Acute Vaso-occlusive Crisis Research Articles

Low-dose methotrexate in sickle-cell disease: a pilot study with rationale borrowed from rheumatoid arthritis

BackgroundInflammation is a major feature of sickle cell disease (SCD). Low-dose methotrexate (MTX) has long been used in chronic inflammatory diseases. This pilot study examined the MTX effect on acute vaso-occlusive pain crises (VOC) in SCD patients.MethodsFourteen adults on hydroxyurea with severe and refractory VOC received one intramuscular injection of 10 mg of MTX per week for 12 weeks. A single weekly dose of 5 mg of leucovorin was administered orally 48 h after each MTX injection. The primary outcome was reduction in number/intensity of acute pain episodes. The secondary outcomes were improvement of quality of life (QOL) and reduction of the inflammatory status.ResultsMTX did not significantly change the median VOC frequency (12 before vs 10.5 during treatment, P = 0.6240) or the median McGill pain index (45 at week 0 vs 39.5 at week 12, P = 0.9311). However, there was a decrease of ≥50% in chronic pain resulting from avascular osteonecrosis (AVN) in 5 out of 7 patients with radiologic evidence of AVN, with the perception of longer pain-free periods. There was a 44.4% median gain in physical function in the SF-36 QOL questionnaire (P = 0.0198). MTX treatment up-regulated two C-X-C motif chemokines (CXCL), CXCL10 (P = 0.0463) and CXCL12 (P < 0.0001), without significant effect on 14 additional plasma inflammatory markers. Adverse events: One individual had fever of unknown origin. Respiratory tract infections were recorded in five patients. Among the latter, one also had dengue fever and another had a central venous line infection and died of pneumonia and septic shock. Three patients with previous history of hydroxyurea-induced hematological toxicity developed low blood platelet counts while receiving simultaneously MTX and hydroxyurea.ConclusionsAlthough MTX did not reduce acute VOC frequency/intensity, it decreased chronic pain and led to QOL improvement.Trial registrationhttp://www.who.int/ictrp/en/ and http://www.ensaiosclinicos.gov.br, RBR-2s9xvn, 19 December 2016, retrospectively registered

Determining the longitudinal validity and meaningful differences in HRQL of the PedsQL\u2122 Sickle Cell Disease Module

BackgroundDetecting change in health status over time and ascertaining meaningful changes are critical elements when using health-related quality of life (HRQL) instruments to measure patient-centered outcomes. The PedsQL™ Sickle Cell Disease module, a disease specific HRQL instrument, has previously been shown to be valid and reliable. Our objectives were to determine the longitudinal validity of the PedsQL™ Sickle Cell Disease module and the change in HRQL that is meaningful to patients.MethodsAn ancillary study was conducted utilizing a multi-center prospective trial design. Children ages 4–21 years with sickle cell disease admitted to the hospital for an acute painful vaso-oclusive crisis were eligible. Children completed HRQL assessments at three time points (in the Emergency Department, one week post-discharge, and at return to baseline (One to three months post-discharge). The primary outcome was change in HRQL score. Both distribution (effect size, standard error of measurement (SEM)) and anchor (global change assessment) based methods were used to determine the longitudinal validity and meaningful change in HRQL. Changes in HRQL meaningful to patients were identified by anchoring the change scores to the patient’s perception of global improvement in pain.ResultsModerate effect sizes (0.20–0.80) were determined for all domains except the Communication I and Cognitive Fatigue domains. The value of 1 SEM varied from 3.8–14.6 across all domains. Over 50% of patients improved by at least 1 SEM in Total HRQL score. A HRQL change score of 7–10 in the pain domains represented minimal perceived improvement in HRQL and a HRQL change score of 18 or greater represented moderate to large improvement.ConclusionsThe PedsQL™ Sickle Cell Disease Module is responsive to changes in HRQL in patients experiencing acute painful vaso-occlusive crises. The study data establish longitudinal validity and meaningful change parameters for the PedsQL™ Sickle Cell Disease Module.Trial RegistrationClinicalTrials.gov (study identifier: NCT01197417). Date of registration: 08/30/2010

Increased Vasoocclusive Crises in "O" Blood Group Sickle Cell Disease Patients: Association with Underlying Thrombospondin Levels.

ObjectivesTo explore the incidence of vaso-occlusive crisis (VOC) in Blood Group “O” sickle cell disease (SCD) patients, and correlate it with the blood group and thrombospondin (TSP) levels.MethodsIn 89 consecutive SCD patients, blood samples were obtained for von Williebrand factor (vWF:Ag) antigen, collagen binding activity (CBA), ristocetin binding activity (RCo), blood group typing, C-reactive protein (CRP), high performance liquid chromatography (HPLC), Serum TSP 1 and TSP 2 levels, complete blood counts (CBC), lactic dehydrogenase (LDH) levels, liver function (LFT) and renal function tests (RFT) during VOC episodes and in steady state conditions.ResultsIn steady state SCD patients (n=72), “O” blood group patients (n=37) showed a significantly higher median serum TSP 1 and TSP 2 levels as compared to non-O blood group patients [n=35] [p <0.05, Mann-Whitney test]; with an inverse relation between vWF:Ag, Factor VIII:C and TSP levels. Furthermore, the serum TSP 1 and TSP 2 levels were significantly higher in patients presenting with acute VOC [n=17], as well as in those with repeated VOC’s (group 1, n=16), especially amongst blood group “O” patients [p, <0.05, Mann-Whitney test].ConclusionsThe study demonstrates an inverse relation between TSP and vWF levels, in blood group “O” SCD patients, with an upregulation of the TSP levels. Expectedly, during active VOC crisis, the TSP 1 and TSP 2 levels were significantly elevated.

Acute vaso-occlusive crisis in patients with sickle cell disease.

Acute vaso-occlusive crisis in patients with sickle cell disease.

The Use of Cannabis by Patients with Sickle Cell Disease Increased the Frequency of Hospitalization due to Vaso-Occlusive Crises.

Introduction: The objective of this study was to determine if patients with sickle cell disease using cannabis had decreased frequency of acute vaso-occlusive crises (VOCs) that required hospitalization.Method: This was a retrospective study in which 270 urine drug screen tests were done on 72 patients: 40 males and 32 females.Results: Cannabinoids were found in 144 urine tests from 37 patients and were negative in 126 tests from 35 patients. Males who used cannabis were significantly younger (p<0.001) than males who did not. Patients who tested positive used benzodiazepines, cocaine, and phencyclidine significantly more often than patients who tested negative. There was no significant difference in the amounts of opioids consumed by users and nonusers of cannabis. The cannabis cohort was seen in the clinic significantly (p<0.05) less often than controls, but hospital admissions were significantly greater in the cannabis group than controls (p<0.05).Conclusion: These data show an unexpected negative effect of cannabis on the frequency of VOCs. This may be due to the effect of cannabis on the brain and/or the severity of the disease in the cannabis users. More controlled studies are needed to clarify these findings.

Nucleated Red Blood Cells in Children with Sickle Cell Disease Hospitalized for Acute Vaso-Occlusive Pain

Background: Acute vaso-occlusive crises (VOC) represent the most common reason for hospitalization among children with sickle cell disease (SCD). Acute chest syndrome (ACS) and severe hemolysis, both of which may require transfusion, are common complications during hospitalizations for VOC. Despite the high morbidity associated with these complications, there are currently no reliable clinical predictors of ACS and transfusion requirement in this setting. Nucleated red blood cells (NRBC), reported as the number of NRBC/100 white blood cells (WBC) on a complete blood count (CBC), may reflect marrow necrosis or increased erythropoetic drive, yet their relationship to VOC and its complications have not been studied. We aimed to evaluate the relationship between admission NRBC and development of ACS or transfusion requirement in children with SCD hospitalized for VOC. We hypothesized that higher admission NRBC is associated with greater risk of ACS/transfusion in this population. Methods: We performed a single-institution, retrospective review of all hospitalizations from 2011 through 2015 for uncomplicated VOC in children with SCD. Hospitalization encounters were identified by ICD-9 codes for SCD and use of parenteral opioids for at least 24 hours in the electronic medical record. Data extracted from encounters meeting inclusion/exclusion criteria included all CBCs and clinical outcomes. Between-group comparisons were performed by Mann-Whitney U test and Pearson's chi-square test for continuous and categorical variables, respectively, as well as by Spearman's rank order correlation (SPSS V24). The relationship of admission and change in NRBC (∆NRBC) to complications was evaluated by binary logistic regression and adjusted for significant co-variates on bivariate analysis. For ∆NRBC, we used the final CBC obtained prior to a complication or prior to discharge for encounters not resulting in a complication. Results: We reviewed 271 encounters for uncomplicated VOC in 96 patients (mean age 13.1 years, 47% male, and 76% Hb SS or S/β0 thalassemia). Overall, 48/271 (18%) encounters for VOC resulted in ACS/transfusion, or both. Median NRBC on admission was 2 /100 WBC (range 0 to 137 /100 WBC) for all encounters. Admission NRBC was significantly correlated with age (r = 0.15, p = 0.02), % reticulocytes (r = 0.18, p &lt; 0.01) and hemoglobin (r = 0.39, p &lt; 0.001). Median admission NRBC was higher among patients on hydroxyurea (3 vs. 1.8 /100 WBC, p &lt; 0.01) but did not differ by sex or genotype. In general, median admission NRBC did not differ between hospitalizations that did or did not result in ACS/transfusion (2 vs 2.5 /100 WBC, p = 0.91). In our logistic regression model that included NRBC, % reticulocytes, WBC, hemoglobin, platelets and genotype as co-variates, only lower hemoglobin (p = 0.02) and higher WBC (p &lt; 0.001) on admission were independently associated with ACS/transfusion. A second CBC was obtained in 110/271 (41%) encounters reviewed. Median ∆NRBC was 0 /100 WBC (range -37 to 116 /100 WBC) in these encounters. ∆NRBC was significantly correlated with age (r = 0.2, p = 0.04) but not with change in other lab values. Median ∆NRBC was higher among patients with a history of hypertension (0 vs. 13 /100 WBC, p &lt; 0.01) but did not differ by hydroxyurea status, sex or genotype. Importantly, we found that median ∆NRBC was significantly higher in hospitalizations that resulted in ACS/transfusion compared to those that did not (0 vs 2 /100 WBC, p &lt; 0.01). By logistic regression, greater ∆NRBC (p &lt; 0.01) and lesser ∆platelets (p = 0.04) were independently associated with higher risk of ACS/transfusion after adjustment for ∆hemoglobin and genotype. A receiver operating characteristic curve constructed from a logistic regression model that included only ∆NRBC, admission WBC, admission hemoglobin and ∆platelets resulted in an area under the curve of 0.82. Conclusions: We found no relationship between admission NRBC and complications during hospitalization for VOC in children with SCD. Greater ∆NRBC, however, was independently associated with development of ACS/transfusion requirement during hospitalization for VOC, suggesting ∆NRBC may represent a useful biomarker for predicting complications in children with SCD hospitalized for VOC. Clinical decision rules that incorporate the careful monitoring of ∆NRBC in this setting should be prospectively studied. Disclosures No relevant conflicts of interest to declare.

Hospitalization for Acute Pain in Sickle Cell Disease: Changes in Clinical Parameters and Factors Predicting Hospital Discharge and Re-Admission

Introduction: Acute vaso-occlusive crisis (VOC) is the hallmark clinical complication of sickle cell disease (SCD). VOC involves several mechanisms, including rigid erythrocytes, adhesive blood cells, activated coagulation, activated and dysregulated endothelium, inflammation, and ischemia-reperfusion injury. Surprisingly few data document clinical and laboratory markers that change from steady state to VOC and during the hospital stay, and that predict subsequent rehospitalization. We characterized such markers in this study.Methods: Data were collected as part of a clinical trial of nitric oxide as potential treatment for VOC (DeNOVO). Participants, age ≥ 10 years requiring hospitalization for VOC, were recruited from 11 academic medical centers at the time of VOC. Length of hospital stay (LOS), change in visual analogue scale (VAS) pain score, and hospital readmission within 30 days were study outcomes. Steady state laboratory values were available in a subgroup of patients for comparison. Differences between admission and steady state laboratory measures and changes in clinical and laboratory parameters from hospital admission to discharge were analyzed by Wilcoxon signed-rank test. Multivariate linear and logistic regression analyses were used to identify the independent predictors of LOS and 30-day readmission risk.Results: 150 recruited patients had a median (interquartile range; IQR) age of 24 (17-33) years; 50% were female, and 91% had HbSS genotype. The median VAS score at time of admission was 7.7 cm which subsided to 3.3 cm at time of discharge (P <0.001). Serum lactate dehydrogenase concentrations (LDH) and neutrophils counts increased from steady state to the time of the VOC (P ≤ 0.001) and then decreased during the hospital stay (P ≤ 0.01) (Figure). The median (IQR) LOS was 3.5 (1.9-6.0) days. Longer LOS was predicted by higher admission VAS and serum alkaline phosphatase level, as well as higher increase in respiratory rate during hospital stay (P ≤ 0.006) (Table). The rate of 30-day hospital/ED readmission was 24%. Longer LOS was associated with lower risk of readmission (OR = 0.65, 95% CI: 0.50-0.86 per day). Other risk factors for readmission were higher serum creatinine at discharge (OR = 5.54, 95% CI: 1.12-27.51 per mg/dL), greater increase in serum alanine aminotransferase (OR = 1.03, 95%CI: 1.01-1.06 per unit increase) and greater cumulative opioid doses (OR = 1.25, 95%CI: 1.11-1.49 per 100 mg) during the index hospitalization.Conclusion: VOC is associated with neutrophilia and increased LDH, consistent with inflammation and hemolysis and/or tissue injury, although of insufficient magnitude to be used as diagnostic criteria. The magnitude of pain and marker of apparent bone involvement predict longer LOS. SCD patients with VOC carry a high burden of readmission risk, which is characterized by higher opioid utilization and earlier discharge from the index hospitalization. These concepts can shape models, especially to reduce readmission rate, to be evaluated in prospective clinical trials.*First two authors contributed equally to this work. [Display omitted] [Display omitted] DisclosuresLanzkron:NKT: Research Funding; Selexys: Research Funding; Prolong: Research Funding; PCORI: Research Funding; NHLBI: Research Funding; HRSA: Research Funding; GBT: Consultancy; Pfizer: Research Funding.

Neutrophil-Platelet Aggregation Enables Vaso-Occlusion in Sickle Cell Disease

Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)1, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution2. Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway. Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to prevent VOC in SS patients.

Use of Marijuana in Patients with Sickle Cell Disease Increased the Frequency of Hospitalization for Acute Painful Vaso-Occlusive Crises

Introduction : Recently, there has been general interest in using marijuana as an analgesic for various types of pain. Cannabis is known to relieve severe nausea and vomiting. Anecdotally, medical marijuana has analgesic effects in patients with chronic and neuropathic pain other than sickle cell pain, decreases the frequency of migraine headache, has anti-spastic effect in multiple sclerosis, and may relieve some of the signs and symptoms of amyotrophic lateral sclerosis and Crohn's disease. Its role in epilepsy is controversial. On the negative side long-term cannabis use is associated with periodontal disease, maternal marijuana use at 20 weeks gestation was strongly associated with spontaneous pre-term birth independent of maternal cigarette smoking and illegal synthetic cannabinoids (K2/spice) were associated with catatonia and myocardial infarction in adolescents. Studies in the transgenic sickle cell mouse showed that cannabinoid receptor-specific mechanisms ameliorate pain via inhibition of mast cell activation and neurogenic inflammation. A questionnaire study from the United Kingdom showed that 36% of the patients with sickle cell disease (SCD) have used cannabis in the previous 12 months to relieve symptoms. Another longitudinal questionnaire study in Jamaica found that marijuana was not related to the clinical severity of SCD. The objective of this study is to report the epidemiological and clinical features of the patients with SCD who were found to be taking marijuana by using random urine drug screening from 1994 to 2009. The frequency of vaso-occlusive crises (VOCs) in these patients was compared to patents with SCD whose urine drug screening showed no marijuana. The terms marijuana and cannabinoid will be used interchangeably in this Abstract. Patients &amp; Methods: A total of 270 random urine drug screen tests were done on 72 patients representing 10% of our patients with SCD during the period of the study. Written consent was obtained from the patients for enrollment in the study that was approved by the IRB. Types of SCD were determined by routine lab tests. Urine samples, collected randomly, were analyzed for the presence of amphetamine, benzodiazepines, opiates, barbiturate, cannabinoid, propoxyphene, methadone and phencyclidine. Samples were classified as either positive or negative for cannabinoid. Statistical analysis included the two-tailed student's t, Fisher exact and the Chi Square tests. Results: Thirty-seven of the 72 patients tested positive for cannabinoid in their urine and 35 patients tested negative. Table 1 shows the major epidemiological and clinical feature of the patients studied. There was no significant difference in ages of men and women who used or did not use marijuana (p &gt; 0.05). However, males who used marijuana were significantly younger (p &lt; 0.001) than males who did not (Table 1). Moreover, the ages of females who tested positive for cannabinoid were not significantly different from women who tested negative (Table 1). The patients who tested positive for cannabinoids also tested positive for other illicit drugs more often than patients who tested negative. Positivity for benzodiazepines, cocaine and phencyclidine were significantly higher in patients who used marijuana than the nonusers. Notably, there was no significant difference in the use of opioids consumed by users and non-users of marijuana. Similarly, there was no significant difference in the frequency of complications of SCD between users and non-users of marijuana (Table 1). The most important aspect of the study is the effect of marijuana use on the frequency of utilization of medical facilities. Patients in the marijuana cohort were admitted to the hospital for VOCs more frequently (Table 1) than the control group: 2,443 versus1,602 admissions respectively (p &lt; 0.05). Conclusion: Together these data suggest that patients in the marijuana cohort were more often in severe pain that required treatment in the hospital for VOCs than non-users. This difference could be due to the quality of the marijuana used, the neuronal effects of marijuana and the severity of pain in the users of marijuana. The role of marijuana in the treatment of sickle cell pain seems more complex than thought. Further controlled trials are needed to clarify this issue. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

Incidence and Risk of Delayed Hemolytic Transfusion Reaction in Sickle Cell Disease Patients Based on a Prospective Study

▪BackgroundDelayed hemolytic transfusion reaction (DHTR) is a life threatening complication of transfusion in sickle cell disease (SCD). This syndrome is underestimated because of a clinical picture that resembles a vaso-occlusive crisis (VOC) and the frequent absence of detectable antibodies. Several retrospective studies have evaluated the frequency of DHTR based on case reports. We conducted a prospective, longitudinal, single center study to determine the incidence of DHTR and the risk of developing DHTR depending on the transfusion regimen: chronic versus punctual.Methods and patientsSCD patients aged over 18 years, undergoing a transfusion, were enrolled in this study. A total of 697 transfusion episodes (TE) in 312 patients were included during 30 months. Some patients had multiple TE during the period. The post transfusion outcome of the patients was assessed up to one month after the included TE. DHTR was confirmed based on the rapid disappearance of HbA (> 50% 15 days post-transfusion) associated with two of the following criteria up to three weeks after transfusion: VOC symptoms, dark urine, worsening anemia, increased LDH. Transfusion episodes were divided into chronic (336 TE in 111 patients) and punctual (361 TE in 201 patients). Chronic transfusions were defined as regular transfusions to treat chronic complications or for primary/secondary prevention of complications. Short transfusion program during pregnancy was considered as punctual transfusions if patients were not previously regularly transfused. The study obtained approval of the local Ethics Committee.ResultsFollow-up of the patients after transfusion showed 15 DHTR during the study. They all developed in punctually transfused patients. Thus, patients who are transfused punctually have a significantly higher risk of developing DHTR than those in a regular transfusion program (p < 0.001). When considering only punctual transfusions, the incidence of DHTR is 4.2% per transfusion episode (IC 95% [2.6; 6.9]) and 7.5% per patient during the 30 months of the study (IC 95% [4.6; 12.4]). In these DHTR cases, the transfusion indication was surgery (n = 6), pregnancy (n = 3), acute chest syndrome (n = 3), stroke (n = 1), profound anemia (n = 1), and VOC prevention before a school exam (n = 1 case). Two patients died of multi-organ failure following severe intravascular hemolysis. The median hemoglobin decrease for all DHTR cases after the triggering transfusion was 4.4 g/dl [IQR 3.6-5.2]; the highest LDH level was 879 [IQR 680-1423]. Ten patients developed newly formed antibodies, but only five among them displayed antibodies with significant serological features (anti-Fya, anti-S, anti-Jka, anti-HI). In the five other cases, the antibodies were of unspecified specificity or auto antibodies in the RH blood group (the genetic RH background was known). Finally, antibodies were undetectable for five cases, confirmed by long-term patient follow up.ConclusionThis prospective study demonstrates, for the first time, that DHTR is a frequent reaction in adult SCD patients, developing only in occasionally transfused patients. This finding highlights that adult patients with regular transfusion who did not previously encountered DHTR are not susceptible to developing this severe reaction. A mechanism linked to acute situations can be suggested as already shown for the induction of allo-immunization. However, many cases developed without detectable antibodies, confirming the complex pathophysiology of this syndrome. A bio-clinical scoring system to predict DHTR, based on this study, is under development and will be presented. DisclosuresMichel:Roche: Research Funding.

Anti-Thrombin III Inhibits Adhesion of Sickle Red Blood Cells and Leukocytes to the Endothelium in a Sickle Cell Mouse Model: Potential Therapeutic Application for Vaso-Occlusive Episodes

Painful vaso-occlusive episodes are a hallmark of sickle cell disease (SCD), an inherited blood disorder caused by a point mutation in the β-globin gene. Vaso-occlusive events account for the majority of SCD-related hospital admissions and contribute importantly to major organ damage and decreased survival. Therapies to prevent and treat acute vaso-occlusive episodes are limited and there is a need for new treatment options. Adhesive interactions between circulating sickle red blood cells (sRBC), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusive episodes. These cell adhesive interactions further manifest with increased local and systemic inflammation and activated coagulation evidenced in patients and mouse models of SCD.Anti-thrombin III (ATIII) is an endogenous inhibitor of thrombin and other serine proteases in the coagulation pathway. Apart from the role in hemostasis, ATIII exerts strong anti-inflammatory properties. Plasma levels of ATIII and ATIII activity are decreased in SCD and further decline during vaso-occlusive crisis. These findings indicate that ATIII deficiency may contribute by multiple mechanisms to the pathophysiology of acute vaso-occlusion and that repletion therapy with ATIII may provide a novel, multi-targeted therapeutic approach to this difficult-to-treat complication.In this study, we examined whether ATIII supplementation is able to reduce adhesion of sRBC and leukocytes to vascular endothelial cells in the calvarial bone marrow microvasculature in a mouse knockout-transgenic mouse model of SCD (Ryan, 1997). Homozygous SCD mice were injected intravenously with 230 U/kg ATIII (Thrombate III; Grifols, USA) or vehicle (saline). After 48 hours, the mice were subjected to 3 hours of hypoxia (8% O2 in medical air) followed by 3 hours of reoxygenation at room air (21% O2). Intravital microscopy analysis of the bone marrow microcirculation was performed to monitor adhesive interactions between blood cells and vascular endothelium. Leukocytes were labeled in vivo with phycoerythrin-conjugated anti-CD45 antibody which was 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 to the recipient mice. Effects of ATIII on sRBC and leukocyte adhesive interactions were compared to vehicle. Administration of ATIII to SCD mice resulted in more than 5 fold decrease in sRBC adhesion (p<0.01) compared to vehicle. Next the effects of ATIII on leukocyte flow dynamics were analyzed quantitatively. Mice pretreated with ATIII, but not those treated with vehicle, showed two-fold decrease in leukocyte rolling flux (p<0.01) and a 3-fold decrease in adhesion (p<0.001) compared with vehicle-treated mice. Furthermore, ATIII improved blood circulation compared to vehicle-treated mice as shown by increased RBC velocity and calculated wall shear rates. The pretreatment with ATIII was associated with decreased plasma levels of IL-1β and a trend to decreased soluble levels of vascular cell adhesion molecule 1 and thrombin-antithrombin complexes.In conclusion, our study demonstrates significant anti-adhesive activities of ATIII, which reduces the adhesion of sRBC and leukocytes to the vascular endothelium in a mouse model of SCD. ATIII may represent a novel and effective therapeutic intervention in the treatment of acute vaso-occlusive crisis in patients with SCD. DisclosuresGutsaeva:Grifols: Research Funding.

Markers of endothelial dysfunction and leucocyte activation in Saudi and non-Saudi haplotypes of sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive inherited hemoglobinopathy, characterized by chronic hemolysis and recurrent vaso-occlusive crisis (VOC). This study investigates changes in leucocyte subsets and the relationship between cell adhesion molecule expression and disease manifestations in patients during steady state and acute VOC. We compared soluble E-selectin and P-selectin levels in 84 SCD patients, in steady state and during VOC to 84 healthy controls. Using immunophenotyping, we also compared lymphocyte subsets in these three groups. Further, we compared E-selectin and P-selectin levels in patients of Saudi ethnicity to non-Saudi patients, in all three groups. Lymphocyte subsets showed high percentages of total T lymphocytes, T helper and suppressor lymphocytes, B lymphocytes as well as NK cells in patients with SCD during steady state, while B lymphocytes and NK cells were significantly higher during acute VOC crisis. High levels of both soluble E-selectin (sE-selectin) and soluble P-selectin (sP-selectin) markers were demonstrated in the serum of patients with SCD during both steady state and acute VOC. Levels of selectins were significantly higher in acute VOC. The immunophenotypic expression of L-selectin, on leucocytes, was high in SCD both during steady state and during acute VOC in comparison to normal control subjects. There was no significant difference in all three study groups between Saudi and non-Saudi patients. These findings suggest that patients with SCD have increased expression of adhesion molecules: E-selectin and P-selectin, which play an important role in the pathogenesis of VOC. Despite the distinct phenotype of Saudi patients with SCD, there was no significant difference in levels of soluble E-selectin and soluble P-selectin between Saudi and non-Saudi patients in all three groups. While sickle cell disease is a well-recognized state of chronic inflammation, the role of specific adhesion molecules is steadily unraveling. Studies are underway to investigate the potential role of selectin antagonists, for prevention and reversal of acute vascular occlusions in SCD patients.

CT abdominal imaging findings in patients with sickle cell disease: acute vaso-occlusive crisis, complications, and chronic sequelae.

Sickle cell disease (SCD) is the most prevalent hemoglobinopathy. Survival in patients with SCD has improved over the past few decades. These patients experience a lifetime of repeated acute pain crises, which are thought to result from sickling and microvascular occlusions; acute abdominal pain is common. Moreover, repeated crises often lead to organ dysfunction, such as asplenia, hepatic failure, and renal failure. The spleen, liver, biliary system, kidneys, and gastrointestinal tract can all be affected. Patients may undergo CT to further direct clinical management. We review the spectrum of CT imaging findings of abdominal manifestations in patients with SCD, from the acute microvascular occlusive pain crisis to the potential complications and chronic sequelae.

Emergency Medicine Management of Sickle Cell Disease Complications: An Evidence-Based Update

BackgroundSickle cell disease (SCD) affects approximately 100,000 individuals in the United States. Due to alterations in the structural conformation of hemoglobin molecules under deoxygenated conditions, patients with SCD are predisposed to numerous sequelae, many of which require acute intervention. ObjectiveOur aim was to provide emergency physicians with an evidence-based update regarding the diagnosis and management of SCD complications. DiscussionSCD patients experience significant morbidity and mortality secondary to cerebrovascular accident, acute chest syndrome, acute vaso-occlusive pain crises, SCD-related multi-organ failure, cholecystitis, acute intrahepatic cholestasis, acute sickle hepatic crisis, acute hepatic sequestration, priapism, and renal disease. Emergency physicians must recognize acute manifestations of SCD in order to deliver timely management and determine patient disposition. ConclusionsA comprehensive review of the emergency department management of acute SCD complications is provided. Comprehensive understanding of these aspects of SCD can assist physicians in expediting patient evaluation and treatment, thus decreasing the morbidity and mortality associated with this hemoglobinopathy.

Targeting Mac-1-mediated leukocyte-RBC interactions uncouples the benefits for acute vaso-occlusion and chronic organ damage.

Vaso-occlusive crisis (VOC) is one of the most common complications of sickle cell disease (SCD). Recurrent episodes of VOC may cause irreversible organ damage and early mortality in patients with SCD. Emerging evidence suggests that VOC arises from a complex cascade that involves interactions among multiple blood and endothelial cells in the vasculature.Previous studies have identified αMβ2 integrin (Mac-1) as a critical molecule that mediatesheterotypic interactions between red blood cells (RBCs) and adherent leukocytes and promotes VOC in SCD mice. Here, we show that RBC-leukocyte interactions are significantly diminished in Mac-1-deficient SCD mice, leading to an improvement of blood flow rates and prolonged survival time in a tumor necrosis factor-alpha and surgical-trauma-induced VOC model. Mac-1-deletion, however, was not sufficient to reduce SCD-related chronic organ damage. Our results thus suggest uncoupled mechanisms between acute VOC benefits and the long-term complications of SCD that should be considered in future clinical trials.

Benefits of Simple Exchange Transfusion in Sickle Cell Disease (HbSS) with Vaso-Occlusive Crisis Not Responding to Standard Therapy

Sickle cell disease is an autosomal recessive genetic disease. Vaso occlusive crisis (VOC) is frequently seen in such patients. Painful VOC is usually recurrent, of variable severity due to many factors and its management poses important challenge in the clinical practice. Few patients do not respond to standard therapies and continue to suffer severe pain for prolonged period or land to serious life threatening situation. The red cell exchange by aphaeresis is presumed to be one efficient alternative in this situation which can reduce the level of HbS below 40% - 50%. However, it is costly and not available everywhere. Both circumstances are common in our state where incidence of sickle cell disease is quite high. In such situations simple red cell exchange i.e. removing 1 unit (350 ml) of blood manually (by phlebotomy) and replacement with one unit normal red cell is effective. All of our four cases of SCA with severe acute VOC, are not responding to standard therapy but responded efficiently to this simple red cell exchange transfusion. Our present observation may pave the way of one simple, affordable, and effective measure to reduce the pain of severe acute VOC not responding to standard therapy. Moderate reduction of HbS by 8% - 14% by simple red cell exchange transfusion was associated with relief of pain of acute VOC; a new observation was reported in all our 4 cases which need to be validated by larger controlled studies.

Resolvin D1 and Resolvin D2 Protect Against Hypoxia/Reoxygenation Induced Lung and Kidney Damage in a Sickle Cell Mouse Model of Acute Vaso-Occlusive Crisis

Sickle cell disease (SCD) is characterized by hemolytic anemia in association with acute and chronic life-threatening clinical complications. Acute vaso-occlusive crisis (VOCs) are the main cause of hospitalization for SCD patients. In VOCs, amplified inflammatory response plays a key role in acute organ damage. Pro-Resolving lipid mediators such as resolvins (Rv) accelerate resolution of acute inflammation in different models, indicating that stimulation of endogenous resolution of inflammatory processes may be an additional strategy in limiting tissue damage. Recent data suggest beneficial effects of Rvs in hypoxia/reoxygenation (H/R) related tissue injury. Here, we study the effects of Rvs on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n =6-7 animals in each group) with RvD1, 7S, 8R, 17S- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid, 17R- RvD1, 7S, 8R, 17R- trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid; RvD2, 7S, 16R, 17S-trihydroxy-docosa-4Z, 8E, 10Z, 12E, 14E, 19Z-hexaenoic acid. Mice were treated with RvD1 and RvD2 at the dose of 2.5 ug/Kg by gavage 1 hour (hr) before H/R stress (10 hrs 8% oxygen followed by 3 hrs reoxygenation), which we have used in the past to mimic acute VOCs. We found that RvD1 and RvD2 significantly reduced the H/R-induced (i) increase in neutrophil count; (ii) lung inflammatory cell infiltrate, mucus and thrombi formation; (iii) glomerular inflammatory cell infiltration, glomerular sclerosis and thrombi formation. In the lung of H/R SCD mice, RvD1 prevented the H/R induced up-regulation of (i) cytokines such as MCP2, IL-6 and ET-1; (ii) vascular endothelial activation markers (VCAM-1 and ICAM-1; (iii) cytoprotective systems such as Prx6 and HO-1. In the kidney of H/R SCD mice, RvD1 significantly reduced H/R induced expression of IL-6 and ET-1 as well as HO-1. Our data indicate that RvD1 and RvD2 modulating inflammatory responses related to H/R in SCD, protect sickle cell target organs, and foster resolution. Thus, RvD1 and RvD2 might represent a novel therapeutic approach for acute VOCs in SCD. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Purified Poloxamer-188 (vepoloxamer) on Sickle Red Blood Cell (RBC) Membrane Function Properties

Background: Sickle erythrocyte adhesion and membrane fragility contribute to vaso-occlusion and downstream tissue and organ ischemia in sickle cell disease (SCD). Vepoloxamer is an amphipathic triblock copolymer with multi-mechanistic properties believed to improve these pathophysiologic consequences, by sealing damaged cell membranes and inhibiting hydrophobic cellular adhesive interactions. Vepoloxamer reduced both acute vaso-occlusive crisis duration and total opioid analgesic requirements in previous clinical studies. A phase 3 clinical trial of vepoloxamer in acute vaso-occlusive crises is ongoing. Currently, there are no standardized clinical assays to assess membrane properties such as adhesion and fragility that vepoloxamer is believed to target. We evaluated if and to what extent vepoloxamer affected adhesion, thrombosis, and membrane fragility in individual patient samples in our standardized microfluidic flow-based whole blood assays.Methods: Blood was obtained from pediatric homozygous sickle cell patients at steady state (n=12). Patients were first characterized by measuring the extent of whole bloodadhesion to vascular cell adhesion molecule (VCAM-1) in a standardized microfluidic flow-based assay. Dynamic adhesive interactions (rolling or sliding) of whole blood and isolated leukocytes to P-selectin were assessed by measuring absolute rolling cell number, cell rolling flux (number of rolling cells per total number of flowing cells), and cell rolling velocity at 0.3dyn/cm2. Detachment assays were performed by exposing adherent cells to vepoloxamer (10 mg/mL) under physiological flow conditions. Erythrocyte membrane fragility was evaluated based on mechanical stress-induced hemolysis at 3 min (Hem3min, %).Results: Vepoloxamer reduced whole blood adhesion to VCAM-1 at 0.1 mg/mL by 18% (p = 0.0015), at 1.0 mg/mL by 69% and at 10.0 mg/mL by 79% (in both cases, p < 0.001), and to HUVECs by >50% 0.1mg/mL (p=0.003, n=10). Vepoloxamer (10mg/mL) did not significantly affect cell-rolling flux in dynamic adhesion assays to P-selectin (p = 0.51 and 0.94, isolated leukocytes and whole blood, respectively). Vepoloxamer reversed established cell adhesion by > 30% in 3 or 10 patient samples tested, although the total average difference in percent detachment did not reach statistical significance (16.3%vs 29.6%, p=0.42, n=10). Finally, vepoloxamer reduced shear-induced hemolysis compared to untreated blood samples (35.2 % vs. 44.6% control, p =0.033).Conclusion: Vepoloxamer can significantly reduce firm cell adhesion to both VCAM-1 and HUVECS under physiologic flow conditions. Shear-induced hemolysis is also reduced in a statistically significant manner, consistent with the proposed membrane protecting effects of vepoloxamer. Interestingly, P-selectin mediated rolling adhesion was not significantly affected. This may suggest that vepoloxamer may have distinct inhibitory effects to VCAM-1 vs. P-selectin. The current findings suggest that combinatory assessment of the response of adhesive properties and membrane fragility to vepoloxamer treatment may facilitate selection of patients most likely to benefit from vepoloxamer therapy. The microfluidic flow-based platform presented in this study may have the potential to predict and monitor individual patient response to anti-adhesive therapies such as vepoloxamer. There are few reliable biomarkers that objectively predict and assess vaso-occlusive potential, thus further clinical study of this microfluidic flow-based platform are warranted. DisclosuresWhite:Functional Fluidics: Employment, Equity Ownership. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment, Equity Ownership. Benjamin:Mast Therapeutics: Consultancy. Gao:Functional Fluidics: Employment, Equity Ownership. Emanuele:Mast Therapeutics: Employment.

Effects of Poloxamer 188 on Red Blood Cells Membrane Properties in Sickle Cell Disease

Introduction: Sickle cell anemia (SCA) is a severe monogenic hereditary disorder characterized by chronic hemolytic anemia and the occurrence of frequent painful vaso-occlusive crises (VOC). SCA classical physiological scheme involves hemoglobin S polymerization under hypoxic conditions, which triggers red blood cells (RBCs) sickling. Recent studies demonstrated that the degree of hemorheological alterations, such as blood hyper-viscosity, determines the risk for VOC. Moreover, sickle RBCs abnormally adhere to the vascular endothelium, triggering microvascular occlusions.Despite extensive research very few drugs are available to efficiently treat VOCs or VOC-like events. A first clinical trial was performed to test the efficacy of poloxamer 188 (P188) in a large sickle cell cohort of adults and children (Orringer et al., 2001). This study demonstrated a significant reduction of pain duration in the children treated with P188. Recently, a new phase III multi-center trial has been started to test the efficacy of this drug during acute VOC in children (Humphries et al., 2015).We conducted in vitro experiments using the commercial formulation named Kolliphor P188 (Sigma-Aldrich) to test the effects of this drug on RBCs biophysical properties of SCA patients.Methods: To measure deformability and mechanical properties of RBCs, we used ektacytometry and microfluidic device mimicking the diameters of the micro vessels. RBCs adhesion assays were performed on HMEC-1 (Human Microvascular Endothelial Cell line) using dynamic flow adhesion platform. RBCs from healthy (AA) and SCA individuals were used for the different experiments.Results: While P188 did not significantly affect blood viscosity in AA, P188 treatment decreased blood viscosity at the lowest shear rates in SCA (Fig 1A). When measured in plasma, RBC aggregation decreased with P188 in SCA patients but not in AA (Fig 1B).RBC deformability assessed by both ektacytometry (not shown) and microfluidic device (Fig 2) was not affected by P188. This is in agreement with the mode of action of P188 suggesting that it binds to hydrophobic surfaces and lowers surface tension without any changes in the organization of the cytoskeleton.We examined the effect of P188 treatment on SCA-RBCs adhesion to the endothelial HMEC-1 cell line. We observed a mean adhesion of 40 cells/ mm2 for the untreated SCA-RBCs versus 20 cells/mm2 in the case of P188 treated RBCs, i.e. a 50% decrease upon P188 treatment (Fig 3). As for RBC aggregation, our findings suggest that the binding of P188 to SCA-RBCs membranes prevent the interaction with endothelial cells. This is of particular importance in the context of SCA since increased RBC adhesiveness has been demonstrated to trigger VOC.Conclusion: In parallel of the phase III clinical trial studying the profit of P188 for sickle cell patients during VOCs, our results bring clarifications regarding its mode of action on RBCs. We show that P188 directly reduces blood viscosity, RBC aggregation and adhesiveness to endothelial cells, making this drug as a potential beneficial therapy in SCA.

Benefits of Patient-Controlled Analgesia without Continuous Infusion for the Treatment of Sickle Cell Disease Vaso-Occlusive Crisis Pain

Introduction: Sickle cell disease (SCD) is a chronic illness characterized by unpredictable episodes of pain, cumulative organ damage, and high-health care utilization rates. Intravenous opioids are considered a mainstay in the management of acute vaso-occlusive pain crisis (VOC). In 2007, van Beers and colleagues demonstrated that the use of patient-controlled analgesia (PCA) results in significant reductions in morphine consumption with equivalent response on measurements of pain and quality of life, and this is largely accepted by SCD providers. There remains controversy regarding the method of administration of opioids via PCA, with studies examining varying ratios of continuous to demand dosing. PCA with basal infusion plus demand dosing seems to be preferred by the hematology community whereas emerging data in other populations, demonstrate adequate pain control and reduced rates of opioid-related adverse events with the use of demand only approaches. In October 2012, as part of a quality improvement initiative at the Children's Hospital at Montefiore, we revised our pain management guidelines to exclude the routine use of basal infusions in our PCA protocol. In addition, pain management consultation was requested to assist in management of PCA, particularly as it related to the appropriate use of basal infusions when indicated and timely transition to oral analgesics. Short-term methadone during the inpatient stay with a short taper post discharge was utilized for improved and long-acting analgesia in patients that did not improve rapidly on the standard regimen.Methods: We conducted a retrospective analysis of patients with SCD < 21 years of age, admitted for VOC 3-7 times/year, comparing patient admissions in 2011 versus 2013, pre- and post-revision of the pain protocol as described above. Baseline variables evaluated include demographic and clinical characteristics outlined in Table 1. Primary outcomes include total opioid utilization during inpatient admission, total hours on intravenous PCA and length of stay (LOS). Secondary outcomes include rates of acute chest syndrome, hypoxia, exchange transfusion and transfer to the pediatric intensive care unit.Results: A total of 144 admissions were included in the analysis, 73 in 2011 and 72 in 2013. Baseline demographics and clinical characteristics were similar in both groups (Table 1). In 2011, 72/73 patient admissions were treated with PCA with basal infusion compared to only 1/72 patient in 2013. Total opioid utilization during inpatient admission, total PCA hours and LOS were significantly reduced in the 2013 group compared to the 2011 group with no significant difference in admit pain to discharge pain (Table 2). Furthermore, patients in the 2013 group demonstrated a significantly lower incidence in episodes of hypoxia and acute chest syndrome (Table 2)Conclusions: In conclusion, we demonstrate the feasibility and benefits of demand only PCA in the management of VOC pain. Superiority of the demand only PCA approach needs to be rigorously evaluated in a randomized prospective study.Table 1Baseline demographic and clinical characteristics by admissions:2011 (n=73)2013 (n=72)P-valueAge (years)16.3 ± 3.515.4 ± 3.30.40Gender M F39 (53.4%) 34 (46.6%)32 (44.4%) 40 (55.6%)0.62Race Black Multiracial60 (82.2%) 13 (17.8%)61 (84.7%) 11 (15.3%)0.87Ethnicity Non-Hispanic Hispanic55 (75.3%) 18 (24.7%)55 (76.4%) 17 (23.6%)0.85Genotype Hbg SS Hgb SC68 (93.2%) 5 (6.8%)59 (81.9%) 13 (18.1%)0.18Weight (kg)57.3 ± 17.654.7 ± 19.30.45Opioid tolerant Y N9 (12.3%) 64 (87.7%)3 (4.2%) 69 (95.8%)0.25Hydroxyurea Y N44 (60.3%) 29 (39.7%)33 (45.8%) 39 (54.2%)0.50Hemoglobin (g/dL)8.7 ± 1.88.6 ± 1.60.85HgbS % HgbF%77.5 ± 10.410.9 ± 6.977.7 ± 15.16.5 ± 5.60.97 0.10Table 2Comparison of primary and secondary outcomes by admissions:2011 (n=73)2013 (n=72)P-valueTotal opioid utilization during inpatient admission* (mg IV ME/kg**)13.3 ± 33.83.6 ± 3.00.0003Total PCA hours153.2 ± 103.280.0 ± 45.4<0.0001Length of stay (days)7.5 ± 5.05.5 ±7.40.002Change in pain score (Admit pain to discharge pain)5.5 ± 3.15.7 ± 3.20.82Hypoxia21/73 (28.8%)5/72 (6.9%)0.0008Acute chest syndrome16/73 (21.9%)2/72 (2.8%)0.0043*all oral and IV opioids were included**mg IV ME/kg = milligrams of intravenous morphine equivalents per kilogram DisclosuresNo relevant conflicts of interest to declare.