Chapter 49: Disorders of Hemoglobin Structure: Sickle Cell Anemia and Related Abnormalities

The red protein hemoglobin (Hb) serves to transport oxygen from the lungs to the tissues and carbon dioxide (CO₂) from the tissues to the lungs. Hb also binds the physiologically important nitric oxide (NO) molecule. The protein has evolved to perform its gas transport functions in a highly efficient manner. The oxygen affinity of Hb permits nearly complete saturation with oxygen in the lungs, as well as efficient oxygen unloading in the tissues because of its sigmoid oxygen dissociation curve. This curve results from the fact that Hb is a four-subunit, allosteric molecule; its conformation, and hence the oxygen affinity, changes as each successive molecule of oxygen is bound. Hb also plays an important role in acid–base balance: deoxyhemoglobin binds protons and oxyhemoglobin releases protons. Regulation of the oxygen dissociation curve to meet the needs of the body is remarkable. Hypoxic tissues become acidotic acutely, and the protons released produce a shift in the oxygen dissociation curve that enables more oxygen to be delivered to the tissue. However, longer-term acidosis or alkalosis (as occurs at high altitudes) is counteracted by modulation of red cell 2,3-bisphosphoglycerate (2,3-BPG), serving to decrease hemoglobin–oxygen affinity (Chap. 47).

Normal mammalian Hbs contain two pairs of related polypeptide chains: one chain of each pair is α or α-like and the other is non-α (β, γ, or δ). The α-chains of all human Hbs encountered after early embryogenesis are the same. The non-α chains include the β-chain of normal adult Hb (α₂β₂), the γ-chain of fetal Hb (α₂γ₂), and the δ-chain of the minor adult Hb (HbA₂ [α₂δ₂]), which accounts for 2.5 percent of the Hb of normal adults. Chapter 48 discusses the regulation of production of the globin chains.

Certain residues in the amino acid sequence of each polypeptide chain appear to be critical to stability and function. Such residues are usually the same (invariant) in α or β chains. The NH₂-terminal valines of the β chains are important in 2,3-BPG interactions. The C-terminal residues are important in the salt bridges that characterize the unliganded molecules. Areas of contact between chains and between heme and globin tend to contain invariant residues. The non-α (β, γ, δ, or ε) chains are all 146 amino acids in length. The γ-chain of fetal Hb (HbF) differs from the β-chain by 39 residues. The γ genes are duplicated: one codes for glycine (^Gγ) and the other for alanine (^Aγ)⁷ at residue 136, giving rise to two kinds of γ chains. In addition, a common polymorphism, the substitution of threonine for isoleucine, is frequently found at residue 75 of the ^Aγ-chain.

Approximately 75 percent of the amino acids in α and β chains are in a helical arrangement. All Hbs studied have a similar helical content (Fig. 49–1A). Eight helical areas, lettered A to H, occur in the β chains. Hb nomenclature specifies that amino acids within helices are designated by the amino acid number and the helix letter, whereas amino acids between helices bear the number of the amino acid and the letters of the two helices. Thus, residue EF3 is the third residue of the segment connecting the E and F helices, whereas residue F8 is the eighth residue of the F helix. Alignment according to helical designation makes homology evident: Residue F8 is the proximal heme linked histidine, and the histidine on the distal side of the heme is E7.

Figure 49–1B show the tertiary structure of the α and β chains. The prosthetic group of Hb is heme (ferroprotoporphyrin IX); Fig. 49–2A shows its structure. The heme group is located in a crevice between the E and F helices in each chain (Fig. 49–2B). The highly polar propionate side chains of the heme are on the surface of the molecule and are ionized at physiologic pH. The rest of the heme is inside the molecule, surrounded by nonpolar residues except for two histidines. The iron atom is linked by a coordinate bond to the imidazole nitrogen (N) of histidine F8. The E7 distal histidine, on the other side of the heme plane, is not bonded to the iron atom, but is very close to the ligand-binding site.

The sigmoid oxygen dissociation curve is a function of the change of the conformation of the molecule from the liganded to the unliganded state (Table 49–1). In the deoxy state, the Hb tetramer is held together by intersubunit salt bonds (Fig. 49–3) and intersubunit hydrophobic contacts (see Fig. 49–1B), in addition to a certain number of hydrogen bonds. In deoxyhemoglobin, 2,3-BPG is situated in the central cavity between the two β chains (see Fig. 49–1B). The change in conformation of the Hb molecule is brought about by a complex, coordinated series of changes in the structure of the molecule as heme binds oxygen. The oxygen dissociation curve can be linearized by a transformation known as the Hill plot:

where K is an empiric overall constant without physicochemical basis. The slope n is taken as a convenient measure of cooperativity. Values of n in noninteracting Hbs that exhibit hyperbolic, not sigmoid, oxygen dissociation curves (e.g., myoglobin) are approximately 1. In a normal tetrameric Hb with four oxygen-reactive sites, the maximum value for n is 4.0; however, n values of 2.7 to 3.0 are found in normal Hb.

The point at which the Hb is one-half saturated with oxygen (P₅₀) is the usual measurement of oxygen affinity. It depends upon pH (the Bohr effect), temperature, and 2,3-BPG concentration. In common practice, P₅₀ is standardized at 37°C and pH 7.20. P₅₀ of freshly drawn blood is approximately 26.7 torr under standard conditions, but the partial pressure of oxygen (P_O2) of Hb from which 2,3-BPG has been removed is only approximately 13 torr. Although fetal and newborn red cells have 2,3-BPG levels similar to those of adults, their oxygen dissociation curve is left shifted (increased oxygen affinity) with a P₅₀ of approximately 23 torr because HbF does not react as strongly with 2,3-BPG as does HbA.

Following the molecular characterization of HbS by Ingram and colleagues in 1956, there has been a rapid and exponential increase in the number of variant or “abnormal” Hbs.¹ This number now exceeds 1000. A detailed description of variant Hbs, their chemical and functional properties, and population distribution can be found on the Globin Gene Server website (http://globin.cse.psu.edu/). Initially, newly described variants were designated by letters of the alphabet (e.g., HbC, HbD, HbE, HbJ). When the letters of the alphabet were exhausted, the practice of naming the variant Hbs after the geographic location where they were first described was adapted (e.g., Hb_Koln, Hb_Zurich). Variants with electrophoretic or functional properties similar to previously described abnormal Hbs were designated with the letter and the geographic location (e.g., HbD_Punjab, HbE_Saskatoon, HbM_{Hyde Park}). Some alphabetic designations were also used to indicate electrophoretic properties of certain variants; for example, there are a number of HbDs (D_Punjab, D_Iran, D_Ibadan). All of these variants share the electrophoretic properties of HbS-like mobility on alkaline (cellulose acetate) electrophoresis, whereas they move with HbA at acidic pH (citrate agar electrophoresis). Similarly, HbEs have HbC-like mobility on alkaline electrophoresis and move with HbA on citrate agar electrophoresis.

The vast majority of Hb variants arise as a result of single nucleotide mutations, leading to an amino acid change in either α-, β-, δ-, or γ-globin subunits of the Hb tetramer resulting in variants of HbA (α or β), HbA2 (δ), or HbF (γ). Other mechanisms include small deletions or insertions, elongated chains, and fusions (for a detailed description of Hb variants and associated clinical syndromes, see “Other Abnormal Hemoglobins” below.

The coinheritance of HbS with some other variant Hbs or β-thalassemia mutations results in a number of sickling syndromes. In the United States, the most common sickling disorder is homozygous HbS (HbSS, sickle cell anemia), which is now commonly referred to as sickle cell disease (SCD). This is followed by sickle cell-HbC disease (HbSC), sickle cell–β⁺-thalassemia (HbS–β⁺-thalassemia), and sickle cell–β⁰-thalassemia (HbS–β⁰-thalassemia). Other rarer forms include HbSD_Punjab, HbSO_Arab, HbS_Lepore, and HbSE diseases. Coinheritance of a large number of β-chain variants with HbS does not result in a symptomatic sickling disorder; rather, they are clinically and hematologically indistinguishable from sickle cell trait (HbAS).

HbC is found in 17 to 28 percent of West Africans, particularly east of the Niger River in the vicinity of North Ghana. The selective factors that account for this high prevalence are unknown at present, but HbC probably confers some resistance to infection with malaria. The prevalence of HbC among Americans of African descent is 2 to 3 percent. Sporadic cases also have been reported in other populations, including Italians and Afrikaners.

HbD_Punjab, which is now recognized to be identical with HbD_{Los Angeles} because both have the structure α₂β₂121 Glu→Gln, also interacts with HbS in forming aggregates in the deoxy conformation. HbD has been found in many parts of the world, including Africa, northern Europe, and India.

HbE is so prevalent that it may be the most common abnormal Hb or second in prevalence only to HbS. HbE is found principally in Burma, Thailand, Laos, Cambodia, Malaysia, and Indonesia. In some areas, HbE is found with a carrier rate of 30 percent. On the other hand, it is not prevalent among Chinese. Studies of restriction length polymorphisms in the β-globin cluster indicate the HbE mutation has arisen several times independently. It, too, probably confers some resistance to infection with malaria.

DEFINITION AND HISTORY

The first case of SCD, reported in 1910, was that of a dental student from Grenada, Walter Clement-Noel, studying in Chicago. Dr. James Herrick and his intern, Dr. Ernest Irons, were in charge of Mr. Noel’s care between 1904 and 1907, during which time he had several bouts of fever and cough and a history of leg ulcers, jaundice, and exercise intolerance. Herrick and Irons made astute clinical observations and prepared blood films and photomicrographs of nucleated red blood cells and of red cells having a “slender sickle shape” (Fig. 49–4).² During the next decade, two more cases of this unusual anemia were reported. In 1915, Cook and Meyer raised the question of a genetic basis for the disorder based on the family history of the third reported case. In 1917, Victor Emmel used in vitro culture to show that sickle red cells represented a physical alteration of morphologically normally appearing red cells and were not released from the marrow as sickle cells.³ He also demonstrated that morphologically normal red cells of the father of a patient became sickle shaped after in vitro culture. Vernon Mason, who reported the fourth case in 1922, coined the term sickle cell anemia after observing the similarities between all the cases reported up to that time. In 1923, Sydenstricker and Huck noted “latent-sicklers” among relatives of the diagnosed patients, confirming and expanding on Emmel’s finding. In 1927, Hahn and Gillespie showed that sickling was related to low oxygen tension and low pH. In 1933, Diggs distinguished the difference of symptomatic cases called sickle cell anemia, from asymptomatic cases that were termed sickle cell trait, and he found that approximately 8 percent of Americans of African descent had the sickle cell trait.⁴

Irving Sherman, while a medical student at Johns Hopkins, showed that sickled red cells were birefringent under a polarizing microscope and that this finding was reversible with oxygenation of the cells. This observation ultimately led Linus Pauling to study sickle Hb after being advised of this property of sickle cell by William Castle, a noted research hematologist. Indeed, in 1949, Pauling and his colleagues demonstrated electrophoretic differences between Hbs from normal, sickle cell trait, and sickle cell anemia subjects and hypothesized that there must be chemical differences, thus establishing sickle cell anemia as the first molecular disease described. In the late 1950s, Hunt and Ingram sequenced the globin peptide and linked the abnormality to a change in the amino acid composition of the β-globin chain (replacement of glutamic acid by valine at residue 6). In 1977, Marotta and coworkers showed that the corresponding change in codon 6 of the β-globin gene was GAG→GTG. The discovery of a variant fragment in HbS versus HbA during restriction endonuclease mapping of amniotic fluid cells by Y. W. Kan paved the way for antenatal diagnosis of SCD and opened the way for modern genetics using recombinant DNA technology.⁵

The history of sickle cell anemia serves as an inspiring reminder of the power of clinical and laboratory observations, and in an era of mechanistic basic science research, serves to highlight the importance of bedside to bench and bench to bedside research integration.^6,7,8,9

EPIDEMIOLOGY

The observation that sickle cell trait may have a survival advantage against some environmental factors was first suggested by Dr. Alan Raper in East Africa in 1949. Drs. Mackey and Vivarelli suggested that the environmental influence might be malaria. It was subsequently noted that blood from sickle cell trait persons contained less malarial parasites and that the sickle trait conferred some protection against malaria in early childhood. Data suggest that sickle trait is maximally protective against severe malaria as opposed to asymptomatic parasitemia or mild disease.¹⁰ The mechanism of such a protection has been the matter of much debate. Plausible mechanisms include selective sickling of parasitized red blood cells, resulting in more effective removal by the monocyte-macrophage system, and inhibitory effect on parasite growth by increased red cell potassium loss, decreased red cell pH, and increased endothelial adherence of parasitized sickle red cells.

Thus, the prevalence of sickle cell anemia closely mirrors the worldwide distribution of falciparum malaria; however, as a result of migration of peoples to the industrialized Western countries, SCD has become more prevalent in areas where malaria is not endemic.

The World Health Organization estimated in 2006 that 5 percent of the world population carries a gene for a hemoglobinopathy. Sickle cell anemia is highly prevalent in sub-Saharan and equatorial Africa with lesser but significant prevalence in the Middle East, India, and the Mediterranean region. Incidence of SCD in sub-Saharan African countries ranges between 1 and 2 percent, which translates into approximately 500,000 cases per year. In the Jamaican cohort study, newborn screening in 100,000 consecutive vaginal deliveries resulted in the finding of sickle cell trait in 10 percent of newborns.¹¹

In the United States, the Centers for Disease Control and Prevention estimates that sickle cell anemia is present in 1 in 500 livebirths among Americans of African descent; 1 in 12 American of African descent have the trait, and approximately 100,000 Americans largely of African descent live with the disease. In Americans of Hispanic descent, the rate of SCD is 1 in 36,000 livebirths. Accurate population statistics of SCD are difficult to obtain in the United States because of a lack of standardized data collection and central reporting.¹²

As of 2002, in the United States, more than one billion dollars are spent per year on hospitalizations for SCD.¹³ Data from a single state Medicaid program estimated a lifetime cost of care of $500,000 per patient with SCD. In this patient population, cost increased with increasing age, including cost of non-SCD health issues. The majority of the costs were for inpatient healthcare utilization.¹⁴

Previously, speculation existed as to whether the sickle mutation arose once and gained worldwide distribution or whether the mutation had arisen independently in different regions of the world. The nonrandom association of restriction endonuclease polymorphisms in the β-globin cluster define the β-globin haplotype. The β-globin gene cluster yields five distinct haplotypes associated with sickle cell mutations (Chap. 9).^15,16,17 Four of the five patterns occur in Africa and are designated as the Senegal, Benin, Bantu, and Cameroon haplotypes, whereas the fifth arose on the Indian subcontinent.¹⁸ These findings indicate that the sickle mutation arose independently at five different times.

PATHOPHYSIOLOGY

The sine qua non of sickle cell anemia is a Glu→Val substitution in the sixth amino acid of the β-globin gene. However, the pathophysiologic processes that result in the clinical phenotype extend beyond the red cell (Fig. 49–5). There is marked clinical heterogeneity from one patient to another and in the same patient over time. The heterogeneity for the same genotypic abnormality therefore implies that a multitude of other factors must contribute to the pathology of sickle cell anemia. The pathology is now far removed from the simplistic theory of hypoxia-induced microvascular occlusion. Sickle cell anemia is a chronic inflammatory state punctuated by acute increase in inflammation wherein the endothelium, neutrophils and monocytes, platelets, coagulation pathways, several plasma proteins, adhesion molecules, and derangements in NO metabolism interact in concert with the abnormality in Hb polymerization described several decades ago (Fig. 49–6). Abnormal adenosine signaling and activation of invariant natural killer T (iNKT) cells have been implicated in disease pathophysiology. Added to that are the complex differences in tissue-specific vascular beds and differences in various parts of the vasculature in the same organ. Also, variation in several genes other than the β-globin gene that modify the milieu in which organ damage occurs may play a role.

The pathophysiology of sickle cell anemia is described in separate sections; however, because no single, dominant pathway explains the multitude of manifestations, no single therapeutic modality serves to abrogate all of the pathology. Most experiments are in isolation in animal models or relatively simplistic experimental conditions with few in vivo studies in humans and thus do not replicate the complexity of this disorder.

Hemoglobin Polymerization

Aggregation of deoxy HbS molecules into polymers occurs when aggregates reach a thermodynamically critical size. This process is termed homogenous nucleation, and the smallest aggregate formed that favors polymer growth is called the critical nucleus.^{19,20,21,22,23,24} Addition of subsequent deoxy HbS molecules to already formed polymers is termed heterogenous nucleation, which results in polymer branching. Polymer growth is, therefore, an exponential process wherein there is a delay time between presence of deoxy HbS molecules and polymer formation. This delay time is inversely proportional to the concentration of HbS molecules. Polymer formation alters the rheologic properties of the red cell.

The quaternary structure of oxy HbS cannot maintain axial and lateral hydrophobic contacts unlike that in the deoxygenated state, thus explaining the unsickling phenomenon upon reoxygenation.^25,26,27,28 The sickling process that is initially reversible with oxygenation of deoxy HbS eventually leads to the formation of sickle-shaped red cells that fail to return to their normal discoid shape with oxygenation because of membrane damage imparted by repeated cycles of sickling and unsickling in the circulation. These cells are then termed irreversibly sickled cells. The rate and extent of polymerization is dependent on several factors, including intracellular Hb concentration, presence of Hbs other than HbS, blood oxygen saturation, pH, temperature, and 2,3-BPG levels.²⁹ Microvascular occlusion by sickle red cells containing polymers is favored by prolonged transit times through the microcirculation, rapid deoxygenation and increased numbers of dense sickle red cells that contain polymers even at oxygen saturation levels found in the arterial circulation.^29,30,31,32 Arguments against HbS polymerization as the major determinant of sickle cell pathophysiology include lack of clinically significant events despite constant sickling of red cells, the association of neutrophilia with vasoocclusive episodes (VOEs), and clinical features that imply macrovascular rather than microvascular perturbation, for example, large-vessel stroke.³³

Cellular Dehydration

Membrane injury in HbSS red cells results in impaired cation homeostasis with decreased ability to maintain intracellular potassium concentrations. The calcium-activated potassium (K⁺) channel (Gardos channel), potassium-chloride cotransport channel, and a sickling-induced nonselective cation leak pathway have been implicated in sickle red cell dehydration. The net result is loss of intracellular potassium and water resulting in cellular dehydration.^{34,35,36,37,38,39} This change effectively increases the red cell Hb concentration, favoring sickling.

Hemolysis and Nitric Oxide Scavenging

NO is a key component of the vascular endothelium that has vasodilatory, antiinflammatory, and antiplatelet properties.⁴⁰ NO is a soluble gas synthesized from L-arginine by endothelial nitric oxide synthase (eNOS).⁴¹ Red cell L-arginase released as a consequence of sickle red cell hemolysis converts arginine to ornithine, thereby limiting L-arginine availability for NO synthesis. Decreased NO production because of elevated levels of endogenous nitric oxide synthase (NOS) inhibitors, especially asymmetric dimethylarginine (ADMA) and reduced L-arginine, have been documented in SCD especially during VOE.^{42,43,44,45,46} Reduced plasma arginine levels and elevated ADMA levels also result in NOS coupling causing production of reactive oxygen species rather than NO.^47,48 Chronic hemolysis with release of plasma free Hb results in scavenging of NO with consequent endothelial dysfunction, which may favor sickle cell adherence.^49,50

Abnormal Cell Adhesiveness

Seminal work by several groups showed that sickle red cells adhere to stimulated endothelium unlike their normal counterparts.^51,52 Newly released red cells, called reticulocytes, express high levels of adhesion molecules, integrin α₄β₁, and CD36, and are more adherent than dense sickle red cells.^53,54 Increased endothelial reticulocyte adhesion as compared to dense red cell adhesion is thought to be secondary to deformable red cells adhering to the endothelium behind which the dense red cells are trapped, leading to microvascular occlusion.²⁹ Other molecules involved in sickle red cell-endothelium interactions include vascular cell adhesion molecule (VCAM)-1, integrin α_Vβ₃, P-selectin, P-selectin glycoprotein ligand (PSGL)-1, E-selectin, Lutheran blood group antigen, and thrombospondin.^{55,56,57,58,59,60} The site of adhesion is purported to be the postcapillary venule at which site sickle red cells appear to interact with white cells adherent to the endothelium rather than engaging the endothelium directly.³¹

Neutrophilia is an adverse prognostic factor in sickle cell anemia. Because of their larger size, adherent leukocytes cause a greater decrease in vessel caliber than red cells. Diapedesis occurs in postcapillary venules, a site of vasoocclusion in sickle cell anemia.^31,61,62,63 Neutrophil integrin α_Mβ₂ microdomains capture sickle red cells causing vascular occlusion in sickle cell mouse models. Monocytes are also highly activated in sickle cell anemia, and they promote increased endothelial activation by increased production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β.⁶⁰ Expression of leukocyte adhesion molecules, L-selectin, and integrin α_Mβ₂, are associated with a severe clinical phenotype.^61,64

Inflammation and Chronic Vasculopathy

Sickle cell anemia is characterized by chronic leukocytosis, abnormal activation of neutrophils and monocytes, and an increase in several proinflammatory mediators including TNF-α, IL-6, and IL-1β. Several adhesion molecules are upregulated, including VCAM, selectins, integrins, the acute phase reactants C-reactive protein, secretory phospholipase A₂ (sPLA₂), and coagulation factors are activated.^64–76 Placenta growth factor (PIGF) released from erythrocytes activates monocytes to produce inflammatory cytokines and upregulates endothelin-1 signaling via the endothelin B receptor. Endothelin-1 is a potent vasoconstrictor and upregulation is associated with adverse outcomes in SCD. Placental growth factor has independently been shown to be correlated with disease severity as well.^77,78 Hemin has been demonstrated to activate PIGF in mice via the erythroid Kruppel-like factor; consequently, PIGF may play an important role in the pathophysiology of iron overload as well.⁷⁹ It is an open question whether inflammation is caused by abnormally adhesive red cells to the vascular endothelium or whether inflammation causes abnormal red cell adhesiveness. It is likely both occur, given that red cell adhesiveness incites endothelial activity, and infection-induced inflammation precipitates clinically significant vascular events in patients.

The vascular beds in sickle cell anemia display changes akin to atherosclerotic vascular disease: large vessel intimal hyperplasia and smooth muscle proliferation.^80,81 However, the characteristic lipid laden plaques of atherosclerotic vascular disease are not present.⁶⁴

Ischemia–Reperfusion Injury

Akin to other disease states, such as myocardial infarction, resolution of vasoocclusion results in reperfusion injury characterized by increased oxygen free radical formation via activation of xanthine oxidase, generation of oxidant stress, lipid peroxidation, upregulation of cellular adhesion molecules, and nuclear factor-κB, a key player in the inflammatory process.^64,82,83 iNKT cells propagate the inflammatory cascade in ischemia reperfusion injury and are increased and activated in patients with SCD. Agonists to adenosine 2A receptor (A_2AR) on iNKT cells downregulate their activation and attenuate inflammation in mouse models of SCD.⁸⁴

Activation of the Coagulation System

The initiator of coagulation, tissue factor (TF), is elevated in patients with sickle cell anemia.^{40,74,85,86,87} Microparticles (MPs) expressing TF derived from monocytes, macrophages, neutrophils and endothelial cells have been described in SCD.^58,68,74,88 Conflicting results exist in the literature on the presence and contribution of TF bearing MPs. There is a lack of correlation between TF bearing MPs and procoagulant activity in SCD. Erythrocyte and platelet MPs are TF-negative and are the major component of MPs in SCD. Activation of the intrinsic pathway of coagulation by TF-negative, red cell, and platelet MPs through a phosphatidylserine-dependent mechanism appears to be the major contributor of MP-dependent coagulation activation in SCD. Perivascular TF interaction with plasma coagulation factors made possible by increased vascular permeability and phosphatidylserine exposure on the surface of red cells secondary to repeated cycles of sickling provide an impetus for the coagulation process.⁸⁹ Heightened thrombin generation, platelet activation, and decreased protein C and S levels favor a procoagulant state.^69,90,91 Increased plasma levels of D-dimers, thrombin–antithrombin complexes, prothrombin fragment 1.2, and plasmin–antiplasmin complexes are indicative of increased thrombin-mediated coagulation with subsequent fibrinolysis.⁹² Plasma from sickle cell patients contains increased ultralarge von Willebrand factor multimers as a result of increased endothelial cell secretion and impaired cleavage by ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif member 13).⁹³

Adenosine Signaling

Cellular stress leads to the degradation of adenine nucleotides, resulting in the generation of adenosine. Adenosine homeostasis is maintained by two enzymes: adenosine kinase, which phosphorylates adenosine to adenosine monophosphate and adenosine deaminase, which converts adenosine to inosine. Adenosine signals through four different receptors that have differing functions. Signaling via the A_2AR expressed on most leukocyte and platelets results in an antiinflammatory effect; however, signaling via the A_2BR was shown to cause priapism in SCD mice via hypoxia-inducible factor (HIF)-1–mediated decrease of phosphodiesterase 5. Signaling via A_2BR also leads to increased 2,3-BPG in red cells causing decreased oxygen binding affinity of Hb, which promotes sickling. Pegylated adenosine deaminase treatment of sickle mice resulted in decreased hemolysis and hypoxia reoxygenation injury.^94,95

SICKLE CELL TRAIT

Inheritance of only one HbS allele is termed sickle cell trait (HbAS). An estimated 300 million people carry the trait worldwide.⁹⁶ The percentage of HbA is always higher (~60 percent) than HbS (~40 percent) in sickle cell trait.

HbAS is considered a generally asymptomatic state with HbA in the cell preventing sickling except in the most unusual circumstances. HbAS cells sickle at O₂ tension of approximately 15 torr.⁹⁷

Plasma myeloperoxidase and red cell sickling have been reported to increase during exercise with fluid restriction in HbAS subjects.⁹⁸ Plasma levels of VCAM-1 are higher in HbAS subjects and remain elevated following exercise compared to normal controls or HbAS with concomitant α-thalassemia, which is suggestive of subtle microcirculatory dysfunction in this population.⁹⁹ Skeletal muscle capillary structures are different in HbAS subjects compared to controls. There is a 30-fold increased risk of sudden death in black army recruits with HbAS.¹⁰⁰ Although controversial, in 2009 the National Collegiate Athletic Association recommended mandatory testing for HbAS for all its student athletes.¹⁰¹

Renal abnormalities are among the most common manifestations of HbAS. Anoxia, hyperosmolarity, and low pH of the renal medulla predisposes to sickling. Microscopic or gross hematuria from renal papillary necrosis is usually painless. Renal neoplasm or stones should be excluded in those with persistent gross hematuria. Isosthenuria may be seen in and may contribute to exercise induced rhabdomyolysis and sudden death.¹⁰² Renal medullary carcinoma is a rare but serious complication of HbAS. Risk of urinary tract infection is higher in females with HbAS, especially during pregnancy. End-stage renal disease occurs at an earlier age for HbAS patients with polycystic kidney disease and HbAS may contribute to erythropoietin resistance.¹⁰³

Splenic infarction occurs under extreme environmental conditions in persons with HbAS; most resolve spontaneously.^104,105 Caution and immediate intervention is also warranted in those HbAS individuals who develop traumatic hyphema.¹⁰⁶ The risk of venous thromboembolism is increased twofold in HbAS subjects compared to those without the trait. The risk appears to be greater for pulmonary embolism than for deep vein thrombosis.^101,105 HbAS patients do not have increased perioperative morbidity or mortality. The life span of patients with HbAS is normal.¹⁰⁷

LABORATORY FEATURES

Sickle cell anemia is characterized by a laboratory profile of evidence of hemolytic anemia with increases in lactate dehydrogenase (LDH), indirect bilirubin, reticulocyte count, and a decrease in serum haptoglobin. Anemia is usually normochromic, normocytic with a steady-state Hb level between 5 and 11 g/dL.^1,108 The red cell density is increased with a normal mean cell Hb concentration (MCHC).¹⁰⁹ Serum erythropoietin level is decreased relative to the degree of anemia.¹¹⁰ Elevated neutrophil and platelet levels are observed even in asymptomatic patients reflective of persistent low-grade inflammation.^111,112,113

Plasma tocopherol and zinc levels are low.^114,115,116 Serum ferritin is increased, especially in iron overloaded patients. Elevated brain natriuretic peptide is seen in patients with pulmonary hypertension (PH) and congestive heart failure. Morphologically, classic sickle red cells are seen on blood film examination, and the marrow shows erythroid hyperplasia.

Sickle cell anemia can be accurately diagnosed with high-performance liquid chromatography (HPLC) and isoelectric focusing.¹¹⁷ Rapid methods, such as solubility testing and sickling of red cells using sodium metabisulfite, are less-reliable tests.¹¹⁸ Polymerase chain reaction is the method of choice for prenatal diagnosis.¹¹⁹ No HbA is found in patients with HbSS, HbSC, or HbSβ⁰ diseases. Varying amounts of HbA (depending on the severity of the β-thalassemia mutation) are found in HbS–β⁺-thalassemia subjects.

COURSE AND PROGNOSIS

Mortality from SCD in the United States has declined since 1968, coinciding with the introduction of pneumococcal polyvalent conjugate 7 (PVC7) vaccine. Comparison of mortality rates between 1979 to 1998 and 1999 to 2009 showed a 61 percent decrease in infants, 67 percent in children ages 1 to 4 years, and 35 percent decrease in children ages 5 to 19 years. Transition from pediatric to adult medical care showed an increased mortality trend with similar rises in rates during the decades of comparison.¹²⁰ Average life expectancy of patients with HbSS disease in the United States is 42 and 48 years for males and females, respectively.¹²¹ In Jamaica, the population has a median survival of 53 years and 58 years for men and women, respectively, with 44 percent of individuals born prior to 1943 still living as of 2009.¹²² As the sickle cell population ages, causes of death change from an infectious etiology to those related to end-organ damage, such as renal failure.

CLINICAL FEATURES AND MANAGEMENT

The reader is referred to the National Institutes of Health, National Heart, Lung and Blood Institute’s guidelines from 2002 for an extensive review on the topic; revised guidelines were released in the fall of 2014 at http://www.nhlbi.nih.gov/health-pro/guidelines/sickle-cell-disease-guidelines/.¹²³ General approaches to SCD management and pain management are described separately (Table 49–2).

Sickle Cell Crises

The typical course for a sickle cell patient is that of periods of relatively normal functioning despite chronic anemia and ongoing vasoocclusion, punctuated by periods of increased pain, and serial changes in various laboratory parameters that is termed “a sickle cell crisis.” Crises have typically been classified as VOEs, aplastic crises, sequestration crises, and hyperhemolytic crises.

Vasoocclusive Crises The hallmark of SCD is the VOE. It is the most common clinical manifestation but occurs with varying frequency in different individuals. It results from increasing vasoocclusion causing tissue hypoxia, which manifests as pain. Vasoocclusion may affect any tissue, but patients typically have pain in the chest, lower back, and extremities. Abdominal pain may mimic acute abdomen from other causes. Different patients display different patterns of painful sites during a VOE, but each patient’s recurrences usually mimic the same pattern of pain. Fever is often present, even in the absence of infection. Episodes may be precipitated by dehydration, infection, and cold weather although in about most cases no precipitating factor is found.¹²⁴

Figure 49–7 illustrates the phases of VOEs.¹²⁵ Crises requiring readmission within 1 week occur in approximately 20 percent of patients after hospital discharge.¹²⁵

The characterization of crisis phases has implications for clinical research, especially in pain management, wherein interventions early in the course of a crisis could result in better outcomes for patients.

Aplastic Crises Aplastic crises in sickle cell anemia result when there is a marked reduction in red cell production in the face of ongoing hemolysis, causing an acute, severe drop in Hb level. The characteristic laboratory finding is a reticulocyte count less than 1 percent. The most common causative agent is parvovirus B19, which attaches to the P antigen receptor on erythroid progenitor cells, causing a temporary arrest in red cell production (Chap. 36). Recurrent aplastic crises by parvovirus B19 are rare because of the development of protective antibodies. Other rare complications associated with parvovirus B19 include acute splenic and/or hepatic sequestration, acute chest syndrome, marrow necrosis, and renal dysfunction. Patients usually recover within 2 weeks; however, those with severe symptomatic anemia need red cell transfusion. Siblings of SCD patients with parvovirus infections should be monitored closely for aplastic crisis given high secondary attack rates (>50 percent). Patients need to be isolated from pregnant individuals given increased risk of hydrops fetalis with parvovirus B19 infection.¹²⁶

Sequestration Crises This type of crisis is characterized by sudden, massive pooling of red cells, typically in the spleen and less commonly in the liver.¹²⁷ Splenic sequestration is typically seen in children (younger than 5 years of age) prior to autoinfarction of the spleen, but can be seen in adults with HbSC disease or HbS–β-thalassemia with persisting splenomegaly.^128,129,130 A minor sequestration episode is usually accompanied by a Hb of more than 7 g/dL, and a major episode usually is one in which the Hb is less than 7 g/dL or the Hb has decreased by 3 g/dL from baseline.¹³¹

Acute splenic and hepatic sequestration crises can present with rapidly enlarging spleen or liver, pain, hypoxemia, and hypovolemic shock. Treatment consists of red cell transfusion. Transfusion carries the risk of hyperviscosity when the sequestration crisis resolves and the sequestered red cells are returned to the general circulation. Splenic sequestration crisis has a high rate of recurrence, especially in children. Splenectomy to prevent recurrence is debated in very young children. Some report chronic red cell exchange transfusion as a means of delaying splenectomy until the child is older while others did not see any benefit to this treatment. Patients younger than 2 years of age can be placed on chronic transfusion until they are older, at which time splenectomy should be considered. Splenectomy is recommended after the first episode of life-threatening splenic sequestration crisis or chronic hypersplenism. Partial splenectomy and emergency splenectomy during a crisis is not recommended. Parental education is important for early recognition of the problem so they can seek medical care promptly.¹²⁶

Hyperhemolytic Crisis The term hyperhemolytic crisis is used to describe the occurrence of episodes of accelerated rates of hemolysis characterized by decreased blood Hb, increasing reticulocytes, and other markers of hemolysis (hyperbilirubinemia, increased LDH). Hyperhemolysis can occur during resolution of a VOE, at which time irreversibly sickled and dense red cells are rapidly destroyed, as well as from an acute or delayed hemolytic transfusion reactions.^126,132

Pain Control

Patients with SCD have acute pain, chronic pain, or both. As a symptom, pain is often underrated in its intensity and undertreated by caregivers, especially inexperienced physicians. Patients are often perceived as drug-seekers or drug addicts, when in fact less than 10 percent of patients are addicted, a number comparable to other disease states. Unsatisfactory relief of pain drives patients to behaviors that appear to healthcare givers as signs of addiction—a state termed pseudoaddiction. A study comparing sickle cell anemia patients who use the emergency department frequently or infrequently found significant impairment in quality of life and increased markers of disease severity in those who use the emergency department frequently, dispelling the myth that frequent emergency department use indicates narcotic-addicted individuals when, in fact, they may have more severe disease.^{3,133,134,135,136,137} The landmark Pain in Sickle Cell Epidemiology Study revealed that adult SCD patients have pain at home approximately 55 percent of the time, which contrasts sharply to pain studies in children, who report at-home pain approximately 9 percent of the time.^138,139

Acute pain is managed with opioids, nonsteroidal antiinflammatory drugs, acetaminophen, or a combination of these medications. Immediate pain assessment and frequent reassessment with appropriate application of medications until pain relief is obtained is important. For adults and children weighing more than 50 kg, morphine can be started at a dose of 0.1 to 0.15 mg/kg. The hydromorphone dose should be 0.015 to 0.020 mg/kg intravenously. These are recommended doses for opioid-naïve patients and are at the lower end of the dosing range.^123,140,141 The use of meperidine has declined because of neurologic side effects, especially in patients with renal failure, who are at risk for the serotonin syndrome in conjunction with use of other medications.^142,143,144 However, the use of morphine is not benign and concerns of increased association of acute chest syndrome, dysphoria, and neuroexcitatory side effects have been raised.¹²⁵ Prior use of opioid therapy should be taken into consideration when deciding initial opioid doses as patients may be tolerant and require higher doses. Caution should be exercised with nonsteroidal antiinflammatory drugs and acetaminophen if there is renal or hepatic dysfunction. Patients with acute pain are better managed in a setting dedicated to sickle cell patients.¹⁴⁵ A multidisciplinary approach is needed for pain management, especially if chronic pain is present.^146,147 Opioid side effects should be anticipated and managed. Antidepressants, anticonvulsants, and clonidine can be used for neuropathic pain. Occasionally, severe, unrelenting pain may require red cell transfusion to decrease sickle Hb below 30 percent in the blood.¹⁴⁸

There is a paucity of data regarding optimal management of pain in SCD. A randomized trial of optimizing patient controlled analgesia strategy was closed because of poor accrual.¹⁴⁹ A trial looking at NO inhalation for treatment of VOE did not show improvement in pain.¹⁵⁰

Pulmonary Manifestations

Acute Chest Syndrome The acute chest syndrome (ACS) is a constellation of signs and symptoms in patients with SCD that includes a new infiltrate on chest radiograph defined by alveolar consolidation but not atelectasis, chest pain, fever, tachypnea, wheezing, or cough, and hypoxia (Fig. 49–8).¹⁵¹ However, respiratory findings on clinical examination in the absence of radiographic findings should trigger high suspicion for ACS and warrants close monitoring. ACS is the leading cause of mortality in patients with SCD.¹²¹ Etiology varies depending on age, with viral and bacterial infections dominating in the pediatric age group and fat embolization resulting from marrow necrosis during VOE dominating in adults.^152,153 Important pathogens include Chlamydia pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, parvovirus B19, respiratory syncytial virus, and influenza. Regardless of the triggering factor, the pathogenesis of ACS involves increased intrapulmonary sickling, intrapulmonary inflammation with increased microvascular permeability, and alveolar consolidation. ACS can rapidly evolve with bilateral infiltrates and consolidation leading to acute respiratory failure requiring intubation and ventilatory assistance.

Independent risk factors for respiratory failure are age older than 20 years, platelet count less than 20 × 10⁹/L, multilobar lung involvement, and a history of cardiac disease.¹⁵² Thrombocytopenia is an independent predictor of neurologic complications during hospitalization for ACS, which was seen in 22 percent of adult patients in the National Acute Chest Syndrome study.¹⁵⁴

The treatment of ACS includes oxygenation, incentive spirometry, adequate pain control to avoid chest splinting, antimicrobial therapy that always covers atypical bacteria and influenza when indicated, avoidance of overhydration, use of bronchodilators, and red cell transfusion to decrease intrapulmonary sickling.^{152,155,156,157,158,159,160} The use of glucocorticoids may attenuate the course of ACS; however, its use is not well established and readmission rates for VOE after ACS resolution are increased.¹⁵³ sPLA₂ has been recognized as a predictor of ACS; however, a clinical trial investigating early transfusion based on sPLA₂ elevation closed because of poor accrual. Hydroxyurea therapy should be offered to all patients with a history of ACS because it reduces the incidence by 50 percent in adults and 73 percent in children.¹⁶¹

Pulmonary Hypertension PH, defined by a resting mean pulmonary arterial pressure of 25 torr or higher on right-heart catheterization, is seen in 6 to 11 percent of SCD patients. An elevated tricuspid regurgitant velocity of 2.5 m/s has a positive predictive value of 25 percent for PH in SCD and is seen in one-third of these patients. PH, as defined by right-heart catheterization, elevated tricuspid regurgitant jet velocity of 2.5 m/s or higher, and a serum N-terminal pro–brain natriuretic peptide (NT-pro-BNP) level of 160 pg/mL or higher, confers an increased mortality risk.¹⁶²

Abnormalities in NO metabolism, hemolysis, and inflammation contribute to the pathophysiology of PH.¹⁶² Parenchymal lung disease from repeated episodes of ACS and thromboembolism are other causal factors.

Clinical symptoms of PH include fatigue, dizziness, and dyspnea on exertion, chest pain, and syncope. These may be unrecognized as being related to PH, as PH is often undiagnosed in patients with SCD.

PH should be treated following guidelines set for the treatment of primary PH unrelated to SCD. Two trials looking at bosentan (endothelin receptor antagonist) in SCD patients closed because of sponsor withdrawal. A trial of sildenafil was halted early because of increased incidence of VOE. Patients who have venous thromboembolism in the setting of PH should be considered for indefinite anticoagulation. Hydroxyurea should be offered to all patients with any of the risk factors for increased mortality described above.¹⁶²

Asthma, Abnormal Pulmonary Function Tests, and Airway Hyperreactivity Asthma is a common comorbidity with higher-than-average prevalence in patients with SCD and is associated with increased risk of ACS, VOE, stroke, and mortality. Airway hyperreactivity as evidenced by a positive bronchodilator response on pulmonary function testing, irrespective of baseline function, and in response to cold air or methacholine challenge, is seen in approximately two-thirds of SCD patients. Inflammation, hypoxemia, and increased oxidative stress associated with asthma may contribute to the vasculopathy of SCD.¹⁶³

Pulmonary function tests collected as part of the Cooperative Study of Sickle Cell Disease (CSSCD) revealed abnormalities in 90 percent of the 310 patients, with the majority having restrictive lung disease.

Asthma treatment follows general treatment guidelines as in the non-SCD populations.^164,165

Cardiac Manifestations

Anemia in SCD results in an elevated cardiac output secondary to an increased stroke volume with minimal increase in heart rate.^166,167 Clinical manifestations of a hyperdynamic circulation include a forceful precordial apical impulse, systolic and diastolic flow murmurs, and tachycardia that may increase during periods of increased hemodynamic stress. Diastolic left ventricular dysfunction may begin in early childhood and is an independent risk factor for death, with even greater risk of mortality in those having PH. Left ventricular hypertrophy is common and progressive with age; left ventricular dysfunction is a late event. Myocardial infarction is an underrecognized problem in SCD. Epicardial coronary artery disease is rare; microvascular ischemia is likely causative. Sudden cardiac death has been reported in 40 percent of patients in an autopsy series.^168,169,170 Previously sudden cardiac death was ascribed to narcotic overdose; currently, it is thought to be secondary to cardiopulmonary causes in the majority of cases. QTc prolongation, atrial and ventricular arrhythmias, nonspecific ST-T wave changes are common in SCD patients. Patients presenting with chest pain should have a thorough evaluation to rule out cardiac disease. Cardiac magnetic resonance may be a good modality to image microvascular flow and quantitate cardiac iron overload.^171,172 Blood pressure in patients with SCD is significantly lower than age-, sex-, and race-matched controls, partly secondary to anemia.¹⁷³ Relative hypertension is associated with end-organ damage. Diuretics may be used, keeping in mind that SCD patients have obligate hyposthenuria and are prone to dehydration, which can precipitate a VOE.

Central Nervous System

Originally thought to be a small vessel disease, stroke in SCD is a macrovascular phenomenon with devastating consequences that affects approximately 11 percent of patients younger than 20 years of age.^174,175 Risk is highest in the first decade of life followed by a second smaller peak after age 29 years. Ischemic stroke is most common in children and older adults, whereas hemorrhagic stroke predominates in the third decade of life.¹⁷⁵ Recurrent stroke is most common in the first 2 years following the primary event.¹⁷⁶ Silent infarcts, defined as an increased T2 signal abnormality on magnetic resonance imaging (MRI), begins in infancy and has a cumulative incidence of 37 percent by age 14 years. They occur in watershed areas of the brain, are not predicted by abnormal transcranial Doppler (TCD) velocity, and may progress despite chronic transfusion.^{177,178,179,180} There is evidence of neurocognitive decline in asymptomatic adults despite having normal brain imaging that is attributed to anemia and hypoxemia.¹⁵⁴

Cerebral blood flow is significantly increased in SCD because of chronic anemia and hypoxemia, but does not increase further in response to increased hypoxic stress, thereby predisposing to ischemia.^181,182 Stenosis of large vessels, especially of the circle of Willis, without the classic atherosclerotic plaque occurs in conjunction with a multitude of other factors, including chronic hemolysis, deranged NO metabolism and impaired vascular autoregulation, and can lead to stroke.¹⁸² Rare causes of cerebral vascular disease include fat embolization and venous sinus thrombosis. Moyamoya type fragile collaterals have been reported in more than one-fifth of patients with prior stroke, possibly leading to hemorrhagic stroke in later life.^{183,184,185,186,187,188}

Risk factors for ischemic stroke include transient ischemic attack, recent or recurrent ACS, nocturnal hypoxemia, silent infarcts, hypertension, elevated lactic dehydrogenase, and leukocytosis, whereas anemia, neutrophilia, the use of glucocorticoids, and recent transfusion are independent risk factors for hemorrhagic stroke, especially in children.^{175,189,190,191,192,193,194,195} Sickle cell genotypes other than HbSS carry a lower risk, as do patients with HbS–α-thalassemia.^175,196,197 The best predictor of stroke risk, however, is an increased blood flow velocity in major intracranial arteries on TCD ultrasonography.¹⁹⁷ Blood flow velocities less than 170 cm/s are considered normal. Velocities between 170 and 200 cm/s are termed conditional, and velocities of greater than 200 cm/s are considered high and are associated with a 10-fold increase in ischemic stroke in children 2 to 16 years of age.

There is an increased frequency of stroke among siblings of patients with SCD than would be expected by chance alone, raising the possibility of other modifier genes contributing to stroke risk.¹⁸³ The TNF (–308) G/A promoter polymorphism is associated with increased large-vessel stroke risk as is the IL-4–receptor gene 503 S/P variant, although it did not reach statistical significance. The clinical features of stroke in SCD encompass the classic findings of stroke in other disorders, including, but not limited to, hemiparesis, seizures, coma, paresthesias, headaches, and cranial nerve palsies. Neurocognitive deficits in IQ, memory, language, and executive function have been demonstrated.^154,198

Imaging approaches for acute stroke are the same as those for non-SCD patients and includes MRI and magnetic resonance angiography.

Prevention of Primary Stroke Based on the results from the Stroke Prevention in Sickle Cell Disease (STOP) Study, it is recommended that asymptomatic children with HbSS disease older than two years of age should be screened for stroke risk using TCD.¹⁹⁷ Those with high TCD velocities should be offered a chronic red cell transfusion program for primary stroke prevention. Repeat TCD screenings should be done every 3 to 12 months even in patients who have normal or conditional baseline velocities, because they can evolve into a higher-risk category. Despite obstacles to TCD screening, clinical practice changes based on the STOP study translated into declining stroke rates since 1991.^199,200

Prevention of Secondary Stroke Patients with SCD who present with a stroke and are not on chronic transfusion should be placed on a transfusion program to prevent secondary strokes. Exchange transfusion may be preferable to periodic red cell transfusion, not only to avoid iron overload, but also to further reduce stroke risk. In a retrospective study, children who received periodic transfusion had a fivefold higher relative risk of a recurrent stroke compared to those on an exchange transfusion regimen.²⁰¹ Despite chronic transfusions, patients may have a recurrent stroke, especially in patients with HbS greater than 30 percent.²⁰² Hydroxyurea was shown to decrease high and conditional TCD velocities in more than 90 percent of patients studied.²⁰³ However, a randomized trial comparing transfusions with iron chelation to hydroxyurea with phlebotomy showed a 10 percent stroke rate in the hydroxyurea arm, thus establishing transfusion as the preferred preventive strategy.²⁰⁴

Anticoagulation therapy has not been studied in patients with SCD and, therefore, no recommendations can be made. Treatment guidelines for intracranial hemorrhage are as those for non-SCD–related intracranial hemorrhage; role of transfusion is less clear in SCD especially when cause of intracranial hemorrhage is unclear. Patients with moyamoya disease who have a particularly poor outcome may benefit from revascularization using encephaloduroarteriosyangiosis.^205,206

Genitourinary Systems

Renal Failure Sickling of HbSS erythrocytes in the hypoxic, acidic, and hypertonic environment of the renal medulla, oxidative stress, increase in prostaglandins and endothelin-1 in the kidney, and abnormalities of the renin angiotensin system contribute to the pathophysiology of renal disease in SCD.²⁰⁷ The incidence of renal failure varies between 4 and 20 percent.^{208,209,210,211} Dehydration is the most common cause of acute renal failure in SCD. Isosthenuria is highly prevalent in SCD, may increase the risk of dehydration, and is irreversible.²¹² Glomerular hypertrophy, focal and segmental glomerular sclerosis, and hemosiderin deposition in proximal renal tubular epithelium have been described; however, no single lesion is pathognomonic of sickle cell nephropathy. Cystatin C is an accurate marker of glomerular filtration and therefore is preferable to serum creatinine in estimating renal function.^213,214 Glomerular hyperfiltration, microalbuminuria, and macroalbuminuria occur sequentially in SCD patients starting in infancy and increasing in frequency with age.^122,161,215 Incidence of microalbuminuria is greater than 60 percent in those over age 35 years.²¹³ End-stage renal disease requiring dialysis carries a poor prognosis and is associated with a median survival of 4 years.²¹⁶

Angiotensin-converting enzyme inhibitors decrease proteinuria and hyperfiltration in SCD; however, large-scale studies are needed to characterize the magnitude of the benefit. Treatment of renal disease follows principles used for non-SCD kidney pathology and includes effective blood pressure control, avoidance of nephrotoxic agents, and treatment of urinary tract infection. A relative decrease in serum erythropoietin levels, proportionate to the degree of anemia is observed; however, erythropoietin treatment, with its resultant increase in Hb may cause an increase in VOEs because of an increase in blood viscosity.²¹³

Renal tubular acidosis type IV, secondary to decreased potassium and hydrogen ion in the distal tubule can cause disproportionate acidosis and hyperkalemia in patients with declining renal function.²¹³

Hematuria is discussed in the section on sickle cell trait.

Priapism Priapism is prevalent in at least 35 percent of male patients with SCD with devastating psychological consequences; true prevalence may be higher as it is often underreported.^217,218,219 The mean age of episodes is 15 years and two-thirds of patients have “stuttering priapism” a term used for episodes that last less than 3 hours.²²⁰ Derangements in NO metabolism and adenosine signaling are thought to be the major contributors to priapism in SCD.⁹⁴ Greater than 95 percent of priapism is the “low-flow” type resulting from ischemia, is painful, and is a medical emergency.²²¹

Aspiration of the corpus cavernosa followed by epinephrine injections, exchange transfusion, and α and β agonists have all been used, but data regarding efficacy are sparse. α-Agonists, etilefrine 50 mg, and ephedrine 15 to 30 mg per day, seem to reduce the incidence of stuttering priapism.²²² Hormonal therapies, including antiandrogens and luteinizing hormone-releasing hormone, reduce nocturnal erections but are associated with loss of libido.²²¹ Transfusion therapy has resulted in neurologic sequelae termed “the ASPEN syndrome” (Association of Sickle Cell Disease, Priapism, Exchange Transfusion) and is thought to be secondary to hyperviscosity; care, therefore, must be taken not to increase the hematocrit beyond 30 percent.²²³ In recalcitrant cases, a shunt is performed but results in permanent impotence.²²² A penile prosthesis is used to ameliorate sexual dysfunction.

Nocturnal Enuresis Nocturnal enuresis is prevalent in 25 to 33 percent of the pediatric sickle cell population, which is higher compared to that of age-matched controls.^224,225,226 It tends to decrease with age but is still prevalent in adults. Social and environmental factors, decreased functional bladder capacity, and decreased arousal during sleep appear to be contributing factors.

Musculoskeletal System

VOE is commonly manifested by marrow infarction causing musculoskeletal pain, swelling at involved sites, fever, and leukocytosis. Marrow hypercellularity is thought to predispose to this phenomenon by causing a decrease in local blood flow and oxygenation.

Dactylitis Dactylitis is a painful swelling of digits of the hands and feet (“hand-foot syndrome”). It occurs early in infancy as hematopoietic marrow is still present in these bones at this age. Most episodes resolve within in 2 weeks.^{227,228,229,230} Epiphyseal infarction can result in joint pain and swelling mimicking septic arthritis. Use of hydroxyurea in the BABY HUG trial resulted in significant reduction of rate of dactylitis.¹⁶¹

Osteomyelitis, Septic Arthritis, and Bone Infarction Impaired cellular and humoral immunity together with infarction of bone contribute to this complication with an estimated prevalence of 12 percent. Atypical serotypes of Salmonella, S. aureus, and Gram-negative bacilli are the principal infectious offenders. No single lab or imaging test reliably differentiates osteomyelitis from infarction.^{227,229,231,232,233,234,235} Culture results may be nondiagnostic as patients usually receive antibiotics on presentation with fever; therefore, the presence of leukocytes in bone and joint aspirates should evoke a high suspicion for osteomyelitis.¹²⁶ Septic arthritis tends to occur in joints involved with avascular necrosis, also seen following hip arthroplasty. Multiple joints may be involved. An elevated C-reactive protein should raise suspicion for septic arthritis and prompt intervention with appropriate antibiotics as needed to prevent joint deterioration and collapse.²²⁷ Vertebral body infarctions with subsequent collapse causes the classic “fish mouth” appearance of vertebrae on radiographs of the spine.

Osteopenia and Osteoporosis Osteopenia and osteoporosis are prevalent (30 to 80 percent) in patients with sickle cell anemia, with a predilection for the lumbar spine. Presence of avascular necrosis with local bone remodeling may lead to false-negative results on a bone mineral density test at the femoral neck.¹²⁶ Fractures of the long bones are commonly underdiagnosed and self-reported rates of fractures in young adults with SCD are high. Etiology of osteoporosis is multifactorial with hypogonadism, hypothyroidism, nutritional deficiencies, and iron overload interfering with osteoblast function being the major causes.^{126,236,237,238} More than 50 percent of patients are vitamin D deficient with the majority (>80 percent) having less-than-optimal levels. High doses of vitamin D supplementation have resulted in improvement in chronic pain and higher levels of physical activity.²³⁹

Avascular Necrosis Vasoocclusion resulting in infarction of articular surfaces of long bone occurs most commonly in the femur followed by the humerus. It was previously thought to occur with increased frequency in HbSC disease as opposed to patients with HbSS. However, with increased longevity of HbSS patients, its prevalence is greater in patients with HbSS.^240,241,242 As per the CSSCD estimates, 50 percent of patients by age 33 years will have avascular necrosis of the femoral head (Fig. 49–9). The presence of concurrent deletional α-thalassemia (–α^3·7) and a history of frequent VOEs are classic risk factors for avascular necrosis. Other risk factors include male gender, higher Hb concentration, low fetal Hb, and vitamin D deficiency.^126,243,244 Polymorphisms in BMP6, ANNEXIN A₂, and KLOTHO genes are associated with avascular necrosis.²⁴⁵

Patients present with chronic joint pain with progressive decrease in range of motion of affected joints. Multiple joints are commonly involved.²⁴⁶ The vast majority of untreated patients will progress to femoral head collapse within 5 years.^247,248

Avascular necrosis has been treated with a number of modalities including core decompression, osteotomy, bone grafting, surface arthroplasty, and joint replacement. Two randomized trials in avascular necrosis compared core decompression and physical therapy versus physical therapy alone and did not show a difference in outcome between the two arms; however, followup was short, a significant number of stage III hip joints were included in one study, and sample size was limited.²⁴⁹ In our experience, core decompression is a useful option in early stage avascular necrosis. Several studies associate total hip replacement in SCD with a higher rate of orthopedic and medical complications. However, other studies show a lower rate of orthopedic complications. Structural bone diseases in SCD make joint replacement challenging.^250,251,252 Hydroxyurea and chronic transfusion therapy have not been shown to reduce the risk of avascular necrosis.²⁴³

Leg Ulcers

Leg ulcers occur in 2 to 40 percent of cases with SCD and varies geographically with the highest rate being reported in Jamaica.^1,253 In the United States, leg ulcers are seen in 4 to 6 percent of patients with SCD and are most common in patients older than 10 years of age.²⁵⁴ They occur on the lower extremities, especially on the malleoli, and cause chronic pain and disability. Venous stasis is a predisposing factor while coinheritance of α-thalassemia appears to have a protective effect. The relationship between hydroxyurea use and increased occurrence of leg ulcers is controversial.²⁵⁵ Polymorphisms in KLOTHO, TEK, and several other genes in the transforming growth factor (TGF)-β and bone morphogenic protein (BMP) pathways are associated with leg ulcers.²⁴⁵ Once established, ulcers are recalcitrant and significantly impair quality of life.²⁵⁶

Treatment of leg ulcers is largely empiric. Leg elevation, bed rest when practical and feasible, wet-to-dry dressings, gentle debridement, Unna boots, and treatment of infection and topical or systemic antibiotics are commonly used. The peptide encoding integrin-interaction site of many extracellular matrix proteins (RGD peptide) enhanced healing of the ulcers in preliminary studies, but, unfortunately, it never came to clinical practice because of nonmedical reasons.²⁵⁷ Increases in HbF and transfusions occasionally hasten healing of leg ulcers.²⁵⁸

Hepatobiliary Complications

Chronic liver abnormalities in SCD are frequent and of different etiologies that include vasoocclusion, transfusional iron overload, pigmented gallstones with bile duct obstruction, acute or chronic cholecystitis, viral hepatitis, and cholestasis.^259,260 Common clinical manifestations include right upper quadrant pain, fever, hepatomegaly, nausea, and vomiting. Bilirubin levels from chronic hemolysis are usually not above 6 mg/dL, with a majority of it being the indirect fraction.²⁶¹ Because some degree of aspartate transaminase elevation is seen with hemolysis, alanine transaminase elevation is a more accurate marker of liver injury.

Vasoocclusion involving the hepatic sinusoids was seen in 39 percent of patients in one study, while previous reports of vasoocclusion involving the liver, termed acute sickle hepatic crisis, has been reported in 10 percent of patients. The differing prevalence is the result of varying criteria used to include biochemical and clinical abnormalities.²⁶² Acute hepatic sequestration crisis characterized by a rapidly enlarging, tender liver and hypovolemia is akin to splenic sequestration but much rarer. It requires prompt treatment with red cell transfusion. Severe intrahepatic cholestasis with serum bilirubin levels as high as 100 mg/dL is a catastrophic situation needing exchange transfusion for resolution; synthetic liver function is lost as characterized by low serum albumin and coagulation protein abnormalities; renal impairment may occur. A more benign form of cholestasis has been described, which resolves with conservative measure.^{263,264,265,266,267,268}

Chronic hemolysis results in an increased burden on the heme catabolic pathway leading to increased unconjugated bilirubin and formation of pigmented gallstones. The incidence of gallstones increases with age, with a reported prevalence of 50 percent at 22 years of age.^269,270,271 The number of uridine diphosphate (UDP) glucuronosyltransferase 1 family (UGT1A1) promoter (TA) repeats (the polymorphism associated with Gilbert syndrome) is strongly associated with increased incidence of gallstones and bilirubin levels while coinherited α-thalassemia (Chap. 48) decreases bilirubin levels in patients with SCD.²⁷² Laparoscopic cholecystectomy is recommended in symptomatic patients with cholelithiasis. The treatment of asymptomatic patients with positive findings on abdominal ultrasonography is more controversial. In the Jamaican cohort study, only 7 percent of patients with positive ultrasonograms had symptoms suggestive of biliary tract disease and needed a cholecystectomy. However, patients in the United States appear to be more symptomatic, with the majority of gallbladders removed after only a positive ultrasonogram have pathologic evidence of cholecystitis.²⁶⁹ Asymptomatic patients with negative screening ultrasonograms should be observed; however, timing and frequency of screening has not been standardized.

Ophthalmic Complications

The microvasculature of the retina with relative hypoxemia facilitates “sickling” akin to several other vascular beds. Microcirculatory obstruction occurs followed by neovascularization and arteriovenous aneurysms. Hemorrhage, scarring, and retinal detachment leading to blindness are the sequelae. Changes occur at the periphery, thereby sparing central vision at earlier stages. The term sickle cell retinopathy encompasses nonproliferative and proliferative changes.

Nonproliferative changes include “salmon-patch” hemorrhages, peripheral retinal lesions termed “black sunbursts,” and iridescent spots, whereas neovascularization is characteristic of proliferative changes, giving a pattern of vascular lesions resembling a marine invertebrate and is termed as “sea fans.”²⁷³

Increased levels of plasma and intraocular vascular endothelial growth factor have been documented in proliferative sickle cell retinopathy, as have angiopoietin-1 and -2 and von Willebrand factor. Pigment epithelium derived factor, an angiogenesis inhibitor, is increased as well, especially in nonviable “sea fans.”^274,275,276

Proliferative sickle cell retinopathy may differ from other proliferative retinopathies in that spontaneous regression of neovascularization can occur in up to 60 percent of cases.^277,278 The Jamaican cohort study reported an annual incidence of 0.5 cases per 100 HbSS subjects versus 2.5 cases per 100 HbSC subjects. Prevalence was greater in HbSC subjects as well, with a 43 percent rate in the third decade versus 14 percent for those with HbSS. However, there was a 32 percent incidence of spontaneous regression. Irreversible visual loss occurred only in 2 percent of HbSC subjects up to 26 years of age observed at time of the study.²⁷⁷

Central retinal artery occlusion is rare in HbSS disease.²⁷⁹ Conjunctival vascularity is decreased in SCD patients compared to controls with further decreased vascularity and decreased conjunctival red cell velocities during vasoocclusion.^{280,281,282,283}

An orbital compression syndrome characterized by fever, headache, orbital swelling, and visual impairment secondary to optic nerve dysfunction can occur in SCD. Orbital marrow infarction is a common cause.²⁸⁴

All patients with sickle hemoglobinopathies should have a yearly ophthalmology examination beginning in childhood. The examination should be carried out by an ophthalmologist and should include slit-lamp examination of the anterior chamber and detailed retinal visualization including a fluorescein angiography in addition to visual acuity.

The evaluation and treatment of proliferative sickle retinopathy is complicated by the fact that spontaneous regression may occur. Laser photocoagulation remains the most commonly performed procedure for this finding. Traumatic hyphema needs urgent optical referral because increased sickle red cells can cause obstruction of outflow channels, resulting in acute glaucoma. This vascular obstruction may cause decrease in retinal and optic nerve perfusion causing further visual problems. Unresolved vitreous hemorrhage and retinal detachment may need surgical intervention. Exchange transfusion to keep HbA at more than 50 percent is recommended. Central retinal artery occlusion needs urgent exchange transfusion and an ophthalmology consultation.^{277,285,286,287} Orbital compression syndrome is treated with glucocorticoids with the addition of antibiotics if concomitant infection cannot be ruled out.¹²⁶

Splenic Complications

Functional asplenia defined as impaired mononuclear phagocyte system functions in the spleen occurs in 86 percent of infants with SCD.²⁸⁸ It is defined by the presence of Howell-Jolly bodies and absence of ^99mTc (99m-technetium) splenic uptake, even in the presence of a palpable spleen. Slow blood flow in the red pulp of the spleen sets the stage for increased red cell sickling. Repeated splenic infarctions lead to “autosplenectomy.” As a consequence, patients are prone to microbial infections, especially with encapsulated microorganisms such as S. pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Hypertransfusion early in childhood, prior to age 7 years, may lead to reversal of functional asplenia. Marrow transplantation and hydroxyurea have resulted in reversal of functional asplenia in some older subjects. Splenic sequestration occurs in young children.^{289,290,291,292,293}

Management during Pregnancy

Differing morbidity and mortality rates have been reported in pregnant women with SCD, some of which is attributed to geographic location and access to healthcare. Although the CSSCD data showed low rates of pregnancy loss and mortality, other studies have shown an increased mortality of 10 to 100 orders of magnitude greater as compared to non-SCD patients.^{285,286,287,288,289,290} Preterm delivery occurs in 30 to 50 percent of SCD patients and two-thirds will have infants with birth weights less than the 50th percentile.^294,295 There is an increased frequency of VOEs reported during pregnancy. In a study looking at pregnancy outcomes in SCD patients compared to non-SCD patients with comorbidities, patients with SCD displayed a significantly increased incidence of venous thromboembolism (VTE), nonhemorrhagic obstetric shock (defined as pulmonary thromboembolism, amniotic fluid embolism, acute uterine inversion, and sepsis), and infection, despite being significantly younger.^296,297 Other studies have shown similar findings, especially the fivefold increased risk of VTE in this population.^295,297,298

Given increased risk of preeclampsia and eclampsia, patients should have close monitoring of blood pressure and proteinuria after 20 weeks of gestation. Fetal nonstress and umbilical artery Doppler studies should be undertaken after 28 weeks to identify patients who might benefit from early delivery. Studies examining prophylactic red cell transfusions in pregnancy have shown mixed results. Patients should be transfused to a Hb concentration of less than 6 g/dL, because abnormal fetal oxygenation and death have been reported below this Hb level in non-SCD populations. Otherwise, patients should be transfused based on guidelines for the nonpregnant patient with SCD.²⁹⁴ Based on data from animal models and small reports of spontaneous abortion or fetal death, the use of hydroxyurea is not recommended during pregnancy and breastfeeding.^299,300 Hydroxyurea may decrease spermatogenesis and therefore male patients may need to stop the drug temporarily when their partners are trying to conceive. Narcotics administered for relief of pain have not been shown to cause fetal harm, but babies of mothers exposed to narcotics during pregnancy should be monitored for the neonatal abstinence syndrome.²⁹⁴

Despite increased concern for VTE, given insufficient data, contraception advice is similar as for women without SCD.³⁰¹

Management of and Prevention of Infection

Patients with SCD are predisposed to infections for a variety of reasons, including functional asplenia and defective neutrophil responses.^{302,303,304,305,306} The magnitude of this problem was highlighted in 1971 in a landmark paper by E. Barrett-Connor.³⁰⁶ Functional asplenia results in susceptibility to encapsulated microorganisms, particularly to S. pneumoniae, especially in children younger than 5 years of age. The CSSCD data reported an eight-per-100-patient-years rate of invasive bacterial infection in children younger than 3 years of age.³⁰⁷

Given the high incidence of infection, especially in childhood, infection prevention and rapid diagnosis of established infections is of paramount importance.^308,309 The pneumococcal vaccine (PCV7) can be administered in infancy with effective immunologic response prior to 2 years of age; the American Academy of Pediatrics recommends it be administered at ages 2, 4, 6, 8, and 12 to 15 months. The PCV7 vaccine decreases invasive pneumococcal disease by as much as 80 to 90 percent.³¹⁰ The pneumococcal polysaccharide vaccine, PPV23, covers more serotypes but is not immunogenic prior to 24 months and response lasts for 3 years. The first dose is recommended at 24 months with additional doses 3 to 5 years later.^{309,311,312,313,314} Nonvaccine covered strains of S. pneumoniae are emerging as important pathogens; therefore, prompt referral of patients with suspected infection to a healthcare facility is important.³¹⁵

Oral penicillin prophylaxis is recommended at a dose of 125 mg twice a day for children between 0 and 3 years of age and at 250 mg twice a day for children between 3 and 5 years of age.³¹⁶ Penicillin prophylaxis beyond 5 years is recommended only for patients with recurrent pneumococcal infections or who have had surgical splenectomy. Patients allergic to penicillin are offered erythromycin.

The meningococcal vaccine covers most invasive isolates of N. meningitidis and is recommended by the American Academy of Pediatrics.³¹⁷ Standard pediatric immunizations protecting against H. influenzae and hepatitis B virus should be given. Influenza virus vaccine should be given annually because viral respiratory infection favors invasive bacterial infection.

Parents and caregivers of children should be educated to recognize infections and to seek medical attention early. Diagnosis of established infections varies by site and offending agent. For invasive pneumococcal disease, ceftriaxone remains the drug of choice despite concerns of immune-mediated hemolysis. Infections seen classically in SCD patients include salmonella osteomyelitis and penumonia caused by atypical bacteria like Chlamydia and M. pneumoniae, which need to be treated with the appropriate antibiotics.

The spectrum of infectious complications in adults may be different. One study reported data on blood infections in adults.³⁰² Pneumococcal infections were rare. S. aureus was the predominant organism. Patients with S. aureus had a predilection for bone-joint infection. Those with indwelling venous catheters and a severe disease course appeared to have a high risk for bloodstream infections.

Although the sickle trait confers resistance to malaria, protection is not complete and severe disease and deaths from malaria have been reported in SCD patients. Malaria chemoprophylaxis is recommended for all patients living in or traveling to endemic regions.^318,319

Management during Anesthesia and Surgery

Patients with SCD should have careful monitoring of Hb concentration, hydration, oxygen, and metabolic studies in the perioperative period. Acute chest syndrome and VOE occur with higher frequency in the perioperative period. Increased age is associated with increased complications.^320,321,322 Transfusion to keep Hb levels approximately 10 g/dL is recommended. Although a prior randomized trial showed no benefit in decreasing SCD-related complications between patients transfused aggressively to a mean HbS of less than 30 percent versus those transfused to a total Hb of 10 g/dL with mean HbS percent of 59, more recent data show reduction in clinically important events, especially serious complications, in the preoperative transfused group prior to low and moderate risk surgery.^202,323 Care should be taken to avoid transfusion-induced hyperviscosity.

MODIFIERS OF DISEASE SEVERITY

Some patients have a mild course with few problems related to SCD, and survive into the sixth or seventh decade. In contrast, some patients have a difficult course with multiple complications, frequent hospitalizations, severe organ damage, and a significantly shortened life expectancy.^324,325 Inheritance of α-thalassemia trait and a high HbF are two factors that ameliorate many complications of SCD. Genome-wide association studies revealed three major loci associated with HbF levels: The β-globin locus on chromosome 11, an intergenic region between HBSIL and MYB genes on chromosome 6, and the BCL11 gene on chromosome 2.³²⁶ Repression of BCL11A results in increased γ-globin gene expression and, consequently, in increased HbF. The exact mechanism of how BCL11A silences γ-globin expression is unclear; its expression seems to be controlled by an erythroid specific transcription factor, KLF1 with decreased expression of BCL11A upon knockdown of KLF1 gene transcript.^326,327

Inheritance of α-thalassemia and HbF level do not account for all of the clinical diversity of SCD. The completion of the human genome project has provided the impetus to study polymorphisms in candidate genes as potential modifiers of disease severity. Association of polymorphisms in candidate genes and different features of SCD such as stroke,^328,329,330 ACS,³³¹ bilirubin levels and cholelithiasis,^{332,333,334,335} avascular necrosis,²⁴⁵ priapism,³³⁶ and leg ulcers,²⁵³ as well as HbF levels,^{337,338,339,340,341,342} and HbF response to hydroxyurea,³⁴³ have been studied in different groups of patients.

Polymorphisms in the TGF-β–BMP pathway, a ubiquitous signaling pathway that is involved in many cellular processes and pathways, have emerged as recurrent findings in many of these studies. Some of the associations have functional consequences; the association of bilirubin levels in polymorphisms in the UGT1A1 gene promoter is such an example. The 7TA repeat in the promoter leads to a decreased activity of this enzyme and hence a decrease in glucuronidation of bilirubin. Thus, the association of this polymorphism with higher bilirubin levels can be understood. On the other hand, the mechanisms by which polymorphisms in the ubiquitous TGF-β–BMP pathway are associated with various complications of SCD are unknown, and thus a causal relationship cannot yet be established. Functional studies of these variants and genomewide association studies are expected to provide a better insight into genetic modulation of the phenotype of SCD.

GENERAL MANAGEMENT OF SICKLE CELL DISEASE

Pharmacotherapeutics to Increase Fetal Hemoglobin Levels

The observation that HbF results in ameliorating the phenotype of SCD led to research focused on HbF modulation as a therapy for SCD. The γ-chains of HbF are excluded from the deoxy HbS polymer; thus the presence of HbF in sickle red cells exerts a potent antisickling effect. This effect has also been supported by clinical observations; the manifestations of SCD do not become apparent in the first few months of life until the switch from γ-chain production to β-chain production is almost complete in the postnatal period. Additionally, the phenotypes of some compound heterozygous states with HbS and other inherited globin disorders that lead to increased expression of HbF in the adult life (δβ-thalassemias, hereditary persistence of HbF) are very mild (Chap. 47). In fact, compound heterozygotes for HbS and deletional hereditary persistence of HbF, in which there is continued high levels of HbF expression (30 to 35 percent) uniformly distributed in all red cells (pancellular), are clinically asymptomatic and hematologically normal. In the late 1970s, further evidence in support of the ameliorating effect of high HbF came from the observation of Saudi Arabian sickle cell anemia patients who had few, if any, symptoms of SCD, had mild anemia, and were not diagnosed until adult age.³⁴⁴ These individuals had HbF levels in the 20 to 25 percent range as opposed to the African patients or American patients of African descent, the majority of whom had HbF levels of approximately 5 percent. Similar patients were reported from India, and this genetic propensity for high HbF production in SCD patients was linked to a unique β-globin gene cluster haplotype (Saudi Arabian–Indian) that is distinct from those found in Africa. These observations paved the way for intense investigations on the cellular and molecular mechanisms of the fetal to adult (γ to β) switch during the perinatal period and the search for “antiswitching” agents, agents that would facilitate retaining elevated HbF levels. The observation that there is a transient increase in HbF production during recovery from marrow aplasia or suppression provided the rationale for the use of myelosuppressive agents as antiswitching therapy (Table 49–3). Antiswitching indicates a mechanism to prevent the switch from γ-globin chains to β-globin chains.

Hydroxyurea Although many myelosuppressive agents have been studied in primates and some have been used in a small number of patients, only one of these, hydroxyurea, has been used, starting in the early 1980s, in large-scale clinical trials. This is largely attributable to its excellent oral bioavailability, relatively short half-life (important from the standpoint of rapid reversibility of toxicity), no evidence that its use leads to an increase in cancer prevalence, and few side effects.

Hydroxyurea is the only FDA-approved agent for the treatment of SCD. It is a ribonucleotide reductase inhibitor and is S-phase specific in the cell cycle. The mechanism by which hydroxyurea increases HbF synthesis is not fully understood; it has been postulated that the myelosuppressive effect leads to the recruitment of early erythroid progenitors that have retained their fetal (γ) globin synthesis capability, giving rise to the production of red cells with a higher HbF content. Some studies show that hydroxyurea acts as a NO donor and increases HbF synthesis via the cyclic guanosine monophosphate (cGMP) pathway.³⁴⁵ Others suggest it works by reducing the neutrophil count, thereby reducing the contributions of neutrophils to the abnormal vascular adhesion of sickle red cells. It has several other actions that explain its efficacy in SCD other than increasing HbF. These include decrease in platelets and reticulocytes, improvement in red cell hydration, and a decrease in red cell adhesiveness to the vascular endothelium (Fig. 49–10).^346,347,348

In the landmark Multicenter Study of Hydroxyurea, hydroxyurea was shown to decrease frequency of painful crises, ACS, hospitalizations, and blood transfusions. Followup showed a 40 percent decrease in mortality in patients randomized to the drug.^160,349 Hydroxyurea is recommended in patients with three or more VOEs or history of ACS. It can be started at a dose of 15 mg/kg given as a single daily dose and escalated by 5 mg/kg per day every 8 weeks until toxicity or a maximum dose of 35 mg/kg is reached. Maximum tolerated dose is defined as the dose that targets an absolute neutrophil count of 2 to 4 × 10⁹/L and absolute reticulocyte count 100 to 200 × 10⁹/L.^350,351 Periodic monitoring of blood cell counts and serum chemistries, especially in the first year of treatment is important. Maximal effect on HbF may not be seen until 6 to 12 months of therapy is completed. The dose should be decreased in renal failure. Although not proven to have teratogenic or leukemogenic potential in SCD patients, it is recommended that it not be used in pregnant or breastfeeding patients. Concerns about detrimental effect on spermatogenesis have also been raised based on studies in mice.^{352,353,354,355}

Patients receiving hydroxyurea who die while on treatment are likely to be older when therapy is initiated, more anemic, likely to have Bantu or Cameron β-globin gene haplotypes, and have impaired renal function.³²⁴

Several studies have now been published on the use of hydroxyurea in infants and children. Therapy can begin between 6 and 9 months of age, is safe and well tolerated with improved growth rates, preserves organ function, and the additional benefits as seen in adults.^{161,351,356,357}

Other Fetal Hemoglobin-Inducing Agents Although significant advances have been made in understanding the basic mechanism(s) of the perinatal switch from γ- to β-globin synthesis, this knowledge is far from complete. Certain epigenetic mechanisms (histone deacetylation and DNA methylation) are involved in the silencing of the γ-globin genes postnatally. This has led to the use of agents that target the two common epigenetic silencing mechanisms: histone deacetylase inhibitors and DNA methyltransferase 1 inhibitors.

The histone deacetylase inhibitors that have been most widely used in early phase small clinical trials in SCD and in some patients with β-thalassemia are butyrate derivatives (arginine butyrate, sodium phenyl butyrate, isobutyramide). Arginine butyrate has to be administered by IV infusion; earlier studies suggested that continuous daily infusions of arginine butyrate were not very effective in leading to a sustained increase in HbF.²⁵⁸ Later, it was shown that daily continuous infusion induced tachyphylaxis and hence the failure to cause a sustained HbF response. An intermittent schedule of administration (4 days, given every 4 weeks) was efficacious in increasing HbF.³⁵⁸ Although orally administered sodium phenyl butyrate was effective in increasing HbF, the daily doses required for maintaining a HbF response required the administration of a large number of tablets and was impractical.³⁵⁹ A phase II trial studying the efficacy of oral 2,2-dimethylbutyrate sodium salt (HQK1001) did not show significant increase in HbF and was associated with a trend for increased VOE.³⁶⁰

The two DNA methyltransferase inhibitors with antiswitching activity are 5′-azacytidine and decitabine. Both of these agents are myelosuppressive when used in higher doses; however, at low doses, they are potent inhibitors of DNA methyltransferase 1 and have been shown to increase HbF synthesis in baboons and in patients with SCD.^{361,362,363,364,365,366,367,368} Unlike 5′-azacytidine, which incorporates into both DNA and RNA, decitabine incorporates only in DNA and is believed to have a better genotoxicity profile. It has been effective in increasing HbF and ameliorating the disease severity in patients with SCD who have been refractory to hydroxyurea.³⁶³

Immunomodulatory agents (thalidomide and derivatives) increase HbF synthesis in erythroid colonies from SCD patients.³⁶⁹ Pomalidomide augments HbF in sickle cell mice.³⁷⁰ Data from use in sickle cell patients is awaited. The finding that the KLF-1–BCL11a axis is the major factor in the switch from β- to γ-globin has made these factors attractive targets for therapy; however, to date, no effective means of targeting these transcription factors has been developed.

Allogeneic Hematopoietic Stem Cell Transplantation

Because SCD is an inherited defect in the hematopoietic stem cell, stem cell transplantation (SCT) is an attractive option to permanently cure the disease rather than managing its sequelae piecemeal. However, the tremendous phenotypic variability that characterizes the disorder combined with lack of an accurate predictive model to foretell which patients are likely to have a catastrophic disease course, make selecting patients for allogeneic hematopoietic stem cell transplantation (AHSCT) challenging. AHSCT should be done in patients who are likely to have a severe disease course, but should be instituted early, prior to end-organ damage. The risk-to-benefit ratio of the morbidity and mortality associated with AHSCT has to be weighed against the disease severity of a nonmalignant hematologic disorder.

AHSCT is an underused treatment modality in SCD even in eligible patients secondary to lack of donor availability and socioeconomic factors.³⁷¹ Human leukocyte antigen (HLA)–matched sibling donor transplant with myeloablative conditioning represents the most common transplant type in SCD. Cerebrovascular disease, recurrent ACS, and frequent VOEs despite adequate hydroxyurea therapy are the most common indications for SCT. Data from approximately 1200 patients worldwide show an overall survival of 95 percent; early or late allograft failure resulting in disease recurrence occurs in 10 to 15 percent of patients.^371,372 The most common myeloablative regimen used is busulfan, cyclophosphamide, and antithymocyte globulin; the addition of antithymocyte globulin resulted in a significant reduction in allograft rejection. Transplant-related mortality ranges between 2 and 8 percent.³⁷² Acute graft-versus-host disease occurs in approximately 10 to 15 percent of patients, whereas chronic graft-versus-host disease has been reported in 12 to 20 percent of patients. Most series have used cyclosporine alone or in combination with methotrexate for graft-versus-host disease prophylaxis (Chap. 21).

Risk of increased incidence of neurologic complications following transplantation has been ameliorated with the use of prophylactic anticonvulsants, strict control of arterial hypertension, correction of hypomagnesemia, and maintenance of Hb greater than 10 g/dL and platelets greater than 50 × 10⁹/L. Long-term toxicity still remains a concern, especially in relation to growth, reproduction, and secondary malignancies. Followup data on AHSCT in children between 1991 and 2000 show significant gonadal toxicity and infertility, especially in females.³⁷³

AHSCT in adults is problematic given toxicity of the conditioning regimen. In an attempt to address this issue, reduced-intensity conditioning has been used but has resulted in an increased rate of graft failure. A small cohort of patients who received blood stem cells from HLA-matched siblings and used low-dose total-body radiation plus alemtuzumab as the conditioning regimen followed by sirolimus for graft-versus-host disease prophylaxis had stable engraftment at 30 months of followup.³⁷⁴

Cord blood and HLA haploidentical transplantation have been used in a small number of patients with SCD, but graft failure remains a significant issue.^371,375,376

Transfusion

Red cell transfusions are used frequently in SCD on an acute or chronic basis. The rationale for transfusion in SCD is twofold. Besides increasing Hb concentration, thereby increasing the oxygen-carrying capacity of the blood, transfusion also decreases the percentage of circulating HbS-containing red cells. Hb level alone should not constitute an indication to transfusion as patients adapt to their level, making it important to know the patient’s baseline Hb concentration. It is also important to calculate whether the reticulocyte count, a measure of marrow red cell production, is adequate or not.

Indications for red cell transfusion include symptomatic anemia, ACS, stroke, aplastic and sequestration crises, other major organ damage secondary to vasoocclusion, and occurrence of unrelenting priapism. Transfusion is also required prior to major surgery or surgery involving critical organs. The best-established indication for chronic transfusion is stroke and an abnormal TCD velocity. Patients with other chronic or recurrent events are sometimes placed on chronic transfusion as well. Inappropriate indications for transfusion include chronic steady-state anemia, uncomplicated VOE, pregnancy, minor surgeries, infection, and avascular necrosis.³⁷⁷

Simple red cell or exchange transfusion can be used.³⁷⁸ Simple transfusion is easier to perform and is generally associated with fewer complications. Exchange transfusion, however, has the advantage of not raising total Hb, and thereby blood viscosity, while decreasing percentage of circulating sickle cells because sickle cell patients transport less oxygen to their tissues beyond a hematocrit of 30 percent as a result of increased blood viscosity.^379,380,381 Exchange transfusion has also the advantage of not causing iron overload.

Alloimmunization occurs in 20 to 50 percent of transfused SCD patients.^382,383,384 In the United States, the majority of blood donors are of European descent, and the majority of SCD patients are of African descent (Chaps. 136 and 138). This results in blood group antigenic disparity, and antibodies to E, C, K, Jkb, S, and Fyb antigens are common. Age at first transfusion, total number of transfusions, transfusion in the context of inflammation, and influence of immunoregulatory genes may affect the rate and extent of alloimmunization.³⁸⁴ Extended antigen phenotyping (Kell, Duffy, Kidd, Lewis, Lutheran, P, and M&S) in addition to the usual ABO and D antigens (Chaps. 136 and 138) and leukodepletion of blood products are recommended.^{378,382,384,385} Delayed hemolytic transfusion reaction complicates 4 to 11 percent³⁸⁶ of transfusions in SCD and may present as a painful crises. It typically occurs a week after transfusion and is caused by alloantibodies to non-ABO antigens. It can cause the Hb to fall lower than the prior pretransfusion Hb and can be associated with a depressed reticulocyte count and autoantibodies. Alloantibody mediated hemolysis will present as a rapid decrease in the percent of HbA as opposed to HbS. A failure to demonstrate a new alloantibody posttransfusion should not exclude the diagnosis of delayed hemolytic transfusion reaction (Chaps. 136 and 138). Patients should be transfused only if symptomatic under such circumstances as further transfusion can exacerbate the problem.^377,384

Iron overload and its attendant complications and infection transmission are the other major complications of transfusion.

Iron Overload

Iron overload (Chap. 43) in SCD is similar to other chronically transfused populations.^169,387,388 The multicenter study of the iron overload research group showed that transfused sickle cell patients had increased morbidity and mortality when compared to transfused thalassemic patients and nontransfused SCD patients.³⁸⁹

Diagnosing significant iron overload accurately and early can be difficult. Serum ferritin is an easy, widely employed method, but is unreliable in SCD as it is an acute phase reactant. Its measurement can result in over- or underestimation and is poorly correlated to liver iron content.³⁹⁰ A serum ferritin value of greater than 1000 mg/mL in the steady-state has been used as an indication of iron overload. Liver iron content is the current accepted standard and a value of 7.7 mg/g dry weight is used as indication for treatment.³⁹¹ However, noninvasive methods of assessment of iron overload, like superconducting quantum interference device (SQUID) or MRI T2^* (Chap. 43), are becoming standard. Transfusion of 120 mL of red blood cells/kg of body weight can also be used as a chelation trigger.³⁸²

Iron chelation (Chap. 43) was typically carried out with desferrioxamine at a dose of 25 to 40 mg/kg per day given over 8 hours subcutaneously.³⁹² Desferrioxamine can reverse cardiac iron overload. A once-daily oral iron chelator, deferasirox, is now approved and available for use in the United States. It is a tridentate ligand that binds iron with a high affinity in a 2:1 ratio. It has a half-life of 8 to 16 hours and is metabolized by glucuronidation and excreted in the feces. In an open-label phase II trial of deferasirox versus desferrioxamine in a 2:1 randomization, safety and tolerability were established. Nausea and vomiting, abdominal pain, rash, reversible increase in liver function tests, and stable increases in serum creatinine were reported. Rare cases of anaphylaxis occurring mostly in the first month of starting treatment have also been reported. Postmarketing reports suggest an increased incidence of renal failure, and caution is to be exercised in a patient population where renal insufficiency may not be readily appreciated prior to starting treatment. Postmarketing experience has also reported cases of fatal hepatotoxicity and agranulocytosis. Auditory and ophthalmic side effects occur in less than 1 percent of patients; however, annual eye and auditory examinations are recommended for deferasirox as they are for desferrioxamine. The recommended daily dose is 20 mg/kg body weight; this dose may be adjusted every 3 to 5 months in increments of 5 to 10 mg/kg if the therapeutic goal is not achieved, although the total dose should not exceed 40 mg/kg. Safety in combination with other iron chelators has not been established.³⁹³ Deferiprone is not available in the United States but has been used in other parts of the world. It is orally administered and is considered a better chelator of cardiac iron because of its ability to cross cell membranes.³⁹⁴ Although iron chelation in SCD follows the general guidelines of iron chelation in other iron overloaded populations, rigorous studies of its effects on morbidity and mortality in SCD are lacking.^394,395

Evolving Therapies

Given the complex pathophysiology of SCD, numerous therapies targeting different pathways have been tried to ameliorate disease manifestations. Many drugs have failed to show efficacy, especially in phase II/III trials, because of failure to choose appropriate end points or because they were too narrowly focused. Table 49–4 is a comprehensive list of trials and their outcomes. A few of the novel and promising studies are with immunomodulatory agents (thalidomide/pomalidomide), E- and P-selectin inhibitors, iNKT agonists, and Aes-103, and all are in trials as of this writing.

The number of Hb variants discovered to the time of writing this chapter totals 1187. The vast majority of these variants are benign, without any significant clinical or hematologic problems, but are of interest to geneticists and biochemists (http://globin.cse.psu.edu). Most of the Hb variants are missense mutations in the globin genes (α, β, γ, or δ) resulting from single nucleotide substitutions. Other uncommon mechanisms include deletion or insertion of one or more nucleotides altering the reading frame and fusion of globin genes with deletion of intergenic DNA sequences (γβ fusion in Hb_Kenya and δβ fusion in Hb_Lepore), mutations of the termination codon leading to the production of elongated globin chains.

Hb variants that significantly alter the structure, stability, synthesis, or function of the molecule have hematologic and/or clinical consequences. These can be classified in certain categories (Table 49–5). HbS and HbC are two examples of mutations on the surface of the Hb molecule that alter both the charge and the physical/chemical properties of the molecule with polymer formation in the case of deoxyhemoglobin S and crystallization in HbC with profound effects on the function, morphology, rheology, and life span of the red cells. Several mechanisms account for the pathogenesis of unstable Hb variants. The common mechanism involves the precipitation of the unstable Hb molecule within the red cell with attachment to the inner layer of the red cell membrane (“Heinz body” formation); red cells containing membrane-attached Heinz bodies (see Chap. 31, Fig. 31–11) have impaired deformability and filterability leading to their premature destruction (congenital Heinz body hemolytic anemia). Mutations in certain residues alter the oxygen affinity of the Hb molecule; a stabilization of the R (relaxed, oxy) state will result in high O₂ affinity variants and erythrocytosis. Conversely, a stabilization of the T (tense, deoxy) configuration will result in a variant with low O₂ affinity with enhanced unloading of O₂ to the tissues with resultant cyanosis and anemia in certain cases (because of the suppression of the O₂ sensing pathway) (Chaps. 32 and 50). Mutations of the heme binding site, particularly those affecting the conserved proximal (F8) and distal (E7) histidine residues, lead to the oxidation of the iron atom in heme from ferrous (Fe²⁺) to ferric (Fe³⁺) state with resultant methemoglobinemia (M Hbs) and cyanosis (Chap. 50). A group of mutations alter both the structure and the synthetic rate of the globin chain leading to a “thalassemic” phenotype (Chap. 48). These include fusion Hbs (e.g., Hb_Lepore, where the 5′ δ-globin sequences are fused to 3′ β-globin sequences with deletion of the intergenic DNA; this puts the δβ-fusion gene under the transcriptional control of the inefficient δ-globin promoter with low expression of the fusion globin (hence the thalassemic phenotype), mutations that cause both a missense mutation and create an aberrant splice site (such as HbE, Hb_Knossos, and Hb_Malay), and “hyperunstable” globins where the nascent globin chains are highly unstable, undergo rapid proteolytic degradation, and result in a reduction in the affected globin.

Except for the commonly occurring variants (HbS, HbC, HbE, and HbD_{Los Angeles}), very few abnormal Hbs have been observed in the homozygous state. Variant Hbs are usually found in the heterozygous state. Although γ-chain variants are expressed in fetal life and their level gradually decreases as the γ-globin to β-globin (fetal to adult) switch progresses during the postnatal period, β- and α-chain variants are expressed throughout life. δ-Globin variants are expressed at very low levels and can be detected only after the switch to adult globin synthesis is complete. Because α-globin chains are present in all of the Hbs expressed after the embryonic stage (HbF-α₂γ₂; HbA-α₂β₂, and HbA₂-α₂δ₂), α-chain variants are associated with the production of variant HbF (α₂^xγ₂) and HbA₂ (α₂^xδ₂) as well. In heterozygous states, β-chain variants constitute 40 to 50 percent of the Hb in red cells; it should, however, be kept in mind that certain factors affect the amount of variant β chains in carriers. These factors include the stability of the variant, the surface charge of the variant β-chain, and the presence of concomitant α- or β-thalassemia (Chap. 48). The more unstable the variant, the lower the quantity. Surface charge of the variant also plays a role in determining the quantity in red cells; this is because the formation of the αβ-dimers (α₁β₁ and α₂β₂ contacts) is the critical first step in Hb tetramer formation, and this step is primarily driven by electrostatic interactions between α and β chains. The α-globin chains have a relatively positive surface charge, they interact more readily with relatively negatively charged β-globin variants to form αβ dimers. This is reflected in the higher percentage of negatively charged β-globin variants such as HbN_Baltimore (β95Lys→Glu), which is found in approximately 50 percent in heterozygotes compared to β-globin variants with a positive surface charge, HbS (β6Glu→Val) or HbC (β6Glu→Lys) whose quantity in the heterozygote is 40 to 45 percent. In the presence of α-thalassemia, negatively charged β-globin variants compete more favorably for the available α-chains; this phenomenon is reflected in even lower percentages of HbS and HbC in heterozygous carriers of these variants in the presence of common deletional forms of α-thalassemia (HbS of 30 to 35 percent in individuals with heterozygous α⁺-thalassemia, –α/αα; and 25 to 30 percent in homozygous α⁺-thalassemia, –α/–α).^396,397 Conversely, the amount of a β-globin variant will increase if there is a β-thalassemia allele in trans; the percentage of the variant will be inversely proportional to the output of the β-thalassemia allele; thus, the higher the variant the lower the output of the β⁺-thalassemia allele. In the case of a β⁰-thalassemia allele in trans, the variant will amount to greater than 90 percent or more of the Hb in red cells, with HbA₂ and HbF constituting the remainder. The quantity of α-globin variants is also variable, depending on the α-globin gene involved, and the presence of concomitant α- or β-thalassemia. Because there are normally four α-globin loci (αα/αα) and the upstream 5′ α-globin gene (α₂) is expressed at a higher level, some of the variation in the level of α-globin variants depends on which α-globin gene carries the mutation; α₂-globin mutations are usually present at 20 to 25 percent of the total Hb, whereas 3′ downstream α₁-globin variants are expressed at a lower level (15 to 20 percent). Concomitant α-thalassemia results in a higher level of expression of α-globin variants. Observations on the different levels of expression of the common α-globin variant, HbG_Philadelphia (α68Asn→Lys), is a case in point.³⁹⁸ Although this variant is found in approximately 25 percent of Northern Italians, its percentages in Americans of African descent can be either 33 or approximately 50 percent. This is clearly related to the different genotypes found in these two distinct populations: In northern Italy and Sardinia, the genotype is α^Gα/αα, with an expression level of approximately 30 percent, whereas in Americans of African descent, the HbG_Philadelphia mutation is commonly found on a hybrid α₂α₁ gene associated with the common 3.7 kb α⁺-thalassemia deletion (–α^G/αα) with approximately 33 percent expression. When there is an α⁺-thalassemia deletion in trans (–α^G/–α genotype), as expected, the level of HbG_Philadelphia will be approximately 50 percent. Coinheritance of α-chain variants with β-thalassemia results in the increase of the α-chain variant.

HEMOGLOBIN C DISEASE

Definition and History

HbC was the second Hb variant described after HbS.³⁹⁹ Homozygous HbC was described by Spaet and colleagues⁴⁰⁰ and Ranney and colleagues.⁴⁰¹ HbC trait is found in 2 percent of Americans of African descent, and approximately one in 6000 have homozygous HbC.⁴⁰² Coinheritance of HbC with HbS results in HbSC disease, which is the second most common form of SCD in the United States. There are also rare cases of HbC-β⁺-thalassemia and HbC–β⁰-thalassemia. HbC is thought to have originated in Central West Africa and in parts of West Africa, where the prevalence of HbC can reach 12.5 percent. The HbC gene was found on three distinct β-globin cluster haplotypes, termed CI, CII, and CIII; the most common is CI, accounting for 70 percent or more of HbC studied.⁴⁰³

Etiology and Pathogenesis

HbC is the result of a GAG→AAG transition in codon 6 of the β-globin gene, which changes the amino acid residue at this position from glutamic acid to lysine (Glu→Lys). The resultant positively charged Hb variant can easily be distinguished from HbA and HbS by electrophoresis and chromatography, including high-performance liquid chromatography. HbC does not differ from HbA in its solubility; however, purified solutions of HbC form tetragonal crystals in high-molarity phosphate buffer. The Hb in red cells from homozygous HbC individuals also form crystals when incubated with hypertonic saline; HbC crystals are also observed in vivo, particularly in the red cells of splenectomized HbCC patients (Fig. 49–11F). Crystal-containing HbCC red cells have impaired deformability and filterability. HbCC red cells have a propensity for potassium (K⁺) loss, which is followed by water loss; unlike in sickle red cells, this K⁺ leak does not appear to be mediated through either the potassium chloride cotransport or the calcium ion activated K⁺ efflux (Gardos) channel; it is thought to be a volume-stimulated K⁺ efflux.⁴⁰² The consequence of this K⁺ loss is dehydrated, often spherocytic, red cells with increased MCHC, and decreased osmotic fragility. These changes result in impaired rheologic properties of HbCC red cells; their life span is reduced to 40 days.

Clinical Features

Mild to moderate splenomegaly is a common feature of homozygous HbC. Like many other chronic hemolytic states, cholelithiasis may be present. HbCC individuals do not suffer from vasoocclusion or episodic pain. Occasionally, abdominal pain may be present and can be a result of splenomegaly and/or cholelithiasis. Pregnancy does not pose an increased risk to women with HbCC. Life expectancy of HbCC individuals is comparable to non-HbC Americans of African descent. In a recent single-institution study, splenomegaly and cholelithiasis occurred in approximately 2.5 percent of patients younger than 8 years of age, but it was far more common (71 percent) in individuals older than 8 years of age.⁴⁰⁴

Laboratory Features

HbCC individuals have a mild to moderate hemolytic anemia. Hb is usually in the 10 to 11 g/dL range. There is associated reticulocytosis usually in the 3 to 4 percent range. There usually is mild microcytosis (mean corpuscular volume [MCV] 70 to 75 fL) and, often, an elevated MCHC. The blood film is characteristic, showing an abundance of target cells, microspherocytes, and HbC red cell crystals, especially in splenectomized patients (see Fig. 49–11F). Indirect bilirubin may be mildly elevated. White cell and platelet counts are normal in the absence of hypersplenism.

Differential Diagnosis

Differential diagnosis is usually achieved by Hb electrophoresis. HbC moves to a cathodic position, comigrating with HbA₂, HbE, and HbO_Arab on alkaline pH (cellulose acetate) electrophoresis. The distinction from these Hbs can be made by electrophoresis on citrate agar in acid pH where HbE and HbA₂ comigrate with HbA; HbO_Arab has a HbS-like mobility, and HbC has a unique migration pattern. Alternatively, newer diagnostic methods can be used; these include isoelectric focusing, where HbC can readily be distinguished from other Hbs with similar mobility on cellulose acetate electrophoresis. In cation exchange HPLC and capillary electrophoresis, HbC has a distinct elution pattern and can be distinguished from HbE and HbO_Arab; these latter methods also have the advantage of separating and quantifying HbA₂ in HbC homozygotes and in HbC trait. This confers the advantage of readily differentiating between HbCC and rare cases of HbC–β⁰–thalassemia (where HbA₂ is significantly higher, ~5 percent).

Therapy

The vast majority of HbCC individuals do not require any therapeutic intervention. Cholecystectomy may be required in individuals who have symptomatic gallstones. Few patients with HbCC develop hypersplenism with a reduction in white cell and platelet counts, and occasionally worsening of anemia. In such instances, splenectomy should be considered. Another indication for splenectomy is pain associated with an enlarged spleen. It is important to apply the usual precautions in patients considered for splenectomy (appropriate vaccinations, prophylactic antibiotic use, and delaying splenectomy in young children). Folic acid supplementation, as usually done in many chronic hemolytic states, is of no proven value.

HEMOGLOBIN DISEASE

Definition and History

HbE (β26Glu→Lys) was the fourth abnormal Hb described.⁴⁰⁵ It is most commonly found in Southeast Asia; in some areas (in the border between Thailand, Laos, and Cambodia, the so-called HbE triangle) the reported gene frequency may reach as high as 0.50.⁴⁰⁶ This high frequency is thought to be from a protective effect against malaria. HbE is also found in other malaria-endemic areas such as Bangladesh, India, and Madagascar. HbE now has a wide distribution as a result of the large population movements from Southeast and South Asia to Western Europe and North America, and may now be the most common Hb variant worldwide.

Etiology and Pathogenesis

The GAG→AAG mutation in codon 26 of the β-globin gene not only leads to a missense mutation (Glu→Lys) at this position, but also activates a cryptic donor splice site at the boundary of exon 1 and intron 1 by increasing the sequence similarity of this site to a consensus splice sequence. The resultant aberrant splicing through this alternate site leads to a decrease in the correctly spliced messenger RNA and hence a β⁺-thalassemic phenotype. This is reflected in the fact that heterozygotes for HbE have 25 to 30 percent of the variant; in the presence of concomitant α-thalassemia, this quantity decreases even further. The coinheritance of HbE with a host of other globin mutants (α-thalassemia, β-thalassemia, other Hb variants), which are also common in the populations where HbE is prevalent, results in a wide spectrum of hemoglobinopathies with varying degrees of severity (HbE disorders or HbE syndromes). The most significant of these is HbE–β-thalassemia syndromes. HbE has also been reported in combination with HbS (HbSE disease).

Clinical Features

Individuals with homozygous HbE are asymptomatic. Most patients do not have hepatosplenomegaly or jaundice. They are usually diagnosed during screening programs or family studies of individuals with severe HbE disorders, or on routine evaluation of a blood film with significant microcytosis without anemia. HbE–β-thalassemia is a rather heterogeneous group of disorders varying from a mild thalassemia intermedia like phenotype to severe transfusion dependent thalassemia major (Chap. 48). Part of this heterogeneity results from the type of coinherited β-thalassemia mutation. Patients who are compound heterozygotes for HbE and one of the mild β⁺-thalassemia mutations (such as the mild promoter mutation, –28A→G) have a mild to moderate anemia, whereas patients with compound heterozygosity for HbE and one of the more severe β⁺-thalassemia mutations (such as IVS I nucleotide 5 or IVS II nucleotide 654 mutations) do have a more severe phenotype with severe anemia and transfusion dependency. There is also a large heterogeneity among patients with HbE–β⁰-thalassemia; these patients do not produce any HbA and have only HbE and varying amounts of HbF. Known factors that influence the phenotype include the ability to produce HbF and the presence of concomitant α-thalassemia. Individuals who have the propensity to synthesize significant amounts of HbF (such as those who have the Xmn I C→T mutation in the ^Gγ-globin promoter) are able to ameliorate the globin chain imbalance and thus have a milder phenotype. Concomitant α-thalassemia also mitigates the course of the disease by decreasing globin chain imbalance. In some cases, there may be nonglobin modifiers that impact on the phenotype. Patients with severe forms of HbE–β⁰-thalassemia have clinical features very similar to β-thalassemia major; they develop complications such as hypersplenism, iron overload, increased susceptibility to infections, thromboembolic complications, and heart failure, and have a shortened life expectancy.⁴⁰⁶ Splenectomized HbE–β-thalassemia patients have more pronounced intravascular hemolysis, markers of endothelia cell activation, and activation of coagulation with increased levels of cell free Hb, sE-selectin, sP-selectin, high-sensitivity C-reactive protein, and thrombin–antithrombin complex compared to nonsplenectomized patients.⁴⁰⁷

Laboratory Features

HbE-trait individuals have a borderline microcytosis (MCV in the lower 80s) but are not anemic. Homozygotes for HbE are usually only borderline or mildly anemic (Hb 11 to 13 g/dL), but they are microcytic (MCV ~70 fL). Blood film shows target cells, hypochromia, and microcytosis (see Fig. 49–11H). Osmotic fragility of the red cells is decreased. Hb electrophoresis shows greater than 90 percent HbE and 5 to 10 percent HbF. Certain chromatography techniques that can separate HbE from HbA₂ reveal elevated levels of HbA₂. Patients with mild forms of HbE–β⁺-thalassemia (Chap. 48) have Hb levels in the 9.0 to 9.5 g/dL range, whereas those with severe HbE–β⁺-thalassemia are more severely anemic (Hb 6.5 to 8.0 g/dL). Individuals with HbE–β⁰-thalassemia have varying degrees of anemia, depending on their ability to produce HbF; these patients have HbE in the 40 to 60 percent range, with the remainder being HbF. Patients with higher HbF values are less anemic.

Therapy

HbE homozygotes do not require any therapy. Patients with severe HbE–β⁰-thalassemia are similar to thalassemia intermedia or major; most of the latter patients should be on a chronic transfusion regimen aiming at Hb levels of approximately 10 g/dL; iron chelation should be a part of standard therapy. Splenectomy should be considered when hypersplenism develops. Patients with a thalassemia intermedia-like phenotype may require sporadic transfusions. Hydroxyurea can increase HbF levels and decrease ineffective erythropoiesis in HbE–β-thalassemia.⁴⁰⁸ AHSCT (including umbilical cord blood–derived stem cells in one patient) has also been used in HbE–β-thalassemia.

Course and Prognosis

The prognosis is dependent upon the clinical phenotype. Patients with milder phenotypes tend to do well. Severe HbE–β-thalassemia patients require chronic red cell transfusion and iron-chelation therapy; this places a great burden on the economies of countries where this disease is prevalent. AHSCT, although potentially curative, will not be available for the vast majority of these patients. Prenatal diagnosis and neonatal screening should be an important part of the strategies to decrease the disease burden and improve care. Long-term use of hydroxyurea and other novel HbF-inducing agents as modifiers of disease (histone deacetylase inhibitors and DNA methyltransferase 1 inhibitors) can be an important addition to therapy.

HEMOGLOBIN D DISEASE

HbD was the third Hb variant identified.⁴⁰⁹ The substitution in HbD is a glutamic acid to glutamine at the 121st amino acid of the β-globin chain (β121Glu→Gln). HbD has an S-like mobility on alkaline electrophoresis, but comigrates with HbA on acid pH. Subsequently, a number of other Hb variants with the same electrophoretic properties were discovered and named HbD (HbD_Ibadan, HbD_Gainesville, etc.). The most common HbD is HbD_{Los Angeles} (β121Glu→Gln), the originally discovered HbD, which is identical to HbD_Punjab. It is most commonly found in Punjab, India where 2 to 3 percent of the population have the HbD gene. Subsequently, it has also been found in a number of other populations including Europeans of Mediterranean region, and Americans of African descent.⁴¹⁰

HbD heterozygotes are asymptomatic, are not anemic, and have normal red cell indices. Homozygotes for HbD_{Los Angeles} are asymptomatic and are hematologically normal with normal red cell indices. Blood films may show target cells (see Fig. 49–11G). Osmotic fragility may be decreased. Compound heterozygotes for HbD_{Los Angeles} and a β⁰-thalassemia mutation have mild microcytic anemia and show minimal hemolysis. Coinheritance of HbD_{Los Angeles} with HbS results in a severe SCD phenotype not different from homozygous HbS.

HbD_{Los Angeles} should be distinguished from HbS. This can be done by a combination of routine alkaline and acid Hb electrophoretic methods. Techniques such as isoelectric focusing, HPLC, and capillary electrophoresis readily provide this distinction. Such methods allow accurate diagnosis of SCD from compound heterozygosity for HbS and HbD_{Los Angeles}.

UNSTABLE HEMOGLOBINS

Unstable Hbs form an important group of clinically significant Hb variants. Several different mechanisms lead to the generation of unstable variants, which result in a congenital hemolytic anemia with inclusion bodies in red cells (Heinz bodies), hence the term congenital Heinz body hemolytic anemia.

Definition and History

Cathie reported a 10-month-old child with hemolytic anemia, jaundice, and splenomegaly in 1952.⁴¹¹ Splenectomy did not result in improvement. The patient’s red cells had large Heinz bodies (Chap. 31). Similar cases were reported from around the world, and the observation that these cases were characterized by the precipitation of their hemolysate upon exposure to heat, suggested a Hb abnormality as the cause. Subsequently, nearly all of similar cases were found to have a variant Hb, and Cathie’s case was found to have Hb-Bristol (β67Val→Asp). To date, 146 unstable variants have been reported; the vast majority is sporadic cases reported only once. Few have been observed repeatedly in different populations.

Etiology and Pathogenesis

Several different mechanisms lead to the instability of the globin molecule with precipitation in the red cell leading to hemolysis. These are summarized below.

Substitutions Near the Heme Pocket Heme is inserted into a hydrophobic pocket in each globin molecule where it is in contact with a number of invariant nonpolar amino acid residues (see Fig. 49–2). Substitution of these invariant nonpolar residues will decrease the stability of heme-globin association and ultimately lead to the instability of the globin moiety. Hb_Zurich (β63His→Arg), Hb_Koln (β98Val→Met), and Hb_Hammersmith (β42Phe→Ser) are examples of this group.

Disruption of Secondary Structure (α-Helix) The secondary structure of globin chains is 75 percent in the conformation of an α helix (see Fig. 49–1). Proline residues cannot participate in an α helical conformation. Thus, the substitution of a proline residue for any other amino acid except for the first three residues of an α helix will disrupt the secondary structure and lead to the disruption and precipitation of the mutant globin chain.

Mutations in α₁β₁ Interface The first step in the assembly of the Hb tetramer is the formation of an αβ dimer. This structure is stabilized by a secondary structure that exposes the charged amino acids (glutamic acid, aspartic acid, lysine, and arginine) on the surface of the molecule in contact with water and stabilizes the interior of the molecule (α₁β₁ interface) with hydrophobic interactions. Substitution of a charged (polar) residue for a nonpolar amino acid involved in α₁β₁ contact will disrupt and destabilize this dimer formation and lead to the precipitation of the Hb molecule.

Amino Acid Deletions Deletion of one or more amino acid residues is expected to disrupt the secondary structure of the globin chains and may lead to instability of the mutant chain. Mutant globins with deletion of one or more residues have been reported. Examples of this type include Hb_Leiden (β6 or β7Glu→0), Hb_{Gun Hill} (β91-95→0), and Hb_Freiburg (β23Val→0).

Elongated Globin Chains Some variants result from either a mutation in the termination codon or a frameshift leading to the synthesis of longer than normal globin chains. These variants tend to be unstable because of the presence of a nonfunctional fragment. Examples include Hb_Cranston and Hb_Tak.

Whatever the underlying mechanism may be, unstable Hb variants precipitate within developing red cell precursors forming hemichromes (intermediate substances in Hb denaturation) and ultimately aggregates that attach to the inner layer of red cell membrane (Heinz bodies). Heinz bodies can be visualized with supravital stains, such as brilliant cresyl blue. Red cells with Heinz bodies have impaired rheologic properties (deformability and filterability) and are trapped in the splenic circulation (Chaps. 6, 34, and 56) with pitting of the membrane attached bodies. Hemolysis ultimately ensues. The degree of hemolysis is proportionate to the quantity and the instability of the variant.

Clinical Features

Patients with unstable Hb variants have varying degrees of hemolytic anemia. This can range from a compensated, asymptomatic hemolytic state to severe, life-threatening hemolysis. Generally, hemolytic anemia is mild to moderate and does not require therapeutic intervention. Typically, hemolysis is exacerbated by increased oxidant stress such as infections and the use of oxidant drugs. Patients may have jaundice and splenomegaly. As is the case with other chronic hemolytic states, gallstones may develop. Hypersplenism can be a problem in some cases. Many unstable Hb variants that are associated with mild, compensated hemolysis are diagnosed fortuitously or during population screening for hemoglobinopathies. Unstable variants are inherited in a mendelian pattern; they usually manifest in the heterozygous state. There are instances of de novo mutations without evidence of the variant in parents of an affected individual. Many of the 146 known unstable variants are found in a single individual or in a limited number of instances. However, some unstable variants like Hb_Koln (β98Val→Met) and Hb_Zurich (β67His→Arg) have been found in many populations around the world.

Laboratory Features

Patients with unstable Hb variants may have varying degrees of anemia. Generally, the anemia is mild and does not require therapeutic intervention. However, exacerbation of anemia during exposure to oxidant stress (such as infections and the use of oxidant drugs) is a common feature. Features of a hemolytic state (reticulocytosis, indirect hyperbilirubinemia, elevated LDH, decreased or undetectable haptoglobin) are present. Red cell morphology shows polychromasia, anisocytosis, poikilocytosis, and occasionally basophilic stippling. A typical feature of this disorder is the presence of Heinz bodies, best visualized with supravital staining with brilliant cresyl blue as membrane attached inclusion bodies in red cells. Hb electrophoresis reveals the presence of an additional abnormal Hb band. The quantity of the variant Hb is variable and inversely proportional to the degree of instability of the abnormal Hb (e.g., the more unstable the variant, the less the quantity). More accurate quantification can be achieved with cation exchange or reversed phase HPLC. The presence of an unstable variant in the hemolysate can be demonstrated by simple tests of stability. The most commonly used tests are heat denaturation and isopropanol precipitation. The heat denaturation test is more cumbersome and time consuming and is seldom used in practice. The isopropanol precipitation test is a simple screening test for unstable variants and involves the incubation of the hemolysate with a 17 percent solution of isopropanol; hemolysates containing unstable Hb variants will form a precipitate, whereas a normal hemolysate will remain clear.

HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY

M Hbs result from mutations around the heme pocket that disrupt the hydrophobic nature of this structure with resultant oxidation of the iron in the heme moiety from ferrous (Fe²⁺) to ferric (Fe³⁺) state and cause methemoglobinemia (Chap. 50). Mutations in certain critical areas of the globin molecule alter the affinity of the globin for oxygen. In general, mutations that stabilize the molecule in the T state lead to low O₂-affinity variants, which can clinically manifest as cyanosis or mild anemia. Mutations that stabilize the R state or destabilize the T state result in high O₂-affinity variants. These variants will cause secondary polycythemia (Chap. 57). The mutations that affect the ligand binding affinity of the Hb molecule are mostly in the α₁β₂ interface. Rarely, mutations in the α₁β₁ interface lead to altered O₂ affinity. Another mechanism in the generation of high O₂ affinity mutants involves mutations that alter the binding of 2,3-BPG.