Bone Marrow, Blood Cells, and the Lymphatic System

CHAPTER 13


Bone Marrow, Blood Cells, and the Lymphatic System*




Bone Marrow and Blood Cells



Structure and Function


In adults, the bone marrow is the primary site of blood cell production, or hematopoiesis, which is a term is derived from haima (Gr., blood) and poiein (Gr., to make). Hematopoiesis may also occur elsewhere (extramedullary hematopoiesis), most commonly in the spleen. Hematopoiesis is also known as hemopoiesis.


Hematopoiesis is first seen as blood islands in the embryonic yolk sac. During gestation, the main site of hematopoiesis shifts from the yolk sac to the liver and spleen and eventually to the bone marrow. The composition of the marrow changes with age. The general pattern is that hematopoietic tissue (red marrow) regresses and is replaced with nonhematopoietic tissue, mainly fat (yellow marrow). Thus, in newborns and very young animals, the bone marrow consists largely of hematopoietically active tissue, with relatively little fat, whereas in geriatric individuals the marrow consists largely of fat. In adults, hematopoiesis occurs primarily in the pelvis, sternum, ribs, vertebrae, and the proximal ends of humeri and femora. Even within these areas of active hematopoiesis, fat may constitute a significant proportion of the marrow volume.


The marrow cavity is interlaced with venous sinuses composed of a luminal layer of specialized endothelial cells and an abluminal layer of specialized fibroblasts known as adventitial reticular cells (Figs. 13-1 and 13-2). Hematopoiesis takes place between these venous sinuses in the so-called hematopoietic spaces. Sinusoidal endothelial cells function as a barrier and regulate traffic of chemicals and particles between the intravascular and extravascular spaces. Reticular cells extend cytoplasmic processes into the hematopoietic spaces, forming a scaffold that supports the hematopoietic cells. Osteoblasts, osteoclasts, and elongated flat cells line the endosteum. Stromal cells, presumably of fibroblast origin, provide structural support and produce extracellular matrix components such as proteoglycans, glycosaminoglycans, collagen, fibronectin, and others. Adipocytes, macrophages, lymphocytes, and plasma cells are also components of the hematopoietic microenvironment.




Hematopoietic cells, supporting connective tissue cells, extracellular matrix components, and soluble factors form the bone marrow hematopoietic microenvironment. There is complex functional interplay among these components. Behavior of hematopoietic cells is influenced by direct cell-to-cell and cell-matrix interactions and by soluble mediators, such as cytokines and hormones, that interact with cells and with matrix proteins. Cells localize to specific niches within the hematopoietic microenvironment via adhesion molecules, such as integrins, immunoglobulins (Igs), lectins, and other receptors, that recognize ligands on other cells or matrix components. Cells also express receptors for soluble molecules such as chemokines (chemoattractant cytokines) and hormones that influence cell trafficking and metabolism.


The following basic concepts provide a framework for understanding the mechanisms of injury and diseases presented later in the chapter.



• 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.”




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.




Coagulation System: Stopping hemorrhage from a damaged blood vessel ultimately depends on the formation of a stable fibrin clot. This process depends on a number of plasma proteins, or clotting factors, that normally circulate in an inactive proenzyme state. These clotting factors, together with other molecules, including nonenzymatic proteins, calcium, and platelet phospholipids, are known collectively as the coagulation system. Activation of the coagulation system initiates a series of linked enzymatic reactions that result in the production of thrombin. Thrombin converts the soluble plasma protein fibrinogen to fibrin, which in turn is enzymatically cross-linked to form an insoluble clot. The coagulation system is conventionally considered to consist of three interrelated pathways for ease of understanding and interpretation of test results. These pathways—the intrinsic, extrinsic, and common pathways—are summarized in Fig. 2-13.


Anticoagulant and fibrinolytic pathways provide critical “checks and balances.” These pathways are initiated simultaneously with activation of the coagulation system. Procoagulant and anticoagulant pathways are regulated at multiple levels and normally maintained in a balance that avoids hypercoagulable or hypocoagulable states. Some key regulators of hemostasis are listed in Web Box 13-1. Counterregulatory mechanisms are critical for the prevention of inappropriate or excessive clotting and the eventual dissolution of the clot (fibrinolysis) after it has formed. Fibrinolysis results from the action of plasmin on fibrin (see Figs. 2-14, 2-16, and 2-17). The inactive proenzyme plasminogen is converted to plasmin by several molecules, including tissue plasminogen activator, factor XII, and thrombin. Note that some of these molecules (e.g., factor XII and thrombin) have both procoagulant and anticoagulant effects. There are also regulators of fibrinolysis, including inhibitors of plasminogen and plasmin. Fibrinolysis results in production of fibrin degradation products (FDPs). D-dimer is a specific type of FDP that results from the degradation of insoluble cross-linked fibrin. Fibrinolytic products (FDPs and D-dimer) in the blood are often measured to detect inappropriate coagulation and are also of clinical importance because they can impair hemostasis by inhibiting fibrin polymerization and platelet function.





Platelets: The basics of platelet function are described in the previous section on Thrombopoiesis. 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, 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 (e.g., phosphatidylserine) expressed on the surface of activated platelets also help to localize the clot to sites of vascular damage.




Defense Mechanisms


The bone marrow is encased by a protective shell of cortical bone, and blood supply to the marrow provides access to systemic humoral and cellular defenses. Of course, leukocytes themselves function as an essential part of inflammation and immune function, as discussed briefly in the Granulopoiesis and Monocytopoiesis section and in greater detail in Chapters 3 and 5.


Biochemical steps in the glycolytic pathway or linked to it generate antioxidant molecules that enable erythrocytes to withstand oxidative insults throughout their many days in circulation. In addition to producing energy in the form of adenosine triphosphate (ATP), glycolysis generates reduced nicotinamide adenine dinucleotide (NADH), which helps convert the oxidized, nonfunctional form of hemoglobin, known as methemoglobin, back to its active reduced state. Another antioxidant erythrocyte metabolic pathway, the pentose shunt or hexose monophosphate shunt pathway, generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) to help maintain glutathione in the reduced state.



Responses to Injury


The main mechanisms of disease in the bone marrow and blood cells are shown in Box 13-2, and are discussed in more detail here.




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.



Abnormal Concentrations of Blood Cells: Cytopenia—from kytos (Gr., hollow vessel) and penia (Gr., poverty)—refers to a deficiency of blood cells in circulation, hence the terms neutropenia, thrombocytopenia, lymphopenia, and so on. Basic mechanisms causing cytopenias include decreased production (hypoplasia), increased destruction, blood loss, consumption, and altered anatomic distribution (e.g., shifts between marginated and circulating compartments in the blood, or between the spleen and the peripheral blood circulation). Most of these basic mechanisms may cause cytopenias of more than one lineage, and some processes affect multiple lineages simultaneously. Sustained decreased production of all three major bone marrow hematopoietic lineages results in pancytopenia (anemia, neutropenia, and thrombocytopenia). Pancytopenia may occur because of myelophthisis, in which normal bone marrow tissue is replaced by abnormal cells or tissue (as in the case of effacement by malignant cells or fibrous tissue) or because of an abnormality of hematopoietic cells themselves. Destruction of hematopoietic stem cells and progenitor cells causes a condition known as aplastic anemia, or aplastic pancytopenia, which is discussed further in the section on specific diseases.


The pattern of development of cytopenias is partly a function of normal blood cell kinetics. The lifespan of different blood cells in circulation varies markedly (neutrophils: hours, platelets: days, erythrocytes: months). Thus severe neutropenia typically develops within 1 week after cessation of granulopoiesis and after the bone marrow storage pool of neutrophils is depleted, whereas severe thrombocytopenia typically develops in the second week after cessation of thrombopoiesis. Anemia develops much more slowly after the cessation of erythropoiesis, if at all, depending in part on how rapidly the marrow recovers from the insult and on species variation in the erythrocyte lifespan.


Increases in the concentration of many blood cells are indicated by the suffix, –osis (erythrocytosis, lymphocytosis, monocytosis, and thrombocytosis). Increases in granulocytes are indicated by the suffix, –philia (neutrophilia, eosinophilia, and basophilia). Basic mechanisms causing increases in the concentration of blood cells vary considerably according to cell type and are discussed in more detail later. Neoplasia can result in increased or decreased numbers of blood cells. In many kinds of hematopoietic neoplasia, especially leukemias, there are readily detectable numbers of neoplastic cells in the blood. Specific types of hematopoietic neoplasia are discussed later in this chapter. Increased concentrations of blood cells can also occur secondary to many forms of neoplasia (i.e., as a paraneoplastic syndrome), including non-hematopoietic neoplasms, because of production of stimulatory cytokines by the tumor.


Veterinary laboratories typically provide species-specific hematology reference values based on a reference population of clinically normal adult animals. However, it is important to note that what is normal may vary not only between species but also as a function of other factors, such as age, breed, geographic location, and differences in laboratory methodology.


Mechanisms of disease causing cytopenias and abnormally increased concentrations of specific types blood cells are covered in more detail in the next section.



Anemia: Anemia refers to subnormal red blood cell mass or hemoglobin concentration. Anemia causes clinical signs referable to decreased oxygen-carrying capacity (pallor of mucous membranes, lethargy, weakness, exercise intolerance) and may also result in detectable abnormalities because of tissue hypoxia (e.g., increased liver enzyme activities as a result of hypoxia-induced damage to hepatocytes). Anemia also causes decreased viscosity of the blood, and in marked cases frequently causes heart murmurs as a result of a decrease in laminar blood flow.


Classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease (Table 13-1). The hallmark of regenerative anemias, except in horses, is reticulocytosis (i.e., increased numbers of circulating reticulocytes [immature erythrocytes]), which is evident as polychromasia on a routinely stained blood smear. In ruminants, reticulocytosis is often accompanied by basophilic stippling (Fig. 13-7).




Reticulocytosis indicates increased bone marrow erythropoiesis (Fig. 13-8) and release of erythrocytes before they are fully mature (further back in the production “pipeline”). Reticulocytosis is an appropriate response to anemia. A strong regenerative response may produce an increased mean cell volume (MCV) and decreased mean cell hemoglobin concentration (MCHC) on the complete blood count (CBC), because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. Horses are an exception to this classification scheme because they do not release reticulocytes into circulation even when their marrow is producing increased numbers of erythrocytes. Horses with a regenerative response may have an increased MCV and red cell distribution width (an index of variation in cell size). But definitive determination of regeneration in a horse requires either bone marrow examination, in which case the evidence of regeneration is erythroid hyperplasia, or sequential CBCs, in which case the evidence of regeneration is an increase in red cell mass over time. Punctate reticulocytes in cats are evident when blood is stained with new methylene blue, but they are not counted as reticulocytes on the CBC and have the same appearance as mature erythrocytes on routine blood smear examination.



Another finding that may accompany regeneration, in addition to reticulocytosis, is the presence of nucleated erythroid cells (nRBCs). However, the presence of circulating nRBCs is not in itself definitive evidence of regeneration and in fact may signify dyserythropoiesis (e.g., because of lead poisoning or bone marrow disease) or splenic dysfunction. When nRBCs are present as part of a regenerative response to anemia, they should be in low numbers relative to the numbers of reticulocytes.


Recall that the stimulus for increased erythropoiesis is increased secretion of Epo in response to hypoxemia. Although the action of Epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. One of the main effects of Epo is to expand the pool of early stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. In a case of acute blood loss, for example, it typically takes 3 to 4 days until reticulocytosis is evident on the CBC, and several more days until the regenerative response peaks. A similar lag occurs in cases of acute hemolysis. The term preregenerative is sometimes used to describe anemia with a regenerative response that is impending but not yet apparent on the CBC. Confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow or emergence of a reticulocytosis on subsequent days.


Regenerative anemia occurs because of hemorrhage or hemolysis. Some find it useful to remember these as the “2 Hs” or alternatively, the “2 Ls” (loss or lysis). In the case of hemorrhage, erythrocytes and the other components of blood escape from the vasculature. Hemorrhage may be acute or chronic or internal or external. Causes of hemorrhage include trauma, abnormal hemostasis, certain forms of parasitism, ulceration, and neoplasia. Thus regenerative anemias usually occur not because of a problem with erythropoiesis but because of a process affecting erythrocytes that have already been released into the blood circulation. However, it is important to note that chronic hemorrhage can deplete the body’s iron stores, leading to iron-deficiency anemia, which may be either regenerative or nonregenerative. A regenerative response may occur when the deficiency is resolving or temporarily compensated (e.g., when hemorrhage ceases, or when a patient suddenly gains access to increased dietary or parenteral iron). Nonregenerative anemias and iron-deficiency anemia specifically are discussed in more detail later in this chapter.


Hemolysis may be intravascular, in which case erythrocytes release their contents, mostly hemoglobin, directly into the blood, or extravascular, in which case macrophages phagocytose erythrocytes, and little or no hemoglobin is released into the blood. Both forms (mostly extravascular hemolysis) occur as part of normal homeostasis and involve pathways to conserve iron and other reusable components in hematopoiesis. However, some diseases are associated with increased destruction of erythrocytes by one, or both, of these mechanisms. The next sections discuss basic mechanisms of intravascular and extravascular hemolysis in health and disease in more detail.


Normal turnover of erythrocytes occurs mainly by extravascular hemolysis, in which senescent erythrocytes are phagocytosed by macrophages in the spleen and to a lesser extent in other organs such as liver (Kupffer cells) and bone marrow. The exact controls are not clear, but factors that likely play a role include the following:



Macrophages degrade erythrocytes into reusable constituents, such as iron and amino acids, and the waste product bilirubin. Intravascular hemolysis normally occurs at only extremely low levels. Hemoglobin is a tetramer that, when released from the erythrocyte into the blood, splits into dimers that bind to a plasma protein called haptoglobin. The hemoglobin-haptoglobin complex is taken up by hepatocytes and macrophages. This is the major pathway for handling free hemoglobin. However, free hemoglobin may also oxidize to form methemoglobin, which dissociates to form metheme and globin. Metheme binds to a plasma protein called hemopexin, which is taken up by hepatocytes and macrophages in a similar manner to hemoglobin-haptoglobin complexes. Free heme in the reduced form binds to albumin, from which it is taken up in the liver and converted into bilirubin.


In hemolytic anemia, erythrocytes are destroyed at an increased rate. Whether the mechanism is intravascular or extravascular, or a combination, depends on the specific disease process (specific diseases are discussed later in this chapter). A classic sequela of hemolytic anemias in general is hyperbilirubinemia, which is an increase in the plasma concentration of bilirubin. Bilirubin is a yellow pigment, which explains why hyperbilirubinemia, if severe enough, causes icterus—the grossly visible yellowing of serum or tissue (Fig. 13-9). Icterus of mucous membranes, skin, and other tissue is known as jaundice. Icterus is usually detectable when the plasma bilirubin concentration exceeds 2 mg/dL. Hemolysis is a cause of prehepatic hyperbilirubinemia, but it is important to note that hyperbilirubinemia can also occur as the result of hepatic or posthepatic conditions causing impaired bile flow (cholestasis).



Laboratory findings and clinical observations may point to a specific mechanism of hemolysis. In patients with extravascular hemolytic anemia, increased destruction of erythrocytes by splenic macrophages often results in splenomegaly (Fig. 13-10). Splenomegaly may also occur because of other conditions, as discussed elsewhere in this chapter. Intravascular hemolysis is grossly evident as hemoglobinemia (pink-tinged plasma or serum), if the concentration of extracellular hemoglobin is greater than approximately 50 mg/dL. Haptoglobin is saturated with dimeric hemoglobin at a concentration of approximately 150 mg/dL. When haptoglobin is saturated, any remaining free hemoglobin has a low enough molecular weight to pass through the renal glomerular filter into the urine (hemoglobinuria), imparting a pink or red discoloration to the urine. Thus extracellular hemoglobin can cause gross discoloration of the plasma, where it is bound to haptoglobin, before becoming grossly visible in urine. The half-life of haptoglobin is markedly decreased when bound to hemoglobin, so when large amounts of haptoglobin-hemoglobin complex are formed, the concentration of haptoglobin in the blood decreases and hemoglobin can pass through the glomerulus at even lower concentrations. Hemoglobinuria is a contributing factor in the renal tubular necrosis (hemoglobinuric nephrosis) that often occurs in cases of acute intravascular hemolysis (see Chapter 11). A similar lesion occurs in the kidneys of individuals with marked muscle damage and resulting myoglobinuria.



Hemoglobinuria cannot be distinguished grossly from hematuria (RBCs in the urine) or myoglobinuria, and both hemoglobin and myoglobin cause a positive reaction for “blood protein” on urine test strips. Comparing the colors of the plasma and the urine may be informative. In contrast to hemoglobin, myoglobin causes gross discoloration of the urine before the plasma is discolored. This is because myoglobin is a low-molecular-weight monomer, freely filtered by the glomerulus, and does not bind plasma proteins to a significant degree. Hematuria can be distinguished from hemoglobinuria on the basis of microscopic examination of urine sediment (RBCs are present in cases of hematuria).


CBC data are often useful in illuminating hemolytic mechanisms. Total hemoglobin concentration is conventionally measured by lysing all the erythrocytes and measuring the hemoglobin in solution spectrophotometrically. The value for mean cell hemoglobin (MCH) is calculated based on the total hemoglobin concentration and the concentration of erythrocytes. The value for MCHC is calculated based on the MCH and MCV. Thus although erythrocytes do not actually contain supranormal concentrations of hemoglobin, an excess of extracellular hemoglobin may cause an artifactual increase in the calculated MCH and MCHC. It is important to remember that similar increases may also occur artifactually because of positive interference with spectrophotometric measurement of hemoglobin such as occurs with lipemia.


Extravascular hemolysis also often produces characteristic CBC abnormalities that may reflect the mechanism of disease. For example, spherocytosis and autoagglutination are hallmarks of IMHA. Spherocytes form when macrophages (mainly in the spleen) phagocytose part of an erythrocyte plasma membrane bound with autoantibody (Fig. 13-11). The remaining portion of the RBC assumes a spherical shape, thus preserving maximal volume. This change in shape results in decreased deformability of the cells. Erythrocytes must be extremely pliable to traverse the splenic red pulp and sinusoidal walls (Fig. 13-12); spherocytes therefore tend to be retained in the spleen in close association with macrophages with risk of further injury and eventual destruction. In the dog, spherocytes appear smaller than normal and have uniform staining (Fig. 13-13, A), in contrast to normal RBCs, which have a region of central pallor imparted by their biconcave shape. This difference in staining between spherocytes and normal RBCs is not consistently discernible in many other domestic animals (including horses, cattle, and cats), whose erythrocytes differ from those of the dog in that they are smaller and have less pronounced biconcavity and therefore less pronounced central pallor. Autoagglutination occurs because of cross-linking of antibodies bound to erythrocytes. Autoagglutination is evident microscopically as clusters of erythrocytes, and macroscopically as blood with a grainy consistency (Fig. 13-13, B). Autoagglutination may also result in a falsely increased MCV and decreased RBC concentration when clustered cells are mistakenly counted as single cells by automated hematology analyzers.





Oxidative damage to erythrocytes occurs when normal antioxidative pathways that generate reducing agents (such as NADH, NADPH, and reduced glutathione) are compromised or overwhelmed and can result in hemolytic anemia, abnormal hemoglobin function, or both. Hemolysis caused by oxidative damage may be extravascular or intravascular, or a combination (predominant forms of hemolysis are discussed further in the section on specific diseases). Evidence of oxidative damage to erythrocytes may be apparent on examination of a blood smear, or even on gross physical examination. Heinz bodies are foci of denatured globin that interact with the erythrocyte membrane. They are usually subtly evident on routine Wright’s-stained blood smears as pale circular inclusions or blunt, rounded protrusions of the cell margin and are readily discernible on smears stained with new methylene blue (Fig. 13-14). Cats are particularly susceptible to Heinz body formation and may have low numbers of small Heinz bodies normally. There is no unanimity of opinion, but some clinical pathologists believe that the presence of Heinz bodies in up to 10% of all RBCs in cats is within normal limits. This predisposition is believed to reflect unique features of the feline erythrocyte, whose hemoglobin has more sulfhydryl groups (preferential sites for oxidative damage) than do erythrocytes of other species and may also have lower intrinsic reducing capacity. It is also possible that the feline spleen, which is nonsinusoidal, does not have as efficient a “pitting” function as does a sinusoidal spleen, such as that of the dog (splenic structure and function are discussed in more detail later in this chapter). Eccentrocytes, evident as erythrocytes in which one side of the cell has increased pallor, are another manifestation of oxidative damage. They form because of cross-linking of membrane proteins, with adhesion of opposing areas of the cell’s inner membrane leaflet, and displacement of most of the hemoglobin toward the other side (Fig. 13-15). Oxidative insult may also result in conversion of hemoglobin (iron in the Fe2+ state) to methemoglobin (iron in the Fe3+ state), which is incapable of binding oxygen. Methemoglobin is produced normally in small amounts but reduced back to oxyhemoglobin by the enzyme cytochrome-b5 reductase (also known as methemoglobin reductase). Methemoglobinemia results when methemoglobin is produced in excessive amounts (because of oxidative insult) or when the normal pathways for maintaining hemoglobin in the Fe2+ state are impaired (as in cytochrome-b5 reductase deficiency). When present in sufficiently high concentration (approximately 10% of total hemoglobin), methemoglobin imparts a grossly discernible chocolate color to the blood.




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Fig. 13-15 Common erythrocyte morphologic abnormalities.
A, Blood from a dog with a microcytic hypochromic iron-deficiency anemia was mixed with an equal volume of blood from a normal dog before blood smear was prepared. Because the hypochromic (pale-staining) cells are leptocytes shown in F, they have diameters similar to normal cells even though they are microcytic cells. Wright-Giemsa stain. B, Echinocytes appear as erythrocytes with scalloped borders; consequently, the old term “crenation” from Latin meaning “notched” is used. Wright-Giemsa stain. C, Three acanthocytes with irregularly spaced, variably sized spicules in blood from a dog with hemangiosarcoma. Wright-Giemsa stain. D, A keratocyte, exhibiting what appears to be a ruptured “vesicle” in blood from a cat with hepatic lipidosis. Wright-Giemsa stain. E, A schistocyte (left), discocyte (top), and echinocyte (bottom) in blood from a dog with disseminated intravascular hemolysis. Wright-Giemsa stain. F, Two thin flat hypochromic-appearing erythrocytes (leptocytes), with increased membrane-to-volume ratios, are present in blood from a dog with severe iron-deficiency anemia. The bottom leptocyte is folded. Wright-Giemsa stain. G, Three codocytes in blood from a Cairn terrier with a regenerative anemia and hepatic hemochromatosis secondary to pyruvate kinase deficiency. These erythrocytes exhibit a central density or “bull’s-eye” and are often referred to as target cells. Wright-Giemsa stain. H, Three eccentrocytes and a discocyte (left) in blood from a dog with oxidant injury induced by the administration of acetaminophen. The cell at top center appears spherical with a small tag of cytoplasm and may be referred to as a pyknocyte. Wright-Giemsa stain. (From Harvey JW: Atlas of veterinary hematology: blood and bone marrow of domestic animals, Philadelphia, 2001, Saunders.)


Nonregenerative anemias are characterized by a lack of reticulocytosis on the CBC; however, reticulocytosis does not occur in horses even in the context of regeneration. Most often this is a result of decreased production in the marrow (i.e., erythroid hypoplasia). Erythrocytes circulate for a long time, so anemias caused by decreased production tend to develop slowly. The most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease. In this form of anemia, RBCs are decreased in number but are typically normal in size and hemoglobin concentration (so-called normocytic, normochromic, anemia). It has long been known that patients with inflammatory or other chronic disease often become anemic and that this condition is associated with increased iron stores in the bone marrow. Sequestration of iron may be a bacteriostatic evolutionary adaptation because many bacteria require iron as a cofactor for growth. In recent years, investigators have begun to elucidate the molecular mechanisms underlying anemia of inflammation. Hepcidin, an acute phase protein synthesized in the liver that was first identified as an antimicrobial peptide, is a key mediator that acts by limiting iron availability. Hepcidin expression increases with inflammation, infection, or iron overload and decreases with anemia or hypoxia. Hepcidin exerts its effects by causing functional iron deficiency. It binds to and causes the degradation of the cell surface iron efflux molecule, ferroportin, thus inhibiting both absorption of dietary iron from the intestinal epithelium and export of iron from macrophages and hepatocytes into the plasma (Fig. 13-16).



Anemia of inflammation involves factors besides decreased iron availability. Inflammatory cytokines are likely to inhibit erythropoiesis by oxidative damage to and triggering apoptosis of developing erythroid cells, by decreasing expression of Epo and stem-cell factor, and by decreasing expression of Epo receptors. In addition, experimentally induced sterile inflammation in cats resulted in shortened erythrocyte survival, indicating that anemia of inflammation is likely also a function of increased erythrocyte destruction.


True iron deficiency has long been recognized as a cause of anemia. In domestic animals, iron deficiency occurs most commonly because of chronic blood loss, and thus loss of iron in the form of hemoglobin, and less frequently because of nutritional deficiency. Although iron deficiency often results in nonregenerative anemia, it is not always so, especially when nutritional iron is not a limiting factor. Many underlying conditions can cause hemorrhage-induced iron deficiency anemia, including primary or secondary gastrointestinal (GI) disease (e.g., hookworms, neoplasia, ulceration) and marked ectoparasitism. The classic hematologic picture with iron deficiency is microcytic, hypochromic anemia. Microcytosis and hypochromasia are indicated on the CBC by abnormally low MCV and MCHC values, respectively. Microcytosis and hypochromasia may also be discernible on review of a blood smear as RBCs that are abnormally small or paler-staining because of their subnormal hemoglobin concentration, respectively. However, microscopic examination is not a reliable means of detection, especially in the case of mild abnormalities. Low hemoglobin concentration is believed to contribute to microcytosis because one of the feedback mechanisms signaling erythroid cells to stop dividing is reaching a threshold hemoglobin concentration. Abnormally low hemoglobin concentration during erythropoiesis thus results in extra cell divisions and smaller erythrocytes. Microcytosis usually develops before hypochromasia.


Other causes of decreased erythropoiesis include the following:



Specific examples of diseases causing nonregenerative anemia by these mechanisms are discussed later in this chapter. It is important to point out that nonregenerative anemia is not always caused by decreased erythropoiesis. For example, IMHA is typically strongly regenerative, but there are also nonregenerative forms of IMHA. Bone marrow findings in dogs with severe nonregenerative IMHA range from a complete absence of erythropoiesis, known as pure red cell aplasia, to erythroid hyperplasia in a majority of patients in one study. The latter situation is an example of ineffective hematopoiesis (in this case, ineffective erythropoiesis), in which cells are produced at abnormal or increased rate but are destroyed, presumably because of an immune-mediated mechanism, before they enter the circulation.



Neutropenia: Neutropenia refers to a decrease in the concentration of neutrophils in circulating blood. Neutropenia may be caused by decreased production, increased destruction, altered distribution, demand for neutrophils in inflamed tissue that exceeds the rate of granulopoiesis, or inherited disease. Decreased production is evident on bone marrow examination as granulocytic hypoplasia. This usually results from an insult that affects multiple hematopoietic lineages, such as chemical insult, radiation, neoplasia, infection, and fibrosis, but may also be caused by a process that preferentially targets granulopoiesis. Immune-mediated neutropenia is a rare but recognized condition in domestic animals. Bone marrow findings range from granulocytic aplasia or hypoplasia to hyperplasia, depending on where the cells under immune attack are in their differentiation programs. Neutropenia with no evidence of decreased production and in which other causes of neutropenia have been excluded may be a result of destruction of neutrophils before they leave the bone marrow, a condition known as ineffective granulopoiesis. Like other forms of ineffective hematopoiesis, this condition is often presumed to be immune mediated; in cats this condition may occur as a result of infection of hematopoietic cells with the feline leukemia virus (FeLV).


In marked contrast to erythrocytes, neutrophils have a very short lifespan in circulation. Once released from the bone marrow, a neutrophil is only in the bloodstream for hours before migrating into the tissues. The fate of neutrophils after they leave the bloodstream in normal conditions (i.e., not in the context of inflammation) is poorly understood. They migrate into the GI and respiratory tracts, liver, and spleen and may be lost through mucosal surfaces or undergo apoptosis and be phagocytosed by macrophages. When neutrophil production ceases, a reserve of mature neutrophils in the bone marrow (discussed in more detail later) may be adequate to maintain normal numbers of circulating neutrophils for a few days; however, after the bone marrow storage pool is depleted, neutropenia rapidly ensues.


Neutrophils within the blood vasculature are in two compartments: a circulating pool, consisting of those cells flowing freely in the blood, and a marginated pool, consisting of those cells transiently associated with the endothelial surface. (In reality, neutrophils are constantly shifting between these two pools, but the proportion of cells in either pool normally remains fairly constant in any given species.) Circulating neutrophils are part of the blood sample collected during routine venipuncture and are thus counted in the CBC, whereas marginated neutrophils are not. Pseudoneutropenia refers to the situation in which there are an increased proportion of neutrophils in the marginated pool. This may occur because of decreased blood flow or in response to stimuli, such as endotoxemia, that increase expression of molecules promoting interaction between neutrophils and endothelial cells.


Neutropenia may also result from increased demand for neutrophils in the tissue. How rapidly such a situation develops depends not only on the magnitude of the inflammatory stimulus but also on the reserve of postmitotic neutrophils in the bone marrow. The size of this reserve, or storage pool, is species dependent. In dogs, this pool contains the equivalent of 5 days normal production of neutrophils. Cattle represent the other extreme in that they have a small storage pool and thus are predisposed to becoming neutropenic more easily than are dogs. Horses and cats are somewhere between the two extremes, closer to cattle and dogs, respectively. It stands to reason that the clinical significance of neutropenia because of a supply and demand imbalance is also species dependent. In dogs, neutropenia as a result of inflammation is an alarming finding because it is evidence of a massive tissue demand for neutrophils that has exhausted the patient’s storage pool and is exceeding the rate of granulopoiesis in the bone marrow, whereas in cows, neutropenia is commonly noted in a wide range of conditions involving acute inflammation and does not necessarily indicate an overwhelming demand.




Thrombocytopenia: Thrombocytopenia refers to a decrease in the concentration of circulating platelets. Mechanisms of thrombocytopenia include decreased production, increased destruction, increased consumption, altered distribution, and hemorrhage. Decreased production may occur because of a condition affecting cells of multiple hematopoietic lineages, including platelets, or to one specifically depressing thrombopoiesis. In either case, decreased thrombopoiesis is evident on examination of a bone marrow sample as megakaryocytic hypoplasia. General causes of decreased hematopoiesis outlined earlier in the sections on anemia and neutropenia also apply to thrombocytopenia. Specific diseases causing thrombocytopenia are covered later in this chapter. Immune-mediated thrombocytopenia is a fairly common disease in dogs and may also occur in other species. Increased consumption of platelets is a hallmark of disseminated intravascular coagulation (DIC), a syndrome in which hypercoagulability leads to increased consumption of both platelets and coagulation factors in the plasma, with subsequent hypocoagulability and susceptibility to bleeding. The spleen normally contains a significant proportion of total platelet mass (up to one-third in some species), and abnormalities involving the spleen may result in changes in the number of circulating platelets. For example, splenic congestion may result in thrombocytopenia, and splenic contraction may result in thrombocytosis. Acute hemorrhage may result in mild to moderate thrombocytopenia. Potential mechanisms of thrombocytopenia caused by hemorrhage include loss and consumption. Of course, thrombocytopenia can also be a cause of hemorrhage. In the absence of other complicating factors, marked to severe thrombocytopenia (less than 50,000 platelets/µL) is more likely to be the cause of rather than the result of bleeding. Megakaryocytic hyperplasia on examination of a bone marrow sample is evidence of a regenerative thrombopoietic response, and an increase in the mean platelet volume (MPV) value on the CBC often accompanies such a response.



Lymphopenia: Lymphopenia refers to a decrease in the concentration of lymphocytes in blood circulation. It is a common CBC finding in sick animals. Usually the precise mechanism of lymphopenia is not clear. It is often presumed to be mediated at least in part by endogenous glucocorticoid excess. Lymphopenia may occur because of various mechanisms, including altered distribution of lymphocytes (increased trafficking of lymphocytes to, and decreased egress from, lymphoid tissues), lymphotoxicity (direct damage to lymphocytes or suppression of lymphopoiesis) of therapeutic or infectious agents, loss of lymphocyte-rich lymphatic fluid, or congenital disorders. Normal lymphocyte trafficking may be altered because of disruption of the normal architecture of lymphoid tissue (e.g., because of neoplasia or inflammation), or in response to cytokine signals. Glucocorticoid excess may cause lymphopenia via redistribution from the blood to lymphoid tissue, or via direct lymphotoxic effects. Anticancer treatments (chemotherapy or radiotherapy) and immunosuppressive drugs may also be lymphotoxic. Some hereditary immunodeficiencies (such as severe combined immunodeficiency or thymic aplasia) can cause lymphopenia.



Erythrocytosis: An increase in the measured red cell mass above the normal range (i.e., the opposite of anemia) is known as erythrocytosis. The term polycythemia is often used interchangeably with erythrocytosis, but technically and for the purposes of this chapter, polycythemia refers to a specific type of leukemia called primary erythrocytosis or polycythemia vera. Relative erythrocytosis occurs most frequently because of dehydration, when the decreased proportion of water in the blood results in hemoconcentration. A similar situation may occur as a result of epinephrine-mediated splenic contraction. Erythrocytosis caused by splenic contraction occurs to the most pronounced degree in the horse and may occur to a lesser extent in other species. In both of these cases, the total RBC mass is not in fact increased but appears to be so because of other factors. Secondary erythrocytosis refers to a true, Epo-mediated increase in red cell mass, either as an appropriate response to hypoxemia (such as may be seen in patients with cardiopulmonary disease and severely impaired oxygenation, or cardiovascular defects causing right-to-left shunting) or in rare cases an Epo-secreting tumor (inappropriate secondary erythrocytosis). Polycythemia vera is diagnosed based on a marked increase in red cell mass (hematocrit in normally hydrated dogs ranges from 65% to >80%), absence of hypoxemia, absence of other tumors, and normal or decreased concentration of plasma erythropoietin concentration. Absolute erythrocytosis, whether primary or secondary, causes increased viscosity of the blood and resulting impairment of blood flow and distention of the microvasculature. Affected individuals are at increased risk of tissue hypoxia and thrombosis or hemorrhage. Related clinical signs (hyperviscosity syndrome) may include erythematous mucous membranes (Fig. 13-17) and prolonged capillary refill time, prominent scleral vessels, evidence of thrombosis or hemorrhage, and secondary signs (e.g., neurologic and cardiovascular) related to specific organ systems affected.




Neutrophilia: Neutrophilia occurs in response to a number of different stimuli, which are not mutually exclusive. Major mechanisms of neutrophilia are shown in Fig. 13-18. Understanding the CBC findings characteristic of these responses is an important part of clinical veterinary medicine. Inflammation can result in neutropenia, as discussed earlier, or neutrophilia, as discussed next. However, before moving on to a discussion of inflammatory neutrophilia and the so-called left shift, it is important to mention two other common causes of neutrophilia.



Glucocorticoid excess, either because of endogenous production or exogenous administration, results in a CBC pattern known as the stress leukogram, characterized by mature neutrophilia (i.e., increased concentration of segmented neutrophils), lymphopenia, and, especially in dogs, monocytosis. Eosinopenia is another feature of the stress leukogram, although in many situations this is inapparent because the normal reference values for eosinophils are so low (in some laboratories, the lower reference value is zero). Mechanisms contributing to glucocorticoid-mediated neutrophilia include the following:



The magnitude of neutrophilia tends to be species dependent, with dogs having the most pronounced response (up to 35,000 cells/µL) and in decreasing order of responsiveness, cats (30,000 cells/µL), horses (20,000 cells/µL), and cows (15,000 cells/µL) less marked responses. With long-term glucocorticoid excess, neutrophil numbers tend to normalize, whereas lymphopenia persists.


Epinephrine release results in a different pattern, known as physiologic leukocytosis, characterized by mature neutrophilia (like the glucocorticoid response) and lymphocytosis (unlike the glucocorticoid response). This phenomenon is short lived (<1 hour). Neutrophilia occurs primarily because of a shift of cells from the marginated to the circulating pool. Physiologic leukocytosis is common in cats (especially when they are highly stressed during blood collection) and horses, less common in cattle, and uncommon in dogs.


Of course, neutrophilia may also indicate inflammation, and inflammatory stimuli of varying magnitude and duration produce different patterns of neutrophilia. A classic hematologic finding in patients with increased demand for neutrophils is the presence of immature forms in the blood, known as a “left shift.” Not all inflammatory responses have a left shift, but the presence of a left shift almost always signifies active demand for neutrophils in the tissue. The magnitude of a left shift is assessed by the number of immature cells and their degree of immaturity. The mildest form is characterized by increased numbers of band neutrophils, the immediate predecessor to the segmented neutrophil normally found in circulation, with increasingly severe forms involving progressively immature predecessors. A left shift is considered orderly if the number of immature neutrophils in circulation decreases as they become progressively immature. The term degenerative left shift is sometimes used to describe cases where the number of immature forms exceeds the number of segmented neutrophils. As with glucocorticoid-mediated neutrophilia, the typical magnitude of neutrophilia caused by inflammation varies by species, with dogs having the most pronounced response.


Marked neutrophilia may also occur because of an inherited condition known as leukocyte adhesion deficiency (LAD), in which leukocytes lack normal expression of an adhesion molecule required for migration from the bloodstream into the tissue. This condition is described further in the section on specific diseases.


It might be useful to think of neutrophil kinetics in terms of a producer-consumer model in which the bone marrow is the factory, and the tissues (where the neutrophils eventually go) are the customers. The bone marrow storage pool is the factory inventory, and the neutrophils in the bloodstream are in delivery to the customer. Within the blood vessels, circulating neutrophils are on the highway, and marginated neutrophils are temporarily pulled off to the side of the road. During health, there is an even flow of neutrophils from the factory to the customer. Thus the system is in steady state, and neutrophil numbers remain relatively constant and within the normal range. However, disease states may perturb this system at multiple levels. Decreased granulopoiesis is analogous to a factory working below normal production level. Ineffective granulopoiesis is analogous to goods produced at a normal to increased rate but that are damaged during manufacturing and never leave the factory. A left shift is analogous to the factory meeting increased customer demand by shipping out unfinished goods. Cases of persistent, established inflammation are characterized by bone marrow granulocytic hyperplasia and mature neutrophilia, analogous to a factory that has had time to adjust to increased demand and is meeting it more efficiently by increasing its output.

Sep 17, 2016 | Posted by in GENERAL | Comments Off on Bone Marrow, Blood Cells, and the Lymphatic System
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