CHAPTER 43 HEREDITARY SPHEROCYTOSIS, ELLIPTOCYTOSIS, AND RELATED DISORDERS
CHAPTER 43 HEREDITARY SPHEROCYTOSIS, ELLIPTOCYTOSIS, AND RELATED DISORDERS
PATRICK G. GALLAGHER
BERNARD G. FORGET
Definition and History
Etiology and Pathogenesis
Therapy and Prognosis
Hereditary Elliptocytosis, Pyropoikilocytosis, and Related Disorders
Definition and History
Etiology and Pathogenesis
Therapy and Prognosis
Hereditary spherocytosis (HS) is a term that refers to a group of disorders characterized by spherically shaped erythrocytes on peripheral blood smear. HS is the most common inherited anemia in individuals of northern European descent, affecting approximately 1 in 2500 individuals. The principal cellular defect in HS is loss of membrane surface area, accounting for the spherical shape and decreased deformability of the erythrocyte. Splenic destruction of nondeformable spherocytes leads to the hemolysis experienced by HS patients. Membrane loss is due to defects in several membrane proteins, including ankyrin, band 3, a spectrin, b spectrin, and protein 4.2. There is significant clinical, laboratory, biochemical, and genetic heterogeneity in HS. It may present at any age, from infancy to late in life, with signs and symptoms due to hemolytic anemia or its complications. In most patients, splenectomy is curative. With a few rare exceptions, HS is due to “private” mutations; that is, each kindred has a unique mutation. Hereditary elliptocytosis (HE) is characterized by the presence of elliptical erythrocytes on peripheral blood smear. It is common in people of African and Mediterranean ancestry and occurs in approximately 1 in 2000 individuals. The principal defect in HE erythrocytes is mechanical weakness or fragility of the membrane skeleton. This is due to abnormalities in the proteins involved in membrane skeleton interactions, including a spectrin, b spectrin, protein 4.1, or GPC. Although the clinical presentation of HE is heterogeneous, the majority of HE patients are asymptomatic, and therapy is rarely necessary. Hereditary pyropoikilocytosis (HPP) is a rare cause of severe hemolytic anemia characterized by erythrocyte morphology similar to that seen in thermal burns. There is a strong relationship between HPP and HE. Approximately one-third of family members of patients with HPP ha ve typical HE, and many of these patients share identical biochemical and genetic defects in spectrin. A wide variety of mutations associated with HE/HPP have been identified, with a few a spectrin mutations responsible for the majority of cases.
Acronyms and abbreviations that appear in this chapter include: 2,3-BPG, 2,3-bisphosphoglycerate; GPC, glycophorin C; GPD, glycophorin D; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; HS, hereditary spherocytosis; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume.
Hemolytic anemias due to defects in the erythrocyte membrane comprise an important group of hereditary anemias. Hereditary spherocytosis, HE, and HPP are the most common disorders among this group. Originally classified by their morphologic presentation, detailed studies have demonstrated considerable overlap between these disorders and significant heterogeneity in their clinical, morphologic, laboratory, and molecular characteristics (Table 43-1). Advances in molecular biology have allowed further characterization of these disorders and, in many cases, detection of the precise genetic defect (see Table 43-1). These molecular analyses have provided additional information on the pathogenesis of these disorders as well as important insights into the structure-function relationships of the proteins of the erythrocyte membrane (Chap. 27).
TABLE 43-1 ERYTHROCYTE MEMBRANE PROTEIN DEFECTS IN INHERITED DISORDERS OF RED CELL SHAPE
DEFINITION AND HISTORY
Hereditary spherocytosis is a term that refers to a group of disorders characterized by spherical, doughnut-shaped erythrocytes with increased osmotic fragility. HS occurs in all racial and ethnic groups. It is the most common inherited anemia in individuals of northern European ancestry, affecting approximately 1 in 2500 individuals in the United States and England. Clinical, laboratory, biochemical, and genetic heterogeneity characterize the spherocytosis syndromes.
Hereditary spherocytosis was first described over 100 years ago by two Belgian physicians, Vanlair and Masius. Twenty years later, the disease was rediscovered by Wilson and Minkowsky, who reported eight cases of HS in three generations of one family. The description of increased erythrocyte osmotic fragility by Chauffard, reports of correction of anemia and hemolysis by splenectomy, and the studies of Ham and Castle implicating the spleen in the conditioning of hereditary spherocytes followed. The early history of HS is elegantly reviewed by Dacie.1
A defect of the erythrocyte membrane was implicated when HS membranes were found to be leaky to sodium and to exhibit a loss of lipids, leading to surface area deficiency. Subsequently, abnormalities of proteins of the erythrocyte membrane have been identified as the etiology of the defect in HS.2,3,4 and 5
ETIOLOGY AND PATHOGENESIS
The hallmark of HS erythrocytes is loss of membrane surface area relative to intracellular volume, accounting for the spheroidal shape and decreased deformability of the red cell. This loss of surface area results from increased membrane fragility due to defects in proteins of the erythrocyte membrane, including ankyrin, band 3, b spectrin, a spectrin, and protein 4.2. Increased fragility leads to membrane vesiculation and surface area loss (Fig. 43-1). Splenic trapping of nondeformable spherocytes, followed by conditioning and destruction of these abnormal erythrocytes, is the cause of hemolysis experienced by HS patients. Thus the spleen plays an important role in hemolysis, secondary to the basic defect of the erythrocyte membrane.
FIGURE 43-1 Pathobiology of HS. The primary defect in HS is a deficiency of membrane surface area, leading to the formation of spherocytes. Decreased surface area may be produced by two different mechanisms: (1) defects of spectrin and ankyrin lead to reduced density of the membrane skeleton, destabilizing the overlying lipid bilayer and releasing band 3-containing microvesicles; or (2) defects of band 3 or protein 4.2 lead to band 3 deficiency and loss of its lipid-stabilizing effect, resulting in the loss of band 3-free microvesicles. Both pathways result in membrane loss, decreased surface area, and formation of spherocytes with decreased deformability. These deformed erythrocytes become trapped in the hostile environment of the spleen, where splenic conditioning inflicts further membrane damage, amplifying the cycle of red cell membrane injury. (From Gallagher PG, Jarolim P: Red cell membrane disorders, in Hematology: Basis Principles and Practice, edited by R Hoffman, EJ Benz, Jr, SJ Shattil, et al. WB Saunders, Philadelphia, 1999. (pp. 576–610), with permission.)
RED CELL MEMBRANE PROTEIN DEFECTS
Study of erythrocyte membranes has revealed quantitative abnormalities of several membrane proteins.6,7 and 8 Combined spectrin and ankyrin deficiency is most commonly observed, followed in frequency by band 3 deficiency, isolated spectrin deficiency, and protein 4.2 deficiency. Multiple genetic loci are involved. Except for a few rare exceptions, HS mutations are private, that is, each kindred has a unique mutation, implying that there is no selective advantage to mutations.
Ankyrin Ankyrin links the spectrin-based membrane skeleton to the lipid bilayer via interactions with band 3. Concomitant spectrin and ankyrin deficiency is the most common finding in HS erythrocyte membranes (Fig. 43-2a). Several mechanisms, including decreased synthesis of ankyrin, decreased ankyrin assembly on the membrane, or assembly of an abnormal ankyrin, could lead to decreased assembly of spectrin on the membrane when spectrin binding sites on ankyrin are decreased, absent, or defective.
FIGURE 43-2 The role of ankyrin and spectrin in HS. (a) Correlation of spectrin and ankyrin deficiencies in 20 dominant HS kindreds. Each point, expressed as a percentage of the control (100%), represents the mean value for a kindred for both red cell spectrin and ankyrin levels. Within experimental error, the degree of spectrin and ankyrin deficiencies is essentially identical in these families with one exception (open circle), an otherwise typical family in which red cells are primarily ankyrin deficient. (From P Savvides et al,7 with permission.) (b) Correlation between red cell spectrin deficiency and unincubated osmotic fragility (a measure of spheroidicity) in HS. Spectrin content, as measured by radioimmunoassay, is shown on the vertical axis; and osmotic fragility, as measured by NaCl concentration producing 50 percent hemolysis of erythrocytes, is shown on the horizontal axis. Circles represent patients with typical autosomal dominant HS, and triangles represent patients with atypical, nondominant HS. Open symbols represent patients who have undergone splenectomy. The right panel shows the hematocrit of every patient at least 4 months after splenectomy. Note that very spectrin-deficient patients have more spherical red cells and an incomplete response to splenectomy. (From P Agre et al,22 with permission.)
Genetic screening has identified a number of ankyrin gene mutations in patients and has demonstrated that ankyrin defects are the most common cause of typical, dominant HS.9,10 and 11 The majority of ankyrin mutations are either frameshift or nonsense mutations that lead to a defective ankyrin molecule, ankyrin deficiency, or both. Missense mutations may disrupt normal ankyrin-protein interactions. One such variant, ankyrinWaldsrode, identified in a kindred whose erythrocyte membranes were deficient in band 3 as well as ankyrin and spectrin, was due to a mutation in the band 3 binding domain of ankyrin that decreased its affinity for band 3.12 With one exception, all ankyrin mutations described to date have been private. The exception, ankyrinFlorianopolis, is a recurrent frameshift mutation associated with severe dominantly inherited HS.13
Genetic variants have been identified in the promoter of the ankyrin gene in a number of patients with recessively inherited HS.9,14 Whether these are disease-causing mutations or are merely polymorphisms in linkage disequilibrium with the as yet unidentified mutation is unknown.
Cytogenetic studies have identified a few ankyrin-deficient HS patients with dysmorphic features, psychomotor retardation, and hypogonadism.15 These patients suffer from a contiguous gene syndrome that includes deletion of the ankyrin gene locus at 8p11.2.
Band 3, the Anion Exchanger Band 3 mediates interactions within the membrane skeleton via ankyrin, protein 4.1, and protein 4.2 via its NH2-terminal cytoplasmic domain and effects anion exchange via its membrane-spanning COOH-terminal domain. A subset of patients with typical dominant HS whose red cells are approximately 20 to 40 percent deficient in band 3 and protein 4.2, but have a normal spectrin content, has been described.4 These patients generally have mild to moderate HS and have pincered spherocytes on peripheral smears.
A variety of band 3 gene mutations associated with HS have been identified, including missense, nonsense, duplication, insertion, deletion, and RNA-processing mutations.16,17,18,19 and 20 The missense mutations include a group of mutations that replace highly conserved arginine residues in the transmembrane domain. These mutant proteins do not fold and fail to insert into the endoplasmic reticulum and, ultimately, into the erythrocyte membrane.17 The nonsense mutations lead to decreased band 3 mRNA accumulation, presumably due to mRNA instability.18 In HS patients with band 3Campinas and band 3Pribram, defects in band 3 mRNA processing, an unexplained renal tubular acidosis has also been observed.19,20
Spectrin Spectrin is composed of two subunits, a and b, which, despite the many similarities, are structurally distinct and are encoded by separate genes. The function of erythrocyte spectrin is to maintain cellular shape, regulate the lateral mobility of integral membrane proteins, and provide structural support for the lipid bilayer.3 Erythrocytes from most patients are spectrin deficient, including both the dominant and recessive forms. The degree of spectrin deficiency correlates with the spheroidicity of erythrocytes, their ability to withstand shear stress, the degree of hemolysis, and the response to splenectomy (Fig. 43-2b).21,22
In humans, a-spectrin synthesis exceeds b-spectrin synthesis by a ratio of about three or four to one.23 Patients who are heterozygous for an a-spectrin defect should still produce enough normal a-spectrin chains to pair with all, or nearly all, of the b-spectrin chains that are synthesized. Thus, patients with a-spectrin defects should only be symptomatic when the defect is found in the homozygous or compound heterozygous state. In a similar manner, deficiency of the limiting b-spectrin chains due to b-spectrin defects should be expressed as a dominantly inherited trait.
a Spectrin The mechanisms of spectrin deficiency are unknown in most HS patients with recessively inherited HS. A number of patients with severe recessively inherited HS and marked spectrin deficiency have a mutant allele, aLEPRA (low-expression Prague), that produces approximately one-sixth of the correctly spliced a-spectrin transcript as the normal allele, due to aberrant mRNA processing.24 In one patient, the combination of the LEPRA allele with another defect of a spectrin in trans, a truncated a-spectrin chain, aPrague, led to severe spectrin deficiency and severe spherocytic anemia.25 Whether aLEPRA is the etiology of many cases of a-spectrin-linked HS is yet to be determined. An amino acid substitution in the aII domain of spectrin, aBug Hill, has been identified in many patients with spectrin-deficient, recessive HS.26 Studies suggest that aBug Hill is not itself responsible for HS, but is likely a polymorphic variant that in some, but not all, cases is in linkage disequilibrium with another uncharacterized a-spectrin gene defect that is the cause of HS.
b Spectrin A group of patients who are heterozygous for defects in the limiting b-spectrin chain associated with spectrin deficiency and dominant HS have been described.27,28 and 29 These patients suffer from typical HS with a subpopulation of acanthocytes.
The majority of b-spectrin mutations have been associated with null alleles, including frameshift, nonsense, and initiator codon mutations. One frameshift mutation of b spectrin, due to a single nucleotide deletion, spectrinHouston, has been found in patients from several unrelated kindreds, suggesting that it might be a common b-spectrin mutation associated with HS.28 Truncated b-spectrin chains due to genomic deletions, exon skipping, and frameshift mutations have also been described. A few missense mutations associated with HS have been reported. One of these, spectrinKissimmee, is an unstable b spectrin that lacks the ability to bind protein 4.1 and binds poorly to actin due to a point mutation in a highly conserved region of b spectrin thought to be involved in protein 4.1 binding.27
Protein 4.2 Protein 4.2–deficient patients with recessively inherited HS have been described, primarily from Japan.16,30 One common variant, protein 4.2Nippon, is due to a point mutation that presumably affects protein 4.2 mRNA processing.30 Other variants are due to homozygosity or compound heterozygosity for frameshift, missense, or mRNA processing mutations of the protein 4.2 gene.
Deficiency of protein 4.2 has also been observed in patients with mutations in the cytoplasmic domain of band 3.31,32 These mutations presumably involve the region of band 3-protein 4.2 interactions.
Cation Content and Membrane Permeability Potassium and water content are diminished in HS red cells, particularly those obtained from splenic pulp. The passive permeability of HS red cells to sodium is increased, presumably secondarily to the underlying skeletal defect.33,34 and 35 The excessive sodium influx activates Na+-K+-ATPase and the monovalent cation pump, and the accelerated pumping increases ATP turnover and glycolysis. The dehydration of HS red cells is likely to be inflicted, at least in part, by the adverse environment of the spleen, since spherocytes from surgically removed spleens are the most dehydrated.
The pathways causing HS red cell dehydration have not been clearly defined. One candidate is increased K-Cl cotransport, which is activated by acid pH. HS red cells, particularly from unsplenectomized subjects, have a low intracellular pH reflecting the low pH of the splenic environment (see below). The K+-Cl––cotransport pathway is also activated by oxidative damage, which is likely to be inflicted by splenic macrophages. Finally, overactivity of Na+-K+-ATPase, triggered by increased intracellular sodium, can dehydrate red cells directly, because three sodium ions are extruded in exchange for only two potassium ions, and the loss of monovalent cations is accompanied by water.
Membrane Lipids The principal lipid abnormality of hereditary spherocytes is a symmetrical loss of each species of membrane lipid as part of the overall loss of membrane surface, the hallmark of HS pathobiology. The relative proportions of cholesterol and the various phospholipids are normal, and the phospholipids show the usual transmembrane asymmetry, even in severe cases.
Role of the Spleen The spleen plays a secondary but important role in the pathophysiology of HS. Splenic destruction of abnormal erythrocytes with decreased deformability is the primary cause of hemolysis. Physical entrapment of spherocytes in the splenic microcirculation and ingestion by phagocytes have been proposed as mechanisms of destruction.
Splenic Trapping of Nondeformable Spherocytes Because of their diminished deformability, spherocytes are unable to traverse the slits between the endothelial and adventitial cells that form a wall separating the splenic cords of the red pulp from the splenic sinuses (see Chap. 5). The decrease in red cell deformability is primarily related to decreased surface area and secondarily to greater internal viscosity that results from mild cellular dehydration. In addition, the splenic environment is hostile to erythrocytes.35 Low pH, low glucose and ATP concentrations, and high local concentrations of toxic free radicals produced by adjacent phagocytes all contribute to membrane damage.
Conditioning and Destruction of Spherocytes in the Spleen Impeded spherocyte passage through the sinus wall fenestrations leads to a markedly engorged red pulp and pulp cords with relatively empty venous sinuses.36 It is here that red cells are “conditioned,” becoming more osmotically fragile and more spherical, with a lower net sodium and potassium content than cells obtained from the systemic circulation.37 Splenic conditioning is a consequence of multiple episodes of splenic stasis. The estimated residence time of HS erythrocytes in the cords is between 10 and 100 min, and only 1 to 10 percent of blood entering the spleen is detained by the congested cords, while more than 90 percent is rapidly shunted into the venous circulation.
Macrophage phagocytosis in the spleen is the final step in the cycle of spherocyte destruction. The stimulus for phagocytosis by the macrophage is unknown.
The genes responsible for HS include ankyrin, b spectrin, band 3 protein, a spectrin, and protein 4.2. In approximately two-thirds to three-quarters of HS patients, inheritance is autosomal dominant. In the remaining patients, dominant inheritance cannot be demonstrated. Inheritance may be autosomal recessive or due to a de novo mutation. Cases with autosomal recessive inheritance are due to defects in either a spectrin or protein 4.2. A surprising number of de novo mutations have been reported in the HS genes.11,27,29 A few cases of “double-dominant” HS due to defects in band 3 or spectrin that result in fetal death or severe hemolytic anemia presenting in the neonatal period have been reported.43,44 In general, affected individuals of the same kindred experience similar degrees of hemolysis. Rarely, members of the same kindred will experience varying degrees of hemolysis. When HS has been identified in one or more siblings whose parents have no identifiable abnormalities or when there is great variability in the clinical severity of affected HS family members, a number of explanations can be sought. These include inheritance of a modifier allele that influences the expression of a membrane protein, leading to the variability in clinical expression, variable penetrance of the genetic defect, a de novo mutation, a mild form of recessively inherited HS, or tissue-specific mosaicism of the defect.11,27,29,38,45,46
The clinical manifestations of the spherocytosis syndromes vary widely. The typical clinical picture of HS combines evidence of hemolysis (anemia, jaundice, reticulocytosis, gallstones, and splenomegaly) with spherocytosis (spherocytes on the blood film and increased osmotic fragility) and a positive family history. Mild, moderate, and severe forms of HS have been defined according to differences in hemoglobin, bilirubin, and reticulocyte counts (Table 43-2), which can be correlated with the degree of compensation for the hemolysis.6,38,39 Initial assessment of a patient with suspected HS should include a family history and questions about history of anemia, jaundice, gallstones, and splenectomy. Physical examination should seek signs such as scleral icterus, jaundice, and splenomegaly.
TABLE 43-2 CLASSIFICATION OF HEREDITARY SPHEROCYTOSIS
TYPICAL HEREDITARY SPHEROCYTOSIS
Hereditary spherocytosis typically presents in infancy or childhood but may present at any age. In children, anemia is the most frequent presenting complaint (50%), followed by splenomegaly, jaundice, or a positive family history.40 No comparable data exist for adults. Two-thirds to three-quarters of HS patients have incompletely compensated hemolysis and mild to moderate anemia. The anemia is often asymptomatic except for fatigue and mild pallor or, with children, nonspecific parental complaints, such as irritability. Jaundice is seen at some time or other in about half of patients, usually in association with viral infections. When present it is acholuric, that is, unconjugated hyperbilirubinemia without detectable bilirubinuria. Palpable splenomegaly is detectable in most (75–95%) older children and adults. Typically the spleen is modestly enlarged (2–6 cm), but it may be massive. There is no proven correlation between the size of the spleen and the severity of HS; however, given the pathophysiology and the response of the disease to splenectomy, such a correlation probably exists. Typical HS is associated with both dominant and recessive inheritance. Although the recessively inherited forms tend to be more severe, there is considerable overlap.
COMPENSATED HEREDITARY SPHEROCYTOSIS
About 20 to 30 percent of HS patients have “compensated hemolysis”; that is, production and destruction are balanced, and the hemoglobin concentration of the blood is normal.4,38 Although the erythrocyte life span may only be about 20 to 30 days, these patients adequately compensate for their hemolysis with increased marrow erythropoiesis. Since they are not anemic, they are usually asymptomatic. In some cases, diagnosis may be difficult because hemolysis, splenomegaly, and spherocytosis are unusually mild. For example, in this group of patients, reticulocyte counts are generally less than 6 percent, and spherocytes are present on smear in only about 60 percent of patients. Many of these individuals escape detection until adulthood when they are being evaluated for unrelated disorders or when complications related to anemia or chronic hemolysis occur. Hemolysis may become severe with illnesses that cause further splenomegaly, such as infectious mononucleosis, or may be exacerbated by other factors, such as pregnancy or sustained, vigorous exercise. Because of the asymptomatic course of HS in these patients, diagnosis of HS should be considered during evaluation of incidentally noted splenomegaly, gallstones at a young age, or anemia resulting from parvovirus B19 infection or other viral infections.
MODERATELY SEVERE AND SEVERE HEREDITARY SPHEROCYTOSIS
Approximately 5 to 10 percent of HS patients have moderately severe to severe anemia. Patients with “moderately severe” disease typically have a hemoglobin level of 6 to 8 g/dl, reticulocytes about 10 percent, bilirubin 2 to 3 mg/dl, and 40 to 80 percent of the normal red cell spectrin content. This category includes patients with both dominant and recessive HS and a variety of molecular defects. Patients with “severe” disease, by definition, have life-threatening anemia and are transfusion dependent. They almost always have recessive HS. Most have isolated, severe spectrin deficiency (<40%), which is thought to be due to a defect in a spectrin.21,22,41 Patients with severe HS often have some irregularly contoured or budding spherocytes or bizarre poikilocytes in addition to typical spherocytes on blood smear. Such cells are rare prior to splenectomy in patients with moderately severe disease, but some may be seen postsplenectomy. In addition to the risks of recurrent transfusions, these patients often suffer from hemolytic and aplastic crises and may develop complications of severe uncompensated anemia including growth retardation, delayed sexual maturation, or aspects of thalassemic facies.
The parents of patients with recessive HS are clinically asymptomatic and do not have anemia, splenomegaly, hyperbilirubinemia, or spherocytosis on the blood films. However, most have subtle laboratory signs of HS, including slight reticulocytosis (»2%), diminished haptoglobin levels, and slightly elevated osmotic fragility. The incubated osmotic fragility test is probably the most sensitive measure of this condition, particularly the 100 pecent lysis point, which is significantly elevated in carriers (0.43 ± 0.05 g NaCl/dl) compared to normal subjects (0.23 ± 0.07).38 However, no single test is sufficient. Carriers can only be detected reliably by considering the results of a battery of tests.42 It has been estimated that at least 1.4 percent of the population are silent carriers.
PREGNANCY AND HEREDITARY SPHEROCYTOSIS
Most patients do well during pregnancy.47 Some experience anemia beyond that expected from expanded plasma volume due to increased hemolysis. A few patients are only symptomatic during pregnancy. Episodes of hemolytic crisis requiring transfusion and cases of folic acid deficiency have been described in pregnant HS patients.
HEREDITARY SPHEROCYTOSIS IN INFANCY
Anemia is the most common finding in neonates with HS, present in about 90 percent of cases.48,49 Some infants have required blood transfusion to treat their anemia. It is interesting to note that the degree of anemia seen in the neonatal period does not predict the severity of the anemia seen in later life. Jaundice occurs in about half of HS neonates and may be severe enough to require phototherapy or exchange transfusion. Jaundice in neonates with HS may be accentuated by the coinheritance of the Gilbert syndrome UDPGT1 gene polymorphism.50 Since kernicterus is a risk,48 exchange transfusions may be necessary, but in most cases the jaundice can be controlled with phototherapy.
Rarely, patients may suffer from severe hemolytic anemia presenting in utero or shortly after birth, continuing through the first year of life. These patients may require regular blood transfusions and, in some cases, early splenectomy. These severe HS patients usually suffer from significant spectrin deficiency, due to presumed homozygosity or compound heterozygosity for a-spectrin gene defects. Several cases of hydrops fetalis in HS patients requiring intrauterine transfusion due to severe anemia have been reported associated with band 3 or spectrin defects.
Gallbladder Disease Chronic hemolysis leads to the formation of bilirubinate gallstones, the most frequently reported complication in HS patients. Although gallstones have been detected in infancy, most appear in adolescents and young adults between 10 and 30 years of age (Fig. 43-3).4,51 Routine management should include interval ultrasonography to detect gallstones, since many patients with cholelithiasis and HS are asymptomatic. This will allow prompt diagnosis and treatment and prevent complications of symptomatic biliary tract disease, including biliary obstruction, cholecystitis, and cholangitis.
FIGURE 43-3 Proportion of normal and HS patients with gallstones as a function of age. Data are from a study of gallbladder disease in 152 consecutive HS patients seen at the Cleveland Clinic before 1952.51 Only patients whose gallbladders were examined at surgery or by cholecystography are included. Data for the general population are from an autopsy series of patients who did not have hemolytic anemia. The prevalence of gallstones rises sharply between the ages of 10 and 30 and parallels that of the general population after 30 years. (From SE Lux and J Palek: Disorders of the red cell membrane, in Blood: Principles and Practice of Hematology, edited by RI Handin, SE Lux, TP Stossel. p 1701. JB Lippincott, Philadelphia, 1995, with permission. Illustration by Joy D. Marlowe.)
Hemolytic, Aplastic, and Megaloblastic Crises Hemolytic crises are usually associated with viral illnesses and typically occur in childhood. They are generally mild and are characterized by jaundice, increased spleen size, a drop in hematocrit, and reticulocytosis. Medical intervention is rarely necessary. When severe hemolytic crises occur, there are marked jaundice, anemia, lethargy, abdominal pain, and tender splenomegaly. Hospitalization and erythrocyte transfusion may be required.
Aplastic crises following virally induced bone marrow suppression are uncommon but may result in severe anemia with serious complications, including congestive heart failure or even death. The most common etiologic agent in these cases is parvovirus B19, the etiologic agent of erythema infectiosum. Parvovirus infection typically presents with fever, chills, lethargy, vomiting, diarrhea, myalgias, and a maculopapular rash on the face (slapped cheek syndrome), trunk, and extremities.
Parvovirus B19 selectively infects erythropoietic progenitor cells and inhibits their growth (see Chap. 32).52 Parvovirus infections are frequently associated with mild neutropenia, thrombocytopenia, or even pancytopenia. During the aplastic phase, the hematocrit level and reticulocyte count fall, marrow erythroblasts disappear, and unused iron accumulates in the serum. Giant pronormoblasts, a hallmark of the cytopathic effects of parvovirus B19, often appear in the marrow. As production of new red cells declines, the cells that remain age, and microspherocytosis and osmotic fragility increase. Bilirubin levels may decrease as the number of abnormal red cells that can be destroyed declines. The return of marrow function is heralded by a fall in the serum iron concentration and the emergence of granulocytes, platelets, and, finally, reticulocytes.
Virally induced aplastic crisis brings many patients to medical attention, particularly asymptomatic HS patients with normally compensated hemolysis.53 As would be expected, because parvovirus may infect multiple members of a family simultaneously, leading to aplastic crises, there have been reports of “epidemics” or “outbreaks” of HS.54 Diagnostic confusion may arise during reemergence of marrow function, when the physician may mistake an aplastic crisis for a hemolytic one. Because aplastic crises usually last 10 to 14 days (about half the life span of HS red cells), the hemoglobin value typically falls to about half its usual level before recovery occurs. In patients with severe HS, the anemia may be profound, requiring hospitalization and transfusion.
Megaloblastic crisis occurs in HS patients with increased folate demands, such as the pregnant patient, growing children, or patients recovering from an aplastic crisis. With appropriate folate supplementation, this complication is preventable.
Other Complications Dermatologic manifestations of HS, including skin ulceration, gouty tophi, and chronic leg dermatitis, are uncommon.55 These dermatologic manifestations usually heal rapidly after splenectomy. The pathogenesis of these manifestations is unknown, but it has been proposed to be related to alterations in erythrocyte deformability, as has been suggested in patients with sickle cell anemia.
In some HS patients, findings attributable to extramedullary hematopoiesis have been described. These include poor growth and deformities of the hand and skull. Extramedullary tumors, particularly along the thoracic and lumbar spine or in the kidney hila, have been described in HS patients, including patients with untreated mild to moderate HS.4,56 Biopsy may be performed, since these masses may be mistaken for a malignant tumor, but because of their composition, it may be complicated by significant hemorrhage. Magnetic resonance imaging appears to be a reliable and safer alternative diagnostic modality. Postsplenectomy, these masses involute and undergo fatty metamorphosis. However, they do not decrease in size.
It has been suggested that HS predisposes patients to hematologic malignancies, including myeloproliferative disorders, particularly multiple myeloma.57 Chronic reticuloendothelial stimulation via splenic clearance of abnormal erythrocytes inducing the proliferation of lymphocytes, plasma cells, and macrophages has been suggested as a possible pathogenic mechanism. Thrombosis has been reported in several HS patients, usually postsplenectomy.
Iron overload has been described in untreated HS patients with coinherited hemochromatosis.58 However, studies clearly demonstrating this association have not been performed. Several of these patients subsequently died of liver disease or hepatoma. Untreated HS may aggravate underlying heart disease, particularly in the elderly. Progressive anemia due to loss of marrow reserve may gradually worsen underlying heart failure.
Angioid streaks have been described in the optic fundi of several adult HS patients.
Nonerythroid Manifestations In most patients with HS, the clinical manifestations are confined to the erythroid lineage. There are a few exceptions. Several HS kindreds have been reported with cosegregating nonerythroid manifestations, particularly neuromuscular abnormalities including cardiomyopathy, slowly progressive spinocerebellar degenerative disease, spinal cord dysfunction, and movement disorders.
The observation that erythrocyte ankyrin and b spectrin are also expressed in muscle, brain, and spinal cord raises the possibility that these HS patients may suffer from defects of one of these proteins.4,35 This hypothesis is further supported by studies of ankyrin-deficient nb/nb mice.59 These mice have almost no detectable ankyrin and suffer from a severe, spherocytic hemolytic anemia and a late-onset cerebellar ataxia that parallels a gradual loss of Purkinje cells. Another possibility is that another, yet to be described gene locus is causative. For example, mice that do not express the junctional complex membrane protein b adducin suffer from a spherocytic anemia and neurologic manifestations.59,60
A few heterozygous defects of band 3 have been described in patients with autosomal dominant distal renal tubular acidosis and normal erythrocytes. This is in contrast to most patients with heterozygous mutations of band 3, who have normal renal acidification and abnormal erythrocytes. Two kindreds with coinherited HS and renal acidification defects due to band 3 mRNA processing mutations, band 3Pribram and band 3Campinas, have been described.19,20
Like the clinical presentation of HS, laboratory findings in HS are heterogeneous.
THE BLOOD FILM
Erythrocyte morphology in HS is quite variable. Typical HS patients have blood films with easily identifiable spherocytes lacking central pallor (Fig. 43-4). Less commonly, patients present with only a few spherocytes on the film or, at the other end of the spectrum, with numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis. Rarely, spherostomatocytes may be seen. Specific morphologic findings have been identified in patients with certain membrane protein defects, such as pincered erythrocytes (band 3) or spherocytic acanthocytes (b spectrin). When examining blood from a patient with suspected spherocytosis, it is important to have a high-quality film with the erythrocytes well separated and some cells with central pallor in the field of examination, since spherocytes are a common artifact.
FIGURE 43-4 Peripheral blood smears from patients with HS of varying severity. (a) Typical HS with a mild deficiency of red cell spectrin and ankyrin. Although many cells have spheroidal shape, some of them retain a central concavity. (b) HS with pincered red cells (arrows), as typically seen in HS associated with band 3 deficiency. Occasionally, spiculated red cells are also present. (c) Severe atypical HS due to a severe combined spectrin and ankyrin deficiency. In addition to spherocytes, there are many cells with irregular contour. (d) HS with isolated spectrin deficiency due to a b spectrin mutation. Some of the spherocytes have prominent surface projections resembling spheroacanthocytes. (Film d courtesy of DL Wolfe.)
Most patients have a mild to moderate anemia with hemoglobin in the 9- to 12-g/dl range (see Table 43-2). The MCHC is increased (between 35 and 38%) due to relative cellular dehydration in approximately 50 percent of patients, but all HS patients have some dehydrated cells. The Technicon H1 blood counter and its successors (Technicon, Tarrytown, NY) provide a histogram of MCHC that has been claimed to be accurate enough to identify nearly all HS patients (Fig. 43-5a).61 Finally, the MCV is usually normal except in cases of severe HS, when it is slightly decreased. Typically, the MCV is relatively low for the age of the cells in most HS patients, reflecting the dehydrated state of the HS erythrocytes.
FIGURE 43-5 Laboratory diagnosis of HS. (a) Histograms of the distribution of (A) MCV and (B) MCHC in red cells of a patient with HS before splenectomy. The vertical lines mark the normal limits of the distributions. The data were collected with a Technicon H1 laser scattering blood counter. The patient has subpopulations of microcytes (low MCV) and dehydrated cells (high MCHC), which presumably represent conditioned microspherocytes. All 21 HS patients in one study had similar subpopulations. (From AR Pati et al,61 with permission.) (b) Osmotic fragility testing. The shaded area is the normal range. Results representative of both typical and severe spherocytosis are shown. A “tail,” representing very fragile erythrocytes that have been conditioned by the spleen, is common in many HS patients prior to splenectomy. (From PG Gallagher et al,35 with permission.)
In the normal erythrocyte, a redundancy of cell membrane gives the cell its characteristic discoid shape and provides it with abundant surface area. In spherocytes, there is a decrease in surface area relative to cell volume, resulting in their abnormal shape. This change is reflected in the increased osmotic fragility found in these cells (Fig. 43-5b). Osmotic fragility is tested by adding increasingly hypotonic concentrations of saline solution to red cells. The normal erythrocyte is able to increase its volume by swelling, but spherocytes, which are already at maximum volume for surface area, burst at higher saline concentrations than normal. Approximately one-quarter of HS individuals will have a normal osmotic fragility on freshly drawn red blood cells, with the osmotic fragility curve approximating the number of spherocytes seen on the blood film. However, after incubation at 37°C (98.6°F) for 24 h, HS red cells lose membrane surface area more readily than normal because their membranes are leaky and unstable. Thus, incubation accentuates the defect in HS erythrocytes and brings out the defect in osmotic fragility, making incubated osmotic fragility the standard test in diagnosing HS. When the spleen is present, a subpopulation of very fragile erythrocytes that have been conditioned by the spleen form the “tail” of the osmotic fragility curve (Fig. 43-5b). This tail disappears after splenectomy. Unfortunately, the osmotic fragility test suffers from poor sensitivity, with as many as 20 percent of mild cases of HS missed after incubation. The osmotic fragility test is unreliable in patients who have small numbers of spherocytes, including those who have been recently transfused. Its results are abnormal in other conditions where spherocytes are present.
Other investigations, such as the autohemolysis test, the hypertonic cryohemolysis test, and the acidified glycerol test, suffer from lack of specificity, are cumbersome to perform, and are not widely used. Specialized testing is available for studying difficult cases or cases where additional information is desired. Useful tests for these purposes include structural and functional studies of erythrocyte membrane proteins, such as protein quantitation, limited tryptic digestion of spectrin, and ion transport. Membrane rigidity and fragility can be examined using an ektacytometer. cDNA and genomic DNA analyses are available when a molecular diagnosis is desired.
Other laboratory manifestations in HS are markers of ongoing hemolysis. Reticulocytosis, increased serum bilirubin, increased lactate dehydrogenase, increased urinary and fecal urobilinogen, and decreased serum haptoglobin reflect increased erythrocyte production or destruction. In many cases of HS, the reticulocyte count appears to be elevated disproportionately relative to the degree of anemia. This has been observed even in HS patients with normal hemoglobin levels. The etiology of this observation is unknown.
Initial laboratory investigation should include a complete blood count with a blood film, reticulocyte count, direct antiglobulin test (Coombs’ test), and serum bilirubin. An incubated osmotic fragility should be obtained. Rarely, additional, specialized testing is required to confirm the diagnosis. In neonates, ABO incompatibility should be considered, but its differentiation from HS becomes clear several months after birth. Other causes of spherocytic hemolytic anemia, such as clostridial sepsis, transfusion reactions, severe burns, and bites from snakes, spiders, bees, and wasps, should be viewed in the appropriate clinical context (see Chap. 53, Chap. 54, and Chap. 140). Occasional spherocytes are also seen in patients with a large spleen (e.g., in cirrhosis or myelofibrosis) or in patients with microangiopathic anemias (see Chap. 51), but the differentiation of these conditions from HS does not usually present diagnostic difficulties.
Hereditary spherocytosis may be obscured in disorders that increase the surface-volume ratio of erythrocytes, such as obstructive jaundice, iron deficiency, b thalassemia trait or hemoglobin SC disease, and vitamin B12 or folate deficiency. In obstructive jaundice, spherocytosis can be obscured by the accumulation of cholesterol and phospholipids in the membrane that characteristically accompanies this condition. While in normal subjects this process leads to target cell formation, hereditary spherocytes acquire a discoidal appearance, and their survival in the circulation is improved. Iron deficiency corrects the abnormal shape but does not improve survival of HS erythrocytes.
THERAPY AND PROGNOSIS
Splenic sequestration is the primary determinant of erythrocyte survival in HS patients. Thus, splenectomy cures or alleviates the anemia in the overwhelming majority of patients, reducing or eliminating the need for red cell transfusions. Elimination of the need for chronic blood transfusions has obvious implications for future iron overload and risk of end organ damage. The incidence of cholelithiasis is also decreased. Postsplenectomy, spherocytosis, and altered osmotic fragility persist, but the “tail” of the osmotic fragility curve, created by conditioning of a subpopulation of spherocytes by the spleen, disappears. Erythrocyte life span nearly normalizes, and reticulocyte counts fall to normal or near-normal levels. Changes typical of the postsplenectomy state, including Howell-Jolly bodies, target cells, siderocytes, and acanthocytes, become evident on the blood film. Postsplenectomy, patients with the most severe forms of HS still suffer from shortened erythrocyte survival and hemolysis, but their clinical improvement is striking.21,22
Complications of Splenectomy Early complications of splenectomy include local infection or bleeding and pancreatitis, presumably due to injury to the tail of the pancreas incurred during removal of the spleen. In general, the morbidity of splenectomy for HS is lower than that of other hematologic disorders. The complications of splenectomy are discussed in Chap. 5.
Indications for Splenectomy In the past, splenectomy, which has a low operative mortality, was considered routine in HS patients. However, the risk of overwhelming post-splenectomy infection (OPSI) and the recent emergence of penicillin-resistant pneumococci have led to a reevaluation of the role of splenectomy in the treatment of HS. Considering the risks and benefits, a reasonable approach would be to splenectomize all patients with severe spherocytosis and all patients who suffer from significant signs or symptoms of anemia, including growth failure, skeletal changes, leg ulcers, and extramedullary hematopoietic tumors. Other candidates for splenectomy are older HS patients who suffer vascular compromise of vital organs.
Whether patients with moderate HS and compensated, asymptomatic anemia should have a splenectomy remains controversial. Patients with mild HS and compensated hemolysis can be followed and referred for splenectomy if clinically indicated. The treatment of patients with mild to moderate HS and gallstones is also debatable, particularly since new treatments for cholelithiasis, including laparoscopic cholecystectomy, endoscopic sphincterotomy, and extracorporal choletripsy, lower the risk of this complication. If such patients have symptomatic gallstones, a combined cholecystectomy and splenectomy can be performed, particularly if acute cholecystitis or biliary obstruction has occurred. There is no evidence that performing cholecystectomy and splenectomy separately, as was done in the past, is of any benefit.
Because the risk of postsplenectomy sepsis is very high in infancy and early childhood, splenectomy should be delayed until the age of 5 to 9 years if possible and to at least 3 years if this is feasible, even if chronic transfusions are required in the interim. There is no evidence that further delay is useful, and it may be harmful, because the risk of cholelithiasis increases dramatically in children after the age of 10 years.
When splenectomy is warranted, laparoscopic splenectomy has become the method of choice in centers where there are surgeons experienced in this technique. If desired, the procedure can be combined with laparoscopic cholecystectomy. Lararoscopic splenectomy results in less postoperative discomfort, a quicker return to preoperative diet and activities, shorter hospitalization, decreased costs, and smaller scars.65 There is an increased risk of bleeding during the operation, and about 10 percent of laparoscopic operations (for all causes) have to be converted to standard splenectomies. Even enormous spleens (>600 g) can be removed laparoscopically, since the spleen is placed in a large bag, diced, and eliminated via suction catheters.
Partial splenectomy via laparotomy has been advocated for infants and young children with significant anemia associated with erythrocyte membrane disorders.66 The goals of this procedure are to allow for the palliation of hemolysis and anemia while maintaining some residual splenic immune function. Long-term follow-up data for this procedure are lacking.
Prior to splenectomy, patients should be immunized with vaccines against pneumococcus, Haemophilus influenzae type b, and meningococcus, preferably several weeks preoperatively. The use of prophylactic antibiotics postsplenectomy to prevent pneumococcal sepsis is controversial. Postsplenectomy, prophylactic antibiotics (penicillin V 125 mg orally twice daily for patients <7 years of age or 250 mg orally twice daily for those >7 years of age, including adults) have been recommended for at least 5 years postsplenectomy by some and for life by others. The optimal duration of prophylactic antibiotic therapy postsplenectomy is unknown. Presplenectomy and, in severe cases, postsplenectomy, HS patients should take folic acid (1 mg/day orally) to prevent folate deficiency.
Splenectomy Failure Splenectomy failure is uncommon. It may be due to an accessory spleen missed during splenectomy, the development of splenunculi resulting from autotransplantation of splenic tissue during surgery, or by another intrinsic red cell defect, such as pyruvate kinase deficiency (see Chap. 45). Accessory spleens occur in 15 to 40 percent of patients and must always be sought. Recurrence of hemolytic anemia years or even decades following splenectomy should raise suspicion of an accessory spleen, particularly if Howell-Jolly bodies are no longer found on blood film. A definitive confirmation of ectopic splenic tissue can be achieved by a radiocolloid liver-spleen scan or a scan using 51Cr-labeled, heat-damaged red cells.
After a patient is diagnosed with HS, family members should be examined for the presence of HS.
HEREDITARY ELLIPTOCYTOSIS, PYROPOIKILOCYTOSIS, AND RELATED DISORDERS
DEFINITION AND HISTORY
Hereditary elliptocytosis (HE) is characterized by the presence of elliptical or oval erythrocytes on the blood films of affected individuals.67,68 The worldwide incidence of HE has been estimated at 1 in 2000 to 1 in 4000 individuals. The true incidence of HE is unknown because its clinical severity is heterogeneous and many patients are asymptomatic. It is common in individuals of African and Mediterranean descent, presumably because elliptocytes confer some resistance to malaria. The incidence of HE is 6 percent in Benin, Africa.69 Genetic haplotyping studies suggest that one HE mutation common in Africa has a “founder effect” with origins in central Africa similar to that attributed to hemoglobin S, Benin-type.
The first description of HE in 1904 was by Dresbach, a physiologist at Ohio State University in Columbus, Ohio, who discovered the condition in a medical student during a laboratory exercise in which the students were examining their own blood.70 This report elicited some controversy, since the student died soon thereafter, leading to speculation that the student actually suffered from pernicious anemia. The demonstration of the disease in three generations of one family by Hunter clearly established the hereditary nature of this disorder.71 The history of HE is reviewed by Dacie.67
Hereditary pyropoikilocytosis (HPP) is a rare cause of anemia first described in three children with severe neonatal anemia with erythrocyte morphology similar to that seen in patients suffering severe burns.72 The erythrocytes from these patients also exhibited increased thermal sensitivity. Subsequently, other patients, mostly of African origin, with similar clinical and laboratory findings have been described.73,74 and 75 There is a strong relationship between HE and HPP. Approximately one-third of parents or siblings of patients with HPP have typical HE, and many of these family members share identical mutations in erythrocyte spectrin. In addition, many patients with HPP proceed to develop typical mild to moderate HE. Patients with HPP tend to experience severe hemolysis and anemia in infancy that gradually improves, evolving toward typical HE later in life.
ETIOLOGY AND PATHOGENESIS
The principal defect in HE and HPP erythrocytes is mechanical weakness or fragility of the erythrocyte membrane skeleton. As in HS, study of erythrocyte membrane proteins in these disorders has identified abnormalities of various erythrocyte membrane proteins. These include a and b spectrin, protein 4.1, and GPC. The majority of defects occur in spectrin, the principal structural protein of the erythrocyte membrane skeleton (see Chap. 27). Most spectrin defects in HE and HPP impair the ability of spectrin dimers to self-associate into tetramers and oligomers, thereby disrupting the membrane skeleton.3,76 Structural and functional defects of protein 4.1, which share similarities in cell shape and membrane stability to abnormalities of spectrin, are primarily due to disruption of the spectrin-actin attachment to the membrane via GPC. The mechanical instability in glycophorin C (GPC) variants appear to be due to secondary protein 4.1 deficiency. In all of these defects, disruption of the membrane skeleton leads to mechanical instability sufficient to cause red cell fragmentation with hemolytic anemia under conditions of normal circulatory shear stress.77
The pathobiology of elliptocytic shape is less clear. Red cell precursors in common HE are round, with the cells becoming progressively more elliptical as they age in vivo. Elliptocytes and poikilocytes may become permanently stabilized in shape because weakened spectrin heterodimer contacts facilitate skeletal reorganization following axial deformation of cells from prolonged or excessive shear stress. This reorganization is likely to involve breakage of the unidirectionally stretched protein connections followed by a formation of new protein contacts that preclude the recovery of normal biconcave shape. This process accounts for the permanent deformation of irreversibly sickle cells.
SPECTRIN AND HEREDITARY ELLIPTOCYTOSIS AND HEREDITARY PYROPOIKILOCYTOSIS
Abnormalities of either a or b spectrin associated with the majority of cases of HE and HPP are due to mutations in the spectrin heterodimer self-association site.76,78 The repeats of spectrin involved in self-association and the locations of reported mutations are shown diagrammatically in Fig. 43-6. Most of these mutations are missense mutations at or very near highly conserved residues of a spectrin. The missense mutations are primarily either a helix-breaking mutations that replace the normal residue with a proline or glycine, or charge-shift mutations. In contrast to HS, the elliptocytosis and pyropoikilocytosis syndromes, while also quite heterogeneous, have been associated with distinct spectrin mutations in persons of similar genetic backgrounds, suggesting a “founder effect” for these mutations.
FIGURE 43-6 Defects of the spectrin self-association site in HE and HPP. A triple helical model of the spectrin repeats that constitute the spectrin self-association site is shown. The symbols denote positions of various genetic defects identified in patients with HE or HP. Limited tryptic digestion of spectrin, followed by two-dimensional gel electrophoresis, identifies abnormal cleavage sites (arrows) in spectrin associated with various mutations. (Modified from PG Gallagher et al,68 with permission.)
HE or HPP phenotype–spectrin mutation genotype correlations are difficult to establish. There is great clinical phenotypic heterogeneity among individuals with the same spectrin mutation. This heterogeneity exists even among individuals from the same kindred. A few general phenotype-genotype correlations can be made. Mutations at the contact sites of a and b spectrin in the spectrin self-association site tend to be more severe.73,74 For example, mutations of codon 28, which is located in this contact site region, are generally associated with phenotypically severe HE or HPP. On the other hand, a common mutation in blacks from West and Central Africa, a leucine insertion at codon 154, is phenotypically very mild, even in the homozygous state.79 Because of the great phenotypic variability described above, the presence of low-expression modifier alleles of spectrin has been postulated (see below).
In contrast to a spectrin mutations, a variety of b spectrin mutations have been identified in HE and HPP patients, including frameshift and splicing mutations that lead to truncated b-spectrin chains lacking the spectrin self-association site.78 Three b-spectrin mutations, spectrinProvidence, spectrinCaligiari, and spectrinBuffalo,80,81 and 82 when inherited in the homozygous state, lead to severe fetal or neonatal anemia and nonimmune hydrops fetalis. Five of 6 homozygotes died; the one survivor remains transfusion dependent.
Protein 4.1 defects associated with HE are much less common than spectrin defects. Protein 4.1 is a multifunctional protein that undergoes complex patterns of tissue- and stage-specific alternative splicing and contains several important functional sites, including a spectrin-actin binding domain and a GPC binding domain. Partial deficiency of protein 4.1 is associated with asymptomatic HE, while complete deficiency leads to hemolytic anemia.77,83,84 Homozygous 4.1 (–/–) erythrocytes fragment more rapidly than normal at moderate sheer stresses, an indication of their intrinsic instability (Fig. 43-7). Membrane mechanical stability can be restored by reconstituting the deficient red cells with protein 4.1 or the protein 4.1-spectrin-actin binding site.85 Homozygous protein 4.1 (–) erythrocytes also lack p55 and have only 30 percent of the normal content of GPC. These 4.1 (–) erythrocytes, as well as GPC (–) Leach erythrocytes (see below) demonstrate decreased invasion and growth of Plasmodium falciparum in vitro.86
FIGURE 43-7 Erythrocyte membrane stability in defects of protein 4.1. Red cell membranes were subjected to shear stress in an ektacytometer, and deformability was measured as a function of time. A fall in deformability occurred as the membranes fragmented. Cells completely lacking protein 4.1 (–/–) have very fragile membranes, and normal fragility can be restored by reconstitution with normal protein 4.1. Heterozygous mutant cells (+/– and 68/65) have intermediate stability. (From N Mohandas and JA Chasis,77 with permission.)
Most patients with protein 4.1– associated elliptocytosis are from certain European and Arab populations. Protein 4.1 utilizes tissue-specific translation start sites, and several HE mutations have involved the downstream initiator codon.4 In one HE mutant lacking the downstream initiator codon, because there is an erythroid stage-specific switch from the upstream initiator codon to the downstream initiator codon, the protein 4.1 HE phenotype does not develop until after the developmentally regulated switch has occurred.87 HE-related protein 4.1 variants due to deletion or duplication of the exons involved in spectrin, actin, and protein 4.1 binding have also been described.88,89
Elliptocytes are present on the blood films of patients whose erythrocytes carry the Leach phenotype (i.e., lacking the Gerbich antigens, Ge-1, -2, -3, and -4) and lack both GPC and glycophorin D (GPD).90 The Leach phenotype is usually due to a deletion of 7 kb of genomic DNA that removes exons 3 and 4 from the GPC/GPD locus.91 A frameshift mutation due to a nucleotide deletion has also been described as the cause of this phenotype. GPC-deficient subjects are also partially deficient in the protein 4.1 and lack p55, presumably because these proteins form a complex and recruit or stabilize each other on the membrane.92 It has been speculated that the protein 4.1 deficiency in Leach erythrocytes is the cause of the elliptocytic shape. In contrast to other forms of HE, which are dominantly inherited, heterozygous carriers are asymptomatic, with normal red blood cell morphology, while homozygous subjects have no anemia, with only mild elliptocytosis as shown on the blood film.
MOLECULAR DETERMINANTS OF CLINICAL SEVERITY
The severity of hemolysis in common HE often varies not only among different kindreds but within a given family as well. Erythrocyte spectrin content and the percentage of dimeric spectrin in crude spectrin extracts are the principal determinants of the severity of hemolysis. The percentage of dimeric spectrin in crude spectrin extracts depends on the degree of dysfunction of the mutant spectrin and by the gene dose (i.e., heterozygote versus homozygote or compound heterozygote) or the presence of other genetic defects in trans. Mutations in the spectrin self-association contact site produce a more severe defect of spectrin function and clinical phenotype than do other elliptocytogenic mutations.
The low-expression a-spectrin allele, aLELY, is the best characterized abnormality affecting spectrin content and clinical severity. This allele is characterized by an amino acid substitution, Leu1857Val, and partial skipping of exon 46.93 These abnormalities are located in the spectrin heterodimer nucleation site (i.e., where spectrin monomers assemble into heterodimers). Alpha-spectrin chains lacking exon 46 are poorly assembled into ab heterodimers and are rapidly degraded.94 Alone, the aLELY allele is clinically silent, even when inherited in the homozygous state, because a spectrin is normally synthesized in three- to fourfold excess.76 When it is present in trans to an elliptocytogenic a-spectrin mutation, it has the effect of increasing the mutant spectrin concentration and worsening the severity of the disease. Conversely, when the aLELY allele is in cis to an a-spectrin mutation, it mutes the elliptocytic phenotype.
Certain acquired factors may affect the clinical severity of HE. In neonatal red cells, the weak binding of 2,3-BPG by fetal hemoglobin leads to an increase in free 2,3-BPG, which in turn induces a superimposed destabilization of the spectrin-actin-protein 4.1 interaction.95 Finally, hemolytic anemia can be worsened by several acquired conditions, including those that alter the microcirculatory stress to the cells.
In most patients, HE is inherited as an autosomal dominant disorder. The clinical severity is highly variable both among different kindreds, reflecting heterogeneous molecular lesions, and, to a lesser extent, in a given kindred, presumably because of other genetic or acquired defects that modify disease expression. Rare cases of de novo mutation have been described,96 as has an HE kindred with a contiguous gene syndrome inherited in an X-linked pattern.97
The clinical presentation of HE is heterogenous, ranging from asymptomatic carriers to patients with severe, life-threatening anemia.73,74,75 and 76 The overwhelming majority of patients with HE are asymptomatic and are diagnosed incidentally during testing for unrelated conditions.
Asymptomatic carriers who possess the same molecular defect as an affected HE relative but who have normal or nearly normal blood films have been identified. The erythrocyte life span is normal, and these patients are not anemic. Asymptomatic HE patients may experience hemolysis in association with infections, hypersplenism, vitamin B12 deficiency, or microangiopathic hemolysis such as disseminated intravascular coagulation or thrombotic thrombocytopenic purpura. In the latter two conditions, worsening hemolysis may be due to microcirculatory damage superimposed on the underlying mechanical instability of red cells. It has been estimated that approximately 12 percent of patients with HE will become symptomatic from their anemia at some time during their lives.
Hereditary elliptocytosis patients with chronic hemolysis experience moderate to severe hemolytic anemia with elliptocytes and poikilocytes on peripheral blood film. Red cell life span is decreased, and patients may develop complications of chronic hemolysis, such as gallbladder disease. In some kindreds, the hemolytic HE has been transmitted through several generations. In others, not all HE subjects have chronic hemolysis; some of them have only mild hemolysis, presumably because another genetic factor modifies disease expression. The blood films of the most severe HE patients with chronic hemolysis exhibit elliptocytes, poikilocytes, and very small microspherocytes. Thus, their clinical presentation is indistinguishable from HPP.
Hereditary pyropoikilocytosis represents a subtype of common HE, as evidenced by the coexistence of both HE and HPP in the same family and the presence of the same molecular defect of spectrin. Unlike HE subjects carrying the spectrin mutation, red cells of the HPP subjects are also partially deficient in spectrin. Typically, one parent of the HPP offspring carries an elliptocytogenic a spectrin mutation, while the other parent is fully asymptomatic and has no detectable biochemical abnormality. In many such patients, the asymptomatic parent carries a silent “thalassemia-like” defect of spectrin synthesis, enhancing the expression of the spectrin mutant and leading to a superimposed spectrin deficiency in the HPP offspring. Some HPP subjects have inherited two structural variants of a spectrin. In these HPP patients, spectrin deficiency may be due to instability of the mutant spectrin. Hereditary pyropoikilocytosis is seen predominantly in subjects with African ancestry, but it has also been diagnosed in those of Arabic and European ancestry.
HEREDITARY ELLIPTOCYTOSIS AND HEREDITARY PYROPOIKILOCYTOSIS IN INFANCY
It is uncommon to find clinical symptoms of elliptocytosis in the neonatal period. Typically, elliptocytes do not appear on the blood film until around 4 to 6 months of age. Occasionally, severe forms of HE may present in the neonatal period with severe, hemolytic anemia with marked poikilocytosis and jaundice. These patients may require red cell transfusion, phototherapy, or even exchange transfusion. Usually, even in severely affected patients, the hemolysis abates between 6 and 12 months of age and the patient progresses to typical HE with mild anemia. Infrequently, patients remain transfusion-dependent beyond the first year of life and require early splenectomy. In cases of suspected neonatal HE or HPP, review of family history and analysis of blood films from the parents are usually of greater diagnostic benefit than other available studies.
A few cases of hydrops fetalis accompanied by fetal or early neonatal death due to unusually severe forms of HE have been described.81,82 One severely affected hydropic infant salvaged by intrauterine transfusions and early exchange transfusion has remained transfusion dependent for over 2 years.82
The hallmark of HE is the presence of cigar-shaped elliptocytes on the blood film (Fig. 43-8). These normochromic, normocytic elliptocytes may number from few to 100 percent. The degree of hemolysis does not correlate with the number of elliptocytes present. Ovalocytes, spherocytes, stomatocytes, and fragmented cells may also be seen. The osmotic fragility is abnormal in severe HE and in HPP. The reticulocyte count generally is less than 5 percent but may be higher when hemolysis is severe. Other laboratory findings in HE are similar to those of other hemolytic anemias and are nonspecific markers of increased erythrocyte production and destruction; for example, increased serum bilirubin, increased urinary urobilinogen, and decreased serum haptoglobin reflect increased erythrocyte destruction.
FIGURE 43-8 Blood films from patients with various forms of HE. (a) Simple heterozygote with mild common HE associated with an elliptogenic spectrin mutation. Note the predominant elliptocytosis, with some rod-shaped cells (arrow) and the virtual absence of poikilocytes. (b) Compound heterozygosity for common HE due to doubly heterozygous state for two spectrin mutations. Both parents have mild HE. There are many elliptocytes as well as numerous fragments and poikilocytes. (c) HPP. The patient is a compound heterozygote for an a spectrin self-association site mutation and a defect characterized by reduced synthesis of this protein. Note prominent microspherocytosis, micropoikilocytosis, and fragmentation. Only a few elliptocytes are present. Some poikilocytes are in the process of budding (arrow). (d) Southeast Asian (Melanesian) ovalocytosis. The majority of cells are oval, some of them containing either a longitudinal slit or a transverse ridge (arrow). See the text for further details.
In HPP, in addition to the blood film findings seen in HE, many HPP erythrocytes are bizarrely shaped, with fragmentation or budding. Microspherocytosis is common, and the MCV is usually low (50–70 fl). Pyknocytes are prominent in smears of neonates with HPP. The thermal instability of erythrocytes, originally reported as diagnostic of HPP, is not unique to this disorder and is also commonly found in HE erythrocytes.
In difficult cases or cases requiring a molecular diagnosis, specialized testing is available. This includes analysis of membrane proteins by one-dimensional gel electrophoresis, limited tryptic digestion of membrane spectrin followed by one- or two-dimensional gel electrophoresis, spectrin dimer self-association assays, ektacytometry, and cDNA and genomic DNA analyses.
Elliptocytes may be seen in association with several disorders, including megaloblastic anemias, hypochromic microcytic anemias (iron deficiency anemia and thalassemia), myelodyplastic syndromes, and myelofibrosis. In these conditions, the elliptocytosis is acquired and generally represents less than a quarter of red cells seen on peripheral smear. History and additional laboratory testing usually clarify the diagnosis of these disorders. Pseudoelliptocytosis is an artifact of blood film preparation. Pseudoelliptocytes are found only in certain areas of the film, usually near its tail, and the long axes of pseudoelliptocytes are parallel, whereas the axes of true elliptocytes are distributed randomly.
THERAPY AND PROGNOSIS
Therapy is rarely needed in patients with HE. In rare cases, occasional red blood cell transfusions may be required. In cases of severe HE and HPP, splenectomy has been palliative, as the spleen is the site of erythrocyte sequestration and destruction. The same indications for splenectomy in HS can be applied to patients with symptomatic HE or HPP. Postsplenectomy, patients with HE or HPP exhibit increased hematocrits, decreased reticulocyte counts, and improvement in clinical symptoms.
Patients should be followed for signs of decompensation during acute illnesses. Interval ultrasonography to detect gallstones should be performed. Patients with significant hemolysis should receive daily folate supplementation.
SOUTHEAST ASIAN OVALOCYTOSIS
Southeast Asian ovalocytosis, also known as Melanesian elliptocytosis or stomatocytic elliptocytosis, is a dominantly inherited trait characterized by the presence of oval red cells, many of which contain one or two transverse ridges or a longitudinal slit (Fig. 43-8d). This condition is widespread in certain ethnic groups of Malaysia, Papua New Guinea, the Philippines, and Indonesia.98 Numerous abnormalities of Southeast Asian ovalocytosis erythrocytes have been reported, including increased red cell rigidity, decreased osmotic fragility, increased thermal stability, resistance to shape change by echinocytic agents, and a reduced expression of many red cell antigens.4 Thus, Southeast Asian ovalocytosis red cells are unique among the elliptocytes in that they are rigid and hyperstable rather than unstable. A remarkable feature of Southeast Asian ovalocytosis erythrocytes is their resistance to in vitro invasion by several strains of malaria parasites, including Plasmodium falciparum and Plasmodium knowlesi.99
The Southeast Asian ovalocytosis phenotype is due to heterozygosity for two band 3 mutations in cis: the deletion of 27 bp encoding amino acids 400 to 408 located at the boundary of the cytoplasmic and membrane domains of band 3 and the amino acid substitution Lys56Glu.100 The latter represents an asymptomatic polymorphism. It has been hypothesized that homozygosity for Southeast Asian ovalocytosis would lead to embryonic lethality.101 Southeast Asian ovalocytosis erythrocytes exhibit increased binding of band 3 to ankyrin, increased tyrosine phosphorylation of band 3, inability to transport sulfate anions, and a markedly restricted lateral and rotational mobility of the band 3 protein in the membrane.
Clinically, the finding of 30 percent or more of oval-shaped red cells on the blood film, some containing a central slit or a transverse ridge, together with a notable absence of clinical and laboratory evicdence of hemolysis in a patient from the above-noted ethnic groups is highly suggestive of the diagnosis. A useful screening test is the demonstration of the resistance of ovalocytes or their ghosts to changes in shape produced by treatments that produce spiculation in normal cells, such as overnight incubation of red cells or exposure of ghosts to salt solutions. Rapid genetic diagnosis can be made by amplifying the region containing the 27-bp deletion from genomic DNA or reticulocyte cDNA and demonstrating a shorter band compared to control after electrophoresis.
In vivo, there is evidence that Southeast Asian ovalocytosis provides some protection against all forms of malaria, particularly against heavy infections and cerebral malaria.102,103 The prevalence of Southeast Asian ovalocytosis increases with age in populations challenged by malaria, suggesting a selective advantage. The mechanism of malaria resistance of Southeast Asian ovalocytosis cells is speculative. Band 3 serves as one of the malaria receptors, as evidenced by inhibition of invasion in vitro by the band 3–containing liposomes.
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