619 Chapter 13 - Red Blood Cell and Bleeding Disorders Jon C. Aster MD, PhD • Chapter 13 - Red Blood Cell and Bleeding Disorders • Normal – Normal Development of Blood Cells » ORIGIN AND DIFFERENTIATION OF HEMATOPOIETIC CELLS • Anatomy of Bone Marrow. • Morphology. • Pathology – Anemias » ANEMIAS OF BLOOD LOSS • Acute Blood Loss • Chronic Blood Loss » HEMOLYTIC ANEMIAS • Morphology. • Hereditary Spherocytosis (HS) • Molecular Pathology. • Morphology. • Clinical Course. • Hemolytic Disease Due to Red Cell Enzyme Defects: Glucose-6-Phosphate Dehydrogenase Deficiency • Sickle Cell Disease • Pathogenesis. • Morphology. • Clinical Course. • Thalassemia Syndromes • β-Thalassemias • Molecular Pathogenesis. • Clinical Syndromes. • Thalassemia Major. • Morphology. • Thalassemia Minor. • α-Thalassemias • Clinical Syndromes. • Silent Carrier State. • α-Thalassemia Trait. • Hemoglobin H Disease. • Hydrops Fetalis. • Paroxysmal Nocturnal Hemoglobinuria • Immunohemolytic Anemia • Warm Antibody Immunohemolytic Anemia. • Cold Agglutinin Immunohemolytic Anemia. • Cold Hemolysin Hemolytic Anemia. • Hemolytic Anemia Resulting from Trauma to Red Cells » ANEMIAS OF DIMINISHED ERYTHROPOIESIS • Megaloblastic Anemias • Morphology. • Anemias of Vitamin B12 Deficiency: Pernicious Anemia • Normal Vitamin B12 Metabolism. • Etiology of Vitamin B12 Deficiency. • Biochemical Functions of Vitamin B12 . • Incidence. • Pathogenesis. • Morphology. • Clinical Course. • Anemia of Folate Deficiency • Etiology. • Iron Deficiency Anemia • Iron Metabolism. • Etiology. • Morphology. • Anemia of Chronic Disease • Aplastic Anemia • Etiology. • Pathogenesis. • Morphology. • Clinical Course. • Pure Red Cell Aplasia • Other Forms of Marrow Failure – Polycythemia – Bleeding Disorders: Hemorrhagic Diatheses » BLEEDING DISORDERS CAUSED BY VESSEL WALL ABNORMALITIES » BLEEDING RELATED TO REDUCED PLATELET NUMBER: THROMBOCYTOPENIA • Immune Thrombocytopenic Purpura (ITP) • Pathogenesis. • Morphology. • Clinical Features. • Acute Immune Thrombocytopenic Purpura •Drug-Induced Thrombocytopenia: Heparin- Induced Thrombocytopenia • HIV-Associated Thrombocytopenia • Thrombotic Microangiopathies: Thrombotic Thrombocytopenic Purpura (TTP) and Hemolytic-Uremic Syndrome (HUS) » BLEEDING DISORDERS RELATED TO DEFECTIVE PLATELET FUNCTIONS » HEMORRHAGIC DIATHESES RELATED TO ABNORMALITIES IN CLOTTING FACTORS • Deficiencies of Factor VIII-vWF Complex • Von Willebrand Disease • Hemophilia A (Factor VIII Deficiency) • Hemophilia B (Christmas Disease, Factor IX Deficiency) » DISSEMINATED INTRAVASCULAR COAGULATION (DIC) • Etiology and Pathogenesis. • Morphology. • Clinical Course. 620 The organs and tissues involved in hematopoiesis have been traditionally divided into myeloid tissue, which includes the bone marrow and the cells derived from it (e.g., erythrocytes, platelets, granulocytes, and monocytes), and lymphoid tissue, consisting of thymus, lymph nodes, and spleen. This subdivision is artificial with respect to both the normal physiology of hematopoietic cells and the diseases affecting them. For example, although bone marrow is not where most mature lymphoid cells are found, it is the source of lymphoid stem cells. Similarly, myeloid leukemias, neoplastic disorders of myeloid stem cells, originate in the bone marrow but secondarily involve the spleen and (to a lesser degree) lymph nodes. Some red cell disorders (hemolytic anemias) result from the formation of autoantibodies, signifying a primary disorder of lymphocytes. Thus, it is not possible to draw neat lines between diseases involving the myeloid and lymphoid tissues. Recognizing this difficulty, we somewhat arbitrarily divide diseases of the hematopoietic tissues into two chapters. In the first, we consider diseases of red cells and those affecting hemostasis. In the second, we discuss white cell diseases and disorders affecting primarily the spleen and thymus. Normal A complete discussion of normal hematopoiesis is beyond our scope, but certain features are helpful to an understanding of the diseases of blood. Normal Development of Blood Cells Blood cells first appear during the third week of fetal embryonic development in the yolk sac, but these cells are generated from a primitive stem cell population restricted to the production of myeloid cells. The origin of definitive hematopoietic stem cells that give rise to lymphoid and myeloid cells is still unsettled. Most studies suggest they arise in the mesoderm of the intraembryonic aorta/gonad/mesonephros (AGM) region, but evidence also exists for an origin within a small subset of yolk sac-derived cells. By the third month of embryogenesis, stem cells derived from the AGM and/or yolk sac migrate to the liver, which is the chief site of blood cell formation until shortly before birth. Beginning in the fourth month of development, stem cells migrate to the bone marrow to commence hematopoiesis at this site. By birth, marrow throughout the skeleton is hematopoietically active and virtually the sole source of blood cells. In fullterm infants, hepatic hematopoiesis dwindles to a trickle, persisting only in widely scattered small foci that become inactive soon after birth. Up to the age of puberty, marrow throughout the skeleton remains red and hematopoietically active. By age 18 only the vertebrae, ribs, sternum, skull, pelvis, and proximal epiphyseal regions of the humerus and femur retain red marrow, the remaining marrow becoming yellow, fatty, and inactive. Thus, in adults, only about half of the marrow space is active in hematopoiesis. Several features of this normal sequence should be emphasized. By birth, the bone marrow is virtually the sole source of all forms of blood cells, including lymphocyte precursors. In the premature infant, foci of hematopoiesis are frequently evident in the liver and, rarely, in the spleen, lymph nodes, or thymus. Significant postembryonic extramedullary hematopoiesis is abnormal in the full-term infant. With an increased demand for blood cells in the adult, the fatty marrow can transform to red, active marrow. For example, in the face of red cell deficiency (anemia), the marrow can increase red cell production (erythropoiesis) as much as eight-fold. If the marrow stem cells and microenvironment are normal and the necessary nutrients are available (e.g., adequate amounts of iron, protein, requisite vitamins), premature loss of red cells (as occurs in hemolytic disorders) produces anemia only when marrow compensatory mechanisms are outstripped. Under these circumstances, extramedullary hematopoiesis can reappear within the spleen, liver, and even lymph nodes. ORIGIN AND DIFFERENTIATION OF HEMATOPOIETIC CELLS The formed elements of blood—red cells, granulocytes, monocytes, platelets, and lymphocytes—have a common origin from pluripotent hematopoietic stem cells sitting at the apex of a complex hierarchy of progenitors ( Fig. 13-1 ). Most of the work supporting this scheme comes from studies conducted in mice, but it is believed hematopoiesis in man proceeds in a highly analogous fashion. The pluripotent stem cell gives rise to two types of multipotent progenitors, the common lymphoid and the common myeloid stem cell. The common lymphoid stem cell in turn gives rise to precursors of T cells (pro-T cells), B cells (pro-B cells), and natural killer cells. The details of lymphoid differentiation are not discussed here, but it is worth pointing out that morphologic distinctions among lymphoid cells at various stages of differentiation are subtle at best. As a result, monoclonal antibodies recognizing differentiation-stage-specific antigens are used widely to define normal lymphocyte subsets ( Chapter 14 ). From the common myeloid stem cell arise at least three types of committed stem cells capable of differentiating along the erythroid/megakaryocytic, eosinophilic, and granulocyte- macrophage pathways. In functional assays the committed stem cells are called colony- forming units (CFU), because each can give rise to colonies of differentiated progeny in vitro (see Fig. 13-1 ). From the various committed stem cells are derived intermediate stages and ultimately the morphologically recognizable precursors of the differentiated cells, such as proerythroblasts, myeloblasts, megakaryoblasts, monoblasts, and eosinophiloblasts, which in turn give rise to mature progeny. The specific characteristics of rare cells lying high up in the hierarchy shown in Figure 13-1 are still debated. What are agreed upon are certain overarching themes that apply to hematopoiesis. Since mature blood elements are terminally differentiated cells with finite life spans, their numbers must be replenished constantly. It follows that stem cells must not only differentiate, but also self-renew, a critical property of stem cells. Pluripotent stem cells have the greatest capacity for self-renewal, but normally most are not in cell cycle. As commitment to particular lines of differentiation proceeds, self-renewal becomes limited, but a greater fraction of committed cells divide actively. For example, few common myeloid stem cells are normally in cell cycle, but up to 50% of CFU-GM 621 Figure 13-1 Differentiation of hematopoietic cells. SCF, stem cell factor; Flt3L, Flt3 ligand; GM-CSF, granulocyte-macrophage colonys-timulating factor; M-CSF, macrophage colony-stimulating factor; G- CSF, granulocyte colony-stimulating factor. (Modified from Wyngaarden JB, et al [eds]: Cecil Textbook of Medicine, 19th ed. Philadelphia, WB Saunders, 1992, p. 820.) 622 (the precursors of granulocytes and macrophages) are actively dividing. This suggests that pools of differentiated cells are replenished mainly by lineage-restricted stem cells. Although the earliest morphologically recognizable precursors (e.g., myeloblasts or proerythroblasts) also actively proliferate, they cannot self-renew, and eventually all of their progeny differentiate and "die." By definition, then, they do not have the properties of stem cells. Many disorders of the marrow, including marrow failure (aplastic anemias) and hematopoietic neoplasms (e.g., leukemias) are caused by stem cell dysfunction, and there is thus great interest in the physiologic mechanisms regulating the proliferation and differentiation of progenitor cells. These processes involve soluble factors and hematopoietic cell-stromal cell interactions in the bone marrow. Among the hematopoietic growth factors, some, such as stem cell factor (also called c-KIT ligand) and FLT3-ligand, act on very early stem cells. Others, such as the granulocyte- macrophage colony-stimulating factor (GM-CSF), act on CFU-GM. Some recombinant factors are currently being used to stimulate hematopoiesis, including erythropoietin, GM-CSF, G-CSF, and thrombopoietin. Bone marrow-derived stem cells have a number of surprising properties. Although residing mainly in the marrow, a subset circulates normally in the peripheral blood. Hence, hematopoiesis occurs in the marrow because its specialized environment fosters stem cell homing, survival, and differentiation, not because stem cells are restricted to this site. The homing of stem cells, which involves surface adhesion molecules, makes it possible to perform bone marrow transplantation by simply infusing donor stem cells into the peripheral blood. More remarkably, circulating marrow-derived stem cells can "seed" other tissues and develop into nonhematopoietic cells as well. The best characterized and most widely accepted of these alternative "fates" is the differentiation of marrow stem cells into endothelial cell precursors (hemangioblasts)  , which in turn give rise to endothelial cells. This capacity is not surprising, given the close functional relationship of blood elements and the cardiovascular system. In fact, many genes involved in the development of hematopoietic cells also participate in the development of blood vessels and endothelial cells. More controversial studies suggest that bone marrow-derived stem cells can also differentiate into hepatocytes, bile duct cells, myocardium, skeletal muscle, endothelial cells, glia, and even neurons directly.  Other explanations for these results, such as fusion of marrow stem cells to these other mature cell types or contamination of hematopoietic stem cells with other types of stem cells,  still need to be excluded ( Chapter 3 ). Nonetheless, it is hoped that marrow-derived stem cells will have sufficient plasticity to permit their use in a variety of stem cell-based therapies. Anatomy of Bone Marrow. The bone marrow provides a unique microenvironment for the orderly proliferation, differentiation, and release of blood cells. Under the electron microscope, the marrow cavity is a vast network of thin-walled sinusoids lined by a single layer of endothelial cells underlaid by a discontinuous layer of basement membrane and adventitial cells. Within the interstitium lie clusters of hematopoietic cells and fat cells. Differentiated blood cells enter sinusoids by transcellular migration through the endothelial cells. The normal marrow is organized anatomically in subtle but important ways. For example, normal megakaryocytes lie next to sinusoids and extend cytoplasmic processes that bud off into the bloodstream to produce platelets. Similarly, normal immature granulocytic myeloid forms are concentrated next to bone trabeculae, while mature granulocytes are located more centrally. Diseases that distort the marrow architecture, such as deposits of metastatic cancer or granulomatous disease, disturb normal function. In such instances, an abnormal release of immature precursors into the peripheral blood can occur that is referred to as "leukoerythroblastosis." Morphology. Although the morphology of hematopoietic cells within the bone marrow is best studied in smears of marrow aspirates, additional complementary information is obtained from bone marrow biopsy specimens. For example, a reasonable estimate of marrow activity is obtained by examining the ratio of fat cells to hematopoietic elements in bone marrow biopsy samples. In normal adults, this ratio is about 1:1, but with marrow hypoplasia (e.g., aplastic anemia), the proportion of fat cells is greatly increased; conversely, fat cells may disappear completely in diseases characterized by increased hematopoiesis (e.g., hemolytic anemias). Also, certain disorders (such as metastatic cancers and granulomatous diseases) induce local marrow fibrosis, rendering the lesional cells "inaspirable"; here too, a biopsy is the examination of choice. The limitation of biopsies is that tissue fixation and decalcification alter the appearance of marrow cells, making them less recognizable than in air-dried aspirate smears. However, it is not always possible to differentiate the various "blast" forms morphologically, even in aspirate smears. Often, tentative identification is based on "the company they keep." Thus, a primitive cell found within a focus of maturing granulocytes is likely a myeloblast. Pluripotent and multipotent stem cells are morphologically inconspicuous lymphocyte- like cells constituting less than 0.1% of the marrow cellularity. Stem cells are identified and purified away from other cell types using antibodies against discriminating markers (e.g., CD34). The relative proportion of hematopoietic precursors is almost always deranged in diseases of the blood and bone marrow, which normally contains about 65% granulocytes and their precursors; 25% erythroid precursors; and 10% lymphocytes and monocytes and their precursors. Thus, the normal myeloid to erythroid ratio is 2 to 3:1. Prevalent cell types in the myeloid compartment include myelocytes, metamyelocytes, and granulocytes. In the erythroid compartment, the most common forms are polychromatophilic and orthochromic normoblasts. Pathology Anemias The function of red cells is to transport oxygen to peripheral tissues. Reduced oxygen- carrying capacity of blood usually results from a deficiency of red cells, or anemia, defined as a reduction below normal limits of the total circulating 623 red cell mass. Measurement of red cell mass is not easy, however, and in routine practice anemia is defined as a reduction below normal in the volume of packed red cells, as measured by the hematocrit, or a reduction in the hemoglobin concentration of the blood. On occasion, fluid retention can expand plasma volume and dehydration can contract plasma volume, creating spurious abnormalities in these values. There are innumerable classifications of anemia. An acceptable one based on underlying mechanisms is presented in Table 13-1 . A second useful approach classifies anemia according to alterations in red cell morphology, which often correlates with the cause of red cell deficiency. Morphologic characteristics providing etiologic clues include red cell size TABLE 13-1 -- Classification of Anemia According to Underlying Mechanism Blood Loss Acute: trauma Chronic: lesions of gastrointestinal tract, gynecologic disturbances Increased Rate of Destruction (Hemolytic Anemias) Intrinsic (intracorpuscular) abnormalities of red cells Hereditary Red cell membrane disorders Disorders of membrane cytoskeleton: spherocytosis, elliptocytosis Disorders of lipid synthesis: selective increase in membrane lecithin Red cell enzyme deficiencies Glycolytic enzymes: pyruvate kinase deficiency, hexokinase deficiency Enzymes of hexose monophosphate shunt: G6PD, glutathione synthetase Disorders of hemoglobin synthesis Deficient globin synthesis: thalassemia syndromes Structurally abnormal globin synthesis (hemoglobinopathies): sickle cell anemia, unstable hemoglobins Acquired Membrane defect: paroxysmal nocturnal hemoglobinuria Extrinsic (extracorpuscular) abnormalities Antibody mediated Isohemagglutinins: transfusion reactions, erythroblastosis fetalis Autoantibodies: idiopathic (primary), drug-associated, systemic lupus erythematosus, malignant neoplasms, mycoplasmal infection Mechanical trauma to red cells Microangiopathic hemolytic anemias: thrombotic thrombocytopenic purpura, disseminated intravascular coagulation Cardiac traumatic hemolytic anemia Infections: malaria, hookworm Chemical injury: lead poisoning Sequestration in mononuclear phagocyte system: hypersplenism Impaired Red Cell Production Disturbance of proliferation and differentiation of stem cells: aplastic anemia, pure red cell aplasia, anemia of renal failure, anemia of endocrine disorders Disturbance of proliferation and maturation of erythroblasts Defective DNA synthesis: deficiency or impaired use of vitamin B12 and folic acid (megaloblastic anemias) Defective hemoglobin synthesis Deficient heme synthesis: iron deficiency Deficient globin synthesis: thalassemias Unknown or multiple mechanisms: sideroblastic anemia, anemia of chronic infections, myelophthisic anemias due to marrow infiltrations (normocytic, microcytic, or macrocytic); degree of hemoglobinization, reflected in the color of red cells (normochromic or hypochromic); and other special features, such as shape. These red cell indices are often judged qualitatively by physicians, but precise quantitation is done in clinical laboratories using special instrumentation. The most useful red cell indices are as follows: • Mean cell volume: the average volume of a red blood cell, expressed in femtoliters (cubic micrometers) • Mean cell hemoglobin: the average content (mass) of hemoglobin per red blood cell, expressed in picograms • Mean cell hemoglobin concentration: the average concentration of hemoglobin in a given volume of packed red blood cells, expressed in grams per deciliter • Red blood cell distribution width: the coefficient of variation of red blood cell volume Adult reference ranges for red cell indices are shown in Table 13-2 . Whatever its cause, anemia leads to certain clinical features when sufficiently severe. Patients appear pale. Weakness, malaise, and easy fatigability are common complaints. The lowered oxygen content of the circulating blood leads to dyspnea on mild exertion. The nails can become brittle, lose their usual convexity, and assume a concave spoon shape (koilonychia). Anoxia can cause fatty change in the liver, myocardium, and kidney. If fatty changes in the myocardium are sufficiently severe, cardiac failure can develop and compound the respiratory difficulty caused by reduced oxygen transport. On occasion, the myocardial hypoxia manifests as angina pectoris, particularly when complicated by preexisting coronary artery disease. With acute blood loss and shock, oliguria and anuria can develop due to renal hypoperfusion. Central nervous system hypoxia can cause headache, dimness of vision, and faintness. ANEMIAS OF BLOOD LOSS Acute Blood Loss The clinical and morphologic reactions to blood loss depend on the rate of hemorrhage and whether the bleeding * TABLE 13-2 -- Adult Reference Ranges for Red Blood Cells Measurement (units) Men Women Hemoglobin (gm/dL) 13.6–17.2 12.0–15.0 Hematocrit (%) 39–49 33–43 Red cell count (106 /µL) 4.3–5.9 3.5–5.0 Reticulocyte count (%) 0.5–1.5 Mean cell volume (µm3 ) 82–96 Mean corpuscular hemoglobin (pg) 27–33 Mean corpuscular hemoglobin concentration 33–37 (gm/dL) RBC distribution width 11.5–14.5 RBC, red blood cell. *Reference ranges vary among laboratories. The reference ranges for the laboratory providing the result should always be used in interpreting the test result. 624 is external or internal. The effects of acute blood loss are mainly due to the loss of intravascular volume, which can lead to cardiovascular collapse, shock, and death. If the patient survives, the blood volume is rapidly restored by shift of water from the interstitial fluid compartment. The resulting hemodilution lowers the hematocrit. Reduction in the oxygenation of renal juxtaglomerular cells triggers increased production of erythropoietin, which stimulates the proliferation of committed erythroid stem cells (CFU-E) in the marrow. It takes about 5 days for the progeny of these CFU-Es to fully differentiate, an event marked by the appearance of increased numbers of newly released red cells (reticulocytes) in the peripheral blood. The iron in hemoglobin is recaptured if red cells are lost internally, as into the peritoneal cavity, but external bleeding leads to iron loss and possible iron deficiency, which can hamper restoration of normal red cell counts. The earliest change in the peripheral blood immediately after acute blood loss is leukocytosis, due to the mobilization of granulocytes from marginal pools. Initially, red cells appear normal in size and color (normocytic, normochromic). However, as marrow production increases, there is a striking increase in the reticulocyte count, reaching 10% to 15% after 7 days. Reticulocytes are recognizable as polychromatophilic macrocytes in the usual blood smear. Early recovery from blood loss is often accompanied by thrombocytosis, which is caused by increased platelet production. Chronic Blood Loss Chronic blood loss induces anemia only when the rate of loss exceeds the regenerative capacity of the marrow or when iron reserves are depleted. Iron deficiency anemia, which has identical features regardless of underlying cause (e.g., bleeding, malnutrition, malabsorption states), will be discussed later. HEMOLYTIC ANEMIAS Hemolytic anemias share the following features: • A shortened red cell life span (normal = 120 days); that is, premature destruction of red cells • Elevated erythropoietin levels and increased erythropoiesis in the marrow and other sites, to compensate for the loss of red cells • Accumulation of the products of hemoglobin catabolism, due to an increased rate of red cell destruction The physiologic destruction of senescent red cells takes place within the mononuclear phagocytic cells of the spleen. In the great majority of hemolytic anemias, the premature destruction of red cells also occurs within the mononuclear phagocyte system (extravascular hemolysis), which undergoes a form of work-related hyperplasia marked by splenomegaly. Much less commonly, lysis of red cells within the vascular compartment (intravascular hemolysis) predominates. Intravascular hemolysis of red cells is caused by mechanical injury, complement fixation, infection by intracellular parasites such as falciparum malaria ( Chapter 8 ), or exogenous toxic factors. Mechanical injury caused by defective cardiac valves, thrombi within the microcirculation, or repetitive physical trauma (marathon running, bongo drum beating) can physically lyse red cells. Complement fixation can occur on antibody-coated cells during transfusion of mismatched blood. Toxic injury is exemplified by clostridial sepsis, which releases toxins that attack the red cell membrane. Whatever the mechanism, intravascular hemolysis is manifested by (1) hemoglobinemia, (2) hemoglobinuria, (3) jaundice, and (4) hemosiderinuria. Free hemoglobin in plasma is promptly bound by an α2 -globulin (haptoglobin), producing a complex that is rapidly cleared by the mononuclear phagocyte system, thus preventing excretion into the urine. Decreased serum haptoglobin is characteristic of intravascular hemolysis. When the haptoglobin is depleted, free hemoglobin is prone to oxidation to methemoglobin, which is brown in color. The renal proximal tubular cells reabsorb and catabolize much of the filtered hemoglobin and methemoglobin, but some passes out with the urine, imparting a red-brown color. Iron released from hemoglobin can accumulate within tubular cells, giving rise to renal hemosiderosis. Concomitantly, heme groups derived from the complexes are catabolized to bilirubin within the mononuclear phagocyte system, leading to jaundice. In hemolytic anemias, the serum bilirubin is unconjugated and the level of hyperbilirubinemia depends on the functional capacity of the liver and the rate of hemolysis. When the liver is normal, jaundice is rarely severe. Excessive bilirubin excreted by the liver into the gastrointestinal tract leads to increased formation and fecal excretion of urobilin ( Chapter 18 ). Extravascular hemolysis takes place whenever red cells are rendered "foreign" or become less deformable. Since extreme alterations in shape are required for red cells to navigate the splenic sinusoids successfully, reduced deformability makes the passage difficult and leads to sequestration within the cords, followed by phagocytosis ( Fig. 13-2 ). This is an important pathogenetic mechanism of extravascular hemolysis in a variety of hemolytic anemias. With extravascular hemolysis, Figure 13-2 Schematic of splenic sinus (electron micrograph). A red cell is in the process of squeezing from the red pulp cords into the sinus lumen. Note the degree of deformability required for red cells to pass through the wall of the sinus. 625 hemoglobinemia and hemoglobinuria are not observed, and its principal features are anemia and jaundice. However, some hemoglobin inevitably escapes from phagocytes, leading to decreases in plasma haptoglobin. The morphologic changes are identical to those in intravascular hemolysis, except that "work" hyperplasia of the mononuclear phagocyte system often leads to splenomegaly. Morphology. Certain morphologic changes are common in hemolytic anemias, regardless of cause or type. Anemia and lowered tissue oxygen tension stimulate increased production of erythropoietin, which leads to the appearance of increased numbers of erythroid precursors (normoblasts) in the marrow ( Fig. 13-3 ). If the anemia is severe, extramedullary hematopoiesis can appear in the liver, spleen, and lymph nodes. The accelerated erythropoiesis leads to a prominent reticulocytosis in the peripheral blood. Elevated biliary excretion of bilirubin promotes the formation of pigment gallstones (cholelithiasis). If chronic, phagocytosis of red cells leads to hemosiderosis, usually confined to the mononuclear phagocyte system. The hemolytic anemias are classified in a variety of ways. One has already been mentioned, namely, division into intravascular and extravascular hemolytic disorders. However, since disorders with predominantly intravascular hemolysis are quite uncommon, this classification is not entirely satisfactory. A second pathogenetic classification is based on whether the underlying cause of red cell destruction is extrinsic (extracorpuscular mechanism) or intrinsic to the red cell (intracorpuscular defect). These anemias can also be divided into hereditary and acquired disorders. In general, hereditary disorders are due to intrinsic defects and the acquired disorders to extrinsic factors such as autoantibodies. Each of the classifications has value. Here we follow the intrinsic- extrinsic outline given in Table 13-1 , limiting our discussion to the more common entities. Figure 13-3 Marrow smear from a patient with hemolytic anemia. The marrow reveals greatly increased numbers of maturing erythroid progenitors (normoblasts). (Courtesy of Dr. Steven Kroft, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Hereditary Spherocytosis (HS) This inherited disorder is caused by intrinsic defects in the red cell membrane that render red cells spheroid, less deformable, and vulnerable to splenic sequestration and destruction. The prevalence of HS is highest in northern Europe, where rates of 1 in 5000 are reported. An autosomal dominant inheritance pattern is seen in three fourths of cases. The remaining patients have a more severe autosomal recessive form of the disease. Molecular Pathology. The remarkable elasticity and durability of the normal red cell are attributable to the physicochemical properties of its specialized membrane skeleton ( Fig. 13-4 ), which lies closely apposed to the internal surface of the plasma membrane. Its chief protein component, spectrin, consists of two polypeptide chains, α and β, which form intertwined (helical) flexible heterodimers. The "head" regions of spectrin dimers self-associate to form tetramers, while the "tails" associate with actin oligomers. Each actin oligomer can bind multiple spectrin tetramers, thus creating a 2-dimensional spectrin-actin skeleton that is connected to the cell membrane by two distinct interactions. The first, involving the proteins ankyrin and band 4.2, binds spectrin to the transmembrane ion transporter, band 3. The second, involving protein 4.1, binds the "tail" of spectrin to another transmembrane protein, glycophorin A. HS is caused by diverse mutations affecting ankyrin, band 3, spectrin, or band 4.2, the proteins involved in the first of these two tethering interactions,  presumably because this complex is particularly important in stabilizing the lipid bilayer. The most common cause of autosomal dominant HS is mutation of red cell ankyrin. Another 20% of autosomal dominant HS cases are caused by mutations in band 3. The remaining cases are associated mostly with mutations in α-spectrin, β-spectrin, or band 4.2. Regardless of the molecular defect, reduced membrane stability leads to loss of membrane fragments during exposure to shear stresses in the circulation (see Fig. 13-4 ). The loss of membrane relative to cytoplasm "forces" the cells to assume the smallest possible diameter for a given volume, namely, a sphere. Although there is much to learn about the molecular defects in HS, the travails of the spherocytic red cells are fairly well defined ( Fig. 13-5 ). In the life of the "portly," inflexible spherocyte, the spleen is the villain. Red cells must undergo extreme deformation to leave the cords of Billroth and enter the sinusoids. Because of their spheroidal shape and reduced membrane plasticity, spherocytes attempting to squeeze out of the cords are like an "obese man attempting to bend at the waist." As spherocytes are trapped in the spleen, the already sluggish circulation of the cords stagnates further, producing a progressively more hostile environment. Lactic acid accumulates and the pH falls, inhibiting glycolysis. The inability to generate adenosine triphosphate impairs the ability of red cells to extrude sodium, adding an element of osmotic injury. Stagnation in the cords also promotes contact with plentiful macrophages, which phagocytose the hapless spherocytes. The cardinal role of the spleen in the premature demise of the spherocytes is proved by the invariably beneficial effect of splenectomy. The spherocytes persist, but the anemia is corrected. 626 Figure 13-4 Schematic representation of the red cell membrane cytoskeleton and alterations leading to spherocytosis and hemolysis. Mutations weakening interactions involving α-spectrin, β-spectrin, ankyrin, band 4.2, or band 3 all cause the normal biconcave red cell to lose membrane fragments and adopt a spherical shape. Such spherocytic cells are less deformable than normal and therefore become trapped in the splenic cords, where they are phagocytosed by macrophages. Morphology. The most outstanding morphologic finding in this disease are spherocytes, apparent on smears as abnormally small, dark-staining (hyperchromic) red cells lacking the normal central zone of pallor ( Fig. 13-6 ). Spherocytosis, although distinctive, is not pathognomonic, as it is also seen in autoimmune hemolytic anemias. Present also are changes associated with all hemolytic anemias, including reticulocytosis, marrow hyperplasia due to increased Figure 13-5 Model of the pathophysiology of hereditary spherocytosis. (Adapted from Wyngaarden JB, et al [eds]: Cecil Textbook of Medicine, 19th ed. Philadelphia, WB Saunders, 1992, p. 859.) erythropoiesis, hemosiderosis, and mild jaundice. Cholelithiasis (pigment stones) occurs in 40% to 50% of the affected adults. Other alterations are fairly distinctive. Moderate splenic enlargement is characteristic (500 to 1000 gm); in few other hemolytic anemias is the spleen enlarged as much or as often. It results from congestion of the cords of Billroth and "work hyperplasia" due to markedly increased erythrophagocytosis. 627 Figure 13-6 Hereditary spherocytosis (peripheral smear). Note the anisocytosis and several dark-appearing spherocytes with no central pallor. Howell-Jolly bodies (small dark nuclear remnants) are also present in red cells of this asplenic patient. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Clinical Course. The characteristic clinical features are anemia, splenomegaly, and jaundice. The severity of the disease varies greatly from one patient to another. In a minority, HS presents at birth with marked jaundice, requiring exchange transfusion. In 20% to 30% of patients, the disease is virtually asymptomatic because the decreased red cell survival is readily compensated for by increased erythropoiesis. In most, however, the compensatory changes are outpaced, producing a chronic hemolytic anemia, usually of mild to moderate severity. The generally stable clinical course is sometimes punctuated by an aplastic crisis, usually triggered by an acute parvovirus infection. Parvovirus infects and kills red cell progenitors, causing red cell production to cease until an effective immune response commences, generally in 1 to 2 weeks. Because the lifespan of red cells in HS is shortened to 10 to 20 days, cessation of erythropoiesis for even short time periods leads to sudden worsening of the anemia accompanied by reticulocytopenia. Transfusions may be necessary to support the patient until the immune response clears the infection. Hemolytic crises are produced by intercurrent events leading to increased splenic destruction of red cells (e.g., infectious mononucleosis); these are clinically less significant than aplastic crises. Gallstones, found in many patients, can also produce symptoms. Diagnosis of HS is based on family history, hematologic findings, and several pieces of laboratory evidence. In two thirds of the patients, the red cells are abnormally sensitive to osmotic lysis when incubated in solutions of hypotonic salt, which induce the influx of water into spherocytes with little margin for expansion. Spherocytes retain most of the cytoplasm they were "born" with and lose sodium and water during conditioning in the circulation, leading to an increased mean cell hemoglobin concentration in most patients. As mentioned earlier, splenectomy is often beneficial. Hemolytic Disease Due to Red Cell Enzyme Defects: Glucose-6-Phosphate Dehydrogenase Deficiency The red cell is vulnerable to injury by exogenous and endogenous oxidants. Abnormalities in the hexose monophosphate shunt or glutathione metabolism resulting from deficient or impaired enzyme function reduce the ability of red cells to protect themselves against oxidative injuries, leading to hemolytic disease. The most important of these enzyme derangements is the hereditary deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. As noted in Figure 13-7 , G6PD reduces NADP to NADPH while oxidizing glucose-6-phosphate. NADPH then provides reducing equivalents needed for conversion of oxidized glutathione to reduced glutathione, which protects against oxidant injury by catalyzing the breakdown of compounds such as H2 O2 . Several hundred G6PD genetic variants are known, but most are harmless. Only two variants, designated G6PD A- and G6PD Mediterranean, cause most clinically significant hemolytic anemias. G6PD A- is present in about 10% of American blacks; G6PD Mediterranean, as the name implies, is prevalent in the Middle East. The high frequency of these variants in each population is believed to stem from a protective effect against Plasmodium falciparum malaria. G6PD variants associated with hemolysis destabilize the enzyme. Compared to the most common normal variant, G6PD B, the half-life of G6PD A- is moderately reduced, whereas that of G6PD Mediterranean is more markedly abnormal. Elucidation of the crystal structure of G6PD has revealed that both disease-associated mutations result in misfolding of the protein, making it more susceptible to proteolytic degradation. Because mature red cells do not synthesize new proteins, G6PD A- or G6PD Mediterranean enzyme activities fall quickly as red cells age to levels inadequate to protect against oxidant stress. G6PD deficiency is a recessive X-linked trait, placing males at highest risk for symptomatic disease. G6PD deficiency manifests in several distinct clinical patterns. Most common Figure 13-7 Role of glucose-6-phosphate dehydrogenase (G6PD) in defense against oxidant injury. The disposal of H2 O2 , a potential oxidant, is dependent on the adequacy of reduced glutathione (GSH), which is generated by the action of NADPH. The synthesis of NADPH is dependent on the activity of G6PD. GSSG, oxidized glutathione. 628 is hemolysis after exposure to oxidant stress. This can occur due to ingestion of certain drugs or foods, or (more commonly) exposure to oxidant free radicals generated by leukocytes in the course of infections. The oxidant drugs implicated are numerous, including antimalarials (e.g., primaquine and chloroquine), sulfonamides, nitrofurantoins, and others. Some drugs cause hemolysis only in those with the more severe Mediterranean variant. Many infections can trigger hemolysis; viral hepatitis, pneumonia, and typhoid fever are among those most likely to do so. Hemolysis can also occur after ingestion of fava beans (favism), which generate oxidants when metabolized. Favism is endemic in the Mediterranean, Middle East, and parts of Africa where consumption of fava beans is prevalent. Uncommonly, G6PD deficiency presents as neonatal jaundice or a chronic low-grade hemolytic anemia in the absence of infection or known environmental triggers. G6PD deficiency causes episodic intravascular and extravascular hemolysis, which seems to involve the following sequence. When G6PD-deficient red cells are exposed to high levels of oxidants, there is oxidation of reactive sulfhydryl groups on globin chains, which become denatured and form membrane-bound precipitates known as Heinz bodies. These are seen within red cells stained with crystal violet as dark inclusions ( Fig. 13-8 ). Heinz bodies can damage the membrane sufficiently to cause intravascular hemolysis. Less severe membrane damage results in decreased red cell deformability. As inclusion- bearing red cells pass through the splenic cords, macrophages pluck out the Heinz bodies. Due to membrane damage, some of these partially devoured cells retain an abnormal shape, appearing to have a bite of cytoplasm removed ("bite cells") (see Fig. 13-8 ). Other less severely damaged cells revert to a spherocytic shape due to loss of membrane surface area. Both bite cells and spherocytes are highly prone to trapping in splenic cords and rapid removal via erythrophagocytosis. Acute intravascular hemolysis marked by anemia, hemoglobinemia, and hemoglobinuria usually begins 2 to 3 days following Figure 13-8 G6PD deficiency: effects of oxidant drug exposure (peripheral blood smear). Inset, Red cells with precipitates of denatured globin (Heinz bodies) revealed by supravital staining. As the splenic macrophages pluck out these inclusions, "bite cells" like the one in this smear are produced. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) exposure of G6PD-deficient individuals to oxidants. The hemolysis tends to be greater in individuals with highly unstable G6PD Mediterranean. Since only older red cells are at risk for lysis, the episode is self-limited, as hemolysis stops when only the younger red cells remain (even if administration of an offending drug continues). The recovery phase is heralded by reticulocytosis. Since hemolytic episodes related to G6PD deficiency occur intermittently, most features of chronic hemolytic anemias (e.g., splenomegaly, cholelithiasis) are absent. Sickle Cell Disease Sickle cell disease is an important hereditary hemoglobinopathy, a type of disease characterized by production of defective hemoglobins. Hemoglobin, as you recall, is a tetrameric protein composed of two like pairs of globin chains, each with its own heme group. Normal adult red cells contain mainly HbA (α2 β2 ) along with small amounts of HbA2 (α2 δ2 ) and fetal hemoglobin (α2 γ2 ). The clinically significant hemoglobinopathies result from mutations in the β-globin gene. Sickle cell anemia is caused by a point mutation at the sixth position of the β-globin chain leading to the substitution of a valine residue for a glutamic acid residue. The abnormal physiochemical properties of the resulting sickle hemoglobin (HbS) are responsible for sickle cell disease. Several hundred other abnormal hemoglobins have been identified containing point mutations or deletions in one of the globin chains. About 8% of black Americans are heterozygous for HbS. If an individual is homozygous for the sickle mutation, almost all the hemoglobin in the red cell is HbS (α2 βs 2 ). In heterozygotes, only about 40% of the hemoglobin is HbS, the remainder being normal hemoglobins. Where malaria is endemic in Africa, as many as 30% of the native population are heterozygous. This high frequency is likely related to protection against falciparum malaria afforded by HbS, particularly in infants. Pathogenesis. When deoxygenated, HbS molecules undergo aggregation and polymerization. Initially, the red cell cytosol converts from a freely flowing liquid to a viscous gel as HbS aggregates form. With continued deoxygenation, aggregated HbS molecules assemble into long needle-like fibers within red cells, producing a distorted sickle or holly-leaf shape ( Fig. 13-9 ). Sickling of red cells is initially a reversible phenomenon; with oxygenation, HbS depolymerizes and the cell shape normalizes. However, with repeated episodes of sickling, membrane damage occurs and cells become irreversibly sickled, retaining their abnormal shape even when fully oxygenated. The precipitation of HbS fibers also causes oxidant damage, not only in irreversibly sickled cells but also in normal-appearing cells. With membrane injury, red cells become loaded with calcium, which is normally excluded rigorously. Calcium ions activate a potassium ion channel, leading to the efflux of potassium and water, intracellular dehydration, and an increase in the mean cell hemoglobin concentration.  In addition, lesions produced by repeated episodes of deoxygenation render sickle red cells abnormally sticky. These membrane changes are important in the pathogenesis of microvascular occlusions, described later. A number of factors affect the rate and degree of sickling. 629 Figure 13-9 Sickle cell anemia (peripheral blood smear). A, Low magnification show sickle cells, anisocytosis, and poikilocytosis. B, Higher magnification shows an irreversibly sickled cell in the center. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) • Perhaps most important is the amount of HbS and its interaction with the other hemoglobin chains in the cell. In heterozygotes, approximately 40% of the hemoglobin is HbS, the rest being HbA, which interacts only weakly with HbS when deoxygenated. Both the relatively low concentration of HbS and the presence of interfering HbA act to prevent efficient HbS aggregation and polymerization, and thus red cells in heterozygous individuals do not sickle except under conditions of severe hypoxia. Such individuals have sickle cell trait, an asymptomatic carrier state. In contrast, homozygous HbS individuals have full- blown sickle cell anemia. Hemoglobins other than the normal HbA also influence the aggregation and polymerization of HbS and thus the severity of sickle cell anemia profoundly. Fetal hemoglobin (HbF) inhibits the polymerization of HbS, and hence newborns do not manifest the disease until they are 5 to 6 months of age, when the amount of HbF in the cells falls close to adult levels. Another hemoglobin modifying the effect of HbS is HbC, which has a point mutation in the β-globin chain leading to substitution of lysine for glutamate at position 6. HbC has a greater tendency to form aggregates with deoxygenated HbS than HbA. As a result, individuals with HbS and HbC have a symptomatic sickling disorder (designated HbSC disease) that is generally milder than sickle cell anemia. About 2% to 3% of American blacks are asymptomatic HbC/HbA heterozygotes, and about 1 in 1250 has HbSC disease. • The rate of HbS polymerization is strongly dependent upon the hemoglobin concentration per cell, that is, the mean corpuscular hemoglobin concentration (MCHC). Higher HbS concentrations increase the probability that aggregation and polymerization will occur during any given period of deoxygenation. Thus, intracellular dehydration, which increases the MCHC, facilitates sickling and vascular occlusion (see later). Conversely, conditions that decrease the MCHC reduce disease severity. This is most clearly illustrated when homozygous sickle cell anemia co-exists with α-thalassemia. These patients have milder disease because thalassemia reduces globin synthesis and limits the total hemoglobin concentration per cell. • A decrease in pH reduces the oxygen affinity of hemoglobin, thereby increasing the fraction of deoxygenated HbS at any given oxygen tension and augmenting the tendency for sickling. • The length of time red cells are exposed to low oxygen tension is an important variable. Normal transit times for red cells passing through capillaries are not sufficient for significant aggregation of deoxygenated HbS to occur. Hence, sickling of red cells is confined to microvascular beds where blood flow is sluggish. This is normally the case in the spleen and the bone marrow, which are prominently affected by sickle cell disease. Two factors play particularly important pathogenic roles in occlusive episodes involving other vascular beds: inflammation and increased red cell adhesion. As you will recall, the exodus of blood from inflamed tissues is slowed, due to the adhesion of leukocytes and red cells to activated endothelium and the transudation of fluid through leaky vessels. As a result, inflamed vascular beds have longer red cell transit times and are prone to induce clinically significant sickling. For reasons that are unclear, sickle red cells also express adhesion molecules at increased levels on their surfaces. In fact, adhesion of sickle red cells to cultured endothelial cells in vitro correlates with clinical severity, presumably because "stickiness" influences transit time in vivo. The clinical manifestations of sickle cell disease are dominated by chronic hemolysis and ischemic tissue damage resulting from occlusion of small blood vessels ( Fig. 13-10 ). Irreversibly sickled cells have rigid, nondeformable cell membranes that lead to difficulty in negotiating the splenic sinusoids, sequestration, and rapid phagocytosis. Some intravascular hemolysis can also occur because of the increased mechanical fragility of severely damaged cells. The red cell survival correlates with the percentage of irreversibly sickled cells in the circulation, supporting the concept that the hemolytic anemia results primarily from premature removal of irreversibly sickled cells. The pathogenesis of microvascular occlusions, a clinically important component of sickle cell anemia, is less certain. There is no correlation between the number of irreversibly sickled cells and the frequency or severity of ischemic episodes, suggesting that reversibly sickled cells initiate microvascular occlusion. As mentioned above, reversibly sickled cells express higher than normal levels of adhesion 630 Figure 13-10 Pathophysiology of sickle cell anemia. molecules and appear abnormally sticky in certain assays. In vivo, it is hypothesized that increased adhesiveness makes reversibly sickled red cells more likely to arrest during transit through the microvasculature, particularly in areas of sluggish flow. This tendency is likely enhanced by inflammation, which up-regulates the expression of adhesion molecules on endothelial cells ( Chapter 2 ), making adherence of granulocytes, monocytes, and reversibly sickled cells more likely. An important role for pro- inflammatory granulocytes is supported by observations showing that leukocytosis correlates with disease severity. The arrest of sickle red cells within hypoxic, inflamed vascular beds results in an extended exposure to a low oxygen tension, causing sickling and vascular obstruction. Indeed it appears that sickled cells, themselves, can induce adhesion molecules on the endothelium. Once this process starts, it is easy to envision how a vicious cycle of sickling, obstruction, hypoxia, and more sickling ensures. In recent years increasing attention is being focused on the role of nitric oxide (NO) in microvascular occlusions. It is thought that in patients with sickle cell anemia, plasma hemoglobin (released from lysed RBC) binds to and inactivates NO. Recall that NO is a potent vasodilator and inhibits platelet aggregation. Such reduction in bioavailable NO predisposes to increased vascular tone (narrowing) and platelet aggregation. These findings provide rationale for NO therapy in sickle cell disease. Morphology. The anatomic alterations are caused by chronic hemolysis, increased formation of bilirubin, and small vessel stasis and thrombosis. The consequences of the increased red cell destruction and anemia have been detailed in the general discussion of all hemolytic anemias. The bone marrow is hyperplastic because of a compensatory hyperplasia of erythroid progenitors. Expansion of the marrow leads to bone resorption and secondary new bone formation, resulting in prominent cheekbones and changes in the skull that resemble a crew-cut in roentgenograms. Extramedullary hematopoiesis can also appear. In children, during the early phase of the disease, the spleen is commonly enlarged up to 500 gm. On histologic examination, there is marked congestion of the red pulp, due mainly to the trapping of sickled red cells in the splenic cords and sinuses ( Fig. 13-11 ). This erythrostasis in the spleen leads to marked tissue hypoxia, thrombosis, infarction, and fibrosis. Continued scarring causes progressive shrinkage of the spleen so that by adolescence or early adulthood only a small nubbin of fibrous tissue is left; this process is called autosplenectomy ( Fig. 13-12 ). Infarction secondary to vascular occlusions and anoxia can occur in many other tissues as well, including the bones, brain, kidney, liver, retina, and pulmonary vessels, the latter sometimes producing cor pulmonale. Vascular stagnation in subcutaneous tissues often leads to leg ulcers in adult patients; this complication is rare in children. As in other hemolytic anemias, increased breakdown of hemoglobin can cause pigment gallstones, and all patients develop hyperbilirubinemia during periods of active hemolysis. 631 Figure 13-11 A, Spleen in sickle cell anemia (low power). Red pulp cords and sinusoids are markedly congested; between the congested areas, pale areas of fibrosis resulting from ischemic damage are evident. B, Under high power, splenic sinusoids are dilated and filled with sickled red cells. (Courtesy of Dr. Darren Wirthwein, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) Clinical Course. From the description of the disease to this point, it is evident that patients are beset with problems stemming from (1) severe anemia, (2) vaso-occlusive complications, and (3) chronic hyperbilirubinemia.  Increased susceptibility to infection with encapsulated organisms, another threat, has at least two causes. First, splenic function is severely impaired, in children because of congestion and poor blood flow, and in adults because of infarction and autosplenectomy. Secondly, defects in the alternative complement pathway impair opsonization of encapsulated bacteria such as pneumococci and Haemophilus influenzae. Septicemia and meningitis caused by these two organisms are the most common causes of death in children with sickle cell anemia. Chronic hemolysis induces moderately severe anemia (hematocrit values between 18% and 30%) associated with striking reticulocytosis and hyperbilirubinemia. Irreversibly sickled cells, ranging in frequency from 5% to 15%, are seen in peripheral smears. The protracted course is frequently exacerbated by a variety of "crises." Vaso-occlusive crises, also called pain crises, represent episodes of hypoxic injury and infarction associated with Figure 13-12 Splenic remnant in sickle cell anemia. (Courtesy of Drs. Dennis Burns and Darren Wirthwein, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) severe pain in the affected region. Although infection, dehydration, and acidosis (all of which favor sickling) sometimes act as triggers, in most instances no predisposing causes are identified. The most commonly involved sites are the bones, lungs, liver, brain, spleen, and penis. In children, painful bone crises are extremely common and often difficult to distinguish from acute osteomyelitis. They frequently manifest as the hand- foot syndrome, a dactylitis of the bones of the hands or feet or both. Particularly dangerous are vaso-occlusive crises involving the lungs, which typically present with fever, cough, chest pain, and a pulmonary infiltrate. Also known as acute chest syndrome, these are sometimes initiated by a simple lung infection.  Due to inflammation, blood flow becomes sluggish and "spleenlike," leading to sickling and vaso-occlusion within pulmonary vascular beds. This further compromises pulmonary function, creating a potentially fatal cycle of worsening pulmonary and systemic hypoxemia, sickling, and vaso-occlusion. Other organs affected by vaso-occlusive crises include the central nervous system, where hypoxia can provoke seizures or strokes, and the cutaneous tissues of the leg, leading to the appearance of ulcers. Although pain crises are the most common cause of patient morbidity and mortality, several other acute events complicate the course of sickle cell disease. Sequestration crises occur in children with intact spleens. Massive sequestration of sickled red cells leads to rapid splenic enlargement, hypovolemia, and sometimes shock. Both sequestration crises and the acute chest syndrome may require treatment with exchange transfusions if the patient is to survive. In aplastic crises, there is a transient cessation of bone marrow erythropoiesis due to an acute infection of erythroid progenitor cells by parvovirus B19. Reticulocytes disappear from the peripheral blood, causing a sudden and rapid worsening of anemia. In addition to these dramatic crises, chronic tissue hypoxia also takes a subtle yet important toll. Chronic hypoxia is responsible for a generalized impairment of growth and development as well as organ damage affecting spleen, heart, kidneys, and lungs. Damage to the renal medulla leads to hyposthenuria (inability to concentrate urine), which causes an increased propensity for dehydration and its attendant 632 risks. It must be emphasized that there is great variation in the clinical manifestations of sickle cell anemia. Some individuals are crippled by repeated vaso-occlusive crises, whereas others have only mild symptoms. The basis for this wide range in disease expression is not understood, but variation in unidentified modifying genes seems likely. The diagnosis is suggested by clinical findings and the presence of irreversibly sickled cells in peripheral blood smears and is confirmed by various tests for sickle hemoglobin. In general, these involve mixing a blood sample with an oxygen-consuming reagent, such as metabisulfite, which induces sickling of red cells if HbS is present. Hemoglobin electrophoresis is also used to demonstrate the presence of HbS and exclude other sickle syndromes, such as HbSC disease. Prenatal diagnosis is possible based on analysis of fetal DNA obtained by amniocentesis or chorionic biopsy. The outlook for patients with sickle cell anemia has improved considerably as a result of better supportive care. Approximately 90% of patients survive to age 20, and close to 50% survive beyond the fifth decade. A major recent advance in sickle cell anemia is treatment with the cancer therapeutic drug hydroxyurea, which has several beneficial effects. Through uncertain mechanisms, hydroxyurea causes a significant increase in the concentration of HbF in red cells, which (as we have discussed) interferes with the polymerization of HbS. However, the therapeutic response to hydroxyurea often precedes the rise in HbF levels, implying that other mechanisms are also important, several of which have been proposed. Firstly, hydroxyurea acts as an anti-inflammatory agent by inhibiting the production of white cells, which may reduce inflammation-related red cell stasis and sickling. Secondly, hydroxyurea increases the mean red cell volume and thereby decreases the concentration of HbS. Thirdly, hydroxyurea can be oxidized by heme groups to produce No. It is hypothesized all of these actions contribute to ability of hydroxyurea to reduce pain crises in children and adults.  Thalassemia Syndromes The thalassemia syndromes are a heterogeneous group of inherited disorders caused by genetic lesions leading to decreased synthesis of either the α- or β-globin chain of HbA (α2 β2 ). β-Thalassemia is caused by deficient synthesis of the β chain, whereas α- thalassemia is caused by deficient synthesis of the α chain. The hematologic consequences of diminished synthesis of one globin chain stem not only from low intracellular Figure 13-13 Diagrammatic representation of the β-globin gene. Arrows denote sites where point mutations giving rise to thalassemia have been identified. hemoglobin (hypochromia), but also from a relative excess of the unimpaired chain. For example, in β-thalassemia, excess free α chains aggregate into insoluble inclusions within red cells and their precursors, leading to premature destruction of maturing erythroblasts in the marrow (ineffective erythropoiesis) and lysis of mature red cells in the spleen (hemolysis). β-Thalassemias The abnormality common to all β-thalassemias is diminished synthesis of structurally normal β-globin chains, coupled with unimpaired synthesis of α chains. The clinical severity of the anemia varies due to heterogeneity in the causative mutations. We begin our discussion with the molecular lesions in β-thalassemia, and then relate the clinical variants to specific underlying molecular defects. Molecular Pathogenesis. Adult hemoglobin (HbA) is a tetramer composed of two α chains and two β chains encoded by a pair of functional α-globin genes on chromosome 16 and a single β-globin gene on chromosome 11. β-Thalassemia syndromes are classified into two categories: (1) β0 -thalassemia, associated with total absence of β-globin chains in the homozygous state, and (2) β+ -thalassemia, characterized by reduced (but detectable) β-globin synthesis in the homozygous state. Sequencing of β-thalassemia genes has revealed approximately 100 different causative mutations.  Most are point mutations; unlike α-thalassemia, gene deletions are uncommon in β-thalassemia. Details of these mutations and their effects on β-globin synthesis are found in specialized texts. A few illustrative examples are cited ( Fig. 13-13 ). • Promoter region mutations. Point mutations within promoter sequences prevent RNA polymerase from binding normally, reducing transcription by 75% to 80%. Some normal β-globin is synthesized, producing β+ -thalassemia. • Chain terminator mutations. Two types of mutations cause premature termination of mRNA translation. One creates a new stop codon within an exon; the second consists of small insertions or deletions that shift the mRNA reading frames and introduce downstream stop codons that terminate protein synthesis (frameshift mutations; see Chapter 5 ). In both cases, synthesis of functional β- globin is prevented by premature chain termination, leading to β0 -thalassemia. • Splicing mutations. Mutations leading to aberrant splicing are the most common cause of β-thalassemia. Most affect 633 introns; others are located within exons. Some of these mutations alter normal splice junctions, such that normal splicing does not occur at all. Unspliced mRNA is degraded within the nucleus, and β0 -thalassemia results. Other mutations occurring in introns create new "ectopic" splice sites at abnormal locations— within an intron, for example. Because normal splice sites remain, both normal and abnormal splicing occurs, giving rise to normal and abnormal β-globin mRNA. These mutations cause β+ -thalassemia. Impaired β-globin synthesis results in anemia by two mechanisms ( Fig. 13-14 ). The deficit in HbA synthesis produces "under-hemoglobinized," hypochromic, microcytic red cells Figure 13-14 Pathogenesis of β-thalassemia major. Note that aggregates of unpaired α-globin chains are not visible in routinely stained blood smears. Blood transfusions are a double-edged sword, correcting the anemia and thereby reducing the stimulus for marrow expansion, but also adding to systemic iron overload. with subnormal oxygen transport capacity. A more important factor is diminished survival of red cells and their precursors, resulting from the imbalance in α- and β-chain synthesis. Free α chains precipitate within the normoblasts, forming insoluble inclusions. These inclusions cause a variety of untoward effects, but cell membrane damage is the proximal cause of most red cell pathology. Many developing normoblasts in the marrow succumb to these membrane lesions, undergoing apoptosis. In severe β-thalassemia, it is estimated that 70% to 85% of normoblasts suffer this fate, leading to ineffective erythropoiesis.  The inclusion-bearing red cells derived from precursors escaping intramedullary death are prone to splenic sequestration and destruction due to cell membrane damage and decreased deformability. 634 In severe β-thalassemia, marked anemia produced by ineffective erythropoiesis and hemolysis leads to several additional problems. Erythropoietin secretion in the setting of severe uncompensated anemia leads to massive erythroid hyperplasia in the marrow and sites of extramedullary hematopoiesis. The expanding mass of erythropoietic marrow invades the bony cortex, impairs bone growth, and produces other skeletal abnormalities, described later. Extramedullary hematopoiesis involves the liver, spleen, and lymph nodes, and in extreme cases produces extraosseous masses in the thorax, abdomen, and pelvis. The metabolically active erythroid progenitors steal nutrients from other tissues that are already oxygen starved, causing severe cachexia in untreated patients. Another disastrous complication seen in severe β-thalassemia (as well as in other causes of ineffective erythropoiesis) is excessive absorption of dietary iron. This, coupled with the iron accumulation due to the repeated blood transfusions required by these patients, leads to a state of severe iron overload. Secondary injury to parenchymal organs, particularly the iron-laden liver, often follows and sometimes induces secondary hemochromatosis ( Chapter 18 ). Clinical Syndromes. Clinical classification of β-thalassemias is based on the severity of the anemia, which in turn depends on the type of genetic defect (β+ or β0 ) and the gene dosage (homozygous or heterozygous). In general, individuals homozygous for β-thalassemia genes (β+ /β+ or β0 /β0 ) have a severe, transfusion-dependent anemia called β-thalassemia major. Heterozygotes with one β-thalassemia gene and one normal gene (β+ /β or β0 /β) usually have a mild microcytic anemia that causes no symptoms. This condition is referred to as β-thalassemia minor or β-thalassemia trait. A third clinical variant of intermediate severity is called β-thalassemia intermedia. β-thalassemia intermedia is genetically heterogeneous. This category includes milder variants of β+ /β+ or β+ /β0 -thalassemia and unusually severe variants of heterozygous β-thalassemia (β0 /? or β+ /?). Ironically, the presence of an α-thalassemia gene defect often decreases the severity of β-thalassemia major, since the imbalance in α- and β-chain synthesis is lessened; this combination can also result in a clinical phenotype resembling β-thalassemia intermedia. The clinical and morphologic features of thalassemia intermedia are not described separately but can be surmised from TABLE 13-3 -- Clinical and Genetic Classification of Thalassemias Clinical Molecular Nomenclature Genotype Disease Genetics β-Thalassemias Thalassemia Homozygous β0 - Severe; requires blood Rare gene deletions major thalassemia (β0 /β0 transfusions in β0 /β0 ) Defects in TABLE 13-3 -- Clinical and Genetic Classification of Thalassemias Clinical Molecular Nomenclature Genotype Disease Genetics Homozygous β+ - transcription, thalassemia (β+ /β+ processing, or ) translation of β- globin mRNA Thalassemia β0 /β Severe, but does not require intermedia regular blood transfusions β+ /β+ Thalassemia β0 /β Asymptomatic with mild or minor absent anemia; red cell abnormalities seen β+ /β α-Thalassemias Hydrops fetails -/- -/- Lethal in utero without transfusions HbH disease -/- -/α Severe; resembles β- thalassemia intermedia α-Thalassemia -/- α/α (Asian) Asymptomatic, like β- Mainly gene trait thalassemia minor deletions -/α -/α (black African) Silent carrier -/α α/α Asymptomatic; no red cell abnormality the following discussions of thalassemia major and minor ( Table 13-3 ). Thalassemia Major. β-Thalassemia is most common in Mediterranean countries and parts of Africa and Southeast Asia. In the United States, the incidence is highest in immigrants from these areas. As indicated in Table 13-3 , the genotype of affected patients can be β+ /β+ , β0 /β0 , or β0 /β+ . With all these genotypes, the anemia manifests 6 to 9 months after birth, as hemoglobin synthesis switches from HbF to HbA. In untransfused patients, hemoglobin levels range between 3 and 6 gm/dL. The peripheral blood smear shows severe red cell morphologic abnormalities, including marked anisocytosis and poikilocytosis (variation in size and shape, respectively), microcytosis (small size), and hypochromia (poor hemoglobinization). Target cells (so called because hemoglobin collects in the center of the cells), basophilic stippling, and fragmented red cells are also common. Inclusions of aggregated α chains are efficiently removed by the spleen and not easily found in peripheral blood smears. The reticulocyte count is elevated, but because of ineffective erythropoiesis is lower than expected for the severity of anemia. Variable numbers of poorly hemoglobinized normoblasts are seen in the peripheral blood due to "stress" erythropoiesis and abnormal release of progenitors from sites of extramedullary hematopoiesis. The red cells can completely lack HbA (β0 /β0 genotype) or contain small amounts (β+ /β+ or β0 /β+ genotypes). HbF is markedly increased and indeed constitutes the major red cell hemoglobin. HbA2 levels may be normal, low, or high. Morphology. The major morphologic alterations, in addition to those found in all hemolytic anemias, involve the bone marrow and spleen. In the untransfused patient, there is striking expansion of hematopoietically active marrow, particularly in facial bones. This erodes existing cortical bone and induces new bone formation, giving rise to a "crew-cut" appearance on X-rays ( Fig. 13-15 ). Both mononuclear phagocytic cell hyperplasia and extramedullary hematopoiesis contribute to enlargement of the spleen, which can weigh up to 1500 gm. 635 Figure 13-15 Thalassemia: x-ray film of the skull showing new bone formation on the outer table, producing perpendicular radiations resembling a crew-cut. (Courtesy of Dr. Jack Reynolds, Department of Radiology, University of Texas Southwestern Medical School, Dallas, TX.) Hemosiderosis and secondary hemochromatosis, the two manifestations of iron overload ( Chapter 18 ), occur in almost all patients due to numerous blood transfusions and increased absorption of dietary iron. Iron deposition often causes damage to several organs, most notably the heart, liver, and pancreas ( Chapter 18 ). The clinical course of β-thalassemia major is brief unless blood transfusions are given. Untreated children suffer from growth retardation and die at an early age from the profound effects of anemia. Blood transfusions not only improve the anemia but also suppress secondary features related to excessive erythropoiesis. In those who survive long enough, the cheekbones and other bony prominences are enlarged and distorted. Hepatosplenomegaly due to extramedullary hematopoiesis is usually present. Cardiac disease resulting from progressive iron overload and secondary hemochromatosis ( Chapter 18 ) is an important cause of death, particularly in heavily transfused patients. Administration of iron chelators can forestall or prevent this complication. With transfusions and iron chelation, survival into the third decade is possible, but the overall outlook remains guarded. Bone marrow transplantation from an HLA-identical sibling is currently the only therapy offering a cure. Prenatal diagnosis is possible by molecular analysis of DNA. Thalassemia Minor. Thalassemia minor is much more common than thalassemia major and understandably affects the same ethnic groups. Most patients are heterozygous carriers of a β+ or β0 gene. Thalassemia trait may offer resistance against falciparum malaria, accounting for its prevalence in parts of the world where malaria is endemic. These patients are usually asymptomatic, and anemia is mild if present. The peripheral blood smear typically shows some red cell abnormalities, including hypochromia, microcytosis, basophilic stippling, and target cells. Mild erythroid hyperplasia is seen in the bone marrow. Hemoglobin electrophoresis characteristically reveals an increase in HbA2 to 4% to 8% of the total hemoglobin (normal, 2.5% ± 0.3%). HbF levels can be normal or slightly increased. Recognition of β-thalassemia trait is important on two counts: (1) differentiation from the hypochromic microcytic anemia of iron deficiency and (2) genetic counseling. Iron deficiency can usually be excluded as the cause of a microcytic anemia through measurement of serum iron, total iron-binding capacity, and serum ferritin (see Iron Deficiency Anemia). Hemoglobin electrophoresis is a very helpful confirmatory test for β-thalassemia trait, particularly in individuals (such as women of childbearing age) at risk for both thalassemia trait and iron deficiency. α-Thalassemias The α-thalassemia disorders are characterized by reduced or absent synthesis of α-globin chains. There are normally four α-globin genes. The severity of α-thalassemia varies greatly depending on the number of α-globin genes affected. As in β-thalassemias, the anemia stems both from lack of adequate hemoglobin and the effects of excess unpaired non-α chains (β, γ, δ). However, the situation is complicated somewhat by synthesis of different non-α chains at varying times of development. Thus, in the newborn with α- thalassemia, excess unpaired γ-globin forms γ4 -tetramers known as hemoglobin Barts, whereas in adults excess β-globin chains form β4 tetramers known as HbH. Since free β and γ chains are more soluble than free α chains and form fairly stable homotetramers, hemolysis and ineffective erythropoiesis are less severe than in β-thalassemias. A variety of molecular lesions result in α-thalassemia, but the most common cause of reduced α- chain synthesis is deletion of α-globin genes. Clinical Syndromes. Clinical syndromes are determined and classified by the number and position of deleted α-globin genes. Each of the four α-globin genes, which occur in two linked pairs on each copy of chromosome 16, normally contributes approximately 25% of the α-globin chains. α-Thalassemia syndromes stem from combinations of deletions that remove one to four α-globin gene copies. Not surprisingly, the severity of the clinical syndrome is proportional to the number of missing α-globin genes. Categories of α-thalassemias are given in Table 13-3 , which lists clinical terms, along with their genetic equivalents and salient clinical features. Silent Carrier State. This occurs if a single α-globin gene is deleted. It is associated with a barely detectable reduction in α-globin chain synthesis that is insufficient to result in anemia. These individuals are completely asymptomatic. α-Thalassemia Trait. This is caused by deletion of two α-globin genes. The two involved genes can be from the same chromosome (α/α -/-), or one α-globin gene can be deleted from each of the two chromosomes (α/- α/-) (see Table 13-3 ). The former genotype is more common in Asian populations, the latter in regions of Africa. Both genotypes produce similar quantitative deficiencies of α-globin chains and are clinically identical, but only matings involving individuals with the α/α -/- genotype are at risk for producing offspring with severe α- thalassemia (HbH disease or hydrops fetalis). As is evident from Table 13-3 , in black African populations, α-thalassemia trait is associated with an α/- α/- genotype, and mating of two such individuals cannot result in progeny with HbH disease or hydrops fetalis. 636 The clinical picture in α-thalassemia trait is identical to that described for β-thalassemia minor, that is, small red cells (microcytosis), minimal or no anemia, and no abnormal physical signs. Hemoglobin H Disease. This is caused by deletion of three α-globin genes. As already discussed, HbH disease is seen most commonly in Asian populations and rarely in those of African origin. With only one normal α-globin gene, the synthesis of α chains is markedly reduced and tetramers of excess β-globin, called HbH, form. HbH has extremely high affinity for oxygen and therefore is not useful for oxygen exchange, leading to tissue hypoxia disproportionate to the level of hemoglobin. Additionally, HbH is prone to oxidation, leading to the formation of intracellular inclusions that can be demonstrated by staining with vital dyes. The instability of HbH is a major cause of anemia, as precipitates of oxidized HbH form in older red cells, which are then removed by splenic macrophages. This produces a moderately severe anemia resembling β-thalassemia intermedia. Hydrops Fetalis. This most severe form of α-thalassemia is caused by deletion of all four α-globin genes. In the fetus, excess γ-globin chains form tetramers (hemoglobin Barts) with such a high affinity for oxygen that they deliver almost no oxygen to tissues. Survival in early development is due to the expression of δ chains, an embryonic globin that pairs with γ chains to form a functional Hb tetramer (δ2 γ2 ). Signs of fetal distress usually become evident by the third trimester of pregnancy. In the past, severe tissue anoxia invariably led to intrauterine fetal death; with intrauterine transfusion, many such infants can be saved. The fetus shows severe pallor, generalized edema, and massive hepatosplenomegaly similar to that seen in erythroblastosis fetalis ( Chapter 10 ). Paroxysmal Nocturnal Hemoglobinuria Despite its rarity, paroxysmal nocturnal hemoglobinuria (PNH) has fascinated hematologists because it is the only hemolytic anemia caused by an acquired intrinsic defect in the cell membrane. Before we describe its molecular basis, it is instructive to recall that proteins are anchored into the lipid bilayer in two ways. Most have a hydrophobic sequence that spans the cell membrane ( Fig. 13-16 ); these are called transmembrane proteins. The remainder are attached to the cell membrane by covalent linkage to a specialized phospholipid called glycosylphosphatidylinositol (GPI). PNH results from acquired mutations in phosphatidylinositol glycan A (PIGA), which is essential for the synthesis of the GPI anchor.  Because PIGA is X-linked and subject to lyonization (see Chapter 5 ), only the active PIGA gene needs to be mutated to produce a functional deficiency. The causative somatic mutations occur in pluripotent stem cells; hence, all its clonal progeny (red cells, white cells, and platelets) are deficient in proteins attached to the cell membrane via GPI. Several GPI-linked proteins inactivate complement. Their absence in PNH renders blood cells unusually sensitive to lysis by complement. Not all blood cells are affected in PNH patients, indicating that the mutant clone exists side by side with progeny of normal stem cells. Three GPI-linked proteins that regulate complement activity—decay-accelerating factor, or CD55; membrane inhibitor of reactive lysis, or CD59; and a C8 binding protein—are deficient in PNH. Of these, CD59 is the most Figure 13-16 Two kinds of membrane proteins: transmembrane and glycosyl phosphatidyl inositol (GPI)- linked. The latter are anchored to cell membranes through a covalent attachment to a glycosyl phospatidyl inositol moiety. In PNH, GPI cannot be synthesized, leading to a global deficiency of GPI-linked membrane proteins. important. It is a potent inhibitor of C3 convertase, and thereby prevents spontaneous activation of the alternative complement pathway in vivo. These defects are not limited to red blood cells, as deficient platelets and granulocytes are also more sensitive to lysis by complement. The intravascular hemolysis is actually paroxysmal and nocturnal in only 25% of cases. Chronic hemolysis without dramatic hemoglobinuria is more common. During the disease course, hemosiderinuria eventually leads to iron deficiency. An inconstant but severe clinical manifestation is episodic venous thrombosis, often involving the hepatic, portal, or cerebral veins; this thrombosis is fatal in 50% of cases. Dysfunction of platelets due to the absence of certain GPI-linked proteins contributes to the prothrombotic state. PNH patients are also at increased risk for developing acute myelogenous leukemia. The basis for this association is unclear. Remarkably, it is now appreciated that normal individuals harbor small numbers of bone marrow cells with PIGA mutations identical to those causing PNH. It is assumed that these cells increase in numbers (thus producing clinically evident PNH) only in very rare instances where they have a selective advantage. Consistent with this view, PNH often arises in the setting of primary bone marrow failure (aplastic anemia), which can be caused by immune-mediated destruction or suppression of marrow stem cells. Based on this scenario, immunosuppression is being evaluated as a therapeutic approach, as it may permit reconstitution of the marrow with the offspring of residual normal stem cells. Immunohemolytic Anemia Hemolytic anemias in this category are caused by extracorpuscular mechanisms. Although these disorders are commonly referred to as autoimmune hemolytic anemias, the designation immunohemolytic anemias is preferred because in some instances the immune reaction is initiated by drug ingestion.  The immunohemolytic disorders are classified in various ways, most centered on the characteristics of the responsible antibody ( Table 13-4 ). The diagnosis of immunohemolytic anemias requires the detection of antibodies and/or complement on patient red cells. This is done using the direct Coombs antiglobulin test. In 637 TABLE 13-4 -- Classification of Immunohemolytic Anemias Warm Antibody Type The antibody is of the IgG type, does not usually fix complement, and is active at 37°C. Primary (idiopathic) Secondary Lymphomas and leukemias Other neoplastic diseases Autoimmune disorder (particularly systemic lupus erythematosus) Drugs Cold Agglutinin Type The antibodies are IgM and most active in vitro at 0° to 4°C. Antibodies dissociate at 30°C or above; agglutination of cells by IgM and complement fixation occurs only in peripheral cool parts of the body (e.g., fingers, ears, and toes). Acute (mycoplasmal infection, infectious mononucleosis) Chronic Idiopathic Associated with lymphoma Cold Hemolysins (Paroxysmal Cold Hemoglobinuria) IgG antibodies bind red cells at low temperature, fix complement, and cause hemolysis when the temperature is raised above 30°C. this test, patient red cells are mixed with heterologous antisera specific for human immunoglobulins or complement. If either is present, red cells are cross-linked by multivalent antibodies, causing clumping or agglutination. The indirect Coombs antiglobulin test, in which patient serum is tested for its ability to agglutinate defined test red cells, can then be used to characterize the target of the autoantibody. The temperature dependence of this reaction also helps to define the type of antibody responsible. Quantitative immunologic tests to measure such antibodies directly are also available. Warm Antibody Immunohemolytic Anemia. This is the most common form (48% to 70%) of immune hemolytic anemia. About 50% of cases are idiopathic (primary); the remainder arise secondarily in the setting of a predisposing condition (see Table 13-4 ) or drug exposure. Most causative antibodies are of the immunoglobulin G (IgG) class; only sometimes are IgA antibodies culpable. Most red cell destruction in this form of hemolytic disease is extravascular. IgG-coated red cells bind Fc receptors on monocytes and splenic macrophages, which results in loss of red cell membrane during "partial" phagocytosis. As in hereditary spherocytosis, the loss of cell membrane converts the red cells to spherocytes, which are sequestered and removed in the spleen, the major site of red cell destruction in this disorder. Thus, moderate splenomegaly is characteristic of this form of anemia. As with other forms of autoimmunity, the cause of autoantibody formation is largely unknown. In many cases, the antibodies are directed against the Rh blood group antigens. The mechanisms of drug-induced hemolysis are better understood. Two predominant immunologic mechanisms have been implicated. • Hapten model. The drugs—exemplified by penicillin and cephalosporins—act as haptens by binding to the red cell membrane. Antibodies directed against the cell-bound drug result in the destructive sequence cited before. This form of hemolytic anemia is usually caused by large intravenous doses of the antibiotic and occurs 1 to 2 weeks after onset of therapy. Sometimes the antibodies bind only to the offending drug, as in penicillin-induced hemolytic anemia. In other cases, such as quinidine-induced hemolysis, the antibodies recognize a complex of the drug and a membrane protein. In drug-induced hemolytic anemias, the destruction of red cells can occur intravascularly after fixation of complement or extravascularly in the mononuclear phagocyte system. • Autoantibody model. These drugs, of which the antihypertensive agent α- methyldopa is the prototype, in some manner initiate the production of antibodies directed against intrinsic red cell antigens, in particular the Rh blood group antigens. Approximately 10% of patients taking α-methyldopa develop autoantibodies, as assessed by the direct Coombs test. However, only 1% develops clinically significant hemolysis. Cold Agglutinin Immunohemolytic Anemia. This form of immunohemolytic anemia is caused by so-called cold agglutinins, IgM antibodies that bind and agglutinate red cells avidly at low temperatures (0° to 4°C). It is less common than warm antibody immunohemolytic anemia, accounting for 16% to 32% of cases of immunohemolytic anemia. Such antibodies appear acutely during the recovery phase of certain infectious disorders, such as mycoplasma pneumonia and infectious mononucleosis. In these settings, the disorder is self-limited and rarely induces clinical manifestations of hemolysis. Other infectious agents associated with this form of anemia include cytomegalovirus, influenza virus, and human immunodeficiency virus (HIV). Chronic cold agglutinin immunohemolytic anemias occur in association with certain lymphoid neoplasms or as an idiopathic condition. Clinical symptoms result from binding of IgM to red cells at sites such as exposed fingers, toes, and ears where the temperature is below 30°C. IgM binding agglutinates red cells and rapidly fixes complement on their surface. As the blood recirculates and warms, IgM is rapidly released, usually before complement-mediated hemolysis can occur. However, the transient interaction with IgM is sufficient to deposit sublytic quantities of C3b, an excellent opsonin, leading to rapid removal of affected red cells by mononuclear phagocytes in the liver and spleen. The hemolysis is of variable severity. Vascular obstruction caused by red cell agglutinates results in pallor, cyanosis of the body parts exposed to cold temperatures, and Raynaud phenomenon ( Chapter 11 ). Cold Hemolysin Hemolytic Anemia. Cold hemolysins are autoantibodies responsible for an unusual entity known as paroxysmal cold hemoglobinuria, characterized by acute intermittent massive intravascular hemolysis, frequently with hemoglobinuria, after exposure to cold temperatures. This is the least common form of immunohemolytic anemia. Lysis is clearly complement dependent. The autoantibodies are IgGs that bind to the P blood group antigen on the red cell surface at low temperatures. Complement-mediated intravascular lysis does not occur until the cells recirculate to warm central regions, as the enzymes of the complement cascade function more efficiently at 37°C. The antibody, also known as the Donath-Landsteiner antibody, was first recognized in association with syphilis. Today, most cases of paroxysmal cold hemoglobinuria follow infections such as mycoplasma pneumonia, 638 measles, mumps, and ill-defined viral and "flu" syndromes. The mechanisms responsible for production of such autoantibodies in these settings are unknown. Hemolytic Anemia Resulting from Trauma to Red Cells Red blood cells can be disrupted by physical trauma in a variety of circumstances. Of these, hemolytic anemias caused by cardiac valve prostheses, or narrowing or obstruction of the microvasculature, are most important clinically. Severe traumatic hemolytic anemia is more frequently associated with artificial mechanical valves than bioprosthetic porcine valves. Hemolysis in both instances stems from shear stresses produced by turbulent blood flow and abnormal pressure gradients. Microangiopathic hemolytic anemia, on the other hand, occurs when red cells are forced to squeeze through abnormally narrowed small vessels. Narrowing is most often caused by fibrin deposition in association with disseminated intravascular coagulation (discussed later in this chapter). Other causes of microangiopathic hemolytic anemia include malignant hypertension, systemic lupus erythematosus, thrombotic thrombocytopenic purpura (TTP), hemolytic-uremic syndrome (HUS), and disseminated cancer, most of which are discussed elsewhere in this book. The common feature among all these disorders is a microvascular lesion that causes mechanical injury to circulating red cells. This damage is evident in peripheral blood smears in the form of red cell fragments (schistocytes), "burr cells," "helmet cells," and "triangle cells" ( Fig. 13-17 ). Except for TTP and HUS, hemolysis is not a major clinical problem in most instances. Figure 13-17 Microangiopathic hemolytic anemia. A peripheral blood smear from a patient with hemolytic-uremic syndrome shows several fragmented red cells. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) ANEMIAS OF DIMINISHED ERYTHROPOIESIS Anemias often result from deficiencies of vital nutrients necessary for red cell formation. Included in this group are the anemias of vitamin B12 and folate deficiency, characterized by defective DNA synthesis (megaloblastic anemias), and iron deficiency anemias, in which heme synthesis is impaired. Other causes of decreased erythropoiesis include anemia of chronic disease, anemia of renal failure, and "marrow stem cell failure," which embraces such conditions as aplastic anemia and pure red cell aplasia. Megaloblastic Anemias The following discussion attempts first to characterize the major features of these anemias and then to discuss the two principal types of megaloblastic anemia: (1) pernicious anemia, the major form of vitamin B12 deficiency anemia, and (2) folate deficiency anemia. The megaloblastic anemias constitute a diverse group of entities, having in common impaired DNA synthesis and distinctive morphologic changes in the blood and bone marrow. As the name implies, erythroid precursors and red cells are abnormally large due to defective cell maturation and division. The precise basis for these changes is not fully understood. Some of the metabolic roles of vitamin B12 and folate are considered later, but for now it suffices that vitamin B12 and folic acid are coenzymes required for synthesis of thymidine, one of the four bases found in DNA. A deficiency of these vitamins or impairment in their metabolism results in defective nuclear maturation due to deranged or inadequate DNA synthesis, with an attendant delay or block in cell division. The synthesis of RNA and protein is relatively unaffected, however, so cytoplasmic maturation proceeds in advance of nuclear maturation, a situation described as nuclear/cytoplasmic asynchrony. Morphology. Certain morphologic features are common to all forms of megaloblastic anemia. A peripheral blood examination usually reveals pancytopenia, as all myeloid lineages are affected. There is marked variation in the size and shape of red cells (anisocytosis), which nonetheless are normochromic. Many red cells are macrocytic and oval (macroovalocytes), with mean corpuscular (cell) volumes above 100 fl (normal, 82 to 98). Because they are thicker than normal and well-hemoglobinized, most macrocytes lack the central pallor of normal red cells and can even appear "hyperchromic," but the MCHC is not elevated. The reticulocyte count is low, and nucleated red cells occasionally appear in the circulating blood with severe anemia. Neutrophils are also larger than normal (macropolymorphonuclear) and hypersegmented; that is, they have five to six or more nuclear lobules ( Fig. 13-18 ). The marrow is usually markedly hypercellular due to increased numbers of all types of myeloid precursors, which may completely replace the fatty marrow. Megaloblastic change is detected in all stages of red cell development. The most primitive cells (promegaloblasts) are large, with a deeply basophilic cytoplasm, prominent 639 Figure 13-18 Megaloblastic anemia. A peripheral blood smear shows a hypersegmented neutrophil with a six-lobed nucleus. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) nucleoli, and a distinctive fine nuclear chromatin pattern ( Fig. 13-19 , cell A). As these cells differentiate and begin to accumulate hemoglobin, the nucleus retains its finely distributed chromatin and thus fails to undergo the chromatin clumping typical of the normoblast. For example, orthochromatic megaloblasts have a large amount of pink, well-hemoglobinized cytoplasm, but the nucleus, instead of becoming pyknotic, remains relatively large and immature. Because DNA synthesis is impaired in all proliferating cells, granulocytic precursors also display nuclear-cytoplasmic asynchrony in the form of giant metamyelocytes and band forms. Megakaryocytes, too, can be abnormally large and have bizarre, multilobate nuclei. The marrow hyperplasia usually seen in megaloblastic anemias is a response to increased levels of growth factors such as erythropoietin. However, due to the derangement in DNA synthesis, most myeloid precursors undergo apoptosis in the marrow (another example of ineffective hematopoiesis), leading to pancytopenia. The anemia is further exacerbated by increased hemolytic destruction of red cells in the periphery. The basis for hemolysis is not entirely clear; both an acquired intracorpuscular defect and a poorly characterized plasma factor have been suggested to contribute. As in other states associated with ineffective erythropoiesis, if the deficiency persists enhanced uptake of iron in the gut can lead to anatomic signs of mild to moderate iron overload after several years ( Chapter 18 ). Anemias of Vitamin B12 Deficiency: Pernicious Anemia The major causes of megaloblastic anemia are listed in Table 13-5 . As mentioned at the outset, pernicious anemia is an important cause of vitamin B12 deficiency. Pernicious anemia is a specific form of megaloblastic anemia caused by atrophic gastritis and an attendant failure of intrinsic factor production that leads to vitamin B12 deficiency. Figure 13-19 Megaloblastic anemia (bone marrow aspirate). A to C, Megaloblasts in various stages of differentiation. Note that the orthochromatic megaloblast (B) is hemoglobinized (as revealed by cytoplasmic color), but in contrast to normal orthochromatic normoblasts, the nucleus is not pyknotic. The granulocytic precursors are also large and have abnormally "immature" chromatin. (Courtesy of Dr. Jose Hernandez, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) We first review vitamin B12 metabolism, as this helps to place pernicious anemia in perspective relative to the other causes of vitamin B12 deficiency anemia. Normal Vitamin B12 Metabolism. Vitamin B12 is a complex organometallic compound known as cobalamin. Under normal circumstances, humans are totally dependent on dietary animal products for their vitamin B12 requirement. Microorganisms are the ultimate origin of cobalamin in the food chain. Plants and vegetables contain little cobalamin save that contributed by microbial contamination; strictly vegetarian or macrobiotic diets, then, do not provide adequate amounts of this essential nutrient. The daily requirement is 2 to 3 mg. A balanced diet contains significantly larger amounts and normally results in accumulation of vitamin B12 in sufficient quantities to last for several years. Absorption of vitamin B12 requires intrinsic factor, which is secreted by the parietal cells of the fundic mucosa ( Fig. 13-20 ). First, vitamin B12 is freed from binding proteins in food through the action of pepsin in the stomach. Free vitamin B12 then binds to salivary proteins called cobalophilins, or R-binders. In the duodenum, cobalophilin-vitamin B12 complexes are broken down by the action of pancreatic proteases, and released vitamin B12 then associates with intrinsic factor. This complex is transported to the ileum, where it is endocytosed by ileal enterocytes that express intrinsic factor-specific receptors on their surfaces. Within ileal cells, vitamin B12 associates with a major carrier protein, transcobalamin II, and is secreted into the plasma. Transcobalamin II delivers vitamin B12 to the liver and other cells of the body, particularly rapidly proliferating cells in the bone 640 TABLE 13-5 -- Causes of Megaloblastic Anemia Vitamin B12 Deficiency Decreased intake Inadequate diet, vegetarianism Impaired absorption Intrinsic factor deficiency Pernicious anemia Gastrectomy Malabsorption states Diffuse intestinal disease, e.g., lymphoma, systemic sclerosis Ileal resection, ileitis Competitive parasitic uptake Fish tapeworm infestation Bacterial overgrowth in blind loops and diverticula of bowel Increased requirement Pregnancy, hyperthyroidism, disseminated cancer Folic Acid Deficiency Decreased intake Inadequate diet—alcoholism, infancy Impaired absorption Malabsorption states Intrinsic intestinal disease Anticonvulsants, oral contraceptives Increased loss Hemodialysis Increased requirement Pregnancy, infancy, disseminated cancer, markedly increased hematopoiesis Impaired use Folic acid antagonists Unresponsive to Vitamin B12 or Folic Acid Therapy Metabolic inhibitors of DNA synthesis and/or folate metabolism, e.g., methotrexate Modified from Beck WS: Megaloblastic anemias. In Wyngaarden JB, Smith LH (eds): Cecil Textbook of Medicine, 18th ed. Philadelphia, WB Saunders, 1988, p. 900. marrow and mucosal lining of the gastrointestinal tract. In addition to the intrinsic-factor dependent pathway, evidence also supports the existence of an alternative mechanism that is not dependent on availability of intrinsic factor or intact terminal ileum. The mechanism involved is not entirely clear but up to 1% of a large oral dose can be absorbed by this pathway, thus making it feasible to treat pernicious anemia by oral vitamin B12 therapy. Etiology of Vitamin B12 Deficiency. With this background, we can consider the various causes of vitamin B12 deficiency (see Table 13-5 ). Inadequate diet is obvious but must be present for many years to deplete reserves. The absorption of vitamin B12 can be impaired by disruption of any one of the steps outlined earlier. With achlorhydria and loss of pepsin secretion (which occurs in some elderly individuals), vitamin B12 is not readily released from proteins in food. With gastrectomy and pernicious anemia, intrinsic factor is not available for transport to the ileum. With loss of exocrine pancreatic function, vitamin B12 cannot be released from R- binder-vitamin B12 complexes. Ileal resection or diffuse ileal disease can remove or damage the site of intrinsic factor-vitamin B12 complex absorption. Tapeworm infestation, by competing for the nutrient, can induce a deficiency state. Under some circumstances, for example, pregnancy, hyperthyroidism, disseminated cancer, and chronic infections, Figure 13-20 Schematic illustration of vitamin B12 absorption. 641 the demand for vitamin B12 can be so great as to produce a relative deficiency, even with normal absorption. Biochemical Functions of Vitamin B12 . Two reactions in humans are known to require vitamin B12 . Methylcobalamin is an essential cofactor for methionine synthase, an enzyme involved in the conversion of homocysteine to methionine ( Fig. 13-21 ). In the process, methylcobalamin yields a methyl group and is regenerated from N5 -methyltetrahydrofolic acid (N5 -methyl FH4 ), the principal form of folic acid in plasma. In the same reaction, N5 -methyl FH4 is converted to tetrahydrofolic acid (FH4 ). FH4 is crucial, since it is required (through its derivative N5,10 -methylene FH4 ) for conversion of deoxyuridine monophosphate to deoxythymidine monophosphate, an immediate precursor of DNA. It has been postulated that the fundamental cause of impaired DNA synthesis in vitamin B12 deficiency is the reduced availability of FH4 , most of which is "trapped" as N5 -methyl FH4 . In addition, the deficit in FH4 can be exacerbated by an "internal" folate deficiency caused by a failure to synthesize metabolically active polyglutamylated forms. This may stem from a requirement for vitamin B12 in synthesis of methionine, which contributes a carbon group needed in the metabolic reactions that create folate polyglutamates (see Fig. 13-21 ). Whatever the mechanism of internal folate deficiency, lack of folate is the proximate cause of anemia in vitamin B12 deficiency, as the anemia inevitably improves with administration of folic acid. The neurologic complications associated with vitamin B12 deficiency are an enigma, as treatment with folate fails to improve neurologic deficits. In addition to the transmethylation reaction discussed previously, the second reaction depending on cobalamin is the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A, which requires adenosylcobalamin as a prosthetic group on the enzyme methylmalonyl- coenzyme A mutase. A deficiency of vitamin B12 thus leads to increased plasma and urine levels of methylmalonic acid. Interruption of the succinyl pathway and consequent Figure 13-21 Relationship of N5 -methyl FH4 , methionine synthase, and thymidylate synthetase. In cobalamin deficiency, folate is sequestered as N5 -methyl FH4 . This ultimately deprives thymidylate synthetase of its folate coenzyme (N5,10 -methylene FH4 ), thereby impairing DNA synthesis. build-up of methylmalonate and propionate (a precursor) could lead to the formation and incorporation of abnormal fatty acids into neuronal lipids. It has been suggested that this biochemical abnormality predisposes to myelin breakdown and thereby produces the neurologic complications of vitamin B12 deficiency ( Chapter 28 ). However, rare individuals with hereditary deficiencies of methylmalonyl-coenzyme A mutase, while having complications related to methylmalonyl acidemia, do not suffer from the neurologic abnormalities seen in vitamin B12 deficiency, casting doubt on this explanation. With this overview of vitamin B12 metabolism, we can now turn our attention to pernicious anemia. Incidence. Although somewhat more prevalent in Scandinavian and "English-speaking" populations, pernicious anemia occurs in all racial groups, including, in the United States, blacks and Hispanics. A disease of older age, it is generally diagnosed in the fifth to eighth decades of life. A genetic predisposition is strongly suspected, but no definable genetic pattern of transmission has been discerned. As described below, these patients probably have a tendency to form antibodies against multiple self-antigens. Pathogenesis. Pernicious anemia is believed to result from immunologically mediated, possibly autoimmune, destruction of gastric mucosa. The resultant chronic atrophic gastritis is marked by a loss of parietal cells, a prominent infiltrate of lymphocytes and plasma cells, and megaloblastic changes in mucosal cells similar to those found in erythroid precursors. A number of immunologic reactions are associated with these morphologic changes. Three types of antibodies are present in many but not all patients with pernicious anemia. About 75% of patients have a type I antibody that blocks binding of vitamin B12 to intrinsic factor. Type I antibodies are found in both plasma and gastric juice. Type II antibodies prevent binding of the intrinsic factor-vitamin B12 complex to its ileal receptor. These immunoglobulins are also found in a large proportion of patients with pernicious anemia. The third type of antibodies, present in 85% to 90% of patients, recognize the α and β subunits of the gastric proton pump, which is normally localized to the microvilli of the canalicular system of the gastric parietal cell. Type III antibodies are not specific for pernicious anemia or other autoimmune diseases, as they are found in up to 50% of elderly patients with idiopathic chronic gastritis not associated with pernicious anemia. As discussed next, they most likely result from gastric injury, rather than cause it. Despite the presence of these autoantibodies, it is not established that they are the primary cause of gastric changes. It is believed that an autoreactive T-cell response initiates gastric mucosal injury, triggering the formation of autoantibodies, which may exacerbate epithelial injury. When the mass of intrinsic factor-secreting cells falls below a threshold (and reserves of stored vitamin B12 are depleted), anemia develops. In an animal model of autoimmune gastritis mediated by CD4+ T cells, a pattern of autoantibodies resembling that seen in pernicious anemia develops, thus supporting the primacy of T-cell autoimmunity. An autoimmune basis is also supported by the association of pernicious anemia with other autoimmune disorders, particularly autoimmune thyroiditis and adrenalitis. Conversely, patients who present with other autoimmune diseases are predisposed to develop antibodies against intrinsic factor. 642 Morphology. The major specific changes in pernicious anemia are found in the bone marrow, alimentary tract, and central nervous system. The changes in the bone marrow and blood are similar to those described earlier for all megaloblastic anemias. In the alimentary system, abnormalities are regularly found in the tongue and stomach. The tongue is shiny, glazed, and "beefy" (atrophic glossitis). The changes in the stomach are those of diffuse chronic gastritis ( Chapter 17 ). The most characteristic histologic alteration is the atrophy of the fundic glands, affecting both chief cells and parietal cells, the latter being virtually absent. The glandular lining epithelium is replaced by mucus- secreting goblet cells that resemble those lining the large intestine, a form of metaplasia referred to as intestinalization. Some of the cells as well as their nuclei may increase to double the normal size, a form of "megaloblastic" change exactly analogous to that seen in the marrow. As will be seen, patients with pernicious anemia have a higher incidence of gastric cancer. The gastric atrophic and metaplastic changes are due to autoimmunity and not vitamin B12 deficiency; hence, parenteral administration of vitamin B12 corrects the bone marrow changes, but gastric atrophy and achlorhydria persist. Central nervous system lesions are found in approximately three fourths of all cases of fulminant pernicious anemia, but in some instances neuronal involvement is seen in the absence of overt megaloblastic anemia. The principal alterations involve the spinal cord, where there is degeneration of myelin in the dorsal and lateral tracts, sometimes followed by loss of axons. These changes give rise to spastic paraparesis, sensory ataxia, and severe paresthesias in the lower limbs. Less frequently, degenerative changes occur in the ganglia of the posterior roots and in peripheral nerves ( Chapter 28 ). Because both sensory and motor pathways are involved, the term "subacute combined degeneration" or "combined system disease" is sometimes used to describe the neurologic changes associated with vitamin B12 deficiency. Clinical Course. Pernicious anemia is insidious in onset, so the anemia is often quite severe by the time the patient seeks medical attention. The course is progressive unless halted by therapy. Diagnostic features include (1) a moderate to severe megaloblastic anemia, (2) leukopenia with hypersegmented granulocytes, (3) mild to moderate thrombocytopenia, (4) mild jaundice due to ineffective erythropoiesis and peripheral hemolysis of red cells, (5) neurologic changes related to involvement of the posterolateral spinal tracts, (6) achlorhydria even after histamine stimulation, (7) inability to absorb an oral dose of cobalamin (assessed by urinary excretion of radiolabeled cyanocobalamin given orally, called the Schilling test), (8) low serum levels of vitamin B12 , (9) elevated levels of homocysteine and methyl malonic acid in the serum (this is more sensitive than serum levels of vitamin B12 ) (10) a striking reticulocytic response and improvement in hematocrit levels beginning about 5 days after parenteral administration of vitamin B12 . Serum antibodies to intrinsic factor are highly specific for pernicious anemia. Their presence attests to the cause of vitamin B12 deficiency, rather than the presence or absence of cobalamin deficiency. As mentioned, serum homocysteine and methylmalonic acid levels are raised in patients with deficiency of vitamin B12 , and high levels of homocysteine are a risk factor for atherosclerosis and thrombosis. It is still not definite whether such an elevation in patients with folate or vitamin B12 deficiency increases the risk of vascular disease. The cytologic aberrations in the gastric mucosa are associated with an increased risk of gastric cancer ( Chapter 17 ). With parenteral or high dose oral vitamin B12 , the anemia can be cured and the peripheral neurologic changes reversed, or at least halted in their progression, but the changes in the gastric mucosa are unaffected. Overall longevity can be restored virtually to normal. Anemia of Folate Deficiency A deficiency of folic acid, more properly pteroylmonoglutamic acid, results in a megaloblastic anemia having the same characteristics as that caused by vitamin B12 deficiency. However, the neurologic changes seen in vitamin B12 deficiency do not occur. Folic acid (or more specifically, tetrahydrofolate [FH4 ] derivatives) acts as an intermediate in the transfer of one-carbon units such as formyl and methyl groups to various compounds ( Fig. 13-22 ). In the process, FH4 also serves as an acceptor of one- carbon fragments from compounds such as serine and formiminoglutamic acid (FIGlu). The FH4 derivatives so generated in turn donate the acquired one-carbon fragments in reactions synthesizing various metabolites. FH4 , then, can be viewed as the biologic "middleman" in a series of swaps involving one-carbon moieties. The most important metabolic processes dependent on such one-carbon transfers are (1) purine synthesis; (2) conversion of homocysteine to methionine, a reaction also requiring vitamin B12 ; and (3) deoxythmidylate monophosphate synthesis. In the first two reactions, FH4 is regenerated from its one-carbon carrier derivatives and is available to accept another one-carbon fragment and re-enter the donor pool. In the synthesis of thymidylate, a dihydrofolate is produced that must be reduced by dihydrofolate reductase for reentry into the FH4 pool. The reductase step is significant, since this enzyme is susceptible to inhibition by various drugs. Among the biologically active molecules whose synthesis is dependent on folates, thymidylate is perhaps the most important. As discussed earlier in relation to pernicious anemia, deoxythymidylate monophosphate is required for DNA synthesis. It should be apparent from our Figure 13-22 Role of folate derivatives in the transfer of one-carbon fragments for synthesis of biologic macromolecules. FH4 , tetrahydrofolic acid; FH2 , dihydrofolic acid; FIGlu, formiminoglutamate; dTMP, deoxythymidylate monophosphate. *Synthesis of methionine also requires vitamin B 12 . 643 discussion that suppressed synthesis of DNA, the common denominator of folic acid and vitamin B12 deficiency, is the immediate cause of megaloblastosis. Etiology. Humans are entirely dependent on dietary sources for their folic acid requirement, which is 50 to 200 mg daily. Most normal diets contain ample amounts. The richest sources are green vegetables such as lettuce, spinach, asparagus, and broccoli. Certain fruits (e.g., lemons, bananas, melons) and animal proteins (e.g., liver) contain lesser amounts. The folic acid in these foods is largely in the form of folylpolyglutamates. Despite their abundance in raw foods, polyglutamates (depending on the specific form) are sensitive to heat; boiling, steaming, or frying of foods for 5 to 10 minutes destroys up to 95% of the folate content. Intestinal conjugases split the polyglutamates into monoglutamates that are readily absorbed in the proximal jejunum. During intestinal absorption, they are modified to 5-methyltetrahydrofolate, the normal transport form of folate. The body's reserves of folate are relatively modest, and a deficiency can arise with months of a negative balance. There are three major causes of folic acid deficiency: (1) decreased intake, (2) increased requirements, and (3) impaired use ( Table 13-5 ). Decreased intake can result from either a nutritionally inadequate diet or impairment of intestinal absorption. A normal daily diet contains folate in excess of the minimal daily adult requirement. Inadequate dietary intakes are almost invariably associated with grossly deficient diets, particularly those lacking vitamins such as the "B group." Such dietary inadequacies are most frequently encountered in chronic alcoholics, the indigent, and the very elderly. In alcoholics with cirrhosis, other mechanisms of folate deficiency such as trapping of folate within the liver, excessive urinary loss, and disordered folate metabolism have also been implicated. Under these circumstances, the megaloblastic anemia is often accompanied by general malnutrition and manifestations of other avitaminoses, including cheilosis, glossitis, and dermatitis. Malabsorption syndromes such as nontropical and tropical sprue can lead to inadequate absorption of this nutrient, as can diffuse infiltrative diseases of the small intestine (e.g., lymphoma). In addition, certain drugs, particularly the anticonvulsant phenytoin and oral contraceptives, interfere with absorption. Despite adequate intake of folic acid, a relative deficiency can be encountered in states of increased requirement, such as pregnancy, infancy, hematologic derangements associated with hyperactive hematopoiesis (hemolytic anemias), and disseminated cancer. In all these circumstances, the demands of active DNA synthesis render normal intake inadequate. Folic acid antagonists, such as methotrexate, inhibit dihydrofolate reductase and lead to a deficiency of tetrahydrofolate. With inhibition of folate metabolism, all rapidly growing cells are affected, thus leading to ulcerative lesions within the gastrointestinal tract as well as megaloblastic anemia. Many other chemotherapeutic drugs damage DNA or inhibit DNA synthesis through other mechanisms; these can also cause megaloblastic changes in rapidly dividing cells. Owing to their growth inhibitory actions, antimetabolites are used in cancer therapy. As mentioned at the outset, megaloblastic anemia resulting from a deficiency of folic acid is identical to that encountered in vitamin B12 deficiency. Thus, the diagnosis of folate deficiency can be made only by demonstration of decreased folate levels in the serum or red cells. As in vitamin B12 deficiency, serum homocysteine levels are increased. Although prompt hematologic response heralded by reticulocytosis follows the administration of folic acid, it should be cautioned that the hematologic symptoms of a vitamin B12 deficiency anemia also respond to folate therapy. However, folate does not prevent (and may even exacerbate) the progression of the neurologic deficits typical of the vitamin B12 deficiency states. It is thus essential to exclude vitamin B12 deficiency in megaloblastic anemia before initiating therapy with folate. Iron Deficiency Anemia Deficiency of iron is probably the most common nutritional disorder in the world. Although the prevalence of iron deficiency anemia is higher in developing countries, this form of anemia is also common in the United States, particularly in toddlers, adolescent girls, and women of childbearing age. The factors underlying the iron deficiency differ somewhat in various population groups and can be best considered in the context of normal iron metabolism. Iron Metabolism. As might be expected given the very high prevalence of iron deficiency in human populations, evolutionary pressures have yielded iron metabolism pathways that are strongly biased toward the retention of iron. There is no regulated pathway for iron excretion, which is limited to the 1 to 2 mg per day lost by shedding of mucosal and skin epithelial cells. Iron balance, therefore, is maintained largely by regulating the absorption of dietary iron. The normal daily Western diet contains approximately 10 to 20 mg of iron, most in the form of heme contained in animal products, with the remainder being inorganic iron in vegetables. About 20% of heme iron (in contrast to 1% to 2% of nonheme iron) is absorbable, so the average Western diet contains sufficient iron to balance fixed daily losses. The total body iron content is normally about 2 gm in women and up to 6 gm in men. As indicated in Table 13-6 , it is divided into functional and storage compartments. Approximately 80% of the functional iron is found in hemoglobin; myoglobin and iron-containing enzymes such as catalase and the cytochromes contain the rest. The storage pool represented by hemosiderin and ferritin contains approximately 15% to 20% of total body iron. Healthy young females have substantially smaller stores of iron than do males, primarily due to blood loss during menstruation. This precarious iron balance is easily tipped into deficiency by excessive losses or increased demands associated with menstruation and pregnancy, respectively. TABLE 13-6 -- Iron Distribution in Healthy Young Adults (mg) Pool Men Women Total 3450 2450 Functional TABLE 13-6 -- Iron Distribution in Healthy Young Adults (mg) Pool Men Women Hemoglobin 2100 1750 Myoglobin 300 250 Enzymes 50 50 Storage Ferritin, hemosiderin 1000 400 644 Free iron is highly toxic, and the pool of storage iron is tightly bound to either ferritin or hemosiderin. Ferritin is a protein-iron complex found in all tissues but particularly in liver, spleen, bone marrow, and skeletal muscles. In the liver, most ferritin is stored within the parenchymal cells; in other tissues, such as spleen and bone marrow, it is mainly in the mononuclear phagocytic cells. Hepatocytic iron is derived from plasma transferrin, whereas storage iron in the mononuclear phagocytic cells (Kupffer cells) is derived from the breakdown of red cells ( Fig. 13-23 ). Intracellular ferritin is located in both the cytosol and lysosomes, in which partially degraded protein shells of ferritin aggregate into hemosiderin granules. With a hematoxylin and eosin stain, hemosiderin appears in cells as golden yellow granules. The iron in hemosiderin is chemically reactive and turns blue-black when exposed to potassium ferrocyanide, which is the basis for the Prussian blue stain. With normal iron stores, only trace amounts of hemosiderin are found in the body, principally in mononuclear phagocytic cells in the bone marrow, spleen, and liver. In iron-overloaded cells, most iron is stored in hemosiderin. Very small amounts of ferritin normally circulate in the plasma. Since plasma ferritin is derived largely from the storage pool of body iron, its levels correlate well with body iron stores. In iron deficiency, serum ferritin is always below 12 µg/L, whereas in iron overload, high values approaching 5000 µg/L can be seen. Of physiologic importance, the storage iron pool can be readily mobilized if iron requirements increase, as may occur after loss of blood. Iron is transported in plasma by an iron-binding glycoprotein called transferrin (see Fig. 13-23 ), which is synthesized in the liver. In normal individuals, transferrin is about 33% saturated with iron, yielding serum iron levels that average 120 µg/dL in men and 100 µg/dL in women. Thus, the total Figure 13-23 The internal iron cycle. Plasma iron bound to transferrin is transported to the marrow, where it is transferred to developing red cells and incorporated into hemoglobin. Mature red blood cells are released into the circulation and, after 120 days, are ingested by macrophages in the reticuloendothelial system (RES). Here iron is extracted from hemoglobin and returned to the plasma, completing the cycle. (From Wyngaarden JB, et al [eds]: Cecil Textbook of Medicine, 19th ed. Philadelphia, WB Saunders, 1992, p. 841.) iron-binding capacity of serum is in the range of 300 to 350 µg/dL. The major function of plasma transferrin is to deliver iron to cells, including erythroid precursors, where iron is required for hemoglobin synthesis. Immature red cells possess high-affinity receptors for transferrin, and iron is transported into erythroblasts by receptor-mediated endocytosis. The absorption of iron and its regulation have become better understood over the last several years. Most iron is absorbed in the duodenum, where uptake of heme and nonheme iron occurs through two distinct pathways ( Fig. 13-24 ). Nonheme iron traverses the apical and basolateral membranes of villus enterocytes through the action of two distinct transporters. After reduction to ferrous (Fe++ ) iron by a membrane-associated cytochrome B, divalent metal transporter 1 (DMT1) first moves nonheme iron across the apical membrane. At least two proteins are then required for the basolateral transfer of iron to transferrin in the plasma: ferroportin, a transporter, and hephaestin, an iron oxidase. Both DMT1 and ferroportin are widely distributed in the body, suggesting their involvement in iron transport in other tissues as well. For example, DMT1 also appears to be important in uptake of iron into erythroblasts. Dietary heme iron is absorbed through a different transporter that is not yet well characterized. The efficacy of enterocyte uptake of heme and nonheme iron differs dramatically. Approximately 25% of the heme iron derived from hemoglobin, myoglobin, and other animal proteins is absorbed. The absorption of nonheme iron is more variable, being influenced by substances in the diet that inhibit (phytates, tannates, and phosphates) or enhance (ascorbic and amino acids) uptake, but frequently less than 5% of that consumed. After absorption, both heme and nonheme iron appear to enter a common pool in the mucosal cell. Normally, a fraction of the iron that enters the cell is rapidly delivered to plasma transferrin. Most, however, is deposited as ferritin, some to be transferred more slowly to plasma transferrin, and some to be lost with exfoliation of mucosal cells. The extent to which the mucosal iron is distributed along these various pathways depends on the body's iron requirements. When the body is replete with iron, formation of ferritin within the mucosal cells is maximal, whereas transport into plasma is enhanced in iron deficiency. Since body losses of iron are limited, iron balance is maintained by regulating absorptive intake. The mechanisms that regulate the absorption of available iron into the mucosal cell are still incompletely understood. Important clues come from observations demonstrating that the rate and level of absorption are dependent on total body iron content and erythropoietic activity, or more specifically the iron needs of the erythroid precursors. As body stores rise, the absorption of iron falls, and vice versa. With ineffective erythropoiesis, as may occur in β-thalassemias, iron absorption is increased despite an excess of stored body iron. Some signal must be delivered to the mucosal cell, modifying its uptake and transfer of iron. An excellent candidate for a negative "iron metabolism regulatory hormone" is hepcidin, a small, liver-derived plasma peptide. Hepcidin inhibits iron uptake in the duodenum and iron release from macrophages. The concentration of hepcidin falls as iron stores become depleted, and hepcidin knockout mice develop hemochromatosis. Conversely, over-expression of hepcidin in transgenic mice causes an iron deficiency 645 Figure 13-24 Diagrammatic representation of iron absorption. Mucosal uptake of heme and nonheme iron is depicted. When the storage sites of the body are replete with iron and erythropoietic activity is normal, most of the absorbed iron is lost into the gut by shedding of the epithelial cells. Conversely, when body iron needs increase or when erythropoiesis is stimulated, a greater fraction of the absorbed iron is transferred into plasma transferrin, with a concomitant decrease in iron loss through mucosal ferritin. anemia. These findings suggest the existence of a hepcidin receptor on duodenal enterocytes. The HFE gene, which encodes an HLA-like transmembrane protein, is also clearly involved in regulation of iron absorption.  As discussed in Chapter 18 , mutation in this gene leads to excessive absorption of dietary iron and eventual hemochromatosis, a disease characterized by systemic iron overload. Etiology. To maintain a normal iron balance, approximately 1 mg of iron must be absorbed from the diet every day. Because only 10% to 15% of ingested iron is absorbed, the daily iron requirement is 7 to 10 mg for adult men and 7 to 20 mg for adult women. Since the average daily dietary intake of iron in the Western world is about 15 to 20 mg, most men ingest more than adequate iron, whereas many women consume marginally adequate amounts of iron. The bioavailability of dietary iron is as important as the overall content. Heme iron is much more absorbable than inorganic iron. The absorption of the latter is influenced by other dietary contents. Ascorbic acid, citric acid, amino acids, and sugars in the diet enhance absorption of inorganic iron, but tannates (as in tea), carbonates, oxalates, and phosphates inhibit its absorption. An iron deficiency can result from (1) dietary lack, (2) impaired absorption, (3) increased requirement, or (4) chronic blood loss. Dietary lack is a rare cause of iron deficiency in industrialized countries having abundant food supplies (including meat) where about two thirds of the dietary iron is in the readily assimilable heme form. The situation is different in developing countries, where food is less abundant and diets are predominantly vegetarian, containing poorly absorbable inorganic iron. Despite the availability of iron, dietary inadequacy still occurs in privileged societies in these groups: • Infants are at high risk because milk diets contain very small amounts of iron. Human breast milk, for example, provides only about 0.3 mg/L of iron. Cow's milk contains about twice as much iron as human breast milk, but the iron in cow's milk has poor bioavailability. • Children, especially during the early years of life, have increased dietary iron needs to accommodate growth, development, and the accompanying expansion of blood volume. • The impoverished, at any age, can have suboptimal diets for socioeconomic reasons. • The elderly often have restricted diets with little meat because of limited income or poor dentition. Impaired absorption is found in sprue, other causes of intestinal steatorrhea, and chronic diarrhea. Gastrectomy impairs iron absorption by decreasing hydrochloric acid and transit time through the duodenum. Specific items in the diet, as is evident from the preceding discussion, can also affect absorption. Increased requirement is an important potential cause of iron deficiency. Growing infants and children, adolescents, and premenopausal (particularly pregnant) women have a much greater requirement for iron than do nonmenstruating adults. Particularly at risk are economically deprived women having multiple, frequent pregnancies. Chronic blood loss is the most common cause of iron deficiency in the Western world. If bleeding occurs into tissues or cavities of the body, the heme iron can be totally recovered and recycled. However, external hemorrhage, as can occur from the gastrointestinal tract (e.g., peptic ulcers, hemorrhagic gastritis, gastric carcinoma, colonic carcinoma, hemorrhoids, or hookworm or pinworm disease), the urinary tract (e.g., renal, pelvic, or bladder tumors), or the genital tract (e.g., menorrhagia, uterine cancer), depletes iron reserves. When all the potential causes of an iron deficiency are taken into consideration, deficiency in adult men and postmenopausal women in the Western world must be attributed to gastrointestinal blood loss until proven otherwise. To prematurely ascribe iron deficiency in such individuals to any other cause is to run the risk of missing an occult gastrointestinal cancer or other bleeding lesion. Whatever its basis, iron deficiency induces a hypochromic microcytic anemia. Simultaneously, depletion of essential iron-containing enzymes in cells throughout the body can cause other changes, including koilonychia, alopecia, atrophic changes in the tongue and gastric mucosa, and intestinal malabsorption. These changes are seen with severe and long-standing iron deficiency. Uncommonly, esophageal webs appear to complete the triad of major findings in the Plummer-Vinson syndrome: (1) microcytic hypochromic anemia, (2) atrophic glossitis, and (3) esophageal webs ( Chapter 17 ). 646 At the outset of chronic blood loss or other states of negative iron balance, reserves in the form of ferritin and hemosiderin may be adequate to maintain normal hemoglobin and hematocrit levels as well as normal serum iron and transferrin saturation. Progressive depletion of these reserves first lowers serum iron and transferrin saturation levels, without producing anemia. In this early stage, there is increased erythroid activity in the bone marrow. Anemia only appears when iron stores are completely depleted, accompanied by low serum iron, serum ferritin, and transferrin saturation. Morphology. The bone marrow reveals a mild to moderate increase in erythroid progenitors (normoblasts). A diagnostically significant finding is the disappearance of stainable iron from mononuclear phagocytic cells in the bone marrow, which is assessed by performing Prussian blue stains on aspirated or sectioned bone marrow. In the peripheral blood smear, red cells are small (microcytic) and pale (hypochromic). Normal well- hemoglobinized red cells have a zone of central pallor measuring about one third of the cell diameter. In established iron deficiency, the zone of pallor is enlarged; hemoglobin may be seen only in a narrow peripheral rim ( Fig. 13-25 ). Poikilocytosis in the form of small, elongated red cells (pencil cells) is also characteristic. The clinical manifestations of the anemia are nonspecific and were detailed earlier. The dominating signs and symptoms Figure 13-25 Hypochromic microcytic anemia of iron deficiency (peripheral blood smear). Note the small red cells containing a narrow rim of peripheral hemoglobin. Scattered fully hemoglobinized cells, present due to recent blood transfusion, stand in contrast. (Courtesy of Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) frequently relate to the underlying cause of the anemia, for example, gastrointestinal or gynecologic disease, malnutrition, pregnancy, and malabsorption. The diagnosis of iron deficiency anemia ultimately rests on laboratory studies. Both the hemoglobin and hematocrit are depressed, usually to moderate levels, in association with hypochromia, microcytosis, and some poikilocytosis. The serum iron and ferritin are low, and the total plasma iron-binding capacity (reflecting transferrin concentration) is high. Low serum iron with increased iron-binding capacity results in a reduction of transferrin saturation levels to below 15%. Reduced iron stores inhibit hepcidin synthesis and its serum levels fall. As mentioned earlier, transferrin receptor, expressed on the surface of many cells, is required for the transport of iron into cells. The level of soluble transferrin receptors, which are mostly derived from erythroid progenitors in the marrow, is elevated in iron deficiency due to a mild expansion of erythroid progenitors and an increased rate of transferrin receptor shedding. Reduced heme synthesis leads to elevation of free erythrocyte protoporphyrin. An alert clinician investigating unexplained iron deficiency anemia will occasionally discover an occult bleed or cancer and thereby save a life. Anemia of Chronic Disease Impaired red cell production associated with chronic diseases is perhaps the most common cause of anemia among hospitalized patients in the United States. It is associated with reduced erythroid proliferation and impaired iron utilization and can therefore mimic iron deficiency. The chronic illnesses associated with this form of anemia can be grouped into three categories: • Chronic microbial infections, such as osteomyelitis, bacterial endocarditis, and lung abscess • Chronic immune disorders, such as rheumatoid arthritis and regional enteritis • Neoplasms, such as Hodgkin lymphoma and carcinomas of the lung and breast The common features characterizing anemia in these diverse clinical settings are low serum iron and reduced total iron-binding capacity in association with abundant stored iron in the mononuclear phagocytic cells. This combination suggests some impediment in the transfer of iron from the storage pool to the erythroid precursors. In addition, marrow erythroid progenitors do not proliferate adequately because erythropoietin levels are inappropriately low for the degree of anemia. The reduction in renal erythropoietin generation is caused by the action of interleukin-1, tumor necrosis factor (TNF), and interferon-γ, secretion of which is triggered by the underlying chronic inflammatory or neoplastic disease. These cytokines also stimulate hepcidin synthesis in the liver, which in turn inhibits the release of iron from the storage pool. The anemia is usually mild, and the dominant symptoms are those of the underlying disease. The red blood cells can be normocytic and normochromic or hypochromic and microcytic as in anemia of iron deficiency. The presence of increased storage iron in the marrow macrophages, a high serum ferritin level, and reduced total iron-binding capacity readily rule out iron deficiency as the cause of anemia. Only successful treatment of the underlying condition reliably corrects the anemia. However, many patients benefit from administration of erythropoietin. 647 Aplastic Anemia Aplastic anemia is a somewhat misleading term applied to a syndrome of marrow failure associated with pancytopenia (anemia, neutropenia, and thrombocytopenia). The marrow failure stems from suppression or disappearance of multipotent myeloid stem cells. Etiology. The major circumstances under which aplastic anemia can appear are listed in Table 13-7 . Most cases of aplastic anemia of "known" etiology follow exposure to chemicals and drugs. With some agents, marrow damage is predictable, dose related and, in most instances, reversible when exposure to the offending agent is stopped. Among the best- known dose-dependent myelotoxins are benzene, chloramphenicol, alkylating agents, and antimetabolites (e.g., 6-mercaptopurine, vincristine, and busulfan). In other instances, however, pancytopenia appears as an apparent idiosyncratic reaction to very small doses of known myelotoxins (e.g., chloramphenicol) or nonmyelotoxic drugs such as phenylbutazone, methylphenylethylhydantoin, streptomycin, and chlorpromazine. In such idiosyncratic reactions, the aplasia can be severe, irreversible, and fatal. Whole-body irradiation is another insult that can destroy hematopoietic stem cells in a dose-dependent fashion. Persons who receive therapeutic irradiation or are exposed to radiation in nuclear accidents (e.g., Chernobyl) are at risk. Aplastic anemia can appear after a variety of viral infections, most commonly viral hepatitis of the non-A, non-B, non-C, and non-G types. Why certain individuals develop this complication is not understood, but it is not related to the severity of infection. Fanconi anemia is a rare autosomal recessive disorder caused by defects in a component of a multiprotein complex TABLE 13-7 -- Major Causes of Aplastic Anemia Acquired Idiopathic Primary stem cell defect Immune mediated Chemical agents Dose related Alkylating agents Antimetabolites Benzene Chloramphenicol Inorganic arsenicals Idiosyncratic Chloramphenicol Phenylbutazone Organic arsenicals Methylphenylethylhydantoin Streptomycin Chlorpromazine Insecticides (e.g., DDT, parathion) Physical agents (e.g., whole-body irradiation) Viral infections Hepatitis (unknown virus) Cytomegalovirus infections Epstein-Barr virus infections Herpes varicella-zoster Miscellaneous Infrequently, many other drugs and chemicals Inherited Fanconi anemia required for DNA repair ( Chapter 7 ). Marrow hypofunction in Fanconi anemia becomes evident early in life and is accompanied by multiple congenital anomalies, such as hypoplasia of the kidney and spleen and hypoplastic anomalies of bone, often involving the thumbs or radii. Despite all these possible causes, no provocative factor can be identified in fully 65% of the cases, which are lumped into the idiopathic category. Pathogenesis. The pathogenesis of aplastic anemia is not fully understood. Indeed, it is unlikely that a single mechanism underlies all cases. Two major etiologies have been invoked: an immunologically mediated suppression and an intrinsic abnormality of stem cells ( Fig. 13-26 ). Recent studies suggest that aplastic anemia results most commonly from suppression of stem cell function by activated T cells. It is postulated that stem cells are first antigenically altered by exposure to drugs, infectious agents, or other unidentified environmental insults. This evokes a cellular immune response, during which activated T cells produce cytokines such as interferon-γ and TNF that prevent normal stem cell growth and development. This scenario is supported by several observations. Immunosuppressive therapy with antithymocyte globulin combined with drugs such as cyclosporine produces responses in 60% to 70% of patients, and successful bone marrow transplantation requires "conditioning" with high doses of myelotoxic drugs or radiation. In both instances, it is hypothesized these therapies work by suppressing or killing autoreactive T-cell clones. The target antigens for T-cell attack are not well defined. In some instances GPI-linked proteins may be the targets of sensitized T cells, Figure 13-26 Pathophysiology of aplastic anemia. Damaged stem cells can produce progeny expressing neo-antigens that evoke an autoimmune reaction, or give rise to a clonal population with reduced proliferative capacity. Either pathway could lead to marrow aplasia. 648 possibly explaining why aplastic anemia sometimes evolves to paroxysmal nocturnal hemoglobinuria. Alternatively, the notion that aplastic anemia results from a fundamental stem cell abnormality is supported by the presence of karyotypic aberrations in many cases and occasional transformation of aplasia to myeloid neoplasms, typically myelodysplasia or acute myelogenous leukemia. Some marrow insult presumably causes genetic damage that limits the proliferative and differentiative capacity of stem cells. If the damage is extensive enough, aplastic anemia results. These two mechanisms are not mutually exclusive, as genetically altered stem cells might also express "neo-antigens" that could serve as targets for T-cell attack. Morphology. The markedly hypocellular bone marrow is largely devoid of hematopoietic cells; often only fat cells, fibrous stroma, and scattered or clustered foci of lymphocytes and plasma cells remain. A marrow aspirate often yields little material (a "dry tap"). Hence, marrow aplasia is best appreciated in a bone marrow biopsy ( Fig. 13-27 ). A number of additional pathologic changes commonly accompanying marrow failure are related to granulocytopenia and thrombocytopenia, such as mucocutaneous bacterial infections and abnormal bleeding, respectively. The toxic drug or agent sometimes injures other tissues as well. Benzene, for example, can cause fatty changes in the liver and kidneys. If the anemia necessitates multiple transfusions, systemic hemosiderosis can appear. Clinical Course. Aplastic anemia can occur at any age and in either sex. The onset is usually insidious. Initial manifestations vary somewhat, depending on which cell line is predominantly affected. Anemia can cause progressive weakness, pallor, and dyspnea. Petechiae and ecchymoses can herald thrombocytopenia. Granulocytopenia can manifest as frequent and persistent minor infections or the sudden onset of chills, fever, and prostration. Splenomegaly is characteristically absent; if present, the diagnosis of aplastic anemia should be seriously questioned. The red cells are typically normocytic and normochromic, although slight macrocytosis is occasionally present. Reticulocytopenia is the rule. Figure 13-27 Aplastic anemia (bone marrow biopsy). Markedly hypocellular marrow contains mainly fat cells. A, Low power. B, High power. (Courtesy of Dr. Steven Kroft, Department of Pathology, University of Texas Southwestern Medical School, Dallas, TX.) The diagnosis rests on examination of bone marrow biopsy and peripheral blood. It is important to distinguish aplastic anemia from other causes of pancytopenia, such as "aleukemic" leukemia and myelodysplastic syndromes ( Chapter 14 ), that can present with identical clinical manifestations. In aplastic anemia, the marrow is hypocellular (and usually markedly so), whereas myeloid neoplasms are associated with hypercellular marrow filled with abnormal myeloid progenitors. The prognosis of aplastic aplasia is unpredictable. As mentioned earlier, withdrawal of toxic drugs can lead to recovery in some cases. Spontaneous remission in idiopathic cases is unfortunately uncommon. In younger patients, allogeneic bone marrow transplantation offers a hope for cure. Older patients or those without suitable donors often respond well to immunosuppressive therapies (antithymocyte globulin and cyclosporine). Pure Red Cell Aplasia Pure red cell aplasia is a rare form of marrow failure characterized by a marked hypoplasia of marrow erythroid elements in the setting of normal granulopoiesis and thrombopoiesis. Pure red cell aplasia can be primary, without any associated disease, or arise secondarily to neoplasms, particularly thymic tumors (thymomas) and large granular lymphocytic leukemia ( Chapter 14 ), drug exposures, or autoimmune disorders. The association with thymoma raises the question of some thymus-related immunologic mechanism; indeed, in about half the patients, resection of the thymic tumor is followed by hematologic improvement. In all likelihood, the primary form is also related to autoimmunity against erythroid precursors, and immunosuppressive therapy can be beneficial in such patients. Plasmapheresis has also been used with some success in refractory cases. Other Forms of Marrow Failure Space-occupying lesions that destroy significant amounts of bone marrow or disturb the marrow architecture depress its productive capacity. This form of marrow failure is referred to as myelophthisic anemia. As you might anticipate, all formed blood elements are affected. Characteristically, immature erythroid and myeloid progenitors appear in the peripheral blood 649 (leukoerythroblastosis). Infiltrative diseases of the marrow typically cause reactive fibrosis and distortion of the marrow architecture; presumably, this disturbs the normal mechanisms that restrict the release of immature erythroid and myeloid cells into the peripheral blood. The most common cause of myelophthisic anemia is metastatic cancer, most often carcinomas arising in the breast, lung, and prostate. However, any tumor or infiltrative process (e.g., granulomatous disease) involving the marrow can produce identical findings. Myelophthisic anemia is also observed in myelofibrosis and other myeloproliferative disorders ( Chapter 14 ), all of which often cause marrow fibrosis. Diffuse liver disease, whether toxic, infectious, or cirrhotic, is associated with an anemia attributed to hypofunction of the marrow. Concomitant folate deficiency and iron deficiency due to gastrointestinal blood loss (varices, hemorrhoids) can also contribute to the anemia. Most often, erythroid progenitors are preferentially affected; depression of the white cell count and platelets is less common, but has been described. The anemia is often slightly macrocytic due to lipid abnormalities associated with liver failure, which cause red cell membranes to acquire phospholipid and cholesterol in the periphery. Chronic renal failure, whatever its cause, is almost invariably associated with anemia that tends to be roughly proportional to the severity of the uremia. The basis of the anemia is multifactorial. There is evidence of an extracorpuscular defect inducing chronic hemolysis. Some patients have an iron deficiency secondary to the bleeding tendency often encountered in uremia. Concomitantly, there is reduced red cell production, related to advanced destruction of the kidneys and inadequate synthesis of erythropoietin, which appears to be the dominant cause of anemia. Not surprisingly, therefore, administration of recombinant erythropoietin results in significant improvement of the anemia associated with renal failure, although optimal response may require aggressive concomitant iron replacement therapy. Polycythemia Polycythemia, or erythrocytosis, denotes an abnormally high concentration of red cells, usually with a corresponding increase in hemoglobin level. The increase in red cells can be relative, when there is hemoconcentration due to decreased plasma volume, or absolute, when there is an increase in total red cell mass. Relative polycythemia results from any cause of dehydration, such as deprivation of water, prolonged vomiting, diarrhea, or excessive use of diuretics. It is also associated with an obscure condition of unknown etiology called stress polycythemia, or Gaisböck syndrome. Affected individuals are usually hypertensive, obese, and anxious ("stressed"). Absolute polycythemia is primary when it results from an intrinsic abnormality of the myeloid stem cells and secondary when the red cell progenitors are responding to increased levels of erythropoietin. Primary polycythemia (polycythemia vera) is one of several neoplasms originating from myeloid stem cells (considered in Chapter 14 ). Another much less common form of "primary" polycythemia results from mutations in the erythropoietin receptor that cause hyperresponsiveness to erythropoietin. Affected individuals have congenital polycythemia. One such individual won an Olympic gold medal in cross- country skiing, having benefited from this natural form of blood doping! Secondary polycythemias can be caused by an increase in erythropoietin secretion that is physiologically appropriate (e.g., chronic hypoxia) or inappropriate (pathologic) ( Table 13-8 ). Bleeding Disorders: Hemorrhagic Diatheses Excessive bleeding can result from (1) increased fragility of vessels, (2) platelet deficiency or dysfunction, (3) derangement of coagulation, and (4) combinations of these. Before discussing these specific bleeding disorders, it is helpful to review normal hemostasis ( Chapter 4 ) and the common laboratory tests used in the evaluation of a bleeding diathesis. It should be recalled from the discussion in Chapter 4 that the normal hemostatic response involves the blood vessel wall, the platelets, and the clotting cascade. Tests used to evaluate different aspects of hemostasis are the following: • Bleeding time. This measures the time taken for a standardized skin puncture to stop bleeding and provides an in vivo assessment of platelet response to limited vascular injury. The reference range depends on the actual method employed and varies from 2 to 9 minutes. Prolongation generally indicates a defect in platelet numbers or function. Bleeding time test is fraught with variability and poor reproducibility. Hence new instrument-based assay systems such as platelet function analyzer-100 (PFA-100) that provide a quantitative measure of platelet function under conditions of high shear stress are being evaluated as replacements for the bleeding time test. • Platelet counts. These are obtained on anticoagulated blood using an electronic particle counter. The reference range is 150 to 300 × 103 /µL. Counts well outside this range need to be confirmed by a visual inspection of a peripheral blood smear, as clumping of platelets can cause spurious "thrombocytopenia" during automated counting, and high counts may be indicative of a myeloproliferative disorder. • Prothrombin time (PT). This assay tests the extrinsic and common coagulation pathways. The clotting of plasma after addition of an exogenous source of tissue thromboplastin (e.g., brain extract) and Ca2+ ions is measured in seconds. A prolonged PT can result from deficiency or dysfunction of factor V, factor VII, factor X, prothrombin, or fibrinogen. • Partial thromboplastin time (PTT). This assay tests the intrinsic and common clotting pathways. The clotting of 650 plasma after addition of kaolin, cephalin, and calcium ions is measured in seconds. Kaolin serves to activate the contact-dependent factor XII, and cephalin substitutes for platelet phospholipids. Prolongation of the PTT can be due to deficiency or dysfunction of factor V, VIII, IX, X, XI, or XII, prothrombin, or fibrinogen. TABLE 13-8 -- Pathophysiologic Classification of Polycythemia Relative Reduced plasma volume (hemoconcentration) Absolute Primary Polycythemia vera, rare erythropoietin receptor mutations (low erythropoietin) Secondary High erythropoietin Appropriate: lung disease, high-altitude living, cyanotic heart disease Inappropriate: erythropoietin-secreting tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, cerebellar hemangioblastoma) More specialized tests are available to measure the levels of specific clotting factors, fibrinogen, fibrin split products, the presence of circulating anticoagulants, and platelet function. With this overview, we can turn to the various categories of bleeding disorders. BLEEDING DISORDERS CAUSED BY VESSEL WALL ABNORMALITIES Disorders within this category, sometimes called nonthrombocytopenic purpuras, are relatively common but do not usually cause serious bleeding problems. Most often, they induce small hemorrhages (petechiae and purpura) in the skin or mucous membranes, particularly the gingivae. On occasion, however, more significant hemorrhages can occur into joints, muscles, and subperiosteal locations or take the form of menorrhagia, nosebleeds, gastrointestinal bleeding, or hematuria. The platelet count, bleeding time, and results of the coagulation tests (PT, PTT) are usually normal. The varied clinical conditions in which hemorrhages can be related to abnormalities in the vessel wall include the following: • Many infections induce petechial and purpuric hemorrhages, but especially implicated are meningococcemia, other forms of septicemia, infective endocarditis, and several of the rickettsioses. The involved mechanism is presumably microbial damage to the microvasculature (vasculitis) or disseminated intravascular coagulation (DIC). Failure to recognize meningococcemia as a cause of petechiae and purpura can be catastrophic for the patient. • Drug reactions sometimes induce cutaneous petechiae and purpura without causing thrombocytopenia. In many instances, the vascular injury is mediated by drug-induced antibodies and deposition of immune complexes in the vessel walls, leading to hypersensitivity (leukocytoclastic) vasculitis ( Chapter 11 ). • Scurvy and the Ehlers-Danlos syndrome are associated with microvascular bleeding resulting from impaired formation of collagens needed for support of vessel walls. The same mechanism may account for spontaneous purpura commonly seen in the very elderly. The predisposition to skin hemorrhages in Cushing syndrome, in which the protein-wasting effects of excessive corticosteroid production cause loss of perivascular supporting tissue, has a similar etiology. • Henoch-Schönlein purpura is a systemic hypersensitivity disease of unknown cause characterized by a purpuric rash, colicky abdominal pain (presumably due to focal hemorrhages into the gastrointestinal tract), polyarthralgia, and acute glomerulonephritis ( Chapter 20 ). All these changes result from the deposition of circulating immune complexes within vessels throughout the body and within the glomerular mesangial regions. • Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder characterized by dilated, tortuous blood vessels with thin walls that bleed readily. Bleeding can occur anywhere in the body but is most common under the mucous membranes of the nose (epistaxis), tongue, mouth, and eyes and throughout the gastrointestinal tract. • Amyloid infiltration of blood vessels. Systemic amyloidosis is associated with perivascular deposition of amyloid and consequent weakening of blood vessel wall. This is most commonly observed in plasma cell dyscrasias ( Chapter 14 ) and is manifested as mucocutaneous petechiae. Bleeding in these conditions is rarely life threatening with the exception of some cases of hereditary telangiectasia. Recognition of the presenting symptoms should prompt further studies to establish a specific diagnosis. BLEEDING RELATED TO REDUCED PLATELET NUMBER: THROMBOCYTOPENIA Reduction in platelet number constitutes an important cause of generalized bleeding. Normal platelet counts range from 150,000 to 300,000/µL. A count below 100,000/µL is generally considered to constitute thrombocytopenia. However, spontaneous bleeding does not become evident until the count falls below 20,000/µL. Platelet counts in the range of 20,000 to 50,000/µL can aggravate post-traumatic bleeding. Bleeding resulting from thrombocytopenia alone is associated with a prolonged bleeding time and normal PT and PTT. The important role of platelets in hemostasis is discussed in Chapter 4 . It hardly needs reiteration that these cells are critical for hemostasis, as they form temporary plugs that quickly stop bleeding and promote key reactions in the clotting cascade. Spontaneous bleeding associated with thrombocytopenia most often involves small vessels. The common sites of such hemorrhage are the skin and the mucous membranes of the gastrointestinal and genitourinary tracts. Intracranial bleeding is a threat to any patient with a markedly depressed platelet count. The many causes of thrombocytopenia can be classified into the four major categories listed in Table 13-9 . • Decreased production of platelets. This can accompany generalized diseases of bone marrow such as aplastic anemia and leukemias or result from diseases that affect the megakaryocytes somewhat selectively. In vitamin B12 or folic acid deficiency, there is poor development and accelerated destruction of megakaryocytes within the bone marrow (ineffective megakaryopoiesis) because DNA synthesis is impaired. • Decreased platelet survival. This important cause of thrombocytopenia can have an immunologic or nonimmunologic etiology. In the immune conditions, platelet destruction is caused by circulating antiplatelet antibodies or, less often, immune complexes. The antiplatelet antibodies can be directed against a self-antigen on the platelets (autoantibodies) or against platelet antigens that differ among different individuals (alloantibodies). Common antigenic targets of both autoantibodies and alloantibodies are the platelet membrane glycoprotein complexes IIb-IIIa and Ib-IX. Autoimmune thrombocytopenias include idiopathic thrombocytopenic purpura, certain drug-induced thrombocytopenias, and HIV- associated thrombocytopenias. All of these are discussed later. Alloimmune thrombocytopenias arise when an individual is exposed to platelets 651 of another person, as may occur after blood transfusion or during pregnancy. In the latter case, neonatal or even fetal thrombocytopenia occurs by a mechanism analogous to erythroblastosis fetalis. Nonimmunologic destruction of platelets may be caused by mechanical injury, in a manner analogous to red cell destruction in microangiopathic hemolytic anemia. The underlying conditions are also similar, including prosthetic heart valves and diffuse narrowing of the microvessels (e.g., malignant hypertension). • Sequestration. Thrombocytopenia, usually moderate in severity, may develop in any patient with marked splenomegaly, a condition sometimes referred to as hypersplenism ( Chapter 14 ). The spleen normally sequesters 30% to 40% of the body's platelets, which remain in equilibrium with the circulating pool. When necessary, hypersplenic thrombocytopenia can be ameliorated by splenectomy. • Dilutional. Massive transfusions can produce a dilutional thrombocytopenia. Blood stored for longer than 24 hours contains virtually no viable platelets; thus, plasma volume and red cell mass are reconstituted by transfusion, but the number of circulating platelets is relatively reduced. TABLE 13-9 -- Causes of Thrombocytopenia Decreased production of platelets Generalized diseases of bone marrow Aplastic anemia: congenital and acquired (see Table 13-7 ) Marrow infiltration: leukemia, disseminated cancer Selective impairment of platelet production Drug-induced: alcohol, thiazides, cytotoxic drugs Infections: measles, human immunodeficiency virus (HIV) Ineffective megakaryopoiesis Megaloblastic anemia Myelodysplastic syndromes Decreased platelet survival Immunologic destruction Autoimmune: idiopathic thrombocytopenic purpura, systemic lupus erythematosus Isoimmune: post-transfusion and neonatal Drug-associated: quinidine, heparin, sulfa compounds Infections: infectious mononucleosis, HIV infection, cytomegalovirus Nonimmunologic destruction Disseminated intravascular coagulation Thrombotic thrombocytopenic purpura Giant hemangiomas Microangiopathic hemolytic anemias Sequestration Hypersplenism Dilutional Immune Thrombocytopenic Purpura (ITP) ITP can occur in the setting of a variety of conditions and exposures (secondary ITP) or in the absence of any known risk factors (primary or idiopathic ITP). There are two clinical subtypes of primary ITP, acute and chronic; both are autoimmune disorders in which platelet destruction results from the formation of antiplatelet autoantibodies. We first discuss the more common chronic form of primary ITP; acute ITP, a self-limited disease of children, is discussed later. Immunologically mediated destruction of platelets (immune thrombocytopenia) occurs in many different settings, including systemic lupus erythematosus, acquired immunodeficiency syndrome (AIDS), after viral infections, and as a complication of drug therapy. These secondary forms of immune thrombocytopenia can sometimes mimic the idiopathic autoimmune variety, and hence the diagnosis of this disorder should be made only after exclusion of other known causes of thrombocytopenia. Particularly important in this regard is systemic lupus erythematosus, a multisystem autoimmune disease ( Chapter 6 ) that can present with thrombocytopenia. Pathogenesis. Chronic ITP is caused by the formation of autoantibodies against platelet membrane glycoproteins, most often IIb-IIIa or Ib-IX. Antibodies reactive with these membrane glycoproteins can be demonstrated in the plasma as well as bound to the platelet surface (platelet-associated immunoglobulins) in approximately 80% of patients. In the overwhelming majority of cases, the antiplatelet antibodies are of the IgG class. The mechanism of platelet destruction is similar to that seen in autoimmune hemolytic anemias. Opsonized platelets are rendered susceptible to phagocytosis by the cells of the mononuclear phagocyte system. About 75% to 80% of patients are remarkably improved after splenectomy, indicating that the spleen is the major site of removal of sensitized platelets. Since it is also an important site of autoantibody synthesis, the beneficial effects of splenectomy may in part derive from removal of the source of autoantibodies. Although destruction of sensitized platelets is the major mechanism responsible for thrombocytopenia, there is some evidence that megakaryocytes may be damaged by autoantibodies, leading to impairment of platelet production. In most cases, however, megakaryocyte injury is not significant enough to deplete their numbers. Morphology. The principal morphologic lesions of thrombocytopenic purpura are found in the spleen and bone marrow but they are not diagnostic. Secondary changes related to the bleeding diathesis may be found in any tissue or structure in the body. The spleen is normal in size. On histologic examination, there is congestion of the sinusoids and hyperactivity and enlargement of the splenic follicles, manifested by the formation of prominent germinal centers. In many instances, scattered megakaryocytes are found within the sinuses and sinusoidal walls. This may represent a very mild form of extramedullary hematopoiesis driven by elevated levels of thrombopoietin. These splenic findings are not sufficiently distinctive to be considered diagnostic. Bone marrow reveals a modestly increased number of megakaryocytes. Some are apparently immature, with large, nonlobulated, single nuclei. These findings are not specific for autoimmune thrombocytopenic purpura but merely reflect accelerated thrombopoiesis, being found in most forms of thrombocytopenia resulting from increased platelet destruction. The importance of bone marrow examination is to rule out thrombocytopenias resulting from bone marrow failure. A decrease in the number of megakaryocytes argues against the diagnosis of ITP. The secondary changes relate to the hemorrhages that are dispersed throughout the body. 652 Clinical Features. Chronic ITP occurs most commonly in adult women younger than age 40 years. The female-to-male ratio is 3:1. This disorder is often insidious in onset and is characterized by bleeding into the skin and mucosal surfaces. Cutaneous bleeding is seen in the form of pinpoint hemorrhages (petechiae), especially prominent in the dependent areas where the capillary pressure is higher. Petechiae can become confluent, giving rise to ecchymoses. Often there is a history of easy bruising, nosebleeds, bleeding from the gums, and hemorrhages into soft tissues from relatively minor trauma. The disease may manifest first with melena, hematuria, or excessive menstrual flow. Subarachnoid hemorrhage and intracerebral hemorrhage are serious consequences of thrombocytopenic purpura but, fortunately, are rare in treated patients. Splenomegaly and lymphadenopathy are uncommon in primary ITP, and their presence should lead one to consider other possible diagnoses. The clinical signs and symptoms associated with ITP are not specific for this condition but rather reflective of thrombocytopenia. Destruction of platelets as the cause of thrombocytopenia is supported by the findings of a low platelet count and normal or increased megakaryocytes in the bone marrow. Accelerated thrombopoiesis often leads to the formation of abnormally large platelets (megathrombocytes), detected easily in a blood smear. The bleeding time is prolonged, but PT and PTT are normal. Tests for platelet autoantibodies are not widely available. Therefore, a diagnosis of ITP should be made only after other causes of platelet deficiencies, such as those listed in Table 13-9 , have been ruled out. Almost all patients respond to immunosuppressive doses of glucocorticoids, but many eventually relapse and come to splenectomy. Most maintain safe platelet counts postsplenectomy and require no further therapy. A significant minority, however, have refractory forms of ITP that can be very difficult to treat. Various immunosuppressive approaches may be effective in such patients. Acute Immune Thrombocytopenic Purpura Like chronic ITP, this condition is caused by antiplatelet autoantibodies, but its clinical features and course are distinct. Acute ITP is a disease of childhood occurring with equal frequency in both sexes. The onset of thrombocytopenia is abrupt and is preceded in many cases by a viral illness. The usual interval between the infection and onset of purpura is 2 weeks. Unlike the adult chronic form of ITP, the childhood disease is self- limited, usually resolving spontaneously within 6 months. Steroid therapy is indicated only if thrombocytopenia is severe. Approximately 20% of the children, usually those without a viral prodrome, have persistent low platelet counts beyond 6 months and appear to have chronic ITP similar in most respects to the adult disease. Drug-Induced Thrombocytopenia: Heparin-Induced Thrombocytopenia Like hemolytic anemia, thrombocytopenia can result from immunologically mediated destruction of platelets after drug ingestion. The drugs most commonly involved are quinine, quinidine, sulfonamide antibiotics, and heparin. Heparin-induced thrombocytopenia (HIT) is of particular importance because this anticoagulant is used widely and failure to make a correct diagnosis can have severe consequences. Thrombocytopenia occurs in approximately 5% of patients receiving heparin. Most develop so-called type I thrombocytopenia, which occurs rapidly after onset of therapy, is modest in severity and clinically insignificant, and may resolve despite continuation of heparin therapy. It most likely results from a direct platelet-aggregating effect of heparin. Type II thrombocytopenia is more severe. It occurs 5 to 14 days after commencement of therapy (or sometimes sooner if the patient has been previously sensitized to heparin) and can, paradoxically, lead to life-threatening venous and arterial thrombosis. HIT is caused by an immune reaction directed against a complex of heparin and platelet factor 4, a normal component of platelet granules that binds tightly to heparin. It appears that heparin binding modifies the conformation of platelet factor 4, making it susceptible to immune recognition.  Binding of antibody to platelet factor 4 produces immune complexes that activate platelets, promoting thrombosis even in the setting of marked thrombocytopenia. The mechanism of platelet activation is not understood. Unless therapy is immediately discontinued, clots within large arteries may lead to vascular insufficiency and limb loss, and emboli from deep venous thrombosis can cause fatal pulmonary thromboembolism. HIV-Associated Thrombocytopenia Thrombocytopenia is perhaps the most common hematologic manifestation of HIV infection. Both impaired platelet production and increased destruction are responsible. CD4, the receptor for HIV on T cells, has also been demonstrated on megakaryocytes, making it possible for these cells to be infected by HIV. Infected megakaryocytes are prone to apoptosis and are impaired in terms of platelet production. HIV infection also causes hyperplasia and dysregulation of B cells, which predispose to the development of immune-mediated thrombocytopenia. Antibodies directed against platelet membrane glycoprotein IIb-III complexes are detected in some patients' sera. These autoantibodies, which sometimes cross-react with HIV-associated gp120, are believed to act as opsonins, thus promoting the phagocytosis of platelets by splenic phagocytes. Some studies also implicate nonspecific deposition of immune complexes on platelets as a factor in their premature destruction by the mononuclear phagocyte system. Thrombotic Microangiopathies: Thrombotic Thrombocytopenic Purpura (TTP) and Hemolytic-Uremic Syndrome (HUS) The term thrombotic microangiopathy encompasses a spectrum of clinical syndromes that includes TTP and HUS. TTP, as originally defined, is associated with the pentad of fever, thrombocytopenia, microangiopathic hemolytic anemia, transient neurologic deficits, and renal failure. HUS is also associated with microangiopathic hemolytic anemia and thrombocytopenia but is distinguished from TTP by the absence of neurologic symptoms, the prominence of acute renal failure, and frequent affliction of children. Recent studies, however, have tended to blur these clinical distinctions. Many adult patients with "TTP" lack one or more of the five criteria, and some patients with "HUS" have fever and neurologic dysfunction. The common fundamental feature in both of these conditions is widespread formation of hyaline thrombi, comprised primarily of platelet aggregates, in the 653 microcirculation. Consumption of platelets leads to thrombocytopenia, and the intravascular thrombi provide a likely mechanism for the microangiopathic hemolytic anemia and widespread organ dysfunction. It is believed the varied clinical manifestations of TTP and HUS are related to differing proclivities for thrombus formation in specific microvascular beds. For many years, the pathogenesis of TTP was enigmatic, although treatment with plasma exchange (initiated in the early 1970s) changed an almost uniformly fatal condition into one that is successfully treated in more than 80% of cases. Recently, the underlying cause of many, but not all, cases of TTP has been elucidated. In brief, symptomatic patients are often deficient in an enzyme called ADAMTS 13. This enzyme is designated "vWF metalloprotease" and it normally degrades very high molecular weight multimers of von Willebrand factor (vWF). (ADAMTS 13 is unrelated to the other tissue metalloproteases that cleave extracellular matrix.) In the absence of this enzyme, very high molecular weight multimers of vWF accumulate in plasma and, under some circumstances, promote platelet microaggregate formation throughout the microcirculation, leading to the symptoms of TTP. Superimposition of endothelial cell injury (caused by some other condition) may further predispose a patient to microaggregate formation, thus initiating or exacerbating clinically evident TTP. The deficiency of ADAMTS 13 may be inherited or acquired. In many patients an antibody that inhibits vWF metalloprotease is detected. Much less commonly the patients have inherited an inactivating mutation in the gene encoding this enzyme. Despite these advances, it is clear that factors other than vWF metalloprotease deficiency must be involved in triggering full-blown TTP, because symptoms are episodic even in those with hereditary deficiency of vWF metalloprotease. It is important to consider the possibility of TTP in any patient presenting with thrombocytopenia and microangiopathic hemolytic anemia, as any delay in diagnosis and treatment can be fatal. Plasma exchange can be life saving by providing the missing enzyme. In contrast to TTP, most patients with HUS have normal levels of vWF metalloprotease, indicating that HUS usually has a different pathogenesis.  One important cause of HUS in children and the elderly is infectious gastroenteritis caused by E. coli strain 0157:H7. This strain elaborates a Shiga-like toxin that is absorbed from the inflamed gastrointestinal mucosa. It binds to and damages endothelial cells in the glomerulus and elsewhere, thus initiating platelet activation and aggregation. Affected children present with bloody diarrhea, and a few days later HUS makes its appearance. With appropriate supportive care, affected children often recover completely, but irreversible renal damage and death can occur in more severe cases. HUS can also be seen in adults following exposures that damage endothelial cells (e.g., certain drugs, radiation therapy). The prognosis of adults with HUS is guarded, as it is most often seen in the setting of other chronic, life-threatening conditions. While DIC and thrombotic microangiopathies share features such as microvascular occlusion and microangiopathic hemolytic anemia, they are pathogenetically distinct. In TTP and HUS (unlike DIC), activation of the coagulation cascade is not of primary importance, and hence results of laboratory tests of coagulation, such as PT and PTT, are usually normal. BLEEDING DISORDERS RELATED TO DEFECTIVE PLATELET FUNCTIONS Qualitative defects of platelet function can be congenital or acquired. Several congenital disorders characterized by prolonged bleeding time and normal platelet count have been described. A brief discussion of these rare diseases is warranted by the fact that they provide excellent models for investigating the molecular mechanisms of platelet function. Congenital disorders of platelet function can be classified into three groups on the basis of the specific functional abnormality: (1) defects of adhesion, (2) defects of aggregation, and (3) disorders of platelet secretion (release reaction). • Bleeding resulting from defective adhesion of platelets to subendothelial matrix is best illustrated by the autosomal recessive disorder Bernard-Soulier syndrome, which is caused by an inherited deficiency of the platelet membrane glycoprotein complex Ib-IX. This glycoprotein is a receptor for vWF and is essential for normal platelet adhesion to subendothelial matrix ( Chapter 4 ). • Bleeding due to defective platelet aggregation is exemplified by Glanzmann's thrombasthenia, which is also transmitted as an autosomal recessive trait. Thrombasthenic platelets fail to aggregate in response to adenosine diphosphate (ADP), collagen, epinephrine, or thrombin owing to deficiency or dysfunction of glycoprotein IIb-IIIa, a protein complex that participates in the formation of "bridges" between platelets by binding fibrinogen and vWF. • Disorders of platelet secretion are characterized by normal initial aggregation with collagen or ADP, but subsequent responses, such as secretion of thromboxanes and release of granule-bound ADP, are impaired. The underlying biochemical defects of these so-called storage pool disorders are varied, complex, and beyond the scope of our discussion. Among the acquired defects of platelet function, two are clinically significant. The first is ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, which significantly prolongs the bleeding time. Aspirin is a potent, irreversible inhibitor of the enzyme cyclooxygenase, which is required for the synthesis of thromboxane A2 and prostaglandins ( Chapter 2 ). These mediators play important roles in platelet aggregation and subsequent release reactions ( Chapter 4 ). The antiplatelet effects of aspirin form the basis for its use in the prophylaxis of thrombosis ( Chapter 12 ). Uremia ( Chapter 20 ) is the second condition exemplifying an acquired defect in platelet function. Although the pathogenesis of bleeding in uremia is complex and not fully understood, several abnormalities of platelet function are found. HEMORRHAGIC DIATHESES RELATED TO ABNORMALITIES IN CLOTTING FACTORS A deficiency of every clotting factor has been reported to be the cause of a bleeding disorder, with the exception of factor XII deficiency, which does not cause bleeding. The bleeding in factor deficiencies differs from platelet deficiencies in that spontaneous petechiae or purpura are uncommon. Rather, the bleeding is manifested by large post- traumatic ecchymoses or hematomas, or prolonged bleeding after a laceration or any form of surgical procedure. Bleeding into the gastrointestinal and urinary tracts, and particularly into weight-bearing joints, is common. Typical stories include the patient who continues to 654 ooze for days after a tooth extraction or who develops a hemarthrosis after relatively trivial stress on a knee joint. The course of history may have been changed by a hereditary coagulation defect present in the intermarried royal families of Great Britain and other parts of Europe. Clotting abnormalities can also be acquired in many different conditions. Acquired disorders are usually characterized by multiple clotting abnormalities. Vitamin K deficiency ( Chapter 9 ) results in impaired synthesis of factors II, VII, IX, and X and protein C. Since the liver makes virtually all the clotting factors, severe parenchymal liver disease can be associated with a hemorrhagic diathesis. Disseminated intravascular coagulation produces a deficiency of multiple coagulation factors. Hereditary deficiencies have been identified for each of the clotting factors. Deficiencies of factor VIII (hemophilia A) and of factor IX (Christmas disease, or hemophilia B) are transmitted as sex-linked recessive disorders. Most others follow autosomal patterns of transmission. These hereditary disorders typically involve a single clotting factor. Deficiencies of Factor VIII-vWF Complex Hemophilia A and von Willebrand disease, two of the most common inherited disorders of bleeding, are caused by qualitative or quantitative defects involving the factor VIII- vWF complex. Before we can discuss these disorders, it is essential to review the structure and function of these proteins.  Plasma factor VIII-vWF is a complex made up of two separate proteins (factor VIII and vWF) that can be characterized according to functional, biochemical, and immunologic criteria. Factor VIII procoagulant protein, or factor VIII ( Fig. 13-28 ; Figure 13-28 Structure and function of factor VIII-von Willebrand factor (vWF) complex. Factor VIII is synthesized in the liver and kidney, and vWF is made in endothelial cells and megakaryocytes. The two associate to form a complex in the circulation. vWF is also present in the subendothelial matrix of normal blood vessels and the alpha granules of platelets. Following endothelial injury, exposure of subendothelial vWF causes adhesion of platelets, primarily via glycoprotein lb platelet receptor. Circulating vWF and vWF released from the alpha granules of activated platelets can bind exposed subendothelial matrix, further contributing to platelet adhesion and activation. Activated platelets form hemostatic aggregates; fibrinogen (and possibly vWF) participate in aggregation through bridging interactions with the platelet receptor GpIIb/III. Factor VIII takes part in the coagulation cascade as a cofactor in the activation of factor X on the surface of activated platelets. also see Chapter 4 ), is an intrinsic pathway component required for activation of factor X. Deficiency of factor VIII gives rise to hemophilia A. Circulating factor VIII is noncovalently associated with very large vWF multimers containing up to 100 subunits; the molecular mass of individual multimers can exceed 20 × 106 daltons. vWF also interacts with several other proteins involved in hemostasis, including collagen, heparin, and platelet membrane glycoproteins (Ib-IX and IIb-IIIa). Glycoprotein Ib-IX serves as the major receptor for vWF. The most important function of vWF in vivo is to promote the adhesion of platelets to subendothelial matrix, which is accomplished in two ways. Some vWF secreted by endothelial cells is normally deposited in the subendothelial matrix, where it promotes platelet adhesion should the endothelial lining be disrupted (see Fig. 13-28 ). Endothelial cells and platelets also release vWF into the circulation, and upon vascular injury this second pool of vWF is adsorbed to exposed subendothelial matrix and further augments platelet adhesion. vWF multimers can also promote platelet aggregation by binding to activated GpIIb/IIIa receptors; this activity may be of particular importance under conditions of high shear stress (such as occurs in small vessels). That vWF is crucial to the normal process of hemostasis ( Chapter 4 ) is supported by the occurrence of a bleeding diathesis known as von Willebrand disease when there is deficiency of this factor. vWF multimers also serve as a carrier for factor VIII and are important for its stability. The half-life of factor VIII in the circulation is 12 hours if vWF levels are normal but only 2.4 hours if it is deficient or abnormal (as in patients with von Willebrand disease). vWF can be assayed by immunologic techniques or by the so-called ristocetin agglutination test. This assay, which can be 655 performed with formalin-fixed platelets, measures the ability of ristocetin (developed as an antibiotic) to promote the interaction between vWF and platelet membrane glycoprotein Ib. Multivalent ristocetin-dependent binding of vWF creates interplatelet "bridges," leading to the formation of platelet clumps (agglutination), an event easily measured in a device called an aggregometer. Thus, the degree of ristocetin-dependent platelet agglutination caused by the addition of patient plasma provides a bioassay for vWF. The two components of the factor VIII-vWF complex are encoded by separate genes and synthesized in different cells. vWF is produced by endothelial cells and megakaryocytes and can be demonstrated in platelet α-granules. Endothelial cells are the major source of subendothelial and plasma vWF. Factor VIII is made in several tissues; sinusoidal endothelial cells and Kupffer cells in the liver and glomerular and tubular epithelial cells in the kidney appear to be particularly important sites of synthesis. To summarize, the two components of factor VIII-vWF complex, synthesized separately, come together and circulate in the plasma as a unit that serves to promote clotting as well as platelet-vessel wall interactions necessary to ensure hemostasis. With this background, we can discuss the diseases resulting from deficiencies of factor VIII-vWF complex. Von Willebrand Disease With an estimated frequency of 1%, von Willebrand disease is believed to be one of the most common inherited disorders of bleeding in humans. Clinically, it is characterized by spontaneous bleeding from mucous membranes, excessive bleeding from wounds, menorrhagia, and a prolonged bleeding time in the presence of a normal platelet count. In most cases, it is transmitted as an autosomal dominant disorder, but several rare autosomal recessive variants have been identified. More than 20 variants of von Willebrand disease have been described, which can be grouped into two major categories: • Type 1 and type 3 von Willebrand disease are associated with a reduced quantity of circulating vWF. Type 1, an autosomal dominant disorder, accounts for approximately 70% of all cases and is relatively mild. Reduced penetrance and variable expressivity characterize this type, and hence clinical manifestations are varied. Type 3 (an autosomal recessive disorder) is associated with extremely low levels of functional vWF, and the clinical manifestations are correspondingly severe. Because a severe deficiency of vWF has a marked affect on the stability of factor VIII, some of the bleeding characteristics resemble those seen in hemophilia. The nature of the mutations in the vast majority of patients with type 1 disease is poorly defined. In some cases missense mutations have been found. In others, it is suspected that both alleles are affected by distinct mutations (compound heterozygotes) producing an apparent dominant inheritance. Type 3 disease is associated with deletions or frameshift mutations. • Type 2 von Willebrand disease is characterized by qualitative defects in vWF; there are several subtypes, of which type 2A is the most common. It is inherited as an autosomal dominant disorder. Because of missense mutations, the vWF formed is abnormal, leading to defective multimer assembly. Large and intermediate multimers, representing the most active forms of vWF, are missing from plasma. Type 2 von Willebrand disease accounts for 25% of all cases and is associated with mild to moderate bleeding. Patients with von Willebrand disease have prolonged bleeding time despite a normal platelet count. The plasma level of active vWF, measured as the ristocetin cofactor activity, is reduced. Because vWF stabilizes factor VIII by binding to it, a deficiency of vWF gives rise to a secondary decrease in factor VIII levels. This may be reflected by a prolongation of the PTT in von Willebrand disease types 1 and 3. To summarize, patients with von Willebrand disease have a compound defect involving platelet function and the coagulation pathway. Even within families in which a single defective allele is segregating, there is often wide variability in the clinical expression of von Willebrand disease. This appears to be due to additional genetic factors that influence circulating levels of vWF, which vary greatly in normal populations. However, except in the most severely affected type 3 patients, adverse complications of factor VIII deficiency, such as bleeding into the joints, are uncommon. Hemophilia A (Factor VIII Deficiency) Hemophilia A is the most common hereditary disease associated with serious bleeding. It is caused by a reduction in the amount or activity of factor VIII. This protein serves as a cofactor for factor IX in the activation of factor X in the coagulation cascade ( Chapter 4 ). Hemophilia A is inherited as an X-linked recessive trait, and thus occurs in males and in homozygous females. However, excessive bleeding has been described in heterozygous females, presumably due to extremely unfavorable lyonization (inactivation of the normal X chromosome in most of the cells). Approximately 30% of patients have no family history; their disease is presumably caused by new mutations. Hemophilia A exhibits a wide range of clinical severity that correlates well with the level of factor VIII activity. Those with less than 1% of normal activity develop severe disease; levels between 2% and 5% of normal are associated with moderate disease; and patients with 6% to 50% of activity develop mild disease. The variable degrees of factor VIII deficiency are largely explained by heterogeneity in the causative mutations. As with β- thalassemias, several genetic lesions (deletions, nonsense mutations that create stop codons, splicing errors) have been documented. Most severe deficiencies result from an unusual inversion involving the X chromosome that completely abolishes the synthesis of factor VIII. Less commonly, severe hemophilia A is associated with point mutations in factor VIII that impair the function of the protein. In such cases, levels of factor VIII appear normal by immunoassay. Mutations permitting some active factor VIII to be synthesized are associated with mild to moderate disease. The disease in such patients may be modified by other genetic factors that influence factor VIII expression levels, which vary widely in normal individuals. In all symptomatic cases, there is a tendency toward easy bruising and massive hemorrhage after trauma or operative procedures. In addition, "spontaneous" hemorrhages frequently occur in regions of the body normally subject to trauma, particularly the joints, where they are known as hemarthroses. Recurrent bleeding into the joints leads to progressive 656 deformities that can be crippling. Petechiae are characteristically absent. Patients with hemophilia A typically have a normal bleeding time, platelet count, and PT, and a prolonged PTT. These tests point to an abnormality of the intrinsic coagulation pathway. Factor VIII-specific assays are required for diagnosis. Given that one arm of the coagulation cascade, the extrinsic pathway, is intact in hemophilia A, it seems reasonable to ask, why do these patients bleed? Obviously, test tube assays of coagulation (discussed briefly below under DIC) are imperfect surrogates for what occurs in vivo, and it must be that in the face of factor VIII deficiency, fibrin deposition is inadequate to achieve hemostasis reliably. It is beyond our scope to discuss this issue in detail, but recent studies suggest the following. First, it appears that the chief role of the extrinsic pathway in hemostasis is to produce a limited initial burst of thrombin activation upon tissue injury. This is reinforced and amplified by a critical feedback loop whereby thrombin activates factors XI and IX of the intrinsic pathway. In addition, high levels of thrombin are required to activate TAFI (thrombin activatable fibrinolysis inhibitor), a factor that augments fibrin deposition by inhibiting fibrinolysis. Thus, both inadequate coagulation (fibrinogenesis) and inappropriate clot removal (fibrinolysis) contribute to the bleeding diathesis in hemophilia. The precise explanation for the tendency of hemophiliacs to bleed at particular sites (joints, muscles, and the central nervous system) remains uncertain. Treatment of hemophilia A involves infusion of recombinant factor VIII. Approximately 15% of patients with low or absent factor VIII develop antibodies that bind to and inhibit factor VIII. Inhibitors are most likely to develop in patients with severe factor VIII deficiency (possibly because the protein is perceived as foreign, having never been "seen" before by the immune system) and represent very difficult therapeutic challenges. There are other hazards of replacement therapy as well, the most serious of which has been the risk of transmission of viral diseases. Until the mid-1980s, before routine screening of blood for HIV antibodies was instituted, thousands of hemophiliacs received plasma- derived factor VIII concentrates containing HIV, and many developed AIDS ( Chapter 6 ). With the availability of recombinant factor VIII, the risk of HIV transmission has been eliminated, but tragically too late for an entire generation of hemophiliacs. Efforts to develop somatic gene therapy for hemophilia are also under way. Hemophilia B (Christmas Disease, Factor IX Deficiency) Severe factor IX deficiency produces a disorder clinically indistinguishable from factor VIII deficiency (hemophilia A). This should not be surprising, given that factor VIII and IX function together to activate factor X. A wide spectrum of mutations involving the factor IX gene are found in hemophilia B. Like hemophilia A, it is inherited as an X- linked recessive trait and shows variable clinical severity. In about 14% of these patients, factor IX is present but nonfunctional. As with hemophilia A, the PTT is prolonged and the PT is normal, as is the bleeding time. Identification of Christmas disease (named after the first patient with this condition and not the holiday) is possible only by assay of the factor levels. Recombinant factor IX is used for treatment. DISSEMINATED INTRAVASCULAR COAGULATION (DIC) DIC is an acute, subacute, or chronic thrombohemorrhagic disorder occurring as a secondary complication in a variety of diseases. It is characterized by activation of the coagulation sequence that leads to the formation of microthrombi throughout the microcirculation of the body, often in a quixotically uneven distribution. Sometimes the coagulopathy is localized to a specific organ or tissue. As a consequence of the thrombotic diathesis, there is consumption of platelets, fibrin, and coagulation factors and, secondarily, activation of fibrinolytic mechanisms. Thus, DIC can present with signs and symptoms relating to tissue hypoxia and infarction caused by the myriad microthrombi or as a hemorrhagic disorder related to depletion of the elements required for hemostasis (hence, the term "consumption coagulopathy" is sometimes used to describe DIC). Activation of the fibrinolytic mechanism aggravates the hemorrhagic diathesis. Etiology and Pathogenesis. At the outset, it must be emphasized that DIC is not a primary disease. It is a coagulopathy that occurs in the course of a variety of clinical conditions. In discussing the general mechanisms underlying DIC, it is useful to briefly review the normal process of blood coagulation and clot removal. Clotting can be initiated by either of two pathways: (1) the extrinsic pathway, which is triggered by the release of tissue factor ("tissue thromboplastin"), and (2) the intrinsic pathway, which involves the activation of factor XII by surface contact with collagen or other negatively charged substances. Both pathways, through a series of intermediate steps, result in the generation of thrombin, which in turn converts fibrinogen to fibrin. Once activated at the site of injury, thrombin further augments local fibrin deposition through feedback activation of the intrinsic pathway and inhibition of fibrinolysis. Remarkably, as excess thrombin is swept away in the blood from sites of tissue injury it is converted to an anticoagulant. Upon binding a surface protein called thrombomodulin on intact endothelial cells, thrombin becomes capable of activating protein C, an inhibitor of the pro-coagulant factors V and VIII. Other important clot-inhibiting factors include the activation of fibrinolysis by plasmin and the clearance of activated clotting factors by the mononuclear phagocyte system and the liver. These and additional checks and balances normally ensure that just enough clotting occurs at the right place and time. From this brief review, it should be clear that DIC could result from pathologic activation of the extrinsic and/or intrinsic pathways of coagulation or impairment of clot-inhibiting influences. Since the latter rarely constitute primary mechanisms of DIC, we focus our attention on the abnormal initiation of clotting.  Two major mechanisms trigger DIC: (1) release of tissue factor or thromboplastic substances into the circulation and (2) widespread injury to the endothelial cells. Tissue thromboplastic substances can be derived from a variety of sources, such as the placenta in obstetric complications ( Table 13-10 ) and the granules of leukemic cells in acute promyelocytic leukemia. Mucus released from certain adenocarcinomas can also act as a thromboplastic substance by directly activating factor X, independent of factor VII. In gram-negative sepsis (an important cause of DIC), bacterial endotoxins cause activated monocytes to release interleukin-1 and TNF, both of which 657 TABLE 13-10 -- Major Disorders Associated with Disseminated Intravascular Coagulation Obstetric Complications Abruptio placentae Retained dead fetus Septic abortion Amniotic fluid embolism Toxemia Infections Gram-negative sepsis Meningococcemia Rocky Mountain spotted fever Histoplasmosis Aspergillosis Malaria Neoplasms Carcinomas of pancreas, prostate, lung, and stomach Acute promyelocytic leukemia Massive Tissue Injury Traumatic Burns Extensive surgery Miscellaneous Acute intravascular hemolysis, snakebite, giant hemangioma, shock, heat stroke, vasculitis, aortic aneurysm, liver disease increase the expression of tissue factor on endothelial cell membranes and simultaneously decrease the expression of thrombomodulin. The net result is a shift in balance toward procoagulation. Endothelial injury, the other major trigger, can initiate DIC by causing release of tissue factor, promoting platelet aggregation, and activating the intrinsic coagulation pathway. TNF is an extremely important mediator of endothelial cell inflammation and injury in septic shock. In addition to the effects previously mentioned, TNF up-regulates the expression of adhesion molecules on endothelial cells and thus favors adhesion of leukocytes, which in turn damage endothelial cells by releasing oxygen-derived free radicals and preformed proteases. Even subtle endothelial injury can unleash procoagulant activity by enhancing membrane expression of tissue factor. Widespread endothelial injury may be produced by deposition of antigen-antibody complexes (e.g., systemic lupus erythematosus), temperature extremes (e.g., heat stroke, burns), or microorganisms (e.g., meningococci, rickettsiae). Several disorders associated with DIC are listed in Table 13-10 . Of these, DIC is most likely to follow obstetric complications, malignant neoplasia, sepsis, and major trauma. The initiating factors in these conditions are often multiple and interrelated. For example, particularly in infections caused by gram-negative bacteria, released endotoxins can activate both the intrinsic and extrinsic pathways by producing endothelial cell injury and release of thromboplastins from inflammatory cells; furthermore, endotoxins inhibit the anticoagulant activity of protein C by suppressing thrombomodulin expression on endothelium. Endothelial cell damage can also be produced directly by meningococci, rickettsiae, and viruses. Antigen-antibody complexes formed during the infection can activate the classical complement pathway, and complement fragments can secondarily activate both platelets and granulocytes. Endotoxins as well as other bacterial products are also capable of directly activating factor XII. In massive trauma, extensive surgery, and severe burns, the major mechanism of DIC is believed to be the release of tissue thromboplastins. In obstetric conditions, thromboplastins derived from the placenta, dead retained fetus, or amniotic fluid may enter the circulation. However, hypoxia, acidosis, and shock, which often coexist with the surgical and obstetric conditions, also cause widespread endothelial injury. Supervening infection can complicate the problems further. Among cancers, acute promyelocytic leukemia and carcinomas of the lung, pancreas, colon, and stomach are most frequently associated with DIC. These tumors release of a variety of thromboplastic substances, including tissue factors, proteolytic enzymes, mucin, and other undefined tumor products. The consequences of DIC are twofold. First, there is widespread deposition of fibrin within the microcirculation. This can lead to ischemia of the more severely affected or more vulnerable organs and to a hemolytic anemia resulting from fragmentation of red cells as they squeeze through the narrowed microvasculature (microangiopathic hemolytic anemia). Second, a hemorrhagic diathesis can dominate the clinical picture. This results from consumption of platelets and clotting factors as well as activation of plasminogen. Plasmin can not only cleave fibrin, but also digest factors V and VIII, thereby reducing their concentration further. In addition, fibrinolysis leads to the formation of fibrin degradation products, which inhibit platelet aggregation and fibrin polymerization and have antithrombin activity. All these influences lead to the hemostatic failure seen in DIC ( Fig. 13-29 ). Morphology. In general, thrombi are found in the following sites in decreasing order of frequency: brain, heart, lungs, kidneys, adrenals, spleen, and liver. However, no tissue is spared, and thrombi are occasionally found in only one or several organs without affecting others. In giant hemangiomas, for example, thrombi are localized to the neoplasm, where they are believed to form due to local stasis and recurrent trauma to fragile blood vessels. The affected kidneys can reveal small thrombi in the glomeruli that may evoke only reactive swelling of endothelial cells or, in severe cases, microinfarcts or even bilateral renal cortical necrosis. Numerous fibrin thrombi may be found in alveolar capillaries, sometimes associated with pulmonary edema and fibrin exudation, creating "hyaline membranes" reminiscent of acute respiratory distress syndrome ( Chapter 15 ). In the central nervous system, fibrin thrombi can cause microinfarcts, occasionally complicated by simultaneous hemorrhage. Such changes are the basis for the bizarre neurologic signs and symptoms sometimes observed in DIC. The manifestations of DIC in the endocrine glands are of considerable interest. In meningococcemia, fibrin thrombi within the microcirculation of the adrenal cortex are the likely basis for the massive adrenal hemorrhages seen in Waterhouse-Friderichsen syndrome ( Chapter 24 ). Similarly, Sheehan postpartum pituitary necrosis ( Chapter 24 ) is a form of DIC complicating labor and delivery. In toxemia of pregnancy ( Chapter 22 ), the placenta exhibits widespread microthrombi, providing a plausible 658 Figure 13-29 Pathophysiology of disseminated intravascular coagulation. explanation for the premature atrophy of the cytotrophoblast and syncytiotrophoblast encountered in this condition. The bleeding manifestations of DIC are not dissimilar to those encountered in the hereditary and acquired disorders affecting the hemostatic mechanisms discussed earlier. Clinical Course. The onset can be fulminant, as in endotoxic shock or amniotic fluid embolism, or insidious and chronic, as in cases of carcinomatosis or retention of a dead fetus. Overall, about 50% of individuals with DIC are obstetric patients having complications of pregnancy. In this setting, the disorder tends to be reversible with delivery of the fetus. About 33% of the patients have carcinomatosis. The remaining cases are associated with the various entities previously listed. It is almost impossible to detail all the potential clinical presentations, but a few common patterns are worthy of description. These include microangiopathic hemolytic anemia; dyspnea, cyanosis, and respiratory failure; convulsions and coma; oliguria and acute renal failure; and sudden or progressive circulatory failure and shock. In general, acute DIC, associated with obstetric complications or major trauma, for example, is dominated by a bleeding diathesis, whereas chronic DIC, such as occurs in cancer patients, tends to present initially with thrombotic complications. Accurate clinical observation and laboratory studies are necessary for the diagnosis. It is usually necessary to monitor fibrinogen, platelets, PT, PTT, and fibrin degradation products. The prognosis is highly variable and depends, to a considerable extent, on the underlying disorder. The management of these cases requires meticulous maneuvering between the Scylla of thrombosis and the Charybdis of bleeding diathesis. Administration of anticoagulants or procoagulants has been advocated in specific settings, but not without controversy. The only definitive treatment is to remove or treat the inciting cause whenever possible. References 1. Dzierzak E: Embryonic beginnings of definitive hematopoietic stem cells. Ann NY Acad Sci 872:256, 1999. 2. Kondo M, et al: Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91:661, 1997. 3. AkashiK, et al: A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193, 2000. SH, Zon LL: Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nature 4. Orkin Immunol 3:323–328, 2002. 5. Clarke D, Frisen J: Differentiation potential of adult stem cells. Curr Opin Genet Dev 11:575, 2001. 6. Vogel G: Stem cell policy. Can adult stem cells suffice? Science 292:1820, 2001. 7. Wang X, et al: Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422:897– 901, 2003. 8. Delaunay J: Molecular basis of red cell membrane disorders. Acta Haematol 108:210, 2002. 9. EberSW, et al: Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis. Nat Genet 13:214, 1996. 10. JarolimP, et al: Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency. Blood 88:4366, 1996. 11. Jandl J, et al: Red cell filtration and the pathogenesis of certain hemolytic anemias. Blood 18:33, 1961. 12. MehtaA, Mason PJ, Vulliamy TJ: Glucose-6-phosphate dehydrogenase deficiency. Bailliers Best Pract Res Clin Hematol 13:21, 2000. 13. Tishkoff SA, et al: Haplotype diversity and linkage disequilibrium at human G6PD: recent origin of alleles that confer malarial resistance. Science 293:455, 2001. 14. Gomez-GallegoF, et al: Structural defects underlying protein dysfunction in human glucose-6- phosphate dehydrogenase A(-) deficiency. J Biol Chem 275:9256, 2000. M, et al: Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 15. Aidoo 359:1311, 2002. 16. Brugnara C, et al: Erythrocyte-active agents and treatment of sickle cell disease. Semin Hematol 38:324, 2001. 17. Hebbel RP: Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest 100:S83, 1997. 659 ST, et al: Prediction of adverse outcomes in children with sickle cell disease. N Engl J Med 18. Miller 342:83, 2000. 19. Zachlederora M, Jarolim, P: Gene expression profile of microvascular endothelial cells after stimuli implicated in pathogenesis of vaso-occlusion. Blood Cell Molecules Dis 30:71, 2003. 20. Frenette PS: Sickle cell vaso-occlusion: multistep and multicellular paradigm. Curr Opin Hematol 9:101, 2002. 21. Liao JC: Blood feud: keeping hemoglobin from nixing NO. Nature Med 8:1350, 2002. 22. Ballas SK: Sickle cell disease: current clinical management. Semin Hematol 38:307, 2001. 23. Smith J: Bone disorders in sickle cell disease. Hematol Oncol Clin North Am 10:1345, 1996. 24. Platt OS: The acute chest syndrome of sickle cell disease. N Engl J Med 342:1904, 2000. 25. Bunn HF: Pathogenesis and treatment of sickle cell disease. N Engl J Med 337:762, 1997. 26. DaviesSC, Gilmore A: The role of hydroxyurea in the management of sickle cell disease. Blood Rev 17:99, 2003. A, et al: Five years of experience with hydroxyurea in children and young adults with sickle cell 27. Ferster disease. Blood 97:3628, 2001. 28. Olivieri NF: The β-thalassemias. N Engl J Med 341:99, 1999. D, Rachmilewitz E: Pathophysiology of α- and β-thalassemia: therapeutic implications. Semin 29. Rund Hematol 38:343, 2001. 30. Rosse WF: New insights into paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol 8:61, 2001. 31. WrightMS: Drug-induced hemolytic anemias: increasing complications to therapeutic interventions. Clin Lab Sci 12:115, 1999. 32. Gehrs BC, Friedberg RC: Autoimmune hemolytic anemia. Am J Hematol 69:258, 2002. 33. Oh RC, Brown DL: Vitamin B12 deficiency. Am Fam Physican 67:979, 2003. 34. Hoffbrand AV, Jackson BF: Correction of the DNA synthesis defect in vitamin B12 deficiency by tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic anaemia in vitamin B12 deficiency. Br J Haematol 83:643, 1993. 35. Chanarin I, et al: Cobalamin and folate: recent developments. J Clin Pathol 45:277, 1992. 36. Wickramasinghe SN: The wide spectrum and unresolved issues of megaloblastic anemia. Semin Hematol 36:3, 1999. MI, et al: Varying neurological phenotypes among mut o and mut- patients with 37. Shevell methylmalonylCoA mutase deficiency. Am J Med Genet 45:619, 1993. 38. Toh BH, et al: Pernicious anemia. N Engl J Med 337:1441, 1997. 39. Looker AC, et al: Prevalence of iron deficiency in the United States. JAMA 277:973, 1997. 40. Andrews, NC: A genetic view of iron homeostasis. Semin Hematol 39:227, 2002. 41. FlemingRE, Sly WS: Mechanisms of iron accumulation in hereditary hemochromatosis. Annu Rev Physiol 64:663, 2002. 42. GanzT: Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 102:783, 2003. 43. MeansRT, Jr: Erythropoietin in the treatment of anemia in chronic infectious, inflammatory, and malignant diseases. Curr Opin Hematol 2:210, 1995. 44. Spivak J: Iron and anemia of chronic disease. Oncology (Huntingt) 16 (Suppl 10):25, 2002. 45. Dokal I: Inherited aplastic anemia. Hematol J 4:3, 2003. 46. Young NS: Acquired aplastic anemia. Ann Intern Med 136:534, 2002. 47. Erslev AJ, Soltan A: Pure red-cell aplasia: a review. Blood Rev 10:20, 1996. 48. Eschbach JW: Current concepts of anemia management in chronic renal failure. Semin Nephrol 20:320, 2000. 49. Gregg XT, Prchal JT: Erythropoietin receptor mutations and human disease. Semin Hematol 34:70, 1997. 50. Longmore GD: Erythropoietin receptor mutations and Olympic glory. Nat Genet 4:108, 1993. 51. Rand ML, Leung R, Packham MA: Platelet function assays. Trans Apher Sci 28:307, 2003. 52. Bussel JB: Alloimmune thrombocytopenia in the fetus and newborn. Semin Thromb Hemost 27:245, 2001. 53. Cines DB, Blanchette VS: Immune thrombocytopenic purpura. N Engl J Med 346:995, 2002. 54. Aster RH: Drug-induced immune thrombocytopenia: an overview of pathogenesis. Semin Hematol 36:2, 1999. 55. Visentin GP, et al: Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/thrombosis. J Lab Clin Med 138:22, 2001. 56. Scaradavou A: HIV-related thrombocytopenia. Blood Rev 16:73, 2002. HM: Deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura. Arterioscler Thromb 57. Tsai Vasc Biol 23:388, 2003. 58. LevyGG, et al: Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413:488, 2001. 59. MoakeJL: Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. Arch Pathol Lab Med 126:1430, 2002. 60. Zoja C, et al: The role of the endothelium in hemolytic uremic syndrome. J Nephrol 14(Suppl 4):S58, 2001. AK: Congenital disorders of platelet function: disorders of signal transduction and secretion. Am J 61. Rao Med Sci 316:69, 1998. 62. BickRL: Platelet function defects associated with hemorrhage or thrombosis. Med Clin North Am 78:577, 1994. PJ, et al: The life cycle of coagulation factor VIII in view of its structure and function. Blood 63. Lenting 92:3983, 1998. 64. RuggeriZM: Structure of von Willebrand factor and its function in platelet adhesion and thrombus formation. Best Pract Res Clin Haematol 14:257, 2001. 65. Sadler JE, et al: Impact, diagnosis and treatment of von Willebrand disease. Thromb Haemost 84:160, 2000. 66. Castman G, et al: von Willebrand disease in year 2003: towards the complete identification of gene defects for correct diagnosis and treatment. Hematologica 88:94, 2003. 67. LevyG, Ginsburg D: Getting at the variable expressivity of von Willebrand disease. Thromb Haemost 86:144, 2001. 68. Bowen DJ: Haemophilia A and haemophilia B: molecular insights. Mol Pathol 55:1, 2002. 69. LensenR, et al: High factor VIII levels contribute to the thrombotic risk in families with factor V Leiden. Br J Haematol 114:380, 2001. 70. Mosnier LO, et al: The defective down-regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration. Thromb Haemost 86:1035, 2001. RL: Disseminated intravascular coagulation: a review of etiology, pathophysiology, diagnosis, and 71. Bick management: guidelines for care. Clin Appl Thromb Hemost 8:1, 2002. 72. Esmon CT, et al: Inflammation, sepsis, and coagulation. Haematologica 84:254, 1999.