CHAPTER 10: Hemolytic Disorders of the Red Cell Membrane and Red Cell Metabolism

MOLECULAR ANATOMY OF THE RED CELL MEMBRANE

As mentioned in Chapter 1, the red cell has two relatively simple yet highly important responsibilities—the transport of oxygen from the lungs to respiring organs and tissues and the transport of carbon dioxide in the reverse direction. Because of its relatively long life span of 120 days, a single red cell makes about 170,000 circuits through the microcirculation and, in doing so, travels roughly 100 miles! The efficiency of both gas transport and flow through narrow channels in the capillary and splenic circulation is enhanced by the ability of the red cell to change shape. Thus, in order for a red cell to fulfill its functions, its membrane must have a high degree of durability and flexibility.

The basis for the remarkable mechanical properties of the red cell is a network of proteins that assemble to form a specialized membrane skeleton, depicted in Figure 10-1. The primary building blocks of the membrane skeleton are alpha (α)-spectrin and beta (β)-spectrin, which bind to each other to form long, stable dimers. Self-association of spectrin dimers and docking of spectrin to a complex of actin filaments, with the aid of protein 4.1, creates a flexible, branching, hexagonal network that covers the entire inner surface of the membrane. The membrane skeleton is tethered to the membrane's lipid bilayer by vertical interactions with a complex composed of the anion exchange channel (band 3), ankyrin, and protein 4.2. These horizontal and vertical interactions within the membrane skeleton and with the lipid bilayer are critical determinants of the pliability and tensile strength of the red cell membrane.

As discussed in the following sections, molecular defects in vertical interactions cause hereditary spherocytosis, whereas defects in horizontal interactions result in the formation of elliptical red cells.

HEREDITARY SPHEROCYTOSIS

Under the microscope, normal red cells appear as biconcave discs. Adoption of this shape requires a significant amount of excess membrane surface area and gives the red cell considerable deformability, allowing it to traverse narrow strictures in the microcirculation, especially within the spleen. In contrast, a cell with a minimal ratio of surface area to volume would assume the shape of a sphere and be non-deformable. A relatively simple laboratory test called osmotic fragility can provide an accurate assessment of the surface area to volume ratio of red cells (Fig. 10-2). When exposed to solutions of decreasing salt (NaCl) concentration, the red cell swells progressively until it becomes a sphere. With further reduction in salt concentration, the non-distensible membrane ruptures, leading to release of the cell's content of hemoglobin. Spherocytes have a low ratio of surface area to volume and undergo lysis at a higher salt concentration than that required for lysis of normal cells. In contrast, target cells, which have a higher surface area to volume ratio, undergo lysis at a lower salt concentration.

In patients with hereditary spherocytosis (HS), the presence of non-deformable spherocytes causes hemolysis of varying severity. It is one of the most commonly encountered inherited hemolytic anemias, with an incidence of about 1 in 5000. In the great majority of cases, an autosomal dominant inheritance pattern is apparent. In most of these patients, the causative mutations involve ankyrin, band 3, or β-spectrin. Those with recessive HS owing to biallelic mutations in α-spectrin have much more severe hemolysis. In contrast, individuals with biallelic mutations in protein 4.2 have mild HS. As shown in Figure 10-1, all of these proteins participate in the "vertical" assembly of a complex that stabilizes the interaction of the membrane skeleton with the lipid bilayer. Mutations

that result in either impaired production or, less commonly, altered function of one of these proteins cause shedding of membrane microvesicles and thus a decrease in the ratio of surface area to volume. These rigid spherocytes are trapped in the spleen, where they are either destroyed or "conditioned" to become even more spherocytic.

The severity of HS varies considerably. Although all affected individuals have hemolysis with increased reticulocyte counts, those with mild anemia (compensated hemolysis) have minimal or no symptoms and often are not diagnosed until later in life. Most patients have a modestly enlarged, palpable spleen. Those with more marked hemolysis have symptoms of severe anemia and are often mildly icteric. Like other patients with chronic hemolysis, patients with HS, even adolescents or young adults, commonly develop gallstones caused by the precipitation of poorly soluble nonconjugated bilirubin, serum levels of which are elevated owing to the enhanced rate of red cell destruction, as explained in Chapter 3. Patients with HS may have sudden worsening of anemia because of either acute suppression of erythropoiesis, such as during parvovirus B19 infection, or enhanced hemolysis, such as during the transient splenic hyperplasia that accompanies infectious mononucleosis.

The diagnosis of HS should be suspected in any patient with life-long hemolytic anemia, especially when family members are also anemic. In moderate or severe cases, spherocytes are readily detected in the peripheral blood film, appearing as small, dark, red cells with no central pallor (Fig. 10-3). When the hemolysis is mild, the spherocytes are less apparent and are often overlooked. Consistent with the dark red color of spherocytes seen on the blood film, the mean cell hemoglobin concentration (MCHC) is increased in a portion of the red cells. Spherocytes are also the prime morphologic feature of an acquired disorder, immune hemolytic anemia (Chapter 11).The diagnosis of HS can be confirmed by the osmotic fragility test, as described in Figure 10-2. The presence of osmotically fragile cells, that is, those with a decreased surface area to volume ratio, can be enhanced by a 24-hour incubation period at 37^°C before the test is performed.

Because spherocytes are trapped by and destroyed in the spleen, splenectomy always greatly decreases the rate of hemolysis and increases the hemoglobin level. Furthermore, the much lower degree of heme catabolism lessens the odds for developing bilirubin gallstones. Thus, splenectomy is recommended in all patients with severe HS. However, especially in very young children, splenectomy increases the risk of sepsis from encapsulated bacteria such as meningococcus and pneumococcus, which are normally opsonized and cleared by the spleen. Therefore, surgery should be avoided in very young children (<3 years) and often is not performed in patients at any age who have compensated hemolysis and little or no anemia. All patients should receive appropriate antimicrobial vaccinations prior to splenectomy, and most physicians treat patients with prophylactic penicillin for at least a few years after the operation. There is controversy as to whether patients with moderate hemolysis should have their spleens removed, because of the concern regarding postsplenectomy sepsis. Partial splenectomy is gaining favor in the treatment of moderately severe HS, because it is assumed that this procedure lessens the risk of sepsis.

HEREDITARY ELLIPTOCYTOSIS

Occasional families have mild hemolysis in association with elongated, elliptical red cells, as shown in Figure 10-4, a condition known as hereditary elliptocytosis (HE). These individuals have inherited, generally in an autosomal dominant manner, a molecular defect in proteins involved in the horizontal interactions depicted in Figure 10-1. As a result, in vitro studies have demonstrated that spectrin αβ-dimers in the HE membrane skeleton fail to assemble into stable hexagonal arrays. Most often HE is due to missense mutations in α-spectrin, especially in the domain through which αβ-spectrin dimers self-associate. Less often, HE is caused by mutations in the self-association domain of β-spectrin or by a deficiency of protein 4.1.

Most individuals with HE have modest hemolysis and either mild or no anemia, and therefore do not require treatment. However, at birth some individuals with HE have severe hemolysis and marked abnormalities of red cell shape (poikilocytosis). During the first year of life, these abnormalities evolve into mild HE. Some individuals whose parents have mild HE are born with even more bizarre deformities of red cell shape, designated hereditary pyropoikilocytosis, a severe, often transfusion-dependent hemolytic anemia that does not abate with time.

OTHER INHERITED RED CELL MEMBRANE DISORDERS

Severe hemolytic anemia can be caused by dominantly inherited defects in the control of red cell cation content. In hereditary xerocytosis, red cells are severely dehydrated, owing to excessive efflux of potassium that exceeds the influx of sodium from the plasma. As a consequence, the osmotic fragility test shows osmotic resistance, the opposite of that seen in HS. Red cell morphology is remarkably normal, with only a modest number of target cells. Xerocytes have a high MCHC and are therefore rigid and rapidly cleared by macrophages in the liver and spleen. The opposite pertains in the very rare hereditary stomatocytosis, in which red cells are overhydrated owing to excess influx of sodium and water. As shown in Figure 10-5, stomatocytes have a mouth-like slit rather than a circular area of central pallor. Patients with hereditary xerocytosis and stomatocytosis have a very high risk of thromboembolism after splenectomy, possibly because the asplenic state prolongs the circulation of damaged red cells that promote coagulation in some way. For this reason, splenectomy should be avoided in these patients whenever possible.

In keeping with the red cell's relatively simple structure and limited function, it has rather modest metabolic capabilities. Devoid of both nucleus and organelles, the red cell subsists on the glycolytic (Embden-Meyerhof) pathway (Fig. 10-6), which supplies only two moles of adenosine triphosphate (ATP) per mole of glucose metabolized. In contrast, cells containing mitochondria can reap 38 moles of ATP per mole of glucose. Nevertheless, this small supply of energy is sufficient for active transport of cations across the red cell membrane, the synthesis of 2,3-diphosphoglycerate (2,3-DPG) to modulate hemoglobin oxygen affinity (as explained in Chapter 3), and the reduction of heme iron from ferric to ferrous, so that hemoglobin can bind with oxygen.

Despite its simple metabolic requirements, the red cell is uniquely vulnerable to mutations that impair the stability of metabolic enzymes. In contrast to most other cell types, during its 120-day life span the red cell does not synthesize any new protein. As a result, mutations causing a modest decrease in enzyme stability can produce a pathogenic metabolic defect in red cells while leaving other cells in the body unaffected.

HEXOSE MONOPHOSPHATE SHUNT AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Normally, a small proportion of glucose is metabolized through the hexose monophosphate shunt, which supplies reducing equivalents in the form of reduced glutathione. When the red cell is challenged by oxidant stress, a much higher fraction of glucose is metabolized through this pathway. As shown in Figure 10-6, glucose-6-phosphate enters the hexose monophosphate shunt and is oxidized to 6-phosphogluconate, transferring reducing electrons on to nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH. This reaction is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). The production of NADPH permits the conversion of oxidized glutathione to reduced glutathione via the enzyme glutathione reductase. Enhanced production of reduced glutathione enables the red cell to detoxify oxidants such as hydrogen peroxide, superoxide, and other reactive oxygen species, which are produced by immune cells in response to infection and by the liver as part of the metabolism of certain drugs and chemicals. If the supply of reduced glutathione is overwhelmed, either by a very large oxidant stress or by enzymatic defects in the hexose monophosphate shunt, the red cell undergoes serious and irreversible damage. Both lipids and red cell membrane proteins become oxidized. Hemoglobin becomes denatured and falls out of solution into dense, intracellular precipitates called Heinz bodies, shown in Figure 10-7. Heinz bodies are also seen in α-thalassemia when three of the four α-globin genes are deleted (hemoglobin H disease, Chapter 8) and in individuals who have inherited mutant hemoglobins that result in improper protein folding and subunit assembly. The presence of these rigid inclusions causes the red cell to be trapped in the narrow interstices of the spleen. Sometimes splenic macrophages surgically excise the portion of the red cell that contains a Heinz body. In these circumstances, the red cell may escape with a concave gap in its perimeter, appearing like a cookie after a large bite has been taken.

Deficiency of G6PD is common in parts of the world where malaria is endemic and accounts for nearly all of the inherited abnormalities of the hexose monophosphate shunt pathway. The gene encoding G6PD is located on the X chromosome, and hemizygous males carrying functionally defective G6PD mutants are accordingly more severely affected. Because of random inactivation of one of their X chromosomes, females who are heterozygous for a functionally defective G6PD mutant have, on average, half normal red cells and half G6PD-deficient red cells. Even when exposed to oxidant stress, these females are free of clinical consequences unless X chromosome inactivation is highly skewed toward the one bearing the deficient gene. Because males have a single X chromosome, half of the sons of a heterozygous mother will be G6PD deficient. Homozygous females are encountered much less frequently but have a clinical phenotype identical to that of affected (hemizygous) males. Figure 10-8 shows family trees that illustrate these points.

G6PD deficiency is probably the most prevalent genetic disorder worldwide, affecting many millions, particularly those living in tropical areas. The African (Gd^A–), Mediterranean (Gd^Med), and Southeast Asian (Gd^Mahidol or Gd^Canton) variants are so common in their respective locales that they can be regarded as polymorphisms. There is convincing epidemiologic evidence and suggestive experimental evidence that these variants confer protection against falciparum malaria and thus become fixed at a high level of gene frequency in susceptible populations. All of these common G6PD mutants are missense mutations that make the enzyme less stable. In addition, over 100 other structurally distinct G6PD mutants have been reported, most in single families.

As shown in Figure 10-9, even normal G6PD loses over half of its enzymatic activity during the red cell's 120-day life span. However "old" normal red cells have more than enough activity for protection against oxidant stress. In contrast, the activity of the African Gd^A– variant decays more rapidly and is nearly absent in red cells older than 60 days. The Mediterranean variant (Gd^Med) is even more unstable; its activity disappears before red cells reach 20 days of age.

Individuals with the common forms of G6PD deficiency do not have hemolysis or any other clinical manifestations unless they are challenged with some type of oxidant stress. Figure 10-10 is a historical account from the 1950s of the clinical course of a Black soldier who participated in a study to evaluate the hematologic effects of primaquine, a drug used to treat malaria. The man had normal initial blood cell counts but, similar to about 10% of other African American soldiers in this study, when given primaquine, he developed acute intravascular hemolysis marked by a sudden drop in hemoglobin and hematocrit and the development of hemoglobinuria and reticulocytosis. Importantly, even with continuation of the drug at the same dose, the hemoglobin and hematocrit subsequently stabilized and then slowly recovered to pretreatment levels. All of these findings can be explained by the decay in G6PD activity as red cells circulate in vivo. As shown in Figure 10-9, exposure of red cells expressing the Gd^A– variant to an oxidant stress results in the abrupt lysis of all cells greater than 60 days old, that is, about half of the person's red cell mass. However, once these old cells are lysed, the remaining cohort of young cells is resistant to oxidants, except for the small fraction of cells that reach 60 days of age with each subsequent day. Thus, as the drug is continued, the red cell life span falls to 60 days, but otherwise healthy individuals easily compensate for this by increasing the production of red cells from the marrow. Once a new equilibration is reached, the person's reticulocyte count stabilizes at about 2%, and the hemoglobin and hematocrit levels are normal.

A variety of drugs can trigger acute hemolysis in G6PD-deficient individuals (Table 10-1). Individuals with the more severe Mediterranean variant are more likely to have drug-induced hemolysis than those with the African variant. In Black individuals with the common Gd^A– variant, bacterial or viral infection is at least as likely as drugs to cause hemolysis, presumably due to release of oxidants from activated macrophages and neutrophils.

GLYCOLYTIC PATHWAY

Because the red cell depends on glycolysis for its energy requirements, it is not surprising that mutations in enzymes in this pathway can cause hemolytic anemia. Most of these glycolytic enzyme defects are inherited in an autosomal recessive manner and generally stem from missense mutations that affect protein stability. All are extremely rare except for pyruvate kinase deficiency. As shown in Figure 10-6, pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate with the concomitant production of ATP. It is likely that the impaired survival of pyruvate kinase–deficient red cells is due to a fall in ATP to levels insufficient to meet the red cell's energy requirements. Like other red cell enzyme defects, abnormalities of red cell morphology in pyruvate kinase deficiency are nonspecific and unimpressive. Diagnosis requires specific enzyme assays. Severely affected patients may benefit modestly from splenectomy.