Hematopoietic and Lymphoid Systems

Pathogenesis

Hereditary spherocytosis is caused by inherited defects in the membrane skeleton, a network of proteins that stabilizes the lipid bilayer of the red cell ( Fig. 10.1 ). The major membrane skeleton protein is spectrin, a long, flexible heterodimer that self-associates at one end and binds short actin filaments at its other end. These contacts create a two-dimensional meshwork that is connected to the intrinsic membrane proteins band 3 and glycophorin via linker proteins like ankyrin and band 4.1. The common feature of the mutations that cause hereditary spherocytosis is that they weaken interactions between the membrane skeleton and intrinsic red cell membrane proteins. This results in the destabilization of the lipid bilayer of red cells, which shed membrane vesicles into the circulation as they age. Little cytoplasm is lost in the process and as a result the surface area-to-volume ratio decreases progressively with time until the cells become spherical ( Fig. 10.1 ).

The floppy discoid shape of normal red cells allows considerable latitude for shape changes. By contrast, spherocytes have limited deformability and are sequestered in the splenic cords, where they are destroyed by the plentiful resident macrophages ( Fig. 10.1 ). The critical role of the spleen in hereditary spherocytosis is illustrated by the beneficial effect of splenectomy; although the red cell defect and spherocytes persist, the anemia is corrected.

Morphology

On peripheral blood smears, spherocytes are dark red and lack central pallor ( Fig. 10.2 ). The excessive red cell destruction and resultant anemia lead to a compensatory hyperplasia of red cell progenitors in the marrow and an increase in red cell production marked by reticulocytosis. Splenomegaly is more common and prominent in hereditary spherocytosis than in other forms of hemolytic anemia. The splenic weight is usually between 500 g and 1000 g (normal, 150 g to 200 g). The enlargement results from marked congestion of the splenic cords and increased numbers of macrophages. Phagocytosed red cells are seen within macrophages lining the sinusoids and within the cords. Other general features of hemolytic anemia may also be seen, including cholelithiasis, which occurs in 40% to 50% of patients with hereditary spherocytosis.

Clinical Features

The characteristic features are anemia, splenomegaly, and jaundice. The anemia is variable in severity, ranging from subclinical to profound; most commonly it is moderate in degree. Because of their spherical shape, red cells in hereditary spherocytosis show increased osmotic fragility when placed in hypotonic salt solutions, a characteristic that can help establish the diagnosis.

The course is generally stable but may be punctuated by aplastic crises, the most severe of which are triggered by parvovirus B19 infection . This virus has a marked tropism for erythroblasts, which undergo apoptosis during viral replication. Until the immune response controls the infection (usually in 10 to 14 days), the marrow may be virtually devoid of red cell progenitors. Because of the shortened life span of red cells in hereditary spherocytosis, a lack of red cell production, even for a few days, results in rapid worsening of the anemia. Blood transfusions may be needed to support patients until the infection is cleared.

There is no specific treatment. Splenectomy improves the anemia by removing the major site of red cell destruction. The benefits of splenectomy must be weighed against the increased risk of serious infections by encapsulated bacteria, particularly in children. Partial splenectomy is gaining favor in young children because this approach produces hematologic improvement while maintaining protection against sepsis. The downside is that because the partially resected spleen eventually regains its size, many patients will need a second resection. The hope is that this can be delayed until later in childhood, when the risk of serious infection is lower.

Pathogenesis

Sickle cell anemia is caused by a single amino acid substitution in β-globin that results in a tendency for deoxygenated HbS to self-associate into polymers. Normal hemoglobins are tetramers composed of two pairs of similar chains. On average, the normal adult red cell contains 96% HbA (α ₂ β ₂ ), 3% HbA2 (α ₂ δ ₂ ), and 1% fetal Hb (HbF, α ₂ γ ₂ ). In patients with sickle cell anemia, HbA is completely replaced by HbS, whereas in heterozygous carriers, only about half is replaced. HbS differs from HbA by having a valine residue instead of a glutamate residue at the 6th amino acid position in β-globin. On deoxygenation HbS molecules undergo a conformational change that allows polymers to form via intermolecular contacts involving the abnormal valine residue. These polymers distort the red cell, which assumes an elongated crescentic or sickle shape ( Fig. 10.3 ).

The sickling of red cells is initially reversible on reoxygenation. However, membrane distortion produced by each sickling episode leads to an influx of calcium, which causes the loss of potassium and water and also damages the membrane skeleton. With time, this cumulative damage creates irreversibly sickled cells that are prone to hemolysis.

Three factors are particularly important in determining whether clinically significant polymerization of HbS occurs in patients:

• The intracellular levels of hemoglobins other than HbS. In heterozygotes approximately 40% of Hb is HbS and the remainder is HbA, which interacts only weakly with deoxygenated HbS. Because HbA greatly retards HbS polymerization, the red cells of HbS heterozygotes have little tendency to sickle in vivo. Such persons are said to have sickle cell trait. Similarly, because fetal hemoglobin (HbF) interacts weakly with HbS, newborns with sickle cell anemia do not manifest the disease until HbF falls to adult levels, generally around the age of 5 to 6 months. Hemoglobin C (HbC), another mutant β-globin, has a lysine residue instead of the normal glutamic acid residue at position 6. Like HbS, HbC confers protection to falciparum malaria and is prevalent in similar populations (sub-Saharan Africa, parts of India, etc.). Due to equatorial African ancestry, about 2.3% of Americans of African descent are heterozygous carriers of HbC, and about 1 in 1250 are compound HbC/HbS heterozygotes.

• The intracellular concentration of HbS. The polymerization of deoxygenated HbS is strongly concentration dependent. Thus, red cell dehydration, which increases the Hb concentration, facilitates sickling. Conversely, the coexistence of α-thalassemia (described later), which decreases the Hb concentration, reduces sickling.

• The time required for red cells to pass through the microvasculature. The normal transit times of red cells through capillary beds are too short for significant polymerization of deoxygenated HbS to occur. Hence, the tissues that are most susceptible to obstruction by sickle cells are those in which blood flow is sluggish, such as the spleen and the bone marrow. However, sickling may occur in other microvascular beds in the presence of factors that retard the passage of red cells, particularly inflammation. Recall that inflammation slows blood flow by increasing the adhesion of leukocytes and red cells to endothelium and by inducing the exudation of fluid through leaky vessels ( Chapter 2 ). In addition, sickle red cells have a greater tendency than normal red cells to adhere to endothelial cells, as repeated bouts of sickling cause membrane damage that makes the red cells abnormally “sticky.” These factors conspire to prolong the transit times of sickle red cells, increasing the probability of clinically significant vascular obstruction.

The sickling of red cells has two major pathologic consequences: chronic moderately severe hemolytic anemia, due to red cell membrane damage; and vascular obstructions, which result in ischemic tissue damage and pain crises ( Fig. 10.4 ). The mean life span of red cells in sickle cell anemia averages only 20 days (one-sixth of normal) and the severity of the hemolysis correlates with the fraction of irreversibly sickled cells that are present in the blood. Vasoocclusion, by contrast, is not related to the number of irreversibly sickled cells and instead appears to be triggered by superimposed factors such as infection, inflammation, dehydration, and acidosis, all of which enhance the tendency of red cells to arrest and sickle within the microvasculature.

Clinical Features

Sickle cell disease is marked by chronic hemolytic anemia and superimposed vasoocclusive crises. It is usually asymptomatic until 6 months of age when the shift from HbF to HbS is complete. The anemia is moderate to severe; most patients have hematocrits of 18% to 30% (normal range, 38% to 48%). The chronic hemolysis is associated with hyperbilirubinemia and compensatory reticulocytosis.

Much more serious are vasoocclusive crises, which are characteristically associated with pain and often lead to tissue damage and significant morbidity and mortality. Among the most common and serious of these crises are the following:

• Hand-foot syndrome, resulting from infarction of bones in the hands and feet, is the most common presenting symptom in young children.

• Acute chest syndrome, in which sluggish blood flow in an inflamed lung (e.g., an area of pneumonia) leads to sickling within hypoxemic pulmonary beds. This exacerbates pulmonary dysfunction, creating a vicious circle of worsening pulmonary and systemic hypoxemia, sickling, and vasoocclusion. Acute chest syndrome may also be triggered by fat emboli emanating from infarcted bone.

• Stroke, which sometimes occurs in the setting of the acute chest syndrome. Stroke and the acute chest syndrome are the two leading causes of ischemia-related death.

• Proliferative retinopathy, a consequence of vasoocclusions in the eye that can lead to loss of visual acuity and blindness.

Another acute event, aplastic crisis, is caused by a sudden decrease in red cell production. As in hereditary spherocytosis, this is usually triggered by infection of erythroblasts by parvovirus B19 and, although severe, is self-limited.

In addition to these crises, patients with sickle cell disease are prone to infections. Both children and adults with sickle cell disease are functionally asplenic, making them susceptible to infections caused by encapsulated bacteria, such as pneumococci. In adults the basis for “hyposplenism” is autoinfarction. In the earlier childhood phase of splenic enlargement, congestion caused by trapped sickled red cells apparently interferes with bacterial sequestration and killing; hence, even children with enlarged spleens are at risk for developing fatal septicemia. Patients with sickle cell disease are also predisposed to bacterial osteomyelitis, which may result from bacterial seeding of infarcted bone. The most common causative organisms are encapsulated bacterial species and gram-negative organisms, particularly E. coli and Salmonella .

In homozygous sickle cell disease, irreversibly sickled red cells are seen in routine peripheral blood smears. In sickle cell trait, sickling can be induced in vitro by exposing cells to hypoxia. In the United States, newborn screening for sickle cell disease is now mandated; HbS and other hemoglobins are identified by methods such as gel electrophoresis in blood obtained by heel-stick. Prenatal diagnosis of sickle cell anemia is performed by analyzing fetal DNA obtained by amniocentesis or biopsy of chorionic villi.

The clinical course of sickle cell disease is highly variable. As a result of improvements in supportive care, approximately 50% of patients now survive beyond the fifth decade. Of particular importance is vaccination and prophylactic treatment with penicillin to prevent pneumococcal infections, especially in children younger than age 5. A mainstay of therapy is hydroxyurea, a “gentle” inhibitor of DNA synthesis. Hydroxyurea reduces pain crises and lessens the anemia through several effects, including (1) an increase in levels of HbF; (2) an anti-inflammatory effect because of the inhibition of white cell production; (3) an increase in red cell size, which lowers the intracellular hemoglobin concentration; and (4) its metabolism to NO, a potent vasodilator and inhibitor of platelet aggregation. More recently, encouraging results have been obtained with allogeneic bone marrow transplantation and corrective gene therapy, both of which are potentially curative.

Pathogenesis

A diverse collection of α-globin and β-globin mutations underlie the thalassemias, which are autosomal codominant conditions. As described previously, adult hemoglobin, or HbA, is a tetramer composed of two α chains and two β chains. The α chains are encoded by two α-globin genes lying in tandem on chromosome 16, whereas the β chains are encoded by a single β-globin gene located on chromosome 11. The clinical features vary widely depending on the specific combination of mutated alleles that are inherited by the patient ( Table 10.3 ).

Clinical Features

β-Thalassemia trait and α-thalassemia trait are typically asymptomatic. There is usually only a mild microcytic hypochromic anemia; these patients have a normal life expectancy. Iron deficiency anemia is associated with a similar red cell appearance and must be excluded by appropriate laboratory tests (described later).

β-Thalassemia major manifests postnatally as HbF synthesis diminishes. Affected children have growth retardation that commences in infancy. They are sustained by blood transfusions, which improve the anemia and reduce the skeletal deformities associated with excessive erythropoiesis. With transfusions alone, survival into the second or third decade is possible, but systemic iron overload gradually develops owing to inappropriate uptake of iron from the gut and the iron load in transfused red cells. The excessive uptake of iron from the gut occurs because the expanded pool of erythroblasts secrete a hormone called erythroferrone, which circulates to the liver and suppresses the release of hepcidin (described later). Unless patients are treated aggressively with iron chelators, cardiac dysfunction from secondary hemochromatosis inevitably develops and often is fatal in the second or third decade of life. When feasible, hematopoietic stem cell transplantation at an early age is the treatment of choice.

HbH disease and β-thalassemia intermedia are not as severe as β-thalassemia major because the imbalance in globin chain synthesis is not as profound and hematopoiesis is more effective. Anemia is of moderate severity, patients usually do not require transfusions, and iron overload is rarely seen.

The diagnosis of β-thalassemia major can be strongly suspected on clinical grounds. Hb electrophoresis shows a profound reduction or absence of HbA and increased levels of HbF. The HbA ₂ level may be normal or increased. Similar but less severe changes are noted in patients affected by β-thalassemia intermedia. Prenatal diagnosis of β-thalassemia is challenging but can be made in specialized centers by DNA analysis. In fact, thalassemia was the first disease diagnosed by DNA-based tests, opening the way for the field of molecular diagnostics. The diagnosis of β-thalassemia minor is made by Hb electrophoresis, which typically shows a reduced level of HbA (α ₂ β ₂ ) and an increased level of HbA ₂ (α ₂ δ ₂ ). HbH disease can be diagnosed by detection of β4 tetramers by electrophoresis.

Pathogenesis

G6PD deficiency is typically associated with transient episodes of intravascular hemolysis caused by exposure to an environmental factor (usually infectious agents or drugs) that produces oxidant stress. Incriminated drugs include antimalarials (e.g., primaquine), sulfonamides, nitrofurantoin, phenacetin, aspirin (in large doses), and vitamin K derivatives. Consumption of certain foods, such as fava beans, may also lead to hemolysis. More commonly, episodes of hemolysis are triggered by infection, which induce phagocytes to generate oxidants as part of the host response. These oxidants, such as hydrogen peroxide, are normally sopped up by GSH, which is converted to oxidized GSH in the process. Because regeneration of GSH is impaired in G6PD-deficient cells, oxidants are free to “attack” other red cell components, including globin chains. Oxidized hemoglobin denatures and precipitates, forming intracellular inclusions called Heinz bodies, which can damage the red cell membrane so severely that intravascular hemolysis results. Other cells with less damage lose their deformability and suffer further injury when splenic phagocytes attempt to “pluck out” the Heinz bodies, creating bite cells ( Fig. 10.6 ). Such cells become trapped on recirculation to the spleen and are destroyed by phagocytes.

Clinical Features

Hemolysis typically develops 2 or 3 days after drug exposure and is of variable severity. Because G6PD is X-linked, the red cells of affected males are uniformly deficient and vulnerable to oxidant injury. By contrast, random inactivation of one X chromosome in heterozygous females ( Chapter 4 ) creates two populations of red cells, one normal and the other G6PD deficient. Most carrier females are unaffected except for those with a large proportion of deficient red cells (a chance situation known as unfavorable lyonization). Susceptibility to hemolysis among different mutated forms of G6PD varies according to the degree of G6PD deficiency. In the case of the G6PD A ^- variant, which is common in areas of Africa where malaria is endemic, the half-life of the variant is only modestly decreased. As a result, only older red cells are susceptible to lysis. Because the marrow compensates for the anemia by increasing its production of new red cells with adequate levels of G6PD, the hemolysis abates even if the drug exposure continues. Other variants such as G6PD Mediterranean, found mainly in the Middle East, produce more marked enzyme deficiency and as a result the hemolysis that occurs on exposure to oxidants is more severe.

Pathogenesis

IgM binding to red cells initiates the fixation of complement, but later steps of the complement cascade occur inefficiently at temperatures lower than 37°C. As a result, red cells with bound IgM are coated with complement C3 fragments C3b and C3d but are not lysed intravascularly. When these cells travel to warmer areas, the weakly bound IgM is released, but the coating of C3b and C3d fragments remains. Because C3b and C3d fragments are opsonins ( Chapter 2 ), the cells are phagocytosed by macrophages, mainly in the spleen and liver; hence, in most cases the hemolysis is mainly extravascular. Because IgM is pentavalent, each molecule can bind to more than one red cell, crosslinking red cells and causing them to clump (agglutinate). Sludging of blood in capillaries because of agglutination of red cells often produces Raynaud phenomenon in the extremities of affected individuals.

Pathogenesis

The life cycle of plasmodia is complex and varies among species; Fig. 10.8 shows the life cycle of P. falciparum . As Anopheles mosquitoes feed on human blood, sporozoites are introduced into the circulation and travel through the blood to the liver, where two sporozoite surface proteins, thrombospondin-related adhesive protein and circumsporozoite protein, bind to factors such as proteoglycans on the surface of hepatocytes. The sporozoites then enter the liver and differentiate into merozoites . After an incubation period of 1 to 4 weeks, the infected hepatocytes rupture and release the merozoites. Next, a lectinlike molecule on the surface of the merozoite binds to sialidated glycophorin, a red cell transmembrane protein, allowing the merozoite to invaginate into red cells within a “digestive” vacuole. Intraerythrocytic organisms then differentiate into trophozoites, which follow two paths. Some trophozoites differentiate into gametocytes, which restart the life cycle in mosquitoes when the infected human is again bitten by another Anopheles mosquito. Most trophozoites differentiate into schizonts, which express an adhesion molecule called PfEMP1 ( Plasmodium falciparum erythrocyte membrane protein 1) that concentrates in knoblike extensions on the red cell surface. Normally, red cells bear negatively charged surfaces that interact poorly with endothelial cells, but PfEMP1 binds to adhesion molecules on the surface of endothelium such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD36, causing the parasitized red cells to arrest in capillary beds. After a period of several days, schizonts differentiate into merozoites, leading to lysis of the infected red cell and another cycle of red cell infection.

Fatal falciparum malaria often involves the small vessels of the brain, a complication known as cerebral malaria. In an unfortunate minority of patients, mainly children, this process involves cerebral vessels, which become engorged and occluded.

Clinical Features

Malaria is associated with episodic shaking chills and fever that coincide with the release of “showers” of merozoites from infected red cells at intervals of approximately 24 hours for P. knowlesi; 48 hours for P. vivax, P. ovale, and P. falciparum; and 72 hours for P. malariae. Hemolytic anemia of varying severity is a constant feature. Cerebral malaria associated with P. falciparum is rapidly progressive; convulsions, coma, and death usually occur within days to weeks. Falciparum malaria more often pursues a chronic course that may be punctuated by blackwater fever , a poorly understood complication marked by massive intravascular hemolysis, hemoglobinemia, hemoglobinuria, jaundice, and renal failure.

With appropriate drug therapy, the prognosis for patients with most forms of malaria is good; however, falciparum malaria is becoming more difficult to treat due to the emergence of drug-resistant strains. Because of the potentially serious consequences of the disease, early diagnosis and treatment are important. An encouraging advance is the recent development of a vaccine containing sporozoite antigens; although the protection afforded by the vaccine is partial, when fully deployed it is expected to prevent thousands of cases of fatal cerebral malaria in children. Currently, the best method of preventing malaria is by public health measures such as removal of stagnant bodies of water (mosquitoes breeding sites) close to living areas, use of insecticide-treated bed nets, and prophylactic intake of antimalarial drugs.

Pathogenesis

Iron deficiency arises in a variety of settings:

• Chronic blood loss is the most important cause of iron deficiency anemia in higher-resource countries. The most common sources of bleeding are the gastrointestinal tract (e.g., peptic ulcers, colon cancer, hemorrhoids) and the female genital tract (e.g., menorrhagia, metrorrhagia, endometrial cancer).

• In lower-resource countries, low intake and poor bioavailability of iron because of predominantly vegetarian diets are the most common causes of iron deficiency. In the United States, low dietary intake is infrequent but is sometimes seen in infants fed exclusively milk, in individuals with food insecurity, and the elderly.

• Increased iron demands not met by normal dietary intake occur worldwide during pregnancy and infancy.

• Malabsorption of iron may occur with celiac disease, various forms of gastritis, or after gastrectomy ( Chapter 13 ).

Regardless of cause, iron deficiency develops insidiously. Iron stores are depleted first, marked by a decline in serum ferritin and the absence of stainable iron in bone marrow macrophages. These changes are followed by a decrease in serum iron and a rise in serum transferrin. Ultimately, the capacity to synthesize hemoglobin, myoglobin, and other iron-containing proteins is diminished, leading to microcytic anemia, impaired work and cognitive performance, and even reduced immunocompetence.

Clinical Features

In most instances iron deficiency anemia is mild and asymptomatic. Nonspecific manifestations, such as weakness, listlessness, and pallor, may be present in severe cases. With long-standing anemia, abnormalities of the fingernails, including thinning, flattening, and “spooning,” may appear. A curious but characteristic neurobehavioral complication is pica, the compunction to consume nonfoodstuffs such as dirt or clay.

In peripheral smears, red cells are microcytic and hypochromic ( Fig. 10.10 ). Characteristic findings include decreased hematocrit; hypochromic, microcytic red cell indices; low serum ferritin and iron levels; low transferrin saturation; increased total iron-binding capacity; and, ultimately, response to iron therapy. For unclear reasons, the platelet count is often high. Erythropoietin levels are elevated, but the marrow response is blunted by the iron deficiency; thus, marrow cellularity is usually only slightly increased.

Persons often die with iron deficiency anemia but virtually never of it. An important point to remember is that in well-nourished persons, microcytic hypochromic anemia is not a disease but rather a symptom of another underlying disorder (e.g., colon cancer leading to chronic blood loss).

Pathogenesis

The anemia of chronic inflammation stems from high levels of plasma hepcidin, which blocks the transfer of iron to erythroid precursors by downregulating ferroportin in marrow macrophages. The elevated hepcidin levels are caused by pro-inflammatory cytokines such as IL-6, which increase hepatic hepcidin synthesis. In addition, chronic inflammation blunts erythropoietin synthesis by the kidney, lowering red cell production by the marrow. The functional advantages of these adaptations in the face of systemic inflammation are unclear; they may serve to inhibit the growth of iron-dependent microorganisms.

Clinical Features

Serum iron levels usually are low in the anemia of chronic disease, and the red cells may be slightly hypochromic and microcytic. In contrast to iron deficiency anemia, however, storage iron in the bone marrow and serum ferritin are increased and the total iron-binding capacity is reduced. Administration of erythropoietin and iron can improve the anemia but only effective treatment of the underlying condition is curative.

Pathogenesis

Megaloblastic anemia stems from metabolic defects that lead to inadequate biosynthesis of thymidine, one of the building blocks of DNA. Folate and vitamin B ₁₂ are both essential for thymidine synthesis, which is required for DNA replication. Thymidine deficiency causes abnormalities in rapidly dividing cells throughout the body, but the hematopoietic marrow is most severely affected. Because the synthesis of RNA and cytoplasmic elements proceeds in parallel at a relatively normal rate and thus outpaces that of DNA, the hematopoietic precursors show nuclear-cytoplasmic asynchrony (described later). This maturational derangement contributes to the anemia in several ways. DNA synthesis is so defective in many red cell progenitors that a DNA damage response is triggered, leading to apoptosis (ineffective hematopoiesis) . Other progenitors mature into red cells but do so after fewer cell divisions, further diminishing the output of red cells. Granulocyte and platelet precursors are also affected (although not as severely), and most patients present with pancytopenia (anemia, thrombocytopenia, and granulocytopenia).

Morphology

Certain morphologic features are common to all megaloblastic anemias. The bone marrow is markedly hypercellular and contains numerous megaloblastic erythroid progenitors. Megaloblasts are larger than normal erythroid progenitors (normoblasts) and have delicate, finely reticulated nuclear chromatin (indicative of nuclear immaturity). As megaloblasts differentiate and acquire hemoglobin, the nucleus retains its finely distributed chromatin and fails to undergo the chromatin clumping typical of normoblasts, a classic example of nuclear-cytoplasmic asynchrony. Granulocytic precursors also demonstrate nuclear-cytoplasmic asynchrony, yielding giant metamyelocytes. Megakaryocytes may also be abnormally large and contain bizarre multilobed nuclei.

In the peripheral blood the earliest change is the appearance of hypersegmented neutrophils ( Fig. 10.11 ), which appear before the onset of anemia. Normal neutrophils have three or four nuclear lobes, but in megaloblastic anemias they often have five or more. The red cells typically include large egg-shaped macroovalocytes; the MCV is often greater than 110 fL (normal, 78–98 fL). Although macroovalocytes appear hyperchromic, in reality their hemoglobin concentration is normal. Large, misshapen platelets also may be seen. Morphologic changes in other systems, especially the gastrointestinal tract, also occur, giving rise to some of the clinical manifestations.

Pathogenesis

Tetrahydrofolate acts as an acceptor and donor of one-carbon units in several reactions that are required for the synthesis of deoxythymidine monophosphate (dTMP), which is used in DNA synthesis. Although the metabolism and functions of folate are complex, here it is sufficient to note that its conversion within cells from dihydrofolate to tetrahydrofolate by dihydrofolate reductase (the target of the drug methotrexate) is particularly important for dTMP synthesis. If intracellular stores of folate fall, insufficient dTMP is synthesized and DNA replication is blocked, leading to megaloblastic anemia.

Clinical Features

The onset of the anemia of folate deficiency is insidious, associated with nonspecific symptoms such as weakness and easy fatigability. The clinical picture may be complicated by coexistent deficiency of other vitamins, especially in those with alcohol use disorder. Because the cells lining the gastrointestinal tract, like the hematopoietic system, turn over rapidly, symptoms referable to the alimentary tract, such as sore tongue, are common. Unlike in vitamin B ₁₂ deficiency, neurologic abnormalities do not occur.

The diagnosis of a megaloblastic anemia is readily made from the examination of smears of peripheral blood and bone marrow. The anemia of folate deficiency is best distinguished from that of vitamin B ₁₂ deficiency by measuring serum and red cell folate and vitamin B ₁₂ levels.

Pathogenesis

The most frequent cause vitamin B ₁₂ deficiency is pernicious anemia, which results from an autoimmune attack on the gastric mucosa that suppresses the production of intrinsic factor ( Chapter 13 ). Histologically, there is a chronic atrophic gastritis marked by a loss of parietal cells, a prominent infiltrate of lymphocytes and plasma cells, and megaloblastic changes in mucosal cells similar to those found in erythroid precursors. The serum of most affected patients contains several types of autoantibodies that block the binding of vitamin B ₁₂ to intrinsic factor or prevent binding of the intrinsic factor–vitamin B ₁₂ complex to cubilin. Autoantibodies are of diagnostic use, but they are not thought to be the primary cause of the gastric pathology; rather, it seems that an autoreactive T-cell response initiates gastric mucosal injury and triggers the formation of autoantibodies. When the mass of intrinsic factor–secreting cells falls below a threshold (and reserves of stored vitamin B ₁₂ are depleted), anemia develops.

Other causes of vitamin B ₁₂ malabsorption include gastrectomy (leading to loss of intrinsic factor–producing cells), ileal resection (resulting in loss of intrinsic factor–B ₁₂ complex–absorbing cells), and disorders that disrupt the function of the distal ileum (such as Crohn disease, environmental enteropathy, and Whipple disease). Particularly in older persons, gastric atrophy and achlorhydria may interfere with the production of acid and pepsin, which are needed to release vitamin B ₁₂ from its bound form in food.

The metabolic defects responsible for the anemia of vitamin B ₁₂ deficiency are intertwined with folate metabolism. Vitamin B ₁₂ is required for recycling tetrahydrofolate, the form of folate needed for DNA synthesis. In keeping with this relationship, the anemia of vitamin B ₁₂ deficiency is reversed by administration of folate. Importantly, however, folate administration does not prevent and may worsen neurologic symptoms that are specific to vitamin B ₁₂ deficiency. The main neurologic lesions associated with vitamin B ₁₂ deficiency are demyelination of the posterior and lateral columns of the spinal cord, sometimes beginning in the peripheral nerves. In time, axonal degeneration may supervene. The severity of the neurologic manifestations is not related to the degree of anemia and neurologic disease may even occur in the absence of overt megaloblastic anemia.

Clinical Features

The manifestations of vitamin B ₁₂ deficiency are nonspecific. As with all anemias, findings include pallor, easy fatigability, and (in severe cases) dyspnea and congestive heart failure. The increased destruction of erythroid progenitors because of ineffective erythropoiesis may give rise to mild jaundice. Megaloblastic changes in the oropharyngeal epithelium may produce a beefy red tongue. The spinal cord disease (subacute combined degeneration) begins with symmetric numbness, tingling, and burning in the feet or hands, followed by unsteadiness of gait and loss of position sense, particularly in the toes. Although the anemia responds dramatically to parenteral vitamin B ₁₂ , the neurologic manifestations often fail to resolve. As discussed in Chapter 13 , patients with pernicious anemia have an increased risk for the development of gastric carcinoma.

Findings supporting the diagnosis of vitamin B ₁₂ deficiency are (1) low serum vitamin B ₁₂ levels, (2) normal or elevated serum folate levels, (3) moderate to severe macrocytic anemia, (4) leukopenia with hypersegmented granulocytes, and (5) a dramatic increase in reticulocytes 2 to 3 days after treatment with vitamin B ₁₂ . Pernicious anemia is associated with all of these findings plus the presence of serum antibodies to intrinsic factor.

Pathogenesis

The pathogenesis of aplastic anemia is not fully understood, but two major etiologies have been invoked: an extrinsic, immune-mediated suppression of marrow progenitors and an intrinsic abnormality of stem cells. Experimental studies have focused on a model in which activated T cells suppress hematopoietic stem cells. It is hypothesized that stem cells are first antigenically altered by exposure to drugs, infectious agents, or other unidentified environmental insults. This provokes a cellular immune response, during which activated Th1 cells produce cytokines that suppress hematopoietic progenitors. In keeping with this scenario, immunosuppressive therapy directed against T cells restores hematopoiesis in 60% to 70% of patients.

Alternatively, the notion that aplastic anemia results from an intrinsic stem cell abnormality is supported by observations showing that from 5% to 10% of patients have inherited defects in telomerase, which is needed for the maintenance and stability of chromosomes. It is hypothesized that telomerase defects lead to premature senescence of hematopoietic stem cells. Of further interest, the bone marrow cells in an additional 50% of cases have unusually short telomeres, possibly stemming from as-yet undiscovered defects in telomerase or excessive replication of hematopoietic stem cells. These two mechanisms are not mutually exclusive, because genetically altered stem cells (e.g., those with abnormal telomeres) also might express “neoantigens” that serve as targets for a T-cell attack.

Clinical Features

Aplastic anemia affects persons of all ages and both sexes. The slowly progressive anemia causes the insidious development of weakness, pallor, and dyspnea. Thrombocytopenia often manifests with petechiae and ecchymoses. Neutropenia may set the stage for serious infections.

It is important to separate aplastic anemia from anemia caused by marrow infiltration (myelophthisic anemia), “aleukemic leukemia,” and granulomatous diseases, which may have similar clinical presentations but are easily distinguished by examination of the bone marrow. Aplastic anemia does not cause splenomegaly; if present, another diagnosis should be sought.

The prognosis is unpredictable. Withdrawal of an inciting drug sometimes leads to recovery, but this is the exception. Hematopoietic stem cell transplantation is often curative, particularly in patients younger than 40 years of age. Transfusion necessary to correct anemia sensitizes patients to alloantigens, producing a high rate of engraftment failure; thus, it must be minimized in persons eligible for transplantation. Successful transplantation requires “conditioning” with immunosuppressive radiation or chemotherapy, reinforcing the notion that autoimmunity has an important role in the disease. As mentioned earlier, patients who are not transplant candidates often benefit from immunosuppressive therapy.