Plasma levels of TNF-alpha in sickle cell patients receiving hydroxyurea

Hydroxyurea (HU), a chemotherapeutic agent, used increasingly in the treatment of sickle cell disease (SCD) stimulates the release of a tumor necrosis factor (TNF-alpha) from human macrophages in vitro and the concentration of TNF-alpha is greater than normal in subjects affected by SCD. It is widely accepted that HU may inhibit vaso-occlusive crisis (VOC) by stimulating the production of fetal hemoglobin (HbF) and nitric oxide (NO) in SCD; however, the beneficial effects of HU in vivo may be counteracted by the release of TNF-alpha and, in turn, the expression of a vascular cell adhesion molecule (VCAM-1) on leukocytes. Previous studies have shown that the severity of SCD increases with the leukocyte count. Therefore, we examined the relationship between plasma levels of TNF-alpha and HbF in SCD patients during steady-state (StSt) conditions (in the absence of VOC) and during VOC conditions after the acute administration of HU. Venous blood was collected in SCD patients over 6 h after administering a single dose of HU. Plasma TNF-alpha was found to be greater in SCD subjects than in reported normal adult controls (p<0.05). TNF-alpha in the StSt group was not significantly different than in the VOC group; however, the plasma TNF-alpha tended to greater in the VOC group (p>0.1). An increase in the HbF concentration after acute administration of HU (p<0.01) was not associated with a significant change in plasma TNF-alpha (p>0.1). Contrary to the results of in vitro studies, HU did not increase the plasma concentration of TNF-alpha. These findings suggest that a HU-induced increase in TNF-alpha does not contribute to VOC and sickle cell patients can be counseled that the HU-induced increase in TNF-alpha does not counteract the beneficial effects of HU in SCD.     

Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy

An elevated erythrocyte arginase activity with a corresponding decrease in nitric oxide (NO) level has been implicated in the pathophysiology of sickle cell disease (SCD). Recent studies have shown that hydroxyurea (HU) increases the production of NO, which increases the soluble guanylate cyclase activity and fetal haemoglobin (HbF) synthesis. To study the effects of HU on the arginase and nitric oxide synthase (NOS) activities in SCD patients, we compared levels of arginase activity and NO metabolites in red blood cells and plasma, respectively, from 23 patients with SCD (HbSS) receiving HU therapy, with those of 12 SCD patients not receiving HU treatment. Patients on HU therapy showed significantly lower arginase activity than that of HbSS patients not on HU therapy (1.36+/-0.2 U/10(8) cells vs. 3.31+/-0.29 U/10(8) cells). NOS activity was higher in patients on HU therapy than in untreated patients (0.72+/-0.4 nmol/ml/min vs. 0.35+/-0.15 nmol/ml/min, P<0.05). Among the HU-treated patients, the decreased level of arginase activity correlated (r=0.71) with HbF level as well as the mean corpuscular haemoglobin content. These data suggest that one of the beneficial effects of HU in vivo may involve the regulation of arginase activity and a concomitant induction of NOS activity, which may lead to an increased production of NO. The outcome of this study may lead to the development of improved NO-based treatments for SCD.     

Hematology Morphology Forum Case 1

Hydroxyurea (HU), a chemotherapeutic agent, used increasingly in the treatment of sickle cell disease (SCD) stimulates the release of a tumor necrosis factor (TNF-) from human macrophages in vitro and the concentration of TNF- is greater than normal in subjects affected by SCD. It is widely accepted that HU may inhibit vaso-occlusive crisis (VOC) by stimulating the production of fetal hemoglobin (HbF) and nitric oxide (NO) in SCD; however, the beneficial effects of HU in vivo may be counteracted by the release of TNF- and, in turn, the expression of a vascular cell adhesion molecule (VCAM-1) on leukocytes. Previous studies have shown that the severity of SCD increases with the leukocyte count. Therefore, we examined the relationship between plasma levels of TNF- and HbF in SCD patients during steady-state (StSt) conditions (in the absence of VOC) and during VOC conditions after the acute administration of HU. Venous blood was collected in SCD patients over 6 h after administering a single dose of HU. Plasma TNF- was found to be greater in SCD subjects than in reported normal adult controls (p<0.05). TNF- in the StSt group was not significantly different than in the VOC group; however, the plasma TNF- tended to greater in the VOC group (p>0.1). An increase in the HbF concentration after acute administration of HU (p<0.01) was not associated with a significant change in plasma TNF- (p>0.1). Contrary to the results of in vitro studies, HU did not increase the plasma concentration of TNF-. These findings suggest that a HU-induced increase in TNF- does not contribute to VOC and sickle cell patients can be counseled that the HU-induced increase in TNF- does not counteract the beneficial effects of HU in SCD.     

Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells

OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.     

Hematologic,biochemical, and cardiopulmonary effects of l‐arginine supplementation or phosphodiesterase 5 inhibition in patients with sickle cell disease who are on hydroxyurea therapy

Objectives: Fetal hemoglobin (HbF) induction involves NO‐cGMP signaling pathways. l ‐arginine, an NO precursor, and the phosphodiesterase (PDE) 5 inhibitor sildenafil, which potentiates cGMP, were studied in adults with sickle cell disease (SCD) who were stably on HU. Methods: Twenty four courses of l ‐arginine (0.1–0.2 g/kg divided TID) or sildenafil (25–100 mg TID), assigned based on gender due to concerns about sildenafil‐related priapism, were successfully completed. Biochemical assays, pulmonary pressures, and cardiopulmonary exercise capacity are reported from patients in whom serial values are available. Hematologic responses are reported in 14 subjects with HbSS who had stable baseline HbF levels. Results: l ‐arginine increased plasma arginine and ornithine, but not citrulline, suggesting diversion by plasma arginase from NO, and citrulline, generation. Glutathione increased only in patients on l ‐arginine. Sildenafil increased plasma cGMP and citrulline, but not other amino acids. Pulmonary pressures and 6‐min walk distances improved only in patients on sildenafil. In subjects with stable baseline HbF levels, HbF levels changed little from a normalized baseline on l ‐arginine, decreasing by 2.9 ± 16.1%, n = 6; P = n.s., but increased on sildenafil, by 7.5 ± 11.7%, n = 8, P < 0.05. Absolute reticulocyte counts initially decreased in patients on sildenafil. Conclusions: l ‐arginine, at doses that increase plasma arginine levels, altered redox potential in red cells. The lack of clinically detectable efficacy of l ‐arginine may be due to increased arginine metabolism in SCD patients. In vivo augmentation of the cyclic nucleotide pathway by PDE inhibition may induce HbF slightly, but strikingly improves hemodynamic and functional status in SCD.     

Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells

Hydroxyurea, a drug widely used for treating myeloproliferative diseases, has also been approved for the treatment of sickle cell disease by raising fetal hemoglobin (HbF). We have shown that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea induction of HbF levels in erythroid progenitor cells (EPCs). We demonstrate now that during erythroid differentiation, endothelial NO synthase mRNA and protein levels decline steadily, as does the production of NO derivatives and cyclic adenosine monophosphate (cAMP) levels, but guanosine 3',5'-cyclic monophosphate (cGMP) levels are stable. Hydroxyurea increased intracellular cGMP levels and cAMP levels in EPCs. The NO donor, DEANONOate, induced much higher cGMP levels, but reduced cAMP levels. Hydroxyurea (1 mM) induced production of approximately 45 pM cGMP/minute/ng of purified sGC, similar to induction by 1 muM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of sGC. Thus, we confirm that hydroxyurea can directly interact with the deoxy-heme of sGC, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the NO/cGMP pathway in EPCs. These mechanisms may also be involved in the cytostatic effects of hydroxyurea, as well as the induction of HbF.     

Insulin-inhibited and stimulated cultured vascular smooth muscle cell migration are related to divergent effects on protein phosphatase-2A and autonomous calcium/calmodulin-dependent protein kinase II

Insulin, in the permissive presence of nitric oxide (NO), stimulates cGMP production which inhibits autonomous calcium/calmodulin-dependent protein kinase II (CaM kinase II) thereby inhibiting cultured vascular smooth muscle cell (VSMC) migration. In the presence of angiotensin II (Ang II), insulin stimulates NAD(P)H oxidase activity leading to increased VSMC migration. We wished to see whether insulin-stimulated cGMP stimulates protein phosphatase-2A (PP-2A) thereby inhibiting autonomous CaM kinase II and migration, and whether insulin, in the presence of Ang II, inhibits PP-2A and stimulates autonomous CaM kinase II in a NAD(P)H oxidase-dependent manner. One nanomole per litre of insulin in the presence of NO, or 50 micromol/L 8-Br-cGMP stimulated PP-2A activity by 46+/-6 and 247+/-23%, respectively (both P<0.05), and 8-Br-cGMP inhibited autonomous CaM kinase II activity by 67+/-9% (P<0.05) by a 10 nmol/L okadaic acid-sensitive pathway. Insulin plus Ang II inhibited PP-2A activity by 57+/-7% (P<0.05) and stimulated autonomous CaM kinase II activity by 120+/-14% (P<0.05), both by an apocynin-sensitive pathway. 8-Br-cGMP-inhibited VSMC migration was blocked by okadaic acid. It is concluded that insulin in the presence of NO stimulates cGMP which stimulates PP-2A activity causing inhibition of autonomous CaM kinase II activity and thus VSMC migration, and that insulin in the presence of Ang II inhibits PP-2A and stimulates autonomous CaM kinase II activities by a NAD(P)H oxidase-dependent mechanism which are associated with insulin-stimulated NAD(P)H oxidase-dependent migration.     

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease

Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an “HbF per red blood cell (HbF/RBC) threshold” requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF - the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti-Human-HbF antibody, was obtained from non-SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso-occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.     

Nitric Oxide Metabolites in Sickle Cell Anemia Patients after Oral Administration of Hydroxyurea; Hemoglobinopathy

The mechanism of action of hydroxyurea (HU) in decreasing the frequency of pain crisis in sickle cell disease (SCD) has not been fully elucidated. In vitro and in vivo studies suggest that nitric oxide (NO), a potent vasodilator, may partly be responsible for the beneficial effect of HU. This study was designed to determine the effect of oral administration of HU on plasma levels of NO metabolites (NO(x) ) in sickle cell patients (SCP). The results indicate that during steady-state plasma levels of NO(x) were significantly higher in HU-treated patients compared to non HU-treated patients or normal controls (p <.05). In five inpatients in mild pain plasma levels of NO(x) increased significantly after 2 h of HU administration (p <.05); however, in three inpatients in persistent pain with significantly lower baseline NO(x) there was a minimal NO(x) response to HU at 2 h (p <.01). These observations indicate that HU administration is associated with the production of NO in some SCP, but that further study of the pharmacodynamics of this effect is necessary.     

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.     

Decreased plasma endothelin-1 levels in children with sickle cell disease treated with hydroxyurea

Plasma endothelin-1 (ET-1) is elevated in patients with sickle cell disease (SCD). Hydroxyurea (HU) is the only drug with demonstrated clinical efficacy in SCD. Here we show that treatment with HU results in a decreased concentration of circulating ET-1 which is not correlated with the HU-induced increase in HbF level. Blunting of the ET-1 vasoconstrictive stimulus could contribute to the beneficial effects of HU.     

Sodium phenyl butyrate downregulates endothelin-1 expression in cultured human endothelial cells: relevance to sickle-cell disease

As hydroxyurea (HU), sodium phenyl butyrate (SPB) is known to induce fetal hemoglobin (HbF) expression and thus shows potentials for sickle-cell disease (SCD) treatment. More recently, few studies suggested that endothelial cells (ECs), a major pathophysiological actor of SCD, are also a target of SPB. Here, we show that SPB, as HU, reduces endothelin-1 mRNA expression and peptide release by human ECs in culture. SPB increases VCAM-1 and ICAM-1 mRNAs and soluble ICAM-1 release. Both drugs have a cumulative effect on ICAM-1 expression. We conclude that SPB, as HU, also affects the expression of molecules important to the pathophysiology of SCD, in addition to its effect on HbF. Its potential as an alternative or adjuvant drug in SCD treatment warrants further investigations.     

Beta-globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon

Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the beta-globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the beta-globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the epsilon-Ggamma-Agamma-psibeta-delta-beta-globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.     

The Effect of Hydroxyurea Therapy in Bahraini Sickle Cell Disease Patients

Hydroxyurea (HU) is used as a disease-modifying agent in sickle cell disease (SCD). Its beneficial effects have been ascribed to inhibition of the sickling process through increase of fetal hemoglobin (HbF) levels and influence on multiple factors affecting adhesion of erythrocytes to vascular endothelium. The present study investigates the effect of HU in SCD patients who were grouped on the basis of association with α- and β-thalassemia using routine laboratory methods. A retrospective cross-sectional chart-review was done of 51 adult Bahraini SCD patients attending Salmaniya Medical Complex, Bahrain. Four sub-groups of cases were identified: (i) homozygous sickle cell anemia, 24 cases; (ii) SCD with microcytosis, 16 cases; (iii) sickle α-thalassemia, seven cases; and (iv) sickle β thalassemia, four cases. Documented laboratory and clinical data included hemoglobin level (Hb), hematocrit (Hct), red cell indices, hemoglobin fractions, hospital admissions (frequency), number of inpatient-days, pain episodes (frequency) and red cell transfusion requirement (number of units). Pre- and post-treatment data were compared. Hydroxyurea treatment led to highly significant reduction of HbS % and pain crisis episodes in all patient groups. Other changes such as increases of total hemoglobin, Hct and HbF and reduction of hospital admissions, inpatient days and red cell units transfused also occurred but with less consistent levels of significance within patient sub-groups. Treatment with HU is beneficial for all subgroups of Bahraini SCD patients, without or with α- and β-thalassemia interactions.     

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.     

Regulation of basal myocardial function by NO.

The effects of exogenous and endogenous. NO on myocardial functions such as contraction, relaxation and heart rate have recently gained considerable scientific interest. .NO stimulates myocardial soluble guanylate cyclase to produce cGMP, which activates two major target proteins. A small increase in cGMP levels predominantly inhibits phosphodiesterase III, while high cGMP levels activate cGMP-dependent protein kinase. Accordingly, submicromolar .NO concentrations improve myocardial contraction, while submillimolar .NO concentrations decrease contractility. The latter action includes direct inhibitory .NO effects on ATP synthesis and voltage-gated calcium channels. Overall, the inotropic effects of exogenous .NO are small and probably of minor importance for myocardial contractility. Cardiomyocytes are capable of expressing eNOS and iNOS. Endogenous .NO has effects on myocardial contraction, similar to that of exogenous .NO. Various NOS inhibitors can substantially reduce myocardial contractility in vitro and in vivo, suggesting that basal endogenous .NO production supports myocardial contractility. There is also evidence for a .NO-dependent cardiodepressive effect of cytokines that is mediated by expression of iNOS. This is consistent with the negative inotropic effects of .NO at high concentrations. Cardiodepressive actions of endogenous .NO production may play a role in certain forms of heart failure. Finally, .NO also has an effect on heart rate. Physiologic .NO concentrations can stimulate heart rate by activating the hyperpolarization-activated inward current (If) and this effect decreases at submillimolar .NO concentrations. In summary, physiological concentrations of .NO increase contractility and heart rate under basal conditions, while high .NO concentrations induce the opposite effects.     

Nitric oxide down-regulates brain-derived neurotrophic factor secretion in cultured hippocampal neurons

The regulation of neurotrophin (NT) secretion is critical for many aspects of NT-mediated neuronal plasticity. Neurons release NTs by activity-regulated secretion pathways, initiated either by neurotransmitters and/or by existing NTs by a positive-feedback mechanism. This process depends on calcium release from intracellular stores. Little is known, however, about potential pathways that down-regulate NT secretion. Here we demonstrate that nitric oxide (NO) induces a rapid down-regulation of brain-derived neurotrophic factor (BDNF) secretion in cultured hippocampal neurons. Similar effects occur by activating a downstream target of intracellular NO, the soluble guanylyl cyclase, or by increasing the levels of its product, cGMP. Furthermore, down-regulation of BDNF secretion is mediated by cGMP-activated protein kinase G, which prevents calcium release from inositol 1,4,5-trisphosphate-sensitive stores. Our data indicate that the NO/cGMP/protein kinase G pathway represents a signaling mechanism by which neurons can rapidly down-regulate BDNF secretion and suggest that, in hippocampal neurons, NT secretion is finely tuned by both stimulatory and inhibitory signals.     

Hereditary persistence of hemoglobin F is protective against red cell sickling. A case report and brief review

Fetal hemoglobin (HbF) is a physiologic protein tetramer that is crucial for a developing fetus to survive in utero. Maternal hemoglobin has a relatively lower affinity for oxygen, and thus allows for an efficient transfer of oxygen from maternal to fetal blood. In addition to fulfilling a critical physiologic role, HbF is also known to alleviate symptoms of sickle-cell disease (SCD). The concentration of HbF depends on several factors. HbF is elevated in inherited conditions, such as hereditary persistence of HbF, hereditary spherocytosis, and thalassemia. The level of HbF is also increased in acquired states, such as pregnancy, aplastic anemia, thyrotoxicosis, hepatoma, myeloproliferative disorders, or hypoplastic myelodysplastic syndrome. It has been identified that some genetic loci have significant influence on HbF levels. The XmnI polymorphism, the HMIP locus, and the BCL11A gene are responsible for 45% of variations in HbF levels. Although SCD has been well described in the subpopulations of Africa, it is less common in the subpopulations of India. We describe a case of SCD, in which a patient with high HbF level presented at a very late age (27 years old). We presume the patient’s inherently elevated HbF levels were able to compensate for the hypoxic episodes associated with SCD. The onset of symptoms was delayed as a result of elevated HbF levels.