RBC Disorders PDF

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R Red Blood Cell and Bleeding Disorders 14...

See TARGETED THERAPY available online at www.studentconsult.com C H A P T E R Red Blood Cell and Bleeding Disorders 14 CHAPTER CONTENTS Anemias 635 Iron Deficiency Anemia 655 Bleeding Disorders Related to Defective Anemias of Blood Loss 636 Anemia of Chronic Inflammation 658 Platelet Function 666 Acute Blood Loss 636 Aplastic Anemia 659 Hemorrhagic Diatheses Related to Chronic Blood Loss 637 Pure Red Cell Aplasia 661 Abnormalities in Clotting Factors 667 Hemolytic Anemias 637 Other Forms of Marrow Failure 661 Factor VIII–vWF Complex 667 Hereditary Spherocytosis 638 Polycythemia 662 Von Willebrand Disease 668 Hemolytic Disease Due to Red Cell Enzyme Bleeding Disorders: Hemorrhagic Hemophilia A (Factor VIII Deficiency) 668 Defects: Glucose-6-Phosphate Dehydrogenase Diatheses 662 Hemophilia B (Christmas Disease, Factor IX Deficiency 640 Bleeding Disorders Caused by Vessel Wall Deficiency) 669 Sickle Cell Disease 641 Abnormalities 663 Disseminated Intravascular Coagulation Thalassemia 644 Bleeding Related to Reduced Platelet (DIC) 669 Paroxysmal Nocturnal Hemoglobinuria 648 Number: Thrombocytopenia 663 Complications of Transfusion 671 Immunohemolytic Anemia 649 Chronic Immune Thrombocytopenic Purpura 664 Allergic Reactions 671 Hemolytic Anemia Resulting From Trauma to Acute Immune Thrombocytopenic Purpura 665 Hemolytic Reactions 671 Red Cells 650 Drug-Induced Thrombocytopenia 665 Transfusion-Related Acute Lung Injury 672 Anemias of Diminished HIV-Associated Thrombocytopenia 665 Infectious Complications 672 Erythropoiesis 651 Thrombotic Microangiopathies: Thrombotic Megaloblastic Anemia 651 Thrombocytopenic Purpura (TTP) and Anemia of Folate Deficiency 654 Hemolytic Uremic Syndrome (HUS) 665 In this chapter, we will first consider diseases of red cells. which often point to particular causes. Morphologic char- By far, the most common and important are the anemias, acteristics that provide etiologic clues include red cell size red cell deficiency states that usually have a nonneoplastic (normocytic, microcytic, or macrocytic); degree of hemo- basis. We will then complete our review of blood diseases globinization, reflected in the color of red cells (normochromic by discussing the major bleeding disorders and complications or hypochromic); and shape. Microcytic hypochromic of blood transfusion. anemias are caused by disorders of hemoglobin synthesis, and macrocytic anemias often stem from abnormalities that impair the maturation of erythroid precursors in the bone ANEMIAS marrow. Normochromic, normocytic anemias have diverse etiologies; in some of these anemias, characteristic abnormali- Anemia is defined as a reduction of the total circulating ties of red cell shape provide an important clue as to the red cell mass below normal limits. Anemia reduces the cause. Red cell shape is assessed through visual inspection oxygen-carrying capacity of the blood, leading to tissue of peripheral smears, whereas as other red cell indices are hypoxia. In practice, the measurement of red cell mass is not determined in clinical laboratories with special instrumenta- easy, and anemia is usually diagnosed based on a reduction tion. The most useful of these indices are as follows: in the hematocrit (the ratio of packed red cells to total blood Mean cell volume: the average volume of a red cell volume) and the hemoglobin concentration of the blood to levels expressed in femtoliters (fL) that are below the normal range. These values correlate with Mean cell hemoglobin: the average content (mass) of the red cell mass except when there are changes in plasma hemoglobin per red cell, expressed in picograms (pg) volume caused by fluid retention or dehydration. Mean cell hemoglobin concentration: the average concentra- There are many classifications of anemia. We will follow tion of hemoglobin in a given volume of packed red one based on underlying mechanisms that is presented in cells, expressed in grams per deciliter (g/dL) Table 14.1. A second clinically useful approach classifies Red cell distribution width: the coefficient of variation of anemia according to alterations in red cell morphology, red cell volume 635 636 C H A P T E R 14 Red Blood Cell and Bleeding Disorders Table 14.1 Classification of Anemia According to Underlying Mechanism Mechanism Specific Examples Blood Loss Acute blood loss Trauma Chronic blood loss Gastrointestinal tract lesions, gynecologic disturbancesa Increased Red Cell Destruction (Hemolysis) Inherited genetic defects Red cell membrane disorders Hereditary spherocytosis, hereditary elliptocytosis Enzyme deficiencies    Hexose monophosphate shunt enzyme G6PD deficiency, glutathione synthetase deficiency deficiencies    Glycolytic enzyme deficiencies Pyruvate kinase deficiency, hexokinase deficiency Hemoglobin abnormalities Deficient globin synthesis Thalassemia syndromes Structurally abnormal globins (hemoglobinopathies) Sickle cell disease, unstable hemoglobins Acquired genetic defects Deficiency of phosphatidylinositol-linked Paroxysmal nocturnal hemoglobinuria glycoproteins Antibody-mediated destruction Hemolytic disease of the newborn (Rh disease), transfusion reactions, drug-induced, autoimmune disorders Mechanical trauma Microangiopathic hemolytic anemias Hemolytic uremic syndrome, disseminated intravascular coagulation, thrombotic thrombocytopenia purpura Cardiac traumatic hemolysis Defective cardiac valves Repetitive physical trauma Bongo drumming, marathon running, karate chopping Infections of red cells Malaria, babesiosis Toxic or chemical injury Clostridial sepsis, snake venom, lead poisoning Membrane lipid abnormalities Abetalipoproteinemia, severe hepatocellular liver disease Sequestration Hypersplenism Decreased Red Cell Production Inherited genetic defects Defects leading to stem cell depletion Fanconi anemia, telomerase defects Defects affecting erythroblast maturation Thalassemia syndromes Nutritional deficiencies Deficiencies affecting DNA synthesis B12 and folate deficiencies Deficiencies affecting hemoglobin synthesis Iron deficiency Erythropoietin deficiency Renal failure, anemia of chronic inflammation Immune-mediated injury of progenitors Aplastic anemia, pure red cell aplasia Inflammation-mediated iron sequestration Anemia of chronic inflammation Primary hematopoietic neoplasms Acute leukemia, myelodysplastic syndrome, myeloproliferative neoplasms (Chapter 13) Space-occupying marrow lesions Metastatic neoplasms, granulomatous disease Infections of red cell progenitors Parvovirus B19 infection Unknown mechanisms Endocrine disorders, hepatocellular liver disease G6PD, Glucose-6-phosphate dehydrogenase. a Most often anemia stems from iron deficiency, not bleeding per se. Adult reference ranges for red cell indices are shown in develop as a result of renal hypoperfusion. Central nervous Table 14.2. system hypoxia can cause headache, dimness of vision, and Whatever its cause, when sufficiently severe anemia leads faintness. to manifestations related to the diminished hemoglobin and oxygen content of the blood. Patients appear pale and often Anemias of Blood Loss report weakness, malaise, easy fatigability, and dyspnea on mild exertion. Hypoxia can cause fatty change in the Acute Blood Loss liver, myocardium, and kidney. On occasion, myocardial The effects of acute blood loss are mainly due to the loss hypoxia manifests as angina pectoris, particularly when of intravascular volume, which if massive can lead to complicated by pre-existing coronary artery disease. cardiovascular collapse, shock, and death. The clinical With acute blood loss and shock, oliguria and anuria can features depend on the rate of hemorrhage and whether  Anemias 637 Table 14.2 Adult Reference Ranges for Red Cellsa Accumulation of hemoglobin degradation products that are Measurement (Units) Men Women created as part of the process of red cell hemolysis Hemoglobin (g/dL) 13.6–17.2 12.0–15.0 The physiologic destruction of senescent red cells takes Hematocrit (%) 39–49 33–43 place within macrophages, which are abundant in the spleen, Red cell count (×106/µL) 4.3–5.9 3.5–5.0 liver, and bone marrow. This process appears to be triggered Reticulocyte count (%) 0.5–1.5 by age-dependent changes in red cell surface proteins, which Mean cell volume (fL) 82–96 lead to their recognition and removal by phagocytes. In the great majority of hemolytic anemias, the premature destruc- Mean cell hemoglobin (pg) 27–33 tion of red cells also occurs within phagocytes, an event Mean cell hemoglobin 33–37 that is referred to as extravascular hemolysis. If persistent, concentration (g/dL) extravascular hemolysis leads to a hyperplasia of phagocytes Red cell distribution width 11.5–14.5 manifested by varying degrees of splenomegaly. a Reference ranges vary among laboratories. The reference ranges for the Extravascular hemolysis is most commonly caused by laboratory providing the result should always be used in interpreting test results. alterations that make red cells less deformable. Extreme changes in shape are required for red cells to navigate the splenic sinusoids successfully. Reduced deformability makes this passage difficult, leading to red cell sequestration and the bleeding is external or internal. If the patient survives, phagocytosis by macrophages located within the splenic the blood volume is rapidly restored by movement of water cords. Regardless of the cause, the principal clinical features of from the interstitial fluid compartment to the intravascular extravascular hemolysis are anemia, splenomegaly, and jaun- compartment. This fluid shift produces hemodilution and dice. Some hemoglobin inevitably escapes from phagocytes, lowers the hematocrit. The resulting reduction in tissue which leads to variable decreases in plasma haptoglobin, oxygenation triggers increased secretion of erythropoietin an α2-globulin that binds free hemoglobin and prevents from the kidney, which stimulates the proliferation of com- its excretion in the urine. Because much of the premature mitted erythroid progenitors (colony-forming unit–erythroid destruction of red cells occurs in the spleen, individuals with [CFU–E]) in the marrow (see Fig. 13.1). It takes about 5 days extravascular hemolysis often benefit from splenectomy. for the progeny of these CFU–Es to mature and appear as Intravascular hemolysis of red cells may be caused by newly released red cells (reticulocytes) in the peripheral mechanical injury, complement fixation, intracellular blood. The iron in hemoglobin is recaptured if red cells parasites (e.g., falciparum malaria, Chapter 8), or exogenous extravasate into tissues, whereas bleeding into the gut or toxic factors. Compared to extravascular hemolysis, it occurs out of the body leads to iron loss and possible iron deficiency, less commonly; sources of mechanical injury include trauma which can hamper the restoration of normal red cell counts. caused by cardiac valves, narrowing of the microcirculation Significant bleeding results in predictable changes in by thrombi, or repetitive physical trauma (e.g., marathon the blood involving not only red cells, but also white cells running and bongo drum beating). Complement fixation and platelets. If the bleeding is sufficiently massive to cause occurs in a variety of situations in which antibodies recognize a decrease in blood pressure, the compensatory release of and bind red cell antigens. Toxic injury is exemplified by adrenergic hormones mobilizes granulocytes from the clostridial sepsis, which results in the release of enzymes intravascular marginal pool and results in leukocytosis. that digest the red cell membrane. Initially, red cells appear normal in size and color (normo- Whatever the mechanism, intravascular hemolysis is cytic, normochromic). However, as marrow production manifested by anemia, hemoglobinemia, hemoglobinuria, increases, there is a striking increase in the reticulocyte count hemosiderinuria, and jaundice. Free hemoglobin released (reticulocytosis), which reaches 10% to 15% after 7 days. from lysed red cells is promptly bound by haptoglobin, Reticulocytes are larger than normal red cells and have producing a complex that is rapidly cleared by mononuclear blue-red polychromatophilic cytoplasm due to the presence phagocytes. As serum haptoglobin is depleted, free hemo- of RNA, a feature that allows them to be identified in the globin oxidizes to methemoglobin, which is brown in color. clinical laboratory. Early recovery from blood loss also is The renal proximal tubular cells reabsorb and break down often accompanied by thrombocytosis, which results from much of the filtered hemoglobin and methemoglobin, but an increase in platelet production. some passes out in the urine, imparting a red-brown color. Iron released from hemoglobin can accumulate within tubular Chronic Blood Loss cells, giving rise to renal hemosiderosis. Concomitantly, heme Chronic blood loss induces anemia only when the rate of groups derived from hemoglobin-haptoglobin complexes loss exceeds the regenerative capacity of the marrow or are metabolized to bilirubin within mononuclear phagocytes, when iron reserves are depleted and iron deficiency anemia leading to jaundice. Unlike in extravascular hemolysis, appears (see later). splenomegaly is not seen. In all types of uncomplicated hemolytic anemia, the excess Hemolytic Anemias serum bilirubin is unconjugated. The level of hyperbiliru- binemia depends on the functional capacity of the liver and Hemolytic anemias share the following features: the rate of hemolysis. When the liver is normal, jaundice is A shortened red cell life span below the normal 120 days rarely severe, but excessive bilirubin excreted by the liver Elevated erythropoietin levels and a compensatory increase in into the biliary tract often leads to the formation of gallstones erythropoiesis derived from heme pigments. 638 C H A P T E R 14 Red Blood Cell and Bleeding Disorders MORPHOLOGY Certain changes are seen in hemolytic anemia regardless of cause or type. Anemia and lowered tissue oxygen tension trigger the production of erythropoietin, which stimulates erythroid differentia- tion and leads to the appearance of increased numbers of erythroid precursors (normoblasts) in the marrow (Fig. 14.1). Compensatory increases in erythropoiesis result in a prominent reticulocytosis in the peripheral blood. The phagocytosis of red cells leads to the accumulation of the iron-containing pigment hemosiderin, particularly in the spleen, liver, and bone marrow. Such iron accumulation is referred to as hemosiderosis. If the anemia is severe, extramedullary hematopoiesis can appear in the liver, spleen, and lymph nodes. With chronic hemolysis, elevated biliary excretion of bilirubin promotes the formation of pigment gallstones (cholelithiasis). Figure 14.1 Marrow aspirate smear from a patient with hemolytic anemia. There is an increased number of maturing erythroid progenitors (normoblasts). (Courtesy Dr. Steven Kroft, Department of Pathology, University of Texas Southwestern Medical School, Dallas, Tex.) The hemolytic anemias can be classified in a variety of ways; here, we rely on the underlying mechanisms (see Table 14.1). We begin by discussing the major inherited closely apposed to the internal surface of the plasma mem- forms of hemolytic anemia, and then move on to the acquired brane. Its chief protein component, spectrin, consists of two forms that are most common or of particular pathophysi- polypeptide chains, α and β, which form intertwined (helical) ologic interest. flexible heterodimers. The “head” regions of spectrin dimers self-associate to form tetramers, and the “tails” associate Hereditary Spherocytosis with actin oligomers. Each actin oligomer can bind multiple Hereditary spherocytosis (HS) is an inherited disorder spectrin tetramers, thus creating a two-dimensional spectrin- caused by intrinsic defects in the red cell membrane actin skeleton that is connected to the cell membrane by skeleton that render red cells spheroid, less deformable, two distinct interactions. The first, involving the proteins and vulnerable to splenic sequestration and destruction. ankyrin and band 4.2, binds spectrin to the transmembrane The prevalence of HS is highest in northern Europe, where ion transporter, band 3. The second, involving protein 4.1, rates of 1 in 5000 are reported. An autosomal dominant binds the “tail” of spectrin to another transmembrane protein, inheritance pattern is seen in about 75% of cases. The glycophorin A. remaining patients have a more severe form of the disease HS is caused by diverse mutations that lead to an that is usually caused by the inheritance of two different insufficiency of membrane skeletal components. As a result defects (a state known as compound heterozygosity). of these alterations, the life span of affected red cells is decreased on average to 10 to 20 days from the normal 120 Pathogenesis days. The pathogenic mutations most commonly affect The remarkable deformability and durability of the normal ankyrin, band 3, spectrin, or band 4.2, the proteins involved red cell are attributable to the physicochemical properties in one of the two tethering interactions. Most mutations of its specialized membrane skeleton (Fig. 14.2), which lies cause frameshifts or introduce premature stop codons, such Spherocyte Band 3 GP Lipid bilayer 4.2 Ankyrin 4.1 β α Normal Actin Spectrin α Splenic β 4.1 macrophage Figure 14.2 Role of the red cell membrane skeleton in hereditary spherocytosis. The left panel shows the normal organization of the major red cell membrane skeletal proteins. Various mutations involving α-spectrin, β-spectrin, ankyrin, band 4.2, or band 3 that weaken the interactions between these proteins cause red cells to lose membrane fragments as they age. To accommodate the resultant change in the ratio of surface area to volume, these cells adopt a spherical shape. Spherocytic cells are less deformable than normal ones and therefore become trapped in the splenic cords, where they are phagocytosed by macrophages. GP, Glycophorin.  Anemias 639 that the mutated allele fails to produce any protein. The resulting deficiency of the affected protein reduces the assembly of the skeleton as a whole, destabilizing the overlying plasma membrane. Young HS red cells are normal in shape, but the destabilized lipid bilayer sheds membrane fragments as red cells age in the circulation. The loss of membrane relative to cytoplasm “forces” the cells to assume the smallest possible diameter for a given volume, namely, a sphere. Compound heterozygosity for two defective alleles understandably results in more profound membrane skeleton deficiency and more severe disease. The invariably beneficial effects of splenectomy prove that the spleen has a cardinal role in the premature demise of spherocytes. The travails of spherocytic red cells are fairly well defined. In the life of the portly, inflexible spherocyte, the spleen is the villain. Normal red cells must undergo Figure 14.4 Hereditary spherocytosis (peripheral smear). Note the extreme deformation to leave the cords of Billroth and enter anisocytosis and several dark-appearing spherocytes with no central pallor. the sinusoids. Because of their spheroidal shape and reduced Howell-Jolly bodies (small, dark nuclear remnants) also are seen in some deformability, the hapless spherocytes are trapped in the of the red cells of this asplenic patient. (Courtesy Dr. Robert W. McKenna, splenic cords, where they are easy prey for macrophages. Department of Pathology, University of Texas Southwestern Medical The splenic environment also exacerbates the tendency of School, Dallas, Tex.) HS red cells to lose membrane along with K+ ions and H2O; prolonged splenic exposure (erythrostasis), depletion of red cell glucose, and diminished red cell pH have all been the central zone of pallor (Fig. 14.4). Spherocytosis is distinctive suggested to contribute to these abnormalities (Fig. 14.3). but not pathognomonic, as spherocytes are also seen in other After splenectomy the spherocytes persist, but the anemia disorders associated with red cell membrane loss, such as in is corrected. autoimmune hemolytic anemia. Other features are common to all hemolytic anemias. These include reticulocytosis, marrow MORPHOLOGY erythroid hyperplasia, hemosiderosis, and mild jaundice. Chole- lithiasis (pigment stones) occurs in 40% to 50% of affected The most specific morphologic finding is spherocytosis, apparent adults. Moderate splenomegaly is characteristic (500 to 1000 g); on smears as small, dark-staining (hyperchromic) red cells lacking in few other hemolytic anemias is the spleen enlarged as much or as consistently. Splenomegaly results from congestion of the cords of Billroth and increased numbers of phagocytes. Primary membrane skeletal defect Clinical Features The diagnosis is based on family history, hematologic find- ings, and laboratory evidence. In two-thirds of cases, the Membrane stability red cells are abnormally sensitive to osmotic lysis when incubated in hypotonic salt solutions, which causes the influx of water into spherocytes with little margin for expansion. Membrane loss HS red cells also have an increased mean cell hemoglobin concentration, due to dehydration caused by the loss of K+ and H2O. Surface to volume ratio The characteristic clinical features are anemia, spleno- (spherocytosis) megaly, and jaundice. The severity varies greatly. In a small Deformability minority (mainly compound heterozygotes), HS presents at birth with marked jaundice and requires exchange transfu- sions. In 20% to 30% of patients, the disease is so mild as Splenic trapping to be virtually asymptomatic; here the decreased red cell survival is readily compensated for by increased erythro- poiesis. In most, however, the compensatory changes are Erythrostasis outpaced, producing a chronic hemolytic anemia of mild Glucose pH to moderate severity. The generally stable clinical course is sometimes punctuated by aplastic crises, usually triggered by an acute parvovirus Phagocytosis infection. Parvovirus infects and kills red cell progenitors, Extravascular hemolysis causing all red cell production to cease until an immune response clears the virus, generally in 1 to 2 weeks. Because of Figure 14.3 Pathophysiology of hereditary spherocytosis. the reduced life span of HS red cells, cessation of erythropoiesis 640 C H A P T E R 14 Red Blood Cell and Bleeding Disorders Glucose-6-phosphate 6-Phosphogluconate G6PD– 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 G6PD population is believed to stem from a protective effect against Plasmodium falciparum malaria (discussed later). G6PD vari- ants associated with hemolysis result in misfolding of the protein, making it more susceptible to proteolytic degrada- NADP NADPH tion. Compared with the most common normal variant, G6PD B, the half-life of G6PD– is moderately reduced, whereas that of G6PD Mediterranean is more markedly Glutathione abnormal. Because mature red cells do not synthesize new reductase proteins, as red cells age G6PD– and G6PD Mediterranean enzyme activities quickly fall to levels that are inadequate to protect against oxidant stress. Thus, older red cells are GSH GSSG much more prone to hemolysis than younger ones. The episodic hemolysis that is characteristic of G6PD deficiency is caused by exposures that generate oxidant Glutathione peroxidase stress. The most common triggers are infections, in which oxygen-derived free radicals are produced by activated leukocytes. Many infections can trigger hemolysis; viral H2O2 H2O hepatitis, pneumonia, and typhoid fever are among those most likely to do so. The other important initiators are drugs Figure 14.5 Role of glucose-6-phosphate dehydrogenase (G6PD) in and certain foods. The drugs implicated are numerous, defense against oxidant injury. Detoxification of H2O2, a potential oxidant, including antimalarials (e.g., primaquine and chloroquine), requires reduced glutathione (GSH), which is generated in a reaction that sulfonamides, nitrofurantoins, and others. Some drugs cause requires reduced nicotinamide adenine dinucleotide (NADPH). The synthesis of NADPH depends on the activity of G6PD. GSSG, Oxidized hemolysis only in individuals with the more severe Mediter- glutathione; NADP, nicotinamide adenine dinucleotide phosphate. ranean variant. The most frequently cited food is the fava bean, which generates oxidants when metabolized. “Favism” is endemic in the Mediterranean, Middle East, and parts of Africa where consumption is prevalent. Uncommonly, G6PD for even short periods leads to sudden worsening of the deficiency presents as neonatal jaundice or a chronic low- anemia. Transfusions may be necessary to support the patient grade hemolytic anemia in the absence of infection or known during the acute phase of the infection. Hemolytic crises are environmental triggers. produced by intercurrent events leading to increased splenic Oxidants cause both intravascular and extravascular destruction of red cells (e.g., infectious mononucleosis and hemolysis in G6PD-deficient individuals. Exposure of G6PD- its attendant increase in spleen size); these are clinically less deficient red cells to high levels of oxidants causes the significant than aplastic crises. Gallstones, found in many cross-linking of reactive sulfhydryl groups on globin chains, patients, may also produce symptoms. Splenectomy treats the which become denatured and form membrane-bound anemia and its complications, but brings with it an increased precipitates known as Heinz bodies. These are seen as dark risk of sepsis because the spleen acts as an important filter inclusions within red cells stained with crystal violet for blood-borne bacteria. (Fig. 14.6). Heinz bodies can damage the membrane suffi- ciently to cause intravascular hemolysis. Less severe Hemolytic Disease Due to Red Cell Enzyme Defects: membrane damage results in decreased red cell deform- Glucose-6-Phosphate Dehydrogenase Deficiency ability. As inclusion-bearing red cells pass through the splenic Abnormalities in the hexose monophosphate shunt or cords, macrophages pluck out the Heinz bodies. As a result glutathione metabolism resulting from deficient or impaired of membrane damage, some of these partially devoured enzyme function reduce the ability of red cells to protect cells retain an abnormal shape, appearing to have a bite themselves against oxidative injuries and lead to hemolysis. taken out of them (see Fig. 14.6). Other less severely damaged The most important of these enzyme derangements is cells become spherocytes due to loss of membrane surface hereditary deficiency of glucose-6-phosphate dehydrogenase area. Both bite cells and spherocytes are trapped in splenic (G6PD) activity. G6PD reduces nicotinamide adenine cords and removed by phagocytes. dinucleotide phosphate (NADP) to NADPH while oxidizing Acute intravascular hemolysis, marked by anemia, glucose-6-phosphate (Fig. 14.5). NADPH then provides hemoglobinemia, and hemoglobinuria, usually begins 2 to reducing equivalents needed for conversion of oxidized 3 days following exposure of G6PD-deficient individuals glutathione to reduced glutathione, which protects against to environmental triggers. Because only older red cells are oxidant injury by participating as a cofactor in reactions at risk for lysis, the episode is self-limited, as hemolysis that neutralize compounds such as H2O2 (see Fig. 14.5). ceases when only younger G6PD-replete red cells remain G6PD deficiency is a recessive X-linked trait, placing (even if exposure to the trigger, e.g., an offending drug, males at much higher risk for symptomatic disease. Several continues). The recovery phase is heralded by reticulocytosis. hundred G6PD genetic variants exist, but most clinically Because hemolytic episodes related to G6PD deficiency occur significant hemolytic anemia is associated with only two intermittently, features related to chronic hemolysis (e.g., variants, designated G6PD– and G6PD Mediterranean. splenomegaly, cholelithiasis) are absent.  Anemias 641 are lacking, two scenarios to explain these observations are favored: Metabolically active intracellular parasites consume oxygen and decrease intracellular pH, both of which promote sickling of AS red cells. These distorted, stiff cells may be cleared more rapidly by splenic and hepatic phagocytes, keeping parasite loads low. Sickling also impairs the formation of membrane knobs containing a protein made by the parasite called PfEMP-1. These membrane knobs are implicated in adhesion of infected red cells to endothelium, which is believed to have an important pathogenic role in the most severe form of the disease, cerebral malaria. It has been suggested that G6PD deficiency and thalas- semia also protect against malaria by increasing the clearance Figure 14.6 Glucose-6-phosphate dehydrogenase deficiency: effects of and decreasing the adherence of infected red cells, possibly oxidant drug exposure (peripheral blood smear). Inset, Red cells with by raising levels of oxidant stress and causing membrane precipitates of denatured globin (Heinz bodies) revealed by supravital damage in the parasite-bearing cells that leads to their rapid staining. As the splenic macrophages pluck out these inclusions, “bite cells” removal from the bloodstream. like the one in this smear are produced. (Courtesy Dr. Robert W. McKenna, Department of Pathology, University of Texas Southwestern Pathogenesis Medical School, Dallas, Tex.) The major pathologic manifestations—chronic hemolysis, microvascular occlusions, and tissue damage—all stem from the tendency of HbS molecules to stack into polymers Sickle Cell Disease when deoxygenated. Initially, this process converts the red Sickle cell disease is a common hereditary hemoglobinopa- cell cytosol from a freely flowing liquid into a viscous gel. thy caused by a point mutation in β-globin that promotes With continued deoxygenation, HbS molecules assemble the polymerization of deoxygenated hemoglobin, leading into long needlelike fibers within red cells, producing a to red cell distortion, hemolytic anemia, microvascular distorted sickle or holly-leaf shape. obstruction, and ischemic tissue damage. Several hundred Several variables affect the rate and degree of sickling: hemoglobinopathies caused by various mutations in globin Interaction of HbS with the other types of hemoglobin. In genes are known, but only those associated with sickle heterozygotes with sickle cell trait, about 40% of the cell disease are prevalent enough in the United States to hemoglobin is HbS and the rest is HbA, which interferes merit discussion. Hemoglobin (Hb) is a tetrameric protein with HbS polymerization. As a result, red cells in het- composed of two pairs of globin chains, each with its own erozygous individuals only sickle if exposed to prolonged, heme group. Normal adult red cells contain mainly HbA relatively severe hypoxia. HbF inhibits the polymerization (α2β2), along with small amounts of HbA2 (α2δ2) and fetal of HbS even more than HbA; hence, infants with sickle hemoglobin (HbF; α2γ2). Sickle cell disease is caused by a cell disease do not become symptomatic until they reach missense mutation in the β-globin gene that leads to the 5 or 6 months of age, when the level of HbF normally replacement of a charged glutamate residue with a hydropho- falls. However, in some individuals HbF expression bic valine residue. The abnormal physiochemical properties remains relatively high, a condition known as hereditary of the resulting sickle hemoglobin (HbS) are responsible for persistence of fetal hemoglobin; in these individuals, sickle the disease. cell disease is much less severe. Another variant hemo- About 8% to 10% of African Americans in the United globin, HbC, also is common in regions where HbS is States are heterozygous for HbS, a largely asymptomatic found; overall, about 2% to 3% of American blacks are condition known as sickle cell trait. The offspring of two HbC heterozygotes, and about 1 in 1250 are compound heterozygotes has a 1 in 4 chance of being homozygous for HbS/HbC heterozygotes. In HbSC red cells, the percent- the sickle mutation, a state that produces symptomatic sickle age of HbS is 50%, as compared with only 40% in HbAS cell disease, which afflicts 70,000 to 100,000 individuals in cells. Moreover, with aging HbSC cells tend to lose salt the United States. In affected individuals, almost all the and water and become dehydrated, an effect that increases hemoglobin in the red cell is HbS (α2βs2). the intracellular concentration of HbS. These factors The high prevalence of sickle cell trait in certain African increase the tendency for HbS to polymerize, and as a populations stems from its protective effects against result compound HbSC heterozygotes have a symptomatic falciparum malaria. Genetic studies have shown that the sickling disorder termed HbSC disease that is somewhat sickle hemoglobin mutation has arisen independently at milder than sickle cell disease. least six times in areas of Africa in which falciparum malaria Mean cell hemoglobin concentration (MCHC). Higher HbS is endemic, providing clear evidence of strong Darwinian concentrations increase the probability that aggregation selection. Parasite densities are lower in infected, heterozy- and polymerization will occur during any given period gous HbAS children than in infected, normal HbAA children, of deoxygenation. Thus, intracellular dehydration, which and AS children are significantly less likely to have severe increases the MCHC, facilitates sickling. Conversely, disease or to die from malaria. Although mechanistic details conditions that decrease the MCHC reduce disease 642 C H A P T E R 14 Red Blood Cell and Bleeding Disorders severity. This occurs when an individual who is homo- zygous for HbS also has coexistent α-thalassemia, which G G reduces Hb synthesis and leads to milder disease. A T C Point C Intracellular pH. A decrease in pH reduces the oxygen G G T mutation A affinity of hemoglobin, thereby increasing the fraction C C of deoxygenated HbS at any given oxygen tension and augmenting the tendency for sickling. Transit time of red cells through microvascular beds. As will HbA HbS be discussed, much of the pathology of sickle cell disease is related to vascular occlusion caused by sickling within microvascular beds. Transit times in most normal micro- RBC vascular beds are too short for significant aggregation of deoxygenated HbS to occur, and as a result sickling Deoxygenation is confined to microvascular beds with slow transit times. Irreversibly Blood flow is sluggish in the normal spleen and bone sickled marrow, which are prominently affected in sickle cell cell Ca2+ K+, H2O disease, and also in vascular beds that are inflamed. The Hemolysis movement of blood through inflamed tissues is slowed Extensive because of the adhesion of leukocytes to activated membrane endothelial cells and the transudation of fluid through damage Oxygenation leaky vessels. As a result, inflamed vascular beds are prone to sickling and occlusion. Additional cycles of Deoxygenation, deoxygenation Sickling causes cumulative damage to red cells through prolonged several mechanisms. As HbS polymers grow, they herniate transit times Microvascular through the membrane skeleton and project from the cell occlusion ensheathed only by the lipid bilayer. This severe derangement in membrane structure causes an influx of Ca2+ ions, which Cell with dehydration induce the cross-linking of membrane proteins and activate and membrane damage an ion channel that leads to the efflux of K+ and H2O. As a Figure 14.7 Pathophysiology of sickle cell disease. HbA, Hemoglobin A; result, with repeated sickling episodes, red cells become HbS, hemoglobin S; RBC, red blood cell. dehydrated, dense, and rigid (Fig. 14.7). Eventually, the most severely damaged cells are converted to nondeformable of vessels) and enhanced platelet aggregation, both of which irreversibly sickled cells that retain a sickle shape, even when may contribute to red cell stasis, sickling, and (in some fully oxygenated. The severity of the hemolysis correlates instances) thrombosis. with the percentage of irreversibly sickled cells, which are rapidly sequestered and removed by mononuclear phagocytes (extravascular hemolysis). Sickled red cells are also mechani- MORPHOLOGY cally fragile, leading to some intravascular hemolysis as well. In sickle cell anemia, the peripheral blood demonstrates variable The pathogenesis of the microvascular occlusions, which numbers of irreversibly sickled cells, reticulocytosis, and target are responsible for the most serious clinical features, is far cells, which result from red cell dehydration (Fig. 14.8). Howell-Jolly less certain. Microvascular occlusions are not related to the bodies (small nuclear remnants) also are present in red cells due to number of irreversibly sickled cells, but instead may be asplenia (see later).The bone marrow is hyperplastic as a result of a dependent on more subtle red cell membrane damage and compensatory erythroid hyperplasia. Marked expansion of the marrow local factors, such as inflammation or vasoconstriction, that leads to bone resorption and secondary new bone formation,producing tend to slow or arrest the movement of red cells through prominent cheekbones and changes in the skull that resemble a microvascular beds (see Fig. 14.7). As mentioned earlier, “crewcut” on radiographic studies. Extramedullary hematopoiesis sickle red cells express higher than normal levels of adhesion may also appear.The increased breakdown of hemoglobin may cause molecules and are sticky. Mediators released from granu- hyperbilirubinemia and formation of pigment gallstones. locytes during inflammatory reactions up-regulate the In early childhood, the spleen is enlarged (up to 500 g) by red expression of adhesion molecules on endothelial cells pulp congestion caused by the trapping of sickled red cells in the (Chapter 3) and further enhance the tendency for sickle red cords and sinuses (Fig. 14.9). With time, however, chronic eryth- cells to arrest during transit through the microvasculature. rostasis leads to splenic infarction, fibrosis, and progressive The stagnation of red cells within inflamed vascular beds shrinkage, so that by adolescence or early adulthood only a small results in extended exposure to low oxygen tension, sickling, nubbin of fibrous splenic tissue is left, a process called autosple- and vascular obstruction. Once started, it is easy to envision nectomy (Fig. 14.10). Infarctions caused by vascular occlusions how a vicious cycle of sickling, obstruction, hypoxia, and may occur in many other tissues as well, including the bones, more sickling ensues. Depletion of nitric oxide (NO) also brain, kidney, liver, retina, and pulmonary vessels, the latter may play a part in the vascular occlusions. Free hemoglobin sometimes producing cor pulmonale. In adult patients, vascular released from lysed sickle red cells can bind and inactivate stagnation in subcutaneous tissues often leads to leg ulcers; this NO, a potent vasodilator and inhibitor of platelet aggregation. complication is rare in children. This in turn may lead to increased vascular tone (narrowing  Anemias 643 A A B B Figure 14.8 Sickle cell disease (peripheral blood smear). (A) Low Figure 14.9 (A) Spleen in sickle cell disease (low power). Red pulp cords magnification shows irreversibly sickled cells as well as target cells and red and sinusoids are markedly congested; between the congested areas, pale cell anisocytosis and poikilocytosis. (B) Higher magnification shows an areas of fibrosis resulting from ischemic damage are evident. (B) Under irreversibly sickled cell in the center. (Courtesy Dr. Robert W. McKenna, high power, splenic sinusoids are dilated and filled with sickled red cells. Department of Pathology, University of Texas Southwestern Medical (Courtesy Dr. Darren Wirthwein, Department of Pathology, University of School, Dallas, Tex.) Texas Southwestern Medical School, Dallas, Tex.) Clinical Features Priapism affects up to 45% of males after puberty and may Sickle cell disease causes a moderately severe hemo- lead to hypoxic damage and erectile dysfunction. Other lytic anemia (hematocrit 18% to 30%) associated with disorders related to vascular obstruction, particularly stroke reticulocytosis, hyperbilirubinemia, and the presence and retinopathy leading to loss of visual acuity and even of irreversibly sickled cells. Its course is punctuated by blindness, can take a devastating toll. Factors proposed to a variety of “crises.” Vaso-occlusive crises, also called pain contribute to stroke include the adhesion of sickle red cells crises, are episodes of hypoxic injury and infarction that cause severe pain in the affected region. Although infection, dehydration, and acidosis (all of which favor sickling) may act as triggers, in most instances no predisposing cause is 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. These frequently manifest as the hand-foot syndrome or dactylitis of the bones of the hands and feet. Acute chest syndrome is a particularly dangerous type of vaso-occlusive crisis involving the lungs that typically presents with fever, cough, chest pain, and pulmonary infiltrates. Pulmonary inflammation (such as may be induced by an infection) may cause blood flow to become sluggish and “spleenlike,” leading to sickling and vaso-occlusion. This compromises pulmonary function, Figure 14.10 “Autoinfarcted” splenic remnant in sickle cell disease. creating a potentially fatal cycle of worsening pulmonary (Courtesy Drs. Dennis Burns and Darren Wirthwein, Department of and systemic hypoxemia, sickling, and vaso-occlusion. Pathology, University of Texas Southwestern Medical School, Dallas, Tex.) 644 C H A P T E R 14 Red Blood Cell and Bleeding Disorders to arterial vascular endothelium and vasoconstriction caused that hematopoietic stem cells produce red cells that express by the depletion of NO by free hemoglobin. fetal hemoglobin instead of sickle hemoglobin. A clinical trial Although occlusive crises are the most common cause testing this approach is ongoing and has produced excellent of patient morbidity and mortality, several other acute events responses. complicate the course. Sequestration crises occur in children with intact spleens. Massive entrapment of sickled red cells Thalassemia leads to rapid splenic enlargement, hypovolemia, and Thalassemia is a genetically heterogeneous disorder sometimes shock. Both sequestration crises and the acute caused by germline mutations that decrease the synthesis chest syndrome may be fatal and sometimes require prompt of either α-globin or β-globin, leading to anemia, tissue treatment with exchange transfusions. Aplastic crises stem hypoxia, and red cell hemolysis related to the imbalance from the infection of red cell progenitors by parvovirus in globin chain synthesis. The two α chains in HbA are B19, which causes a transient cessation of erythropoiesis encoded by an identical pair of α-globin genes on chromo- and a sudden worsening of the anemia. some 16, and the two β chains are encoded by a single In addition to these dramatic crises, chronic tissue hypoxia β-globin gene on chromosome 11. β-thalassemia is caused takes a subtle but important toll. Chronic hypoxia is responsible by deficient synthesis of β chains, whereas α-thalassemia is for a generalized impairment of growth and development, as caused by deficient synthesis of α chains. The hematologic well as organ damage affecting the spleen, heart, kidneys, and consequences of diminished synthesis of one globin chain lungs. Sickling provoked by hypertonicity in the renal medulla stem not only from hemoglobin deficiency but also from causes damage that eventually leads to hyposthenuria (the a relative excess of the other globin chain, particularly in inability to concentrate urine), which increases the propensity β-thalassemia (described later). for dehydration and its attendant risks. Thalassemia is endemic in the Mediterranean basin Increased susceptibility to infection with encapsulated (indeed, thalassa means “sea” in Greek) as well as the Middle organisms is another threat. This is due in large part to East, tropical Africa, the Indian subcontinent, and Asia, and altered splenic function, which is severely impaired in in aggregate is among the most common inherited disorders children by congestion and poor blood flow, and completely of humans. As with sickle cell disease and other common absent in adults because of splenic infarction. Defects of inherited red cell disorders, its prevalence seems to be uncertain etiology in the alternative complement pathway explained by the protection it affords heterozygous carriers also impair the opsonization of bacteria. Pneumococcus against malaria. Although we discuss thalassemia with other pneumoniae and Haemophilus influenzae septicemia and inherited forms of anemia associated with hemolysis, it is meningitis are common, particularly in children, but can important to recognize that the defects in globin synthesis be reduced by vaccination and prophylactic antibiotics. that underlie these disorders cause anemia through two It must be emphasized that there is great variation in mechanisms: decreased red cell production, and decreased the clinical manifestations of sickle cell disease. Some red cell lifespan. individuals suffer repeated vaso-occlusive crises, whereas others have only mild symptoms. The basis for this wide β-Thalassemia range in disease expression is not understood; both modifying β-thalassemia is caused by mutations that diminish the genes and environmental factors are suspected. synthesis of β-globin chains. Its clinical severity varies The diagnosis is suggested by the clinical findings and widely due to heterogeneity in the causative mutations. We the presence of irreversibly sickled red cells and is confirmed will begin our discussion with the molecular lesions in by various tests for sickle hemoglobin. Prenatal diagnosis β-thalassemia and then relate the clinical variants to specific is possible by analysis of fetal DNA obtained by amniocen- underlying molecular defects. tesis or chorionic biopsy. Newborn screening for sickle hemoglobin is now routinely performed in all 50 states, Molecular Pathogenesis typically using samples obtained by heel stick at birth. The causative mutations fall into two categories: (1) β0 The outlook for patients with sickle cell disease has mutations, associated with absent β-globin synthesis, and improved considerably over the past 10 to 20 years. About (2) β+ mutations, characterized by reduced (but detectable) 90% of patients survive to 20 years of age, and close to 50% β-globin synthesis. Sequencing of β-thalassemia genes has survive beyond the fifth decade. The mainstay of treatment revealed more than 100 different causative mutations, mostly is an inhibitor of DNA synthesis, hydroxyurea, which has consisting of point mutations, which fall into three major several beneficial effects. These include (1) an increase in red classes: cell HbF levels, which occurs by unknown mechanisms; and Splicing mutations. These are the most common cause of (2) an anti-inflammatory effect, which stems from an inhibition β+-thalassemia. Some of these mutations destroy normal of leukocyte production. These activities (and possibly others) RNA splice junctions and completely prevent the produc- are believed to act in concert to decrease crises related to tion of normal β-globin mRNA, resulting in β0-thalassemia. vascular occlusions in both children and adults. When added Others create an “ectopic” splice site within an intron. to hydroxyurea, L-glutamine has been shown to decrease Because the flanking normal splice site remains, both pain crises; the mechanism is uncertain, but it may involve normal and abnormal splicing occurs and some normal changes in metabolism that decrease oxidant stress in red cells. β-globin mRNA is made, resulting in β+-thalassemia. Hematopoietic stem cell transplantation offers a chance at cure Promoter region mutations. These mutations reduce and is increasingly being explored as a therapeutic option. transcription by 75% to 80%. Some normal β-globin is Another exciting new approach involves using gene editing synthesized; thus, these mutations are associated with (CRISPR technology) to reverse hemoglobin switching, so β+-thalassemia.  Anemias 645 Chain terminator mutations. These are the most common it is estimated that 70% to 85% of red cell precursors cause of β0-thalassemia. They consist of either nonsense suffer this fate, which leads to ineffective erythropoiesis. mutations that introduce a premature stop codon or small Those red cells that are released from the marrow also insertions or deletions that shift the mRNA reading frames contain inclusions and have membrane damage, leaving (frameshift mutations; Chapter 5). Both block translation theme prone to splenic sequestration and extravascular and prevent the synthesis of any functional β-globin. hemolysis. In severe β-thalassemia, ineffective erythropoiesis creates Impaired β-globin synthesis results in anemia by two several additional problems. Erythropoietic drive in the mechanisms (Fig. 14.11). The deficit in HbA synthesis setting of severe uncompensated anemia leads to massive produces “underhemoglobinized” hypochromic, microcytic erythroid hyperplasia in the marrow and extensive extra- red cells with subnormal oxygen transport capacity. Even medullary hematopoiesis. The expanding mass of red cell more important is the diminished survival of red cells precursors erodes the bony cortex, impairs bone growth, and their precursors, which results from the imbalance in and produces skeletal abnormalities (described later). α- and β-globin synthesis. Unpaired α chains precipitate Extramedullary hematopoiesis involves the liver, spleen, within red cell precursors, forming insoluble inclusions. and lymph nodes, and in extreme cases produces extraos- These inclusions cause a variety of untoward effects, but seous masses in the thorax, abdomen, and pelvis. The membrane damage is the proximal cause of most red cell metabolically active erythroid progenitors steal nutrients pathology. Many red cell precursors succumb to membrane from other tissues that are already oxygen-starved, causing damage and undergo apoptosis. In severe β-thalassemia, severe cachexia in untreated patients. NORMAL β-THALASSEMIA Reduced β-globin synthesis, with relative excess of α-globin Insoluble α-globin aggregate HbA (α2β2) HbA Normal erythroblast Abnormal erythroblast α-globin aggregate Few abnormal red cells leave Normal HbA Hypochromic red cell Normal red blood cells Extravascular hemolysis Ineffective erythropoiesis Destruction of Most erythroblasts aggregate-containing Suppression of die in bone marrow red cells in spleen Liver hepcidin Dietary iron ANEMIA Increased iron absorption by Blood gut enterocytes transfusions Tissue hypoxia Reduce Erythropoietin increase Marrow expansion Systemic iron overload (secondary hemochromatosis) Skeletal deformities Figure 14.11 Pathogenesis of β-thalassemia major. Note that the aggregates of unpaired α-globin chains, a hallmark of the disease, are not visible in routinely stained blood smears. Blood transfusions are a double-edged sword, diminishing the anemia and its attendant complications, but also adding to the systemic iron overload. HbA, Hemoglobin A. 646 C H A P T E R 14 Red Blood Cell and Bleeding Disorders Another serious complication of ineffective erythropoiesis INEFFECTIVE is excessive absorption of dietary iron. Erythroid precursors NORMAL ERYTHROPOIESIS secrete a hormone called erythroferrone that inhibits produc- tion of hepcidin, a key negative regulator of iron uptake in the gut (described later in this chapter). In thalessemia, the Anemia, hypoxia marked expansion of erythroid precursors leads to increased absorption of iron from the gut (Fig. 14.12), and this together Normal Increased with repeated blood transfusions inevitably lead to severe erythropoietin erythropoietin iron accumulation (secondary hemochromatosis) unless preven- levels levels tive steps are taken. Injury to parenchymal organs, particu- larly the heart and liver, often follows (Chapter 18). Normal red cell Increased red cell Clinical Syndromes progenitors progenitors The relationships of clinical phenotypes to underlying genotypes are summarized in Table 14.3. Clinical classifica- tion of β-thalassemia is based on the severity of the anemia, which in turn depends on the genetic defect (β+ or β0) and Normal Normal Increased red cell erythroferrone the gene dosage (homozygous or heterozygous). In general, production erythroferrone individuals with two β-thalassemia alleles (β+/β+, β+/β0, or β0/β0) have a severe, transfusion-dependent anemia called β-thalassemia major. Heterozygotes with one β-thalassemia Liver Liver Reduced gene and one normal gene (β+/β or β0/β) usually have a iron mild asymptomatic microcytic anemia. This condition is utliization due referred to as β-thalassemia minor or β-thalassemia trait. A to defective Iron third genetically heterogeneous variant of moderate severity incorporation Normal Decreased red cell is called β-thalassemia intermedia. This category includes hepcidin hepcidin production into red cells milder variants of β+/β+ or β+/β0-thalassemia and unusual forms of heterozygous β-thalassemia. Some patients with β-thalassemia intermedia have two defective β-globin genes Iron Increased iron absorption absorption and an α-thalassemia gene defect, which improves the effectiveness of erythropoiesis and red cell survival by lessening the imbalance in α- and β-chain synthesis. In other rare but informative cases, affected individuals have a single Iron β-globin defect and one or two extra copies of normal overload α-globin genes (stemming from a gene duplication event), which worsens the chain imbalance. These unusual forms Figure 14.12 Mechanism of iron overload due to ineffective of the disease emphasize the cardinal role of unpaired hematopoiesis. In the setting of ineffective erythropoiesis, such as in those with severed thalassemia, increased release of erythroferrone from the α-globin chains in the pathology. The clinical and morpho- expanded mass of erythroid progenitors suppresses hepcidin production, logic features of β-thalassemia intermedia are not described leading to increased iron uptake from the gut. separately but can be surmised from the following discus- sions of β-thalassemia major and β-thalassemia minor. Table 14.3 Clinical and Genetic Classification of Thalassemia Clinical Syndrome Genotype Clinical Features Molecular Genetics β-Thalassemia β-Thalassemia major Homozygous β-thalassemia (β0/β0, β+/β+, β0/β+) Severe; requires blood transfusions Mainly point mutations that β-Thalassemia intermedia Variable (β0/β+, β+/β+, β0/β, β+/β) Severe but does not require lead to defects in the regular blood transfusions transcription, splicing, or β-Thalassemia minor Heterozygous β-thalassemia (β0/β, β+/β) Asymptomatic with mild or absent translation of β-globin anemia; red cell abnormalities mRNA seen α-Thalassemia Silent carrier −/α α/α Asymptomatic; no red cell Mainly gene deletions abnormality α-Thalassemia trait −/− α/α (Asian) Asymptomatic, like β-thalassemia −/α −/α (black African, Asian) minor HbH disease −/− −/α Severe; resembles β-thalassemia intermedia Hydrops fetalis −/− −/− Lethal in utero without transfusions  Anemias 647 β-Thalassemia Major. β-Thalassemia major is most β-Thalassemia Minor. β-Thalassemia minor is much more common in Mediterranean countries, parts of Africa, and common than β-thalassemia major and understandably Southeast Asia. In the United States, the incidence is highest affects the same ethnic groups. Most patients are heterozy- in immigrants from these areas. The anemia manifests 6 to gous carriers of a β+ or β0 allele. These patients are usually 9 months after birth as hemoglobin synthesis switches from asymptomatic. Anemia, if present, is mild. The peripheral HbF to HbA. In untransfused patients, hemoglobin levels blood smear typically shows hypochromia, microcytosis, are 3 to 6 g/dL. The red cells may completely lack HbA basophilic stippling, and target cells. Mild erythroid hyper- (β0/β0 genotype) or contain small amounts (β+/β+ or β0/β+ plasia is seen in the bone marrow. Hemoglobin electropho- genotypes). The major red cell hemoglobin is HbF, which resis usually reveals an increase in HbA2 (α2δ2) to 4% to 8% is markedly elevated. HbA2 levels are sometimes high but of the total hemoglobin (normal, 2.5% ± 0.3%), reflecting more often are normal or low. an elevated ratio of δ-chain to β-chain synthesis. HbF levels are generally normal or occasionally slightly increased. Recognition of β-thalassemia trait is important for two reasons: (1) it may be mistaken for iron deficiency, and (2) it MORPHOLOGY has implications for genetic counseling. Iron deficiency (the most common cause of microcytic anemia) can usually be Blood smears show severe red cell abnormalities, including marked excluded by measurement of serum iron, total iron-binding variation in size (anisocytosis) and shape (poikilocytosis), capacity, and serum ferritin (see the Iron Deficiency Anemia microcytosis, and hypochromia. Target cells (so called because section later in this chapter). The increase in HbA2 is diag- hemoglobin collects in the center of the cell), basophilic stippling, nostically useful, particularly in individuals (such as women and fragmented red cells also are common. Inclusions of aggregated of childbearing age) who are at high risk of iron deficiency. α chains are efficiently removed by the spleen and not easily seen.The reticulocyte count is elevated, but is lower than expected α-Thalassemia for the severity of anemia because of ineffective erythropoiesis. α-Thalassemia is caused by inherited deletions that result Variable numbers of poorly hemoglobinized nucleated red cell in reduced or absent synthesis of α-globin chains. Normal precursors (normoblasts) are seen in the peripheral blood as a individuals have four α-globin genes, and the severity of result of “stress” erythropoiesis and abnormal release of red cell α-thalassemia depends on how many α-globin genes are precursors from sites of extramedullary hematopoiesis. affected. As in β-thalassemias, the anemia stems both from Other major alterations involve the bone marrow and spleen. inadequate hemoglobin synthesis and the presence of excess, In untransfused patients, there is a striking expansion of hemato- unpaired β, γ, and δ globin chains, which vary in type at dif- poietically active marrow. In the bones of the face and skull, the ferent ages. In newborns with α-thalassemia, excess unpaired burgeoning marrow erodes existing cortical bone and induces γ-globin chains form γ4 tetramers known as hemoglobin Barts, new bone formation, giving rise to a “crewcut” appearance on whereas in older children and adults excess β-globin chains radiographic studies (Fig. 14.13). Both phagocyte hyperplasia and form β4 tetramers known as HbH. Because free β and γ chains extramedullary hematopoiesis contribute to enlargement of the are more soluble than free α chains and form fairly stable spleen, which can weigh as much as 1500 g.The liver and the lymph homotetramers, hemolysis and ineffective erythropoiesis nodes also may be enlarged by extramedullary hematopoiesis. are less severe than in β-thalassemia. A variety of molecular Hemosiderosis and secondary hemochromatosis, the two lesions give rise to α-thalassemia, but gene deletion is the manifestations of iron overload (Chapter 18), inevitably occur most common cause of reduced α-chain synthesis. unless chelation therapy is given.The deposited iron often damages organs, most notably the heart, liver, and pancreas. 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 effects of anemia. In those who survive long enough, the cheekbones and other bony prominences are enlarged and distorted. Hepatosplenomegaly due to extramedullary hematopoiesis is usually present. Although blood transfusions improve the anemia and suppress complications related to excessive erythropoiesis, they lead to complications of their own. Cardiac disease resulting from progressive iron overload and secondary hemochromatosis (Chapter 18) is an important cause of death, particularly in heavily transfused patients, who must be treated with iron chelators to prevent this complication. With transfusions and iron chelation, survival into the third decade is possible, but the overall outlook remains guarded. Hematopoietic stem cell transplantation Figure 14.13 β-Thalassemia major. X-ray film of the skull showing new is the only therapy offering a cure and is being used increas- bone formation on the outer table, producing perpendicular radiations ingly. Prenatal diagnosis is possible by molecular analysis resembling a crewcut. (Courtesy Dr. Jack Reynolds, Department of of DNA. Radiology, University of Texas Southwestern Medical School, Dallas, Tex.) 648 C H A P T E R 14 Red Blood Cell and Bleeding Disorders dependence on blood transfusions for survival, with the Clinical Syndromes associated risk of iron overload. Hematopoietic stem cell The clinical syndromes are determined and classified by transplantation can be curative. the number of α-globin genes that are deleted. Each of the four α-globin genes normally contributes 25% of the total Paroxysmal Nocturnal Hemoglobinuria α-globin chains. α-Thalassemia syndromes stem from Paroxysmal nocturnal hemoglobinuria (PNH) is a disease combinations of deletions that remove one to four α-globin that results from acquired mutations in the phosphati- genes. Not surprisingly, the severity of the clinical syndrome dylinositol glycan complementation group A gene (PIGA), is proportional to the number of α-globin genes that are an enzyme that is essential for the synthesis of certain deleted. The different types of α-thalassemia and their salient membrane-associated complement regulatory proteins. clinical features are listed in Table 14.3. PNH has an incidence of 2 to 5 per million in the United States. Despite its rarity, it has fascinated hematologists Silent Carrier State. Silent carrier state is associated with because it is the only hemolytic anemia caused by an acquired the deletion of a single α-globin gene, which causes a barely genetic defect. Recall that proteins are anchored into the detectable reduction in α-globin chain synthesis. These lipid bilayer in two ways. Most have a hydrophobic region individuals are completely asymptomatic but have slight that spans the cell membrane; these are called transmembrane microcytosis. proteins. The others are attached to the cell membrane through a covalent linkage to a specialized phospholipid α-Thalassemia Trait. α-Thalassemia trait is caused by the called glycosylphosphatidylinositol (GPI). In PNH, these deletion of two α-globin genes from a single chromosome GPI-linked proteins are deficient because of somatic muta- (α/α −/−) or the deletion of one α-globin gene from each tions that inactivate PIGA. PIGA is X-linked and subject to of the two chromosomes (α/− α/−) (see Table 14.3). The lyonization (random inactivation of one X chromosome in former genotype is more common in Asian populations, cells of females; Chapter 5). As a result, a single acquired the latter in regions of Africa. Both genotypes produce similar mutation in the active PIGA gene of any given cell is sufficient deficiencies of α-globin, but they have different implications to produce a deficiency state. Because the causative mutations for the children of affected individuals, who are at risk of occur in a hematopoietic stem cell, all of its clonal progeny clinically significant α-thalassemia (HbH disease or hydrops (red cells, white cells, and platelets) are deficient in GPI- fetalis) only when at least one parent has the −/− haplotype. linked proteins. Typically, only a subset of stem cells acquires As a result, symptomatic α-thalassemia is relatively common the mutation, and the mutant clone coexists with the progeny in Asian populations and rare in African populations. The of normal stem cells that are not PIGA deficient. clinical picture in α-thalassemia trait is identical to that Remarkably, most normal individuals harbor small described for β-thalassemia minor, that is, small red cells numbers of bone marrow cells with PIGA mutations identical (microcytosis), minimal or no anemia, and no abnormal to those that cause PNH. It is hypothesized that these cells physical signs. HbA2 levels are normal or low. increase in numbers (thus producing clinically evident PNH) only in rare instances where they have a selective advantage, Hemoglobin H (HbH) Disease. HbH disease is caused by such as in the setting of autoimmune reactions against deletion of three α-globin genes. It is most common in Asian GPI-linked antigens. Such a scenario might explain the populations. With only one normal α-globin gene, the frequent association of PNH and aplastic anemia, a marrow synthesis of α chains is markedly reduced, and tetramers failure syndrome (discussed later) that has an autoimmune of β-globin, called HbH, form. HbH has an extremely high basis in many individuals. affinity for oxygen and therefore is not useful for oxygen PNH blood cells are deficient in three GPI-linked proteins delivery, leading to tissue hypoxia disproportionate to the that regulate complement activity: (1) decay-accelerating level of hemoglobin. Additionally, HbH is prone to oxidation, factor, or CD55; (2) membrane inhibitor of reactive lysis, or which causes it to precipitate and form intracellular inclu- CD59; and (3) C8-binding protein. Of these factors, the most sions that promote red cell sequestration and phagocytosis important is CD59, a potent inhibitor of C3 convertase that in the spleen. The result is a moderately severe anemia prevents the spontaneous activation of the alternative resembling β-thalassemia intermedia. complement pathway. Red cells deficient in GPI-linked factors are abnormally Hydrops Fetalis. Hydrops fetalis, the most severe form of susceptible to lysis or injury by complement. This manifests α-thalassemia, is caused by deletion of all four α-globin as intravascular hemolysis, which is caused by the C5b-C9 genes. In the fetus, excess γ-globin chains form tetramers membrane attack complex. The hemolysis is paroxysmal (hemoglobin Barts) that have such a high affinity for oxygen and nocturnal in only 25% of cases; chronic hemolysis that they deliver little to tissues. Survival in early develop- without dramatic hemoglobinuria is more typical. The ment is due to the expression of ζ chains, an embryonic tendency for red cells to lyse at night is explained by a slight globin that pairs with γ chains to form a functional ζ2γ2 Hb decrease in blood pH during sleep, which increases the tetramer. Signs of fetal distress usually become evident by activity of complement. The anemia is variable but usually the third trimester of pregnancy. In the past, severe tissue mild to moderate in severity. The loss of heme iron in the anoxia led to death in utero or shortly after birth; with urine (hemosiderinuria) eventually leads to iron deficiency, intrauterine transfusion many affected infants are now saved. which can exacerbate the anemia if untreated. The fetus shows severe pallor, generalized edema, and Thrombosis is the leading cause of disease-related death massive hepatosplenomegaly similar to that seen in hemolytic in individuals with PNH. About 40% of patients suffer from disease of the new