Chapter 13: Erythrocyte Membrane Disorders

• The erythrocyte membrane plays a critical role in the maintenance of the biconcave shape and integrity of the red cell.

• It provides flexibility, durability, and tensile strength, enabling erythrocytes to undergo extensive and repeated distortion during their passage through the microvasculature.

• It consists of a lipid bilayer with embedded transmembrane proteins and an underlying membrane protein skeleton that is attached to the bilayer via linker proteins.

• The integrity of the membrane relies on vertical interactions between the skeleton and the bilayer, as well as on horizontal interactions within the membrane skeletal network.

• Inherited membrane protein defects disrupt the membrane architecture and alter the shape of the cell resulting in hemolytic anemia as illustrated in Figure 13–1.

• 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.

• Protein defects affecting horizontal protein interactions within the membrane skeletal network disrupt the skeleton, resulting in defective shape recovery and elliptocytes.

• Red cell membrane disorders exhibit significant heterogeneity in their clinical, morphologic, laboratory, and molecular characteristics.

Table 13–1 summarizes the relationship between red cell membrane proteins and disease phenotype.

Definition and Epidemiology

• Hereditary spherocytosis (HS) is characterized by osmotically fragile, spherical erythrocytes.

• HS occurs in all race groups but is the most common inherited hemolytic anemia in patients of northern European descent.

Etiology and Pathogenesis

• HS is typically caused by a red cell membrane protein deficiency that compromises vertical interactions between the membrane skeleton and the lipid bilayer.

• Defects in spectrin, ankyrin, band 3, and protein 4.2 are common (see Table 13–1).

• The underlying molecular mutations are heterogeneous and may be family-specific.

• The membrane protein deficiency destabilizes the lipid bilayer, causing microvesicles to bud off from weakened areas, which leads to spherocyte formation.

• Spherocytes exhibit a decreased surface area-to-volume ratio and are dehydrated, which decreases their deformability.

• The passage of spherocytes through the spleen is impeded, and during erythrostasis they are engulfed by splenic macrophages and destroyed.

Inheritance

• HS is typically inherited in an autosomal dominant fashion.

• In approximately 25% of cases, HS is due to autosomal recessive inheritance or de novo mutations.

• Recessive HS is often caused by mutations in α spectrin or protein 4.2.

Clinical Features

• The typical clinical picture of HS combines evidence of hemolysis with spherocytosis and positive family history.

• The clinical manifestations of HS vary widely. Mild, moderate, and severe forms of HS have been defined according to differences in blood hemoglobin, bilirubin, and reticulocyte counts, which can be correlated with the degree of compensation for hemolysis as shown in Table 13–2.

• Severe cases may be diagnosed in infancy or childhood, but mild cases may escape detection until adulthood or may remain undetected.

• The majority of HS patients (60%–70%) have moderate disease with a variable degree of hemolytic anemia.

• Approximately 20% to 30% of HS patients have mild disease with compensated hemolysis where red blood cell production and destruction are balanced.

• As many as 10% of HS patients have severe disease in infancy. A small number of these, typically with autosomal recessive HS, present with life-threatening, transfusion-dependent anemia.

• An asymptomatic carrier state has been suggested in the case of clinically asymptomatic parents whose children present with typical HS.

• In the majority of HS cases, the clinical findings are limited to the erythroid lineage. However, a few kindred have cosegregating nonerythroid manifestations, particularly neuromuscular abnormalities and inherited distal renal tubular acidosis.

Complications

• Chronic hemolysis leads to the development of bilirubin gallstones in approximately 50% of patients.

• Hemolytic crises are usually associated with viral illnesses and typically occur in childhood.

• Parvovirus B19 infection can precipitate an aplastic crisis with coexistent reticulocytopenia.

• Megaloblastic crises may occur in patients with increased folate demands such as during pregnancy.

• Lower leg ulcers or dermatitis develop in some patients but tend to heal quickly after splenectomy.

• In severe cases, extramedullary hematopoiesis from masses of erythroblasts simulating a tumor is seen.

• Severely affected individuals may develop iron overload, often but not entirely, due to frequent transfusions (see Chap. 9).

Laboratory Features

• Spherocytes on the blood film are the hallmark of the disease and are characterized by a smaller diameter, darker staining, and a decreased or absent central pallor, compared to normal red cells as depicted in Figure 13–1B.

• HS erythrocyte morphology is not uniform and ranges from very few spherocytes to large numbers of dense microspherocytes and in some cases poikilocytosis.

• “Pincered” red cells are often seen in band 3–deficient individuals, whereas spherocytic acanthocytes are associated with β-spectrin mutations.

• Erythrocyte indices (Table 13–2) reflect a mild to moderate decrease in hemoglobin in most patients and an increased mean (red) cell hemoglobin concentration (MCHC) in approximately 50% of cases.

• Markers of hemolysis include increased serum lactate dehydrogenase and unconjugated bilirubin, decreased haptoglobin concentration, and increased urobilinogen in the urine.

• HS red cells are osmotically fragile, and this has been exploited in various laboratory tests, including the glycerol lysis test and the cryohemolysis test. The standard osmotic fragility test measures the premature lysis of HS red cells in hypotonic salt solutions. Incubation of cells for 24 hours prior to measuring osmotic fragility improves sensitivity of the test (Figure 13–2). However, the disadvantage of this test that not all cases are detected and that it does not distinguish HS from other conditions with secondary spherocytes.

• Eosin 5′-maleimide is a fluorescent dye that binds to erythrocyte transmembrane proteins, and HS patients exhibit decreased fluorescence, although the sensitivity and specificity of the test vary, depending on the cutoff value.

• Biochemical and molecular diagnostics involve an initial analysis of the red cell membrane proteins by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to identify the underlying defective protein. A simple DNA test as a part of the workup to diagnose HS is not feasible because HS is caused by mutations in several different genes and there are very few common mutations.

Differential Diagnosis

• Clinical features and family history should accompany an initial laboratory investigation comprising a complete blood count with a blood film, reticulocyte count, and serum bilirubin. Children, parents, and siblings of the proband should have blood counts, a reticulocyte count, and blood film examined to identify affected family members.

• HS should be considered in patients with incidentally noted splenomegaly, gallstones at a young age, and parvovirus infections.

• Other causes of spherocytic hemolytic anemia should be excluded, particularly autoimmune hemolytic disease, by performing a direct antiglobulin test (Coombs test).

• HS may be obscured in disorders that increase the surface-to-volume ratio of erythrocytes, such as obstructive jaundice.

Therapy and Prognosis

• Patients with aplastic crises or severe hemolysis may require transfusion.

• Splenectomy cures or alleviates the anemia in the overwhelming majority of patients because splenic sequestration is the primary determinant of erythrocyte survival in HS.

• Patients with severe disease are good candidates for splenectomy, but in other cases the risk of overwhelming postsplenectomy infection, especially the emergence of penicillin-resistant pneumococci, has to be taken into consideration and weighed against the benefits.

• Splenectomy should be delayed until age 5 to 9 years, if possible, because of increased susceptibility to infection in younger children.

• Laparoscopic splenectomy has become the method of choice in centers with surgeons experienced in the technique.

• Occasionally, splenectomy may not correct the anemia, usually due to an accessory spleen.

Definition and Epidemiology

• Hereditary elliptocytosis (HE) is a heterogeneous disease characterized by the presence of elliptical or oval erythrocytes on the blood film (Figure 13–1D).

• Children, parents, and siblings of the proband should have blood counts, a reticulocyte count, and blood film examined to identify affected family members.

• HE occurs in all race groups but is more prevalent in individuals of African descent, possibly because elliptocytes may confer some resistance to malaria.

Etiology and Pathogenesis

• The primary abnormality in HE erythrocytes is defective horizontal interactions between protein components of the membrane skeleton that compromise its ability to maintain the biconcave disk-shape of the red cell during circulatory shear stress.

• Spectrin defects that impair self-association into tetramers and a deficiency of protein 4.1 are the most common underlying causes of HE (see Table 13–1).

• The molecular mutations are heterogeneous and but family-specific.

• In severe HE, red cell fragmentation may occur.

Inheritance

• HE is typically inherited as an autosomal dominant disorder, and de novo mutations are rare.

Clinical Features

• The clinical presentation of HE is heterogeneous, ranging from asymptomatic carriers to patients with severe, life-threatening anemia.

• The majority of HE patients are asymptomatic.

• Occasionally, severe forms of HE requiring red cell transfusion may present in the neonatal period, but hemolysis abates by 12 months of age, and the patient progresses to HE with mild anemia.

Laboratory Features

• The hallmark of HE is the presence of normochromic, normocytic elliptocytes on blood films as depicted in Figure 13–1D.

• Poikilocytes may be present in severe HE.

• The degree of hemolysis does not correlate with the number of elliptocytes.

• The reticulocyte count generally is less than 5% but may be higher when hemolysis is severe.

• Nonspecific markers of increased erythrocyte production and destruction are present.

• Specialized biochemical and molecular diagnostic tests involve analysis of the red cell membrane proteins by quantitative SDS-PAGE, as well as spectrin analysis to evaluate the spectrin dimer-to-tetramer ratio and to identify the abnormal spectrin domain. The defective gene may then be analyzed to elucidate the mutation.

Differential Diagnosis

• Acquired elliptocytes are associated with several disorders, including megaloblastic anemias, hypochromic microcytic anemias (iron-deficiency anemia and thalassemia), myelodysplastic syndromes, and myelofibrosis.

• Family history and the presence of other clinical features associated with the above diseases usually clarify the diagnosis.

• Specialized biochemical and molecular testing may additionally be used to establish a diagnosis of HE.

Therapy and Prognosis

• Therapy is rarely needed in HE patients.

• In severe HE cases, occasional red blood cell transfusions may be required and splenectomy has been palliative.

• Hereditary pyropoikilocytosis (HPP) is part of the HE spectrum of disorders.

• It is a rare autosomal recessive disorder typically found in patients of African origin.

• HPP is characterized by severe hemolytic anemia with marked microspherocytes and micropoikilocytes, and very few elliptocytes on the blood film.

• The mean (red) cell volume (MCV) is very low, ranging between 50 and 70 fL.

• HPP patients are often transfusion-dependent, and splenectomy is beneficial because the spleen is the site of erythrocyte sequestration and destruction.

• The molecular defects in HPP patients are a combination of horizontal (severely impaired spectrin tetramer formation) and vertical (spectrin deficiency) abnormalities, with the latter causing microspherocytes and exacerbating the hemolytic anemia.

• Southeast Asian ovalocytosis (SAO) is widespread in certain ethnic groups of Southeast Asia.

• SAO is characterized by the presence of large, oval red cells, many of which contain one or two transverse ridges or a longitudinal slit (see Figure 13–1C).

• Typically, there is no clinical or laboratory evidence of hemolysis.

• SAO is a dominantly inherited disorder and homozygosity is postulated to be embryonic lethal.

• SAO erythrocytes are rigid and resistant to infection by several species of malaria parasites.

• SAO is caused by a nine–amino acid deletion in the hinge region of the band 3 protein.

• 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.

• Acanthocytes (spiculated red cells with multiple, irregular projections) and echinocytes (spiculated red cells with small uniform projections) occur in various inherited disorders and acquired conditions, as well as postsplenectomy.

Severe Liver Disease

• The anemia in patients with liver disease is often called “spur cell anemia.”

• 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.

• Spur cell anemia is most common in patients with advanced alcoholic cirrhosis and characterized by rapidly progressive hemolytic anemia.

• Splenectomy is not advised because of severe liver disease.

Neuroacanthocytosis

• This is 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 and pantothenate kinase-associated neurodegeneration.

Abetalipoproteinemia

• This rare autosomal recessive condition is characterized by progressive ataxic neurologic disease.

• It is caused by a failure to synthesize or secrete lipoproteins containing products of the apolipoprotein B (apoB) gene.

• Patients exhibit mild anemia, and 50% to 90% of red cells are acanthocytic (Figure 13–1F).

• Steatorrhea develops early in life; retinitis pigmentosa and other progressive neurologic abnormalities lead to death in the second or third decade of life.

• Patients are treated with dietary restriction of triglycerides and supplementation with fat-soluble vitamins.

Chorea-Acanthocytosis Syndrome

• This rare autosomal recessive movement disorder is characterized by atrophy of the basal ganglia and progressive neurodegenerative disease with acanthocytosis.

• It is caused by an absence or markedly reduced levels of chorein, a protein involved in trafficking of membrane proteins.

• Lipoproteins are normal, and patients are not anemic.

McLeod Phenotype

• This is a rare X-linked defect of the Kell blood group system.

• It is caused by a deficiency of the XK protein, an integral membrane transporter component.

• Male hemizygotes who lack XK have up to 85% acanthocytes on the blood film with mild, compensated hemolysis and normal membrane lipids.

• Patients develop late-onset multisystem myopathy.

• Large deletions of the XK locus result in other coexisting disorders, such as Duchenne muscular dystrophy.

• Stomatocytes are cup-shaped red cells characterized by a central hemoglobin-free area (Figure 13–1E).

• A net increase in cations causes water to enter the cells, resulting in overhydrated cells or stomatocytes, whereas a net loss of cations dehydrates the cells and forms xerocytes.

• Very rare conditions such as cryohydrocytosis show features intermediate between the two extreme phenotypes.

• Erythrocyte volume homeostasis is linked to monovalent cationic permeability, and this is disrupted in the hereditary stomatocytosis syndromes.

• These disorders of red cell cation permeability are very rare conditions that are inherited in an autosomal dominant fashion with marked clinical and biochemical heterogeneity.

Hereditary Stomatocytosis/Hydrocytosis

• This autosomal dominant disease is characterized by moderate to severe hemolytic anemia.

• It is caused by a marked passive sodium leak into the cell.

• Up to 50% stomatocytes are present and the osmotic fragility is increased.

• Red cell indices show decreased MCHC and a highly elevated MCV up to 150 fL.

Hereditary Xerocytosis (Desiccocytosis)

• This autosomal dominant disease is characterized by mild to moderate compensated hemolytic anemia.

• There is an efflux of potassium and red cell dehydration.

• The MCHC is increased, and red cells are resistant to osmotic lysis.

Other Stomatocytic Disorders

Rh Deficiency Syndrome

• The Rh complex is either absent or markedly reduced in patients with Rh deficiency syndrome.

• Patients present with mild to moderate hemolytic anemia.

• Stomatocytes and occasional spherocytes are seen on the blood film.

• Red cells have cation transport abnormalities, which cause dehydration.

• Splenectomy improves the anemia.

Familial Deficiency of High-Density Lipoproteins

• This rare condition with severe deficiency or absence of high-density lipoproteins leads to accumulation of cholesteryl esters in many tissues.

• Patients exhibit moderately severe hemolytic anemia with stomatocytosis.

Acquired Stomatocytosis

• Normal individuals have up to 3% stomatocytes on blood films.

• Acquired stomatocytosis is common in alcoholics and in patients with leukemias and lymphomas who have been treated with vinca alkaloids.