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Red Blood Cell and Bleeding Disorders
In this chapter, we will first consider diseases of red cells. By far, the most common and important are the anemias, red cell deficiency states that usually have a nonneoplastic basis. We will then complete our review of blood diseases by discussing the major bleeding disorders and complications of blood transfusion.
Anemias
Anemia is defined as a reduction of the total circulating red cell mass below normal limits. Anemia reduces the oxygen-carrying capacity of the blood, leading to tissue hypoxia. In practice, the measurement of red cell mass is not easy, and anemia is usually diagnosed based on a reduction in the hematocrit (the ratio of packed red cells to total blood volume) and the hemoglobin concentration of the blood to levels that are below the normal range. These values correlate with the red cell mass except when there are changes in plasma volume caused by fluid retention or dehydration.
There are many classifications of anemia. We will follow one based on underlying mechanisms that is presented in Table 14.1 . A second clinically useful approach classifies anemia according to alterations in red cell morphology, which often point to particular causes. Morphologic characteristics that provide etiologic clues include red cell size (normocytic, microcytic, or macrocytic); degree of hemoglobinization, reflected in the color of red cells (normochromic or hypochromic); and shape. Microcytic hypochromic 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 marrow. Normochromic, normocytic anemias have diverse etiologies; in some of these anemias, characteristic abnormalities of red cell shape provide an important clue as to the cause. Red cell shape is assessed through visual inspection of peripheral smears, whereas as other red cell indices are determined in clinical laboratories with special instrumentation. The most useful of these indices are as follows:
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Mean cell volume: the average volume of a red cell expressed in femtoliters (fL)
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Mean cell hemoglobin: the average content (mass) of hemoglobin per red cell, expressed in picograms (pg)
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Mean cell hemoglobin concentration: the average concentration of hemoglobin in a given volume of packed red cells, expressed in grams per deciliter (g/dL)
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Red cell distribution width: the coefficient of variation of red cell volume
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 disturbances a |
| Increased Red Cell Destruction (Hemolysis) | |
| Inherited genetic defects | |
| Red cell membrane disorders | Hereditary spherocytosis, hereditary elliptocytosis |
| Enzyme deficiencies | |
| Hexose monophosphate shunt enzyme deficiencies | G6PD deficiency, glutathione synthetase deficiency |
| 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 glycoproteins | Paroxysmal nocturnal hemoglobinuria |
| 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 | B 12 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 Table 14.2 .
Table 14.2
Adult Reference Ranges for Red Cells a
| Measurement (Units) | Men | Women |
|---|---|---|
| Hemoglobin (g/dL) | 13.6–17.2 | 12.0–15.0 |
| Hematocrit (%) | 39–49 | 33–43 |
| Red cell count (×10 6 /µL) | 4.3–5.9 | 3.5–5.0 |
| Reticulocyte count (%) | 0.5–1.5 | |
| Mean cell volume (fL) | 82–96 | |
| Mean cell hemoglobin (pg) | 27–33 | |
| Mean cell hemoglobin concentration (g/dL) | 33–37 | |
| Red cell distribution width | 11.5–14.5 |
a Reference ranges vary among laboratories. The reference ranges for the laboratory providing the result should always be used in interpreting test results.
Whatever its cause, when sufficiently severe anemia leads to manifestations related to the diminished hemoglobin and oxygen content of the blood. Patients appear pale and often report weakness, malaise, easy fatigability, and dyspnea on mild exertion. Hypoxia can cause fatty change in the liver, myocardium, and kidney. On occasion, myocardial hypoxia manifests as angina pectoris, particularly when complicated by pre-existing coronary artery disease. With acute blood loss and shock, oliguria and anuria can develop as a result of renal hypoperfusion. Central nervous system hypoxia can cause headache, dimness of vision, and faintness.
Anemias of Blood Loss
Acute Blood Loss
The effects of acute blood loss are mainly due to the loss of intravascular volume, which if massive can lead to cardiovascular collapse, shock, and death. The clinical features depend on the rate of hemorrhage and whether the bleeding is external or internal. If the patient survives, the blood volume is rapidly restored by movement of water from the interstitial fluid compartment to the intravascular compartment. This fluid shift produces hemodilution and lowers the hematocrit. The resulting reduction in tissue oxygenation triggers increased secretion of erythropoietin from the kidney, which stimulates the proliferation of committed erythroid progenitors (colony-forming unit–erythroid [CFU–E]) in the marrow (see Fig. 13.1 ). It takes about 5 days for the progeny of these CFU–Es to mature and appear as newly released red cells (reticulocytes) in the peripheral blood. The iron in hemoglobin is recaptured if red cells extravasate into tissues, whereas bleeding into the gut or out of the body leads to iron loss and possible iron deficiency, which can hamper the restoration of normal red cell counts.
Significant bleeding results in predictable changes in the blood involving not only red cells, but also white cells and platelets. If the bleeding is sufficiently massive to cause a decrease in blood pressure, the compensatory release of adrenergic hormones mobilizes granulocytes from the intravascular marginal pool and results in leukocytosis. 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 (reticulocytosis), which reaches 10% to 15% after 7 days. Reticulocytes are larger than normal red cells and have blue-red polychromatophilic cytoplasm due to the presence of RNA, a feature that allows them to be identified in the clinical laboratory. Early recovery from blood loss also is often accompanied by thrombocytosis, which results from an increase in 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 and iron deficiency anemia appears (see later).
Hemolytic Anemias
Hemolytic anemias share the following features:
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A shortened red cell life span below the normal 120 days
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Elevated erythropoietin levels and a compensatory increase in erythropoiesis
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Accumulation of hemoglobin degradation products that are created as part of the process of red cell hemolysis
The physiologic destruction of senescent red cells takes place within macrophages, which are abundant in the spleen, liver, and bone marrow. This process appears to be triggered by age-dependent changes in red cell surface proteins, which lead to their recognition and removal by phagocytes. In the great majority of hemolytic anemias, the premature destruction of red cells also occurs within phagocytes, an event that is referred to as extravascular hemolysis. If persistent, extravascular hemolysis leads to a hyperplasia of phagocytes manifested by varying degrees of splenomegaly .
Extravascular hemolysis is most commonly caused by 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 phagocytosis by macrophages located within the splenic cords. Regardless of the cause, the principal clinical features of extravascular hemolysis are anemia, splenomegaly, and jaundice. Some hemoglobin inevitably escapes from phagocytes, which leads to variable decreases in plasma haptoglobin, an α 2 -globulin that binds free hemoglobin and prevents its excretion in the urine. Because much of the premature destruction of red cells occurs in the spleen, individuals with extravascular hemolysis often benefit from splenectomy.
Intravascular hemolysis of red cells may be caused by mechanical injury, complement fixation, intracellular parasites (e.g., falciparum malaria, Chapter 8 ), or exogenous toxic factors. Compared to extravascular hemolysis, it occurs less commonly; sources of mechanical injury include trauma caused by cardiac valves, narrowing of the microcirculation by thrombi, or repetitive physical trauma (e.g., marathon running and bongo drum beating). Complement fixation occurs in a variety of situations in which antibodies recognize and bind red cell antigens. Toxic injury is exemplified by clostridial sepsis, which results in the release of enzymes that digest the red cell membrane.
Whatever the mechanism, intravascular hemolysis is manifested by anemia, hemoglobinemia, hemoglobinuria, hemosiderinuria, and jaundice. Free hemoglobin released from lysed red cells is promptly bound by haptoglobin, producing a complex that is rapidly cleared by mononuclear phagocytes. As serum haptoglobin is depleted, free hemoglobin oxidizes to methemoglobin, which is brown in color. The renal proximal tubular cells reabsorb and break down much of the filtered hemoglobin and methemoglobin, but some passes out in 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 hemoglobin-haptoglobin complexes are metabolized to bilirubin within mononuclear phagocytes, leading to jaundice. Unlike in extravascular hemolysis, splenomegaly is not seen.
In all types of uncomplicated hemolytic anemia, the excess serum bilirubin is unconjugated. 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, but excessive bilirubin excreted by the liver into the biliary tract often leads to the formation of gallstones derived from heme pigments.
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 differentiation 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).
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 forms of hemolytic anemia, and then move on to the acquired forms that are most common or of particular pathophysiologic interest.
Hereditary Spherocytosis
Hereditary spherocytosis (HS) is an inherited disorder caused by intrinsic defects in the red cell membrane skeleton 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 about 75% of cases. The remaining patients have a more severe form of the disease that is usually caused by the inheritance of two different defects (a state known as compound heterozygosity).
Pathogenesis
The remarkable deformability and durability of the normal red cell are attributable to the physicochemical properties of its specialized membrane skeleton ( Fig. 14.2 ), 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, and the “tails” associate with actin oligomers. Each actin oligomer can bind multiple spectrin tetramers, thus creating a two-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 that lead to an insufficiency of membrane skeletal components . As a result of these alterations, the life span of affected red cells is decreased on average to 10 to 20 days from the normal 120 days. The pathogenic mutations most commonly affect ankyrin, band 3, spectrin, or band 4.2, the proteins involved in one of the two tethering interactions. Most mutations cause frameshifts or introduce premature stop codons, such 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 extreme deformation to leave the cords of Billroth and enter the sinusoids. Because of their spheroidal shape and reduced deformability, the hapless spherocytes are trapped in the splenic cords, where they are easy prey for macrophages. The splenic environment also exacerbates the tendency of HS red cells to lose membrane along with K + ions and H 2 O; prolonged splenic exposure (erythrostasis), depletion of red cell glucose, and diminished red cell pH have all been suggested to contribute to these abnormalities ( Fig. 14.3 ). After splenectomy the spherocytes persist, but the anemia is corrected.
Morphology
The most specific morphologic finding is spherocytosis, apparent on smears as small, dark-staining (hyperchromic) red cells lacking the central zone of pallor ( Fig. 14.4 ). Spherocytosis is distinctive but not pathognomonic, as spherocytes are also seen in other disorders associated with red cell membrane loss, such as in autoimmune hemolytic anemia. Other features are common to all hemolytic anemias. These include reticulocytosis, marrow erythroid hyperplasia, hemosiderosis, and mild jaundice. Cholelithiasis (pigment stones) occurs in 40% to 50% of affected adults. Moderate splenomegaly is characteristic (500 to 1000 g); 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.
Clinical Features
The diagnosis is based on family history, hematologic findings, and laboratory evidence. In two-thirds of cases, the 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. HS red cells also have an increased mean cell hemoglobin concentration, due to dehydration caused by the loss of K + and H 2 O.
The characteristic clinical features are anemia, splenomegaly, and jaundice. The severity varies greatly. In a small minority (mainly compound heterozygotes), HS presents at birth with marked jaundice and requires exchange transfusions. In 20% to 30% of patients, the disease is so mild as to be virtually asymptomatic; here 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 of mild to moderate severity.
The generally stable clinical course is sometimes punctuated by aplastic crises, usually triggered by an acute parvovirus infection. Parvovirus infects and kills red cell progenitors, causing all red cell production to cease until an immune response clears the virus, generally in 1 to 2 weeks. Because of the reduced life span of HS red cells, cessation of erythropoiesis for even short periods leads to sudden worsening of the anemia. Transfusions may be necessary to support the patient during the acute phase of the infection. Hemolytic crises are produced by intercurrent events leading to increased splenic destruction of red cells (e.g., infectious mononucleosis and its attendant increase in spleen size); these are clinically less significant than aplastic crises. Gallstones, found in many patients, may also produce symptoms. Splenectomy treats the anemia and its complications, but brings with it an increased risk of sepsis because the spleen acts as an important filter for blood-borne bacteria.
Hemolytic Disease Due to Red Cell Enzyme Defects: Glucose-6-Phosphate Dehydrogenase Deficiency
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 and lead to hemolysis. The most important of these enzyme derangements is hereditary deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH while oxidizing glucose-6-phosphate ( Fig. 14.5 ). NADPH then provides reducing equivalents needed for conversion of oxidized glutathione to reduced glutathione, which protects against oxidant injury by participating as a cofactor in reactions that neutralize compounds such as H 2 O 2 (see Fig. 14.5 ).
G6PD deficiency is a recessive X-linked trait, placing males at much higher risk for symptomatic disease. Several hundred G6PD genetic variants exist, but most clinically significant hemolytic anemia is associated with only two variants, designated G6PD – and G6PD Mediterranean. 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 population is believed to stem from a protective effect against Plasmodium falciparum malaria (discussed later). G6PD variants associated with hemolysis result in misfolding of the protein, making it more susceptible to proteolytic degradation. 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 abnormal. Because mature red cells do not synthesize new 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 much more prone to hemolysis than younger ones.
The episodic hemolysis that is characteristic of G6PD deficiency is caused by exposures that generate oxidant stress. The most common triggers are infections, in which oxygen-derived free radicals are produced by activated leukocytes. Many infections can trigger hemolysis; viral hepatitis, pneumonia, and typhoid fever are among those most likely to do so. The other important initiators are drugs and certain foods. The drugs implicated are numerous, including antimalarials (e.g., primaquine and chloroquine), sulfonamides, nitrofurantoins, and others. Some drugs cause hemolysis only in individuals with the more severe Mediterranean 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 deficiency presents as neonatal jaundice or a chronic low-grade hemolytic anemia in the absence of infection or known environmental triggers.
Oxidants cause both intravascular and extravascular hemolysis in G6PD-deficient individuals. Exposure of G6PD-deficient red cells to high levels of oxidants causes the cross-linking of reactive sulfhydryl groups on globin chains, which become denatured and form membrane-bound precipitates known as Heinz bodies. These are seen as dark inclusions within red cells stained with crystal violet ( Fig. 14.6 ). 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. As a result of membrane damage, some of these partially devoured cells retain an abnormal shape, appearing to have a bite taken out of them (see Fig. 14.6 ). Other less severely damaged cells become spherocytes due to loss of membrane surface area. Both bite cells and spherocytes are trapped in splenic cords and removed by phagocytes.
Acute intravascular hemolysis, marked by anemia, hemoglobinemia, and hemoglobinuria, usually begins 2 to 3 days following exposure of G6PD-deficient individuals to environmental triggers. Because only older red cells are at risk for lysis, the episode is self-limited, as hemolysis ceases when only younger G6PD-replete red cells remain (even if exposure to the trigger, e.g., an offending drug, continues). The recovery phase is heralded by reticulocytosis. Because hemolytic episodes related to G6PD deficiency occur intermittently, features related to chronic hemolysis (e.g., splenomegaly, cholelithiasis) are absent.
Sickle Cell Disease
Sickle cell disease is a common hereditary hemoglobinopathy caused by a point mutation in β-globin that promotes the polymerization of deoxygenated hemoglobin, leading to red cell distortion, hemolytic anemia, microvascular obstruction, and ischemic tissue damage. Several hundred hemoglobinopathies caused by various mutations in globin genes are known, but only those associated with sickle cell disease are prevalent enough in the United States to merit discussion. Hemoglobin (Hb) is a tetrameric protein composed of two pairs of globin chains, each with its own heme group. Normal adult red cells contain mainly HbA (α 2 β 2 ), along with small amounts of HbA 2 (α 2 δ 2 ) and fetal hemoglobin (HbF; α 2 γ 2 ). Sickle cell disease is caused by a missense mutation in the β-globin gene that leads to the replacement of a charged glutamate residue with a hydrophobic valine residue. The abnormal physiochemical properties of the resulting sickle hemoglobin (HbS) are responsible for the disease.
About 8% to 10% of African Americans in the United States are heterozygous for HbS, a largely asymptomatic condition known as sickle cell trait. The offspring of two heterozygotes has a 1 in 4 chance of being homozygous for the sickle mutation, a state that produces symptomatic sickle cell disease, which afflicts 70,000 to 100,000 individuals in the United States. In affected individuals, almost all the hemoglobin in the red cell is HbS (α 2 β s 2 ).
The high prevalence of sickle cell trait in certain African populations stems from its protective effects against falciparum malaria . Genetic studies have shown that the sickle hemoglobin mutation has arisen independently at least six times in areas of Africa in which falciparum malaria is endemic, providing clear evidence of strong Darwinian selection. Parasite densities are lower in infected, heterozygous HbAS children than in infected, normal HbAA children, and AS children are significantly less likely to have severe disease or to die from malaria. Although mechanistic details are lacking, two scenarios to explain these observations are favored:
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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.
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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 thalassemia also protect against malaria by increasing the clearance and decreasing the adherence of infected red cells, possibly by raising levels of oxidant stress and causing membrane damage in the parasite-bearing cells that leads to their rapid removal from the bloodstream.
Pathogenesis
The major pathologic manifestations—chronic hemolysis, microvascular occlusions, and tissue damage—all stem from the tendency of HbS molecules to stack into polymers when deoxygenated. Initially, this process converts the red cell cytosol from a freely flowing liquid into a viscous gel. With continued deoxygenation, HbS molecules assemble into long needlelike fibers within red cells, producing a distorted sickle or holly-leaf shape.
Several variables affect the rate and degree of sickling:
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Interaction of HbS with the other types of hemoglobin. In heterozygotes with sickle cell trait, about 40% of the hemoglobin is HbS and the rest is HbA, which interferes with HbS polymerization. As a result, red cells in heterozygous individuals only sickle if exposed to prolonged, relatively severe hypoxia. HbF inhibits the polymerization of HbS even more than HbA; hence, infants with sickle cell disease do not become symptomatic until they reach 5 or 6 months of age, when the level of HbF normally falls. However, in some individuals HbF expression remains relatively high, a condition known as hereditary persistence of fetal hemoglobin; in these individuals, sickle cell disease is much less severe. Another variant hemoglobin, HbC, also is common in regions where HbS is found; overall, about 2% to 3% of American blacks are HbC heterozygotes, and about 1 in 1250 are compound HbS/HbC heterozygotes. In HbSC red cells, the percentage of HbS is 50%, as compared with only 40% in HbAS cells. Moreover, with aging HbSC cells tend to lose salt and water and become dehydrated, an effect that increases the intracellular concentration of HbS. These factors increase the tendency for HbS to polymerize, and as a result compound HbSC heterozygotes have a symptomatic sickling disorder termed HbSC disease that is somewhat milder than sickle cell disease.
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Mean cell 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. Conversely, conditions that decrease the MCHC reduce disease severity. This occurs when an individual who is homozygous for HbS also has coexistent α-thalassemia, which reduces Hb synthesis and leads to milder disease.
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Intracellular pH. 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.
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Transit time of red cells through microvascular beds. As will 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 microvascular beds are too short for significant aggregation of deoxygenated HbS to occur, and as a result sickling is confined to microvascular beds with slow transit times. Blood flow is sluggish in the normal spleen and bone marrow, which are prominently affected in sickle cell disease, and also in vascular beds that are inflamed. The movement of blood through inflamed tissues is slowed because of the adhesion of leukocytes to activated endothelial cells and the transudation of fluid through leaky vessels. As a result, inflamed vascular beds are prone to sickling and occlusion.
Sickling causes cumulative damage to red cells through several mechanisms. As HbS polymers grow, they herniate through the membrane skeleton and project from the cell ensheathed only by the lipid bilayer. This severe derangement in membrane structure causes an influx of Ca 2+ ions, which induce the cross-linking of membrane proteins and activate an ion channel that leads to the efflux of K + and H 2 O. As a result, with repeated sickling episodes, red cells become dehydrated, dense, and rigid ( Fig. 14.7 ) . Eventually, the most severely damaged cells are converted to nondeformable irreversibly sickled cells that retain a sickle shape, even when fully oxygenated. The severity of the hemolysis correlates with the percentage of irreversibly sickled cells, which are rapidly sequestered and removed by mononuclear phagocytes (extravascular hemolysis). Sickled red cells are also mechanically fragile, leading to some intravascular hemolysis as well.
The pathogenesis of the microvascular occlusions, which are responsible for the most serious clinical features, is far less certain. Microvascular occlusions are not related to the number of irreversibly sickled cells, but instead may be dependent on more subtle red cell membrane damage and local factors, such as inflammation or vasoconstriction, that tend to slow or arrest the movement of red cells through microvascular beds (see Fig. 14.7 ). As mentioned earlier, sickle red cells express higher than normal levels of adhesion molecules and are sticky. Mediators released from granulocytes during inflammatory reactions up-regulate the expression of adhesion molecules on endothelial cells ( Chapter 3 ) and further enhance the tendency for sickle red cells to arrest during transit through the microvasculature. The stagnation of red cells within inflamed vascular beds results in extended exposure to low oxygen tension, sickling, and vascular obstruction. Once started, it is easy to envision how a vicious cycle of sickling, obstruction, hypoxia, and more sickling ensues. Depletion of nitric oxide (NO) also may play a part in the vascular occlusions. Free hemoglobin released from lysed sickle red cells can bind and inactivate NO, a potent vasodilator and inhibitor of platelet aggregation. This in turn may lead to increased vascular tone (narrowing of vessels) and enhanced platelet aggregation, both of which may contribute to red cell stasis, sickling, and (in some instances) thrombosis.
Morphology
In sickle cell anemia, the peripheral blood demonstrates variable numbers of irreversibly sickled cells, reticulocytosis, and target cells, which result from red cell dehydration ( Fig. 14.8 ). Howell-Jolly bodies (small nuclear remnants) also are present in red cells due to asplenia (see later). The bone marrow is hyperplastic as a result of a compensatory erythroid hyperplasia. Marked expansion of the marrow leads to bone resorption and secondary new bone formation, producing prominent cheekbones and changes in the skull that resemble a “crewcut” on radiographic studies. Extramedullary hematopoiesis may also appear. The increased breakdown of hemoglobin may cause hyperbilirubinemia and formation of pigment gallstones.
In early childhood, the spleen is enlarged (up to 500 g) by red pulp congestion caused by the trapping of sickled red cells in the cords and sinuses ( Fig. 14.9 ). With time, however, chronic erythrostasis leads to splenic infarction, fibrosis, and progressive shrinkage, so that by adolescence or early adulthood only a small nubbin of fibrous splenic tissue is left, a process called autosplenectomy ( Fig. 14.10 ). Infarctions caused by vascular occlusions may occur in many other tissues as well, including the bones, brain, kidney, liver, retina, and pulmonary vessels, the latter sometimes producing cor pulmonale. In adult patients, vascular stagnation in subcutaneous tissues often leads to leg ulcers; this complication is rare in children.
Clinical Features
Sickle cell disease causes a moderately severe hemolytic anemia (hematocrit 18% to 30%) associated with reticulocytosis, hyperbilirubinemia, and the presence of irreversibly sickled cells. Its course is punctuated by a variety of “crises.” Vaso-occlusive crises, also called pain 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, creating a potentially fatal cycle of worsening pulmonary and systemic hypoxemia, sickling, and vaso-occlusion. Priapism affects up to 45% of males after puberty and may lead to hypoxic damage and erectile dysfunction. Other disorders related to vascular obstruction, particularly stroke and retinopathy leading to loss of visual acuity and even blindness, can take a devastating toll. Factors proposed to contribute to stroke include the adhesion of sickle red cells to arterial vascular endothelium and vasoconstriction caused by the depletion of NO by free hemoglobin.
Although occlusive crises are the most common cause of patient morbidity and mortality, several other acute events complicate the course. Sequestration crises occur in children with intact spleens. Massive entrapment of sickled red cells leads to rapid splenic enlargement, hypovolemia, and sometimes shock. Both sequestration crises and the acute chest syndrome may be fatal and sometimes require prompt treatment with exchange transfusions. Aplastic crises stem from the infection of red cell progenitors by parvovirus B19, which causes a transient cessation of erythropoiesis and a sudden worsening of the anemia.
In addition to these dramatic crises, chronic tissue hypoxia takes a subtle but important toll. Chronic hypoxia is responsible for a generalized impairment of growth and development, as well as organ damage affecting the spleen, heart, kidneys, and lungs. Sickling provoked by hypertonicity in the renal medulla causes damage that eventually leads to hyposthenuria (the inability to concentrate urine), which increases the propensity for dehydration and its attendant risks.
Increased susceptibility to infection with encapsulated organisms is another threat. This is due in large part to altered splenic function, which is severely impaired in children by congestion and poor blood flow, and completely absent in adults because of splenic infarction. Defects of uncertain etiology in the alternative complement pathway also impair the opsonization of bacteria. Pneumococcus pneumoniae and Haemophilus influenzae septicemia and meningitis are common, particularly in children, but can be reduced by vaccination and prophylactic antibiotics.
It must be emphasized that there is great variation in the clinical manifestations of sickle cell disease. Some individuals suffer repeated vaso-occlusive crises, whereas others have only mild symptoms. The basis for this wide range in disease expression is not understood; both modifying genes and environmental factors are suspected.
The diagnosis is suggested by the clinical findings and the presence of irreversibly sickled red cells and is confirmed by various tests for sickle hemoglobin. Prenatal diagnosis is possible by analysis of fetal DNA obtained by amniocentesis or chorionic biopsy. Newborn screening for sickle hemoglobin is now routinely performed in all 50 states, typically using samples obtained by heel stick at birth.
The outlook for patients with sickle cell disease has improved considerably over the past 10 to 20 years. About 90% of patients survive to 20 years of age, and close to 50% survive beyond the fifth decade. The mainstay of treatment is an inhibitor of DNA synthesis, hydroxyurea, which has several beneficial effects. These include (1) an increase in red cell HbF levels, which occurs by unknown mechanisms; and (2) an anti-inflammatory effect, which stems from an inhibition of leukocyte production. These activities (and possibly others) are believed to act in concert to decrease crises related to vascular occlusions in both children and adults. When added to hydroxyurea, L-glutamine has been shown to decrease pain crises; the mechanism is uncertain, but it may involve changes in metabolism that decrease oxidant stress in red cells. Hematopoietic stem cell transplantation offers a chance at cure and is increasingly being explored as a therapeutic option. Another exciting new approach involves using gene editing (CRISPR technology) to reverse hemoglobin switching, so that hematopoietic stem cells produce red cells that express fetal hemoglobin instead of sickle hemoglobin. A clinical trial testing this approach is ongoing and has produced excellent responses.
Thalassemia
Thalassemia is a genetically heterogeneous disorder caused by germline mutations that decrease the synthesis of either α-globin or β-globin, leading to anemia, tissue hypoxia, and red cell hemolysis related to the imbalance in globin chain synthesis . The two α chains in HbA are encoded by an identical pair of α-globin genes on chromosome 16, and the two β chains are encoded by a single β-globin gene on chromosome 11. β-thalassemia is caused by deficient synthesis of β chains, whereas α-thalassemia is caused by deficient synthesis of α chains. The hematologic consequences of diminished synthesis of one globin chain stem not only from hemoglobin deficiency but also from a relative excess of the other globin chain, particularly in β-thalassemia (described later).
Thalassemia is endemic in the Mediterranean basin (indeed, thalassa means “sea” in Greek) as well as the Middle East, tropical Africa, the Indian subcontinent, and Asia, and in aggregate is among the most common inherited disorders of humans. As with sickle cell disease and other common inherited red cell disorders, its prevalence seems to be explained by the protection it affords heterozygous carriers against malaria. Although we discuss thalassemia with other inherited forms of anemia associated with hemolysis, it is important to recognize that the defects in globin synthesis that underlie these disorders cause anemia through two mechanisms: decreased red cell production, and decreased red cell lifespan.
β-Thalassemia
β-thalassemia is caused by mutations that diminish the synthesis of β-globin chains. Its clinical severity varies widely due to heterogeneity in the causative mutations. We will begin our discussion with the molecular lesions in β-thalassemia and then relate the clinical variants to specific underlying molecular defects.
Molecular Pathogenesis
The causative mutations fall into two categories: (1) β 0 mutations, associated with absent β-globin synthesis, and (2) β + mutations, characterized by reduced (but detectable) β-globin synthesis. Sequencing of β-thalassemia genes has revealed more than 100 different causative mutations, mostly consisting of point mutations, which fall into three major classes:
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Splicing mutations. These are the most common cause of β + -thalassemia. Some of these mutations destroy normal RNA splice junctions and completely prevent the production of normal β-globin mRNA, resulting in β 0 -thalassemia. Others create an “ectopic” splice site within an intron. Because the flanking normal splice site remains, both normal and abnormal splicing occurs and some normal β-globin mRNA is made, resulting in β + -thalassemia.
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Promoter region mutations. These mutations reduce transcription by 75% to 80%. Some normal β-globin is synthesized; thus, these mutations are associated with β + -thalassemia.
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Chain terminator mutations. These are the most common cause of β 0 -thalassemia. They consist of either nonsense mutations that introduce a premature stop codon or small insertions or deletions that shift the mRNA reading frames (frameshift mutations; Chapter 5 ). Both block translation and prevent the synthesis of any functional β-globin.
Impaired β-globin synthesis results in anemia by two mechanisms ( Fig. 14.11 ). The deficit in HbA synthesis produces “underhemoglobinized” hypochromic, microcytic red cells with subnormal oxygen transport capacity. Even more important is the diminished survival of red cells and their precursors, which results from the imbalance in α- and β-globin synthesis. Unpaired α chains precipitate within red cell precursors, forming insoluble inclusions. These inclusions cause a variety of untoward effects, but membrane damage is the proximal cause of most red cell pathology. Many red cell precursors succumb to membrane damage and undergo apoptosis. In severe β-thalassemia, it is estimated that 70% to 85% of red cell precursors suffer this fate, which leads to ineffective erythropoiesis . Those red cells that are released from the marrow also contain inclusions and have membrane damage, leaving theme prone to splenic sequestration and extravascular hemolysis.
In severe β-thalassemia, ineffective erythropoiesis creates several additional problems. Erythropoietic drive in the setting of severe uncompensated anemia leads to massive erythroid hyperplasia in the marrow and extensive extramedullary hematopoiesis. The expanding mass of red cell precursors erodes the bony cortex, impairs bone growth, and produces 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 serious complication of ineffective erythropoiesis is excessive absorption of dietary iron. Erythroid precursors secrete a hormone called erythroferrone that inhibits production of hepcidin, a key negative regulator of iron uptake in the gut (described later in this chapter). In thalessemia, the marked expansion of erythroid precursors leads to increased absorption of iron from the gut ( Fig. 14.12 ), and this together with repeated blood transfusions inevitably lead to severe iron accumulation (secondary hemochromatosis) unless preventive steps are taken. Injury to parenchymal organs, particularly the heart and liver, often follows ( Chapter 18 ).
Clinical Syndromes
The relationships of clinical phenotypes to underlying genotypes are summarized in Table 14.3 . Clinical classification of β-thalassemia is based on the severity of the anemia, which in turn depends on the genetic defect (β + or β 0 ) and the gene dosage (homozygous or heterozygous). In general, individuals with two β-thalassemia alleles (β + /β + , β +/ β 0 , or β 0 /β 0 ) have a severe, transfusion-dependent anemia called β-thalassemia major. Heterozygotes with one β-thalassemia gene and one normal gene (β +/ β or β 0 /β) usually have a mild asymptomatic microcytic anemia. This condition is referred to as β-thalassemia minor or β-thalassemia trait. A third genetically heterogeneous variant of moderate severity is called β-thalassemia intermedia. This category includes milder variants of β + /β + or β + /β 0 -thalassemia and unusual forms of heterozygous β-thalassemia. Some patients with β-thalassemia intermedia have two defective β-globin genes 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 β-globin defect and one or two extra copies of normal α-globin genes (stemming from a gene duplication event), which worsens the chain imbalance. These unusual forms of the disease emphasize the cardinal role of unpaired α-globin chains in the pathology. The clinical and morphologic features of β-thalassemia intermedia are not described separately but can be surmised from the following discussions of β-thalassemia major and β-thalassemia minor.
Table 14.3
Clinical and Genetic Classification of Thalassemia
β-Thalassemia Major
β-Thalassemia major is most common in Mediterranean countries, parts of Africa, and Southeast Asia. In the United States, the incidence is highest in immigrants from these areas. The anemia manifests 6 to 9 months after birth as hemoglobin synthesis switches from HbF to HbA. In untransfused patients, hemoglobin levels are 3 to 6 g/dL. The red cells may completely lack HbA (β 0 /β 0 genotype) or contain small amounts (β + /β + or β 0 /β + genotypes). The major red cell hemoglobin is HbF, which is markedly elevated. HbA 2 levels are sometimes high but more often are normal or low.
Morphology
Blood smears show severe red cell abnormalities, including marked variation in size (anisocytosis) and shape (poikilocytosis), microcytosis, and hypochromia. Target cells (so called because hemoglobin collects in the center of the cell), basophilic stippling, and fragmented red cells also are common. Inclusions of aggregated α chains are efficiently removed by the spleen and not easily seen. The reticulocyte count is elevated, but is lower than expected for the severity of anemia because of ineffective erythropoiesis. Variable numbers of poorly hemoglobinized nucleated red cell precursors (normoblasts) are seen in the peripheral blood as a result of “stress” erythropoiesis and abnormal release of red cell precursors from sites of extramedullary hematopoiesis.
Other major alterations involve the bone marrow and spleen. In untransfused patients, there is a striking expansion of hematopoietically active marrow. In the bones of the face and skull, the burgeoning marrow erodes existing cortical bone and induces new bone formation, giving rise to a “crewcut” appearance on radiographic studies ( Fig. 14.13 ). Both phagocyte hyperplasia and extramedullary hematopoiesis contribute to enlargement of the spleen, which can weigh as much as 1500 g. The liver and the lymph nodes also may be enlarged by extramedullary hematopoiesis.
Hemosiderosis and secondary hemochromatosis, the two manifestations of iron overload ( Chapter 18 ), inevitably occur 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 is the only therapy offering a cure and is being used increasingly. Prenatal diagnosis is possible by molecular analysis of DNA.
β-Thalassemia Minor
β-Thalassemia minor is much more common than β-thalassemia major and understandably affects the same ethnic groups. Most patients are heterozygous carriers of a β + or β 0 allele. These patients are usually asymptomatic. Anemia, if present, is mild. The peripheral blood smear typically shows hypochromia, microcytosis, basophilic stippling, and target cells. Mild erythroid hyperplasia is seen in the bone marrow. Hemoglobin electrophoresis usually reveals an increase in HbA 2 (α 2 δ 2 ) to 4% to 8% of the total hemoglobin (normal, 2.5% ± 0.3%), reflecting 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 has implications for genetic counseling. Iron deficiency (the most common cause of microcytic anemia) can usually be excluded by measurement of serum iron, total iron-binding capacity, and serum ferritin (see the Iron Deficiency Anemia section later in this chapter). The increase in HbA 2 is diagnostically useful, particularly in individuals (such as women of childbearing age) who are at high risk of iron deficiency.
α-Thalassemia
α-Thalassemia is caused by inherited deletions that result in reduced or absent synthesis of α-globin chains. Normal individuals have four α-globin genes, and the severity of α-thalassemia depends on how many α-globin genes are affected. As in β-thalassemias, the anemia stems both from inadequate hemoglobin synthesis and the presence of excess, unpaired β, γ, and δ globin chains, which vary in type at different ages. In newborns with α-thalassemia, excess unpaired γ-globin chains form γ 4 tetramers known as hemoglobin Barts, whereas in older children and adults excess β-globin chains form β 4 tetramers known as HbH . Because 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-thalassemia. A variety of molecular lesions give rise to α-thalassemia, but gene deletion is the most common cause of reduced α-chain synthesis.
Clinical Syndromes
The clinical syndromes are determined and classified by the number of α-globin genes that are deleted. Each of the four α-globin genes normally contributes 25% of the total α-globin chains. α-Thalassemia syndromes stem from combinations of deletions that remove one to four α-globin genes. Not surprisingly, the severity of the clinical syndrome is proportional to the number of α-globin genes that are deleted. The different types of α-thalassemia and their salient clinical features are listed in Table 14.3 .
Silent Carrier State
Silent carrier state is associated with the deletion of a single α-globin gene, which causes a barely detectable reduction in α-globin chain synthesis. These individuals are completely asymptomatic but have slight microcytosis.
α-Thalassemia Trait
α-Thalassemia trait is caused by the deletion of two α-globin genes from a single chromosome (α/α −/−) or the deletion of one α-globin gene from each of the two chromosomes (α/− α/−) (see Table 14.3 ). The former genotype is more common in Asian populations, the latter in regions of Africa. Both genotypes produce similar deficiencies of α-globin, but they have different implications for the children of affected individuals, who are at risk of clinically significant α-thalassemia (HbH disease or hydrops fetalis) only when at least one parent has the −/− haplotype. As a result, symptomatic α-thalassemia is relatively common in Asian populations and rare in African populations. 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. HbA 2 levels are normal or low.
Hemoglobin H (HbH) Disease
HbH disease is caused by deletion of three α-globin genes. It is most common in Asian populations. With only one normal α-globin gene, the synthesis of α chains is markedly reduced, and tetramers of β-globin, called HbH, form. HbH has an extremely high affinity for oxygen and therefore is not useful for oxygen delivery, leading to tissue hypoxia disproportionate to the level of hemoglobin. Additionally, HbH is prone to oxidation, which causes it to precipitate and form intracellular inclusions that promote red cell sequestration and phagocytosis in the spleen. The result is a moderately severe anemia resembling β-thalassemia intermedia.
Hydrops Fetalis
Hydrops fetalis, the 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) that have such a high affinity for oxygen that they deliver little to tissues. Survival in early development is due to the expression of ζ chains, an embryonic globin that pairs with γ chains to form a functional ζ 2 γ 2 Hb tetramer. Signs of fetal distress usually become evident by the third trimester of pregnancy. In the past, severe tissue anoxia led to death in utero or shortly after birth; with intrauterine transfusion many affected infants are now saved. The fetus shows severe pallor, generalized edema, and massive hepatosplenomegaly similar to that seen in hemolytic disease of the newborn ( Chapter 10 ). There is a lifelong dependence on blood transfusions for survival, with the associated risk of iron overload. Hematopoietic stem cell transplantation can be curative.
Paroxysmal Nocturnal Hemoglobinuria
Paroxysmal nocturnal hemoglobinuria (PNH) is a disease that results from acquired mutations in the phosphatidylinositol glycan complementation group A gene (PIGA), an enzyme that is essential for the synthesis of certain membrane-associated complement regulatory proteins. PNH has an incidence of 2 to 5 per million in the United States. Despite its rarity, it has fascinated hematologists because it is the only hemolytic anemia caused by an acquired genetic defect. Recall that proteins are anchored into the lipid bilayer in two ways. Most have a hydrophobic region that spans the cell membrane; these are called transmembrane proteins. The others are attached to the cell membrane through a covalent linkage to a specialized phospholipid called glycosylphosphatidylinositol (GPI). In PNH, these GPI-linked proteins are deficient because of somatic mutations that inactivate PIGA. PIGA is X-linked and subject to lyonization (random inactivation of one X chromosome in cells of females; Chapter 5 ). As a result, a single acquired mutation in the active PIGA gene of any given cell is sufficient to produce a deficiency state. Because the causative mutations occur in a hematopoietic stem cell, all of its clonal progeny (red cells, white cells, and platelets) are deficient in GPI-linked proteins. Typically, only a subset of stem cells acquires the mutation, and the mutant clone coexists with the progeny of normal stem cells that are not PIGA deficient.
Remarkably, most normal individuals harbor small numbers of bone marrow cells with PIGA mutations identical to those that cause PNH. It is hypothesized that these cells increase in numbers (thus producing clinically evident PNH) only in rare instances where they have a selective advantage, such as in the setting of autoimmune reactions against GPI-linked antigens. Such a scenario might explain the frequent association of PNH and aplastic anemia, a marrow failure syndrome (discussed later) that has an autoimmune basis in many individuals.
PNH blood cells are deficient in three GPI-linked proteins that regulate complement activity: (1) decay-accelerating factor, or CD55; (2) membrane inhibitor of reactive lysis, or CD59; and (3) C8-binding protein. Of these factors, the most important is CD59, a potent inhibitor of membrane attack complex that helps to prevent intravascular hemolysis of red cells by complement.
Red cells deficient in GPI-linked factors are abnormally susceptible to lysis or injury by complement. This manifests as intravascular hemolysis, which is caused by the C5b-C9 membrane attack complex. The hemolysis is paroxysmal and nocturnal in only 25% of cases; chronic hemolysis without dramatic hemoglobinuria is more typical. The tendency for red cells to lyse at night is explained by a slight decrease in blood pH during sleep, which increases the activity of complement. The anemia is variable but usually mild to moderate in severity. The loss of heme iron in the urine (hemosiderinuria) eventually leads to iron deficiency, which can exacerbate the anemia if untreated.
Thrombosis is the leading cause of disease-related death in individuals with PNH . About 40% of patients suffer from venous thrombosis, often involving the hepatic, portal, or cerebral veins . How complement activation leads to thrombosis in patients with PNH is not clear; the absorption of NO by free hemoglobin (discussed in the Sickle Cell Disease section earlier in this chapter) may be one contributing factor, and a role for endothelial damage caused by the C5-9 membrane attack complex is also suspected.
About 5% to 10% of patients eventually develop acute myeloid leukemia or a myelodysplastic syndrome, indicating that PNH may arise in the context of genetic damage to hematopoietic stem cells.
PNH is diagnosed by flow cytometry, which provides a sensitive means for detecting red cells that are deficient in GPI-linked proteins such as CD59 ( Fig. 14.14 ). The cardinal role of complement activation in PNH pathogenesis has been proven by therapeutic use of a monoclonal antibody called Eculizumab that prevents the conversion of C5 to C5a. This inhibitor not only reduces the hemolysis and attendant transfusion requirements, but also lowers the risk of thrombosis by up to 90%. The drawbacks to C5 inhibitor therapy are its high cost and an increased risk of serious or fatal meningococcal infection (as is true in individuals with inherited complement defects). Immunosuppressive drugs are sometimes beneficial for those with evidence of marrow aplasia. The only cure is hematopoietic stem cell transplantation.
Immunohemolytic Anemia
Immunohemolytic anemia is caused by antibodies that recognize red cells and lead to their premature destruction. Although these disorders are commonly referred to as autoimmune hemolytic anemias, the designation immunohemolytic anemia is preferred because the immune reaction is initiated in some instances by an ingested drug. Immunohemolytic anemia can be classified based on the characteristics of the responsible antibody ( Table 14.4 ).
Table 14.4
Classification of Immunohemolytic Anemia
| Warm Antibody Type (IgG Antibodies Active at 37°C) |
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| Cold Agglutinin Type (IgM Antibodies Active Below 37°C) |
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| Cold Hemolysin Type (IgG Antibodies Active Below 37°C) |
|
The diagnosis of immunohemolytic anemia requires the detection of antibodies and/or complement on red cells from the patient. This is done using the direct Coombs antiglobulin test, in which the patient's red cells are mixed with sera containing antibodies that are specific for human immunoglobulin or complement. If either immunoglobulin or complement is present on the surface of the red cells, the antibodies cause agglutination, which is appreciated visually as clumping. In the indirect Coombs antiglobulin test, the patient's serum is tested for its ability to agglutinate commercially available red cells bearing particular defined antigens. This test is used to characterize the antigen target and temperature dependence of the responsible antibody. Quantitative immunologic tests to measure such antibodies directly also are available.
Warm Antibody Type
This form constitutes approximately 80% of cases of immunohemolytic anemia. It is caused by antibodies that bind stably to red cells at 37°C. About 50% of cases are idiopathic (primary); others are related to a predisposing condition (see Table 14.4 ) or exposure to a drug. Most causative antibodies are of the IgG class; less commonly, IgA antibodies are the culprits. The red cell hemolysis is mostly extravascular. IgG-coated red cells bind to Fc receptors on phagocytes, which remove red cell membrane during “partial” phagocytosis. As in hereditary spherocytosis, the loss of membrane converts the red cells to spherocytes, which are sequestered and destroyed in the spleen. Moderate splenomegaly due to hyperplasia of splenic phagocytes is usually seen.
As with other autoimmune disorders, the cause of primary immunohemolytic anemia is unknown. In cases that are idiopathic, the antibodies are directed against red cell surface proteins, often components of the Rh blood group complex. In drug-induced cases, two mechanisms have been described.
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Antigenic drugs. In this setting hemolysis usually follows large, intravenous doses of the offending drug and occurs 1 to 2 weeks after therapy is initiated. These drugs, exemplified by penicillin and cephalosporins, bind to the red cell membrane and create a new antigenic determinant that is recognized by antibodies. The responsible antibodies sometimes fix complement and cause intravascular hemolysis, but more often they act as opsonins that promote extravascular hemolysis within phagocytes.
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Tolerance-breaking drugs. These drugs, of which the antihypertensive agent α-methyldopa is the prototype, break tolerance in some unknown manner that leads to the production of antibodies against red cell antigens, particularly the Rh blood group antigens. About 10% of patients taking α-methyldopa develop autoantibodies, as assessed by the direct Coombs test, and roughly 1% develop clinically significant hemolysis.
Treatment of warm antibody immunohemolytic anemia centers on the removal of initiating factors (i.e., drugs); when this is not feasible, immunosuppressive drugs and splenectomy are the mainstays.
Cold Agglutinin Type
This type of immunohemolytic anemia is caused by IgM antibodies that bind to red cells avidly at low temperatures (0°C to 4°C) but not at 37°C. It accounts for 15% to 20% of cases. Cold agglutinin antibodies sometimes appear transiently following certain infections, such as with Mycoplasma pneumoniae, Epstein-Barr virus, cytomegalovirus, influenza virus, and human immunodeficiency virus (HIV). In these settings, the disorder is self-limited and the antibodies rarely induce clinically important hemolysis. Chronic cold agglutinin immunohemolytic anemia occurs in association with certain B-cell neoplasms or as an idiopathic condition.
Clinical symptoms result from binding of IgM to red cells in vascular beds where the temperature may fall below 30°C, such as in exposed fingers, toes, and ears. IgM binding agglutinates red cells and fixes complement rapidly. As the blood recirculates and warms, IgM is released, usually before complement-mediated hemolysis can occur; therefore, intravascular hemolysis is usually not seen. However, the transient interaction with IgM is sufficient to deposit sublytic quantities of C3b, an excellent opsonin, which leads to the removal of red cells by phagocytes in the spleen, liver, and bone marrow (extravascular hemolysis). The hemolysis is of variable severity. Vascular obstruction caused by agglutinated red cells may produce pallor, cyanosis, and Raynaud phenomenon ( Chapter 11 ) in parts of the body that are exposed to cold temperatures. Chronic cold agglutinin immunohemolytic anemia caused by IgM antibodies may be difficult to treat. The best approach, when possible, is avoidance of cold temperatures.
Cold Hemolysin Type
Cold hemolysins are autoantibodies responsible for an unusual entity known as paroxysmal cold hemoglobinuria. This rare disorder may cause substantial, sometimes fatal, intravascular hemolysis and hemoglobinuria. The autoantibodies are IgGs that bind to the P blood group antigen on the red cell surface in cool, peripheral regions of the body. Complement-mediated lysis occurs when the cells recirculate to the body's warm core, where the complement cascade functions more efficiently. Most cases are seen in children following viral infections; in this setting the disorder is transient, and most of those affected recover within 1 month.
Hemolytic Anemia Resulting From Trauma to Red Cells
The most significant hemolysis caused by trauma to red cells is seen in individuals with cardiac valve prostheses and microangiopathic disorders. Artificial mechanical cardiac valves are more frequently implicated than are bioprosthetic porcine or bovine valves. The hemolysis stems from shear forces produced by turbulent blood flow and pressure gradients across damaged valves. Microangiopathic hemolytic anemia is most commonly seen with disseminated intravascular coagulation (DIC), but it also occurs in thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), malignant hypertension, systemic lupus erythematosus, and disseminated cancer. The common pathogenic feature in these disorders is microvascular lesions that result in luminal narrowing, often due to the deposition of thrombi, producing shear stresses that mechanically injure passing red cells. Regardless of the cause, traumatic damage leads to intravascular hemolysis and the appearance of red cell fragments (schistocytes), “burr cells,” “helmet cells,” and “triangle cells” in blood smears ( Fig. 14.15 ).
Key Concepts
Hereditary Spherocytosis
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Autosomal dominant disorder caused by mutations that affect the red cell membrane skeleton, leading to loss of membrane and eventual conversion of red cells to spherocytes, which are phagocytosed and removed in the spleen
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Manifested by anemia and splenomegaly
Thalassemias
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Autosomal codominant disorders caused by mutations in α- or β-globin that reduce hemoglobin synthesis, resulting in a microcytic, hypochromic anemia.
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In β-thalassemia, unpaired α-globin chains form aggregates that damage red cell precursors and further impair erythropoiesis.
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Ineffective erythropoesis increases iron absorption and can lead to systemic iron overload.
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