Conditional Transcranial Doppler Research Articles

Transcranial Doppler Screening of Children with Sickle Cell Disease for a Large, Multinational Interventional Study: Experience from the Phase 3 HOPE-Kids 2 Trial Investigating the Effect of Voxelotor Treatment on Transcranial Doppler Flow Velocity

Introduction: Transcranial Doppler (TCD) ultrasound is an effective screening tool for identifying children with sickle cell disease (SCD) who are at high risk of stroke. Implementing TCD screening, along with appropriate therapies, for the purpose of primary stroke prevention in resource-constrained settings remains challenging. HOPE-KIDS 2 (NCT04218084) is an ongoing, phase 3, multicenter, double-blind, placebo-controlled trial of voxelotor in children with SCD and elevated arterial cerebral blood flow (measured by time-averaged maximum of the mean velocity [TAMMV]). Our aim is to describe the findings from successful implementation of a standardized TCD screening protocol that was conducted in a multinational setting. Methods: The purpose of HOPE-KIDS 2 is to evaluate the effect of voxelotor treatment on reducing the risk of stroke in participants aged 2 to <15 years with SCD who have conditional TAMMV (170 to <200 cm/s) at baseline. Children aged 2 to <15 years with SCD (HbSS/HbSβ 0) were screened after local sonographers were trained and certified on standardized TCD examination protocol, using the same model of Multi-Dop T digital machine (Compumedics DWL, Germany). TCD assessments were sent for central quality review and interpretation by 2 independent reviewers. STOP criteria were used to classify stroke risk: normal, <170 cm/s; conditional, 170 to <200 cm/s; or abnormal, ≥200 cm/s. Patient baseline characteristics, including clinical measurements of markers of anemia and hemolysis, were measured during study screening. The reasons for patient screen failure were summarized. Results: Between Nov 2020 and Feb 2023, 708 participants consented at 29 sites in Nigeria (n=250; 5 sites), Kenya (n=241; 4 sites), Egypt (n=145; 4 sites), Ghana (n=28; 2 sites), the US (n=17; 8 sites), Italy (n=9; 3 sites), Oman (n=9; 1 site), Saudi Arabia (n=8; 1 site), and the UK (n=1; 1 site). Of these patients, 92.1% (652/708) completed TCD screening examinations (mean [SD] age 7.6 [3.24] years; range 2.0-14.0 years; 50.8% male; 23.0% receiving hydroxyurea). Of the patients who completed TCD screening, the mean (SD) TAMMV was 163.0 (31.26) cm/s, and 47.1% (307/652) had conditional TCD at baseline (29.0% low conditional, 18.1% high conditional). The main reason for screen failure was index TAMMV outside of conditional range (abnormal, n=39 [6.0%]; normal, n=306 [46.9%]). Elevated TAMMV on screening TCD was more common in younger children (2 to ≤8 years vs >8 to <15 years); patients aged 2-8 years comprised 66.8% and 82.1% of the conditional and abnormal TCD categories, respectively. There were 39 assessments that required adjudication, but only 9 were deemed unreadable. Of all patients screened, 36.2% (236/652) fulfilled eligibility criteria for randomization; the characteristics at screening of these patients are summarized (Table). The mean (SD) Hb level and TAMMV at screening for randomly assigned patients was 7.7 (1.06) g/dL and 182.7 (7.53) cm/s, respectively. Conclusions: Clinical sites for the HOPE-KIDS 2 study successfully implemented a standardized, local TCD screening protocol supported by central quality review. For a large interventional trial aimed at reducing the risk of stroke in children with SCD, African and Middle Eastern sites presented relatively few limitations with respect to participant screening.

Hydroxyurea Improves Intelligence Quotient Scores in Children with Sickle Cell Anemia and Elevated Transcranial Doppler Velocity

Introduction: Structural brain abnormalities resulting from sickle cell disease because of strokes, silent cerebral infarcts (SCI) and other biophysical characteristics of parenchymal damage from repeated sickling affect cognitive functioning. Hydroxyurea therapy reduces transcranial Doppler (TCD) velocities and improves SCI and other neurological outcomes in children with sickle cell anaemia. Hence the objectives of EXpanding Treatment for Existing Neurological Disease (EXTEND, NCT02556099), a phase 2 clinical trial, was to determine whether hydroxyurea titrated to maximum tolerated dose was effective in lowering transcranial Doppler (TCD) velocities and improving cognitive function in Jamaican children with sickle cell anemia (SCA) with increased stroke risk. Methods: Full-Scale Intelligence Quotient (FSIQ), verbal and nonverbal IQ were measured with the Wechsler Abbreviated Scale of Intelligence (WASI-II) at baseline and after 18 months of hydroxyurea treatment in 30 of the 43 enrolled children. Verbal Comprehension Index (VCI) was assessed using the Vocabulary and Similarities subtests. Perceptual Reasoning Index (PRI) was scored with the Block Design and Matrix Reasoning subtests. Members of the team conducting the tests were trained and test results were verified by an experienced member of the Epidemiology Research Unit, Caribbean Institute for Health Research. Participants were enrolled in the EXTEND trial based on a pre-treatment conditional or abnormal TCD velocity. They were assigned to one of three risk categories according to treatment status and stroke history at study enrollment: Group 1 (low stroke risk: no previous stroke, on hydroxyurea treatment n= 12), Group 2 (medium stroke risk: no previous stroke, no hydroxyurea, n=10) and Group 3 (High stroke risk: previous stroke, no hydroxyurea n=8). Anthropometric variables, including height and weight, were recorded as well as oxygen saturation and hematological variables. Differences in post treatment outcome were modelled using mixed linear regression. A p-value of <0.05 was considered statistically significant. Results: There was no difference in mean anthropometric values or gender distribution at baseline by stroke risk category groups (Table 1). The mean(sd) age in years of participants in Group 1 was 8.2(1.7), while for Group 2 the average age was 7.7(2.1) and for Group 3 it was 10.5(2.8). The mean age of Group 3 was significantly greater than Group 1. After 18 months of hydroxyurea treatment, participants were taking an average dose of 25 mg/kg/d, range 13.7 to 34.9 mg/kg/d. On treatment TCD velocities decreased by mean(sd) of 29.5 cm/sec (31.5). Hemoglobin (Hb) concentration was lowest in Group 3 but all groups showed increases during treatment with the rate of increase in Groups 2 and 3 being greater than Group 1 (p<0.001). Absolute neutrophil counts and absolute reticulocyte counts were not different by group, and both decreased significantly post treatment (p<0.001) (Table 1). At both time points, the VCI, PRI and FSIQ mean scores in Group 3 were low [Overall means(sd); 71(11.7), 71(12.6),69(11.6), respectively] and were significantly lower compared with Group 1 (p<0.001) (Table 1). While there was no significant effect of treatment on VCI and PRI scores, the mean FSIQ score increased on average by 2.9 units (95%CI 0.3, 5.4) post treatment among participants. Adjusting for age at enrollment had no effect on the improvement on FSIQ, however adjusting for changes in hemoglobin resulted in an increase in FSIQ by 3.3 units (95%CI -0.4, 7.0) which approached statistical significance (p=0.079). Conclusion: Participants treated with hydroxyurea at maximum tolerated dose for 18 months with or without a previous stroke had significant improvement in hematological variables and neuropsychological scores. Children with sickle cell anemia with high risk of stroke may benefit from routine neuropsychological testing, hydroxyurea treatment in addition to educational interventions.

Socioeconomic Status and Risk of Stroke As Assessed By Transcranial Doppler: A Displace Study Secondary Analysis

Background: Transcranial doppler (TCD) screening (Sickle Stroke Screening) identifies children ages 2-16 years with sickle cell disease (SCD) at an increased risk for stroke. Significant barriers to undergoing annual screening exist for patients with SCD. Previous studies have shown that individuals from households on public insurance with limited neighborhood opportunity have increased barriers to receiving adequate healthcare, including routine screening; however it is unknown whether low socioeconomic status (SES) influences TCD results. Methods: We conducted a secondary analysis of the DISPLACE study, which included a cohort of 3243 TCD-eligible individuals aged 2-16 who had at least one TCD screen performed during their year of study enrollment. We utilized insurance type (Medicaid/CHIP or public versus all other insurances) and overall childhood opportunity index (COI) group (Very Low versus all other COI levels [Low, Medium, High, and Very High]) to proxy household- and neighborhood-level SES. We then examined the association between household- and neighborhood-level SES and conditional and/or abnormal TCD screening results. Baseline characteristics of the overall cohort (Table 1) were described by COI group and insurance type. The primary outcome was TCD result at baseline (i.e. a patient's first TCD in the database); categorized as abnormal, high-conditional, and normal (reference group). Multinomial logistic regression analysis was used to assess whether clinical (age, sex [female vs. male], hemoglobin (Hgb) level, white blood cell [WBC] count, presence of clinical hypertension [yes, no], hydroxyurea (HU) prescription at baseline [yes, no]) or socioeconomic factors (insurance type and COI group) were independently associated with TCD result. The multinomial regression accounted for age, Hgb level, WBC count, HU prescription, COI group, and insurance type. All tests were two-tailed and, an alpha level of <0.05 and <0.1 (to reduce type II error in logistic regression) were used to determine statistical significance. Results: The analytic cohort included 3243 children (Table 1), with 1346 (41.5%) children living in Very Low COI neighborhoods and 2180 (67.2%) of households with public insurance. HU was prescribed to 60.7% of individuals, and this varied by COI level (p=0.021), but not insurance type (p=0.758). WBC count was significantly higher in the Very Low COI group (p=0.004) and public insurance group (p<0.001). Hgb level was noted to be significantly lower in the Very Low COI (p=0.031) and public insurance (p<0.001) groups. Bivariate logistic regression analysis revealed that COI group (OR 1.53, p=0.08) and insurance type (OR=1.76, p=0.056) were significantly associated at p<0.1 with abnormal TCD outcome individually. Lack of HU prescription was significantly associated at p<0.05 with abnormal TCD result (Odds Ratio [OR]=20.36, p<0.001), and WBC count (OR=1.05, p=0.055) was significantly associated at p<0.1 with abnormal TCD result individually. In the multinomial logistic regression model (included variables listed above), only lack of HU prescription remained significantly associated at p<0.05 with abnormal TCD result (OR=19.09, p<0.001). Conclusion: The most robust predictive factor determining abnormal or conditional TCD status in this cohort was lack of HU prescription. Public insurance and COI group as proxies for SES were not independently associated with abnormal or conditional TCD results. As such, the disparities seen by individuals from proxied lower SES are likely mediated by utilization of HU. Notably, HU prescription rates differed by COI group, but not insurance type, suggesting possible neighborhood level factors (such as availability of pharmacies dispensing HU) influencing HU prescription that wouldn't be reflected by insurance type. The limitations of using socioeconomic proxies such as COI group and insurance type must be noted, as these proxies assign group characteristics to an individual based on a snapshot in time, which may not be representative of the dynamic nature of one's SES. Nevertheless, these findings highlight the need to better understand the possible association between SES and HU prescription, as limited HU access and utilization could result in significant health disparities in both treatment and outcomes.

Microemboli on Transcranial Doppler in Patients with Sickle Cell Disease: A Single Center Case Series

Background: Stroke remains a major cause of morbidity in patients with sickle cell disease (SCD). Current guidelines recommend that patients with Hemoglobin SS and Hemoglobin S beta zero thalassemia have annual transcranial doppler (TCD) screening and that patients with abnormal velocities should have regular blood transfusions to maintain a maximum HbS level <30% and maintain a hemoglobin level >9 g/dL for at least one year to decrease risk of stroke (DeBaun et al., 2020). While research has shown that patients with velocities >200cm/sec are at increased risk of cerebral infarct, there is no literature to discuss the follow up and management of patients with microemboli on TCD. In the past microemoli were unable to be detected, however TCD is now able to detect such findings (Wan et al., 2022). Studies have been completed on patients with ischemic stroke without SCD and have shown that the presence of microemboli on TCD is associated with a higher rate of recurrence (Das et al., 2020). The presence of emboli has also been shown to recognize patients with asymptomatic carotid stenosis and increased risk for stroke (Markus et al., 2010). Although noted in the general population, the significance of such a finding on an annual TCD screening in patients with sickle cell disease is unknown. Methods:At the Medical University of South Carolina, we performed a retrospective review of all patients with SCD who have had TCDs from 2017 to February 2023 and were found to have microemboli to evaluate management and outcome. Results: We have had 5 patients with an incidental finding of microemboli on their annual TCD since 2017. Three were male with median age of 3 (range 2 to 15 years). Four individuals are HbSS genotype. Mean hemoglobin at the time of TCD was 8.26 ± 1.4 g/dL. TCD velocity was normal in four patients at the time of the TCD, while the remaining individual had a low conditional TCD. Two have a previous history of conditional or abnormal TCDs. The location of the microemboli was variable among patients (Table 1). At the time of microemboli findings on TCD, discussions were held with family regarding options for treatment including chronic partial exchange transfusions and optimization of other disease modifying therapies. Management of all patients included MRI at the time of microemboli, which was normal in only two patients. Three patients were started on chronic manual exchange transfusions for 10 months to 3 years. One out of the 3 continued home HU while receiving transfusions while the remainder were transitioned to HU once chronic transfusions were complete. One patient was not started on chronic transfusions but monitored closely with TCDs due to family choice. On follow up, 4 have had repeat TCDs and all have normalized without any subsequent episodes of microemboli. One patient was found to have silent infarct on MRI at time of TCD and 1 has had follow up MRI 4 years after original TCD with a new silent infarct. Of note, this patient had inconsistent follow up with chronic transfusions after initial abnormal TCD. Importantly, none of our 5 patients had an acute symptomatic ischemic event since the microemboli were noted. Overall, among these patients, 2 have had concern for silent infarct and 3 of 5 started on chronic transfusions (Table 1). Conclusion: This is the first report within the literature of microemboli on annual TCD screening in patients with SCD. As this is a new phenomenon, the significance within the disease course is not known for those with sickle cell disease. Identifying patients with microemboli has been used to help navigate the need for early intervention to prevent stroke. Given that individuals with SCD are already at an increased risk for cerebrovascular events, these findings of microemboli could indicate further risk in these individuals as well and deserve further investigation. Further research is warranted as the significance of such a finding is unknown and further understanding could impact the standard of care of this patient population.

Relationship between hemoglobin, hemolysis, and transcranial Doppler velocities in children with sickle cell disease: Results from a long-term natural history study in Italy in the era of multimodal therapy.

Stroke and cerebral vasculopathy are leading causes of morbidity and mortality in patients with sickle cell disease (SCD). Transcranial Doppler (TCD) is a reliable and validated predictor of stroke risk. Children with conditional or abnormal TCD are at an increased risk for stroke, which can be mitigated by red blood cell transfusion or hydroxyurea. Elucidating the relationship between cerebral hemodynamics and hemolytic anemia can help identify novel therapeutic approaches to reduce stroke risk and transfusion dependence. This long-term, real-world study was designed to evaluate the prevalence of TCD imaging (TCDi)-assessed flow velocities in children and to interrogate their relationship with markers of anemia and hemolysis. In total, 155 children (median follow-up 79.8 months, 1358.44 patient-years) had 583 evaluable TCDi results. Only patients with HbSS or HbSβ0 had abnormal (1.6%) or conditional (10.9%) TCDi. Children with abnormal or conditional TCDi had lower hemoglobin (Hb) and higher hemolysis markers. A linear correlation was detected between TCD velocity and Hb: an Hb increase of 1 g/dL corresponded to decreases in velocity in the internal carotid and middle cerebral arteries (6.137 cm/s and 7.243 cm/s). Moreover, patients with Hb >9 g/dL presented a lower risk of TCDi-associated events. These results support the need to optimize disease-modifying treatments that increase Hb and reduce hemolysis for stroke prevention in young children with SCD.

5616493 MACHINE LEARNING TO IDENTIFY CHILDREN WITH SICKLE CELL DISEASE AT VERY LOW HIGH RISK OF ABNORMAL OR CONDITIONAL TRANSCRANIAL DOPPLER SCREENING.

Background: Annual Transcranial doppler (TCD) screening is an important strategy to mitigate the risk of stroke in children with sickle cell anemia (SCA). While an abnormal TCD is strongly associated with increased chance of stroke, the risk factors for an abnormal doppler study are not well understood. Identifying children at very low risk could help to ensure appropriate focus on screening children at the highest risk. Machine learning presents an opportunity to develop a risk prediction model, incorporating complex interactions between variables. Aim: Identify potential predictive features for normal TCD screens by applying machine learning algorithms to clinical data collected for a multi-site collaboration, Dissemination and Implementation of Stroke Prevention Looking at the Care Environment (DISPLACE).1 Methods: We analyzed clinical data collected for the 28-site DISPLACE study, which included 5116 patients in whom a TCD was indicated.1 For the current study, we included patients, aged 2-16 years, who had both hemoglobin and TCD results reported in the same year between 2012-2016 (N = 2234); after excluding patients who had prior studies with abnormal or conditional results, our analysis dataset included 22 predictive features (maximum missingness: 25.7%) for 1825 children (N = 30 with abnormal, N = 122 with conditional results). We applied traditional logistic regression, logistic regression with LASSO, and tree-based classifiers to 19 features including historical, biometric, vital sign, and laboratory data, to predict the risk of abnormal or conditional TCD. Results: In univariate logistic regression models, all 19 features had a statistically significant relationship with TCD outcome. Eight features (age, hemoglobin, heart rate, systolic blood pressure, oxygen saturation, weight, mean corpuscular volume, and percent reticulocytes) were shown to be of consistently high importance across different machine learning models. Conclusion: Next steps include development of ensemble models to optimize predictive performance and internal validation on our hold-out test set. Machine learning shows promise for identifying low risk children with SCD who may not require annual TCD screening, such that TCD resources can be appropriately directed to higher risk children to mitigate the risk of stroke.Reference 1. Kanter et al, J Pediatr Hematol Oncol 2021; 43:e1062-8

Optimizing the Frequency of Transcranial Doppler (TCD) Exams in Children with Sickle Cell Anemia

Background Transcranial Doppler (TCD) screening of the large intracranial arteries can detect elevated blood velocity and is a strong predictor of stroke risk in children with sickle cell anemia (SCA, HbSS and HbSβ0-thalassemia). Initiation of chronic blood transfusion for patients with confirmed abnormal TCD velocities (time-averaged mean velocity, TAMV≥200 cm/sec) is shown to effectively reduce primary stroke. Current guidelines recommend annual TCD screening for children with SCA ages 2-16 years. Screening intervals were developed following evidence from the STOP studies1 (Figure 1). The estimated prevalence of normal TCD velocities is 83%2 and these children will have received 15 TCD exams from ages 2 to 16 if all results remain within normal range. The cumulative incidence of conversion from normal to conditional TCD velocities is dependent on age, whereby the risk is four times lower in children >10 years, compared to those <10 years of age3. Although TCD screening is non-invasive and safe, it consumes time and requires specialized equipment and trained examiners. Identifying sub-populations for whom TCD screening frequency could be safely reduced could lessen time burden on patients and caregivers and could be cost saving. Main objective To estimate the risk of TAMV conversion from normal to conditional or abnormal categories among children with SCA relative to age. Design/Method We included all participants with SCA from the Sickle Cell Clinical Research and Intervention Program, a longitudinal lifetime cohort of patients with SCD with at least one TCD measurement available both before and after age 10 years. We classified participants into two groups based on TCD screening results before 10 years of age; one group with all normal TCDs, and the other group with at least one non-normal TCD. We then compared the time to first non-normal study after ten years of age between the two groups using the log rank test. Time to first conversion was defined as time from age 10, to time of conversion if conversion occurred, or time to last follow up if no conversion occurred. We correlated the number of TCD exams before age 10 with time to first conversion using the Cox proportional regression model. Exposure to chronic transfusion or hydroxyurea before age 10 and age at first TCD measurement were covariates and were correlated with the time to first conversion. Results Of the 614 patients with TCD screening, 297 had SCA and at least 1 screening before and after age 10 (Table 1). Of these, 174 patients had only normal TCD screening before age 10 and the remaining 123 patients had at least 1 non-normal TCD screening before age 10. During the time they were followed,no patients with only normal TCD screenings before age 10, regardless of exposure to hydroxyurea or transfusions, had a conditional or abnormal TCD exam. Conversely, 41.5% of patients with at least one non-normal TCD before age 10 had a conditional or abnormal TCD screening after age 10. Conclusion In a large retrospective cohort study analysis of children with SCA, patients with TCD exams consistently within the normal range under age 10 years were not at risk of conversion to conditional or abnormal TAMV. These results suggest that children with SCA with all normal TCD velocities before age 10 years may not require additional TCD screening exams after age 10. These results translate to 6 fewer TCD exams per each child with SCA over age 10 with all prior normal TCD exams, potentially conserving resources and reducing burden on patients, families, and the health system. De-implementation studies to reduce the use of TCD screening exams in low-risk cases are needed

Implementation Of Transcranial Doppler Ultrasonography Screening and Primary Stroke Prevention In Urban and Rural Sickle Cell Disease Populations

Transcranial doppler (TCD) ultrasonography has been used successfully to identify children with sickle cell disease (SCD) at increased risk of stroke. A large randomized trial (STOP trial) demonstrated that initiation of chronic transfusions as primary stroke prevention in children with an abnormal TCD significantly reduced stroke risk. Based on these findings, annual TCD screening and primary stroke prevention for patients with abnormal findings are considered standard of care (SOC) for children with SCD. We report here results describing the implementation of TCD screening and primary stroke prevention in both urban and rural clinical practice.A retrospective chart review was conducted to identify children ages 2-16 years with Hgb SS or Sß0 thalassemia and no history of stroke. Patients are followed in the local urban comprehensive sickle cell program at Georgia Regents University of in 4 outreach clinics in rural Georgia. Starting in 2010 TCDs conducted by a STOP trained sonographer were offered three times annually in the rural clinics. All TCD results were recorded before and after the implementation of the rural TCD program. SOC is defined as having one TCD performed annually between January 1, 2010 and December 31,2012 starting at age 2 years in patients with no history of abnormal TCD.A total of 213 children were identified who met inclusion criteria. Table 1 describes demographic and TCD information since 2010. Prior to 2010 20% of patients had no history of TCD; following implementation of the rural TCD program 7% of patients never had a TCD (p≤ 0.01). The percentage of patients with conditional or normal TCD results since 2010 is comparable between urban and rural clinics, whereas the proportion of patients with abnormal TCDs is higher in the rural clinics (p=0.01). Overall 41% of patients achieved SOC screening. There was no difference in SOC rates between urban and rural clinics (Table 1, p=0.12). Furthermore, there was no difference in SOC rates in the urban and rural clinics in patients with a history of normal (33% and 42% respectively, p=0.21) or conditional TCDs (50% and 58% respectively, p=0.72). Among the group of patients with a history of abnormal TCDs (n=18) the majority of patients were on chronic transfusions (83%) and none have experienced a stroke. While rural patients were able to receive transfusions locally, monitoring of transfusions was difficult with 38% and 31% of rurally transfused patients lacking documentation of Hgb S% and ferritin levels respectively in the past year. [Display omitted] We report here data describing implementation of the STOP trial recommendations in clinical practice with unique data on rural patients. Our data demonstrates that rural TCD screening is feasible and can achieve SOC equal to that in an urban setting. We did find a higher number of abnormal TCDs in the rural population, perhaps secondary to differences in TCD sonographer training or an unrecognized disparity in care. While the majority of children (93%) have received at least one TCD, a significant number of patients are not obtaining annual SOC screening. Chart review indicated common barriers include adherence with clinic appointments, refusal of screening, and loss to follow up care. Unlike screening, implementation of primary stroke prevention in rural patients is difficult with minimal laboratory data being followed by the hematologist. In conclusion, our data suggest that practice and physician level barriers exist that impact the ability to provide SOC primary stroke prevention. Future research should target quality measures to improve practice level barriers, provide parental education on the importance of screening and novel interventions to enhance patient adherence. Disclosures:No relevant conflicts of interest to declare.

Silent Cerebral Infarct Pathophysiology Study in Sickle Cell Anemia Children By a Multimodal Approach

▪Context and objectives:Infra clinical cerebral infarcts have emerged as a central problem in sickle cell anemia (SCA) since the effective reduction of clinical cerebral infarcts following systematic vasculopathy screening and prevention. They are found in up to 37% of SCA children at 14 years old, and correlate with neurocognitive deficit. The tendency of silent infarcts to occur in the border zone areas of the brain, mainly the deep subcortical white matter, highly suggests that they could be due to hypoperfusion or hypoxic events but no clear pathophysiological process has been demonstrated. The aim of this study was to better decipher the mechanisms involved in infra clinical cerebral injury by analyzing the determinants of cerebral oxygenation and perfusion at the microcirculatory level in children with SCA and without cerebral or cervical vasculopathy. We conducted a multimodal prospective pilot study combining conventional brain exploration and laboratory biomarkers as well as novel brain investigational tools such as Arterial Spin Labeling (ASL) perfusion Magnetic Resonance imaging (MRI) and brain Near Infra-Red Spectroscopy (NIRS).Patients and Methods:Following parental approval, children with SCA aged 5-17 years, with no past history of abnormal or conditional transcranial Doppler (TCD), at steady state (> 3 months from any vaso occlusive event, transfusion or infection) were enrolled in two investigating centers in Ile de France. Relevant medical history and biological parameters were recorded and all investigation was performed during an out-patient visit.Results:Fifty-nine SCA patients (11.4± 3.9 yrs) at steady state were included between February 2015 and August 2016. Eight (13%) children had infra clinical lesions on MRI (3-5 mm: n= 6; 5-15 mm: n=4; > 15mm: n=0), predominating in the middle cerebral artery border zone in five children. The two groups of patients with lesions (MRI+) and without lesions (MRI-) were comparable in terms of age, hydroxycarbamide (HC) treatment, G6PD or alpha thalassemia status. Conversely, the MRI+ group had a lower annual VOC rate (0.2 ± 0.2 vs 0.7 ± 0.6/year, p< 0.05) and lower mean arterial pressure (76 ± 8 vs 83 ± 9mmHg, p< 0.05), hematocrit (23.0 ± 3.0 vs 25.3 ± 3.2 %, p < 0.05), RBC deformability (Elongation index: 0.44 ± 0.07 vs 0.50 ± 0.08, p < 0.05) but higher P selectin level (73.5 ± 21.7 vs 57.1 ± 20.7ng/mL, p < 0.05) and RBC aggregate strength (491 ± 236 vs 358 ± 190 s-1, p < 0.05), when compared to the MRI- group.ASL perfusion in all cerebral territories showed no major asymmetry. High ASL perfusion was significantly inversely correlated with Hb level, RBC deformability, cerebral oximetry, HbF level and blood viscosity. High ASL perfusion correlated positively with vasomotion, hemolysis markers (AST, LDH, absolute reticulocyte count), platelets, P-and E- selectin levels, RBC aggregate strength, neutrophil count. Furthermore and importantly, silent lesions in the right anterior border zone were associated with high ASL perfusion (p<0.05).Discussion and conclusion:Among this cohort of highly selected SCA children with no vasculopathy, we found a prevalence of infra clinical cerebral injury of 13.5%. Our results altogether suggest that marked hemolysis, anemia and hypoxia result in an increased compensatory cerebral perfusion, as well as endothelial lesions as evidenced by elevated E and P selectins. Our results comfort the hypothesis that infra clinical cerebral lesions in the border zone territories occur when the compensatory perfusion of the brain, which is increased at steady state, is unable to further adapt. Our results argue for early treatment of SCA children with marked hemolytic anemia to avoid cerebral injury in case of acute hypoxia, anemia or increased metabolic demand. DisclosuresBrousse:Add Medica: Membership on an entity's Board of Directors or advisory committees. De Montalembert:Addmedica: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Distribution of sickle cell disease and assessment of risk factors based on transcranial Doppler values in the Gulf region

ABSTRACT Background/Objective: Stroke is a potentially fatal complication of sickle cell disease (SCD). Transcranial Doppler (TCD) is useful at identifying increased risk of stroke in children with SCD and vasospasm after subarachnoid hemorrhage. The main aim of this study was to determine the proportion of patients with SCD in the Gulf region who are at a high risk of stroke, as determined by TCD. Methods: This multicenter (Oman, Qatar, and UAE), descriptive, cross-sectional study in patients (aged 2–16 years) with SCD included a baseline visit, 1 follow-up visit for patients with conditional TCD, and 3-year retrospective data analysis for all patients. Results: Of the 410 eligible patients (Oman, 86.5%; Qatar, 8.2%; UAE, 5.1%), most had a TCD finding (left side, 91.7%; right side, 92.0%) of normal velocity (<155 cm/s) at baseline. For 6 of 7 patients with conditional velocity (155–179 cm/s) and 1 patient with high velocity (≥180 cm/s), baseline TCD results were not confirmed at follow-up. As per bivariate linear regression, age, race, transfusion type, and transfusion frequency were significant predictors of the TCD velocities. Multivariate logistic regressions revealed that TCD velocities were significantly correlated with sex, race, and type of transfusion. No patients reported any adverse events at follow-up. No deaths occurred during the study. Discussion/Conclusions: The study results show that far fewer patients with SCD in the Gulf have abnormal TCD findings than the internationally reported. Larger studies are needed to identify the factors underlying this observation.

Stroke Avoidance for Children in República Dominicana (SACRED):a Prospective Trial to Reduce Stroke in Children with Sickle Cell Anemia

Introduction. The burden of sickle cell anemia (SCA) is high across the Caribbean, including the Dominican Republic where the morbidity of SCA is substantial due to limited resources. Approximately 10% of affected children will develop primary stroke, with serious medical and neurocognitive sequelae. In the US, transcranial Doppler (TCD) ultrasonography is an effective screening tool to identify children at risk for primary stroke, which then allows preventive therapy with either hydroxyurea or blood transfusions. To date, however, no routine TCD screening and treatment program for stroke prevention has been established in the Dominican Republic. We designed a prospective screening and treatment study (SACRED, NCT02769845) for prevention of primary stroke in children with SCA using open-label hydroxyurea at maximum tolerated dose (MTD). Methods. Children with SCA between age 3-15 years and receiving medical care at Robert Reid Cabral Children's Hospital in Santo Domingo, Dominican Republic were recruited over a 12-month period. Treatment depended on the baseline TCD examination: children with abnormal time-averaged mean velocities (≥200 cm/sec, high stroke risk) were recommended to receive monthly transfusions for primary stroke prevention, as per standard treatment preferences at the Robert Reid Hospital; those with conditional velocities (170-199 cm/sec, moderate stroke risk) were offered hydroxyurea study treatment unless they were currently receiving chronic transfusions for other clinical indications. Oral open-label hydroxyurea using 500mg capsules was prescribed at fixed-dose (20 mg/kg/day) for 6 months, followed by dose escalation to MTD. TCD examinations were repeated every 6 months, with the main study endpoints being changes in laboratory parameters and TCD velocities after 18 months of study treatment. Results. A total of 283 children were enrolled with the following baseline TCD velocities: 200 (70.7%) normal, 63 (22.3%) conditional, 11 (3.9%) abnormal, and 9 (3.1%) inadequate velocities. Among 50 children eligible for hydroxyurea study treatment, the average age was 6.8 ± 2.8 years and included 27 males/23 females with evidence of substantial SCA-related morbidity (88% with previous transfusions, 60% with &gt;5 hospitalizations). A total of 48 children initiated hydroxyurea at 20.2 ± 1.2 mg/kg/day, which was maintained for 6 months followed by dose escalation, reaching MTD at 24.1 ± 6.0 mg/kg/day. Laboratory changes from baseline to Month 18 included significant increases in hemoglobin (7.5 ± 0.9 to 8.9 ± 1.3 g/dL, P&lt;0.0001) and fetal hemoglobin (16.4 ± 7.4 to 30.9 ± 13.6%, P&lt;0.0001). Serial TCD examinations revealed a significant decline over the treatment period, with the average TCD velocity decreasing from 180 ± 8 cm/sec at baseline to 159 ± 21 cm/sec at Month 18, P&lt;0.0001. At Month 18, 68% of the originally conditional TCD examinations had reverted to a normal velocity, while only 4% had converted to abnormal velocity. No clinical strokes occurred in the hydroxyurea treatment cohort. Conclusion. Conditional TCD velocities are common (&gt;20%) in the Dominican Republic, which documents an elevated stroke risk for children with SCA. The SACRED trial established a local TCD screening program and provided open-label hydroxyurea treatment for children with conditional TCD velocities. Hydroxyurea treatment at MTD led to improved hematological parameters, lower TCD velocities, and decreased stroke risk. SACRED represents an important international partnership and prospective collaborative research trial providing capacity building and valuable epidemiological data regarding TCD screening, stroke risk, and hydroxyurea effects for children with SCA. Hydroxyurea at MTD is a suitable treatment to reduce primary stroke risk for children with SCA living in the Dominican Republic or other low-resource settings. Disclosures Jeste: Jannsen: Employment. Ware:Novartis: Other: DSMB; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees.

Prospective longitudinal follow‐up of children with sickle cell disease treated with hydroxyurea since infancy

Hydroxyurea (HU) increases fetal hemoglobin (HgbF) and ameliorates sickle cell disease (SCD) symptoms. Studies have demonstrated the safety and efficacy of HU in infants and children. Initiation of HU in infancy for children with SCD needs to be implemented in community practice. Starting in 2011, the Pediatric Sickle Cell Program of Northern Virginia initiated HU in infants with SCD. A prospective longitudinal database tracked the clinical course and outcomes. Twenty-four children with HgbSS who started HU by age 1 were continuously followed for a total of 95 person-years. Age at the time of analysis ranged from 2 to 7 years. Average hemoglobin at 6-month intervals ranged from 9.5 + 1.9 to 10.7 + 0.8g/dL, and average HgbF ranged from 27.8 + 5.0% to 34.1 + 6.6%. Twenty-seven hospitalizations occurred (0.28/person-year), all before age 3, including 19 (70%) for fever or infection, five (19%) for splenic sequestration, and one (4%) for pain in an infant prior to starting HU. The treat-and-release emergency department visits totaled 68 (0.72/person-year), including 62 visits (91%) for fever, infection, or viral illness, and two visits (3%) for pain/dactylitis in infants before HU initiation. Splenic sequestration accounted for all five transfusions. No pain episodes requiring medical attention were documented after HU initiation. No complicated acute chest syndrome, no abnormal or conditional transcranial Doppler ultrasound, and no overt strokes occurred. Implementation of HU in infancy for patients with SCD in community practice is feasible and is highly effective in preventing disease complications.

Practice Patterns in the Use of MRI/MRA and Chronic Transfusion Therapy for Monitoring and Treatment of Stroke in Pediatric Patients with Sickle Cell Anemia

Introduction: Current stroke prevention guidelines in pediatric sickle cell anemia (SCA) specify the use of transcranial Doppler (TCD) and chronic red cell transfusion therapy (CRCT) for detection and prevention of stroke. Guidance on the use of MRI/MRA in the context of stroke prevention, screening, and monitoring is less clear. While overt stroke is able to be identified clinically, silent stroke can be more difficult to diagnose and screening guidelines have not been developed. The DISPLACE Consortium (Dissemination and Implementation Looking at the Care Environment) is a multi-center study funded to evaluate optimal implementation of stroke prevention guidelines. The purpose of the present study was to provide information on practice patterns across the DISPLACE Consortium as they relate to the use of MRI/MRA and CRCT for monitoring and treating stroke in pediatric patients with SCA.Methods: DISPLACE is a multi-site study in SCA including 28 centers across the United States. A practice patterns survey was sent to the principal investigator (PI) for each DISPLACE site through RedCap. All PIs are specialty care providers in hematology/oncology who provide care to pediatric patients with SCA. The sites were chosen to represent a range of characteristics, including both urban and rural regions, large and small academic institutions, and community-based institutions. The survey was developed by the study investigators and questions were provided predominantly in a multiple choice format. The following types of questions were asked about MRI/MRA: indication for pediatric patients with SCA in general and for those on CRCT for stroke prevention. The following types of questions were asked about CRCT: indication and frequency of CRCT and target hemoglobin S level.Results: All 28 providers completed the survey (100%). About half of providers were female (53.5%). Most providers identified as White (77.8%), followed by Asian (11.1%) and Black or African American (7.4%). Two identified as Hispanic or Latino (7.4%). MRI was used for the following indications: abnormal TCD (85.7%), conditional TCD (46.4%), recurrent headaches (78.6%), and behavior issues (53.6%). A few sites also reported obtaining an MRI if a child has had poor school performance (10.7%) or if there was concern for developmental delay (3.7%). Indications for MRA were similar. Notably, many sites reported the use of a screening or baseline MRI in the absence of other indications (25.0%). The most common indications for initiating CRCT were abnormal TCD (96.4%) and overt stroke (92.9%), followed by silent stroke (53.6%) and abnormal MRA (50.0%). The majority of sites reported scheduling CRCT at a frequency of every 4 weeks (63.0%), though many (37.0%) described other more frequent transfusions intended to either more rapidly reduce hemoglobin S levels or maintain a level under 30%. Most sites used a target of 30% hemoglobin S, though a few sites noted alternatives including 40% (3.7%) and 50% (3.7%). The frequency of MRI for patients receiving CRCT for stroke prevention was as follows: annually (44.4%), every 2 years (14.8%), and every 5 years (3.7%). Several sites reported obtaining MRIs only if there were new changes or concerns (14.8%) or reported alternative practices (14.8%) that depended on patient characteristics. Frequency for MRA was similar for patients on CRCT.Conclusions: The results of the DISPLACE practice patterns survey suggested nearly universal adoption of CRCT for children with abnormal TCD and prior overt stroke across sites. The use of MRI/MRA in detecting potential cerebrovascular abnormalities in the absence of clinical neurologic symptoms and for monitoring patients on CRCT was much more variable, which likely reflects the minimal amount of guidance for the use of MRI/MRA in SCA. Notably, a number of sites were using MRI/MRA to screen pediatric patients for cerebrovascular abnormalities and to monitor patients on CRCT. The variability in responses for MRI/MRA highlights the importance of future studies evaluating best neuroimaging practices for detecting cerebrovascular abnormalities (apart from TCD) and for using MRI/MRA to monitor patients on CRCT. DisclosuresSchlenz:NHLBI: Research Funding. Mueller:NHLBI: Research Funding. Phillips:NHLBI: Research Funding. Melvin:NHLBI: Research Funding. Adams:NHLBI: Research Funding. Kanter:bluebird bio: Membership on an entity's Board of Directors or advisory committees, Research Funding; NHLBI: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Apopharma: Research Funding; Sancilio: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees.

Children with HbSβ0 thalassemia have higher hemoglobin levels and lower incidence rate of acute chest syndrome compared to children with HbSS.

Based on the presumed clinical similarity between the two most severe sickle cell disease (SCD) genotypes, hemoglobin (Hb) Sβ0 thalassemia and HbSS, randomized controlled trials (RCTs) have included both genotypes. Our group has demonstrated that healthcare providers inadequately distinguish the two diagnoses through clinical and laboratory parameters. In a retrospective cohort study utilizing data from the Silent Cerebral Infarct Transfusion trial (NCT00072761), we tested the hypothesis that children with genotypic diagnoses of HbSβ0 thalassemia, when compared to HbSS, have significantly different rates of SCD comorbidities. Exclusion criteria included those with previous overt stroke or treatment with hydroxyurea or regular blood transfusion therapy. Among children with HbSβ0 thalassemia (n=22) and HbSS (n=786), the mean hemoglobin was higher in children with HbSβ0 thalassemia (9.2g/dl) compared to HbSS (8.1g/dl, P<0.001). In children with HbSβ0 thalassemia, when compared to HbSS, the incidence rate of acute chest syndrome (ACS) was 3.0 and 14.4 events per 100 patient-years (P=0.028), and mean transcranial Doppler (TCD) velocities were 112.6 and 135.6cm/sec, respectively (P=0.026). The number of children with HbSβ0 thalassemia and HbSS with conditional TCD velocities were zero (0%) and 26 (4.9%), respectively (P=1.00), and the number with silent cerebral infarcts were five (27.8%) and 209 (30.8%), respectively (P=0.78). We have provided preliminary evidence that clinically relevant differences occur in ACS rates and TCD velocities between children with HbSβ0 thalassemia and HbSS. Future SCD RCTs should consider balanced allocation of these SCD genotypes, particularly when ACS and abnormal TCD velocities are primary outcome measures.

Hydroxyurea Therapy Prevents High Risk Trans-Cranial Doppler Development in Children with Sickle Cell Disorders

▪Annual evaluation by Trans-Cranial Doppler (TCD) of cerebral artery blood flow is useful for determining stroke risk in children with sickle cell disease. Since the publication of the Stroke Prevention Trial in Sickle Cell Anaemia (STOP) results in 2006 it has been standard practice at our centre to perform annual TCD imaging scans on all children with sickle cell disease to identify individuals with high risk features. Data from the STOP trial demonstrated that the incidence of stroke was reduced by 90% in children with high risk TCDs when they were managed on a regular transfusion programme.In more recent years, the use of hydroxyurea as a disease modifying therapy has increasingly become our practice, particularly so since the publication of the BABY-HUG trial data in 2014. Hydroxyurea has significantly altered the clinical course of sickle cell disease in children and prior to this era, 11% of patients would experience a stroke before the age of 20 years.In light of the changing management strategies for children with sickle cell disease, we reviewed the past 10 years TCD results from our patients.Annual TCD evaluation of cerebral artery blood flow was performed by radiologists and radiographers accredited to the Kings College national training programme. Scans were performed using Philips iU22 scanners with dedicated F5-1 transducers as per the UK National Screening protocol. Velocities were measured in the middle (MCA), anterior (ACA) and posterior cerebral arteries (PCA), internal carotid artery (ICA) and bifurcation on right and left sides (10 measurements per scan). The TCD service at our centre has had a positive external peer review.Over the past 10 years, 303 TCD imaging scans have been performed on 53 children aged 2-18 years. The number of abnormal, high-risk results (cerebral blood flow velocity (CBF-V) >200 cm/s) has been zero. 137 scans were performed in children prior to those individuals commencing hydroxyurea therapy, and the results of 3 scans were conditional, based on an increased CBF-V (171-200 cm/s) detected in 1 or more arteries. The other 168 scans were performed on children who were receiving hydroxyurea, of which 4 results were conditional. 49 of the 53 children have continuously had a normal TCD with no conditional or abnormal results. 4 children developed transient conditional TCD.The 3 conditional results recorded prior to commencing hydroxyurea therapy relate to 2 patients. One case in which a child with severe phenotype already on regular blood transfusions developed a conditional TCD which subsequently normalised without adjustment to their regular transfusion programme. Transfusions were then stopped and hydroxyurea commenced and this patient has since maintained normal TCD features (8 years follow up). The other 2 conditional scans relate to one patient where a conditional TCD developed in a young child not receiving any therapy. This feature normalised without treatment by the time of the following annual scan but subsequently reoccurred the following year. Hydroxyurea was commenced because of the second conditional TCD result, which then normalised and has remained normal (7 years follow up).The 4 conditional results that occurred in individuals receiving hydroxyurea relate to 2 patients. Both children developed conditional TCDs and the increased CBF-V persisted until their next annual scan. In both cases hydroxyurea was escalated to maximum tolerated dose following the second conditional TCD result. TCDs in both patients normalised by the time of their next annual scan and have remained normal since (6 years and 2 years follow up).Our hydroxyurea practice has resulted in not a single child being started on transfusion therapy from our TCD programme. Importantly, at the end of this observation period all 53 children have normal TCD features and no child is managed on a transfusion programme. In the era of high dose hydroxyurea therapy, our experience is that we have not seen any abnormal TCD results. We need to consider whether TCD remains relevant or whether alternative strategies to predict and prevent stroke need to be developed. DisclosuresNo relevant conflicts of interest to declare.

Changes in Transcranial Doppler Flow Velocities in Children with Sickle Cell Disease: The Impact of Hydroxyurea Therapy

Hydroxyurea (HU) was recently described as a substitute for chronic transfusion for children with sickle cell disease (SCD) and abnormal transcranial Doppler (TCD) velocities who have received at least 1 year of transfusions. However, the role of HU in reverting elevated TCD velocities in patients not treated with transfusion is still debatable. The objective of the study was to examine whether HU influences the progression of TCD velocities in children with SCD. Children with SCD with at least 2 TCDs not less than 6 months apart were evaluated over 51 months. Time-averaged maximum mean (TAMM) velocities for the initial and the last transcranial Doppler examinations were noted and differences compared between HU and HU-naive groups. Overall, 68.8% of the HU-group with elevated TCD velocities compared with 40.0% of the HU-naive experienced TCD reversal (P = .047). A higher proportion of the HU-naive group, 7 (14.3%) versus 9.8% of the HU group experienced TCD conversion. Those with initial conditional velocities in the HU-group experienced a significant reduction in TAMM velocities (from 176.8 ± 5.3 to 162.7 ± 13.9 cm/s, difference of 14.1 cm/s; P = .001) unlike those in the HU-naive group (176.3 ± 5.3 to 170.0 ± 18.6 cm/s, difference of 6.3 cm/s; P = .148). The change in the TAMM velocities was also significantly higher among the HU-group (14.1 ± 12.4 cm/s versus 6.3 ± 18.5 cm/s, P = .015). Our data suggest a beneficial role of HU in TCD velocity reduction in patients not treated with chronic transfusions, particularly among those with initial conditional TCD velocities.

Stroke Avoidance for Children in REpública Dominicana (SACRED): Protocol for a Prospective Study of Stroke Risk and Hydroxyurea Treatment in Sickle Cell Anemia

BackgroundIn the Dominican Republic, where the burden of sickle cell anemia (SCA) is high, many children lack access to routine screening and preventative care. Children with SCA are at risk for stroke, an event that leads to significant morbidity and mortality. In the United States, screening via transcranial Doppler (TCD) identifies children with SCA at highest stroke risk, allowing early intervention with blood transfusions. The need for indefinite transfusions for primary stroke prevention limits their practicality in limited-resource countries. Hydroxyurea has been shown to lower TCD velocities and to prevent conversion from conditional (170 to 199 cm/sec) to abnormal (greater than or equal to 200 cm/sec) velocities. In resource-limited settings, implementation of a TCD screening program, coupled with hydroxyurea therapy, could reduce the burden of SCA and stroke.ObjectiveThe aims of the Stroke Avoidance for Children in REpública Dominicana (SACRED) trial are (1) to screen children with SCA for stroke risk using TCD and to determine the prevalence of elevated velocities in a cross-sectional sample; (2) to identify clinical and laboratory correlates of elevated velocities; and (3) to obtain longitudinal data on the natural history of TCD velocities and to measure therapeutic effects of hydroxyurea.MethodsThis prospective trial, designed and conducted by Cincinnati Children’s Hospital Medical Center (CCHMC) and Hospital Infantil Robert Reid Cabral (HIRRC) with Centro de Obstetricia y Ginecología, includes a baseline cross-sectional epidemiological survey of the distribution of TCD velocities across a large cohort of children with SCA in the Dominican Republic. Children with conditional velocities are eligible to begin protocol-directed hydroxyurea if laboratory criteria are met. The treatment schedule begins with a fixed-dose of approximately 20 mg/kg/day for 6 months, after which it escalates to maximum tolerated dose (MTD). All participants undergo longitudinal annual TCD evaluation, while those on hydroxyurea have semi-annual evaluations during the 3-year study period. Data are collected using an Internet-based Research Electronic Data Capture (REDCap) system with forms translated into Spanish; both remote and on-site monitoring are used.ResultsTo date, 122 children with SCA have enrolled in SACRED including 85 (69.7%, 85/122) with normal, 29 (23.8%, 29/122) with conditional, 5 (4.1%, 5/122) with abnormal, and 3 (2.5%, 3/122) with inadequate TCD velocities. Of the 29 children with conditional TCD velocities, 17 (59%, 17/29) have initiated hydroxyurea per protocol, with plans for escalation to MTD.ConclusionsThe SACRED trial will provide novel epidemiologic data about the prevalence of children with SCA and increased stroke risk in the Dominican Republic. The study also includes an investigation of the impact of hydroxyurea at MTD on elevated TCD velocities, as well as clinical and laboratory parameters. The design and implementation of SACRED reflect a successful international institutional partnership, one that features local capacity building and training in research methods and clinical care. The trial’s results have important implications for screening and prevention of primary stroke in children with SCA living in resource-limited settings.Trial RegistrationClinicalTrials.gov NCT02769845; https://www.clinicaltrials.gov/ct2/show/NCT02769845 (Archived by WebCite at http://www.webcitation.org/6qf6n0Egh)

Genome Wide Association Study of Sickle Cell Disease Individuals with Stroke Risk

INTRODUCTIONSickle cell disease (SCD) is characterized by the hemoglobin S (HbS) presence associated with other hemoglobin variants, as found in the hemoglobin SC disease (HbSC), or with globin chain synthesis defects, and sickle cell anemia (SCA) is the most severe form of SCD. Among clinical manifestations in SCD it has been highlight the stroke, which is the main cause of death among patients. Recent studies have associated the presence of gene polymorphisms with the stroke risk in SCA individuals. Several genetic markers have been previously associated with stroke risk, but many of the results remain controversial once they could not completely elucidate the effect of individuals' genetic heterogeneity in the stroke development. Thus, we sought to investigate genetic polymorphisms through genome-wide association among SCD patients with stroke risk. METHODSWe selected four SCA individuals and four HbSC individuals based on the transcranial Doppler (TCD) values in according to the velocities described by Adams et al and Deane et al respectively. Patients were matched by sex and age and are followed in the Pediatric Cerebrovascular Disease Outpatient Center at the Hospital Universitario Professor Edgard Santos of the Universidade Federal da Bahia. This study was approved by the Research Board of the Secretaria de Saúde do Estado da Bahia, and all experimental protocol were performed in accordance with the ethical standards of national research committee and with the Helsinki Declaration of 1975 and its later amendments. TCD was performed in all subjects included in the study, and the highest velocity found was assessed in the middle cerebral arteries and distal intracranial internal carotid. Genomic DNA was extracted from peripheral blood and the DNA concentration was evaluated and used for SNP analysis in an Illumina Human Omni5-4 v.1.1 BeadChip kit, and raw intensities were analyzed in GenomeStudio Software. We select SNPs showing different genotypes when individuals were pairwise compared according to TCD velocities such is showed in the Figure 1. We evaluate the SNPs using SNPnexus online tool and the UCSC, SIFT and POLYPHEN categories. The next step consisted of running these selected SNPs in the NHGRI-EBI Catalog of published genome-wide association studies with p-values < 1.0 x 10-5. Additionally, we used STRING database to construct a network of protein-protein interactions. RESULTSUsing the UCSC category, we selected 3698, 3851 and 3528 unique SNPs in coding regions in non-synonymous for corresponding amino acid change for the three comparisons of SCA individuals respectively: abnormal TCD vs normal TCD, abnormal TCD vs conditional TCD and abnormal TCD vs conditional high TCD. Using the SIFT and POLYPHEN categories we selected the SNPs with a highly confident prediction to be damaging and SNPs predicted to be probably damaging, and we found 551, 505 and 598 unique SNPs and 424, 393 and 425 unique SNPs, in TCD-SCA individuals respectively. The next step was to select the common SNPs to perform Genome-wide association study (GWAS) classification. The same strategy used for TCD-SCA was performed to TCD-HbSC groups of individuals. We identified 3654, 3722 and 3847 as unique non-synonymous SNPs predicted by UCSC to be located in genomic coding regions respectively. Using the SIFT category 429, 447 and 418 unique SNPs were found in the TCD-HbSC groups of individuals respectively. PolyPhen classification was used and we found 517, 539 and 508 unique SNPs in the TCD-HbSC groups respectively. The next step was to perform GWAS classification. Results obtained utilizing the GWAS showed that each pair of individuals had a different number of SNPs identified (Table 1). The network of protein-protein interactions for the three conditions of SCA individuals and HbSC individuals are shown in the Figure 2. CONCLUSIONSWe suggest that the DOCK6 rs2278426, TYR rs1042602, CYP4F2 rs2108622, MST1 rs3197999, OR51B5/6 rs5006884, THADA rs7578597, FUT2 rs602662, MTHFR rs1801133, TSEN15 rs1046934, CFB rs12614 and ABCG5 rs6756629SNPs may be candidate variants to be further investigated in HbSC individuals with high speed of cerebral blood flow. We suggest that the SLCO1B1 rs4149056, PRIM1 rs2277339, APOB rs676210, TYK2 rs12720356, TSEN15 rs1046934, CYP4F2 rs2108622 and MST1 rs3197999 as candidate SNPs to be further investigated in SCA with high cerebral blood flow velocities. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

EXpanding Treatment for Existing Neurological Disease (EXTEND): An Open-Label Phase II Clinical Trial of Hydroxyurea Treatment in Sickle Cell Anemia.

BackgroundCerebral vasculopathy in sickle cell anemia (SCA) begins in childhood and features intracranial arterial stenosis with high risk of ischemic stroke. Stroke risk can be reduced by transcranial doppler (TCD) screening and chronic transfusion therapy; however, this approach is impractical in many developing countries. Accumulating evidence supports the use of hydroxyurea for the prevention and treatment of cerebrovascular disease in children with SCA. Recently we reported that hydroxyurea significantly reduced the conversion from conditional TCD velocities to abnormal velocities; whether hydroxyurea can be used for children with newly diagnosed severe cerebrovascular disease in place of starting transfusion therapy remains unknown.ObjectiveThe primary objective of the EXpanding Treatment for Existing Neurological Disease (EXTEND) trial is to investigate the effect of open label hydroxyurea on the maximum time-averaged mean velocity (TAMV) after 18 months of treatment compared to the pre-treatment value. Secondary objectives include the effects of hydroxyurea on serial TCD velocities, the incidence of neurological and non-neurological events, quality of life (QOL), body composition and metabolism, toxicity and treatment response, changes to brain magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA), genetic and serologic markers of disease severity, and cognitive and pulmonary function.MethodsThis prospective Phase II trial will enroll children with SCA in Jamaica, between the ages of 2 and 17 years, with either conditional (170-199 cm/sec) or abnormal (≥ 200 cm/sec) TCD velocities. Oral hydroxyurea will be administered daily and escalated to the maximum tolerated dose (MTD). Participants will be seen in the Sickle Cell Unit (SCU) in Kingston, Jamaica monthly until achieving MTD, and then every 3 months. TCD will be performed every 6 months.ResultsCurrently, 43 participants have been enrolled out of a projected 50. There was one withdrawal due to immigration, with no permanent screen failures. Of the 43 enrolled, 37 participants have initiated study treatment.ConclusionsThis trial investigates the effects of hydroxyurea treatment at MTD in children with conditional or abnormal TCD velocities before transfusion therapy and may represent an important advance towards establishing a suitable non-transfusion protocol for stroke prevention in children with SCA. The trial outcomes will have profound significance in developing countries where the disease burden is highest.ClinicalTrialClinicalTrials.gov NCT02556099; https://clinicaltrials.gov/ct2/show/NCT02556099 (Archived by WebCite at http://www.webcitation.org/6k1yMAa9G)

Early Reticulocytosis and Anemia Are Associated with Abnormal and Conditional Transcranial Doppler Velocities in Children with Sickle Cell Anemia

To improve prediction of sickle cell anemia severity at an early age, we evaluated whether absolute reticulocyte count (ARC) or hemoglobin (Hb) levels during early infancy (2-6 months of age) in patients with sickle cell anemia predict the risk of later developing an abnormal (abTCD) or conditional (cdTCD) Transcranial Doppler (TCD). We used chart review to identify 121 consecutive patients who underwent TCD screening and had steady state ARC and Hb levels recorded between 2 and 6 months of age. Cox regression analysis was used to determine the relationship between ARC, Hb levels, and risk of developing cdTCD/abTCD over time. Mean ARC in early infancy was highest and mean Hb lowest in those children with abTCDs and cdTCDs. Cox regression analysis revealed that those subjects with an ARC ≥200 K/μL in early infancy had nearly 3 times the risk of having an abTCD/cdTCD than the group with an ARC <200 K/μL, and patients with a Hb <8.5 g/dL had 2.7 times the risk of having an abTCD/cdTCD. These data suggest that both elevated ARC and low baseline Hb during early infancy are associated with an increased risk of developing a cdTCD or abTCD later in childhood.