CHAPTER 9: Sickle Cell Disease

In 1910, Dr. James Herrick, while teaching a course in laboratory medicine, noted that a student from the West Indies had blood with normal-appearing red cells along with a population of "thin sickle-shaped and crescent-shaped red cells" similar to what is shown in Figure 9-1. Dr. Herrick found that this student was anemic and had breathlessness, palpitations, and occasional bouts of icterus. During the ensuing decade, a number of similar cases were reported, nearly all of them individuals of African ancestry. Most of these patients complained of intermittent attacks of severe pain. In vitro studies demonstrated that when blood from these patients was deoxygenated, all of the red cells were transformed into irregular and elongated "sickle cells."

In the late 1940s, the era of molecular medicine was launched with the discovery by Linus Pauling that the hemoglobin from sickle cell anemia patients had an abnormal electrophoretic mobility, indicating that its structure was different from that of normal hemoglobin. Moreover, Pauling found that healthy relatives of these patients often had a 50:50 mixture of the normal and abnormal hemoglobins.

In 1957, Vernon Ingram showed that hemoglobin (Hb) S was identical to normal Hb A except for the replacement of glutamic acid, the sixth amino acid in beta (β)-globin, with valine. This was the first demonstration of a human disease arising from a single structural mutation.

Careful observations of families showed that sickle cell anemia is inherited in an autosomal recessive manner, as shown in Figure 9-2.

SICKLE TRAIT

About 10% of black Americans are heterozygotes, inheriting a sickle globin gene (βS) from one parent and a normal (βA) gene from the other parent. As shown in Table 9-1, these individuals with sickle cell trait (Hb AS or AS) have no significant clinical problems unless they are subjected to severe stress, such as marked dehydration or hypoxia. Rarely AS individuals sustain splenic infarction, stroke, or sudden death following strenuous military or athletic training, particularly at high altitude. As discussed later in this chapter, the renal medulla is particularly susceptible to sickling. Many AS individuals have impaired ability to form concentrated urine (hyposthenuria), and a few have recurrent episodes of painless hematuria as a result of medullary infarction. However, damage to other organs is extremely uncommon in sickle trait individuals. They have normal life expectancy.

The gene frequency for Hb S is highest in Africa (Fig. 9-3), particularly in regions where malaria is endemic. Extensive analysis of polymorphisms adjacent to the β-globin gene has shown that the β6 valine mutation has arisen independently at four different times and places. This finding and the remarkably high frequency of the Hb S allele in central African populations (in which the prevalence of heterozygosity may be as high as 30%) indicate that the AS genotype confers a substantial selective advantage in this part of the world. There is strong epidemiologic evidence and somewhat less compelling experimental support for the notion that AS heterozygotes are less likely to succumb to falciparum malaria, particularly during infancy before immune defense becomes fully developed.

SICKLE CELL DISEASE: Hb SS, Hb S/β-THALASSEMIA, Hb SC

According to simple Mendelian genetics, about one-fourth of the children of parents with sickle trait (about 1 in 400 babies born to African American parents) will be homozygotes and have red cells containing primarily Hb S and no Hb A. As discussed in detail in this chapter, these individuals have severe hemolytic anemia and episodes of vaso-occlusion that cause acute bouts of pain and progressive organ damage. A similar clinical phenotype is encountered in individuals who are compound heterozygotes—inheriting the sickle gene from one parent and, from the other, either a β-thalassemia gene (Fig. 9-2B) or a gene that encodes Hb C, another β-globin structural mutant. Patients with S/β⁰ thalassemia¹ are unable to make any Hb A and are as severely affected as those with Hb SS (or SS) disease. In contrast, patients with Hb S/β⁺-thalassemia or with Hb SC disease have less morbidity and a longer life span (Table 9-1).

An overview of the pathogenesis of sickle cell disease is presented in Figure 9-4. The overarching question, which continues to defy easy answers, is how replacement of one of the 146 amino acids in β-globin causes a disease that can wreak havoc on nearly every organ system and display a wide range of clinical phenotypes. A partial understanding has come from rigorous study of 1) the structure of the sickle hemoglobin polymer, 2) the kinetics of polymerization, 3) the impact of recurrent episodes of sickling on the red cell membrane, and 4) the influence of Hb F on disease severity.

STRUCTURE OF THE SICKLE CELL FIBER

Electron micrographs of deoxygenated SS red cells reveal the presence of elongated parallel fibers of about 20 nm diameter that are oriented parallel to the long axis of sickling. Each fiber consists of a helical polymer composed of seven double strands (Fig. 9-5), each containing deoxyhemoglobin S molecules that interact through lateral and axial contacts (Fig. 9-5). Importantly, the double strand is stabilized by non-covalent hydrophobic bonding between β6 valine and a complementary "acceptor" site on another portion of the β-chain on the partner strand. In addition, the double strand is stabilized by other non-covalent contacts between molecules. When the hemoglobin is reoxygenated, conformational changes disrupt these contacts, and the polymer "melts" into individual molecules. The potential to form polymer is greatly affected by the presence of non-S hemoglobin in the red cell. Red cells of SS patients contain 2% to 20% Hb F (α₂γ₂), compartmentalized in a subset of cells (F cells). Because of amino acid sequence differences in gamma (γ)-globin, it cannot provide a proper hydrophobic acceptor site for binding to the β6 valine site, and therefore Hb F inhibits the polymerization of Hb S.

KINETICS OF FIBER FORMATION

The rate at which polymerization and sickling occur depends primarily on intracellular concentration of Hb S and the extent of deoxygenation. These two variables are the major determinants of the time required for the formation of the polymer. During this "delay time," deoxygenated Hb S aggregates to form a critical nucleus of about 15 molecules. As depicted in Figure 9-6, once this occurs there is a rapid and explosive propagation of polymer extending throughout the cell, deforming it into irregular sickle or holly leaf shapes. If polymer formation occurs before the red cells exit the narrow lumen of the microcirculation, obstruction to flow may occur, resulting in local tissue hypoxia, deoxygenation of red cells perfusing neighboring tissue, and further sickling. Amplification of this vicious cycle may result in a larger area of ischemia and infarction.

PERTURBATIONS OF THE RED CELL MEMBRANE

Recurrent hypoxia-dependent sickling stretches and deforms the red cell membrane, leading to the accumulation of intracellular calcium and the loss of potassium and water. As SS red cells become progressively dehydrated, the increasing intracellular hemoglobin concentration greatly lowers the delay time, thereby enhancing sickling. The most dehydrated cells appear on blood films as dense, irreversibly sickled forms, as shown in Figure 9-1. Other acquired structural alterations in the stressed membranes of SS red cells increase their adherence to endothelial cells lining the vessels in the microcirculation. In addition, leukocytes, which roll on the surface of endothelial cells, can serve as docking sites for sickle cells (Fig. 9-7). Red cell–endothelial cell adhesion slows the transit time of the red cell in the microcirculation and therefore increases the probability that sickling will occur in narrow-lumen vessels. Acute inflammation causes up-regulation of adhesive molecules on the surface of endothelial cells and leukocytes. This response may contribute to the higher frequency of vaso-occlusive events among patients with acute bacterial or viral infections.

EFFECT OF Hb F

The most important variable affecting the clinical severity of sickle cell disease is the level of Hb F. As mentioned earlier, Hb F is distributed unevenly among SS red cells. Because Hb F is a potent inhibitor of polymerization, F cells are resistant to sickling and survive much longer in the circulation than non-F SS cells. The ability to produce Hb F varies considerably among different populations. Among American patients with SS disease, clinical severity is inversely proportional to Hb F levels. Among populations in the eastern region of the Arabian peninsula and India (Fig. 9-3), SS patients have high Hb F levels and relatively mild disease. These epidemiological observations have been a powerful incentive for development of pharmacological agents for induction of γ-globin expression and Hb F production. Currently, genome-wide scanning is being actively pursued in search of gene polymorphisms that may modulate sickle cell severity and complications.

A number of methods are available to test for sickle cell hemoglobin. Simple inexpensive screening tests take advantage of the low solubility of deoxyhemoglobin S and provides a sensitive and reasonably specific way of detecting Hb S. These tests are suitable for large field studies, as they require only a drop of blood and use stable reagents, but do not distinguish between homozygotes and heterozygotes. Newborn screening is mandatory in most states in the United States. It is generally done by transfer of a heel-stick blood specimen to a piece of filter paper, which is then mailed to a central laboratory. A positive result on one of these screening tests needs to be pursued with a follow-up blood specimen for determination of hemoglobin, hematocrit, and red cell indices as well as an electrophoresis of the patient's hemoglobin (Fig. 9-8). Individuals with Hb AS have Hb S and Hb A in nearly equal proportions. Hb SS homozygotes and individuals with S/β⁰-thalassemia have predominantly Hb S along with a variable amount of Hb F. As shown in Figure 9-8, those with S/β⁺-thalassemia also have a small amount of Hb A. Hb SC individuals have nearly equal proportions of Hb S and Hb C. Because of the high level of Hb F in newborns, those with Hb SS, Hb S/β-thalassemia, and Hb SC have no clinical or hematologic abnormalities. They do not become anemic or develop signs and symptoms of sickle cell disease until they are at least 6 months of age, around the time that the switch from γ-globin expression to β-globin expression is complete. Nevertheless, accurate diagnosis of neonates is important in identifying those who are homozygotes or compound heterozygotes, enabling them to benefit from parental counseling and enrollment in a health care network, in anticipation of the complex medical problems that lie ahead.

PRENATAL DIAGNOSIS

When both parents carry the β^S gene, they are understandably concerned about whether future children will have SS disease. This is especially true if they already have experienced the stresses and challenges of caring for a child with the disease. Prenatal diagnosis can be made safely and with nearly 100% accuracy on DNA from a chorionic villus biopsy, usually taken at the end of the first trimester of gestation. If the fetus is SS, the parents face the larger question of whether to interrupt the pregnancy, one that involves moral, religious, and economic issues that are well beyond the scope of this book.

Patients with homozygous sickle cell disease as well as those with Hb S/β-thalassemia and Hb SC disease have a variety of clinical problems, outlined in Table 9-2. Among the constitutional manifestations are delayed growth and development. In addition, sickle cell patients are at increased risk of developing serious infections, due to pneumococcus as well as a broad range of other bacterial pathogens. Repeated splenic infarctions lead to loss of organ function and therefore defective clearance of circulating bacteria. Occasionally infarcts in the spleen or other sites of vaso-occlusion become infected, leading to abscess formation.

ANEMIA

Hb SS homozygotes have severe hemolytic anemia with hemoglobin values between 6 and 9 g/dL and elevated reticulocyte counts. The mean red cell lifespan is 10 to 15 days. As a result of accelerated red cell destruction, sickle cell patients have laboratory test findings characteristic of hemolysis, as described in Chapter 3. Patients with Hb S/β⁺-thalassemia and Hb SC disease have less severe hemolysis. In Hb SS patients, infection will suppress erythropoiesis and cause a fall in hemoglobin level (Table 9-2). In particular, parvovirus B19 has a tropism for erythroid progenitors and therefore can trigger a drop in the hemoglobin levels of sickle cell patients to extremely low levels.

VASO-OCCLUSION

The morbidity and mortality of sickle cell disease are due primarily to recurrent vaso-occlusive phenomena. As outlined in Table 9-2, these can be divided into microinfarctions, which manifest as acute, episodic attacks of pain, and macroinfarctions, which cause progressive and usually irreversible damage to a wide range of organs.

Pain Crises

Throughout their lives, most sickle cell patients are plagued with recurrent attacks of acute pain caused by vaso-occlusion. These episodes account for most of the hospital admissions of sickle cell patients. Pain crises are sporadic and highly unpredictable. Usually, they appear suddenly and are localized to a limited area, particularly the abdomen, chest, back, or joints. As mentioned earlier and in Table 9-2, pain crises are often preceded by a viral or bacterial infection. Most patients are able to distinguish sickle cell pain from pain due to some other type of acute process such as biliary colic, a perforated viscus, or abscess.

The acute chest syndrome, a pulmonary vaso-occlusive crisis, is the most common cause of death in both children and adults with Hb SS disease. The diagnosis is based on the combination of acute chest pain, pulmonary infiltrate(s) and arterial hypoxemia. Because it is often difficult to distinguish between pulmonary vaso-occlusion and pneumonia, all patients should be treated with antibiotic therapy. If there is rapid expansion in the size of the infiltrate(s) or deterioration of pulmonary function, progressive vaso-occlusion is likely, and prompt intervention is necessary. As shown in Figure 9-9, replacing at least half of the patient's Hb SS red cells with normal (Hb AA) red cells (so-called exchange transfusion) can result in surprisingly rapid clinical improvement and resolution of pulmonary infiltrates. In some patients, acute chest syndrome is triggered by fat emboli shed into the circulation from infarcted bone marrow, a common site of painful vaso-occlusion.

Chronic Organ Damage

Over time, most sickle cell patients sustain damage to various organs due to the cumulative effect of recurrent vaso-occlusive episodes.

Neurologic

Roughly one-fourth of sickle cell patients develop a central neurologic complication at some point in their lifetimes. Noninvasive studies in children have revealed a surprising and sobering frequency of abnormalities of cerebral blood flow that are associated with subclinical impairment of cognition along with increased risk of vaso-occlusive stroke. As patients get older, hemorrhagic strokes may occur. A rigorous and sustained program of red cell transfusions has proven effective in preventing strokes during childhood. It is not known yet whether hydroxyurea therapy (discussed later in the prevention Sickling section) will be as effective.

Cardiopulmonary

Sickle cell patients commonly have impairment of pulmonary function. Resting arterial PO₂ is usually low, in part because of intrapulmonary arterial-venous shunting. Repetitive bouts of acute chest syndrome, described earlier, and chronic hypoxemia can lead to pulmonary hypertension and occasionally to cor pulmonale. In addition, many sickle cell patients have left ventricular dilatation and decompensation, probably due to a combination of chronic anemia, hypoxia, and iron overload.

Hepatobiliary

Like other patients with chronic hemolytic anemia, those with sickle cell disease are very likely to develop gallstones. Nearly all Hb SS patients and many with Hb S/β-thalassemia and Hb SC disease have abnormal liver function, owing to a combination of hepatic vaso-occlusion, hepatitis transmitted via blood transfusions, and iron overload.

Genitourinary

The hypertonic milieu of the renal medulla dehydrates red cells and increases the intracellular hemoglobin concentration, thereby promoting sickling. Therefore, all individuals with Hb S, even Hb AS heterozygotes, develop hyposthenuria (the inability to concentrate urine) and some have episodes of painless hematuria. Hyposthenuria has little deleterious effect on its own, but makes patients (and thus their red cells) prone to dehydration, which can increase the risk for vaso-occlusion. In addition, Hb SS patients develop progressive impairment of glomerular function. As they survive into the fifth and sixth decades of life, renal failure becomes an important contributor to morbidity and mortality. Males with sickle cell disease occasionally develop episodes of priapism, acute painful engorgement of the penis, due to vaso-occlusion in the sinuses of the corpus cavernosum.

Skeletal

Vaso-occlusion within a bony matrix leads to aseptic necrosis, particularly of the hips and shoulders, and may cause sufficient disability to require joint replacement. Figure 9-10 shows the characteristic deformity of the lumbar spine due to vaso-occlusion in the interior of the vertebral body.

Dermal

Many sickle cell patients are plagued by chronic ulceration of the skin overlying the ankle bones (Fig. 9-11). These lesions probably arise from a combination of minor trauma, local infection, and circulatory compromise by vaso-occlusion. These ulcers are often extremely refractory to treatment.

Ocular

Sickle cell disease is the leading cause of monocular blindness among people of African ancestry. The retina can be damaged by a variety of events including arterial occlusion, hemorrhage, and proliferation of blood vessels in response to ischemia. Figure 9-12 shows the retina of a patient who sustained a localized hemorrhage, the resolution of which led to the accumulation of hemosiderin-laden macrophages. Proliferative retinopathy is the most commonly encountered ocular complication, surprisingly affecting patients with Hb SC disease and Hb S/β⁺-thalassemia about twice as often as those with Hb SS and Hb S/β⁰-thalassemia.

SUPPORTIVE

Pain Crises

The management of acute pain episodes remains a very challenging problem. The mainstays of treatment have changed very little in the last 50 years. Nearly all patients experiencing severe pain should receive nasal administration of oxygen to minimize hypoxia and deoxygenation-induced sickling. Close attention must be paid to fluid, electrolyte, and acid-base balances. Pain treatment requires considerable skill and experience, as it is important to give the patient the benefit of analgesia without depressing the central nervous system and suppressing ventilation. Avoidance of chronic narcotic dependency is another important objective.

Febrile Episodes

This must be evaluated and treated promptly and effectively because sickle cell patients are at increased risk of overwhelming sepsis. Nevertheless, with good judgment, at least half of febrile episodes can be managed on an outpatient basis. Because sickle cell patients are at higher risk of developing life-threatening bacterial infections, physicians should have a low threshold for the administration of antibiotics.

PREVENTION OF SICKLING

Induction of Hb F Synthesis

As mentioned earlier, the presence of even a minor fraction of Hb F in a Hb SS red cell greatly inhibits Hb polymerization. In an attempt to pharmacologically increase the production of Hb F, anti-leukemia agents were tested largely on an empirical basis. Although several agents were effective, hydroxyurea was shown to be the safest and most predictable. It blocks DNA synthesis by inhibiting the enzyme ribonucleotide reductase. Hydroxyurea has been given to thousands of patients with Hb SS disease. In most patients, it produces a significant, often marked, increase in the Hb F level. The mechanism of γ-globin induction is unclear. Patients treated with hydroxyurea develop increased hemoglobin levels, have less hemolysis, and show a marked reduction in irreversibly sickled cells in the peripheral blood (Fig. 9-1). As exemplified in Figure 9-13, hydroxyurea treatment reduces the frequency and severity of vaso-occlusive events.

Reduction in Intracellular Hemoglobin Concentration

Based on the realization that Hb S polymerization is markedly concentration-dependent, clinical trials are investigating the safety and efficacy of drugs that inhibit sickling-induced K⁺ efflux and Hb SS cell dehydration. The efficacy of one promising agent has been demonstrated in transgenic sickle cell mice and recently in a few patients.

Stem Cell Transplantation

Hematopoietic stem cell transplantation is the only curative therapy available for patients with sickle cell anemia. However, as explained in detail in Chapter 26, this formidable therapy poses serious risks of graft rejection, graft-versus-host disease, and life-threatening infections. A number of complex independent factors need to be carefully weighed in making a decision as to whether the patient is an appropriate candidate for stem cell transplantation. Thus far, stem cell transplantation has been restricted to young Hb SS adults with histories of stroke, acute chest syndrome, or frequent acute pain crises. It is anticipated that during the next decade advances in stem cell transplantation will result in a significant reduction in morbidity and mortality, thereby making this therapy more widely available to sickle cell patients.

Because of improvements in therapy, morbidity and mortality among sickle cell patients have decreased markedly in the last two decades. A major contributor has been the use of prophylactic penicillin, which has been effective in preventing pneumococcal and meningococcal sepsis. It is too early to know whether hydroxyurea therapy will have a positive impact on life expectancy. It is hoped that in the future safe and effective gene therapy can be developed, perhaps using recent technology that allows point mutations to be corrected more efficiently and precisely. This challenging goal is commanding a worldwide research effort.