypoalbuminemia, hyperglobulinemia, anemia, thrombocytopenia, and neoplastic cells in circulation. In all tumors, normal nodal architecture was diffusely effaced. Extensive necrosis was common. Many of the tumors were composed of morphologically heterogeneous cells, and a smaller subset had marked infiltration with histiocytes and multinucleated giant cells. Most T lymphocyte tumors were composed of cells of small to medium size, and most B lymphocyte tumors were classified as T lymphocyte-rich B lymphocyte neoplasms.

In cattle, lymphoma is primarily a multicentric B lymphocyte disease of adults and is strongly associated with BLV infection and BLV-related persistent lymphocytosis. Approximately 30% of cattle infected with BLV develop nonneoplastic persistent lymphocytosis and of these, less than 5% develop lymphoma. The BLV-induced form of lymphoma is known as bovine enzootic lymphoma. BLV is a retrovirus that persists within lymphocytes for the life of the infected animal. Transmission of the virus is mostly horizontal caused by transmission of infected lymphocytes as opposed to free virus in secretions. Blood-sucking arthropods or other mechanical means of transferring small numbers of infected lymphocytes (such as contaminated needles) are the principal means of spread. BLV infection is more common in dairy cattle than in beef cattle, presumably because dairy cattle husbandry favors viral transmission. Incidence of infection varies widely between regions and between herds. Antibodies against viral antigen can be detected by an agarose gel immunodiffusion test, and some herds remain free of BLV by testing and culling infected animals. In addition to lymph nodes, lymphoma in cattle often involves other locations, such as abomasum, vertebral canal, kidney, heart, retro-orbital space, and uterus (see Fig. 13-86). Enlargement of superficial lymph nodes is common, and enlarged pelvic and abdominal lymph nodes are often found on rectal palpation. Involvement of the digestive tract and heart may cause vagal indigestion or diarrhea and congestive heart failure, respectively. Lymphoma of the central nervous system is most frequently manifested as posterior paresis or paralysis because of pressure by the extradural mass on the cauda equina or on the lumbar cord. Other sporadic forms of lymphoma not associated with BLV infection also occur in cattle, including thymic, multicentric, and cutaneous forms affecting young animals. The thymic form of sporadic bovine lymphoma is characterized by large cranial thoracic and lower cervical masses, respiratory distress, and weight loss in cattle less than 2 years of age. The sporadic multicentric form is characterized by disseminated disease in calves 3 to 6 months of age. Affected calves frequently have widespread lymph node enlargement in addition to hepatic, splenic, and renal involvement. Marked blood and bone marrow involvement may also occur. The cutaneous form is rare, occurs in young cattle, and consists of discrete cutaneous plaques or large scabby lesions.

In dogs, lymphoma is a common form of neoplasia, representing approximately 7% to 9% of all malignancies and the large majority of hematopoietic malignancies. No retroviral or other cause is known. Lymphoma is typically a disease of middle-aged to older dogs. Multicentric lymphoma with generalized lymph node enlargement is the most common form (80% to 85% of all cases of canine lymphoma), and the majority of these (approximately 80%) are intermediate- or high-grade tumors. Other less common forms of canine lymphoma include alimentary, thymic, and cutaneous forms, and sporadically occurring forms in virtually every conceivable location. Approximately 70% to 80% of canine lymphomas are of B lymphocyte origin. Although the remaining cases are mostly of T lymphocyte origin, “null” (non-B, non-T, possibly of natural killer cell origin) lymphomas also occur. Clinical signs may be referable to the specific organ systems affected but are usually absent or nonspecific when lymphoma is first diagnosed. Approximately 15% of dogs with lymphoma and approximately 40% of those with mediastinal lymphoma, are hypercalcemic. In the majority of canine lymphoma cases, significant blood or bone marrow involvement is not detected by routine methods on initial diagnosis but may be seen with advanced disease. Splenic and hepatic involvement is common in cases of canine multicentric lymphoma.

In cats, lymphoma is among the many hematopoietic abnormalities caused by FeLV, and the epidemiology of feline lymphoma has changed with the development of routine vaccination and testing for FeLV. Cats infected with FeLV are likely to develop lymphoma at a younger age, and are more likely to develop certain types of lymphoma, especially mediastinal and multicentric T lymphocyte forms, than FeLV-negative cats. These predispositions still apply, but the percentage of cats infected with FeLV has decreased dramatically. As a result, mediastinal (thymic) and multicentric forms of lymphoma, which used to represent the majority of cases, have become relatively uncommon. At present, approximately 80% to 90% of cats with lymphoma are FeLV-negative, and the majority of lymphoma cases are the alimentary form. Other less common locations in cats include mediastinal, multicentric, renal, and other extranodal sites. FeLV-positive cats with lymphoma still tend to be younger, and their tumors more often of T lymphocyte origin, than FeLV-negative cats. The large majority (>80%) of cats with mediastinal lymphoma, and approximately one-third of cats with multicentric lymphoma, are FeLV-positive. Mediastinal and multicentric forms, although less common now, are still more likely to occur in younger cats, whereas the alimentary form typically develops in older cats (>10 years of age). The neoplastic cell type also tends to vary by anatomic location. Alimentary lymphoma in cats is predominantly a B lymphocyte disease, whereas mediastinal lymphoma is predominantly a T lymphocyte disease (consistent with thymic origin). A subtype of intestinal lymphoma in cats, large granular lymphocyte (LGL) lymphoma, is predominantly a T lymphocyte disease with a highly aggressive biologic behavior. Unlike dogs, cats are usually clinically ill when lymphoma is first diagnosed. In addition to nonspecific signs, such as weight loss, anorexia, and poor grooming habits, cats often have signs referable to the affected organ systems. For example, animals with alimentary lymphoma often have chronic diarrhea and vomiting and may have palpable abdominal masses, whereas those with mediastinal lymphoma are often dyspneic.

Lymphoma is the most commonly reported malignancy in pigs and may be more likely to affect females than males. Multicentric lymphoma is the most common form of the disease in pigs. Lymph node enlargement is more common in visceral than peripheral nodes. Other commonly affected organs include spleen, liver, kidney, and bone marrow. Lymphoma often affects animals less than 1 year of age, and the mediastinal form of lymphoma tends to affect younger pigs more commonly than the multicentric form. A viral cause (C type viruses) for lymphoma in pigs has been suggested, but transmission studies are lacking. A form of hereditary multicentric lymphoma has also been reported in inbred herds.

Plasma Cell Neoplasia

There are two main forms of plasma cells tumors recognized in veterinary species: multiple myeloma (MM) and plasmacytoma.

MM is a malignant tumor of plasma cells that arises in the bone marrow and usually secretes large amounts of Ig. Blood involvement of the neoplastic cells is not a feature of the disease. MM is a rare disease in animals. Dogs are affected more frequently than other species, but MM has also been reported in horses, cattle, cats, and pigs. The hallmark laboratory finding in patients with MM is hyperglobulinemia, which occurs because of production of large amounts of Ig or Ig subunit by the neoplastic cells. This homogeneous protein fraction is often called paraprotein or M protein. Concentrations of other Igs are often decreased.

Diagnosis of MM is based on finding a minimum of two or three (opinions vary) of the following abnormalities:

Other pathologic findings in patients with MM may include the following:

MM typically has a slowly progressive clinical course. Common sites of metastasis include the spleen, liver, lymph nodes, and kidneys.

Cutaneous plasmacytomas are solid tumors of plasma cell origin that involve the skin or mucous membranes. Masses may be single or multiple. These tumors are usually benign, and excision is usually curative, but more aggressive forms may occur. Cutaneous plasmacytoma is discussed in more detail in Chapter 17. The tumor type known as extramedullary plasmacytoma (EMP) is a malignant solid tissue tumor of plasma cells arising from sites other than the bone marrow. The tumor is rare in animals, occurring most often in dogs and also reported in horses and cats. In one study, there was a disproportionately high percentage of EMP cases in cocker spaniels. The tumors occur most frequently in the GI tract but may also occur in the trachea, spleen, kidney, uterus, central nervous system, and elsewhere. Grossly, the tumors may be multinodular or cause thickening of the intestinal wall. Metastasis to regional lymph nodes is common. As with MM, the neoplastic cells composing the tumor may be well-differentiated to poorly differentiated (Fig. 13-33). EMPs produce monoclonal immunoglobulins, and monoclonal gammopathy has been reported in some cases of EMP. If there is bone or bone marrow involvement of a malignant plasma cell tumor, it is considered to be MM. Amyloidosis is associated with EMP in many reports and may be useful in distinguishing extramedullary plasmacytoma from other tumors. The distinction between cutaneous plasmacytoma and extramedullary plasmacytoma is not always clear, and benign plasmacytomas of the skin have also been referred to as cutaneous extramedullary plasmacytomas.

Histiocytic Neoplasia

Histiocytic sarcoma (HS) is an uncommon malignant neoplasm of histiocytic (macrophage or dendritic cell) origin. It occurs most frequently in the dog but has also been reported in the cat. There is a general belief that Rottweiler and Bernese mountain dogs are at increased risk for HS, although good epidemiologic data is thus far lacking. Some have called the disseminated form of this disease, with multiple organ involvement, by the name malignant histiocytosis, reserving the term histiocytic sarcoma for cases of single solid tumors, but there is an emerging consensus that the latter term encompasses both forms of the disease. The disseminated form of the disease has a rapid, aggressive clinical course. Sites commonly involved include the spleen, lung, lymph nodes, bone marrow, skin, and subcutis. Liver involvement occurs secondary to disease in the spleen. The solitary form of HS can occur at any of the aforementioned sites and also in joints (in close association with the subsynovium) or brain. Most cases of HS are of dendritic antigen-presenting cell origin, with a similar immunophenotype to (but dramatically different biologic behavior than) cutaneous histiocytoma. Fewer cases of HS are of macrophage cell origin. These malignant cells have an immunophenotype characteristic of resident macrophages in the splenic red pulp and the bone marrow, and frequently show marked phagocytosis of erythrocytes (hemophagocytosis). Dogs with this form of HS frequently have a hemophagocytic syndrome characterized by severe nonregenerative to mildly regenerative anemia (presumably a result largely of destruction of RBCs by the neoplastic cells), splenomegaly, hepatomegaly, and extramedullary hematopoiesis in the spleen and elsewhere. Microscopically, HS tumor cells are large, round-to-spindloid in shape and vary from relatively well-differentiated histiocytic morphology to cells with notable features of malignancy (Fig. 13-34).

Cutaneous histiocytosis (CH) and systemic histiocytosis (SH), nonneoplastic canine immunoregulatory disorders that share some clinical features of histiocytic malignancies, are covered in the section on canine immune-mediated diseases, and CH is discussed in more detail in Chapter 17.

Canine cutaneous histiocytoma, a benign neoplasm of epidermal Langerhans cell origin, and feline progressive histiocytosis, an initially indolent cutaneous neoplasm that is probably of dendritic cell origin, are discussed in detail in Chapter 17.

Mast Cell Neoplasia

Mast cell tumors (MCTs) of the skin and other organs are relatively common in animals, especially in dogs, and are covered in Chapter 6. Primary leukemia of mast cell origin is a rare form of myeloid leukemia. Mast cells normally are not present in circulation, but the finding of mast cells in the blood (mastocytemia) does not necessarily indicate myeloid neoplasia. In fact, one study found that severity of mastocytemia in dogs was frequently higher in animals without MCTs than those with MCTs and that random detection of mast cells in blood smears usually is not a result of underlying MCT.

Granulocytic Sarcoma

There are rare reports in animals of extramedullary solid tumors of granulocytic origin, known as granulocytic or myeloid sarcomas. These tumors are poorly characterized.

Flavin Adenine Dinucleotide Deficiency

Flavin adenine dinucleotide (FAD) is a cofactor for cytochrome b₅ reductase, the enzyme that maintains hemoglobin in its functional reduced state, and for the glutathione reductase reaction that also helps protect erythrocytes from oxidative damage. Deficiency of erythrocyte FAD caused by abnormal erythrocyte riboflavin metabolism was recently reported in a Spanish mustang mare with methemoglobinemia and eccentrocytosis and has also been recognized in a Kentucky mountain saddle horse gelding.

Equine Infectious Anemia Virus

EIAV is a lentivirus that infects cells of the mononuclear phagocyte system in horses (also ponies, donkeys, and mules) throughout the world. Natural transmission is by arthropods, and the virus can also be transmitted transplacentally. Disease occurs in both an acute, potentially fatal form after initial infection, as well as a chronic intermittent form associated with recurrent viremia. Recurrent episodes happen mostly in the first year after infection, and tend to decrease in frequency and severity with time. Infection is lifelong and animals may become asymptomatic carriers. Viral antigen is found mainly within liver, spleen, and serum of infected animals, and also in bone marrow, lymph nodes, thymus, circulating mononuclear cells, and other tissues. EIAV causes anemia by both immune-mediated hemolysis and decreased erythropoiesis. Hemolysis is typically extravascular but may have an intravascular component during the acute phase. Decreased erythropoiesis may result from direct suppression of early-stage erythroid cells by the virus, as well as anemia of inflammation. Thrombocytopenia, likely caused by secondary immune-mediated destruction, is also a classic feature of acute EIAV infection and recurring febrile episodes. Clinical findings associated with viremic episodes include fever, depression, icterus, petechial hemorrhages, lymph node enlargement, and dependent edema. EIA infection is diagnosed on the basis of the Coggin’s test, an agarose gel immunodiffusion test for the presence of the antibody against the virus.

Animals dying during hemolytic crises have icterus, anemia, and widespread hemorrhages. The spleen and liver are enlarged, dark and turgid, and they and other organs have superficial subcapsular hemorrhages. Petechiae are evident beneath the renal capsule and throughout the cortex and medulla. The bone marrow is dark red as a result of replacement of fat by hematopoietic tissue; the extent of replacement is an indication of the duration of the anemia. The severity of microscopic lesions is dependent on the chronicity of the disease, and they are most significant in the spleen, liver, and bone marrow. As would be anticipated, microscopic findings of the spleen are predominantly influenced by the number and activity of macrophages, which is a reflection of the duration of the disease and the frequency of hemolytic episodes. Hemosiderin-laden macrophages persist for months to years; therefore, large numbers are consistent with chronicity. Kupffer cell hyperplasia with hemosiderin stores and periportal infiltrates of lymphocytes are the most significant changes in the liver. Bone marrow histologic findings vary depending on the duration of the disease. In most animals, the marrow is cellular because of the replacement of fat by intense, orderly erythropoiesis. Granulocytes are relatively less numerous and plasma cells are increased. As in the spleen, hemosiderin-laden macrophages are present in large numbers in chronic cases. In more chronic cases, emaciated animals have serous atrophy of fat (see Web Fig. 13-1).

Congenital Dyserythropoiesis in Polled Herefords

A syndrome of congenital dyserythropoiesis and alopecia occurs in polled Hereford calves. The disease is often fatal. Hematologically, the condition is characterized by anemia that is nonregenerative to mildly regenerative (poorly regenerative given the degree of anemia). Bone marrow findings include marked erythroid predominance and morphologic abnormalities, consistent with ineffective erythropoiesis. The specific defect has not been identified.

Erythrocyte Band 3 Deficiency in Japanese Black Cattle

Japanese black cattle lacking band 3, an erythrocyte integral membrane protein that connects to the cytoskeleton, have moderate hemolytic anemia and retarded growth.

Hypophosphatemic Hemolytic Anemia

Marked hypophosphatemia is recognized as a cause of intravascular hemolytic anemia in postparturient dairy cows. Hypophosphatemia develops in these animals because of increased loss of phosphorus in their milk. Biochemical studies suggest that the mechanism of hemolysis involves decreased erythrocyte production of ATP, which may lead to compromised membrane and cytoskeletal integrity. An accompanying decrease in reducing capacity and increase in methemoglobin concentration have also been noted in experimental studies of hypophosphatemic hemolytic anemia in dairy cattle, suggesting that oxidative mechanisms may also contribute to anemia. Affected cows are anemic and hemoglobinuric. Gross postmortem findings include pallor, decreased viscosity of the blood, and discolored pale yellow and swollen liver and kidney. Renal tubular necrosis and hemoglobin pigment within the tubules is evident microscopically. Hemolysis has also been reported in association with hypophosphatemia in dogs and cats.

Water Intoxication

Calves with sporadic access to water sometimes drink excessively when water is available to the point where their plasma becomes hypotonic and osmotic intravascular hemolysis occurs. Hemoglobinuria is commonly observed in affected animals. The condition is rarely fatal.

Bovine Leukemia Virus

BLV is discussed in the earlier section on lymphoma.

Bovine Viral Diarrhea Virus

Thrombocytopenia, often severe, has been reported in association with acute bovine viral diarrhea virus (BVDV) infection in calves and adult cattle. Infection with type II BVDV has been specifically associated with a thrombocytopenic hemorrhagic syndrome. Calves infected with type II BVDV have also been shown to have impaired platelet function. Investigations of the mechanism of BVDV-induced thrombocytopenia have resulted in varying, sometimes conflicting, conclusions. More than one study has shown viral antigen associated both with bone marrow megakaryocytes and with circulating platelets. Evidence of impaired thrombopoiesis (megakaryocyte necrosis, megakaryocyte pyknosis, and degeneration) and increased thrombopoiesis (megakaryocytic hyperplasia, increased numbers of immature megakaryocytes) in the bone marrow has been reported in type II BVDV-infected animals, including concurrent megakaryocyte necrosis and hyperplasia in some experimental subjects. To our knowledge, antibody-mediated destruction of platelets has not been shown.

Cyclic Hematopoiesis

Cyclic hematopoiesis is an inherited disorder of pluripotent hematopoietic stem cells, recognized in dogs and people. In dogs, the condition has an autosomal recessive inheritance pattern and is associated with dilute hair coat color. In dogs, cyclic hematopoiesis (also known as lethal gray collie disease) is characterized by predictable fluctuations in concentrations of blood cells that occur in 14-day cycles. The pattern is cyclic marked neutropenia and in a different phase, cyclic reticulocytosis, monocytosis, and thrombocytosis. Production of key cytokines involved in regulation of hematopoiesis is also cyclic. The specific lesion in cyclic hematopoiesis is believed to involve defective intracellular signaling but remains to be explained on the molecular level. Neutropenia predisposes affected animals to infection, and many die of infectious causes. Other related clinical manifestations include bleeding tendency, attributable at least in part to defective platelet function, and systemic amyloidosis, which occurs because of cyclic increases in concentration of acute phase proteins during phases of monocytosis.

Phosphofructokinase Deficiency

Inherited autosomal recessive deficiency of the erythrocyte glycolytic enzyme, phosphofructokinase (PFK), is described in English springer spaniel, American cocker spaniel, and mixed-breed dogs. The enzyme is also deficient in muscle tissue of affected dogs. Erythrocytes in PFK-deficient dogs have decreased ATP and 2,3-diphosphoglycerate production, and increased fragility under alkaline conditions. The disease is characterized by chronic, extravascular hemolysis with marked reticulocytosis. The marked regenerative response may compensate for the ongoing hemolysis, therefore affected animals are not necessarily anemic. However, acute intravascular hemolytic episodes may occur with hyperventilation and resulting alkalemia. There are three genes encoding PFK enzymes, designated M in muscle and erythrocytes, L in liver, and P in platelets. A point mutation in the gene coding for the M enzyme results in an unstable, truncated molecule. A single DNA-based test is available to detect the common mutation.

Erythrocyte Structural Abnormalities

A heterogeneous group of conditions, known as hereditary stomatocytosis because of the characteristic slit-shaped area of central pallor in RBCs on stained blood smears, has been described in several dog breeds. The clinical manifestations vary in affected animals, and the specific underlying defects have not been identified. In all cases, however, erythrocytes have increased osmotic fragility and decreased survival.

Other (presumably heritable) erythrocyte abnormalities in dogs that are not associated with clinical signs include elliptocytosis caused by band 4.1 deficiency or β-spectrin mutation, and familial macrocytosis and dyshematopoiesis in Poodles.

Scott-Like Syndrome

An inherited thrombopathy resembling Scott syndrome in humans, in which platelets lack normal procoagulant activity, has been recognized in a family of German shepherd dogs. Affected dogs have a mild to moderate clinical bleeding tendency characterized by epistaxis, hyphema, intramuscular hematoma formation, and increased hemorrhage associated with surgery. The specific defect in these dogs has not been identified on the molecular level, but involves impaired expression of phosphatidylserine on the platelet surface.

Breed-Related Platelet Abnormalities

Cavalier King Charles spaniels often have a lower than normal concentration of platelets, many of which are abnormally large (a condition known as macrothrombocytopenia). The molecular basis for this trait is a mutation in the gene encoding β1 tubulin. In general, affected dogs are asymptomatic. Cavalier King Charles spaniels have been shown to have abnormal platelet aggregation in vitro in some studies, although the clinical significance of these findings, including a possible causal relationship between platelet abnormalities and mitral valve disease in this breed, is not clear.

Greyhounds tend to have lower concentrations of circulating platelets than other dog breeds, although this is not of any known clinical significance. Other Greyhound traits include lower neutrophil and plasma protein concentrations and higher hematocrits than other dog breeds.

Cutaneous and Systemic Histiocytoses

CH, a rare immunoregulatory disorder caused by proliferation of activated dermal dendritic cells, is discussed in more detail in Chapter 17. SH is a familial disorder of Bernese mountain dogs (also found sporadically in other breeds) characterized by large, dense proliferations of activated interstitial dendritic cells in multiple tissues, including skin, peripheral lymph nodes, and ocular and nasal mucosa. The proliferating cells are immunophenotypically identical to those in CH, and the angiocentric skin lesions in SH are histologically identical to those in CH. Lesions of SH in locations other than skin are also angiocentric infiltrates consisting mostly of histiocytes and lymphocytes.

Increased Erythrocyte Osmotic Fragility

A condition characterized by increased erythrocyte osmotic fragility has been described in Abyssinian and Somali cats. The specific defect has not been identified, but pyruvate kinase deficiency (which has been reported in these breeds) was excluded as the cause. Affected cats have chronic intermittent severe hemolytic anemia and often have other lesions secondary to hemolytic anemia (e.g., splenomegaly and hyperbilirubinemia).

Cytauxzoonosis

Cytauxzoon felis is a protozoal organism causing severe, often fatal, disease in cats. Cytauxzoonosis in domestic cats is relatively common in the South Central United States, particularly during summer months. Bobcats (Lynx rufus), and perhaps other wild felids, are believed to be the reservoir host. They are usually asymptomatic, with persistent parasitemia and transient schizogony. Cytauxzoon felis is transmitted by a tick vector, Dermacentor variabilis, which is probably essential for infectivity of the organism. Cytauxzoonosis has a schizogenous phase involving macrophages throughout the body (especially liver, spleen, lung, lymph nodes, and bone marrow) that causes systemic illness, and an erythrocytic phase that causes anemia of variable severity. Schizont-containing macrophages (Fig. 13-35) enlarge and accumulate within the walls of veins, eventually causing occlusion of the vessels. Merozoites are released and enter erythrocytes. Parasitemia develops relatively late in the course of infection. The signet ring–shaped erythrocytic inclusions (piroplasmas) of Cytauxzoon felis closely resemble small form Babesia (see Fig. 13-23, A) and some Theileria organisms. Affected cats typically become acutely ill with fever, pallor, and icterus and usually die within 2 to 3 days. For many years, cytauxzoonosis was considered to be almost always fatal. However, a recent report, in which numerous cats from a subregion of the endemic area in the United States survived infection with an organism with greater than 99% homology to Cytauxzoon felis, suggests the emergence of a less virulent strain. In affected cats, erythrophagocytosis is also often a prominent finding in tissue with the schizogenous phase.

Feline Leukemia Virus

FeLV is an oncogenic, immunosuppressive lentivirus associated with hematologic abnormalities of widely varying type and severity, including anemia in most infected cats. Manifestations of disease caused by FeLV infection vary depending on dose, viral genetics, and host factors, but normal hematopoiesis is probably suppressed to some degree in all cases. The virus infects hematopoietic precursor cells soon after the animal is exposed and continues to replicate in hematopoietic and lymphatic tissue of animals that remain persistently viremic. Persistently viremic cats are immunosuppressed and are prone to developing other diseases, including infectious diseases, bone marrow disorders, and lymphoma (lymphosarcoma).

FeLV-induced anemia is usually nonregenerative, presumably caused by direct effects of the virus on infected erythroid cells. However, macrocytosis and metarubricytosis (the presence of nucleated RBC precursors in circulation) are often noted in the absence of significant reticulocytosis—findings consistent with dyserythropoiesis, although the exact mechanism is not clear. Bone marrow of cats with FeLV-induced anemia often has evidence of arrested or disordered maturation of hematopoietic precursors. The relatively uncommon subgroup C viruses are associated with erythroid aplasia, probably because of infection of early stage erythroid precursors. Regenerative anemia may also occur with FeLV infection, often because of coinfection with Mycoplasma haemofelis.

FeLV infects hematopoietic cells and can produce a wide array of hematologic and other disease manifestations. FeLV may be detected in megakaryocytes and platelets in infected cats, and may result in platelet abnormalities, including thrombocytopenia, thrombocytosis, increased platelet size, and decreased function. Proposed mechanisms of FeLV-induced thrombocytopenia include direct cytopathic effects, myelophthisis, and immune-mediated destruction. Platelet life span and function have been shown to be decreased in FeLV-positive cats.

Feline Immunodeficiency Virus

FIV, another feline lentivirus, is associated with anemia in a minority of infected cats. Immunosuppressive effects of FIV from thymic depletion are discussed elsewhere. It is generally accepted that anemia does not result directly from FIV infection but instead develops because of concurrent disease such as co-infection with FeLV or hemotropic mycoplasma, other infection, or malignancy. The severity and type of anemia in FIV-infected cats depends on the other specific disease processes involved.

Structure and Function

The thymus is essential for the development and function of the immune system, specifically for the differentiation, selection, and maturation of T lymphocytes. This is described in detail in Chapter 5, and the interrelationship of the T lymphocytes with the components of the thymus are described in this section. The shape and location of the thymus vary among young domestic animals. In ruminants and pigs, the thymus has two lobes: cervical and thoracic. The cervical lobe is large and extends along the lateral surfaces of the cervical trachea. The size of the cervical lobe varies in cats and horses but is usually small. Dogs do not have a cervical lobe. The thoracic lobe is present in all domestic animals and lies in the cranial mediastinum, ventrally in the horse, pig, and dog and dorsally in ruminants.

The thymus has also been called a lymphoepithelial organ because besides lymphocytes it has an epithelial component. Histologically, it is divided into two portions, the stromal portion and the thymocyte portion (T lymphocytes at different stages of maturation and differentiation). All T lymphocytes in the thymus are derived from progenitor T lymphocytes in the bone marrow. The capsule, trabeculae, and blood vessels arise from the mesoderm of a branchial arch. Some stroma is designated “epithelial reticulum,” reflecting its embryonic origin from branchial pouches and the third pharyngeal cleft. It produces no reticular fibers and thus is different from the reticulum composed of a meshwork of specialized fibroblast-like cells with long interdigitating filamentous projections (reticular cells), through which are dispersed macrophages and occasional myoid cells (smooth muscle-like).

The lymphoid component is composed of differentiating lymphocytes, which during gestation arise from hematopoietic progenitor cells, initially in the fetal liver, then spleen and later in the fetal bone marrow. The epithelial component consists of individual cells in the cortex and medulla and aggregates of epithelial cells in the medulla. The latter, called Hassall’s corpuscles, are a characteristic histologic feature of the thymus. Cortical and medullary epithelial cells contribute to the microenvironment and are essential for differentiation, selection, and maturation of T lymphocytes in neonate and adult thymuses.

Microscopically, the thymus is divided into incomplete lobules, each of which has a cortex and a medulla, with the medullas being confluent centrally (Fig. 13-36). On the basis of functional and epithelial components, three zones are recognized: a subcapsular zone, a cortex, and a medulla. Bone marrow–derived T lymphocytes enter the circulation, travel to the thymus, and enter at the subcapsular zone (Fig. 13-37). Here, they begin differentiation and selection processes and develop into mature naïve T lymphocytes as they traverse the thymic cortex to the medulla. In the cortex, T lymphocytes that recognize self-molecules (i.e., major histocompatibility complex [MHC] molecules) but not self-antigens are permitted to mature by a process called positive selection. Cells that do not recognize MHC molecules are removed by apoptosis. Those T lymphocytes that recognize both MHC molecules and self-antigens are removed by macrophages at the corticomedullary junction, a process called negative selection. Because of the rigid differentiation requirements attributable to MHC restriction and tolerance (positive and negative selection, respectively) only a small fraction (<5%) of the developing T lymphocytes that arrive at the thymus from the bone marrow survive. Mature naïve T lymphocytes exit the thymus through postcapillary venules in the corticomedullary region, enter the circulation, and recirculate through secondary lymphoid tissues in a highly regulated manner. The T lymphocyte regions of secondary lymphoid organs are specialized sites where these circulating mature naïve T lymphocytes (T lymphocytes that have not been exposed to their T lymphocyte receptor-specific antigen) are activated. On exposure to their specific antigens, they undergo additional phases of development and differentiate into effector and memory cells, some of which remain localized in lymph nodes and others migrate to sites of infection. The balance between the rates of production and distribution of T lymphocytes is critically important in maintaining immune homeostasis. In fact, immunodeficiency or autoimmunity may result from inadequate or overly zealous activity, respectively, of a particular subset of T lymphocytes.

The thymus attains its maximal mass relative to body weight at birth and involutes after sexual maturity. The lymphoid and epithelial components are gradually replaced by loose connective tissue and fat, although remnants remain histologically, even in aged animals.

Responses to injury

The responses of the thymus to injury are listed in Box 13-4, and the most common ones are lymphoid atrophy, inflammation, and neoplasia. The most common change is lymphoid atrophy caused by physical and physiologic stresses, toxins, drugs, and viral infections.

Structure

The spleen, suspended in the gastrosplenic ligament and covered by peritoneum, is located in the left cranial hypogastric region of the abdominal cavity between the diaphragm, stomach, and the body wall, except in domestic ruminants, where it is closely adhered to the left dorsolateral aspect of the rumen. The gross morphology (shape and size) of the spleen varies markedly among domestic animals, but generally it is a flattened, elongated organ. The spleen is covered by a thick fibromuscular capsule from which numerous intertwining fibromuscular trabeculae extend into the parenchyma and which along with the reticulum form the supporting stroma for the parenchymal components. The splenic parenchyma, unlike that of many organs, does not have a cortex and medulla but is divided into two distinct structural and functional components: the red pulp and white pulp. The red pulp consists of cells of the monocyte-macrophage system in the splenic cords and PAMS and red pulp vascular spaces and associated stroma (reticular cells and fibroblasts and myocytes in the trabeculae). The white pulp consists lymphocytes of the B lymphocyte regions (splenic follicles), T lymphocyte regions (PALS), and the marginal zone in which are macrophages, antigen-presenting cells, and trafficking B and T lymphocytes. There are minor differences in some of the splenic components of the domestic animals (e.g., the cat has a small marginal sinus but a well-developed PAMS).

The arterial and venous vasculature have minor differences among the domestic animals. The following account is correct for the canine spleen, and if the sinusoids, which are only in the canine spleen, are omitted, it applies in general to the nonsinusal spleens of the other domestic animals. The artery supplying the spleen is the splenic artery, a branch of the celiac artery, which is a major branch of the abdominal aorta. The splenic artery penetrates the splenic capsule at the hilus, branches, and enters the fibromuscular trabeculae as the trabecular artery (Fig. 13-38). As it leaves the trabecula, the trabecular artery becomes the central arteriole of the white pulp and is surrounded by a cuff of T lymphocytes (PALS). Adjacent to or eccentrically embedded in the PALS is a splenic follicle (B lymphocytes). A branch of the central artery goes to the marginal sinus, which surrounds the splenic follicle and carries blood-borne antigens and trafficking B and T lymphocytes into close contact with the follicle (Fig. 13-39). In the dog, the marginal sinus drains into the sinusoids, but in other domestic animals, it drains into the red pulp vascular spaces. It is significant that the first branch of the central artery goes to the marginal sinus. As a result, antigens, bacteria, particles, and other material are delivered to the marginal sinus first, before either the sinusoids (in the dog) or red pulp vascular spaces. After emerging from the PALS, the arteriole is surrounded by a sheath of macrophages known as PAMS. The PAMSs are very prominent in pigs. It then becomes the penicillary arteriole, and in horses, cattle, pigs, and cats, its terminal branches empty into the red pulp vascular space lined by reticular cells, which lie in and between the splenic cords. The term red pulp vascular spaces used here has the advantage of being descriptive and correct and clearly differentiates them from venous sinuses and sinusoids. In dogs and humans, branches of the central arteriole of the white pulp enter into venous sinusoids, which are lined by a discontinuous endothelium and these empty into splenic venules. The anatomic arrangement of arterioles opening into sinusoids is known as a closed circulation because the blood flow is through arterioles, capillaries, sinusoids, and venules, all of which are vessels lined by endothelium. The other route is through the red pulp vascular spaces, which are not lined by endothelium but by reticular cells and is designated an “open system” (see Fig. 13-38). Because the dog has sinusoids and red pulp vascular spaces, it has both open and closed splenic circulations. Blood flowing through either the sinusoids or red pulp vascular spaces is exposed to the surveillance of macrophages. These are located perivascularly around sinusoids, but their pseudopodia project into the sinusoidal lumen through the spaces in the discontinuous endothelium. In the red pulp, macrophages are present in the vascular spaces, attached to the reticular walls, and in the splenic cords. Blood then drains into the splenic venules, splenic veins, and ultimately the portal vein, which empties into the liver.