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Tricuspid regurgitant (TR) jet velocity and its relationship to pulmonary hypertension has been controversial in sickle cell disease (SCD). Plasma free hemoglobin is elevated in SCD patients and acutely impairs systemic vascular reactivity. We postulated that plasma free hemoglobin would be negatively associated with both systemic and pulmonary endothelial function, assessed by flow-mediated dilation (FMD) of the brachial artery and TR jet velocity, respectively. Whole blood viscosity, plasma free hemoglobin, TR jet, and FMD were measured in chronically transfused SCD pre- and posttransfusion (N = 25), in nontransfused SCD (N = 26), and in ethnicity-matched control subjects (N = 10). We found increased TR jet velocity and decreased FMD in nontransfused SCD patients compared with the other 2 groups. TR jet velocity was inversely correlated with FMD. There was a striking nonlinear relationship between plasma free hemoglobin and both TR jet velocity and FMD. A single transfusion in the chronically transfused cohort improved FMD. In our patient sample, TR jet velocity and FMD were most strongly associated with plasma free hemoglobin and transfusion status (transfusions being protective), and thus consistent with the hypothesis that intravascular hemolysis and increased endogenous erythropoiesis damage vascular endothelia.

Introduction: Sickle cell disease (SCD), an inherited blood disorder, is characterized by episodes of painful vaso-occlusive crises (VOC). Peripheral vasoconstriction may contribute to VOC by prolonging the transit time of red blood cells through the microvasculature. We recently demonstrated that heat-induced pain produces stronger vasoconstriction in SCD subjects versus controls, suggesting abnormal autonomic regulation of regional blood flow in SCD. Thus, although pain is generally thought to be a consequence of VOC, it is possible that pain itself may trigger a cascade of events that leads to large-scale VOC by promoting regional peripheral vasoconstriction. Objective: To determine if the underlying functional mechanisms of the vasoconstriction response to heat-induced pain in SCD differ from normal controls. Experimental Protocols: 22 SCD and 23 control (healthy and sickle cell trait) subjects were recruited at Children's Hospital of Los Angeles. Quasi-periodic pulses of pain were delivered on the right forearm using TSA-II neuro analyzer heating thermode. Electrocardiogram, respiration, continuous blood pressure and photoplethysmogram (PPG) were recorded. Both blood pressure and PPG were measured on the ring finger and thumb on the contralateral hand. Reductions in PPG amplitude were taken to represent vasoconstriction. Analysis: A mathematical model was applied to the data to extract the key parameters relating the thermal (pain) pulses, blood pressure and respiration to vasoconstriction. The model enabled the vasoconstriction response to pain to be decomposed into: 1) a neurogenic component, measuring the direct effect of the thermal pain pulse on vasoconstriction; 2) a local vascular component, relating fluctuations in blood pressure, induced by pain, to vasoconstriction; 3) a neurogenic-vascular interaction component, reflecting the modulation of the vascular component by the pain stimulus; and 4) a respiratory coupling component. Results: The model predicted stronger vasoconstriction responses to heat pain in SCD than controls (p = 0.002), consistent with the previous results reported by Khaleel et al. (Blood 126 (23):67, 2015). The pattern of the neurogenic response in both groups was similar but stronger in SCD (Fig 1a). The time-course of the local vascular component was quite pronounced in SCD (Fig 1b) - increases in blood pressure promoted stronger and more rapid vasoconstriction in SCD relative to controls. The neurogenic-vascular interaction (not shown) caused further peripheral vasoconstriction in SCD, but tended to reduce vasoconstriction in controls. The respiratory contribution was small in both groups. Discussion & Conclusions: Heat-induced pain produces peripheral vasoconstriction via direct autonomic nervous system activation, as well as indirectly through the local vascular response to pain-triggered blood pressure fluctuations. The complex dynamics of the vasoconstriction response can only be understood by application of a mathematical model incorporating several relevant simultaneously measured and frequently sampled physiological signals. The stronger neurogenic response in SCD suggests elevated sympathetic activity compared to controls. The local vascular component in the SCD subjects exhibits a higher reactivity to increases in local blood pressure that promotes vasoconstriction, consistent with underlying endothelial dysfunction. The contribution of neurogenic-vascular interaction derived from the model likely represents the degree to which sympathetic overactivity leads to vascular dysfunction in SCD. These results suggest that dysautonomia and its interaction with peripheral vascular dysregulation participate in the genesis of vaso-occlusive crisis. Figure 1 (a) average neurogenic response (% change from baseline) to a heat pain pulse (20 °C, 10-sec long) in SCD (N = 22) and controls (N = 23); (b) average vascular response (% change from baseline) to a blood pressure pulse induced by pain (10 mmHg, 10-sec long) in SCD and controls. Figure 1. (a) average neurogenic response (% change from baseline) to a heat pain pulse (20 °C, 10-sec long) in SCD (N = 22) and controls (N = 23); (b) average vascular response (% change from baseline) to a blood pressure pulse induced by pain (10 mmHg, 10-sec long) in SCD and controls. Disclosures Wood: Biomed Informatics: Consultancy; AMAG: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Biomed Informatics: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy.

Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy in which hemoglobin polymerizes when deoxygenated, causing increased red blood cell (RBC) stiffness and RBC sickling. This leads to microcirculatory slowing and occlusion, in turn causing vaso-occlusive crises acutely and microcirculatory damage chronically. RBC are known to signal blood vessels in the microcirculation, regulating vessel tone. Nitric oxide (NO) is a potent vasodilator and key regulator of vascular smooth muscle tone throughout the vasculature. However, the possibility that RBC make NO and that this might contribute to vascular tone, particularly in arterioles is controversial. Modulation of RBC NO production due to deformation of RBC by shear stress could have important physiological consequences in SCD where RBC deformability is decreased and dependent on the polymerization state of hemoglobin S. Objective: To determine if shear stress induced deformation induces change in NO production in individual RBC and if the magnitude and time course of this NO differs between RBC from patients with sickle cell disease and normal controls. Methods: NO production was measured using imaging microscopy of fluorescently labeled RBC exposed to shear after being allowed to adhere to a flow chamber attached to the microscope stage. Four SCD patients on simple chronic transfusion therapy, 4 SCD patients not on chronic transfusion therapy and 4 healthy control subjects were recruited from the hematology clinics at Children's Hospital Los Angeles under an IRB approved protocol. RBC underwent histopaque separation then washed 3x and resuspended in KRP buffer. The RBC were then incubated with 4 micromolar DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate, ThermoFisher Scientific), a fluorescent indicator related to NO production. RBC were then exposed to three experimental conditions: 1. buffer alone; 2. 1 milimolar arginine (substrate for NOS NO production); 3. 1 milimolar arginine and 1 milimolar L-NAME (a non-specific NOS inhibitor). The RBC were placed in poly L-lysine coated microfluidic chambers and placed under no shear conditions for 30 minutesk, then sheared at 0.5Pa for 30minutes. Individual cell analysis was performed using Image J analysis software (FIJI). Results: There is a slow baseline rise of NO fluorescent signal without exposure to shear and there is a large, acute increase in NO fluorescent signal with exposure to shear stress. (figure 1) The baseline fluorescent rate of rise is increased in non-transfused sickle cell subjects (p Discussion: The results here indicate that RBC produce NO upon shear and the basal rate of NO production is higher in non-transfused patients with sickle cell disease. Addition of arginine increases both the magnitude and rate of NO production in all three groups. SCD patients on chronic transfusion therapy demonstrate a larger increase in NO response to shear. The increased response of NO related fluorescence with the addition of arginine suggests NOS has a role in shear mediated RBC NO production; however, L-NAME blockade does not significantly blunt the response in the presence of arginine. Shear induced RBC NO production may play an important role in regulation of microvascular profusion and would likely be modulated by the RBC deformability differences due to HbS polymerization. Conclusion: There is basal and shear dependent NO production in both AA and SCD RBC, and RBC NOS is differentially regulated in SCD and AA RBC. Figure 1 Response of RBC NO related fluorescence to shear stress Figure 1. Response of RBC NO related fluorescence to shear stress Figure 2 Basal rate of NO related DAF fluorescence. Figure 2. Basal rate of NO related DAF fluorescence. Disclosures Wood: Ionis Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Vifor: Consultancy; World Care Clinical: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Celgene: Consultancy; AMAG: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; Biomed Informatics: Consultancy; Biomed Informatics: Consultancy.

BACKGROUND: Sickle cell disease is an inheritable hemoglobinopathy that causes increased red blood cell (RBC) stiffness due to hemoglobin polymerization at low oxygen tension. This leads to microcirculatory occlusion as well as chronic hemolysis and chronic inflammation, resulting in a diffuse vasculopathy of bothmacrovascular and microvascular beds. Chronic transfusion therapy is used for both primary and secondary prevention of ischemic stroke, amacrovascular complication; however, there is limited knowledge of its effect on microvascular function. OBJECTIVE: To determine the effects of chronic transfusion therapy on microvascular function and tissue oxygenation. DESIGN/METHODS: We utilized a forearm, arterial and venous occlusion model, commonly used for flow-mediated dilation of the brachial artery, to simultaneously assess post occlusive reactive hyperemia (PORH) in the microcirculation and post hyperemic change in tissue oxygenation. PORH was measured using laser Doppler flowmetry placed over the third finger, proximal to the nailbed. Regional tissue oxygenation (rSO2) was measured using near infrared spectroscopy (NIRS) placed on the dorsal surface of the hand. FMD was measured using a linear ultrasound probe. Transfused patients were measured before and after a single transfusion. Kruskal-Wallis was used to test between group differences. Multivariate linear regression was used to model predictors of vascular function. RESULTS: We enrolled 18 control (CTL) patients, 75 non-transfused (NTR) SCD patients, and 26 chronically transfused (TR) SCD patients. Consistent with our previously published data, FMD was improved but not normalized by chronic transfusion and it was predicted by ln[plasma hgb], age and sex. PORH was reduced in both SCD patient groups when compared to the control group while resting flow by laser Doppler was elevated in both SCD groups (figure 1). PORH was also inversely related to resting flow, suggesting that basal conditions in the microcirculation are significant predictors of their response to forearm occlusion. Percent change in rSO2 was not different between the three groups. Lower baseline rSO2 was inversely associated with percent rSO2 increase following hyperemia; and, the baseline rSO2 was significantly decreased in the non-transfused SCD patients but normalized in the transfused SCD patients (Figure 2). Resting hypoxia was best predicted by RBC deformability in the non-transfused patients, even after correction for hemoglobin level, whereas resting hemoglobin levels predicted hypoxia in the transfused and healthy patients. DISCUSSION: With the goal to better understand the mechanism by which transfusion therapy decreases the risk of stroke, we tested the response to arterial and venous occlusion, simultaneously, at three different functional levels of the vascular system: 1.Conduit artery, 2.Pre-capillary arteriolar and capillary, 3.Tissue oxygen exchange. We confirm our previous findings that conduit vessel dysfunction is highly dependent on free hemoglobin, which is consistent with the hemolysis paradigm as a mechanism for sickle cell vasculopathy. This study extends our model to the microcirculation. Resting microcirculatory flow is increased in both transfused and non-transfused SCD patients; and while the resting rSO2 is near normal in the transfused SCD patients it is significantly lower in the non-transfused subjects. This creates the potential for severe hypoxia in the setting of a limited ability to augment flow through the microcirculation during periods of high demand, as in PORH. If there is also decreased conduit vessel reactivity upstream this creates the scenario over which large areas of tissue may rapidly become ischemic. This is a novel system, with simultaneous measurements in multiple vascular beds, allowing us to evaluate supply-demand matching. CONCLUSIONS: Chronic transfusion therapy improves both brachial artery endothelial function and regional tissue oxygenation but it does not significantly improve microcirculatory function, which may be near its maximal flow at rest. Hemoglobin determines resting tissue saturation in healthy and transfused SCD patients, but high shear RBC deformability determines rSO2 in non-transfused SCD patients. Figure 1. Laser Doppler resting flow is higher but post occlusive hyperemia is lower in SCD Figure 1. Laser Doppler resting flow is higher but post occlusive hyperemia is lower in SCD Figure 2. NIRS repsonse to forearm occlusion. Figure 2. NIRS repsonse to forearm occlusion. Disclosures Wood: World Care Clinical: Consultancy; Vifor: Consultancy; AMAG: Consultancy; Ionis Pharmaceuticals: Consultancy; Biomed Informatics: Consultancy; World Care Clinical: Consultancy; Ionis Pharmaceuticals: Consultancy; Apopharma: Consultancy; Vifor: Consultancy; Apopharma: Consultancy; AMAG: Consultancy; Biomed Informatics: Consultancy; Celgene: Consultancy; Celgene: Consultancy.

This study was designed to assess the binding of glycophorin A–specific antibodies to polyethylene glycol (PEG)-modified red blood cells (RBCs) and evaluate their resistance to invasion byPlasmodium falciparum malaria parasites. RBCs were conjugated with a range of concentrations (0.05 to 7.5 mM) of activated PEG derivatives of either 3.35 or 18.5 kd molecular mass. The binding of glycophorin A–specific antibodies was assessed by hemagglutination and flow cytometry. PEG-modified RBCs were assessed for their ability to form rosettes around Chinese hamster ovary (CHO) cells transiently expressing the glycophorin A binding domain of EBA-175, a P falciparum ligand crucial to RBC invasion. PEG-RBCs were also tested for their ability to be invaded by the malaria parasite. RBCs coated with 3.35 and 18.5 kd PEG demonstrated a dose-dependent inhibition of glycophorin A–specific antibody binding, CHO cell rosetting, and P falciparum invasion. These results indicate that glycophorin A epitopes responsible for antibody and parasite binding are concealed by PEG coating, rendering these cells resistant to P falciparum invasion. These studies confirm the effectiveness of PEG modification for masking RBC-surface glycoproteins. This may provide a means to prevent alloimmunization in the setting of RBC transfusion and suggests a novel method to enhance the effectiveness of exchange transfusion for the treatment of cerebral malaria.

Hemoglobinopathies, such as β-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34+ hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of β-globin production, with negligible γ-globin synthesis. To test the gene therapy potential using this model, CD34+ cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34+ cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

The vasoocclusive process in patients with sickle cell disease (SCD) is complex and involves interactions among sickle erythrocytes (SS-RBC), vascular endothelium, and plasma and cellular components. The role of neutrophils (PMN) in vasoocclusion has not been examined. Patients with SCD appear to have chronically activated PMN. Because the first step in PMN activation is particle recognition, we explored whether normal PMN recognize SS-RBC and whether this recognition results in PMN monolayers, significantly more SS-RBC adhered to the PMN than did normal erythrocytes (AA-RBC; P < .001). Preincubation of erythrocytes with autologous plasma significantly increased the adherence of SS-RBC to PMN but had no effect on AA-RBC (P < .001). When adhesion of density fractionated SS-RBC was performed, dense SS-RBC showed greater adherence to the PMN monolayers than did light SS-RBC (P < .001). To determine mechanisms of this adhesion, IgG and Arg-Gly-Asp-Ser (RGDS) receptor sites on PMN were saturated. IgG inhibited adherence of dense SS-RBC, whereas RGDS inhibited adherence in both fractions, although to a greater extent in the light fraction. We measured SS-RBC activation of PMN by incubating SS-RBC with 2′, 7′-Dichloro-fluroescin Diacetate (DCF)-labeled PMN. Incubation of PMN with SS-RBC resulted in a significant increase in fluorescence compared to AA-RBC. We show here that PMN recognize SS-RBC through multiple mechanisms and that this recognition results in activation of PMN. These findings contribute to the understanding of vasoocclusive crisis in patients with SCD and may have therapeutic implications.

Acute chest syndrome (ACS) is a potentially fatal complication of sickle cell disease (SCD) and is characterized by opacification of the chest x-ray (CXR) and progressive pulmonary failure due, in part, to intra-pulmonary sickling. The ACS process can proceed very rapidly from a small area of lung involvement in one lobe to total opacification of the lung and pulmonary failure within 12 to 24 hours. In the early phases of this process, oxygenation and pulmonary function may be preserved. In the face of rapidly progressing CXR changes, the ACS process may be reversed if diagnosed early and managed by emergent transfusion to decrease the percent of sickle red blood cells (SRBC). A 10 years old African American child with hemoglobin SC type SCD was transferred to our institution with fever and right upper lobe consolidation. Her respiratory rate was 23 breaths/min, SpO2 was 95% breathing room air. Serial CXR showed opacification of the entire right lung and part of the left lower lobe over a 12-hour period (Panel A). Because of the rapid progression, transfusion was recommended. However, because of the family's Jehovah's Witness religious faith, transfusion was refused. PEG-COHb is in clinical development for the treatment of SCD and is designed to deliver preloaded carbon monoxide (CO), pick up O2, and deliver O2 to hypoxic tissue. PEG-COHb serves as a vasodilator and anti-inflammatory agent. It has been shown to have anti-sickling properties in vitro (ASH Abstract 1372, 2014). The agent was obtained from Prolong Pharmaceuticals via an emergency IND (16432) from the FDA. The agent was acceptable to the family and church elders. After written consent was obtained, 500 cc were infused according to dosing information obtained from Prolong Pharma. The CXR (Panel A) 3 hours before infusion shows opacification of the right lung and the left lower lobe. A CXR obtained one hour after infusion showed no worsening, and the CXR (Panel B) obtained 29 hours after Panel A shows significant improvement in the opacification of the lower lobes. The right upper lobe consolidation was likely bacterial pneumonia, and would not be expected to clear rapidly. The patient was mildly hypertensive for age (138/72 mmHg) prior to PEG-COHb infusion. Her blood pressure rose to 153/85 mmHg during infusion; the infusion was stopped and anti-hypertensives were administered. The infusion was restarted at a lower infusion rate and completed in 6 hours instead of the planned 4 with no untoward effects. She was discharged 4 days after the infusion. There were no other serious adverse events clearly related to the drug. There were significant laboratory abnormalities and transaminases that were most likely falsely elevated due to interference of the PEG-COHb with the laboratory methods. Continuous non-invasive monitoring of carboxyhemoglobin showed basal levels of 7% rose to 24% during infusions and returned to normal prior to discharge. Continual recording of SpO2, methemoglobin, heart-rate variability and blood rheological measures showed no significant abnormalities. The rapid reversal of radiographic features consistent with progressive "pure ACS" secondary to the right upper lobe infectious process suggests that PEG-COHb may be an effective treatment for sickle cell related ACS. SHAPE Figure 1. Panel A demonstrates the chest x-ray 3 hours prior to PEG-COHb with right upper lobe consolidation and evolving bilateral lower lobe consolidation and Panel B 29 hours following administration of PEG-COHb demonstrating improvement in the lower lobes. Figure 1. Panel A demonstrates the chest x-ray 3 hours prior to PEG-COHb with right upper lobe consolidation and evolving bilateral lower lobe consolidation and Panel B 29 hours following administration of PEG-COHb demonstrating improvement in the lower lobes. Disclosures Off Label Use: SANGUINATE (pegylated carboxyhemoglobin bovine) is 40 mg/mL of purified bovine hemoglobin that has been pegylated, saturated with carbon monoxide, and dissolved in a buffered saline solution.. Abuchowski:Prolong Pharmaceuticals: Employment. Parmar:Prolong Pharmaceuticals: Employment.

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

Abstract 4773 Background: The pathophysiology of sickle cell disease (SCD) is commonly thought to be in part a result of sickled red blood cells obstructing blood flow leading to hypoxic cellular injury. The most common SCD complication is painful vaso-occlusive crisis (VOC). However, the physiologic changes that occur at the site of VOC are poorly understood and there are no objective measurements of VOC. We hypothesized that regional oxygenation at the site of pain would be decreased from the patient's asymptomatic baseline and could be used as an objective measurement of the VOC process. Methods: Subjects were enrolled at the time of hospitalization for unilateral painful VOC. The subjects completed the adolescent pediatric pain tool (APPT) to assess the location, severity and quality of their pain. Regional oxygenation (RSO2) was measured by near infrared spectroscopy (NIRS) at the site that the subject identified as where they experienced the most severe VOC related pain. NIRS measurements were also made on the contralateral body site. NIRS measurements were made for 15 minutes with the subject in the supine position. Measurements were made in the first 24 hours of hospitalization. Blood pressure, heart rate, pulse oximetry, hematologic parameters and use of oxygen were determined. The subject returned 4 weeks after discharge for repeat testing to determine their physiologic post VOC RSO2 baseline. Mean values for the NIRS testing period were determined and the change in RSO2 during VOC from post VOC baseline were calculated. Results: 5 female subjects with SCD were studied, mean age 15.8±3.0years. There was an average pain score of 7.4/10+1.9. Pain occurred in the back of 2 subjects and the lower extremities in the others. The RSO2% in the painful and not painful sites was increased from baseline by an average of 10.6±4.0% and 16.6±5.6%, respectively. The painful site had a decreased RSO2% in comparison to the contralateral side in 2 of 5 subjects (see figure). The RSO2% VOC baseline at the painful site post was higher in 3 of 5 subjects. There was no difference from baseline in blood pressure, heart rate, or arterial oxygen saturations during VOC. There were changes in the hematologic parameters consistent with VOC and active hemolysis. Conclusions: The SCD subjects demonstrated increased regional oxygenation at both the painful and contralateral not painful sites during VOC compared to the post VOC baseline. During VOC there was no predictable difference in regional oxygenation at the site of VOC pain contrary to our original hypothesis. Disclosures: Wood: Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding. Coates:Novartis: Speakers Bureau; Apopharma: Consultancy.

Abstract 1001 Introduction: SCT occurs in 8% of African Americans and is not commonly associated with clinical disease. Nonetheless, the United States Armed Forces has reported that SCT conveys a 30-fold risk of sudden cardiac arrest and a 200-fold risk from exertional rhabdomyolysis. In fact, rhabdomyolysis in athletes with SCT has been the principal cause of death in NCAA football players in the last decade, leading to recently mandated SCT testing in all Division-1 players. In SCT, RBC sickle only under extreme conditions and with slow kinetics. Therefore, rhabdomyolysis most likely occurs in SCT when a “perfect storm” of factors converges to critically imbalance oxygen supply and demand in muscles. We hypothesize that in SCT subjects, abnormal RBC rheology, particularly aggregation and deformability, play an important role in abnormal muscle blood flow supply and distribution to exercising muscle. To test this hypothesis, we examined whole blood viscosity, RBC aggregation, and RBC deformability in 11 SCT and 10 control subjects prior to and following maximum handgrip exercise. Methods: Maximum voluntary contraction (MVC) was assessed by handgrip dynamometer in the dominant arm. Baseline blood was collected for CBC, whole blood viscosity, RBC aggregation, and RBC deformability. Patients then maintained 60% MVC exercise until exhaustion. Following 8 minutes of recovery, a venous blood gas and blood for repeat viscosity assessments was collected from the antecubital fossa of the exercising limb. Whole blood viscosity over a shear rate range of 1–1, 000 1/s was determined by an automated tube viscometer, RBC deformability from 0.5–50 Pa via laser ektacytometry (LORCA) and RBC aggregation in both autologous plasma and 3% dextran 70 kDa using an automated cone-place aggregometer (Myrenne). Aggregation measurements included extent at stasis (M), strength of aggregation (GT min) and kinetics (T ½). Results: Baseline CBC and aggregation values are summarized in Table 1. Both static RBC aggregation in plasma and RBC aggregation in dextran (aggregability) were significantly increased in SCT (Table 1). The rate of aggregation formation trended higher in SCT but the strength of aggregation was not different between the two groups. In SCT subjects, red cell deformability was impaired at low shear stress but greater than controls at higher shear stress (Figure 1). Red cell deformability was completely independent of oxygenation status states in both SCT and control subjects. Whole blood viscosity did not different between the two groups whether oxygenated or deoxygenated and prior to or following handgrip exercise. Discussion: Three important hemorheological differences were observed for SCT subjects versus controls: a) RBC deformability was below control at low stress levels yet greater than control at higher stress; b) The extent of RBC aggregation in autologous plasma was about 40% greater; c) The extent of RBC aggregation for washed RBC re-suspended in an aggregating medium (i.e., 3% dextran 70 kDa) was about 30% higher. RBC deformability is a major determinant of in vivo blood flow dynamics, especially in the microcirculation; decreased deformability adversely affects tissue perfusion. RBC aggregation is also an important determinant since it affects both resistance to blood flow and RBC distribution in a vascular bed (e.g., plasma skimming). The finding of greater aggregability (i.e., higher aggregation in the defined dextran medium) indicates that RBC in SCT have an altered membrane surface in which the penetration of this polymer into the glycocalyx is abnormal. The combined effects of these three rheological parameters is likely to impair in vivo blood flow in SCT, perhaps to a degree resulting in pathophysiological changes of the cardiovascular system. Disclosures: Coates: Novartis: Speakers Bureau; Apopharma: Consultancy. Wood:Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding.

Abstract 4764 Background: Sickle cell disease (SCD) is a genetic condition resulting from a homozygous hemoglobin S mutation of the beta globin gene. HbS polymerizes in the deoxygenated state, causing the red cell membrane to become rigid and obstruct blood flow. Ischemia results leading to early organ damage, impaired quality of life and short life expectancy. Patients with SCD die from many causes including infection, splenic sequestration, stroke, and pulmonary emboli, however; a number of deaths in this population are acute and unexplained. The increased risk of sudden death is hypothesized to be associated with autonomic dysfunction. Heart rate variability (HRV) is a measurement of autonomic function and predictor of risk of sudden death. Measures of HRV can be compared to other factors associated with autonomic dysfunction. Individuals with obstructive sleep apnea have increased sympathetic tone. We hypothesized that SCD subjects that experienced nighttime hypoxemia will have autonomic dysfunction. Methods: Subjects were fitted with an ambulatory electrocardiogram device (Acitheart, CamNTech, Inc) and monitored for 24 hours. High frequency power (HFP), a measure of cardiac parasympathetic tone, and low:high ratio (LHR), a measure of sympathovagal balance, were calculated from the HRV by the product software. During sleep, baseline oxyhemoglobin saturation (SpO2) and number of desaturations below 88% was monitored using an ambulatory pulse oximeter (WristOX, Respironics, Inc). Bivariate analysis of the median HFP and LHR values over a 24 hour period were compared to baseline SpO2and number of desaturation events. Sex, age and hematologic values including hemoglobin, CRP and lactate dehydrogenase values were also considered. Results: 30 SCD subjects (mean age= 26.1 years+10.96, 36% male) were evaluated. Baseline nighttime SpO2 was 95.5+2.8% with 1 subject with a baseline SpO2 < 90%. Baseline SpO2 did not correlate with HF with R2 = 0.03 but did correlate with LHR with R2 = 0.46. There was a mean number of desaturation events of 16.6+38.3 with 4 subjects greater than 25 desaturation events. The number of desaturation events did not correlate with HF with R2 = 0.027 but did correlate with LHR with R2= 0.63. Stepwise multivariate analysis was performed including the baseline SpO2, number of desaturation events, sex, age and hematologic parameters showed that the number of desaturation events was the best predictor of LHR. Conclusions: SCD subjects that experienced nighttime hypoxemia and increased number of desaturation events during sleep have higher LHR ratios consistent with increased sympathovagal tone. Disclosures: Coates: Novartis: Speakers Bureau; Apopharma: Consultancy. Wood:Ferrokin Biosciences: Consultancy; Shire: Consultancy; Apotex: Consultancy, Honoraria; Novartis: Honoraria, Research Funding.

Abstract 2138 Chronic transfusion therapy (CTT) is a mainstay for stroke prophylaxis in sickle cell anemia. Long-term changes with transfusion include decreased hemoglobin S% and hemolysis resulting in decreased plasma free hemoglobin. Long-term benefits are well documented, however, patients on CTT continue to suffer from acute crises. The acute effects of each transfusion are not well known but might include improved oxygen carrying capacity secondary to increased hematocrit, reducing demands for high cardiac output. But, the increased hematocrit and oxygen carrying capacity is at the cost of increased viscosity and resistance to blood flow. Despite long-term benefits, acute complications continue to plague this patient population and could be due to acute rheologic changes with transfusion. We hypothesized that transfusion would acutely improve tissue oxygen delivery despite increasing blood viscosity and vascular resistance. To test this hypothesis, we prospectively examined patients on CTT immediately pre transfusion and again 12–120 hours post transfusion. Hemodynamics were tested by measuring blood pressure, heart rate and cardiac function by echocardiography. Tissue oxygen delivery was assessed using echocardiographic estimates of cardiac output, pulse oximetry and oxygen carrying capacity as well as near infrared spectroscopy (NIRS). We obtained basic hematology and metabolic labs in addition to markers of inflammation, hemolysis and amino acid profile at both visits. Male and female patients were equally represented with similar average age. The reasons for starting transfusion as well as medication profiles were similar between sexes. Comparable changes in hemoglobin, hematocrit, reticulocyte count and hemoglobin S with transfusion were observed in all patients. However, when pre transfusion levels of free hemoglobin, hemoglobin S%, platelet count and reticulocyte count were examined with regard to timing from the previous transfusion, males had faster recovery of endogenous marrow activity and increased hemolysis, producing higher average hemoglobin S%, reticulocyte count, platelet count and free hemoglobin levels (figure 1). In males, transfusion decreased heart rate, stroke volume, and cardiac index while estimates for pulmonary and systemic vascular resistance rose, culminating in decreased oxygen delivery. In contrast, stroke volume and cardiac index were preserved in women following transfusion, while systemic and pulmonary vascular resistance did not change such that oxygen delivery improved 16%. NIRS measurements assessing tissue oxygenation confirmed the differences seen using echocardiography and oxygen carrying capacity estimates(figure 2).Figure 1Figure 1. Figure 2Figure 2. Increased endogenous marrow activity in males, resulting in higher pre-transfusion reticulocyte count, hemoglobin S%, platelet count and free hemoglobin might account for the exaggerated reduction in cardiac index, as well as increased systemic and pulmonary vascular resistance with transfusion. In addition, the faster recovery of hemoglobin S% and hemolysis likely confers increased risk of vascular complications in male patients despite chronic transfusion therapy. Disclosures: Wood: Novartis: Research Funding; Ferrokin Biosciences: Consultancy; Cooleys Anemia Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

Abstract 1259 The ability for hemoglobin S to polymerize causes increased viscosity, decreased RBC deformability and increased RBC aggregation. Chronic transfusion therapy (CTT) in patients with sickle cell disease (SCD) decreases the percentage of hemoglobin S in the bloodstream. The hematocrit to viscosity ratio (HVR) has been used to estimate red cell oxygen transport effectiveness, a property of blood flow. There are undeniable benefits from CTT, however, there are patients on CTT who still suffer from acute crises. Acute hemorheologic changes after transfusion might contribute to these episodes. We hypothesize that viscosity and aggregation will increase post transfusion. Furthermore, we believe that these changes will result in lower oxygen transport effectiveness as measured by HVR. To test this hypothesis we enrolled 26 patients on chronic transfusion therapy in a prospective study to evaluate blood viscosity and aggregation changes with transfusion. We measured both oxygenated and deoxygenated whole blood viscosity at shear rates from 1s−1 to 1000s−1 and RBC aggregation at native hematocrit using a Rheolog viscometer (Rheologics Co). We also obtained pre and post transfusion blood counts, chemistry panels and markers of inflammation and hemolysis. 14 females and 12 males were enrolled with one patient who was excluded due to incomplete data. The ages and reasons for starting transfusion were similar for male and female patients. As expected, transfusion resulted in significant increases of hemoglobin and hematocrit with a concomitant decrease of percent hemoglobin S and reticulocyte count. There was a trend toward decreased platelet count. Male patients had a significantly higher percent hemoglobin S, reticulocyte count, plasma free hemoglobin and platelet count compared to females. Viscosity increased significantly across all shear rates with transfusion and with deoxygenation. There was no sex difference in viscosity. Deoxygenation and transfusion increased the aggregation index. Change in viscosity positively correlated with increased hematocrit and aggregation index. There was no correlation of viscosity change with change in hemoglobin S, markers of inflammation or hemolysis. Deoxygenation lowered HVR at all shear rates. HVR was significantly lower post transfusion at low shear rates of 1s−1, 2s−1 and 5s−1 (Figure 1). At mid to high shear rates there was no difference in HVR, although as the shear rate increased, the HVR curves crossed with apparently increasing improvement in HVR post transfusion at the highest shear rates. The significant decrease in HVR at low shear correlated with increased aggregation index but not increased hematocrit. In this population of patients on CTT, blood viscosity and RBC aggregation increased post transfusion, thus predisposing them to impaired perfusion in low flow vascular beds. The decrease in HVR at low shear rates correlated with RBC aggregation, providing further evidence for post-transfusion risk of impaired perfusion. These results support our hypothesis and indicate potential adverse effects of transfusion on microvascular blood flow.Figure 1Figure 1. Disclosures: Wood: Novartis: Research Funding; Ferrokin Biosciences: Consultancy; Cooleys Anemia Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 2655 Sickle cell disease (SCD) is characterized by repeated episodes of vaso-occlusion leading to painful episodes, acute chest syndrome, stroke, end organ damage and death. Blood flow changes that result in decreased perfusion of tissues and organs increase the risk for vaso-occlusion by increasing the residence time of deoxygenated and deformed sickled red blood cells in the capillary bed. Patients with SCD are more likely to have transient decreases in perfusion in response to sighs (unpublished data). We thus hypothesized that SCD patients would have an enhanced vasoconstriction response to other autonomic stimuli, such as the cold face stimulation test, when compared to normal controls and SCD patients who are chronically transfused. Using an IRB approved protocol, we studied 13 normal, ethnic matched, subjects (mean age 30.2 + 14 yrs), 12 SCD subjects (mean age 22.9 + 13.1 yrs) and 7 chronically transfused SCD subjects (mean age 22.5 + 11.5 yrs). Dermal capillary perfusion (DCP) measured by laser Doppler and near infrared regional oxygen saturation (rSO2) of the hand were monitored as continuous measures of peripheral blood flow. Physiologic data including systemic vascular resistance (SVR), respiratory rate, tidal volume, heart rate and mean arterial blood pressure, as well as DCP and rSO2, were continuously monitored and recorded digitally. A cold pack was applied to the forehead for 60 seconds and the perfusion and physiological data were recorded. Baseline physiologic values were calculated as the median from 5 min to 1 min prior to the cold face stimulation and compared to median values during the cold face stimulation. In all three populations (normal controls, chronically transfused SCD, SCD) there was a rapid decrease of DCP from baseline (−38.2%, −26.6%, −22.5% respectively) and rSO2 (−8.8%, −4.8%, −3.3% respectively) during the cold face stimulation. Thus, compared to controls, the vascular response was blunted in both SCD and transfused SCD. The blood flow changes in response to the cold face stimulation in SCD improved with chronic transfusion but did not normalize. SVR increased in all populations (17%, 16%, 9% respectively) but the response was blunted in the non-transfused SCD group. Bradycardia was observed in the control and chronically transfused SCD groups (−5.9%, −3.7%) but not in the non-transfused SCD (−0.21%). Our results indicate that, as expected, there is a decrease in peripheral blood flow during cold exposure in normal controls which also occurs in SCD and chronically transfused SCD; SVR is increased consistent with peripheral vasoconstriction. Interestingly, the peripheral blood flow response to cold face stimulation is significantly blunted in both SCD and SCD with chronic transfusion, and thus transfusion does not correct the abnormality. However, chronic transfusion in SCD normalizes the bradycardiac/parasympathetic response to cold face stimulation. In overview, these findings indicate that chronic transfusion partially improves autonomic function in SCD patients, but does not correct the abnormality in peripheral microvascular reactivity. Disclosures: Coates: Novartis: Research Funding, Speakers Bureau.

Abstract 1645 Introduction: Vascular disease in both pulmonary and systemic vessels underlies most complications of sickle cell anemia (SCA). Multiple mechanisms contribute to this process, including inflammation, oxidative stress and decreased nitric oxide bioavailability. Chronic transfusion therapy (CTT) is thought to ameliorate some of these factors. Our goal was to determine the relationship between systemic vascular function, as indexed by flow mediated dilation (FMD), and elevated tricuspid regurgitation jet (TRJ) as a surrogate for pulmonary hypertension (PH), in chronically transfused SCA patients. Methods: 25 SCA patients on CTT were enrolled in a prospective cross sectional study between August 2008 and June 2010; the protocol was approved by our IRB. Patients were studied on the day of transfusion prior to receiving blood and then again between 24 and 120 hours post transfusion. Echocardiography, brachial artery FMD and blood sampling for CBC, reticulocyte count, LDH, plasma hemoglobin and hs-CRP were performed during each study visit. A TRJ of 2.5 m/s or higher was defined as “pulmonary hypertension” and an FMD of Results: Table 1 summarizes pre and post transfusion results. A TRJ > 2.5 m/s was seen in 20% of patients pre-transfusion and in 12% post-transfusion, but this difference did not reach statistical significance. None of the patients with a TRJ < 2.5m/s pre transfusion had a TRJ > 2.5m/s post transfusion. FMD was significantly improved consequent to transfusion (5.4% to 6.8%, p 2.5 m/s exhibited blunted FMD response. Abnormal TRJ and FMD correlated with elevated reticulocyte count, LDH, %Hb S, and prolonged left ventricular ejection time and isovolumic contraction time. FMD was directly correlated with hct and Hb and inversely correlated with stroke volume and cardiac output. There was a trend towards an inverse correlation of FMD with plasma hemoglobin, arginine/ ornithine ratio and high sensitivity CRP. Discussion: CTT offers incomplete protection from vascular disease in both the systemic and pulmonary beds. Patients with elevated TRJ > 2.5m/s also had abnormal FMD: both correlated with markers of hemolysis, increased %HbS as well as prolonged cardiac time intervals. Abnormal FMD, but not TRJ, was correlated with decreased Hb and hct and increased cardiac output and stroke volume, suggesting that worsening anemia affects FMD to a greater degree than TRJ. These data support the concept that pulmonary hypertension in SCA is only one manifestation of pan-systemic endothelial disease. Disclosures: Coates: Novartis: Research Funding, Speakers Bureau.

Abstract 1516 Poster Board I-539 Introduction Patients with sickle cell anemia (SCA) who are at risk for stroke or develop recurrent episodes of acute chest are often placed on chronic transfusion therapy. The acute hemodynamic and vascular consequences of transfusion are poorly characterized. In order to better characterize chronic changes in these patients, it is important to understand the acute changes involved with this intervention. Methods We enrolled SCA patients who are on chronic transfusion therapy in a prospective study to determine the cardiovascular effects of a single transfusion. The patients were older than 10 years of age, received chronic transfusions for greater than one year, and were crisis free for more than four weeks. All patients were studied immediately prior to transfusion and repeated within five days. Brachial artery relaxivity, laser doppler capillary blood flow monitoring, and near infrared spectroscopy of the hand was done in the setting of post occlusive reactive hyperemia. Studies included complete cardiac echocardiogram as well as laboratory markers of hemolysis, iron levels, blood count, viscosity and inflammation were obtained pre and post transfusion. Statistics utilized paired t-tests for paired data and for correlative data t-tests and chi square analysis was used. Results There are 15 patients enrolled in the study with an average age of 18.7yrs. There are 8 females and 7 males with average ages of 20.6 and 16.4 yrs respectively. With transfusion, hemoglobin increased from 9.4 g/dl to 11.6 g/dl post (p Discussion Transfusion therapy predictably increased oxygen carrying capacity, lowered reticulocyte count and %hemoglobin S. While these changes might improve tissue oxygen delivery, parallel changes in vascular resistance served to maintain relatively constant oxygen delivery. The rise in vascular resistance could result from increased blood viscosity or changes in vascular tone; we are currently conducting studies to try to separate these mechanisms. Despite the 24% reduction in cardiac index, no significant change was observed in microvascular perfusion or its response to forearm occlusion. The vascular system of SCA patients demonstrates chronic endothelial dysfunction but was not acutely influenced by transfusions, consistent with the lack of acute improvement in surrogates for hemolysis or nitric oxide metabolism. Further work is necessary to determine whether transfusion-mediated changes in tissue oxygen delivery vary among different organs and vascular beds. Disclosures Wood: Novartis: Research Funding.

Abstract 1518 Poster Board I-541 Introduction Chronic blood transfusions are commonly used as therapy for sickle cell disease (SCD, HbSS) in order to improve oxygen delivery and minimize complications such as stroke in high-risk children. Vaso-occlusive crises can occur in regions of high shear flow (e.g., major cerebral artery occlusions) or regions of low shear flow (e.g., marrow infarct) leading to acute ischemia and, if severe, to necrosis of affected tissues. Transfusion with normal (AA) RBC causes an increase of hematocrit (H) that is complicated by two opposing factors: increased hematocrit (H) causes a linear increase of oxygen carrying capacity and also an exponential increase of blood viscosity (η). As a consequence, the calculated oxygen transport effectiveness, defined as the ratio of H to η (H/η), is a biphasic function of hematocrit: H/η initially increases with H, reaches a maximum at an optimal H value, and then declines with further increases of H. At equal H and shear rate, sickle (SS) blood has significantly higher viscosity than AA and hence part of the strategy for transfusing SCD patients is to reduce η so as to improve H/η. Viscosity studies at high shear rates indicate that an optimum H can be demonstrated for AA-SS RBC mixtures prepared by adding AA RBC to SS blood to simulate transfusion. In marked contrast, low shear rate results for AA-SS mixtures indicate that there is no optimum hematocrit and H/η always decreases with increasing H (Transfusion 46:912-918, 2006). In order to extend these previous in vitro observations to SCD patients, we have measured blood viscosity and hematocrit using whole blood samples acquired prior to and following routine therapeutic transfusion; H/η was calculated over a wide, physiologically relevant shear rate range. Methods All subjects (n= 8, mean age =18.7 years) had homozygous HbSS disease, were crisis-free for > 4 weeks, and were enrolled in a chronic transfusion protocol designed to yield < 30% HbS and a post-transfusion H of 30-35%. Blood samples were obtained pre- and within 120 hours post-transfusion. A computer-controller tube viscometer was used to determine blood viscosity (37 °C, 40 mm Hg oxygen tension) over a shear rate range of 1 – 1,000 1/s. Results 1) As anticipated, blood viscosity and the degree of non-Newtonian flow behavior increased with H (24.7% pre-transfusion, 34.6% post-transfusion); 2) the change of H/η from pre- to post- transfusion was markedly affected by shear rate (Figure). As indicated, there is a large adverse effect at low shear (i.e., H/η reduced by 20-25% following transfusion), a neutral effect at about 50-100 1/s, and an improved H/η at high shear (Figure). That is, transfusion with AA RBC to obtain a lower percent SS RBC and a higher H actually impairs oxygen transport effectiveness at low shear and is only beneficial at high shear. Conclusions Clinical experience suggests that transfusion regimens aimed a keeping HbS at 30-50% are effective in preventing recurrent strokes in high-risk children. However, our new in vivo transfusion data suggest that at low shear rates, %HbS must be reduced further for H/η to surpass pre-transfusion levels. We interpret these findings as being consistent with our previous data for AA-SS RBC mixtures. They are also consistent with clinical results indicating lack of efficacy for transfusion in low flow areas (e.g., bone marrow during acute crisis) but highly beneficial effects in high flow regions (e.g., cerebral arteries). Our results thus suggest that benefits of transfusion may vary depending on local flow rates (i.e., shear rates) and organ-specific hemodynamics. Disclosures No relevant conflicts of interest to declare.

Abstract 5084 Introduction Discordant monozygotic twins with sickle cell anemia (SCA) are rare. In the present study we describe current clinical and laboratory data in a set of 49 year old discordant monozygotic twins with coexistent SCA and α-/αα thalassemia. We compare the current findings to their previously described data (Hemoglobin 1991;15:247-56). Methods Evaluations of painful episode frequency, episode severity, frequency of the acute chest syndrome, standard blood counts and chemistries, hemolysis, pulmonary hypertension, irreversibly sickled cells (ISC) and RBC rheology were obtained. DNA from peripheral blood was genotyped using Illumina 610K SNP (single nucleotide polymorphism) beadchip allowing analysis of SNP genotypes and DNA copy number variations (CNVs). Results Twin I is right handed and Twin II is left handed. Frequency of hospitalized painful episodes examined over 34 years were similar (approximately 1 episode per year) and were asynchronous. Prior to approximately 25-27 years of age, the painful episodes in Twin I were moderately severe with decreasing severity as ageing occurred. In contrast, Twin II, who also had moderately severe painful episodes during early years, showed marked worsening of severity as ageing occurred. Episodes of acute chest syndrome were asynchronous with similar frequency (Twin I had 2 episodes in 18 years; Twin II had 2 episodes in 21 years). Systemic blood pressures were similar. Osteonecrosis, leg ulcers, priapism, stroke were absent. Concordance of SNP genotypes confirmed that the twins are monozygotic. Analysis of the entire genome for CNVs revealed a number of candidate loci that differ. Most differences in CNVs occurred in Twin II. Conclusions Clinical and laboratory findings in these twins include differences and similarities. Clinical differences have been observed in handedness, asynchronism of painful episodes and differences in episode severity with ageing. Similarity was observed in the frequency of the painful episodes. Laboratory differences indicate that the degree of intravascular hemolysis is greater in Twin II than in Twin I, as shown by the differences in plasma Hb and RBC-derived vesicle levels (Brit J Haematol 2008;142:126-35). In contrast, the current pulmonary pressures and the current and previous rheologic studies are similar. Ageing contributes to the extent of intravascular hemolysis, as noted when the current findings are compared to those observed in the earlier study. Levels of ISC,which are related to the life span of sickle RBC (Am J Med 1978;64:253-7, Brit J Haemat 1969;17:527-533), appear to be inversely associated with indicators of intravascular hemolysis, such as plasma Hb and RBC-derived vesicles (Twin I, ISC 24.4%, Pl Hb 3.72, RBC-vesicles 2.98; Twin II, ISC 17.5%, Pl Hb 7.79, RBC-vesicles 6.32). The difference in CNVs between the twins, which is greater in Twin II, may explain the phenotypic discordance (Amer J Human Genetics 2008;82:763-71). The loci showing the differences will require further study. To what extent genetic differences in handedness in the twins are associated with the phenotypic differences have not been examined (Mol Psychiatry 2007;12:1129-39). Disclosures No relevant conflicts of interest to declare.

Abstract 1523 Poster Board I-546 Sickle cell anemia (SCA) is a genetic disorder characterized by recurring episodes of vaso-occlusive crisis (VOC) that can lead to hospitalization or sudden death. Hypoxia is an accepted trigger of sickling and degrees of nighttime hypoxia correlate with strokes and frequency of VOC. To better understand the mechanism of events leading to VOC, we simulated the occurrence of nocturnal hypoxia in SCA patients by administration of five breaths of 100% nitrogen. Tidal volume (Vt), arterial oxygen saturation, electrocardiogram (ECG), and microvascular perfusion (PU) by Laser-Doppler were continuously recorded. We had anticipated a drop in PU after each controlled episode of hypoxia. However, we observed multiple prominent drops in PU in SCA subjects (n=8) that were not as clearly evident in controls (CTL; n=9), and found no direct relationship between hypoxia and change in PU (p = NS). As deep breaths or sighs can trigger reflex peripheral vasoconstriction, we examined Vt respiratory tracings obtained simultaneously and observed that PU drops frequently followed sighs (see Figure) in SCA subjects, but rarely in CTL. A statistical algorithm was used to find all sighs and vasoconstrictive events (PU drops) during each 40-minute experimental session. PU drops were associated with sighs in 7 of 8 SCA patients and in 4 of 9 CTL subjects (P < 0.001, Poisson regression analysis). Five CTL and 1 SCA subjects had infrequent sighs and no association between sighs and PU drops. The likelihood ratio of sigh-associated PU drops was significantly higher in SCA than CTL subjects (median = 59.9 % vs. < 1 % for SCA vs. CTL, P = 0.008, rank-sum test) whereas the frequency of sighs was not significantly different between the two groups (median = 2.2 % vs. 1.3 % for SCA vs. CTL, P = 0.16, rank-sum test), indicating that SCA patients are much more likely to have sigh-associated peripheral vasoconstriction. Since the sigh-vasoconstrictor response is controlled by the autonomic nervous system (ANS), we measured heart rate variability (HRV) which is an accepted index of sympathetic/parasympathetic balance. These studies showed substantial reduction of parasympathetic modulation of HRV during hypoxia in SCA but not in CTL subjects (p < 0.01), indicating a marked abnormality of the ANS in SCA. In overview, the likelihood of coupling between spontaneous sighs and subsequent vasoconstrictive events (PU drops) is much higher in SCA patients than in CTL. Thus, we speculate that a drop in perfusion secondary to increased neural coupling between the lung and vasculature may be an initiating event in VOC. Hypoxia may secondarily promote VOC by altering ANS sensitivity and increasing the probability that a sigh will in turn lead to reflex peripheral vasoconstriction. In a background of HbS, transient decreases in perfusion may prolong red cell residence time in the microvasculature, leading to HbS polymerization, sickling and vascular occlusion. Disclosures No relevant conflicts of interest to declare.

While genetic delivery of recombinant anti-sickling β-globin genes have been shown to correct murine sickle cell anemia (SCA), correction of SCA by delivery of a natural hemoglobin, fetal hemoglobin (HbF), the proportion of genetically modified hematopoietic stem cells (HSC), or amount of HbF necessary to correct the disease is unknown. We designed a lentivirus vector carrying γ-globin exons with β-globin regulatory elements and non-coding sequences, GbG. First, GbG or mock transduced Berkeley sickle HSC were transplanted using a myeloablative (lethal irradiation) transplant model, to acheive full donor chimerism. GbG mice showed high HbF expression (HbF 41 ± 5% measured by HPLC) that was sustained in primary (6 mo) and secondary (7.5 mo) transplant recipients, and resulted in effective correction of hematological and functional RBC parameters, and reduction of inflammation that results from sickle cell disease. We found significantly reduced irreversibly sickled cells (2.3 ± 0.7% in GbG versus 10.2 ± 0.3% in mock mice; p30% HbF/F-cell and >60% F-cells consistently corrected SCA. The mean HSC transduction (assessed by secondary HbF+ CFU-S at 6 months post transplant) was 50% and 30% in the myeloablative and reduced intensity transplant models, respectively, with 1–3 GbG copies/ cell. Furthermore, three GbG mice showed correction of SCA with 20% HSC transduction, a clinically achievable goal. Taken together, this study is the first demonstration of correction of SCA with gene therapy using γ-globin, and defines critical determinants for effective gene therapy of this disease. Mouse Model Hb (g/dl) RBC 106/ul) Reticulocyte (%) WBC (K/ul) *p

Background: Rapid clearance of poly(ethylene glycol)-asparaginase (PEG-asparaginase) has been reported for up to one third of patients treated for acute lymphoblastic leukemia, potentially rendering their treatment ineffective [Hempel G, Lanvers-Kaminsky C, Mueller, HJ, Würtwein G, Boos J. Blood. 2004;11:751A]. Poly(ethylene glycol) is an inert, biocompatible synthetic polymer and has long been claimed to be non-immunogenic; several PEG-conjugated drugs are FDA approved and many other PEGylated agents are in development. However, we have previously reported a 25% occurrence of an antibody against PEG (anti-PEG) in healthy blood donors, which suggests that PEG is an immunogenic agent. [Garratty G. Trans Med Rev. 2004;18:245–56]. Objective: To determine whether an anti-PEG could account for the rapid clearance of PEG-asparaginase. Methods: This study re-analyzed stored serum samples from pediatric patients who were enrolled in the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 studies. Samples were selected to include 15 subjects who showed undetectable asparaginase activity after receiving PEG-asparaginase (Oncaspar®) and 13 with normal sustained levels of asparaginase activity after treatment with PEG-asparaginase. Sixteen subjects treated with unmodified asparaginase (Medac®) were also included, 8 with negligible asparaginase activity. Sera were tested for the presence of an anti-PEG using two techniques: 1) Serology, by agglutination of PEG-coated red blood cells; 2) Flow cytometry, by analysis of 10 μm PEG beads (Tentagel®-OH) pre-incubated with sera and stained for bound immunoglobulins with fluorescein-anti-human IgG and R-phycoerythrin-anti-human IgM. Testing for anti-PEG was performed in a blinded manner. Results: For the PEG-asparaginase treated patients, of the 15 who showed undetectable asparaginase activity, 9 tested positive for anti-PEG by serology and 13 tested positive for anti-PEG by flow cytometry. All PEG-asparaginase treated patients with normal sustained asparaginase activity tested negative for anti-PEG. Four sera yielded somewhat unusual results: One weak IgM anti-PEG positive sample showed measurable asparaginase activity and three anti-PEG negative patients had low asparaginase activity, two of which were positive for anti-asparaginase. No relationship was observed between the presence of anti-PEG and serum asparaginase activity for the patients treated with unmodified asparaginase (Medac®). Conclusions: The presence of anti-PEG was very closely associated with rapid clearance of PEG-asparaginase. Screening and monitoring for anti-PEG would allow for identification of a subset of patients for whom a modified dosing strategy or use of a non-PEGylated drug would be appropriate.

Background: Studies of the circulatory time of red blood cells (RBCs) or drug delivery vehicles such as liposomes require a radiolabel (e.g., 51Cr), a fluorescent probe or an affinity tag (e.g., biotin). These techniques may be complicated by progressive loss of the probe from the labeled cells, transfer to other cells, and sometimes by the development of an immune response to the label. Long chain dialkylcarbocyanines (Vybrant DiO, DiI and DiD) are bright, stable lipophilic membrane fluorescent labels that can exhibit fluorescence resonance energy transfer (FRET) and are easily observed by flow cytometry using standard filters (FL1, FL2 and FL4 detection channels respectively). The staining is very stable in vivo and is dose-dependent, thus populations of labeled cells may have discrete fluorescence intensities. We recognized that use of these dyes in combination could permit the simultaneous observation of several different populations of cells within the same subject, together with one or more internal controls. To evaluate this technique, we developed procedures suitable for the fluorescent labeling up to 14 discrete populations of human or rabbit RBCs. The properties of the rabbit RBCs were then evaluated in vivo. Methods: Washed human RBCs were suspended at a 10% hematocrit in 20mM HEPES buffer, and added to an equal volume of freshly prepared 1, 4 or 8 μM DiO, DiI, DiD, or mixtures of two labels; 36 different combinations in total. The cells were incubated with the dye for 60 minutes at 37°C, washed twice in HEPES with 0.1% albumin and examined by flow cytometry. Aliquots from 6 to 15 differently-labeled samples were then combined, and the mixtures re-examined. For in vivo studies, the labeling procedure was repeated with autologous rabbit RBCs at dye concentrations of 1.5, 6, and 12 μM. The labeled rabbit RBCs were washed, suspended in saline and reinfused via the ear vein. Small blood samples were drawn daily for 4 days and weekly for 5 weeks, and the number of fluorescently labeled cells of each color remaining was determined by flow cytometry. Results: Of the 36 possible combinations, 14 discrete populations could be clearly distinguished from “dot-plots” of FL1 vs. FL2, FL1 vs. FL4 and FL2 vs. FL4. FRET was observed for all cells stained with two labels. In vivo evaluation of 8 different labeled rabbit RBC populations showed normal survival. A 10–20% reduction in fluorescence intensity was observed over the study period; this reduction did not compromise the clear identification of each discrete population. Rabbits that were repeatedly exposed to Vybrant-labeled autologous RBCs (up to 15 exposures over 3 years) showed no evidence of accelerated clearance, suggesting that these fluorescent labels do not exhibit significant immunogenicity. Conclusion: The use of two long-chain dialkylcarbocyanines in combination is ideal for studies which require multiple cell populations to be followed within the same experiment, and additionally allows for one or more internal controls to correct for any variability of circulation times. The technique is presently being used for studies of RBC senescence in which multiple discrete density populations of RBCs are individually labeled. If these dyes can be shown to have an acceptably low toxicity, the ability to track multiple populations could also be useful for some clinical applications, e.g., to simultaneously evaluate the survival of RBCs from many different donors in patients with multiple alloantibodies, to identify compatible units for transfusion.

The addition of malonyldialdehyde (5–20 micro M) to human erythrocytes results in a marked decrease in cellular deformability as measured with a counter-rotating, cone plate Rheoscope when low shear forces (2.5–25 dynes/sq cm) are applied. At high shear forces (125–500 dynes/sq cm), malonyldialdehyde at 5 micro M had no effect on deformability, although at concentrations of 10 and 20 micro M a small but statistically significant decrease was evident. These effects of this crosslinking agent are observed in the absence of alterations in cell volume and intracellular viscosity. The results obtained are in accord with the view that the polymerization of membrane constituents may contribute to the events that lead to the removal from the circulation of either aging cells or cells exposed to peroxidation-initiating agents.

Although there is evidence that the deformability of the entire red blood cell (RBC) decreases during aging, reports on changes in relevant specific properties associated with the aging process are limited and not in total agreement. The purpose of this study was to evaluate some of the factors that might contribute to this decreased deformability. Geometric, osmotic, and membrane mechanical properties of unfractionated, top (“young”) and bottom (“old”) RBC from 5 healthy adult donors were measured using micropipette techniques. Surface area, volume, and diameter of RBC were measured at osmolalities of 297, 254, 202, and 153 mosm/kg. Two membrane mechanical properties, surface shear modulus of elasticity (mu) and time constant (tc) of viscoelastic recovery, were studied only in isotonic media. At each of the osmolalities, volume and surface area of the bottom cells were about 25% lower than those of the top cells. Bottom cells showed smaller increases in volume with decreasing osmolality than top cells; the surface area remained constant with changing osmolality for all three groups. The surface area-to-volume ratio and the minimum cylindrical diameter of the bottom cells were essentially identical to the top cells. However, both the surface area index (actual are of RBC divided by area of a sphere of same volume) and the swelling index (maximal volume divided by actual volume) of the bottom cells were significantly lower than top RBC. The shear modules of elasticity (mu) was about 0.006 dyne/cm in all 3 RBC populations, indicating that the forces necessary to deform a portion of the membrane did not change with RBC aging. The viscoelastic time constant (tc) was 0.148 +/- 0.020 (SD) sec for the bottom RBC and 0.099 +/- 0.017 sec for the top cells. This difference indicates that shape recovery following membrane deformation is delayed in old RBC. The membrane surface viscosity (eta), calculated as the product of tc times mu was 0.95 +/- 0.22 x 10(-3) dyne-sec/cm for the bottom cells and 0.54 +/- 0.15 x 10(-3) for the top RBC. These data indicate that the relative deficit in membrane surface area and the increased membrane viscosity of old RBC may be important determinants for their decreased deformability and their eventual removal from the circulation.

Although the relations between the metabolic state and the mechanical properties of human red blood cells (RBC) continue to be of current interest, literature reports in this area are not in agreement. The present investigation was designed to determine several intrinsic mechanical properties of human RBC membranes before and after metabolic depletion via incubation at 37 degrees C for 24 hr. Using micropipette and flow channel techniques, three properties were measured: (1) mu, surface shear modulus of elasticity; (2) K, elastic area compressibility modulus; (3) etap, shear viscosity in the plastic domain. Our results indicate no significant differences in these parameters between fresh and ATP-depleted human RBC membranes. These present data are thus in disagreement with other literature reports indicating large changes in membrane mechanical properties consequent to metabolic depletion. A brief discussion of the possible reasons for this disagreement is presented.

A recently developed technique for deforming red blood cells (RBC) in which they are centrifuged through buffer and into a glutaraldehyde solution was evaluated as a method of assessing cellular deformability (i.e., the ability of the entire RBC to form a new configuration). To accomplish this, RBC populations of differing cellular deformability were tested, using three generally accepted techniques to obtain these differences: partial fixation with low concentrations of glutaraldehyde, density fractionation, and suspension of RBC in nonisotonic media. Our results indicate that at a constant deforming force the mean deformed length of the RBC decreased under conditions where cellular deformability is known to decrease, thus suggesting the usefulness of this centrifugal method for the estimation of this cellular property.

Little data exist for the mechanical properties of individual irreversible or reversible sickle cells (ISC and RSC, respectively), nor is the process of ISC formation well understood. For oxygenated ISC and density-fractionated RSC, we have used micropipette techniques to measure cell surface area (SA) and volume (V), membrane shear elastic modulus (mu), time constant for viscoelastic shape recovery (tc), and hence to calculate membrane surface viscosity (eta = mu X tc). Volume loss associated with increasing cell density was accompanied by a proportionately smaller surface area decrease; SA/V ratio thus increased for denser cells, with ISC having the highest values. Membrane area loss by fragmentation must thus be accompanied by an accelerated decrease in cell volume. ISC had relatively rigid membranes (mu 130% above normal controls) and tc close to normal values, so that their effective membrane viscosity was more than double control. RSC had viscoelastic properties close to control, but showed wider variation between sickle cell donors and within samples. Measurements on density-separated RSC showed that, on average, mu was nearly constant, but that tc was longer for the densest cells, with their eta approaching ISC levels. A small subpopulation of RSC were found that had mu close to ISC values. Hypotonically swollen ISC (with internal hemoglobin concentration decreased to normal levels) retained their increased membrane stiffness but had markedly decreased tc, so that their eta approached normal values. The results show that elevated hemoglobin concentration influences the viscoelastic behavior of ISC and RSC, but that an irreversible change in membrane elasticity also occurs for ISC. These data suggest that ISC formation occurs via a two- stage process: (1) accelerated volume loss leading to increased cytoplasmic and effective membrane viscosity; (2) a sharp rise in membrane rigidity, presumably linked to membrane structural alteration.

Whole cell and membrane deformability are essential for red blood cell (RBC) survival and for effective blood flow. Neonatal RBCs display several specific properties (eg, large size, high hemoglobin F) that could influence their deformation characteristics and contribute to their shortened life span. The present study was designed to compare selected rheologic properties (cellular deformability, pressure required to aspirate RBC into micropipettes, static and dynamic viscoelastic material properties) of neonatal and adult RBCs. RBC deformability, as studied by a rheoscope, was similar for neonates and adults over a shear stress range of 2.5 to 500 dyn/cm2. The pressure required to aspirate RBCs completely into 3.3-micron diameter pipettes was 129 +/- 87 dyn/cm2 for neonatal RBCs and 71 +/- 37 dyn/cm2 for adult RBCs. The aspiration pressure for neonatal and adult RBCs increased with increasing RBC volume, suggesting that the increased mean aspiration pressure for neonatal RBCs resulted from their larger volume. When RBCs with same volume and diameter were compared, the aspiration pressure tended to be smaller for neonatal RBCs than for adult cells. To characterize material properties determining RBC deformability, we measured membrane extensional (shear) and bending elastic moduli, the time constant for elastic recovery from extensional deformation and hemoglobin viscosity (ie, cytoplasmic viscosity) of neonatal and adult RBCs. Membrane surface viscosity and time constant for recovery from bending deformation were calculated. The extensional and bending moduli of neonatal RBCs were slightly smaller (10% and 16%, respectively) compared with adult cells. This suggests that the static resistance of neonatal RBC membrane to deformation and failure in response to a given force is slightly smaller. The time constant for recovery from extensional deformation of neonatal RBCs was larger by 14%, compared with adult cells. The time constant for bending deformation related to the RBC diameter and surface area was increased by 18% in the neonates. Membrane surface viscosity and hemoglobin viscosity were similar for both cell types. These results indicate that the deformability and viscoelastic properties of neonatal RBCs deviate only slightly from those of adult RBCs and that the increased aspiration pressure of neonatal RBCs is solely due to their large size. Some of the specific deformation characteristics observed in this study (increased aspiration pressure, decreased resistance to elastic deformation) may contribute to the shortened life span of neonatal RBCs.