Vaso-occlusive Episodes In Sickle Cell Disease Research Articles

CRISPR/Cas9 System as a Promising Therapy in Thalassemia and Sickle Cell Disease: A Systematic Review of Clinical Trials.

Clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is a new gene editing tool that represents a revolution in gene therapy. This study aimed to review the clinical trials conducted to evaluate the efficacy and safety of the CRISPR/Cas9 system in treating thalassemia and sickle cell disease (SCD). We searched relevant literature using "CRISPR Cas", "thalassemia", "sickle cell" and "clinical trial" as subject terms in PubMed, Cochrane, Web of Science, and Google Scholar up to December 3rd, 2023. Following the PIO format (Patients, Intervention, Outcome), PRISMA guidelines were followed in the study selection, data extraction, and quality assessment processes. Out of 110 publications, 6 studies met our eligibility criteria with a total of 115 patients involved. CRISPR/Cas9 system was used to disrupt BCL11A gene enhancer in 4 studies and to disrupt γ-globin gene promoters in 2 studies. Patients demonstrated significant activation of fetal hemoglobin, elevated total hemoglobin, transfusion independence in thalassemia, and repression of vaso-occlusive episodes in SCD. Using CRISPR/Cas9 system to directly disrupt genes provides a safe and potential one-time functional cure for thalassemia and SCD, suggesting CRISPR/Cas9 as a potential therapeutic tool for the treatment of inherited hematological disorders.

Influence of Weather on Sickle Cell Disease Vaso-Occlusive Episodes and Acute Chest Syndrome: A Nationwide Sample Analysis.

The clinical manifestations of sickle cell disease (SCD) result in significant morbidity and healthcare costs. The effect of weather as a risk factor for the development of vaso-occlusive episodes (VOEs) has been previously studied, although with variable results. The aim of our retrospective, nationwide study was to determine the association between weather patterns and pediatric VOE and acute chest syndrome (ACS). Demographic and clinical data were obtained between 2015 and 2022 from the Pediatric Health Information System and merged with weather data from the National Climatic Data Center. We observed a higher incidence of VOE and ACS admissions during the colder months. Results also revealed a negative association between VOE admissions and average temperature, whereas a positive association was found for ACS admissions. Medical providers should continue to counsel patients with SCD to take appropriate precautions during cold weather.

Lactated Ringer vs Normal Saline Solution During Sickle Cell Vaso-Occlusive Episodes

Sickle cell disease (SCD), a clinically heterogenous genetic hemoglobinopathy, is characterized by painful vaso-occlusive episodes (VOEs) that can require hospitalization. Patients admitted with VOEs are often initially resuscitated with normal saline (NS) to improve concurrent hypovolemia, despite preclinical evidence that NS may promote erythrocyte sickling. The comparative effectiveness of alternative volume-expanding fluids (eg, lactated Ringer [LR]) for resuscitation during VOEs is unclear. To compare the effectiveness of LR to NS fluid resuscitation in patients with SCD and VOEs. This multicenter cohort study and target trial emulation included inpatient adults with SCD VOEs who received either LR or NS on hospital day 1. The Premier PINC AI database (2016-2022), a multicenter clinical database including approximately 25% of US hospitalizations was used. The analysis took place between October 6, 2023, and June 20, 2024. Receipt of LR (intervention) or NS (control) on hospital day 1. The primary outcome was hospital-free days (HFDs) by day 30. Targeted maximum likelihood estimation was used to calculate marginal effect estimates. Heterogeneity of treatment effect was explored in subgroups. A total of 55 574 patient encounters where LR (n = 3495) or NS (n = 52 079) was administered on hospital day 1 were included; the median (IQR) age was 30 (25-37) years. Patients who received LR had more HFDs compared with those who received NS (marginal mean difference, 0.4; 95% CI, 0.1-0.6 days). Patients who received LR also had shorter hospital lengths of stay (marginal mean difference, -0.4; 95% CI, -0.7 to -0.1 days) and lower risk of 30-day readmission (marginal risk difference, -5.8%; 95% CI, -9.8% to -1.8%). Differences in HFDs between LR and NS were heterogenous based on fluid volume received: among patients who received less than 2 L, there was no difference in LR vs NS; among those who received 2 or more L, LR was superior to NS. This cohort study found that, compared with NS, LR had a small but significant improvement in HFDs and secondary outcomes including 30-day readmission. These results suggest that, among patients with VOEs in whom clinicians plan to give volume resuscitation fluids on hospital admission, LR should be preferred over NS.

Comparison of Measures of Pain Intensity During Sickle Cell Disease Vaso-Occlusive Episodes

We aimed to determine the minimal clinically important difference (MCID) in pain severity and agreement between the visual analog scale (VAS) and the verbal numeric rating scale (NRS) in people with sickle cell disease (SCD) experiencing an acute vaso-occlusive episode in the emergency department. In the COMPARE-VOE trial (NCT03933397), participants were administered the VAS (0–100), NRS (0–100), and descriptor scale (a lot better, a little better, same, a little worse, much worse) every 30 minutes while in the emergency department. We analyzed data from 100 participants (mean age 30.2 years; 61% female). We calculated the mean differences and 95% confidence intervals (CIs) between current and preceding scores when the participant reported a little worse or a little better pain for each scale (255 VAS and 150 NRS observations) to assess the MCID for the VAS and NRS. Pearson correlation and the Bland-Altman method were used to assess the agreement among 411 paired VAS and NRS observations. Our results indicated that the MCID for the VAS was 8.77 mm (95% CI: 7.43 mm, 10.83 mm) and the NRS was 8.29 (95% CI: 6.47, 11.60). The VAS and NRS scales had a correlation of .88 (P < .001). The Bland-Altman method indicated a mean difference of −4.6 ± 1.96 and the 95% limits of agreement ranged from 20 to −29. Despite high correlation, there was considerable variability of agreement between the VAS and NRS scales, indicating that these scales are not interchangeable to assess pain during a vaso-occlusive event. PerspectiveThe MCID in pain severity for individuals with a SCD vaso-occlusive episode using the VAS (8.77 mm) is lower than previously reported, and the MCID for NRS was 8.29. The agreement between the VAS and NRS was determined and the scales cannot be used interchangeably to measure SCD pain intensity.

Impact of an individualized pain plan to treat sickle cell disease vaso-occlusive episodes in the emergency department

AbstractTo address acute vaso-occlusive episodes (VOEs), the leading cause of emergency department (ED) visits among individuals with sickle cell disease (SCD), we conducted the clinical study, ALIGN (An Individualized Pain Plan with Patient and Provider Access for Emergency Department care of SCD), across 8 sites. We hypothesized an improvement of 0.5 standard deviations in perceived quality of ED pain treatment of a VOE after implementing individualized pain plans (IPPs) accessible to both patients and providers. Patients with SCD were aged 18 to 45 years, owned a cell phone, and had an ED VOE visit within 90 days prior. Patients completed the perceived quality of care surveys at baseline and within 96 hours after an ED VOE visit. Providers completed surveys regarding comfort managing VOEs at baseline and after managing an enrolled patient. Most of the 153 patients were African American (95.4%), female (64.7%), and had Hb SS/Sβ0 genotype (71.9%). The perceived quality of ED pain treatment was high at both baseline and after implementation of IPPs; our primary outcome hypothesis was not met, because no statistically significant change in the patient-perceived quality of ED treatment occurred. A total of 135 providers completed baseline and follow-up surveys. On a scale of 1 to 7, with 7 being extremely comfortable managing VOEs, 60.5% reported a score ≥6 after IPP implementation vs 57.8% at baseline. Almost all (97.6%) ordered the recommended medication, and 94.7% intended to use IPPs. In this implementation protocol, all sites successfully implemented IPPs. Patients and ED providers both endorsed the use of IPPs. This trial was registered at www.ClinicalTrials.gov as # NCT04584528.

Delayed Management of Sickle Cell Disease Vaso-Occlusive Episodes and the Impact on Prolonged Hospitalizations at a Single Academic Center

Introduction: Sickle Cell Disease (SCD) affects nearly 100,000 people in the United States (Centers for Disease Control and Prevention, 2023). Vaso-occlusive episode (VOE) is the most common reason for hospitalization in SCD. Many providers are unfamiliar with the pain management timing and dosages required to adequately treat these episodes (Jang et al., J Transl Med 2021). Nationally, areas with comprehensive sickle cell centers have shown decreased hospital utilization, fewer VOEs, and improved quality of life (Oyedeji et al. JAMA 2020). Even in areas without a comprehensive center, implementing individualized pain care plans for SCD has reduced admission rates of high-utilizing patients while not impacting their pain rating scores (Welch-Coltrane et al. Pain Medicine 2021). Our academic hospital, Oregon Health and Science University (OHSU), lacks a comprehensive SCD center and VOE treatment plans are not easily accessible for these patients. We conducted a retrospective analysis on a cohort of patients with SCD to investigate the factors influencing the length of emergency department (ED) and hospital stays for VOE. We aim to identify if implementing individualized pain plans via a standardized template within the electronic health record (EHR) for these patients will decrease hospital admission, length of stay and time to ED discharge. We also aim to integrate individualized pain plans into the EHR that would allow for direct exposure to the care team by creating plans that are easily accessible, reviewed, and routinely utilized for clinical decision-making. Methods: We performed a preliminary retrospective analysis of encounters (n=107) from 2008-2023 at OHSU, where the primary diagnosis was either a VOE or an equivalent diagnosis. Based on data demonstrating earlier maximum opioid doses leading to shortened hospital stays in frequent pain patients, we recorded the timing of the first opioid and morphine milliequivalents per hour in the first 24 hours (Payne et al. Pediatr Blood Cancer 2018). Additionally, we recorded the length of stay, admission rates, time to first IV fluid, and the type of IV fluid used. We performed a linear regression analysis on the collected data with the dependent variable as the length of stay across the independent variables of time to first opioid, time to first intravenous fluid, and morphine milliequivalents per hour during the first 24 hours after presentation. Results: Eighteen patients accounted for the 107 analyzed encounters. Thirteen patients (72%) had Hgb-SS disease, 2 (11%) had Hgb-SC disease, and 3 (17%) had Hgb-S-beta thalassemia. Sixty-two admissions (58%) were ED discharges, and 45 (42%) were ED to hospital admissions. The length of stay and time to the first opioid positively correlated with p=0.005 (Figure 1a). The length of stay and time to first intravenous fluid administration was also positively correlated with p=0.01. The length of stay and morphine milliequivalents per hour in the first 24 hours were negatively correlated with p&amp;lt;0.0001 (Figure 1b). Conclusions: Longer time to administration of opioids and intravenous fluids correlated with a longer length of stay in both the ED and the hospital. Additionally, lower morphine milliequivalents in the first 24 hours of presentation were associated with a prolonged hospital stay at our institution. We hypothesize that these three factors are secondary to delays in care and inappropriate initial treatment of VOE. To address these factors, we aim to implement standardized pain plans in the EHR of our institution. Implementation of individualized pain plans, particularly in the ED, has led to the earlier initiation of opioids (Della-Moretta et al. Ann Emerg Med 2020). We anticipate similar effectiveness for timely and appropriate fluid administration and adequate opioid dosing. We plan to continue retrospective analysis and compare this data to VOE encounters post-implementation. By implementing individualized plans that are integrated within the EHR and efficiently utilized by the care team, we hope to improve VOE management at our institution with decreased length of stay, less ED to hospital admissions, and improved patient satisfaction.

Assessment of Novel Erythroid Signaling Pathways Modulating Hydroxyurea-Mediated Reduction of Vaso-Occlusive Adhesive Interactions in Early Precursors of Sickle Red Blood Cells (RBCs)

Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). In addition to fetal hemoglobin induction, hydroxyurea (HU) reduces the frequency of VOEs by increasing nitric oxide (NO) bioavailability and decreasing adhesion receptor expression and RBC-endothelial interactions. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on circulating reticulocytes (immature RBCs) and white blood cells (WBCs) during VOEs and in SCD patients with severe disease phenotypes and, decreased in response to HU therapy. Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands that are elevated in the SCD micro-environment. There is highly intriguing evidence showing HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity however specific mechanisms remain unclear. The objective of this study was to assess rapid cell signaling pathways involved in HU-mediated reduction in VLA-4 binding to endothelial vascular cell adhesion molecule-1 (VCAM-1). Whole blood (WB) was obtained from patients with SCD at benign clinic visits according to IRB-approved protocols. WB density-gradient centrifugation was used to isolate hematopoietic stem cells (HSCs) and WBCs from the buffy coat layer and, a CD71 monoclonal antibody (Ab) coupled with magnetic beads to isolate reticulocytes from the packed RBC layer. HSCs were matured to erythroblasts (EBs) in appropriate cell culture media and flow cytometric analyses assessed purity of blood samples using CD235a (exclusive WBC marker) and CD45 (exclusive RBC marker) Abs . Blood samples were pretreated with and without a therapeutically relevant hydroxyurea concentration (50mcM) followed by perfusion through micro-fluidic channels coated with VCAM-1 during pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). Additionally, EBs were treated with HU combined and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ; NO-dependent guanylate cyclase inhibitor). Adhered cells were quantified to establish an adhesion index. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value &amp;lt; 0.05 was considered statistically significant. There was a significant decrease in WB (p=0.01) and reticulocyte (p&amp;lt;0.0001) adhesion to VCAM-1 in HU-treated samples however, there was no effect on WBC (p&amp;gt;0.99) adhesion suggesting reticulocytes reduce the adhesive burden of blood cells in WB adhesion assays and modulation of VLA-4 binding differs in sickle RBCs when compared to WBCs. More recently, preliminary data from my lab demonstrated that EBs pretreated with HU reduces VLA-4 binding to VCAM-1 (from 126.5 ± 29.42 to 109.5 ± 29.09) by cGMP-dependent mechanisms (HU + ODQ; from 109.5 ± 29.09 to 120.2 ± 42.80). Laboratory-based static and flow adhesion assays have been used for decades to describe adhesion in SCD since earlier studies established that sickle RBCs are more adherent than non-sickle blood cells and, adhesion correlates with SCD severity. For example, the adhesion assay described above has been used to demonstrate that VLA-4 binding can stratify SCD patients based on disease severity and predict impending VOEs. Additionally, reticulocyte and HbF levels strongly correlate with VLA-4 binding to VCAM-1 and, VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. This is the first SCD study demonstrating the role of HU in modulating VLA-4 binding to endothelial VCAM-1 through rapid erythroid signaling pathways. There is also compelling evidence involving downregulation of VLA-4 activity/binding affinity through NO/cGMP dependent mechanisms and enhancement of VLA-4 binding by kinase-dependent pathways. HU has also been shown to modulate adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity. Collectively, these data suggest that VLA-4 mechanisms may be targeted to therapeutically inhibit vaso-occlusive adhesive events that promote VOEs in SCD.

Analysis of Volatile Organic Compounds in Exhaled Air in Patients with Sickle Cell Disease during Vaso-Occlusive Episodes

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder characterized by hemolytic anemia, inflammation and vaso-occlusion. Due to the process of vaso-occlusion patients with SCD experience unpredictable and painful vaso-occlusive episodes (VOE), causing stress and disruption of daily life. To date, we lack adequate markers to predict the development of a VOE before it occurs. In other conditions such as asthma, exhaled air analyses have shown to be of diagnostic value and may predict exacerbations. In the human body, metabolic processes generate volatile organic compounds, that are first released into the blood, and then exhaled by the lungs. As a result, exhaled air consists of several volatile, organic compounds that may reflect pathophysiological disease processes. As we presently lack urgently needed biomarkers to predict VOEs, the aim of this study was to identify distinctive volatile organic compounds in exhaled air (eVOC) during VOE in patients with SCD, that may serve as predictors of VOE when detected in low quantities as a VOE is evolving. Methods: In this longitudinal, observational single center cohort study, patients with SCD aged 6 years or older were eligible if they were hospitalized for a VOE at Amsterdam University Medical Centers between October 2021 and March 2023. Following informed consent, an exhaled air sample was taken during hospitalization, and one at least 4 weeks after discharge from the hospital (steady state). Clinical data were collected from medical files including demographics, medical history, medication use and laboratory values during steady state, and during hospitalization for VOE. Patients were instructed not to eat, drink or take medication orally 2 hours prior to the measurements. Exhaled air samples were collected in Nalophan bags, and the eVOCs were transferred to stainless steel thermal desorption tubes filled with Tenax GR for analysis by gas chromatography-mass spectrometry. The Wilcoxon signed-rank test was used to test for differences in eVOCs between VOE and steady state. Significantly different eVOCs were tentatively identified by linking raw chromatograms to corresponding metabolites based on NIST-library matching. The significance level was set to 0.05. All analyses were performed using R Studio (version 1.3.1093) software. Results: In total, 25 patients with SCD were included in this study, providing 71 exhaled air samples. The mean age at inclusion was 25.7 years (SD ±10.2). The majority of the participants had SCD genotype HbSS (60%). At time of exhaled air measurement during hospitalization, participants had symptoms for the median number of 1 day (IQR 0.5-3), and the median pain score was 6 out of 10 (IQR 3-7). A total of 58 different fragments could be identified between VOE and steady state (Wilcoxon rank p&amp;lt; 0.05), representing 26 unique eVOCs. The majority of these eVOCs were alkanes, alkenes, and esters. A representing compound for each chemical group is shown in Figure 1. In the majority of the patients, cyclohexane,1,2,4-trimethyl and propanoic acid, ethyl ester were upregulated during VOE, while 1-propene, 2-methyl- was downregulated. Conclusion: This pilot study identified 26 compounds in exhaled air that differentiated VOE from steady state in patients with SCD. Thus, analysis of exhaled air could serve as a promising, non-invasive and patient-friendly biomarker to predict VOE in SCD. Further identification of the discriminative compounds in exhaled air could provide valuable insights into the metabolic processes during VOE.

Influence of Weather Patterns and Changes on Sickle Cell Disease Vaso-Occlusive Episodes and Acute Chest Syndrome: A Nationwide Sample Analysis

Introduction: Vaso-occlusive episodes (VOE) in sickle cell disease (SCD) contribute to significant morbidity and healthcare costs due to repeated emergency department (ED) visits and hospital stays. Recurrent VOE's also often trigger serious sequelae, such as acute chest syndrome (ACS). Known risk factors for VOE include dehydration and infection. The effect of weather on the incidence of VOE has been studied in specific geographic locations with inconsistent results. Our study aims to determine the correlation between change in weather patterns and VOE nationwide using a large dataset. Methods: After IRB approval, the Pediatric Health Information System (PHIS) database was queried for SCD patients younger than 21 years of age evaluated in the ED or admitted for VOE and ACS from 2016-2021. The PHIS is a comparative database with clinical and resource utilization data for inpatient, ambulatory surgery, ED, and observation unit encounters for more than 49 children's hospitals nationwide. Patients with type HbSS, HbSC, Hb Sβ+, and Sβ(0) SCD were included. Demographic and clinical information was collected, including age, sex, race, geographic location, and inpatient length of stay. Weather information was collected via the National Climatic Data Center database. The weather variables included average temperature (Tavg), delta in temperature (TΔ), average precipitation, and average wind speed for every city in which a pediatric hospital within the PHIS database is located. Descriptive statistics were used to describe the demographics and weather characteristics. Population-averaged GEE analysis with negative binomial distribution with log link was performed with weekly counts of VOE admissions as the outcome, weather parameters as predictors, and hospitals as clusters. The weather parameters used were the averages of the daily units by week. Results from the regression analyses were reported as incidence rate ratios with 95% confidence intervals. A two-sided p-value less than 0.05 was considered as statistically significant for the analysis. Results: Of the total 79, 872 encounters included, 86% were diagnosed with VOE and 14% with ACS. Approximately, 52% of patients presented to hospitals in the southern region (Table 1). Regions (number of participating hospitals) include midwest (MW, 8), northeast (NE, 4), south (S, 11), and west (W, 5). Information from both the PHIS and weather database were available for 11,547 weeks. The south had the highest Tavg, TΔ, and precipitation. The MW had the highest wind speed and lowest Tavg. The NE had the lowest TΔ and Tavg. The west had the lowest wind speed and precipitation. The weather variables remained non-significant for ACS admissions. Overall, Tavg, TΔ, and wind speed were significant for VOE admissions while Tavg, precipitation, and wind speed were significant for VOE ED visits. Table 2 highlights the associations between overall VOE visits, weather variables, and regions. Conclusion: Our results show average temperature and delta in temperature (TΔ) is associated with the incidence of VOE admissions and ED visits. A positive association is observed in the midwest and northeast region for Tavg and negative association in the northeast region for TΔ. Future prospective studies will need to assess the effect of weather on VOE in a larger population, so specific weather-related guidelines can be developed for SCD patients and families. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

320 Trends in Acute Care of Vaso-Occlusive Episodes in the Emergency Department Following the 2014 National Heart Lung Blood Institute Guidelines

Patients presenting to the Emergency Department (ED) with sickle cell disease (SCD) vaso-occlusive episodes (VOE) encounter variability in triage acuity level designation and pain management practices. Our study aims to describe national trends in Emergency Severity Index (ESI) level designation and the rate of narcotic administration for ED patients with SCD VOE. We additionally compare both of these variables before and after publication of the 2014 National Heart Lymph Blood Institute (NHLBI) guidelines for the management of patients with VOE.

Crizanlizumab Therapy Is Associated with Lower Levels of Circulating Extracellular Vesicles in Sickle Cell Disease Patients

Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity.Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD.This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab.We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019).We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively).Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation.Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug. DisclosuresBenites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Validation of Discharge Diagnosis of Sickle Cell Disease Vaso-Occlusive Episodes in the French Hospital Electronic Database

Validation of Discharge Diagnosis of Sickle Cell Disease Vaso-Occlusive Episodes in the French Hospital Electronic Database

Managing Sickle Cell Disease Patients with Acute Pain Crisis in the Most Appropriate Setting

Introduction: Pain is the top concern of individuals with sickle cell disease (SCD). Acute painful vaso-occlusive episodes in SCD are the leading cause of emergency department (ED) encounters and frequent hospital admissions. Well-documented disparities include significant delay and under treatment of SCD patients with acute pain crisis in the ED. An acute care observation unit (ACOU) staffed with SCD specialists can help to address these disparities. SCD patients treated in a dedicated ACOU have a 40% lower admission rate than patients treated in the ED. An expedited transfer and treatment program at our dedicated sickle ACOU at the University of Illinois Hospital (UIH) was implemented with the goal of improving overall care and decreasing the hospital admission rate for SCD patients. Method: This is an outcome study of individuals with SCD &gt;16 years of age who presented with an acute painful episode to UIH. A quality improvement project used the Plan-Do-Study-Act translation method. The following key areas were identified for intervention: 1) established criteria for direct ACOU admission, 2) expedited transfer to the ACOU from the ED, 3) addition of a third provider to expand hours, and 4) establishing a consistent but individualized pain treatment plan across the ED and ACOU. The number of admissions to hospital of patients with SCD was examined from September 2018 through August 2019. Applying the Donabedian triad of Structure, Process, and Outcomes, we demonstrated improved outcome and decrease hospitalization. Results: There were 877 admissions to the ACOU from January to July of 2019, which is an increase of 37% compared to the same period in 2018. Of the 877 admissions, 793 were discharged home (90.4%) as compared to 88.6 % in 2018. The average time to first dose of opioids in the ACOU in 2019 was 55 minutes with and average decrease in the pain score of 2.62 during an average length of stay of 4:18 hours. Conclusions: Expedited care and treatment with a focus on improving quality and improving access resulted in increased volume of patients treated and decreased rate of admission to the hospital. Allocating resources to a dedicated sickle acute care observation unit can significantly decrease inpatient hospitalizations. Table. Disclosures No relevant conflicts of interest to declare.

Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM

Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.

Temperature and Humidity Effects on Acute Vaso-Occlusive Pain Episodes in Sickle Cell Disease

Background: Sickle cell patients suffer from painful vaso-occlusive episodes (VOE) that interrupt patients' lives and productivity and lead to emergency department visits and hospitalizations and, on occasion, death. The VOE in sickle cell disease (SCD) accounts for 90% of hospital admissions for patients with SCD and constitute a financial burden on both patients and hospitals. Efforts to prevent pain crises have failed to establish a causal relationship in about 40% of reported cases. The remaining 60% of known causes of VOE are related to dehydration, febrile illness, and infections. The Emory University Sickle Cell Center at Grady Memorial hospital has been providing specialized services for SCD patients for 30 years. The center includes a 24/7 acute care unit (ACU) that is staffed by SCD providers who specialize in the management of VOE. The patients are started on intravenous narcotics and fluids within 30 minutes of presenting to the ACC. After eight hours of management, the patient is then either discharged home if the VOE is controlled or admitted to the hospital for continued management. Annually, around 3000 ACU visits are recorded with a 17% hospital admission rate. When patients are questioned on the reason for their presentation, “weather change” is frequently reported as the trigger of the VOE. A review of the literature reveals scant data to support the hypothesis that weather-related changes trigger sickle cell pain crises.Methods: In a retrospective evaluation of patient visits over the last 4 years, we identified temperature and humidity measurements for that period of time and attempted to correlate them with the frequency of ACU visits. We used the “weatherspark” website that records weather changes for the city of Atlanta because > 90% of our patients reside in the city.Results: The four-year review of weather data and ACU visits did not show a direct correlation between graphs of temperature and humidity, and the number of ACU visits. The annual number of visits to the ACU was 2930, 2467, 3195 and 3370 for the years 2011, 2012, 2013 and 2014 respectively. The average admission rate was 16.6% overall, and the admission rate in the years 2011, 2012, 2013 and 2014 was 12.4%, 14.9%, 19.6% and 19.6% respectively. The attached figure has an example of the year 2013 charts for temperature, humidity, sickle cell acute care visits and hospital admission graphs.Conclusion: The retrospective analysis does not reflect a direct correlation between the temperature and humidity graphs and the number of ACU visits and hospital admissions. Given the volume of data analyzed, it is unlikely that there is any correlation between temperature and humidity variations and either acute care visits or admissions.Atlanta, GA- ACU visits, Hospital admissions, Temperature and Humidity Charts for 2013: DATA from weatherspark.com [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A-Kinase Anchoring Proteins Mediate Healthy and Sickle Red Blood Cell Adhesion To Endothelial Laminin-5

IntroductionHealthy and pathological human red blood cells (RBCs) express surface adhesion receptors which mediate interactions with the endothelium, platelets, and white blood cells. The adhesive properties of RBCs are defined by the density and distribution of adhesion receptors, and the binding force to specific ligands. In sickle cell disease (SCD), cytoadherence of RBCs to the vascular endothelium and subendothelial matrix is thought to be a major contributor to and possibly the primary cause of vasculopathy in SCD1. Painful vaso-occlusive episodes (VOEs), the hallmark of SCD, result from the blockage of small vessels. Here, we establish a molecular-level technique, based on atomic force microscopy (AFM), to measure the binding force between a specific ligand and its corresponding receptor on a single RBC. Using AFM, we can compare the frequency and localization of adhesion receptors present on the membrane of RBCs collected from healthy human volunteers and SCD patients. We focused on the interaction between basal cell adhesion molecule and its isoform Lutheran (BCAM/Lu) on the RBC and the subendothelial matrix protein laminin-5. Sickle RBCs (SS RBCs) overexpress BCAM/Lu and are more adherent to laminin-5 than healthy RBCs. Importantly, by using a flow adhesion assay, Hines et al2 demonstrated that the interaction between BCAM/Lu and laminin-5 is mediated by cyclic adenosine monophosphate (cAMP) which activates protein kinase A (PKA) and BCAM/Lu receptors. Because PKA binding can be mediated by A-kinase anchoring proteins (AKAPs), which sequester PKA to subcellular sites3, here we investigate whether AKAPs play a role in BCAM/Lu receptor activation on RBCs from healthy volunteers and SCD patients. MethodsPeripheral blood RBCs were collected from consenting healthy volunteers (n=5) and individuals with SCD (n=4). RBCs were separated from whole blood using centrifugation. AFM was employed to quantify the frequency of active BCAM/Lu receptors on a single RBC. Molecular interactions were quantified by recording force v. distance curves between a laminin-5 functionalized probe and a 1μm2 area of the RBC surface with a lateral resolution of 30nm. Detected binding forces represent adhesive events between BCAM/Lu and laminin-5, which translate to the percentage of active BCAM/Lu receptors on the RBC surface. To detect the presence of AKAPs, RBCs were treated with St-Ht31 inhibitor peptide (83nM bath), which inhibits the interaction between the regulatory subunits of PKA and AKAP in cells. As a negative control, RBCs were treated with St-Ht31P control peptide (83nM bath), which is the inactive form of the St-Ht31 inhibitor peptide. ResultsOur experiments reveal that following treatment with St-Ht31, the frequency of active BCAM/Lu receptors is significantly lower on healthy RBCs (from 5.71±1.23% to 3.15±1.09%; p<0.05) and even more in SS RBCs (from 7.19±1.42% to 2.02±0.37%; p<0.0001). Treatment with St-Ht31P control peptide did not change the frequency of active BCAM/Lu receptors on healthy RBCs (5.92±1.09%) or SS RBCs (6.49±1.27%) as compared to baseline measurements. ConclusionsImportantly, using St-Ht31, we confirm the presence of AKAPs as scaffold proteins which can modulate the adhesion of BCAM/Lu adhesion receptors on the SS RBC to laminin-5. PKA binding can be mediated by AKAPs, which sequester PKA to specific subcellular sites through binding to regulatory subunits3. Each AKAP contains two classes of binding sites: an anchoring motif, which binds the regulatory subunit of PKA, and a targeting domain, which directs the subcellular localization of the PKA-AKAP complex through association with structural proteins, membranes, or other components. This finding provides evidence for a new paradigm in AKAP adhesive signaling in RBCs and a potential new pharmacologic target for the prevention and treatment of VOEs in SCD.

Effects of GMI-1070, a Pan-Selectin Inhibitor, on Leukocyte Adhesion In Sickle Cell Disease: Results From a Phase 1/2 Study

AbstractAbstract 262Adhesion molecules are critically involved in the pathophysiology of sickle cell disease (SCD). A growing body of evidence from animal models and humans supports the role of selectin-mediated cell adhesion in the pathophysiology of vaso-occlusion. Leukocyte adhesion has been demonstrated to reduce microvascular blood flow in a sickle cell mouse model, and there is evidence to suggest leukocyte adhesion in humans contributes to vaso-occlusion (Stuart et al, Lancet 2004; Turhan et al, PNAS 2002). GMI-1070 is a pan-selectin inhibitor that targets E-, P-, and L-selectins and has previously been shown to restore blood flow and improve survival in a mouse model of vaso-occlusion (Chang et al, Blood 2010). Also, it is potent inhibitor of adhesion of human neutrophils to immobilized E-selectin and ICAM-1 under flow conditions in vitro. As part of a Phase 1/2 study of GMI-1070 in patients with SCD we determined the effects of the drug on biomarkers of adhesion in vivo and ex vivo. Methods: An open-label phase 1/2 study of the safety, PK, and activity of GMI-1070 was performed, enrolling adults with SCD at steady state. Here we are reporting data on GMI-1070 anti-adhesion activity. GMI-1070 was administered in two IV doses given on the same day: 20 mg/kg as a loading dose, followed 10 hours later by 10 mg/kg. Serial WBC count with differential (all 15 subjects), computer-assisted intravital microscopy (CAIM) (4 subjects), and ex vivo activity of GMI-1070 in plasma (4 subjects) were measured. CAIM is a non-invasive technique for quantitative measurement of microvascular blood flow in vessels of the bulbar conjunctiva. In this study it was used to measure RBC velocity before and after dosing (at 30 minutes, 2, 4, 8, and 24 hours) with GMI-1070. Ex vivo evaluation of plasma GMI-1070 activity in a cell adhesion assay was also performed, with samples taken at 0, 8, 24, and 48 hours after first infusion. Results: Adults were enrolled at three centers: 13 with HbSS, 2 with HbSB0thal. All were African-American, and 9 were male. All subjects received both doses of study drug. The t1/2 was 7.7 hours. Mean baseline WBC was 10 K/mm3, and baseline absolute neutrophil count (ANC) was 5.4 K/mm3. The mean WBC was 10.4, 11.6, 10.2, and 8.7 K/mm3 at 8, 24, 48 hours and 7 days, respectively. The ANC mean was 5.5, 7.5, 5.6, and 4.2 K/mm3 at the same time points. ANC % change from baseline was significant at 24 and 48 hours (p=0.001, 0.025 mixed effects model) (Figure). There were no significant changes in absolute monocyte or lymphocyte counts. There was no correlation with any clinical adverse events. In one subject, the WBC rose from 10.4 to a peak of 28, with no clinical symptoms or significant changes in other lab values. CAIM (n=4) evaluating microvascular blood flow in the bulbar conjunctiva (measured in screen pixels/sec), showed mean RBC velocity at baseline was 335 (SD 70) pixels/sec, with mean values at 30 min., 2, 4, and 8 hours of 368 (59), 345 (63), 341 (59), and 338 (83) pixels/sec respectively, and returned to baseline of 317 (82) pixels/sec at 24 h. These differences did not reach statistical significance (mixed effects model). In ex vivo evaluation of neutrophil adhesion to matrix proteins (as quantified by # of bound neutrophils per 50× field) from samples 0, 4, 8, 24, and 48 hours after the first infusion revealed reduction in adhesions at 4 and 8 hours; mean adhered neutrophils were 33 (17), 20 (11), 18 (9), 47 (49), and 42 (15) respectively. However, these differences did not reach statistical significance. In conclusion, GMI-1070 infusion resulted in neutrophilia, a trend towards increased RBC velocity in the vessels of the bulbar conjunctiva immediately after infusion, and reduced leukoctye adhesion in an ex vivo assay despite neutrophilia. All of these findings are consistent with an anti-adhesive effect on leukocyte adhesion in vivo and suggest that the findings in sickle mouse studies can be translated into SCD patients. This study supports further evaluation of GMI-1070 for the treatment of vaso-occlusive episodes in SCD. [Display omitted] Disclosures:Wun:GlycoMimetics: Clinical Trial Sponsorship, Consultancy; Eli Lilly: Clinical Trial Sponsorship, Consultancy. Off Label Use: This drug (GMI-1070) has not been approved for any clinical indication. De Castro:GlycoMimetics: clinical trial sponsorship. Styles:GlycoMimetics: Clinical Trial Sponsorship, Consultancy. Cheung:GlycoMimetics: clinical trial sponsorship. Chase:GlycoMimetics: clinical trial sponsorship. Simon:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership.

Single-Stranded Oligonucleotide Binding to P-Selectin Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to Endothelial Cells in Sickle Cell Model Mice: Novel Therapeutics for Vaso-Occlusive Episodes.

Abstract 1545Poster Board I-568To date, a variety of therapeutic approaches to sickle cell disease (SCD) have been explored, however, knowledge about the efficacy of anti-adhesive agents in this disorder is limited. In SCD, P-selectin expressed on endothelial cells plays a key role in leukocyte recruitment as well as in the adhesion of sickle red blood cells (sRBC) to the endothelium. The interaction of P-selectin and its ligands is thus likely to contribute to impairment of the microvascular flow and thereby to the development of painful vaso-occlusive episodes. Studies with anti-P-selectin antibody and P-selectin-deficient mice support the notion that P-selectin-directed interventions may be a potential approach to the treatment of vaso-occlusive episodes in SCD. Aptamers, short single-stranded oligonucleotides, have been developed for a wide range of therapeutic targets (Keefe & Schaub, 2008). Aptamers bind molecular targets with high affinity and specificity, do not elicit immune responses, and can be readily delivered to the vascular compartment intravenously or subcutaneously. Although anti-P-selectin aptamers have been shown to inhibit leukocyte rolling in vitro and to display efficacy in mouse models for inflammation, the anti-adhesion activity of anti-P-selectin aptamers has never been evaluated in SCD. The purpose of this study was to determine in vivo whether the anti-P-selectin aptamer ARC5690 can inhibit adhesion of sickle RBC and leukocytes to vascular endothelial cells in the bone marrow microvasculature of SCD model mice. Knockout-transgenic SCD mice generated by Ryan et al. (Ryan et al., 1997) were used in this study. SCD mice were injected with 20 mg/kg ARC5690, scrambled aptamer ARC5694 (negative control), or vehicle (saline). Anti-P-selectin monoclonal antibody was also used as a positive control. After 2.5 hours, the mice were subjected to 1 hour of hypoxia (12% O2 in air) followed by 1 hour of reoxygenation at room air. Intravital observations of the bone marrow microcirculation were performed to monitor adhesive interactions between blood cells and endothelial cells. Leukocytes were labeled in vivo with rat anti mouse CD45 antibody conjugated with PE which were infused via the carotid artery. Sickle RBC were obtained from donor mice, fluorescently labeled in vitro with 2,7-bis-(carboxyethyl)-5-(and-6) carboxyfluorescein, and infused through the carotid artery. Effects of ARC5690 on sRBC and leukocyte adhesive interactions were compared to those of ARC5694 and vehicle. Administration of the anti-P-selectin aptamer ARC5690 significantly reduced sRBC adhesion (p<0.05) in SCD mice compared to those treated with vehicle. The anti-adhesive activity of ARC5690 for sRBC was comparable to anti-P-selectin monoclonal antibody, whereas the scrambled aptamer did not show any anti-adhesive activity. We also studied the effects of the anti-P-selectin-specific ARC5690 on leukocyte flow dynamics. Mice pretreated with ARC5690, but not those treated with scrambled aptamer ARC5694, showed a greater than four-fold decrease in leukocyte rolling (p<0.001) and a six-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Again, the anti-adhesive activity of ARC5690 and its effect on leukocyte rolling and adhesion were similar to those of the anti-P-selectin antibody. Furthermore, ARC5690 improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and d wall shear rates. In conclusion, our study demonstrates significant anti-adhesive activities of ARC5690, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in SCD model mice. ARC5690 may represent a novel therapeutic strategy that can be used to treat vaso-occlusive episodes in SCD DisclosuresGutsaeva:Archemix Corporation: Research Funding. Parkerson:Archemix Corporation: Research Funding. Schaub:Archemix: Research Funding. Kurz:Archemix Corporation: Employment. Head:Archemix Corporation: Research Funding.