Hemolytic anemias resulting from defects in the erythrocyte membrane comprise an important group of hereditary anemias. The disorders are characterized by altered red cell morphology, which is reflected in the nomenclature of HS, HE, hereditary pyropoikilocytosis (HPP) and southeast Asian ovalocytosis (SAO), which are the most common disorders in this group. Protein studies have identified the underlying membrane abnormalities and advances in molecular biology have enabled further characterization of these disorders and, in many cases, identification of the causative mutations. These molecular analyses have provided additional information on the pathogenesis of these disorders and important insights into the structure–function relationships of erythrocyte membrane proteins.
As predicted in 1984 by Jiri Palek61 and confirmed by subsequent studies, protein defects that compromise vertical interactions between the membrane skeleton and the lipid bilayer result in destabilization of the bilayer, loss of membrane microvesicles and spherocyte formation; whereas mutations affecting horizontal protein interactions within the membrane skeletal network disrupt the skeleton resulting in defective shape recovery and elliptocytes (Table 46–2). Red cell membrane disorders exhibit significant heterogeneity in their clinical, morphologic, laboratory and molecular characteristics.
Table 46–2.Erythrocyte Membrane Protein Defects in Inherited Disorders of Red Cell Shape ||Download (.pdf) Table 46–2. Erythrocyte Membrane Protein Defects in Inherited Disorders of Red Cell Shape
|Protein ||Disorder ||Comment |
|Ankyrin ||HS ||Most common cause of typical dominant HS |
|Band 3 ||HS, SAO, NIHF, HAc ||“Pincered” HS spherocytes seen on blood film presplenectomy; SAO results from 9-amino-acid deletion |
|β-Spectrin ||HS, HE, HPP, NIHF ||“Acanthocytic” spherocytes seen on blood film presplenectomy; location of mutation in β-spectrin determines clinical phenotype |
|α-Spectrin ||HS, HE, HPP, NIHF ||Location of mutation in α-spectrin determines clinical phenotype; α-spectrin mutations most common cause of typical HE |
|Protein 4.2 ||HS ||Primarily found in Japanese patients |
|Protein 4.1 ||HE ||Found in certain European and Arab populations |
|GPC ||HE ||Concomitant protein 4.1 deficiency is basis of HE in GPC defects |
Hereditary spherocytosis is characterized by the presence of osmotically fragile spherical red blood cells on the blood film (Fig. 46–10B). The disorder was first described in 1871 as microcythemia in a case history by two Belgian physicians.62
Blood films from patients with erythrocyte membrane disorders. A. Normal blood film. B. HS with dense spherocytes. C. SAO with large ovalocytes exhibiting a transverse ridge. D. HE with elongated elliptocytes and some poikilocytes. E. HSt with cup-shaped stomatocytes. F. Hereditary abetalipoproteinemia with acanthocytes. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
HS occurs in all racial and ethnic groups. It is the most common inherited hemolytic anemia in individuals of northern European ancestry, affecting approximately 1 in 2000 individuals in North America and Europe.63 It is also common in Japan and in Africans from southern Africa. Males and females are affected equally.
Etiology and Pathogenesis
The hallmark of HS erythrocytes is loss of membrane surface area relative to intracellular volume, which accounts for the spherical shape and loss of central pallor of the cell (Figs 46-10B and 46-11C). Spherocytes exhibit decreased deformability and are thus selectively retained, damaged and ultimately destroyed in the spleen, which causes the hemolysis experienced by HS patients. The HS red cell membrane is destabilized by a deficiency of critical membrane proteins, including spectrin, ankyrin, band 3 and protein 4.2, which decreases the vertical interactions between the skeleton and the bilayer, resulting in the release of microvesicles and loss of surface area (Fig. 46–12). It is hypothesized that two mechanisms underlie the membrane loss: (1) in cells with spectrin/ankyrin deficiency, sections of the lipid bilayer and band 3 are not in contact with the skeleton, which will increase the lateral and rotational mobility of band 3, allowing lipid microvesicles containing band 3 to be generated, and (2) in cells with decreased amounts of band 3/protein 4.2, the stabilizing effect of the transmembrane section of band 3 on the lipid bilayer is lost, facilitating the formation of band 3-free microvesicles.13
Scanning electron micrographs of erythrocytes with abnormal morphology due to membrane defects. A. Normal discocyte. B. Echinocyte. C. Spherocyte. D. Stomatocytes. E. Ovalocytes. F. Elliptocytes. G. Acanthocytes. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
Pathobiology of hereditary spherocytosis (HS). The primary defect in HS is a deficiency of one of the membrane proteins, which destabilizes the lipid bilayer and leads to a loss of membrane in the form of microvesicles. This reduces the surface area of the cell and leads to spherocyte formation. Red cells with a deficiency of spectrin or ankyrin produce microvesicles containing band 3, whereas a reduced amount of band 3 or protein 4.1R gives rise to band 3–free microvesicles. Spherocytes have decreased deformability and are trapped in the spleen where the membrane is further damaged by splenic conditioning, which ultimately results in hemolysis.
Red Cell Membrane Protein Defects
Analysis of HS red cell membrane proteins by several research groups has revealed quantitative abnormalities of spectrin, ankyrin, band 3, and protein 4.2 in 70 to 90 percent of the cases.13,63,64 This spectrum of defects is found worldwide in all the HS cohorts that have been studied; however, the relative frequency of each defect varies with the geographical area and ethnic group. In the United States, parts of Europe, and in Korea, the most common defect is ankyrin deficiency (30 to 60 percent),63,65,66 whereas it is relatively uncommon elsewhere in the world (<15 percent). In other parts of Europe64,67 and in South Africa (unpublished), band 3 deficiency is the main defect. In Japan, almost half of the HS cases are caused by a decreased amount of protein 4.2, and in Korea and South Africa, this defect is the second most common, but in other populations it is rare (<6 percent).63,64,65 The underlying gene mutations have not been investigated in all HS subjects, but the limited research that has been conducted on the defective genes has identified more than 140 different mutations, which are often unique to a family.
Concomitant ankyrin and spectrin deficiency was first described in two patients with severe atypical HS and the primary defect was identified as an ankyrin abnormality.68 Subsequent DNA analysis of the ANK1 gene in patients with typical HS identified several mutations,69 and numerous other studies have shown that ankyrin/spectrin deficiency is a common cause of HS. Ankyrin binds to spectrin with high affinity and attaches it to the membrane, which stabilizes the molecule. Because ankyrin is present in limiting amounts, a deficiency of ankyrin causes an equivalent loss of spectrin.
Different types of ankyrin mutations have been identified throughout the gene, indicating that there are several mechanisms that ultimately result in a decreased amount of ankyrin in the membrane. Interestingly, the majority of these mutations are frameshift and nonsense mutations that either result in unstable transcripts that are destroyed by nonsense-mediated mRNA decay or else produce a truncated defective ankyrin molecule.66 More than 50 mutations have been documented and they are typically family-specific, although a few recurrent mutations have been described69,70 and 15 to 20 percent of mutations are de novo.63 Missense mutations have been documented in all the ankyrin domains and are thought to disrupt normal ankyrin–protein interactions. A few splicing mutations have been identified, including a mutation in intron 16, which created a new splice acceptor site and a complex pattern of aberrant splicing.71 Both parents were heterozygous for this mutation and the proband was homozygous, indicating that homozygosity for an ankyrin mutation is compatible with life.
Mutations in the erythroid-specific promoter of the ANK1 gene are common in recessive HS. A dinucleotide deletion impairs the binding of a transcription factor complex, which leads to a reduced number of ankyrin transcripts.72 Point mutations in a barrier insulator element of the promoter also decrease transcription of the gene.73
Cytogenetic studies have identified a few ankyrin-deficient HS patients with a contiguous gene syndrome that includes deletion of the ankyrin gene locus at 8p11.2. These patients additionally suffer from dysmorphic features, psychomotor retardation, and hypogonadism.74
A subset of HS patients present with a band 3 deficiency, typically accompanied by a secondary decrease in protein 4.2, a result of the reduction in protein 4.2 binding sites in the cytoplasmic domain of band 3. The extent of band 3 deficiency in heterozygous patients ranges between 20 and 50 percent, depending on the severity of the mutation, and the compensatory effect of the in trans normal allele. Mushroom-shaped “pincered” cells are commonly seen on the blood film of HS patients with a band 3 abnormality.
More than 55 underlying mutations have been described; they are variable and occur throughout the band 3 gene.13,66 Null mutations are typically family-specific and are caused by frameshift or nonsense mutations, or, in a few cases, by abnormal splicing, all of which result in truncated nonfunctional proteins or unstable transcripts that are not translated into protein. Missense mutations are common and often occur in several kindred. Highly conserved arginine residues at the internal boundaries of the transmembrane segments of the protein (see Fig. 46–2) are frequently mutated, including residues 490, 518, 760, 808, and 870.66,75 The mutations probably interfere with the cotranslational insertion of band 3 into the endoplasmic reticulum and ultimately into the red cell membrane. Short in-frame insertions or deletions have been documented and presumably also impair insertion of the mutant protein into the lipid bilayer.
Mutations in the cytoplasmic domain of band 3 impact on the interaction of band 3 with proteins in the membrane skeleton, or may alter the conformation of the protein rendering it unstable and prone to degradation prior to insertion into the membrane. Some cytoplasmic mutations, such as band 3 Cape Town and band 3 Mondega, are silent in the heterozygous state, but exacerbate the clinical presentation when inherited in trans to another mutation.76,77
Erythrocytes from HS patients with defects in spectrin or ankyrin are deficient in spectrin. The degree of deficiency correlates with the severity of hemolysis, the response to splenectomy and the ability to withstand mechanical shear stress.78,79 Visualization of the membrane skeleton of these red cells revealed a decreased density of the spectrin filaments connecting the junctional complexes.80 The causative mutations occur in either α- or β-spectrin genes.
Defects in α-spectrin are rare and are associated with severe recessive HS. During erythropoiesis α-spectrin is synthesized in a two- to fourfold excess over β-spectrin and heterozygotes thus still produce sufficient α-spectrin to form heterodimers with all the β-spectrin molecules, which will not result in spectrin deficiency. The defect will only be manifested in individuals who are homozygous or doubly heterozygous for mutations in α-spectrin. The mechanism underlying spectrin deficiency has not been fully elucidated, but a low-expression allele or a polymorphism inherited in trans to a causative null mutation plays a role. An example of a low-expression allele is αLEPRA (low-expression Prague), which produces less than 20 percent of the normal amount of α-spectrin transcripts as a result of a splicing and mRNA processing defect, but does not cause any symptoms even in the homozygous state. However, in combination with another mutation on the other α-spectrin allele, which produces a nonfunctional truncated protein, it causes severe spectrin deficiency and anemia.81 A polymorphic missense mutation in the αII domain in spectrin Bug Hill has been identified in several patients with spectrin-deficient, recessive HS who carry another uncharacterized α-spectrin gene defect that causes the disease.82 Extensive analysis of the α-spectrin gene in a proband with severe nondominant HS revealed a partial maternal isodisomy of chromosome 1, resulting in homozygosity of the 1q23 region containing the maternal SPTA1 gene, which carried an R891X nonsense mutation.83 Uniparental disomy therefore unmasked a recessive mutation in the mother, which caused severe clinical symptoms in the child.
The production of β-spectrin polypeptides is the limiting factor in spectrin heterodimer formation and one mutant allele is sufficient to cause spectrin deficiency in autosomal dominant HS. The blood films of these patients typically show a subpopulation of spiculated cells (acanthocytes and echinocytes) in addition to spherocytes.63 Mutations in β-spectrin are found throughout the gene and are mainly null mutations caused by frameshift, nonsense, splicing, and initiator codon defects, which silence the mutant allele.84 With a few exceptions the mutations are all kindred-specific. Truncated β-spectrin chains have also been described and are caused by frameshift mutations, in-frame deletions, or exon skipping. These mutations lead to, for example, reduced synthesis of an unstable protein,85 or they impair the interaction with ankyrin and thereby the insertion of spectrin into the membrane.86 A few missense mutations have been identified, including β-spectrinKissimmee, which is caused by a mutation in the 4.1R/actin–binding domain of the protein.87 The mutant protein is unstable and does not bind to 4.1R, and thus it only interacts weakly with actin, which may explain why these red cells are deficient in spectrin.87
Protein 4.2 deficiency is common in Japanese patients with recessively inherited HS who exhibit almost a complete absence of the protein.63 Defects in this protein also occur in whites and other population groups, and 13 mutations have been described in the 4.2 gene of individual kindred, including missense mutations and in-frame deletion and insertion of nucleotides. Nonsense, frameshift, and splicing defects result in premature termination of translation and these mutant truncated proteins are not detected on the membrane, indicating that they are unstable and presumably degraded.47 Amino acids 306 to 320 are highly conserved and five of the known mutations (three missense and two nonsense) occur in this region, which is adjacent to the hairpin that binds to band 3 in the predicted tertiary structure of protein 4.2.88 The only recurrent and most common mutation, protein 4.2 Nippon, is caused by a point mutation that affects mRNA processing.89 Patients are either homozygous for this mutation or heterozygous for a second mutation on the other allele.47 Mutations have also been identified in individual patients with recessive HS from Europe, Tunisia, and Pakistan. South African kindred with autosomal dominant HS from a deficiency of protein 4.2 have been noted, but the underlying mutations have not been investigated.
Secondary Membrane Defects
The decreased membrane surface area in hereditary spherocytes involves a symmetrical loss of each species of membrane lipid. The relative proportions of cholesterol and phospholipids are therefore normal and the asymmetrical distribution of phospholipids is maintained.
HS red cells exhibit increased cation permeability, presumably secondary to the underlying membrane defect.90 The excessive sodium influx activates the Na+-K+ ATPase cation pump, which increases ATP turnover and glycolysis. Spherocytes are dehydrated, especially cells obtained from the splenic pulp, but the underlying mechanism has not been clearly defined. The acidic environment of the spleen and oxidative damage by splenic macrophages increase the activity of the K+Cl– cotransporter, which may play a role in dehydration. The hyperactive Na+-K+ ATPase pump may also contribute as three sodium ions are extruded in exchange for two potassium ions, and this loss of monovalent cations is accompanied by the loss of water. Dehydration may also be related to loss of surface area.
Molecular Determinants of Clinical Severity
Affected individuals of the same kindred typically experience similar degrees of hemolysis. However, in some families the clinical expression is variable and this may be influenced by several factors. Low-expression alleles decrease transcription of the gene or influence the expression or incorporation of the protein into the membrane, but there is no phenotypic effect in the heterozygous state because the normal allele compensates for the deleterious effect. However, when inherited with a mutant allele that causes HS, it exacerbates the clinical expression of the disease. Examples of low-expression alleles that influence HS include band 3 Genas, band 3 Mondego, and two α-spectrin alleles, αLELY and αLEPRA.77,81,91,92,93,94
Variable penetrance of the defective gene, a de novo mutation or a mild form of recessively inherited HS may also influence the clinical severity. Double heterozygosity for two mild band 3 mutations can have an additive effect76 and rare cases caused by homozygous defects in band 3 result in severe transfusion-dependent hemolytic anemia or fetal death.91,95,96 Coinheritance of other hematologic disorders or Gilbert syndrome, caused by homozygosity for a polymorphism in the promoter of the uridine diphosphate-glucuronosyltransferase (UGT1) gene, can also alter the clinical symptoms.63,97,98
The spleen plays a secondary but important role in the pathophysiology of HS. Spherocytes are retained and ultimately destroyed in the spleen and this is the primary cause of the chronic hemolysis experienced by HS patients (see Fig. 46–12). The reduced deformability of spherocytes impedes their passage through the interendothelial slits separating the splenic cords of the red pulp from the splenic sinuses. The decrease in red cell deformability is primarily related to a loss of surface area and, to a lesser extent, to an increase in internal viscosity as a result of mild cellular dehydration. Ex vivo experiments using perfused human spleens and red cells treated with lysophosphatidylcholine to induce spherocytosis revealed that the degree of splenic retention correlated with the reduction in the surface-area-to-volume ratio.99
The spleen is a metabolically hostile environment with a decreased pH, low concentrations of glucose and ATP, and increased oxidants, all of which are detrimental to the red cell. Spherocytes are “conditioned” during erythrostasis in the spleen and become more osmotically fragile and increasingly spherocytic.100 Exposure to macrophages in the spleen eventually leads to erythrophagocytosis and destruction.
In approximately 75 percent of HS patients, inheritance is autosomal dominant. In the remaining patients, the disorder may be autosomal recessive or result from de novo mutations, which is relatively common.101,102 Mutations in α-spectrin or protein 4.2 are often associated with recessive HS.
The clinical manifestations of HS vary widely. The typical clinical picture combines evidence of hemolysis (anemia, jaundice, reticulocytosis, gallstones, 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 46–3), which can be correlated with the degree of compensation for hemolysis. 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 46–3.Classification of Hereditary Spherocytosis ||Download (.pdf) Table 46–3. Classification of Hereditary Spherocytosis
|Laboratory Findings ||HS Trait or Carrier ||Mild Spherocytosis ||Moderate Spherocytosis ||Moderately Severe Spherocytosis* ||Severe Spherocytosis† |
|Hemoglobin (g/dL) ||Normal ||11–15 ||8–12 ||6–8 ||<6 |
|Reticulocytes (%) ||1–2 ||3–8 ||± 8 ||≥10 ||≥10 |
|Bilirubin (mg/dL) ||0–1 ||1–2 ||± 2 ||2–3 ||≥3 |
|Spectrin content (% of normal)‡ ||100 ||80–100 ||50–80 ||40–80§ ||20–50 |
|Blood film ||Normal ||Mild spherocytosis ||Spherocytosis ||Spherocytosis ||Spherocytosis and poikilocytosis |
|Osmotic fragility || || || || || |
|Fresh blood ||Normal ||Normal or slightly increased ||Distinctly increased ||Distinctly increased ||Distinctly increased |
|Incubated blood ||Slightly increased ||Distinctly increased ||Distinctly increased ||Distinctly increased ||Markedly increased |
Typical Hereditary Spherocytosis
Approximately 60 to 70 percent of HS patients have moderate disease, which typically presents in infancy or childhood but may present at any age. In children, anemia is the most frequent finding (50 percent of cases), followed by splenomegaly, jaundice, or a positive family history.13,63 No comparable data exist for adults. Hemolysis may be incompletely compensated with mild to moderate anemia (see Table 46–3). The moderate anemia may often be asymptomatic; however, fatigue and mild pallor or both may be present. Jaundice may be intermittent and is seen in about half of patients, usually in association with viral infections. When present, jaundice is acholuric, characterized by unconjugated hyperbilirubinemia without detectable bilirubinuria. Palpable splenomegaly is evident in most (>75 percent) older children and adults. Typically the spleen is modestly enlarged (2 to 6 cm below the costal margin), but it may be massive. No proven correlation exists between the spleen size and the severity of HS. However, given the pathophysiology and response of the disease to splenectomy, such a correlation probably exists.
Mild Hereditary Spherocytosis
Approximately 20 to 30 percent of HS patients have mild disease with “compensated hemolysis,” that is, red blood cell production and destruction are balanced, and the hemoglobin concentration of the blood is normal (see Table 46–3).63,103 The life span of spherocytes is decreased, but patients adequately compensate for hemolysis with increased marrow erythropoiesis. These patients are usually asymptomatic. Splenomegaly is mild, reticulocyte counts are generally less than 6 percent, and spherocytes on the blood film may be minimal, which complicates the diagnosis. 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 further increase 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 disease, as evidenced by indicators of anemia that are more pronounced than in typical moderate HS, and an intermittent requirement for transfusions (see Table 46–3). This category includes patients with dominant and recessive HS. A small number (<5 percent) of patients have severe disease with life-threatening anemia and are transfusion-dependent. They almost always have recessive HS. Most have severe spectrin deficiency, which is thought to result from a defect in α-spectrin,78,79 but defects in ankyrin or band 3 have also been identified.91,96 Patients with severe HS often have irregularly contoured or budding spherocytes or bizarre poikilocytes in addition to typical spherocytes and microspherocytes on the blood film. Added to the risks of recurrent transfusions, patients often suffer from hemolytic and aplastic crises and may develop complications of severe uncompensated anemia, including growth retardation, delayed sexual maturation, and aspects of thalassemic facies.
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 (see Table 46–3), including slight reticulocytosis, diminished haptoglobin levels, and slightly elevated incubated osmotic fragility, particularly the 100 percent red cell lysis point, which occurs at a higher sodium chloride concentration in carriers compared to normal subjects.103 The acidified glycerol lysis test may also be useful to detect carriers. In North America and parts of Europe, approximately 1 percent of the population is estimated to be silent carriers.63
Pregnancy and Hereditary Spherocytosis
Most patients do well during pregnancy104 although anemia may be exacerbated by plasma volume expansion and increased hemolysis. A few patients are symptomatic only during pregnancy. Transfusions are rarely required.
Hereditary Spherocytosis in the Neonate
Jaundice is the most common finding in neonates with HS, present in approximately 90 percent of cases. It may be accentuated by coinheritance of Gilbert syndrome, caused by homozygosity for a polymorphism in the promoter of the UGT1 gene (Chaps. 33 and 47).63,97,98 Less than half of infants are anemic and severe anemia is rare. A few cases of hydrops fetalis from homozygosity or compound heterozygosity for band 3 or spectrin defects have been reported.91,105,106
Chronic hemolysis leads to formation of bilirubinate gallstones, the most frequently reported complication in up to half of HS patients. Coinheritance of Gilbert syndrome markedly increases the risk of gallstone formation. Although gallstones have been detected in children, they mainly occur in adolescents and young adults.13,63 Routine management should include interval ultrasonography to detect gallstones because many patients with cholelithiasis and HS are asymptomatic. Interval ultrasonography allows prompt diagnosis and treatment and prevents complications of symptomatic biliary tract disease, including biliary obstruction, cholecystitis, and cholangitis.
Hemolytic, Aplastic and Megaloblastic Crises
Hemolytic crises are the most common and are usually associated with viral illnesses and typically occur in childhood.13,63 They are generally mild and characterized by jaundice, splenomegaly, anemia and reticulocytosis. Medical intervention is seldom necessary. During rare severe hemolytic crises, red cell transfusion may be required.
Aplastic crises following virally induced marrow suppression are uncommon but may result in severe anemia requiring hospitalization and transfusion with serious complications, including congestive heart failure or even death.13,63 The most common etiologic agent in these cases is parvovirus B19 (Chap. 36). The virus selectively infects erythropoietic progenitor cells and inhibits their growth leading to the characteristic finding of a low number of reticulocytes despite severe anemia. Aplastic crises usually last for 10 to 14 days and may bring asymptomatic, undiagnosed HS patients with compensated hemolysis to medical attention.63
Megaloblastic crises may occur in HS patients with increased folate demands, such as pregnant patients, growing children, or patients recovering from an aplastic crisis. This complication can be prevented with appropriate folate supplementation.
Leg ulcers, chronic dermatitis on the legs and gout are rare manifestations of HS, which usually heal rapidly after splenectomy. In severe cases, skeletal abnormalities resulting from expansion of the marrow can occur. Extramedullary hematopoiesis can lead to tumors, particularly along the thoracic and lumbar spine or in the kidney hila, in nonsplenectomized patients with mild to moderate HS.13,63 Postsplenectomy, the masses involute and undergo fatty metamorphosis.
HS has been suggested to predispose patients to hematologic malignancies, including myeloproliferative disorders, particularly myeloma, but cause and effect have not been proven. Thrombosis has been reported in several HS patients, usually postsplenectomy. Untreated HS may aggravate other underlying diseases, such as congestive heart disease and hemochromatosis.13,63
Clinical manifestations are confined to the erythroid lineage in the majority of patients with HS, but a few exceptions have been observed. Several HS kindred have been reported with cosegregating nonerythroid manifestations, particularly neuromuscular abnormalities including cardiomyopathy, slowly progressive spinocerebellar degenerative disease, spinal cord dysfunction, and movement disorders. Erythrocyte ankyrin and β-spectrin are also expressed in muscle, brain, and spinal cord, which raises the possibility that these HS patients suffer from defects of one of these proteins.13
An isoform of band 3 is expressed in the kidney and heterozygous defects of band 3 have been described in patients with inherited distal renal tubular acidosis and normal erythrocytes. This finding is in contrast to most patients with heterozygous mutations of band 3, who have normal renal acidification and abnormal erythrocytes. Kindred with HS and renal acidification defects resulting from band 3 mRNA processing mutations, band 3Pribram and band 3Campinas, have been described.107,108 Homozygosity for band 3Coimbra, a V488M missense mutation, resulted in the absence of band 3 and renal tubular acidosis in a severely affected HS infant.91
Laboratory findings in HS are variable, which correlate with the heterogeneous clinical presentation.
Erythrocyte morphology in HS is not uniform. Typical HS patients have blood films with easily identifiable spherocytes lacking central pallor (see Fig. 46–10B and 46-11C). Patients with mild HS may present with only a few spherocytes, and at the other end of the spectrum, severely affected patients exhibit numerous dense microspherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis. Blood films from patients with band 3 defects often exhibit “pincered” or mushroom-shaped red cells, whereas spherocytic acanthocytes are associated with β-spectrin mutations. When examining blood from a patient with suspected spherocytosis, a high-quality film with the erythrocytes properly separated and some cells with central pallor in the field of examination are important because spherocytes can be an artifact.
Most patients have mild to moderate anemia with hemoglobin in the 9 to 12 g/dL range (see Table 46–3). Mean corpuscular hemoglobin concentration (MCHC) is increased (>36 g/dL) because of relative cellular dehydration in approximately half of patients, but all HS patients have some dehydrated cells. Some automated hematology analyzers measure the hemoglobin concentration of individual red cells and a demonstration of a population of hyperdense erythrocytes can be useful as a screening test for HS, especially when combined with an increased red cell distribution width. Mean corpuscular volume (MCV) is usually normal except in cases of severe HS, when MCV is slightly decreased.
Other laboratory features of HS are markers of ongoing hemolysis. Reticulocytosis, variably increased lactate dehydrogenase, increased urinary and fecal urobilinogen, unconjugated hyperbilirubinemia, and decreased serum haptoglobin reflect hemolysis and increased erythropoiesis (Chaps. 32 and 33). The reticulocyte count may appear to be elevated disproportionately relative to the degree of anemia.
Erythrocyte Fragility Tests
Spherocytes have a decreased surface area relative to cell volume and this renders them osmotically fragile. Several laboratory tests exploit this characteristic and are used to diagnose HS. The most common osmotic fragility (OF) test measures lysis of red cells, either from freshly drawn blood or after incubation of the sample at 37°C for 24 hours, in a range of hypotonic concentrations of sodium chloride. Spherocytes typically swell and burst much more readily than normal biconcave disk-shaped red cells. Other tests based on the same principle measure the rate and extent of cell lysis in buffered glycerol solutions and include the glycerol lysis test (GLT) and the acidified glycerol lysis test (AGLT). These tests, however, have relatively poor sensitivity and do not detect all cases of mild HS or those with small numbers of spherocytes, including patients who had recent blood transfusions.63,67,109 These tests may also be unreliable and give normal results in the presence of iron deficiency, obstructive jaundice, or during the recovery phase of an aplastic crisis.63 In addition, these tests do not differentiate HS from other disorders with secondary spherocytosis, such as the autoimmune hemolytic anemias (Chap. 54).
Other fragility tests include the cryohemolysis test, based on the sensitivity of HS red cells to cooling at 0°C in hypertonic conditions, and the autohemolysis test, but these tests also do not detect all cases of HS.13 The reduced surface area of spherocytes can be measured by osmotic gradient ektacytometry, but the highly specialized equipment required for this procedure is only available in a few research-oriented laboratories.
Eosin 5′-Maleimide Flow Cytometry Test
Eosin 5′-maleimide (EMA) is a fluorescent dye that binds to the transmembrane proteins, band 3, Rh protein, Rh glycoprotein, and CD47.110 Patients with HS exhibit decreased fluorescence compared to controls, irrespective of the underlying defective membrane protein, although not all patients with HS are detected. In addition, lower fluorescence values are also observed in patients with HE, HPP, some red cell enzymopathies, and other abnormalities of band 3, such as congenital dyserythropoietic anemia type II (CDAII; Chap. 39). The sensitivity and specificity of the test vary, depending on the cutoff value of the fluorescence, which differs between laboratories.109,111,112,113
Because HS can be caused by mutations in several different genes and because there are very few common mutations, a simple DNA test to diagnose HS is not feasible. Initial analysis of the red cell membrane proteins by quantitative SDS-PAGE is required to identify the underlying defective protein. The sensitivity of this test varies between laboratories and different patient populations, but typically an abnormality is defined in 75 to 93 percent of cases.63,64,67 Patients with clinically identified HS and normal SDS-PAGE results may have a slight decrease of 10 to 15 percent in one of the membrane proteins, which may be missed by the densitometric analysis, or they may have an abnormality in a protein that is currently not quantified and not linked to HS, for example, adducin.
Knowledge of the defective protein facilitates subsequent DNA/RNA investigations to characterize the gene defect, although this approach is challenging as the genes causing HS are large and contain many exons. Polymorphisms may be used to identify reduced expression from one allele or loss of heterozygosity because of a null mutation. In families with variable clinical expression of HS, a molecular investigation into low-expression alleles and other modifying genes is useful. A molecular diagnosis is informative in patients with atypical features; severe disease; unclear or recessive inheritance; de novo mutations; or undiagnosed hemolytic anemia. Identification of silent carriers and prenatal diagnosis also require molecular testing.
Clinical features and family history should accompany an initial laboratory investigation comprising a complete blood count with a blood film, reticulocyte count, direct antiglobulin test (Coombs test), and serum bilirubin. Other causes of anemia should be excluded, particularly autoimmune hemolytic anemia, CDAII and HSt. Further diagnostic tests (discussed in “Laboratory Features” earlier), are not standardized as reflected by a European survey of 25 centers.114 A consistent finding was that all the laboratories used at least two tests to make a final diagnosis, as none of the currently available methods have 100 percent sensitivity. The EMA test was most commonly used. Recent guidelines from the British Committee for Standards in Haematology (BCSH)115 advocate the use of the EMA test or cryohemolysis, but OF is not recommended for routine use.
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 autoimmune hemolysis, clostridial sepsis, transfusion reactions, severe burns, and bites from snakes, spiders, bees, and wasps (Chaps. 52 to 54), should be viewed in the appropriate clinical context. Occasional spherocytes are seen in patients with a large spleen (e.g., in cirrhosis or myelofibrosis) or in patients with microangiopathic anemias (Chap. 51), but differentiation of these conditions from HS does not usually present diagnostic difficulties.
HS may be obscured in disorders that increase the surface-to-volume ratio of erythrocytes, such as obstructive jaundice, iron deficiency (Chap. 43), β-thalassemia trait, or hemoglobin SC disease (Chaps. 48 and 49), and vitamin B12 or folate deficiency (Chap. 41).
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, which has obvious implications for future iron overload and hemochromatosis-related end-organ damage. The incidence of cholelithiasis is decreased. Postsplenectomy, spherocytosis, and altered OF persist, but the “tail” of the OF 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, Pappenheimer bodies (siderocytes), and acanthocytes (Chaps. 2 and 31), 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.79
Complications of Splenectomy
Early complications of splenectomy include local infection, thrombotic complications and in particular hepatic and mesenteric thrombosis, bleeding, and pancreatitis, presumably resulting from injury to the tail of the pancreas incurred during spleen removal. In general, the morbidity of splenectomy for HS is lower than the morbidity of other hematologic disorders. Chapters 5 and 6 discuss the complications of splenectomy.
Indications for Splenectomy
In the past, splenectomy, which has a low operative mortality, was considered routine in HS patients. However, the risk of overwhelming postsplenectomy infection and the emergence of penicillin-resistant pneumococci have led to reevaluation of the role of splenectomy in the treatment of HS.116 Considering the risks and benefits, a reasonable approach is to splenectomize all patients with transfusion-dependent severe spherocytosis and all patients suffering 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 suffering from vascular compromise of vital organs.
Whether patients with moderate HS and compensated, asymptomatic anemia should undergo splenectomy is controversial. Patients with mild HS and compensated hemolysis can be followed and referred for splenectomy if clinically indicated. Treatment of patients with mild to moderate HS and gallstones is debatable, particularly because new treatments for cholelithiasis, including laparoscopic cholecystectomy, and endoscopic sphincterotomy, 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. No evidence indicates any benefit to performing cholecystectomy and splenectomy separately, as performed in the past.
Because the risk of postsplenectomy sepsis is very high during infancy and early childhood, splenectomy should be delayed until age 5 to 9 years if possible and to at least 3 years if feasible, even if chronic transfusions are required in the interim. No evidence indicates further delay is useful. In fact, further delay may be harmful because the risk of cholelithiasis increases dramatically in children older than 10 years.
When splenectomy is warranted, laparoscopic splenectomy has become the method of choice in centers with surgeons experienced in the technique.117 If desired, the procedure can be combined with laparoscopic cholecystectomy. Laparoscopic splenectomy results in less postoperative discomfort, a quicker return to preoperative diet and activities, shorter hospitalization, decreased costs, and smaller scars. The risk of bleeding increases during the operation and approximately 10 percent of laparoscopic operations (for all causes) must be converted to standard splenectomies. Even very large spleens (>600 g) can be removed laparoscopically because 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.118 The goal of this procedure is to allow for palliation of hemolysis and anemia while maintaining some residual splenic immune function. Long-term followup data for this procedure have been variable.
Prior to splenectomy, patients should be immunized with vaccines against pneumococcus, Haemophilus influenzae type B, and meningococcus, preferably several weeks preoperatively. Use of prophylactic antibiotics postsplenectomy for prevention of pneumococcal sepsis is controversial. Prophylactic antibiotics (penicillin V 125 mg orally twice daily for patients younger than 7 years or 250 mg orally twice daily for those older than 7 years, 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 is uncommon. Failure may result from an accessory spleen missed during splenectomy, from development of splenunculi as a consequence of autotransplantation of splenic tissue during surgery, or from another intrinsic red cell defect, such as pyruvate kinase deficiency (Chap. 47). 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 (Chaps. 2 and 31). 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. A history, physical examination for splenomegaly, complete blood count, examination of the blood film for spherocytes, and a reticulocyte count should be obtained for parents, children, and siblings, if available.
HEREDITARY ELLIPTOCYTOSIS AND PYROPOIKILOCYTOSIS
HE is characterized by the presence of elliptical or oval erythrocytes on the blood films of affected individuals (Figs 46-10D and 46-11F). In 1904, Dresbach, a physiologist at Ohio State University in Columbus, Ohio, published the first description of elliptical red blood cells in one of his students, noticed during a laboratory exercise in which the students were examining their own blood.119 The report elicited controversy because the student died soon thereafter, leading to speculation that he had actually suffered from pernicious anemia. The demonstration of elliptocytosis in three generations of one family established the hereditary nature of this disorder.120 A related disorder, HPP is a rare disease first described in 1975 in children with severe neonatal anemia with abnormal poikilocytic red cell morphology reminiscent of that seen in patients suffering from severe burns (Figure 46-13).121 The erythrocytes from these patients exhibited increased thermal sensitivity.
Epidemiology and Inheritance
HE has a worldwide distribution but the true incidence is unknown because the disease is heterogeneous and many patients are asymptomatic. In the United States, the incidence is estimated to be 1 in 2000 to 4000 individuals.13,122 HE occurs in all racial groups but is more prevalent in individuals of West African descent, possibly because elliptocytes may confer some resistance to malaria.123,124 HPP is typically found in patients of African origin, but it has also been diagnosed in subjects of European and Arabic descent.122,125,126
Etiology and Pathogenesis
The primary abnormality in HE and HPP erythrocytes is defective horizontal interactions between components of the membrane skeleton, which weakens the skeleton and compromises its ability to maintain the biconcave disk shape of the red cell during circulatory shear stress. Investigations of erythrocyte membrane proteins in these disorders have identified abnormalities in α- and β-spectrin, protein 4.1, and GPC.122 The most common defects occur in spectrin, the main structural protein of the erythrocyte membrane skeleton, and they impair the ability of spectrin dimers to self-associate into tetramers and oligomers, thereby disrupting the skeletal lattice.55 Abnormalities in 4.1R diminish the interaction between the tail ends of spectrin tetramers in the junctional complex and thus destabilize the skeleton. Deficiency of GPC/GPD is associated with reduced levels of 4.1R, which presumably is responsible for the elliptocytosis.
When the integrity of the skeleton is compromised, the capacity of the erythrocyte to undergo flow-induced deformation and rearrangement of the skeleton is reduced. Disruption of the dynamic dissociation and reassociation of spectrin tetramers causes mechanical instability of the membrane, which precludes the recovery of the normal biconcave disk shape of the cell after prolonged and repeated unidirectional axial distortion in the microcirculation.127 HE reticulocytes have a normal shape when released into the circulation but the mature red cells become progressively more elliptical as they age and ultimately the abnormal shape becomes permanent.13,122 As the severity of the defect increases, poikilocytes are formed and the cells become prone to fragmentation. HPP patients exhibit a combination of horizontal (impaired spectrin tetramer formation) and vertical (spectrin deficiency) defects, with the latter causing microspherocytes and exacerbating the hemolytic anemia.128,129
Red Cell Membrane Protein Defects
Mutations that affect spectrin heterodimer self-association are found in the majority of HE patients and in all patients with HPP. This functional defect results in an increased percentage of spectrin dimers relative to tetramers,130 which is reflected on a structural level by an abnormal tryptic digest pattern of the protein, whereby the normal peptide is decreased with a concomitant increase in an abnormal peptide of lower molecular weight. Most of the defects affect the 80-kDa αI domain of α-spectrin and of the nine structural variants the most common are SpαI/74, SpαI/65, and SpαI/46 or 50a.128
More than 50 mutations have been identified in either α- or β-spectrin genes. The majority of the mutations are missense mutations that substitute highly conserved amino acids or those in close proximity. The abnormal amino acids typically have a different charge, or in the case of glycine or proline substitutions, they disrupt the helical structure of the spectrin repeats, which alter the interactions between α and β subunits. Interestingly, mutations in α-spectrin primarily occur in helix C of the repeats, which highlights the importance of this helix in the triple helical bundle (see Fig. 46–3). Several mechanisms have been identified by which the mutations impair spectrin tetramer formation.
SpαI/74 mutations are mostly missense mutations found at the self-association site, which consists of helix C of the α0 partial spectrin repeat that interacts with helices B and C of β-spectrin partial repeat 17 to form a complete triple helical bundle.34 In vitro studies on missense mutations in α0 revealed that the mutant peptides were stable folded structures, similar to wild type, but their binding affinities to β-spectrin peptides were variable. This suggested that their effect on tetramer formation was exerted through defective molecular recognition and disruption of protein-protein interactions at the contact site, rather than an altered structure.131 These findings contrasted with mutations in the β17 repeat of β-spectrin, which perturbed the structural conformation of this partial repeat and the adjacent β16 repeat.132 Codon 28 in helix C of α0 has been identified as a mutation “hotspot” since four different point mutations occur in this position, resulting in different amino acid substitutions, and the mutations have also been found in several unrelated kindred.133 Arginine 28 is a highly conserved amino acid and any changes in this position are typically associated with severe HE or HPP.133,134 An interesting case of HE SpαI/74 involving an intragenic crossover in the α-spectrin gene and uniparental disomy, together with an underlying R34P mutation, was recently described in a Utah family.126
SpαI/74 defects are also caused by mutations in β-spectrin, which presumably expose the αI domain of spectrin to increased tryptic digestion. These abnormalities are all located in partial repeat 17. Missense mutations are found in both helices A and B of the β17 repeat, but some in helix A are particularly severe, including spectrinProvidence, spectrinCagliari, and spectrinBuffalo, which cause severe fetal or neonatal anemia and nonimmune hydrops fetalis when inherited in the homozygous state.105,106,135 Frame-shift mutations and splicing defects predominate in helix B, resulting in truncated spectrin molecules lacking the self-association site.13,122,136
SpαI/65 is a mild defect, even in the homozygous state, because of a duplication of leucine 154 in helix C of the α1 repeat.137 It is very common in blacks from West and Central Africa, as well as Arabs in North Africa, suggesting genetic selection, possibly by protecting carriers against P. falciparum malaria.13,122,123
SpαI/46 or 50a mutations are distal from the self-association site and usually occur close to the helical linker regions between individual repeats and often involve the substitution of an amino acid with a proline residue, which is a helix breaker.13,122 In vitro studies on Q471P between repeats 4 and 5 of α-spectrin showed that the mutation uncoupled the repeats and caused cooperative unfolding, which abolished the stabilizing influence of the helical linker on adjacent repeats.138 Because β-spectrin has fewer repeats than α-spectrin, the alignment of the heterodimers places α4 and α5 in contact with β16 and β17, suggesting that unfolding of the mutant spectrin repeats interferes with the self-association site and prevents tetramer formation.139 The L260P mutation is in a similar position to Q471P, but is between repeats α2 and α3 of spectrin. When heterodimers are aligned, repeats α03 are not in contact with β-spectrin and they represent an open dimer configuration, which facilitates tetramer formation. Open dimers are in equilibrium with closed dimers whereby α0 to α3 are folded onto β16 and β17 of the same dimer, thus preventing bivalent tetramer formation.139 In vitro experiments on the L260P mutation revealed a conformational change, which stabilized the mutant spectrin in the closed dimer configuration and reduced tetramer assembly.140
Mutations in the αII domain of spectrin implicated in HE are rare. SpectrinSt Claude is caused by a single point mutation in intron 19 of α-spectrin,141,142 which creates complex splicing events that ultimately impair the function of both α- and β-spectrin, resulting in decreased binding to ankyrin, defective spectrin self-association and spectrin deficiency.141 These membrane abnormalities have profound effects on red blood cell morphology and survival, manifesting as severe HE.
Defects in the erythrocyte isoform of protein 4.1 associated with HE are relatively common in some Arab and European populations.13 Heterozygotes exhibit partial deficiency of 4.1R, manifesting as mild or asymptomatic HE, whereas homozygotes lack 4.1R and p55, have a reduced content of GPC, and present with severe HE. These red blood cells are mechanically unstable and fragment at moderate shear stress, but the stability can be restored by reconstituting the deficient red cells with 4.1R or the 4.1R spectrin–actin binding domain.143 The 4.1R null erythrocytes demonstrate decreased invasion and growth of P. falciparum parasites in vitro.144
Mutations in the 4.1R gene often affect the erythroid-specific initiation codon, which abolishes transcription, or else they tend to cluster in the spectrin-actin binding domain where exon deletions or duplications result in mutant proteins that are smaller or larger than normal.122
GPC and GPD carry the Gerbich antigens and rare patients with the Leach phenotype are Gerbich-negative and lack both GPs. The underlying mutations are either a 7-kb deletion of genomic DNA or a frameshift mutation.145 Heterozygous carriers are asymptomatic, with normal red blood cell morphology, whereas homozygous subjects exhibit elliptocytes on the blood film and present with mild HE, presumably as a result of the concomitant partial deficiency of 4.1R.13,145
Molecular Determinants of Clinical Severity
HE patients exhibit marked clinical heterogeneity ranging from asymptomatic carrier to severe, transfusion-dependent anemia. In patients with spectrin heterodimer self-association defects, the resultant increase in spectrin dimers and concomitant decrease in spectrin tetramers, weakens the membrane skeleton and facilitates the formation of elliptocytes under circulatory shear stress. The most important determinants of the severity of hemolysis in these patients are the percentage of spectrin dimers and the spectrin content of the membrane skeleton. These parameters are influenced by the degree of dysfunction of the mutant spectrin, and the gene dose (heterozygote versus homozygote or compound heterozygote).128 Genotype–phenotype correlations indicate that the order of clinical severity of αI domain defects is SpαI/74 > SpαI/46–50a > SpαI/65 and it depends on the position of the mutations within the proteins, as well as the type of mutation. Defects in the spectrin dimer self-association contact site leading to SpαI/74 mutants are the most severe128 and, for example, codon 28 mutations, which affect a highly conserved and critical arginine residue, are generally associated with phenotypically severe HE or HPP.133 A more distal mutation such as the duplication of leucine 154, which causes SpαI/65, is phenotypically very mild, even in the homozygous state.137 Proline or glycine helix-breaking mutations resulting in SpαI/46 or 50a are more severe even though they are further away from the self-association site.138
The clinical expression of HE often varies within the same kindred, despite all the affected individuals carrying the same causative mutation. This heterogeneity is a result of the inheritance of modifier alleles or additional defects. The low-expression αLELY is the most common polymorphism affecting spectrin content and clinical severity. The allele is characterized by an L1857V amino acid substitution, and partial skipping of exon 46 in 50 percent of the α-spectrin mRNA.94 The six amino acids encoded by exon 46 are essential for spectrin heterodimer assembly and therefore SpαLELY results in a reduced amount of spectrin, as monomers are rapidly degraded.146 The SpαLELY allele is clinically silent, even when homozygous, because α-spectrin is normally synthesized in three- to fourfold excess.147 Inheritance of SpαLELY in cis to an elliptocytogenic α-spectrin mutation ameliorates symptoms,148 whereas inheritance in trans causes a relative increase in the mutant spectrin and therefore exacerbates the disease.94
Coinheritance of other molecular defects also plays a role in modifying the clinical expression. HPP patients are very severely affected because they are homozygous or doubly heterozygous for spectrin self-association mutations and are also deficient in spectrin.129 Several molecular mechanisms have been identified that underlie the spectrin deficiency, including an RNA processing defect149; reduced α-spectrin mRNA and protein synthesis150; abnormal splicing resulting in a premature stop codon151; and degradation of α-spectrin.150 A recent study revealed the complexity of genotype–phenotype interactions in two large Utah families of northern European descent in whom a novel R34P mutation in α-spectrin was associated with three morphologic phenotypes.126 This heterogeneity was caused by an intricate interplay and coinheritance of other factors, including SpαLELY in trans, reduced transcription from the α-spectrin gene and intragenic crossover.126
In neonates the clinical severity of HE can be affected by the weak binding of BPG to fetal hemoglobin leading to an increase in free BPG, which, in turn, destabilizes the spectrin–actin–protein 4.1 interaction.152 Finally, hemolytic anemia can be exacerbated by several acquired conditions, including those that alter microcirculatory stress to the cells.
HE is typically inherited as an autosomal dominant disorder. De novo mutations are rare.134 The severity of clinical symptoms is highly variable reflecting heterogeneous molecular abnormalities, as well as the coinheritance of other genetic defects or polymorphisms that modify disease expression. A strong genetic relationship exists between HE and HPP, and parents or siblings of patients with HPP often have typical HE.
The clinical presentation of HE is heterogeneous, ranging from asymptomatic carriers to patients with severe, life-threatening anemia. The overwhelming majority of patients with HE are asymptomatic and are diagnosed incidentally during testing for unrelated conditions. HPP patients present in infancy or early childhood with a very severe hemolytic anemia.
Asymptomatic carriers who possess the same molecular defect as an affected HE relative but who have normal or near-normal blood films have been identified. The erythrocyte life span is normal, and the 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, increased hemolysis may result from microcirculatory damage superimposed on the underlying mechanical instability of red cells.
HE patients with chronic hemolysis experience moderate to severe hemolytic anemia with elliptocytes and poikilocytes on the 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 other kindreds, not all HE subjects have chronic hemolysis; some 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, fragments and small microspherocytes, reminiscent of HPP.
HPP represents a subtype of common HE, as evidenced by the coexistence of HE and HPP in the same family and the presence of the same molecular defects of spectrin.130 HE relatives are heterozygous for an elliptocytogenic spectrin mutation, whereas HPP patients are homozygous or doubly heterozygous and are also partially deficient in spectrin.128,129
Hereditary Elliptocytosis and Pyropoikilocytosis in Infancy
Clinical symptoms of elliptocytosis are uncommon in the neonatal period. Typically, elliptocytes do not appear on the blood film until the patient is 4 to 6 months old. Occasionally, severe forms of HE present in the neonatal period with severe, hemolytic anemia with marked poikilocytosis and jaundice. These patients may require red cell transfusion, phototherapy, or exchange transfusion. Usually, even in severely affected patients, the hemolysis abates between 9 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 usually are of greater diagnostic benefit than other available studies.
A few cases of hydrops fetalis accompanied by fetal or early neonatal death as a result of unusually severe forms of HE have been described.105 A severely affected hydropic infant salvaged by intrauterine transfusions (Chap. 55) and early exchange transfusion has remained transfusion dependent for more than 2 years.
The hallmark of HE is the presence of cigar-shaped elliptocytes on blood films Figs. 46-10D and 46-11F. These normochromic, normocytic elliptocytes may number from a few to 100 percent. The degree of hemolysis does not correlate with the number of elliptocytes present. Spherocytes, stomatocytes, and fragmented cells may be seen. 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.
HPP blood films exhibit similar features to severe HE, but in addition, they reveal extreme poikilocytosis, some bizarre-shaped cells with fragmentation or budding and often only very few or no elliptocytes Fig. 46-13. Microspherocytosis is common and MCV is usually low, ranging between 50 to 70 fL. Pyknocytes are prominent on blood films of neonates with HPP. The thermal instability of erythrocytes, originally reported as diagnostic of HPP, is not unique to this disorder because it is also commonly found in HE erythrocytes.
Specialized testing has been used in difficult cases or cases requiring a molecular diagnosis. Tests on isolated membrane proteins include analysis and quantitation of the proteins by SDS-PAGE; extraction of spectrin from the membranes to evaluate the spectrin-dimer-to-tetramer ratio on nondenaturing gels, as well as limited tryptic digestion of spectrin followed by SDS-PAGE or two-dimensional gel electrophoresis to identify the defective domain. Ektacytometry may be used to measure membrane stability and deformability. Genomic DNA and/or complementary DNA analyses are used to determine the underlying mutation.
Elliptocytes may be seen in association with several disorders, including megaloblastic anemias, hypochromic microcytic anemias (iron-deficiency anemia and thalassemia), myelodysplastic syndromes, and myelofibrosis. In these conditions, elliptocytosis is acquired and generally represents less than one-quarter of red cells seen on the blood film. History and additional laboratory testing usually clarify the diagnosis of these disorders. Pseudoelliptocytosis is an artifact of blood film preparation and these cells are found only in certain areas of the film, usually near its tail. The long axes of pseudoelliptocytes are parallel, whereas the axes of true elliptocytes are distributed randomly.
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 hematocrit, decreased reticulocyte counts, and improved clinical symptoms.
Patients should be followed for signs of decompensation during acute illnesses, characterized by acute decrease of hematocrit from nonspecific suppression of erythropoiesis by a concurrent acute event. HE and particularly HPP patients are at increased risk for parvovirus infection generally requiring short-lasting transfusion support (Chap. 36).153 Interval ultrasonography to detect gallstones should be performed. Patients with significant hemolysis should receive daily folate supplementation.
SOUTHEAST ASIAN OVALOCYTOSIS
SAO, also known as Melanesian elliptocytosis or stomatocytic elliptocytosis, is widespread in certain ethnic groups of Malaysia, Papua New Guinea, the Philippines, and Indonesia,123 but is also common in the Cape Coloured population in South Africa.154 It is characterized by the presence of large oval red cells, many of which contain one or two transverse ridges or a longitudinal slit Figs 46-10C and 46-11E.
SAO erythrocytes are rigid and hyperstable because of a structurally and functionally abnormal band 3. SAO band 3 binds tightly to ankyrin, forms oligomers, exhibits restricted lateral and rotational mobility155,156 and is unable to transport anions.157 The underlying molecular abnormality is an in-frame deletion of 27 bp in the band 3 gene resulting in the loss of amino acids 400 to 408 located at the boundary of the cytoplasmic and membrane domains of band 3.158 The defective SLC4A1 allele also carries a linked band 3Memphis polymorphism, L56E.
SAO is a dominantly inherited trait and homozygosity is postulated to be lethal during embryonic development.159 A recent case of homozygous SAO has been described where the fetus was kept alive by two intrauterine transfusions and since birth he has been on a monthly transfusion program.160 Distal renal tubular acidosis was diagnosed at 3 months as a result of the inability of the SAO band 3 to transport anions.
A remarkable feature of SAO erythrocytes is their resistance to infection by several species of malaria parasites. This has been demonstrated by numerous in vitro studies, as well as in vivo evidence indicating that SAO provides protection against severe malaria and cerebral malaria.123,161 Epidemiologic data and the increased prevalence of SAO in populations challenged by malaria suggest a selective advantage of the gene.123 Numerous factors have been implicated in the protective effect, but the precise mechanism of malaria resistance of SAO red cells has not been fully elucidated.
Clinically, the presence on the blood film of at least 20 percent ovalocytic red cells, some containing a central slit or a transverse ridge, and the notable absence of clinical and laboratory evidence of hemolysis are highly suggestive of SAO. Rapid genetic diagnosis can be made by amplifying the defective region of the band 3 gene and demonstrating heterozygosity for the SAO allele containing the 27 bp deletion.
Spiculated red cells are classified into two types: acanthocytes and echinocytes. Acanthocytes are contracted, dense cells with irregular projections from the red cell surface that vary in width and length (Figs. 46-10F and 46-11G). Echinocytes have small, uniform projections spread evenly over the circumference of the red cell Fig. 46-11B. Diagnostically, the distinction is not critical, and disorders of spiculated red cells are generally classified together. Normal adults may have up to 3 percent spiculated erythrocytes, but care should be taken when preparing and examining the blood film, because spiculated cells, particularly echinocytes, are common artifacts of blood film preparation and blood storage.
Blood films from a patient with HPP. A. Pre-splenectomy. B. Post-splenectomy. Note the prominent micropoikilocytosis, microspherocytosis, and fragmentation especially after splenectomy. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
Acanthocytes/echinocytes are found in various inherited disorders and acquired conditions. Spiculated cells can occur transiently in several instances, such as after transfusion with stored blood, ingestion of alcohol and certain drugs, exposure to ionizing radiation or certain venoms, and during hemodialysis.13 Spiculated cells are commonly seen on the blood films of patients with functional or actual splenectomy, severe liver disease, severe uremia, abetalipoproteinemia, certain inherited neurologic disorders and abnormalities of the Kell blood group. Occasionally acanthocytes and/or echinocytes may be present in patients with glycolytic enzyme defects, myelodysplasia, hypothyroidism, anorexia nervosa, vitamin E deficiency, and in premature infants.13 Individuals with suppressed expression of Lua and Lub, the major antigens of the Lutheran blood group system, may also exhibit acanthocytes.13
The molecular mechanisms whereby acanthocytes are generated have not been fully elucidated. However, alterations in band 3 have emerged as a pivotal causative factor. The abnormal red cell membrane lipid composition and altered lipid distribution between the inner and outer leaflets of the bilayer are only found in some, but not all, of these disorders, implying that they may play a secondary role.162
ACANTHOCYTOSIS IN SEVERE LIVER DISEASE
The anemia in patients with liver disease is often called “spur cell anemia” because of the projections on the red cells. Although only a small number of patients with end-stage liver disease acquire spur cell anemia, these individuals typically account for the majority of cases of acanthocytosis seen in clinical practice.
Etiology and Pathogenesis
The anemia in patients with liver disease is of complex etiology. Common causes include blood loss, iron or folate deficiency, hypersplenism, and marrow suppression from alcohol, malnutrition, hepatitis infection, or other factors. Acquired abnormalities of the red cell membrane may contribute to the anemia in some patients.163
In vivo acanthocyte formation in spur cell anemia is a two-step process involving accumulation of free (nonesterified) cholesterol in the red cell membrane and remodeling of abnormally shaped red cells by the spleen.13,163 The diseased liver of the patient produces abnormal lipoproteins with excess cholesterol, which is acquired by circulating erythrocytes, increasing their cholesterol content. The cholesterol preferentially partitions into the outer leaflet, increasing the surface area to volume ratio and forming scalloped edges. In the spleen, membrane fragments are lost and the cells develop the characteristic projections of acanthocytes (see Fig. 46–13). Cholesterol interacts with band 3 and changes its conformation, which may affect the membrane skeleton and reduce the deformability of the cell,162 causing it to be trapped and eventually destroyed in the narrow sinusoids of the spleen.
Spur cell anemia is characterized by rapidly progressive hemolytic anemia with large numbers of acanthocytes on the blood film. Splenomegaly and jaundice become more prominent and are accompanied by severe ascites, bleeding diatheses, and hepatic encephalopathy. Spur cell anemia is most common in patients with alcoholic liver disease, but similar clinical syndromes have been described in association with advanced metastatic liver disease, cardiac cirrhosis, Wilson disease, fulminant hepatitis, and infantile cholestatic liver disease.13
Most patients have moderate anemia with a hematocrit of 20 to 30 percent, marked indirect hyperbilirubinemia, and laboratory evidence of severe hepatocellular disease. Blood films reveal significant acanthocytosis and in some patients, echinocytes, target cells and microspherocytes, many with very fine spicules, are visible (see Fig. 46–13).
Spur cell hemolytic anemia should be distinguished from other hemolytic syndromes associated with liver disease, including congestive splenomegaly, in which patients exhibit chronic, mild hemolysis and occasional spherocytes, and patients with transient hemolytic episodes.
Therapy, Course, and Prognosis
The anemia of spur cell anemia usually is not a significant clinical problem, but it can aggravate pre-existing anemia resulting from, for example, gastrointestinal bleeding, to the point that erythrocyte transfusion is required. The life span of spur cells is markedly decreased because of splenic sequestration, and, as expected, hemolysis abates after splenectomy. However, splenectomy is a dangerous and potentially fatal procedure in these critically ill patients and is generally not recommended.
The term neuroacanthocytosis describes a heterogeneous group of rare disorders with variable clinical phenotypes and inheritance. The common features are a degeneration of neurons and abnormal acanthocytic erythrocyte morphology. These syndromes may be divided into: (1) lipoprotein abnormalities, which cause peripheral neuropathy, such as abetalipoproteinemia and hypobetalipoproteinemia, (2) neural degeneration of the basal ganglia resulting in movement disorders with normal lipoproteins, such as chorea-acanthocytosis and McLeod syndrome, and (3) movement abnormalities in which acanthocytes are occasionally seen, such as Huntington disease-like 2 (HDL2) and pantothenate kinase-associated neurodegeneration (PKAN).
Abetalipoproteinemia or Bassen-Kornzweig syndrome is a rare autosomal recessive disorder characterized by progressive ataxic neurologic disease, dietary fat malabsorption, retinitis pigmentosa, and acanthocytosis found in people of diverse ethnic backgrounds.164
Etiology and Pathogenesis
This disorder is caused by a failure to synthesize or secrete lipoproteins containing products of the apolipoprotein B (apoB) gene and this leads to changes in the plasma lipid profile.164 The primary molecular defect is a lack of the microsomal triglyceride transfer protein, which performs an essential step in apoB-containing lipoprotein synthesis.165 The relative distribution of erythrocyte membrane phospholipids is altered and the phosphatidylcholine content is decreased with a corresponding increase in sphingomyelin. The excess sphingomyelin is preferentially confined to the outer leaflet of the membrane bilayer, where it presumably causes an expansion of this layer and modifies the conformation of band 3, which contributes to the irregularities in cell surface contour.162 Red cell precursors and reticulocytes have a normal shape and acanthocytosis only becomes apparent as the red cells mature in the circulation, worsening with increasing red cell age.166
The disorder manifests in the first month of life by steatorrhea. Atypical retinitis pigmentosa, which often results in blindness, and progressive neurologic abnormalities characterized by ataxia and intention tremors develop between 5 and 10 years of age and progress to death in the second or third decade.166
Patients usually have mild anemia with normal red cell indices and normal or slightly increased reticulocyte counts.166 Acanthocytosis is prominent, ranging from approximately 50 to 90 percent of red cells. Despite the red cell lipid abnormalities, the hemolysis is mild and the spleen is normal in patients with abetalipoproteinemia, in contrast to spur cell anemia. There is marked vitamin E deficiency (Chap. 44), which is thought to be a primary stimulus for the neuropathy. Coagulopathy may be observed.164
The related disorders hypobetalipoproteinemia, normotriglyceridemic abetalipoproteinemia, and chylomicron retention disease are associated with partial production of apoB-containing lipoproteins or with secretion of lipoproteins containing truncated forms of apoB. Patients with these disorders may experience neurologic disease and acanthocytosis, depending on the severity of the underlying defect.
Therapy, Course, and Prognosis
Treatment includes dietary restriction of triglycerides and supplementation with high doses of vitamins A, K, D, and E.166 Chronic administration of vitamin E can delay or prevent the neurologic symptoms.
Chorea-acanthocytosis is a rare autosomal recessive movement disorder characterized by atrophy of the basal ganglia and progressive neurodegenerative disease with onset in adolescence or adult life.167 In some patients, acanthocytosis may precede the onset of neurologic symptoms. The lipoproteins are normal.
Molecular studies have identified approximately 100 mutations in the VPS13A gene, which codes for chorein, a protein ubiquitously expressed in the brain and also found in mature red cells.168,169,170 It is a member of a conserved protein family involved in trafficking of membrane proteins between cellular compartments, but its role in red cells and the pathogenesis of the disorder and acanthocytes is unknown. The mutations result in the absence or markedly reduced levels of chorein and founder mutations have been identified in Japanese and French-Canadian families.167
Patients are not anemic, and red cell survival is only slightly decreased. Plasma and erythrocyte membrane lipids, as well as membrane protein composition and content, are normal, but electron microscopy studies revealed structural abnormalities in the skeleton and an uneven distribution of intra-membrane particles. Red cell membrane fluidity is decreased. Increased serine-threonine and tyrosine phosphorylation of band 3, β-spectrin, and β-adducin has been documented.171 In particular, abnormal activation of Lyn kinase results in increased tyrosine phosphorylation of band 3, which alters the association of band 3 with β-adducin and the junctional complex of the skeleton.171 This may lead to localized disruption of the skeleton–membrane interaction, facilitating the formation of protrusions. In one chorea-acanthocytosis kindred a point mutation near the C terminus of band 3 has been identified, which may influence the interaction of band 3 with the skeleton.172
The McLeod phenotype is a rare X-linked defect of the Kell blood group system, whereby cells react poorly with Kell antisera. The XK protein is an integral membrane transport channel protein that is covalently linked to the Kell antigen by disulphide bonds, and mutations in the XK gene cause a deficiency of the XK protein.167,170 Male hemizygotes who lack XK have up to 85 percent acanthocytes on the blood film with mild, compensated hemolysis and develop late-onset multisystem myopathy or chorea known as the McLeod syndrome. Female heterozygous carriers may have occasional acanthocytes as a result of mosaicism in X chromosome inactivation. Large deletions involving not only the XK locus at Xp21.1, but also contiguous genes, result in the McLeod syndrome being associated with other diseases, such as chronic granulomatous disease of childhood, retinitis pigmentosa, Duchenne muscular dystrophy, and ornithine transcarbamylase deficiency.
Red cell membrane protein and lipid composition are normal, but the distribution of intramembrane particles is altered and increased phosphorylation of membrane proteins, notably band 3, has been noted, which again implicates band 3 as a key player in the generation of acanthocytes.
Other Neuroacanthocytosis Syndromes
The HDL2 disorder is caused by expanded CGT/CAG trinucleotide repeat mutations in the junctophilin-3 gene, which encodes a protein involved in junctional membrane structures and calcium regulation.167 The disease is autosomal dominant and presents with late-onset chorea, parkinsonism, and progressive cognitive defects. Acanthocytes are present in some patients. In one unusual kindred autosomal dominant inheritance of chorea-acanthocytosis with polyglutamine neuronal inclusions was described in association with HDL2. Proteolysis of band 3 was also noted, which could contribute to the altered red cell morphology.170,173
Acanthocytes have been noted in some patients with PKAN (formerly known as Hallervorden-Spatz syndrome) with features of dystonia, dysarthria, and rigidity in childhood, and in HARP syndrome (hypobetalipoproteinemia, acanthocytosis, retinitis pigmentosa and pallidal degeneration). Both conditions are caused by mutations in pantothenate kinase 2, which is involved in synthesis of coenzyme A and phospholipids.167,170,174
Differential Diagnosis of Neuroacanthocytosis with Normal Lipoproteins
Chorea-acanthocytosis, McLeod syndrome, HDL2, and pantothenate kinase disorders present with overlapping neurologic symptoms and clinical phenotypes and also resemble Huntington disease, which renders the clinical diagnosis difficult. Identification of the underlying gene defects and the availability of molecular tests have markedly improved the diagnostic accuracy. This also provides insight into the underlying pathogenesis and suggests that the affected proteins, which are all linked to membrane structure, may participate in a common pathway that ultimately causes degeneration of the basal ganglia.
HEREDITARY STOMATOCYTOSIS SYNDROMES
The intracellular concentration of the monovalent cations, Na+ and K+, contribute to erythrocyte volume homeostasis. A net increase in these cations causes water to enter the cells resulting in overhydrated cells or stomatocytes, whereas a net loss dehydrates the cells and forms xerocytes. Disorders of red cell cation permeability are very rare conditions that are inherited in an autosomal dominant fashion with marked clinical and biochemical heterogeneity (Table 46–4).175
Table 46–4.Heterogeneity of the Hereditary Stomatocytosis Syndromes ||Download (.pdf) Table 46–4. Heterogeneity of the Hereditary Stomatocytosis Syndromes
| ||Stomatocytosis (Hydrocytosis) ||Intermediate Syndromes |
| ||Severe Hemolysis ||Mild Hemolysis ||Cryohydrocytosis ||Stomatocytic Xerocytosis ||Xerocytosis with High Phosphatidylcholine ||Xerocytosis |
|Hemolysis ||Severe ||Mild–moderate ||Moderate ||Mild ||Moderate ||Moderate |
|Anemia ||Severe ||Mild–moderate ||Mild–moderate ||None ||Mild ||Moderate |
|Blood film ||Stomatocytes ||Stomatocytes ||Stomatocytes ||Stomatocytes ||Targets ||Targets, echinocytes |
|MCV (80–100 fL)* ||110–150 ||95–130 ||90–105 ||91–98 ||84–92 ||100–110 |
|MCHC (32–36%) ||24–30 ||26–29 ||34–40 ||33–39 ||34–38 ||34–38 |
|Unincubated osmotic fragility ||Markedly increased ||Increased ||Normal ||Decreased ||Markedly decreased ||Markedly decreased |
|RBC Na+5–12† ||60–100 ||30–60 ||40–50 ||10–20 ||10–15 ||10–20 |
|RBC K+90–103 ||20–55 ||40–85 ||55–65 ||75–85 ||75–90 ||60–80 |
|RBC Na++K+95–110 ||110–140 ||115–145 ||100–105 ||87–103 ||93–99 ||75–90 |
|Phosphatidylcholine content ||Normal ||± Increased ||Normal ||Normal ||Increased ||Normal |
|Cold autohemolysis ||No ||No ||Yes ||No ||No ||? |
|Effect of splenectomy‡ ||Good ||Good ||Fair ||? ||? ||? Poor |
|Inheritance ||Autosomal dominant?, autosomal recessive ||Autosomal dominant ||Autosomal dominant ||Autosomal dominant ||Autosomal dominant ||Autosomal dominant |
Stomatocytes are cup-shaped red cells characterized by a central hemoglobin-free area (Figs. 46-10E and 46-11D). The molecular mechanism of stomatocyte formation has not been elucidated, but several theories have been postulated. The lipid bilayer hypothesis predicts that agents or abnormalities that expand the inner leaflet will tend to form stomatocytes.176 Other theories relegate lipids to a secondary role and propose that membrane proteins, specifically band 3, play a major role in regulating the structure of the red cell.162 Band 3 tetramers are attached to the spectrin skeleton and different configurations of band 3 that either face inward or outward can influence the topography of the skeleton and the shape of the cell.
Hereditary xerocytosis, also known as dehydrated HSt, is the most common form of the cation permeability defects. It is an autosomal dominant hemolytic anemia characterized by an efflux of K+ and red cell dehydration. Hereditary xerocytosis is part of a pleiotropic syndrome and patients may also exhibit pseudohyperkalemia and perinatal edema.177
Etiology and Pathogenesis
The underlying membrane permeability defect is complex and involves a net loss of potassium from the red cells that is not accompanied by a proportional gain of sodium. Consequently, the net intracellular cation content and cell water content are decreased. In some cases, erythrocytes exhibit an increase in phosphatidylcholine and reduced BPG content.13
The genetic locus for this disorder was mapped to 16q23–q24.177 Subsequent refinement of the locus and exome sequencing of several large unrelated multigenerational kindred identified numerous missense mutations in the gene encoding the PIEZO1 protein.178,179 Cosegregation of some of the mutations in families with multiple disease phenotypes suggested a correlation between PIEZO and perinatal edema.178 PIEZO proteins were recently identified as mechanosensory molecules that form part of stretch-activated cation channels. The PIEZO1 protein is present in red cell membranes and two PIEZO1 mutations, R2456H and R2488Q, were demonstrated to regulate a mechanosensitive transduction channel, leading to increased cation transport in erythrocytes.178,179
Patients may present with symptoms of compensated hemolytic anemia, including jaundice, splenomegaly, and gallstones. Some patients may also exhibit pseudohyperkalemia and perinatal edema and even hydrops fetalis.13,177 Variable penetrance is present in this disorder, with significant disparity in clinical symptoms between affected individuals in the same kindred. Patients display a strong tendency to iron overload (Chap. 43).175
The hematologic picture is that of mild to moderate compensated hemolytic anemia (see Table 46–4) with an elevated reticulocyte count. The K+ content is decreased and the Na+ content is increased, but the total monovalent cation content is reduced. The MCHC is increased reflecting cellular dehydration and the MCV is frequently mildly increased.175 Erythrocytes are resistant to osmotic lysis and the bell-shaped curve obtained by osmotic gradient ektacytometry is shifted to the left. Stomatocytes are not a prominent feature on blood films, but some target cells and spiculated cells are seen. In some of the cells, hemoglobin is concentrated (“puddled”) in discrete areas on the cell periphery.
Therapy, Course, and Prognosis
Most patients experience only mild anemia and therapy is not required. The patients should receive folate supplementation and be monitored for complications of hemolysis. Splenectomy does not significantly improve the anemia, which suggests that xerocytes are detected and eliminated in other areas of the reticuloendothelial system. Because of a markedly high risk of hypercoagulability and life-threatening thrombotic episodes after splenectomy, the procedure is contraindicated.13
Hereditary stomatocytosis, also known as hereditary hydrocytosis or overhydrated stomatocytosis, is characterized by a marked passive sodium leak, which causes red cell overhydration and macrocytosis. It is an autosomal dominantly inherited hemolytic anemia. The syndrome was first described in a girl with dominantly inherited hemolytic anemia whose blood film contained stomatocytes.180 The hallmarks of abnormal cation transport and overhydration of the red cells were discovered subsequently.181
Etiology and Pathogenesis
The red cell membrane of stomatocytes has enhanced permeability toward monovalent cations, especially sodium ions. This marked passive sodium leak into the cell represents the principal lesion in this disorder. The Na+-K+-ATPase pump, which normally maintains low intracellular sodium and high potassium concentrations, is stimulated but this increase in active transport, coupled to enhanced glycolysis to provide ATP, is insufficient to overcome the leak.175,182
The overhydrated red cells of some patients lack stomatin, a 31-kDa integral membrane protein, but no gene mutations have been found implying that the absence of the protein is a secondary phenomenon.13,175 Stomatin interacts with GLUT-1 and converts it to a dehydroascorbic acid transporter, suggesting that it might be beneficial to inhibit this interaction in stomatocytes, because they require additional glucose for their increased ATP needs.175
In some stomatocytosis patients, missense mutations causing amino acid substitutions of conserved residues in the transmembrane domain of the RhAG protein, a component of the band 3–Rh–RhAG multiprotein complex in the membrane, have been described.183 RhAG is a transport protein that may function as a gas and/or ammonium channel through pore-like structures. The mutations are thought to widen the pores allowing cations to leak through the membrane. A de novo missense mutation in the transmembrane domain of band 3 has been described in one patient with stomatocytosis associated with dyserythropoiesis.184 This changed the transport function of band 3 from an anion exchanger to a cation channel. The tyrosine phosphorylation profile of the stomatocyte membranes revealed increased phosphorylation of band 3 and stomatin, as a result of enhanced activity of the Syk and Lyn tyrosine kinases, suggesting that phospho-signaling pathways involved in cell volume regulation may be perturbed.184
Moderate to severe anemia is present. Jaundice and splenomegaly are common, as are complications of chronic hemolysis, such as cholelithiasis. Patients exhibit a tendency for iron overload, independent of transfusion status or splenectomy. No other organ system abnormalities have been noted.13,175 A dyserythropoietic phenotype was noted in one patient with mild anemia.184
The blood film reveals striking stomatocytosis and up to 50 percent of red cells may have abnormal morphology (Figs. 46-10E and 46-11D). In addition to the anemia, red cell indices show decreased MCHC and marked macrocytosis, as reflected by an elevated MCV, which can reach 150 fL in some severely affected patients (see Table 46–4). The K+ content is decreased and the Na+ content is markedly increased, leading to elevated total monovalent cation content. The OF of stomatocytes is markedly increased because many of the swollen red cells approach their critical hemolytic volume, which causes a shift of the osmotic gradient ektacytometer curve to the right. Red cell deformability is decreased.
Therapy, Course, and Prognosis
The majority of hydrocytosis patients suffer from significant lifelong anemia. They should be monitored for complications of hemolysis, such as cholelithiasis and parvovirus infection, and should receive folate supplementation. The outcome of splenectomy has been variable, but typically it has been beneficial and improved the hemolytic anemia in severely affected patients.13 This is expected because stomatocytes expend large amounts of ATP to pump cations in an attempt to avoid osmotic lysis and are, therefore, vulnerable in the metabolically challenging environment of the spleen. However, splenectomy should be carefully considered in patients with this disorder, since they are at high risk of developing hypercoagulability after splenectomy, leading to catastrophic thrombotic episodes.13
The clinical phenotype and biochemical features of some patients with stomatocytes are intermediate between the extremes of hereditary hydrocytosis and hereditary xerocytosis. One of these disorders is cryohydrocytosis in which the mild cation leak is markedly enhanced at low temperatures. It is a very rare condition associated with mild to moderate hemolytic anemia and splenectomy appears to be beneficial.175 Missense mutations have been found in the transmembrane section of band 3 that cluster between membrane span eight and the last two membrane-spanning domains.184,185,186 In vitro studies indicated that the mutant proteins have lost their anion exchange capability and are converted to a nonselective cation channel.175,186,187
Two cases of cryohydrocytosis and stomatin deficiency have been described with mutations in GLUT-1, which abolish the glucose transport function of the protein and create a cation leak.188
OTHER STOMATOCYTIC DISORDERS
Rh-deficiency syndrome designates rare individuals who either lack all Rh antigens (Rhnull) or exhibit markedly reduced (Rhmod) Rh antigen expression. Rh antigens are carried on RhCE and RhD proteins that associate with RhAG and enable the formation of the Rh multiprotein complex in the red cell membrane. The Rh complex is either absent or markedly reduced in patients with Rh deficiency syndrome and they present with mild to moderate hemolytic anemia. Stomatocytes and occasional spherocytes are seen on the blood film and the cells have cation transport abnormalities, which cause dehydration. Hemolytic anemia is improved by splenectomy.13,175 Chapter 136 reviews the structure, localization, and functions of the Rh antigens.
Familial deficiency of high-density lipoproteins is a rare condition that leads to accumulation of cholesteryl esters in many tissues, resulting in clinical findings of large orange tonsils and hepatosplenomegaly. Hematologic manifestations include moderately severe hemolytic anemia with stomatocytosis. Red cell membrane lipid analyses revealed a low cholesterol content and a relative increase in phosphatidylcholine at the expense of sphingomyelin.13
Normal individuals have up to 3 percent stomatocytes on blood films. Acquired stomatocytosis is common in alcoholics particularly those with acute alcoholism. Vinca alkaloids, such as vincristine and vinblastine, may induce hemolysis with increased sodium permeability and stomatocytosis at the doses used for chemotherapy of leukemias and lymphomas.189 Transient stomatocytosis has been observed in long distance runners immediately after a race. The molecular basis of acquired stomatocytosis is unknown.13