ALBERT

Description: 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.

Description: 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.

Description: Electrocardiographic abnormalities are common in Thalassemia Major patients as well as in animal models of iron overload. The relationship between these ECG abnormalities and the degree of cardiac iron overload has not been well characterized. Hypothesis: Standard 12-lead ECG identifies preclinical cardiac iron deposition. Methods: Fifty-four patients with Thalassemia Major underwent MRI quantification of cardiac iron within one month of a standard 12-lead ECG. Cardiac T2* measurements to quantify iron were performed using a validated multiecho gradient-echo sequence performed on a 1.5 Tesla General Electric CVi scanner. The PR, QRS, QT, and QTc durations as well as P-wave, T-wave and QRS axes and heart rates were measured. Normative data was derived from 20 control patients without detectable cardiac iron (T2* 〉 20 ms) and age-appropriate norms were created for each ECG criteria. Z-scores for the ECG parameters were then calculated for 34 patients having detectable cardiac iron (T2* 〈 20ms). Results: ECG parameters varied significantly with age, with correlation coefficients ranging from 0.39 (QTc interval) to 0.66 (PR interval). Cardiac iron was associated with lower heart rates (Z=−0.44, p=0.06), QTc prolongation (Z=0.48, p=0.04) and leftward shift of the P and T wave axes [P-Axis (Z = −1.53, p

Description: Blood transfusions are given for acute and chronic illness including cardiothoracic surgery, acute stabilization, and chronic transfusion therapy (CTT) in patients with thalassemia and sickle cell disease (SCD). Increased age of stored red blood cells and the associated storage lesion has been implicated in poor cardiovascular outcomes, increased length of ICU stay, multiorgan failure and increased use of inotropes after cardiac surgery. Red cell aging, during storage, is thought to alter the metabolic profile of the red cell in a manner that causes decreased nitric oxide bioavailability. Aged red cells demonstrate vasoinhibitory activity of aortic ring preparations. S-nitrosohemoglobin is decreased in stored blood but when replete, tissue oxygen delivery and renal damage were ameliorated in an animal blood loss model. We aimed to determine whether the age of the stored blood relates to vascular function in-vivo, using a population of chronically transfused patients with SCD. We did prospective study to examine transfusion effects on vascular function, in which we enrolled 26 patients with SCD on CTT and tested flow mediated dilation (FMD) of the brachial artery, cardiac output and blood viscosity changes with transfusion. We measured both oxygenated and deoxygenated whole blood viscosity at shear rates from 1s-1 to 1000s-1 at native hematocrit using a Rheolog viscometer (Rheologics Co). We also obtained pre and post transfusion measures of cardiac output, flow mediated dilation of the brachial artery, blood counts, chemistry panels, markers of inflammation and hemolysis. 14 females and 12 males were enrolled. The ages and reasons for starting transfusions were similar for male and female patients. As expected, transfusion resulted in significant increases of hemoglobin and hematocrit (hct) with concomitant decrease of hemoglobin S% (HbS%) and reticulocyte count. Male patients had a significantly higher HbS%, 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. FMD was significantly improved following transfusion with an average increase of 1.4% (P=0.01). By univariate analysis, elevated body mass index (BMI), small change in hematocrit to viscosity ratio (HVR) at 2s-1 (low shear), and the lower age of the stored blood were significantly associated with improved FMD. By multivariate analysis, using only two variables at a time due to small sample size, elevated BMI was the best predictor of improved FMD. (Figure 1) There was a confounding effect between the age of the blood and BMI in our study; there was a negative correlation between age of the blood given and BMI, whereby smaller patients received older blood in our cohort.(R2 0.20, P=0.03). BMI did not correlate with pre-transfusion FMD. Most patients with SCD on CTT had improved FMD following transfusion and given the dependence of FMD on shear stress, changes in rheologic factors such as viscosity and hematocrit would be expected to cause significant changes in FMD. The increase in hct, and viscosity and the decrease in HbS% did not correlate with improvement in FMD. Decrease in low shear HVR did correlate with improved FMD, however, FMD is thought to correlate with higher shear rates, whereas the HVR at low shear rate would be found in the venous system. This could provide a link between endothelial function and the low shear venous system, particularly due to its FMD correlation with “deoxygenated” HVR. BMI and age of the stored red cells correlated whereby our smaller patients received older blood resulting in confounding between these variables. Despite the confounding effect, when blood age was locked in the model, BMI still exerts an independent effect on FMD. Whether the effect of BMI is chronic or acute could not be determined in our study; however, nutritional factors, adipose tissue and hormones might play a role in the set point of FMD via eNOS expression in the endothelium. Older blood age was associated with worsening FMD, which is consistent with decreased nitric oxide bioavailability as a piece of the red cell storage lesion puzzle. Future studies should attempt to control for BMI and blood age to minimize confounding effects. Disclosures: Coates: Novartis Inc.: Honoraria, Speakers Bureau; Apopharma: Honoraria, Speakers Bureau; Shire: Speakers Bureau. Wood:Shire: Consultancy, Research Funding; Apopharma: Honoraria, Patents & Royalties; Novartis: Honoraria.

Description: 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

Description: Introduction: Vaso-occlusive pain crisis (VOC) is a significant contributor to the morbidity of Sickle Cell Disease (SCD) and cold exposure has long been associated with increased frequency and intensity of VOC. However, the mechanism by which cold exposure causes the transition from steady state to vaso-occlusion has not been well elucidated. Decreased regional blood flow results in red blood cells spending a longer period of time in the deoxygenated state in the capillaries, increasing the likelihood of hemoglobin S polymerization in the microvasculature and subsequent vaso-occlusion. Regional blood flow is primarily regulated by the autonomic nervous system (ANS) and recent literature shows evidence of cardiovascular autonomic dysfunction in SCD and of its role as a disease modulating factor. We hypothesized that cold exposure triggers a central autonomic response leading to vasoconstriction in the microvasculature and that SCD subjects will have a stronger response when compared to controls. Methods: 17 SCD and 16 control (healthy or sickle trait) subjects aged 13 to 39 years were exposed to thermal stimuli via a computer-controlled thermode (TSA-II) placed on the thenar eminence of the right hand. Predetermined individual threshold temperatures for heat detection, cold detection, heat pain and cold pain were applied, and changes in the microvascular blood flow (MBF) were measured at a sub-second sampling rate on the contralateral thumb using photo-plethysmography (PPG). Mean MBF was derived from the amplitude of the PPG signal, a greater decrease in mean MBF from baseline signifies stronger vasoconstriction. The vasoconstriction response within the PPG signal, and the time (in sec) to vasoconstriction were determined from the cross-correlation function of the pain stimulus signal and the vascular response. The R-to-R interval (RRI) derived from electrocardiogram was used to evaluate cardiac autonomic balance. Standard deviation of RRI represents total heart rate variability, Spectral indices of the RRI represent parasympathetic activity (high frequency power; HFP) and sympatho-vagal balance (Low to high Ratio; LHR = low frequency power(LFP)/HFP). Prior to the study, subjects were administered measures of current anxiety and general level of anxiety (STAI Y1 and Y2, respectively) as well as a measure of pain related anxiety (PASS). Results: All the thermal stimulation tasks caused a significant decrease in MBF from baseline (p values for cold detection, heat detection, heat pain and cold pain=0.027,

Description: INTRODUCTION Chronic red blood cell (RBC) transfusion therapy can prevent new and progressive stroke in Sickle Cell Disease (SCD), as shown by multiple clinical trials in SCD including the STOP 1&2, SIT, and SWITCH trials. Middle cerebral artery time-averaged mean velocity (MCA velocity), measured by Trans-cranial Doppler Ultrasound (TCD), is a clinical biomarker utilized to initiate chronic RBC transfusion to reduce stroke risk in children with SCD. However, MCA velocity measured by TCD lacks sensitivity and specificity in predicting stroke. Most SCD patients with high MCA velocity do not experience overt stroke, while 10-20% of SCD children will have new strokes on chronic RBC transfusion in spite of normalized MCA velocity. Prior studies showed an association with high MCA velocity and MCA narrowing when TCD and angiography were performed after RBC transfusion to target a hematocrit (HCT) 〉 30%. HCT adjustments addressed effects of anemia and higher cardiac output (Adams et al, 1992; Stroke PMID 1636180). Nevertheless, HCT adjustments may not always be used for routine TCD. Thus, despite the substantial decrease in strokes with TCD screening, there may be significant lack of specificity that leads to unnecessary RBC transfusion resulting from TCD screening in non-optimal conditions. Due to these concerns, we undertook a detailed study assessing MCA velocity by TCD immediately pre- and post-transfusion in SCD patients. METHODS The study was approved by the Children's Hospital Los Angeles Institutional Review Board. SCD and thalassemia patients who receive chronic transfusion had pre- and post-transfusion blood laboratory tests and MCA velocity measurements by TCD (GE Vivid I, FE Medical Systems Ultrasound Israel Ltd., Israel). The TCD ultrasound probe was placed on left and right trans-temporal window, landmarks were visualized, and MCA velocity data were collected in 1.7mm depth increments from 65mm to 35mm depth. Magnetic resonance angiography was not done at the time of these measurements. Data was recorded and analyzed in JMP Pro Version 13.0. RESULTS SCD patients (n=24) had a median and interquartile range of 15 years & 14-18 years, with 18 females & 6 males, received 90 transfusions, and 70% of the patients had 3 transfusion events with repeat studies. Seventeen patients received simple RBC transfusion and 7 received red cell exchange. The thalassemia control population received simple transfusion (n=11; 48 transfusion events). Figure 1 shows the relation of MCA velocity compared to HCT in all subjects. There is a highly significant negative correlation showing lower HCT is associated with higher MCA velocity (Dots = TAMV measurements; lines = average TAMV measurements at different TCD depths; R2=0.12, F(1,4270)=556, p 30%. Quantifiable blood, patient, and measurement factors can impact TCD MCA velocity and clinical decisions that commit children to years of RBC transfusions. Disclosures Coates: ApoPharma: Consultancy, Honoraria; Celgene Corp.: Consultancy; Vifor Pharma: Consultancy; Sangamo: Consultancy, Honoraria.

Description: Introduction Over the last two decades, sickle cell disease has become a model of diffuse endotheliopathy. In 2004, Gladwin and colleagues demonstrated that elevated tricuspid regurgitation jet velocity (TRJ) was an independent predictor of mortality and that chronic red blood cell hemolysis underlies pulmonary vascular disease (Gladwin et. al. NEJM, PMID:14985486). Belhassen and colleagues demonstrated that flow-mediated dilation of the brachial artery (FMD) was decreased in patients with sickle cell disease, specifically shear-mediated vasodilation (Belhassen et. al. Blood, PMID:11238095). Our group demonstrated that plasma free hemoglobin links decreased FMD and elevated TRJ in patients with sickle cell disease and that chronic transfusion therapy improves FMD (Detterich et. al. Blood, PMID:26036801). We aimed to determine whether markers of vascular function predict the risk of mortality in a cohort of patients with sickle cell disease. Methods This was a single center, prospective cohort study of non-transfused and chronically transfused sickle cell anemia patients at Children's Hospital of Los Angeles between 2009 and 2012 aiming to assess vascular function in sickle cell disease. This study was approved by the Children's Hospital Los Angeles Institutional Review Board. We used FMD, a test of shear-mediated endothelial nitric oxide release as the model for vascular function. We simultaneously measured the regional hemoglobin oxygen saturation in the hand using near infrared spectroscopy (NIRS). We also measured microcirculatory perfusion using laser doppler flowmetry (LDF) over the skin of the nailbed. Echocardiography was performed on the same day to assess tricuspid regurgitation jet velocity and cardiac function. Markers of inflammation, cell count and hemolysis were also measured on the day of the study. We prospectively followed them until 2018 to assess our primary outcome, survival. All of the measurements were made pre-transfusion for chronically transfused subjects. Kaplan Meier and Cox-Proportional Hazards multivariate model was used to determine independent predictors of survival. All statistics were performed using Stata/IC version 14.0. Results We enrolled 26 chronically transfused patients with SCD and 63 non-transfused SCD patients in a study assessing the effects of hemolysis on endothelial function. Overall mortality in our cohort was 20% with a median follow up time of 7.8 years (7.1-8.3 IQR). Median age was 24 years at the time of study (17-36 IQR). Median age at the time of death was 36 years(28-51 IQR) and at the last follow up for the alive patients was 30 years (25-41 IQR), p=0.01. In the patients who died, they had higher TRJ (median 270 vs 235, p=0.03) and had elevated plasma Hgb, median 26 vs 14, p=0.01. FMD, regional oxygen saturation and microcirculatory perfusion were not different between alive and deceased patients. There was no difference for inflammatory markers (white blood cell count, platelet count, high sensitivity C-reactive protein), hemoglobin S%/transfusion, or anemia (hemoglobin/hematocrit/reticulocytes) between alive or deceased patients. By Kaplan Meier analysis individual factors associated with mortality were TRJ〉250 (p=0.04) and age〉21 (p=0.02) (Fig 1 and Fig 2). By Cox-Proportional Hazards Model, both TR jet velocity and plasma hemoglobin remain significant after correcting for age (Table 1). Conclusions In our cohort, there are multiple abnormal markers of vascular function and elevated markers of hemolysis; however, the strongest predictors of mortality were age, plasma free hemoglobin and tricuspid regurgitation jet velocity. These findings support the association between hemolysis, pulmonary vascular disease and mortality, a controversial topic in sickle cell disease. Disclosures Coates: Vifor Pharma: Consultancy; Sangamo: Consultancy, Honoraria; Celgene Corp.: Consultancy; ApoPharma: Consultancy, Honoraria.

Description: Introduction: Sickle cell disease (SCD) is a genetic disorder characterized by sudden onset of painful vaso-occlusive episodes (VOC) which can be triggered by stress as reported by sickle cell patients. The exact mechanism of VOC origin is not well understood; however, it could result as a progression of microvascular blockade with rigid sickled red blood cells (RBC). Anything that decreases regional blood flow (RBF) will increase the transit time that it takes the RBC to escape the microvasculature before it gets rigid and entrapped, thus increasing the chances of vaso-occlusion that may progress to VOC. The RBF is controlled by the autonomic nervous system (ANS) which is modulated by stress, and as we have previously shown, SCD patients have augmented autonomic mediated vasoconstriction response (VCR) to sigh and pain. Our studies showed significant VCR when subjects were told they were about to experience pain, suggesting that VCR might be the physiologic link between common VOC triggers like stress and sickled RBC retention in the microvasculature. Objectives: To study the effect of mental stress on autonomic parameters peripheral blood flow (PBF) and heart rate variability (HRV) in SCD. Methods: 19 SCD and 16 control (healthy and sickle cell trait) subjects were studied. Two standard mental stress tasks with graded levels of difficulty (N-back test and Stroop test) were presented to subjects using E-prime (Psychological software). Subjects were also exposed to a novel pain anticipation task, previously shown to induce VCR. PBF was measured using photo-plethysmography (PPG) on the left thumb. Reduction in PPG amplitude indicates vasoconstriction. Cardiac beat-to-beat variability (R-to-R interval;RRI) was extracted from electrocardiogram. ANS balance was derived from the following spectral indices of the RRI: high frequency power (HFP) ≈ parasympathetic activity, low frequency power (LFP), and the low/high ratio (LFP/HFP) representing sympathovagal balance. Average changes in PBF and HRV from the baseline were taken as the responses to the mental tasks. Results: There was a significant decrease in mean PBF, RRI and HFP during all the mental stressors (N-back test, Stroop test and pain fear) compared to the baseline (p