90_02

Sickle Cell Syndromes

The sickle cell syndromes are caused by a mutation in the -globin gene that changes the sixth amino acid from glutamic acid to valine. HbS (226 GluVa1) polymerizes reversibly when deoxygenated to form a gelatinous network of fibrous polymers that stiffen the erythrocyte membrane, increase viscosity, and cause dehydration due to potassium leakage and calcium influx (Fig. 91-3). These changes also produce the characteristic sickle shape. Sickled cells lose the pliability needed to traverse small capillaries. They possess altered sticky membranes (especially reticulocytes) that are abnormally adherent to the endothelium of small venules. These abnormalities provoke unpredictable episodes of microvascular vasoocclusion and premature RBC destruction (hemolytic anemia). Hemolysis occurs because the abnormal erythrocytes are destroyed by the spleen. The rigid adherent cells also clog small capillaries and venules, causing tissue ischemia, acute pain, and gradual end-organ damage. This venoocclusive component usually dominates the clinical course. Prominent manifestations include episodes of ischemic pain (i.e., painful crises) and ischemic malfunction or frank infarction in the spleen, central nervous system, bones, liver, kidneys, and lungs (Fig. 91-3).


Figure 91-3 Pathophysiology of sickle cell crisis.


Several sickle syndromes occur as the result of inheritance of HbS from one parent and another hemoglobinopathy, such as thalassemia or HbC (226 GluLys), from the other parent. The prototype disease, sickle cell anemia, is the homozygous state for HbS (Table 91-2).

Clinical Manifestations of Sickle Cell Anemia

Most patients with sickling syndromes suffer from hemolytic anemia, with hematocrits from 15 to 30%, and significant reticulocytosis. Anemia was once thought to exert protective effects against vasoocclusion by reducing blood viscosity. However, natural history and drug therapy trials suggest that an increase in the hematocrit and feedback inhibition of reticulocytosis might be beneficial, even at the expense of increased blood viscosity. The role of adhesive reticulocytes in vasoocclusion might account for these paradoxical effects.

Granulocytosis is common. The white count can fluctuate substantially and unpredictably during and between painful crises, infectious episodes, and other intercurrent illnesses.

Vasoocclusion causes protean manifestations. Intermittent episodes of vasoocclusion in connective and musculoskeletal structures produce painful ischemia manifested by acute pain and tenderness, fever, tachycardia, and anxiety. These recurrent episodes, called painful crises, are the most common clinical manifestation. Their frequency and severity vary greatly. Pain can develop almost anywhere in the body and may last from a few hours to 2 weeks. Repeated crises requiring hospitalization (>3 per year) correlate with reduced survival in adult life, suggesting that these episodes are associated with accumulation of chronic end-organ damage. Provocative factors include infection, fever, excessive exercise, anxiety, abrupt changes in temperature, hypoxia, or hypertonic dyes.

Repeated microinfarction can destroy tissues having microvascular beds that promote sickling. Thus, the spleen is frequently lost within the first 18 to 36 months of life, causing susceptibility to infection, particularly by pneumococci. Acute venous obstruction of the spleen (splenic sequestration crisis), a rare occurrence in early childhood, may require emergency transfusion and/or splenectomy to prevent trapping of the entire arterial output in the obstructed spleen. Occlusion of retinal vessels can produce hemorrhage, neovascularization, and eventual detachments. Renal papillary necrosis invariably produces isosthenuria. More widespread renal necrosis leads to renal failure in adults, a common late cause of death. Bone and joint ischemia can lead to aseptic necrosis, especially of the femoral or humeral heads; chronic arthropathy; and unusual susceptibility to osteomyelitis, which may be caused by organisms, such as Salmonella, rarely encountered in other settings. The hand-foot syndrome is caused by painful infarcts of the digits and dactylitis. Stroke is especially common in children, a small subset of whom tend to suffer repeated episodes; stroke is less common in adults and is often hemorrhagic. A particularly painful complication in males is priapism, due to infarction of the penile venous outflow tracts; permanent impotence is a frequent consequence. Chronic lower leg ulcers probably arise from ischemia and superinfection in the distal circulation.

Acute chest syndrome is a distinctive manifestation characterized by chest pain, tachypnea, fever, cough, and arterial oxygen desaturation. It can mimic pneumonia, pulmonary emboli, bone marrow infarction and embolism, myocardial ischemia, or in situ lung infarction. Acute chest syndrome is thought to reflect in situ sickling within the lung producing pain and temporary pulmonary dysfunction. It is frequently difficult or impossible to distinguish among other possibilities. Pulmonary infarction and pneumonia are the most frequent underlying or concomitant conditions in patients with this syndrome. Repeated episodes of acute chest pain correlate with reduced survival. Acutely, reduction in arterial oxygen saturation is especially ominous because it promotes sickling on a massive scale. Chronic acute or subacute pulmonary crises lead to pulmonary hypertension and cor pulmonale, an increasingly common cause of death as patients survive further into adult life.

Sickle cell syndromes are remarkable for their clinical heterogeneity. Some patients remain virtually asymptomatic into or even through adult life, while others suffer repeated crises requiring hospitalization from early childhood. Patients with sickle thalassemia and sickle-HbE tend to have similar, slightly milder, symptoms, perhaps because of the ameliorating effects of production of other hemoglobins within the RBC. Hemoglobin SC disease, one of the more common variants of sickle cell anemia, is frequently marked by lesser degrees of hemolytic anemia and a greater propensity for the development of retinopathy and aseptic necrosis of bones. In most respects, however, the clinical manifestations resemble sickle cell anemia. Some rare hemoglobin variants actually aggravate the sickling phenomenon.

Clinical Manifestations of Sickle Cell Trait

Sickle cell trait is usually asymptomatic. Anemia and painful crises are exceedingly rare. An uncommon, but highly distinctive, symptom is painless hematuria often occurring in adolescent males, probably due to papillary necrosis. Isosthenuria is a more common manifestation of the same process. Sloughing of papillae with ureteral obstruction has been reported, as have isolated cases of massive sickling or sudden death due to exposure to high altitudes or extraordinary extremes of exercise and dehydration.

Diagnosis

Sickle cell syndromes are readily suspected on the basis of characteristic hemolytic anemia, red cell morphology (Fig. 91-4), and intermittent episodes of ischemic pain. Diagnosis is confirmed by hemoglobin electrophoresis and the sickling tests already discussed. Thorough characterization of the exact hemoglobin profile of the patient is important, because sickle thalassemia and hemoglobin SC disease are correlated with alterations in prognosis or clinical features. Diagnosis is usually established in childhood, but occasional patients, often with compound heterozygous states, do not develop symptoms until the onset of puberty, pregnancy, or early adult life. Genotyping of family members and potential parental partners is critical for genetic counseling. Details of the childhood history establish prognosis and eligibility for aggressive or experimental therapies. Factors associated with increased morbidity and reduced survival are more than three crises requiring hospitalization per year, a chronic neutrophilia, a history of splenic sequestration or hand-foot syndrome, and second episodes of acute chest syndrome. Patients with a history of cerebrovascular accidents are at higher risk for repeated episodes and require especially close monitoring.


Figure 91-4 Sickle cell anemia. The elongated and crescent-shaped red blood cells seen on this smear represent circulating irreversibly sickled cells. Target cells and a nucleated red blood cell are also seen.


Treatment

Patients with sickle cell syndromes require ongoing continuity of care. Familiarity with the pattern of symptoms provides the best safeguard against excessive use of the emergency room, hospitalization, and habituation to addictive narcotics. Additional preventive measures include regular slit-lamp examinations to monitor development of retinopathy; antibiotic prophylaxis appropriate for splenectomized patients during dental or other invasive procedures; and vigorous oral hydration during or in anticipation of periods of extreme exercise, exposure to heat or cold, emotional stress, or infection. Pneumococcal and Haemophilus influenzae vaccines are less effective in splenectomized individuals. Thus, patients with sickle cell anemia should be vaccinated early in life.

The management of acute painful crisis includes vigorous hydration, thorough evaluation for underlying causes (such as infection), and aggressive analgesia administered by a standing order and/or patient-controlled analgesia (PCA) pump. Morphine (0.1 to 0.15 mg/kg every 3 to 4 h) or meperidine (0.75 to 1.5 mg/kg every 2 to 4 h) should control severe pain. Meperidine should be used only for acute short-term pain control; as a chronic analgesic, it is unsuitable. Bone pain may respond as well to ketorolac (30 to 60 mg initial dose, then 15 to 30 mg every 6 to 8 h). Inhalation of nitrous oxide can provide short-term pain relief, but great care must be exercised to avoid hypoxia and respiratory depression. Nitrous oxide also elevates O2 affinity, reducing O2 delivery to tissues. Its use should be restricted to experts. Many crises can be managed at home with oral hydration and oral analgesia. Use of the emergency room should be reserved for especially severe symptoms or circumstances in which other processes, e.g., infection, are strongly suspected. Nasal oxygen should be employed as appropriate to protect arterial saturation. Most crises resolve in 1 to 7 days. Use of blood transfusion should be reserved for extreme cases: no evidence exists for a beneficial effect in shortening the duration of the crisis.

No tests are definitive to diagnose acute painful crisis. Critical to good management is an approach that recognizes that most patients reporting crisis symptoms do indeed have crisis or another significant medical problem. Diligent diagnostic evaluation for underlying causes is imperative, even though these are found infrequently. In adults, the possibility of aseptic necrosis or sickle arthropathy must be considered, especially if pain and immobility become repeated or chronic at a single site. Nonsteroidal anti-inflammatory agents are often effective for sickle cell arthropathy.

Acute chest syndrome is a medical emergency that may require management in an intensive care unit. Hydration should be monitored carefully to avoid the development of pulmonary edema, and oxygen therapy should be especially vigorous for protection of arterial saturation. Diagnostic evaluation for pneumonia and pulmonary embolism should be especially thorough, since these may occur with atypical symptoms. Critical interventions are transfusion to maintain a hematocrit >30, and emergency exchange transfusion if arterial saturation drops to <90%. As patients with sickle cell syndrome increasingly survive into their fifth and sixth decades, end-stage renal failure and pulmonary hypertension are becoming increasingly prominent causes of end-stage morbidity. Anecdotal evidence suggests that a sickle cell cardiomyopathy and/or premature coronary artery disease may compromise cardiac function in later years. Sickle cell patients have received kidney transplants, but they often experience an increase in the frequency and severity of crises, possibly due to increased infection as a consequence of immunosuppression.

The most significant advance in the therapy of sickle cell anemia has been the introduction of hydroxyurea as a mainstay of therapy for patients with severe symptoms. Hydroxyurea (10 to 30 mg/kg per day) increases fetal hemoglobin and may also exert beneficial affects on red cell hydration, vascular wall adherence, and suppression of the granulocyte and reticulocyte counts; indeed, dosage is titrated to maintain a white count between 5000 and 8000 cells/L. White cells and reticulocytes may play a major role in the pathogenesis of sickle cell crisis, and their suppression may be an important benefit of hydroxyurea therapy.

Hydroxyurea should be considered in patients experiencing repeated episodes of acute chest syndrome or with more than three crises per year requiring hospitalization. The utility of this agent for reducing the incidence of other complications (priapism, retinopathy) is under evaluation, as are the long-term side effects. The preponderance of evidence, however, is that hydroxyurea offers broad benefits to most patients whose disease is severe enough to impair their functional status. One long-term study suggests that hydroxyurea may improve survival. A number of experts are advocating more widespread use of this agent. HbF levels increase in most patients within a few months.

The antitumor drug, 5-azacytidine, was the first agent found to elevate HbF. It never achieved widespread use because of concerns about acute toxicity and carcinogenesis. However, low doses of the related agent, 5-deoxyazacytidine (decitabine) can elevate HbF with acceptable toxicity.

Bone marrow transplantation can provide definitive cures but is known to be effective and safe only in children. Prognostic features justifying bone marrow transplant are the presence of repeated crises early in life, a high neutrophil count, or the development of hand-foot syndrome. Children at risk for stroke can now be identified through the use of Doppler ultrasound techniques. Prophylactic exchange transfusion appears to substantially reduce the risk of stroke in this population. Children who do suffer a cerebrovascular accident should be maintained for at least 3 to 5 years on a program of vigorous exchange transfusion, since the risk of second strokes is extremely high in this population.

Gene therapy for sickle cell anemia is being intensively pursued, but no safe measures are currently available. Agents blocking RBC dehydration or vascular adhesion, such as clotrimazole or magnesium, may have value as an adjunct to hydroxyurea therapy, pending the completion of ongoing trials. Combinations of clotrimazole and magnesium are being evaluated in clinical trials.

Unstable Hemoglobins

Amino acid substitutions that reduce solubility or increase susceptibility to oxidation produce unstable hemoglobins that precipitate, forming inclusion bodies injurious to the red cell membrane. Representative mutations are those that interfere with contact points between the and subunits [e.g., Hb Philly (35TyrPhe)], alter the helical segments [e.g., Hb Genova (28LeuPro)], or disrupt interactions of the hydrophobic pockets of the globin subunits with heme [e.g., Hb Koln (98ValMet)] (Table 91-3). The inclusions, called Heinz bodies, are clinically detectable by staining with supravital dyes such as crystal violet (Heinz body test). Removal of these inclusions by the spleen generates pitted, rigid cells that have shortened life spans, producing hemolytic anemia of variable severity, sometimes requiring chronic transfusion support. Splenectomy may be needed to correct the anemia. Leg ulcers and premature gallbladder disease due to bilirubin turnover are frequent stigmata.

Unstable hemoglobins occur sporadically, often by spontaneous new mutations. Heterozygotes are often symptomatic because a significant Heinz body burden can develop even when the unstable variant accounts for a portion of the total hemoglobin. Symptomatic unstable hemoglobins tend to be -globin variants, because sporadic mutations affecting only one of the four globins would generate only 20 to 30% abnormal hemoglobin.

Hemoglobins with Altered Oxygen Affinity

High-affinity hemoglobins[e.g., Hb Yakima (99 AspHis)] bind oxygen more readily but deliver less O2 to tissues at normal capillary PO2 levels (Fig. 91-2). Mild tissue hypoxia ensues, stimulating RBC production and erythrocytosis (Table 91-3). In extreme cases, the hematocrits can rise to 60 to 65%, increasing blood viscosity and producing typical symptoms (headache, somnolence, or dizziness). Phlebotomy may be required. Typical mutations alter interactions within the heme pocket or disrupt the Bohr effect or salt-bond site. Mutations that impair the interaction of HbA with 2,3-BPG can increase O2 affinity because 2,3-BPG binding lowers O2 affinity.

Low-affinity hemoglobins[e.g., Hb Kansas (102AsnLys)] bind sufficient oxygen in the lungs, despite their lower oxygen affinity, to achieve nearly full saturation. At capillary oxygen tensions, they lose sufficient amounts of oxygen to maintain homeostasis at a low hematocrit (Fig. 91-2) (pseudoanemia). Capillary hemoglobin desaturation can also be sufficient to produce clinically apparent cyanosis. Despite these findings, patients usually require no specific treatment.

Methemoglobinemias

Methemoglobin is generated by oxidation of the heme iron moieties to the ferric state, causing a characteristic bluish-brown muddy color resembling cyanosis. Methemoglobin has such high oxygen affinity that virtually no oxygen is delivered to tissues. Levels >50 to 60% are often fatal.

Congenital methemoglobinemia arises from globin mutations that stabilize iron in the ferric state [e.g., HbM Iwata (87 HisTyr), Table 91-3] or from mutations that impair the enzymes that reduce methemoglobin to hemoglobin (e.g., methemoglobin reductase, NADP diaphorase). Acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds.

Diagnosis and Management of Patients with Unstable Hemoglobins, High-Affinity Hemoglobins, and Methemoglobinemia

Unstable hemoglobin variants should be suspected in patients with nonimmune hemolytic anemia, jaundice, splenomegaly, or premature biliary tract disease. Severe hemolysis usually presents during infancy as neonatal jaundice or anemia. Milder cases may present in adult life with anemia or only as unexplained reticulocytosis, hepatosplenomegaly, premature biliary tract disease, or leg ulcers. Because spontaneous mutation is common, family history of anemia may be absent. The peripheral blood smear often shows anisocytosis, abundant cells with punctate inclusions, and irregular shapes (i.e., poikilocytosis).

The two best tests for diagnosing unstable hemoglobins are the Heinz body preparation and the isopropanol or heat stability test. Many unstable Hb variants are electrophoretically silent. A normal electrophoresis does not rule out the diagnosis.

Severely affected patients may require transfusion support for the first 3 years of life, because splenectomy before age 3 is associated with a significantly higher immune deficit. Splenectomy is usually effective thereafter, but occasional patients may require lifelong transfusion support. Even after splenectomy, patients can develop cholelithiasis and leg ulcers. Splenectomy can also be considered in patients exhibiting severe secondary complications of chronic hemolysis, even if anemia is absent. Precipitation of unstable hemoglobins is aggravated by oxidative stress, e.g., infection, antimalarial drugs.

High-O2 affinity hemoglobin variants should be suspected in patients with erythrocytosis. The best test for confirmation is measurement of the P50. A high-O2 affinity Hb causes a significant left shift (i.e., lower numeric value of the P50); confounding conditions, e.g., tobacco smoking or carbon monoxide exposure, can also lower the P50.

High-affinity hemoglobins are often asymptomatic; rubor or plethora may be telltale signs. When the hematocrit reaches to 55 to 60%, symptoms of high blood viscosity and sluggish flow (headache, lethargy, dizziness, etc.) may be present. These persons may benefit from judicious phlebotomy. Erythrocytosis represents an appropriate attempt to compensate for the impaired oxygen delivery by the abnormal variant. Overzealous phlebotomy may stimulate increased erythropoiesis or aggravate symptoms by thwarting this compensatory mechanism. The guiding principle of phlebotomy should be to improve oxygen delivery by reducing blood viscosity and increasing blood flow rather than restoration of a normal hematocrit. Modest iron deficiency may aid in control.

Low-affinity hemoglobins should be considered in patients with cyanosis or a low hematocrit with no other reason apparent after thorough evaluation. The P50 test confirms the diagnosis. Counseling and reassurance are the interventions of choice.

Methemoglobin should be suspected in patients with hypoxic symptoms who appear cyanotic but have a PaO2 sufficiently high that hemoglobin should be fully saturated with oxygen. A history of nitrite or other oxidant ingestions may not always be available; some exposures may be unapparent to the patient, and others may result from suicide attempts. The characteristic muddy appearance of freshly drawn blood can be a critical clue. The diagnostic test of choice is measurement of the methemoglobin content, which is usually available on an emergency basis.

Methemoglobinemia often causes symptoms of cerebral ischemia at levels >15%; levels >60% are usually lethal. Intravenous injection of 1 mg/kg of methylene blue is effective emergency therapy. Milder cases and follow-up of severe cases can be treated orally with methylene blue (60 mg three to four times each day) or ascorbic acid (300 to 600 mg/d).

Thalassemia Syndromes

The thalassemia syndromes are inherited disorders of - or -globin biosynthesis. The reduced supply of globin diminishes production of hemoglobin tetramers, causing hypochromia and microcytosis. Unbalanced accumulation of and subunits occurs because the synthesis of the unaffected globins proceeds at a normal rate. Unbalanced chain accumulation dominates the clinical phenotype. Clinical severity varies widely, depending on the degree to which the synthesis of the affected globin is impaired, altered synthesis of other globin chains, and coinheritance of other abnormal globin alleles.

Clinical Manifestations of -Thalassemia Syndromes

Mutations causing thalassemia can affect any step in the pathway of globin gene expression: transcription, processing of the mRNA precursor, translation, and posttranslational metabolism of the -globin polypeptide chain. The most common forms arise from mutations that derange splicing of the mRNA precursor or prematurely terminate translation of the mRNA.

Hypochromia and microcytosis characterize all forms of thalassemia because of the reduced amounts of hemoglobin tetramers (Fig. 91-5). In heterozygotes (-thalassemia trait), this is the only abnormality seen. Anemia is minimal. In more severe homozygous states, unbalanced - and -globin accumulation causes accumulation of highly insoluble unpaired chains. They form toxic inclusion bodies that kill developing erythroblasts in the marrow. Few of the proerythroblasts beginning erythroid maturation survive. The few RBCs surviving bear a burden of inclusion bodies that are detected in the spleen, shortening the RBC life span and producing severe hemolytic anemia. The resulting profound anemia stimulates erythropoietin release and compensatory erythroid hyperplasia, but the marrow response is sabotaged by ineffective erythropoiesis. Anemia persists. Erythroid hyperplasia can become exuberant and produce masses of extramedullary erythropoietic tissue in the liver and spleen.


Figure 91-5 -Thalassemia intermedia. Microcytic and hypochromic red blood cells are seen that resemble the red blood cells of severe iron deficiency anemia. Many elliptical and teardrop-shaped red blood cells are noted.


Massive bone marrow expansion deranges growth and development. Children develop characteristic chipmunk facies due to maxillary marrow hyperplasia and frontal bossing, thinning and pathologic fracture of long bones and vertebrae due to cortical invasion by erythroid elements, and profound growth retardation. Hemolytic anemia causes hepatosplenomegaly, leg ulcers, gallstones, and high-output congestive heart failure. The conscription of caloric resources to support erythropoiesis leads to inanition, susceptibility to infection, endocrine dysfunction, and in the most severe cases, death during the first decade of life. Chronic transfusions with RBCs improves oxygen delivery, suppresses the excessive ineffective erythropoiesis, and prolongs life, but the inevitable side effects, notably iron overload, usually prove fatal by age 30.

Severity is highly variable. Known modulating factors are those that ameliorate the burden of unpaired -globin inclusions. Alleles associated with milder synthetic defects and coinheritance of -thalassemia trait reduce clinical severity by reducing accumulation of excess globin. HbF persists to various degrees in thalassemias. -Globin gene chains can substitute for chains, simultaneously generating more hemoglobin and reducing the burden of -globin inclusions. The terms -thalassemia major and -thalassemia intermedia are used to reflect the clinical heterogeneity. Patients with -thalassemia major require intensive transfusion support to survive. Patients with -thalassemia intermedia have a somewhat milder phenotype and can survive without transfusion. The terms -thalassemia minor and -thalassemia trait describe asymptomatic heterozygotes for -thalassemia.

-Thalassemia Syndromes

The four classic -thalassemias, most common in Asians, are -thalassemia-2 trait, in which one of the four -globin loci is deleted; -thalassemia-1 trait, with two deleted loci; HbH disease, with three loci deleted; and hydrops fetalis with Hb Bart's, with all four loci deleted (Table 91-4). Nondeletion forms of -thalassemia also exist.

-Thalassemia-2 trait is an asymptomatic, silent carrier state. -Thalassemia-1 trait resembles -thalassemia minor. Offspring doubly heterozygous for -thalassemia-2 and -thalassemia-1 exhibit a more severe phenotype called HbH disease. Heterozygosity for a deletion that removes both genes from the same chromosome (cis deletion) is common in Asians and Mediterranean individuals, as is homozygosity for -thalassemia-2 (trans deletion). Both produce asymptomatic hypochromia and microcytosis.

In HbH disease, HbA production is only 25 to 30% of normal. Fetuses accumulate some unpaired chains. In adults, unpaired chains accumulate and are soluble enough to form 4 tetramers called HbH. HbH forms few inclusions in erythroblasts but does not precipitate in circulating red cells. Patients with HbH disease have thalassemia intermedia characterized by moderately severe hemolytic anemia but milder ineffective erythropoiesis. Survival into mid-adult life without transfusions is common.

The homozygous state for the -thalassemia-1 cis deletion (hydrops fetalis) causes total absence of -globin synthesis. No physiologically useful hemoglobin is produced beyond the embryonic stage. Excess globin forms tetramers called Hb Barts (4), which has an extraordinarily high oxygen affinity. It delivers almost no O2 to fetal tissues, causing tissue asphyxia, edema (hydrops fetalis), congestive heart failure, and death in utero. -Thalassemia-2 trait is common (15 to 20%) among people of African descent. The cis-thalassemia-1 deletion is almost never seen, however. Thus, -thalassemia-2 and the trans form of -thalassemia-1 are very common, but HbH disease and hydrops fetalis are almost never encountered.

It has been known for some time that some patients with myelodysplasia or erythroleukemia produce red cell clones containing HbH. It now appears that this phenomenon is due to mutations in the ATRX pathway that affect the LCR of the -globin gene cluster.

Diagnosis and Management of Thalassemias

The diagnosis of -thalassemia major is readily made during childhood on the basis of severe anemia accompanied by the characteristic signs of massive ineffective erythropoiesis: hepatosplenomegaly, profound microcytosis, a characteristic blood smear (Fig. 91-5), and elevated levels of HbF, HbA2, or both. Many patients require chronic hypertransfusion therapy designed to maintain a hematocrit of at least 27 to 30% so that erythropoiesis is suppressed. Splenectomy is required if the annual transfusion requirement (volume of RBCs per kilogram of body weight per year) increases by >50%. Folic acid supplements may be useful. Vaccination with Pneumovax in anticipation of eventual splenectomy is advised, as is close monitoring for infection, leg ulcers, and biliary tract disease. Early endocrine evaluation is required for glucose intolerance, thyroid dysfunction, and delayed onset of puberty or secondary sexual characteristics. Many patients develop endocrine deficiencies as a result of iron overload.

Patients with -thalassemia intermedia exhibit similar stigmata but can survive without chronic hypertransfusion. Management is particularly challenging because a number of factors can aggravate the anemia, including infection, onset of puberty, and development of splenomegaly and hypersplenism. Some patients may eventually benefit from splenectomy. The expanded erythron can cause absorption of excessive dietary iron and hemosiderosis, even without transfusion.

-Thalassemia minor (i.e., thalassemia trait) usually presents as profound microcytosis and hypochromia with target cells, but only minimal or mild anemia. The mean corpuscular volume is rarely >75 fL; the hematocrit is rarely <30 to 33%. Hemoglobin electrophoresis classically reveals an elevated HbA2 (3.5 to 7.5%), but some forms are associated with normal HbA2 and/or elevated HbF. Genetic counseling and patient education are essential. Patients with -thalassemia trait should be warned that their blood picture resembles iron deficiency and can be misdiagnosed. They should eschew routine use of iron but know that iron deficiency requiring supplementation can develop, as in other persons, during pregnancy or from chronic bleeding.

Persons with -thalassemia trait may exhibit mild hypochromia and microcytosis usually without anemia. HbA2 and HbF levels are normal. Affected individuals usually require only genetic counseling. HbH disease resembles -thalassemia intermedia, with the added complication that the HbH molecule behaves like a moderately unstable hemoglobin. Patients with HbH disease should undergo splenectomy if excessive anemia or a transfusion requirement develops. Oxidative drugs should be avoided. Iron overload leading to death can occur in more severely affected patients.

Prevention

Antenatal diagnosis of thalassemia syndromes is now widely available. DNA diagnosis is based on PCR amplification of fetal DNA, obtained by amniocentesis or chorionic villus biopsy followed by hybridization to allele-specific oligonucleotides probes. The probes can be designed to detect simultaneously the subset of mutations that account for 95 to 99% of the - or -thalassemias that occur in a particular ethnic group.

Thalassemic Structural Variants

Thalassemic structural variants are characterized by both defective synthesis and abnormal structure.

Hemoglobin Lepore

Hb Lepore [2()2] arises by an unequal crossover and recombination event that fuses the proximal end of the -gene with the distal end of the closely linked -gene. The resulting chromosome contains only the fused gene. The Lepore () globin is synthesized poorly because the fused gene is under the control of the weak -globin promoter. Hb Lepore alleles have a phenotype like -thalassemia, except for the added presence of 2 to 20% Hb Lepore. Compound heterozygotes for Hb Lepore and a classic -thalassemia allele may also have severe thalassemia.

Hemoglobin E

HbE (i.e., 2226GluLys) is extremely common in Cambodia, Thailand, and Vietnam. The gene has become far more prevalent in the United States as a result of immigration of Asian persons, especially in California, where HbE is the most common variant detected. HbE is mildly unstable but not enough to affect RBC life span significantly. The high frequency of the HbE gene may be a result of the thalassemia phenotype associated with its inheritance. Heterozygotes resemble individuals with mild -thalassemia trait. Homozygotes have somewhat more marked abnormalities but are asymptomatic. Compound heterozygotes for HbE and a -thalassemia gene can have -thalassemia intermedia or -thalassemia major, depending on the severity of the coinherited thalassemic gene.

The E allele contains only a single base change, in codon 26, that causes the amino acid substitution. However, this mutation activates a cryptic RNA splice site generating a structurally abnormal globin mRNA that cannot be translated from about 50% of the initial pre-mRNA molecules. The remaining 40 to 50% that are normally spliced generate functional mRNA that is translated into E-globin because the mature mRNA carries the base change that alters codon 26.

Genetic counseling of the persons at risk for HbE should be concerned with the interaction of HbE with thalassemia rather than HbE homozygosity, a condition associated with asymptomatic microcytosis, hypochromia, and hemoglobin levels rarely <10 g/dL.

Hereditary Persistence of Fetal Hemoglobin

HPFH is characterized by continued synthesis of high levels of HbF in adult life. No deleterious effects are apparent, even when all of the hemoglobin produced is HbF. These rare patients demonstrate convincingly that prevention or reversal of the fetal to adult hemoglobin switch would provide efficacious therapy for sickle cell anemia and thalassemia.

Acquired Hemoglobinopathies

The two most important acquired hemoglobinopathies are carbon monoxide poisoning and methemoglobinemia, which is covered elsewhere in this chapter. Carbon monoxide has a higher affinity for hemoglobin than does oxygen; it can replace oxygen and diminish O2 delivery. Chronic elevation of carboxyhemoglobin levels to 10 or 15%, as occurs in smokers, can lead to secondary polycythemia. Carboxyhemoglobin is cherry red in color and masks the development of cyanosis usually associated with poor O2 delivery to tissues.

Abnormalities of hemoglobin biosynthesis have also been described in blood dyscrasias. In some patients with myelodysplasia, erythroleukemia, or myeloproliferative disorders, a mild form of HbH disease may also be seen. The abnormalities are not severe enough to alter the course of the underlying disease.

Management of Transfusional Hemosiderosis

Chronic blood transfusion can lead to bloodborne infection, alloimmunization, febrile reactions, and lethal iron overload. A unit of packed RBCs contains 250 to 300 mg iron (1 mg/mL). The iron assimilated by single transfusion of two units of packed RBCs is thus equal to a 1- to 2-year intake of iron. Iron accumulates in chronically transfused patients because no mechanisms exist for increasing iron excretion: an expanded erythron causes especially rapid development of iron overload because accelerated erythropoiesis promotes excessive absorption of dietary iron. Vitamin C should not be supplemented because it generates free radicals in iron excess states.

Patients who receive >100 units of packed RBCs usually develop hemosiderosis. The ferritin level rises, followed by early endocrine dysfunction (glucose intolerance and delayed puberty), cirrhosis, and cardiomyopathy. Liver biopsy shows both parenchymal and reticuloendothelial iron. The superconducting quantum-interference device (SQUID) is accurate at measuring hepatic iron but not widely available. Cardiac toxicity is often insidious. Early development of pericarditis is followed by dysrhythmia and pump failure. The onset of heart failure is ominous, often presaging death within a year (Chap. 336).

The decision to start long-term transfusion support should also prompt one to institute therapy with iron-chelating agents. The only approved and available iron chelator, desferoxamine (Desferal), is expensive and poorly absorbed from the gastrointestinal tract. Its iron-binding kinetics require chronic slow infusion via a metering pump. The constant presence of the drug improves the efficiency of chelation and protects tissues from occasional releases of the most toxic fraction of iron—low-molecular-weight iron—which may not be sequestered by protective proteins. Oral iron-chelating agents such as deferiprone showed initial promise, but long-term trials have raised serious doubts about their efficacy and safety. Newer oral agents are in clinical trials.

Desferoxamine is relatively nontoxic. Occasional cataracts, deafness, and local skin reactions, including urticaria, occur. Skin reactions can usually be managed with antihistamines. Negative iron balance can be achieved, even in the face of a high transfusion requirement, but this alone does not prevent long-term morbidity and mortality in chronically transfused patients. Irreversible end-organ deterioration develops at relatively modest levels of iron overload, even if symptoms do not appear for many years thereafter. To enjoy a significant survival advantage, chelation must begin before 5 to 8 years of age in thalassemia major.

Experimental Therapies

Bone Marrow Transplantation, Gene Therapy, and Manipulation of HbF

Bone marrow transplantation provides stem cells able to express normal hemoglobin; it has been used in a large number of patients with thalassemia and a smaller number of patients with sickle cell anemia. Early in the course of disease, before end-organ damage occurs, transplantation is curative in 80 to 90% of patients. In highly experienced centers, the treatment-related mortality is <10%. Since survival into adult life is possible with conventional therapy, the decision to transplant is best made in consultation with specialized centers.

Gene therapy of thalassemia and sickle cell disease has proved to be an exceptionally elusive goal. Uptake of gene vectors into the nondividing hematopoietic stem cells has been disappointingly inefficient. Lentiviral-type vectors that can transduce nondividing cells may solve this problem.

Reestablishing high levels of fetal hemoglobin synthesis should ameliorate the symptoms of thalassemia. Cytotoxic agents such as hydroxyurea and cytarabine promote high levels of HbF synthesis, probably by stimulating proliferation of the primitive HbF-producing progenitor cell population (i.e., F cell progenitors). Unfortunately, no regimen has yet been identified that ameliorates the clinical manifestations of thalassemia. Butyrates stimulate HbF production, but only transiently. Pulsed or intermittent administration has recently been found to sustain HbF induction in the majority of patients with sickle cell disease. It is unclear whether butyrates will have similar activity in patients with thalassemia.

Aplastic and Hypoplastic Crisis in Patients with Hemoglobinopathies

Patients with hemolytic anemias sometimes exhibit an alarming decline in hematocrit during and immediately after acute illnesses. Bone marrow suppression occurs in almost everyone during acute inflammatory illnesses. In patients with short RBC life spans, suppression can affect RBC counts more dramatically. These hypoplastic crises are usually transient and self-correcting before intervention is required.

Aplastic crisis refers to a profound cessation of erythroid activity in patients with chronic hemolytic anemias. It is associated with a rapidly falling hematocrit. Episodes are usually self-limited. Aplastic crises are caused by infection with a particular strain of parvovirus, B19A. Children infected with this virus usually develop permanent immunity. Aplastic crises do not often recur and are rarely seen in adults. Management requires close monitoring of the hematocrit and reticulocyte count. If anemia becomes symptomatic, transfusion support is indicated. Most crises resolve spontaneously within 1 to 2 weeks.

Megaloblastic Anemias: Introduction

The megaloblastic anemias are disorders caused by impaired DNA synthesis. Cells primarily affected are those having relatively rapid turnover, especially hematopoietic precursors and gastrointestinal epithelial cells. Cell division is sluggish, but cytoplasmic development progresses normally, so megaloblastic cells tend to be large, with an increased ratio of RNA to DNA. Megaloblastic erythroid progenitors tend to be destroyed in the marrow. Thus, marrow cellularity is often increased but production of red blood cells (RBC) is decreased, an abnormality termed ineffective erythropoiesis (Chap. 52).

Most megaloblastic anemias are due to a deficiency of cobalamin (vitamin B12) and/or folic acid. The various clinical entities associated with megaloblastic anemia are listed in Table 92-1.

Physiologic and Biochemical Considerations

Folic Acid

Folic acid is the common name for pteroylmonoglutamic acid. It is synthesized by many different plants and bacteria. Fruits and vegetables constitute the primary dietary source of the vitamin. Some forms of dietary folic acid are labile and may be destroyed by cooking. The minimum daily requirement is normally about 50 g, but this may be increased severalfold during periods of enhanced metabolic demand such as pregnancy.

The assimilation of adequate amounts of folic acid depends on the nature of the diet and its means of preparation. Folates in various foodstuffs are largely conjugated to a chain of glutamic acid residues. This highly polar side chain impairs the intestinal absorption of the vitamin. However, conjugases (-glutamyl carboxypeptidases) in the lumen of the gut convert polyglutamates to mono- and diglutamates, which are readily absorbed in the proximal jejunum.

Plasma folate is primarily in the form of N5-methyltetrahydrofolate, a monoglutamate, which is transported into cells by a carrier that is specific for the tetrahydro forms of the vitamin. Once in the cell, the N5-methyl group is removed in a cobalamin-requiring reaction (see below), and the folate is then reconverted to the polyglutamate form. Conjugation to polyglutamate may be useful for retention of folate within the cell.

A folate-binding protein occurs in plasma, milk, and other body fluids. The function of this folate binder and its membrane-bound precursor is unknown. Neither the binder nor its precursor is related to the tetrahydrofolate carrier.

Normal individuals have about 5 to 20 mg folic acid in various body stores, half in the liver. In light of the minimum daily requirement, it is not surprising that a deficiency will occur within months if dietary intake or intestinal absorption is curtailed.

The prime function of folate compounds is to transfer 1-carbon moieties such as methyl and formyl groups to various organic compounds (Fig. 92-1). The sources of these 1-carbon moieties is usually serine, which reacts with tetrahydrofolate to produce glycine and N5,10-methylenetetrahydrofolate. An alternative source is formiminoglutamic acid, an intermediate in histidine catabolism, which gives up its formimino group to tetrahydrofolate to yield N5-formiminotetrahydrofolate and glutamic acid. These derivatives provide entry into an interconvertible donor pool consisting of tetrahydrofolate derivatives carrying various 1-carbon moieties. The constituents of this pool can donate their 1-carbon moieties to appropriate acceptor compounds to form metabolic intermediates, which are ultimately converted to building blocks used in the synthesis of macromolecules. The most important building blocks are (1) purines, in which the C-2 and C-8 atoms are introduced in folate-dependent reactions; (2) deoxythymidylate monophosphate (dTMP), synthesized from N5,10-methylenetetrahydrofolate and deoxyuridylate monophosphate (dUMP); and (3) methionine, formed by the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine (two of these three reactions are shown in Fig. 92-1).


Figure 92-1 Folate metabolism. Folate is essential for the de novo synthesis of purines, deoxythymidylate monophosphate (dTMP), and methionine, serving as an intermediate carrier of 1-carbon fragments used in the biosynthesis of these compounds. Its active form is tetrahydrofolate (THF). THF acquires the 1-carbon fragment principally from serine, which is converted to glycine in the course of the reaction. For purine synthesis, the 1-carbon fragment is first oxidized to the level of formic acid, then transferred to substrate. For methionine synthesis, a cobalamin-requiring reaction, the 1-carbon fragment is first reduced to the level of a methyl group, then transferred to homocysteine. In these reactions the cofactor is released as THF, which can immediately participate in another 1-carbon transfer cycle. During the production of dTMP from dUMP, however, the 1-carbon fragment is reduced from formaldehyde to a methyl group in the course of the transfer reaction. The hydrogen atoms used for this reduction come from the cofactor, which is therefore released, not as THF, but as dihydrofolate (DHF). To participate further in the 1-carbon transfer cycle, the DHF has to be re-reduced to THF, a reaction catalyzed by dihydrofolate reductase.


In all but one of the 1-carbon transfer reactions, tetrahydrofolate is produced. It can immediately accept a 1-carbon moiety and reenter the donor pool. The single exception is the thymidylate synthase reaction (dUMPdTMP), in which dihydrofolate is the product (Fig. 92-1). This must be reduced to tetrahydrofolate by the enzyme dihydrofolate reductase before it can reenter the donor pool. A number of drugs are able to inhibit dihydrofolate reductase (Table 92-1), thereby diverting folate from the donor pool and producing what amounts to a state of folate deficiency in the face of normal tissue folate concentrations.

Cobalamin

This vitamin is a complex organometallic compound in which a cobalt atom is situated within a corrin ring, a structure similar to the porphyrin from which heme is formed. Unlike heme, however, cobalamin cannot be synthesized in the human body and must be supplied in the diet. The only dietary source of cobalamin is animal products: meat and dairy foods. The minimum daily requirement for cobalamin is about 2.5 g.

During gastric digestion, cobalamin in food is released and forms a stable complex with gastric R binder, one of a closely related group of glycoproteins of unknown function that are found in secretions (e.g., saliva, milk, gastric juice, bile), phagocytes, and plasma. On entering the duodenum, the cobalamin–R binder complex is digested, releasing the cobalamin, which then binds to intrinsic factor (IF), a 50-kDa glycoprotein produced by the parietal cells of the stomach. The secretion of IF generally parallels that of hydrochloric acid. The cobalamin-IF complex is resistant to proteolytic digestion and travels to the distal ileum, where specific receptors on the mucosal brush border bind and absorb the cobalamin-IF complex. Thus, IF, like iron-binding transferrin, is a cell-directed carrier protein. The receptor-bound cobalamin-IF complex is taken into the ileal mucosal cell, where the IF is destroyed and the cobalamin is transferred to another transport protein, transcobalamin (TC) II. The cobalamin–TC II complex is then secreted into the circulation, from which it is rapidly taken up by the liver, bone marrow, and other cells. The pathway of cobalamin absorption is shown in Fig. 92-2. Normally, about 2 mg cobalamin is stored in the liver, and another 2 mg is stored elsewhere in the body. In view of the minimum daily requirement, about 3 to 6 years would be required for a normal individual to become deficient in cobalamin if absorption were to cease abruptly.


Figure 92-2 The assimilation of cobalamin. On entering the stomach, dietary cobalamin (Cbl) forms a complex with R binding protein. As this protein is digested in the small intestine, cobalamin is transferred to intrinsic factor (IF). This complex passes through the intestine until it reaches specific receptors on the mucosa of the distal ileum. The internalized Cbl is then transferred to transcobalamin II (TC II), which circulates in the plasma until it binds to receptors on cells throughout the body and is internalized.


Although TC II is the acceptor for newly absorbed cobalamin, most circulating cobalamin is bound to TC I, a glycoprotein closely related to gastric R binder. TC I appears to be derived in part from leukocytes. The paradox that most circulating cobalamin is bound to TC I rather than TC II, even though TC II initially carries all the cobalamin that is absorbed by the intestine, is explained by the fact that cobalamin bound to TC II is rapidly cleared from the blood (t1/2 about 1 h), while clearance of cobalamin bound to TC I requires many days. The function of TC I is unknown.

Cobalamin is an essential cofactor for two enzymes in human cells: methionine synthase and methylmalonyl–coenzyme A (CoA) synthase. Cobalamin exists in two metabolically active forms, identified by the alkyl group attached to the sixth coordination position of the cobalt atom: methylcobalamin and adenosylcobalamin. The vitamin preparation that is used therapeutically is cyanocobalamin (also called vitamin B12). Cyanocobalamin has no known physiologic role and must be converted to a biologically active form before it can be used by tissues.

Methylcobalamin is the form required for methionine synthase, which catalyzes the conversion of homocysteine to methionine (Fig. 92-1). When this reaction is impaired, folate metabolism is deranged; it is this derangement that underlies the defect in DNA synthesis and the megaloblastic maturation pattern in patients who are deficient in cobalamin. In cobalamin deficiency, the unconjugated N5-methyltetrahydrofolate newly taken from the bloodstream cannot be converted to other forms of tetrahydrofolate by methyl transfer. This is the so-called folate trap hypothesis. Because N5-methyltetrahydrofolate is a poor substrate for the conjugating enzyme, it largely remains in the unconjugated form and slowly leaks from the cell. Tissue folate deficiency therefore develops, and this results in megaloblastic hematopoiesis. This hypothesis explains why tissue folate stores in cobalamin deficiency are substantially reduced, with a disproportionate reduction in conjugated, as compared with unconjugated, folates, despite normal or supranormal serum folate levels. It also explains why large doses of folate can produce a partial hematologic remission in patients with cobalamin deficiency.

Megaloblastic changes in both cobalamin and folate deficiency as well as in methotrexate treatment are related to a deficiency in production of dTMP. In addition, the excess deoxyuridylate that accumulates can be phosphorylated and mistakenly incorporated into DNA in place of thymidylate; base pairing can be affected by this U-for-T substitution.

Plasma homocysteine levels are elevated in both folate and cobalamin deficiency, and high levels of plasma homocysteine appear to be a risk factor for venous and arterial thrombosis. It is not yet known if hyperhomocysteinemia due to folate or cobalamin deficiency predisposes to thrombosis or alters its response to treatment.

Impairment in the conversion of homocysteine to methionine may also contribute to the neurologic complications of cobalamin deficiency (see below). The methionine formed in this reaction is needed for the production of choline and choline-containing phospholipids. Nervous system damage is postulated to result at least in part from interference with these processes due to decreased methionine production in cobalamin deficiency.

Adenosylcobalamin is required for the conversion of methylmalonyl CoA to succinyl CoA. Lack of this cofactor leads to large increases in the tissue levels of methylmalonyl CoA and its precursor, propionyl CoA. As a consequence, nonphysiologic fatty acids containing an odd number of carbon atoms are synthesized and incorporated into neuronal lipids. This biochemical abnormality may also contribute to the neurologic complications of cobalamin deficiency (see Clinical Disorders, below).

Clinical Disorders

Classification of Megaloblastic Anemias

Table 92-1. The cause of megaloblastic anemia varies in different parts of the world. In temperate zones, folate deficiency in alcoholics and cobalamin deficiency due to pernicious anemia or achlorhydria are the common types of megaloblastic anemias. In certain areas close to the equator, tropical sprue is endemic and an important cause of megaloblastic anemia, while in Scandinavia, infestations by the fish tapeworm, Diphyllobothrium latum, may be a cause.

The dietary intake of cobalamin is more than adequate for the body's requirements, except in complete vegetarians (vegans) and their breast-fed infants. Thus deficiency of cobalamin is almost always due to malabsorption. Malabsorption can occur at several levels. In contrast, the dietary intake of folic acid is marginal in many parts of the world. Furthermore, because the body's stores of folate are relatively low, folic acid deficiency can arise rather suddenly during periods of decreased dietary intake or increased metabolic demand. Finally, folic acid deficiency may be due to malabsorption. Often two or more of these factors coexist in a given patient.

Combined deficiencies of cobalamin and folic acid are not uncommon. Patients with tropical sprue are often deficient in both vitamins. The biochemical lesion that results in megaloblastic maturation of bone marrow cells also causes structural and functional abnormalities of the rapidly proliferating epithelial cells of the intestinal mucosa. Thus severe deficiency of one vitamin can lead to malabsorption of the other. Furthermore, as discussed above, a deficiency of cobalamin causes a secondary reduction in cellular folic acid.

Finally, megaloblastic anemias may occasionally be induced by factors unrelated to a vitamin deficiency. Most such cases are caused by drugs that interfere with DNA synthesis. Less commonly, megaloblastic maturation is a feature of certain acquired hematopoietic stem cell defects. Rarest of all are specific congenital enzyme deficiencies.

Cobalamin Deficiency

The clinical features of cobalamin deficiency involve the blood, the gastrointestinal tract, and the nervous system.

The hematologic manifestations are almost entirely the result of anemia, although very rarely purpura may appear, due to thrombocytopenia. Symptoms of anemia may include weakness, light-headedness, vertigo, and tinnitus, as well as palpitations, angina, and the symptoms of congestive failure. On physical examination, the patient with florid cobalamin deficiency is pale, with slightly icteric skin and eyes. Elevated bilirubin levels are related to high erythroid cell turnover in the marrow. The pulse is rapid, and the heart may be enlarged; auscultation will usually reveal a systolic flow murmur.

The gastrointestinal manifestations reflect the effect of cobalamin deficiency on the rapidly proliferating gastrointestinal epithelium. The patient sometimes complains of a sore tongue, which on inspection will be smooth and beefy red. Anorexia with moderate weight loss may also be evident, possibly accompanied by diarrhea and other gastrointestinal symptoms. These latter manifestations may be caused in part by megaloblastosis of the small intestinal epithelium, which results in malabsorption.

The neurologic manifestations often fail to remit fully on treatment. The initial pathology is demyelination, followed by axonal degeneration and eventual neuronal death; the final stage, of course, is irreversible. Sites of involvement include peripheral nerves; the spinal cord, where the posterior and lateral columns undergo demyelination; and the cerebrum itself. Signs and symptoms include numbness and paresthesia in the extremities (the earliest neurologic manifestations), weakness, and ataxia. There may be sphincter disturbances. Reflexes may be diminished or increased. The Romberg and Babinski's signs may be positive, and position and vibration senses are usually diminished. Disturbances of mentation will vary from mild irritability and forgetfulness to severe dementia or frank psychosis. It should be emphasized that neurologic disease may occur in a patient with a normal hematocrit and normal RBC indices. Although it has many benefits, folate supplementation of food may increase the likelihood of neurologic presentations of cobalamin deficiency.

In the classic patient, in whom hematologic problems predominate, the blood and bone marrow show characteristic megaloblastic changes (described under Diagnosis, below). The anemia may be very severe—hematocrits of 15 to 20 are not infrequent—but is surprisingly well tolerated by the patient because it develops slowly.

Defective Release of Cobalamin from Food

Cobalamin in food is tightly bound to enzymes in meat and is split from these enzymes by hydrochloric acid and pepsin in the stomach. People >70 years commonly have achlorhydria. Therefore, they are unable to release cobalamin from food sources but retain the ability to absorb crystalline B12, the form most commonly found in multivitamins. The exact incidence of the defect in cobalamin release from food has not been well defined; estimates vary from 10 to >50% of those over age 70 years. Only a minority of these persons go on to develop frank cobalamin deficiency, but many have biochemical changes, including low levels of cobalamin bound to TC II and elevated homocysteine levels, that augur cobalamin deficiency (see below).

Similarly, patients on drugs that suppress gastric acid production, such as omeprazole, may also fail to release cobalamin from food. However, the proton pump inhibitors do not inhibit IF secretion by parietal cells.

Pernicious Anemia

Pernicious anemia, considered the most common cause of cobalamin deficiency, is caused by the absence of IF, due to either atrophy of the mucosa or autoimmune destruction of parietal cells. It is most frequently seen in individuals of northern European descent and African Americans and is much less common in southern Europeans and Asians. Men and women are equally affected. It is a disease of the elderly, the average patient presenting near age 60; it is rare under age 30, although typical pernicious anemia can be seen in children under age 10 (juvenile pernicious anemia). Inherited conditions in which a histologically normal stomach secretes either an abnormal IF or none at all will induce cobalamin deficiency in infancy or early childhood.

The incidence of pernicious anemia is substantially increased in patients with other diseases thought to be of immunologic origin, including Graves' disease, myxedema, thyroiditis, idiopathic adrenocortical insufficiency, vitiligo, and hypoparathyroidism. Patients with pernicious anemia also have abnormal circulating antibodies related to their disease: 90% have antiparietal cell antibody, which is directed against the H+,K+-ATPase, while 60% have anti-IF antibody. Antiparietal cell antibody is also found in 50% of patients with gastric atrophy without pernicious anemia, as well as in 10 to 15% of an unselected patient population, but anti-IF antibody is usually absent from these patients. Relatives of patients with pernicious anemia have an increased incidence of the disease, and even clinically unaffected relatives may have anti-IF antibody in their serum. Finally, treatment with glucocorticoids may reverse the disease.

Cytotoxic T cells may also contribute to the destruction of parietal cells in pernicious anemia. Pernicious anemia is unusually common in patients with agammaglobulinemia, supporting a role for the cellular immune system in its pathogenesis. In contrast, Helicobacter pylori does not cause parietal cell destruction in pernicious anemia.

The most characteristic finding in pernicious anemia is gastric atrophy affecting the acid- and pepsin-secreting portion of the stomach; the antrum is spared. Other pathologic changes are secondary to the deficiency of cobalamin; these include megaloblastic alterations in the gastric and intestinal epithelium and the neurologic changes described above. The abnormalities in the gastric epithelium appear as cellular atypia in gastric cytology specimens, a finding that must be carefully distinguished from the cytologic abnormalities seen in gastric malignancy.

The clinical manifestations are primarily those of cobalamin deficiency, as described above. The disease is of insidious onset and progresses slowly. Laboratory examination will reveal hypergastrinemia and pentagastrin-fast achlorhydria as well as the hematologic and other laboratory abnormalities discussed under Diagnosis.

Through appropriate replacement therapy, patients with pernicious anemia should experience complete and lifelong correction of all abnormalities that are due to cobalamin deficiency, except to the extent that irreversible changes in the nervous system may have occurred before treatment. These patients, however, are unusually subject to gastric polyps and have about twice the normal incidence of cancer of the stomach. Thus, patients should be followed with frequent stool guaiac examinations and endoscopy when indicated.

Postgastrectomy

Following total gastrectomy or extensive damage to gastric mucosa as, for example, by ingestion of corrosive agents, megaloblastic anemia will develop because the source of IF has been removed. In all such patients, the absorption of orally administered cobalamin is impaired. Megaloblastic anemia may also follow partial gastrectomy, but the incidence is lower than after total gastrectomy. The cause of cobalamin deficiency after partial gastrectomy is not clear; defective release of cobalamin from food and intestinal overgrowth of bacteria have been suggested, but response to antibiotics is not common.

Intestinal Organisms

Megaloblastic anemia may occur with intestinal stasis due to anatomic lesions (strictures, diverticula, anastomoses, "blind loops") or pseudoobstruction (diabetes mellitus, scleroderma, amyloid). This anemia is caused by colonization of the small intestine by large masses of bacteria that consume intestinal cobalamin before absorption. Steatorrhea may also be seen under these circumstances because bile salt metabolism is disturbed when the intestine is heavily colonized with bacteria. Hematologic responses have been observed after administration of oral antibiotics such as tetracycline and ampicillin. Megaloblastic anemia is seen in persons harboring the fish tapeworm, D. latum, due to competition by the worm for cobalamin. Destruction of the worm eliminates the problem.

Ileal Abnormalities

Cobalamin deficiency is common in tropical sprue, while it is an unusual complication of nontropical sprue (gluten-sensitive enteropathy; Chap. 275). Virtually any disorder that compromises the absorptive capacity of the distal ileum can result in cobalamin deficiency. Specific entities include regional enteritis, Whipple's disease, and tuberculosis. Segmental involvement of the distal ileum by disease can cause megaloblastic anemia without any other manifestations of intestinal malabsorption such as steatorrhea. Cobalamin malabsorption is also seen after ileal resection. The Zollinger-Ellison syndrome (intense gastric hyperacidity due to a gastrin-secreting tumor) may cause cobalamin malabsorption by acidifying the small intestine, retarding the transfer of the vitamin from R binder to IF and impairing the binding of the cobalamin-IF complex to the ileal receptors. Chronic pancreatitis may also cause cobalamin malabsorption by impairing the transfer of the vitamin from R binder to IF. This abnormality can be detected by tests of cobalamin absorption (see below, Schilling test), but it is invariably mild and never causes clinical cobalamin deficiency. Finally, a rare congenital disorder, Imerslund-Gräsbeck disease, involves a selective defect in cobalamin absorption accompanied by proteinuria. Affected individuals have a mutation in cubulin, a receptor that mediates intestinal absorption of the cobalamin-IF complex.

Nitrous Oxide

Inhalation of nitrous oxide as an anesthetic destroys endogenous cobalamin. As ordinarily used, the magnitude of the effects are not sufficient to cause clinical cobalamin deficiency, but repeated or protracted exposure (>6 h), particularly in older patients with borderline cobalamin stores, can lead to severe megaloblastic anemia and/or acute neurologic deficits.

Folic Acid Deficiency

Since January 1998, folic acid has been added to all enriched grain products by order of the U.S. Food and Drug Administration; accordingly, the incidence of folic acid deficiency has fallen markedly. Patients with folic acid deficiency are more often malnourished than those with cobalamin deficiency. The gastrointestinal manifestations are similar to but may be more widespread and more severe than those of pernicious anemia. Diarrhea is often present, and cheilosis and glossitis are also encountered. However, in contrast to cobalamin deficiency, neurologic abnormalities do not occur.

The hematologic manifestations of folic acid deficiency are the same as those of cobalamin deficiency. Folic acid deficiency can generally be attributed to one or more of the following factors: inadequate intake, increased demand, or malabsorption.

Inadequate Intake

Alcoholics may become folate deficient because their main source of caloric intake is alcoholic beverages. Distilled spirits are virtually devoid of folic acid, while beer and wine do not contain enough of the vitamin to satisfy the daily requirement. In addition, alcohol may interfere with folate metabolism. Narcotic addicts are also prone to become folate deficient because of malnutrition. Many indigent and elderly individuals who subsist primarily on canned foods or "tea and toast" and occasional teenagers whose diet consists of "junk food" develop folate deficiency.

Increased Demand

Tissues with a relatively high rate of cell division such as the bone marrow or gut mucosa have a large requirement for folate. Therefore, patients with chronic hemolytic anemias or other causes of very active erythropoiesis may become deficient. Pregnant women formerly were at risk to become deficient in folic acid because of the high demand of the developing fetus. Deficiency in the first weeks of pregnancy can cause neural tube defects in newborns. Often the pregnancy was not detected until the defect had developed; thus, provision of folate supplementation to women after they learned they were pregnant was ineffective. However, folate food supplementation has decreased neural tube defects by >50%. Folate deficiency may also occur during the growth spurts of infancy and adolescence. Patients on chronic hemodialysis may require supplementary folate to replace that lost in the dialysate.

Malabsorption

Folic acid deficiency is a common accompaniment of tropical sprue. Both the gastrointestinal symptoms and malabsorption are improved by the administration of either folic acid or antibiotics by mouth. Patients with nontropical sprue (gluten-sensitive enteropathy) may also develop significant folic acid deficiency that parallels other parameters of malabsorption. Similarly, folate deficiency in alcoholics may be due in part to malabsorption. In addition, other primary small-bowel disorders are sometimes associated with folate deficiency (Chap. 275).

Drugs

Next to deficiency of folate or cobalamin, the most common cause of megaloblastic anemia is drugs. Agents that cause megaloblastic anemia do so by interfering with DNA synthesis, either directly or by antagonizing the action of folate. They can be classified as follows:

1. Direct inhibitors of DNA synthesis. They include purine analogues (6-thioguanine, azathioprine, 6-mercaptopurine), pyrimidine analogues (5-fluorouracil, cytosine arabinoside), and other drugs that interfere with DNA synthesis by a variety of mechanisms (hydroxyurea, procarbazine). The antiviral agent zidovudine (AZT), used for treating HIV, often causes severe megaloblastic anemia.

2. Folate antagonists. The most toxic of these is methotrexate, a powerful inhibitor of dihydrofolate reductase, which is used in the treatment of certain malignancies and rheumatologic disorders. Much less toxic but still capable of inducing a megaloblastic anemia are several weak dihydrofolate reductase inhibitors used to treat a variety of nonmalignant conditions. These drugs include pentamidine, trimethoprim, triamterene, and pyrimethamine.

3. Others. A number of drugs antagonize folate by mechanisms that are poorly understood but are thought to involve an effect on absorption of the vitamin by the intestine. In this category are the anticonvulsants phenytoin, primidone, and phenobarbital. Megaloblastic anemia induced by these agents is mild.


Other Mechanisms

Hereditary

Megaloblastic anemia may be seen in several hereditary disorders. Orotic aciduria is a deficiency of orotidylic decarboxylase and phosphorylase, leading to a defect in pyrimidine metabolism and characterized by retarded growth and development as well as by the excretion of large amounts of orotic acid. Congenital folate malabsorption causes megaloblastic anemia, accompanied by ataxia and mental retardation. A thiamine-responsive megaloblastic anemia accompanied by nerve deafness and diabetes mellitus has been reported in several children. Megaloblastic changes as well as multinuclearity of RBC precursors are seen in the marrow of certain patients with congenital dyserythropoietic anemia, a group of inherited disorders characterized by mild to moderate anemia and a benign course.

TC II deficiency, like the congenital abnormalities in cobalamin absorption, causes pronounced deficiency in cobalamin in infancy or early childhood. Megaloblastic anemia is not seen in hereditary TC I deficiency.

Refractory Megaloblastic Anemia

Megaloblastic erythropoiesis may sometimes be seen in myelodysplasia. Megaloblastic changes are restricted to the RBC series (see below). Myelodysplasia often produces a distinct morphologic picture most apparent in orthochromatic normoblasts in which a megaloblastic nucleus is associated with severely hypochromic cytoplasm. This variant has been called "megaloblastoid" and refers to the presence of both nuclear and cytoplasmic maturation defects. "Megaloblastoid" does not mean "mildly megaloblastic." As with other forms of myelodysplasia, refractory megaloblastic anemia is associated with an increased incidence of acute leukemia.

Megaloblastic changes are seen in erythremic myelosis and acute erythroleukemia, where RBC precursors are prominently involved. Here, the marrow is characterized by bizarre erythroid maturation, with multinuclearity and multipolar mitotic figures in the RBC precursors (Chap. 96).

Megaloblastic Disease Without Anemia

Megaloblastic disease is easily overlooked in nonanemic patients. It can present in one of two ways.

Acute Megaloblastic Disease

Occasionally, a full-blown megaloblastic state can develop over the course of just a few days. This is usually seen following nitrous oxide anesthesia but may occur in any patient with a serious illness requiring intensive care, especially a patient receiving multiple transfusions, dialysis, or total parenteral nutrition. An acute megaloblastic state can also be precipitated by the administration of a weak antifolate (e.g., trimethoprim) to a patient with marginal tissue folate stores.

The condition resembles an immune cytopenia, with a rapidly developing thrombocytopenia and/or leukopenia in the absence of anemia. The blood smear may be completely normal, but the marrow is floridly megaloblastic. Acute megaloblastic anemia responds rapidly to treatment with folate plus cobalamin in the usual therapeutic doses.

Cobalamin Deficiency Without Anemia

Cobalamin deficiency without hematologic abnormalities is surprisingly common, especially in the elderly. The risk of a nonhematologic presentation for cobalamin deficiency is increased by the folate food fortification because folate can mask the hematologic effects of cobalamin deficiency. Between 10 and 30% of persons over age 70 years have metabolic evidence of cobalamin deficiency, either elevated homocysteine levels, low cobalamin-TC II levels, or both. Only 10% of these patients have defective production of IF, and the remainder often have atrophic gastritis and cannot release cobalamin from their food (see above). Serum cobalamin levels may be normal or low, but serum levels of methylmalonic acid are almost invariably increased due to a deficiency of cobalamin at the tissue level. The neuropsychiatric abnormalities tend to improve, and serum methylmalonic acid levels generally return to normal after treatment with cobalamin. Neurologic defects do not always reverse with cobalamin supplementation.

Diagnosis

The finding of significant macrocytosis [mean corpuscular volume (MCV) > 100 fL] suggests the presence of a megaloblastic anemia. Other causes of macrocytosis include hemolysis, liver disease, alcoholism, hypothyroidism, and aplastic anemia. If the macrocytosis is marked (MCV > 110 fL), the patient is much more likely to have a megaloblastic anemia. Macrocytosis is less marked with concurrent iron deficiency or thalassemia. The reticulocyte index is low, and the leukocyte and platelet count may also be decreased, particularly in severely anemic patients. The blood smear (Fig. 92-3) demonstrates marked anisocytosis and poikilocytosis, together with macroovalocytes, which are large, oval, fully hemoglobinized erythrocytes typical of megaloblastic anemias. There is some basophilic stippling, and an occasional nucleated RBC may be seen. In the white blood cell series, the neutrophils show hypersegmentation of the nucleus (Fig. 92-4). This is such a characteristic finding that a single cell with a nucleus of six lobes or more should raise the immediate suspicion of a megaloblastic anemia. A rare myelocyte may also be seen. Bizarre, misshapen platelets are also observed. The bone marrow is hypercellular with a decreased myeloid/erythroid ratio and abundant stainable iron. RBC precursors are abnormally large and have nuclei that appear much less mature than would be expected from the development of the cytoplasm (nuclear-cytoplasmic asynchrony). The nuclear chromatin is more dispersed than expected, and it condenses in a peculiar fenestrated pattern that is very characteristic of megaloblastic erythropoiesis. Abnormal mitoses may be seen. Granulocyte precursors are also affected, many being larger than normal, including giant bands and metamyelocytes. Megakaryocytes are decreased and show abnormal morphology.


Figure 92-3 Megaloblastic anemia. Oval macrocytes, well filled with hemoglobin, are admixed with lesser numbers of large red blood cells, some of which are teardrop-shaped. Note also hypersegmented granulocyte.


Figure 92-4 This marrow section demonstrates nuclear-cytoplasmic dissociation. Nuclei of late stage (orthochromatic) erythroblasts have loose chromatin more characteristic of more immature cells while their cytoplasms are nearly filled with hemoglobin. Slow nuclear maturation is related to a decrese in DNA synthesis related to an insufficient supply of thymidylate. An inadequate supply of reduced folate (usually folate or B12 deficiency) or drugs that inhibit DNA synthesis can produce this picture.


Megaloblastic anemias are characterized by ineffective erythropoiesis (Chap. 52). In a severely megaloblastic patient, as many as 90% of the RBC precursors may be destroyed before they are released into the bloodstream, compared with 10 to 15% in normal individuals. Enhanced intramedullary destruction of erythroblasts results in an increase in unconjugated bilirubin and lactic acid dehydrogenase (isoenzyme 1) in plasma.

In evaluating a patient with megaloblastic anemia, it is important to determine whether there is a specific vitamin deficiency by measuring serum cobalamin and folate levels. The normal range of cobalamin in serum is 300 to 900 pg/mL; values <200 pg/mL indicate clinically significant deficiency. Measurements of cobalamin bound to TC II would be a more physiologic measure of cobalamin status, but such assays are not yet routinely available. The normal serum concentration of folic acid ranges from 6 to 20 ng/mL; values 4 ng/mL are generally considered to be diagnostic of folate deficiency. Unlike serum cobalamin, serum folate levels may reflect recent alterations in dietary intake. Measurement of RBC folate level provides useful information because it is not subject to short-term fluctuations in folate intake and is better than serum folate as an index of folate stores.

Once cobalamin deficiency has been established, its pathogenesis can be delineated by means of a Schilling test. A patient is given radioactive cobalamin by mouth, followed shortly thereafter by an intramuscular injection of unlabeled cobalamin. The proportion of the administered radioactivity excreted in the urine during the next 24 h provides an accurate measure of absorption of cobalamin, assuming that a complete urine sample has been collected. Because cobalamin deficiency is almost always due to malabsorption (Table 92-1), this first stage of the Schilling test should be abnormal (i.e., small amounts of radioactivity in the urine). The patient is then given labeled cobalamin bound to IF. Absorption of the vitamin will now approach normal if the patient has pernicious anemia or some other type of IF deficiency. If cobalamin absorption is still decreased, the patient may have bacterial overgrowth (blind loop syndrome) or ileal disease (including an ileal absorptive defect secondary to the cobalamin deficiency itself). Cobalamin malabsorption due to bacterial overgrowth can frequently be corrected by the administration of antibiotics. The Schilling test can provide equally reliable information after the patient has had adequate therapy with parenteral cobalamin.

A normal Schilling test in a patient with documented cobalamin deficiency may indicate poor absorption of the vitamin when mixed with food. This can be established by repeating the Schilling test with radioactive cobalamin scrambled with an egg.

Serum methylmalonic acid and homocysteine levels are also useful in the diagnosis of megaloblastic anemias. Both are elevated in cobalamin deficiency, while elevated levels of homocysteine but not methylmalonic acid are seen in folate deficiency. These tests measure tissue vitamin stores and may demonstrate a deficiency even when the more traditional but less reliable folate and cobalamin levels are borderline or even normal. Patients (particularly older patients) without anemia and with normal serum cobalamin levels but elevated levels of serum methylmalonic acid may develop neuropsychiatric abnormalities. Treatment of patients with this "subtle" cobalamin deficiency will usually prevent further deterioration and may result in improvement.

Treatment

Cobalamin Deficiency

Apart from specific therapy related to the underlying disorder (e.g., antibiotics for intestinal overgrowth with bacteria), the mainstay of treatment for cobalamin deficiency is replacement therapy. Because the defect is nearly always malabsorption, patients are generally given parenteral treatment, specifically in the form of intramuscular cyanocobalamin. Parenteral treatment begins with 1000 g cobalamin per week for 8 weeks, followed by 1000 g cyanocobalamin intramuscularly every month for the rest of the patient's life. Cobalamin deficiency can also be managed very effectively by oral replacement therapy with 2 mg crystalline B12 per day; however, compliance is a greater concern with oral than intramuscular treatment.

The response to treatment is gratifying. Shortly after treatment is begun, and several days before a hematologic response is evident in the peripheral blood, the patient will experience an increase in strength and an improved sense of well-being. Marrow morphology begins to revert toward normal within a few hours after treatment is initiated. Reticulocytosis begins 4 to 5 days after therapy is started and peaks at about day 7 (Fig. 92-5), with subsequent remission of the anemia over the next several weeks. If a reticulocytosis does not occur, or if it is less brisk than expected from the level of the hematocrit, a search should be made for other factors contributing to the anemia (e.g., infection, coexisting iron and/or folate deficiency, or hypothyroidism). Hypokalemia and salt retention may occur early in the course of therapy. Thrombocytosis may also be seen.


Figure 92-5 Hematologic response of a patient with pernicious anemia to an intramuscular injection of 100 g cobalamin on day 0. Retics, reticulocytes; RBC, red blood cell; Hb, hemoglobin.


In most cases, replacement therapy is all that is needed for the treatment of cobalamin deficiency. Occasionally, however, a patient with a severe anemia will have such a precarious cardiovascular status that emergency transfusion is necessary. This must be done with great care, because such patients may develop heart failure from fluid overload. Blood must be administered slowly in the form of packed RBCs, with very close observation. A small volume of packed RBCs will frequently be enough to ameliorate the acute cardiovascular problems. If necessary, blood may be administered by exchanging patient blood (mostly plasma) for packed cells.

With lifelong treatment, patients should experience no further manifestations of cobalamin deficiency, although neurologic symptoms may not be fully corrected even by optimal therapy. The potential for late development of gastric carcinoma in pernicious anemia necessitates careful follow-up of the patient.

Folate, particularly in large doses, can correct the megaloblastic anemia of cobalamin deficiency without altering the neurologic abnormalities. The neurologic manifestations may even be aggravated by folate therapy. Cobalamin deficiency can thus be masked in patients who are taking large doses of folate. For this reason, a hematologic response to folate must never be used to rule out cobalamin deficiency in a given patient; cobalamin deficiency can be excluded only by appropriate laboratory evaluation.

In light of the high frequency of defective cobalamin absorption in older people and the possible increased risk that overt cobalamin deficiency will present with neurologic rather than hematologic symptoms (because of folate food fortification), some experts have recommended the use of 0.1 mg oral crystalline cobalamin prophylaxis daily in people over age 65 years.

Folate Deficiency

Like cobalamin deficiency, folate deficiency is treated by replacement therapy. The usual dose of folic acid is 1 mg/d, by mouth, but higher doses (up to 5 mg/d) may be required for folate deficiency due to malabsorption. Parenteral folate is rarely necessary. The hematologic response is similar to that seen after replacement therapy for cobalamin deficiency, i.e., a brisk reticulocytosis after about 4 days, followed by correction of the anemia over the next 1 to 2 months. The duration of therapy depends on the basis of the deficiency state. Patients with a continuously increased requirement (such as patients with hemolytic anemia) or those with malabsorption or chronic malnutrition should continue to receive oral folic acid indefinitely. In addition, the patient should be encouraged to maintain an optimal diet containing adequate amounts of folate.

Other Causes of Megaloblastic Anemia

Megaloblastic anemia due to drugs can be treated, if necessary, by reducing the dose of the drug or eliminating it altogether. The effects of folate antagonists that inhibit dihydrofolate reductase can be counteracted by folinic acid [5-formyl tetrahydrofolate (THF)] in a dose of 100 to 200 mg/d (Fig. 92-1), which circumvents the block in folate metabolism by providing a form of folate that can be converted to 5,10-methylene THF. For the megaloblastic forms of sideroblastic anemia, pyridoxine in pharmacologic doses (as high as 300 mg/d) should be tried. Simple supportive measures are all that appear to be in order for treatment of refractory megaloblastic anemia. Acute erythroleukemia is treated like other types of acute myeloid leukemia (Chap. 96).

Hemolytic Anemias and Acute Blood Loss: Introduction

The loss of red cells either through hemorrhage or, less commonly, through premature destruction of the red cells (hemolysis) may cause anemia. Hemolysis or blood loss normally leads to an increase in red cell production, detected by an increase in reticulocyte index.

Hemolytic Anemias

Red blood cells (RBC) normally survive 90 to 120 days in the circulation. The life span of RBC may be shortened in a number of disorders, often resulting in anemia if the bone marrow is not able to replenish adequately the prematurely destroyed RBC.

In all patients with hemolytic anemia, a careful history and physical examination provide important clues to the diagnosis. The patient may complain of fatigue and other symptoms of anemia (Chap. 52). Less commonly, jaundice and even red-brown urine (hemoglobinuria) are reported. A complete drug and toxin exposure history and the family history often provide crucial information. The physical examination may show jaundice of skin and mucosae. Splenomegaly is encountered in a variety of hemolytic anemias. Other historic and physical findings are associated with specific hemolytic anemias (see below).

Laboratory tests may be used initially to demonstrate the presence of hemolysis (Table 93-1) and define its cause. An elevated reticulocyte count in the patient with anemia is the most useful indicator of hemolysis, reflecting erythroid hyperplasia of the bone marrow; biopsy of the bone marrow is often unnecessary. Reticulocytes are also elevated in patients with active blood loss, those with myelophthisis, and those who are recovering from suppression of erythropoiesis (Chap. 52). The morphology of the RBC may provide evidence both of hemolysis and of its cause; the characteristic abnormalities and their associated causes and syndromes are listed in Table 93-2. While the findings on the peripheral blood smear alone are rarely pathognomonic, they may provide important clues to the presence of hemolysis and to diagnosis.

Hemolysis results in increased heme catabolism and enhanced formation of unconjugated bilirubin. The plasma level of unconjugated bilirubin may be high enough to produce readily apparent jaundice (detectable usually when serum bilirubin is >34 mol/L or 2 mg/dL). The unconjugated (indirect) bilirubin level can be further elevated by a commonly encountered defect in conjugation of bilirubin (Gilbert's syndrome) (Chap. 284). In patients with hemolysis, the level of unconjugated bilirubin never exceeds 70 to 85 mol/L (4 to 5 mg/dL) unless liver function is impaired.

In the absence of tissue damage in other organs, serum enzyme levels can be useful in the diagnosis and monitoring of patients with hemolysis. Lactate dehydrogenase (LDH), particularly LDH-2, is elevated by accelerated RBC destruction. Serum AST (SGOT) may be somewhat elevated, whereas ALT (SGPT) is not.

Haptoglobin is an globulin that is present in high concentration (1.0 g/L) in the serum. It binds specifically to the globin in hemoglobin. The hemoglobin-haptoglobin complex is cleared rapidly by the mononuclear phagocyte system. Thus patients with significant hemolysis, either intravascular or extravascular, have low or absent levels of serum haptoglobin. The fact that haptoglobin synthesis is decreased in patients with hepatocellular disease and increased in inflammatory states must be considered in the interpretation of serum haptoglobin.

Intravascular hemolysis (which is uncommon) results in the release of hemoglobin into the plasma. In these cases, plasma hemoglobin is increased in proportion to the degree of hemolysis (plasma hemoglobin may be falsely elevated due to lysis of RBC in vitro). If the haptoglobin-binding capacity of the plasma is exceeded, free hemoglobin tetramer dissociates into dimers that pass through renal glomeruli. This filtered hemoglobin is reabsorbed by the proximal tubule, where it is catabolized in situ, and the heme iron is incorporated into storage proteins (ferritin and hemosiderin). The presence of hemosiderin in the urine, detected by staining the sediment with Prussian blue, indicates that a significant amount of circulating free hemoglobin has been filtered by the kidneys. When the absorptive capacity of the tubular cells is exceeded, hemoglobinuria ensues and indicates severe intravascular hemolysis. Hemoglobinuria must be distinguished from hematuria (in which case RBC are seen on urine examination) and from myoglobinuria due to rhabdomyolysis; in all three cases, the urine is positive with the benzidine reaction, commonly used in analysis of urine. After centrifugation of an anticoagulated blood specimen, the plasma of patients with hemoglobinuria has a reddish-brown color, whereas that of patients with myoglobinuria is normal in color. Because of its higher molecular weight, hemoglobin has lower glomerular permeability than myoglobin and is less rapidly cleared by the kidneys.

Classification

The hemolytic anemias can be grouped into three categories (Table 93-3). Accelerated RBC destruction can be caused by (1) a molecular defect (hemoglobinopathy or enzymopathy) inside the red cell, (2) an abnormality in membrane structure and function, or (3) an environmental factor such as mechanical trauma or an autoantibody. In intracorpuscular types of hemolysis, the patient's RBC have an abnormally short life span in a normal recipient (with a compatible blood type), while compatible normal RBC survive normally in the patient. The opposite is true in extracorpuscular types of hemolysis. Finally, hemolytic disorders can be either inherited or acquired.

Inherited Hemolytic Anemias

The inherited hemolytic anemias are due to inborn defects in one of three main components of red cells: the membrane, enzymes, or hemoglobin. These defects are often known at the genomic level, but their identification still largely depends on their clinical and laboratory manifestations.

Red Cell Membrane Disorders

These are usually readily detected by morphologic abnormalities of the RBC on the blood film. The three inherited RBC membrane abnormalities are hereditary spherocytosis, hereditary elliptocytosis (including hereditary pyropoikilocytosis), and hereditary stomatocytosis.

Hereditary Spherocytosis

Hereditary spherocytosis is characterized by spherical RBC due to a molecular defect in one of the proteins in the cytoskeleton of the RBC membrane; this leads to a loss of membrane and hence decreased ratio of surface area to volume and consequently spherocytosis. Usually an autosomal dominant trait, this disorder has an incidence of 1:1000 to 1:4500. In 20% of patients, the absence of hematologic abnormalities in family members suggests either autosomal recessive inheritance or a spontaneous mutation. The disorder is sometimes clinically apparent in early infancy but often escapes detection until adult life.

Clinical Manifestations

The major clinical features of hereditary spherocytosis are anemia, splenomegaly, and jaundice. Jaundice may be intermittent and tends to be less pronounced in early childhood. Because of the increased bile pigment production, pigmented gallstones are common, even in childhood. Compensatory erythroid hyperplasia of the bone marrow occurs, with the extension of red marrow into the midshafts of long bones and occasionally with extramedullary erythropoiesis, at times leading to the formation of paravertebral masses visible on chest x-ray. Because the bone marrow's capacity to increase erythropoiesis nearly matches the rate of hemolysis, anemia is usually mild or moderate and may even be absent in an otherwise healthy individual. Compensation may be temporarily interrupted by episodes of relative erythroid hypoplasia precipitated by infections, particularly parvovirus. Splenomegaly is very common. The hemolytic rate may increase transiently during systemic infections, which induce further splenic enlargement. Chronic leg ulcers, similar to those observed in sickle cell anemia, occur occasionally.

The characteristic erythrocyte abnormality is the spherocyte (Fig. 93-1). The mean corpuscular volume (MCV) is usually normal or slightly decreased, and the mean corpuscular hemoglobin concentration (MCHC) is increased to 350 to 400 g/L. Spheroidicity may be quantitatively assessed by measurement of the osmotic fragility of the RBC on exposure to hyposmotic solutions causing a net influx of water (Fig. 93-2). On microscopic examination, spherocytes are usually detected as small cells without central pallor.


Figure 93-1 Hereditary spherocytosis Small, densely staining red blood cells are seen that have lost their central area of pallor (microspherocytes). Microspherocytes may also be found in other hemolytic disorders (Fig. 93-5).


Figure 93-2 Osmotic fragility of RBC in hereditary spherocytosis (HS). The results from two patients are compared to those from a normal individual.


Pathogenesis

The molecular abnormality in hereditary spherocytosis primarily involves the proteins responsible for tethering the lipid bilayer to the underlying cytoskeletal network. About 50% of patients have a defect in ankyrin, the protein that forms a bridge between protein 3 and spectrin (Fig. 93-3). Homozygotes who have a recessive inheritance pattern for ankyrin deficiency have more severe anemia than heterozygotes with the more common dominant form. About 25% of patients have a mutation of protein 3, resulting in a deficiency of that protein and mild anemia with dominant inheritance. Most of the remaining 25% have mutations of spectrin, leading to impaired synthesis or self-association. -spectrin mutants are generally mild, with dominant inheritance, while -spectrin deficiency is severe, with a recessive inheritance pattern. Less often, deficiency of palladin (protein 4.2) is a cause of hereditary spherocytosis. Because the lipid bilayer is not well anchored when these proteins are defective, part of it is lost by vesiculation, resulting in a more spherical and less deformable cell. Because of their shape and rigidity, spherocytes are trapped in the spleen where their increased metabolic rate cannot be sustained, causing a further loss of surface membrane. This "conditioning" produces a subpopulation of hyperspheroidal RBC in the peripheral blood.


Figure 93-3 Diagram of a cross-section of the RBC membrane. Spectrin, actin, tropomyosin, adducin, and protein 4.1 form a meshwork that laminates the inner surface of the membrane. In contrast, other proteins such as the glycophorins (GPA and GPC) and protein 3 (anion transport channel) traverse the lipid bilayer. Long polysaccharide chains are covalently attached to these proteins on the outer surface of the cell and also to glycolipid. Ankyrin and protein 4.2 form a bridge between spectrin and a fraction of the anion transport proteins. Protein 4.1 binds to GPC.


Diagnosis

Hereditary spherocytosis must be distinguished primarily from the spherocytic hemolytic anemias associated with RBC antibodies. The family history of anemia and/or splenectomy is helpful, when present. The diagnosis of immune spherocytosis is usually readily established by a positive direct Coombs test (see below). Spherocytes are also seen in association with hemolysis induced by splenomegaly in patients with cirrhosis, in clostridial infections, and in certain snake envenomations (due to the action of phospholipases on the membrane). A few spherocytes are seen in the course of a wide variety of hemolytic disorders, particularly glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Treatment

Splenectomy is recommended in patients with moderate or severe hemolysis. Although the RBC defect and its consequent morphology persist, anemia is ameliorated. The operative risk is low, particularly if performed by laparoscopy. RBC survival after splenectomy is normal or nearly so; if it is not, an accessory spleen or another diagnosis should be sought. Because of the potential for gallstones and for episodes of bone marrow hypoplasia or hemolytic crises, splenectomy should be performed in symptomatic individuals; cholecystectomy should not be performed without splenectomy, as intrahepatic gallstones may result. Splenectomy in children should be postponed until age 4, if possible, to minimize the risk of severe infections with gram-positive encapsulated organisms. Pneumococcal, meningococcal, and Haemophilus influenzae vaccines should be administered at least 2 weeks before splenectomy. In patients with severe hemolysis, folic acid (1 mg/d) should be administered prophylactically.

Hereditary Elliptocytosis and Hereditary Pyropoikilocytosis

Hereditary elliptocytosis is an autosomal dominant trait and affects 1 per 4000 to 5000 people, a frequency similar to that of hereditary spherocytosis (rarely, patients with myelodysplastic disorders of the bone marrow may have acquired elliptocytosis). In most affected individuals, a structural abnormality of erythrocyte spectrin leads to impaired assembly of the cytoskeleton. In some families, affected individuals have a deficiency of erythrocyte membrane protein 4.1, which stabilizes the interaction of spectrin and actin in the cytoskeleton (Fig. 93-3). In Southeast Asia, the incidence of hereditary ovalocytosis is high; a small internal deletion of protein 3 makes the membrane rigid and confers resistance against malaria.

The great majority of patients manifest only mild hemolysis, with little or no anemia. RBC destruction occurs predominantly in an enlarged spleen. Hemolysis is corrected by splenectomy.

The blood smear reveals elongated or oval red cells (elliptocytes). Patients with marked hemolysis have microovalocytes, bizarre-shaped RBC, and RBC fragments, all of which increase in number after splenectomy. The degree of hemolysis does not correlate with the percentage of elliptocytes.

Hereditary pyropoikilocytosis is a rare disorder related to hereditary elliptocytosis and is characterized by bizarre-shaped, microcytic RBC that undergo disruption at temperatures of 44 to 45°C (in contrast, normal RBC are stable up to 49°C). This condition results from a deficiency of spectrin and an abnormality of spectrin self-assembly. Hemolysis is usually severe, is recognized in childhood, and is partially responsive to splenectomy.

Hereditary Stomatocytosis

Stomatocytes are cup-shaped RBC that have a slitlike central zone of pallor on blood smears. The disorder is inherited in an autosomal dominant pattern. RBC have an increased permeability to sodium and potassium, which is compensated for by an increased active transport of these cations. In some patients, the RBC are swollen with an excess of ions and water and a decreased mean corpuscular hemoglobin concentration (overhydrated stomatocytes, "hydrocytosis"); many of these patients lack the RBC membrane protein 7.2 (stomatin). RBC lacking Rh proteins (Rhnull cells) are also stomatocytic and have a shortened life span. In other patients, the RBC are shrunken, with a decreased ion and water content, appearing as target cells on blood smears. Most patients have splenomegaly and mild anemia. Splenectomy decreases but does not totally correct the hemolytic process.

Red Cell Enzyme Defects

During its maturation, the RBC loses its nucleus, ribosomes, and mitochondria and thus its capability for protein synthesis and oxidative phosphorylation. The mature circulating RBC has a relatively simple pattern of intermediary metabolism (Fig. 93-4) in keeping with its modest metabolic obligations. ATP must be generated from the Embden-Meyerhof pathway to drive the cation pump that maintains the ionic milieu in the RBC. Smaller amounts of energy are needed for the preservation of hemoglobin iron in the ferrous (Fe2+) state and perhaps for the renewal of the lipids in the RBC membrane. About 10% of the glucose consumed by the RBC is metabolized via the hexose-monophosphate shunt (Fig. 93-4), which protects both hemoglobin and the membrane from oxidants, including certain drugs.


Figure 93-4 RBC metabolism. The Embden-Meyerhof pathway (glycolysis) generates ATP for energy and membrane maintenance. The generation of NADPH maintains hemoglobin in a reduced state. The hexose monophosphate shunt generates NADPH that is used to reduce glutathione, which protects the red cell against oxidant stress. Regulation of 2,3-bisphosphoglycerate levels is a critical determinant of oxygen affinity of hemoglobin. Enzyme deficiency states in order of prevalence: glucose-6-phosphate dehydrogenase (G6PD) >>> pyruvate kinase > glucose-6-phosphate isomerase > rare deficiencies of other enzymes in the pathway. The more common enzyme deficiencies are encircled.


Defects in the Embden-Meyerhof Pathway

Most of the glycolytic enzyme defects are inherited in an autosomal recessive pattern. Since the gene frequency for this group of defects is low, true heterozygotes are often the offspring of a consanguineous mating. More often, affected individuals are compound homozygotes. Patients with severe hemolysis usually present during early childhood with anemia, jaundice, and splenomegaly. The RBC are often relatively deficient in ATP, resulting in a leak of potassium ion out of these cells. These RBC are rigid and thus more readily sequestered by the mononuclear phagocyte system.

Some of these glycolytic enzyme deficiencies such as pyruvate kinase (PK) deficiency and hexokinase deficiency are localized to the RBC, with no apparent metabolic abnormality in other cells. In other disorders, the enzyme deficiency is more widespread.

About 95% of the clinically significant defects in the glycolytic pathway are due to PK deficiency, and about 4% are due to glucose phosphate isomerase deficiency. The remainder, shown in Fig. 93-4, are extremely rare. Most have been encountered in isolated families; clinical manifestations are variable.

Laboratory Findings

Patients have a normocytic (or slightly macrocytic), normochromic anemia with reticulocytosis. In those with PK deficiency, bizarre erythrocytes, including spiculated cells, are noted on the peripheral smear, especially after splenectomy. Spherocytes are usually absent; the term congenital nonspherocytic hemolytic anemia has been applied to these disorders. The diagnosis of this group of anemias depends on specific enzymatic assays. Abnormalities in enzymatic properties may be useful in distinguishing among enzyme mutants. DNA sequencing is the only definitive way to identify a mutant.

Treatment

Most patients do not require therapy. Those with severe hemolysis should be given folic acid (1 mg/d). Blood transfusions may be necessary during a hypoplastic crisis. Women with PK deficiency may become very anemic during pregnancy, sometimes leading to the diagnosis for the first time. Patients with PK or glucose phosphate isomerase deficiency may benefit from splenectomy.

Defects in the Hexose-Monophosphate Shunt

Normal RBC are well protected against oxidant stress. When the cells are exposed to a drug or toxin that generates oxygen radicals, glucose metabolism via the hexose-monophosphate shunt is normally increased severalfold. Reduced glutathione is regenerated, protecting the sulfhydryl groups of hemoglobin and the RBC membrane from oxidation. Individuals with an inherited defect in the hexose-monophosphate shunt are unable to maintain an adequate level of reduced glutathione in their RBC, hemoglobin sulfhydryl groups become oxidized, and the hemoglobin precipitates within the RBC, forming Heinz bodies.

G6PD Deficiency

This is by far the most common congenital shunt defect, affecting more than 200 million people throughout the world; like hemoglobin S, it partially protects the patient from malaria by providing a defective home for the merozoite. Over 400 variants of G6PD have been described, resulting in considerable clinical heterogeneity among affected individuals. Most are missense mutations resulting in altered enzymatic properties.

The normal G6PD is designated as type B. About 20% of individuals of African descent have a G6PD (designated A+) that differs by a single amino acid and is electrophoretically distinguishable but functionally normal. Among the clinically significant G6PD variants, the most common, the so-called A– type, is due to two base substitutions and is encountered primarily in individuals of central African descent. The A– G6PD has the same electrophoretic mobility as the A+ type, but it is unstable and has abnormal kinetic properties. This variant is found in about 11% of African-American males. A second relatively common G6PD variant is encountered among groups of Mediterranean origin, particularly Sardinians and Sephardic Jews; this variant is more severe than the A– variant and may result in nonspherocytic hemolytic anemia in the absence of known oxidative stress. A third relatively common and slightly less severe variant occurs in southern Chinese populations.

The G6PD gene is located on the X chromosome; thus the deficiency state is a sex-linked trait. Affected males (hemizygotes) inherit the abnormal gene from their mothers who are usually carriers (heterozygotes). Because of inactivation of one of the two X chromosomes (Lyon hypothesis: Chap. 56), the heterozygote has two populations of RBC: normal and deficient in G6PD. Most female carriers have no problems. Those who happen to have a high proportion of deficient cells resemble the male hemizygotes. G6PD activity normally declines 50% during the 120-day life span of the RBC. This decay is moderately accelerated in A– RBC and markedly so in RBC containing the Mediterranean variant. Individuals with the A– variant normally have a slightly shortened RBC survival time, but they are not anemic. Clinical problems arise only when the affected individual is subjected to some type of environmental stress. Most often, hemolytic episodes are triggered by viral and bacterial infections. The mechanism is unknown. In addition, drugs or toxins that pose an oxidant threat to the RBC (most commonly sulfa drugs, antimalarials, and nitrofurantoin) cause hemolysis in individuals deficient in G6PD (Table 93-4). Although aspirin is frequently mentioned as a likely offender, it has no deleterious effect in A– individuals. Accidental ingestion of toxic compounds such as naphthalene (moth balls) may cause severe hemolysis. Metabolic acidosis can precipitate an episode of hemolysis in individuals deficient in G6PD.

Clinical and Laboratory Features

The patient may experience an acute hemolytic crisis within hours of exposure to the oxidant stress, leading to hemoglobinuria and peripheral vascular collapse in severe cases. Since only the older population of RBC is rapidly destroyed, the hemolytic crisis is usually self-limited, even if the exposure to the oxidant continues. Among black males with the A– variant, the RBC mass decreases by a maximum of 25 to 30%. The oxidation of hemoglobin leads to the formation of Heinz bodies, visualized by means of a supravital stain such as crystal violet. However, Heinz bodies are usually not seen after the first day or so, since these inclusions are readily removed by the spleen. Their removal leads to the formation of "bite cells" (RBC that have lost a peripheral portion of the cell). Multiple bites cause the formation of fragments. A few spherocytes also may be present. Individuals with the Mediterranean type G6PD have much lower overall enzyme activity than those with the A– variant and, therefore, have more severe clinical manifestations. A minority of patients are exquisitely sensitive to fava beans and develop a fulminant hemolytic crisis after exposure. The oxidants in Vicia fava are two -glycosides whose aglycones, when autooxidized, produce oxygen free radicals. The incidence of favism is highly variable due to variations in concentration, in absorption, or in metabolism of the aglycones. Favism is seldom encountered in individuals with the A– variant.

The diagnosis of G6PD deficiency should be considered in any individual, particularly a male of African or Mediterranean descent, who experiences an acute hemolytic episode. The patient should be questioned about possible exposure to oxidant agents. The diagnosis can be established by a number of tests that assess either the enzyme activity or the effects of its deficiency. However, the test may yield a false-negative result during a hemolytic episode when the old RBC deficient in the enzyme have already lysed.

Treatment

Since hemolysis in patients deficient in A– G6PD is usually self-limited, no specific treatment is necessary. Splenectomy does not benefit Mediterranean patients with chronic hemolysis. Blood transfusions are rarely indicated. Adequate urine flow should be maintained if hemoglobinuria develops during an acute hemolytic episode.

Hemolytic episodes can be prevented by warning patients about risks posed by oxidant drugs and fava beans and by prompt treatment of infections.

Other Defects of the Hexose-Monophosphate Shunt

A few kindreds have been found to have congenital deficiency in RBC glutathione due to a defect in either of the two enzymes responsible for the synthesis of this tripeptide. Affected individuals have a hemolytic anemia with Heinz bodies that is aggravated by oxidant drugs.

Other Enzyme Defects

Hemolytic anemia may sometimes be caused by abnormalities in enzymes of nucleotide metabolism. Individuals with pyrimidine 5'-nucleotidase deficiency have marked coarse basophilic stippling in their RBC because the mRNA of the cell is not properly metabolized. Hemolytic anemia also has been noted in individuals whose RBC have supranormal levels of adenosine deaminase and relatively low levels of ATP.

Hemoglobinopathies

Hemolysis is a component of anemias related to some hemoglobinopathies (Chap. 91).